i 200808833 九、發明說明： 本申請案宣稱2001年1 〇月11日申請之美國臨時申請 案弟60/328,6245虎的盈處’將其完整地以引用的方气 併入本文中。 【發明所屬之技術領域】 本發明係關於認得並與血管生成素_2 (Ang-2)結合的專 一結合劑，更特定而言，本發明係關於該專一結合劑及其 片段的產製、診斷用途和治療用途，其專一地與結 合。 【先前技術】 血管生成作用，從現存的血管中形成新的血管，對於許 多生理學和病理學過程而言是必要的。通常，血管生成作 用是緊密地由前-和抗-血管生成之因素調節，但在疾病的 情況下，像是癌症、眼睛的新生血管疾病、關節炎和牛皮 癬，該過程可能錯誤地進行。Folkman, L，Nat. Med.，1: 27-31 (1995)。 已知許多疾病與取消管制或不想要的血管生成作用有關 。這些疾病包括’但不限於眼睛的新血管形成，像是視網 膜病（包括糖尿病性的視網膜病）、與年齡有關的黃斑變性 、牛皮癬、血管母細胞瘤、血管瘤、動脈粥樣硬化、炎症 疾病像疋類風濕或風濕性的炎症疾病，尤其是關節炎（ 包括風濕性關節炎），或其他的慢性炎症疾病，像是慢性 氣喘、動脈或移植後的動脈粥樣硬化、子宮内膜異位症和i 200808833 IX. INSTRUCTIONS: This application claims that the US provisional application filed on January 11th, 2001, is filed in the text of the 60/328, 6245 Tigers' case. TECHNICAL FIELD OF THE INVENTION The present invention relates to a specific binding agent that recognizes and binds to angiopoietin-2 (Ang-2), and more particularly, the present invention relates to the production of the specific binding agent and its fragment, Diagnostic use and therapeutic use, which are specifically combined with. [Prior Art] Angiogenesis, the formation of new blood vessels from existing blood vessels, is essential for many physiological and pathological processes. Generally, angiogenesis is closely regulated by pre- and anti-angiogenic factors, but in the case of diseases such as cancer, neovascular disease of the eye, arthritis and psoriasis, the process may be performed erroneously. Folkman, L, Nat. Med., 1: 27-31 (1995). Many diseases are known to be associated with deregulated or unwanted angiogenic effects. These diseases include, but are not limited to, neovascularization of the eye, such as retinopathy (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, atherosclerosis, inflammatory disease An inflammatory disease such as rheumatoid or rheumatoid arthritis, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory diseases such as chronic asthma, arterial or post-transplant atherosclerosis, endometriosis And
O:\121\121929.DOC 200808833 贅生物的疾病，例如 田 瘤(像是白血、广“ 腫瘤和液體(或造血的)腫 (像疋病和淋巴瘤）。其他與不想要之血管生成作用 有關的疾病，對熟諸此藝者而言將是明顯的。 雖然血管生成作用的 ^ ^ . 用的凋即，已經涉及許多信號轉導系統 ，但最具特色和最主亜沾& | 芏要的内皮細胞-選擇系統，涉及Tie_2 受體赂胺酸激酶（稱為，iTie_2 "或"Tie_2R，，（亦稱為"〇RK")，· 亦將老鼠的Tie-2稱為"tek”)及其配體’血管生成素(Gaie， N.W.^Yancopoulos, G.D., Genes Dev. 13: l〇55- 1066 [1999])。有4個已知@血管生成素；血管生成素](”Ang_ 1")到血管生成素-4 ("Ang_4")。亦將這些血管生成素稱為O:\121\121929.DOC 200808833 Diseases of neoplasms, such as tumors (like white blood, wide tumors and fluid (or hematopoietic) tumors (like rickets and lymphomas). Other and unwanted angiogenesis The relevant diseases will be obvious to those who are familiar with this art. Although the use of angiogenesis is already involved, many signal transduction systems have been involved, but the most characteristic and most important 亜 && A desirable endothelial cell-selection system involving the Tie_2 receptor glycosyl kinase (called iTie_2 " or "Tie_2R, (also known as "〇RK"), · also refers to the mouse Tie-2 ""tek") and its ligand 'Angiopoietin (Gaie, NW^Yancopoulos, GD, Genes Dev. 13: l〇55-1066 [1999]). There are 4 known @angiogenin; angiogenesis素]("Ang_ 1") to Angiopoietin-4 ("Ang_4"). These angiogenins are also called
Tie-2配體”。（Davis，S·等人，Cell，87: 1161-1169 [1996] ，Grosios，K·等人 ’ Cytogenet Cell Genet，84: 118-120 [1999] ’ Holash，J.等人，investigative OphUmoiogy & Visual Science，42·· 1617-1625 [1999] ; Koblizek，T.I·等人 ，Current Biology，8:529-532 [1998] ; Lin，P.等人，ProcTie-2 ligand". (Davis, S. et al., Cell, 87: 1161-1169 [1996], Grosios, K. et al. 'Cytogenet Cell Genet, 84: 118-120 [1999] ' Holash, J. Et al, investigative OphUmoiogy & Visual Science, 42·1617-1625 [1999] ; Koblizek, TI et al., Current Biology, 8: 529-532 [1998]; Lin, P. et al., Proc
Natl Acad Sci USA，95: 8829-8834 [1998] ; Maisonpierre， P.C·等人，Science，277: 55-60 [1997] ; Papapetropoulos， A.等人，Lab Invest，79:213-223 [1999] ; Sato, Τ·Ν·等人， Nature，375: 70-74 [1998] ; Shyu，K.G.等人，Circulation， 98: 2081-2087 [1998] ; Suri，C·等人，Cell，87: 1171-1180 [1996] ; Suri，C.等人，Science，282: 468-471 [1998];Natl Acad Sci USA, 95: 8829-8834 [1998] ; Maisonpierre, PC et al., Science, 277: 55-60 [1997]; Papapetropoulos, A. et al., Lab Invest, 79: 213-223 [1999] Sato, Τ·Ν· et al., Nature, 375: 70-74 [1998] ; Shyu, KG et al., Circulation, 98: 2081-2087 [1998] ; Suri, C. et al., Cell, 87: 1171 -1180 [1996] ; Suri, C. et al., Science, 282: 468-471 [1998];
Valenzuela, D.M.等人，Proceedings of the National Academy of Sciences of the USA, 96: 1904-1909 [1999] > Witzenbichler，B.等人 ’ J Biol Chem，273: 18514-18521Valenzuela, D.M., et al., Proceedings of the National Academy of Sciences of the USA, 96: 1904-1909 [1999] > Witzenbichler, B. et al. 'J Biol Chem, 273: 18514-18521
O:\121\121929.DOC 200808833 [1998] )。然而，Ang-l與Tie-2結合，在培養的内皮細胞中 ，刺激受體的填酸化作用，已經觀察到Ang-2激動並拮抗O:\121\121929.DOC 200808833 [1998]). However, Ang-1 binds to Tie-2, and in the cultured endothelial cells, stimulates the filling of the receptor, and Ang-2 has been observed to agonize and antagonize.
Tie_2受體的磷酸化作用（Davis，S.等人，[1996]，在前； Maisonpierre，P.C.等人，[1997]，在前；Kim，I.，J.H. Kim， 等人，〇11〇〇£6116 19(39):4549-4552 (2000);丁61(：]161：1>Phosphorylation of the Tie_2 receptor (Davis, S. et al., [1996], supra; Maisonpierre, PC et al., [1997], supra; Kim, I., JH Kim, et al., 〇11〇〇 £6116 19(39): 4549-4552 (2000); Ding 61(:]161:1>
Kuliszewska，K·，P.C. Maisonpierre等人，Cardiovascular Research 49(3): 659-70 (2001)) 〇 老鼠Tie-2和Ang-1基因剔除的表現型是類似的，並提出 Ang-Ι刺激之Tie-2磷酸化作用，經由維持内皮細胞-支持細 胞的附著，傳達了在子宮中正在發育之血管的改造和穩定 化作用（Dumont，D.J·#、，Genes&Development，8:1897· 1909 [1994] ; Sato, Τ·Ν·等人，Nature，376: 70-74 [1995]; Suri，C.等人，[1996]，在前）。認為在成年時仍保留Ang-1 在血管穩定化作用中的角色，在那裏被廣泛地並在組成上 表現（Hanahan，D.等人，Science，277: 48-50 [1997];Kuliszewska, K., PC Maisonpierre et al., Cardiovascular Research 49(3): 659-70 (2001)) The phenotypes of Tie-2 and Ang-1 gene knockout in scorpion mice are similar, and Ang-Ι stimulation Tie is proposed. -2 phosphorylation, through the maintenance of endothelial cell-supporting cell attachment, conveys the transformation and stabilization of the developing blood vessels in the uterus (Dumont, DJ·#,, Genes & Development, 8:1897·1909 [1994 Sato, Τ·Ν· et al., Nature, 376: 70-74 [1995]; Suri, C. et al., [1996], supra). It is believed that the role of Ang-1 in vascular stabilization remains in adulthood, where it is widely and compositionally expressed (Hanahan, D. et al., Science, 277: 48-50 [1997];
Zagzag，D.等人，Experimental Neurology, 159: 391-400 [1999] )。相反的，Ang_2的表現主要限於血管改造的地方 ，認為它在那裏阻斷了 Ang-1的功能，藉此誘導有助於血 管生成作用之血管可塑性的狀態（Hanahan，D·等人’ [1997] ，在前；Holash，J.等人，Science，284: 1994-1998 [1999] ;Maisonpierre，P.C·等人，[1997]，在前）。 許多已發表的研究，可能已經證實血管-選擇性Ang-2在 疾病狀態中的表現，與血管生成作用有關。這些病理學的 狀況包括，例如牛皮癬、黃斑變性和癌症（Bunone，G.等人Zagzag, D. et al., Experimental Neurology, 159: 391-400 [1999]). Conversely, the performance of Ang_2 is mainly limited to the area of vascular modification, where it is thought to block the function of Ang-1, thereby inducing the state of vascular plasticity that contributes to angiogenesis (Hanahan, D. et al. [1997] ], in the front; Holash, J. et al., Science, 284: 1994-1998 [1999]; Maisonpierre, PC et al., [1997], former). Many published studies may have demonstrated the performance of vascular-selective Ang-2 in disease states and are associated with angiogenesis. These pathological conditions include, for example, psoriasis, macular degeneration, and cancer (Bunone, G. et al.
O:\121\121929.DOC 200808833 ，American Journal of Pathology，1 5 5: 1967-1976 [1999]； Etoh，Τ·等人，Cancer Research，61: 2145-2153 [2001]; Hangai，M·等人，Investigative Ophthalmology & Visual Science，42: 1617-1625 [2001] ; Holash，J.等人，[1999]， 在前 ；Kuroda， K.等人， Journal of Investigative Dermatology，1 16: 713-720 [2001] ; Otani，A·等人，O:\121\121929.DOC 200808833, American Journal of Pathology, 1 5 5: 1967-1976 [1999]; Etoh, Τ· et al., Cancer Research, 61: 2145-2153 [2001]; Hangai, M. et al. Investigative Ophthalmology & Visual Science, 42: 1617-1625 [2001] ; Holash, J. et al., [1999], supra; Kuroda, K. et al., Journal of Investigative Dermatology, 1 16: 713-720 [2001] ; Otani, A· et al.
Investigative Ophthalmology & Visual Science, 40: 1912-1920 [1999] ; Stratmann，A.等人，American Journal of Pathology，153: 1459-1466 [1998] ; Tanaka，S.等人，J Clin Invest， 103: 34-345 [1999] ; Yoshida， Y.等人， International Journal of Oncology，15: 1221-1225 [1999]; Yuan，K.等人，Journal of Periodontal Research，35: 165-171[2000] ; Zagzag，D·等人，[1999]，在前）。這些研究中 ，大多數集中於癌症，其中許多腫瘤類型似乎展現血管的 Ang-2表現。與其在病理學之血管生成作用中的表現相反 ，Ang_2在正常組織中的表現是極度受限的（Maisonpierre， P.C.等人，[1997]，在前；Mezquita，J.等人，Biochemical and Biophysical Research Communications, 260: 492-498 [1999])。在正常的成人中，三個主要的血管生成作用位置 為卵巢、胎盤和子宮；在已經在其中檢測到Ang-2 mRNA 的正常（也就是非-癌性的）組織中，這些是主要的組織。 某些功能性的研究提出Ang-2可能涉及腫瘤的血管生成 作用。Ahmad 等人（Cancer Res·，61: 1255-1259 [2001])描述 Ang-2過度表現，並顯示其在老鼠異種移植的模式中，可 O:\121\121929.DOC -9- 200808833 能與在腫瘤生長上的增加有關。亦參見Etoh等人，在前和 Tanaka等人，在前，其中提交的資料，可能使Ang-2過度 表現與腫瘤血管過多化發生關連。然而，相反的，Yu等人 (Am. J· Path·，158: 563-570 [2001])報告的資料則顯示 Ang-2在Lewis肺癌和TA3乳癌細胞中的過度表現，可能延長注 射相對應之轉移感染物（transfectant)之老鼠的存活。 在過去數年中，各種出版物建議以Ang-1、Ang-2及/或 Tie-2作為抗癌治療的可能標靶。例如，美國專利第 6，166，185 號、5,650,490 號和 5,814,464 號，分別揭示抗-Tie-2配體抗體和受體本體的觀念。Lin等人（Proc. Natl. Acad· Sci USA，95: 8 829-8 834 [1998])將腺病毒表現的可溶 性Tie-2注射到老鼠内；可溶性Tie_2可能降低了老鼠發展 之腫瘤的數目和尺寸。在相關的研究中，Lin等人（j· Clin.Investigative Ophthalmology & Visual Science, 40: 1912-1920 [1999] ; Stratmann, A. et al., American Journal of Pathology, 153: 1459-1466 [1998] ; Tanaka, S. et al., J Clin Invest, 103: 34-345 [1999] ; Yoshida, Y. et al., International Journal of Oncology, 15: 1221-1225 [1999]; Yuan, K. et al., Journal of Periodontal Research, 35: 165-171 [2000]; Zagzag , D. et al., [1999], former). Most of these studies focus on cancer, and many of these tumor types appear to exhibit Ang-2 manifestations of blood vessels. Contrary to its performance in pathological angiogenesis, the performance of Ang_2 in normal tissues is extremely limited (Maisonpierre, PC et al, [1997], prior; Mezquita, J. et al., Biochemical and Biophysical Research Communications, 260: 492-498 [1999]). In normal adults, the three major angiogenic sites are the ovary, placenta, and uterus; these are the major tissues in normal (ie, non-cancerous) tissues in which Ang-2 mRNA has been detected. . Some functional studies suggest that Ang-2 may be involved in tumor angiogenesis. Ahmad et al. (Cancer Res., 61: 1255-1259 [2001]) describe the overexpression of Ang-2 and show that it can be used in mouse xenograft mode, O:\121\121929.DOC -9- 200808833 It is related to an increase in tumor growth. See also Etoh et al., formerly and Tanaka et al., previously submitted information that may correlate Ang-2 overexpression with tumor vascular hyperplasia. However, on the contrary, the information reported by Yu et al. (Am. J. Path., 158: 563-570 [2001]) shows that Ang-2 is overexpressed in Lewis lung cancer and TA3 breast cancer cells, possibly prolonging the injection. The survival of mice that transferred transfectants. In the past few years, various publications have suggested Ang-1, Ang-2 and/or Tie-2 as potential targets for anticancer therapy. For example, U.S. Patent Nos. 6,166,185, 5,650,490 and 5,814,464 disclose the concept of anti-Tie-2 ligand antibodies and receptor bodies, respectively. Lin et al. (Proc. Natl. Acad. Sci USA, 95: 8 829-8 834 [1998]) injected soluble Tie-2 expressed by adenovirus into mice; soluble Tie_2 may reduce the number of tumors developed in mice and size. In related research, Lin et al. (j. Clin.
Invest·，100: 2072-2078 [1997])將 Tie-2 的可溶形式注射到 大鼠内；該化合物可能降低了大鼠中腫瘤的尺寸。Invest·, 100: 2072-2078 [1997]) Injects a soluble form of Tie-2 into rats; this compound may reduce the size of the tumor in rats.
Siemeister等人（Cancer Res·，59: 3 185-3 189 [1999])產製表 現Tie_2之細胞外功能部位的人類黑色素瘤細胞株，將這些 細胞株注射到裸鼠内，並認定可溶性的Tie_2可能導致腫瘤 生長和腫瘤血管生成的”明顯抑制作用”。回顧該資訊，並 得到Ang-1和Ang-2兩者均與Tie-2結合，從這些研究中， 不清楚Ang-1、Ang-2或Tie_2是否將是抗-癌治療之有吸引 力的標把。 已經在例如2000年5月4曰發表之pct出版物WO 00/24782 中，描述了將某些肽與穩定的血漿蛋白質y像是Ig恆定區Siemeister et al. (Cancer Res., 59: 3 185-3 189 [1999]) produced human melanoma cell lines exhibiting extracellular functional sites of Tie_2, which were injected into nude mice and identified as soluble Tie_2. It may cause "significant inhibition" of tumor growth and tumor angiogenesis. Reviewing this information and obtaining both Ang-1 and Ang-2 binding to Tie-2, from these studies, it is not clear whether Ang-1, Ang-2 or Tie_2 will be attractive for anti-cancer therapy. Header. It has been described, for example, in the pct publication WO 00/24782, published May 4, 2000, that certain peptides and stable plasma proteins y are Ig constant regions.
O:\121\121929.DOC -10 - 200808833 融合，而改善這些分子的半衰期。 已經多方面地描述了蛋白質或其片段與穩定之血漿蛋白 質’像是ig恒定區的融合，改善了這些分子的半衰期（參見 ’例如美國專利第5,480,981號；Zheng等人，j. Immun〇1.， 154: 5590-5600，（1995) ; Fisher等人，Ν· Engl· J. Med.， 334: 1697-1702,（1996) ; Van Zee，Κ·等人，j· Immun〇1.， 156: 2221-2230,（1996) ; 1998年9月15日發證的美國專利 苐 5,808,029號，Capon等人，Nature，337: 525-531 (1989) ;Harvill 等人，Immunotech·，1: 95-105，（1995) ; 1997年 7 月3日發表之WO 97/23614 ; 1997年12月11日申請之 PCT/US 97/23183 ; Linsley，J· Exp. Med.，174: 561-569, (1991) ; 1995年 8 月 10 日發表的 w〇 95/21258)。 有效的抗-Ang-2治療可能有益於龐大族群的癌症患者， 因為大多數的固體腫瘤需要新血管形成，以便生長至直徑 1_2耄米以上。這類治療在其他與血管生成作用有關的疾 病上，可能具有廣大的應用，像是視網膜病、關節炎和牛 皮癖。 對於確認專一地認得並與Ang-2結合的新製劑，有尚未 發展的需求。類製劑將可用於診斷篩選，並治療干涉與 Ang_2活性有關之疾病狀態。 因此本&明的目軚是提供調節Ang-2活性的Ang-2之專 -結合劑。本發明的這類製劑採用肽體之形式，也就是與 其他分子融合的肤，像是抗體的&功能部位，其中肽部分 專一地與Ang-2結合。O:\121\121929.DOC -10 - 200808833 Fusion, which improves the half-life of these molecules. The fusion of proteins or fragments thereof with stable plasma proteins, such as the ig constant region, has been described in many ways to improve the half-life of these molecules (see, e.g., U.S. Patent No. 5,480,981; Zheng et al., j. Immun. , 154: 5590-5600, (1995); Fisher et al., Ν Engl J. Med., 334: 1697-1702, (1996); Van Zee, Κ· et al., j. Immun〇1., 156 : 2221-2230, (1996); U.S. Patent No. 5,808,029 issued September 15, 1998, Capon et al, Nature, 337: 525-531 (1989); Harvill et al., Immunotech, 1:95- 105, (1995); WO 97/23614, published on July 3, 1997; PCT/US97/23183, filed on December 11, 1997; Linsley, J. Exp. Med., 174: 561-569, ( 1991); published on August 10, 1995, w〇95/21258). Effective anti-Ang-2 treatment may be beneficial for a large group of cancer patients, as most solid tumors require new blood vessel formation to grow to a diameter of more than 1 2 耄. Such treatments may have a wide range of applications in other diseases associated with angiogenesis, such as retinopathy, arthritis, and porcine sputum. There is an undeveloped need to identify new formulations that are specifically recognized and combined with Ang-2. The class of agents will be useful for diagnostic screening and for the treatment of disease states associated with Ang 2 activity. Therefore, the aim of this & Ming is to provide a specific binding agent for Ang-2 which modulates Ang-2 activity. Such preparations of the invention are in the form of peptibodies, i.e., peptides fused to other molecules, such as & functional sites of antibodies, wherein the peptide moiety specifically binds to Ang-2.
O:\121\121929.DOC 200808833 【發明内容】 在與Ang-2結合之肽（在本文中亦稱為多肽）的具體實施 例中指不本發明。在本發明中亦包含這類肽的變體和衍生 物。 在其他的具體實施例中，本發明之肽及其變體和衍生物 與媒介附接。 在其他的具體實施例中，肽可與Fc功能部位融合，藉此 提供肽體。肽體可視需要包括至少一個，例如序列識^ : 3旎至序列識別：6號，或序列識別：76號至序列識別： 157號的肽，以及其變體和衍生物。此外，肽亦可包括至 少一個根據在序列識別：65號至序列識別·· 75號和序列識 別：158號中陳述之公式的肽。 在另一個具體實施例中，本發明提供編碼該專一結合劑 的核酸分子，以及其變體和衍生物。 在另一個具體實施例中，本發明提供編碼肽體的核酸分 子’以及其變體和衍生物。這類核酸分子可視需要包含序 列識別·· 33號至序列識別：53號。 在另一個具體實施例中，本發明提供藉著對需要其之個 體投與有效含量的本發明之專一結合劑，來減少腫瘤的方 法。本發明亦提供在個體中抑制血管生成的方法，包括對 需要其之個體投與有效含量的本發明之專一結合劑。本發 明更提供在個體中治療癌症的方法，包括對需要其之個體 投與有效含量的本發明之專一結合劑。 本發明亦關於能夠與Ang-2結合的多肽，其中該多肽包 O:\121\121929.DOC -12- 200808833 括胺基酸序列WDPWT (序列識別：65號），且其中該多肽 之長度是從5至50個胺基酸，及其在生理學上可接受的鹽 類。該多肽亦可包括胺基酸序列： WDPWTC (序列識別：66號） 及其在生理學上可接受的鹽類。此外，該多肽可包括胺基 酸序列：O: \121\121929.DOC 200808833 SUMMARY OF THE INVENTION In a specific embodiment of a peptide that binds to Ang-2 (also referred to herein as a polypeptide), it is not the invention. Variants and derivatives of such peptides are also included in the present invention. In other specific embodiments, the peptides of the invention, and variants and derivatives thereof, are attached to a vehicle. In other embodiments, the peptide can be fused to an Fc functional site, thereby providing a peptibody. Peptibodies may include at least one, for example, sequence recognition: sequence recognition: number 6, or sequence recognition: 76 to sequence recognition: peptide number 157, as well as variants and derivatives thereof. In addition, the peptide may also include at least one peptide according to the formula set forth in Sequence Identification: No. 65 to Sequence Recognition No. 75 and Sequence Identification: No. 158. In another embodiment, the invention provides nucleic acid molecules encoding the specific binding agents, as well as variants and derivatives thereof. In another embodiment, the invention provides nucleic acid molecules' encoding propeptides, as well as variants and derivatives thereof. Such nucleic acid molecules may optionally include sequence identification··33 to sequence identification: 53. In another embodiment, the invention provides a method of reducing tumors by administering to a subject in need thereof an effective amount of a specific binding agent of the invention. The invention also provides a method of inhibiting angiogenesis in an individual comprising administering to the individual in need thereof an effective amount of a specific binding agent of the invention. The invention further provides a method of treating cancer in an individual comprising administering to the individual in need thereof an effective amount of a specific binding agent of the invention. The invention also relates to a polypeptide capable of binding to Ang-2, wherein the polypeptide comprises O:\121\121929.DOC-12-200808833 comprises the amino acid sequence WDPWT (sequence recognition: No. 65), and wherein the length of the polypeptide is From 5 to 50 amino acids, and their physiologically acceptable salts. The polypeptide may also include an amino acid sequence: WDPWTC (sequence recognition: No. 66) and its physiologically acceptable salts. Furthermore, the polypeptide may comprise an amino acid sequence:
Cz2WDPWT (序列識別· 6 7號） 其中z2為酸性或中性的極性胺基酸殘基，及其在生理學上 可接受的鹽類。該多肽尚可包括胺基酸序列：Cz2WDPWT (SEQ ID NO: 6 7) wherein z2 is an acidic or neutral polar amino acid residue, and a physiologically acceptable salt thereof. The polypeptide may also include an amino acid sequence:
Cz2WDPWTC (序列識別：68號） 其中z2為酸性或中性的極性胺基酸殘基，及其在生理學上 可接受的鹽類。 在其他的具體實施例中，本發明係關於能夠與Ang-2結 合的多肽，其包括下式的胺基酸序列： a^^^a^DPWTCa^a^a14 (序列識別：69號） 其中： a1、a2和a3分別為胺基酸殘基； a5為胺基酸殘基； a12缺少或是胺基酸殘基； a13缺少或是中性疏水性的、中性極性的，或鹼性的胺基 O:\121\121929.DOC -13- 200808833 酸殘基； 或中性極性的胺基酸殘基； a14為中性疏水性的 在較佳的具體實施例 及其在生理學上可接受的鹽類 中： 2 W GS、Q、N、E、K、R或 Η ; a:為V、P、M、G、S、Q、D、Ε、κ、R或 Η; a3為A、V、P、M、F、T、G、D、E、K或 Η; a5 為 A、V、G、Q、ν、D 或 Ε ; a 為S、Q、N、D、E、K*R; a13為L、T或Η ;且 a 為 v、L、I、W或 Μ。 在更佳的具體實施例中 或 Ε ; a12為 D*E ; 為 Η ; a1為 Q ; a2 為 Ε ; a3 為 Ε ; ^為1) 且a14為Μ。 ^應瞭解在本文中使用帶有肩上數字之小寫字母（像是&1和 二企圖確認胺基酸位置，而非意圖代表特定胺基酸的單 -字母縮寫。在本文中以大寫字母提供單一字母的胺基酸 縮寫。 包括下式之胺 本發明更關於能夠與Ang_2結合的多肽 基酸序列： b1b2b3b4b5b6Cb8WDPWTCb15b16b17b18b19b20 (序列識別：70號） 其中： b1缺少或是胺基酸殘基； b2缺少或是中性疏水性的、中性極性的或驗性的胺基酸Cz2WDPWTC (SEQ ID NO: 68) wherein z2 is an acidic or neutral polar amino acid residue, and a physiologically acceptable salt thereof. In other specific embodiments, the invention relates to a polypeptide capable of binding to Ang-2, comprising an amino acid sequence of the formula: a^^^a^DPWTCa^a^a14 (SEQ ID NO: 69) wherein : a1, a2 and a3 are each an amino acid residue; a5 is an amino acid residue; a12 is absent or an amino acid residue; a13 is absent or neutrally hydrophobic, neutrally polar, or alkaline Amino group O: \121\121929.DOC -13- 200808833 acid residue; or neutral polar amino acid residue; a14 is neutral hydrophobic in preferred embodiments and physiologically Among the acceptable salts: 2 W GS, Q, N, E, K, R or Η; a: V, P, M, G, S, Q, D, Ε, κ, R or Η; a3 is A, V, P, M, F, T, G, D, E, K or Η; a5 is A, V, G, Q, ν, D or Ε; a is S, Q, N, D, E, K*R; a13 is L, T or Η; and a is v, L, I, W or Μ. In a more specific embodiment or Ε; a12 is D*E; Η; a1 is Q; a2 is Ε; a3 is Ε; ^ is 1) and a14 is Μ. ^ It should be understood that the use of lowercase letters with shoulder numbers (such as &1 and two attempts to confirm the position of the amino acid, rather than the one-letter abbreviation intended to represent a particular amino acid) is used herein in capital letters. A single-letter amino acid abbreviation is provided. An amine comprising the following formula The present invention relates more specifically to a polypeptide-based acid sequence capable of binding to Ang-2: b1b2b3b4b5b6Cb8WDPWTCb15b16b17b18b19b20 (SEQ ID NO: 70) wherein: b1 is absent or an amino acid residue; b2 is absent Neutral hydrophobic, neutral polar or atrial amino acid
O:\121\121929.DOC -14- 200808833 殘基； b3、b4、b5和b6分別缺少或是胺基酸殘基； b8為胺基酸殘基； b15缺少或是胺基酸殘基； b16缺少或是中性疏水性的、中性極性的或鹼性的胺基酸 殘基； b17缺少或是中性疏水性的或中性極性的胺基酸殘基； b 、b和b分別缺少或是胺基酸殘基； 及其在生理學上可接受的鹽類。 b1缺少或是A、V、l、P、W 、R^H ;O: \121\121929.DOC -14- 200808833 Residue; b3, b4, b5 and b6 are respectively missing or amino acid residues; b8 is an amino acid residue; b15 is absent or an amino acid residue; B16 lacks a neutral or hydrophobic, neutral polar or basic amino acid residue; b17 lacks a neutral or neutral polar amino acid residue; b, b and b respectively Lack of or amino acid residues; and physiologically acceptable salts thereof. B1 is missing or A, V, l, P, W, R^H;
在較佳的具體實施例中： 、F、T、G、S、Q、N、K b2缺少或是A、V、l 、K、R或 Η ;In a preferred embodiment: F, T, G, S, Q, N, K b2 are absent or A, V, l, K, R or Η;
P、W、m、T、G、S、Y、N b3缺少或是A、L、 、尺或11 ; P、W、m、t、g、s、q、n、e b4為V、i、p、w、g、s、q'n b5為v、p、m、g、s、q、d、e b6為A、V、P、M、f、t、g、d b8為 A、V、G、Q、N、D*E ; E、K、R或 H ; K、R或 H ; E、K或 H ; b15為 S、Q、N、E)、E、K*R ; b16為 L、T或 H ; b17為V、L、I、w或 m; 七18缺少或是八、乂、[、？、\^、1? 或R ;P, W, m, T, G, S, Y, N b3 are missing or A, L, , ruler or 11; P, W, m, t, g, s, q, n, e b4 are V, i , p, w, g, s, q'n b5 are v, p, m, g, s, q, d, e b6 are A, V, P, M, f, t, g, d b8 is A, V, G, Q, N, D*E; E, K, R or H; K, R or H; E, K or H; b15 is S, Q, N, E), E, K*R; b16 Is L, T or H; b17 is V, L, I, w or m; 718 is missing or 八, 乂, [,? , \^, 1? or R;
T、G、Y、Q、D、ET, G, Y, Q, D, E
O:\121\121929.DOC 15- 200808833 b19缺少或是V、L、I、P、T、G、S、Y、q、n、d、E 或R ;且 1>20缺少或是\^、1^、？、1^、1^、丁、〇、8、丫、（）、^^、 D、K或 R。 在更佳的具體實施例中，b1缺少或是P或T; b2缺少或是I 或N ; b3缺少或是R或I ; b4為Q ; b5為E ; b6為e ; b8為D或E ，b 5為〇或e; b16為H; b17為M; b18缺少或為w或p; b19缺 》或為G或E，且b20缺少或為v或K。 亦應瞭解本發明最好係關於包括至少一個選自由序列識 別· 4唬和序列識別：76號至序列識別：ιΐ8號所組成之群 的胺基酸序列之多月大，其中該多肽能夠與Ang-2結合，及 其在生理子上可接受的鹽類。在下文中陳述肽序列：O:\121\121929.DOC 15- 200808833 b19 is missing or V, L, I, P, T, G, S, Y, q, n, d, E or R; and 1 > 20 is missing or \^ , 1^,? , 1^, 1^, D, 〇, 8, 丫, (), ^^, D, K or R. In a more preferred embodiment, b1 is absent or P or T; b2 is absent or I or N; b3 is absent or R or I; b4 is Q; b5 is E; b6 is e; b8 is D or E b 5 is 〇 or e; b16 is H; b17 is M; b18 is absent or is w or p; b19 is absent or is G or E, and b20 is absent or is v or K. It will also be appreciated that the present invention is preferably directed to a multi-monthly large amino acid sequence comprising at least one selected from the group consisting of sequence recognition and sequence recognition: 76 to sequence recognition: ι ΐ8, wherein the polypeptide is capable of Ang-2 binds to its physiologically acceptable salts. The peptide sequence is stated below:
O:\121\121929.DOC -16· 200808833 表1O:\121\121929.DOC -16· 200808833 Table 1
肽 序列識別號 肽序列 Con4-44 76 PIRQEECDWDPWTCEHMWEV Con4-40 77 TNIQEECEWDPWTCDHMPGK Con4-4 78 WYEQDACEWDPWTCEHMAEV Con4-31 79 NRLQEVCEWDPWTCEHMENV Con4-C5 80 AATQEECEWDPWTCEHMPRS Con4-42 81 LRHQEGCEWDPWTCEHMFDW Con4-35 82 VPRQKDCEWDPWTCEHMYVG Con4-43 83 SISHEECEWDPWTCEHMQVG Con4-49 84 WAAQEECEWDPWTCEHMGRM Con4-27 85 TWPQDKCEWDPWTCEHMGST Con4-48 86 GHSQEECGWDPWTCEHMGTS Con4-46 87 QHWQEECEWDPWTCDHMPSK Con4-41 88 NVRQEKCEWDPWTCEHMPVR Con4-36 89 KSGQVECNWDPWTCEHMPRN Con4-34 90 VKTQEHCDWDPWTCEHMREW Con4-28 91 AWGQEGCDWDPWTCEHMLPM Con4-39 92 PVNQEDCEWDPWTCEHMPPM Con4-25 93 RAPQEDCEWDPWTCAHMDIK Con4-50 94 HGQNMECEWDPWTCEHMFRY Con4-38 95 PRLQEECVWDPWTCEHMPLR Con4-29 96 RTTQEKCEWDPWTCEHMESQ Con4-47 97 QTSQEDCVWDPWTCDHMVSS Con4-20 98 QVIGRPCEWDPWTCEHLEGL Con4-45 99 WAQQEECAWDPWTCDHMVGL Con4-37 100 LPGQEDCEWDPWTCEHMVRS Con4-33 101 PMNQVECDWDPWTCEHMPRS AC2-Con4 102 FGWSHGCEWDPWTCEHMGST Con4-32 103 KSTQDDCDWDPWTCEHMVGP Con4-17 104 GPRISTCQWDPWTCEHMDQL Con4-8 105 STIGDMCEWDPWTCAHMQVD AC4-Con4 106 VLGGQGCEWDPWTCRLLQGW Con4-l 107 VLGGQGCQWDPWTCSHLEDG O:\121\121929.DOC -17- 200808833Peptide sequence identification number peptide sequence Con4-44 76 PIRQEECDWDPWTCEHMWEV Con4-40 77 TNIQEECEWDPWTCDHMPGK Con4-4 78 WYEQDACEWDPWTCEHMAEV Con4-31 79 NRLQEVCEWDPWTCEHMENV Con4-C5 80 AATQEECEWDPWTCEHMPRS Con4-42 81 LRHQEGCEWDPWTCEHMFDW Con4-35 82 VPRQKDCEWDPWTCEHMYVG Con4-43 83 SISHEECEWDPWTCEHMQVG Con4-49 84 WAAQEECEWDPWTCEHMGRM Con4-27 85 TWPQDKCEWDPWTCEHMGST Con4-48 86 GHSQEECGWDPWTCEHMGTS Con4-46 87 QHWQEECEWDPWTCDHMPSK Con4-41 88 NVRQEKCEWDPWTCEHMPVR Con4-36 89 KSGQVECNWDPWTCEHMPRN Con4-34 90 VKTQEHCDWDPWTCEHMREW Con4-28 91 AWGQEGCDWDPWTCEHMLPM Con4-39 92 PVNQEDCEWDPWTCEHMPPM Con4-25 93 RAPQEDCEWDPWTCAHMDIK Con4-50 94 HGQNMECEWDPWTCEHMFRY Con4-38 95 PRLQEECVWDPWTCEHMPLR Con4-29 96 RTTQEKCEWDPWTCEHMESQ Con4-47 97 QTSQEDCVWDPWTCDHMVSS Con4-20 98 QVIGRPCEWDPWTCEHLEGL Con4-45 99 WAQQEECAWDPWTCDHMVGL Con4-37 100 LPGQEDCEWDPWTCEHMVRS Con4-33 101 PMNQVECDWDPWTCEHMPRS AC2-Con4 102 FGWSHGCEWDPWTCEHMGST Con4-32 103 KSTQDDCDWDPWTCEHMVGP Con4-17 104 GPRISTCQWDPWTCEHMDQL Con4-8 105 STIGDMCEWDPWTCAHMQVD AC4-Con4 106 VLGGQGCEWDPWTCRLLQGW Con4-l 107 VLGGQGCQWDPWTCSHLEDG O:\121\121929.DOC -17- 200808833
Con4-Cl 108 TTIGSMCEWDPWTCAHMQGG Con4-21 109 TKGKSVCQWDPWTCSHMQSG Con4-C2 110 TTIGSMCQWDPWTCAHMQGG Con4-18 111 WVNEVVCEWDPWTCNHWDTP Con4-19 112 VVQVGMCQWDPWTCKHMRLQ Con4-16 113 AVGSQTCEWDPWTCAHLVEV Con4-ll 114 QGMKMFCEWDPWTCAHIVYR Con4-C4 115 TTIGSMCQWDPWTCEHMQGG Con4-23 116 TSQRVGCEWDPWTCQHLTYT Con4-15 117 QWSWPPCEWDPWTCQTVWPS Con4-9 118 GTSPSFCQWDPWTCSHMVQG TN8-Con4* 4 QEECEWDPWTCEHM 應瞭解某些肽及/或肽體可含有字首的ΠΤΝ”、ΠΤΝ8Π或 ΠΤΝ12Π，並可以或可以不提供特定肽體該字首。因此，在 本文中可交替使用例WnTN8-Con4’，，，Con4n—詞。 在另一個具體實施例中，本發明係關於具有下式之物質 的組合物： 及其多聚體，其中： F1為媒介； X1和X2分別選自 -(L^c-P1 ； (L1)c-P1-(L2)d-P2 ； -(L^e-P^CL^d-P'-CL^e-P^CL^rP4 ； 其中一或多個P1、P2、P3和P4分別包括如同在本文中描 述之多肽。例如，在較佳的具體實施例中，P1、P2、P3和 O:\121\121929.DOC -18 - 200808833 P4可分別包括4列識Μ : 3號至4列識別：6號，及/或序列 識別：76號至序列識別：157號的多肽。 在另一個具體實施例中，物質之組合物是式： X^F1 或 F!-X2 及其在生理學上可接受的鹽類，其中χ1、ρ丨和χ2如同在本 文中之定義。在另一個具體實施例中，物質之組合物是式：Con4-Cl 108 TTIGSMCEWDPWTCAHMQGG Con4-21 109 TKGKSVCQWDPWTCSHMQSG Con4-C2 110 TTIGSMCQWDPWTCAHMQGG Con4-18 111 WVNEVVCEWDPWTCNHWDTP Con4-19 112 VVQVGMCQWDPWTCKHMRLQ Con4-16 113 AVGSQTCEWDPWTCAHLVEV Con4-ll 114 QGMKMFCEWDPWTCAHIVYR Con4-C4 115 TTIGSMCQWDPWTCEHMQGG Con4-23 116 TSQRVGCEWDPWTCQHLTYT Con4-15 117 QWSWPPCEWDPWTCQTVWPS Con4-9 118 GTSPSFCQWDPWTCSHMVQG TN8-Con4* 4 QEECEWDPWTCEHM It should be understood that certain peptides and/or peptibodies may contain ΠΤΝ", ΠΤΝ8Π or ΠΤΝ12Π of the prefix and may or may not provide the prefix of the particular peptide. Therefore, in this paper The WnTN8-Con4',, Con4n-word can be used interchangeably. In another embodiment, the invention relates to a composition having a substance of the formula: and a multimer thereof, wherein: F1 is a medium; And X2 are respectively selected from -(L^c-P1; (L1)c-P1-(L2)d-P2; -(L^eP^CL^d-P'-CL^eP^CL^rP4; one of them Or a plurality of P1, P2, P3 and P4 respectively comprise a polypeptide as described herein. For example, in a preferred embodiment, P1, P2, P3 and O: \121\121929.DOC -18 - 200808833 P4 may comprise 4 columns of identification: 3 to 4 column identification: No. 6, and/or sequence identification: 76 to sequence identification: polypeptide number 157. In another embodiment, the composition of matter Formula: X^F1 or F!-X2 and physiologically acceptable salts thereof, wherein χ1, ρ丨 and χ2 are as defined herein. In another embodiment, the composition of matter is formula:
FhLVp1 學上可接受的鹽類’其中Ll、F>pi如同在 本文中之定義。在另-個具體實施例中，物質之化合物 是式： F^(Ll)c^^(L2)d^2 及其在生理學上可接受的鹽類，其中1^1、沪、1>1、"和〇與 d，如同在本文中之定義。在另一個具體實施例中，物〜質 之組合物是式： P^VF^O^d-P2 在生理學上可接受的鹽類。在較佳的具體實施例中， F1是Fc功能部位或其片段。 本發明更關於能夠與Ang_2結合的多肽，包括 、^ 基酸序列： 气FhLVp1 a scientifically acceptable salt' wherein Ll, F>pi are as defined herein. In another embodiment, the compound of the substance is of the formula: F^(Ll)c^^(L2)d^2 and a physiologically acceptable salt thereof, wherein 1^1, Shanghai, 1> 1, " and 〇 and d, as defined in this article. In another embodiment, the composition of the substance is of the formula: P^VF^O^d-P2 is a physiologically acceptable salt. In a preferred embodiment, F1 is an Fc functional site or a fragment thereof. The invention further relates to a polypeptide capable of binding to Ang_2, comprising: an acid sequence: gas
Pc2Dc4Lc6c7c8LY (序列識別：71號） 其中Pc2Dc4Lc6c7c8LY (sequence identification: 71)
O:\121\121929.DOC -19- 200808833 C2為中性疏水性的胺基酸殘基； c4 為 A、D 或 E ; c6為酸性的胺基酸殘基； c7為胺基酸殘基；且 c8為中性疏水性的、中性極性的或鹼性的胺基酸殘基； 及其在生理學上可接受的鹽類。在較佳的具體實施例中 ，c2為L或Μ。在另一個較佳的具體實施例中，c6為〇或£。 本發明更關於能夠與Ang-2結合的多肽，包括下式之胺 基酸序列： d1d2d3d4Pd6Dd8Ld10d1 Μ12ίΥά15(116(117(118(119(12()ά21(122 (序列識別：72號） 其中， d1缺少或是胺基酸殘基； d2缺少或是中性極性的、酸性的或鹼性的胺基酸殘基； d3缺少或是中性疏水性的或中性極性的胺基酸殘基； d4缺少或是胺基酸殘基； d6為中性疏水性的胺基酸殘基； d8 為 A、D 或 E ; d1G為酸性的胺基酸殘基； d11為胺基酸殘基； d12為中性疏水性的、中性極性的或驗性的胺基酸殘基； d15缺少或是中性極性的、酸性的或鹼性的胺基酸殘基； d16缺少或是中性極性的、酸性的或驗性的胺基酸殘基； d17缺少或是中性疏水性的或中性極性的胺基酸殘基； d18缺少或是中性疏水性的或中性極性的胺基酸殘基； O:\121\121929.DOC -20- 200808833 d19缺少或是中性疏水性的、中性極性的或驗性的胺基酸 殘基； d2G缺少或是胺基酸殘基； 缺上或疋中性極性的、酸性的或鹼性的胺基酸殘基； ci缺少或是中性疏水性的、中性極性的或驗性的胺基酸 殘基； 及其在生理學上可接受的鹽类頁。在較佳的具體實施例中： d1為 T、S、Q、R或 η ; d2 為 Τ、Q、Ν 或 Κ ; d3 為 F ; d4 為 Μ、Q、E 或 K ; d6為L或Μ ; d8為D或Ε ; d10為 E ; d11為Q或E ; d12為T或R ; d15為 Y、D、E或 K ; d16為 Q ; d17為W或F ; d18為L、I、Μ或 T ; d19為 L、F或 Υ ; d20 為 Q、D 或 E ; d21缺少或是Q或Η ; d22缺少或是A、L、G、s或R。 在較佳的具體實施例中，該多肽包括至少一個選自由序O:\121\121929.DOC -19- 200808833 C2 is a neutral hydrophobic amino acid residue; c4 is A, D or E; c6 is an acidic amino acid residue; c7 is an amino acid residue And c8 is a neutral hydrophobic, neutral polar or basic amino acid residue; and a physiologically acceptable salt thereof. In a preferred embodiment, c2 is L or Μ. In another preferred embodiment, c6 is 〇 or £. The present invention relates more specifically to a polypeptide capable of binding to Ang-2, comprising the amino acid sequence of the formula: d1d2d3d4Pd6Dd8Ld10d1 Μ12ίΥά15 (116(117(118(119(12())21(122 (SEQ ID NO:72)), wherein d1 is absent Or an amino acid residue; d2 lacks a neutral or acidic, basic or acidic amino acid residue; d3 lacks a neutral or neutral polar amino acid residue; d4 Lack of amino acid residues; d6 is a neutral hydrophobic amino acid residue; d8 is A, D or E; d1G is an acidic amino acid residue; d11 is an amino acid residue; d12 is Neutral hydrophobic, neutral polar or atrial amino acid residue; d15 lacks a neutral or acidic, basic or acidic amino acid residue; d16 lacks or is neutral, Acidic or amphoteric amino acid residues; d17 lacks a neutral or neutral polar amino acid residue; d18 lacks a neutral or neutral polar amino acid residue O;\121\121929.DOC -20- 200808833 d19 lacks or neutral hydrophobic, neutral polar or atrial amino acid residues; d2G is absent or An amino acid residue; an amino acid residue that is devoid of or neutral to a neutral polarity; an acidic or basic amino acid residue; a ci lacking or a neutral hydrophobic, neutrally polar or a test amino acid residue And its physiologically acceptable salt sheet. In a preferred embodiment: d1 is T, S, Q, R or η; d2 is Τ, Q, Κ or Κ; d3 is F; d4 Is Μ, Q, E or K; d6 is L or Μ; d8 is D or Ε; d10 is E; d11 is Q or E; d12 is T or R; d15 is Y, D, E or K; d16 is Q D17 is W or F; d18 is L, I, Μ or T; d19 is L, F or Υ; d20 is Q, D or E; d21 is absent or Q or Η; d22 is absent or A, L, G Or s or R. In a preferred embodiment, the polypeptide comprises at least one selected from the group consisting of
O:\121\121929.DOC -21 - 200808833 列識別：6號和序列識別：119號至序列識別：142號所組 成之群的胺基酸序列，其中該多肽能夠與Ang-2結合。在 下文中陳述序列識別：6號和序列識別：119-142號：O: \121\121929.DOC -21 - 200808833 Column identification: No. 6 and sequence identification: No. 119 to sequence identification: Amino acid sequence of the group consisting of No. 142, wherein the polypeptide is capable of binding to Ang-2. The sequence identification is stated below: No. 6 and sequence identification: No. 119-142:
肽 序列識別號 肽序列 LM 119 QNYKPLDELDATLYEHFIFHYT L1-2 120 LNFTPLDELEQTLYEQWTLQQS L1-3 121 TKFNPLDELEQTLYEQWTLQHQ L1-4 122 VKFKPLDALEQTLYEHWMFQQA L1-5 123 VKYKPLDELDEILYEQQTFQER L1-7 124 TNFMPMDDLEQRLYEQFILQQG L1-9 125 SKFKPLDELEQTLYEQWTLQHA L1-10 126 QKFQPLDELEQTLYEQFMLQQA L1-11 127 QNFKPMDELEDTLYKQFLFQHS L1-12 128 YKFTPLDDLEQTLYEQWTLQHV LM3 129 QEYEPLDELDETLYNQWMFHQR LM4 130 SNFMPLDELEQTLYEQFMLQHQ L1-15 131 QKYQPLDELDKTLYDQFMLQQG LM6 132 QKFQPLDELEETLYKQWTLQQR L1-17 133 VKYKPLDELDEWLYHQFTLHHQ L1-18 134 QKFMPLDELDEILYEQFMFQQS L1-19 135 QTFQPLDDLEEYLYEQWIRRYH L1-20 136 EDYMPLDALDAQLYEQFILLHG L1-21 137 HTFQPLDELEETLYYQWLYDQL L1-22 138 YKFNPMDELEQTLYEEFLFQHA AC6-L1 139 TNYKPLDELDATLYEHWILQHS L1-C1 140 QKFKPLDELEQTLYEQWTLQQR L1-C2 141 TKFQPLDELDQTLYEQWTLQQR L1-C3 142 TNFQPLDELDQTLYEQWTLQOR L1 6 KFNPLDELEETLYEQFTFQQ O:\121\121929.DOC -22- 200808833 本發明亦關於能夠與Ang-2結合的多肽，包括下式的胺 基酸序列： RPeWe6e7G (序列識別：73號） 其中 e3為中性極性的胺基酸殘基； e4為酸性的胺基酸殘基； e5為中性極性的或酸性的胺基酸殘基； e6為中性疏水性的胺基酸殘基； e7為中性疏水性的胺基酸殘基； 及其在生理學上可接受的鹽類。在較佳的具體 収貫施例中 ，e3為Y或C。在另一個較佳的具體實施例中，y為 在另一個較佳的具體實施例中，e6為I或Μ。 包括下式的胺 本發明更關於能夠與Ang-2結合的多肽 基酸序列： fVfYRPfWfiOfUGf^WWWYo (序列識別：74號） 其中， f1為中性疏水性的或中性極性的胺基酸殘基； f2為中性疏水性的或中性極性的胺基酸殘基； f為中性極性的或酸性的胺基酸殘基； f4為中性疏水性的或中性極性的胺基酸殘基； f7為中性極性的胺基酸殘基； f8為酸性的胺基酸殘基；Peptide SEQ ID NO Peptide Sequence LM 119 QNYKPLDELDATLYEHFIFHYT L1-2 120 LNFTPLDELEQTLYEQWTLQQS L1-3 121 TKFNPLDELEQTLYEQWTLQHQ L1-4 122 VKFKPLDALEQTLYEHWMFQQA L1-5 123 VKYKPLDELDEILYEQQTFQER L1-7 124 TNFMPMDDLEQRLYEQFILQQG L1-9 125 SKFKPLDELEQTLYEQWTLQHA L1-10 126 QKFQPLDELEQTLYEQFMLQQA L1-11 127 QNFKPMDELEDTLYKQFLFQHS L1 -12 128 YKFTPLDDLEQTLYEQWTLQHV LM3 129 QEYEPLDELDETLYNQWMFHQR LM4 130 SNFMPLDELEQTLYEQFMLQHQ L1-15 131 QKYQPLDELDKTLYDQFMLQQG LM6 132 QKFQPLDELEETLYKQWTLQQR L1-17 133 VKYKPLDELDEWLYHQFTLHHQ L1-18 134 QKFMPLDELDEILYEQFMFQQS L1-19 135 QTFQPLDDLEEYLYEQWIRRYH L1-20 136 EDYMPLDALDAQLYEQFILLHG L1-21 137 HTFQPLDELEETLYYQWLYDQL L1-22 138 YKFNPMDELEQTLYEEFLFQHA AC6- L1 139 TNYKPLDELDATLYEHWILQHS L1-C1 140 QKFKPLDELEQTLYEQWTLQQR L1-C2 141 TKFQPLDELDQTLYEQWTLQQR L1-C3 142 TNFQPLDELDQTLYEQWTLQOR L1 6 KFNPLDELEETLYEQFTFQQ O:\121\121929.DOC -22- 200808833 The present invention also relates to a polypeptide capable of binding to Ang-2, including the following formula Amino acid sequence: RPeWe6e7G (sequence recognition :73) where e3 is a neutral polar amino acid residue; e4 is an acidic amino acid residue; e5 is a neutral polar or acidic amino acid residue; e6 is a neutral hydrophobic amine a base acid residue; e7 is a neutral hydrophobic amino acid residue; and a physiologically acceptable salt thereof. In a preferred specific embodiment, e3 is Y or C. In another preferred embodiment, y is in another preferred embodiment, e6 is I or Μ. Amines Included in the Invention The present invention relates more specifically to polypeptide-based acid sequences capable of binding to Ang-2: fVfYRPfWfiOfUGf^WWWYo (SEQ ID NO: 74) wherein, f1 is a neutral hydrophobic or neutral polar amino acid residue ; f2 is a neutral hydrophobic or neutral polar amino acid residue; f is a neutral polar or acidic amino acid residue; f4 is a neutral hydrophobic or neutral polar amino acid residue a base; f7 is a neutral polar amino acid residue; f8 is an acidic amino acid residue;
O:\121\121929.DOC -23- 200808833 f為中性極性的或酸性的胺基酸殘基； f1G為中性疏水性的胺基酸殘基； fU為中性疏水性的胺基酸殘基； f為中性疏水性的或中性極性的胺基酸殘基； f14為中性疏水性的或中性極性的胺基酸殘基； f15為中性極性的胺基酸殘基； f16為中性極性的胺基酸殘基； f17為中性極性的或酸性的胺基酸殘基； f18為中性疏水性的或鹼性的胺基酸殘基； f19為中性疏水性的或中性極性的胺基酸殘基；且 f2G為中性疏水性的或中性極性的胺基酸殘基； 及其在生理學上可接受的鹽類。在較佳的具體實施例 中·· f1為 S、A或 G ; f2 為 G、Q 或 P ; f3 為 Q、G 或 D ; f4 為 L、Μ 或 Q ; f7為C或Υ ; f8為E或D ; f9 為 E、G 或 D ; f1Q為I或Μ ; f11為F或L ; f13為C或W ; f14為G或P ; O:\121\121929.DOC -24 - 200808833 f15為T或N ; f16為 Q、Y或 K ; f17為 Ν、D或 Q ; f18為L、V、W或 R ; f19為 A、Q、Y或I ;且 f20 為 L、A、G 或 V。 在更佳的具體實施例中，本發明係關於包括至少一個選 自由序列識別：3號和序列識別：143號至序列識別·· 148 號之胺基酸序列的多肽所組成之群，其中該多肽能夠與 Ang-2結合，及其在生理學上可接受的鹽類。序列識別：3 號和序列識別：143號至序枸識別：148號如下。O:\121\121929.DOC -23- 200808833 f is a neutral polar or acidic amino acid residue; f1G is a neutral hydrophobic amino acid residue; fU is a neutral hydrophobic amino acid Residue; f is a neutral hydrophobic or neutral polar amino acid residue; f14 is a neutral hydrophobic or neutral polar amino acid residue; f15 is a neutral polar amino acid residue F16 is a neutral polar amino acid residue; f17 is a neutral polar or acidic amino acid residue; f18 is a neutral hydrophobic or basic amino acid residue; f19 is neutral hydrophobic A neutral or neutral polar amino acid residue; and f2G is a neutral hydrophobic or neutral polar amino acid residue; and a physiologically acceptable salt thereof. In a preferred embodiment, f1 is S, A or G; f2 is G, Q or P; f3 is Q, G or D; f4 is L, 或 or Q; f7 is C or Υ; f8 is E or D; f9 is E, G or D; f1Q is I or Μ; f11 is F or L; f13 is C or W; f14 is G or P; O:\121\121929.DOC -24 - 200808833 f15 is T or N; f16 is Q, Y or K; f17 is Ν, D or Q; f18 is L, V, W or R; f19 is A, Q, Y or I; and f20 is L, A, G or V . In a more preferred embodiment, the invention relates to a population comprising at least one polypeptide selected from the group consisting of sequence recognition: number 3 and sequence recognition: 143 to sequence identification 148, wherein The polypeptide is capable of binding to Ang-2, and its physiologically acceptable salts. Sequence identification: No. 3 and sequence identification: No. 143 to serial identification: No. 148 is as follows.
肽 序列識別& 肽序列 Conl-1 143 AGGMRPYDGMLGWPNYDVQA Con 1-2 144 QTWDDPCMHILGPVTWRRCI Coni -3 145 APGQRPYDGMLGWPTYQRIV Coni-4 146 SGQLRPCEEIFGCGTQNLAL Cdnl-5 147 FGDKRPLECMFGGPIQLCPR Con 1-6 148 GQDLRPCEDMFGCGTKDWYG Coni 3 KRPCEEIFGGCTYO 在其他方面，本發明係關於能與Ang-2結合的多肽，包 括下式的胺基酸序列：Peptide Sequence Recognition & Peptide Sequence Conl-1 143 AGGMRPYDGMLGWPNYDVQA Con 1-2 144 QTWDDPCMHILGPVTWRRCI Coni -3 145 APGQRPYDGMLGWPTYQRIV Coni-4 146 SGQLRPCEEIFGCGTQNLAL Cdnl-5 147 FGDKRPLECMFGGPIQLCPR Con 1-6 148 GQDLRPCEDMFGCGTKDWYG Coni 3 KRPCEEIFGGCTYO In other respects, the present invention relates to A polypeptide capable of binding to Ang-2, including an amino acid sequence of the formula:
Cg2Gg4g5DPFTg10GCg13 (序列識別：75號） 其中 g2為酸性的胺基酸殘基； O:\121\121929.DOC -25- 200808833 g4為中性疏水性的胺基酸殘基； g5為 E、D或 Q ; g1()為中性疏水性的或中性極性的胺基酸殘基； g13為酸性的殘基； 及其在生理學上可接受的鹽類。在較佳的具體實施例中’ g2為E或D。在另一個較佳的具體實施例中，g4為V或Μ。 在另一個具體實施例中，gio為F或Q。在另一個具體實施 例中，g13為D或E。 本發明更關於能夠與Ang-2結合的多肽，包括下式的胺 基酸序列： h1h2h3h4Ch6Gh8h9DPFTh14GCh17h18h19h20 (序列識別：158號） 其中， h1缺少或是中性疏水性的、中性極性的或鹼性的胺基酸 殘基； h2為中性疏水性的或中性極性的胺基酸殘基； h3為酸性的胺基酸殘基； h4為中性疏水性的或中性極性的胺基酸殘基； h6為酸性的胺基酸殘基； h8為中性疏水性的胺基酸殘基； h9 為 E、D 或 Q ; h14為中性疏水性的或中性極性的胺基酸殘基； h17為酸性的胺基酸殘基； h18為中性疏水性的、中性極性的或鹼性的胺基酸殘基； O:\121\121929.DOC -26- 200808833 h19為中性疏水性的或中性極性的胺基酸殘基；且 h2()缺少或是胺基酸殘基； 及其在生理學上可接受的鹽類。在較佳的具體實施例中： h1 缺少或是 A、L、M、G、K4H; h2 為 L、F 或 Q ; h3為D或E ; h4為W或Y ; h6為D或E ; h8為V或Μ ; h14為F或Q ; h17為D或E ; h18為Μ、Y、N或 K ; h19為L或Q ;且 h20 缺少或是Μ、T、G、S、D、K或 R。 在更佳的具體實施例中，本發明係關於包括至少一個選 自由序列識別：5號和序列識別：149號至序列識別：157 號之胺基酸序列的多肽所組成之群，其中該多肽能夠與 Ang-2結合，及其在生理學上可接受的鹽類。在下文中陳 述序列識別：5號和序列識別：149號至序列識別：15 7 號0 O:\121\121929.DOC 27- 200808833Cg2Gg4g5DPFTg10GCg13 (SEQ ID NO: 75) wherein g2 is an acidic amino acid residue; O: \121\121929.DOC -25- 200808833 g4 is a neutral hydrophobic amino acid residue; g5 is E, D or Q; g1() is a neutral hydrophobic or neutral polar amino acid residue; g13 is an acidic residue; and a physiologically acceptable salt thereof. In a preferred embodiment 'g2 is E or D. In another preferred embodiment, g4 is V or Μ. In another specific embodiment, gio is F or Q. In another embodiment, g13 is D or E. The invention further relates to a polypeptide capable of binding to Ang-2, comprising an amino acid sequence of the formula: h1h2h3h4Ch6Gh8h9DPFTh14GCh17h18h19h20 (SEQ ID NO: 158) wherein h1 is absent or neutrally hydrophobic, neutrally polar or alkaline Amino acid residue; h2 is a neutral hydrophobic or neutral polar amino acid residue; h3 is an acidic amino acid residue; h4 is a neutral hydrophobic or neutral polar amino acid residue H6 is an acidic amino acid residue; h8 is a neutral hydrophobic amino acid residue; h9 is E, D or Q; h14 is a neutral hydrophobic or neutral polar amino acid residue H17 is an acidic amino acid residue; h18 is a neutral hydrophobic, neutral polar or basic amino acid residue; O:\121\121929.DOC -26- 200808833 h19 is neutral hydrophobic A neutral or neutral polar amino acid residue; and h2() is absent or an amino acid residue; and a physiologically acceptable salt thereof. In a preferred embodiment: h1 is absent or A, L, M, G, K4H; h2 is L, F or Q; h3 is D or E; h4 is W or Y; h6 is D or E; h8 Is V or Μ; h14 is F or Q; h17 is D or E; h18 is Μ, Y, N or K; h19 is L or Q; and h20 is absent or Μ, T, G, S, D, K or R. In a more preferred embodiment, the invention relates to a population comprising at least one polypeptide selected from the group consisting of: sequence recognition: number 5 and sequence recognition: number 149 to sequence identification: 157 amino acid sequence, wherein the polypeptide It is capable of binding to Ang-2 and its physiologically acceptable salts. Sequence identification is identified below: No. 5 and sequence identification: No. 149 to sequence identification: 15 7 No. 0 O: \121\121929.DOC 27- 200808833
肽 序列識別號 肽序列 12-9-1 149 GFEYCDGMEDPFTFGCDKQT 12-9-2 150 KLEYCDGMEDPFTQGCDNOS 12-9-3 151 LQEWCEGVEDPFTFGCEKOR 12-9-4 152 AQDYCEGMEDPFTFGCEMQK 12-9-5 153 LLDYCEGVQDPFTFGCENLD 12-9-6 154 HQEYCEGMEDPFTFGCEYQG 12-9-7 ,155 MLDYCEGMDDPFTFGCDKQM 12-9-C2 156 LQDYCEGVEDPFTFGCENQR 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR 12-9 5 FDYCEGVEDPFTFGCDNH 在極佳的具體實施例中，本發明係關於具有下式之物質 的組合物： 及其多聚體，其中： F1為媒介； X1和X2分別選自 -(L^s-P1 ； -(L^s-P'-CL^t-P2 ； -(L^s-P^CL^t-P^CL^u-P3 ； ^ -(L1)s-P1-(L2)t-P2-(L3)u-P3-(L4)v.P4 ； 其中一或多個P1、p2、P3和P4分別包括選自由下列所組 成之群的多肽： (a) 胺基酸序列WDPWT (序列識別·· 65號），其中該多 肽之長度是從5至50個胺基酸； (b) 胺基酸序列WDPWTC (序列識別·· 66號）； (c) 胺基酸序列Cz2WDPWT (序列識別：67號），其中z2 為酸性或中性極性的胺基酸殘基； O:\121\121929DOC -28- 200808833 (d) 胺基酸序列Cz2WDPWTC (序列識別：68號），其中 Z2為酸性或中性極性的胺基酸殘基； (e) 胺基酸序列pc2Dc4Lc6c7c8LY (序列識別：71號），其 中c2為中性疏水性的胺基酸殘基；c4為A、D或E ; c6為酸 性的胺基酸殘基；c7為胺基酸殘基；且c8為中性疏水性的 、中性極性的或鹼性的胺基酸殘基； (f) 胺基酸序列RPe3e4e5e6e7G (序列識別：73號），其中 e3為中性極性的胺基酸殘基；e4為酸性的胺基酸殘基；e5 為中性極性的或酸性的胺基酸殘基；e6為中性疏水性的胺 基酸殘基；且e7為中性疏水性的胺基酸殘基； (g) 胺基酸序列 Cg2Gg4g5DPFTg1()GCg13 (序列識別：75 號），其中g2為酸性的胺基酸殘基；g4為中性疏水性的胺基 酸殘基；g5為中性極性的或酸性的胺基酸殘基；gio為中性 疏水性的或中性極性的胺基酸殘基；且^3為酸性的殘基； (h) 序列識別：1號之多肽； (i) 序列識別：2號之多肽；以及 (j) 序列識別：7號之多肽； 其中L1、L2、L3和L4分別為聯結子；且q、r、s、t、^口 v分別為0或1，其限制條件為c^〇r中至少有一個是丨；及其 在生理學上可接受的鹽類。 應瞭解本發明更關於融合多肽，包括至少一個如 文中描述的肽和媒介，纟中該融合多肽能夠與Ang-2U ’及其在生理學上可接受的鹽類。在融合多肽中，媒= 好是至少-個Fc功能部位、&乙二醇、脂質、膽固醇群、 碳水化合物和寡.。熟諳此藝者應瞭解 像是白蛋白及其類似物，亦包括右太ιΒη a田的媒"， J I栝在本發明的範圍内。Peptide SEQ ID NO: 12-9-1 149 GFEYCDGMEDPFTFGCDKQT 12-9-2 150 KLEYCDGMEDPFTQGCDNOS 12-9-3 151 LQEWCEGVEDPFTFGCEKOR 12-9-4 152 AQDYCEGMEDPFTFGCEMQK 12-9-5 153 LLDYCEGVQDPFTFGCENLD 12-9-6 154 HQEYCEGMEDPFTFGCEYQG 12- 9-7,155 MLDYCEGMDDPFTFGCDKQM 12-9-C2 156 LQDYCEGVEDPFTFGCENQR 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR 12-9 5 FDYCEGVEDPFTFGCDNH In an excellent embodiment, the invention relates to a composition having the following formula: a polymer, wherein: F1 is a medium; X1 and X2 are respectively selected from -(L^s-P1; -(L^s-P'-CL^t-P2; -(L^sP^CL^tP^CL^ u-P3; ^ -(L1)s-P1-(L2)t-P2-(L3)u-P3-(L4)v.P4; wherein one or more of P1, p2, P3 and P4 respectively comprise A polypeptide consisting of: (a) the amino acid sequence WDPWT (SEQ ID NO: 65), wherein the polypeptide is from 5 to 50 amino acids in length; (b) the amino acid sequence WDPWTC (sequence) Identification · · 66); (c) Amino acid sequence Cz2WDPWT (SEQ ID NO: 67), wherein z2 is an acidic or neutral polar amino acid residue; O: \121\121929DOC -28-2 00808833 (d) Amino acid sequence Cz2WDPWTC (SEQ ID NO: 68), wherein Z2 is an acidic or neutral polar amino acid residue; (e) Amino acid sequence pc2Dc4Lc6c7c8LY (SEQ ID NO: 71), wherein c2 Is a neutral hydrophobic amino acid residue; c4 is A, D or E; c6 is an acidic amino acid residue; c7 is an amino acid residue; and c8 is neutral hydrophobic, neutral polarity Or basic amino acid residue; (f) amino acid sequence RPe3e4e5e6e7G (SEQ ID NO: 73), wherein e3 is a neutral polar amino acid residue; e4 is an acidic amino acid residue; E5 is a neutral polar or acidic amino acid residue; e6 is a neutral hydrophobic amino acid residue; and e7 is a neutral hydrophobic amino acid residue; (g) amino acid sequence Cg2Gg4g5DPFTg1 () GCg13 (sequence recognition: No. 75), wherein g2 is an acidic amino acid residue; g4 is a neutral hydrophobic amino acid residue; and g5 is a neutral polar or acidic amino acid residue; Gio is a neutral hydrophobic or neutral polar amino acid residue; and ^3 is an acidic residue; (h) sequence recognition: polypeptide number 1; (i) sequence recognition: 2 And (j) sequence recognition: polypeptide of No. 7; wherein L1, L2, L3 and L4 are respectively a linker; and q, r, s, t, ^ port v are respectively 0 or 1, the limitation condition is At least one of c^〇r is 丨; and its physiologically acceptable salts. It will be appreciated that the invention further relates to fusion polypeptides comprising at least one peptide and vehicle as described herein, wherein the fusion polypeptide is capable of interacting with Ang-2U' and physiologically acceptable salts thereof. In the fusion polypeptide, the medium = preferably at least - an Fc functional site, & ethylene glycol, lipids, cholesterol groups, carbohydrates and oligos. Those skilled in the art should be aware of albumin and its analogs, as well as media of the right Β Β a field, J I 栝 within the scope of the present invention.
O:\121\121929.DOC -29- 200808833 熟諳此藝者將承認可將各種分子插入專一結合劑的結構 内。因此，可在例如專一結合劑的肽和媒介部分之間插入 特定的分子，或是在肽部分本身内插入，同時仍保留該專 一結合劑的想要活性。可輕易地插入例如分子，像是F c功 能部位或其片段、聚乙二醇或其他相關的分子，像是葡聚 醣、脂肪酸、脂質、膽固醇群、小的碳水化合物、肽、細 胞毒性製劑、化學治療劑、如同在本文中描述的可檢測部 分（包括螢光劑、放射性標記，像是放射性同位素）和寡醣 、寡核苷酸、多核苷酸、干涉（或其他的）RNA、酵素、荷 爾蒙或其類似物。熟諳此藝者應瞭解其他適合以此方式插 入的分子，並包括在本發明的範圍内。這包括例如在兩個 連續的胺基酸之間插入想要的分子，可視需要藉著適當的 聯結子連接。舉例來說，在Con4(C)肽體序列中： M-Fc-GGGGGAQQEECEWDPWTCEHMLE(序列識別：23號） 熟諳此藝者可輕易地在例如兩個相鄰的穀胺醯胺（nQQ’’) 殘基之間插入想要的分子，獲得想要的結構及/或功能， 同時仍保留該肽與Ang-2結合的能力。因此，可如下修改 該序列：O:\121\121929.DOC -29- 200808833 Those skilled in the art will recognize that various molecules can be inserted into the structure of a specific binder. Thus, a particular molecule can be inserted between, for example, the peptide and the vector portion of the specific binding agent, or inserted within the peptide portion itself while still retaining the desired activity of the specific binding agent. It is easy to insert, for example, molecules such as F c functional sites or fragments thereof, polyethylene glycol or other related molecules such as dextran, fatty acids, lipids, cholesterol groups, small carbohydrates, peptides, cytotoxic agents Chemotherapeutic agents, detectable moieties as described herein (including fluorescent agents, radioactive labels such as radioisotopes) and oligosaccharides, oligonucleotides, polynucleotides, interference (or other) RNA, enzymes , hormones or the like. Those skilled in the art will recognize other molecules suitable for insertion in this manner and are included within the scope of the invention. This includes, for example, the insertion of the desired molecule between two consecutive amino acids, optionally via a suitable linker. For example, in the Con4(C) peptidic sequence: M-Fc-GGGGGAQQEECEWDPWTCEHMLE (sequence recognition: No. 23) skilled in this art can easily be afflicted, for example, in two adjacent glutamine (nQQ'') The desired molecule is inserted between the bases to obtain the desired structure and/or function while still retaining the ability of the peptide to bind to Ang-2. Therefore, the sequence can be modified as follows:
M_Fc-GGGGGAQ-[分子]-QEECEWDPWTCEHMLE 如果需要可加入適當的聯結子分子。更知道可將該分子 插入在分子上的許多位置，包括在適當的側鏈上，在媒介 和肽序列之間，如下：M_Fc-GGGGGAQ-[Molecule]-QEECEWDPWTCEHMLE The appropriate linker molecule can be added if desired. It is further known that the molecule can be inserted at a number of positions on the molecule, including on the appropriate side chain, between the vector and the peptide sequence, as follows:
M-Fc-[分子]-GGGGGAQQEECEWDPWTCEHMLE 或是在任何熟諳此藝者想要的其他位置。熟諳此藝者應 瞭解其他適當的具體實施例。 在另一個具體實施例中，本發明係關於編碼如同在本文 O:\121\121929.DOC -30- 200808833 中描述之本發明專一結合劑（包括但不限於肽及/或肽體）的 多核苷酸。熟諳此藝者應瞭解在已知胺基酸序列之處，可 使用已知的技術，輕易地判定相對應的核苷酸序列（們）。 參見，例如 Suzuki，D。An Introduction to Genetic Analysis，W.H. Freeman Pub. Co· (1986)。在下文中陳述編 碼本發明之肽的代表性核苷酸序列。熟諳此藝者將承認有 一個以上的密碼子可編碼特定的胺基酸，並因此本發明係 關於任何編碼本發明之肽及/或肽體的核苷酸序列。 肽 序列 識別 號 肽序列 代表性的 dna序列 Con4-44 76 PIRQEECDWDPWTCEHMWEV ccgatccgtcaggaagaatgcga ctgggacccgtggacctgcgaac acatgtgggaagtt(房列制： 159«) Con4-40 77 TNIQEECEWDPWTCDHMPGK accaacatccaggaagaatgcga atgggacccgtggacctgcgacc acatgccgggtaaa(房列HfcW : 160 號） Con4-4 78 WYEQDACEWDPWTCEHMAEV tggtacgaacaggacgcttgcga atgggacccgtggacctgcgaac acatggctgaagtt (序列城别·· 161¾) Con4-31 79 NRLQEVCEWDPWTCEHMENV aaccgtctgcaggaagtttgcgaa tgggacccgtggacctgcgaaca catggaaaacgtt (房列 1M*J ·· 162ft) Con4- C5 80 AATQEECEWDPWTCEHMPRS gctgctacccaggaagaatgcga atgggacccgtggacctgcgaac acatgccgcgttcc (序列 ·· 163¾) Con4-42 81 LRHQEGCEWDPWTCEHMFDW ctgcgtcaccaggaaggttgcga atgggacccgtggacctgcgaac acatgttcgactgg(序列識别： 164 號） O:\121\121929.DOC -31- 200808833M-Fc-[Molecule]-GGGGGAQQEECEWDPWTCEHMLE Or at any other location that is familiar to the artist. Those skilled in the art should be aware of other suitable embodiments. In another embodiment, the invention relates to a multinuclear encoding a specific binding agent (including but not limited to peptides and/or peptibodies) of the invention as described in the above: O:\121\121929.DOC -30-200808833 Glycosylate. Those skilled in the art will appreciate that where amino acid sequences are known, the corresponding nucleotide sequences can be readily determined using known techniques. See, for example, Suzuki, D. An Introduction to Genetic Analysis, W.H. Freeman Pub. Co. (1986). Representative nucleotide sequences encoding the peptides of the invention are set forth below. Those skilled in the art will recognize that more than one codon can encode a particular amino acid, and thus the invention relates to any nucleotide sequence encoding a peptide and/or peptidide of the invention. Peptide SEQ ID NO peptide sequence representative dna sequence Con4-44 76 PIRQEECDWDPWTCEHMWEV ccgatccgtcaggaagaatgcga ctgggacccgtggacctgcgaac acatgtgggaagtt (manufactured by column room: 159 «) Con4-40 77 TNIQEECEWDPWTCDHMPGK accaacatccaggaagaatgcga atgggacccgtggacctgcgacc acatgccgggtaaa (Room column HfcW: No. 160) Con4-4 78 WYEQDACEWDPWTCEHMAEV tggtacgaacaggacgcttgcga atgggacccgtggacctgcgaac acatggctgaagtt (sequence not City ·· 161¾) Con4-31 79 NRLQEVCEWDPWTCEHMENV aaccgtctgcaggaagtttgcgaa tgggacccgtggacctgcgaaca catggaaaacgtt (column housing 1M * J ·· 162ft) Con4- C5 80 AATQEECEWDPWTCEHMPRS gctgctacccaggaagaatgcga atgggacccgtggacctgcgaac acatgccgcgttcc (sequence ·· 163¾) Con4-42 81 LRHQEGCEWDPWTCEHMFDW ctgcgtcaccaggaaggttgcga atgggacccgtggacctgcgaac acatgttcgactgg ( Sequence identification: No. 164) O:\121\121929.DOC -31- 200808833
Con4-35 82 VPRQKDCEWDPWTCEHMYVG gttccgcgtcagaaagactgcga atgggacccgtggacctgcgaac acatgtacgttggt (房列識別·· 165ft) Con4-43 83 SISHEECEWDPWTCEHMQVG tccatctcccacgaagaatgcgaa tgggacccgtggacctgcgaaca catgcaggttggt (序列識別： 360號） Con4-49 84 WAAQEECEWDPWTCEHMGRM tgggctgctcaggaagaatgcga atgggatccgtggacttgcgaaca catgggtcgtatg (房列識利： 166 號） Con4-27 85 TWPQDKCEWDPWTCEHMGST acttggccgcaggacaaatgcga atgggatccgtggacttgcgaaca catgggttctact (序列_ : 167 號） Con4-48 86 GHSQEECGWDPWTCEHMGTS ggtcactcccaggaagaatgcgg ttgggacccgtggacctgcgaac acatgggtacgtcc (房列細： 168 號） Con4-46 87 QHWQEECEWDPWTCDHMPSK cagcactggcaggaagaatgcga atgggacccgtggacctgcgacc acatgccgtccaaa(岸列·· 169 號） Con4-41 88 NVRQEKCEWDPWTCEHMPVR aacgttcgtcaggaaaaatgcgaa tgggacccgtggacctgcgaaca catgccggttcgt (岸列細： no號） Con4-36 89 KSGQVECNWDPWTCEHMPRN aaatccggtcaggttgaatgcaac tgggacccgtggacctgcgaaca catgccgcgtaac (房列識利： m號）· Con4-34 90 VKTQEHCDWDPWTCEHMREW gttaaaacccaggaacactgcga ctgggacccgtggacctgcgaac acatgcgtgaatgg (片列勒J: 172 號） Con4-28 91 AWGQEGCDWDPWTCEHMLPM gcttggggtcaggaaggttgcga ctgggacccgtggacctgcgaac acatgctgccgatg(序列識利： 173 號） Con4-39 92 PVNQEDCEWDPWTCEHMPPM ccggttaaccaggaagactgcga atgggacccgtggacctgcgaac acatgccgccgatg(房列: Π4號） O:\121\121929.DOC -32- 200808833Con4-35 82 VPRQKDCEWDPWTCEHMYVG gttccgcgtcagaaagactgcga atgggacccgtggacctgcgaac acatgtacgttggt (Room sequence identification ·· 165ft) Con4-43 83 SISHEECEWDPWTCEHMQVG tccatctcccacgaagaatgcgaa tgggacccgtggacctgcgaaca catgcaggttggt (SEQ ID: No. 360) Con4-49 84 WAAQEECEWDPWTCEHMGRM tgggctgctcaggaagaatgcga atgggatccgtggacttgcgaaca catgggtcgtatg (column room identification Lee: No. 166) Con4- 27 85 TWPQDKCEWDPWTCEHMGST acttggccgcaggacaaatgcga atgggatccgtggacttgcgaaca catgggttctact (_ sequence: No. 167) Con4-48 86 GHSQEECGWDPWTCEHMGTS ggtcactcccaggaagaatgcgg ttgggacccgtggacctgcgaac acatgggtacgtcc (room fine column: No. 168) Con4-46 87 QHWQEECEWDPWTCDHMPSK cagcactggcaggaagaatgcga atgggacccgtggacctgcgacc acatgccgtccaaa (shore column ·· No. 169) Con4-41 88 NVRQEKCEWDPWTCEHMPVR Aacgttcgtcaggaaaaatgcgaa tgggacccgtggacctgcgaaca catgccggttcgt (shore series: no) Con4-36 89 KSGQVECNWDPWTCEHMPRN aaatccggtcaggttgaatgcaac tgggacccgtggacctgcgaaca catgccgcgtaac (Rooms: m) · Con4-34 90 VKTQEHCDWDPWTCEHMREW g ttaaaacccaggaacactgcga ctgggacccgtggacctgcgaac acatgcgtgaatgg (column sheet Le J: No. 172) Con4-28 91 AWGQEGCDWDPWTCEHMLPM gcttggggtcaggaaggttgcga ctgggacccgtggacctgcgaac acatgctgccgatg (Lee identification sequence: No. 173) Con4-39 92 PVNQEDCEWDPWTCEHMPPM ccggttaaccaggaagactgcga atgggacccgtggacctgcgaac acatgccgccgatg (Room column: No. Π4) O: \ 121 \ 121929. DOC -32- 200808833
Con4-25 93 RAPQEDCEWDPWTCAHMDIK cgtgctccgcaggaagactgcga atgggacccgtggacctgcgctc acatggacatcaaa (序列狱J: 175¾) Con4-50 94 HGQNMECEWDPWTCEHMFRY cacggtcagaacatggaatgcga atgggacccgtggacctgcgaac acatgttccgttac (序列: 176 號） Con4,38 95 PRLQEECVWDPWTCEHMPLR ccgcgtctgcaggaagaatgcgtt tgggacccgtggacctgcgaaca catgccgctgcgt (外列識别： 177¾) Con4-29 96 RTTQEKCEWDPWTCEHMESQ cgtaccacccaggaaaaatgcga atgggacccgtggacctgcgaac acatggaatcccag (4 列細： 178 號） Con4-47 97 QTSQEDCVWDPWTCDHMVSS cagacctcccaggaagactgcgtt tgggacccgtggacctgcgacca catggtttcctcc (序列拟ij ·· 179 號） Con4-20 98 QVIGRPCEWDPWTCEHLEGL caggttatcggtcgtecgtgcgaa tgggacccgtggacctgcgaaca cctggaaggtctg (外列識利： 180 號） Con4-45 99 WAQQEECAWDPWTCDHMVGL tgggctcagcaggaagaatgcgc ttgggacccgtggacctgcgacc acatggttggtctg (序列識利： :181 號） Con4-37 100 LPGQEDCEWDPWTCEHMVRS ctgccgggtcaggaagactgcga atgggacccgtggacctgcgaac acatggttcgttcc (年列_ : 182¾) · Con4-33 101 PMNQVECDWDPWTCEHMPRS ccgatgaaccaggttgaatgcga ctgggacccgtggacctgcgaac acatgccgcgttcc (序列渺J: 183¾) AC2- Con4 102 FGWSHGCEWDPWTCEHMGST ttcggttggtctcacggttgcgaat gggatccgtggacttgcgaacac atgggttctacc《房列細： 184 號） Con4-32 103 KSTQDDCDWDPWTCEHMVGP aaatccacccaggacgactgcga ctgggacccgtggacctgcgaac acatggttggtccg丨(岸列識利： 185¾) O:\121\121929.DOC -33- 200808833Con4-25 93 RAPQEDCEWDPWTCAHMDIK cgtgctccgcaggaagactgcga atgggacccgtggacctgcgctc acatggacatcaaa (sequence prison J: 175¾) Con4-50 94 HGQNMECEWDPWTCEHMFRY cacggtcagaacatggaatgcga atgggacccgtggacctgcgaac acatgttccgttac (sequence: No. 176) Con4,38 95 PRLQEECVWDPWTCEHMPLR ccgcgtctgcaggaagaatgcgtt tgggacccgtggacctgcgaaca catgccgctgcgt (outer columns identified: 177¾) Con4-29 96 RTTQEKCEWDPWTCEHMESQ cgtaccacccaggaaaaatgcga atgggacccgtggacctgcgaac acatggaatcccag (4 fine column: No. 178) Con4-47 97 QTSQEDCVWDPWTCDHMVSS cagacctcccaggaagactgcgtt tgggacccgtggacctgcgacca catggtttcctcc (ij ·· intended sequence number 179) Con4-20 98 QVIGRPCEWDPWTCEHLEGL caggttatcggtcgtecgtgcgaa tgggacccgtggacctgcgaaca cctggaaggtctg (Lee identification outer columns: No. 180) Con4-45 99 WAQQEECAWDPWTCDHMVGL tgggctcagcaggaagaatgcgc Ttgggacccgtggacctgcgacc acatggttggtctg (sequence recognition: :181) Con4-37 100 LPGQEDCEWDPWTCEHMVRS ctgccgggtcaggaagactgcga atgggacccgtggacctgcgaac acatggttcgttcc (year column _ : 1823⁄4) · Con4-33 101 PMNQVECDWDPWTCEHMPRS ccg atgaaccaggttgaatgcga ctgggacccgtggacctgcgaac acatgccgcgttcc (sequence Miao J: 183¾) AC2- Con4 102 FGWSHGCEWDPWTCEHMGST ttcggttggtctcacggttgcgaat gggatccgtggacttgcgaacac atgggttctacc "Room Fine column: No. 184) Con4-32 103 KSTQDDCDWDPWTCEHMVGP aaatccacccaggacgactgcga ctgggacccgtggacctgcgaac acatggttggtccg Shu (Shore column know Lee: 185¾) O: \ 121 \ 121929. DOC -33- 200808833
Con4-17 104 GPRISTCQWDPWTCEHMDQL ggtccgcgtatctccacctgccag tgggacccgtggacctgcgaaca catggaccagctg (序列·· 186 號） Con4-8 105 STIGDMCEWDPWTCAHMQVD tccaccatcggtgacatgtgcgaa tgggacccgtggacctgcgctca catgcaggttgac (庠列識利： 187ft) AC4_ Con4 106 VLGGQGCEWDPWTCRLLQGW gttctgggtggtcagggttgcgaa tgggacccgtggacctgccgtctg ctgcagggttgg (序列識利： 188%) Con4-l 107 VLGGQGCQWDPWTCSHLEDG gttctgggtggtcagggttgccag tgggacccgtggacctgctccca cctggaagacggt (序列識: 189¾) Con4- C1 108 TTIGSMCEWDPWTCAHMQGG accaccatcggttccatgtgcgaa tgggacccgtggacctgcgctca catgcagggtggt (4 列朗： 190¾) Con4-21 109 TKGKSVCQWDPWTCSHMQSG accaaaggtaaatccgtttgccag tgggacccgtggacctgctccca catgcagtccggt (序列識利： 191 號） Con4- C2 110 TTIGSMCQWDPWTCAHMQGG accaccatcggttccatgtgccag tgggacccgtggacctgcgctca catgcagggtggt (序列朗： 192¾) Con4-18 111 WYNEVVCEWDPWTCNHWDTP tgggttaacgaagttgtttgcgaat gggacccgtggacctgcaaccac tgggacaccccg (序列識利： 193¾) Con4-19 112 VVQVGMCQWDPWTCKHMRLQ gttgttcaggttggtatgtgccagt gggacccgtggacctgcaaacac atgcgtctgcag (序列識别： 194 號） O:\121\121929.DOC 34- 200808833Con4-17 104 GPRISTCQWDPWTCEHMDQL ggtccgcgtatctccacctgccag tgggacccgtggacctgcgaaca catggaccagctg (·· serial number 186) Con4-8 105 STIGDMCEWDPWTCAHMQVD tccaccatcggtgacatgtgcgaa tgggacccgtggacctgcgctca catgcaggttgac (Xiang Li knowledge column: 187ft) AC4_ Con4 106 VLGGQGCEWDPWTCRLLQGW gttctgggtggtcagggttgcgaa tgggacccgtggacctgccgtctg ctgcagggttgg (recognition sequence Lee: 188%) Con4-l 107 VLGGQGCQWDPWTCSHLEDG gttctgggtggtcagggttgccag tgggacccgtggacctgctccca cctggaagacggt (recognition sequence: 189¾) Con4- C1 108 TTIGSMCEWDPWTCAHMQGG accaccatcggttccatgtgcgaa tgggacccgtggacctgcgctca catgcagggtggt (4 Long column: 190¾) Con4-21 109 TKGKSVCQWDPWTCSHMQSG accaaaggtaaatccgtttgccag tgggacccgtggacctgctccca catgcagtccggt (Lee identification sequence: No. 191) Con4- C2 110 TTIGSMCQWDPWTCAHMQGG accaccatcggttccatgtgccag tgggacccgtggacctgcgctca catgcagggtggt ( Sequence RAN: 1923⁄4) Con4-18 111 WYNEVVCEWDPWTCNHWDTP tgggttaacgaagttgtttgcgaat gggacccgtggacctgcaaccac tgggacaccccg (sequence literacy: 1933⁄4) Con4-19 112 VVQVGMCQWDPWTCKHMRLQ gttgttc Aggttggtatgtgccagt gggacccgtggacctgcaaacac atgcgtctgcag (sequence identification: No. 194) O:\121\121929.DOC 34- 200808833
Con4-16 113 AVGSQTCEWDPWTCAHLVEV gctgttggttcccagacctgcgaat gggacccgtggacctgcgctcac ctggttgaagtt (岸列識: 195ft) Con4-l 1 114 QGMKMFCEWDPWTCAHIVYR cagggtatgaaaatgttctgcgaat gggacccgtggacctgcgctcac atcgtttaccgt (房列識别·· 196¾) Con4- C4 115 TTIGSMCQWDPWTCEHMQGG accaccatcggttccatgtgccag tgggacccgtggacctgcgaaca catgcagggtggt (序列識W : 197¾) Con4-23 116 TSQRVGCEWDPWTCQHLTYT acctcccagcgtgttggttgcgaat gggacccgtggacctgccagcac ctgacctacacc (4 列勒J: 198ft) Con4-15 117 QWSWPPCEWDPWTCQTVWPS cagtggtcctggccgccgtgcga atgggacccgtggacctgccaga ccgtttggccgtcc(房列識利： 199 號） Con4-9 118 GTSPSFCQWDPWTCSHMVQG ggtacctecccgtccttctgccagt gggacccgtggacctgctcccac atggttcagggt (岸列·· 200號） TN8- Con4 4 QEECEWDPWTCEHM caggaagaatgcgaatgggaccc atggacttgcgaacacatg (序列識別：201號） Ll-1 119 QNYKPLDELDATLYEHFIFHYT cagaactacaaaccgctggacga actggacgc^ccctgtacgaaca cttcatcttccactacacc (序列識別：202號） Ll-2 120 LNFTPLDELEQTLYEQWTLQQS ctgaacttcaccccgctggacgaa ctggaacagaccctgtacgaaca gtggaccctgcagcagtcc K序列識別：203號） Ll-3 121 TKFNPLDELEQTLYEQWTLQHQ accaaattcaacccgctggacga actggaacagaccctgtacgaac agtggaccctgcagcaccag (痔列識別：204號） O:\121\121929.DOC 35- 200808833 LI-4 122 VKFKPLDALEQTLYEHWMFQQA gttaaattcaaaccgctggacgct ctggaacagaccctgtacgaaca ctggatgttccagcaggct (序列識別：20$號） LI - 5 123 VKYKPLDELDEILYEQQTFQER gttaaatacaaaccgctggacgaa ctggacgaaatcctgtacgaacag cagaccttccaggaacgt (序列識別：206號） Ll-7 124 TNFMPMDDLEQRLYEQFILQQG accaacttcatgccgatggacgac ctggaacagcgtctgtacgaaca gttcatcctgcagcagggt (庠列識別：207號） Ll-9 125 SKFKPLDELEQTLYEQWTLQHA tccaaattcaaaccgctggacgaa ctggaacagaccctgtacgaaca gtggaccctgcagcacgct (序列識別：208tt) Ll-10 126 QKFQPLDELEQTLYEQFMLQQA cagaaattccagccgctggacga actggaacagaccctgtacgaac agttcatgctgcagcaggct (序列識別：209») Ll-11 127 QNFKPMDELEDTLYKQFLFQHS cagaacttcaaaccgatggacga attggaagacaccctgtacaaaca gttcctgttccagcactcc (序列識別：210號） LM2 128 YKFTPLDDLEQTLYEQWTLQHV tacaaattcaccccgctggacgac ctggaacagaccctgtacgaaca gtggaccctgcagcacgtt (房列識別：211號） Ll-13 129 QEYEPLDELDETLYNQWMFHQR caggaatacgaaccgctggacga actggacgaaaccctgtacaacc agtggatgttCcaccagcgt (房列識別：212號） LI -14 130 SNFMPLDELEQTLYEQFMLQHQ tccaacttcatgccgctggacgaa ctggaacagaccctgtacgaaca gttcatgctgcagcaccag (專列識別：213號） Ll-15 131 QKYQPLDELDKTLYDQFMLQQG * cagaaataccagccgctggacga actggacaaaaccctgtacgatca gttcatgctgcagcagggt (序列識別：214號） 36-Con4-16 113 AVGSQTCEWDPWTCAHLVEV gctgttggttcccagacctgcgaat gggacccgtggacctgcgctcac ctggttgaagtt (Shore column knowledge: 195ft) Con4-l 1 114 QGMKMFCEWDPWTCAHIVYR cagggtatgaaaatgttctgcgaat gggacccgtggacctgcgctcac atcgtttaccgt (Room sequence identification ·· 196¾) Con4- C4 115 TTIGSMCQWDPWTCEHMQGG accaccatcggttccatgtgccag tgggacccgtggacctgcgaaca catgcagggtggt (recognition sequence W: 197¾) Con4-23 116 TSQRVGCEWDPWTCQHLTYT acctcccagcgtgttggttgcgaat gggacccgtggacctgccagcac ctgacctacacc (4 columns Le J: 198ft) Con4-15 117 QWSWPPCEWDPWTCQTVWPS cagtggtcctggccgccgtgcga atgggacccgtggacctgccaga ccgtttggccgtcc (column room identification Lee: No. 199) Con4-9 118 GTSPSFCQWDPWTCSHMVQG ggtacctecccgtccttctgccagt gggacccgtggacctgctcccac atggttcagggt (shore column ·· No. 200) TN8- Con4 4 QEECEWDPWTCEHM caggaagaatgcgaatgggaccc atggacttgcgaacacatg (sequence identification: 201) Ll-1 119 QNYKPLDELDATLYEHFIFHYT cagaactacaaaccgctggacga actggacgc^ccctgtacgaaca cttcatcttccactacacc (sequence identification: 202) Ll-2 120 LNFTPLDELEQTLYEQWTLQQS ctgaacttcaccccgctggacg aa ctggaacagaccctgtacgaaca gtggaccctgcagcagtcc K SEQ ID: No. 203) Ll-3 121 TKFNPLDELEQTLYEQWTLQHQ accaaattcaacccgctggacga actggaacagaccctgtacgaac agtggaccctgcagcaccag (hemorrhoids sequence identification: No. 204) O: \ 121 \ 121929.DOC 35- 200808833 LI-4 122 VKFKPLDALEQTLYEHWMFQQA gttaaattcaaaccgctggacgct ctggaacagaccctgtacgaaca ctggatgttccagcaggct (SEQ ID: 20 $ No.) LI - 5 123 VKYKPLDELDEILYEQQTFQER gttaaatacaaaccgctggacgaa ctggacgaaatcctgtacgaacag cagaccttccaggaacgt (SEQ ID: No. 206) Ll-7 124 TNFMPMDDLEQRLYEQFILQQG accaacttcatgccgatggacgac ctggaacagcgtctgtacgaaca gttcatcctgcagcagggt (Xiang sequence identification: No. 207) Ll-9 125 SKFKPLDELEQTLYEQWTLQHA tccaaattcaaaccgctggacgaa ctggaacagaccctgtacgaaca gtggaccctgcagcacgct (SEQ ID: 208tt) ll- 10 126 QKFQPLDELEQTLYEQFMLQQA cagaaattccagccgctggacga actggaacagaccctgtacgaac agttcatgctgcagcaggct (sequence identification: 209») Ll-11 127 QNFKPMDELEDTLYKQFLFQHS cagaacttcaaaccgatggacga attggaagacaccctgtacaaaca gttcctgttccagcactcc (sequence identification: 210) L M2 128 YKFTPLDDLEQTLYEQWTLQHV tacaaattcaccccgctggacgac ctggaacagaccctgtacgaaca gtggaccctgcagcacgtt (Room sequence identification: No. 211) Ll-13 129 QEYEPLDELDETLYNQWMFHQR caggaatacgaaccgctggacga actggacgaaaccctgtacaacc agtggatgttCcaccagcgt (Room sequence identification: No. 212) LI -14 130 SNFMPLDELEQTLYEQFMLQHQ tccaacttcatgccgctggacgaa ctggaacagaccctgtacgaaca gttcatgctgcagcaccag (train identification: No. 213) Ll-15 131 QKYQPLDELDKTLYDQFMLQQG * cagaaataccagccgctggacga actggacaaaaccctgtacgatca gttcatgctgcagcagggt (sequence identification: 214) 36-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Ll-16 132 QKFQPLDELEETLYKQWTLQQR cagaaattccagccgctggacga actggaagaaaccctgtacaaac agtggaccctgcagcagcgt (序列識別：2〗5號） LM7 133 VKYKPLDELDEWLYHQFTLHHQ gttaaatacaaaccgctggacgaa ctggacgaatggctgtaccacca gttcaccctgcaccaccag i(序列識別：216號） Ll-18 134 QKFMPLDELDEILYEQFMFQQS cagaaattcatgccgctggacgaa ctggacgaaatcctgtacgaacag ttcatgttccagcagtccc (序列識別：217號） LM9 135 QTFQPLDDLEEYLYEQWIRRYH cagaccttccagccgctggacga cctggaagaatacttgtacgaaca gtggatccgtcgttaccac i庠列鏃別：218ft) Ll-20 136 EDYMPLDALDAQLYEQFILLHG gaagactacatgccgctggacgc tctggacgctcagctgtacgaaca gttcatcctgctgcacggt (序列識別：219號） Ll-21 137 HTFQPLDELEETLYYQWLYDQL cacaccttccagccgctggacga actggaagaaaccctgtactacca gtggctgtacgaccagctg (庠列識別：220ft) Ll-22 138 YKFNPMDELEQTLYEEFLFQHA tacaaattcaacccgatggacgaa ctggaacagaccctgtacgaaga attcctgttccagcacgct (庠列識別：221號） AC6-L1 139 TNYKPLDELDATLYEHWILQHS accaactacaaaccgctggacga actggacgctaccctgtacgaaca ctggatcctgcagcactcc (序列識別：222號） Ll-Cl 140 QKFKPLDELEQTLYEQWTLQQR cagaaattcaaaccgctggacga actggaacagaccctgtacgaac agtggaccctgcagcagcgt (房列識別：223號） L1-C2 141 TKFQPLDELDQTLYEQWTLQQR accaaattccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt (房列識別：224號） O:\121\121929.DOC 37- 200808833 L1-C3 142 TNFQPLDELDQTLYEQWTLQQR accaacttccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt i序列識別：225號） LI 6 KFNPLDELEETLYEQFTFQQ aaattcaacccgctggacgagctg gaagagactctgtacgaacagttt acttttcaacag (房列: 226號） Conl-1 143 AGGMRPYDGMLGWPNYDVQA gctggtggtatgcgtccgtacgac ggtatgctgggttggccgaactac gacgttcaggct (序列識利：. 227號） Con 1-2 144 QTWDDPCMHILGPVTWRRCI cagacttgggacgatccgtgcatg cacattctgggtccggttacttggc gtcgttgcatc i(岸列識利·· 228號） Con 1-3 145 APGQRPYDGMLGWPTYQRIV gctccgggtcagcgtccgtacga cggtatgctgggttggccgaccta ccagcgtatcgtt (岸列HUM : 229號） Con 1-4 146 SGQLRPCEEIFGCGTQNLAL tccggtcagctgcgtccgtgcgaa gaaatcttcggttgcggtacccag aacctggctctg (岸列拟》j : 230ft) Con 1-5 147 FGDKRPLECMFGGPIQLCPR ttcggtgacaaacgtccgctggaa tgcatgttcggtggtccgatccag ctgtgcccgcgt (岸列酬： 231«) Coni-6 148 GQDLRPCEDMFGCGTKDWYG ggtcaggacctgcgtccgtgcga agacatgttcggttgcggtaccaa agactggtacggt (年列細： 232¾) 12-9-1 149 GFEYCDGMEDPFTFGCDKQT ggtttcgaatactgcgacggtatg gaagacccgttcaccttcggttgc gacaaacagacc ^序列 : 233¾) 12-9-2 150 KLEYCDGMEDPFTQGCDNQS aaactggaatactgcgacggtatg gaagacccgttcacccagggttg cgacaaccagtcc (序列戴别： 234¾) 12-9-3 151 LQEWCEGVEDPFTFGCEKQR ctgcagRaatggtgcgaaggtgtt O:\121\121929.DOC -38- 200808833 gaagacccgttcaccttcggttgc gaaaaacagcgt (序列iftJW : 2355ft) 12-9-4 152 AQDYCEGMEDPFTFGCEMQK gctcaggactactgcgaaggtatg gaagacccgttcaccttcggttgc gaaatgcagaaa (序列拟d ·· 236號） 12-9-5 153 LLDYCEGVQDPFTFGCENLD ctgctggactactgcgaaggtgtt caggacccgttcaccttcggttgc gaaaacctggac (序列: 237ft) 12-9-6 154 HQEYCEGMEDPFTFGCEYQG caccaggaatactgcgaaggtat ggaagacccgttcaccttcggttg cgaataccagggt (序列蛾利： 238«) 12-9-7 155 MLDYCEGMDDPFTFGCDKQM atgctggactactgcgaaggtatg gacgacccgttcaccttcggttgc gacaaacagatg (岸列齡J : 239¾) 12-9-C2 156 LQDYCEGVEDPFTFGCENQR ctgcaggactactgcgaaggtgtt gaagacccgttcaccttcggttgc gaaaaccagcgt (岸列拟: 240號） 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR ctgcaggactactgcgaaggtgtt gaagacccgttcaccttcggttgc gaaaaacagcgt (外列·· 241ft) 12·9 5 FDYCEGVEDPFTFGCDNH ttcgactactgcgaaggtgttgaa gacccgttcactttcggctgtgata accac (房列識別：242號） Z在另一個具體實施例中，本發明係關於包括至少一個 本發明之多核甞酸的表現載體。在其他的具體實施例中’ 本發明係關於包括該表現載體的宿主細胞。應瞭解該宿主 細胞最好是原核生物的細胞（例如大腸桿菌細胞）或真核生 物的細胞。 本發明亦關於包括有效含量的如同在本文中描述之組合 物，與在藥學上可接受之載劑混合的醫藥組合物。 本發明亦關於在哺乳動物中，抑制不想要的血管生成作 用的方法，包括投與在治療上有效之含量的如同在本文中 -39-Ll-16 132 QKFQPLDELEETLYKQWTLQQR cagaaattccagccgctggacga actggaagaaaccctgtacaaac agtggaccctgcagcagcgt (SEQ ID: No. 2〗 5) LM7 133 VKYKPLDELDEWLYHQFTLHHQ gttaaatacaaaccgctggacgaa ctggacgaatggctgtaccacca gttcaccctgcaccaccag i (SEQ ID: No. 216) Ll-18 134 QKFMPLDELDEILYEQFMFQQS cagaaattcatgccgctggacgaa ctggacgaaatcctgtacgaacag ttcatgttccagcagtccc (SEQ ID: No. 217) LM9 135 QTFQPLDDLEEYLYEQWIRRYH cagaccttccagccgctggacga cctggaagaatacttgtacgaaca gtggatccgtcgttaccac i Xiang column arrowhead respectively: 218ft) Ll-20 136 EDYMPLDALDAQLYEQFILLHG gaagactacatgccgctggacgc tctggacgctcagctgtacgaaca gttcatcctgctgcacggt (SEQ ID: No. 219) Ll-21 137 HTFQPLDELEETLYYQWLYDQL cacaccttccagccgctggacga actggaagaaaccctgtactacca gtggctgtacgaccagctg (Xiang sequence identification: 220ft) Ll-22 138 YKFNPMDELEQTLYEEFLFQHA tacaaattcaacccgatggacgaa ctggaacagaccctgtacgaaga attcctgttccagcacgct (Xiang Column identification: No. 221) AC6-L1 139 TNYKPLDELDATLYEHWILQHS accaactacaaaccgctggacga actggacgctaccctgtacgaaca ctggatcctgcagcactcc (SEQ ID: No. 222) Ll-Cl 140 QKFKPLDELEQTLYEQWTLQQR cagaaattcaaaccgctggacga actggaacagaccctgtacgaac agtggaccctgcagcagcgt (Room sequence identification: No. 223) L1-C2 141 TKFQPLDELDQTLYEQWTLQQR accaaattccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt (Room sequence identification: No. 224) O: \ 121 \ 121929.DOC 37- 200808833 L1-C3 142 TNFQPLDELDQTLYEQWTLQQR accaacttccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt i SEQ ID: No. 225) LI 6 KFNPLDELEETLYEQFTFQQ aaattcaacccgctggacgagctg gaagagactctgtacgaacagttt acttttcaacag (room column: No. 226) Conl-1 143 AGGMRPYDGMLGWPNYDVQA gctggtggtatgcgtccgtacgac ggtatgctgggttggccgaactac gacgttcaggct (Lee recognition sequence: No. 227) Con 1-2 144 QTWDDPCMHILGPVTWRRCI cagacttgggacgatccgtgcatg cacattctgggtccggttacttggc gtcgttgcatc i (Lee ·· land identification number column 228) Con 1-3 145 APGQRPYDGMLGWPTYQRIV gctccgggtcagcgtccgtacga cggtatgctgggttggccgaccta ccagcgtatcgtt (shore column HUM: No. 229) Con 1-4 146 SGQLRPCEEIFGCGTQNLAL tccggtcagctgcgtccgtgcgaa gaaatcttcggtt gcggtacccag aacctggctctg (Shore column Quasi "j: 230ft) Con 1-5 147 FGDKRPLECMFGGPIQLCPR ttcggtgacaaacgtccgctggaa tgcatgttcggtggtccgatccag ctgtgcccgcgt (Shore pay columns: 231«) Coni-6 148 GQDLRPCEDMFGCGTKDWYG ggtcaggacctgcgtccgtgcga agacatgttcggttgcggtaccaa agactggtacggt (in small columns: 232¾) 12-9-1 149 GFEYCDGMEDPFTFGCDKQT ggtttcgaatactgcgacggtatg gaagacccgttcaccttcggttgc gacaaacagacc ^ sequence: 233¾) 12-9-2 150 KLEYCDGMEDPFTQGCDNQS aaactggaatactgcgacggtatg gaagacccgttcacccagggttg cgacaaccagtcc (not wearing sequence: 234¾) 12-9-3 151 LQEWCEGVEDPFTFGCEKQR ctgcagRaatggtgcgaaggtgtt O: \ 121 \ 121929.DOC -38- 200808833 gaagacccgttcaccttcggttgc gaaaaacagcgt (sequence iftJW : 2355ft) 12-9-4 152 AQDYCEGMEDPFTFGCEMQK gctcaggactactgcgaaggtatg gaagacccgttcaccttcggttgc gaaatgcagaaa (d ·· intended sequence No. 236) 12-9-5 153 LLDYCEGVQDPFTFGCENLD ctgctggactactgcgaaggtgtt caggacccgttcaccttcggttgc gaaaacctggac (sequence: 237ft) 12-9-6 154 HQEYCEGMEDPFTFGCEYQG caccaggaatactgcgaaggtat ggaagacccgttcaccttcggttg cgaatacca Gggt (sequence moth: 238«) 12-9-7 155 MLDYCEGMDDPFTFGCDKQM atgctggactactgcgaaggtatg gacgacccgttcaccttcggttgc gacaaacagatg (shore age J: 2393⁄4) 12-9-C2 156 LQDYCEGVEDPFTFGCENQR ctgcaggactactgcgaaggtgtt gaagacccgttcaccttcggttgc gaaaaccagcgt (shore list: 240) 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR ctgcaggactactgcgaaggtgtt gaagacccgttcaccttcggttgc gaaaaacagcgt (external · 241ft) 12·9 5 FDYCEGVEDPFTFGCDNH ttcgactactgcgaaggtgttgaa gacccgttcactttcggctgtgata accac (Room Identification: No. 242) Z In another embodiment, the invention relates to at least one polynuclear citric acid of the invention Performance carrier. In other specific embodiments, the invention relates to host cells comprising the expression vector. It is to be understood that the host cell is preferably a cell of a prokaryote (e.g., an E. coli cell) or a cell of a eukaryotic organism. The invention also relates to pharmaceutical compositions comprising an effective amount of a composition as described herein in admixture with a pharmaceutically acceptable carrier. The invention also relates to a method of inhibiting unwanted angiogenesis in a mammal, comprising administering a therapeutically effective amount as herein-39-
O:\121\121929.DOC 200808833 描，之多肽或組合物。本發明亦關於在哺乳動物中，調『 血官生成作用的方法，包括投與在治療上.有效之含量的: 同在本文中描述之多肽或組合物。本發明更有關於在哺乳 動物中，抑制其特徵為不想要的血管生成作用之腫瘤生長 用的方法，包括投與在治療上有效之含量的如同在本文^ 描述之多肽或組合物。此外，本發明亦關於在哺乳動物中 治療癌症的方法，包括投與在治療上有效之含量的如同在 本文中描述之多肽或組合物’以及化學治療劑。在較佳的 具體實施财’該化學治療劑為至少—種咖、^仙和 剋癌易(taX0tere)。然而’應瞭解亦可使用其他適當的化風 治療劑和癌症治療劑。 予 本發明亦關於在哺乳動物中’調節至少一種血管通透性 或血㈣出的方法’包括投與在治療上有效之含量的如同 在本文中描述之多肽或組合物。本發明更有關於在哺乳動 物中，治療至少一種眼睛的新生血管疾病、肥胖、血管母 細胞瘤、血管瘤、動脈粥樣硬化、炎症疾病、炎症病症、 動脈粥樣硬化、子宮内膜異位症、贅生物的疾病、與骨絡 有關的疾病或牛皮癖，包括招* |太、Zx rt 一 Μ群I括技與在治療上有效之含量的如 同在本文中描述之多肽或組合物。 應瞭解本發明之專-結合劑，可用來治療許多與取消管 制或不想要的Α管生成作用有關的疾病。這類疾病包括， f不限於眼睛的新血管形成，像是視網膜病（包括糖尿病 性的視網臈病和與年齡有關的黃斑變性）、牛皮癖、血其 母細胞瘤、血管瘤、動脈粥樣硬化、炎症疾病，像是類^O:\121\121929.DOC 200808833, a polypeptide or composition. The invention also relates to a method of modulating the production of blood in a mammal, comprising administering a therapeutically effective amount of: a polypeptide or composition as described herein. More particularly, the present invention relates to a method of inhibiting tumor growth characterized by unwanted angiogenic effects in a mammal, comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. Furthermore, the invention also relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein and a chemotherapeutic agent. In a preferred embodiment, the chemotherapeutic agent is at least - a type of coffee, a scent, and a taX0tere. However, it should be understood that other suitable chemotherapeutic agents and cancer therapeutics may also be used. The invention also relates to a method of modulating at least one vascular permeability or blood (four) in a mammal' comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. The invention further relates to the treatment of neovascular diseases, obesity, hemangioblastoma, hemangioma, atherosclerosis, inflammatory diseases, inflammatory conditions, atherosclerosis, endometriosis in at least one eye in a mammal. Diseases, neoplastic diseases, skeletal-related diseases or psoriasis, including polypeptides or compositions as described herein, in a therapeutically effective amount. It will be appreciated that the specific binding agents of the present invention can be used to treat a number of conditions associated with the elimination of tuberculosis or unwanted fistula formation. Such diseases include, f is not limited to the formation of new blood vessels in the eye, such as retinopathy (including diabetic visual rickets and age-related macular degeneration), psoriasis, blood blastoma, hemangioma, arterial porridge Sclerotherapy, inflammatory disease, like class ^
O:\121\121929.DOC -40- 200808833 :二風濕性的炎症疾病’尤其是關節炎(包括風濕性關節 二或其他的慢性炎症疾病，像是慢性氣喘、動脈或移 '的動脈粥樣硬化、子宮内膜異位症和贅生物的疾病， 2所1胃的固體腫瘤和液體腫繼白血病)。對熟諸此 二而δ可猎著投與專一結合劑治療的額外疾病是顯而 見的k類額外的疾病包括，但不限於肥胖、血管通透 水漏出，以及與骨骼有關的疾病’包括骨質疏鬆症 ,因此’本發明更有關於治療這些與取消管制或不想要之 血苔生成作用有關的疾病的方法。 從在本文中提供的揭示内容中，將更容易瞭解本發明的 其他具體實施例。 【實施方式】 在本文中使用的段落開頭，僅為了組織化的目的，而不 可解釋成以任何方式限制所描述的主題物質。 關於重組的DNA分子、蛋白質和抗體的產製，以及組織 培養和細胞轉化，均可使用標準技術。酵素反應和純化技 術，通常根據製造者的說明書來進行，或在此項技藝中， 使用傳統程序，像是在Sambr〇〇k等人（M〇lecuiar 叫： A Laboratory Manual. Cold Spring Harbor Laboratory Press，Cold Spring Harbor，NY [i989])中陳述的那些，或 按照在本文t描述的來完成。除非提供特定的定義，與在 本文中描述之實驗室程序和分析化學技術、合成有機化學 ，以及醫學和藥理化學一起使用的術語，是那些在此項技 藝中已熟知並普遍使用的。關於化學合成、化學分析、藥O:\121\121929.DOC -40- 200808833 : Two rheumatic inflammatory diseases 'especially arthritis (including rheumatoid joints II or other chronic inflammatory diseases such as chronic asthma, arteries or arteries) Sclerosing, endometriosis and neoplasms, 2 solid tumors and liquid swelling of the stomach followed by leukemia). For additional diseases that are accustomed to δ and can be hunted for treatment with a specific combination of agents, it is obvious that class K additional diseases include, but are not limited to, obesity, vascular permeable water leakage, and bone-related diseases' including Osteoporosis, therefore, the present invention is more concerned with methods of treating these diseases associated with deregulated or unwanted moss production. Other embodiments of the invention will be more readily apparent from the disclosure provided herein. [Embodiment] The paragraphs used herein are initially for organizational purposes only and are not to be construed as limiting the subject matter described in any way. Standard techniques can be used for the production of recombinant DNA molecules, proteins and antibodies, as well as tissue culture and cell transformation. Enzyme reaction and purification techniques are usually performed according to the manufacturer's instructions, or in the art, using traditional procedures, such as in Sambr〇〇k et al. (M〇lecuiar: A Laboratory Manual. Cold Spring Harbor Laboratory Press , those described in Cold Spring Harbor, NY [i989], or as described in t. Unless specific definitions are provided, the terms used in connection with the laboratory procedures and analytical chemistry techniques, synthetic organic chemistry, and medical and pharmacological chemistry described herein are those well known and commonly employed in the art. About chemical synthesis, chemical analysis, medicine
O:\121\121929.DOC -41 - 200808833 理學製備、調配和遽送，以及患者的處理，均可使用標準 技術。 定義 如下定義在本說明書中使用的名詞，除非另行以特定的 例子來限制之。 ’’Ang-2，，一詞意指在美國專利第6，166，185號之圖6中陳述 的多肽（Tie-2配體-2，，）或其片段，以及相關的多肽，包括 對偶基因變體、接合變體、衍生物、取代、刪除及/或插 入變體、融合肽和多肽，以及物種間的同系物。Ang_2多 肽可以或可以不含額外的終端殘基’例如前導序列、瞒準 序列、胺基終端之甲硫胺酸、胺基終端之甲硫胺酸和離胺 酉文殘基，及/或標籤或融合蛋白質序列，視製備其之方式 而定。 "在生物學上有活性的"一詞，當與Ang_24Ang_2專一結 合劑一起使用，意指該肽或多肽具有至少一種Ang_2或O:\121\121929.DOC -41 - 200808833 Standard techniques can be used for the preparation, formulation and delivery of physiology, as well as for the treatment of patients. Definitions The nouns used in this specification are defined as follows unless otherwise limited by specific examples. The term ''Ang-2,') means a polypeptide (Tie-2 ligand-2,,) or a fragment thereof as set forth in Figure 6 of U.S. Patent No. 6,166,185, and related polypeptides, including dual Gene variants, ligation variants, derivatives, substitutions, deletions and/or insertion variants, fusion peptides and polypeptides, and homologs between species. Ang_2 polypeptide may or may not contain additional terminal residues such as a leader sequence, a quasi-sequence, an amine-terminated methionine, an amine-terminated methionine and an amidoxime residue, and/or a tag Or fusion protein sequences, depending on the manner in which they are prepared. "Biologically active", when used in conjunction with Ang_24Ang_2 specific binder, means that the peptide or polypeptide has at least one Ang_2 or
Ang-2專一結合劑特有的活性。Ang_2的專一結合劑，可能 對於至少-種Ang_2的生物活性，具有激動劑、拮抗劑， 或中和或阻斷活性。 寻一結合劑"一詞，意指一分子，最好是蛋白質的分子 ’專-地與Ang_2結合’及其變體和料物，㈣在本文 中定義的。專—結合劑可以是蛋白質、肽、核酸、碳水化 合物：脂質’ 4小分子量的化合物’其優先與A㈣結合 。在較佳的具體實施例中，根據本發明之專—結合劑是肤 或肽體，以及其^、變體或衍生物，單獨或舆其他的胺Ang-2 is uniquely specific to the binding agent. A specific binding agent for Ang_2 may have an agonist, antagonist, or neutralizing or blocking activity for at least the biological activity of Ang_2. The term "binding agent" means a molecule, preferably a protein molecule, 'specifically bound to Ang_2' and its variants and materials, (iv) as defined herein. The specific binder may be a protein, a peptide, a nucleic acid, a carbohydrate: a lipid '4 small molecular weight compound' which preferentially binds to A (d). In a preferred embodiment, the specific binding agent according to the present invention is a peptide or a peptide, and a compound, derivative or derivative thereof, alone or in addition to other amines.
O:\12W121929.OOC -42- 200808833 基酸序列結合，由已知的技術提供。這類技術包括，但不 限於酵素切開、化學切開、肽合成或重組技術。本發明之 抗-Ang-2專一結合劑能夠與Ang-2的結合部分結合，調節 ’例如抑制或促進Ang-2的生物活性及/或其他的Ang-2-相 關活性。 在本文中使用的”變體”一詞，包括其中將胺基酸殘基插 入結合劑之天然存在的（或至少一個已知的）胺基酸序列内 ’從其中刪除胺基酸殘基，及/或在其内取代的那些肽和 多肽。本發明之變體包括如同下述的融合蛋白質。 π衍生物，，包括那些已經以化學方式修改，以一些與插入 、刪除或取代變體不同的方式修改的結合劑。 ”專一地與Ang-2結合”，意指本發明之專一結合劑（像是 肽體或其肽部分）’認出並與成熟的、全長或部分長度的 人類Ang-2多肽或其異物種同源基因（〇rth〇i〇g)的能力，使 得其親和力（藉著例如如同在本文中描述的親和力ELISA或 BIAcore測定來判定），或其中和能力（藉著例如如同在本文 中描述的中和ELISA測定，或類似的測定來判定），為相同 物對於任何其他的血管生成素，或其他肽或多肽的親和力 或中和能力的至少10倍那樣大，但可視需要至少5〇倍那樣 大，100、25 0或500倍那樣大，或甚至至少1〇〇〇倍那樣大 ，其中該肽體的肽部分首先與人類以部分融合，以便在這 類測定中進行評估。 ，’抗原決定位”一詞意指任何分子中，能夠在一或多個結 合劑的抗原結合區，被專一結合劑，例如肽體認出並被其 O:\121\121929.DOC -43- 200808833 結合的部分。抗原決定位通常包括具有化學活性的分子之 表面基團，像是例如胺基酸或碳水化合物側鏈，並具有特 定之三度空間的結構特徵，以及特定的電荷特徵。在本文 中使用的抗原決定位可以是相鄰或不-相鄰的。 11抑制並/或中和抗原決定位”一詞，為一抗原決定位，當 它被諸如肽體之類的專一結合劑結合時，結果在活體内、 在活體外或就地喪失了（或至少減少了）含有這類抗原決定 位之分子、細胞或生物的生物活性。在本發明的前後文中 ，中和抗原決定位係位在Ang-2之具有生物活性的區域上 ，或與其結合。或者，”激活抗原決定位，，一詞為一抗原決 定位，當它被諸如抗體之類的本發明之專一結合劑結合時 ，結果激活，或至少維持Ang-2的具有生物活性之構形。 ••肽體片段"一詞意指肽或多肽，其包括少於完全、完整 的肽體。 ’·天然存在的”一詞，當與諸如核酸分子、多肽、宿主細 胞及其類似物的生物學物質一起使用時，意指在自然界中 發現，且未被人類修改的那些。 ’’經過分離’’一詞，當與Ang-2或Ang-2的專一結合劑一起 使用時’意指不含至少一種在其天然環境中找到之污染多 肽或化合物的化合物，且最好實質上不含任何其他污染的 哺乳動物多肽，其將干擾它的治療或診斷用途。 π成熟π—詞，當與Ang-2肽體或其片段，或任何其他蛋 白質的Ang-2之專一結合劑一起使用時，意指缺乏前導或 信號序列的肽或多肽。當例如在原核生物宿主細胞中表現 O:\121\121929DOC -44 - 200808833 本發明之結合劑時，”成熟的”肽或多肽亦可包含額外的胺 基酸殘基（但仍缺乏前導序列），像是胺基終端的甲硫胺酸 ’或一或多個甲硫胺酸和離胺酸殘基。可利用以此方式產 製’有或無已經移除這些額外胺基酸殘基的肽或多肽。 π有效含量π或π在治療上有效之含量”，當與Ang-2之專 一結合劑一起使用時，意指該專一結合劑的含量，對於在 一或多種Ang-2之生物活性的程度上，支持可觀察的改變 是有用或必需的。該改變可以是增加或降低Ang-2活性的 程度。該改變最好是降低Ang-2之活性。 π肽體π —詞意指包括與至少一個肽附接之抗體FC功能部 位的分子。肽體的產製通常描述在2〇〇〇年5月4日發表之 PCT 出版物 WO 00/24782 中。 在本文中使用的”變體”一詞，包括諸如肽或肽-媒介組合 的那些分子，像是本發明之肽體，其中將胺基酸殘基插入 這類分子之胺基酸序列中，從其中刪除胺基酸殘基，及/ 或在其内取代。變體具有一或多個被插入的胺基酸，包括 如同下述的融合蛋白質。 ’’衍生物’’包括那些肽及/或肽-媒介組合，像是已經以化 學方式修改，以一些與插入、刪除或取代變體不同的方式 修改的肽體。 •，片段”一詞意指肽或肽-媒介組合，其包括少於這類肽及/ 或肽-媒介組合的全長胺基酸序列。這類片段可起因於例 如在胺基終端截短、在羧基-終端截短，及/或從該肽或肽-媒介組合的胺基酸序列中刪除内部的殘基（們）。片段可以 O:\121\121929.DOC -45- 200808833 疋另一種RNA接合，及/或在活體内或在活體外之蛋白酶 活性的結果。亦可藉著化學肽合成方法，或藉著修改編碼 肽、肽-媒介組合，或肽體之Fc部分及/或肽部分的多核苷 酸’來建構這類片段。 一詞意指一種類型的本發明之媒介，且包括抗體之 非-抗原-結合片段的序列，起因於完整抗體的蛋白水解消 化作用，無論是以單體或多聚體的形式。在本發明中， 之來源最好是完整的人類Fc，並可以是任何的免疫球蛋白 ，雖然IgGl和IgG2是較佳的。然而，在本文中亦包括一部 分是人類，或得自非_人類物種的Fc分子。Fc，s由單體的多 肽構成，可藉著共價（也就是二硫鍵）和非-共價的結合，將 其連接成二聚體或多聚體的形式。在天然Fc分子的單體亞 單元之間，分子間二硫鍵的數目範圍從丨至4，係視類別（ 例如 IgG、IgA、IgE)或亞類（例如 IgG1、IgG2、Ig(}3、O:\12W121929.OOC -42- 200808833 Base acid sequence binding, provided by known techniques. Such techniques include, but are not limited to, enzyme incision, chemical incision, peptide synthesis or recombinant techniques. The anti-Ang-2 specific binding agent of the present invention is capable of binding to a binding moiety of Ang-2, and modulating, for example, inhibiting or promoting the biological activity of Ang-2 and/or other Ang-2-related activities. The term "variant" as used herein, includes the insertion of an amino acid residue into a naturally occurring (or at least one known) amino acid sequence of a binding agent to 'deletion of an amino acid residue therefrom, And/or those peptides and polypeptides substituted therein. Variants of the invention include fusion proteins as described below. π derivatives, including those that have been chemically modified, modified in a manner different from the insertion, deletion or substitution variants. "Specially binding to Ang-2" means that a specific binding agent (such as a peptibody or a peptide portion thereof) of the present invention recognizes and is mature, full length or partial length of human Ang-2 polypeptide or a heterologous species thereof. The ability of a homologous gene (〇rth〇i〇g) to confer its affinity (by, for example, an affinity ELISA or BIAcore assay as described herein), or a neutralizing ability (by, for example, as described herein) A neutralization ELISA assay, or a similar assay, is as large as at least 10 times the affinity or neutralization ability of the same for any other angiopoietin, or other peptide or polypeptide, but may be at least 5 fold as desired Large, 100, 25 or 500 times as large, or even as large as at least 1 fold, wherein the peptide portion of the peptibody is first partially fused to humans for evaluation in such assays. The term 'antigenic epitope' means any molecule that is capable of being recognized by a specific binding agent, such as a peptide, in the antigen binding region of one or more binding agents and is O:\121\121929.DOC-43 - 200808833 The binding moiety. Antigenic epitopes typically include surface groups of chemically active molecules, such as, for example, amino acids or carbohydrate side chains, and have structural features of a particular tertiary space, as well as specific charge characteristics. The epitope used herein may be contiguous or non-adjacent. The term "inhibit and/or neutralize an epitope" is an epitope that is specifically bound by a peptide or the like. When the agents are combined, the biological activity of the molecule, cell or organism containing such epitopes is lost (or at least reduced) in vivo, in vitro or in situ. In the context of the present invention, the neutralizing epitope is located on or in association with the biologically active region of Ang-2. Alternatively, "activating an epitope, the term is an epitope, when it is bound by a specific binding agent of the invention, such as an antibody, the result is activated, or at least maintains a biologically active configuration of Ang-2. • • Peptide fragment " refers to a peptide or polypeptide that includes less than a complete, intact peptibody. The term 'naturally occurring', when used with, for example, nucleic acid molecules, polypeptides, host cells, and the like When used together with biological material, it means those found in nature and not modified by humans. ''Separated', when used with a specific binding agent for Ang-2 or Ang-2, 'meaning a compound that does not contain at least one contaminating polypeptide or compound found in its natural environment, and preferably the essence There are no other contaminating mammalian polypeptides that would interfere with its therapeutic or diagnostic use. π mature π-word, when used with an Ang-2 peptibody or a fragment thereof, or a specific binding agent for Ang-2 of any other protein, means a peptide or polypeptide lacking a leader or signal sequence. A "mature" peptide or polypeptide may also comprise additional amino acid residues (but still lacking a leader sequence) when, for example, a binding agent of the invention is expressed in a prokaryotic host cell; O: \121\121929DOC -44 - 200808833 , such as the amino terminal methionine' or one or more methionine and lysine residues. Peptides or polypeptides with or without the removal of these additional amino acid residues can be produced in this manner. π effective content π or π is a therapeutically effective amount", when used together with a specific binding agent for Ang-2, means the content of the specific binding agent, to the extent of biological activity of one or more Ang-2 It is useful or necessary to support an observable change. The change may be to increase or decrease the degree of Ang-2 activity. The change is preferably to reduce the activity of Ang-2. π Peptide π - the word means to include and at least one The molecule of the FC functional site of the peptide-attached antibody. The production of the peptibody is generally described in PCT publication WO 00/24782, published May 4, 2000. The term "variant" is used herein. Included are molecules such as peptide or peptide-media combinations, such as the peptibodies of the invention, wherein an amino acid residue is inserted into the amino acid sequence of such a molecule from which the amino acid residues are deleted, and / Or substituted therein. The variant has one or more inserted amino acids, including fusion proteins as described below. ''Derivatives'' include those peptides and/or peptide-media combinations, as already chemistry Way to modify some to insert, delete or replace Different ways to modify the body peptibody •, fragment "is intended to mean a peptide or peptide - medium composition, which comprises less than such peptides and / or peptide - amino acid sequence of the full length of the medium combination. Such fragments may result from, for example, truncation at the amino terminus, truncation at the carboxy-terminus, and/or deletion of internal residues from the amino acid sequence of the peptide or peptide-media combination. The fragment can be O:\121\121929.DOC -45- 200808833 疋 another RNA junction, and/or the result of protease activity in vivo or in vitro. Such fragments can also be constructed by chemical peptide synthesis methods, or by modifying polynucleotides encoding peptides, peptide-vehicle combinations, or Fc portions of peptides and/or peptide portions. The term means a type of vector of the invention and includes the sequence of the non-antigen-binding fragment of the antibody, resulting from proteolytic digestion of the intact antibody, whether in the form of a monomer or a multimer. In the present invention, the source is preferably intact human Fc, and may be any immunoglobulin, although IgG1 and IgG2 are preferred. However, a Fc molecule derived from humans or from non-human species is also included herein. Fc,s consists of a monomeric polypeptide which can be linked into a dimeric or multimeric form by covalent (i.e., disulfide bond) and non-covalent binding. Between monomeric subunits of a native Fc molecule, the number of intermolecular disulfide bonds ranges from 丨 to 4, depending on the class (eg, IgG, IgA, IgE) or subclass (eg, IgG1, IgG2, Ig(}3,
IgAl、IgA2)而定。天然Fc的一個實例是二硫_鍵結的二聚 體’起因於IgG的木瓜蛋白酶消化作用[參見E1Hs〇n等人 (1982), Nucl· Acids· Res· 10: 4071-9]。在本文中使用的"天 然的Fc’’一詞’通常是單體、二聚體和多聚體的形式。 f’Fc功能部位’’一詞，包括天然的Fc和Fc變體分子，以及 如同上文定義之序列。因為有Fc變體和天然的Fc，s，，，以功 能部位” 一詞包括單體或多聚體形式的分子，無論是從完 整抗體中消化或藉著其他方法產製的。 當應用在Fc功能部位或包括Fc功能部位之分子上時，”多 聚體” 一詞意指具有二或多個以共價、非共價之方式，IgAl, IgA2). An example of a native Fc is a disulfide-bonded dimer' resulting from papain digestion of IgG [see E1 Hs〇n et al. (1982), Nucl Acids Res 10: 4071-9]. The term "natural Fc'' as used herein is generally in the form of monomers, dimers, and multimers. The term f'Fc functional site'' includes natural Fc and Fc variant molecules, as well as sequences as defined above. Because there are Fc variants and natural Fc, s,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, When the Fc functional site or a molecule comprising an Fc functional site, the term "multimer" means having two or more in a covalent, non-covalent manner,
O:\121\121929.DOC -46- 200808833 或藉著共價和非共價之交互作㈣合的多肽鏈的 ⑽分子通常形成二聚體；IgM，五聚體；啡 。 而1gA，單體、二聚體、三聚體或四聚體。可藉著利： 之天然Ig來源的序列和所得的活性，或藉著衍生(C 之定義）天然的Fc，而形成多聚體。 又 當應用在Fc功能部位或包括氏功能部位之分子上時，” 二聚體’’-詞意指具有兩個以共價或非-共價之方式結 多肽鏈的分子。 ^ "媒介"-詞意指防止降解並/或增加半衰期，降低毒性， 降低免疫原性，或增加治療性蛋白f之生物活性的分子。 代表性的媒介包括Fc功能部位，以及直線的聚合物(例如 聚乙二醇（PEG)、聚離胺酸、葡聚膽等等）；支鏈的聚合物 (參見’例如Denkenwaher等人，1981年9月15曰發證之美 國專利第4,289,872號；Tam，1993年7月2()日發證之美國專 利第5,229,490號；Frechet等人，1993年1〇月28日發表之 WO 93/21259)，脂質；膽固醇群（像是類固醇）；碳水化合 物或寡醣；或任何與補救受體結合的天然或合成的蛋白質 、多肽或肽。在後文中進一步說明媒介。 衍生、衍生的或M經過衍生的，，一詞，分別包括過程 和所得的化合物，其中（1)具有環狀部分的化合物；例如， 在該化合物内的半胱胺醯基殘基之間交聯；（2)將該化合物 父聯’或具有父聯位置；例如該化合物具有半胱胺醯基殘 基，並因此在培養時或在活體内形成交聯的二聚體；（3)藉 著非肽鍵結置換一或多個肽鍵結；（4)藉著_NRR][、 O:\121\121929.DOC -47- 200808833O:\121\121929.DOC -46- 200808833 Or (10) molecules of the (4) polypeptide chain by covalent and non-covalent interactions usually form dimers; IgM, pentamer; And 1 gA, monomer, dimer, trimer or tetramer. The multimer can be formed by a sequence derived from the natural Ig source and the resulting activity, or by derivatization (as defined by C) a native Fc. Also when applied to a Fc functional site or a molecule comprising a functional site, a "dimer"' word means having two molecules that bind the polypeptide chain in a covalent or non-covalent manner. ^ "Media "-word means a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases the biological activity of therapeutic protein f. Representative media include Fc functional sites, as well as linear polymers (eg Polyethylene glycol (PEG), polylysine, glucosamine, etc.; branched polymers (see, for example, Denkenwaher et al., U.S. Patent No. 4,289,872 issued September 15, 1981; Tam, U.S. Patent No. 5,229,490 issued July 2, 1993; Frechet et al., WO 93/21259, published on January 28, 1993, lipids; cholesterol groups (like steroids); carbohydrates or oligosaccharides a sugar; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor. The medium is further described hereinafter. Derivatized, derived or M-derived, the term includes the process and the resulting compound, respectively. (1) has a ring a compound; for example, cross-linking between cysteamine residues in the compound; (2) parenting of the compound or having a paternal position; for example, the compound having a cysteamine-based residue, And thus forming cross-linked dimers during culture or in vivo; (3) replacing one or more peptide bonds by non-peptide bonds; (4) by _NRR][, O:\121\ 121929.DOC -47- 200808833
-NRC(0)R1、_NRC(0)0R1、_NRS(0)2R1、_NHC(0)NHR 、玻珀醯亞胺基基團’或經取代或未經取代之芊氧魏基_ NH-置換N-終端，其中以和！^及環取代基如同在後文中之 疋義’（5)藉者_C(0)R2或-NR3R4置換C -終端，其中R2、 R3和R4如同在後文中之定義；以及（6)其中個別的胺基酸 部分在整個處理之中，利用能夠與選出之側鏈或終端殘基 反應的製劑修改的化合物。在後文中進一步說明衍生物。 ”肽” 一詞意指大約3到大約75個胺基酸的分子，較佳的 疋大約5至50個胺基酸，更佳的是8至4〇個胺基酸，而大約 10至2 5個胺基酸的那些是最佳的。肽可以是天然存在或人 造的（也就是非-天然存在的）胺基酸序列。可藉著在本文中 陳述的任何方法來產製代表性的肽，像是在肽庫（例如噬 菌體展示庫）中所具有的、藉著化學合成來產製、藉著蛋 白質之消化作用而衍生，或使用重組DNA技術來產製。 ’’在藥理學上具有活性的"一詞，意指判定如此描述之物 夤具有衫響醫學參數（例如血壓、血球細胞計數、膽固 酉予合ϊ )或疾病狀態（例如癌症、自體免疫病症等等）的活性。 ’’拮抗劑肽’’或”抑制劑肽，，一詞，意指阻斷或以某些方式 干擾感興趣之相關蛋白質的生物活性，或具有可與感興趣 之相關蛋白質的已知拮抗劑或抑制劑相比擬之生物活性的 肽。因此，”Ang-2-拮抗劑肽”一詞，包括可視為或衍生成 具有Ang-2-拮抗特徵的肽。 此外’在本文中亦包括本發明化合物之在生理學上可接 受的鹽類。”在生理學上可接受的鹽類，，意指任何已知或稍-NRC(0)R1, _NRC(0)0R1, _NRS(0)2R1, _NHC(0)NHR, a boroinium iodide group' or a substituted or unsubstituted oxirane group _NH-substitution N-terminal, which is and! ^ and a ring substituent as in the following ' '(5) borrower _C(0)R2 or -NR3R4 replaces a C-terminus, wherein R2, R3 and R4 are as defined hereinafter; and (6) The individual amino acid moieties are utilized throughout the treatment, using compounds that are modified by the formulation that reacts with the selected side chain or terminal residue. The derivatives are further described later. The term "peptide" means a molecule of from about 3 to about 75 amino acids, preferably about 5 to 50 amino acids, more preferably 8 to 4 amino acids, and about 10 to 2 Those of the five amino acids are optimal. The peptide may be a naturally occurring or artificial (i.e., non-naturally occurring) amino acid sequence. Representative peptides can be produced by any of the methods set forth herein, such as in peptide libraries (eg, phage display libraries), by chemical synthesis, and by protein digestion. Or using recombinant DNA technology to produce. The term 'pharmacologically active' means determining that the substance so described has a medical parameter (eg blood pressure, blood cell count, cholesteric sputum) or disease state (eg cancer, self) Activity of a body immune disorder, etc.). ''antagonist peptide'' or 'inhibitor peptide,' means the biological activity that blocks or otherwise interferes with the protein of interest, or has a known antagonist that is associated with a protein of interest. Or a biologically active peptide compared to an inhibitor. Thus, the term "Ang-2-antagonist peptide" includes peptides that can be considered or derived as having Ang-2-antagonistic characteristics. Further, the invention is also included herein. a physiologically acceptable salt of a compound." A physiologically acceptable salt, meaning any known or slightly
O:\121\121929.DOC -48- 200808833 2發現是在藥學上可接受的鹽類…些特定的實例為：乙 酉文息，二氟乙酸鹽；氫卣化物，像是鹽酸鹽和氫溴酸鹽； 硫酸鹽；擰檬酸鹽；酒石酸鹽；乙醇酸鹽；和草酸鹽、甲 石黃酸鹽和罐酸鹽。 肽體 本發明一方面係關於Ang_2肽體的發展。蛋白質配體與 八又體的父互作用，通常在相對上較大的界面處發生。然 而，如同對於人類生長荷爾蒙與其反應所證實的，在界面 處僅有少數的關鍵殘基貢獻大多數的結合能。Clackson等 人Science 267:383·6 (1995)。龐大的蛋白質配體主要以 正確的拓樸學展示結合的抗原決定位，或提供與結合無關 的功能。因此，僅有”肽”長度的分子（通常是2至4〇個胺基 酸）可與特定的大蛋白質配體之受體蛋白質結合。這類肽 可模仿大蛋白質配體的生物活性（”肽激動劑”），或經由競爭 性結合作用，抑制大蛋白質配體的生物活性（，，肽拮抗劑"）。 已經顯現噬菌體展示技術，在確認這類肽激動劑和拮抗 劑時’是有效的方法。參見，例如Sc〇tt等人，Science 249. 386 (1990)，Devlin等人，Science 249:404 (1990); 1993年6月29日發證之美國專利第5,223,409號；1998年3月 31曰發證之美國專利第5,733,731號；1996年3月12日發證 之美國專利第5,4 98,530號；1995年7月11日發證之美國專 利第5,432,0 18號；1994年8月16日發證之美國專利第 5,3 38,665號；1999年7月13日發證之美國專利第5,922,545 號；1996年12月19日發表之w〇 96/40987 ;和1998年4月16 O:\121\121929.DOC -49- 200808833 曰發表之WO 98/15833 (分別以引用的方式併入本文中）。 在肽噬菌體展示庫中，可藉著與絲狀噬菌體的外殼蛋白融 合，來展示任意的肽序列。如果想要，可對固定-抗體的 受體之細胞外功能部位，以親和力-洗脫展示的肽。可藉 著連續循環的親和力純化作用和再繁殖，增強保留的噬菌 體。可疋序敢佳結合的肽，在一或多個在結構上相關的肽 家族中，確認出關鍵性的殘基。參見，例如Cwirla等人， Science 276: 1696-9 (1997)，其中確認出兩個不同的家族 。亦可提出可藉著丙胺酸掃描或藉著在DNA層面之突變生 成作用，安全地置換其殘基的肽序列。可創造並篩選突變 庫’以便更充份地運用最佳結合劑之序列。L〇wrnan，Ann.O:\121\121929.DOC -48- 200808833 2 found to be a pharmaceutically acceptable salt...some specific examples are: acetaminophen, difluoroacetate; hydroquinones such as hydrochloride and hydrogen Bromate; sulphate; sulphate; tartrate; glycolate; and oxalate, formate and pot. Peptibodies One aspect of the invention relates to the development of Ang-2 peptibodies. Protein ligands interact with the octagonal father and usually occur at relatively large interfaces. However, as evidenced by human growth hormones and their responses, only a few key residues contribute most of the binding energy at the interface. Clackson et al. Science 267: 383. 6 (1995). Large protein ligands display the antigenic epitopes of binding in the correct topology, or provide functions unrelated to binding. Thus, only molecules of "peptide" length (usually 2 to 4 amino acids) can bind to receptor proteins of specific large protein ligands. Such peptides can mimic the biological activity of large protein ligands ("peptide agonists") or inhibit the biological activity of large protein ligands via competitive binding (, peptide antagonists "). Phage display technology has emerged as an effective method in identifying such peptide agonists and antagonists. See, for example, Sc〇tt et al., Science 249. 386 (1990), Devlin et al., Science 249: 404 (1990); U.S. Patent No. 5,223,409 issued June 29, 1993; March 31, 1998 U.S. Patent No. 5,733,731, issued on Jan. 12, 1996, and U.S. Patent No. 5,432,018, issued on Jul. 11, 1995; U.S. Patent No. 5,3,38,665 issued to U.S. Patent; U.S. Patent No. 5,922,545, issued on Jul. 13, 1999, and issued on December 19, 1996, the number of s. 96/40,987; and April 16, 2006: WO 121/15833 (hereby incorporated by reference herein in its entirety). In the peptide phage display library, any peptide sequence can be displayed by fusion with the coat protein of the filamentous phage. If desired, the displayed peptide can be eluted with affinity to the extracellular functional site of the immobilized-antibody receptor. The retained phage can be enhanced by continuous cycling of affinity purification and repopulation. Peptides that bind well to one another, in one or more structurally related peptide families, identify key residues. See, for example, Cwirla et al, Science 276: 1696-9 (1997), in which two different families are identified. Peptide sequences which can be safely replaced by alanine scanning or by mutation at the DNA level can also be proposed. The mutation library can be created and screened to more fully utilize the sequence of the optimal binder. L〇wrnan, Ann.
Rev· Biophys· Biomol· Struct. 26: 401-24 (1997) 〇 亦可使用蛋白質-蛋白質之交互作用的結構分析，提出 模仿大蛋白質配體之結合活性的肽。在這類分析中，結晶 結構可暗示大蛋白質配體之決定性殘基的身分和相關方位 便了攸其中來设計肽。參見，例如Takasaki等人，Rev· Biophys· Biomol· Struct. 26: 401-24 (1997) 肽 A peptide that mimics the binding activity of a large protein ligand can also be proposed using structural analysis of protein-protein interactions. In this type of analysis, the crystalline structure can suggest the identity and related orientation of the decisive residues of the large protein ligand to design the peptide. See, for example, Takasaki et al.
Nature Biotech 15·· 1266-70 (1997)。這些分析方法亦可用 來调查在受體蛋白質和藉著噬菌體展示而選出的肽之間的 父互作用，其可建議進一步修改肽，以便增加結合親和力。 其他的方法在肽研究中與噬菌體展示競爭。可將肽庫與 lac阻遏物的羧基終端融合，並在大腸桿菌中表現。其他以 大腸桿菌為基礎的方法，容許藉著與肽多醣-結合之脂蛋 白（PAL)融合，在細胞的外膜上展示。在後文中，將這些 和相關的方法集體地稱為”大腸桿菌展示"。在其他方法^Nature Biotech 15·· 1266-70 (1997). These assays can also be used to investigate the parental interaction between the receptor protein and the peptide selected by phage display, which may suggest further modification of the peptide to increase binding affinity. Other methods compete with phage display in peptide studies. The peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Other E. coli-based methods allow for display on the outer membrane of cells by fusion with peptidoglycan-bound lipoprotein (PAL). In the following, these and related methods are collectively referred to as "E. coli display". In other methods ^
O:\121\121929.DOC •50- 200808833 ，在核糖體釋放之前，使任意RNA的轉譯停止，導致多肽 庫將附加其相關的RNA。在後文中，將該方法及相關方法 集體地稱為”核糖體展示”。其他方法使用肽與RNA的化學 鍵結。參見’例如R〇berts 和 Szostak，Proc Natl Acad Sci USA’ 94: 12297-3 03 (1997)。在後文中，將該方法和相關 方法集體地稱為”RNA-肽篩選”。已經發展出以化學方式衍 生的肤庫’其中將肽固定在穩定的、非_生物學的材料上 ’像是聚乙烯棒或溶劑可通透的樹脂。其他以化學方式衍 生的肽庫，使用照相平版法，掃描固定在玻片上的肽。在 後文中，將该方法及相關方法集體地稱為"化學-肽篩選"。 化學-肽篩選可能是有利的，其容許使用D_胺基酸和其他 非天然的類似物，以及非-肽的元件。在WeUs*L〇wman，O:\121\121929.DOC •50- 200808833, stopping the translation of any RNA prior to ribosome release, causing the peptide library to attach its associated RNA. Hereinafter, the method and related methods are collectively referred to as "ribosome display". Other methods use chemical bonding of peptides to RNA. See, for example, R〇berts and Szostak, Proc Natl Acad Sci USA' 94: 12297-3 03 (1997). Hereinafter, the method and related methods are collectively referred to as "RNA-peptide screening". A chemically derived skin library has been developed in which the peptide is immobilized on a stable, non-biological material such as a polyethylene rod or a solvent permeable resin. Other chemically derived peptide libraries were scanned for peptides immobilized on slides using photolithography. Hereinafter, the method and related methods are collectively referred to as "chemical-peptide screening". Chemical-peptide screening may be advantageous, which allows the use of D-amino acids and other non-natural analogs, as well as non-peptide elements. At WeUs*L〇wman,
Curr· 〇pin· Bi〇techn〇1·，3: 355_62 (1992)中回顧生物學和 化學方法。 在概念上，可使用噬菌體展示和其他上文提及的方法， 發現任何蛋白質的肽模仿物。已經為了抗原決定位作圖、 在蛋白質-蛋白質之交互作用中確認決定性的胺基酸而使 用這些方法，並作為發現新治療劑的指導。參見，例如 Cortese等人，Curr_ 〇pin Bi〇tech 7· 616 2i (1996)。目前 最吊在免疫學的研究中使用肽庫，像是抗原決定位作圖。 參見 Kreeger，The Scientist 10(13): 19_20 (1996)。 已經將藉著噬菌體展示庫篩選所確認的肽，視為發展治 療劑的，，指導”，而非治療劑本身。像其他的蛋白質和肽一 樣，將可能迅速地在活體内藉著腎臟過濾、藉著在網狀内Review of biological and chemical methods in Curr·〇pin· Bi〇techn〇1·, 3: 355_62 (1992). Conceptually, peptide mimetics of any protein can be found using phage display and other methods mentioned above. These methods have been used for mapping antigenic epitopes, confirming decisive amino acids in protein-protein interactions, and as a guide for discovering new therapeutic agents. See, for example, Cortese et al., Curr_ 〇pin Bi〇tech 7·616 2i (1996). Peptide libraries are currently used in immunology research, such as epitope mapping. See Kreeger, The Scientist 10(13): 19_20 (1996). The peptides identified by screening the phage display library have been regarded as the development of therapeutic agents, and are not the therapeutic agents themselves. Like other proteins and peptides, they may be rapidly filtered in the living body by the kidneys. By being in the mesh
O:\121\121929.DOC -51 - 200808833 皮系統中的細胞清_制，或藉著蛋白水解的降解作用 [Frrcis，（在前）]移除它們。結果，該技藝目前使用肽，批 准樂物標㈣作為設計有機化合物㈣架其可能不像經由 化學庫筛選來確認的那樣容易或那樣快[Lowman，（在前）； W等人’（在前）]。該項技藝將獲益於過程，經由該過程 ，适類肤可更容易產㈣抗血管生成作用的治療劑。 肽艘的結構 ' 在根據本發明製備之物質的組合物中，可㈣肽的N'终 端或C'終端’將肽與媒介附接。因此，可藉著下列的五個 化學式及其多聚體，來描述本發明之媒介·肤分子：O:\121\121929.DOC -51 - 200808833 Cells in the dermal system are cleared, or they are removed by proteolytic degradation [Frrcis, (previous)]. As a result, the art currently uses peptides, which are approved as the design of organic compounds (4). They may not be as easy or as fast as confirmed by chemical library screening [Lowman, (previous); W et al. before)]. This skill will benefit from the process through which the appropriate skin can be more easily produced (4) anti-angiogenic therapeutic agents. Structure of the peptide boat 'In the composition of the substance prepared according to the present invention, the peptide can be attached to the medium by the N' terminal or C' terminal' of the (d) peptide. Therefore, the medium and skin molecules of the present invention can be described by the following five chemical formulas and their multimers:
[馮媒介（最好是Fc功 x>x2 分別選自、（从_Ρι· (L2)d-P2-(L3)e-P3 » ^•(L-)c-P1-(L2)d-P2-(L3)e.p3.(L4)f_p4 , ^2、？3和?4分別為具有藥學活性的肤， 中描述的；[Feng Media (preferably Fc work x) x2 are respectively selected from (from _Ρι· (L2)d-P2-(L3)e-P3 » ^•(L-)c-P1-(L2)d- P2-(L3)e.p3.(L4)f_p4, ^2, ?3 and ?4 are respectively described as pharmaceutically active peptides;
Li、L2、L3和L4分別為聯結子； 且"a”、”b"、"c"、"d"、"e"和”f 件為"a”和” b"中至少有一個是卜 ⑷或1 ’其限制條Li, L2, L3, and L4 are junctions, respectively; and "a", "b", "c", "d", "e" and "f are ""a" and "b" At least one of them is a (4) or 1 'limit strip
O:\121\121929.DOC -52- 200808833 肽 本發明期待選擇性地結合或專一地結合Ang-2的肽。任 何數目的這類肽，可連同本發明一起使用。在產製可在本 發明中使用的肽時，噬菌體展示是特別有用的，因為已經 顯示可使用來自任意肽之庫的親和力選擇，來確認任何基 因產物之任何位置的肽配體。Dedman等人，j. Bi〇1.O: \121\121929.DOC -52- 200808833 Peptide The present invention contemplates a peptide that selectively binds or specifically binds Ang-2. Any number of such peptides can be used in conjunction with the present invention. Phage display is particularly useful in the production of peptides that can be used in the present invention, as affinity ligand selection from libraries of any peptides has been shown to confirm peptide ligands at any position of any of the gene products. Dedman et al., j. Bi〇1.
Chem· 268: 23025-30 (1993)。 可藉著任何在此項技藝中揭示的方法，來製備本發明之 肽。使用單一字母的胺基酸縮寫。在任何序列中（且在本 說明書中，除非另行特別舉例指定）的，，χ”，意指2〇個天然 存在之胺基酸殘基中的任一個，或任何可出現之非-天然 存在的胺基酸（在下文”變體”中說明）。這些肽的任一個均 可以縱列連接（也就是連續地），有或無聯結子，並在表中 提供縱列_連接的實例。以”L”列舉聯結子，並可以是在本 文中描述的任何聯結子。為了清楚起見，以破折號分開所 示的縱列重覆段和聯結子。可使任何含有半胱胺酸殘基的 肽與其他含有Cys的肽交聯’其中任一或兩者皆可與媒介 連接。任何具有一個以上Cys殘基的肽，均可形成肽内的 二硫鍵結。這些肽中的任一個均可按照在本文中的描述衍 生。關於其中可利用胺基基團加帽之羧基終端的衍生物， 加帽之胺基基團為-ΝΑ。至於其中胺基酸殘基被胺基酸殘 基以外的部分取代的衍生物，以S代表取代作用，其表示 在 Bhatnagar 等人，J· Med. Chem· 39: 3814-9 (1996)和 Cuthbertscm等人，J· Med_ Chem. 40: 2876-82 (1997)中描 O:\121\121929.DOC -53- 200808833 除非另行 述的任何部分，將其以引用的方式併入本文中 指示’所有的肽均經由肽鍵結連接。 媒介 ▲在一個具體實施財，轉明提供至少一個月太，其經由 «亥肽（們）之N_終端、C·終端或—個胺基酸殘基之側鍵與至 少:個媒介（F,，F2)附接。亦可使用多個媒介，例如在每個 終端的Fc’s，或在一終端為Fc，且在另一個終端或側鏈處 為PEG基團。Chem. 268: 23025-30 (1993). The peptides of the present invention can be prepared by any of the methods disclosed in the art. A single letter amino acid abbreviation is used. In any sequence (and in the present specification, unless otherwise specified by way of example), "," means any one of two naturally occurring amino acid residues, or any non-naturally occurring one that may occur. The amino acids (described below) are described. Any of these peptides can be linked in series (i.e., continuously), with or without a linker, and provides an example of a column-linkage in the table. The linker is listed as "L" and may be any of the linkers described herein. For clarity, the column repeats and linkers are shown separated by a dash. Any cysteine-containing residue may be made. The peptide is cross-linked with other Cys-containing peptides. Either or both of them can be linked to a vector. Any peptide having more than one Cys residue can form a disulfide bond in the peptide. Any of these peptides It can be derivatized as described herein. Regarding the derivative of the carboxyl terminal in which the amine group can be capped, the capped amine group is -ΝΑ. The amino acid residue is dehydrated by the amino acid. Partially substituted derivatives other than the base, Table substitution, which is indicated in Bhatnagar et al, J. Med. Chem. 39: 3814-9 (1996) and Cuthbertscm et al, J. Med_Chem. 40: 2876-82 (1997) O:\121\ 121929.DOC-53-200808833 Unless otherwise stated, it is incorporated herein by reference to indicate that 'all peptides are linked via peptide linkages. Medium ▲ In a specific implementation, illuminate for at least one month Too, it is attached to at least one medium (F, F2) via a side bond of the N-terminal, C. terminal or -amino acid residue of the [Hyper Peptide]. Multiple media may also be used. For example, Fc's at each terminal, or Fc at one terminal, and a PEG group at another terminal or side chain.
Fc功犯部位是一種較佳的媒介。可將。功能部位與該肽 的N或C終端，或終端兩者融合。 如同上文提及的，在本發明之範圍内，Fc變體是適當的 變體。可廣泛地修改天然的^，形成根據本發明之以變體 ’其條件為仍維持與補救受體結合。參見，例如w〇 97/34631和WO 96/32478。在這類Fc變體中，可移除天然The Fc site is a preferred medium. Can be. The functional site is fused to both the N or C terminal of the peptide, or the terminal. As mentioned above, the Fc variant is a suitable variant within the scope of the invention. The natural ones can be extensively modified to form variants according to the invention', provided that the binding to the salvage receptor is still maintained. See, for example, w〇 97/34631 and WO 96/32478. In such Fc variants, natural removable
Fc的一或多個位置，其提供本發明之融合分子不需要的結 構特徵或功能活性。例如，可藉著取代或删除殘基、在該 位置插入殘基，或截短含有該位置的部分，移除這些位置 。插入或取代的殘基亦可以是經過更改的胺基酸，像是肽 模仿物或D-胺基酸。因為許多理由，可能想要&變體，在 下文中描述數個理由。代表性的F c變體包括分子和序列， 其中： 1·移除涉及二硫鍵形成的位置。這類移除可避免與出 現在用來產製本發明之分子的宿主細胞中，其他含有半胱 胺酸之蛋白質的反應。為了該目的，可截短在N-終端含有 O:\121\121929.DOC -54- 200808833 半脱胺酸的斷片’或可刪除半胱胺酸殘基，或以其他的胺 基&L (例如丙胺醯基、絲胺醯基）取代。甚至在移除半胱胺 酉文殘基^ ’單鍵的&功能部位仍可形成二聚體的R功能部 位，其以非-共價之方式維持在一起。 2·修改天然的Fc，使其較可與選出之宿主細胞相容。 例如可移除接近典型天然Fc之N-終端的PA序列，其可 被在大腸桿菌中之消化酵素認出，像是脯胺酸亞胺基肽酶 。亦可加入N-終端的甲硫胺醯基殘基，尤其是在以重組方 式在諸如大腸桿菌之類的細菌細胞中表現該分子時。 3·移除天然Fc的一部分]^_終端，以防止在選出之宿主 細胞中表現時，N-終端的異質性。為了該目的，可刪除任 何在N-終端前的20個胺基酸殘基，特別是在位置1、2、3 、4和5處的那些。 4·可移除一或多個糖基化作用位置。通常糖基化的殘 基（例如天冬醯胺），可賦與細胞溶解的反應。可刪除這類 殘基’或以未糖基化的殘基（例如丙胺酸）取代之。 5·移除涉及補體交互作用的位置，像是Clq結合位置。 例如，可刪除或取代人類IgG1的EKK序列。補體的徵募對 於本發明之分子可能不是有利的，故可利用這類F 〇變體避 免。 6·移除影響與補救受體以外之Fc受體的結合的位置。 天然的Fc可具有與某些白血球細胞產生交互作用的位置， 是本發明之融合分子不需要的，故可將其移除。 7.移除ADCC位置。ADCC位置是此項技藝中已知的。 O:\121\121929.DOC -55- 200808833 參見，例如 Molec· Immunol· 29(5): 033-9 (1992)，關於在 IgGl中的ADCC位置。該位置是本發明之融合分子完全不 需要的’故可將其移除。 8· g天然的Fc衍生自非_人類抗體時，可將該天然的Fc 人類化。通常，欲人類化天然的Fc，將在非-人類的天然 Fc中，以正常在人類天然Fc中發現的殘基，取代選出的殘 基。抗體人類化的技術是此項技藝中已熟知的。 另類的媒介將是能夠與補救受體結合的蛋白質、多肽、 肽、抗體、抗體片段或小分子（例如肽模仿化合物）。例如 ，可使用在Presta等人，於1998年4月14日發證的美國專利 第5,739,277號中描述的多肽作為媒介。亦可藉著噬菌體展 示’針對與FcRn補救受體的結合來選擇肽。這類補救受 體-結合化合物，亦被包括在，，媒介”的意義内，並在本發明 的範圍内。應針對增加半衰期（例如藉著避免被蛋白酶認 出之序列）和降低免疫原性（例如藉著偏愛非_免疫原性的序 列，如同在抗體人類化中揭示的）來選擇這類媒介。 如同上文提及的，關於^和匕亦可使用聚合物媒介。目 前可獲得各種附接可用來作為媒介之化學部分的方法，參 見，例如專利合作條約（”PCT”）國際出版物第w〇 96/1 1953 號，標題為”以化學方式修改終端的蛋白質組合物和方 法（N-Terminally Chemically Modified Protein CompositionsOne or more positions of the Fc that provide structural features or functional activities that are not required for the fusion molecules of the invention. For example, these positions can be removed by substituting or deleting residues, inserting residues at the position, or truncating the portion containing the position. The inserted or substituted residue may also be a modified amino acid such as a peptide mimetic or a D-amino acid. For many reasons, you may want & variants, which are described below for several reasons. Representative F c variants include molecules and sequences, wherein: 1. The position involved in the formation of disulfide bonds is removed. Such removal avoids the reaction with other cysteine-containing proteins in the host cells that are used to produce the molecules of the invention. For this purpose, a fragment containing O:\121\121929.DOC-54-200808833 semi-deaminic acid at the N-terminus can be truncated or the cysteine residue can be deleted, or with other amine groups &L Substituted (for example, alaninyl, serinyl). Even at the & functional site where the cysteamine residue ' single bond is removed, the R functional moiety of the dimer can be formed, which is maintained together in a non-covalent manner. 2. Modify the native Fc to be more compatible with the selected host cell. For example, a PA sequence close to the N-terminus of a typical native Fc can be removed, which can be recognized by digestive enzymes in E. coli, such as prolyl iminopeptidase. It is also possible to add an N-terminal methylthioguanidine residue, especially when the molecule is expressed in a bacterial cell such as E. coli in a recombinant manner. 3. Remove a portion of the native Fc]^ terminal to prevent heterogeneity of the N-terminus when expressed in the host cell of choice. For this purpose, any of the 20 amino acid residues preceding the N-terminus can be deleted, particularly those at positions 1, 2, 3, 4 and 5. 4. One or more glycosylation sites can be removed. Usually glycosylated residues (e.g., asparagine) can confer a lytic reaction. Such residues can be deleted or replaced with unglycosylated residues such as alanine. 5. Remove the location involved in the complement interaction, such as the Clq binding position. For example, the EKK sequence of human IgGl can be deleted or replaced. The recruitment of complement may not be advantageous for the molecules of the invention, and such F 〇 variants may be avoided. 6. Remove the location that affects binding to Fc receptors other than the salvage receptor. Natural Fc can have a position to interact with certain white blood cells, which is not required for the fusion molecules of the present invention and can be removed. 7. Remove the ADCC position. The ADCC position is known in the art. O:\121\121929.DOC-55-200808833 See, for example, Molec. Immunol 29(5): 033-9 (1992) for ADCC positions in IgG1. This position is completely unnecessary for the fusion molecule of the present invention, so it can be removed. When the native Fc is derived from a non-human antibody, the native Fc can be humanized. Typically, in order to humanize a native Fc, the selected residue will be replaced in a non-human native Fc with a residue normally found in human native Fc. Techniques for humanizing antibodies are well known in the art. An alternative medium would be a protein, polypeptide, peptide, antibody, antibody fragment or small molecule (eg, a peptide mimetic compound) that is capable of binding to a salvage receptor. For example, the polypeptide described in U.S. Patent No. 5,739,277, issued to thess. Peptides can also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of, and are within the scope of the invention, should be directed to increasing half-life (eg, by avoiding sequences recognized by proteases) and reducing immunogenicity. (For example, by preferring non-immunogenic sequences, as revealed in antibody humanization), such media are selected. As mentioned above, polymer media can also be used for ^ and 匕. A method of attaching a chemical moiety that can be used as a medium, see, for example, the Patent Cooperation Treaty ("PCT") International Publication No. WO 96/1 1953, entitled "Chemical Modification of Terminal Protein Compositions and Methods ( N-Terminally Chemically Modified Protein Compositions
and Methods)”，完整地以引用的方式併入本文中。該pcT 出版物中，揭示水溶性聚合物與蛋白質之N_終端的選擇性 附接。And Methods)", which is incorporated herein by reference in its entirety in its entirety, in the disclosure of the <RTIgt;
O:\121\121929.DOC -56- 200808833 車又挂的聚合物媒介是聚乙二醇（PEG)。PEG基團可具有 任何便利的分子量，並可以是直線或分支的。pEG的平均 分子置最好將是在大約千道爾吞（，，kDan)到大約1〇〇 kDa的 範圍内，更佳的是從大約5 kDa到大約50 kDa，最好是從 大約5 kDa到大約10 kDa。pEG基團通常將經由醯化作用或 逛原性烷基化作用，經由在PEG部分上的反應性基團（例如 醛、胺基、硫醇或酯基團）與在本發明化合物上的反應性 基團（例如醛、胺基或酯基團），與本發明之化合物附接。 合成肽之PEG化作用的有用策略，包括經由在溶液中形 成共軛鍵結，混合肽和PEG部分，其分別攜帶特殊的官能 度，互相對另一個反應。可利用此項技藝中已知的傳統固 相合成，輕易地製備肽。利用適當的官能基團，在指定的 位置預先激活"肽。純化前驅物，並在與pEG部分反應之 前，先完全定出特徵。肽與PEG的連接作用，通常在含水 相中發生，並可輕易地藉著逆相分析HPLC監視之。可藉 著製備HPLC輕易地純化pEG化的肽，並藉著分析HpLC、 胺基酸分析和激光解吸質譜分析定出特徵。 多醣聚合物是另一類的水溶性聚合物，其可用來進行蛋 白質修改。葡聚醣是多醣聚合物，包括主要藉著丨_6鍵結 連接的葡萄糖之個別亞單元。可利用很多分子量範圍的葡 聚酶本身，並可輕易地利用從大約1 kDa到大約7〇让£^的 分子量。對於在本發明中用來作為媒介而言，葡聚醣本身 或與其他媒介（例如Fc)混合，均是適當的水溶性聚合物。 參見’例如WO 96/1 1953和WO 96/05309。已經報告與治O:\121\121929.DOC -56- 200808833 The polymer medium that is attached to the car is polyethylene glycol (PEG). The PEG group can have any convenient molecular weight and can be straight or branched. The average molecular arrangement of pEG will preferably be in the range of from about 1,000 Torr (, kDan) to about 1 〇〇 kDa, more preferably from about 5 kDa to about 50 kDa, and most preferably from about 5 kDa. To about 10 kDa. The pEG group will typically react via reactive or alkylation via a reactive group (eg, an aldehyde, amine, thiol or ester group) on the PEG moiety to the compound of the invention. A moiety (such as an aldehyde, amine or ester group) is attached to a compound of the invention. A useful strategy for the PEGylation of synthetic peptides involves mixing the peptide and PEG moieties via a conjugated bond in solution, each carrying a particular functionality, and reacting to each other. Peptides can be readily prepared using conventional solid phase synthesis as known in the art. The "peptide is pre-activated at the designated position using the appropriate functional group. The precursor is purified and the characteristics are fully determined prior to reaction with the pEG moiety. The attachment of the peptide to PEG typically occurs in the aqueous phase and can be readily monitored by reverse phase analytical HPLC. The pEGylated peptide was readily purified by preparative HPLC and characterized by analysis of HpLC, amino acid analysis and laser desorption mass spectrometry. Polysaccharide polymers are another class of water soluble polymers that can be used to modify protein. Glucan is a polysaccharide polymer that includes individual subunits of glucose that are primarily linked by a 丨6 bond. A wide range of molecular weight ranges of the ligase itself can be utilized and the molecular weight of from about 1 kDa to about 7 Å can be readily utilized. For use as a vehicle in the present invention, glucan itself or a mixture with other vehicles (e.g., Fc) is a suitable water-soluble polymer. See, for example, WO 96/1 1953 and WO 96/05309. Reported and treated
O:\121\121929.DOC -57- 200808833 療性或診斷用的免疫球蛋白共輛之葡聚醣的用途；參見， 例如歐洲專利出版物第0 315 456號’將其以引用的方式併 入本文中。當根據本發明使用葡聚醣作為媒介時，最好是 大約1 kDa至大約20 kDa的葡聚醣。 聯結子 任何”聯結子，，基團均是任意的。當出現時，其化學結構 不具決定性，因為其主要擔任間隔基。聯結子最好是由胺 基酸組成，藉著肽鍵結連接在一起。因此，在較佳的具體 實施例中，聯結子由從丨至20個胺基酸組成，藉著肽鍵結 連接，其中該胺基酸係從20個天然存在的胺基酸中選出。 可將這些胺基酸中的一或多個糖基化，如同熟諳此藝者已 完全瞭解的。在更佳的具體實施例中，這丨至2〇個胺基酸 可從甘胺酸、丙胺酸、脯胺酸、天冬醯胺、榖胺醯胺和離 月女fee中選出。更佳的疋，聯結子大多是由無空間位阻的胺 基酸組成’像是甘胺酸和丙胺酸。因此，較佳的聯結子是 聚甘胺酸（特別是（Gly)5、（Gly)8)、聚（Gly_Ala)和聚丙胺酸 。Gly和Ala的組合亦是較佳的，像是在本文中稱為K1，並 具有在本文實例中陳述之胺基酸序列的聯結子。 亦可能使用非-肽聯結子。例如可使用烷基聯結子，像 疋·ΝΗ·((：Η2)8-(：(0)- ’其中s=2_20。這些烧基聯結子可進 一步被任何非-空間位阻的基團取代，像是低碳數烷基（例 如Ci_C6)低碳數醯基、鹵素（例如ci、Br)、CN、NH2、苯 基等等。代表性的非-肽聯結子是PEG聯結子，並具有1〇〇 kDa至5000 kDa之分子量，較佳的是1〇〇至5〇〇 kDa。可以 O:\121\121929.DOC -58- 200808833 形成衍生物。 與上述相同的方式更改肽聯結子 變趙和衍生物 專一結合劑的變體和衍生物，亦包 。/+ ^ j匕栝在本發明的範圍内 在，交體中，包括插入、刪除和取代 n夂體。應瞭解本發明 、疋的專〜合劑可含有一、二或所有三種類型的變體。 插入和取代變體可含有天_胺基酸、非傳統的胺基 如下文陳述），或兩者。 在一個實例中，提供其中一或多個胺基酸殘基，天然存 在的或㈣統的胺基酸，補充肽或肽體之胺基酸序列的插 入變體。插人可位在蛋白f的任_或兩端，或可位在肤體 胺基酸序㈣内部區域巾。在任—或兩端帶有額外殘基的 插入隻體’可包括例如融合蛋白冑，以及包括胺基酸標籤 或標記的蛋白質。包括肽和肽體的插入變體，丨中將一或 多個胺基酸殘基加至肽或肽體的胺基酸序列，或其片段。 本發明之變體產物亦包括成熟的肽和肽體，其中移除前 導或信號序列，且所得的蛋白質具有額外的胺基終端序列 ，該胺基酸可以是天然的或非-天然的。期待在胺基酸位 置-1處帶有額外甲硫胺醯基殘基的專一結合劑（像是肽體） (Met -肽體），像是在位置_2和_丨處帶有額外之甲硫胺酸和 離胺酸殘基的專一結合劑（Met'Lys — L)。帶有額外之Met 、Met-Lys、Lys殘基的變體（或一或多個鹼性殘基，一般 而a)’對於在細菌宿主細胞中提高重組蛋白質之產製是 特別有用的。 本發明亦包括具有起因於使用特定表現系統之額外胺基O:\121\121929.DOC -57- 200808833 The use of immunoglobulins for therapeutic or diagnostic use of glucan; see, for example, European Patent Publication No. 0 315 456 'by reference Into this article. When dextran is used as a medium according to the present invention, it is preferably a dextran of about 1 kDa to about 20 kDa. Any "linker" of a linker is arbitrary. When present, its chemical structure is not decisive because it acts primarily as a spacer. The linker is preferably composed of an amino acid, linked by a peptide bond. Thus, in a preferred embodiment, the linker consists of from hydrazine to 20 amino acids joined by peptide linkages wherein the amino acid is selected from 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated, as is well understood by those skilled in the art. In a more preferred embodiment, the guanidine to 2 amino acids can be derived from glycine. , alanine, lysine, aspartame, indoleamine and exfoliate fee. Better 疋, the linker is mostly composed of sterically hindered amino acid 'like glycine And alanine. Therefore, preferred linkers are polyglycine (especially (Gly) 5, (Gly) 8), poly (Gly_Ala) and polyalanine. Combinations of Gly and Ala are also preferred, Like a linker referred to herein as K1 and having the amino acid sequence set forth in the examples herein. Use a non-peptide linker. For example, an alkyl linker can be used, such as 疋·ΝΗ·((:Η2)8-(:(0)- ' where s=2_20. These alkyl linkers can be further protected by any non- Substituted sterically hindered groups such as lower alkyl (eg Ci_C6) lower fluorenyl, halogen (eg ci, Br), CN, NH2, phenyl, etc. Representative non-peptide linkers It is a PEG linker and has a molecular weight of from 1 〇〇 kDa to 5000 kDa, preferably from 1 〇〇 to 5 〇〇 kDa. A derivative can be formed from O:\121\121929.DOC -58- 200808833. Modifications of peptide linker variants and derivatives of derivative-specific binders, also included. /+ ^ j匕栝 Within the scope of the invention, including, insertion, deletion and substitution of n-steroids It will be understood that the invention may contain one, two or all three types of variants. Insertion and substitution variants may contain a-amino acid, an unconventional amine group as set forth below, or two In one example, one or more amino acid residues, naturally occurring or (tetra) amino acids, supplemental peptides or peptibodies are provided. An insertion variant of an amino acid sequence which can be inserted at any or both ends of the protein f, or can be placed in the internal region of the amino acid sequence of the dermaton. Intercalation with additional residues at any or both ends The body ' can include, for example, a fusion protein raft, and a protein including an amino acid tag or label. Insertion variants including peptides and peptibodies in which one or more amino acid residues are added to the peptide or peptibody Amino acid sequence, or a fragment thereof. The variant product of the invention also includes mature peptides and peptibodies, wherein the leader or signal sequence is removed, and the resulting protein has an additional amine-based terminal sequence, which may be Natural or non-native. A specific binder (like a peptide) (Met-peptide) with an additional methylthioamine thiol residue at position -1 of the amino acid is expected, as in position _2 And a specific binding agent (Met'Lys-L) with additional methionine and lysine residues. Variants with additional Met, Met-Lys, Lys residues (or one or more basic residues, generally a) are particularly useful for increasing the production of recombinant proteins in bacterial host cells. The invention also encompasses additional amine groups that result from the use of a particular performance system
O:\121\121929.DOC -59- 200808833 酸殘基的專一結合劑。例如，使用以穀胱甘肽_s_轉移酶 (GST)融合蛋白質的一部分，來表現想要多肽的市售載體 使付在攸想要的多妝中切開G S T組份之後，該想要的多 肽在胺基酸位置-1處帶有額外的甘胺酸。亦期待在其他載 體系統中表現而產生的變體，包括其中將聚_組胺酸標籤 併入胺基酸序列内的那些，通常是在該序列的羧基及/或 胺基終端。 插入變體亦包括融合蛋白質，其中使肽或肽體之胺基及/ 或羧基終端與其他多肽、其片段或胺基酸融合，通常不認 為它是任何特定蛋白質序列的一部分。這類融合蛋白質的 實例為免疫原性的多肽、具有長循環半衰期的蛋白質，像 疋免疫球蛋白恆定區、標記蛋白質、有助於純化想要之肽O:\121\121929.DOC -59- 200808833 A specific binder for acid residues. For example, using a portion of a glutathione_s_transferase (GST) fusion protein to express a desired carrier of a polypeptide, after the GST component is cut in a multi-makeup desired, the desired The polypeptide carries additional glycine at position -1 of the amino acid. Variants that are also expected to behave in other vector systems, including those in which a poly-histidine tag is incorporated into an amino acid sequence, are typically terminated at the carboxyl and/or amine end of the sequence. Insertion variants also include fusion proteins in which the amino and/or carboxyl terminus of the peptide or peptibody is fused to other polypeptides, fragments thereof or amino acids, and is generally not considered to be part of any particular protein sequence. Examples of such fusion proteins are immunogenic polypeptides, proteins with long circulating half-lives, such as the immunoglobulin constant region, labeled proteins, and the purification of the desired peptide.
主題）。 這類型的插入變體通常具有全部或實質上一部分的天然 分子，在N-或C-終端與第二個多肽的全部或一部分連接。 一部分的天然theme). Insertion variants of this type typically have all or a substantial portion of the native molecule linked to all or a portion of the second polypeptide at the N- or C-terminus. Part of the natural
’像是得自酵素的活性位置、 仍逆按具有功能的功能部位 糖基化作用功能部位、細胞'like the active site derived from the enzyme, still reversed the functional part of the function, glycosylation function site, cells
O:\121\121929.DOC -60- 200808833 瞄準信號或穿透膜區域。 有各種市售的融合蛋白質表現系統，可在本發明中使用 。特別有用的系統包括，但不限於穀胱甘肽_s_轉移酶 (GST)系統（Pharmacia)，麥芽糖結合蛋白質系統（neb，O:\121\121929.DOC -60- 200808833 Aiming at the signal or penetrating the membrane area. There are various commercially available fusion protein expression systems which can be used in the present invention. Particularly useful systems include, but are not limited to, the glutathione_s_transferase (GST) system (Pharmacia), the maltose binding protein system (neb,
Beverley，MA)、FLAG 系統（IBI，New Haven，CT)和 6xHis 系統（Qiagen，Chatsworth，CA)。這些系統能夠產生重組的 肽及/或肽體，僅攜帶少量的額外胺基酸，其不可能明顯 地影響該肽或肽體的活性。例如，FLAG系統和6xHis系統 兩者僅加入短的序列，已知它們兩個均是不佳抗原性的， 而不會不利地影響多肽折疊成其天然的構形。另一個預期 有用的N-終端融合，是在蛋白質或肽之N_終端區域的Met_Beverley, MA), FLAG system (IBI, New Haven, CT) and 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing recombinant peptides and/or peptibodies, carrying only a small amount of additional amino acids, which are unlikely to significantly affect the activity of the peptide or peptibosome. For example, both the FLAG system and the 6xHis system incorporate only short sequences, both of which are known to be poorly antigenic without adversely affecting the folding of the polypeptide into its native conformation. Another expected useful N-terminal fusion is Met_ in the N_terminal region of a protein or peptide.
Lys二肽融合。這類融合在蛋白質表現或活性上，可產生 有益的增加。 其他的融合系統產生多肽雜化物，其中想要從想要的肽 或肽體中，切除融合夥伴。在一個具體實施例中，融合夥 伴藉著含有對蛋白酶專一認知之序列的肽序列，與重組的 月太體連接。適當序列的實例，是被Tobacco Etch virus protease (Life Technologies，Gaithersburg，MD)或 Factor Xa (New England Biolabs，Bevedey，MA)承認的那些。 本發明亦提供融合多肽，其包括全部或一部分的本發明 之肽體或肽，與截短之組織因子（tTF)混合。tTF是血管瞄 準劑，由截短形式的人類凝固-誘導蛋白質所組成，其擔 任腫瘤血管凝固劑，如同美國專利第5,877,289號； M〇4,555 號；6，132,729 號；6，132,730 號；6 156 321 號；Lys dipeptide fusion. Such fusions can produce a beneficial increase in protein performance or activity. Other fusion systems produce polypeptide hybrids in which it is desired to excise the fusion partner from the desired peptide or peptibody. In a specific embodiment, the fusion partner is linked to the reconstituted celestial body by a peptide sequence comprising a sequence specifically recognized by the protease. Examples of suitable sequences are those recognized by Tobacco Etch virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Bevedey, MA). The invention also provides fusion polypeptides comprising all or a portion of a peptidomimetic or peptide of the invention, mixed with truncated tissue factor (tTF). tTF is a vascular targeting agent consisting of a truncated form of human coagulation-inducing protein, which acts as a tumor vascular coagulant, as in U.S. Patent No. 5,877,289; M. 4,555; 6,132,729; 6,132,730; 6 156 No. 321;
O:\121\121929.DOC -61 - 200808833 以及歐洲專利第EP 0988056號的描述。tTF與抗_Ang_2肽 體或肽，或其片段的融合，有助於將抗-Ang-2遞送至標靶 細胞。 另一方面，本發明提供刪除變體，其中移除一或多個在 肽或肽體中的胺基酸殘基。可在肽體的一或兩端完成刪除 ，或移除一或多個在肽體胺基酸序列内的殘基。刪除變體 必然包括肽或肽體所有的片段。 另一方面’本發明提供本發明之肽和肽體的取代變體。 取代變體包括其中移除一或多個胺基酸殘基，並以一或多 個另類胺基酸殘基置換的那些肽和肽體，該胺基酸可以是 天然存在或非-天然存在的。取代變體所產生的肽或肽體 ，’f類似”原始的肽或肽體，在這兩個分子中具有某些百分 比的胺基酸是相同的。取代變體在肽或肽體中，包括1、2 、3、4、5、6、7、8、9、10、15、20、25、30 個胺基酸 的取代’其中取代的數目可高達1〇%肽或肽體的胺基酸或 更多。一方面，取代在性質上可以是保留性的，然而，本 發明包括非-保留性的取代作用，亦包括非_傳統的胺基酸。 可藉著已知的方法迅速地計算相關之肽和肽體的同一性 和類似性。這類方法包括’但不限於在ComputationalO:\121\121929.DOC-61 - 200808833 and the description of European Patent No. EP 0988056. Fusion of tTF with an anti-Ang_2 peptide or peptide, or a fragment thereof, facilitates delivery of anti-Ang-2 to the target cell. In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a peptide or peptidic body are removed. Deletion can be accomplished at one or both ends of the peptibody, or one or more residues within the peptidic amino acid sequence can be removed. Deletion variants necessarily include all fragments of the peptide or peptide. In another aspect, the invention provides substituted variants of the peptides and peptibodies of the invention. Substitutional variants include those in which one or more amino acid residues are removed and replaced with one or more additional amino acid residues, which may be naturally occurring or non-naturally occurring. of. Substituting a peptide or peptibody produced by a variant, 'f is similar to the original peptide or peptibosome, in which a certain percentage of the amino acid is the same. The substitution variant is in a peptide or peptide, Including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 amino acid substitutions, wherein the number of substitutions can be as high as 1% by weight of the peptide or peptide amine The acid or more. In one aspect, the substitution may be retentive in nature, however, the invention includes non-retentive substitutions, as well as non-traditional amino acids. It can be rapidly known by known methods. Calculate the identity and similarity of related peptides and peptibodies. Such methods include, but are not limited to, in Computational
Molecular Biology，Lesk，A.Μ·編輯，Oxford UniversityMolecular Biology, Lesk, A.Μ·Editor, Oxford University
Press, New York (1988); Biocomputing: Informatics andPress, New York (1988); Biocomputing: Informatics and
Genome Projects，Smith，D.W·編輯，Academic Press，NewGenome Projects, Smith, D.W. Editor, Academic Press, New
York (1993); Computer Analysis of Sequence Data, Part 1? Griffin，A.M·，和 Griffin，H.G.編輯，Humana Press，New O:\121\121929.DOC -62- 200808833York (1993); Computer Analysis of Sequence Data, Part 1? Griffin, A.M., and Griffin, H.G. Editor, Humana Press, New O:\121\121929.DOC -62- 200808833
Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje，G·，Academic Press (1987); Sequence Analysis Primer，Gribskov，M·和 Devereux，J.編輯，M. Stockton Press，New York (1991);以及 Carillo 等人，SIAM J. Applied Math·，48:1073 (1988)中描述的那些。 設計判定兩個肽或多肽，或多肽和肽之關係或同一性百 分比的較佳方法，以便提供在受試序列之間的最大相配。 在可公開獲得的電腦程式中，描述了判定同一性的方法。 在兩個序列之間判定同一性的較佳電腦程式方法，包括但 不限於GCG套裝程式，包括GAP (Davereux等人，Nucl· Acid. Res·， 12:387 (1984); Genetics Computer Group， University of Wisconsin，Madison，WI，BLASTP，BLASTN, and FASTA (Altschul等人，J. Mol. Biol·，215 :403-410 (1990))。從 National Center for Biotechnology Information (NCBI)和其他來源（BLAST Manual, Altschul 等人， NCB/NLM/NIH Bethesda，MD 20894; Altschul等人，在前 (1990))公開獲得BLASTX程式。亦可使用已熟知的Smith Waterman演算法來判定同一性。 某些將兩個胺基酸序列排列成一直線的排列計劃，可導 致僅將兩個序列的短區域配對，而這個小的排列區可能具 有極高的序列同一性，即使在這兩個全長的序列之間沒有 顯著的關係。因此，在某些具體實施例中，選出的排列方 法（GAP程式）將導致跨越待比較之標靶至少10%全長的排 列，也就是在欲比較至少400個胺基酸的序列處，為至少 O:\121\121929.DOC -63- 200808833 40個連續的胺基酸，在待比較至少300至大約400個胺基酸 的序列處，為30個連續的胺基酸，在待比較至少2〇〇至大 約300個胺基酸的序列處，為至少2〇個連續的胺基酸，且 在待比較大約1 〇 〇至2 0 0個胺基酸的序列處，為至少1 〇個連 續的胺基酸。 例如’使用電腦演算法GAP (Genetics Computer Group， University of Wisconsin，Madison，WI)，將兩個欲判定其 序列同一性百分比的多肽排成一直線，以便使其各自的胺 基酸最為相配（由演算法決定，，配對跨幅"）。在某些具體實 施例中，連同演算法一起使用間隙開放罰點（其通常按照 3X平均對角線來計算；"平均對角線”是欲使用之比較矩陣 對角線的平均值；”對角線”是由特定之比較矩陣指派給每 個最佳胺基酸配對的分數或數目），和間隙延伸罰點（其經 常是間隙開放罰點的1/10倍），以及比較矩陣，像是PAM 25 0或BLOSUM 62。在某些具體實施例中，演算法亦使用 標準比較矩陣（關於PAM 250比較矩陣，參見Dayhoff等人 ，Atlas of Protein Sequence and Structure, 5(3)(1978)；關 於BLOSUM 62比較矩陣，參見Henikoff等人，proc. Natl. Acad· Sci USA，89:10915-10919 (1992)。 在某些具體實施例中，多肽序列比較的參數，包括下列： 演算法：Needleman等人，J. Mol· Biol·，48:443-453 (1970)； 比較矩陣：BLOSUM 62得自Henikoff等人，在前（199 2); 間隙罰點：12 O:\121\121929.DOC -64· 200808833 間隙長度罰點：4 類似性之閾值：〇 利用以上的參數，GAP程式可能是有用的。在某些具體 只施例中’上文提及的參數是使用GAp演算法之多肤比較 的預設參數（連同末端間隙無罰點）。 在某些具體實施射’多料酸分子序列（與胺基酸序 列相反）比較的參數包括下列： 演算法：Needleman等人，在前（1970); 比車父矩陣·相配=+ 1 〇，誤配=〇 間隙罰點：50 間隙長度罰點·· 3 利用以上的參數，GAP程式亦可能是有用的。上文提及 的參數是多核甞酸分子比較的預設參數。 可使用其他代表性的演算法、間隙開放罰點、間隙延伸 罰點、比較矩陣、類似性之閾值等等，包括在卜叫以㈤Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J. ed., M. Stockton Press, New York (1991); Carillo et al., those described in SIAM J. Applied Math., 48: 1073 (1988). A preferred method of determining the relationship or identity of two peptides or polypeptides, or polypeptides and peptides, is designed to provide maximum agreement between the sequences tested. In a publicly available computer program, a method of determining identity is described. Preferred computer program methods for determining identity between two sequences, including but not limited to GCG suites, including GAP (Davereux et al, Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University Of Wisconsin, Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al, J. Mol. Biol., 215: 403-410 (1990)). From the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual) Altschul et al., NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra (1990), publicly available BLASTX programs. The well-known Smith Waterman algorithm can also be used to determine identity. Arrangement of amino acid sequences in a straight line arrangement can result in pairing only short regions of two sequences, and this small alignment region may have very high sequence identity, even if there is no significant between the two full-length sequences Therefore, in some embodiments, the selected alignment method (GAP program) will result in an arrangement that spans at least 10% of the full length of the target to be compared, ie, at least 400 The sequence of the amino acid is at least O:\121\121929.DOC-63-200808833 40 consecutive amino acids, 30 consecutive in the sequence to be compared with at least 300 to about 400 amino acids The amino acid is at least 2 consecutive amino acids at a sequence to be compared of at least 2 to about 300 amino acids, and is about 1 to 200 amino acids to be compared. At the sequence, there are at least 1 contiguous amino acid. For example, 'Use the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI) to arrange two polypeptides whose sequence identity is determined to be in line. In order to best match their respective amino acids (determined by the algorithm, paired spans). In some embodiments, the gap opening penalty is used along with the algorithm (which is usually in accordance with the 3X average diagonal) Line to calculate; "average diagonal" is the average of the diagonals of the comparison matrix to be used; "diagonal" is the fraction or number assigned to each optimal amino acid pair by a particular comparison matrix) , and gap extension penalty points (which often It is 1/10 times the gap opening penalty) and the comparison matrix, such as PAM 25 0 or BLOSUM 62. In some embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al, Atlas of Protein Sequence and Structure, 5(3) (1978); for the BLOSUM 62 comparison matrix, see Henikoff Et al., proc. Natl. Acad. Sci USA, 89: 10915-10919 (1992). In some embodiments, the parameters of the polypeptide sequence comparison include the following: Algorithm: Needleman et al, J. Mol Biol ·, 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., former (199 2); gap penalty: 12 O: \121\121929.DOC -64· 200808833 gap length penalty :4 Threshold of similarity: G Using the above parameters, the GAP program may be useful. In some specific examples, the parameters mentioned above are preset parameters for comparing the skin using the GAp algorithm (along with There is no penalty for the end gap.) In some specific implementations, the parameters of the multi-acid acid sequence (as opposed to the amino acid sequence) include the following: Algorithm: Needleman et al., former (1970); · Matching = + 1 〇, mismatch = 〇 gap penalty :50 Gap length penalty point · 3 Using the above parameters, the GAP program may also be useful. The parameters mentioned above are preset parameters for the comparison of polynuclear citrate molecules. Other representative algorithms can be used, gaps can be used. Penalty points, gap extension penalty points, comparison matrices, similarity thresholds, etc., included in the call (5)
Manual，Wisconsin Package，第 9版，1997年 9月中陳述的 那些。進行特定的選擇，將是熟諳此藝者所瞭解的，並將 視待進行的特定比較而定，像是DNA-對-DNA，蛋白質_ 對-蛋白質，蛋白質-對-DNA;此外，是否是在特定的序列 對之間（在此情況下，GAP或BestFit通常是較佳的），或是 在一個序列與大的序列資料庫之間（在此情況下faSTA或 BLASTA是較佳的）進行比較。 在本文中使用時，20個傳統的胺基酸及其縮寫，依循慣Manual, Wisconsin Package, 9th Edition, those stated in September 1997. Specific choices will be known to those skilled in the art and will depend on the particular comparison being made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA; Between a particular pair of sequences (in this case, GAP or BestFit is usually preferred), or between a sequence and a large sequence library (in which case faSTA or BLASTA is preferred) Comparison. When used in this article, 20 traditional amino acids and their abbreviations, used to follow
例使用。參見 Immunology—A Synthesis (第 2 版，e S O:\121\121929.DOC •65- 200808833Example use. See Immunology - A Synthesis (2nd Edition, e S O:\121\121929.DOC •65- 200808833
Golub和 D.R. Gren編輯，Sinauer Ass〇ciates，Edited by Golub and D.R. Gren, Sinauer Ass〇ciates,
Mass. (1991))，為了任何目的將其以引用的方式併入本文 中〇 胺基酸可具有L或D立體化學（除了 Gly之外，它不是乙也 不是D)，而本發明之多肽和組合物可包括立體化學的組合 。然而，L立體化學是較佳的。本發明亦提供相反分子， 其中胺基酸之胺基終端至羧基終端的序列是相反的。例如 ，具有正常序列Xi-XrX3之分子的相反將是H2_Xi。本 發明亦提供逆-相反分子，其中，如同上文，胺基酸之胺 基終端至羧基終端的序列是相反的，並將正常是”L”對映 體的殘基更改為"D”立體異構物之形式。 20個傳統胺基酸之立體異構物（例如胺基酸）、非天然 的胺基酸，像是α-，α-二經取代之胺基酸、N_烷基胺基酸 、乳酸和其他非傳統的胺基酸，亦可以是本發明之多肽的 適當組份。非傳統胺基酸的實例包括，但不限於：胺基己 二酸、β-丙胺酸、β-胺基丙酸、胺基丁酸、六氫吡啶酸、 胺基己酸、胺基庚酸、胺基異丁酸、胺基庚二酸、二胺基 丁酸、鎖連素、二胺基庚二酸、二胺基丙酸、Ν-乙基甘胺 酸、Ν-乙基天冬醯胺、羥基離胺酸、別-羥基離胺酸、羥 基脯胺酸、異鎖連素、別-異亮胺酸、Ν-甲基甘胺酸、肌 胺酸、Ν-甲基異亮胺酸、Ν-甲基纈胺酸、正纈胺酸、正亮 胺酸、鳥胺酸、4-羥基脯胺酸、γ-羧基榖胺酸、ε-Ν，Ν，Ν-三甲基離胺酸、ε-Ν-乙醯基離胺酸、〇-磷酸絲胺酸、Ν-乙 醯基絲胺酸、Ν-甲醯基曱硫胺酸、3-曱基組胺酸、5-羥基 O:\121\121929.DOC 66- 200808833 -妝…-N-甲基精胺酸，以及其他類似的胺基酸和胺 基酸（例如4-羥基脯胺酸）。 同樣的’除非另行指定’單股多核甞酸序列的左-手端 為5,端；雙股多核#酸序列的左_手方向則意指$，方向。將 初生RNA轉錄本的5,至3,方向添加，稱為轉錄方向；在具 有與RNA相同之序列的DNA股上的序列區，且它是” RNA轉錄本的5,末端，將其稱為，，上游序列"；在具有與 RNA相同之序列的DNA股上的序列區，且它是3，對rna轉 錄本的3,末端，則將其稱為”下游序列，，。 應瞭解可以其共同的側鏈特性為基礎，將胺基酸殘基分 類： 1·中性疏水性的：丙胺酸（Ala; A);纈胺酸（Val; V); 冗fe酸（Leu; L);異壳胺酸（ne; I);脯胺酸（Pr〇; p);色胺 酸（Trp; W);苯丙胺酸（Phe; F)和甲硫胺酸（Met; M)。 2·中性極性的：甘胺酸（Gly; G);絲胺酸（Ser; s);蘇胺 酸（Thr; T);酪胺酸（Tyr; Y);半胱胺酸（Cys; c);縠胺醯 胺（Gin; Q);天冬胺酸（Asn; N)和正亮胺酸。 3·酸性的：天冬胺酸（Asp;D)，穀胺酸（Glu;E); 4·鹼性的：離胺酸（Lys; K)，精胺酸（Arg; R)，組胺酸 (His; Η) 〇 參見 Lewin，B·，Genes V，Oxford University Press (1994)， 第11頁。 保留性胺基酸置換可包括非傳統的胺基酸殘基，其通常 是藉著化學肽合成，而非藉著在生物學系統中的合成併入 O:\121\121929.DOC -67- 200808833 。這些包括，但不限於肽模仿物和胺基酸部分的其他相反 或逆向形式。非-保留性置換可涉及交換這些種類之一的 成員與得自其他種類之成員。 在進行這類改變時，根據某些具體實施例，可考慮胺基 酸的疏水指數。已經以其疏水性和電荷特徵為基礎，來指 派每個胺基酸的疏水指數。其為：異亮胺酸（ + 4·5);纈胺 Μ+4.2),壳胺酸（+3.8);苯丙胺酸（+2·8);半胱胺酸/胱胺 酸（+2.5);甲硫胺酸（+1·9);丙胺酸（+1·8);甘胺酸㈠.4); 蘇胺酸（-0.7);絲胺酸（_〇·8);色胺酸（-〇·9);酪胺酸（_13) ;脯胺酸（-1.6);組胺酸〇3·2);榖胺酸卜35);穀胺醯胺 (_3.5);天冬胺酸（-3.5);天冬醯胺g3.5);離胺酸（_3.9); 和精胺酸（-4.5)。 在此項技藝中瞭解疏水胺基酸指數，在賦與蛋白質交互 作用之生物學功能上的重要性。Kyte等人，j. Mol· Bi〇l.， 157:105-131 (1982)。已知某些胺基酸可被其他具有類似疏 水指數或分數的胺基酸取代，並仍保留類似的生物學活性 。在以疏水指數為基礎來進行改變時，在某些具體實施例 中’胺基酸的取代作用包括其疏水指數在±2内的胺基酸。 在某些具體實施例中，包括在土 1内的那些，而在某些具體 實施例中，包括在±0·5内的那些。 在此項技藝中，亦瞭解類似胺基酸的取代作用，可以親 水性為基礎有效地進行，特別是在藉此創造想要用在免疫 學具體實施例中之具有生物功能的肽體或肽之處。在某些 具體實施例中，蛋白質的最大局部平均親水性，由其相鄰 O:\121\121929.DOC -68 - 200808833 胺基酸之親水性支配，與其免疫原性和抗原性，也就是該 蛋白質的生物學特性有關係。 已經對這些胺基酸殘基指派了下列的親水性值：精胺酸 (+3.0);離胺酸（+3.0);天冬胺酸（+3.〇±1);穀胺酸（ + 3.0土 1);絲胺酸（ + 0.3);天冬醯胺（+〇·2);榖胺醯胺（+0·2);甘 胺酸（0);蘇胺酸（-0.4);脯胺酸（_〇·5± 1);丙胺酸（_〇.5); 組胺酸（-0.5);半胱胺酸（_ι·〇);甲硫胺酸（_13);纈胺酸（_ 1.5);亮胺酸（_1.8);異亮胺酸（-u);酪胺酸（_2·3);苯丙 胺酸（-2.5)和色胺酸（—3.4)。在以類似的親水性值為基礎進 行改變時’在某些具體實施例中，胺基酸之取代作用包括 其親水性值在±2内的胺基酸，在某些具體實施例中，包括 在±1内的那些，而在某些具體實施例中，包括在土〇5内的 那些。亦可以親水性為基礎，確認得自原始胺基酸序列的 抗原決定位。亦將這些區域稱為，，抗原決定位核心區，，。 在下文表2中陳述了代表性的胺基酸取代作用。 表2 胺基酸取代作用 原始殘基 代表的取代 較佳取代 Ala Val，Leu，lie Val Arg Lys，Gln，Asn Lys Asn Gln，Glu，Asp Gin Asp Glu, Gin, Asn Glu Cys Ser，Ala Ser Gin Asn, Glu, Asp Asn Glu Asp, Gin, Asn AspMass. (1991)), which is incorporated herein by reference for any purpose. The amic acid may have L or D stereochemistry (other than Gly, it is not B or D), and the polypeptide of the present invention The composition and composition can include a combination of stereochemistry. However, L stereochemistry is preferred. The present invention also provides opposite molecules in which the amino terminal termination to carboxyl terminal sequence of the amino acid is reversed. For example, the opposite of the molecule with the normal sequence Xi-XrX3 would be H2_Xi. The present invention also provides an inverse-opposite molecule wherein, as above, the amino acid terminal to carboxyl terminal sequence of the amino acid is reversed, and the residue which is normally the "L" enantiomer is changed to "D" a form of a stereoisomer. 20 stereoisomers of a conventional amino acid (eg, an amino acid), a non-natural amino acid, such as an alpha-, alpha-disubstituted amino acid, N-alkane The amino amino acid, lactic acid and other non-conventional amino acids may also be suitable components of the polypeptide of the present invention. Examples of non-conventional amino acids include, but are not limited to, amino adipic acid, β-alanine , β-aminopropionic acid, aminobutyric acid, hexahydropyridyl acid, aminocaproic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, lockin, Diaminopimelic acid, diaminopropionic acid, Ν-ethylglycine, Ν-ethyl aspartate, hydroxy cis acid, bis-hydroxy lysine, hydroxyproline, hetero-lock , bis-isoleucine, Ν-methylglycine, sarcosine, Ν-methyl isoleucine, Ν-methylproline, n-proline, leucine, avian Acid, 4-hydroxyguanamine Acid, γ-carboxy glutamic acid, ε-Ν, Ν, Ν-trimethyl lysine, ε-Ν-acetamido lysine, 〇-phosphoric acid, Ν-ethionine , Ν-methionine thiol acid, 3-mercapto histidine, 5-hydroxy O: \121\121929.DOC 66- 200808833 - makeup...-N-methyl arginine, and other similar amines Acid and amino acid (eg 4-hydroxyproline). The same 'unless otherwise specified' single-stranded polynuclear acid sequence has a left-hand end of 5, the end; double-stranded multinuclear acid sequence of the left-hand direction Means $, direction. Adds 5, to 3, directions of the nascent RNA transcript, called the direction of transcription; in the sequence region on the DNA strand having the same sequence as the RNA, and it is "RNA transcript 5, The end, which is called, the upstream sequence "; the sequence region on the DNA strand having the same sequence as the RNA, and it is 3, and the 3, end of the rna transcript, which is called the "downstream sequence," It should be understood that amino acid residues can be classified based on their common side chain properties: 1. Neutral hydrophobic: alanine (Ala; A); proline (Val; V); (Leu; L); isocyanic acid (ne; I); proline (Pr〇; p); tryptophan (Trp; W); phenylalanine (Phe; F) and methionine (Met; M). 2. Neutral polarity: glycine (Gly; G); serine (Ser; s); threonine (Thr; T); tyrosine (Tyr; Y); cysteine (Cys; c); amidoxime (Gin; Q); aspartic acid (Asn; N) and norleucine. 3. Acidic: aspartic acid (Asp; D), glutamic acid (Glu; E); 4. alkaline: lysine (Lys; K), arginine (Arg; R), histidine (His; Η) 〇 See Lewin, B., Genes V, Oxford University Press (1994), p. 11. Reserved amino acid substitutions can include non-traditional amino acid residues, which are typically synthesized by chemical peptides rather than by synthesis in biological systems. O:\121\121929.DOC-67- 200808833. These include, but are not limited to, peptide mimetics and other opposite or reverse forms of the amino acid moiety. Non-reserved permutations may involve exchanging members of one of these categories with members of other classes. In making such changes, the hydrophobicity index of the amino acid can be considered in accordance with certain embodiments. The hydrophobicity index of each amino acid has been assigned based on its hydrophobicity and charge characteristics. It is: isoleucine ( + 4 · 5); amidoxime + 4.2), chiral acid (+3.8); phenylalanine (+2 · 8); cysteine / cystine (+2.5) Methionine (+1·9); alanine (+1·8); glycine (I).4); threonine (-0.7); serine (_〇·8); tryptophan (-〇·9); tyrosine (_13); proline (-1.6); histidine 3·2); valine 35; glutamine (_3.5); Amino acid (-3.5); aspartame g3.5); lysine (-3.9); and arginine (-4.5). Understanding the hydrophobic amino acid index in this technique is important in conferring the biological function of protein interaction. Kyte et al., j. Mol. Bi〇l., 157: 105-131 (1982). It is known that certain amino acids can be substituted by other amino acids having a similar hydrophobic index or fraction and still retain similar biological activities. In the case of a change based on the hydrophobic index, in some embodiments the substitution of the amino acid includes an amino acid having a hydrophobicity index within ±2. In some embodiments, those included within the soil 1 are included, and in some specific embodiments, those within ± 0. 5 are included. In this art, it is also understood that the substitution of a similar amino acid can be carried out efficiently on the basis of hydrophilicity, in particular by creating a biologically functional peptibody or peptide which is desired to be used in a specific embodiment of immunology. Where. In some embodiments, the maximum local average hydrophilicity of the protein is governed by the hydrophilicity of its adjacent O:\121\121929.DOC-68-200808833 amino acid, with its immunogenicity and antigenicity, ie The biological properties of this protein are related. These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 〇±1); glutamic acid (+ 3.0 soil 1); serine acid (+0.3); aspartame (+〇·2); amidoxime (+0·2); glycine (0); threonine (-0.4); Proline (_〇·5± 1); alanine (_〇.5); histidine (-0.5); cysteine (_ι·〇); methionine (_13); proline (_ 1.5); leucine (_1.8); isoleucine (-u); tyrosine (_2·3); phenylalanine (-2.5) and tryptophan (-3.4). In the case of a change based on a similar hydrophilicity value, 'in some embodiments, the substitution of the amino acid includes an amino acid having a hydrophilicity value within ±2, in certain embodiments, including Those within ±1, and in some embodiments, those included within the soil 5. It is also possible to confirm the epitope determined from the original amino acid sequence based on hydrophilicity. These regions are also referred to as, the antigenic decision core region, . Representative amino acid substitutions are set forth in Table 2 below. Table 2 Amino acid substitution The substitution represented by the original residue is preferably substituted for Ala Val, Leu, lie Val Arg Lys, Gln, Asn Lys Asn Gln, Glu, Asp Gin Asp Glu, Gin, Asn Glu Cys Ser, Ala Ser Gin Asn, Glu, Asp Asn Glu Asp, Gin, Asn Asp
O:\121\121929.DOC -69· 200808833 原始殘基 代表的取代 較佳取代 Gly Pro, Ala Ala His Asn，Gln，Lys，Arg Arg lie Leu，Val，Met，Ala，Phe，正亮胺酸 Leu Leu 正亮胺酸，lie，Val，Met，Ala，Phe lie Lys Arg，1，4-二胺基-丁酸，Gln，Asn Arg Met Leu，Phe，He Leu Phe Leu, Val, lie, Ala, Tyr Leu Pro Ala Gly Ser Thr，Ala，Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp，Phe，Thr，Ser Phe Val lie，Met，Leu，Phe，Ala，正亮胺酸 Leu 熟諳此藝者將能夠使用已熟知的技術，判定在本文中陳 述之多肽的適當變體。在某些具體實施例中，熟諳此藝者 可藉著瞄準不相信其對於活性是很重要的區域，來確認分 子中可加以改變，但不破壞活性的適當區域。在某些具體 實施例中，可確認分子被保留在類似之肽或多肽中的殘基 和部分。在某些具體實施例中，即使對生物活性或結構可 能很重要的區域，亦可使其經歷保留性胺基酸置換，而不 破壞生物活性，或對多肽結構有不利的影響。 此外，熟諳此藝者可回顧結構-功能的研究，確認在類 似多肽中對於活性或結構很重要的殘基。從這類比較來看 ，可預期在蛋白質中，與在類似蛋白質中對於活性或結構 很重要的胺基酸殘基一致之胺基酸殘基的重要性。熟諳此 藝者可為這類預期很重要的胺基酸殘基，選擇在化學上類 O:\121\121929.DOC -70- 200808833 似的胺基酸取代作用。 熟諳此藝者亦可分析三維結構和胺基酸序列，與在類似 多肽中之結構的關係。從這類資訊來看，熟諳此藝者可預 期抗體之胺基酸殘基的排列，與其三維結構的關係。在某 些具體實施例中，熟諳此藝者可選擇對於預期是在蛋白質 表面上的胺基酸殘基，不進行徹底的改變，因為這類殘基 可能涉及與其他分子的重要交互作用。此外，熟諳此藝者 可產製測試變體，其在每個想要的胺基酸殘基處，含有單 一胺基酸取代。然後可使用熟諳此藝者已知的活性測定來 篩選變體。可使用這類變體收集有關於適當變體的資訊。 例如，如果發現對特定胺基酸殘基的改變，導致受到破壞 、不想要地降低，或不適當的活性，便可避免這類改變。 換句話說，以收集自這類例行實驗的資訊為基礎，熟諳此 藝者可輕易地判定應避免單獨或與其他突變組合，進一步 取代的胺基酸。 許多科學出版物已經致力於二級結構的預測。參見 Moult J.? Curr. Op. in Biotech., 7(4):422-427 (1996), Chou 等人，Biochemistry，13(2):222-245 (1974); Chou等人， Biochemistry，113(2)··211-222 (1974); Chou等人，八心· Enzymol· Relat. Areas Mol. Biol·，47:45-148 (1978); Chou 等人，Ann· Rev. Biochem·，47:251-276 和 Chou 等人， Biophys· J·，26:3 67-3 84 (1979)。此外，目前可利用電腦程 式來幫助預測二級結構。一種預測二級結構的方法是以同 種性塑型為基礎。例如，具有超過3 〇 %之序列同一性，或 O:\121\121929.DOC -71 - 200808833 超過40%之類似性的兩個多肽或蛋白質，通常具有類似的 結構拓樸學。蛋白質結構資料庫（pDB)最近的成長，已經 提供了增強的二級結構可預測性，包括在多肽或蛋白質之 結構中，可能的折疊次數。參見Holm等人，Nucl. Acid.O:\121\121929.DOC -69· 200808833 The substitution represented by the original residue is preferably substituted for Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg lie Leu, Val, Met, Ala, Phe, leucine Leu Leu leucine, lie, Val, Met, Ala, Phe lie Lys Arg, 1,4-diamino-butyric acid, Gln, Asn Arg Met Leu, Phe, He Leu Phe Leu, Val, lie, Ala , Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val lie, Met, Leu, Phe, Ala, Leleucine Leu Suitable variants of the polypeptides set forth herein will be able to be determined using well-known techniques. In some embodiments, those skilled in the art can identify areas of the molecule that can be altered without disrupting activity by targeting areas that are not believed to be important for activity. In certain embodiments, residues and moieties in which a molecule is retained in a similar peptide or polypeptide can be identified. In certain embodiments, even regions that may be important for biological activity or structure may undergo undergoing retention amino acid substitution without disrupting biological activity or adversely affecting polypeptide structure. In addition, those skilled in the art can review structure-function studies to identify residues that are important for activity or structure in a similar polypeptide. From such comparisons, the importance of amino acid residues consistent with amino acid residues important for activity or structure in similar proteins can be expected in proteins. Those skilled in the art can choose amino acid substitutions such as chemically O:\121\121929.DOC-70-200808833 for such amino acid residues that are expected to be important. Those skilled in the art can also analyze the relationship between the three-dimensional structure and the amino acid sequence, as well as structures in similar polypeptides. From this kind of information, those skilled in the art can predict the arrangement of the amino acid residues of the antibody and its three-dimensional structure. In some embodiments, those skilled in the art may choose to base the amino acid residues expected to be on the surface of the protein without radical changes, as such residues may involve important interactions with other molecules. In addition, those skilled in the art can produce test variants which contain a single amino acid substitution at each desired amino acid residue. Variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about appropriate variants. For example, such changes can be avoided if changes to specific amino acid residues are found to result in damage, undesired reduction, or inappropriate activity. In other words, based on information gathered from such routine experiments, those skilled in the art can readily determine that amino acids that are further substituted, either alone or in combination with other mutations, should be avoided. Many scientific publications have focused on the prediction of secondary structures. See Moult J.? Curr. Op. in Biotech., 7(4): 422-427 (1996), Chou et al, Biochemistry, 13(2): 222-245 (1974); Chou et al., Biochemistry, 113 (2)··211-222 (1974); Chou et al., Baxin·Enzymol·Relat. Areas Mol. Biol·, 47:45-148 (1978); Chou et al., Ann· Rev. Biochem·, 47 :251-276 and Chou et al., Biophys J., 26:3 67-3 84 (1979). In addition, computer programs are currently available to help predict secondary structure. One method of predicting secondary structure is based on isomorphic modeling. For example, two polypeptides or proteins with more than 3 % % sequence identity, or O: \121 \121929.DOC -71 - 200808833 over 40% similarity, usually have similar structural topologies. Recent growth in the protein structure database (pDB) has provided enhanced secondary structure predictability, including the number of possible folds in the structure of a polypeptide or protein. See Holm et al., Nucl. Acid.
Res·，27(1):244-247 (1999)。已經暗示（Brenner等人，Curr· Op· Struct· Biol·，7(3):369-376 (1997))在特定的多肽或蛋 白質中，有限制次數的折疊，且一旦已經解決結構的決定 性次數’結構的預測便變成戲劇化地更精確。 預測二級結構的其他方法，包括”穿線（threading)，， (Jones，D·，Curr· Opin. Struct. Biol·，7(3):377-87 (1997)·Res., 27(1): 244-247 (1999). It has been suggested (Brenner et al., Curr Op. Struct Biol., 7(3): 369-376 (1997)) that there are a limited number of folds in a particular polypeptide or protein, and once the structural number of structures has been resolved 'The prediction of the structure becomes dramatic and more precise. Other methods of predicting secondary structure include "threading," (Jones, D., Curr Opin. Struct. Biol., 7(3): 377-87 (1997).
Sippl等人，Structure，4(1):15-19 (1996))、”輪廓分析” (Bowie 等人，Science，253:164-170 (1991); Gribskov 等人 ，Meth. Enzym·，183:146-159 (1990); Gribskov等人，proc· Nat· Acad· Sci·，84(13):4355-4358 (1987))，以及”進化連鎖" (參見 Holm，在前（1999)和 Brenner,在前（1997)) 〇 在某些具體實施例中，肽體變體包括糖基化作用變體， 其中已經將一或多個糖基化作用位置，像是Ν_連接的糖基 化作用位置，加至該肽體中。Ν_連接的糖基化作用位置是 序列：Asn-X-Ser或Asn-X-Thr所特有的，其中以X表示的 胺基酸殘基可以是脯胺酸以外任何的胺基酸殘基。胺基酸 殘基的取代或添加創造該序列，提供可能加入連接之碳 水化合物鏈的新位置。或者，排除該序列的取代作用，將 移除現存的N-連接之碳水化合物鏈。亦提供N-連接之碳水 化合物鍵的重新排列，其中排除一或多個N -連接之糖基化 O:\121\121929.DOC 72- 200808833 作用位置（通常是天然存在的那些），並創造一或多個新的 N-連接位置。 本發明亦提供”衍生物”，其包括攜帶不同的修改，或胺 基酸殘基之添加、插入、刪除或取代的肽體。該修改在性 質上最好是共價的，並包括例如與聚合物、脂質、其他有 機和無機部分的化學結合。可製備本發明的衍生物，以便 增加肽讜體的循環半衰期，或可設計以便改良該肽體對想 要之細胞、組織或器官的瞄準能力。 代表性的衍生物包括其中已經進行一或多個下列修改的 部分： •已經藉著非-肽基鍵結，像是_CH2_胺基甲酸酯鍵結[ CH2-0C(0)NR-];膦酸酯鍵結；磺酸胺[_CH2_ S(0)2NR-]鍵結；脲[_NHC⑼NH_]鍵結；_ch2二級 胺鍵結；或烷基化了的肽基鍵結，其中R6 為烧基]，置換一或多個肽基[/⑼顺·]鍵結（鍵）； •其中N-終端被衍生成_NRR1基團；_NRC(〇)R基團；-NRC(0)OR基團；_NRS(〇)2R* 團；·nhc(〇)nhr* 團（其中R和R1為氫或低碳數烷基，其限制條件為r和 R並非兩者都是氫）；琥珀醯亞胺基團；苄氧羰基一 NH-(CBZ-NH-)基團；或芊氧羰基基團（在苯基環 上具有從1至3個取代基，選自由低碳數烷基、低碳 數烧氧基、氯和溴所組成之群）的肽； •其中自由C終端被衍生為-C(0)R2 (其中R2選自由低碳 數烷氧基所組成之群）和_NR3r4 (其中R3和R4分別選自 O:\121\121929.DOC -73- 200808833 由括氫和低碳數烷基所組成之群）的肽。"低碳數，，意 指具有從1至6個碳原子的基團。 此外，亦可藉著使肽中被瞄準之胺基酸殘基與能夠與選 出之側鏈或終端殘基反應的有機衍生劑反應，將個別胺基 酸的修改導入本發明之多肽或組合物中。下列的是典型 的： 離胺醯基和胺基終端的殘基可與琥珀酸或其他的羧酸酐 反應。利用這些製劑的衍生作用，具有逆轉離胺醯基殘基 之電荷的效果。其他適合衍生含有-胺基之殘基的製劑 ’包括醯亞胺酯，像是甲基P比咬醢亞胺甲酯；鱗酸P比哆盤 ;峨哆醛；氯硼氫化物；三硝基苯磺酸；〇_甲基異脲； 2,4-戊二酮；以及利用乙醛酸之轉胺基酶催化的反應。 可藉著與一或數個傳統試劑的反應，來修改精胺醯基殘 基’其中有苯甲醯曱醛、2,3-丁二酮、1，2-環己二酮和茚 二酮。精胺酸殘基的衍生作用需要在驗性的條件下進行反 應，因為胍官能基的高pKa。此外，這些試劑可與離胺酸 之基團，以及精胺酸的胍基團反應。 酪胺醯基殘基的特定修改，本身已經被廣泛地研究，而 特別感興趣的是藉著與芳香族之重氮化合物或四硝基曱烷 的反應，將光譜標記導入酪胺醯基殘基内。最常見的是， 可使用N-乙醯基咪唑和四硝基曱烷，分別形成〇_乙醯基酪 胺醯基物種和3-硝基衍生物。 可藉著與碳化二醯亞胺（11，_]^=；(：>：^尺，）的反應，像是卜環 己基-3-(2-嗎啉基_(4_乙基）碳化二醯亞胺或丨_乙基_3兴4_氮Sippl et al., Structure, 4(1): 15-19 (1996)), "Profile Analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym, 183: 146-159 (1990); Gribskov et al., proc· Nat·Acad·Sci·, 84(13): 4355-4358 (1987)), and “Evolution Chain” (see Holm, former (1999) and Brenner Previous (1997)) In certain embodiments, peptibody variants include glycosylation variants in which one or more glycosylation sites, such as Ν-linked glycosylation, have been The position of action is added to the peptibosome. The position of the glycosylation of the Ν_ linkage is unique to the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue represented by X may be 脯Any amino acid residue other than an amino acid. Substitution or addition of an amino acid residue creates the sequence, providing a new position that may be added to the linked carbohydrate chain. Alternatively, excluding the substitution of the sequence will remove the existing one. N-linked carbohydrate chain. Also provides rearrangement of N-linked carbohydrate bonds, excluding one or more N-linked glycosylation O:\121\ 121929.DOC 72- 200808833 The position of action (usually those that occur naturally) and the creation of one or more new N-linked positions. The invention also provides "derivatives" which include carrying different modifications, or amino acids Peptide bodies added, inserted, deleted or substituted for residues. The modification is preferably covalent in nature and includes, for example, chemical binding to polymers, lipids, other organic and inorganic moieties. Derivatives of the invention can be prepared , in order to increase the circulating half-life of the peptide steroid, or may be designed to improve the targeting ability of the peptide to the desired cell, tissue or organ. Representative derivatives include those in which one or more of the following modifications have been made: • Has been bonded by a non-peptidyl group, such as _CH2_carbamate linkage [CH2-0C(0)NR-]; phosphonate linkage; sulfonate amine [_CH2_S(0)2NR -] linkage; urea [_NHC(9)NH_] linkage; _ch2 secondary amine linkage; or alkylated peptidyl linkage, wherein R6 is alkyl], replacing one or more peptidyl groups [/(9) cis] Bonding (bond); • wherein the N-terminal is derivatized into a _NRR1 group; _NRC(〇)R group; -NRC(0)OR group ;_NRS(〇)2R* 团;·nhc(〇)nhr* group (wherein R and R1 are hydrogen or a lower alkyl group, the restrictions are that r and R are not both hydrogen); amber quinone imine a group; a benzyloxycarbonyl-NH-(CBZ-NH-) group; or a fluorenyloxy group (having from 1 to 3 substituents on the phenyl ring, selected from a lower alkyl group, a lower carbon number) a peptide of a group consisting of alkoxy groups, chlorine and bromine; wherein the free C terminal is derivatized as -C(0)R2 (wherein R2 is selected from the group consisting of a lower alkoxy group) and _NR3r4 (wherein R3 and R4 are each selected from the group consisting of O:\121\121929.DOC-73-200808833 a group consisting of hydrogen and a lower alkyl group. "low carbon number, meaning a group having from 1 to 6 carbon atoms. Alternatively, modifications of the individual amino acids can be introduced into the polypeptide or composition of the invention by reacting the targeted amino acid residue in the peptide with an organic derivatizing agent capable of reacting with the selected side chain or terminal residue. in. The following are typical: residues from the amine and amine end groups can be reacted with succinic acid or other carboxylic anhydrides. The derivatization of these preparations has the effect of reversing the charge of the amine sulfhydryl residue. Other preparations suitable for deriving residues containing -amino groups include quinones such as methyl P to dimethyl imidate; squaric acid P to sputum; furfural; chloroborohydride; Alkylbenzenesulfonic acid; hydrazine-methylisourea; 2,4-pentanedione; and a reaction catalyzed by a transaminase of glyoxylic acid. The arginyl residue can be modified by reaction with one or several conventional reagents, including benzaldehyde, 2,3-butanedione, 1,2-cyclohexanedione and anthracenedione. . The derivatization of arginine residues requires a reaction under the conditions of the test, because of the high pKa of the oxime functional group. Further, these reagents can react with a group derived from an amine acid and a sulfonium group of arginine. The specific modification of the tyramine sulfhydryl residue itself has been extensively studied, and it is of particular interest to introduce the spectral marker into the tyramine thiol residue by reaction with an aromatic diazonium compound or tetranitrononane. Base. Most commonly, N-ethinylimidazole and tetranitrononane can be used to form the oxime-acetamidoguanidine species and the 3-nitro derivative, respectively. By reaction with carbodiimide (11, _]^=; (:>:^,), like cyclohexyl-3-(2-morpholinyl-(4-ethyl) Carbonized diimine or 丨_ethyl_3 兴4_nitrogen
O:\121\121929.DOC •74· 200808833 鏘-4,4-二甲戊基）碳化二醯亞胺，選擇性地修改魏基側鍵 基團（天冬胺醯基或榖胺醯基）。此外，亦可藉著與銨離子 的反應，將天冬胺醯基和穀胺醯基殘基轉變為天冬醯胺醯 基和穀胺醯胺醯基殘基。 通常將穀胺醯胺醯基和天冬醯胺醯基殘基脫醯胺化，成 為相對應的榖胺醯基和天冬胺醯基殘基。或者，可在弱酸 性的條件下將這些殘基脫醯胺化。這些殘基的任一個形式 均在本發明的範圍内。 利用雙重功能之製劑的衍生作用，對於將肽或其功能衍 生物與不溶於水的支撐矩陣或其他大分子載劑交聯是有用 的。常用的聯結子包括，例如1，1_雙（重氮乙醯基）_2_苯基 乙烧、戊一备、N-經基琥轴醢亞胺醋，例如帶有4_疊氮水 楊酸的酯、高雙重功能的醯亞胺酯，包括二琥珀醯亞胺基 酉曰’像疋3，3 ’ -二硫雙（琥轴醯亞胺基丙酸g旨），以及雙重功 能的順丁烯二醯亞胺，像是雙順丁烯二醯亞胺基4,8-辛烷。諸如甲基-3-[(對-疊氮苯基）二硫代]丙醯亞胺酯之類 的衍生劑，產生光可激活的中間物，其能夠在光的存在下 形成交聯。或者，為了固定蛋白質，可使用反應性的不溶 於水之矩陣，像是在美國專利第3,969,287號；3,691，016號 ’ 4，195，128 號；4,247,642 號；4,229,537 號和 4,330,440 號 中描述的溴化氰激活之碳水化合物和反應性受質。 其他可能的修改包括脯胺酸和離胺酸的羥化作用，絲胺 酿基或蘇胺醯基殘基之羥基基團的磷酸化作用，在Cys中 &原子的氧化作用，離胺酸、精胺酸和組胺酸侧鏈之α-胺O:\121\121929.DOC •74· 200808833 锵-4,4-dimethylpentyl)carbodiimide, selectively modifying the Weiyl side bond group (aspartame or amidoxime group) ). In addition, aspartame and glutamine residues can be converted to aspartic acid sulfhydryl groups and glutamine amidoxime residues by reaction with ammonium ions. The glutamine amidoxime and the aspartame sulfhydryl residues are typically deaminated to form the corresponding amidoxime and aspartame residues. Alternatively, these residues can be deaminated under weak acid conditions. Any form of these residues is within the scope of the invention. Derivatization with dual-functional formulations is useful for crosslinking peptides or functional derivatives thereof with water-insoluble support matrices or other macromolecular carriers. Commonly used linkers include, for example, 1,1_bis(diazonyi)-2-phenylene bromide, pentacene, N-trans-succinimide, for example, with 4_azido-salt Acidic esters, highly dual-functional sulfhydryl esters, including disuccinimide 酉曰 'like 疋 3,3 '-dithio bis (succinimide iminopropionic acid g), and dual function Maleimide, such as bis-n-butylenediamine 4,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propanimide produce photoactivatable intermediates which are capable of forming crosslinks in the presence of light. Alternatively, in order to immobilize the protein, a reactive water-insoluble matrix can be used, such as the bromine described in U.S. Patent Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440. Cyanide activated carbohydrates and reactive substrates. Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of serine or sulfhydryl residues, oxidation of atoms in Cys, and lysine , arginine and histidine side chain α-amine
O:\121\121929.DOC -75- 200808833 基基團的甲基化作用（Creighton，T.E·，Proteins: Structure and Molecule Properties, W.H. Freeman & Co.? SanO:\121\121929.DOC -75- 200808833 Methylation of the group (Creighton, T.E., Proteins: Structure and Molecule Properties, W.H. Freeman & Co.? San
Francisco，第79_86頁（1983))，n-終端胺的乙醯化作用， 以及在一些例子中，C-終端羧基基團的醯胺化作用。 攻類衍生的部分最好改善一或多個特徵，包括化合物的 抗血g生成之活性、溶解性、吸收、生物學的半衰期， 及其類似者。或者，衍生的部分可導化合物具有與未衍 生之化合物相同，或基本上相同的特徵及/或特性。該部 分可另行排除或減少該化合物任何不想要的副作用，及其 類似者。 ~ 另外也可在DNA層面改變本發明之化合物。可將化合物 之任何部分的DNA序列，改變成更可與選出之宿主細胞相 容的密碼子。關於大腸桿菌，其為最佳的宿主細胞，最適 切之密碼子為此項技藝中已知的。可取代密碼子，以便排 除限制位置，4包括不活動的限制位置，其可幫助祖在 選出之宿主細胞中的加工。可修改媒介、聯結子和肤而八 序列，使其包括任何前述的序列改變。因此，所有在本文 中討論的修改、取代、衍生作料等，同等地適用於本發 明的所有觀點’包括但不限於肽、肽二聚體和多聚體、聯 結子和媒介。 此外，熟諳此藝者可回顧結構·功能研究，確認在類似 肽中對活性或結構报重要的殘基。從這類比較來看，可預 期在肽中’與在類似肽中對於活性或結構报重要的胺基酸 殘基一致之胺基酸殘基的重要性。熟諳此藝者可為這類預Francisco, pp. 79-86 (1983)), acetamylation of n-terminal amines, and, in some instances, amide amination of C-terminal carboxyl groups. The fraction derived from the attack preferably improves one or more of the characteristics, including the activity of the compound against blood g production, solubility, absorption, biological half-life, and the like. Alternatively, the derivatized moiety can have the same or substantially the same characteristics and/or characteristics as the underived compound. This moiety may additionally exclude or reduce any unwanted side effects of the compound, and the like. ~ The compounds of the invention can also be altered at the DNA level. The DNA sequence of any portion of the compound can be altered to a codon that is more compatible with the host cell of choice. With regard to E. coli, which is the best host cell, the most suitable codons are known in the art. Codons can be substituted to exclude restricted positions, 4 including inactive restricted positions, which can aid in the processing of progenitors in selected host cells. The media, linker and skin-eight sequences can be modified to include any of the aforementioned sequence changes. Accordingly, all modifications, substitutions, derivatives, and the like discussed herein are equally applicable to all aspects of the present invention including, but not limited to, peptides, peptide dimers and multimers, linkers and media. In addition, those skilled in the art can review structural and functional studies to identify residues that are important for activity or structure in similar peptides. From such comparisons, the importance of amino acid residues in the peptide that are consistent with amino acid residues that are important for activity or structure in similar peptides can be expected. Those who are familiar with this artist can make this kind of pre-
〇：\121\121929.DOC -76- 200808833 期很重要之肽的胺基g线基，選擇在化學上類似的胺基酸 取代作用。 热諳此藝者亦可分析三維結構和胺基酸序列，與在類似 多肽中之結構的關係。從這類資訊來看，熟諳此藝者可預 期肽之胺基酸殘基的排列，與其三維結構的關係。熟諳此 藝者可選擇對於預期是在蛋白質表面上的胺基酸殘基，不 進打徹底的改變，因為這類殘基可能涉及與其他分子的重 要父互作用。此外，熟諳此藝者可產製測試變體，其在每 個想要的胺基酸殘基處，含有單一胺基酸取代。然後可使 用熟諳此藝者已知的活性測定來篩選變體。可使用這類變 體收集有關於適當變體的資訊。例如，如果發現對特定胺 基酸殘基的改變，導致受到破壞、不想要地降低，或不適 當的活性’便可避免這類改變。換句話說，以收集自這類 例 <于實驗的資訊為基礎’熟諳此藝者可輕易地判定應避免 單獨或與其他突變組合，進一步取代的胺基酸。 許多科學出版物已經致力於二級結構的預測。參見 Moult J.? Curr. Op. in Biotech., 7(4):422-427 (1996), Chou 等人，Biochemistry，13(2)··222-245 (1974); Chou等人， Biochemistry, 113(2):21 1-222 (1974); Chou #A，Adv· Enzymol· Relat· Areas Mol. Biol·，47:45-148 (1978); Chou 等人，Ann· Rev. Biochem·，47:251-276 和 Chou 等人， Biophys· J·，26:3 67-3 84 (1979)。此外，目前可利用電腦程 式來幫助預測二級結構。一種預測二級結構的方法是以同 種性塑型為基礎。例如，具有超過30%之序列同一性，或 O:\121\121929.DOC -77- 200808833 超過40%之類似性的兩個多肽或蛋白質，通常具有類似的 結構拓樸學。蛋白質結構資料庫（PDB)最近的成長，已經 提供了增強的二級結構可預測性，包括在多肽或蛋白質之 結構中，可能的折疊次數。參見Holm等人，Nucl. Aei(L Res·，27(1):244-247 (1999)。已經暗示（Brenner等人，〇11〇·· Op· Struct. Biol·，7(3):369-376 (1997))在特定的多肽或蛋 白質中，有限制次數的折疊，且一旦已經解決結構的決定 性次數，結構的預測便變成戲劇化地更精確。 預測一級結構的其他方法，包括’’穿線’’（jones，D·，Curr〇: \121\121929.DOC-76- 200808833 The amino group of the peptide, which is important, is selected for chemically similar amino acid substitution. Those skilled in the art can also analyze the relationship between the three-dimensional structure and the amino acid sequence, as well as structures in similar polypeptides. From this kind of information, those skilled in the art can predict the arrangement of the amino acid residues of the peptide and its three-dimensional structure. Those skilled in the art may choose to make radical changes to the amino acid residues expected to be on the surface of the protein, as such residues may involve interaction with important parents of other molecules. In addition, those skilled in the art can produce test variants which contain a single amino acid substitution at each desired amino acid residue. Variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about appropriate variants. For example, if a change to a particular amino acid residue is found, resulting in damage, undesired reduction, or inappropriate activity, such changes can be avoided. In other words, based on information collected from such cases <experimental information', those skilled in the art can readily determine that amino acids that are further substituted, alone or in combination with other mutations, should be avoided. Many scientific publications have focused on the prediction of secondary structures. See Moult J.? Curr. Op. in Biotech., 7(4): 422-427 (1996), Chou et al, Biochemistry, 13(2) 222-245 (1974); Chou et al, Biochemistry, 113(2): 21 1-222 (1974); Chou #A, Adv· Enzymol· Relat· Areas Mol. Biol·, 47:45-148 (1978); Chou et al., Ann· Rev. Biochem·, 47 :251-276 and Chou et al., Biophys J., 26:3 67-3 84 (1979). In addition, computer programs are currently available to help predict secondary structure. One method of predicting secondary structure is based on isomorphic modeling. For example, two polypeptides or proteins having more than 30% sequence identity, or more than 40% similarity of O:\121\121929.DOC-77-200808833, typically have similar structural topologies. Recent growth in the Protein Structure Database (PDB) has provided enhanced secondary structure predictability, including the number of possible folds in the structure of a polypeptide or protein. See Holm et al., Nucl. Aei (L Res., 27(1): 244-247 (1999). already suggested (Brenner et al., 〇11〇·· Op·Struct. Biol·, 7(3): 369 -376 (1997)) There are a limited number of folds in a particular polypeptide or protein, and once the critical number of structures has been resolved, the prediction of the structure becomes dramatic and more precise. Other methods of predicting primary structure, including '' Threading ''(jones,D·, Curr
Opin. Struct· Biol.，7(3):377-87 (1997); Sippl 等人，Opin. Struct· Biol., 7(3): 377-87 (1997); Sippl et al.
Structure，4(1):15-9 (1996))、"輪廓分析，’（B〇wie等人， Science， 253:164-170 (1991); Gribskov 等人，Meth Enzym·，183:146-159 (1990); Gribskov等人，Proc· Nat Acad· Sci·，84(13):4355-8 (1987))，以及，，進化連鎖”（參見 Holm，在前和Brenner，在前）。 本發明尚包括衍生的專一結合劑，例如肽體，共價修改 以便包括一或多個水溶性聚合物附接，像是聚乙二醇、聚 氧乙二醇，或聚丙二醇，如同美國專利第4,64〇,835號； 4,496,689 號；4,301，144 號；4,67M17 號；4,79i，i92 號和 4，179,337號的描述。此項技藝中已知其他有用的聚合物， 二醇、葡聚醣、纖維素或其他以碳水Structure, 4(1): 15-9 (1996)), "profile analysis, '(B〇wie et al, Science, 253: 164-170 (1991); Gribskov et al, Meth Enzym, 183: 146 -159 (1990); Gribskov et al., Proc. Nat Acad. Sci., 84(13): 4355-8 (1987)), and, evolutionary linkages (see Holm, formerly and Brenner, supra). The invention also includes derivatized specific binding agents, such as peptibodies, covalently modified to include one or more water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as in the U.S. patent. Nos. 4,64, 835; 4,496,689; 4,301,144; 4,67M17; 4,79i, i92 and 4,179,337. Other useful polymers, diols are known in the art. , dextran, cellulose or other carbon water
以及這些聚合物 O:\121\121929.DOC 包括早曱乳基-聚乙二醇、 > -78 - 200808833 的混合物。特佳的是利用聚乙二醇（PEG)亞單元共價修改 的肽體。可在特定的位置與水溶性聚合物結合，例如，在 肽體的胺基終端處，或隨機地附接在多肽的一或多個側鏈 上使用PEG，以便改善專一結合劑，例如月太體的治療效 力，並將抗體人類化，特別在⑸…“以等人，2〇〇〇年丨〇月 17日發證之美國專利第6，133,426號中描述之。 本發明亦企圖衍生化合物的肽及/或媒介部分。這類衍 生物可改善化合物的溶解性、吸收、生物學的半衰期，及 其類似者。該部分可另行排除或減少化合物任何不想要的 副作用，及其類似者。代表性的衍生物包括化合物，其中： 1 ·化合物或其某些部分是環狀的。例如，可修改肽部 分’使其含有二或多個Cys殘基（例如以聯結子），可藉著 二硫鍵的形成將其環化。 2·化合物在分子之間交聯，或使其成為能夠交聯的。 例如，可修改肽部分，使其含有一個Cys殘基，並藉著能 夠與類似分子形成分子間的二硫鍵。化合物亦可經由其c-終端交聯。 3.藉著非，肽基鍵結置換一或多個肽基卜c(〇)nr_]鍵結 (鍵）。代表性的非-肽基鍵結為-CH2-胺基曱酸酯[-CH2-〇C(0)NR-]、膦酸酯、-CH2-績醯胺[-CH2-S(0)2NR-]、脲 [-NHC(0)NH-]、-CH2-二級胺和烷基化 了的肽[-C(0)NR6-，其中R6為低碳數烷基]。 4 ·將N-終端衍生。通常，可將終端醯基化或修改成 經取代之胺。代表性的N-終端衍生基團包括-NRl (-NH2 O:\121\121929.DOC -79- 200808833 以外）、-NRC^CORi、-NRC^COORi、-NRSWhK、-NHC(O) NHRi、琥珀醯亞胺，或芊氧羰基-NH-(cbz_nh-)，其中R 和Ri分別為氫或低碳數烷基，且其中苯基環可利用1至3個 選自由CrC4烧基、CrC#烧氧基、氯和漠所組成之群的取 代基取代。 5·將自由的C-終端衍生。通常，可將c-終端酯化或醯 胺化。例如，可使用在此項技藝中描述的方法，在終端 處將（NH-CH2_CH2_NH2)2加至本發明之化合物上。同樣的 ，可使用在此項技藝中描述的方法，在c_終端處將_NH2M 至本發明之化合物上。代表性的c-終端衍生基團包括，例 如-C(0)R2，其中R2為低碳數烷氧基，或-NR3R4，其中I 和R4分別為氫或C「C8烷基（最好*C「C4烷基）。 6·以其他的，最好是更穩定的交聯部分（例如伸烷基）來 置換二硫鍵。參見，例如Bhatnagar (在前）；Alberts等人 ，Thirteenth Am. Pep. Symp·，357_9 (1993) 〇 7.修改一或多個各別的胺基酸殘基。已知各種專一地 與所選擇之側鍵或終端殘基反應的衍生劑，如同在下文中 詳細說明的。 離胺醯基殘基和胺基終端的殘基，可與琥珀酸或其他的 羧酸酐反應，其逆轉離胺醯基殘基的鼋荷。其他適合衍生 含有α-胺基之殘基的製劑，包括醯亞胺酯，像是甲基p比咬 醯亞胺甲酯；磷酸吡哆醛；吡哆醛；氯硼氫化物；三確基 苯磺酸；0-甲基異脲；2,4-戊二酮；以及利用乙醛酸之轉 胺基酶催化的反應。 O:\121\121929.DOC -80· 200808833 可藉著與任一或數個傳統試劑之組合的反應，來修改精 胺酿基殘基，包括苯甲醯甲醛、2,3_丁二酮、U2_環己二 酉同和雖三顯I。精胺酸殘基的衍生作用需要在鹼性的條件下 進行反應，因為脈官能基的高pKa。此外，這些試劑可與 離胺酸之基團，以及精胺酸ε -胺基基團反應。 酪胺醯基殘基的特定修改，已經被廣泛地研究，而特別 感興趣的是藉著與芳香族之重氮化合物或四硝基甲烷的反 應’將光譜標記導入酪胺醯基殘基内。最常見的是，可使 用Ν-乙醯基咪唑和四硝基甲烷，分別形成〇_乙醯基酪胺醯 基物種和3 -確基衍生物。 可藉著與碳化二醯亞胺（r，_N=C=N-R，）的反應，像是1_環 己基-3-(2-嗎琳基- (4 -乙基）碳化二醯亞胺或ι_乙基_3_(4•氮 鑕-4,4-二甲戊基）碳化二醯亞胺，選擇性地修改羧基側鏈 基團（天冬胺醯基或穀胺醯基）。此外，亦可藉著與銨離子 的反應，將天冬胺醯基和穀胺醯基殘基轉變為天冬醯胺醯 基和穀胺醯胺醯基殘基。 可將穀胺醯胺醯基和天冬醯胺醯基殘基脫醯胺化，成為 相對應的縠胺醯基和天冬胺醯基殘基。或者，可在弱酸性 的條件下將這些殘基脫醯胺化。這些殘基的任一個形式均 在本發明的範圍内。 可藉著胺基酸殘基或其他部分置換半胱胺醯基殘基，排 除二硫鍵結，或相反地穩定交聯作用。參見，例如 Bhatnagar (在前）〇 利用雙重功能之製劑的衍生作用，對於將肽或其功能衍And these polymers O:\121\121929.DOC include a mixture of early-milk-polyethylene glycol, >-78 - 200808833. Particularly preferred are peptibodies covalently modified using polyethylene glycol (PEG) subunits. The PEG can be used at a specific position in combination with a water soluble polymer, for example, at the amine terminus of the peptibody, or randomly attached to one or more side chains of the polypeptide, in order to improve the specific binding agent, such as The therapeutic efficacy of the body, and the humanization of the antibody, in particular, is described in U.S. Patent No. 6,133,426 issued to et al. Peptides and/or mediators. Such derivatives may improve the solubility, absorption, biological half-life of the compound, and the like. This moiety may additionally exclude or reduce any unwanted side effects of the compound, and the like. Representative derivatives include compounds wherein: 1 - the compound or some portion thereof is cyclic. For example, the peptide moiety can be modified to contain two or more Cys residues (eg, in a linker), The formation of a disulfide bond cyclizes it. 2. The compound crosslinks between molecules or makes it crosslinkable. For example, the peptide moiety can be modified to contain a Cys residue, and by being able to molecule Intermolecular disulfide bond. The compound can also be cross-linked via its c-terminal. 3. By means of a non-peptidyl bond, one or more peptidyl groups are c(〇)nr_] linkages (bonds). The non-peptidyl linkage is -CH2-amino phthalate [-CH2-〇C(0)NR-], phosphonate, -CH2- decylamine [-CH2-S(0)2NR- ], urea [-NHC(0)NH-], -CH2-subamine, and alkylated peptide [-C(0)NR6-, wherein R6 is a lower alkyl group]. Terminal derivatization. Typically, the terminal can be thiolated or modified to a substituted amine. Representative N-terminal derivatization groups include -NRl (-NH2 O: \121\121929.DOC-79-200808833), - NRC^CORi, -NRC^COORi, -NRSWhK, -NHC(O) NHRi, amber succinimide, or oxime carbonyl-NH-(cbz_nh-), wherein R and Ri are each hydrogen or a lower alkyl group, And wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of CrC4 alkyl, CrC# alkoxy, chlorine and desert. 5. Derivatization of a free C-terminal. Usually, c - terminal esterification or guanidation. For example, (NH-CH2_CH2_NH2)2 can be added to the compound of the invention at the terminal using the method described in the art. Similarly, _NH2M can be added to the compound of the invention at the c-terminal using methods described in the art. Representative c-terminal derivatization groups include, for example, -C(0)R2 Wherein R2 is a lower alkoxy group, or -NR3R4, wherein I and R4 are each hydrogen or C"C8 alkyl (preferably *C "C4 alkyl"). 6. Others, preferably more stable The cross-linking moiety (e.g., alkylene) is substituted for the disulfide bond. See, for example, Bhatnagar (previously); Alberts et al, Thirteenth Am. Pep. Symp, 357_9 (1993) 〇 7. Modification of one or more individual amino acid residues. Derivatives that specifically react with selected side or terminal residues are known, as will be described in detail below. Residues from the amine sulfhydryl residue and the amine terminal may be reacted with succinic acid or other carboxylic anhydride to reverse the loading of the amine sulfhydryl residue. Other preparations suitable for deriving residues containing an α-amino group, including quinone imide, such as methyl p to butyl imine methyl ester; pyridoxal phosphate; pyridoxal; chloroborohydride; Benzenesulfonic acid; 0-methylisourea; 2,4-pentanedione; and a reaction catalyzed by a transaminase of glyoxylic acid. O:\121\121929.DOC -80· 200808833 The spermine-based residue, including benzamidine formaldehyde, 2,3-butanedione, can be modified by reaction with any one or several conventional reagents. , U2_ ring has two identical and although three shows I. Derivatization of arginine residues requires reaction under basic conditions due to the high pKa of the pulse functional groups. In addition, these reagents can react with the leucine group and the arginine ε-amino group. Specific modifications of tyramine sulfhydryl residues have been extensively studied, and it is of particular interest to introduce spectral markers into tyramine thiol residues by reaction with aromatic diazonium compounds or tetranitromethane. . Most commonly, ruthenium-ethenyl imidazole and tetranitromethane can be used to form the oxime-acetamid oxime species and the 3-de radical derivative, respectively. By reaction with carbodiimide (r, _N=C=NR,), such as 1-cyclohexyl-3-(2-morphinyl-(4-ethyl)carbodiimide or Io_ethyl_3_(4•azepine-4,4-dimethylpentyl)carbodiimide, selectively modifying a carboxyl side chain group (aspartame or glutamine) , by reacting with ammonium ions, converting aspartame and glutamine residues to aspartic acid sulfhydryl and glutamine amidoxime residues. And the amidoxime residue is deaminated to become the corresponding amidoxime and aspartame residues. Alternatively, these residues can be deaminated under weakly acidic conditions. Any form of the residue is within the scope of the invention. The cysteamine sulfhydryl residue may be replaced by an amino acid residue or other moiety, excluding the disulfide bond, or conversely stabilizing the cross-linking effect. For example, Bhatnagar (previously) uses a dual-function formulation to derivatize peptides or their functional derivatives.
O:\121\121929.DOC -81 - 200808833 生物與不溶於水的支撐矩陣或其他大分子載劑交聯是有用 的。常用的聯結子包括，例如^•雙（重氮乙醯基）_2_苯基 乙烷、戊二醛、羥基琥珀醯亞胺酯，例如帶有4-疊氮水 揚酸的酯、高雙重功能的醯亞胺酯，包括二琥珀醯亞胺基 酉曰 像疋3,3 · 一^硫雙（玻ίό酿亞胺基丙酸g旨），以及雙重功 月b的順丁稀一醯亞胺’像是雙順丁稀二醢亞胺基_ι，8_ 辛烧。諸如甲基-3-[(對-疊氮苯基）二硫代]丙醯亞胺酯之類 的衍生劑，產生光可激活的中間物，其能夠在光的存在下 形成交聯。或者，為了固定蛋白質，可使用反應性的不溶 於水之矩陣’像是在美國專利第3,969,287號；3,691，016號 ;4，195，128 號；4,247,642 號；4,229,537 號和 4,330,440 號 中描述的 >臭化氣激活之碳水化合物和反應性受質。 可便利地將碳水化合物（寡醣）附接在蛋白質中已知是糖 基化作用位置的位置上。通常，將〇_連接之寡醣類附接在 絲胺酸（Ser)或蘇胺酸（Thr)殘基上，同時將N-連接之寡醣 類附接在天冬醢胺（Asn)殘基上，此時它們是序列Asn_x_ Ser/Thr的一部分，其中X可以是脯胺酸以外的任何胺基酸 。X最好脯胺酸以外19個天然存在的胺基酸之一。N_連接 和〇·連接之募醣類的結構，以及在每種類型中找到的糖殘 基都是不同的。一種經常在兩者中找到之糖的類型是N_乙 醯基神經氨糖酸（稱為唾液酸）。唾液酸經常是N_連接和〇_ 連接之寡醣類的終端殘基，且憑藉著它的負電荷，可賦與 糖基化之化合物酸性的性質。可將這類位置（們）併入本發 明之化合物的聯結子中，且最好是在多肽化合物之重組產 O:\121\121929.DOC -82 - 200808833 製期間，被細胞糖基化（例如在哺乳動物細胞中，像是 CHO、BHK、COS)。然而，尚可藉著此項技藝中已知的合 成或半-合成程序，將這類位置糖基化。 其他可能的修改，包括脯胺酸和離胺酸的羥化作用，絲 胺酿基或蘇胺醯基殘基之羥基基團的磷酸化作用，在Cys 中石1原子的氧化作用，離胺酸、精胺酸和組胺酸側鏈之心 胺基基團的甲基化作用[；Creight〇n，Pr〇teins: structure andO:\121\121929.DOC -81 - 200808833 It is useful to crosslink biologically with water-insoluble support matrices or other macromolecular carriers. Commonly used linkers include, for example, bis(diazonium)-2-phenylethane, glutaraldehyde, hydroxy amber ylide, such as esters with 4-azide salicylic acid, high double Functional quinones, including di-succinimide-based ruthenium 疋 3,3 · sulphur bis (glass ό 亚 亚 亚 旨 , , , , , , , , , , , , , , , The imine is like a double-butadiene diimide imine group _ι, 8_ octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propanimide produce photoactivatable intermediates which are capable of forming crosslinks in the presence of light. Alternatively, in order to immobilize the protein, a reactive water-insoluble matrix can be used, as described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440. Stinky gas activated carbohydrates and reactive substrates. Carbohydrates (oligosaccharides) can be conveniently attached to positions in the protein known to be glycosylation sites. Typically, 〇-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues Above this, they are now part of the sequence Asn_x_ Ser/Thr, where X can be any amino acid other than valerine. X is preferably one of the 19 naturally occurring amino acids other than proline. The structure of the N_linked and 〇·linked sugars, and the sugar residues found in each type are different. One type of sugar often found in both is N-ethyl-neuraminic acid (referred to as sialic acid). Sialic acid is often the terminal residue of the N-linked and 〇-linked oligosaccharides, and by virtue of its negative charge, can impart the acidic nature of the glycosylated compound. Such positions may be incorporated into the linker of the compounds of the invention, and are preferably glycosylated by the cell during the recombinant production of the polypeptide compound O: \121\121929.DOC -82 - 200808833 ( For example, in mammalian cells, such as CHO, BHK, COS). However, such positions can be glycosylated by synthetic or semi-synthetic procedures known in the art. Other possible modifications include the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl group of the serine or sulfhydryl residue, the oxidation of the stone 1 atom in Cys, the lysine , methylation of arginine and histidine acid side chain amine groups [;Creight〇n,Pr〇teins: structure and
Molecular Properties (W.H. Freeman & Co.5 San Francisco) ，第 79-86 頁（1983)]。 另外亦可在DNA層面改變本發明之化合物。可將化合物 之任何部分的DNA序列，改變成更可與選出之宿主細胞相 容的密碼子。關於大腸桿菌，其為最佳的宿主細胞，最適 切之密碼子為此項技藝中已知的。可取代密碼子，以便排 除限制位置，或包括不活動的限制位置，其可幫助Dna在 選出之宿主細胞中的加工。可修改媒介、聯結子和肽1^^八 序列’使其包括任何前述的序列改變。 親和力成熟 本發明的一個具體實施例包括"親和力成熟的"肽和肽體 。該過程企圖使用噬菌體展示或其他選擇技術，增加本發 明之肽和肽體的親和力或生物-活性。以一致序列（為了相 關肽的集合而產製)為基礎’可產製指引的二級嗤菌體展 示庫，其中"核心”胺基酸（從一致序列來判定）不變地受到 保留’或傾向於頻繁地發生。或者’可使用各別的肤序列 來產製傾向一方、指引之噬菌體展示庫。淘洗這類的庫，Molecular Properties (W.H. Freeman & Co. 5 San Francisco), pp. 79-86 (1983)]. Alternatively, the compounds of the invention may be altered at the DNA level. The DNA sequence of any portion of the compound can be altered to a codon that is more compatible with the host cell of choice. With regard to E. coli, which is the best host cell, the most suitable codons are known in the art. Codons can be substituted to exclude restricted positions, or include inactive restricted positions, which can aid in the processing of Dna in selected host cells. The vector, linker and peptide can be modified to include any of the aforementioned sequence changes. Affinity maturation A particular embodiment of the invention includes "affinity mature" peptides and peptibodies. This process attempts to increase the affinity or bio-activity of the peptides and peptibodies of the present invention using phage display or other selection techniques. Based on a consistent sequence (produced for the collection of related peptides), a second-stage microbial display library that can be produced, in which the "core" amino acid (determined from a consensus sequence) is invariably retained' Or tend to occur frequently. Or 'you can use a different skin sequence to produce a phage display library that favors one side and guides.
O:\121\121929.DOC -83 - 200808833 可產生提高對Ang-2之結合作用，或具有提高之生物活性 的肽（可將其轉變為肽體）。 非-肽類似物/蛋白質模仿物 此外，亦期待肽的非-肽類似物，其提供穩定化之結構 或減少的生物降解作用。可以選出的抑制肽為基礎，藉著 以非肽部分置換一或多個殘基，來製備肽模仿類似物。非 肽部分最好容許該肽保留其天然的證實，或穩定較佳的， 例如具有生物活性之證實，其保留認出並與入叩_2結合的 能力。一方面，所得的類似物/模仿物對八^_2顯示出增加 的結合親和力。在Nachman等人，Regul· Pept 57:359_37〇 (1995)中，描述了一種從肽來製備非肽模仿類似物之方法 的實例。如果想要，可修改本發明之肽，例如藉著糖基化 作用、胺化作用、缓基化作用或填酸化作用，或藉著創 本發明之肽的酸加成鹽、醯胺、酯，特別是C_終端的西旨 ，以及N-醯基衍生物。亦可修改肽體，藉著與其他部分形 成共價或非共價的複合物，來創造肽衍生物。可藉著使化 學部分與在包括肽體之胺基酸側鏈上，或冰或^終端處的 官能基交聯，來製備共價-結合的複合物。 特別期待可與報告者基團共軛的肽，該報告者基團包括 ，但不限於放射性標記、螢光標記、酵素（例如催化比色 或螢光反應）、受質、固相矩陣或載劑（例如生物素或抗生 物素蛋白）°因此，本發明提供包括肽體分子之分子，其 中該分子最好尚包括報告者基團，選自由放射性標記、螢 光標記、酵素、受質、固相矩陣和載劑所組成之群。這類O:\121\121929.DOC -83 - 200808833 A peptide that increases binding to Ang-2 or has enhanced biological activity (which can be converted to a peptide). Non-peptide analogs/protein mimetics In addition, non-peptide analogs of peptides are also contemplated which provide stabilized structures or reduced biodegradation. Based on the selected inhibitory peptide, a peptide mimetic analog can be prepared by replacing one or more residues with a non-peptide moiety. Preferably, the non-peptide portion allows the peptide to retain its natural identity, or is stable, such as having a biologically active confirmation that retains the ability to recognize and bind to 叩_2. In one aspect, the resulting analog/mimetic exhibits increased binding affinity for 八_2. An example of a method for preparing a non-peptide mimetic analog from a peptide is described in Nachman et al., Regul Pept 57:359_37 (1995). If desired, the peptides of the invention may be modified, for example by glycosylation, amination, slow-acting or acidification, or by acid addition salts, guanamines, esters of the peptides of the invention. , especially the C-terminal, and N-mercapto derivatives. Peptibodies can also be modified to create peptide derivatives by forming covalent or non-covalent complexes with other moieties. Covalent-bound complexes can be prepared by crosslinking the chemical moiety with a functional group on the amino acid side chain including the peptidic body, or at the end of the ice or the terminal. Particularly contemplated are peptides that are conjugated to a reporter group, including, but not limited to, radioactive labels, fluorescent labels, enzymes (eg, catalytic colorimetric or fluorescent reactions), substrates, solid phase matrices, or Agent (eg biotin or avidin). Accordingly, the invention provides a molecule comprising a peptidomimetic molecule, wherein the molecule preferably further comprises a reporter group selected from the group consisting of radiolabels, fluorescent labels, enzymes, receptors, A group of solid phase matrices and carriers. This type
O:\121\121929.DOC -84- 200808833 標記是熟諳此藝者已熟知的，例如特別期待生物素標記。 這類標記的使用為熟諳此藝者已熟知的，並描述在例如美 國專利第 3,817,837 號；3,850,752 號；3,996,345 號和 4,277,437號中。其他有用的標記包括，但不限於放射性標 記、螢光標記和化學發光標記。關於使用這類標記的美國 專利，包括例如美國專利第3,817,837號；3,850,752號； 3,939,350號和3,996,345號。任何本發明之肽體均可包括一 、二或多個這些標記的任一個。 製造肽的方法 可使用各式各樣在此項技藝中已知的技術，來產製本發 明的肽。例如，可根據傳統的技術，在溶液中，或在固相 支撐物上合成這類肽。有各種市售的自動合成器，並可根 據已知的草案使用之。參見，例如Stewart和Young (在前） ;Tam等人，J. Am. Chem. Soc.，105:6442 (1983); Merrifield， Science 232:341-347 (1986); Barany 和 Merrifield，TheO:\121\121929.DOC-84-200808833 Marking is well known to those skilled in the art, for example, biotin labeling is particularly expected. The use of such indicia is well known to those skilled in the art and is described in, for example, U.S. Patent Nos. 3,817,837; 3,850,752; 3,996,345 and 4,277,437. Other useful labels include, but are not limited to, radioactive labels, fluorescent labels, and chemiluminescent labels. U.S. Patents for the Use of Such Marks include, for example, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350 and 3,996,345. Any of the peptibodies of the invention may comprise any one of one, two or more of these labels. Methods of Making Peptides The peptides of the present invention can be produced using a wide variety of techniques known in the art. For example, such peptides can be synthesized in solution or on a solid support according to conventional techniques. There are various commercially available automatic synthesizers that can be used according to known drafts. See, for example, Stewart and Young (formerly); Tam et al, J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The
Peptides，Gross 和 Meienhofer 編輯，Academic Press，New York，1_284; Barany等人，Int· J. Pep. Protein Res·，30:705-739 (1987);以及美國專利第5,424,398號，分別以引用的 方式併入本文中。 固相肽合成法使用含有0.1-1.0 mM胺/克聚合物的共聚（ 苯乙烯-二乙烯苯）。這些合成肽的方法，使用乙氧羰基（t-BOC)或9-苐基甲氧羰基（FMOC)保護的α-胺基基團。兩種 方法均涉及逐步合成，藉此在每個步驟中，從肽之C-終端 開始加入單一的胺基酸（參見，Coligan等人，Curr. Prot. O:\121\121929.DOC -85 - 200808833Peptides, Editing by Gross and Meienhofer, Academic Press, New York, 1_284; Barany et al, Int J. Pep. Protein Res, 30: 705-739 (1987); and U.S. Patent No. 5,424,398, each by reference Incorporated herein. The solid phase peptide synthesis method uses a copolymer (styrene-divinylbenzene) containing 0.1 to 1.0 mM amine per gram of polymer. These methods of synthesizing peptides use an ethoxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (FMOC) protected a-amino group. Both methods involve stepwise synthesis whereby a single amino acid is added from the C-terminus of the peptide in each step (see, Coligan et al., Curr. Prot. O:\121\121929.DOC-85 - 200808833
Immunol” Wiley Interscience，1991 ’ 單元9)。在完成化學 合成時，可將合成的肽脫保護，以便移除t_B〇c或fm〇c 胺基酸阻斷基團，並藉著在降低的溫度下以酸處理，從聚 合物中切開（例如液態HF-10%茴香醚，在〇°C下大約〇·25至 大約1小時）。在蒸發試劑之後，利用1。/。乙酸溶液，從聚合 物中萃取肽，然後冷凍乾燥，產生粗製的物質。可藉著諸 如在交聯葡聚糖（}_15上，使用5%乙酸作為溶劑的凝膠過 濾之類的技術，正常地純化該粗製物質。管柱之適當溶離 份的冷凍乾燥，將產生均質的肽或肽衍生物，然後可藉著 諸如胺基酸分析、薄層層析、高效率液相層析、紫外線吸 收光谱、莫耳旋光、溶解性之類的標準技術，定出特徵， 並藉著固相Edman降解作用定量。 亦了利用其他方法，像是從嗟菌體展示庫中選擇肽。可 從按照在本文中之描述的胺基酸組，來製備庫。噬菌體展 示在確認可根據本發明使用的肽上，可能是特別有效的。 簡言之，製備噬菌體庫（使用例如ml 13以或λ噬菌體），展 示從4至大約80個胺基酸殘基的插入物。該插入物可代表 ，例如一個完全退化或偏愛的陣列。然後可選擇攜帶噬菌 體的插入物，其與想要的抗原結合。可經由數次再選擇與 想要抗原結合之噬菌體的回合，重覆該方法。重覆的回合 導致富含攜帶特殊序列的噬菌體。可進rDna序列分析， 以便確認經表現之肽的序列。可判定與想要抗原結合之序 列的最小直線部分。可使用含有插人物之偏愛的庫重覆該 過程，該插入物含有部分或全部的最小直線料，加一或 O:\121\121929.DOC -86 - 200808833 多個額外的退化殘基上游’或其下游。這些技術可確認本 發明之肚，仍對Ang-2具有比業已在本文中確認之製劑更 大的結合親和力。 與製備肽的方式無關，可使用標準重組DNA程序，來產 製編碼每個這類肽和肽體的核酸分子。可適當地操縱這類 DNA分子的核苷酸序列，不需改變其編碼的胺基酸序列， 以便說明核酸密碼的簡併，並說明在特定宿主細胞中的密 碼子優勢。 重組的DNA技術是製備本發明之全長肽體和其他大蛋白 質專一結合劑，或其片段的便利方法。可將編碼肽體或片 段的DNA分子插入表現載體内，其可依序再被插入宿主細 胞内，來產製抗體或片段。 一般而言，可使用在本文中，在實例中描述的程序，獲 得編碼肽或肽體的DNA分子。探針和典型的雜交條件，是 諸如在Ausubei等人（Current Pr〇t〇c〇ls沁心丨⑽心Immunol" Wiley Interscience, 1991 'Unit 9). Upon completion of chemical synthesis, the synthetic peptide can be deprotected in order to remove the t_B〇c or fm〇c amino acid blocking group and at a reduced temperature Under acid treatment, cut from the polymer (for example, liquid HF-10% anisole, about 2525 to about 1 hour at 〇 ° C). After evaporation of the reagent, use 1 / / acetic acid solution, from the polymerization The peptide is extracted and then lyophilized to give a crude material. The crude material can be purified normally by a technique such as gel filtration using 5% acetic acid as a solvent on a cross-linked dextran (}-15). Freeze drying of the appropriate fraction of the column will result in a homogeneous peptide or peptide derivative, which can then be analyzed by, for example, amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, and moiré. Standard techniques such as solubility, characterizing, and quantifying by solid phase Edman degradation. Also using other methods, such as selecting peptides from the sputum display library, can be as described in this article. Amino acid group to prepare Phage display may be particularly effective in identifying peptides that can be used in accordance with the present invention. Briefly, a phage library (using, for example, ml 13 or lambda phage) is displayed, displaying from 4 to about 80 amino acid residues. The insert may represent, for example, a fully degraded or favored array. The insert carrying the phage may then be selected to bind to the desired antigen. The phage that binds to the desired antigen may be reselected several times. Round, repeat the method. Repeated rounds result in enrichment of phage carrying specific sequences. RDna sequence analysis can be performed to confirm the sequence of the expressed peptide. The smallest linear portion of the sequence to which the antigen is desired can be determined. Repeat the process with a library containing the favorite of the person, the insert containing some or all of the smallest straight line, plus one or O: \121\121929.DOC -86 - 200808833 multiple additional degenerate residues upstream' or Downstream. These techniques confirm the belly of the present invention and still have greater binding affinity for Ang-2 than the formulations already identified herein. A standard recombinant DNA program can be used to produce a nucleic acid molecule encoding each of these peptides and peptibodies. The nucleotide sequence of such a DNA molecule can be appropriately manipulated without changing the amino acid sequence encoded thereby, so that Describe the degeneracy of the nucleic acid code and indicate the codon usage in a particular host cell. Recombinant DNA technology is a convenient method for preparing the full length peptibody and other large protein specific binding agents of the present invention, or fragments thereof. The DNA molecule of the body or fragment is inserted into the expression vector, which can be inserted into the host cell in sequence to produce the antibody or fragment. In general, the coding peptide can be obtained using the procedures described herein, in the examples. Peptide DNA molecules. Probes and typical hybridization conditions are such as in Ausubei et al. (Current Pr〇t〇c〇ls沁心丨(10) heart
Biology，Current Protocols Press [1994])中陳述的那些。在 雜父之後，可在適當的嚴格度下沖洗探測到的墨點，將視 諸如探針尺寸、預期之探針對純種系的同種性、待篩選之 庫的類型，以及待篩選之純種系的數目之類的因素而定。 高嚴格度篩選的實例為在50_65r之間的溫度下，〇 ι X SSC和 0.1% SDS 〇 亦可使用酵母菌兩個-雜化物的篩選方法，來確認與 ^ng-2結合的本發明之肽。因此，可使用抗原或其片段來 篩k肽庫，包括噬菌體展示庫，以便確認和選擇A吨_2結Biology, those stated in Current Protocols Press [1994]). After the uncle, the detected ink spots can be washed under appropriate stringency, depending on the size of the probe, the expected homology of the probe to the pure germline, the type of library to be screened, and the pure species to be screened. Depending on factors such as the number of departments. An example of high stringency screening is that at temperatures between 50_65 rpm, 〇ι X SSC and 0.1% SDS 〇 can also be screened using yeast two-hybrids to confirm the combination of the invention with ^ng-2 Peptide. Thus, antigens or fragments thereof can be used to screen k-peptide libraries, including phage display libraries, to confirm and select A-ton-2 junctions.
O:\121\121929.DOC -87- 200808833 合劑，例如本發明之肽體。 或者，可利用各種表現載體/宿主系統，容納並表現本 發明之肽。這些系統包括但不限於微生物，像是以重組噬 fe體、吳體或接合質體DNA表現載體轉化的細菌；以酵母 菌表現載體轉化的酵母菌；以病毒表現載體（例如桿狀病 毒）轉移感染的昆蟲細胞系統；以病毒表現載體（例如花椰 菜花葉病毒，CaMV ;煙草花葉病毒，TMV)轉移感染，以 及以細菌表現載體（例如丁丨或邱以^質體）轉移感染的植物 細胞系統；或哺乳動物細胞。在重組蛋白質產製中有用的 哺乳動物細胞，包括但不限於VER〇細胞、HeLa細胞、中 國倉鼠卵巢（CHO)細胞株、c〇S細胞（像是COS_7)、W138 、BHK、HepG2、3T3、RIN、MDCK、A549、PC12、 K562和293細胞。在後文中描述重組表現肽的代表性草案。 f•表現載體，，一詞意指質體、噬菌體、病毒或載體，以便 從DNA (RNA)序列中表現多肽。表現載體可包括轉錄單元 ，包括（1)在基因表現中具有調節角色的遺傳元件或元件（ 們），例如啟動基因或促進序列，（2)編碼結合劑的結構或 序列，將其轉錄成mRNA，再轉譯成蛋白質，以及（3)適當 之轉錄開始和終止序列的集合。想要在酵母菌或真核生物 表現系統中使用的結構單位，最好包括能夠由宿主細胞將 所轉譯之蛋白質分泌至細胞外的前導序列。或者，在無前 導或運送序列，表現重組蛋白質之處，可包括胺基終端的 甲硫胺醯基殘基。後續可以或可以不從所表現之重組蛋白 質中切開該殘基，提供最終的肽產物。O: \121\121929.DOC -87- 200808833 A mixture, such as a peptibody of the invention. Alternatively, various expression vector/host systems can be utilized to house and characterize the peptides of the invention. These systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage, corpus callosum or plastid DNA expression vectors; yeasts transformed with yeast expression vectors; and viral expression vectors (eg, baculovirus) Infected insect cell system; transfer infection with viral expression vectors (eg, cauliflower mosaic virus, CaMV; Tobacco mosaic virus, TMV), and transfer of infected plant cells with bacterial expression vectors (eg, Ding or Qiu) System; or mammalian cells. Mammalian cells useful in recombinant protein production, including but not limited to VER〇 cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, c〇S cells (like COS_7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. A representative draft of recombinant expression peptides is described later. f•Expression vector, the term means a plastid, bacteriophage, virus or vector to express a polypeptide from a DNA (RNA) sequence. A performance vector can include a transcription unit comprising (1) a genetic element or element that has a regulatory role in gene expression, such as a promoter or promoter sequence, and (2) a structure or sequence encoding the binding agent, which is transcribed into mRNA. , then translated into proteins, and (3) a collection of appropriate transcriptional start and stop sequences. The structural unit to be used in the yeast or eukaryotic expression system preferably includes a leader sequence capable of secreting the translated protein to the outside of the cell by the host cell. Alternatively, in the absence of a leader or delivery sequence, where the recombinant protein is expressed, an amine-based terminal methionine residue can be included. This residue may or may not be cleaved from the expressed recombinant protein to provide the final peptide product.
O:\121\121929.DOC -88 - 200808833 例如可使用市售的表現糸統’例如Pichia ExpressionO:\121\121929.DOC -88 - 200808833 For example, a commercially available performance system such as Pichia Expression can be used.
System (Invitrogen，San Diego，CA)，依據製造者的指示， 在酵母菌中以重組的方式表現肽。該系統亦依賴先（pre)_ 鈾（pro)-oc系統來指揮分泌，但插入物的轉錄作用，則在藉 著甲醇誘導時由醇氧化酶（alc〇h〇l 〇xidase)(A〇xl)啟動基 因來推動。 藉著例如用來從細菌和哺乳動物細胞上清液中，純化肽 的方法，從酵母菌生長培養基中純化已分泌的肽。 或者，亦可將編碼肽的cDNA選殖到桿狀病毒表現載體 PVL1393 (PharMingen，San Diego, CA)内。可根據製造者 的指弓丨（PharMingen)使用該載體，在不含仆9蛋白質之培養 基中感染秋黏蟲（Spodoptera frugiperda)細胞，並產生重組 的蛋白質。可使用肝素_瓊脂糖管柱（Pharmacia)，從培養 基中純化並濃縮該重組蛋白質。 或者’可在昆蟲系統中表現肽。可供蛋白質表現之昆蟲 系統是熟諳此藝者已熟知的。在一種這類系統中，可使用 ΐ 蓿尺虫筻顆粒體病毒（Autographa California nuclear polyhedrosis virUS)(AcNPV)作為載體，在秋黏蟲細胞或夜 蛾（Trichoplusia)幼蟲中表現外來基因。可將肽密碼序列選 殖到病毒的非必要區域内，像是多面素（p〇lyhedrin)基因， 並放在多面素啟動基因的控制之下。該肽的順利插入，將 使夕面素基因成為無活性的，並產生缺乏外殼蛋白殼的重 組病毒。可使用該重組病毒來感染秋黏蟲細胞或夜蛾幼蟲 ’在其中表現該肽。Smith等人，J. Virol. 46:584 (1983); O:\121\121929.DOC -89- 200808833System (Invitrogen, San Diego, CA), expressed in recombinant form in yeast according to the manufacturer's instructions. The system also relies on the pre- (pre) uranium (pro)-oc system to direct secretion, but the transcription of the insert is induced by methanol by alcohol oxidase (alc〇h〇l 〇xidase) (A〇 Xl) Start the gene to drive. The secreted peptide is purified from the yeast growth medium by, for example, a method for purifying the peptide from bacterial and mammalian cell supernatants. Alternatively, the cDNA encoding the peptide can be cloned into the baculovirus expression vector PVL1393 (PharMingen, San Diego, CA). The vector can be used according to the manufacturer's finger licking (PharMingen), and the Spodoptera frugiperda cells are infected in a medium containing no servoprotein 9 and a recombinant protein is produced. The recombinant protein can be purified and concentrated from the medium using a heparin-agarose column (Pharmacia). Alternatively, the peptide can be expressed in an insect system. Insect systems for protein expression are well known to those skilled in the art. In one such system, Autographa California nuclear polyhedrosis virUS (AcNPV) can be used as a vector to express foreign genes in fall beetle cells or Trichoplusia larvae. The peptide coding sequence can be cloned into a non-essential region of the virus, such as the p〇lyhedrin gene, and placed under the control of a polyhedrin promoter. The smooth insertion of the peptide will render the primamycin gene inactive and produce a recombinant virus lacking the coat protein shell. The recombinant virus can be used to infect the autumn mucus cells or the larvae of the larvae' in which the peptide is expressed. Smith et al., J. Virol. 46: 584 (1983); O:\121\121929.DOC -89- 200808833
Engelhard等人，Proc. Nat. Acad. Sci. (USA) 91:3224-7 (1994)。 在其他實例中，可藉著PCR擴大編碼肽的DNA序列，並 選殖到適當的載體内，例如pGEX-3X (Pharmacia)。設計 pGEX載體，產生包括由該載體編碼之榖胱甘肽-S-轉移酶 (GST)，以及由插入載體之選殖位置内的DNA片段編碼之 蛋白質的融合蛋白質。可產製PCR用的引子，包括例如適 當的切開位置。在單獨使用融合部分，以便促進表現，或 另外不想要附接感興趣的肽之處，可從融合蛋白質之GST 部分中切開重組的融合蛋白質。將PGEX-3X/專一結合劑 肽構築體轉化至大腸桿菌XL-1 Blue細胞（Stratagene，La Jolla CA)内，分離各別的轉化物，並使其生長。可純化得 自各別轉化物的質體DNA，並使用自動定序器部分地定序 ，以便證實編碼想要之專一結合劑的核酸插入物，以適當 的方位出現。 本發明的某些肽組合物是其中肽體與任何抗-腫瘤肽， 像是腫瘤壞死因子（TNF)共軛的那些。在特佳的方法中， 在架構中將編碼肽之序列與編碼TNF (Novagen，Madison， WI)之序列融合，以重組體融合來產製TNF-專一結合劑肽 嵌合體。可將肽_TNF cDNA選殖到pET-llb載體（Novagen) 内，並可根據pETllb製造者的指示，誘導在BL21大腸桿 菌中表現TNF-肽。可從細菌的溶胞產物中，藉著硫酸銨製 備、在苯基··瓊脂糖6速流上的疏水性交互作用層析法、在 DEAL·-瓊月旨糖速流上的離子交換層析法，和在Sephacryl- O:\121\121929.DOC -90- 200808833 S-300 HR上的凝膠過濾層析法，來純化可溶性的TNF-肽。 可如下純化可在細菌中，以不溶包涵體之形式產製的融 合蛋白質。可藉著離心犧牲宿主細胞；以0.15 M NaCl，10 mM Tris，pH 8，1 mM EDTA沖洗；並在室溫下以0.1毫克/ 毫升溶菌酶（Sigma，St. Louis，MO)處理15分鐘。可藉著超 音波振盪使溶胞產物澄清，並藉著以12,000 X g離心10分 鐘，使細胞碎屑形成小球。可將含有小球之融合蛋白質再 懸浮於50 mM Tris，pH 8和10 mM EDTA中，在50%甘油上 分層，並以6000 X g離心30分鐘。可將小球再懸浮於不含 Mg++和Ca++的標準磷酸緩衝生理鹽水溶液（PBS)中。可藉 著在變性之SDS-PAGE中，分級分離再懸浮的小球 (Sambrook等人，在前），進一步純化融合蛋白質。可將凝 膠浸於0.4 M KC1中，使蛋白質呈像，可將其切下，並在 缺乏SDS之跑凝膠的緩衝溶液中進行電洗脫。如果在細菌 中產生的GST/融合蛋白質為可溶的蛋白質，則可使用GST 純化基本單位（Pharmacia)純化之。 可消化融合蛋白質，從本發明之肽中切開GST。可在室 溫下培養消化反應（20-40毫克融合蛋白質，20-30單位人類 凝血酶（4000單位/毫克，Sigma)，在0.5毫升PBS中）16-48 小時，並裝入變性的SDS-PAGE凝膠中，分級分離反應產 物。可將凝膠浸於0.4 M KC1中，使蛋白質譜帶呈像。可 藉著胺基酸序列分析，使用自動定序器（Applied Bio systems 473 A型，Foster City，C A)，證實與肽之預期分 子量一致的蛋白質譜態之身分。或者，可藉著進行肽的 O:\121\121929.DOC -91 - 200808833 HPLC及/或質譜分析，來證實其身分。 或者，可將編碼肽的DNA序列選殖到含有想要之啟動基 因，可視需要還有前導序列的質體内[Better等人，Sdence 240:1041-43 (1988)]。可藉著自動定序證實該構築體之序 列。然後可使用標準程序，使用細菌的CaC12培養和熱休 克處理（Sambrook等人，在前），將質體轉化到大腸桿菌品 系MC1061内。可使經過轉化的細菌生長在補充有羧苄青 黴素的LB培養基中，並可藉著使其在適當培養基中生長， 誘導表現蛋白質的產製。如果出現前導序列，便可完成肽 的分泌’並在分泌期間切開。 可藉著在下文中描述的方法，從細菌培養基中純化已經 分泌的重組蛋白質。 表現重組蛋白質之哺乳動物宿主系統，是熟諳此藝者已 4知的。彳針對加工經表現之蛋白冑，或產生某些在提供 蛋白質活性時有用的轉譯-後修改之特殊能力，選擇宿主 細胞品系。這類蛋白質之修改包括，但不限於乙酿化作用 、羧化作、糖基化作用、磷酸化作用、脂質化作用和醯 化作用。不同的宿主細胞，像是CHO、HeLa、MDCK、 293、WI38及其類似物，對於這類轉譯-後活性，具有特定 的細胞機構和獨特的機制，並可選擇以便確保導人之外來 蛋白質的正確修改和加工。 希望所使用的轉化細胞，最好可供長期、高產量的蛋白 貝產製且本身穩定的表現。一旦利用含有可選擇標記， 連同心要表現之卡匣的載體轉化這類細胞，便可容許該細Engelhard et al., Proc. Nat. Acad. Sci. (USA) 91:3224-7 (1994). In other examples, the DNA sequence encoding the peptide can be amplified by PCR and colonized into a suitable vector, such as pGEX-3X (Pharmacia). The pGEX vector is designed to produce a fusion protein comprising a glutathione-S-transferase (GST) encoded by the vector, and a protein encoded by a DNA fragment inserted into the selection site of the vector. Primers for PCR can be produced, including, for example, suitable incision sites. The recombinant fusion protein can be cleaved from the GST portion of the fusion protein where the fusion moiety is used alone to promote performance, or where it is not desired to attach the peptide of interest. The PGEX-3X/specific binding agent peptide construct was transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla CA), and the respective transformants were isolated and grown. The plastid DNA from the individual transformants can be purified and partially sequenced using an automated sequencer to confirm that the nucleic acid insert encoding the desired specific binding agent is present in the appropriate orientation. Certain peptide compositions of the invention are those in which the peptibodies are conjugated to any anti-tumor peptide, such as tumor necrosis factor (TNF). In a particularly preferred method, the sequence encoding the peptide is fused to a sequence encoding TNF (Novagen, Madison, WI) in a framework to produce a TNF-specific binder peptide chimera by recombinant fusion. The peptide_TNF cDNA can be cloned into the pET-llb vector (Novagen) and the TNF-peptide can be induced to be expressed in BL21 E. coli according to the instructions of the manufacturer of pETllb. It can be prepared from the lysate of bacteria by ammonium sulfate, hydrophobic interaction chromatography on phenyl agarose 6-speed flow, ion exchange layer on DEAL·-Joongue sugar flow The soluble TNF-peptide was purified by gel filtration chromatography on Sephacryl-O: \121\121929.DOC-90-200808833 S-300 HR. The fusion protein which can be produced in the form of insoluble inclusion bodies in bacteria can be purified as follows. Host cells can be sacrificed by centrifugation; rinsed with 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. The lysate was clarified by ultrasonic oscillation and pelletized by centrifugation at 12,000 X g for 10 minutes. The fusion protein containing the pellets can be resuspended in 50 mM Tris, pH 8 and 10 mM EDTA, layered on 50% glycerol, and centrifuged at 6000 Xg for 30 minutes. The pellets can be resuspended in standard phosphate buffered saline solution (PBS) without Mg++ and Ca++. The fusion protein can be further purified by fractionation of resuspended pellets (Sambrook et al., supra) in denatured SDS-PAGE. The gel can be immersed in 0.4 M KC1 to visualize the protein, which can be cut and electroeluted in a buffer solution lacking the SDS running gel. If the GST/fusion protein produced in bacteria is a soluble protein, it can be purified using GST Purification Base Unit (Pharmacia). The GST can be cleaved from the peptide of the present invention by digesting the fusion protein. The digestion reaction (20-40 mg fusion protein, 20-30 units human thrombin (4000 units/mg, Sigma) in 0.5 ml PBS) can be incubated at room temperature for 16-48 hours and loaded into denatured SDS- The reaction product was fractionated in a PAGE gel. The gel can be immersed in 0.4 M KC1 to visualize the protein band. The identity of the protein profile consistent with the expected molecular weight of the peptide can be confirmed by amino acid sequence analysis using an automatic sequencer (Applied Biosystem Model 473 A, Foster City, CA). Alternatively, the identity of the peptide can be confirmed by HPLC and/or mass spectrometry analysis of the peptide: O: \121\121929.DOC -91 - 200808833. Alternatively, the DNA sequence encoding the peptide can be cloned into a plastid containing the desired promoter and, if desired, a leader sequence [Better et al, Sdence 240: 1041-43 (1988)]. The sequence of the structure can be confirmed by automatic sequencing. The plastids can then be transformed into E. coli strain MC1061 using standard procedures using bacterial CaC12 culture and heat shock treatment (Sambrook et al., supra). The transformed bacteria can be grown in LB medium supplemented with carbenicillin and can be induced to produce protein by growing in a suitable medium. If a leader sequence is present, the secretion of the peptide is completed and cleaved during secretion. The recombinant protein that has been secreted can be purified from the bacterial culture medium by the method described below. Mammalian host systems that express recombinant proteins are known to those skilled in the art. The host cell line is selected for the processing of the expressed peptone, or for the specific ability to produce some translation-post-modification useful in providing protein activity. Modifications of such proteins include, but are not limited to, B-stuffing, carboxylation, glycosylation, phosphorylation, lipidation, and deuteration. Different host cells, such as CHO, HeLa, MDCK, 293, WI38 and their analogs, have specific cellular mechanisms and unique mechanisms for such translation-post-activity, and can be selected to ensure the introduction of foreign proteins. Correct modification and processing. It is desirable that the transformed cells used are preferably produced in a long-term, high-yield protein shell and are inherently stable. Once the cells are transformed with a vector containing a selectable marker, along with a cardinal representation, the cell can be tolerated.
O:\121\121929.DOC •92· 200808833 胞在增強的培養基中生長丨-2天’隨後再將它們轉至選擇 性培養基。設計可選擇標記，賦與對選擇的抵抗力，而它 的出現容許成功表現導入序列之細胞的生長和回收。可使 用適合該細胞的組織培養技術，使具有抵抗力之穩定轉化 的細胞叢增殖。 可使用許多選擇系統，回收已經為了重組蛋白質產製而 轉化的細胞。這類選擇系統包括，但不限於在、h卯… 或aprt-細胞中，分別為HSV胸腺核甞激酶、次黃嘌呤一鳥 嘌呤轉磷酸核糖基酶，和腺嘌呤轉磷酸核糖基酶基因。亦 可使用抗-代謝產物的抗藥性，作為選擇DHFR的基礎，其 賦與對胺甲碟呤的抵抗力；gpt，其賦與對黴酚酸的抵抗 力；neo，其賦與對胺基糖-G418的抵抗力，並賦與對氣 硫昂（chl〇rSUlfuron)的抵抗力；以及hygr〇,其賦與對潮黴 素的抵抗力。其他可能有用的可選擇基因，包括trpB，其 容許細胞使用吲哚代替色胺酸，或hisD，其容許細胞使用 組胺醇（histinol)代替組胺酸。對於確認轉化物提供可見指 示的標記，包括花青素苷類、p_尿甘酸酶及其受質GUS， 和蟲螢光素酶及其受質蟲榮光素。 專一結合劑的純化和再折曼 在一些案例中，專一結合劑，像是本發明之肽及/或肽 體，可能需要”再折疊”並氧化成適當的三級結構，並產生 二硫鍵結，以便成為有生物活性的。可使用許多此項技藝 中已熟知的程序完成再折疊。這類方法包括，例如在促溶 劑的存在下，使加溶多肽製劑暴露在通常大於7的pH值下O:\121\121929.DOC •92· 200808833 Cells were grown in enhanced medium for 丨-2 days' and then transferred to selective medium. The selectable marker is designed to confer resistance to selection, and its presence allows for successful growth and recovery of cells expressing the introduced sequence. A tissue culture technique suitable for the cell can be used to proliferate a stable transformed cell cluster. Many selection systems can be used to recover cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine to phosphoribosyltransferase genes, respectively, in h卯... or aprt- cells. Anti-metabolite resistance can also be used as the basis for the selection of DHFR, which confers resistance to the amine sputum; gpt, which confers resistance to mycophenolic acid; neo, which confers an amino group The resistance of sugar-G418 is given to the resistance to chl〇rSUlfuron; and hygr〇, which confers resistance to hygromycin. Other potentially useful alternative genes, including trpB, allow cells to use guanidine instead of tryptophan, or hisD, which allows cells to use histamine instead of histidine. Markers for confirming that the transformant provides visible indications include anthocyanins, p-uronic acid and its receptor GUS, and luciferase and its receptor glory. Purification and Refolding of Specific Binding Agents In some cases, specific binding agents, such as the peptides and/or peptibodies of the invention, may need to be "refolded" and oxidized to the appropriate tertiary structure and produce disulfide bonds. Knot in order to become biologically active. Refolding can be accomplished using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide formulation to a pH of typically greater than 7 in the presence of a solubilizing agent.
O:\121\121929.DOC -93- 200808833 。促溶劑的選擇類似包涵體溶解所使用的選擇法， 通常以低濃度使用促溶劑。 …、 ㈠代表性的促溶劑為胍。在大多 數的情況下，再折疊/氧化作 作用的洛液亦將含有還原劑， 加上其氧化形式，按昭特宏沾 … …、特疋的比例，以便產生特定的氧化 遇原電位’容許二硫化物的洗牌，而發生半胱胺酸橋的形 成书用的氧化還原對包括半脱胺酸/耽胺、穀耽甘 太/爪雙GSH、氣化銅、二硫蘇糖醇dtt/二4烧dtt , 以及2-疏基乙醇（bME)/二硫掏£。在許多例子中，可使用 共-溶劑’增加再折疊的效率。常用的共溶劑包括甘油、 各種分子量的聚乙二醇，和精胺酸。 可此想要純化本發明的肽和肽體。蛋白f純化技術是熟 諳此藝者已熟知的。在一個標準上，這些技術涉及蛋白質 和非-蛋白質溶離份的粗略分級分離。已經從其他蛋白質 中分離出的肽及/或肽體，可使用層析和電泳技術進一步 純化感興趣的肽或肽體，達成部分或完全的純化（或同質 性的純化）。特別適合製備本發明之肽或肽體的分析方法 ，是離子交換層析法、排阻層析法；聚丙烯醯胺凝膠電泳 ;等電聚焦。 本發明在某些方面係關於純化作用，且在特定的具體實 施例中’為本發明之肽體或肽的實質純化作用。在本文中 使用的，，經過純化的肽體或肽” 一詞，企圖意指可從其他組 伤中分離的化合物，其中相對於其天然可獲得的狀態，將 月太體或肽純化成任何等級。因此，經過純化的肽或肽體， 亦意指不含其可能天然存在之環境的肽體或肽。 O:\121\121929.DOC 94- 200808833 一般而g ’”經過純化的”將意指已經接受分級分離，移 除各種其他組分的肽或肽體組合物，且該組合物實質上仍 保留其表現的生物活性。在使用”實質上經過純化的，，一詞 之處，該稱呼將意指肽或肽體組合物，其中肽體或肽形成 組合物的主要組份，像是構成組合物中大約5〇%、大約 60/〇、大約70%、大約8〇%、大約9〇%、大約95%或更多的 蛋白質。 各種定量肽或肽體之純化程度的方法，從本揭示内容來 看，將是熟諳此藝者已知的。這些包括，例如判定活性溶 離份的專一結合活性，或藉著SDS/PAGE*析評估肽或肽 體在溶離份中的含量。評估肽或肽體溶離分之純度的較佳 方法’疋计异該溶離份的結合親和力，將其與原始萃取物 之結合活性作比較，並如此計算出純化的程度，在本文中 係藉著倍純化數”來評估。用來代表結合活性之量的實際 單位，當然將視在純化之後選用的特定測定技術，以及肽 體或肽是否顯示出可檢測的結合活性而定。 適用於純化作用的各種技術，將是熟諳此藝者已熟知的 。這些包括，例如利用硫酸銨、PEG、抗體（免疫沉澱）及 其類似物的沉澱作用，或藉著熱變性，接著離心；層析步 驟，像是親和力層析法（例如蛋白質_A_瓊脂糖）、離子交換 减膠過濾、逆相、羥基磷灰石和親和力層析法；等電聚 焦；凝膠電泳；以及這類及其他技術的組合。如同在此項 技☆中音遍已知的，咸相信可改變進行各種純化步驟的順 序，或可省略某些㈣，而仍可產㈣合製備冑質上純的O:\121\121929.DOC -93- 200808833. The choice of solubilizing agent is similar to the selection method used for the dissolution of inclusion bodies, and the solubilizing agent is usually used in a low concentration. ..., (a) A representative solvent is hydrazine. In most cases, the refolding/oxidizing effect will also contain a reducing agent, plus its oxidized form, according to the ratio of Zhao Tehong, in order to produce a specific oxidation potential. The shredding of disulfide is allowed, and the redox pair for the formation of cysteine bridges includes semi-deaminating/decylamine, glutathione/claw double GSH, vaporized copper, dithiothreitol. Dtt/two 4 burning dtt, and 2-mercaptoethanol (bME) / dithizone. In many instances, co-solvent can be used to increase the efficiency of refolding. Common cosolvents include glycerin, polyethylene glycols of various molecular weights, and arginine. It is thus desirable to purify the peptides and peptibodies of the invention. Protein f purification techniques are well known to those skilled in the art. In one standard, these techniques involve a rough fractionation of protein and non-protein fractions. Peptides and/or peptibodies that have been isolated from other proteins can be further purified by chromatography and electrophoresis techniques to achieve partial or complete purification (or homogenous purification). An analytical method particularly suitable for preparing the peptide or peptibody of the present invention is ion exchange chromatography, exclusion chromatography; polypropylene guanamine gel electrophoresis; isoelectric focusing. The present invention is in some respects related to the purification, and in a particular embodiment, is the substantial purification of the peptibody or peptide of the invention. The term "purified peptibody or peptide" as used herein, is intended to mean a compound that can be isolated from other group injuries, wherein the celestial body or peptide is purified to any relative to its naturally available state. Grade. Therefore, a purified peptide or peptidate also means a peptide or peptide that does not contain the environment in which it may naturally occur. O:\121\121929.DOC 94- 200808833 Generally and g '"purified" Means that a peptide or peptidic composition has been subjected to fractionation to remove various other components, and that the composition retains substantially the biological activity of its expression. In the use of "substantially purified," The term will mean a peptide or peptidic composition wherein the peptibody or peptide forms a major component of the composition, such as about 5%, about 60/〇, about 70%, about 8%, of the composition. About 9%, about 95% or more protein. A variety of methods for quantifying the degree of purification of peptides or peptibodies will be apparent to those skilled in the art from this disclosure. These include, for example, determining the specific binding activity of the active fraction, or assessing the amount of the peptide or peptide in the fraction by SDS/PAGE* analysis. A preferred method for assessing the purity of a peptide or peptidic fraction is to compare the binding affinity of the dissolving component, compare it to the binding activity of the original extract, and calculate the degree of purification in this context. The number of purifications is evaluated. The actual unit used to represent the amount of binding activity will of course depend on the particular assay technique chosen after purification and whether the peptide or peptide exhibits detectable binding activity. Various techniques will be well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation) and the like, or by thermal denaturation followed by centrifugation; chromatography steps, Such as affinity chromatography (eg protein _A_ agarose), ion exchange gel reduction filtration, reverse phase, hydroxyapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and such and other techniques Combination. As is known in the art ☆ whistle, it is believed that the order of various purification steps can be changed, or some (4) can be omitted, and the enamel can still be produced. pure
O:\121\121929.DOC -95- 200808833 專一結合劑的方法。 通常不需要總是以其最純的狀態來提供本發明之肽或肽 體。確實，預期實質上較少的專一結合劑產物，在某些具 體實施例中仍具有利用性。可藉著在組合中使用較少:純 化步驟，或藉著使用相同之共同純化計畫的不同形式，來 凡成部分純化作用。例如，瞭解使用HPLC装置來進行的 陽離子-交換管柱層析法，通常將產生比使用低_壓力層析 系統的相同技術更多倍"的純化。顯示出較低相對純化程 度的方法，可能有利於肽或肽體的總回收，或有利於維持 月太或肤體的結合活性。 已知可利用SDS/PAGE的不同條件，有時明顯地改變肽或 肽體的移動[Capaldi等人，Biochem. Biophys. Res. Comm. 76:425 (1977)]。因此應瞭解在不同的電泳條件下，可改變 經過純化或部分純化之專一結合劑表現產物的表面分子量。 結合測定 免疫結合測定通常使用專一地結合，且經常固定分析物 標靶抗原的捕捉劑。捕捉劑是與分析物專一結合的部分。 在一個本發明的具體實施例中，捕捉劑是與Ang-2專一地 結合的肽或肽體或其片段。這些免疫結合測定是此項技藝 中已熟知的[Asai，編輯，Methods in Cell Biology，第 37冊 ’ Antibodies in Cell Biology，Academic Press，Inc., New York (1993)]。 免疫結合測定經常使用標示劑，其將發送由捕捉劑和抗 原形成之已結合複合物出現的信號。標示劑可以是一個包 0:\121\121929.D0C -96- 200808833 =已結合複合物的分子，也就是說，它可以是已標示之專 -結合劑或已標示之抗·專一結合劑的抗體。或者，標示 劑可以是第三個分子’通常是其他抗體，其與已結合之複 合物結合。標示劑可以是，例如攜帶標記之抗-專一結合 劑的抗體。第二個抗體’對已結合之複合物是專一的，可 ，缺乏標記，但可被對第二個抗體所屬之抗體物種專一的 弟四個分子結合。例如’可利用可檢測部分，像是生物素 ，來修改第二個抗體然後使其與第四個分子結合，像是 以酵素標示之鏈黴菌抗生物素蛋白。亦可使用能夠專一地 與免疫球蛋白恆定區結合的其他蛋白質作為標示劑，像是 蛋白質A或蛋白質G。這些結合蛋白質是鏈球菌細菌之細 胞壁的正常成分’並對得自各種物種之免疫球蛋白恆定區 顯示出強的非-免疫原反應性。Akem_，j imm_u 135:2589-2542 (1985); Chaubert, Mod. Pathol., l〇：585-591 (1997) 〇 在測定之中，可能在每次混合試劑之後，需要培養及/ 或沖洗步驟。培養步驟可從大約5秒變化至數小時，最好 是從大約5分鐘至大約24小時。然而，培養時間將視測定 格式、分析物、溶液體積、濃度及其類似者而定。通常， 測定將在周圍溫度下進行，雖然亦可在一定範圍的溫度下 進行。 A.非_競爭性結合測定： 免疫結合測定可以是非·競爭性的類型。這些測定直接 測量被捕捉之分析物的含量。例如，在一種較佳的"三明 O:\121\121929.DOC -97- 200808833 冶測定巾，捕捉劑（抗體或肽體）可直接與固體受質結合， :被固定在那裏。然後這些已經固定的捕捉劑再;捉。(結 口)出現在受試試樣中的抗原。然後如此固定的蛋白質再 與標示劑結合’像是具有標記的第二個抗體。在另一種較 佳的”三明治"測定中，第二個抗體缺乏標記，但可盥對從 其中衍生第二個抗體之抗體物種專一的已標示抗體::合。 亦可：用可檢測部分’像是生物素，來修改第二個抗體， 而使第三個已標示分子，像是鏈黴菌抗生物素蛋白愈立專 〇 Harlows Lanej Antibodies, A LaboratoryO:\121\121929.DOC -95- 200808833 The method of specific bonding agent. It is generally not necessary to always provide the peptide or peptide of the present invention in its purest state. Indeed, substantially less specific binder product is expected to be useful in certain embodiments. It can be partially purified by using less in the combination: the purification step, or by using different forms of the same common purification program. For example, understanding cation-exchange column chromatography using an HPLC apparatus will typically result in more purification than the same technique using a low-pressure chromatography system. A method that exhibits a relatively low degree of purification may be beneficial for the overall recovery of the peptide or peptibody or for maintaining the binding activity of the moon or the skin. It is known that different conditions of SDS/PAGE can be utilized, sometimes significantly altering the movement of peptides or peptibodies [Capaldi et al, Biochem. Biophys. Res. Comm. 76: 425 (1977)]. It is therefore understood that under different electrophoresis conditions, the surface molecular weight of the product represented by the purified or partially purified specific binder can be altered. Binding Assays Immunological binding assays typically employ a capture reagent that specifically binds and often immobilizes the analyte target antigen. The capture agent is the part that is specifically combined with the analyte. In a particular embodiment of the invention, the capture agent is a peptide or peptibody or fragment thereof that specifically binds to Ang-2. These immunological binding assays are well known in the art [Asai, ed., Methods in Cell Biology, Vol. 37' Antibodies in Cell Biology, Academic Press, Inc., New York (1993)]. Immunological binding assays often use a labeling agent that will signal the presence of bound complexes formed by the capture reagent and the antigen. The labeling agent can be a package of 0:\121\121929.D0C -96- 200808833 = the molecule of the complex has been bound, that is, it can be a labeled specific-binding agent or a labeled anti-specific binding agent. antibody. Alternatively, the labeling agent can be a third molecule 'generally other antibodies that bind to the bound complex. The labeling agent can be, for example, an antibody carrying a labeled anti-specific binder. The second antibody' is specific for the bound complex, and may lack labeling, but may be bound by four molecules that are specific to the antibody species to which the second antibody belongs. For example, a detectable moiety, such as biotin, can be used to modify the second antibody and then bind it to a fourth molecule, such as Streptomyces avidin labeled with an enzyme. Other proteins that specifically bind to the immunoglobulin constant region, such as protein A or protein G, can also be used. These binding proteins are normal components of the cell wall of streptococcal bacteria' and exhibit strong non-immunogen reactivity to immunoglobulin constant regions from various species. Akem_,j imm_u 135:2589-2542 (1985); Chaubert, Mod. Pathol., l〇:585-591 (1997) 〇In the assay, it may be necessary to culture and/or rinse steps after each reagent mixing . The culturing step can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend on the assay format, analyte, solution volume, concentration, and the like. Usually, the measurement will be carried out at ambient temperature, although it can also be carried out at a range of temperatures. A. Non-competitive binding assay: The immunological binding assay can be of a non-competitive type. These assays directly measure the amount of analyte captured. For example, in a preferred "Sanming O:\121\121929.DOC-97-200808833, the capture agent (antibody or peptidomimetic) can be directly bound to the solid substrate: it is immobilized there. Then these already fixed capture agents are again; catch. (Estration) The antigen that appears in the test sample. The protein so immobilized is then bound to the labeling agent as if it were a second antibody with a label. In another preferred "sandwich" assay, the second antibody lacks a label, but can be labeled with an antibody that is specific to the antibody species from which the second antibody is derived:: can also: use a detectable moiety 'Like biotin, to modify the second antibody, so that the third labeled molecule, like Streptomyces avidin, is specialized in Harlows Lanej Antibodies, A Laboratory
MailUal ’ 第 14 章，Cold SPring 肠b〇r Laboratory，ny (1988)，以引用的方式併入本文中。 B ·競爭性結合測定：MailUal' Chapter 14, Cold SPring, 〇 (1988), incorporated herein by reference. B · Competitive binding assay:
免疫結合測定可以是競爭性的類型。藉著測量所加入之 分析物’㈣現在試樣中之分析物取代，或競爭遠離捕捉 劑（抗體^肽體）的含量，間接地測量出現在試樣中之分析 物的含置。在一種較佳的競爭性結合測定中，在試樣中加 入通常已標示的已知含量之分析物’然後使試樣與捕捉劑 接觸。已標*分析物與抗體結合的量，與A現在試樣中的 刀析物/辰度成反比（參見Harl〇w* Lane，A^b〇dies，AImmunological binding assays can be of a competitive type. The inclusion of the analyte present in the sample is indirectly measured by measuring the analyte added to the analyte (4), the analyte in the sample, or competing away from the capture agent (antibody). In a preferred competitive binding assay, a commonly indicated known amount of analyte is added to the sample' and the sample is then contacted with a capture reagent. The amount of analyte bound to the antibody is inversely proportional to the knife/density in the current sample (see Harl〇w* Lane, A^b〇dies, A).
Laboratory Manua卜第 14章，第 579_583 頁，在前）。 在其他較佳的競爭性結合測定中，將捕捉劑固定在固體 文質上。可藉著測量出現在蛋白質/抗體複合物中之蛋白 質的量，或另外藉著測量仍未複合之蛋白質的量，來判定 〃捕捉β結合之蛋白質的量。可藉著提供已標示的蛋白質Laboratory Manua, Chapter 14, page 579_583, in the former). In other preferred competitive binding assays, the capture reagent is immobilized on a solid text. The amount of protein that captures β-binding can be determined by measuring the amount of protein present in the protein/antibody complex, or otherwise by measuring the amount of protein that has not yet been complexed. By providing labeled proteins
O:\121\121929.DOC -98· 200808833 ’來檢測蛋白質之含量。Harlow和Lane (在前）。 另一種較佳的競爭性結合測定，利用半抗原的抑制作用 。在那裏，將已知的分析物固定在固體受質上。在試樣中 加入已知含量的抗體，並使試樣與已經固定的分析物接觸 。與已固定分析物結合之抗體的量，與出現在試樣中之分 析物的含量成反比。可藉著檢測已固定之抗體溶離份，i 仍留在溶液中之溶離份的量，來檢測已固定之抗體的量。 在標示抗體之處，可直接檢測，或藉著後續加入已標示部 分，其按照上述專一地與抗體結合，間接地檢測之。 C•競爭性結合測定的利用： 可為了交又_反應性的判定，使用競爭性結合測定，容 許热Μ此藝者判定被本發明之肽體認出的蛋白質或酵素複 '物疋否疋想要的蛋白質，而不是交又-反應的分子， 或判定該肽體是對抗原專一的，且不與無關的抗原結合。 在這類型的測定中，可將抗原固定在固相支撐物上，並在 测定中加入未知的蛋白質混合物，其將與已固定之蛋白質 贶爭與該肽體的結合。競爭性分子亦與一或多個與該抗原 無關的抗原結合。將蛋白質與已固定之抗原競爭該肽體結 口的此力’與被固定在固相支撐物上之相同蛋白質的結合 做比較，來判定蛋白質混合物的交叉-反應性。 D ·其他的結合測定： 本么月亦長：供西方墨點法，來檢測或定量Ang_2在試樣 中的存在。該技術通常包括藉著凝膠電泳，以分子量為基 刀離"式樣蛋白質，並將該蛋白質移至適當的固相支撐O:\121\121929.DOC -98· 200808833 ' to detect the protein content. Harlow and Lane (in the first place). Another preferred competitive binding assay utilizes the inhibition of haptens. There, known analytes are immobilized on a solid substrate. A known amount of antibody is added to the sample and the sample is contacted with the analyte that has been immobilized. The amount of antibody bound to the immobilized analyte is inversely proportional to the amount of analyte present in the sample. The amount of immobilized antibody can be detected by detecting the amount of immobilized antibody-dissociated fraction, i remaining in solution. Where the antibody is labeled, it can be detected directly, or by subsequent addition of the labeled moiety, which is indirectly detected by binding to the antibody as described above. C• Use of Competitive Binding Assay: Competitive binding assays can be used for the determination of cross-reactivity, allowing the artist to determine whether the protein or enzyme complex recognized by the peptibody of the present invention is defective. The desired protein, rather than the cross-reactive molecule, or the peptide is determined to be antigen-specific and does not bind to an unrelated antigen. In this type of assay, the antigen can be immobilized on a solid support and an unknown protein mixture added to the assay will compete with the immobilized protein for binding to the peptide. Competitive molecules also bind to one or more antigens that are not associated with the antigen. The ability of the protein to compete with the immobilized antigen for the peptone junction is compared to the binding of the same protein immobilized on the solid support to determine the cross-reactivity of the protein mixture. D · Other binding assays: This month is also long: for Western blotting to detect or quantify the presence of Ang_2 in the sample. This technique typically involves the use of molecular weight-based gel-gel electrophoresis to move the protein to the appropriate solid support.
O:\121\121929.DOC -99- 200808833 物上，像是硝基纖維素濾紙、尼龍濾紙，或衍生的尼龍遽 紙。將試樣與專一地結合Ang-2的肽體或其片段一起培養 ’並檢測所得的複合物。可直接標示這些肽體，或另行使 用已標示之抗體，其專一地與該肽體結合，進行後續的檢 測。 診斷測定 衍生的結合劑，像是本發明之肽和肽體或其片段，可用 來診斷其特徵為Ang-2或亞單元之表現的病況或疾病，或 用於監視待利用Ang-2之誘導劑、其片段、Ang-2活性之激 動劑或抑制劑治療之患者的測定。Ang-2的診斷測定，包 括使用肽體和標記，來檢測在人類體液或細胞或組織之萃 取物中的Ang-2的方法。可使用有或無修改之本發明的肽 體。在較佳的診斷測定中，將藉著附接，例如標記或報告 者分子，來標不肽體。各種標記和報告者分子均是已知的 ，已經在本文中描述了其中的一些。本發明對於診斷人類 疾病是特別有用的。 *各種使用對各個蛋白質專一之肽體，來測量Ang-2蛋白 質的草案’為此項技藝中已知的。實例包括酵素連結之免 疫吸附測定（ELISA)、放射性免疫測定（RIA)和螢光激活的 細胞分_ACS)。兩個位置、以單株為基礎的免疫測定是 較佳的，其利用與在Ang_2上兩個不-抵觸之抗原決定位起 反應的單株抗體，但亦可使用競爭性結合測定。在例如 等人]· Exp.胸·，158:12u (1983)中描述 了這些O:\121\121929.DOC -99- 200808833 On the object, it is like nitrocellulose filter paper, nylon filter paper, or derivative nylon crepe paper. The sample was cultured with a peptibody or a fragment thereof specifically bound to Ang-2 and the resulting complex was detected. These peptibodies can be directly labeled or otherwise labeled with an antibody that specifically binds to the peptibody for subsequent detection. Diagnostic assays Derivatized binding agents, such as the peptides and peptibodies of the invention, or fragments thereof, can be used to diagnose conditions or diseases characterized by the expression of Ang-2 or subunits, or to monitor the induction of Ang-2 to be utilized. Determination of a patient, a fragment thereof, an agonist of Ang-2 activity, or a patient treated with an inhibitor. Diagnostic assays for Ang-2, including the use of peptibodies and markers, to detect Ang-2 in human body fluids or cells or tissue extracts. Peptides of the invention with or without modification can be used. In a preferred diagnostic assay, the peptibodies will be labeled by attachment, such as a label or reporter molecule. A variety of labeling and reporter molecules are known, some of which have been described herein. The invention is particularly useful for diagnosing human diseases. * Various drafts for measuring Ang-2 protein using peptibosome specific for each protein are known in the art. Examples include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescently activated cell fractions (ACS). Two-site, single-strain-based immunoassays are preferred that utilize monoclonal antibodies that react with two non-contradictory epitopes on Ang_2, but competitive binding assays can also be used. These are described in, for example, et al., Exp. Chest, 158: 12u (1983).
O:\121\121929.DOC -100· 200808833 為了 ^供#斷的基礎，通當奢 I吊建立人類Ang-2的正常或標 準值。可猎者在適合複合物 物心成的條件下，這在此項技蓺 中已知的，將得自正常個妒 " ^最好是人類的體液或細胞萃 取物’與對抗Ang-2的肚I#、、曰人 + 二 S旧肽體此合，來完成該判定。可藉著 比較控制和疾病試樣兩者， ^ 狀體與已知含量之Ang-2蛋白 貝的結合作用，來定量擇集 里铋旱禝合物形成的量。然後，可將 k正常試樣中獲得的標準， 干徂興攸來自可能受疾病影響之 個體的試樣中獲得的值作+ & . ^ 、 I㈣值作比車父。在#準和個體值之間的偏 差，暗示Ang-2在疾病狀態中的角色。 、為了 β斷應用’在某些具體實施例中，通常將利用可檢 測部分標示本發日月之肢#々 知Λ之肽體或肽。可檢測部分可以是任何能 夠直接或間接產生可檢測作號 似叫L就的部分。例如，可檢測部分 可以是放射性同位素，像是3H、MC、32p、35s或125ι，榮光 或化學發光化合物’像是螢光素異硫代氰酸醋、若丹明或 蟲螢光素’·或酵素，像是驗性磷酸酶、卜半乳糖菩酶或辣 根過氧化酶。Bayer等人，祕^肩：咖⑹⑽ 疾病 本發明提供與Ang-2結合的結合劑，像是肽、肽體，或 其片段、變體或衍生物’其可用來治療人類疾病和病理學 狀況。調節Ang-2結合活性或其他細胞活性的製劑，可與O:\121\121929.DOC -100· 200808833 In order to provide the basis for the break, the normal or standard value of human Ang-2 is established. The hunter is known to be suitable for the composition of the complex, which is known in the art and will be derived from a normal 妒" ^ preferably a human body fluid or cell extract' with anti-Ang-2 This is determined by the combination of the belly I#, the sputum + the two S old peptides. The amount of aglycone complex formation can be quantified by comparing the control and disease samples with the binding of known amounts of Ang-2 protein shell. Then, the value obtained in the normal sample of k can be obtained from the samples obtained from the samples of individuals who may be affected by the disease as + & ^, I (four) values as the parent. The deviation between the quasi- and individual values suggests the role of Ang-2 in the disease state. For beta application, in some embodiments, a peptibody or peptide that is identifiable by the identifiable portion of the genus will be indicated. The detectable portion can be any portion that can directly or indirectly produce a detectable number L. For example, the detectable moiety can be a radioisotope such as 3H, MC, 32p, 35s or 125ι, a glory or chemiluminescent compound such as luciferin isothiocyanate, rhodamine or luciferin. Or an enzyme, such as an assay phosphatase, galactose or horseradish peroxidase. Bayer et al., Secrets: Coffee (6) (10) Diseases The present invention provides binding agents that bind to Ang-2, such as peptides, peptibodies, or fragments, variants or derivatives thereof, which are useful for treating human diseases and pathologies. . a preparation for modulating Ang-2 binding activity or other cellular activity,
其他治療劑混合使儲，以便提高其治療效果或降低可茫、 的副作用。 I 本發明一方面提供可用來治療疾病和病況的試劑和方法 ’該疾病和病況之特徵為在細胞中有不想要或越軌程度的Other therapeutic agents are mixed to allow for their therapeutic effect or reduced side effects. I In one aspect, the invention provides agents and methods useful for treating diseases and conditions. The disease and condition are characterized by unwanted or deviant levels in the cell.
O:\121\121929.DOC -101 - 200808833 ng 2活。攻些疾病包括癌症，以及其他過度增殖的狀況 像疋增生、牛皮癬、接觸性皮膚炎、免疫學的病症，以 及不孕。 本發明亦提供在包括人類的動物中，治療癌症的方法， 包括對該動物投與有效含量的專一結合劑，像是抑制或降 低Ang-2活性的肽體。本發明尚針對抑制癌細胞生長的方 法，包括在生物系統中之細胞增殖、侵入性和轉移的過程 '法包括使用本發明之化合物作為癌細胞±長的抑制劑 。最好是在活動物中’像是哺乳動物，使用該方法來抑制 或降低癌細胞生長、侵入性、轉移或腫瘤發生率。亦可迅 速地改編本發明之方法，以便使用在測定系統中，例如， 測定癌細胞生長及其特性，以及確認影響癌細胞生長的化 合物。 可藉著本發明之方法治療的癌症，最好是發生在哺乳動 物中。哺乳動物包括，例如人類和其他靈長類，以及寵物 或同伴動物’像是狗和|苗，實驗室動物，像是大鼠、老鼠 和兔子，還有農場動物，像是馬、豬、綿羊和牛。 腫瘤和贅生物包括其中細胞的增加是不受控制並前進的 組織細胞生長。-些這類的生長是良性的’但將其他的稱 為惡性的，並可導致生物的死亡。惡性費生物或癌症與良 性生長的區別在於除了顯示攻擊性的細胞增殖之外，它們 可能侵犯周圍的組織並轉移。此外，惡性贅生物之特徵為它 們相對於彼此及其周圍組織，顯示出喪失較多的分化（較多 的反分化）和它們的組織化。亦將該特性稱為"退行發育,，。O:\121\121929.DOC -101 - 200808833 ng 2 live. Attacks on these diseases include cancer, as well as other hyperproliferative conditions such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility. The invention also provides a method of treating cancer in an animal comprising a human comprising administering to the animal an effective amount of a specific binding agent, such as a peptibosome that inhibits or reduces Ang-2 activity. The present invention is also directed to a method of inhibiting the growth of cancer cells, including the process of cell proliferation, invasiveness, and metastasis in a biological system. The method includes the use of the compound of the present invention as a cancer cell ± long inhibitor. Preferably, this method is used in living animals, such as mammals, to inhibit or reduce cancer cell growth, invasion, metastasis or tumor incidence. The method of the present invention can also be rapidly adapted for use in assay systems, for example, to measure cancer cell growth and its properties, and to identify compounds that affect cancer cell growth. The cancer which can be treated by the method of the present invention preferably occurs in mammals. Mammals include, for example, humans and other primates, as well as pets or companions 'like dogs and | seedlings, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, and sheep. Wagyu. Tumors and neoplasms include tissue cells in which the increase in cells is uncontrolled and advanced. - Some of these growths are benign' but others are described as malignant and can lead to the death of living things. Malignant organisms or cancers differ from benign growth in that they may invade surrounding tissues and metastasize in addition to showing aggressive cell proliferation. In addition, malignant neoplasms are characterized by their loss of more differentiation (more dedifferentiation) and their organization relative to each other and their surrounding tissues. This feature is also called "regressive development,,.
O:\121\121929.DOC -102- 200808833 可藉著本發明治療的贅生物亦包括固體腫瘤，也就是癌 和肉瘤。癌包括衍生自上皮細胞的那些惡性贅生物，其浸 潤(佼犯)周圍組織’並引起轉移。腺癌是衍生自腺體組織 的癌，或其形成可認出的腺體結構。其他廣泛的種類或癌 症，包括肉瘤，其為腫瘤，它的細胞被埋入原纖維或均一 的物質中，像是胚胎的結締組織。本發明亦能夠治療骨髓 或淋巴系統的癌症，包括白Α病、淋巴瘤和其他通常不是 以腫瘤團塊之形式出現，但是被散布在血管或淋巴網狀系 統中的癌症。 τ根據本發明服從治療之癌症或腫瘤細胞的類型，包括 例如產生ACTH的腫瘤、急性淋巴細胞白血病、急性非淋 巴細胞白血病、腎上腺皮質的癌症、膀胱癌、腦癌、乳癌 、子宮頸癌、慢性淋巴細胞白血病、慢性骨髓細胞白血病 、結直腸癌、皮膚的Τ_細胞淋巴瘤、子宮内膜癌、食道癌 、尤英氏（Ewing’s)肉瘤、膽囊癌、毛細胞白血病、頭和頸 部的癌症、霍奇金氏（H〇dgkin，s)淋巴瘤、卡波西氏 (Kaposi’s)肉瘤、腎癌、肝癌、肺癌（小細胞和非_小細胞） 、惡性腹腔積液、惡性胸腔積液、黑色素瘤、間皮瘤、多 發性骨婕瘤、神經胚細胞瘤、神經膠質瘤、非_霍奇金氏 淋巴瘤、骨肉瘤、卵巢癌、卵巢（生殖細胞）癌、胰臟癌、 陰莖癌、前列腺癌、視網膜胚細胞瘤、皮膚癌、軟組織肉 瘤、鱗狀細胞癌、胃癌、睪丸癌、甲狀腺癌、滋養層的贅 生物、子宮癌、陰道癌、外陰的癌症和腎母細胞瘤。 關於某些類型以實驗定義之癌症的治療，在本文中特別 O:\121\121929.DOC -103- 200808833 解釋本發明。在這此解釋 士 —鮮釋性的治療中，已經使用標準的現 有之最尚技術的在活體外和在 、 在活體内杈式。可使用這些方 法來確認預期它在活體内 λ 円之/口療攝生法中是有效的製劑。 然而，應瞭解本發明之太、土 π ^ 方法不限於這些腫瘤類型的治療， 申至來自任何器吕系統的任何固體腫瘤。其侵入性或 轉移與Ang_2表現或活性有關的癌症，特別容易受到本發 明的抑制，或甚至引起其退行。 亦可猎著包括本發明化合物，像是肽體，與其他抗-癌 症之化學治療劑混合，像是任何傳統的化學治療劑，來實 行本發明。專—結合劑與這類其他製劑的混合，可產生化 學治療草案的效力。許多化學治療草案將依熟諳此藝者的 想法呈現它們自己’而能夠併人本發明之方法中。可使用 任何化學治療劑，包括烧基化製劑、抗_代謝產物、荷爾 蒙和拮抗劑、放射性同位素，以及天然的產物。例如，可 將本發明之化合物與抗生素混合，像是阿黴素和其他氨菌 環黴素類似物，氮芥’像是環磷醯胺，嘯咬類似物，像是 Μ尿㈣、順氣氨始（cisplatin)、經基脲、紫杉醇⑽叫 ，及其天然和合成的衍生物，及其類似物。像其他實例一 樣，在混合腫瘤的案例中，像是乳房的腺癌，其中腫瘤包 括促性腺激素-依賴性和促性腺激素_獨立性的細胞，可連 同亮丙里德（leuprolide)或戈舍瑞林（g〇sereUn)(LH_RH之合 成的肽類似物）一起投與化合物。其他的抗贅生物草案包 括使用四環素化合物與其他的治療用藥程式，例如手術、 放射線等等，在本文中亦稱為"輔助的抗贅生物用藥程式,, 〇：\121\121929.DOC -104· 200808833 。因此，本發明之方法可與這類傳統攝生法一起使用，具 有降低副作用和提高效力的優點。 口此本务明&供可用來治療各種癌症的組合物和方法 ，包括固體腫瘤和白血病。可治療之癌症的類型，包括但 不限於：乳房、前列腺和結腸的腺癌；所有形式的肺臟之 支氣官源的癌症；骨髓的；黑色素瘤；肝腫瘤；神經胚細 胞瘤；乳頭狀瘤；胺前體攝取與脫羧細胞瘤；迷行瘤丨鰓 原瘤；惡性的類癌徵候群；類癌心臟病；癌（例如沃克 (Walker)、基底細胞、基底鱗狀細胞、布朗_皮爾斯 (Brown-Pearce)、導管的、艾瑞區（Ehrlich)腫瘤、克瑞伯 茲（Krebs) 2、默寇（Merkel)細胞、黏蛋白的、非_小細胞肺 、燕麥細胞、乳頭狀的、硬癌的、細支氣管的、支氣管源 的、鱗狀細胞和移行細胞）；組織細胞的病症；白血病； 惡性的組織細胞增生；霍奇金氏症；免疫增殖性小肺臟細 胞癌；非-霍奇金氏的淋巴瘤；漿細胞瘤；網狀内皮增殖 症；黑色素瘤；軟骨母細胞瘤；軟骨瘤；軟骨肉瘤；纖維 瘤；纖維肉瘤；巨細胞腫瘤；組織細胞瘤；脂肪瘤；脂肉 瘤；間皮瘤；黏液瘤；黏液肉瘤；骨瘤；骨肉瘤；軟骨瘤 ;顱咽瘤；無性細胞瘤；錯構瘤；間葉瘤；中腎瘤；肌肉 瘤；釉質母細胞瘤；齒堊質瘤；齒瘤；畸胎瘤；胸腺瘤； 痛風石胚的腫瘤（tophoblastic tumor)。此外，亦可、、Λ療下 列類型的癌症：腺瘤；膽管瘤；膽硬脂瘤；圓栓瘤；囊腺 癌；囊腺瘤；粒層細胞腫瘤；兩性胚細胞瘤；肝腫瘤·汗 腺腺瘤；胰島細胞瘤；萊狄吉（Leydig)細胞瘤；乳頭狀瘤 O:\121\121929.DOC -105- 200808833 ;賽托利（Sertoli)細胞瘤；卵泡膜細胞瘤；平滑肌瘤；平 滑肌肉瘤；肌胚細胞瘤；肌瘤；肌肉瘤；橫紋肌瘤；橫紋 肌肉瘤；室管瘤；神經節瘤；神經膠質瘤；神經管胚細胞 瘤；腦膜瘤；神經鞘瘤；神經母細胞瘤；神經上皮瘤；神 經纖維瘤；神經瘤；副神經節瘤；非嗜鉻性副神經節瘤； 血管角化瘤；血管淋巴樣增生伴嗜曙紅細胞增多症；硬化 性血管瘤；血管瘤病；血管球瘤；血管内皮瘤；血管瘤； 血管外皮細胞瘤；血管肉瘤；淋巴管瘤；淋巴管肌瘤；淋 巴官肉瘤；松果體瘤；癌肉瘤；軟骨肉瘤；葉狀囊肉瘤； 纖維肉瘤；血管肉瘤；平滑肌肉瘤；白血病性肉瘤；脂肉 瘤；淋巴管肉瘤；肌肉瘤；黏液肉瘤；卵巢癌；橫紋肌肉 瘤；肉瘤；贅生物；多發性神經纖維病和子宮頸發育不全。 本發明的其他、觀點是使用本發明的材料和方法，預防及/ 或治療任何皮膚之過度增殖的狀況，包括牛皮癖和接觸性 皮膚炎’或其他過度增殖的疾病。已證實罹患牛皮癖和接 觸性皮膚炎的患者’在這些病變中具有升高的Ang_2活性 [Ogosln等人，J. inv. Dermat〇1，11〇:818 23 (Η%)]。最好 是’將對Ang-2專一的專一結合劑與其他的藥理學製劑混 合使用，來治療表現這些臨床症狀的人。可使用各種載劑 的任-種’經由在本文中描述的，以及其他熟諳此藝者已 熟知的投藥途徑，來遞送專一結合劑。 本發明的其他觀點包括治療各種涉及血管生成作用 網膜病變（包括糖尿病性I㈣病變和與年齡有關 變性）’以及雌性生殖道的病症/疾病，像是子宮内膜里位O:\121\121929.DOC-102-200808833 Neoplasms which can be treated by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Cancers include those malignant neoplasms derived from epithelial cells that infiltrate and cause metastases. Adenocarcinoma is a cancer derived from glandular tissue, or it forms a recognizable glandular structure. Other broad categories or cancers, including sarcomas, are tumors whose cells are embedded in fibrils or homogeneous materials, such as the connective tissue of an embryo. The invention is also capable of treating cancers of the bone marrow or lymphatic system, including rickets, lymphomas, and other cancers that are not normally found in the form of tumor masses but are dispersed in blood vessels or lymphatic network systems. τ according to the present invention, the type of cancer or tumor cell to be treated, including, for example, ACTH-producing tumor, acute lymphocytic leukemia, acute non-lymphocytic leukemia, adrenal cortical cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic Lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, skin Τ cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer , H〇dgkin, s) lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small cells and non-small cells), malignant ascites, malignant pleural effusion, Melanoma, mesothelioma, multiple osteosarcoma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovarian (germ cell) cancer, pancreatic cancer, penile cancer , prostate cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasm, uterine cancer, yin Cancer, vulvar cancer, and Wilms' tumor. With regard to the treatment of certain types of experimentally defined cancers, the invention is explained in particular herein: O: \121\121929.DOC-103-200808833. In this explanation, the treatment of fresh-release, the standard existing and the most advanced techniques have been used in vitro and in vivo. These methods can be used to confirm that it is expected to be an effective preparation in the in vivo λ 円 / oral therapy regimen. However, it should be understood that the method of the present invention is not limited to the treatment of these tumor types, and is applied to any solid tumor from any of the Erlu systems. Cancers that are invasive or metastatic in relation to the performance or activity of Ang2 are particularly susceptible to inhibition by the present invention or even cause regression. The present invention can also be practiced by including a compound of the present invention, such as a peptibosome, in combination with other anti-cancer chemotherapeutic agents, such as any conventional chemotherapeutic agent. The combination of a specific binder and such other preparations can produce the efficacy of a chemical treatment draft. Many chemotherapeutic drafts will present themselves in a way that is familiar to the artist's ideas. Any chemotherapeutic agent can be used, including an alkylating agent, an anti-metabolite, a hormone and an antagonist, a radioisotope, and a natural product. For example, the compound of the present invention may be mixed with an antibiotic such as doxorubicin and other ampicillin analogs, such as cyclophosphamide, which is like a sputum, such as urinary (four), shunqi Cisplatin, transurea, paclitaxel (10), and natural and synthetic derivatives thereof, and analogs thereof. Like other examples, in the case of mixed tumors, such as breast adenocarcinoma, where the tumor includes gonadotropin-dependent and gonadotropin-independent cells, it can be combined with leuprolide or Gosher. The compound (g〇sereUn) (peptide analog of the synthesis of LH_RH) is administered together. Other anti-tuberculosis drafts include the use of tetracycline compounds and other therapeutic regimens, such as surgery, radiation, etc., also referred to herein as "assisted anti-caries biologics, 〇:\121\121929.DOC - 104· 200808833. Therefore, the method of the present invention can be used together with such conventional breeding methods, and has the advantages of reducing side effects and improving efficacy. The present invention is directed to compositions and methods useful for treating various cancers, including solid tumors and leukemias. Types of treatable cancer, including but not limited to: breast, prostate, and colon adenocarcinoma; all forms of lung-associated cancer; bone marrow; melanoma; liver neoplasms; neuroblastoma; papilloma Amine precursor uptake and decarboxylated cell tumors; sputum tumors; malignant carcinoid syndrome; carcinoid heart disease; cancer (eg Walker, basal cells, basal squamous cells, Brown-Pearce ( Brown-Pearce), ductal, Ehrlich tumor, Krebs 2, Merkel cells, mucin, non-small cell lung, oat cells, papillary, hard Cancerous, bronchiole, bronchial, squamous and transitional cells; disorders of histiocytes; leukemia; malignant histiocytosis; Hodgkin's disease; immunoproliferative small lung squamous cell carcinoma; non-Hodge Lymphoma of Kim; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; Tumor; mesothelioma; myxoma; mucinous sarcoma; osteoma; osteosarcoma; chondroma; craniopharynx; dysgerminoma; hamartoma; mesenchymal; middle renal tumor; muscle tumor; enamel blastoma; Gonoma; odontoma; teratoma; thymoma; tophoblastic tumor. In addition, the following types of cancer can also be treated: adenoma; cholangiocarcinoma; cholesteatoma; round plug tumor; cystadenocarcinoma; cystadenoma; granulosa cell tumor; amphoblastic blastoma; liver tumor Adenoma; islet cell tumor; Leydig cell tumor; papilloma O: \121\121929.DOC -105- 200808833; Sertoli cell tumor; ovarian cytoma; leiomyomas; Leiomyosarcoma; myoblastoma; fibroids; muscle tumor; rhabdomyosarcoma; rhabdomyosarcoma; ependymoma; ganglionoma; glioma; bronchial blastoma; meningiomas; schwannomas; neuroblastoma; Neuroepithelial neoplasia; neurofibromatosis; neuroma; paraganglioma; non-chromophobic paraganglioma; vascular keratomas; vascular lymphoid hyperplasia with eosinophilia; sclerosing hemangioma; Hemangioma; hemangioendothelioma; hemangiomas; angioendothelioma; angiosarcoma; lymphangioma; lymphangioma; lymphoid sarcoma; pineal tumor; carcinosarcoma; chondrosarcoma; phyllodes cystosarcoma; Angiosarcoma; Leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; muscle tumor; mucinous sarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasm; multiple neurofibrosis and cervical hypoplasia. Other aspects of the invention are the use of the materials and methods of the invention to prevent and/or treat any hyperproliferative condition of the skin, including psoriasis and contact dermatitis' or other hyperproliferative diseases. Patients with psoriasis and contact dermatitis have been shown to have elevated Ang 2 activity in these lesions [Ogosln et al., J. inv. Dermat〇 1, 11: 818 23 (Η%)]. It is best to use an exclusive combination of Ang-2 specific agents in combination with other pharmacological agents to treat those who exhibit these clinical symptoms. Any of a variety of carriers can be used to deliver a specific binding agent via the routes of administration described herein, as well as other routes of administration well known to those skilled in the art. Other aspects of the invention include the treatment of various vascular-related retinal lesions (including diabetic I(s) lesions and age-related degeneration) and the female genital tract disorders/diseases, such as endometrial linings.
O:\121\121929.DOC -106- 200808833 症、子宮纖維樣，以及在雌性生殖周期中，其他與血管增 殖功此障礙有關的這類病況（包括子宮内膜微血管生長）。 本發明的其他觀點係關於治療異常的血管生長，包括大 腦動靜脈畸型（AVMs)、胃腸道黏膜傷害和修補、在具有 消化性潰瘍疾病之病史的患者中，胃十二指腸黏膜的潰瘍 ’包括起因於猝發的局部缺血，在罹患非·肝臟門靜脈壓 過高的患者中，在肝病和門靜脈壓過高時的廣泛肺血管病 症。 本發明的其他觀點是使用由本發明提供之組合物和方法 來預防癌症。這類試劑將包括專一結合劑，像是對抗 2的肽體。 醫藥組合物O:\121\121929.DOC -106- 200808833 Disease, uterine fibrosis, and other conditions associated with vascular proliferation in the female reproductive cycle (including endometrial microvascular growth). Other aspects of the present invention relate to the treatment of abnormal blood vessel growth, including cerebral arteriovenous malformations (AVMs), gastrointestinal mucosal injuries and repairs, in patients with a history of peptic ulcer disease, ulcers of the gastroduodenal mucosa, including Ischemia in the hair, in patients with non-hepatic portal hypertension, extensive pulmonary vascular disease in patients with liver disease and portal hypertension. Another aspect of the invention is the use of the compositions and methods provided by the present invention to prevent cancer. Such agents will include a specific binding agent, such as a peptide against 2. Pharmaceutical composition
Ang-2專一結合劑，像是肽體的醫藥組合物，是在本發 明的範圍内。醫藥組合物包括在例如Lam等人，於2〇〇1年 1月9日發證之美國專利第6,171，586號中詳細描述的抗體。 這類組合物包括在治療上或在預防上有效之含量的專一結 合劑’像是如同在本文中描述的抗體或其片段、變體、衍 生物或融合蛋白質，並與在藥學上可接受的製劑混合。在 較佳的具體實施例中，醫藥組合物包括拮抗的專一結合劑 ’其部分或全部地調節至少一種Ang-2的生物活性，並與 在藥學上可接受的製劑混合。通常，為了投與動物，將充 份地純化該專一結合劑。 醫藥組合物可含有調配物質，以便修改、維持或保留例 如該組合物的pH值、滲透性、黏度、澄清度、顏色、等張 O:\121\121929.DOC -107- 200808833Ang-2 specific binding agents, such as pharmaceutical compositions of peptibodies, are within the scope of the invention. The pharmaceutical compositions include the antibodies described in detail in U.S. Patent No. 6,171,586, issued to Jan. Such compositions include a therapeutically or prophylactically effective amount of a specific binding agent, such as an antibody, or a fragment, variant, derivative or fusion protein thereof, as described herein, and in pharmaceutically acceptable form. The preparation is mixed. In a preferred embodiment, the pharmaceutical composition comprises an antagonistic, specific binding agent that partially or fully modulates the biological activity of at least one Ang-2 and is combined with a pharmaceutically acceptable formulation. Typically, the specific binding agent will be fully purified for administration to the animal. The pharmaceutical composition may contain a formulation to modify, maintain or retain, for example, the pH, permeability, viscosity, clarity, color, isotonicity of the composition. O: \121\121929.DOC -107- 200808833
性、味道、無菌性、穩定性、溶解或釋放、吸收或穿透的 速率。適當的調配物質包括，但不限於胺基酸（像是甘胺 酸、穀胺醯胺、天冬醯胺、精胺酸或離胺酸）；抗微生物 製劑；抗氧化劑（像是抗壞血酸、亞硫酸鈉或亞硫酸氫鈉） ;緩衝溶液（像是硼酸鹽、碳酸氫鹽、Tris_Hcl、檸檬酸鹽 、磷酸鹽·，其他的有機酸類）；填充劑（像是甘露糖醇或甘 胺酸）；螯合劑[像是乙二胺四乙酸（EDTA)];錯合劑（像是 咖啡因、聚乙烯吡咯烷酮、β-環糊精或羥丙基_β_環糊精） ;填料；單醣類；雙醣類和其他的碳水化合物（像是葡萄 糖、甘露糖或糊精），蛋白質（像是血清白蛋白、明膠或免 疫球蛋白）；色素；調味劑和稀釋劑；乳化劑；親水性聚 合物（像是聚乙烯吡咯烷酮）；低分子量的多肽；形成鹽的 抗衡離子（像是納）；防腐劑（像是氯化苯甲烴銨、苯甲酸、 水杨酸、乙基采硫代水楊酸納、苯乙醇、對經苯曱酸甲西旨 、對羥苯甲酸丙酯、氯己定（chlorhexidine)、山梨酸或過 氧化氫）；溶劑（像是甘油、丙二醇或聚乙二醇）；糖醇類（ 像是甘露糖醇或山梨糖醇）；懸浮劑；表面活性劑或濕潤 劑（像是普魯尼克（pluronics)、PEG、脫水山梨糖醇酯、多 乙氧基鱗類，像是多乙氧基鱗20、多乙氧基醚8〇、三通、 三甲醇氨基甲烷（tromethamine)、卵填脂、膽固醇、泰洛 沙泊（tyloxapal));穩定性促進劑（蔗糖或山梨糖醇）；張力 促進劑（像是驗金屬_化物（最好是氯化鈉或鉀）、甘露糖醇 、山梨糖醇）；遞送媒劑；稀釋劑；賦形劑及/或藥學佐劑 。（Remington’s Pharmaceutical Sciences，第 18 版，a.R O:\121\121929.DOC -108· 200808833Rate of sex, taste, sterility, stability, dissolution or release, absorption or penetration. Suitable formulations include, but are not limited to, amino acids (such as glycine, glutamine, aspartame, arginine or lysine); antimicrobial agents; antioxidants (like ascorbic acid, sodium sulfite) Or sodium bisulfite); buffer solutions (such as borate, bicarbonate, Tris_Hcl, citrate, phosphate, other organic acids); fillers (like mannitol or glycine); Mixtures [like ethylenediaminetetraacetic acid (EDTA)]; complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl_β_cyclodextrin); fillers; monosaccharides; Carbohydrates and other carbohydrates (like glucose, mannose or dextrin), proteins (like serum albumin, gelatin or immunoglobulin); pigments; flavoring and diluents; emulsifiers; hydrophilic polymers ( Such as polyvinylpyrrolidone); low molecular weight peptides; salt-forming counterions (like nano); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, ethyl thiosalicylic acid) Na, phenylethyl alcohol, benzoic acid, Propyl hydroxybenzoate, chlorhexidine, sorbic acid or hydrogen peroxide; solvents such as glycerol, propylene glycol or polyethylene glycol; sugar alcohols (like mannitol or sorbitol) Suspending agent; surfactant or wetting agent (such as pluronics, PEG, sorbitan ester, polyethoxy scales, like polyethoxy scale 20, polysorbate 8 Bismuth, tee, tromethamine, egg fat, cholesterol, tyloxapal; stability promoter (sucrose or sorbitol); tension promoter (like metallization) (preferably sodium chloride or potassium), mannitol, sorbitol; delivery vehicle; diluent; excipient and/or pharmaceutical adjuvant. (Remington’s Pharmaceutical Sciences, 18th edition, a.R O:\121\121929.DOC -108· 200808833
Gennaro編輯，Mack Publishing Company，1990) 〇 最適切的醫藥組合物將由熟諳此藝者依據例如期待的投 藥途徑、遞送格式，和想要的劑量來判定。參見，例如Gennaro, ed., Mack Publishing Company, 1990) 〇 The most appropriate pharmaceutical composition will be judged by the skilled artisan based on, for example, the desired route of administration, the delivery format, and the desired dosage. See, for example
Remington’s Pharmaceutical Sciences，在前。這類組合物 可影響專一結合劑的物理狀態、穩定性、在活體内的釋放 速率，和在活體内的清除速率。 在醫藥組合物中，主要的媒劑或載劑之性質可以是含水 的或不-含水的。例如，適當的媒劑或載劑可以是注射用 水、生理鹽水溶液或人造的腦脊髓液，可能補充有其他經 常在非經腸投藥之組合物中使用的物質。中性緩衝之鹽水 或與血清白蛋白混合的鹽水，是更具代表性的媒劑。其他 代表性的醫藥組合物包括大約pH 7.0_8.5的加緩衝溶液， 或pH 4.0-5.5的醋酸緩衝溶液，其尚可包括山梨糖醇或其 適當之取代物。在本發明的一個具體實施例中，藉著將= 選擇、具有想要純度的組合物與任意的調配劑 (Remington’s Pharmaeeutical Sciences，在前）混合，以冷 象乾燥餅或含水溶液之形式，製備可供儲存的結:劑^ 物。此外，可使用適當的賦形劑，像是絲，將結合劑產 物調配成冷凍乾燥物。 可選擇醫藥組合物以供非經腸遞送。或者，可選擇組合 物以供吸入或經腸遞送，像是口服、經耳、經眼、經 技藝的技術範圍内 或經陰道。這類在藥學上可接受之組合物的製傷，在此項 例如，使用緩 調配組份以投藥部位可接受的濃度存在Remington’s Pharmaceutical Sciences, formerly. Such compositions can affect the physical state, stability, rate of release in vivo, and rate of clearance in vivo. In pharmaceutical compositions, the nature of the primary vehicle or carrier can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials which are often used in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin is a more representative vehicle. Other representative pharmaceutical compositions include a buffered solution having a pH of about 7.0 to about 8.5, or an acetic acid buffered solution having a pH of from 4.0 to 5.5, which may also include sorbitol or a suitable replacement thereof. In a particular embodiment of the invention, the composition is prepared by mixing the composition of the desired purity with any of the formulating agents (Remington's Pharmaeeutical Sciences, prior) in the form of a cold-dried cake or an aqueous solution. A knot that can be stored: a substance. In addition, the binding agent product can be formulated into a lyophilizate using a suitable excipient such as silk. The pharmaceutical composition can be selected for parenteral delivery. Alternatively, the composition can be selected for inhalation or enteral delivery, such as oral, otic, transocular, technical, or transvaginal. Such wounding in pharmaceutically acceptable compositions, for example, is carried out using a slow-adjusting component at a concentration acceptable to the site of administration.
O:\121\121929.DOC -109- 200808833 衝溶液二純合物維持在生理學的PH值，或稍低的pH值 下通_疋在從大約5到大約8的pH值範圍内。 、：企圖非經腸投藥時，在本發明中使用的治療組合物可 、…、…、原非經知可接受之含水溶液的形式，在在藥學 上Y接叉的媒劑中，包括想要的專一結合劑。特別適合非 經腸注射的媒劑為無菌的蒸餾水，在其中將結合劑調配成 適口保存的無菌、等張之溶液。另一種製品可涉及與諸如 可庄射的中心體、生物可腐蝕的顆粒、聚合化合物（聚乳 酸、聚乙醇酸）、小珠或微脂粒之類的製劑，一起調配想 要的刀子，其&供產物的控制或持續釋放，然後可經由積 儲注射遞送它們。亦可使用透明質酸，而這具有在循環中 增進持續期間的效果。其他導入想要分子的適當方法，包 括植入藥物遞送裝置。 在其他方面，可在含水溶液中調配適合非經腸投藥的醫 藥調配物，最好是在生理學上可相容的緩衝溶液中，像是 漢克氏（Hank’s)溶液、林格氏液或在生理學上經過緩衝的 溶液。含水的注射懸浮液可含有增加懸浮液黏度的物質， 像是羧甲基纖維素鈉、山梨糖醇或葡聚醣。或者，可將活 性化合物的懸浮液製備成適當的油性注射懸浮液。適當的 親脂性溶劑或媒劑，包括脂油，像是芝麻油，或合成的脂 肪酸酯，像是油酸乙酯、三酸甘油酯或微脂粒。亦可使用 非-脂質聚陽離子的胺基聚合物來進行遞送。該懸浮液亦 可視需要含有適當的穩定劑，或增加化合物溶解度的製劑 ，並容許製備極高濃度的溶液。 O:\121\121929.DOC -110- 200808833 在其他的具體實施例中，可調配吸人料醫藥组合物。 例如’可將結合劑調配成吸人用的乾粉。亦可為了氣溶膠 的遞送，利用推進劑來調配多肽或核酸分子的吸入溶液。 在另一個具體實施例中，可將該溶液霧化。在pcT申請案 第pct/US94/001875號中’進一步描述了肺臟投藥，^描 述以化學方式修改之蛋白質的肺臟遞送。 亦嘗試可口服投與某些調配物。在本發明的一個具體實 施例中，以該方式投與的結合劑分子，可與或不與習慣上 在混合固體劑量形式，像是錠劑和膠囊時使用的那些載劑 一起調配。例如，可將膠囊設計成在胃腸道中某處釋放調 配物之活性部份，此時生物利用性最高，而前_全身性的 降解作用最低。可含有額外的製劑，以便促進結合劑分子 的吸收。亦可使用稀釋劑、調味劑、低熔點的蠟、植物油 、潤滑劑、懸浮劑、錠劑崩解劑和黏合劑。 亦可使用在此項技藝中已熟知的在藥學上可接受之載劑 ’以適合口服投藥之劑量，來調配可供口服投藥的醫藥組 合物。這類載劑得以將醫藥組合物調配成錠劑、藥丸、糖 衣錠'膠囊、液體、凝膠、糖漿'淤漿、懸浮液及其類似 物，以供患者攝取。 可經由將活性化合物與固體賦形劑混合，並加工所得的 顆粒混合物（可視需要在磨碎之後），獲得錠劑或糖衣旋核 心’而獲得口服使用的醫藥組合物。如果想要，可加入適 當的辅助劑。適當的賦形劑包括碳水化合物或蛋白質填料 ’像是糖類，包括乳糖、蔗糠、甘露糠醇和山梨糠醇丨得 O:\121\121929.DOC -111- 200808833 自玉米、小麥、稻米、馬鈴著或其他植物的澱粉；纖維素 ’像是甲基纖維素、羥丙基曱美德喜 ^丨JT丞_纖維素或羧甲基纖維素 鈉；樹膠類，包括阿拉伯樹膠和黃蓍膠；以及蛋白質，像 疋明膠和勝原蛋白。如要相亚 π ^ . Τ ^ ^如果想要，可加入崩解劑或加溶劑， 像疋父聯的聚乙烯-吡咯烷酮、瓊脂和藻酸，或其鹽類， 像是藻酸銅。 可連同適當的塗膜一起使用糖衣錠核心，像是濃縮的糖 溶液，其亦可含有阿拉伯樹膠、滑石、聚乙烯吡咯烷酮、 卡波醇（carbopol)凝膠、聚乙二醇及/或二氧化鈦、漆溶液 和適當的有機溶劑或溶劑混合物。可在錠劑或糖衣錠塗膜 中加入染料或色素，用來確認產物，或用來顯示活性化合 物的含|特性，也就是劑量。 可口服使用的醫藥製品亦包括推入配合的明膠製造之膠 囊’以及以明膠製造之軟的密封膠囊，和塗料，像是甘油 或山梨糖醇。推入配合的膠囊可含有活性成分，與填料或 枯合劑，像是乳糖或澱粉，潤滑劑，像是滑石或硬脂酸鎂 混合，並可視需要還有穩定劑。在軟膠囊中，可將活性成 分溶解或懸浮於適當的液體中，像是脂油、液體，或液體 聚乙二醇，可有或無穩定劑。 其他的醫藥組合物可涉及在帶有適合製造旋劑的無毒性 賦形劑之混合物中的有效含量之結合劑。藉著將該錠劑溶 解於無菌的水，或其他適當的媒劑中，可製備單位劑量形 式的溶液。適當的賦形劑包括，但不限於惰性稀釋劑，像 是碳酸鈣、碳酸鈉或碳酸氫鈉、乳糖或磷酸弼；或枯合劑 O:\121\121929.DOC -112- 200808833 ，像是澱粉、明膠或阿拉伯樹膠； 鎂、硬脂酸或滑石。 閑滑劑，像是硬脂酸 額外的醫藥組合物對於熟諳此蓺 括涉及以持續·或控制_遞送之方：★而言將是明顯的’包 物。調配久鐘盆你杜洁 周配、纟〇合劑分子的調配 ^ ^ ^ 別-釋放方法的技術，像是 斂月曰粒載劑、生物可腐蝕的微顆粒， 射，介9/夕孔小珠和積儲注 射亦疋熱钿此藝者已知的。束f w、+、& 乂見例如 PCT/US93/00829 ，描述用來遞送醫藥化合物之控 ,.^ 4制擇放的多孔聚合微顆粒 。持釋放之製品的其他實例， n ” 包括+透性的聚合物基 質’以成形物件的形式，像是薄膜戎 %疋得胰或试膠囊。持續釋放的 基質可包括聚酿、水膠體、聚交醋(美國專利第3,773,919 號，歐洲專利5M8m)、L-穀胺酸^乙基_毅胺酸醋的共 聚物[Sidman 等人，Biopolymers，22:547_556 (1983)]、聚 (2-羥乙基_異丁烯酸酯）[Langer等人，jO:\121\121929.DOC -109- 200808833 The dip solution is maintained at a physiological pH, or at a slightly lower pH, at a pH ranging from about 5 to about 8. In the case of attempting parenteral administration, the therapeutic composition used in the present invention may be in the form of an aqueous solution which is not known to be acceptable, in the form of a pharmaceutically acceptable Y-containing medium, including A specific combination of ingredients. A vehicle which is particularly suitable for parenteral injection is sterile distilled water in which the binding agent is formulated into a sterile, isotonic solution which is palatable. Another article may involve blending a desired knife with a formulation such as a smudged centrosome, bioerodible particles, polymeric compound (polylactic acid, polyglycolic acid), beads or vesicles, & Control or sustained release of the product, which can then be delivered via a reservoir injection. Hyaluronic acid can also be used, which has the effect of enhancing the duration during the cycle. Other suitable methods of introducing the desired molecule include implanting a drug delivery device. In other aspects, pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffer solutions such as Hank's solution, Ringer's solution or A physiologically buffered solution. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Alternatively, a suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or vesicles. Amino-based polymers of non-lipid polycations can also be used for delivery. The suspension may also contain suitable stabilizers as needed, or a formulation which increases the solubility of the compound, and allows for the preparation of very high concentrations of solutions. O: \121\121929.DOC-110- 200808833 In other embodiments, an inhalable pharmaceutical composition can be formulated. For example, the binding agent can be formulated into a dry powder for inhalation. Propellants can also be used to formulate inhalation solutions of polypeptides or nucleic acid molecules for aerosol delivery. In another embodiment, the solution can be atomized. Lung administration is further described in pcT application No. pct/US94/001875, which describes the pulmonary delivery of a chemically modified protein. It has also been attempted to orally administer certain formulations. In a particular embodiment of the invention, the binding agent molecules administered in this manner may or may not be formulated with those carriers conventionally used in the mixing of solid dosage forms, such as lozenges and capsules. For example, the capsule can be designed to release the active portion of the formulation somewhere in the gastrointestinal tract, with the highest bioavailability and the lowest pre-systemic degradation. Additional formulations may be included to promote absorption of the binding agent molecules. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used. A pharmaceutical composition for oral administration can also be formulated using a pharmaceutically acceptable carrier, as is well known in the art, at a dosage suitable for oral administration. Such carriers are capable of formulating pharmaceutical compositions into tablets, pills, dragee capsules, liquids, gels, syrups, slurries, suspensions and the like for ingestion by a patient. The pharmaceutical composition for oral use can be obtained by mixing the active compound with a solid excipient and processing the resulting mixture of granules (after pulverization as needed) to obtain a lozenge or dragee core. If necessary, add the appropriate adjuvant. Suitable excipients include carbohydrates or protein fillers such as sugars, including lactose, sugarcane, mannitol and sorbitol. O:\121\121929.DOC -111- 200808833 From corn, wheat, rice, horse bells Or starch of other plants; cellulose such as methyl cellulose, hydroxypropyl hydrazine, 丨JT 丞 cellulose or sodium carboxymethyl cellulose; gums, including gum arabic and tragacanth; Proteins, like gelatin and ginseng proteins. If you want to add π ^ . Τ ^ ^ If you want, you can add disintegrants or solubilizers, like the father-linked polyethylene-pyrrolidone, agar and alginic acid, or its salts, such as copper alginate. A sugar-coated core may be used in conjunction with a suitable coating, such as a concentrated sugar solution, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer A solution and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablet or dragee coating to confirm the product or to indicate the containing properties of the active compound, i.e., the dosage. Pharmaceutical products which can be used orally also include gelatin capsules made by push gel and 'soft capsules made of gelatin, and coatings such as glycerin or sorbitol. The push-fit capsules may contain the active ingredient in admixture with a filler or a carrier such as lactose or starch, a lubricant such as talc or magnesium stearate, and a stabilizing agent if desired. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid, or liquid polyethylene glycol, with or without a stabilizer. Other pharmaceutical compositions may involve an effective level of binding agent in a mixture with non-toxic excipients suitable for the manufacture of a syringe. A solution in unit dosage form can be prepared by dissolving the lozenge in sterile water or other suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or strontium phosphate; or dry agents O:\121\121929.DOC-112-200808833, such as starch , gelatin or gum arabic; magnesium, stearic acid or talc. A slippery agent, such as stearic acid, is an additional pharmaceutical composition for those skilled in the art that are involved in continuous or controlled delivery: ★ will be apparent. Mixing the long-term pots, you can arrange the molecules of the sputum, and mix the molecules of the sputum. ^ ^ 别 - The technique of the release method, such as the sputum granule carrier, the bio-corrosive micro-particles, the shot, the medium 9/ 夕孔小珠 and the accumulation Injections are also known to the artist. The bundles f w, +, & see, for example, PCT/US93/00829, describe the use of controlled delivery of pharmaceutical compounds, porous polymeric microparticles. Other examples of articles that are released, n" include a +permeable polymeric matrix' in the form of a shaped article, such as a film of pancreatic or test capsules. The sustained release matrix may include agglomerates, hydrocolloids, poly Co. vinegar (U.S. Patent No. 3,773,919, European Patent 5M8m), L-glutamic acid ethyl-acetic acid vinegar copolymer [Sidman et al, Biopolymers, 22: 547_556 (1983)], poly(2-hydroxyl) Ethyl-methacrylate) [Langer et al., j
Res·，15:167-277，（1981)]和[Langer 等人，Chem. Tech·， 12:98· 105 (1982)]，乙烯乙酸乙烯酯（Langer等人，在前）或 聚-D(-)-3-羥基丁酸（歐洲專利133,988號）。持續-釋放之組 合物亦包括微脂粒，可藉著任何此項技藝中已知的數種方 法來製備之。參見，例如Eppstein等人，Proc. Natl. Acad. Sci· (USA)，82:3688-3692 (1985);歐洲專利 36,676號；歐 洲專利88,046號；歐洲專利143,949號。 欲用於活體内投藥的醫藥組合物，通常必須是無菌的。 這可藉著通過無菌過濾膜過濾而達成。在組合物被冷凍乾 燥之處，可在冷凍乾燥和重建之前或之後，進行使用該方 O:\121\121929.DOC 113- 200808833 法之滅菌作用。可以冷凍 非經腸投藥的組合物。此外:、=非：以溶液形式儲存 有滅菌接頭的容器中的、工腸組合物放在具 皮下注射針頭刺穿之塞子的小瓶中。4具有可被 膠、乳心ΓΓ組合物，便可以溶液、懸浮液、凝 存在滅菌的小瓶中：：二冷; 東乾燥散劑的形式，將其館 t f t # (m ^ ^ 使用之形式，或在投藥之 例如冷康乾燥）的形式，錯存這類調配物。 於—疋的具體實施例中，本發明針對產生單-·劍量之 二樂:位的套組。套組可分別含有第一個具有脫心 範圍第二個具有含水調配物的容器。在本發明的 射器二括合有單一和多個分開隔間之預先裝填的注 、口歹1 /夜體注射器和溶解注射器（1州灿⑼的套組。 …療上使用之醫藥組合物的有效含量，將視例如治療 旦二，關係和目標而定。熟諳此藝者將瞭解適合治療之劑 曰-曰因此邛刀將依據遞送之分子、欲使用結合劑分 子的適應症、投藥的途徑，以及患者的尺寸（體重、體表 積或器g尺寸）和狀況（年齡和一般的健康）而改變。因此 ’臨床醫師可滴定劑量，並修改投藥的途徑，以便獲得最 佳的^療效果。代表性的劑量，範圍可從大狀i毫克/公 斤到高達大約100毫克/公斤或更多，視上文提及的因素而 疋。在其他的具體實施例中，劑量範圍可從ϋ·1毫克/公斤 同達大約1〇0毫克/公斤；或1毫克/公斤高達大約1〇〇毫克/ 公斤，或5毫克/公斤高達大約1〇〇毫克/公斤。Res., 15: 167-277, (1981)] and [Langer et al., Chem. Tech., 12: 98. 105 (1982)], ethylene vinyl acetate (Langer et al., supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No. 133,988). The sustained-release composition also includes vesicles which can be prepared by any of a number of methods known in the art. See, for example, Eppstein et al., Proc. Natl. Acad. Sci. (USA), 82: 3688-3692 (1985); European Patent No. 36,676; European Patent No. 88,046; European Patent No. 143,949. Pharmaceutical compositions intended for in vivo administration must generally be sterile. This can be achieved by filtration through a sterile filtration membrane. Where the composition is lyophilized, the sterilization of the method using O:\121\121929.DOC 113-200808833 may be carried out before or after lyophilization and reconstitution. Compositions for parenteral administration can be frozen. In addition: = = non: stored in solution The container in the container with the sterilized joint is placed in a vial with a stopper pierced by a hypodermic needle. 4 has a gelatinous, milky heart sputum composition, it can be solution, suspension, coagulation in the sterilized vial:: two cold; East dry powder form, its pavilion tft # (m ^ ^ use form, or In the form of administration such as cold-drying, such formulations are erroneously present. In a particular embodiment of the invention, the present invention is directed to a set of two music: bits that produce a single--sword. The kits may each contain a first container having a second dewatering range with an aqueous formulation. The ejector of the present invention comprises a prefilled injection of a single and a plurality of separate compartments, a mouthpiece 1 / a night body syringe and a dissolution syringe (a kit of 1 can (9). The effective amount will depend on, for example, the treatment, the relationship and the target. Those who are familiar with this art will know the appropriate treatment agent. Therefore, the sickle will be based on the molecule to be delivered, the indication of the partner molecule to be used, and the drug to be administered. The route, as well as the size of the patient (body weight, body surface or g-size) and condition (age and general health). Therefore, 'the clinician can titrate the dose and modify the route of administration to get the best treatment. Effect. Representative dosages can range from i mg/kg to as high as about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage range can be from ϋ • 1 mg/kg is about 1 mg/kg; or 1 mg/kg is up to about 1 mg/kg, or 5 mg/kg is up to about 1 mg/kg.
O:\121\121929.DOC -114- 200808833 對於任何化合物，一開始可先在 語‘土 〇 口養測定中，或在 ，建立：、大氣、兔子、狗、豬或猴子之類的動物模式中 nfm之劑4 °亦可使用動物模式來判定適 田勺/辰度乾圍和投藥途徑。然後可 從用k類資訊來判定在 “員中有用的劑量和投藥途徑。 將從與需要治療之個體有關的因素，來判定精確的劑量 。調整劑量和投藥，以便提供充分含量的活性化合物，或 維持想要的效果。可考慮的因素包括疾病狀態的嚴重性、 個體的-般健康狀況、個體的年齡、體重和性別、投藥的 時間和頻率、藥物之組合（們）、反應敏感性和對治療之反 應。長期作用的醫藥組合物，可每隔3至4天、每週或兩週 一次投與，將視特定調配物之半衰期和清除速率而定^ 投藥的頻率將視在調配物中使用之結合劑分子的藥物動 力學參數而定。通常投與組合物，直到達到獲得想要效果 的劑量為止。因此，可在一段時間内，以單一劑量或多個 劑量（以相同或不同的濃度/劑量），或以連續輸液之方式投 與組合物。例行地更詳細地討論適當的劑量。可經由使用 適當的劑量-反應資料，來探查適當的劑量。 醫藥組合物的投藥途徑，係根據已知的方法，例如口服 、經由靜脈内注射、腹腔内、大腦内（實質内）、腦室内、 肌肉内、眼内、動脈内、門脈内（intraportal)、病灶内的路 徑、脊髓内、鞘内、心室内、經皮、皮下、腹腔内、鼻内 、經腸、局部、舌下、尿道、陰道或直腸之方式，藉著持 續釋放的系統，或藉著植入裝置。在想要之處，可藉著團 O:\121\121929.DOC -115- 200808833 塊主射或連續輸液，或藉著植入裝置投與該組合物。 另外或額外地’亦可經由將膜、海綿，或其他已經在其 中吸附或包封想要分子的適當物質植入，局部投與該組合 物。在使用植入裝置之處，可將該裝置植入任何適當的組 織或器宫中，並可經由擴散、按時釋放之團塊，或連續投 藥’來遞送想要的分子。 在某些h況下，可能想要以在活體外之方式，使用醫藥 組合物。在這類例子中，將已經從患者中移出的細胞、組 織或器官暴露在醫藥組合物下，隨後再將該細胞、組織及/ 或器官移植回患者内。 在其他的案例中，可藉著植入某些已經使用像是在本文 中描述的那些方法，以遺傳之方式設計，以便表現和分泌 該多肽的細胞，來遞送本發明之結合劑，像是肽體。這類 細胞可以是動物或人類的細胞，並可以是自體的、異種的 或異種的。該細胞可視需要為永存不死的。為了降低免疫 學反應的改變，可將細胞包膠，以避免周圍組織的浸潤。 包膠的材料通常是生物可相容的、半-透性的聚合封入物 或膜’其容許蛋白質產物（們）的釋放，但防止該細胞被患 者的免疫系統，或被其他來自周圍組織的有害因素破壞。 混合治療 本發明之專一結合劑，像是肽體，可與其他治療與Ang-2表現有關之疾病的治療劑一起使用。這些其他的治療劑 包括，例如放射線治療、化學治療，以及瞄準治療，像是 HerceptinTM、RituxanTM、GleevecTM及其類似物。在本文 O:\121\121929.DOC -116- 200808833 中未特別列舉其他的混合治療，亦在本發明的範圍内。 化學治療可使用抗-贅生物製劑，包括例如烷基化製劑 ’包括氮芥，像是氣芥（mechlorethamine)、環鱗酸胺、異 環磷醯胺（ifosfamide)、苯丙胺酸氮芬（melphalan)和苯丁酸 氮芥（chlorambucil);亞石肖基脲，像是亞琐基脲氮芥 (BCNU)、洛莫司汀（lomustine)(CCNU)和西莫司汀 (semustine)(曱基-CCNU);氮丙啶/甲基蜜胺，像是三伸乙 基蜜胺（TEM)、三伸乙基硫代填醯胺（p塞替派（thiotepa))、 六甲基蜜胺（HMM，六曱蜜胺）；烷基磺酸酯，像是白消安 (busulfan);三畊，像是甲嗪咪唑胺（dacarbazine)(DTIC); 抗代謝產物，包括葉酸類似物，像是胺曱碟呤和三曱曲沙 (trimetrexate)，嘧咬類似物，像是5-氟尿嘧唆、氟脫氧尿 嘗、吉西他濱（gemcitabine)、阿拉伯糖胞嘗（AraC，胞η密唆 糖苷）、5-氮胞甞、2,2’-二氟脫氧胞苷，嘌呤類似物，像是 6-疏基嗓呤、6-硫代鳥嗓呤、硝基脒嗤硫嗓呤、2’-脫氧助 間型黴素（喷司他丁（pentostatin))、紅經基壬基腺嘌吟 (erythrohydroxynonyladenine)(EHNA)、填酸氟達拉濱 (Fludarabine phosphate)和2-氯脫氧腺誓（克拉君賓 (cladribine)，2-CdA);天然產物，包括抗-有絲分裂藥物 ，像是紫杉醇（paclitaxel)、長春花生物鹼，包括長春花鹼 (VbB)、長春新驗和長春瑞賓（vinorelbine)、姓癌易、雌莫 司丁（estramustine)和雌莫司丁磷酸酯；表鬼臼素 (epipodophyllotoxins)，像是依托泊菩（etoposide)和替尼泊 甞（teniposide);抗生素，像是放線菌素D、道諾黴素（紅比 O:\121\121929.DOC -117- 200808833 黴素）、阿黴素、米托蒽臟（mitoxantrone)、伊達比星 (idarubicin)、博菜黴素、普卡黴素（plicamycin)(光神黴素） 、絲裂黴素C和放線菌素；酵素，像是L-天冬醯胺酶；生 物反應修改劑，像是干擾素-a、IL-2、G-CSF和GM-CSF ; 雜項的製劑，包括翻配位錯合物，像是順氯氨舶和卡麵 (carboplatin)，蒽二酮，像是米托慈g昆，經取代之脲，像 是羥基脲，甲基肼衍生物，包括N-甲基肼（MIH)和丙卡巴 月井（procarbazine)，腎上腺皮質抑制劑，像是米托坦 (mitotane) (o，p’-DDD)和氨魯米特（aminoglutethimide);荷 爾蒙和拮抗劑，包括腎上腺皮質類固醇拮抗劑，像是脫氫 可的松和相等物，地塞米松和氨魯米特；黃體製劑，像是 己酸經孕酮（hydroxyprogesterone caproate)、醋酸甲經孕酮 (medroxyprogesterone acetate)和醋酸甲地孕酮（megestrol acetate);雌激素，像是己浠雌醇（diethylstilbestrol)和炔雌 醇（ethinyl estradiol)相等物；抗雌激素，像是他莫昔芬 (tamoxifen);雄激素，包括睪固酮丙酸S旨和氟曱睪酮 (fluoxymesterone)/相等物；抗雄激素，像是氟他胺 (flutamide)、促性腺激素-釋放荷爾蒙類似物和亮丙里德； 以及非-類固醇的抗雄激素，像是氟他胺。 利用生長因子之混合治療，包括細胞激動素、淋巴細胞 活素、生長因子或其他造血因子，像是M-CSF、GM-CSF 、TNF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、 IL-16、IL-17、IL-18、IFN、TNF0、TNF1、TNF2、G- O:\121\121929.DOC -118 - 200808833 CSF、Meg:CSF ' GM_CSF、血小板生成素、幹細胞因子和 紅血球生成素。其他的組合物可包括已知的血管生成素， 例如—-^、-^，及/或人類的類八叩多肤’及/或 血管内皮生長因子（VEGF)。生長因子包括促企管生長辛 ⑽giogenin)、骨形態發生蛋白七骨形態發生蛋白、骨 形態發生蛋白·3、骨形態發生蛋白-4、骨形態發生蛋白_5 月形’ϋ生蛋白-6、骨形態發生蛋白_7、骨形態發生蛋 白_8、骨形II發生蛋白_9、㈣態發生蛋白.iq、骨形態發 生蛋白-11、骨形態發生蛋白_12、骨形態發生蛋白…、骨 形態發生蛋白-14、骨形態發生蛋白·15、骨形態發生蛋白 受體IA、骨形態發生蛋白受體IB、腦衍生之神經營養因子 、纖毛的嗜中性因+、纖毛的嗜中性因子受體、細胞激動 素誘導的曰中j·生白血球趨化因子^、細胞激動素-誘導的嗜 中性白血球趨化因+ ?^ 千2細胞激動素-誘導的嗜中性白血球 趨化因子2、内皮細胞生長因子、内皮肽（end〇theHn)卜 上皮生長因子、上念：A, 皮_何生的嗜中性白血球吸引劑、纖維 母胞生長因子4、纖維母細胞生長因子5、纖維母細胞生 長因子6、纖維母細胞生長因子7、纖維母細胞生長因子8 、纖維母細胞生長因^ 、義維母細胞生長因子8 c、纖維 母細胞生長因子O:\121\121929.DOC -114- 200808833 For any compound, you can start with the word 'study' in the soil, or establish an animal model such as: atmosphere, rabbit, dog, pig or monkey. In the medium of nfm 4 ° animal model can also be used to determine the appropriate spoon / Chen dry circumference and route of administration. The dose and route of administration that are useful in the clerk can then be determined from the k-type information. The precise dose will be determined from the factors associated with the individual in need of treatment. The dosage and administration are adjusted to provide a sufficient level of active compound, Or maintain the desired effect. Factors to consider include the severity of the disease state, the general health of the individual, the age, weight and sex of the individual, the time and frequency of administration, the combination of drugs, the sensitivity of the response, and Reaction to treatment. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, weekly or biweekly, depending on the half-life and clearance rate of the particular formulation. The frequency of administration will depend on the formulation. Depending on the pharmacokinetic parameters of the binding agent molecule used, the composition is usually administered until the dose at which the desired effect is achieved is achieved. Thus, it can be administered in a single dose or in multiple doses (with the same or different) over a period of time. Concentration/dose), or administration of the composition as a continuous infusion. Routinely discuss the appropriate dosage in more detail. - Reaction data to probe the appropriate dose. The pharmaceutical composition is administered according to known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (intrinsic), intraventricular, intramuscular, intraocular , intraarterial, intraportal, path within the lesion, intraspinal, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal or rectal Way, by means of a sustained release system, or by implanting a device. Where desired, by means of a group O:\121\121929.DOC -115- 200808833 block main or continuous infusion, or by implantation The device is administered with the composition. Additionally or additionally, the composition may also be administered topically by implanting a membrane, sponge, or other suitable substance in which the desired molecule has been adsorbed or encapsulated. Where the device can be implanted in any appropriate tissue or device, and the desired molecule can be delivered via diffusion, on-time release of the mass, or continuous administration. In some cases, it may be desirable To be in a way that is in vitro, In a pharmaceutical composition, in such an instance, the cells, tissues or organs that have been removed from the patient are exposed to the pharmaceutical composition, and the cells, tissues and/or organs are subsequently transplanted back into the patient. The binding agents of the invention, such as peptibodies, can be delivered by implanting certain cells that have been genetically engineered to express and secrete the polypeptide, such as those described herein. The cytocell can be an animal or human cell and can be autologous, xenogeneic or xenogeneic. The cell can be immortalized as needed. To reduce the change in immunological response, the cells can be encapsulated to avoid surrounding tissue. The infiltrated material is usually a biocompatible, semi-permeable polymeric encapsulant or membrane that allows the release of the protein product, but prevents the cell from being affected by the patient's immune system, or by others. Harmful damage to surrounding tissues. Mixed Therapy The specific binding agents of the present invention, such as peptibodies, can be used with other therapeutic agents for treating diseases associated with Ang-2 expression. These other therapeutic agents include, for example, radiation therapy, chemotherapy, and targeted therapy such as HerceptinTM, RituxanTM, GleevecTM, and the like. Other mixed treatments are not specifically recited herein in O:\121\121929.DOC-116-200808833 and are also within the scope of the present invention. Chemotherapy may use anti-caries biological agents including, for example, alkylating agents 'including nitrogen mustards such as mechlorethamine, cyclosporin, ifosfamide, melphalan And chlorambucil; succinyl urea, such as sulphonic acid mustard (BCNU), lomustine (CCNU), and semustine (sulfhydryl-CCNU) Aziridine/methyl melamine, such as tri-ethyl melamine (TEM), tri-ethyl thioacetate (ptethiopa), hexamethyl melamine (HMM, six Acrylamine); alkyl sulfonate, like busulfan; three tillage, such as dacarbazine (DTIC); antimetabolite, including folic acid analogs, such as amines Trimetrexate, a pyrimidine analog, such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, arabinose (AraC, cytosine), 5- Nitrosine, 2,2'-difluorodeoxycytidine, purine analogs, such as 6-mercaptopurine, 6-thioguanine, nitroguanidine, 2'-deoxygenation Phenomycin (pentostatin), erythrohydroxynonyladenine (EHNA), Fludarabine phosphate, and 2-chlorodeoxy agglutination Cladribine), 2-CdA); natural products, including anti-mitotic drugs, such as paclitaxel, vinca alkaloids, including vinblastine (VbB), Changchun new test and vinorelbine, surname cancer Easy, estramustine and estramustine phosphate; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, like actinomycin D , daunorubicin (red ratio O: \121 \121929.DOC -117- 200808833 mycin), doxorubicin, mitoxantrone, idarubicin, borschine, puka Plexamycin (mitomycin), mitomycin C and actinomycin; enzymes, such as L-aspartate; biological response modifiers, such as interferon-a, IL-2, G-CSF and GM-CSF; miscellaneous preparations, including fitting dislocation complexes, such as cisplatin and carboplatin Anthraquinones, such as mitoxantine, substituted ureas, such as hydroxyurea, methyl hydrazine derivatives, including N-methyl hydrazine (MIH) and procarbazine, adrenal cortical inhibitors , such as mitotane (o, p'-DDD) and aminoglutethimide; hormones and antagonists, including adrenal corticosteroid antagonists, such as dehydrocortisone and equivalents, Dexamethasone and aminoglutethimide; corpus luteum preparations, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogen, like 浠Diethylstilbestrol and ethinyl estradiol equivalent; antiestrogens, such as tamoxifen; androgens, including sterolone propionate and fluoxymesterone/equivalent; Antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and anti-androgens of non-steroids, such as flutamide. A combination of growth factors, including cytokinin, lymphokine, growth factors, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL- 4. IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-O: \121\121929.DOC-118 - 200808833 CSF, Meg: CSF 'GM_CSF, thrombopoietin, stem cell factor and erythropoietin. Other compositions may include known angiopoietins, such as -^, -^, and/or human scorpion-like polypeptides and/or vascular endothelial growth factor (VEGF). Growth factors include angiogenesis (10) giogenin), bone morphogenetic protein seven bone morphogenetic proteins, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein _5 moon-shaped axillin-6, bone Morphogenetic protein _7, bone morphogenetic protein _8, bone morphogenetic protein _9, (four) morphogenetic protein. iq, bone morphogenetic protein-11, bone morphogenetic protein -12, bone morphogenetic protein..., bone morphology Protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, neutrophilic factor of cilia +, neutrophilic factor of cilia Somatic, cytokinin-induced sputum j. white blood cell chemotactic factor, cytokinin-induced neutrophil chemotaxis + ^ 2 cytokinin-induced neutrophil chemokine 2 , endothelial cell growth factor, endothelin (end〇theHn) epithelial growth factor, upper reading: A, _hesheng neutrophil leukorrhea attractor, fibroblast growth factor 4, fibroblast growth factor 5, fiber Maternal growth factor 6, blastoma growth factor 7, fibroblast growth factor 8, fibroblast growth factor, virgin cell growth factor 8 c, fibroblast growth factor
纖維母細胞生長因子丨〇、纖維母細胞 生長因子酸性的'纖維母細胞生長因子驗性的、神經膠質 細胞株·何生之嗜中性因子受體」、神經谬質細胞株-街生 之嗜中性因子受體-2、與生長有關的蛋白質、與 的蛋白質-卜與生長有關的蛋白質_2、與生長有關的JFibroblast growth factor 丨〇, fibroblast growth factor acidic 'fibroblast growth factor test, glial cell line · Hesheng neutrophil receptor', sputum cell line - street birth Neutrophil receptor-2, growth-related protein, protein-related growth-related protein-2, growth-related J
O:\121\121929.DOC -119- 200808833 f3、肝素結合之上皮生長因子、肝細胞生長因子、肝細 :生長因子受冑、類胰島素生長因子I、類胰島素生長因 子：體、類胰島素生長因子„、類胰島素生長因子結合蛋 貝角化細胞生長因子、白血病抑制因子、白血病抑制 因子受體-卜神經生長因子、神經生長因子受體、神經營 養因子小神經營養因子_4、胎盤生長因+、胎盤生長因 子2、血小板衍生之内皮細胞生長因+、血小板衍生之生 長因子、血小板衍生之生長因子Α鏈、血小板衍生之生長 口子AA血小板衍生之生長因子ab、血小板衍生之生長 口子B鏈A小板衍生之生長因子BB、血小板衍生之生長 因子文體-1、血小板衍生之生長因子受體·2、前_B細胞生 長刺激因子、幹細胞因子、幹細胞因子受體、轉化生長因 子-1、轉化生長因子_2、轉化生長因子_丨、轉化生長因子_ 1.2、轉化生長因子_2、轉化生長因子_3、轉化生長因子巧 、潛伏轉化生長因子-1、轉化生長因子-1結合蛋白質I、轉 化生長因子_1結合蛋白質π、轉化生長因子_丨結合蛋白質 III、腫瘤壞死因子受體第、腫瘤壞死因子受體第π型、 尿激酶-型血纖維蛋白溶酶原激活劑受體、血管内皮生長 因子’以及飯合型蛋白質，及其在生物學或免疫學上具有 活性的片段。 免疫治療 免疫治療通常依賴瞄準並破壞癌細胞之免疫效應物細胞 和为子的使用。免疫效應物可以是，例如本發明之肽體， 其認得在標靶細胞表面上的一些標記。肽體可單獨作為治O:\121\121929.DOC -119- 200808833 f3, heparin-binding epithelial growth factor, hepatocyte growth factor, liver fine: growth factor receptor, insulin-like growth factor I, insulin-like growth factor: body, insulin-like growth Factor „, insulin-like growth factor binding to egg keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor-b nerve growth factor, nerve growth factor receptor, neurotrophic factor small neurotrophic factor _4, placental growth factor +, placental growth factor 2, platelet-derived endothelial cell growth factor +, platelet-derived growth factor, platelet-derived growth factor Α chain, platelet-derived growth mouth AA platelet-derived growth factor ab, platelet-derived growth port B chain A small plate derived growth factor BB, platelet-derived growth factor strepto-1, platelet-derived growth factor receptor 2. pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor-1, Transforming growth factor-2, transforming growth factor_丨, transforming growth factor _ 1.2, transforming growth factor _2, transforming growth factor _3, transforming growth factor, latent transforming growth factor-1, transforming growth factor-1 binding protein I, transforming growth factor _1 binding protein π, transforming growth factor 丨 丨 binding protein III, tumor necrosis factor Receptor, tumor necrosis factor receptor type π, urokinase-type plasminogen activator receptor, vascular endothelial growth factor', and rice-type protein, and their biological or immunological activity Fragments. Immunotherapy Immunotherapy typically relies on the use of immune effector cells and targets for targeting and destroying cancer cells. The immune effector can be, for example, a peptibody of the invention that recognizes some of the markers on the surface of the target cells. Body alone
O:\121\121929.DOC -120- 200808833 療的效應⑯，或$可徵募其他實際完成殺死細㈣細胞。 亦可使肽體與藥物或毒素(化學治療劑、放射性核素、蓖 麻蛋白Α鏈、雈亂弧菌毒素、百日咳毒素等等）結合，而因 此主要擔任瞄準劑。 根據本發明’可藉著免疫治療，以本發明之肽體或肽體 共軛物瞄準Ang-2的突變形式。特別期待在連同Ang_2瞄準 治療的混合治療方法中，使用本發明的肽體組合物。 已Ie實被動免疫治療，對許多癌症是特別有效的。參 見，例如 WO 98/39027。 下列的實例僅為了解釋之目的，不應以任何方式解釋為 限制本發明之範圍。 實例1 在病理學和正常組織中的Ang_2表現 使用就地雜交作用，在正常和病理學組織中檢查八％_2 的表現。藉著逆轉錄酶-PCR，從人類或老鼠胎兒肺臟 cDNA中，擴大人類（Genbank登錄編號·· AF〇〇4327，核芸 酸1274- 1726)和老鼠（Genbank登錄編號：AF〇〇4326，核甞 酸1 135-1588) Ang-2序列的片段，選殖到pGEM_T質體内， 並藉著定序證實。從直線化的質體模板中，使用33ρ_υτρ 和RNA聚合扭，轉錄33Ρ-標示之反義RNA探針。將曱醛固 定、石蠟包埋的組織塊切成5微米，並收集在帶電的玻片 上。在就地雜交之前，先使〇·2Μ 11(：1透過組織，接著以蛋 白酶κ消化，並利用三乙醇胺和乙酸酐將其乙醯化。在55 1下，使切片與放射性標示的探針雜交過夜，然後接受 O:\121\121929.DOC -121 - 200808833 RNA酶消化，以及在55°C下，在大約〇·1 X SSC中的高嚴 格沖洗。將玻片浸在Kodak NTB2乳劑中，在4°C下暴露2-3 週，顯影並對比染色。以暗場和標準照明檢查切片，容許 同時評估組織形態學和雜交信號。 結果指出在正常的出生後人類中，Ang-2的表現受限於 含有血管生成之血管分布的少數組織，像是卵巢、胎盤和 子宮。在正常成人的心臟、腦、腎臟、肝臟、肺臟、胰臟 、脾臟、肌肉、扁桃腺、胸腺、闌尾、淋巴結、膽囊、前 列腺或睪丸中，沒有可檢測到的Ang_2表現。在5•週齡的 老鼠中（但在成年猴子或人類中沒有），腎臟在直管中展現 出顯著的Ang-2表現。欲判定該表現是否為胚胎發育的遺 物，在何生自年齡範圍高達丨歲大之老鼠的腎臟上，使用 老鼠Ang_2探針和上述的條件，重覆該表現。觀察到八叫_2 的表現’在新生兒發育的期間降低，但在續大老鼠的腎 臟中仍然是明顯的。 亦在幾乎所有的受試腫瘤類型中’檢測AM。表現，包 括原發性人類腫瘤，像是結腸癌(5個案例)、乳癌(10個案 例)、肺癌(8個案例)、神經膠質母細胞瘤(ι個案例)、轉移 :人類腫瘤，像是乳癌(2個案例)、肺癌㈣案例 癌(2個案例）’其已經轉移 果 a Λ及囑齒類腫瘤模式，像 疋6 (大"神經膠質瘤)、⑽(人類結腸癌)、c〇1〇_2 人類結腸癌）、HCΤ Π 6 f Λ 从 6 (人類結腸癌）、Α431 (人類表㈣ 癌）、Α673 (人類橫紋肌肉瘤 7 PC 3 f人^ 』屬）HT1_ (人類纖維肉瘤）、 PC-3 (人頰前列腺癌）、m6Fi〇 (老鼠黑色素瘤）、MethA (O:\121\121929.DOC-120- 200808833 The effect of treatment 16, or $ can be recruited to actually kill the fine (four) cells. It is also possible to combine a peptide with a drug or a toxin (a chemotherapeutic agent, a radionuclide, a ricin chain, a Vibrio cholera toxin, a pertussis toxin, etc.), and thus mainly serves as a targeting agent. According to the present invention, a mutant form of Ang-2 can be targeted by the peptibody or peptidomimetic conjugate of the present invention by immunotherapy. It is particularly desirable to use the peptidomimetic compositions of the present invention in a combination therapy with Ang 2 targeting therapy. Ie has been passively immunotherapy and is particularly effective against many cancers. See, for example, WO 98/39027. The following examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. Example 1 Ang 2 expression in pathology and normal tissues The in situ hybridization was used to examine the performance of 8% in normal and pathological tissues. Humans (Genbank accession number·· AF〇〇4327, nuclear acid 1274- 1726) and mice (Genbank accession number: AF〇〇4326, nuclear) were amplified from human or mouse fetal lung cDNA by reverse transcriptase-PCR. A fragment of the Ang-2 sequence of tannic acid 1 135-1588 was cloned into the pGEM_T plastid and confirmed by sequencing. From the linear plastid template, the 33Ρ-labeled antisense RNA probe was transcribed using 33ρ_υτρ and RNA polymerase. The furfural-fixed, paraffin-embedded tissue block was cut into 5 microns and collected on a charged slide. Prior to in situ hybridization, 〇·2Μ 11 (:1 was permeated through tissue, then digested with protease κ, and acetylated with triethanolamine and acetic anhydride. At 55 1 , sections were probed with radiolabeled probes. Hybrid overnight, then subjected to O:\121\121929.DOC-121 - 200808833 RNase digestion, and high stringency wash in approximately 〇·1 X SSC at 55 ° C. Soak the slide in Kodak NTB2 emulsion Exposure to 2-3 weeks at 4 ° C, development and contrast staining. Examination of sections with dark field and standard illumination allowed simultaneous assessment of histomorphology and hybridization signals. The results indicated that in normal postnatal humans, Ang-2 Performance is limited to a small number of tissues containing vascular blood vessels, such as the ovary, placenta, and uterus. In normal adult heart, brain, kidney, liver, lung, pancreas, spleen, muscle, tonsils, thymus, appendix, There was no detectable Ang-2 expression in lymph nodes, gallbladder, prostate or testicles. In 5 weeks old mice (but not in adult monkeys or humans), the kidneys exhibited significant Ang-2 performance in straight tubes. Desire Whether the performance is a relic of embryonic development, in the kidney of the mouse of the age range up to the age of the mouse, using the mouse Ang_2 probe and the above conditions, repeat the performance. Observed the performance of 八叫_2' in the newborn The period of development of the child is reduced, but it is still evident in the kidneys of the reintroduced mice. It also detects AM in almost all types of tumors tested, including primary human tumors, such as colon cancer (5 cases) ), breast cancer (10 cases), lung cancer (8 cases), glioblastoma (1 case), metastasis: human tumors, such as breast cancer (2 cases), lung cancer (4) case cancer (2 cases) 'It has transferred fruit a Λ and caries tumor models like 疋6 (large " glioma), (10) (human colon cancer), c〇1〇_2 human colon cancer), HCΤ Π 6 f Λ from 6 (human colon cancer), Α 431 (human (4) cancer), Α 673 (human rhabdomyosarcoma 7 PC 3 f human ^ genus) HT1_ (human fibrosarcoma), PC-3 (human buccal prostate cancer), m6Fi 〇 (mouse) Melanoma), MethA (
〇：\121\121929.DOC -122- 200808833 老鼠肉瘤）和路易斯（Lewis)肺癌。此外，亦在反應VEGF而 正生長至Matrigel塞子内的新血管中，並在早熟之視網膜 病的老鼠低氧症模式中，檢測Ang-2表現。 實例2 評估Ang_2肽體的分子測定 發展分子測定（親和力ELISA，中和ELISA和BIAcore)來 評估直接與Ang-2和相關之家族成員結合的肽體，以及肽 體對Ang-2 : Tie-2交互作用的影響。描述這些在活體外的 測定，如下。〇: \121\121929.DOC -122- 200808833 mouse sarcoma) and Lewis lung cancer. In addition, Ang-2 expression was also detected in the neovascularization in the Matrigel plug in response to VEGF and in the hypoxic model of precocious retinopathy in mice. Example 2 Evaluation of Molecular Assays for Ang_2 Peptide Development Molecular assays (affinity ELISA, Neutralization ELISA and BIAcore) to evaluate peptibodies directly binding to Ang-2 and related family members, and peptibodies against Ang-2: Tie-2 The impact of interaction. Describe these in vitro assays as follows.
親和力ELISA 關於候選之抗-Ang-2肽體的最初篩選，使用經過純化的 人類 Ang-2 (R&D Systems，Inc·;目錄第 623-AN號；以 2種 截短版本之混合物提供的ANg-2)，或老鼠Ang-2多肽（按照 上述製備）。為了證實性的結合測定，以全長人類Ang-2 DNA轉移感染獲自人類293T細胞之調理培養基的人類Ang-2，並在不含血清、含有大約50微克/毫升牛血清白蛋白 (BSA)的杜貝可氏（Dulbecco*s)經過修改的鷹式（Eagle)培養 基（DMEM)中培養。 使用微量滴定盤，在每孔中加入大約100微升的Ang-2， 並培養該盤大約2小時，隨後以含有大約0.1%吐溫-20的磷 酸缓衝之生理鹽水（PBS)沖洗培養盤四次。然後使用大約 250微升/孔的大約5%在PBS中之BSA阻斷各孔，並在室溫 下培養該盤大約2小時。在培養之後，拋棄過量的阻斷溶 液，並以從大約40毫微莫耳之濃度開始，然後以含有大約 O:\121\121929.DOC -123- 200808833 1% BSA之PBS連續稀釋4-倍的連續稀釋，在孔中加入大約 100微升的每種候選的抗-Ang_2肽體。然後在室溫下培養 δ亥盤過仪。在培養之後’以含有大約1 %吐溫_2〇之pBs沖 洗該盤。重覆沖洗四次，隨後以1〇〇微升/孔，加入預先按 照1 · 5000，以含有1% BSA之PBS稀釋的山羊抗人類Affinity ELISA For the initial screening of candidate anti-Ang-2 peptibodies, purified human Ang-2 (R&D Systems, Inc.; catalogue 623-AN; provided as a mixture of 2 truncated versions) ANg-2), or mouse Ang-2 polypeptide (prepared as described above). For confirmatory binding assays, human Ang-2 obtained from human 293T cell conditioned medium was transfected with full length human Ang-2 DNA and contained no serum, containing approximately 50 μg/ml bovine serum albumin (BSA). Dulbecco*s is cultured in modified Eagle medium (DMEM). Using a microtiter plate, add approximately 100 microliters of Ang-2 to each well and incubate the plate for approximately 2 hours, then rinse the plate with phosphate buffered saline (PBS) containing approximately 0.1% Tween-20. Four times. Each well was then blocked using approximately 250 liters/well of approximately 5% BSA in PBS and the plate was incubated for approximately 2 hours at room temperature. After incubation, the excess blocking solution was discarded and started at a concentration of approximately 40 nanomoles, and then serially diluted 4-fold with PBS containing approximately O:\121\121929.DOC-123-200808833 1% BSA. Serial dilutions were made by adding approximately 100 microliters of each candidate anti-Ang 2 peptibody to the well. The δ hai plate was then incubated at room temperature. After the incubation, the disk was washed with pBs containing about 1% Tween 2 Torr. Repeat the rinsing four times, then add 1 5000 5,000 liters of goat anti-human diluted with 1% BSA in PBS at 1 〇〇 microliter/well.
IgG(Fc)- HRP (Pierce Chemical Co.，目錄#31416)。在室 溫下培養该盤大約1小時。然後以含有大約〇·丨％吐溫_2〇的 PBS沖洗5次，隨後加入大約1〇〇微升/孔的tmb (3,3,5,5，-四 曱聯苯胺液體受質系統；Sigma Chemical Company，St. Louis，MO，目錄第T8665號）受質，並培養大約5_15分鐘， 直到藍色展現為止。然後在分光光度計大約37〇毫微米處 ，讀取吸光度。IgG (Fc) - HRP (Pierce Chemical Co., catalog #31416). The plate was incubated at room temperature for about 1 hour. Then rinsed 5 times with PBS containing approximately 〇·丨% Tween_2〇, followed by addition of approximately 1 μL/well of tmb (3,3,5,5,-tetrazine benzidine liquid receptor system; Sigma Chemical Company, St. Louis, MO, Catalog No. T8665) was incubated and cultured for approximately 5-15 minutes until blue appeared. The absorbance is then read at a spectrophotometer of approximately 37 Å nm.
中和ELISA 按照親和力ELIS A的描述，製備已經結合人類Ang-2多 肽的微量滴定盤。滴定候選的抗_Ang_2肽體，從1〇〇〇 nM 到〇·2 pM，以含有大約1% BSA和大約1 nM Tie-2 (以Tie-2-Fc分子之形式提供，其中Tie-2部分僅含有分子的可溶性細 胞外部分；R&D Systems，目錄第313-TI號）的PBS溶液4-倍稀釋。在每孔中加入1〇〇微升抗體/Tie-2溶液之後，在室 溫下培養該盤過夜，然後以含有大約〇.ρ/。吐溫-2〇的PBs沖 洗5次。在沖洗之後，加入大約1〇〇微升/孔的抗-Tie_2抗體 (Pharmingen Inc·，S錄#557039)，至大約1微克/毫升的終 濃度，並在室溫下培養該盤大約1小時。接下來，以利用 含有大約1% BSA之PBS的1:10,000稀釋，加入大約100微 O:\121\121929.DOC -124- 200808833 升/孔的山羊抗-老鼠-IgG-HRP (Pierce Chemical Co·，目錄 #31432)。在室溫下培養該盤大約1小時，隨後以含有大約 0. 1%吐溫-20的PBS沖洗5次。然後加入大約100微升/孔的 TMB受質（上述），並容許顏色展現。然後在分光光度計 370毫微米處，讀取吸光度。 親和力BIAcore 在 BIAcore@ 2000 (Biacore，Inc·，Piscataway，NJ)上進行 每個候選Ang-2肽體的親和力分析，利用PBS和0.005% P20 表面活性劑（Biacore，Inc.)作為執行緩衝溶液。將重組的蛋 白質G (Repligen，Needham，MA)，經由一級胺基團，使用 胺偶聯套組（Biacore，Inc.)，根據製造者建議的草案，固定 在研究等級的CM5感應晶片（Biacore，Inc.)上。 藉著首先捕捉每種候選的抗-Ang-2肽體大約100 Ru，至 已經固定之蛋白質G上，隨後以50微升/分鐘之流速，將各 種濃度（〇-1 〇〇 nM)的huAng-2或mAng-2注射至已結合的抗 體表面上3分鐘，來進行結合測定。使用BIA估計3.1電腦 程式（Biacore，Inc.)判定肽體結合的動力學參數，包括ka( 締合速率常數）、kd (解離速率常數）和KD (解離平衡常數） 。解離平衡常數越低，表示肽體對Ang-2的親和力越大。 實例3Neutralization ELISA A microtiter plate that has been bound to the human Ang-2 polypeptide is prepared as described by affinity ELIS A. Titrate candidate anti-Ang_2 peptibodies from 1〇〇〇nM to 〇·2 pM to contain approximately 1% BSA and approximately 1 nM Tie-2 (provided as Tie-2-Fc molecule, where Tie-2 Part of the soluble extracellular fraction containing only the molecule; R&D Systems, catalog No. 313-TI) was diluted 4-fold in PBS. After adding 1 μL of the antibody/Tie-2 solution to each well, the plate was incubated at room temperature overnight, and then contained approximately 〇.ρ/. Tween-2〇 PBs were washed 5 times. After rinsing, approximately 1 μL/well of anti-Tie 2 antibody (Pharmingen Inc., S. #557039) was added to a final concentration of approximately 1 μg/ml and the plate was incubated for approximately 1 hour at room temperature. . Next, to a 1:10,000 dilution with PBS containing approximately 1% BSA, add approximately 100 micro O: \121\121929.DOC -124 - 200808833 liters/well of goat anti-mouse-IgG-HRP (Pierce Chemical Co ·, directory #31432). The plate was incubated at room temperature for about 1 hour and then washed 5 times with PBS containing about 0.1% Tween-20. Approximately 100 microliters/well of TMB substrate (described above) was then added and color rendering was allowed. The absorbance was then read at a spectrophotometer of 370 nm. Affinity BIAcore Affinity analysis of each candidate Ang-2 peptibody was performed on BIAcore@2000 (Biacore, Inc., Piscataway, NJ) using PBS and 0.005% P20 surfactant (Biacore, Inc.) as the execution buffer solution. The recombinant protein G (Repligen, Needham, MA) was immobilized on a research grade CM5 sensor wafer (Biacore, via a primary amine group using an amine coupling kit (Biacore, Inc.) according to the manufacturer's proposed draft. Inc.). By first capturing approximately 100 Ru of each candidate anti-Ang-2 peptibody to the already immobilized protein G, then at various concentrations (〇-1 〇〇nM) of huAng at a flow rate of 50 μl/min. Binding assays were performed by injection of -2 or mAng-2 onto the surface of the bound antibody for 3 minutes. The BIAcore 3.1 computer program (Biacore, Inc.) was used to determine the kinetic parameters of peptidomimetic binding, including ka (association rate constant), kd (dissociation rate constant), and KD (dissociation equilibrium constant). The lower the dissociation equilibrium constant, the greater the affinity of the peptibody for Ang-2. Example 3
Ang-2結合肤的破認 1. 製備塗覆Ang-2的磁性小珠 A.將Ang-2固定在磁性小珠上 為了非-專一的洗脫，針對所有三個選擇回合，以每100 O:\121\121929.DOC -125- 200808833 微升得自製造者的小珠母液含大約4微克生物素基化之 Ang_2蛋白質的濃度，將生物素基化的Ang_2蛋白質（生物 素基化的重組人類血管生成素·2，R&D Systems，Ine ;目 錄第BT623號）固定在鏈黴菌抗生物素蛋白Dynabeads (Dynal，Lake Success，Νγ)上。為 了抗原（Ang_2)和受體 (Tie-2)的洗脫，針對兩個選擇回合，將2微克生物素基化 的Ang-2蛋白質固定在50微升鏈黴菌抗生物素蛋白 Dynabeads上。對於第三個選擇回合，將塗覆濃度降低至 每50微升小珠母液，含大約！微克的生物素基化之八叩_2蛋 白質。藉著使用磁鐵將小珠吸引至試管的一側，並吸移掉 液體，以磷酸緩衝之生理鹽水（PBS)沖洗5次，並再懸浮於 PBS中。以上文的濃度將生物素基化的Ang_2蛋白質加至 經過沖洗的小珠中，並在室溫下培養丨小時，並加以旋轉 接者在4 C下過仪培養數小時，並加以旋轉。然後藉著 加入BSA至大約1%之終濃度，阻斷Ang_2塗覆的小珠，並 在4 C下培養，並加以旋轉。然後在接受選擇程序之前， 先以PBS沖洗所得的Ang-2塗覆小珠5次。 B.製備陰性選擇用的小珠 亦為了陰性選擇製備額外的小珠。針對每個淘洗條件， 使500微升得自製造者的小珠母液接受以上的程序（段落 1A)，除了省略培養生物素基化之Ang_2的步驟。在最後的 沖洗步驟中，將小珠分成5個1 〇〇微升的等分。 2·選擇Ang_2結合嗤菌體 A.全面的策略 O:\121\121929.DOC -126- 200808833 使用三個絲狀噬菌體庫，命名為”TN8-IX”（5X109個獨立 的轉化物）、ΠΤΝ12-Γ (1.4Χ109個獨立的轉化物）和”直線” (2.3X109個獨立的轉化物）（全都得自Dyax Corp.)來選擇 Ang-2結合噬菌體。然後使每個庫接受非-專一的洗脫、 Ang-2洗脫和受體洗脫（Tie-2)。對於Ang-2，使用9種不同 的淘洗條件（使用非-專一之洗脫方法的TN8-IX，使用Ang-2洗脫方法的TN8-IX，使用Tie-2洗脫方法的TN8-IX，使用 非-專一之洗脫方法的TN12-I，使用Ang-2洗脫方法的 TN12_I，使用Tie-2洗脫方法的TN12-I，使用非-專一之洗 脫方法的直線，使用Ang-2洗脫方法的直線，和使用Tie-2 洗脫方法的直線）。對於全部的三個庫而言，僅以非-專一 的方式，洗脫得自第一回合選擇的噬菌體，進行下一回合 的選擇。在第二和第三回合的選擇中，使用Ang-2和Tie-2 洗脫。至於直線庫，僅進行第二回合Ang_2和Tie-2洗脫的 選擇。 B.陰性選擇 關於每種淘洗條件，從庫母液中，將TN8-IX和TN12-I庫 的大約100個隨機庫相等物（TN8_IX為大約5 X 10" pfu，而 TN12-I為大約ι·4 χ 1〇ii pfu以及直線庫的大約1〇個隨機庫 相等物（大約1 X l〇upfu)分成等分，並以pBST (帶有〇 〇5% 吐/夏-20的PBS)稀釋成大約4〇〇微升。在最後一次沖洗之後 ’從最初100微升等分的為了陰性選擇而製備的小珠（段落 iB)中吸出液體’在小珠中加入大約4〇〇微升經過稀釋的庫 母液。在室溫下培養所得的混合物大約1〇分鐘，並加以旋Breaking of Ang-2 Binding Skin 1. Preparation of Magnetic Beads Coated with Ang-2 A. Fixing Ang-2 on Magnetic Beads For non-specific elution, select rounds for all three to 100 per 100 O:\121\121929.DOC -125- 200808833 Microliters of the bead mother liquor from the manufacturer contains approximately 4 μg of biotinylated Ang 2 protein at a concentration of biotinylated Ang 2 protein (biotinylated) Recombinant Human Angiopoietin-2, R&D Systems, Ine; Catalog No. BT623) was immobilized on Streptomyces avidin Dynabeads (Dynal, Lake Success, Νγ). For the elution of antigen (Ang_2) and receptor (Tie-2), 2 μg of biotinylated Ang-2 protein was immobilized on 50 μl of streptavidin Dynabeads for two selection rounds. For the third selection round, reduce the coating concentration to 50 μl of bead mother liquor, containing approximately! Micrograms of biotinylated gossip 2 protein. The beads were attracted to one side of the tube by using a magnet, and the liquid was aspirated, washed 5 times with phosphate buffered saline (PBS), and resuspended in PBS. The biotinylated Ang2 protein was added to the washed beads at the above concentrations and incubated for a few hours at room temperature, and incubated at 4 C for several hours with a spinner and rotated. The Ang_2 coated beads were then blocked by the addition of BSA to a final concentration of approximately 1% and cultured at 4 C and spun. The resulting Ang-2 coated beads were then rinsed 5 times with PBS before accepting the selection procedure. B. Preparation of beads for negative selection Additional beads were also prepared for negative selection. For each panning condition, 500 microliters of the bead mother liquor obtained from the manufacturer was subjected to the above procedure (paragraph 1A) except that the step of culturing the biotinylated Ang_2 was omitted. In the final rinse step, the beads are divided into five 1 〇〇 microliter aliquots. 2. Select Ang_2 combined with bacillus A. Comprehensive strategy O:\121\121929.DOC -126- 200808833 Use three filamentous phage libraries, named "TN8-IX" (5X109 independent transformants), ΠΤΝ12 - Γ (1.4 Χ 109 independent transformants) and "straight line" (2.3 X 109 independent transformants) (all from Dyax Corp.) to select Ang-2 binding phage. Each library was then subjected to non-specific elution, Ang-2 elution, and receptor elution (Tie-2). For Ang-2, 9 different panning conditions were used (TN8-IX using non-specific elution method, TN8-IX using Ang-2 elution method, TN8-IX using Tie-2 elution method) TN12-I using non-specific elution method, TN12_I using Ang-2 elution method, TN12-I using Tie-2 elution method, straight line using non-specific elution method, using Ang- 2 straight line of elution method, and straight line using Tie-2 elution method). For all three pools, the phage selected from the first round was eluted only in a non-specific manner for the next round of selection. In the second and third rounds of selection, Ang-2 and Tie-2 were used for elution. As for the linear library, only the selection of the second round of Ang_2 and Tie-2 elution is performed. B. Negative selection For each panning condition, approximately 100 random pool equivalents of the TN8-IX and TN12-I pools from the stock solution (TN8_IX is approximately 5 X 10" pfu, while TN12-I is approximately ι · 4 χ 1〇ii pfu and approximately 1 random pool equivalent of the linear library (approximately 1 X l〇upfu) are divided into equal parts and diluted with pBST (PBS with 〇〇 5% spit / summer-20) Approximately 4 μL. After the last rinse, 'Aspirate liquid from the initial 100 μl aliquot of the beads prepared for negative selection (Section iB)' Add about 4 μL of the beads to the beads. Diluted stock solution. The resulting mixture is incubated at room temperature for about 1 minute and swirled.
O:\121\121929.DOC -127- 200808833 轉。使用磁鐵，吸出噬菌體上清液，並為了另外的陰性選 擇步驟，加至第二個100微升的等分中。這樣子，進行5個 陰性選擇步驟。 C.使用Ang-2蛋白質塗覆之小珠的選擇 在最後一次陰性選擇步驟（段落1B)之後，將噬菌體上清 液加至Ang-2塗覆的小珠（段落ία)中。在室溫下培養該混 合物1到2小時，並加以旋轉，容許噬菌體與標靶蛋白質結 合。在拋棄上清液之後，以PBST沖洗小珠大約1〇次，接 著以PBS沖洗2次。 D·非-專一的洗脫 在吸出最後的沖洗液體（段落2C)之後，在小珠中加入大 約 1 毫升的 Min A鹽溶液（60 mM Κ2ΗΡ04，33 mM KH2P〇4 ’ 7.6 mM (NH4)S04和1.7 mM檸檬酸鈉）。直接將該小珠混 合物加至濃縮的細菌試樣中進行感染（參見段落3 a和3 B)。 E.已結合之噬菌體的抗原（Ang_2)洗脫 關於第2回合，在最後的沖洗步驟（段落2C)之後，藉著 連續加入100微升1 ρΜ ’ 0·1 nM和10 nM重組的Ang-2蛋白 質（重組的人類血管生成素-2，R&D Systems，Inc. Minneapolis，Minnesota)，每種條件各培養3〇分鐘，從磁 性小珠中洗脫出已結合的噬菌體。剩下的噬菌體以非-專 一的方式洗脫（段落2D)。混合從1〇 nM和非-專一之洗脫中 洗脫出的嗤菌體’並使其接受第3回合的選擇（參見下文的 段落4)。 關於第3回合，在最後的沖洗步騾（段落2C)之後，藉著 O:\121\121929.DOC -128- 200808833 連續加入大約1 nM重組的Ang_2蛋白質和10 nM重組的 Ang-2蛋白質，每種條件各培養3〇分鐘，從磁性小珠中洗 脫出已結合的噬菌體。此外，在旋轉器上，利用i毫升i 〇〇 mM的三乙胺溶液（Sigma，St· L〇uis，Miss〇u⑴洗脫噬菌體 1〇分鐘。利用0.5毫升1 M THs_Hcl (ρΗ 7·5)中和含有噬菌 體之三乙胺溶液的pH值。在最後一次以1〇〇 mM三乙胺溶 液洗脫之後，藉著將小珠加至細菌中，洗脫出剩下的噬菌 體（段落2D)。 F·已結合之噬菌體的報告者（Tie_2)洗脫 關於第2回合’在最後的沖洗步驟（段落2C)之後，藉著 連績加入大約1〇〇微升i pM，〇1 nM和ίο nM重組的Tie-2 蛋白質（重組的人類Tie-2-Fc嵌合體，r&d Systems，Inc· Minneapolis，Minnesota)，每種條件各培養3〇分鐘，從磁 f生小珠中洗脫出已結合的嗟菌體。剩下的嗟菌體以非·專 的方式洗脫（段落2D)。混合從1 〇 nM和非-專一之洗脫中 洗脫出的噬菌體’並使其接受第3回合的選擇（參見下文的 段落4)。 關於第3回合，在最後的沖洗步驟（段落2C)之後，藉著 連續加入大約1 nM重組的Ang-2蛋白質和1〇 nM重組的Tie-2蛋白質，每種條件各培養3〇分鐘，從磁性小珠中洗脫出 已結合的噬菌體。此外，在旋轉器上，利用1毫升丨〇〇 mM 的二乙胺溶液（Sigma，St· Louis，Missouri)洗脫噬菌體10分 鐘。利用0·5毫升1 M Tris-HCl (pH 7.5)中和含有噬菌體之三 乙胺溶液的pH值。在最後一次以1〇〇 mM三乙胺溶液洗脫之 O:\121\121929.DOC -129- 200808833 後，藉著將小珠加至細菌中先 元贶出剩下的噬菌體（段落 2D)。 3·擴大作用 A.製備平舖的細胞 在含有大約12.5微克/毫升四環素的LB培養基中，使新 鮮的大腸桿菌（XL-1 Blue瓣，）培養物生長至大約〇5的 〇D0GG。針對每個淘洗條件，在冰上冷卻大約汕毫升的該 培養物，並離心之。將細菌小球再懸浮於大約丨毫升的Min A鹽溶液中。 B·轉導作用 將得自上述每個不同洗脫方法（段落2D、2£和217)的混合 物，分別加至濃縮的細菌試樣（段落3A)中，並在大約^ 下培養大約1 5分鐘。在每種混合物中加入大約2毫升的 NZCYM培養基（2XNZCYM ’ 50微克/毫升氨爷青黴素），並 在大約37°C下培養15分鐘。將所得的4毫升溶液平舖在含 有大約50微克/毫升氨芊青黴素的大NZCYM瓊脂板上，並 在37 °C下培養過夜。 C.收獲噬菌體 使每種細菌/噬菌體混合物在大的NZCYM瓊脂板上生長 過夜（段落3B)，隨後將其刮到大約35毫升的LB培養基中。 以另外3 5毫升的LB培養基，進一步清洗瓊脂板。離心所得 的在LB培養基中之細菌/噬菌體混合物，去掉細菌形成的 小球。然後將大約50毫升的噬菌體上清液移至新的試管中 ，並加入大約12.5毫升的PEG溶液（20% PEG8000，3.5M乙 O:\121\121929.DOC -130- 200808833 酸錢），在冰上培養2小時，使㈣體沉殿。將沉澱的㈣ 體離心’並再懸浮於6毫升㈣體再懸浮緩衝溶液（25〇碰 NaC1，HK)mMTrispH8，lmMEDTA)中。藉著離心去掉 剩：的細菌’進一步純化該嗟菌體溶液，並藉著加入大約 1.5宅升的PEG溶液’使噬菌體沉澱兩次。在離心步驟之後 ’將嗟菌體小球再懸浮於大约彻微升的咖中。使該溶液 Μ㈣的離4' ’除去任何殘餘細菌碎屬的溶液。使用標 準溶菌斑形成測定，滴定所得的噬菌體製品。 4.另外的選擇和擴大作用 在第2回合中，使用得自第1回合的擴大噬菌體製品(大 約l〇1G pfu)作為輸入噬菌體，來進行選擇和擴大步驟（段落 2和3)。至於Ang_2和Tie_2溶液，混合得自ι〇 和非專一 之洗脫的嗟菌體，並針對第3回合的選擇擴大。再轉而使 用得自第2回合的擴大噬菌體製品（大約1〇9pfu)作為輸入噬 菌體，進行第3回合的選擇和擴大（段落2和3)。在第3回合 的洗脫步驟（段落2D、2E和2F)之後，平舖洗脫噬菌體的小 溶離份，進行溶菌斑形成測定（段落3C)。挑選出個別的溶 菌斑，並放在每孔含有100微升TE緩衝溶液的96孔微量滴 定盤内。在4°C下培養這些主要培養盤過夜，容許噬菌體 洗脫到TE緩衝溶液内。 5·選殖分析 藉著嗤菌體ELIS A和DNA定序，分析噬菌體純種系。以 得自這兩個測定的綜合結果為基礎來排列序列。O:\121\121929.DOC -127- 200808833 Turn. The phage supernatant was aspirated using a magnet and added to the second 100 microliter aliquot for additional negative selection steps. In this way, 5 negative selection steps are performed. C. Selection of beads coated with Ang-2 protein After the last negative selection step (paragraph 1B), the phage supernatant was added to the Ang-2 coated beads (paragraph ία). The mixture was incubated for 1 to 2 hours at room temperature and spun to allow phage to bind to the target protein. After discarding the supernatant, the beads were rinsed with PBST approximately 1 time, followed by rinsing twice with PBS. D·Non-Specific Elution After aspirating the final rinse liquid (Section 2C), add approximately 1 mL of Min A salt solution (60 mM Κ2ΗΡ04, 33 mM KH2P〇4 ' 7.6 mM (NH4)S04 to the beads. And 1.7 mM sodium citrate). The bead mixture was added directly to a concentrated bacterial sample for infection (see paragraphs 3 a and 3 B). E. Adhered to the phage antigen (Ang_2) for the second round, after the final rinse step (paragraph 2C), by continuously adding 100 μl of 1 ρΜ '0·1 nM and 10 nM recombinant Ang- 2 Protein (recombinant human angiopoietin-2, R&D Systems, Inc. Minneapolis, Minnesota), each condition was cultured for 3 minutes, and the bound phage was eluted from the magnetic beads. The remaining phage eluted in a non-specific manner (paragraph 2D). Mix the sputum bacteria eluted from 1 〇 nM and non-specific elution and accept the selection of round 3 (see paragraph 4 below). For the third round, after the final rinse step (paragraph 2C), approximately 1 nM recombinant Ang 2 protein and 10 nM recombinant Ang-2 protein were continuously added by O:\121\121929.DOC-128-200808833, Each condition was cultured for 3 minutes each, and the bound phage was eluted from the magnetic beads. In addition, on a rotator, the phage was eluted with i ml of i mM mM triethylamine solution (Sigma, St. L〇uis, Miss〇u (1) for 1 min. Using 0.5 ml of 1 M THs_Hcl (ρΗ 7·5) Neutralize the pH of the phage-containing triethylamine solution. After the last elution with 1 mM mM triethylamine solution, the remaining phage is eluted by adding the beads to the bacteria (paragraph 2D). F. The reporter of the combined phage (Tie_2) elutes about the second round 'after the final rinsing step (paragraph 2C), by adding about 1 〇〇 microliter i pM, 〇 1 nM and ίο nM recombinant Tie-2 protein (recombinant human Tie-2-Fc chimera, r&d Systems, Inc. Minneapolis, Minnesota), each condition incubated for 3 min, eluted from magnetic f beads The combined sputum bacteria. The remaining sputum bacteria are eluted in a non-specific manner (paragraph 2D). Mix the phage eluted from 1 〇nM and non-specific elution and make it accept 3 rounds of choice (see paragraph 4 below). Regarding round 3, after the final rinse step (paragraph 2C), by About 1 nM of recombinant Ang-2 protein and 1〇nM recombinant Tie-2 protein were continuously added, and each condition was cultured for 3 minutes each, and the bound phage was eluted from the magnetic beads. Further, on the rotator The phage was eluted with 1 ml of mM mM diethylamine solution (Sigma, St. Louis, Missouri) for 10 minutes. The phage-containing triethylamine was neutralized with 0.5 mL of 1 M Tris-HCl (pH 7.5). The pH of the solution. After the last elution of O:\121\121929.DOC -129-200808833 with 1 mM mM triethylamine solution, the remaining ones were removed by adding the beads to the bacteria. Phage (Section 2D) 3. Expansion (A. Preparation of tiled cells) Fresh E. coli (XL-1 Blue flap) cultures were grown to approximately 〇5 in LB medium containing approximately 12.5 μg/ml tetracycline. 〇D0GG. For each panning condition, approximately 汕ml of this culture was cooled on ice and centrifuged. The bacterial pellet was resuspended in approximately 丨ml of Min A salt solution. B·Transduction will a mixture obtained from each of the different elution methods described above (paragraphs 2D, 2 and 217), Do not add to the concentrated bacterial sample (Section 3A) and incubate for approximately 15 minutes at approximately 2. Add about 2 ml of NZCYM medium (2XNZCYM '50 μg/ml ampicillin) to each mixture, and Incubate for 15 minutes at approximately 37° C. The resulting 4 ml solution was plated on large NZCYM agar plates containing approximately 50 μg/ml ampicillin and incubated overnight at 37 °C. C. Harvesting of phage Each bacterial/phage mixture was grown overnight on large NZCYM agar plates (Section 3B) and subsequently scraped into approximately 35 mL of LB medium. The agar plates were further washed with an additional 35 ml of LB medium. The resulting bacterial/phage mixture in LB medium was centrifuged to remove the pellet formed by the bacteria. Then transfer approximately 50 ml of the phage supernatant to a new tube and add approximately 12.5 ml of PEG solution (20% PEG 8000, 3.5 M E:\121\121929.DOC-130-200808833 acid), at Cultivate on ice for 2 hours to make the (four) body sink. The precipitated (tetra) was centrifuged' and resuspended in 6 ml of (four) body resuspension buffer (25 Na NaC1, HK) mM Tris pH 8, lmMEDTA). The remaining bacteria were removed by centrifugation 'further purification of the bacillus solution, and the phage was precipitated twice by adding about 1.5 liters of PEG solution. After the centrifugation step, the sputum pellets were resuspended in approximately a liter of coffee. A solution of any residual bacterial genus was removed from the 4'' of the solution 四(iv). The resulting phage preparation was titrated using a standard plaque assay. 4. Additional selection and expansion In the second round, the selection and expansion steps (paragraphs 2 and 3) were carried out using the expanded phage preparation (about lG gfu) obtained from the first round as the input phage. As for the Ang_2 and Tie_2 solutions, the mixed bacteria obtained from 〇 and non-specific were expanded and expanded for the selection of the third round. Further, the expanded phage product (about 1 〇 9 pfu) obtained from the second round was used as the input phage, and the selection and expansion of the third round were carried out (paragraphs 2 and 3). After the elution step of the third round (paragraphs 2D, 2E and 2F), the small soluble fraction of the eluted phage was tiled and assayed for plaque formation (paragraph 3C). Individual plaques were selected and placed in 96-well microtiter plates containing 100 microliters of TE buffer solution per well. These primary plates were incubated overnight at 4 °C, allowing the phage to elute into the TE buffer solution. 5. Selection analysis The phage pure lineage was analyzed by sputum cell ELIS A and DNA sequencing. The sequences are arranged based on the combined results obtained from these two assays.
A.噬菌體ELISA O:\121\121929.DOC -131 - 200808833 使XL-l Blue MRF’的培養物生長，直到〇〇6()()達到大約 0·5為止在96-孔微夏滴定盤的各孔中，將該培養物分成 大約30微升的等分。在各孔中加入大約10微升的洗脫噬菌 體（段落4)，並容許在室溫下感染細菌大約15分鐘。在各孔 中分別加入大約1〇〇微升，含有大約12·5微克/毫升四環素 和大約50微克/毫升氨苄青黴素的[Β培養基。然後在大約 3 7 C下’振盈培養微量滴定盤過夜。在大約4它下，容許 重組的Ang-2蛋白質（大約1微克/毫升，在pBS中）與96孔A. Phage ELISA O: \121\121929.DOC -131 - 200808833 The culture of XL-l Blue MRF' is grown until 〇〇6()() reaches approximately 0.5·5 in a 96-well micro-sodium titration tray In each well, the culture was divided into approximately 25 microliter aliquots. Approximately 10 microliters of eluted phage (paragraph 4) was added to each well and allowed to infect the bacteria for approximately 15 minutes at room temperature. About 1 μL of microliters, containing approximately 12·5 μg/ml tetracycline and approximately 50 μg/ml ampicillin were added to each well. The microtiter plate was then grown overnight at approximately 3 7 C. Under about 4, allow recombinant Ang-2 protein (approximately 1 μg/ml in pBS) with 96 wells
Maxisorp培養盤（NUNC)結合過夜。以在pbs中大約2微克/ 毫升’將鏈黴菌抗生物素蛋白塗覆在另一個Maxisorp盤上 ，作為對照組。 在第二天時，拋棄在塗覆蛋白質之Maxisorp盤中的液體 ’並在大約4°C下，利用大約300微升的5%牛奶溶液過夜 阻斷每個孔（或者在室溫下i小時）。然後拋棄牛奶溶液，並 以PBST溶液沖洗各孔3次。在最後的沖洗步驟之後，在塗 覆蛋白質之Maxisorp盤的各孔中，分別加入大約50微升 PBST-4%牛奶。將大約50微升、得自在96孔微量滴定盤中 之各孔的過夜培養物移至Ang-2塗覆之培養盤，和對照組 塗覆鏈黴菌抗生物素蛋白之培養盤的對應孔中。在室溫下 ，培養在每種培養盤中的1〇〇微升混合物大約i小時。拋棄 得自Maxisorp盤的液體，並以PBST沖洗各孔大約3次。將 HRP-共幸厄的抗 _M13 抗體（Amersham Pharmacia Biotech)稀 釋成大約1:7,500，並將大約100微升的稀釋溶液加至 Maxisorp盤的各孔中，在室溫下培養大約1小時。再度拋 O:\121\121929.DOC -132- 200808833 棄液體，並以PBST沖洗各孔大約5次。然後將大約100微 升的TMB受質（Sigma)加至各孔中，並利用大約50微升的 5N H2S04溶液中止該反應。在分光光度計（Molecular Devices)上讀取 OD45〇。 B.噬菌體純種系的定序 針對每個噬菌體純種系，使用PCR製備定序模板。使用 下列的寡核甞酸對來擴大大約500個核甞酸的片段。 引子1 : S’-CGGCGCAACTATCGGTATCAAGCTGG* (序 列識別：54號） 弓1 子2 : 5，-CATGTACCGTAACACTGAGTTTCGTC-3,（序 列識別：55號） 為每個純種系製備下列的混合物： 試劑 體積(微升)/試管 dH20 26.25 50%甘油 10 10 X PCR緩衝溶液(無MgCl2) 5 25 mM MgCl2 4 10 mM dNTP混合物 1 ΙΟΟμΜ引子1 0.25 100 μΜ引子2 0.25 Taq聚合酶 0.25 在TE中之噬菌體(段落4) 3 終反應體積 50 關於 PCR ’ 使用熱循環器（GeneAmp PCR System 9700, Applied Biosystems)執行下列的程式：94°C 5分鐘；（94°C 30秒，55°C 30秒，72°C 45秒）x 30個回合；72°C 7分鐘； 冷卻至 4°C。使用 QIAquick Multiwell PCR 純化套組（Qiagen) O:\121\121929.DOC -133- 200808833 ，依據製造者的草案，純化得自每個反應的PCR產物。然 後藉著使PCR反應混合物各大约10微升，與1微升染料（10 X BBXS瓊脂糖裝載染料）跑1%瓊脂糖凝膠，分析經過純化 的PCR產物。然後使用ABI 377定序器（Perkin Elmer)，依 據製造者建議的草案，定序剩下的產物。 6.序列排列和一致序列的判定 A. 序列排列與分析 將從可變核甞酸序列（段落5B)中轉譯的肽序列，與 ELISA資料連貫。給與在塗覆Ang-2之孔中顯示出高OD450 值，且在塗覆鏈黴菌抗生物素蛋白之孔中顯示出低od45〇 值的純種系，較優先的排列。亦給與出現多次的序列較優 先的排列。以這些標準為基礎，為了進一步為肽或肽體的 分析，選擇候選的序列。 B. —致序列的判定 從TN8-IX庫中產製三個不同種類的一致主題，如下·· KRPCEEXWGGCXYX (序列識別:56號） KRPCEEXFGGCXYX (序列識別·· 57號) XXXCXDXYWYCXXX (序列識別:61 號） XXXCXDXYTYCXXX (序列識別:62號） XXXCXDXFWYCXXX (序列識別:63號） XXXCXDXFTYCXXX (序列識別：64號） XXXCX^E^XC^XM* (序列識別·· 58號） 從TN12-I庫產製一個一致主題： WSXCAWFXGXXXXXCRRX (序列識別·· 59號） O:\121\121929.DOC •134- 200808833 針對所有的一致主題序列，藉著判定在每個位置中最常 出現的胺基酸，獲得得自每個一致序列之加下標線的”核 心胺基酸序列”。’’X’’意指任何天然存在的胺基酸。在TN8-IX和TN12-I庫中，與核心序列相鄰的兩個半胱胺酸是固定 的胺基酸。 在下文的表3中陳述確認出與Ang-2結合的肽。 表3 : Ang-2結合肽Maxisorp plates (NUNC) were combined overnight. Streptomyc avidin was coated on another Maxisorp disk at approximately 2 μg/ml in pbs as a control group. On the second day, discard the liquid in the protein coated Maxisorp dish and block each well with approximately 300 microliters of 5% milk solution overnight at approximately 4 °C (or i hour at room temperature) ). The milk solution was then discarded and the wells were rinsed 3 times with PBST solution. After the final rinse step, approximately 50 microliters of PBST-4% milk was added to each well of the protein coated Maxisorp disk. Approximately 50 microliters of the overnight culture from each well in a 96-well microtiter plate was transferred to an Ang-2 coated plate and the corresponding well of a control plate coated with streptavidin. . One 〇〇 microliter of the mixture in each plate was incubated for approximately one hour at room temperature. The liquid from the Maxisorp disk was discarded and the wells were rinsed approximately 3 times with PBST. HRP-co-soyler anti-_M13 antibody (Amersham Pharmacia Biotech) was diluted to approximately 1:7,500, and approximately 100 microliters of the diluted solution was added to each well of a Maxisorp disk and incubated at room temperature for approximately one hour. Throw again O:\121\121929.DOC -132- 200808833 Discard the liquid and rinse each well approximately 5 times with PBST. Approximately 100 microliters of TMB substrate (Sigma) was then added to each well and the reaction was stopped with approximately 50 microliters of 5N H2SO4 solution. OD45 读取 was read on a spectrophotometer (Molecular Devices). B. Sequencing of Phage Pure Lines For each phage pure line, a sequencing template was prepared using PCR. Approximately 500 nucleotide fragments were amplified using the following oligonucleotides. Introduction 1: S'-CGGCGCAACTATCGGTATCAAGCTGG* (Sequence recognition: No. 54) Bow 1 Sub 2 : 5,-CATGTACCGTAACACTGAGTTTCGTC-3, (sequence recognition: No. 55) The following mixture was prepared for each pure line: Reagent volume (microliter ) / test tube dH20 26.25 50% glycerol 10 10 X PCR buffer solution (no MgCl2) 5 25 mM MgCl2 4 10 mM dNTP mixture 1 ΙΟΟμΜ primer 1 0.25 100 μΜ primer 2 0.25 Taq polymerase 0.25 phage in TE (paragraph 4) 3 Final reaction volume 50 for PCR 'The following procedure was performed using a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems): 94 ° C for 5 minutes; (94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 45 seconds) ) x 30 rounds; 72 ° C for 7 minutes; cooled to 4 ° C. The QIAquick Multiwell PCR Purification Kit (Qiagen) O:\121\121929.DOC-133-200808833 was used to purify the PCR product from each reaction according to the manufacturer's draft. The purified PCR product was then analyzed by running a 1% agarose gel with 1 microliter of dye (10 X BBXS agarose loaded dye) for approximately 10 microliters each of the PCR reaction mixture. The remaining products were then sequenced using the ABI 377 sequencer (Perkin Elmer) according to the manufacturer's proposed draft. 6. Sequence alignment and determination of consensus sequences A. Sequence alignment and analysis The peptide sequences translated from the variable nucleotide sequence (paragraph 5B) were coherent with the ELISA data. A pure line showing a high OD450 value in the wells coated with Ang-2 and a low od45〇 value in the pores coated with Streptomyces avidin was preferred. It is also given a preferred arrangement of sequences that appear multiple times. Based on these criteria, candidate sequences are selected for further analysis of peptides or peptibodies. B. - Determination of Sequences Three different kinds of consistent subjects were produced from the TN8-IX library, as follows: · KRPCEEXWGGCXYX (sequence recognition: No. 56) KRPCEEXFGGCXYX (sequence recognition · · 57) XXXCXDXYWYCXXX (sequence identification: No. 61) XXXCXDXYTYCXXX (sequence identification: 62) XXXCXDXFWYCXXX (sequence identification: 63) XXXCXDXFTYCXXX (sequence identification: 64) XXXCX^E^XC^XM* (sequence recognition · · 58) Produce a consistent theme from the TN12-I library : WSXCAWFXGXXXXXCRRX (Sequence Identification··59) O:\121\121929.DOC •134- 200808833 For all consistent subject sequences, by deciding the most frequently occurring amino acids in each position, The "core amino acid sequence" of the consensus sequence added to the underline. ''X'' means any naturally occurring amino acid. In the TN8-IX and TN12-I libraries, the two cysteine acids adjacent to the core sequence are immobilized amino acids. Peptides that bind to Ang-2 were identified in Table 3 below. Table 3: Ang-2 binding peptide
肽 序列識別號 序列 TN8-8 1 KRPCEEMWGGCNYD TN8-14 2 HQICKWDPWTCKHW TN8-Conl 3 KRPCEEIFGGCTYQ TN8-Con4 4 QEECEWDPWTCEHM TN12-9 5 FDYCEGVEDPFTFGCDNH LI 6 KFNPLDELEETLYEQFTFQQ C17 7 QYGCDGFLYGCMIN 實例4Peptide SEQ ID NO: TN8-8 1 KRPCEEMWGGCNYD TN8-14 2 HQICKWDPWTCKHW TN8-Conl 3 KRPCEEIFGGCTYQ TN8-Con4 4 QEECEWDPWTCEHM TN12-9 5 FDYCEGVEDPFTFGCDNH LI 6 KFNPLDELEETLYEQFTFQQ C17 7 QYGCDGFLYGCMIN Example 4
建構編碼肽體的DNA 選擇有可能抑制Ang-2:Tie-2結合之經過修改的肽（參見 表3)，用來建構融合蛋白質，其中每個肽的單體或每個肽 的縱列二聚體（在單體單元之間有聯結子），在架構中與編 碼聯結子，接著是人類IgGl之Fc區的DNA融合。藉著黏接 成對的寡核苷酸（募），產生編碼該肽，連同聯結子的多核 苷酸雙股，建構每個經過修改的肽，將依據肽而包括5個 甘胺酸殘基、8個甘胺酸殘基或1個離胺酸殘基；以Ndel至 Xhol片段之形式產生這些構築體。將這些雙股多核甞酸分 子連接到含有人類Fc基因的載體（pAMG21-Fc N-終端，在 下文中進一步說明）内，先前已經利用Ndel和Xhol將其消 O:\121\121929.DOC -135- 200808833 化過了。藉著電穿透作用，使用標準程序，將所得的連接 混合物轉化至大腸桿菌品系2596細胞（GM221，在下文中 進-步說明）内。針對產生重組蛋白質產物，並具有正確 核替酸序列之基因融合的能力，纟篩選純種系。對每個經 過修改的肽，選出單一的這類純種系（也就是Fc_肽融合^ 物）。 建構pAMG21-Fc N-終端載體pAMG21 表現質體？人]\4021(八1^(：第98113號）係衍生自表現載體 pCFM1656 (ATCC 第 69576號），並在美國專利第 4,71〇,473 號中杬述了表現載體系統，遵循在已發表之國際專利申請 案WO 00/24782中描述的程序（參見其中實例2的部分，從 第100至103頁，以及圖17A和17B)。Construction of DNA encoding peptibodies It is possible to select modified peptides that bind Ang-2:Tie-2 (see Table 3) to construct fusion proteins in which the monomer of each peptide or the column of each peptide is The polymer (with a linker between the monomer units) is fused in the framework to the DNA encoding the linker followed by the Fc region of human IgG1. By binding a pair of oligonucleotides (producing), generating a polynucleotide double-stranded with the peptide, together with the linker, constructs each modified peptide, which will include 5 glycine residues depending on the peptide. , 8 glycine residues or 1 lysine residue; these constructs are produced as Ndel to Xhol fragments. These double-stranded polynucleic acid molecules were ligated into a vector containing the human Fc gene (pAMG21-Fc N-terminus, as further explained below), which had previously been eliminated by Ndel and Xhol: O: \121\121929.DOC-135 - 200808833 has passed. The resulting ligation mixture was transformed into E. coli strain 2596 cells (GM221, described further below) by electroporation using standard procedures. The pure lineage is screened for the ability to produce recombinant protein products with the correct fusion of the acid sequence of the acid sequence. For each of the modified peptides, a single such pure line (i.e., Fc-peptide fusion) was selected. Construction of pAMG21-Fc N-terminal vector pAMG21 expression plastid? Person] \4021 (8 1^(: No. 98113) is derived from the performance vector pCFM1656 (ATCC No. 69576), and the performance carrier system is described in U.S. Patent No. 4,71,473, The procedure described in the published international patent application WO 00/24782 (see the section of Example 2, from pages 100 to 103, and Figures 17A and 17B).
Fc N-終端載體 使用大腸桿菌品系3788，pAMG21 Τρο一Gly5—Fc單體作 為模板，創造Fe N-終端載體。可在w〇 00/24782中找到選 殖該品系之資訊（參見在本文中的實例2和圖1 〇)。設計 5’PCR引子（在後文中進一步說明），移除在pAMG Tpo Gly5 中的Top肽序列，並以含有ApaLI和Xhol位置的多聯結子取 代匕。使用σα糸3 7 8 8作為模板’利用延伸長聚合酶 (Expand Long Polymerase)進行PCR，使用下文序列識別： 8號的募核嘗酸作為51引子，以及萬能的3’引子，下文的序 列識別：9號。以凝膠純化所得的PCR產物，並以限制酵 素 Ndel 和 BsrGI消化。使用 Qiagen (Chatsworth，CA)凝膠純 化自旋管柱，以凝膠純化編碼感興趣之肽及其聯結子的多 核苷酸和質體兩者。然後使用標準連接程序，連接質體和 插入物，並將所得的連接混合物轉化到大腸桿菌細胞（品 系2596)内。選擇單一的純種系，並進行DNA定序。確認 O:\121\121929.DOC -136- 200808833 正確的純種系，並用來作為在本文中描述的經過修改之肽 的載體來源。 53丨子：Fc N-terminal vector E. coli strain 3788, pAMG21 Τρο-Gly5-Fc monomer was used as a template to create a Fe N-terminal vector. Information on the selection of this line can be found in w〇 00/24782 (see Example 2 and Figure 1 in this article). The 5' PCR primer (described further below) was designed to remove the Top peptide sequence in pAMG Tpo Gly5 and replace the 匕 with a multi-linker containing the ApaLI and Xhol positions. Using σα糸3 7 8 8 as a template for PCR using Expand Long Polymerase, using the following sequence recognition: No. 8 nucleotic acid as 51 primer, and versatile 3' primer, sequence recognition below :No.9. The resulting PCR product was gel purified and digested with restriction enzymes Ndel and BsrGI. The Qiagen (Chatsworth, CA) gel was used to purify the spin column to gel-purify both the polynucleotide and the plastid encoding the peptide of interest and its linker. The plastids and inserts were then ligated using standard ligation procedures and the resulting ligation mix was transformed into E. coli cells (line 2596). A single pure line is selected and subjected to DNA sequencing. Confirmation O:\121\121929.DOC -136- 200808833 The correct pure lineage and used as a carrier source for the modified peptides described herein. 53 丨子:
ACAAACAAACATATGGGTGCACAGAAAGCGGCCGCA AAAAAACTCGAGGGTGGAGGCGGTGGGGACA (序列識 別：8號） 3’引子： GGTCATTACTGGACCGGATC (序列識別：9號） 除了使這些經過修改的肽成為N-終端，與Fc融合（N-終 端的肽體）之外，亦可使其中一些成為C-終端融合產物（C-終端的肽體）。在下文中描述用來進行C-終端融合的載體。 建構Fc C-終端的載體 使用大腸桿菌品系3728，pAMG21 Fc_Gly5_Tpo單體作 為模板，產製經過修改之肽的Fc C-終端載體。可在WO 00/24782中找到選殖該品系的資訊（參見在本文中的實例2 和圖7)。設計3’ PCR引子（序列識別：10號），移除Τρο肽序 列，並以含有ApaLI和Xhol位置的多聯結子取代它。使用 品系3728作為模板，利用延伸長聚合酶進行PCR，使用萬 能的5,引子（序列識別：Π號），以及上文提及的3,引子。以 凝膠純化所得的PCR產物，並以限制酵素BsrGI和BamHI消 化。經由Qiagen凝膠純化自旋管柱’以凝膠純化編碼每個 感興趣之肽及其聯結子的質體和多核苷酸兩者。然後使用 標準連接草案，連接質體和插入物，並將所得的連接混合 物轉化至大腸桿菌（品系2596)的細胞内。選出單一的純種 系，並進行DNA定序。確認正確的純種系，並用來作為在 本文中描述的經過修改之肽的載體來源。 5’引子： O:\121\121929.DOC -137- 200808833 CGTACAGGTTTACGCAAGAAAATGG (序列識別：10號） 3f引子： TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGCCGCT TTCTGTGCACCACCACCTCCACCTTTAC (序列識別：11 號） GM221 (#2596)。用來表現Fc-肽融合蛋白質的宿主品系 #2596，是修改而使其含有lux啟動基因，並在早期ebg區 域含有感溫性λ阻遏物cI857s7，且在晚期ebg區域含有 lacIQ阻遏物的大腸桿菌K-12品系。這兩個阻遏物基因的出 現，容許該宿主使用各式各樣的表現系統。ATCC將該品 系命名為202174。 實例5 肽體的產製 在大腸桿菌中表現。在37°C下，使每個在大腸桿菌中之 pAMG21-Fc融合構築體的培養物生長在Terrific肉湯培養 基中（參見Tartof和Hobbs，”使質體和黏接質體純種系生長 的改良培養基（Improved media for growing plasmid and cosmid clones)”，Bethesda Research Labs Focus，第 9冊， 第l2頁，l987，在前文中提及的Sambrook等人，參考）。 在培養基中加入合成的自動誘導劑，N-(3_氧基己醯基）_ DL-高絲胺酸内酯，至每毫升20毫微克（毫微克/毫升）之終 濃度，完成從luxPR啟動基因中誘導基因產物的表現。在 37°C下培養該培養物另外6小時。然後藉著顯微鏡，針對 包涵體的存在來檢查細菌培養物，並藉著離心收集。在經 過誘導之培養基中觀察到折射的包涵體，表示Fc-融合在 大腸桿菌中最有可能以不溶性的片段來產製。藉著再懸浮 於含有10% β-疏基乙醇的Laemmli試樣緩衝溶液中，直接 O:\121\121929.DOC -138- 200808833 溶解細胞小球，然後藉著SDS-PAGE分析。在大多數的案 例中，在SDS-PAGE凝膠上觀察以正考馬斯-染色之適當分 子量的譜帶。 純化。在水（1/10)中使用高壓均質化作用（以14,000磅/平 方英吋通過兩次）打破細胞，並藉著離心（在J-6B離心機中 以4000 RPM，1小時）收獲包涵體。以1/10之比例，將包涵 體溶解於6 Μ胍，50 mM Tris，10 mM DTT，pH 8.5 中 1小 時。針對與Fc融合之直線肽，在2 Μ脲，50 mM Tris，160 mM精胺酸，2 mM半胱胺酸，pH 8.5中，將加溶混合物稀 釋25倍。容許在4°C下進行氧化作用2天，容許形成二硫-連接的化合物（也就是Fc-肽同二聚體）。至於與Fc融合的環 狀肽，則依據相同的草案，並加入下列三個折疊條件：（1) 2 Μ脲，50 mM Tris，160 mM精胺酸，4 mM半胱胺酸，1 mM胱胺，pH 8.5 ; (2) 4 Μ脲，20%甘油，50 mM Tris， 160 mM精胺酸，2 mM半胱胺酸，pH 8.5 ;和（3) 4 Μ脲， 20%甘油，50 mM Tris，160 mM精胺酸，4 mM半胱胺酸 ，1 mM胱胺，pH 8.5。將再折疊的蛋白質對1.5 Μ脲，50 mM NaCl，50 mM Tris，pH 9.0進行透析。利用醋酸將該 混合物的pH值降至pH 5。藉著離心移除沉澱物，並依據每 個融合產物的等電點，將上清液調整成pH 5至6.5。過濾 蛋白質，並在4°C下裝至以20 mM NaAc，50 mM NaCl，在 對每個構築體判定之pH值下平衡的SP-瓊脂糖HP管柱中。 使用20-管柱體積，範圍從50 mM NaCl至500 mM NaCl之 相同緩衝溶液的直線梯度，洗脫蛋白質。收集高峰並過濾 之。 在以下的表4中陳述使用以上之程序產製的肽體。 O:\121\121929.DOC -139- 200808833 表4 肽體 肽體序列 LI (N) MGAQKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (序列識別：12號） LI (N)WT MKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (序列識別：13號） LI (N)1KWT mkfnpldeleetlyeqftfwgsgsatggsgstassgs GSATHLEGGGGG-Fc (序列識別：14ft) 2xLl (N) MGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPL DELEETLYEQFTFQQLEGGOGG-Fc (序列識別：15號） 2xLl (N) WT MKFNPLDELEETLYEQFTFQQGGGGGGGKFNPLDELEE TLYEQFTF〇〇LEGGGGG-Fc (序列識別：16號） Con4 (N) MGAQQEECEWDPWTCEHMLEGGGGG-Fc (序列識別：17號） Con4(N) 1K-WT MQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATH LEGGGGG-Fc (序列識別：18號） 2xCon4 (N) IK MGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGS ATHQEECEWDPWTCEHMLEGGGGG-Fc (序列識別：19號） LI (C) M-Fc-GGGGGAOKFNPLDELEETLYEQFTFQQLE (序列識別：20號）ACAAACAAACATATGGGTGCACAGAAAGCGGCCGCA AAAAAACTCGAGGGTGGAGGCGGTGGGGACA (Sequence recognition: No. 8) 3' primer: GGTCATTACTGGACCGGATC (sequence recognition: No. 9) In addition to making these modified peptides N-terminal, fusion with Fc (N-terminal peptide), Some of them become C-terminal fusion products (C-terminal peptibodies). A vector for performing C-terminal fusion is described below. Construction of Fc C-terminal vector The Escherichia coli line 3728, pAMG21 Fc_Gly5_Tpo monomer was used as a template to produce an Fc C-terminal vector of the modified peptide. Information on the selection of this line can be found in WO 00/24782 (see Example 2 and Figure 7 herein). The 3' PCR primer (SEQ ID NO: 10) was designed, the Τρο peptide sequence was removed, and it was replaced with a multi-linker containing the ApaLI and Xhol positions. Using strain 3728 as a template, PCR was carried out using an extended polymerase, using a universal 5, primer (sequence recognition: nickname), and the above mentioned 3, primer. The resulting PCR product was gel purified and digested with restriction enzymes BsrGI and BamHI. Purification of the spin column by Qiagen gel' gel purification of both the plastid and the polynucleotide encoding each peptide of interest and its linker. The plastids and inserts were then ligated using standard ligation sequences and the resulting ligation mix was transformed into cells of E. coli (line 2596). A single pure line is selected and subjected to DNA sequencing. The correct pure line is confirmed and used as a carrier source for the modified peptides described herein. 5' primer: O:\121\121929.DOC-137- 200808833 CGTACAGGTTTACGCAAGAAAATGG (sequence recognition: No. 10) 3f primer: TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGCCGCT TTCTGTGCACCACCACCTCCACCTTTAC (sequence identification: No. 11) GM221 (#2596). Host line #2596, which is used to represent the Fc-peptide fusion protein, is modified to contain the lux promoter gene and contains the thermosensitive lambda repressor cI857s7 in the early ebg region and the lacIQ repressor in the late ebg region. K-12 strain. The emergence of these two repressor genes allows the host to use a wide variety of expression systems. The ATCC named the line 202174. Example 5 Production of peptibodies was performed in E. coli. Cultures of each of the pAMG21-Fc fusion constructs in E. coli were grown in Terrific Broth medium (see Tartof and Hobbs, at 37 ° C) to grow plastid and adherent pure lines. "Improved media for growing plasmids and cosmid clones", Bethesda Research Labs Focus, Vol. 9, p. 12, l987, mentioned in the aforementioned Sambrook et al., reference). Add the synthetic autoinducer, N-(3-methoxyhexyl)_DL-homoxolide, to a final concentration of 20 ng/mL per ml to complete the boot from luxPR The gene is induced to express the gene product. The culture was incubated at 37 ° C for an additional 6 hours. The bacterial culture is then inspected for the presence of inclusion bodies by means of a microscope and collected by centrifugation. Inclusion bodies were observed to be refracted in the induced medium, indicating that Fc-fusion is most likely to be produced in insoluble fragments in E. coli. The cells were lysed by resuspending in a Laemmli sample buffer solution containing 10% β-mercaptoethanol, directly by O:\121\121929.DOC-138-200808833, and then analyzed by SDS-PAGE. In most cases, the band of the appropriate molecular weight of Coomassie-stained was observed on an SDS-PAGE gel. purification. The cells were disrupted by high pressure homogenization (passed at 14,000 psig twice) in water (1/10) and harvested by centrifugation (4000 RPM in a J-6B centrifuge for 1 hour). . The inclusion bodies were dissolved in 6 Torr, 50 mM Tris, 10 mM DTT, pH 8.5 for 1 hour at a ratio of 1/10. For the linear peptide fused to Fc, the solubilized mixture was diluted 25-fold in 2 guanidine, 50 mM Tris, 160 mM arginine, 2 mM cysteine, pH 8.5. Oxidation was allowed to proceed at 4 ° C for 2 days, allowing the formation of disulfide-linked compounds (i.e., Fc-peptide homodimers). As for the cyclic peptide fused to Fc, according to the same draft, the following three folding conditions were added: (1) 2 guanidine, 50 mM Tris, 160 mM arginine, 4 mM cysteine, 1 mM cyst Amine, pH 8.5; (2) 4 guanidine, 20% glycerol, 50 mM Tris, 160 mM arginine, 2 mM cysteine, pH 8.5; and (3) 4 guanidine, 20% glycerol, 50 mM Tris, 160 mM arginine, 4 mM cysteine, 1 mM cystamine, pH 8.5. The refolded protein was dialyzed against 1.5 guanidine urea, 50 mM NaCl, 50 mM Tris, pH 9.0. The pH of the mixture was lowered to pH 5 using acetic acid. The precipitate was removed by centrifugation and the supernatant was adjusted to pH 5 to 6.5 based on the isoelectric point of each fusion product. The protein was filtered and loaded into an SP-Sepharose HP column equilibrated at 20 mM NaAc, 50 mM NaCl at a pH determined for each construct at 4 °C. The protein was eluted using a 20-column volume, a linear gradient of the same buffer solution ranging from 50 mM NaCl to 500 mM NaCl. Collect peaks and filter them. Peptides produced using the above procedure are set forth in Table 4 below. O:\121\121929.DOC -139- 200808833 Table 4 Peptide Peptide Sequence LI (N) MGAQKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (Sequence Recognition: No. 12) LI (N)WT MKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (Sequence Recognition: No. 13) LI ( N) 1KWT mkfnpldeleetlyeqftfwgsgsatggsgstassgs GSATHLEGGGGG-Fc (SEQ ID: 14ft) 2xLl (N) MGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPL DELEETLYEQFTFQQLEGGOGG-Fc (SEQ ID: No. 15) 2xLl (N) WT MKFNPLDELEETLYEQFTFQQGGGGGGGKFNPLDELEE TLYEQFTF〇〇LEGGGGG-Fc (SEQ ID: No. 16) Con4 (N MGAQQEECEWDPWTCEHMLEGGGGG-Fc (SEQ ID NO: 17) Con4(N) 1K-WT MQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATH LEGGGGG-Fc (sequence recognition: 18) 2xCon4 (N) IK MGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGS ATHQEECEWDPWTCEHMLEGGGGG-Fc (sequence identification: 19) LI (C) M -Fc-GGGGGAOKFNPLDELEETLYEQFTFQQLE (Sequence Identification: No. 20)
O:\121\121929.DOC 140- 200808833 LI (C) 1Κ M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELE ETLYEQFTFQQLE (序列識別：21 猇） 2xLl (C) M-Fc- GGGGGAQKFNPLDELEETLYEOFTFOOGGGGGGGGKF npldeleetlyeqftfqqle (序列識別：22H) Con4 (C) M-Fc-GGGGGAQQEECEWDPWTCEHMLE (序列識別：23號） Con4 (C) IK M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHQEECEWDP WTCEHMLE (序列識別：24號） 2xCon4 (C) IK M-Fc- GGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASS GSGSATHQEECEWDPWTCEHMLE (序列識別：25號） Con4-Ll (N) MGAQEECEWDPWTCEHMGGGGGGGGKFNPLDELEET LYEOFTFOOGSGSATGGSGSTASSGSGSATHLEGGGGG-Fc (序列識別：26號） Con4-Ll (C) M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELE ETLYEQFTFQQGGGGGQEECEWDPWTCEHMLE (序列識別：27號） TN-12-9 (N) MGAQ-FDYCEGVEDPFTFGCDNHLE-GGGGG-Fc (序列識別：28號） C17(N) MGAQ-QYGCDGFLYGCMINLE-GGGGG-Fc (序列識別：29號） TN8-8 (N) Vi G AQ-KRPCEEM W GGCNYDLEGGGGG-Fc (序列識別·· 30號） TN8-14(N) MGAQ-HQICKWDPWTCKHWLEGGGGG-Fc (序列識別：31號） Coni (N) MGAQ-KRPCEEIFGGCTYQLEGGGGG-Fc (序列識別：32號） 在表4中，，，Fcn意指人類Fc IgGl序列。欄位2陳述肽體的 胺基酸序列。其Fc部分以，，Fc，，標示’並如同在下文中序列 識別·· 60號中的陳述。應暸解使用標記之處’例如nCon4" 或，，Con-4，，，這意指Con-4肽，但是在其上使用字尾的"C” 、，，（C)，，或"-C，，；或"N，，、，，(N)，，或”·Ν"，表示該分子為如同 在本文中描述的肽體。在肽體名中的字尾’’Ν’’、Μ(Ν)’’或 -141-O:\121\121929.DOC 140- 200808833 LI (C) 1Κ M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELE ETLYEQFTFQQLE (Sequence recognition: 21 猇) 2xLl (C) M-Fc- GGGGGAQKFNPLDELEETLYEOFTFOOGGGGGGGGKF npldeleetlyeqftfqqle (sequence identification: 22H) Con4 (C) M -Fc-GGGGGAQQEECEWDPWTCEHMLE (SEQ ID NO: 23) Con4 (C) IK M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHQEECEWDP WTCEHMLE (Sequence recognition: No. 24) 2xCon4 (C) IK M-Fc- GGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASS GSGSATHQEECEWDPWTCEHMLE (Sequence recognition: 25) Con4-Ll (N) MGAQEECEWDPWTCEHMGGGGGGGGKFNPLDELEET LYEOFTFOOGSGSATGGSGSTASSGSGSATHLEGGGGG-Fc (SEQ ID NO: 26) Con4-Ll (C) M-Fc- GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELE ETLYEQFTFQQGGGGGQEECEWDPWTCEHMLE (Sequence recognition: 27) TN-12-9 (N) MGAQ-FDYCEGVEDPFTFGCDNHLE-GGGGG-Fc (sequence Identification: No. 28) C17(N) MGAQ-QYGCDGFLYGCMINLE-GGGGG-Fc (Sequence recognition: No. 29) TN8-8 (N) Vi G AQ-KRPCEEM W GGCNYDLEGGGGG-Fc (sequence recognition · · 30) TN8-14 ( N) MGAQ-HQICKWDPWTCKHWLEGGGGG-Fc (sequence recognition: 31) Con i (N) MGAQ-KRPCEEIFGGCTYQLEGGGGG-Fc (SEQ ID NO: 32) In Table 4, Fcn means a human Fc IgG1 sequence. Column 2 states the amino acid sequence of the peptibodies. Its Fc portion is denoted by ,, Fc,, and is as described in the sequence below. It should be understood that where the mark is used 'eg nCon4" or, Con-4,, this means the Con-4 peptide, but the suffix 'C', , (C), or "-C,,; or "N,,,,, (N), or "·Ν", means that the molecule is a peptibody as described herein. The suffix '’Ν’, Μ(Ν)’’ or -141- in the peptidic name
O:\121\121929.DOC 200808833 Π-Νπ，表示Ang-2-結合肽（或肽們）是N-終端對Fc功能部位 ，而字尾”C”、"（C)”或，’-C"，表示Ang-2-結合肽（或肽們）是 C-終端對Fc功能部位。此外，2xCon4 (C) 1K，如同在序 列識別：25號中之定義，在本文中亦可以無Π1ΚΠ2字尾表 示0 每個肽體之Fc部分的胺基酸序列如下（從胺基終端到羧 基終端）：O:\121\121929.DOC 200808833 Π-Νπ, indicating that the Ang-2-binding peptide (or peptide) is the N-terminal to the Fc functional part, and the suffix "C", "(C)" or, ' -C", indicating that the Ang-2-binding peptide (or peptide) is a C-terminal to Fc functional site. In addition, 2xCon4 (C) 1K, as defined in Sequence Identification: No. 25, may also be absent herein. Π1ΚΠ2 尾尾00 The amino acid sequence of the Fc portion of each peptibody is as follows (from the amine end to the carboxyl end):
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRYVSVLTVLSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRYVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (序列識別：60號） 在下文中陳述分別編碼相當於肽體序列識別：12-32號（ 在表4中）之肽體的DNA序列（序列識別：33-53號）： 序列識別：33號DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (Sequence recognition: No. 60) The DNA sequences encoding peptibodies corresponding to peptidic sequence recognition: 12-32 (in Table 4) are reported below (sequence recognition: No. 33-53): Sequence recognition: No. 33
ATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCT
GTACGAACAGTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGGGTACGAACAGTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGG
ACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT
CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGACTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACGCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAGACGCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGACCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT ggacaagaGcaggtggcagcaggggaacgtcttctcatgctccgtgatCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT ggacaagaGcaggtggcagcaggggaacgtcttctcatgctccgtgat
GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGTAAATAATGGATCC O:\121\121929.DOC -142- 200808833 序列識別：34號TCCGGGTAAATAATGGATCC O:\121\121929.DOC -142- 200808833 Sequence Identification: No. 34
ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACA
GTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCAGTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCA
CACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
TTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGACCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA
GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGTCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC
AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAATCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA
ATAA 序列識別：35號ATAA Sequence Identification: No. 35
ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACA
GTTCACTTTCCAGCAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCGTTCACTTTCCAGCAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCC
ACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCTCGAGGGTGGAGGCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCTCGAGGGTGGAGGC
GGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTC
CTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACC
CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCATGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACAT
CGCCGTGGAGTGGGAGAGCAATGGQCAGCCGGAGAACAACTACAAGACGCCGTGGAGTGGGAGAGCAATGGQCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC
TCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC 143- 200808833 序列識別：36號TCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC 143- 200808833 Sequence Identification: No. 36
ATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCT GTACGAACAGTTCACTTTCCAGCAGGGTGGTGGTGGTGGTGGCGGTGG TAAGTTCAACCCACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTT CACTTTCCAGCAACTCGAGGGTGGAGGCGGTGGGGACAAAACJCACA CATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTT TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TAA 序列識別：37號ATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCT GTACGAACAGTTCACTTTCCAGCAGGGTGGTGGTGGTGGTGGCGGTGG TAAGTTCAACCCACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTT CACTTTCCAGCAACTCGAGGGTGGAGGCGGTGGGGACAAAACJCACA CATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTT TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TAA SEQ ID: No. 37
ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACA GTTCACTTTCCAGCAGGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCC ACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCA ACTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTT GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC •144- 200808833 序列識別：38號ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACA GTTCACTTTCCAGCAGGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCC ACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCA ACTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTT GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O: \ 121 \ 121929.DOC • 144- 200808833 SEQ ID: 38
ATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGA ACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTC CACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA 序列識別：39號ATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGA ACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTC CACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA SEQ ID: 39
ATGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGG ATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCA GGCAGTGCGACTCATCTCGAGGGTGGAGGCGGTGGGGACAAAACTCA CACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT TTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA ATAA O:\121\121929.DOC -145- 200808833 序列識別：40號ATGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGG ATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCA GGCAGTGCGACTCATCTCGAGGGTGGAGGCGGTGGGGACAAAACTCA CACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT TTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA ATAA O: \ 121 \ 121929.DOC -145- 200808833 SEQ ID: 40
ATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGA
ACACATGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGACACATGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAG
CTCTGGTTCAGGCAGTGCGACTCATCAGGAAGAATGCGAATGGGACCCCTCTGGTTCAGGCAGTGCGACTCATCAGGAAGAATGCGAATGGGACCC
ATGGACTTGCGAACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAAATGGACTTGCGAACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAA
CTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAATACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGACTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC
TGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACATGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATAA 序列識別：41號GTAAATAA Sequence Identification: No. 41
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG
GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGACACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCC
GCTGGACGAGCTGGAAGAGACTCTGTACGAACAGTTTACTTTTCAACAGCTGGACGAGCTGGAAGAGACTCTGTACGAACAGTTTACTTTTCAACA
GCTCGAGTAA O:\121\121929.DOC -146- 200808833 序列識別：42號GCTCGAGTAA O:\121\121929.DOC -146- 200808833 Sequence Identification: No. 42
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTC TGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGC GACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGA ACAGTTCACTTTCCAGCAACTCGAGTAA 序列識別：43號ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTC TGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGC GACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGA ACAGTTCACTTTCCAGCAACTCGAGTAA SEQ ID: 43
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC acaggtgtacaccctgcccccatcccgggatgagctgaccaAgaacca GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACGA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCC GCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCA GGGTGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCCACTGGATGAGCT GGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCAACTCGAGTAA O:\121\121929.DOC -147- 200808833 序列識別·· 44號ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC acaggtgtacaccctgcccccatcccgggatgagctgaccaAgaacca GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACGA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCC GCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCA GGGTGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCCACTGGATGAGCT GGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCAACTCGAGTAA O: \ 121 \ 121929.DOC -147- 200808833 SEQ ID NO · 44
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG
GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGACACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGCAGGAAGAATCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGCAGGAAGAAT
GCGAATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA 序列識別·· 45號GCGAATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA Sequence Identification·· 45
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG
GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGACACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGTGGATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAGGTGGATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTC
TGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGC
GACTCATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACAGACTCATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACA
TGCTCGAGTAA 148-TGCTCGAGTAA 148-
O:\121\121929.DOC 200808833 序列識別：46號O:\121\121929.DOC 200808833 Sequence Identification: No. 46
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGCAGGAAGAAT GCGAATGGGACCCATGGACTTGCGAACACATGGGATCCGGTTCTGCTA C 丁 GGTGGTTCCGGC 丁 (XACCGCAAGCTCTGGTTCAGGCAGCGCGAC 丁 C ATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTC GAGTAA 序列識別：47號ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGCAGGAAGAAT GCGAATGGGACCCATGGACTTGCGAACACATGGGATCCGGTTCTGCTA C D D GGTGGTTCCGGC (XACCGCAAGCTCTGGTTCAGGCAGCGCGAC D C ATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTC GAGTAA SEQ ID: 47
ATGGGTGCACAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACA CATGGGTGGTGGTGGTGGTGGCGGTGGTAAATTCAACCCGCTGGACGA ACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGATCCGG TTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGT GCGAGTCATCTCGAGGGTGGAGGCGGTGGgGACAAAACTCACACATGT CCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC -149- 200808833 序列識別：48號ATGGGTGCACAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACA CATGGGTGGTGGTGGTGGTGGCGGTGGTAAATTCAACCCGCTGGACGA ACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGATCCGG TTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGT GCGAGTCATCTCGAGGGTGGAGGCGGTGGgGACAAAACTCACACATGT CCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O: \ 121 \ 121929.DOC -149- 200808833 SEQ ID: 48
ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTC TGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGC GACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGA ACAGTTCACTTTCCAGCAGGGTGGTGGCGGTGGTCAGGAAGAATGCGA ATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA 序列識別：49號ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTC TGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGC GACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGA ACAGTTCACTTTCCAGCAGGGTGGTGGCGGTGGTCAGGAAGAATGCGA ATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA SEQ ID: 49
ATGGGTGCACAGTTCGACTACTGCGAAGGTGTTGAAGACCCGTTCACT TTCGGTTGCGACAACCACCTCGAGGGTGGAGGCGGTGGGGACAAAAC TCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCGTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATAA O:\121\121929.DOC 150- 200808833 序列識別：50號ATGGGTGCACAGTTCGACTACTGCGAAGGTGTTGAAGACCCGTTCACT TTCGGTTGCGACAACCACCTCGAGGGTGGAGGCGGTGGGGACAAAAC TCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCGTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATAA O: \ 121 \ 121929.DOC 150- 200808833 SEQ ID: No. 50
ATGGGTGCACAGCAGTACGGTTGCGACGGTTTTCTGTACGGTTGCATG ATCAACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCC ACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA 序列識別：51號ATGGGTGCACAGCAGTACGGTTGCGACGGTTTTCTGTACGGTTGCATG ATCAACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCC ACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA SEQ ID: 51
ATGGGTGCACAGAAACGCCCATGCGAAGAAATGTGGGGTGGTTGCAA CTACGACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTC CACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA.GTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC 151 - 200808833 序列識別：52號ATGGGTGCACAGAAACGCCCATGCGAAGAAATGTGGGGTGGTTGCAA CTACGACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTC CACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA.GTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O: \ 121 \ 121929.DOC 151 - 200808833 SEQ ID: 52
ATGGGTGCACAGCACCAGATCTGCAAATGGGACCCGTGGACCTGCAAATGGGTGCACAGCACCAGATCTGCAAATGGGACCCGTGGACCTGCAA
ACACTGGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCACACTGGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTC
CACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCACCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA 序列識別：53號AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA Sequence Identification: No. 53
ATGGGTGCACAGAAACGTCCATGCGAAGAAATCTTCGGTGGTTGCACCATGGGTGCACAGAAACGTCCATGCGAAGAAATCTTCGGTGGTTGCACC
TACCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCTACCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCC
ACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAGAAAGCCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAGAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGCCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC
CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAACGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC 152- 200808833 實例6 肽醴測定 使用中和ELISA測試14個肽體，並使用親和力Elisa測 試3個肽體。在表5中陳述其結果。 表5 hAng-2 mAng-2 hAng-1 肽體 IC 50 (nM) EC 50 (nM) IC 50 (nM) EC 50 (nM) IC 50 (nM) EC 50 (nM) 2xCon4 (C) 1K 0.04 0.02 Con4-Ll (C) 0.05 0.04 Con4 (C) 0.20 0.30 2xLl (N) 0.65 0.80 Con4 (N) 0.85 0.03 0.72 0.07 無抑制作用 無結合作用 2xLl (C) 0.90 1.0 Con4 (N) 1K-WT 1.9 L1(N) 6 11 無抑制作用 C17(N) 9 13 無抑制作用 12-9 (N) 21 7.7 無抑制作用 Coni (N) 26 〜200 無抑制作用 8-14 (N) 45 33 無抑制作用 L1(C) 65 37 8-8 (N) 80 〜700 無抑制作用 陰性對照組 肽體4883 無抑制 作用 無結合 作用 無抑制 作用 無結合 作用 無抑制作用 無結合作用 陰性對照組肽體4883的胺基酸序列如下（在Fc部分加下 標線，聯結子為nGGGGGn，且肽部分以粗體表示）： MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVnAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA O:\121\121929.DOC 152-200808833 Example 6 Peptone 醴 assay 14 peptibodies were tested using a neutralization ELISA and 3 peptibodies were tested using affinity Elisa. The results are stated in Table 5. Table 5 hAng-2 mAng-2 hAng-1 Peptide IC 50 (nM) EC 50 (nM) IC 50 (nM) EC 50 (nM) IC 50 (nM) EC 50 (nM) 2xCon4 (C) 1K 0.04 0.02 Con4-Ll (C) 0.05 0.04 Con4 (C) 0.20 0.30 2xLl (N) 0.65 0.80 Con4 (N) 0.85 0.03 0.72 0.07 No inhibition No binding 2xLl (C) 0.90 1.0 Con4 (N) 1K-WT 1.9 L1 ( N) 6 11 No inhibition C17(N) 9 13 No inhibition 12-9 (N) 21 7.7 No inhibition Coni (N) 26 ~ 200 No inhibition 8-14 (N) 45 33 No inhibition L1 ( C) 65 37 8-8 (N) 80 ~700 no inhibition negative control peptibody 4883 no inhibition no binding no inhibition no binding no inhibition no binding negative control peptide 4883 amino acid The sequence is as follows (the reticle is added to the Fc portion, the linker is nGGGGGn, and the peptide portion is shown in bold): MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVn
VSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVL
HODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPQVYTTPPSHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPQVYTTPPS
RDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVT.DRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVT.D
SDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSr.SLS PGK-GGGGG-CTAGYHWNSDCECCRRN (序列識別·· 243號） 應瞭解在本文中使用”無抑制作用” 一詞，並非企圖表示 O:\121\121929.DOC -153- 200808833 該化合物沒有抑制性質。在本文中使用的，·無抑制作用，，一 詞，寧可意指那些化合物在使用中和eusa測定測試時， 在本文中描述的條件，μ -,, 千下顯不出比1000 ηΜ更大的1(：50值 ’其為篩選這些化合物時的最高濃度。雖然對於以"無抑 制作用"標示之分子’並未觀察到顯著的抑制性質，但應 瞭解事實上可在不同的敎條件τ，或在不同的測定中广 證實那些分子的抑制性質。在較佳的具體實施例中，應瞭 解本發明係關於在使用在本文巾料之敎時，具有抑制 性質的肽體。 使用親和力BIAcore測定測試兩個肽體（如同在實例2中 描述的）。在下文表6中陳述其結果。 表6 肽體（Pb)對hAng_2和mAng_2的親和力SDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSr.SLS PGK-GGGGG-CTAGYHWNSDCECCRRN (Sequence Identification·· No. 243) It should be understood that the term “no inhibition” is used herein and is not intended to mean O:\121\121929.DOC-153- 200808833 . As used herein, the term "non-inhibiting," is used to mean those conditions in which the compounds are described in the eusa assay, and the conditions described herein, μ -, , thousand are less than 1000 ηΜ The 1 (:50 value' is the highest concentration at the time of screening these compounds. Although no significant inhibitory properties are observed for the "no inhibition"labeled molecule', it should be understood that in fact different 敎The conditions τ, or the inhibitory properties of those molecules, are widely confirmed in different assays. In a preferred embodiment, it is to be understood that the present invention relates to peptide bodies having inhibitory properties when used herein. Affinity BIAcore assays tested two peptibodies (as described in Example 2.) The results are presented in Table 6 below. Table 6 Affinity of pBII (Pb) for hAng_2 and mAng_2
實例7 全身性投與Aug-2肽體的治療效力研究 以每天1次的計畫，在腫瘤攻毒之後72小時，皮下投與 Ang-2肽體，TN8-Con4-C。所使用之肽體的劑量為1〇〇〇、 200、40和8微克/老鼠/天。所有的動物均給與總共2〇個劑 量。每週三次記錄腫瘤體積和體重。在研究結束時，犧牲 動物，並收集其血清，藉著ELISA測量肽體的含量。從所 有組別中收集腫瘤和名單中的正常組織。 O:\121\121929.DOC -154- 200808833 在圖1出示其結果。如同所見，在Ang-2肽體處理組和媒 劑對照組之間，觀察到腫瘤生長上的顯著差異。與媒劑對 照組相比較，所有四種劑量的Ang-2肽體均抑制腫瘤生長 (ρ<0·0001對媒劑對照組，使用重覆測量ANOVA)。相反的 ，在對照組中的腫瘤，以高很多的速率持續生長。利用肽 體的處理，對於以上述劑量處理之動物的最後體重、器官 重量或血液學參數，並無顯著的影響。 實例8 1·建構Ang_2二級肽庫 A. 電反應潛能的大腸桿菌細胞 從 Stratagene (Stratagene Cloning Systems，La Jolla，CA) 購買Epicurian Coli® XLl-Blue MRF'電穿透反應潛能細胞 (Stratagene #200158) ° B. pCESl載體的修改 使用延伸長模板PCR系統（Roche Diagnostics Corp·， Indianapolis，IN)進行 PCR，利用 1 微克 pCESl 載體 (TargetQuest Inc.)作為模板。PCR混合物體積為100微升， 其含有lxPCR缓衝溶液，兩種引子 ：5’-CAAACGAATGGATCCTCATTAAAGCCAGA-3’ 序列識另U : 244號） 和 SLGGTGGTGCGGCCGCACTCGAGACTGTTGAAAGTTGnTAGCAJ ( 序列識別：245號）各200 nM，200 nM dNTP和3單位（U)的 Tag DNA聚合酶。如下執行 TRIO-Thermoblock (Biometra)Example 7 Therapeutic Efficacy Study of Systemic Administration of Aug-2 Peptide The Ang-2 peptide, TN8-Con4-C, was administered subcutaneously 72 hours after tumor challenge on a daily basis. The doses of the peptibodies used were 1 〇〇〇, 200, 40 and 8 μg/mouse/day. All animals were given a total of 2 doses. Tumor volume and body weight were recorded three times a week. At the end of the study, the animals were sacrificed and their serum was collected and the peptone content was measured by ELISA. Tumors and normal tissues in the list were collected from all groups. O:\121\121929.DOC -154- 200808833 The results are shown in Figure 1. As can be seen, a significant difference in tumor growth was observed between the Ang-2 peptidial treatment group and the vehicle control group. All four doses of Ang-2 peptibody inhibited tumor growth compared to the vehicle control group (ρ < 0·0001 vs. vehicle control group, using repeated measures ANOVA). In contrast, tumors in the control group continued to grow at a much higher rate. Treatment with the peptide has no significant effect on the final body weight, organ weight or hematological parameters of the animals treated at the above doses. Example 8 1· Construction of Ang_2 secondary peptide library A. Electroreactive potential of E. coli cells Purchased Epicurian Coli® XLl-Blue MRF' electroporation response potential cells from Stratagene (Stratagene Cloning Systems, La Jolla, CA) (Stratagene #200158 ° B. Modification of pCES1 vector PCR was carried out using an extended-length template PCR system (Roche Diagnostics Corp., Indianapolis, IN) using 1 μg of pCES1 vector (TargetQuest Inc.) as a template. The PCR mixture has a volume of 100 μl, which contains lx PCR buffer solution, two primers: 5'-CAAACGAATGGATCCTCATTAAAGCCAGA-3' sequence identification U: 244) and SLGGTGGTGCGGCCGCACTCGAGACTGTTGAAAGTTGnTAGCAJ (sequence identification: 245) each 200 nM, 200 nM dNTP And 3 units (U) of Tag DNA polymerase. Perform TRIO-Thermoblock (Biometra) as follows
PCR系統：94°C 5 分鐘；94°C 30 秒、50°C 30 秒、72°C 45秒的30個回合；以及72°C 10分鐘；冷卻至4°C。 O:\121\121929.DOC -155- 200808833 然後在1 %瓊脂糖凝膠上，跑P C R產物，並利用 QIAGEN 自旋管柱（QIAGEN Inc·，Valencia，CA)，根據製 造者的草案純化。利用5微升PCR產物，和兩種引子51-CAAACGAATGGATCCTCATTAAAGCCAGA-3,（序列識別：246號） 和 5，-AACACAAAAGTGCACAGGGTGGAGGTGGTGGTGCGGCCGCACT-3,（ 序列識別：247號）各200 nM，在與上述相同的PCR條件下 ，進行第二個PCR反應。 然後在37°C下，含有1χΝΕΒ2缓衝溶液、60單位ApaLI (New England Biolabs，Beverly，ΜΑ)、60單位 BamHI (New England Biolabs)的100微升反應中，分別消化PCR產物和 原始的pCESl載體1小時。然後使用QIAGEN自旋管柱，純 化經過消化的DNA，並在室溫下，在含有1χ連接緩衝溶液 和40單位T4 DNA連接酶（New England Biolabs)的40微升反 應中過夜，將其連接在一起。 將載體轉移感染到大腸桿菌内，並在37°C下培養過夜。 選出經過分離的單一菌落，然後使用QIAGEN自旋管柱純 化質體。藉著DNA定序證實正確的插入物。PCR system: 94 ° C for 5 minutes; 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 45 seconds for 30 rounds; and 72 ° C for 10 minutes; cooled to 4 ° C. O:\121\121929.DOC -155- 200808833 The P C R product was then run on a 1% agarose gel and purified using a QIAGEN spin column (QIAGEN Inc., Valencia, CA) according to the manufacturer's draft. Using 5 μl of PCR product, and two primers 51-CAAACGAATGGATCCTCATTAAAGCCAGA-3, (sequence recognition: No. 246) and 5,-AACACAAAAGTGCACAGGGTGGAGGTGGTGGTGCGGCCGCACT-3, (sequence recognition: No. 247) each 200 nM, in the same PCR conditions as above Next, a second PCR reaction is performed. The PCR product and the original pCES1 vector were then separately digested in a 100 μl reaction containing 1 χΝΕΒ 2 buffer solution, 60 units of ApaLI (New England Biolabs, Beverly, ΜΑ), 60 units of BamHI (New England Biolabs) at 37 °C. 1 hour. The digested DNA was then purified using a QIAGEN spin column and incubated overnight at room temperature in a 40 microliter reaction containing 1 χ ligation buffer and 40 units of T4 DNA ligase (New England Biolabs). together. The vector was transferred into E. coli and incubated overnight at 37 °C. A single colony that was isolated was selected and the plastid was purified using a QIAGEN spin column. Confirm the correct insert by DNA sequencing.
C.製備載體DNA 使用設定為2500伏特，25微法拉第和200歐姆的Gene Pulser II (BIO-RAD，Hercules，CA)，將 1微克經過修改的 pCESl載體DNA (得自上文段落1B)轉化至40微升電反應潛 能的XL 1-blue大腸桿菌（得自上文段落ία)内。然後立刻將 經過轉化的細菌試樣移至含有960微升SOC (2%胰蛋白酶 水解產生的酶，0.5%酵母菌萃取物，1〇 mM NaCl，2.5 O:\121\121929.DOC -156- 200808833 mM KC1，20 mM葡萄糖，10 mM MgS〇4，10 mM MgCl2) 的試管中，並容許該培養物在37°C下生長，並振盈1小時。 然後將細胞散播在2xYTAGT (2xYT，含有1〇〇微克/毫升 氨苄青黴素，12·5微克/毫升四環素和2%葡萄糖）瓊脂培養 盤上，並在37°C下培養過夜。藉著定序證實單一的菌落， 並用來接種2公升的2xYTAGT培養基，並在37°C下振盪過 夜。利用QIAGEN質體Maxi套組，根據製造者的草案，純 化質體載體DN A。C. Preparation of vector DNA Using a Gene Pulser II (BIO-RAD, Hercules, CA) set at 2500 volts, 25 microfarads and 200 ohms, 1 microgram of modified pCES1 vector DNA (from paragraph 1B above) was converted to 40 microliters of electroreactive potential of XL 1-blue E. coli (obtained from paragraph ία above). The transformed bacterial sample was then immediately transferred to contain 960 microliters of SOC (2% trypsin hydrolyzed enzyme, 0.5% yeast extract, 1 mM mM NaCl, 2.5 O: \121\121929.DOC-156- 200808833 mM KC1, 20 mM glucose, 10 mM MgS 4 , 10 mM MgCl 2 ) in a test tube and allowed to grow at 37 ° C and shake for 1 hour. The cells were then spread on a 2x YTAGT (2xYT, containing 1 μg/ml ampicillin, 12·5 μg/ml tetracycline and 2% glucose) agar plates and incubated overnight at 37 °C. A single colony was confirmed by sequencing and used to inoculate 2 liters of 2xYTAGT medium and shaken overnight at 37 °C. The QAAGEN plastid Maxi kit was used to purify the plastid vector DN A according to the manufacturer's draft.
D.消化載艎DNA 在37°C下，在含有2xNEB緩衝溶液2，300單位ApaLI和 300單位Xhol的5000微升反應中，總共消化大約2000微克 的載體DNA (得自上文段落1C)過夜。在37°C下培養該限制 消化反應過夜，並在預先-製造的0.8%瓊脂糖凝膠（Embi Tec，San Diego，CA)中分析之。然後從凝膠中切下直線化 的載體DNA，並根據製造者的指示，以QIAquick凝膠萃取 套組（QIAGEN Inc.)萃取。 Ε·庫募核苷酸的製備 以衍生自上述結果的序列為基礎，設計六個庫寡核苷酸 (1個固定的和5個麻醉的）。一個固定的庫募核苷酸為： 5，-CACAGTGCACAGGGTNNKNNKNNKNNKNNKNNKNNKS ARTGGGATCCGTGGASCNNKNNKNNKNNKNNKNNKN NKCATT CTCTCGAGATCA-3,（庫 20號）（序列識別：248號） 而兩個70%麻醉的庫寡核甞酸如下： 5，-CACAGTGCACAGGGTNNKNNKNNKaaKcgKccKNNKga O:\121\121929.DOC -157- 200808833D. Digestion of the contained DNA A total of approximately 2000 micrograms of vector DNA (obtained from paragraph 1C above) was digested overnight at 37 ° C in a 5000 μl reaction containing 2, NEB buffer solution of 2,300 units of ApaLI and 300 units of Xhol. . The restriction digestion reaction was incubated overnight at 37 ° C and analyzed in a pre-made 0.8% agarose gel (Embi Tec, San Diego, CA). The linearized vector DNA was then excised from the gel and extracted with a QIAquick Gel Extraction Kit (QIAGEN Inc.) according to the manufacturer's instructions. Preparation of nucleotides from the library. Six library oligonucleotides (1 fixed and 5 anesthetized) were designed based on the sequences derived from the above results. A fixed library of nucleotides is: 5,-CACAGTGCACAGGGTNNKNNKNNKNNKNNKNNKNNKS ARTGGGATCCGTGGASCNNKNNKNNKNNKNNKNNKN NKCATT CTCTCGAGATCA-3, (Library No. 20) (sequence identification: No. 248) and two 70% anesthetized oligodeoxynucleotides are as follows: 5,-CACAGTGCACAGGGTNNKNNKNNKaaKcgKccKNNKga O:\121\121929.DOC -157- 200808833
KgaKatKttKggKNNKacKtaKcaKNNKNNKNNKCATTCTC TCGAGATCA-3’（庫 27 號）（序列識別：249 號） 5’-CACAGTGCACAGGGTNNKaaKttKaaKccKctKgaKgaKctKgaKga KacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATCA-31 (庫99號）(序列識別·· 250號）； 小寫字母代表具有70%指定鹼基，其他三種核甞酸各 10%的混合物。另外3個91 %麻醉的庫寡核甞酸如下： 5，-CACAGTGCACAGGGTNNKNNKNNKcaKgaKgaKTGCgaKtg KgaKccKtgKacKTGCgaKcaKatKNNKNNKNNKCATTCTCTCGAGA TCA-3,（庫94號）(序列識別：251號）； 5，-CACAGTGCACAGGGTNNKttKgaKtaKNNKgaKggKgtKgaKgaKcc KttKacKttKggKNNKgaKaaKcaKNNKCATTCTCTCGAGATCA-3, (庫25號）（序列識別：252號）； 和 5f-CACAGTGCACAGGGTNNKaaKttKaaKccKctKgaKgaKctKgaKga KacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATCA-3, (庫26號）(序列識別：253號）； 關於以上的募，熟諳此藝者應瞭解ΠΝΠ表示在寡合成期 間，相等地代表四個核苷酸（A、Τ、C和G)中的每一個， 而”Κ"表示在寡合成期間，相等地代表核甞酸G和Τ。小寫 字母代表具有91%指定鹼基，其他三種核苷酸各3%的混合 物。在PCR中分別使用這些寡核甞酸作為模板。 至於PCR反應，使用延伸高忠實性的PCR系統套組 (Roche Diagnostics Corp.)。在含有 InM庫寡核替酸， O:\121\121929.DOC -158- 200808833 1XPCR緩衝溶液，引子： 5，-CACAGTGCACAGGGT-3,（序列識別：254號）； 和 5，-TGATCTCGAGAGAATG-3,（序歹識別·· 255號）； 各 300 nM ; 200 μΜ dNTP，1.5 mM MgCl2和 350單位延 伸聚合酶的96孔50微升之PCR反應中，擴大每個庫寡。使 用熱循環器（GeneAmp PCR System 9700， Applied Biosystems)執行下列的程式：94°C 5分鐘；（94°C 30秒， 52.5〇C 60秒，72°C 30秒）x 25個回合；72°C 10分鐘；冷 卻至4°C。然後使用QIAquick PCR純化套組（QIAGEN Inc. 目錄#28 104)，根據製造者的草案，移除自由的核苷酸。 F.消化庫募核苷酸 在37°C下，在含有2xNEB緩衝溶液2，750單位ApaLI和 750單位Xhol的1200微升反應中，消化每個庫的PCR產物（ 段落1E)過夜。在預先-製造的3%瓊脂糖凝膠（Embi Tec)上 分離經過消化的DNA。從凝膠上切下得自每個反應的感興 趣DNA譜帶，並利用COSTAR自旋_χ離心管濾紙，0.22微 米的乙酸纖維素（Corning Inc.，目錄#8 160)萃取。 G·將載體與庫募核甞酸連接 使450微升的連接反應在16°C下過夜，其含有按照i:5之 莫耳比例的直線化載體（段落1D)和每個經過消化的庫pcR 產物（段落IF)，1 X NEB連接緩衝溶液和2〇,〇〇〇單位的T4 DNA連接酶。在65 C下培養該連接產物2〇分鐘，使Τ4 DNA連接酶失活，並在37°C下與100單位NotI再培養2小時 O:\121\121929.DOC -159- 200808833 ’將載體的自我連接減至最少。然後藉著標準紛/氯仿萃 取作用（Molecular Cloning: A Laboratory Manual，Maniatis 等人，第3版，Cold Spring Harbor Laboratory Press，2000) ’純化連接產物，並再懸浮於120微升H20中。 H·電穿透轉化作用 針對每個庫，進行12個電穿透反應。對於每個轉化作用 ，在0.2-公分的比色杯（BIO-RAD)中，混合1〇微升的連接 載體DNA (段落1G)和300微升的XL1-BLUE MRF，細胞（段 落1A)。藉著設定為2500伏特、25微法拉第和2〇〇歐姆的 Gene Pulserll，脈衝所得的混合物。然後混合得自12個電 穿透反應的轉化細菌，並移至含有26毫升SOC的燒瓶中， 在37°C下培養1小時。將細胞加至450毫升2xYTAG中，並 使其在37 C下振盪生長5小時。在4°C下，以4000 rpm離心 該細胞15分鐘。然後將細胞小球再懸浮於12毫升的15%甘 油/2xYT中’並儲存在_8〇°c下。這是庫的原始母液。滴定 顯示庫的尺寸為5·0χ109 (庫20號）、3·3χ1〇1()(庫94號）、 4·7χ109 (庫 25號）、5·0χ109 (庫 26號）、3.0Χ109 (庫 27號）， 和4·2χ109(庫99號）個獨立的轉化物。 2.庫的擴大作用 Α·製造二級的庫母液 從原始的庫細胞母液（得自上文的段落1 Η)中，使用包括 10Χ過量之每個庫尺寸的足夠細胞，來接種(帶 有100微克/毫升氨苄青黴素、12·5微克/毫升四環素和2%葡 萄糖的2ΥΤ)培養基，使得起始〇d0⑽為〇1。容許培養物在KgaKatKttKggKNNKacKtaKcaKNNKNNKNNKCATTCTC TCGAGATCA-3' (Library No. 27) (sequence identification: No. 249) 5'-CACAGTGCACAGGGTNNKaaKttKaaKccKctKgaKgaKctKgaKga KacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATCA-31 (Library No. 99) (sequence identification · · 250); lowercase letters represent 70% of designated bases, the other three A 10% mixture of each of the nucleosides. Further three oligonucleotide library anesthesia Chang acid 91% are as follows: 5, (library number 94) -CACAGTGCACAGGGTNNKNNKNNKcaKgaKgaKTGCgaKtg KgaKccKtgKacKTGCgaKcaKatKNNKNNKNNKCATTCTCTCGAGA TCA-3 (SEQ ID: No. 251); 5, -CACAGTGCACAGGGTNNKttKgaKtaKNNKgaKggKgtKgaKgaKcc KttKacKttKggKNNKgaKaaKcaKNNKCATTCTCTCGAGATCA-3, (library number 25) ( Sequence identification: 252); and 5f-CACAGTGCACAGGGTNNKaaKttKgaKccKctKgaKgaKctKgaKga KacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATCA-3, (Library No. 26) (sequence identification: No. 253); Regarding the above recruitment, those skilled in the art should understand that ΠΝΠ indicates equal representation during the oligo synthesis. Each of the nucleotides (A, Τ, C, and G), and "Κ" indicates equal representation of nucleotides G and Τ during oligo synthesis. Lowercase letters represent 91% of the specified bases, and the other three A mixture of 3% of each nucleotide. These oligonucleotides were used as templates in PCR, respectively. As for the PCR reaction, a highly faithful PCR system kit (Roche Diagnostics Corp.) was used. Acid, O:\121\121929.DOC -158- 200808833 1XPCR buffer solution, primer: 5,-CACAGTGCACAGGGT-3, (sequence recognition: No. 254); and 5,-TGATCTCGAGAGAATG-3, (preface identification · · 255); each 300 nM; 200 μΜ dNTP, 1.5 mM MgCl2 Each library was expanded in a 96-well 50 μl PCR reaction with 350 units of extended polymerase. The following procedure was performed using a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems): 94 ° C for 5 minutes; (94 ° C 30 seconds, 52.5 ° C 60 seconds, 72 ° C 30 seconds) x 25 rounds; 72 ° C for 10 minutes; cooled to 4 ° C. Then use QIAquick PCR purification kit (QIAGEN Inc. catalog # 28 104), According to the manufacturer's draft, free nucleotides were removed. F. Digested nucleotides were digested at 37 ° C in a 1200 μl reaction containing 2, NEB buffer solution of 2,750 units of ApaLI and 750 units of Xhol. The PCR product of each pool (paragraph 1E) was used overnight. The digested DNA was separated on a pre-made 3% agarose gel (Embi Tec). The DNA bands of interest derived from each reaction were excised from the gel and extracted using a COSTAR spin-χ centrifuge tube filter paper, 0.22 μm of cellulose acetate (Corning Inc., catalog #8 160). G. Linking the vector to the library for nucleating acid to allow 450 microliters of ligation reaction at 16 ° C overnight, containing a linearized vector according to the molar ratio of i: 5 (paragraph 1D) and each digested library The pcR product (paragraph IF), 1 X NEB ligation buffer solution and 2 〇, 〇〇〇 unit of T4 DNA ligase. The ligation product was incubated at 65 C for 2 min to inactivate Τ4 DNA ligase and incubated with 100 units of NotI for 2 h at 37 ° C. O:\121\121929.DOC -159- 200808833 'The vector Self-connection is minimized. The ligation product was then purified by standard chloroform/chloroform extraction (Molecular Cloning: A Laboratory Manual, Maniatis et al., 3rd edition, Cold Spring Harbor Laboratory Press, 2000) and resuspended in 120 μl of H20. H·Electrical Penetration Transformation For each pool, 12 electroporation reactions were performed. For each transformation, 1 〇 microliter of ligation vector DNA (paragraph 1G) and 300 μl of XL1-BLUE MRF, cells (segment 1A) were mixed in a 0.2-cm cuvette (BIO-RAD). The resulting mixture was pulsed by a Gene Pulserll set to 2500 volts, 25 microfarads and 2 ohms. The transformed bacteria obtained from the 12 electroporation reactions were then mixed and transferred to a flask containing 26 ml of SOC, and cultured at 37 ° C for 1 hour. The cells were added to 450 ml of 2xYTAG and allowed to grow at 37 C for 5 hours with shaking. The cells were centrifuged at 4000 rpm for 15 minutes at 4 °C. The cell pellet was then resuspended in 12 ml of 15% glycerol/2xYT and stored at _8 〇 °c. This is the original mother liquor of the library. The size of the titration display library is 5·0χ109 (Library 20), 3·3χ1〇1() (Library 94), 4·7χ109 (Library 25), 5·0χ109 (Library 26), 3.0Χ109 (Library No. 27), and 4·2χ109 (Library 99) independent transformants. 2. Enlargement of the library Α Manufacture of secondary stock solution from the original stock cell mother liquor (from paragraph 1 above), using sufficient cells including 10 Χ excess of each size of the library to inoculate (with A medium of 100 μg/ml ampicillin, 12·5 μg/ml tetracycline and 2% glucose was used to make the starting 〇d0(10) 〇1. Allow cultures at
O:\121\121929.DOC 160- 200808833 37°C下生長，並加以振盪數小時，直到OD6GG==〇 5為止。 從每個庫中取出十分之一的等分，並在37°c下在不同的燒 觀中生長另外2小時。然後在4°C下，使用Beckman JA-14 旋轉子，以4000 rpm離心10分鐘，並將細菌小球再懸浮於 7·〇毫升（每個庫）15°/〇甘油/2xYT中，儲存在-80°C下。 B·噬菌體誘導 將M13K07協助者噬菌體等分（Amersham PharmaciaO:\121\121929.DOC 160- 200808833 Grow at 37 ° C and shake for several hours until OD6GG == 〇 5. One tenth of an aliquot was taken from each pool and grown for an additional 2 hours at 37 ° C in different burns. Then, using a Beckman JA-14 rotator at 4 ° C, centrifuge at 4000 rpm for 10 minutes, and resuspend the bacterial pellets in 7 · 〇 ml (each library) 15 ° / 〇 glycerol / 2 x YT, stored in -80 ° C. B. Phage induction aliquoting of M13K07 facilitator phage (Amersham Pharmacia
Biotech)加至〇D6go = 〇·5之剩下的細菌培養物（得自上文的段 落2A)中，至3x109 pfu/毫升之終濃度。容許該協助者噬菌 體在37C下感染細邊30分鐘’不需振盈’再慢慢地振盈30 分鐘。在4°C下，以5000 rpm離心該感染細胞15分鐘。將 細胞小球再懸浮於相同體積（得自上文的段落2A)的 2xYTAK培養基（帶有1〇〇微克/毫升氨苄青黴素和4〇微克/毫 升康黴素的2YT)中。容許在30°C下發生噬菌體質體的產製 過夜，同時加以振盪。 C·收獲噬菌體 在4°C下，以5000 rpm離心得自上文段落2B的細菌培養 物15分鐘。然後將上清液移至新的瓶子中，並加入〇·2倍 體積的20% PEG/2.5M NaCl ’在冰上培養1小時，使嗤菌體 質體沉澱。在4°C下，以10,〇〇〇 rpm離心已經沉澱的噬菌體 質體30分鐘，並小心地再懸浮於1 〇〇毫升冰冷的pBs中。藉 著在4°C下以4000 rpm離心10分鐘，去掉剩下的細胞，進 一步純化噬菌體質體溶液’並藉著加入〇·2倍體積的2〇0/〇 PEG/2·5MNaCl’使嗤¾體質體沉〉殿。在4。c下，以loooo O:\121\121929.DOC -161 - 200808833 rpm離心嗤菌體質體3 0分鐘，並將嗤菌體質體小球再懸浮 於18毫升冰冷的PBS中。將6毫升60%甘油溶液加至噬菌體 質體的溶液中，並儲存在-80 °C下。藉著標準程序 (Molecular Cloning，Maniatis等人，第3版）判定嗤菌體質體 的力價。 3.擇Ang-2結合噬菌鱧 A·將Ang-2固定在磁性小珠上 以每100毫升得自製造者之小珠母液含2000微克Ang-2蛋 白質的濃度，將生物素基化的Ang-2 (得自上文的段落3 A) 固定在Dynabead M-280鏈黴菌抗生物素蛋白（DYNAL， Lake Success，NY)上。在使用磁鐵將小珠吸引至試管的一 邊’並吸掉液體之後，以填酸緩衝之生理鹽水（PB S)沖洗 小珠兩次，並再懸浮於PBS中。按上述的濃度，將生物素 基化的Ang-2蛋白質加至沖洗過的小珠中，並在室溫下培 養並旋轉1小時。然後藉著加入BSA至2%之終濃度，阻斷 塗覆Ang-2的小珠，並在4。(：下培養過夜，並加以旋轉。然 後在接受選擇程序之前，先以PBST (帶有〇〇5%吐溫·2〇的 PBS)沖洗所得的塗覆Ang-2之小珠兩次。 B·使用塗覆Ang-2之小珠來選擇 利用1毫升含有2% BSA的PBS’阻斷大約1〇〇〇_倍庫相等 物嗟菌體質體（得自上文的段落2C) !小時。藉著將其加至 空白小珠（與段落3八相同的小珠，但無塗覆Αήρ?蛋白質） 中，並在室溫下培養該混合物15分鐘’並加以旋轉，使铖 過阻斷的則體質料樣接受三個陰性選擇步^使用^Biotech) was added to the remaining bacterial culture of 〇D6go = 〇·5 (from paragraph 2A above) to a final concentration of 3 x 109 pfu/ml. The helper phage was allowed to infect the fine side at 37 C for 30 minutes 'without vibration' and then slowly oscillate for 30 minutes. The infected cells were centrifuged at 5000 rpm for 15 minutes at 4 °C. The cell pellet was resuspended in 2 x YTAK medium (2YT with 1 〇〇 microgram/ml ampicillin and 4 〇 microgram/ml ampinomycin) in the same volume (paragraph 2A above). Production of phage plastids was allowed to occur overnight at 30 ° C while shaking. C. Harvesting phage The bacterial culture from paragraph 2B above was centrifuged at 5000 rpm for 15 minutes at 4 °C. The supernatant was then transferred to a new bottle and incubated with 2 volumes of 20% PEG/2.5 M NaCl' for 1 hour on ice to precipitate the plastid body. The precipitated phage bodies were centrifuged at 10, rpm for 10 minutes at 4 ° C and carefully resuspended in 1 ml of ice-cold pBs. After centrifugation at 4000 rpm for 10 minutes at 4 ° C, the remaining cells were removed, and the phage plastid solution was further purified 'by 〇·2 volumes of 2〇0/〇PEG/2·5MNaCl'. 3⁄4 Physique sinks the temple. In; 4. Under c, centrifuge the plastid body for 30 minutes at loooo O:\121\121929.DOC -161 - 200808833 rpm, and resuspend the plastid pellet in 18 ml of ice-cold PBS. 6 ml of a 60% glycerol solution was added to the phage plastid solution and stored at -80 °C. The price of the plastid body was determined by standard procedures (Molecular Cloning, Maniatis et al., 3rd edition). 3. Select Ang-2 binding phage A. Fix Ang-2 on magnetic beads to biotinylated at a concentration of 2000 micrograms of Ang-2 protein per 100 milliliters of mother beads from the manufacturer. Ang-2 (from paragraph 3 A above) was immobilized on Dynabead M-280 Streptomyces avidin (DYNAL, Lake Success, NY). After the beads were attracted to one side of the test tube using a magnet and the liquid was aspirated, the beads were washed twice with acid buffered saline (PB S) and resuspended in PBS. Biotinylated Ang-2 protein was added to the washed beads at the above concentrations and incubated at room temperature for 1 hour. The Ang-2 coated beads were then blocked by the addition of BSA to a final concentration of 2% and at 4. (: Incubate overnight and rotate. Then, before receiving the selection procedure, the resulting Ang-2 coated beads were washed twice with PBST (PBS with 〇〇 5% Tween 2 。). • Use an Ang-2 coated bead to select about 1 liter of PBS containing 2% BSA to block the plastid body (obtained from paragraph 2C above)! By adding it to a blank bead (the same bead as in paragraph 3-8, but without coating the ?ρ? protein) and culturing the mixture for 15 minutes at room temperature' and rotating it to block the sputum Then the body material sample accepts three negative selection steps ^ use ^
O:\121\121929.DOC -162- 200808833 鐵吸出含有上清液的嗤菌體質體，並移至第二個含有空白 小珠（與在上文段落3 A中之描述相同的小珠，但其上無塗 覆Ang-2蛋白質）的試管中，並在室溫下培養該混合物15分 鐘，並加以旋轉。 重覆邊程序。使用磁鐵吸出含有上清液的嗤菌體質體， 並移至含有塗覆Ang-2蛋白質之小珠（得自段落3A)的新試 管中，並在室溫下培養該混合物1小時，並加以旋轉。在 拋棄上清液之後，以2%牛奶_PBS沖洗與噬菌體質體結合 的小珠10次；以2% BSA-PBS沖洗10次；以PBST沖洗1〇次 ;並以PBS沖洗2次。容許在旋轉器上，以i毫升10〇瓜“三 乙胺溶液（Sigma，St· Louis，MO)洗脫該噬菌體質體1〇分鐘 。藉著加入0.5毫升1 M Tris_HCl (pH 7.5)中和含有噬菌體 質體之溶液的pH值。使用所得的噬菌體質體，在37^下感 染10毫升新近生長的XL 1-Blue MRF，細菌（〇D_大約0.5) 3 0分鐘，不需振盪，再慢慢地振盪3 〇分鐘。然後將所有被O:\121\121929.DOC -162- 200808833 The iron sucks out the plastid body containing the supernatant and moves to the second one containing the blank beads (the same beads as described in paragraph 3 A above). However, in the test tube without the Ang-2 protein coated thereon, the mixture was incubated at room temperature for 15 minutes and rotated. Repeat the edge program. The plastid body containing the supernatant was aspirated using a magnet and transferred to a new tube containing the coated Ang-2 protein beads (obtained from paragraph 3A), and the mixture was incubated at room temperature for 1 hour. Rotate. After discarding the supernatant, the beads bound to the phage plastids were washed 10 times with 2% milk_PBS; 10 times with 2% BSA-PBS; 1 wash with PBST; and washed twice with PBS. The phage plastids were allowed to elute on a rotator with 1 ml of 10 guan melon "Triethylamine solution (Sigma, St. Louis, MO) for 1 min. by adding 0.5 ml of 1 M Tris_HCl (pH 7.5) to neutralize. The pH of the solution containing the phage plastid. Using the resulting phage plastid, infect 10 ml of newly grown XL 1-Blue MRF at 37 °, bacteria (〇D_about 0.5) for 30 minutes, without shaking, and then Slowly oscillate for 3 〇 minutes. Then all will be
感的XL 1 -BLUE MRF’細胞平舖在15x15公分的2xYTAG 盤上，並在30°C下培養過夜。 C·誘導和收獲噬菌體 將10毫升等分的2XYTAGT培養基加至培養盤（得自段落 3B)中，再懸浮XL1-BLUE MRF，細胞。將所有的XL1_ BLUE MRF’細胞收集在試管中，並將25〇微升等分的這些 細胞加至25毫升2xYTAGT中，使其在37°c下生長，直到 OD_ = 0.5為止。加入M13K07協助者噬菌體，至3 χ 1〇9 Pfu/毫升之終濃度，並在37。(：下培養3〇分鐘，不需振盪， O:\121\121929.DOC -163 - 200808833 再〖又〖又地振盈3 0分鐘。在4 °C下，以5 000 rpm離心該細胞 10分鐘，並再懸浮於25毫升2χΥΤΑΚ中。容許這些細菌在 30 C下生長過夜，並加以振盪。按照段落2c，收獲並純化 經過誘導的噬菌體質體。 D·第2回合選擇 按照在段落3B至3C中概述的進行第2回合的選擇，除了 以下的之外。使用得自段落3C之大約100-倍庫相等物噬菌 體貝體作為輸入嗟菌體質體。將塗覆在Dynabead M-280鏈 黴菌抗生物素蛋白上的生物素基化之Ang-2蛋白質的含量（ 段落3A)減少至20毫微克。然後以2°/。牛奶-PBS沖洗與嗤菌 體質體結合的小珠10次；以2% BSA-PBS沖洗1〇次；以 PBST沖洗1 〇次，在此處最後的沖洗涉及在室溫下，在 PB ST中培養60分鐘。以PB S沖洗小珠兩次。洗脫條件與第 1回合（段落3B)相同。 E·第3回合選擇 按照在段落3B至3C中概述的進行第3回合的選擇，除了 以下的之外。使用得自段落3D之大約10-倍庫相等物嗟菌 體質體作為輸入噬菌體質體。使用大約20毫微克的生物素 基化之Ang_2蛋白質（得自段落3A)，來塗覆Dynabea(i 280鏈黴菌抗生物素蛋白。以2%牛奶-PBS沖洗與噬菌體質 體結合的小珠10次；以2% BSA-PBS沖洗10次；以PBST沖 洗10次，在此處最後的沖洗涉及在室溫下，在PBST中培 養60分鐘。以PBS沖洗小珠兩次。洗脫條件與第1回合（段 落3B)相同。 O:\121\121929.DOC -164- 200808833 F.第4回合選擇 按照在段落3B至3C中概述的進行第4回合的選擇，除了 以下的之外。使用得自段落3E之庫相等物噬菌體質體作為 輸入噬菌體質體。對於庫25、26和27，將塗覆在Dynabead M-280鏈黴菌抗生物素蛋白上的生物素基化之Ang-2蛋白質 的含量（段落3 A)減少至0.4毫微克。對於庫20和94，塗覆含 量保持為第3回合的2毫微克。庫99不進行第4回合的選擇 步驟。洗脫條件與第1回合（段落3B)相同。 4.選殖分析 A. 製備主要的培養盤 挑選得自第2回合選擇的單一菌落，並接種在每孔含有 120微升2xYTAGT的96孔培養盤中。在30°C振盪器中，培 養該96孔培養盤過夜。在每孔中加入40微升60%甘油，儲 存在-80°C下。The XL 1 -BLUE MRF' cells were plated on a 15 x 15 cm 2x YTAG plate and incubated overnight at 30 °C. C. Induction and harvesting of phage A 10 ml aliquot of 2XYTAGT medium was added to the plate (from paragraph 3B) and the XL1-BLUE MRF, cells were resuspended. All XL1_BLUE MRF' cells were collected in tubes and 25 μL aliquots of these cells were added to 25 ml 2xYTAGT and grown at 37 ° C until OD_ = 0.5. Add M13K07 facilitator phage to a final concentration of 3 χ 1〇9 Pfu/ml and at 37. (: culture for 3 minutes, no need to oscillate, O:\121\121929.DOC-163 - 200808833 and then 〖also oscillate for 30 minutes. Centrifuge the cell at 5 000 rpm at 4 °C. Minutes, and resuspend in 25 ml of 2 。. Allow these bacteria to grow overnight at 30 C and shake them. Harvest and purify the induced phage plastids according to paragraph 2c. D·2nd round is selected according to paragraph 3B The selection of the second round outlined in 3C, except for the following, uses approximately 100-fold equivalent phage shells from paragraph 3C as input sputum plastids. Will be coated on Dynabead M-280 Streptomyces The amount of biotinylated Ang-2 protein on avidin (paragraph 3A) was reduced to 20 ng. The beads bound to the plastid body were then washed 10 times with 2°/milk-PBS; Rinse 1% BSA-PBS 1 time; rinse 1 time with PBST, where the final rinse involves incubation in PB ST for 60 minutes at room temperature. Wash the beads twice with PB S. The first round (paragraph 3B) is the same. E. The third round is selected in accordance with paragraphs 3B to 3C. The selection of the third round was carried out except that the following 10-fold library equivalents of phage plastids from paragraph 3D were used as input phage plastids. About 20 ng of biotinylated Ang 2 protein was used. (from paragraph 3A) to coat Dynabea (i 280 Streptomyces avidin. The beads bound to the phage plastids were washed 10 times with 2% milk-PBS; 10 times with 2% BSA-PBS; The PBST was rinsed 10 times, where the final rinse involved incubation in PBST for 60 minutes at room temperature. The beads were rinsed twice with PBS. The elution conditions were the same as in the first round (paragraph 3B). O:\121\ 121929.DOC-164-200808833 F. Round 4 selection was carried out in accordance with the selection of the fourth round as outlined in paragraphs 3B to 3C, except for the following: using the library equivalent phage plastid from paragraph 3E as the input phage Platinum. For pools 25, 26 and 27, the content of biotinylated Ang-2 protein (paragraph 3 A) coated on Dynabead M-280 Streptomyces avidin was reduced to 0.4 nanograms. Pools 20 and 94, coating content maintained at 2 ng for the third round. Library 99 The selection step of the fourth round is carried out. The elution conditions are the same as in the first round (paragraph 3B). 4. Selection analysis A. Preparation of the main culture plate The single colonies selected from the second round are selected and inoculated in each well. 120 microliters of 2x YTAGT in 96 well plates. The 96 well plates were incubated overnight in a 30 °C shaker. 40 microliters of 60% glycerol was added to each well and stored at -80 °C.
B. 噬菌髏質體ELISA 將得自主要培養盤的大約2微升等分的細胞（得自上文的 段落4A)，接種在新鮮的Costar® 96孔培養盤（Corning incorporated，Corning，NY，目錄#9794)中，其每孔含 100 微升2xYTAGT，並使該新培養盤的細胞在37°C下生長，直 到大約Ο D 6 〇 〇=0.5為止。 將40微升含有M13K07協助者噬菌體（1.5 X 1013cfu/毫升 )的2xYTAGT加至各孔中，並在37°C下培養96孔培養盤30 分鐘，不需振盪，再慢慢地振盪30分鐘。在4 °C下，以 2000 rpm (Beckman CS-6R桌上型離心機）離心培養盤10分 O:\121\121929.DOC -165- 200808833 鐘。從孔中移除上清液，並使用每孔150微升2xYTAK，再 懸浮每個細胞小球。為了噬菌體質體表現，在30°C下培養 該盤過夜。 在4°C下，以1微克/毫升在lxPBS中，將人類Ang-2蛋白 質塗覆在96孔Maxisorp培養盤（NUNC)上過夜。在第二天 時，在4°C下，以2000 rpm離心過夜的細胞培養物10分鐘 。將得自每孔的各10微升上清液移至新的、含有BSA/PBS 溶液的96孔培養盤中，以1:1〇稀釋上清液。在室溫下培養 所得的混合物1小時，並加以振盪，以便阻斷噬菌體質體 。在此時，在室溫下以每孔400微升2% BSA/PBS溶液阻斷 塗覆Ang-2蛋白質的培養盤1小時，同時加以振盪。拋棄 BSA溶液，並以PBS溶液沖洗各孔3次。在最後一次的沖洗 步驟之後，在塗覆Ang-2蛋白質之培養盤和對照組培養盤 的各孔中，加入100微升經過阻斷的噬菌體質體溶液，並 在室溫下培養1小時，並加以攪拌。拋棄液體，並以PBST 溶液沖洗各孔三次。以15,000之稀釋度，在塗覆Ang-2蛋 白質和對照組培養盤的各孔中，加入1 00微升HRP-共軛之 抗-M13 mAb (Amersham Pharmacia Biotech)，並在室溫下 培養這些培養盤1小時，並加以攪拌。再度拋棄液體，並 以PBST溶液沖洗各孔三次。在孔中加入1〇〇微升LumiGLO 化學發光受質（Kirkegaard & Perry Laboratories， Gaithersburg，MD)，並藉著Luminoskan Ascent DLRearly機 器（Labsystems，Franklin, MA)讀取各孔。 C.噬菌體純種系的定序 O:\121\121929.DOC -166- 200808833 使用1微升得自主要培養盤（段落4A)之各孔的細菌作為 模板。每個PCR混合物的體積為50微升，其含有1 X PCR缓 衝溶液、兩個引子： 5,-GTTAGCTCACTCATTAGGCAC-3,（序列識別：256號）和 5，-GTACCGTAACACTGAGTTTCG_3,（序列識另J : 257號）；B. Phage plastid ELISA Approximately 2 microliter aliquots of cells from the primary culture plate (obtained from paragraph 4A above) were seeded in fresh Costar® 96-well plates (Corning incorporated, Corning, NY) , catalog #9794), containing 100 microliters of 2x YTAGT per well, and allowing the cells of the new plate to grow at 37 ° C until approximately Ο D 6 〇〇 = 0.5. 40 μl of 2xYTAGT containing M13K07 helper phage (1.5 X 1013 cfu/ml) was added to each well, and the 96-well culture plate was incubated at 37 ° C for 30 minutes without shaking, and then slowly shaken for 30 minutes. Centrifuge the plate at 2000 rpm (Beckman CS-6R tabletop centrifuge) at 10 °C for 10 minutes O:\121\121929.DOC -165- 200808833. The supernatant was removed from the wells and each cell pellet was resuspended using 150 microliters of 2x YTAK per well. For phage plastid expression, the plate was incubated overnight at 30 °C. Human Ang-2 protein was plated on a 96-well Maxisorp plate (NUNC) overnight at 1 °C in 1 μg/ml in lxPBS. On the next day, the cell culture was centrifuged at 2000 rpm for 10 minutes at 4 °C. Ten microliters of supernatant from each well was transferred to a new 96-well plate containing BSA/PBS solution and the supernatant was diluted 1:1. The resulting mixture was incubated at room temperature for 1 hour and shaken to block phage plastids. At this time, the plate coated with Ang-2 protein was blocked with 400 μl of 2% BSA/PBS solution per well at room temperature for 1 hour while shaking. The BSA solution was discarded and the wells were rinsed 3 times with PBS solution. After the last rinsing step, 100 μl of the blocked phage plastid solution was added to each well of the culture plate coated with Ang-2 protein and the control plate, and incubated at room temperature for 1 hour. And stir. Discard the liquid and rinse each well three times with PBST solution. 100 μl of HRP-conjugated anti-M13 mAb (Amersham Pharmacia Biotech) was added to each well of the Ang-2 protein and control culture plates at a dilution of 15,000, and these were cultured at room temperature. The plate was incubated for 1 hour and stirred. The liquid was again discarded and the wells were rinsed three times with PBST solution. One microliter of LumiGLO chemiluminescent substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the wells and each well was read by a Luminoskan Ascent DLRearly machine (Labsystems, Franklin, MA). C. Sequencing of the phage pure lineage O: \121\121929.DOC -166- 200808833 A microliter of bacteria obtained from each well of the main culture dish (Section 4A) was used as a template. Each PCR mixture has a volume of 50 μl containing 1 X PCR buffer solution, two primers: 5,-GTTAGCTCACTCATTAGGCAC-3, (sequence recognition: 256) and 5,-GTACCGTAACACTGAGTTTCG_3, (sequence identification J: No. 257);
各 300 nM ； 200uM dNTP，2mM MgCl2 和 2.5 單位 taq DNA 聚合酶（Roche Molecular Biochemicals)。使用 GeneAmp PCR 系統 9700 (Applied Biosystems)，執行下列 的程序：94°C 5 分鐘；（94°C 45 秒，55°C 45 秒，72°C 90秒）的40個回合；72 °C 10分鐘；冷卻至4 °C。利用 QIAquick 96 PCR純化套組（QIAGEN Inc.)，根據製造者的 指示，純化 PCR產物。利用引子 5’-TTACACTTTATGCTTCCG-3’（序列識別：258 號），使用 ABI 3770定序器（Perkin Elmer)，根據製造者的指示，定出 所有經過純化之PCR產物的序列。 5.序列排列 將已經從核苷酸序列轉譯的肽序列（得自上文的段落4C) ，與ELISA資料連貫起來。認為在塗覆Ang-2之孔中顯示 出高OD讀數，並在塗覆BSA之孔中顯示出低OD讀數的純 種系是較重要的。亦認為出現多次的序列是重要的。從庫 20中選出24個肽序列，從庫94中選出26個肽序列，從庫25 中選出7個肽序列，從庫26中選出18個肽序列，從庫27中 選出6個肽序列，並從庫99中選出4個肽序列，進行進一步 的分析和肽體產製。此外，亦從庫20和94中推衍出11個一 O:\121\121929.DOC -167- 200808833 致序列，從庫26和99中推衍出3個一致序列，並從庫25中 推衍出2個一致序列，用來產製肽體。使用在本文之實例 10中描述的中和ELISA草案，來評估表7中的肽體。在表7 中出示該結果。 表7300 nM each; 200 uM dNTP, 2 mM MgCl2 and 2.5 units of taq DNA polymerase (Roche Molecular Biochemicals). Using GeneAmp PCR System 9700 (Applied Biosystems), the following procedure was performed: 94 ° C for 5 minutes; (94 ° C for 45 seconds, 55 ° C for 45 seconds, 72 ° C for 90 seconds) 40 rounds; 72 ° C for 10 minutes ; Cool to 4 °C. The PCR product was purified using a QIAquick 96 PCR Purification Kit (QIAGEN Inc.) according to the manufacturer's instructions. Using the primer 5'-TTACACTTTATGCTTCCG-3' (SEQ ID NO: 258), the sequence of all purified PCR products was determined using the ABI 3770 sequencer (Perkin Elmer) according to the manufacturer's instructions. 5. Sequence alignment The peptide sequence (obtained from paragraph 4C above) that has been translated from the nucleotide sequence is contiguous with the ELISA data. It is believed that a high OD reading is shown in the wells coated with Ang-2 and a pure line showing a low OD reading in the BSA coated well is more important. It is also considered that the occurrence of multiple sequences is important. 24 peptide sequences were selected from library 20, 26 peptide sequences were selected from library 94, 7 peptide sequences were selected from library 25, 18 peptide sequences were selected from library 26, and six peptide sequences were selected from library 27. Four peptide sequences were selected from library 99 for further analysis and peptidomimetic production. In addition, 11 O-\121\121929.DOC-167-200808833 sequences were derived from the libraries 20 and 94, and three consistent sequences were derived from the libraries 26 and 99, and pushed from the library 25. Two consensus sequences were derived for the production of peptibodies. The peptibodies in Table 7 were evaluated using the neutralizing ELISA draft described in Example 10 herein. The results are shown in Table 7. Table 7
Co4衍生之親和 力-成熟的Pbs hAng-2:Tie2 IC5〇 (nM) 肽體序列(序列識別號） C〇n4-44 (C) 0.09 M-Fc-GGGGGAQ-PIRQEECDWDPWTCEHMWEV-LE (序列識別：259號） Con4-40 (C) 0.10 M-Fc-GGGGGAQ-TNIQEECEWDPWTCDHMPGK-LE (序列識別：260號） Con4-4 (C) 0.12 M-Fc-GGGGGAQ- WYEQDACEWDPWTCEHMAEV-LE (序列識別：261號） Con4-31 (C) 0.16 M-Fc-GGGGGAQ-MRLQEVCEWDPWTCEHMENV-LE (序列識別：262號） Con4-C5 (C) 0.16 M-Fc-GGGGGAQ-AATQEECEWDPWTCEHMPRS-LE (序列識別：263號） Con4-42 (C) 0.17 M-Fc-GGGGGAQ-LRHQEGCEWDPWTCEHMFDW-LE (序列識別：264號） O:\121\121929.DOC 168- 200808833Co4-derived Affinity-Mature Pbs hAng-2: Tie2 IC5〇(nM) Peptide Sequence (SEQ ID NO: C〇n4-44 (C) 0.09 M-Fc-GGGGGAQ-PIRQEECDWDPWTCEHMWEV-LE (Sequence Recognition: No. 259 Con4-40 (C) 0.10 M-Fc-GGGGGAQ-TNIQEECEWDPWTCDHMPGK-LE (Sequence recognition: No. 260) Con4-4 (C) 0.12 M-Fc-GGGGGAQ- WYEQDACEWDPWTCEHMAEV-LE (Sequence recognition: No. 261) Con4-31 (C) 0.16 M-Fc-GGGGGAQ-MRLQEVCEWDPWTCEHMENV-LE (SEQ ID NO: 262) Con4-C5 (C) 0.16 M-Fc-GGGGGAQ-AATQEECEWDPWTCEHMPRS-LE (Sequence recognition: No. 263) Con4-42 (C) 0.17 M-Fc-GGGGGAQ-LRHQEGCEWDPWTCEHMFDW-LE (Sequence Identification: No. 264) O:\121\121929.DOC 168- 200808833
Con4-35 (C) 0.18 M-Fc-GGGGGAQ-VPRQKDCEWDPWTCEHMYVG-LE (序列議別：265號） Con4‘43 (C) 0.18 M-Fc-GGGGGAQ- , SISHEECEWDPWTCEHMQVG-LE (序列識別：266¾) Con4-49 (C) 0.19 M-Fc-GGGGGAQ- WAAOEECEWDPWTCEHMGRM-LE (序列識別：267號） Con4-27 (C) 0.22 M-Fc-GGGGGAQ-TWPQDKCEWDPWTCEHMGST-LE (序列識別：268號） Con4-48 (C) 0.26 M-Fc-GGGGGAQ-GHSQEECGWDPWTCEHMGTS-LE (序列識別：269ife) Con4-46 (C) 0.26 M-Fc-GGGGGAQ-QHWQEECEWDPWTCDHMPSK-LE (序列識別·· 270號） Con4-41 (C) 0.26 M-Fc-GGGGGAQ-NVRQEKCEWDPWTCEHMPVR-LE (序列議別·· 271號） Con4-36 (C) 0.28 M-Fc-GGGGGAQ-KSGOVECNWDPWTCEHMPRN-LE (寺列識別：272號） Con4-34 (C) 0.28 M-Fc-GGGGGAQ- VKTQEHCDWDPWTCEHMREW-LE (序列識別：273號） Con4-28 (C) 0.30 M-Fc-GGGGGAQ- AWGQEGCDWDPWTCEHMLPM^LE (序列識別：274號） Con4-39 (C) 0.30 M-Fc-GGGGGAQ-PVNQEDCEWDPWTCEHMPPM-LE (序列議別：275號） Con4-25 (C) 0.31 M-Fc-GGGGGAQ-RAPQEDCEWDPWTCAHMDIK-LE (寺列議別：276號） O:\121\121929.DOC 169- 200808833Con4-35 (C) 0.18 M-Fc-GGGGGAQ-VPRQKDCEWDPWTCEHMYVG-LE (sequence discrimination: 265) Con4'43 (C) 0.18 M-Fc-GGGGGAQ- , SISHEECEWDPWTCEHMQVG-LE (sequence recognition: 2663⁄4) Con4-49 (C) 0.19 M-Fc-GGGGGAQ-WAAOEECEWDPWTCEHMGRM-LE (SEQ ID NO: 267) Con4-27 (C) 0.22 M-Fc-GGGGGAQ-TWPQDKCEWDPWTCEHMGST-LE (Sequence recognition: No. 268) Con4-48 (C) 0.26 M-Fc-GGGGGAQ-GHSQEECGWDPWTCEHMGTS-LE (SEQ ID NO: 269ife) Con4-46 (C) 0.26 M-Fc-GGGGGAQ-QHWQEECEWDPWTCDHMPSK-LE (Sequence Recognition·· No. 270) Con4-41 (C) 0.26 M-Fc- GGGGGAQ-NVRQEKCEWDPWTCEHMPVR-LE (Sequence No. 271) Con4-36 (C) 0.28 M-Fc-GGGGGAQ-KSGOVECNWDPWTCEHMPRN-LE (Temple Column Identification: No. 272) Con4-34 (C) 0.28 M-Fc-GGGGGAQ - VKTQEHCDWDPWTCEHMREW-LE (Sequence recognition: No. 273) Con4-28 (C) 0.30 M-Fc-GGGGGAQ- AWGQEGCDWDPWTCEHMLPM^LE (Sequence recognition: No. 274) Con4-39 (C) 0.30 M-Fc-GGGGGAQ-PVNQEDCEWDPWTCEHMPPM-LE (Sequence Disclosure: No. 275) Con4-25 (C) 0.31 M-Fc-GGGGGAQ-RAPQEDCEWDPWTCAHMDIK-LE (Temple Column: No. 276) O:\121\121929.DOC 169- 200808833
Con4-50 (C) 0.38 M-Fc-GGGGGAQ· HGONMECEWDPWTCEHMFRY-LE (序列識別：277號） Con4-38 (C) 0.40 M-Fc-GGGGGAQ-PRLQEECVWDPWTCEHMPLR-LE (序列識別：278號） Con4-29 (C) 0.41 M-Fc-GGGGGAQ-RTTOEKCEWDPWTCEHMESQ-LE (序列識別：279ft) Con4-47 (C) 0.44 M-FoGGGGGAQ-OTSQEDCVWDPWTCDHMVSS-LE (序列識別：280ft) Con4-20 (C) 0.48 M-Fc-GGGGGAQ-ΟνίΟΚΡΟΕ^ΡΨΊΌΕΗίΕΟΙ^Ε (序列識別：281號） Con4-45 (C) 0.48 M-Fc-GGGGGAQ- WAOOEECAWDPWTCDHMVGL-LE (房列識別：282號） Con4-37 (C) 0.49 M-Fc-GGGGGAQ-Γ PGOEDCEWDP WTCEHMVRS-LE (序列識別：283號） Con4-33 (C) 0.52 M-Fc-GGGGGAQ-PMN〇VECDWDPWTCEHMPRS-LE (序列識別：284») AC2-Con4 (C) 0.52 M-Fc-GGGGGAQ-FGWSHGCEWDPWTCEHMGST-LE (外列識別：285號） Con4-32 (C) 0.75 M-Fc-GGGGGAQ-KSTQDDCDWDPWTCEHMVGP-LE (序列識別：286») Con4-17 (C) 0.96 M-Fc-GGGGGAQ- * GPRISTCQWDPWTCEHMDQL-LE (序列識別：287號） Con4-8 (C) 1.20 M-Fc-GGGGGAQ-STIGDMCEWDPWTCAHMQVD-LE (序列識別：288號） AC4-Con4 (C) 1.54 M-Fc-GGGGGAQ-VLGGOGCEWDPWTCRLLQGW-LE (序列識別：289號） Con4-l (C) 2.47 M-Fc-GGGGGAQ-VLGGQGCQWDPWTCSHLEDG-LE (序列識別：290號） Con4-Cl (C) 2.75 M-Fc-GGGGGAQ- TTIGSMCEWDPWTCAHMQGG-LE O:\121\121929.DOC -170- 200808833 (吃列識別：291號） Con4-21 (C) 3.21 M-Fc-GGGGGAQ-TKGKSVCQWDPWTCSHMQSG-LE (序列識別·· 292號） Con4-C2 (C) 3.75 M-F c-GGGGG AQ-TTIGSMCQWDPWTCAHMQGG-LE (涔列識別：293¾) Con4-18 (C) 4.80 M-Fc-GGGGGAQ- WVNEVVCEWDPWTCNHWDTP-LE (序列識別：294ft) Con4-19 (C) 5.76 * M-Fc-GGGGGAQ- VVQVGMCQWDPWTCKHMRLQ-LE (序列識別：295 ft) Con4-16 (C) 6.94 M-Fc-GGGGGAQ-AVGSQTCEWDPWTCAHLVEV-LE (序列議別：296就） Con4-ll (C) 9.70 M-Fc-GGGGGAQ-QGMKMFCEWDPWTCAHIVYR-LE (序列識別：297號） Con4-C4 (C) 9.80 M-Fc-GGGGGAQ-TTIGSMCQWDPWTCEHMQGG-LE (序列識別：298猇） Con4-23 (C) 9,88 M-Fc-GGGGGAQ-TSQRVGCEWDPWTCQHLTYT-LE (寺列識別：299*) Con4-15 (C) 15.00 M-Fc-GGGGGAQ-QWSWPPCEWDPWTCQTVWPS-LE (序列識別：300¾) Con4-9 (C) 20.11 M-Fc-GGGGGAQ-GTSPSFCQWDPWTCSHMVQG-LE (序列識別：3014*;) Con4-10 (C) 86.61 M-Fc-GGGGGAQ-TQGLHQCEWDPWTCKVLWPS-LE (序列識別·· 302號） Con4-22 (C) 150,00 M-Fc-GGGGGAQ- VWRSQVCQWDPWTCNLGGDW-LE (序列識別：303號） Con4-3 (C) 281.50 M-Fc-GGGGGAQ-DKILEECOWDPWTCQFFYGA-LE (序列識別：304«) O:\121\121929.DOC 171 - 200808833Con4-50 (C) 0.38 M-Fc-GGGGGAQ· HGONMECEWDPWTCEHMFRY-LE (Sequence recognition: No. 277) Con4-38 (C) 0.40 M-Fc-GGGGGAQ-PRLQEECVWDPWTCEHMPLR-LE (Sequence recognition: No. 278) Con4-29 ( C) 0.41 M-Fc-GGGGGAQ-RTTOEKCEWDPWTCEHMESQ-LE (sequence recognition: 279 ft) Con4-47 (C) 0.44 M-FoGGGGGAQ-OTSQEDCVWDPWTCDHMVSS-LE (sequence recognition: 280 ft) Con4-20 (C) 0.48 M-Fc-GGGGGAQ -ΟνίΟΚΡΟΕ^ΡΨΊΌΕΗίΕΟΙ^Ε (Sequence recognition: No. 281) Con4-45 (C) 0.48 M-Fc-GGGGGAQ- WAOOEECAWDPWTCDHMVGL-LE (Room identification: No. 282) Con4-37 (C) 0.49 M-Fc-GGGGGAQ- Γ PGOEDCEWDP WTCEHMVRS-LE (SEQ ID NO: 283) Con4-33 (C) 0.52 M-Fc-GGGGGAQ-PMN〇VECDWDPWTCEHMPRS-LE (Sequence recognition: 284») AC2-Con4 (C) 0.52 M-Fc-GGGGGAQ- FGWSHGCEWDPWTCEHMGST-LE (external identification: No. 285) Con4-32 (C) 0.75 M-Fc-GGGGGAQ-KSTQDDCDWDPWTCEHMVGP-LE (sequence identification: 286») Con4-17 (C) 0.96 M-Fc-GGGGGAQ- * GPRISTCQWDPWTCEHMDQL- LE (Sequence Identification: No. 287) Con4-8 (C) 1.20 M-Fc-GGGGGAQ-STIGDMCEWDPWTCAHMQVD-LE (Sequence Identification: No. 288) AC4-Con4 (C) 1.54 M-Fc-GGGGGAQ-VLGGOGCEWDPWTCRLLQGW-LE (SEQ ID NO: 289) Con4-l (C) 2.47 M-Fc-GGGGGAQ-VLGGQGCQWDPWTCSHLEDG-LE (Sequence recognition: No. 290) Con4-Cl (C) 2.75 M-Fc -GGGGGAQ- TTIGSMCEWDPWTCAHMQGG-LE O:\121\121929.DOC -170- 200808833 (eat column identification: 291) Con4-21 (C) 3.21 M-Fc-GGGGGAQ-TKGKSVCQWDPWTCSHMQSG-LE (sequence identification · · No. 292) Con4-C2 (C) 3.75 MF c-GGGGG AQ-TTIGSMCQWDPWTCAHMQGG-LE (column identification: 2933⁄4) Con4-18 (C) 4.80 M-Fc-GGGGGAQ- WVNEVVCEWDPWTCNHWDTP-LE (sequence identification: 294ft) Con4-19 (C 5.76 * M-Fc-GGGGGAQ- VVQVGMCQWDPWTCKHMRLQ-LE (sequence recognition: 295 ft) Con4-16 (C) 6.94 M-Fc-GGGGGAQ-AVGSQTCEWDPWTCAHLVEV-LE (sequence resolution: 296) Con4-ll (C) 9.70 M-Fc-GGGGGAQ-QGMKMFCEWDPWTCAHIVYR-LE (SEQ ID NO: 297) Con4-C4 (C) 9.80 M-Fc-GGGGGAQ-TTIGSMCQWDPWTCEHMQGG-LE (Sequence recognition: 298猇) Con4-23 (C) 9,88 M- Fc-GGGGGAQ-TSQRVGCEWDPWTCQHLTYT-LE (Temple identification: 299*) Con4-15 (C) 15.00 M-Fc-GGGGGAQ-QWSWPPCEWDPWTCQTVWPS-LE (Sequence recognition: 3003⁄4) Con4-9 (C) 2 0.11 M-Fc-GGGGGAQ-GTSPSFCQWDPWTCSHMVQG-LE (sequence recognition: 3014*;) Con4-10 (C) 86.61 M-Fc-GGGGGAQ-TQGLHQCEWDPWTCKVLWPS-LE (sequence identification · · 302) Con4-22 (C) 150, 00 M-Fc-GGGGGAQ- VWRSQVCQWDPWTCNLGGDW-LE (Sequence recognition: No. 303) Con4-3 (C) 281.50 M-Fc-GGGGGAQ-DKILEECOWDPWTCQFFYGA-LE (Sequence identification: 304«) O:\121\121929.DOC 171 - 200808833
Con4-5 (C) 無抑制作用 M-Fc-GGGGGAQ- ATFARQCQWDPWTCALGGNW-LE (序列識別：305號） Con4-30 (C) 無抑制作用 M-Fc-GGGGGAQ-GPAQEECEWDPWTCEPLPLM-LE (序列識別·· 306») Con4-26 (C) 無抑制作用 M-Fc-GGGGGAQ- RPEDMCSQWDPWTWHLQGYC-LE (序列識別：3〇7«t) Con4-7 (C) 無抑制作1 M-Fc-GGGGGAQ- LWQLAVC〇WDP〇TCDHMGAL-LE (务列識別：308统 Con4-12 (C) 無抑制作用 M-Fc-GGGGGAQ-TOLVSLCEWDPWTCRLLDGW-LE (序列識別·· 309號） Con4-13 (C) 無抑制作用 M-Fc-GGGGGAQ· MGGAGRCEWDPWTCQLLQGW-LE (房列識別：310號） Con4-14 (C) 無抑制作用 M-Fc-GGGGGAQ-MFLPNECQWDPWTCSNLPEA-LE (岸列鐵別：311號） Con4-2 (C) 無抑制作用 M-Fc-GGGGGAQ-FGWSHGCEWDPWTCRLLQGW-LE (房列識別：312號） Con4-6 (C) 無抑制作用 _ M-Fc-GGGGGAQ- WP〇TEGC〇WDPWTCRLLHGW-LE (房列識別：313號） Con4-24 (C) 無抑制作用 M-FoGGGGGAQ- PDTRQGCQWDPWTCRLYGMW-LE (片列識別·· 314號） ACl-Con4 (C) 無抑制作用 M-Fc-GGGGGAQ- · TWPODKCEWDPWTCRLLQGW-LE (存列識別：315號） AC3-Con4 (C) 無抑制作Λ M-Fc-GGGGGAQ-DKILEECEWDPWTCRLLQGW-LE (序列識別：316號） AC5-Con4 (C) 無抑制作用 M-Fc-GGGGGAQ-AATQEECEWDPWTCRLLQGW-LE (序列識別：317號）Con4-5 (C) No inhibition M-Fc-GGGGGAQ- ATFARQCQWDPWTCALGGNW-LE (Sequence recognition: No. 305) Con4-30 (C) No inhibition M-Fc-GGGGGAQ-GPAQEECEWDPWTCEPLPLM-LE (Sequence recognition·· 306» Con4-26 (C) no inhibition M-Fc-GGGGGAQ- RPEDMCSQWDPWTWHLQGYC-LE (sequence recognition: 3〇7«t) Con4-7 (C) no inhibition 1 M-Fc-GGGGGAQ- LWQLAVC〇WDP〇TCDHMGAL -LE (question identification: 308 system Con4-12 (C) no inhibition M-Fc-GGGGGAQ-TOLVSLCEWDPWTCRLLDGW-LE (sequence identification · · No. 309) Con4-13 (C) no inhibition M-Fc-GGGGGAQ· MGGAGRCEWDPWTCQLLQGW-LE (Room Identification: No. 310) Con4-14 (C) No inhibition M-Fc-GGGGGAQ-MFLPNECQWDPWTCSNLPEA-LE (Ashore Heilongjiang: No. 311) Con4-2 (C) No inhibition M-Fc -GGGGGAQ-FGWSHGCEWDPWTCRLLQGW-LE (Rate identification: No. 312) Con4-6 (C) No inhibition _ M-Fc-GGGGGAQ- WP〇TEGC〇WDPWTCRLLHGW-LE (Room identification: No. 313) Con4-24 (C No inhibition M-FoGGGGGAQ- PDTRQGCQWDPWTCRLYGMW-LE (Slice identification · · No. 314) ACl-Con4 (C) No inhibition M-Fc-GGGGGAQ- · TWPODKCEWDPWTCRLLQGW-LE (Stock identification: No. 315) AC3-Con4 (C) No inhibition Λ M-Fc-GGGGGAQ-DKILEECEWDPWTCRLLQGW-LE (Sequence recognition: No. 316) AC5-Con4 (C) No inhibition M-Fc-GGGGGAQ-AATQEECEWDPWTCRLLQGW -LE (sequence identification: 317)
O:\121\121929.DOC 172- 200808833 L1衍生之親和 力-成熟的Pbs hAng-2:Tie2 IC5〇(nM) 肽體序列（序列識別號：） Ll-7 (N) 0.03 MGAQ- . TNFMPMDDLEQRLYEQFILQQG-LEGGGGG-Fc (序列識別：318號） AC6-L1 (N) 0.03 MGAQ- TNYKPLDELDATLYEHWILQHS LEGGGGG-Fc (序列識別：319號） L1-15(N) 0.04 MGAQ- QKYQPLDELDKTLYDQFMLQQG LEGGGGG-Fc (序列識別：320號） Ll-2 (N) 0.04 MGAQ-LNFTPLDELEQTLYEQWTLQQS LEGGGGG-Fc (序列識別：321號） LM0(N) 0.05 MGAQ- QKFQPLDELEQTLYEQFMLQQA LEGGGGG-Fc (房列識別：322號） Ll-13 (N) 0.05 MGAQ- QEYEPLDELDETLYNQWMFHQR LEGGGGG-Fc (序列識別：323號） Ll-5 (N) 0.05 MGAQ-VKYKPLDELDEILYEQQTFQER LEGGGGG-Fc (序列識別：324號） L1-C2 (N) 0.05 MGAQ- TKFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (序列識別：325號） L1-C3 (N) 0.06 MGAQ- TNFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (序列_識別：326號) Ll-11 (N) 0.07 MGAQ- QNFKPMDELEDTLYKQFLFQHS LEGGGGG-Fc (序列識別：327號） O:\121\121929.DOC -173 - 200808833O:\121\121929.DOC 172- 200808833 L1-derived affinity-mature Pbs hAng-2: Tie2 IC5〇(nM) Peptide sequence (SEQ ID NO:) Ll-7 (N) 0.03 MGAQ- . TNFMPMDDLEQRLYEQFILQQG- LEGGGGG-Fc (SEQ ID NO: 318) AC6-L1 (N) 0.03 MGAQ- TNYKPLDELDATLYEHWILQHS LEGGGGG-Fc (SEQ ID NO: 319) L1-15(N) 0.04 MGAQ- QKYQPLDELDKTLYDQFMLQQG LEGGGGG-Fc (Sequence recognition: No. 320) Ll-2 (N) 0.04 MGAQ-LNFTPLDELEQTLYEQWTLQQS LEGGGGG-Fc (Sequence recognition: No. 321) LM0(N) 0.05 MGAQ- QKFQPLDELEQTLYEQFMLQQA LEGGGGG-Fc (Room identification: No. 322) Ll-13 (N) 0.05 MGAQ- QEYEPLDELDETLYNQWMFHQR LEGGGGG -Fc (SEQ ID NO: 323) Ll-5 (N) 0.05 MGAQ-VKYKPLDELDEILYEQQTFQER LEGGGGG-Fc (SEQ ID NO: 324) L1-C2 (N) 0.05 MGAQ- TKFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (Sequence recognition: No. 325) L1 -C3 (N) 0.06 MGAQ- TNFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (sequence_identification: No. 326) Ll-11 (N) 0.07 MGAQ- QNFKPMDELEDTLYKQFLFQHS LEGGGGG-Fc (sequence identification: 327) O:\121\121929.DOC -173 - 200808833
Ll-17 (N) 0.08 MGAQ- VKYKPLDELDEWLYHQFTLHHQ LEGGGGG-Fc (4列識別：328號) LM2 (N) 0.08 MGAQ- YKFTPLDDLEQTLYEQWTLQHV LEGGGGG-Fc (序列誠別：329號） LM (N) 0.08 MGAQ-QNYKPLDELDATLYEHFIFHYT LEGGGGG-Fc (房列識別：330號） Ll-4 (N) 0.08 MGAQ- VKFKPLDALEQTLYEHWMFQQA LEGGGGG-Fc (序列識別：331號） Ll-20 (N) 0.09 MGAQ- EDYMPLDALDAQLYEQFILLHG LEGGGGG-Fc (序列識別：332號） LI-22 (N) 0.09 MGAQ- YKFNPMDELEQTLYEEFLFQHA LEGGGGG-Fc (序列識別·· 333號） Ll-14 (N) 0.11 MGAQ- SNFMPLDELEQTLYEQFMLQHQ LEGGGGG-Fc (序列_識別：334號） LM6(N) 0.11 MGAQ- QKFQPLDELEETLYKQWTLQQR LEGGGGG-Fc (序列識別：335號） · LM8(N) 0.16 MGAQ-QKFMPLDELDEILYEQFMFQQS LEGGGGG-Fc (序列識別：336號） Ll-3 (N) 0.16 MGAQ- TKFNPLDELEQTLYEQWTLQHQ LEGGGGG-Fc (序列識別：337號） Ll-21 (N) 0.17 MGAQ - HTFQPLDELEETLYYQWLYDQL LEGGGGG-Fc (序列識別：338號） O:\121\121929.DOC -174- 200808833Ll-17 (N) 0.08 MGAQ- VKYKPLDELDEWLYHQFTLHHQ LEGGGGG-Fc (4 columns identification: 328) LM2 (N) 0.08 MGAQ- YKFTPLDDLEQTLYEQWTLQHV LEGGGGG-Fc (sequence: 329) LM (N) 0.08 MGAQ-QNYKPLDELDATLYEHFIFHYT LEGGGGG- Fc (Ring ID: 330) Ll-4 (N) 0.08 MGAQ- VKFKPLDALEQTLYEHWMFQQA LEGGGGG-Fc (Sequence recognition: No. 331) Ll-20 (N) 0.09 MGAQ- EDYMPLDALDAQLYEQFILLHG LEGGGGG-Fc (Sequence recognition: No. 332) LI -22 (N) 0.09 MGAQ- YKFNPMDELEQTLYEEFLFQHA LEGGGGG-Fc (Sequence Recognition·· No. 333) Ll-14 (N) 0.11 MGAQ- SNFMPLDELEQTLYEQFMLQHQ LEGGGGG-Fc (Sequence_Identification: No. 334) LM6(N) 0.11 MGAQ- QKFQPLDELEETLYKQWTLQQR LEGGGGG -Fc (SEQ ID NO: 335) · LM8(N) 0.16 MGAQ-QKFMPLDELDEILYEQFMFQQS LEGGGGG-Fc (SEQ ID NO: 336) Ll-3 (N) 0.16 MGAQ- TKFNPLDELEQTLYEQWTLQHQ LEGGGGG-Fc (Sequence recognition: No. 337) Ll- 21 (N) 0.17 MGAQ - HTFQPLDELEETLYYQWLYDQL LEGGGGG-Fc (SEQ ID NO: 338) O:\121\121929.DOC -174- 200808833
Ll-Cl (N) 0.56 MGAQ- QKFKPLDELEQTLYEQWTLQQR LEGGGGG-Fc (序列識別：339號） Ll-19 (N) 126 MGAQ- 、 QTFQPLDDLEEYLYEQWIRRYH LEGGGGG-Fc (序列識別·· 340號） LI-9 ⑽ 1.62 MGAQ- SKFKPLDELEQTLYEQWTLQHA LEGGGGG-Fc (序列識別：341號) Coni衍生之親和 力-成熟的Pbs hAng-2:Tie2 ICso(nM) 肽體序列（序列識別號：） Conl-4 (C) 1.68 M-Fc-GGGGGAQ· SGQLRPCEEIFGCGTQNLAL-LE (序列锇別：342號） Conl-1 (C) 3.08 M-Fc-GGGGGAQ- AGGMRPYDGMLGWPNYDVQA-LE (房列識別：343號） Coni-6 (C) 8.60 M-Fc-GGGGGAQ-GODLRPCEDMFGCGTKDWYG-LE (序列識別：344ft) Coni-3 (C) 16.42 M-Fc-GGGGGAQ-APGQRPYDGMLGWPTYQRIV-LE (序列識別：345ft) Coni-2 (C) 無抑制作甩 M-Fc-GGGGGAQ-QTWDDPCMHILGPVTWRRCI-LE (房列識別：346號） · Coni-5 (C) 無抑制作用 M-Fc-GGGGGAQ-FGDKRPLECMFGGPIQLCPR-LE (序列熾別：347號) 親代::Coni (C) 26.00 M-Fc-GGGGGAQ-KRPCEEIFGGCTYQ- LE (序列識別：348號） 0:\121\121929.D0C 175- 200808833 12-9衍生之親和 力-成熟的Pbs hAng-2:Tie2 ICso(nM) 肽體序列（序列識別號：） 12-9-3 (C) 0.81 M-Fc-GGGGGAQ-LOEWCEGVEDPFTFGCEKQR-LE (序列識別：349號) 12-9-7 (C) 0.93 M-Fc-GGGGGAQ-MLDYCEGMDDPFTFGCDKQM-LE (序列識別：350號） 12-9-6 (C) 0.95 M-Fc-GGGGGAQ-HOEYCEGMEDPFTFGCEYQG-LE (年列識別·· 351號} 12-9-C2 (C) 1.41 M-Fc-GGGGGAQ-LODYCEGVEDPFTFGCENQR 七 E (序列識別：352號） 12-9-5 (C) 1.56 M-Fc-GGGGGAQ-LLDYCEGVQDPFTFGCENLD-LE (序列識別：353號） 12-9-1 (C) 1.84 M-Fc-GGGGGAQ-GFEYCDGMEDPFTFGCDKQT-LE (序列識別：354號） 12-9-4 (C) 2.05 M-Fc-GGGGGAQ-AODYCEGMEDPFTFGCEMQK-LE (序列識別：355ft) 12-9-C1 (C) 2.68 M-FoGGGGGAQ-LQDYCEGVEDPFTFGCEKQR-LE (痔列識別·· 356ft) 12-9-2 (C) 8,42 M-Fc-GGGGGAQ-KLEYCDGMEDPFTQGCDNQS-LE (务列識別：357號） 親代：12-9(C) 15.00 M-Fc-GGGGGAQ- # FDYCEGVEDPFTFGCDNH-LE (序列識別：358號） 實例9 測試六個抗-Ang2肽體之試樣，在BIAcore上與huAng2 (R&D Systems，BNO12103A)結合的活性。根據標準胺-偶 聯草案（BIAcore Inc.)，將蛋白質G固定在CM5晶片上’然 後將肽體注射到蛋白質G表面上進行捕捉（RL約100 Ru) ° 欲測試在hAng2和捕捉肽體之間的結合，將〇·3 nM至40 nM 的huAng2注射到捕捉肽體的表面上’並使用ΒΙΑ a平估3 ·0 -176-Ll-Cl (N) 0.56 MGAQ- QKFKPLDELEQTLYEQWTLQQR LEGGGGG-Fc (Sequence recognition: No. 339) Ll-19 (N) 126 MGAQ-, QTFQPLDDLEEYLYEQWIRRYH LEGGGGG-Fc (sequence identification · · No. 340) LI-9 (10) 1.62 MGAQ- SKFKPLDELEQTLYEQWTLQHA LEGGGGG-Fc (SEQ ID NO: 341) Coni-derived affinity - mature Pbs hAng-2: Tie2 ICso (nM) Peptide sequence (SEQ ID NO:) Conl-4 (C) 1.68 M-Fc-GGGGGAQ· SGQLRPCEEIFGCGTQNLAL -LE (sequence identification: No. 342) Conl-1 (C) 3.08 M-Fc-GGGGGAQ- AGGMRPYDGMLGWPNYDVQA-LE (Room identification: No. 343) Coni-6 (C) 8.60 M-Fc-GGGGGAQ-GODLRPCEDMFGCGTKDWYG-LE (sequence recognition: 344ft) Coni-3 (C) 16.42 M-Fc-GGGGGAQ-APGQRPYDGMLGWPTYQRIV-LE (sequence recognition: 345ft) Coni-2 (C) no suppression as M-Fc-GGGGGAQ-QTWDDPCMHILGPVTWRRCI-LE (Room Identification: No. 346) · Coni-5 (C) No inhibition M-Fc-GGGGGAQ-FGDKRPLECMFGGPIQLCPR-LE (Sequence: 347) Parental:: Coni (C) 26.00 M-Fc-GGGGGAQ-KRPCEEIFGGCTYQ-LE (Sequence identification: No. 348) 0:\121\121929.D0C 175- 200808833 12-9 Affinity derived - mature Pbs hAng-2: Tie2 ICso (nM) Peptide sequence (SEQ ID NO:) 12-9-3 (C) 0.81 M-Fc-GGGGGAQ-LOEWCEGVEDPFTFGCEKQR-LE (Sequence recognition: No. 349) 12-9-7 (C ) 0.93 M-Fc-GGGGGAQ-MLDYCEGMDDPFTFGCDKQM-LE (Sequence Recognition: No. 350) 12-9-6 (C) 0.95 M-Fc-GGGGGAQ-HOEYCEGMEDPFTFGCEYQG-LE (Yearly Recognition·· No. 351) 12-9-C2 (C) 1.41 M-Fc-GGGGGAQ-LODYCEGVEDPFTFGCENQR Seven E (sequence recognition: 352) 12-9-5 (C) 1.56 M-Fc-GGGGGAQ-LLDYCEGVQDPFTFGCENLD-LE (sequence recognition: 353) 12-9-1 (C) 1.84 M-Fc-GGGGGAQ-GFEYCDGMEDPFTFGCDKQT-LE (SEQ ID NO: 354) 12-9-4 (C) 2.05 M-Fc-GGGGGAQ-AODYCEGMEDPFTFGCEMQK-LE (Sequence recognition: 355ft) 12-9-C1 ( C) 2.68 M-FoGGGGGAQ-LQDYCEGVEDPFTFGCEKQR-LE (column identification · · 356ft) 12-9-2 (C) 8,42 M-Fc-GGGGGAQ-KLEYCDGMEDPFTQGCDNQS-LE (question identification: 357) Parent: 12 -9(C) 15.00 M-Fc-GGGGGAQ- # FDYCEGVEDPFTFGCDNH-LE (SEQ ID NO: 358) Example 9 Test samples of six anti-Ang2 peptibodies on BIAcore with huAng2 (R&D Systems, BNO12103A) Binding activity. Protein G was immobilized on a CM5 wafer according to the standard amine-coupling draft (BIAcore Inc.) and the peptide was then injected onto the surface of protein G for capture (RL approximately 100 Ru) ° to be tested in hAng2 and capture peptibodies Inter-injection, hu3 nM to 40 nM huAng2 was injected onto the surface of the capture peptide body' and used ΒΙΑ a flat estimate 3 · 0 - 176-
O:\121\121929.DOC 200808833 (BIAcore Inc.)分析結合感應器圖。表8概述該實驗的結果。 表8 肽體 批號 KD(M) ka (1/Ms) kd (1/s) Con4-44 (C) 011702 2.1E-10 2.9E+05 5.9E-05 L1-7(N) 022102 2.4E-10 3.7E+05 8.7E-05 L1-10(N) 021302 7.7E-10 1.5E+05 1.1E-04 Ll-21 (N) 021802 2.4E-10 5.6E+05 1.4E-04 Con4 (C) 33456-77 3.8E-10 5.3E+05 2.0E-04 2xCon4 (C) IK 092501 3.4E-10 4.8E+05 1.6E-04 實例10O:\121\121929.DOC 200808833 (BIAcore Inc.) analyzes the combined sensor map. Table 8 summarizes the results of this experiment. Table 8 Peptide lot number KD(M) ka (1/Ms) kd (1/s) Con4-44 (C) 011702 2.1E-10 2.9E+05 5.9E-05 L1-7(N) 022102 2.4E- 10 3.7E+05 8.7E-05 L1-10(N) 021302 7.7E-10 1.5E+05 1.1E-04 Ll-21 (N) 021802 2.4E-10 5.6E+05 1.4E-04 Con4 (C 33456-77 3.8E-10 5.3E+05 2.0E-04 2xCon4 (C) IK 092501 3.4E-10 4.8E+05 1.6E-04 Example 10
中和ELISA 以DMEM/50微克/毫升BSA如下稀釋人類、老鼠、犬和 大鼠Ang-2，以及人類和老鼠Ang-Ι的調理培養基·· hAng-2-1:64稀釋；mAng-2-l:64稀釋；大鼠Ang-2-不稀釋；犬 Ang-2-l:32 稀釋；hAng-l_l:4 稀釋；和 mAng-l-l:4 稀釋。 藉著其在50%可達成之最大結合時（也就是高原期），與1 nM hTie2-Fc (以Tie-2_Fc分子提供，其中Tie-2部分僅含有 該分子之可溶的細胞外部分；R&D Systems，目錄第3 13-TI號）結合的能力，來判定這些調理培養基每一個的稀釋 程度。以100微升經過稀釋的調理培養基塗覆微量滴定盤 。關於Ang-2中和ELISA，以含有大約1% BSA和大約1 nM Tie-2 (以Tie-2-Fc分子提供，其中Tie-2部分僅含有該分子 之可溶的細胞外部分；R&D Systems，目錄第313-TI號）的 PBS溶液4-倍稀釋，從62.5 nM至0.015 pM，滴定候選的 抗-Ang-2肽體。至於Ang-2中和ELISA，以含有大約1% BSA和大約1 nM Tie-2 (以Tie-2-Fc分子提供，其中Tie-2部 O:\121\121929.DOC -177- 200808833 分僅含有該分子之可溶的細胞外部分；R&D Systems，目 錄第313-TI號）的PBS溶液4-倍稀釋，從1000 nM至0·2 pM ，滴定候選的抗-Ang-2肽體。 在各孔中加入大約100微升肽體/Tie-2溶液之後，在室溫 下培養該盤過夜，然後以含有大約1%吐溫-20的PBS沖洗5 次。在沖洗之後，加入每孔大約100微升的抗-Tie-2抗體 (Pharmingen Inc.，目錄#557039)至大約每毫升1微克之終 濃度，並在室溫下培養該盤大約1小時。接著，以在含有 大約1% BSA之PBS中1:10,000的稀釋度，加入每孔大約 100微升的山羊抗-老鼠-IgG_HRP (Pierce Chemical Co.，目 錄#31432) 〇 在室溫下培養該盤大約1小時，隨後以含有大約0.1%吐 溫-20的PBS沖洗5次。然後加入每孔大約100微升的ΤΜΒ溶 液（SIGMA，目錄#Τ8665)，並容許藍色顯現。然後在分光 光度計的370毫微米處，讀取吸光度。在下文表9中陳述其 結果。 表9 肽體-調節之血管生成素：Tie2交互作用的中和作用 hAng-2 mAng-2 rAng-2 cAng-2 hAng-1 mAng-2 肽體 IC5〇 (nM) IC5〇 (nM) IC5〇 (nM) IC5〇 (nM) IC5〇 (nM) IC5〇 (nM) 2xCon4 (C) 0.026 0.035 0.024 0.047 3.0 3.2 Con4 (C) 0.197 0.298 0.236 0.540 200 300 C〇n4_44 (C) 0.08 0.16 0.22 — 43 — Con4-40 (C) 0.20 0.27 0.35 ---- > 1000 — L1-7(N) 0.046 0.063 0.035 0.108 > 1000 > 1000 Ll-21 (N) 0.179 0.249 0.204 0.608 > 1000 > 1000 Ll-10 (N) 0.06 0.06 0.06 — > 1000 — O:\121\121929.DOC -178- 200808833 實例11 PK研究 研究設計 將重20-30克的雄性CD-1老鼠隨機分到每個肽體處理組 (2xCon4-C，L1-7-N和L1-21-N)内。動物接受5〇微克肽體 的單一 IV團塊（n=38/組）或單一 SC投藥（n=34/組）。關於iv 和SC投藥，分別經由尾靜脈和在肩膀皮膚下進行注射。 血液採樣和分析方法 針對每個抗- Ang2肤體濃度測量之預定劑量，並在SC和 IV組的投藥後 1、2、4、8、16、24、48、72 ' 96、120、 144、168、216、264、3 12和336小時，收集血液試樣。對 於IV組，在投藥後5和3 0分鐘時收集額外的試樣。每個時 間點對兩隻動物採血，並在採樣後犧牲動物。從心臟穿刺 ，將血液（大約0.50毫升）收集至聚丙烯microtainer@血清分 離管中。將減樣放在冰上大約2 0分鐘’或直到發生凝塊形 成為止。精者在2-8C下離心大約10分鐘，從企液試樣中 分離血清’並儲存在大約_7〇。〇下，直到進行測定為止。 使用經過校準、具有1〇〇毫微克/毫升之定量下限（LL〇Q)的 時間分辨螢光（TRF)測定，來測量試樣。以重組的老鼠 Ang-2蛋白質塗覆NUNC fluoroMaxisorp微量滴定盤。然後 利用蛋白質溶液阻斷該盤，以便減少非專一性的結合。在 10%老鼠血清測定缓衝溶液中，製備標準物、品質控制組 和未知試樣，並吸移至微量滴定盤的孔中。肽體與已經固 定之Ang-2專一地結合。在沖洗掉任何未結合的受質Neutralization ELISA Human, mouse, canine and rat Ang-2 were diluted as follows with DMEM/50 μg/ml BSA, and human and mouse Ang-Ι conditioning medium··hAng-2-1:64 dilution; mAng-2- l: 64 dilution; rat Ang-2-not diluted; canine Ang-2-l:32 dilution; hAng-l_l:4 dilution; and mAng-ll: 4 dilution. By virtue of its 50% achievable maximum binding (ie, the plateau phase), with 1 nM hTie2-Fc (provided as a Tie-2_Fc molecule, wherein the Tie-2 portion contains only the soluble extracellular portion of the molecule; R&D Systems, Catalog No. 3 13-TI) combines the ability to determine the degree of dilution of each of these conditioning media. The microtiter plate was coated with 100 microliters of diluted conditioning medium. Regarding the Ang-2 Neutralization ELISA, containing approximately 1% BSA and approximately 1 nM Tie-2 (provided as a Tie-2-Fc molecule, wherein the Tie-2 moiety contains only soluble extracellular portions of the molecule; R& Candidate anti-Ang-2 peptibodies were titrated 4-fold dilutions of PBS solution from D Systems, catalog No. 313-TI, from 62.5 nM to 0.015 pM. As for the Ang-2 neutralization ELISA, containing approximately 1% BSA and approximately 1 nM Tie-2 (provided as Tie-2-Fc molecule, wherein Tie-2 part O:\121\121929.DOC-177-200808833 only A soluble extracellular portion containing the molecule; R&D Systems, catalog No. 313-TI) PBS solution 4-fold dilution, titer candidate anti-Ang-2 peptibody from 1000 nM to 0·2 pM . After adding approximately 100 microliters of peptibody/Tie-2 solution to each well, the plate was incubated overnight at room temperature and then washed 5 times with PBS containing approximately 1% Tween-20. After rinsing, approximately 100 microliters of anti-Tie-2 antibody (Pharmingen Inc., catalog #557039) per well was added to a final concentration of approximately 1 microgram per milliliter and the plate was incubated for approximately one hour at room temperature. Next, approximately 100 microliters of goat anti-mouse-IgG-HRP (Pierce Chemical Co., catalog #31432) per well was added at a dilution of 1:10,000 in PBS containing approximately 1% BSA and incubated at room temperature. The plate was incubated for approximately 1 hour and then washed 5 times with PBS containing approximately 0.1% Tween-20. Then add approximately 100 microliters of hydrazine solution (SIGMA, catalog #Τ8665) per well and allow blue to appear. The absorbance was then read at 370 nm of the spectrophotometer. The results are set forth in Table 9 below. Table 9 Peptide-regulated angiopoietin: Neutralization of Tie2 interaction hAng-2 mAng-2 rAng-2 cAng-2 hAng-1 mAng-2 Peptide IC5〇(nM) IC5〇(nM) IC5〇 (nM) IC5〇(nM) IC5〇(nM) IC5〇(nM) 2xCon4 (C) 0.026 0.035 0.024 0.047 3.0 3.2 Con4 (C) 0.197 0.298 0.236 0.540 200 300 C〇n4_44 (C) 0.08 0.16 0.22 — 43 — Con4-40 (C) 0.20 0.27 0.35 ---- > 1000 — L1-7(N) 0.046 0.063 0.035 0.108 > 1000 > 1000 Ll-21 (N) 0.179 0.249 0.204 0.608 > 1000 > 1000 Ll -10 (N) 0.06 0.06 0.06 — > 1000 — O:\121\121929.DOC -178- 200808833 Example 11 PK Study Study Design Male CD-1 mice weighing 20-30 grams were randomly assigned to each peptibody Within the treatment group (2xCon4-C, L1-7-N and L1-21-N). Animals received a single IV mass of 5 micrograms of peptide (n=38/group) or single SC (n=34/group). For iv and SC administration, injections were made via the tail vein and under the skin of the shoulder, respectively. Blood sampling and analysis methods are based on predetermined doses measured for each anti-Ang2 skin concentration, and 1, 2, 4, 8, 16, 24, 48, 72' 96, 120, 144 after administration in the SC and IV groups. Blood samples were collected at 168, 216, 264, 3 12 and 336 hours. For group IV, additional samples were collected at 5 and 30 minutes after administration. Two animals were bled at each time point and sacrificed after sampling. From the heart puncture, blood (approximately 0.50 ml) was collected into a polypropylene microtainer@serum separation tube. Place the sample on ice for approximately 20 minutes' or until a clot is formed. The sperm was centrifuged at 2-8 C for about 10 minutes, and the serum was separated from the sample of the liquid solution and stored at about _7 Torr. Kneel down until the measurement is taken. Samples were measured using a time-resolved fluorescence (TRF) assay that was calibrated to have a lower limit of quantitation (LL〇Q) of 1 〇〇 ng/ml. A NUNC fluoroMaxisorp microtiter plate was coated with recombinant mouse Ang-2 protein. The disk is then blocked with a protein solution to reduce non-specific binding. Standards, quality control groups, and unknown samples were prepared in a 10% rat serum assay buffer and pipetted into the wells of a microtiter plate. The peptibodies are specifically bound to the already fixed Ang-2. Rinse off any unbound substrate
O:\121\121929.DOC -179- 200808833 (Kirkegaard & Perry Laboratories Inc·)之後，在孔中加入 生物素基化的山羊抗-人類IgG (H+L)單株抗體（Jacks〇n ImmunoResearch Laboratories Inc.)。在移除任何未結合之 生物素基化單株抗體的沖洗步驟之後，在孔中加入銪標示 之鏈黴菌抗生物素蛋白。在沖洗掉未結合的鏈黴菌抗生物 素蛋白銪之後，利用將酸性溶液吸移到各孔中，使已結合 的銪從鏈黴菌抗生物素蛋白中釋放出來。產生螢光信號， 並在Wallac’s螢光讀取器中讀取。在老鼠血清中分析抗_ Ang-2肽體的測定範圍是〇·〇78-5微克/毫升。 藥物動力學分析 使用 WinNonlin Professional (3.3 版，Pharsight Corp·，O:\121\121929.DOC -179- 200808833 (Kirkegaard & Perry Laboratories Inc.), after adding biotinylated goat anti-human IgG (H+L) monoclonal antibody to the well (Jacks〇n ImmunoResearch Laboratories Inc.). After the rinsing step of removing any unbound biotinylated monoclonal antibodies, sputum labeled streptavidin was added to the wells. After the unbound streptavidin peptone is washed away, the bound quinone is released from the streptavidin by pipetting the acidic solution into each well. A fluorescent signal is generated and read in a Wallac's fluorescent reader. The assay range for analysis of anti-Ang-2 peptibodies in mouse serum was -5·〇78-5 μg/ml. Pharmacokinetic Analysis Using WinNonlin Professional (version 3.3, Pharsight Corp.,
Mountain View，CA)，使每組的綜合平均濃度_時間數據接 受無隔間的分析。PK分析所使用的票面採樣時間，在票面 時間的10%内收集試樣。在PK分析之前，將所有低於 LLOQ的濃度值設定為〇 〇估計下列的pK參數： •以（^==111(2)/1^計算終點半衰期，其中kei為經由終點 對數-線性蜆變期的線性回歸，來評估的一級終點速 率常數。 •使用線性/對數梯形方法，從時間〇至最後，最後可定 篁之濃度的時間（Clast)，來評估在血清濃度-時間曲線 下的面積（AUC(().last))。 以相對應之AUC^-Ust；)和預測〇1^1/]^1值的總和，來評 估在從時間0至無限大之曲線下的面積（AUC(q 〇〇)):Mountain View, CA), allows the combined average concentration_time data for each group to be analyzed without compartments. The coupon sampling time used in the PK analysis was collected within 10% of the face time. Prior to PK analysis, all concentration values below LLOQ were set to 〇〇 estimate the following pK parameters: • Calculate the endpoint half-life with (^==111(2)/1^, where kei is the log-linear transformation through the endpoint Linear regression of the period to estimate the first-order endpoint rate constant. • Use the linear/logarithmic trapezoidal method to estimate the area under the serum concentration-time curve from time 〇 to the end and the final concentration of the concentration (Clast). (AUC(().last)). The area under the curve from time 0 to infinity is evaluated by the sum of the corresponding AUC^-Ust;) and predicted 〇1^1/]^1 values (AUC) (q 〇〇)):
O:\121\121929.DOC -180- 200808833 AUC(0_〇〇 )=AUC(0-last)+ ㈣ @ Clast kel •如下計算在sc投藥之後的絕對生物利用性（F) ·· F= + ⑺)sc x 100 AUC ro. 〇〇 uv 在圖2中陳述其結果。 實例12 在研究第0天，以lxlO7個A431細胞皮下注射雌性的裸鼠 。在第3天，以200微克/老鼠/天之劑量，皮下投與Ang-2肽 體2xCon4-C。以規律的間隔記錄腫瘤體積和體重，如同圖 中所示。在Ang-2肽體-處理組對媒劑對照組和對照組肽體 之間，觀察到腫瘤生長的明顯差異（ρ<〇·〇〇〇1對每個對照組 ，使用重覆測量ANOVA，與Scheffe，s post hoc檢定）。利 用該肽體的處理，對體重沒有顯著的影響。在圖3中陳述 其結果。 實例13 A431在活體外的生長曲線 以每孔2000個細胞，在200微升補充有10%胎牛血清 (FBS)之DMEM中，將A431細胞播種在96孔的組織培養盤 上。在播種之後’對培養基抽氣16小時。然後將下列的物 質加回孔中’並建立一式三份：每孔1〇〇微升DMEM，10% FBS ’ 1宅克/宅升陰性對照組肽體4883，或肽體TN8-Con4 。在5個培養盤上重覆相同的組合。在處理後24、48、72 、96和120小時，對得自一個培養盤的培養基抽氣。然後 O:\121\121929.DOC -181- 200808833 在各孔中加入100微升10%三氯乙酸（TCA)，然後將培養盤 儲存在4 C下。收集所有的培養盤，此時最後一個培養盤 已經在10% TCA中至少4小時。甩乾1〇% TCA，並以自來 水沖洗各孔5次。然後在室溫下，以ι〇〇微升在乙酸 (Sigma A-6283)中之 〇·4〇/0 硫若丹明B (sigma S-9〇12)，將細 胞染色10分鐘，並以1 %乙酸沖洗5次。然後風乾培養盤。 在方疋轉振盈器上’利用300微升20 mM未緩衝之Tris (pH>10)加溶染料2小時。然後在微量滴定盤讀取器54〇毫 微米處’讀取光密度（OD)。在圖4中陳述其結果。 實例14 在研究第0天’以2xl06個Colo-205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。在第3天，以14微克/老鼠之劑量，一 週兩次皮下投與Ang_2肽體L1-7-N、L1-21_N、Con4-C和 2xCon4-C。一週三次投與抗-Ang-2抗體，Ab536，47微克/ 老鼠’作為陽性對照組。按照規律的間隔，記錄腫瘤體積 和體重。 在每一種Ang-2肽體處理組對媒劑對照組和對照組肽體 之間，觀察到腫瘤生長的顯著差異（ρ<0·0001對每種對照組 ’使用重覆測量ANOVA，與Scheffe’s post h〇c檢定）。利 用這些肽體的處理，對體重沒有顯著的影響（結果未顯示） 。在圖5中陳述其結果。 實例15 在研究第0天，以2xl06個C〇1〇_205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。在第3天，以14、2.8和〇·56微克/老鼠 O:\121\121929.DOC •182- 200808833 之劑量，一週兩次皮下投與Ang-2肽體2xCon4-C。按照所 示的規律間隔’記錄腫瘤體積和體重。在兩種較高劑量之 Ang-2肽體處理組對媒劑對照組和對照組肽體之間，觀察 到腫瘤生長的顯著差異（中間劑量的p=〇 〇〇3，而高劑量的 p<0_0001，使用重覆測量 ANOVA，與 ScheffVs post hoc 檢 定）。利用這些肽體的處理，對體重沒有顯著的影響。破 折線代表組別之總η值，從1〇隻減少至9隻老鼠，歸因於一 隻老鼠不明原因地死亡。在圖6中陳述其結果。 實例16 抗-Ang-2肽體對Colo-205異種移植之腫瘤 在研究第0天，以2xl06個C〇1〇_205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。在第3天，以3 5 0微克/天之劑量，皮 下投與Ang-2肽體2xCon4-C或對照組肽體。在第14天（尺 寸-配合對照組）或第18天（時間-配合對照組），收獲得自以 對照組肽體處理之組別的腫瘤（如同在表5中描述的）。然後 在第18天，收獲得自2xCon4(C)的腫瘤按照所示的規律間 隔，記錄腫瘤體積。在時間-配合對照組和2xC〇n4_c處理 組之間，觀察到腫瘤生長的顯著差異（ρ = 0·01 54，藉著重覆 測量AN0VA ’與Scheffe’s post hoc檢定）。利用這些肽體 的處理，對體重沒有顯著的影響。 將為了影像分析準備的腫瘤從頂部分切為二，並將一半 猛然冷束於 OCT (Sakura Finetek USA Inc.，Torrance，CA) 中。使用帶有作為色原之DAB的2微克/毫升之抗-老鼠 CD31 (目錄 #553370，BD PharMingen，San Diego，CA)，以 O:\121\121929.DOC •183- 200808833 免疫組織化學之方式染色冷凍_切片。以2〇χ物鏡放大率， 拍攝腫瘤切片的數位照片。捕捉每個腫瘤的四個”包圍_點，， 領域，每種處理組有1〇個腫瘤。對於在影像内被CD31染 色之血管的門檻’使用MetaMorph (Universal Imaging Corporation，Downington，PA)影像分析系統。以在每個領 域内的總腫瘤組織之比例，表現CD3 1陽性染色的面積。 在圖7中陳述其結果。 實例17 在研究第0天，以2xl06個Colo-205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。從研究第3、1〇或15天開始，以35〇微 克/老鼠，皮下，一週兩次的Ang-2肽體2xCon4-C，或以相 等的對照組肽體處理。按照規律的間隔，記錄腫瘤體積和 體重。在所有的Ang-2肽體處理組對媒劑對照組之間，觀 察到腫瘤生長的顯著差異（第15天組，p = 0_〇89，而第3和第 10天組，ρ<〇.〇〇〇1，使用重覆測量ANOVA，與Scheffe，s post hoc檢定）。利用這些肽體的處理，對體重沒有顯著的 影響。在圖8中陳述其結果（未顯示體重）。 實例18 在A43 1和Colo-205異種移植的模式中，使用以47微克/ 雌性裸鼠之劑量的抗體Ab536，一週三次腹腔内投與，或 按照在不同的長期研究（210週投藥）中之多次投藥時間表 ’給與肽體2xCon4(C) ’獲得完全反應（CR)率的概要。在 本文中使用的CR，意指在治療之後，其中沒有剩下可測 量之腫瘤的成果。在圖9中陳述其結果。 O:\121\121929.DOC -184- 200808833 實例19 a) 在Colo-205腫瘤模式中，混合抑與剋癌易 在研究第0天，以2xl〇6個Colo-205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。在研究第14天，開始以a) 350微克/老 鼠’皮下，一週兩次的Ang-2肽體2xCon4-C，b) 20毫克/公 斤，一週三次，腹腔内的剋癌易，或c)兩者混合處理。 才女照規律的間隔，記錄腫瘤體積和體重。在所有的處理組 對媒劑對照組之間，觀察到腫瘤生長的顯著差異（ρ<0β0001 ’使用重覆測量ANOVA，與Scheffe’s post hoc檢定）。此 外，混合治療組與任一個單一治療劑有顯著的差異 (ρ<0·0001對2xCon4-C，且ρ = 〇·〇122對剋癌易）。破折線代 表組別之總η值，從1 〇隻減少至9隻老鼠，歸因於一隻老鼠 不明原因地死亡。利用這些肽體的處理，對體重沒有顯著 的影響。在圖10a中陳述其結果。O:\121\121929.DOC -180- 200808833 AUC(0_〇〇)=AUC(0-last)+ (4) @Clast kel • Calculate the absolute bioavailability after sc administration as follows (F) ·· F= + (7)) sc x 100 AUC ro. 〇〇uv The results are presented in Figure 2. Example 12 On day 0 of the study, female nude mice were injected subcutaneously with lxlO7 A431 cells. On day 3, the Ang-2 peptide 2xCon4-C was administered subcutaneously at a dose of 200 μg/mouse/day. Tumor volume and body weight were recorded at regular intervals as shown. A significant difference in tumor growth was observed between the vehicle control group and the control peptibosome in the Ang-2 peptid-treated group (ρ<〇·〇〇〇1 for each control group, using repeated measures ANOVA, With Scheffe, s post hoc accreditation). Treatment with this peptibody had no significant effect on body weight. The result is stated in Figure 3. Example 13 Growth curve of A431 in vitro A431 cells were seeded on 96-well tissue culture dishes in 2000 microliters per well in 200 microliters of DMEM supplemented with 10% fetal bovine serum (FBS). The medium was evacuated for 16 hours after sowing. The following substances were then added back to the wells' and triplicate was established: 1 mM microliter DMEM per well, 10% FBS' 1 housew/home-negative control peptibody 4883, or peptidomimetic TN8-Con4. Repeat the same combination on 5 plates. The medium from one plate was evacuated at 24, 48, 72, 96 and 120 hours after the treatment. Then O: \121\121929.DOC -181- 200808833 100 μl of 10% trichloroacetic acid (TCA) was added to each well, and the plate was stored at 4 C. All plates were collected and the last plate was at least 4 hours in 10% TCA. Dry 1% TCA and rinse each well 5 times with tap water. The cells were then stained with 〇〇·4〇/0 sulphur rhodamine B (sigma S-9〇12) in acetic acid (Sigma A-6283) at room temperature for 10 minutes and Rinse 1% acetic acid 5 times. The plate is then air dried. On the square-turn vibrator, 300 μl of 20 mM unbuffered Tris (pH > 10) was used to solubilize the dye for 2 hours. The optical density (OD) was then read at the microtiter plate reader 54 〇 nanometer. The result is stated in Figure 4. Example 14 On day 0 of the study, female nude mice were injected subcutaneously with 2 x 106 Colo-205 cells plus Matrigel (2:1). On day 3, Ang 2 peptides L1-7-N, L1-21_N, Con4-C and 2xCon4-C were administered subcutaneously twice a week at a dose of 14 μg/mouse. Anti-Ang-2 antibody, Ab536, 47 μg/mouse' was administered as a positive control group three times a week. Tumor volume and body weight were recorded at regular intervals. Significant differences in tumor growth were observed between the vehicle control group and the control peptone in each of the Ang-2 peptoid treatment groups (ρ<0·0001 for each control group using repeated measures ANOVA, with Scheffe's Post h〇c check). Treatment with these peptibodies had no significant effect on body weight (results not shown). The result is stated in Figure 5. Example 15 On day 0 of the study, female nude mice were injected subcutaneously with 2 x 106 C〇1〇_205 cells plus Matrigel (2:1). On day 3, Ang-2 peptidomimetic 2xCon4-C was administered subcutaneously twice a week at doses of 14, 2.8 and 〇·56 μg/mouse O:\121\121929.DOC •182-200808833. Tumor volume and body weight were recorded at regular intervals as shown. Significant differences in tumor growth were observed between the vehicle control group and the control peptibodies in the two higher doses of the Ang-2 peptoid treatment group (intermediate dose p=〇〇〇3, and high dose p< ; 0_0001, using repeated measures ANOVA, with ScheffVs post hoc check). Treatment with these peptibodies did not have a significant effect on body weight. The broken line represents the total η value of the group, which decreased from 1 to 9 mice, due to a mouse dying for unknown reasons. The result is stated in Figure 6. Example 16 Anti-Ang-2 Peptide vs. Colo-205 Xenograft Tumor On day 0 of the study, female nude mice were injected subcutaneously with 2 x 106 C〇1〇_205 cells plus Matrigel (2:1). On day 3, Ang-2 peptidomimetic 2xCon4-C or control peptibody was administered subcutaneously at a dose of 350 μg/day. Tumors from the group treated with the control peptids were obtained on day 14 (size-match control group) or day 18 (time-match control group) (as described in Table 5). Then on day 18, tumors obtained from 2xCon4(C) were recorded at the regular intervals shown, and tumor volumes were recorded. Significant differences in tumor growth were observed between the time-matched control group and the 2xC〇n4_c treated group (ρ = 0·01 54, with emphasis on AN0VA' and Scheffe's post hoc assay). Treatment with these peptides had no significant effect on body weight. The tumor prepared for image analysis was cut from the top portion to two, and half of the tumor was suddenly cooled in OCT (Sakura Finetek USA Inc., Torrance, CA). 2 μg/ml anti-mouse CD31 (catalog #553370, BD PharMingen, San Diego, CA) with DAB as chromogen, using O:\121\121929.DOC •183- 200808833 immunohistochemistry Dyeing frozen_slices. Digital images of tumor sections were taken at a magnification of 2 〇χ objective lens. Capturing each of the four "encirclement_points, fields, one tumor per treatment group. For the threshold of blood vessels that were stained with CD31 in the image, use MetaMorph (Universal Imaging Corporation, Downington, PA) image analysis System. The area of CD3 1 positive staining was expressed as the proportion of total tumor tissue in each field. The results are presented in Figure 7. Example 17 On study day 0, 2xl06 Colo-205 cells plus Matrigel (2 :1) Subcutaneous injection of female nude mice. Starting from study day 3, 1 or 15 days, 35 μg/mouse, subcutaneous, twice a week of Ang-2 peptibody 2xCon4-C, or equivalent control peptide Body volume. Tumor volume and body weight were recorded at regular intervals. Significant differences in tumor growth were observed between all Ang-2 peptidic treated groups and vehicle control groups (Day 15 group, p = 0_〇) 89, and the 3rd and 10th day groups, ρ<〇.〇〇〇1, using repeated measures ANOVA, with Scheffe, s post hoc assay.) Treatment with these peptides had no significant effect on body weight. The results are shown in Figure 8 (weight is not shown). Example 18 In the A43 1 and Colo-205 xenograft mode, antibody Ab536 was administered at a dose of 47 μg/female nude mice, administered intraperitoneally three times a week, or according to different long-term studies (210 weeks of dosing). A multi-administration schedule 'giving the peptibody 2xCon4(C)' to obtain a summary of the complete response (CR) rate. The CR used herein means that after treatment, there is no measurable tumor remaining. The results are set forth in Figure 9. Example: a) In the Colo-205 tumor model, mixed inhibition and gram cancer were easily studied on day 0, with 2xl 〇 6 Colo- 205 cells plus Matrigel (2:1) were injected subcutaneously into female nude mice. On day 14 of the study, start with a) 350 μg/mouse' subcutaneous, twice a week of Ang-2 peptibo 2xCon4-C, b) 20 mg/ Kg, three times a week, gram-free in the abdominal cavity, or c) mixed treatment. The tumor volume and body weight were recorded at regular intervals. Tumor growth was observed between all treatment groups and the vehicle control group. Significant difference (ρ<0β0001' using repeated measures ANOVA, with Scheffe's post hoc In addition, there was a significant difference between the mixed treatment group and any single therapeutic agent (ρ < 0·0001 versus 2xCon4-C, and ρ = 〇·〇122 for gram cancer). The dashed line represents the total η of the group. The value, from 1 〇 to only 9 mice, was attributed to a mouse dying for unknown reasons. Treatment with these peptibodies did not have a significant effect on body weight. The result is set forth in Figure 10a.
b) 在Colo-205腫瘤模式中，混合Pb與5-FU 在研究弟0天’以2xl06個Colo-205細胞加Matrigel (2:1) 皮下注射雌性裸鼠。在研究第14天，開始以a) 350微克/老 鼠，皮下，一週兩次的Ang-2肽體2xCon4-C，b) 50毫克/公 斤，一週5次，腹腔内的5-FU，或c)兩者混合處理。按照 所示的規律間隔，記錄腫瘤體積和體重。 在所有的處理組對媒劑對照組之間，觀察到腫瘤生長的 顯著差異（ρ<〇·〇〇〇1，使用重覆測量AN0VA，與Scheffe，s post hoc檢定）。此外，混合治療組與任一個單一治療劑有 顯著的差異（ρ=〇·〇375 對 2xCon4-C，且 ρ=〇·〇453 對 5_FU)。 O:\121\121929.DOC • 185 - 200808833 在5-FU處理組（在研究第2〇天時18%)和混合治療組（在研究 第20天時16%)中’觀察到體重的暫時降低，隨後完全恢復 體重。在圖10b中陳述其結果。 實例20 佐劑關節炎模式 在12-小時亮/暗循環的環境控制室（溫度23±2t，相對濕 度50±20%)中，將雄性1^*大鼠（12〇_13〇克，（：1^1以 Hiver·，Wilmington MA)飼養在兩個分別以濾紙加蓋的籠子 中。银食動物市售的嚅齒動物食物（配方8640; Tek Lab， Madison，WI)，並可無限制地收到濾紙_純化的自來水。飼 糧中鈣和磷的含量分別為1·2〇/〇和1.0〇/〇。 藉著在尾根部，皮内單次注射懸浮於〇 〇5毫升石蠟油 (Crescent Chemical Co·，Hauppauge，ΝΥ)中之 0.5 毫克熱-殺 死的結核才干鹵 H37Ra (Difco Laboratories，Detroit，MI)，誘 導佐劑關節炎。在第9天時，出現藉由後腳掌腫脹和行走 困難所代表的關節炎之臨床症狀。除了 2xc〇n4(C)處理組（ 從免疫後第1天起處理）之外，在免疫後第9天開始，給與 每曰皮下注射的處理（在關節炎發作之前），並持續到第18 天。 佐劑關節炎的臨床監視 在臨床上藉著間歇地測量後腳掌體積，使用根據由b) In the Colo-205 tumor model, mixed Pb and 5-FU were injected subcutaneously into female nude mice at 2 x 106 Colo-205 cells plus Matrigel (2:1) on day 0 of study. On study day 14, start with a) 350 μg/mouse, subcutaneous, twice weekly Ang-2 peptibody 2xCon4-C, b) 50 mg/kg, 5 times a week, intraperitoneal 5-FU, or c Both are mixed. Tumor volume and body weight were recorded at regular intervals as shown. Significant differences in tumor growth were observed between all treatment groups versus vehicle control groups (ρ<〇·〇〇〇1, using repeated measures ANOV, with Scheffe, s post hoc assay). In addition, there was a significant difference between the mixed treatment group and any single therapeutic agent (ρ=〇·〇375 versus 2xCon4-C, and ρ=〇·〇453 versus 5_FU). O:\121\121929.DOC • 185 - 200808833 In the 5-FU treatment group (18% at study day 2) and the mixed treatment group (16% at study day 20), a temporary observation of body weight was observed. Reduce, then fully restore weight. The result is stated in Figure 10b. Example 20 Adjuvant arthritis model In a 12-hour light/dark cycle environmental control room (temperature 23 ± 2 t, relative humidity 50 ± 20%), male 1 ^ * rats (12 〇 _13 〇, ( : 1^1 is housed in two cages covered with filter paper by Hiver·, Wilmington MA). Carnivorous animal food (formula 8840; Tek Lab, Madison, WI) commercially available from silver food animals, and unlimited The filter paper was obtained as purified tap water. The contents of calcium and phosphorus in the diet were 1.2 〇/〇 and 1.0 〇/〇, respectively. By injecting a single injection into the 〇〇 5 ml of paraffin oil (in the tail root) 0.5 mg of heat-killed tuberculosis halogen H37Ra (Difco Laboratories, Detroit, MI) in Crescent Chemical Co., Hauppauge, ΝΥ), induced adjuvant arthritis. On day 9, there was swelling of the hind paw and The clinical symptoms of arthritis represented by difficulty in walking. Except for the 2xc〇n4(C) treatment group (treated from the first day after immunization), the treatment of subcutaneous injection per sputum was started on the 9th day after immunization ( Before the onset of arthritis) and continued until day 18. Clinical surveillance of adjuvant arthritis By foot bed volume after intermittently measured, according to the use of
Feige等人，Cellular Molec· Life Sci·，57:1457-1470 (2000) 描述之方法的水體積描記法，來評估炎症的進行。以在曲 線下之面積（AUC)為基礎，使用根據下式的梯形規則，來 O:\121\121929.DOC -186 - 200808833 叶异腳掌炎症的抑制作用： [1-{(經過處理的AdA)-正常值)/(未經處理之AdA-正常值)}]χ100 此外’在9-天的治療攝生法期間内，每天判定總體重， 作為補充的終點’因為在關節炎模式中，體重喪失已經顯 不關節炎症的平行進行。在第18天時，在co2下犧牲動物。 在驗屍時（免疫後第i 8天），檢查骨礦物質密度（BMD)的 喪失。在軟毛分界處（正好在足踝（後腳踝）附近）移除後腳 掌，浸泡在70。/〇乙醇中，然後以水平方向使用扇形光束χ-射線光密度計（QDR-4500A型；Hologic，Waltham，ΜΑ)掃 描。參見Feige等人，在前。在掃描之後，放好中心位置 在跟骨（calcaneus)的長方形盒子（29χ25毫米），以便描繪出 待分析之位置的輪廓，並以有專利的演算&(H〇1〇gic軟體） 計算骨面積、骨礦物質内含量和骨礦物質密度。 所有的結果均以平均值士標準差來表示。使用〇 〇5iP值 ’描述在組別之間的顯著差異。使用市售的統計軟體 (StatSoft 3.0版；StatSoft，Tulsa，OK)，對臨床數據（連續變 數）進行 Kruskal-Wallis AN0VA和 Mann-Whitney U·檢定。 分別在圖11 a、11 b和11 c中陳述其結果。 實例21 角膜血管生成模式 在大鼠中Con4(C)對VEGF·誘導之血管生成的影響 在大鼠的血管生成之角膜模式中，評估Ang_2肽體 Con4(C)。藉著將VEGF-(或BSA對照組）浸泡的尼龍圓盤植 入角膜基質内，誘導血管生成作用（n=8/組）。藉著皮下注 O:\121\121929.DOC -187- 200808833 射投與1.0或0·1毫克/大氣/天的肽體丁N8c〇n4_c，持續7天 。另兩組動物則以相同劑量的陰性對照組肽體4883處@理。 所有組別均以單一負荷劑量的3.0或0.3毫克進行前-處理， 其為維持劑量1.0或O.i毫克的3倍（參見圖式）。在處理7天 之後，從每個大鼠角膜的數位照片中，判定兩個血管終點 .在圓盤與角膜鞏膜連結部之間的中_點處交又的血管數 目，以及血管面積。以TN8CON4-C處理，以劑量·依賴性 的方式’明顯地抑制了 VEGF-誘導的血管生成作用 (ρ<0·04) ’而以對照組肽體處理’對任_終點均無顯著影 響。以接受處理之動物的體重為基礎，並沒有明顯毒性的 證據。在圖12中陳述其結果。 實例22 抗原決定位作圖 將全長（胺基酸1-495)、Ν-終端（胺基-終端（ 胺基酸255-495)的人類Ang_2 (hAng-2)蛋白質，選殖到帶 有C-終端6xHis標籤之CMV推動的哺乳動物表現載體内。 暫時性地在293T細胞中表現三個所得的構築體，加載體對 照組。然後從經過轉移感染的細胞中，收集調理培養基， 並藉著抗-6xhis ELISA和西方墨點法，評估在培養基中 Ang-2的表現程度。 經由ELISA，根據下列的草案，藉著其與三種版本之人 類hAng-2結合的能力，判定抗_Ang-2抗體和肽體的結合抗 原決定位：以100微升調理培養基塗覆高_結合之96_孔測定 盤的各孔’並在37°C下培養1小時。吸出調理培養基，並 O:\121\121929.DOC -188- 200808833 在室溫下，以每孔200微升在PBS中的5% BSA阻斷該盤1小 時。然後吸出阻斷溶液。以1微克/毫升（在在PBS中之1% BSA中），加入每孔100微升抗體、肽體或Tie2-Fc，並在室 溫下培養1小時。以200微升在PBS中的0.1%吐溫沖洗該孔 4次。每孔加入100微升HRP-共軛的山羊抗-人類IgG或山羊 抗-老鼠IgG，並在室溫下培養45分鐘。然後以200微升在 PBS中的0.1%吐溫沖洗該孔4次。然後每孔加入100微升 TMB受質。在370毫微米處讀取O.D.值。 分別在圖13a、圖13b和圖13c中陳述其結果。 實例23 因為在BiaCore測定中有某些敏感性的限制，亦使用 Sepidyne KinExA測定來評估結合親和力。 在 KinExA (Sapidyne，Boise，ID)上測試 2xCON4-C (Pb5714)與huAng_2的結合作用。預先以huAng_2塗覆 Reacti-Gel 6x 小珠（Pierce，Rockford，IL)，並以BSA 阻斷。 在跑過塗覆huAng-2的小珠之前，先將10 pM和30 pM的 2xCON4-C試樣與各種濃度（0·3 pM-3 nM)的huAng-2—起在 室溫下培養8小時。藉著螢光（Cy5)標示之山羊抗-人類-Fc 抗體（Jackson Immuno Research，West Grove，PA)，定量與 小珠結合之肽體的量。結合信號與平衡時自由肽體的濃度 成比例。 使用雙-曲線單一-位置均一結合模式（KinExTM軟體），從 競爭性曲線的非線性回歸中獲得解離平衡常數（KD)。然後 判定KD為大約2 pM的2xCON4-C與HuAng-2結合。 O:\121\121929.DOC -189- 200808833 如同在圖14中所示的，使用KinExA測定，顯示肽體 2xCon4對hAng-2具有大約2pM之親和力。 實例24 聚乙二醇化的肽 以431 ABI合成器，使用標準偶聯草案以及雙重偶聯， 從殘基14(met)至N-終端殘基l(Cys)(從N-終端至C-終端編 號），合成L1-7肽。 L1-7肽與甲氧基-聚（乙二醇）-順丁烯二醯亞胺的共輛作用 ;分子量：5KDa ;稱為”mPEG5K-(Ll-7肽）” 利用13.5毫克甲氧基-聚（乙二醇）-順丁烯二醯亞胺（分子 量=5KDa ; Shearwater Corp_)(0.27毫升在緩衝溶液1中的 50.0毫克/毫升溶液），處理在400微升緩衝溶液1 (20 mM磷 酸鹽，5 mM EDTA，pH 6.5)中之0.8毫克L1-7肽的溶液。 在4°C下培養該反應混合物過夜，然後以1.6毫升缓衝溶液 A (20 mM Tris鹽酸鹽，pH 7.2)稀釋，並在 Slide-A-Lyzer卡 匣（3 5 00 MWCO, Pierce)中，對相同的緩衝溶液透析。藉著 離子交換層析法，在1 ·0毫升HiTrap Q瓊脂糖HP管柱 (Amersham Biosciences Corp·)上，純化經過透析的反應混 合物。經由從100%緩衝溶液A至100%緩衝溶液B (緩衝溶 液A+0.5 M NaCl)的梯度，超過40份管柱體積，在兩個^ 毫升的溶離份中洗脫出產物高峰。利用Microsep 1K離心 裝置（Pall Life Sciences)，將混合的產物溶離份濃縮至250 微升，含有〇·23毫克蛋白質/毫升。 L1-7肤與1，11-雙-順丁烯二醯连胺基四乙二醇的共概作用 O:\121\121929.DOC -190- 200808833 ;稱為，，PE04 (L1-7肽）2，， 以0.03 75毫克的1，11-雙-順丁烯二醯亞胺基四乙二醇 (Pierce)(0.375毫升在缓衝溶液1中的0.1毫克/毫升溶液）， 處理在500微升缓衝溶液1 (20 mM磷酸鹽，5 mM EDTA， pH 6·5)中之1.0毫克L1-7肽的溶液。在4°C下培養該反應混 合物 3.33小時，然後在 Slide-A-Lyzer 卡匣（3500 MWCO, Pierce)中，對緩衝溶液A (20 mM THs鹽酸鹽，pH 7.2)透 析。藉著離子交換層析法，在1.0毫升HiTrap Q瓊脂糖HP 管柱（Amersham Biosciences Corp.)上，純化經過透析的反 應混合物。經由從100%緩衝溶液A至100%緩衝溶液B (緩 衝溶液A+0.5 M NaCl)的梯度，超過40份管柱體積，在三 個1 ·0毫升的溶離份中洗脫出二聚體的產物高峰。利用 Microsep 1K離心裝置（Pall Life Sciences)，將混合的產物 溶離份濃縮至550微升，含有0.12毫克蛋白質/毫升。 L1-7肽與聚（乙二醇）·雙-順丁烯二醯亞胺的共輊作用；分 子量：3.4KDa;稱為”PEG3.4K(Ll-7肽）2’， 以1.125毫克的聚（乙二醇雙-順丁烯二醯亞胺（分子量 = 3.4 KDa，Shearwater Corp.)(0.563 毫升在緩衝溶液1 中的 2.0毫克/毫升溶液），處理在1.5毫升缓衝溶液1 (20 mM磷 酸鹽，5 mM EDTA，pH 6.5)中之3.0毫克L1-7肽的溶液。 在4°C下培養該反應混合物過夜，然後在Slide-A-Lyzer卡 匣（3500 MWCO，Pierce)中，對緩衝溶液 A (20 mM Tris 鹽 酸鹽，pH 7·2)透析。藉著離子交換層析法，在5.0毫升 HiTrap Q瓊脂糖HP管柱（Amersham Biosciences Corp.)上， O:\121\121929.DOC -191 - 200808833 純化經過透析的反應混合物。經由從100%缓衝溶液A至 100%緩衝溶液B (緩衝溶液Α+0·5 M NaCl)的梯度，超過40 份管柱體積，在三個3.0毫升的溶離份中洗脫出產物高峰 。利用兩個 Microsep 1K離心裝置（Pall Life Sciences)，將 混合的產物溶離份濃縮至850微升，含有0.24毫克蛋白質/ 毫升。 MALDI-TOF質譜分析的結果如下： 試樣編號 身分 MS實驗值 MS觀察值 1 Ll-7(未PEG化的肽） 3,545 3,538.7 2 mPEG5K-(Ll-7 肽） 8,500 8,851 3 PE04(L1_7 肽)2 7,443 7,446.29 4 PEG3.4K(Ll-7 肽)2 10,550 10,552 6,882.61 3,550.13 應瞭解PEG3.4K(Ll-7肽）和PE04(Ll-7肽）的下標，代 表每個聚合物鏈有兩個肽，位在聚合物的兩端。 ic5〇的判定 藉著如同在實例2中描述之中和ELISA，判定自由的L1-7和PEG化的肽，對hAng-2:hTie2-Fc交互作用之抑制作用 的IC5〇。關於中和ELISA，按照在實例2中關於親和力 ELISA的描述，製備已結合人類Ang-2多肽的微量滴定盤 。滴定候選的抗-Ang-2之PEG化的L1-7和自由肽，從1000 nM至0.2 pM，以含有大約1% BSA和大約1 nM Tie-2 (以 Tie-2-Fc分子的形式提供，其中Tie-2部分僅含有該分子的 可溶性細胞外部外分；R&D Systems，目錄第313-TI號）的 PBS溶液4-倍稀釋。在每孔中加入大約100微升抗體/Tie-2 O:\121\121929.DOC -192- 200808833 溶液之後，在室溫下培養該盤過夜，然後以含有大約0.1% 吐溫-20的PBS沖洗5次。在沖洗之後，加入大約100微升/ 孔的抗-Tie-2抗體（Pharmingen Inc.，目錄#557039)，至大 約1微克/毫升的終濃度，並在室溫下培養該盤大約1小時 。接下來，以利用含有大約1°/。BSA之PBS的1:10,000稀釋 ，加入大約1〇〇微升/孔的山羊抗-老鼠-IgG-HPR (Pierce Chemical Co.，目錄#3 1432)。在室溫下培養該盤大約1小 時，隨後以含有大約〇·1%吐溫-20的PBS沖洗5次。然後加 入大約100微升/孔的TMB受質（上述），並容許顏色展現。 然後在分光光度計370毫微米處，讀取吸光度。 L1-7肽（OGGGGG-AQ-TNFMPMDDLEQRLYEQFILQQG-LE)(序列識別：359號）包括：與PEG偶聯的N-終端半胱胺 酸；以及5Gly聯結子。AQ和LE側面序列兩者均出現在原 始的嗟菌 體純種系和該肽體中。hAng_2:Tie2抑制IC50結果如下： 肽 IC5〇 (nM) L1-7 肽 0.49 mPEG5K-(Ll-7 肽） 11.7 PE04(Ll-7 肽)2 0.064 PEG3.4K(Ll-7 肽)2 0.058 【圖式簡單說明】 圖1敘述在利用本發明之肽體TN8-Con4-C，或以磷酸缓 衝之生理鹽水（PBS)處理的帶有A-431腫瘤之小鼠中，腫瘤 體積（y-軸）對時間（x_轴）的圖。在實例中描述其細節。Feige et al., Cellular Molec· Life Sci., 57: 1457-1470 (2000) describe the method of water plethysmography to assess the progression of inflammation. Based on the area under the curve (AUC), use the trapezoidal rule according to the following formula to O:\121\121929.DOC -186 - 200808833 Inhibition of leaf-to-foot inflammation: [1-{(Processed AdA) ) - normal value) / (untreated AdA - normal value)}] χ 100 In addition, during the 9-day treatment regimen, the total weight is judged daily as a supplementary end point 'because in arthritis mode, weight Loss has been carried out in parallel with the development of joint inflammation. On day 18, the animals were sacrificed under co2. At the time of autopsy (day i8 post immunization), the loss of bone mineral density (BMD) was examined. Remove the hind paw at the soft hair boundary (near the ankle (after the ankle)) and soak at 70. /〇 in ethanol, then scanned in a horizontal direction using a fan beam χ-ray densitometer (Model QDR-4500A; Hologic, Waltham, ΜΑ). See Feige et al., formerly. After scanning, place the rectangular box (29χ25 mm) centered on the calcaneus to depict the contour of the location to be analyzed and calculate the bone with a patented calculation & (H〇1〇gic software) Area, bone mineral content and bone mineral density. All results are expressed as mean ± standard deviation. A significant difference between the groups is described using 〇 〇 5iP value ‘. Kruskal-Wallis AN0VA and Mann-Whitney U. assays were performed on clinical data (continuous variables) using commercially available statistical software (StatSoft version 3.0; StatSoft, Tulsa, OK). The results are presented in Figures 11 a, 11 b and 11 c, respectively. Example 21 Corneal Angiogenesis Pattern Effect of Con4(C) on VEGF-Induced Angiogenesis in Rats Ang_2 peptibody Con4 (C) was evaluated in the angiogenic cornea model of rats. Angiogenesis was induced by implantation of a nylon disc soaked with VEGF- (or BSA control) into the corneal stroma (n=8/group). By the skin bet O:\121\121929.DOC -187- 200808833 Shoot with 1.0 or 0.1 mg/atmosphere/day of the peptide body N8c〇n4_c for 7 days. The other two groups of animals were treated with the same dose of the negative control peptibody at 4883. All groups were pre-treated with a single loading dose of 3.0 or 0.3 mg, which was 3 times the maintenance dose of 1.0 or 0.5 mg (see figure). After 7 days of treatment, the two vascular end points were determined from the digital photograph of each rat cornea. The number of blood vessels at the midpoint between the disc and the corneal sclera junction, and the blood vessel area. Treatment with TN8CON4-C significantly inhibited VEGF-induced angiogenesis (ρ<0·04)' in a dose-dependent manner and had no significant effect on the _ end point of treatment with the control peptone. Based on the body weight of the treated animals, there is no evidence of significant toxicity. The result is set forth in FIG. Example 22 Epitope mapping The full length (amino acid 1-495), guanidine-terminal (amino-terminal (amino acid 255-495) human Ang_2 (hAng-2) protein, was colonized with C - Terminal 6xHis tagged CMV-driven mammalian expression vector. Temporarily expressed three resulting constructs in the 293T cells, the loading control group. Then, the culture medium was collected from the transfected cells, and Anti--6xhis ELISA and Western blotting method were used to evaluate the degree of expression of Ang-2 in the medium. According to the following draft, the anti-Ang-2 was judged by its ability to bind to three versions of human hAng-2 according to the following draft. Binding epitopes of antibodies and peptibodies: Coating each well of a high-binding 96-well assay disk with 100 microliters of conditioning medium and incubating for 1 hour at 37° C. Aspirate the conditioned medium and O:\121 \121929.DOC -188- 200808833 Block the plate for 1 hour at room temperature with 200 μl of 5% BSA in PBS. Then aspirate the blocking solution to 1 μg/ml (in PBS) 1% BSA), add 100 μl of antibody, peptibody or Tie2-Fc per well and at room temperature Incubate for 1 hour. Rinse the well 4 times with 200 μl of 0.1% Tween in PBS. Add 100 μl of HRP-conjugated goat anti-human IgG or goat anti-mouse IgG per well and at room temperature Incubate for 45 minutes. Then rinse the well 4 times with 200 μl of 0.1% Tween in PBS. Then add 100 μl of TMB substrate per well. Read the OD value at 370 nm. Figure 13a, Figure The results are set forth in 13b and Figure 13c.Example 23 The Sepidyne KinExA assay was also used to assess binding affinity due to certain sensitivity limitations in the BiaCore assay. 2xCON4-C was tested on KinExA (Sapidyne, Boise, ID) (Pb5714) Binding with huAng_2. Reacti-Gel 6x beads (Pierce, Rockford, IL) were pre-coated with huAng_2 and blocked with BSA. Before running the beads coated with huAng-2, first 10 pM and A 30 pM 2xCON4-C sample was incubated with various concentrations (0·3 pM-3 nM) of huAng-2 for 8 hours at room temperature. Goat anti-human-Fc antibody by fluorescent (Cy5) (Jackson Immuno Research, West Grove, PA), quantifying the amount of peptibosome bound to beads. Freedom of binding signal and balance The concentration of the peptibody is proportional. Using the double-curve single-position uniform binding mode (KinExTM software), the dissociation equilibrium constant (KD) is obtained from the nonlinear regression of the competitive curve. It was then determined that 2xCON4-C with a KD of about 2 pM bound to HuAng-2. O: \121\121929.DOC -189- 200808833 As shown in Figure 14, using the KinExA assay, it was shown that the peptibody 2xCon4 has an affinity of about 2 pM for hAng-2. Example 24 PEGylated peptide with a 431 ABI synthesizer using standard coupling draft and double coupling, from residue 14 (met) to N-terminal residue l (Cys) (from N-terminal to C-terminal No.), synthesizing L1-7 peptide. Co-action of L1-7 peptide with methoxy-poly(ethylene glycol)-maleimide; molecular weight: 5KDa; called "mPEG5K-(Ll-7 peptide)" Using 13.5 mg methoxy - Poly(ethylene glycol)-m-butyleneimine (molecular weight = 5 KDa; Shearwater Corp_) (0.27 ml of 50.0 mg/ml solution in buffer solution 1), treated in 400 μl of buffer solution 1 (20 mM) A solution of 0.8 mg of L1-7 peptide in phosphate, 5 mM EDTA, pH 6.5). The reaction mixture was incubated at 4 ° C overnight, then diluted with 1.6 ml of buffer solution A (20 mM Tris hydrochloride, pH 7.2) and in a Slide-A-Lyzer cassette (3 5 00 MWCO, Pierce). , dialysis against the same buffer solution. The dialyzed reaction mixture was purified by ion exchange chromatography on a 1.0 mL HiTrap Q Sepharose HP column (Amersham Biosciences Corp.). A product peak eluted in two ^ ml of the fraction over a column volume of more than 40 column volumes via a gradient from 100% buffer solution A to 100% buffer solution B (buffer solution A + 0.5 M NaCl). The mixed product fractions were concentrated to 250 microliters using a Microsep 1K centrifuge (Pall Life Sciences) containing 〇23 mg protein/ml. The co-acting effect of L1-7 peptide with 1,11-bis-m-butylenediamine tetraethylene glycol O:\121\121929.DOC-190- 200808833; called, PE04 (L1-7 peptide 2,, with 0.03 75 mg of 1,11-bis-maleimidoiminotetraethylene glycol (Pierce) (0.375 ml of 0.1 mg/ml solution in buffer solution 1), treated at 500 A solution of 1.0 mg of L1-7 peptide in microliter buffer solution 1 (20 mM phosphate, 5 mM EDTA, pH 6.5). The reaction mixture was incubated at 4 ° C for 3.33 hours and then dialyzed against buffer A (20 mM THs hydrochloride, pH 7.2) in a Slide-A-Lyzer cassette (3500 MWCO, Pierce). The dialyzed reaction mixture was purified by ion exchange chromatography on a 1.0 ml HiTrap Q Sepharose HP column (Amersham Biosciences Corp.). Dimer eluted in three 1.0 mL of the fraction over a volume of more than 40 column volumes via a gradient from 100% buffer solution A to 100% buffer solution B (buffer solution A + 0.5 M NaCl) Product peak. The mixed product fractions were concentrated to 550 μl using a Microsep 1K centrifuge (Pall Life Sciences) containing 0.12 mg protein/ml. Co-quinone effect of L1-7 peptide with poly(ethylene glycol)·bis-maleimide; molecular weight: 3.4KDa; called “PEG3.4K (Ll-7 peptide) 2′, with 1.125 mg Poly(ethylene glycol bis-m-butyleneimine (molecular weight = 3.4 KDa, Shearwater Corp.) (0.563 ml of 2.0 mg/ml solution in buffer solution 1), treated in 1.5 ml buffer solution 1 (20 a solution of 3.0 mg of L1-7 peptide in mM phosphate, 5 mM EDTA, pH 6.5). The reaction mixture was incubated overnight at 4 ° C, then in a Slide-A-Lyzer cassette (3500 MWCO, Pierce), Dialysis against buffer solution A (20 mM Tris hydrochloride, pH 7.2) by ion exchange chromatography on a 5.0 ml HiTrap Q Sepharose HP column (Amersham Biosciences Corp.), O:\121\ 121929.DOC -191 - 200808833 Purification of the dialyzed reaction mixture. Over a volume of more than 40 column volumes via a gradient from 100% buffer solution A to 100% buffer solution B (buffer solution Α +0·5 M NaCl) Product peaks eluted from three 3.0 ml fractions. The mixed product was dissolved using two Microsep 1K centrifuges (Pall Life Sciences). Reduced to 850 μl, containing 0.24 mg protein/ml. The results of MALDI-TOF mass spectrometry are as follows: Sample number identity MS experimental value MS observation 1 Ll-7 (unPEGylated peptide) 3,545 3,538.7 2 mPEG5K-(Ll -7 peptide) 8,500 8,851 3 PE04 (L1_7 peptide) 2 7,443 7,446.29 4 PEG3.4K (Ll-7 peptide) 2 10,550 10,552 6,882.61 3,550.13 It should be understood that PEG3.4K (Ll-7 peptide) and PE04 (Ll-7 peptide) The subscript, which represents two peptides per polymer chain, is located at both ends of the polymer. The determination of ic5〇 was determined by the same as described in Example 2 and ELISA to determine free L1-7 and PEGylated peptides. , IC5 抑制 for inhibition of hAng-2:hTie2-Fc interaction. For neutralization ELISA, microtiter plates bound to human Ang-2 polypeptide were prepared as described for affinity ELISA in Example 2. Titration candidates Anti-Ang-2 PEGylated L1-7 and free peptide, from 1000 nM to 0.2 pM, containing approximately 1% BSA and approximately 1 nM Tie-2 (provided as a Tie-2-Fc molecule, where Tie -2 part contains only the soluble external exogenous fraction of the molecule; R&D Systems, catalogue No. 313-TI) PBS solution 4-fold thin . After adding approximately 100 microliters of antibody/Tie-2 O:\121\121929.DOC-192-200808833 solution to each well, the plate was incubated overnight at room temperature and then in PBS containing approximately 0.1% Tween-20. Rinse 5 times. After rinsing, approximately 100 μl/well of anti-Tie-2 antibody (Pharmingen Inc., catalog #557039) was added to a final concentration of approximately 1 μg/ml, and the plate was incubated at room temperature for approximately 1 hour. Next, to take advantage of containing about 1 ° /. A 1:10,000 dilution of BSA in PBS was added to approximately 1 μL/well of goat anti-mouse-IgG-HPR (Pierce Chemical Co., catalog #3 1432). The plate was incubated at room temperature for about 1 hour, and then washed 5 times with PBS containing about 0.1% Tween-20. Then about 100 microliters/well of TMB substrate (described above) was added and color rendering was allowed. The absorbance was then read at 370 nm in a spectrophotometer. The L1-7 peptide (OGGGGG-AQ-TNFMPMDDLEQRLYEQFILQQG-LE) (sequence recognition: No. 359) includes: N-terminal cysteine coupled to PEG; and 5Gly linker. Both the AQ and LE side sequences are present in the original sputum pure line and the peptide. The results of hAng_2:Tie2 inhibition IC50 are as follows: peptide IC5〇(nM) L1-7 peptide 0.49 mPEG5K-(Ll-7 peptide) 11.7 PE04(Ll-7 peptide)2 0.064 PEG3.4K(Ll-7 peptide)2 0.058 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts tumor volume (y-axis in mice bearing A-431 tumors treated with the peptide body TN8-Con4-C of the present invention or phosphate buffered saline (PBS). ) A graph of time (x_axis). The details are described in the examples.
圖2敘述在以50微克劑量之2xCon4-C、L1-7-N或L1-21-N O:\121\121929.DOC -193- 200808833 肤體處理的野外型老鼠中，肽體濃度（y_軸）對投藥後時間 (X-軸）的圖。在實例中描述其細節。 圖3敘述在利用根據本發明之肽體2xc〇n4-C，或以填酸 緩衝之生理鹽水（PBS)，或對照組肽體處理的帶有A431腫 瘤之小鼠中，腫瘤體積（y_軸）對時間（x-軸）的圖。在實例中 描述其細節。 圖4敘述代表以根據本發明之肽體con4_c、對照組肽體 處理’或未處理的經過培養之A43 1細胞，在活體外生長的 圖。在實例中描述其細節。 圖5敘述在利用根據本發明之肽體C0n4-c、肽體L1-7-N 、肽體L1-21-N，或肽體2xCon4_C，或以填酸緩衝之生理 鹽水（PBS)、抗-Ang-2抗體（Ab53 6)，或Fc處理的C〇1〇205腫 瘤細胞中，腫瘤體積（y-軸）對時間（x_軸）的圖。在實例中描 述其細節。 圖6敘述在利用各種劑量之根據本發明的肽體2xC〇n4-C ’或以磷酸緩衝之生理鹽水（PBS)或Fc處理的帶有C〇i〇2〇5 異種移植腫瘤的小鼠中，腫瘤體積（y_軸）對時間（χ_轴）的圖 。在實例中描述其細節。 圖7敘述在利用根據本發明之肽體2xC〇n4-C，或以對照 組肽體處理的帶有C〇1〇205異種移植腫瘤的小鼠中，腫瘤 體積（y-軸）對時間（X-軸）的圖。圖7亦敘述這些肽體的CD31 染色面積/總腫瘤面積的圖。在實例中描述其細節。 圖8敘述在利用根據本發明之肽體2xc〇n4-C，或以構酸 緩衝之生理鹽水（PBS)，或對照組肽體處理的帶有c〇l〇2〇5 O:\121\121929JDOC -194- 200808833 異種移植腫瘤的小鼠中，腫瘤體積（y_軸）對時間（χ_軸）的圖 。在實例中描述其細節。該圖顯示抗_Ang_2肽體能夠抑制 C〇1〇205腫瘤生長，不考慮何時開始投藥。 圖9敘述在A431*c〇i〇_2〇5異種移植模式兩者中，在雌 性裸鼠中，使用抗體Ab536或利用肽體2xC〇n4_cm獲得之 完全反應（CR)速率的概要。在實例中描述其細節。 圖10A敘述在利用根據本發明之肽體2xC〇n4_c，或 2xCon4-C與剋癌易的組合，或以磷酸緩衝之生理鹽水 (PBS)，或以PBS加剋癌易處理的帶有c〇1〇2〇5異種移植腫 瘤的小鼠中，腫瘤體積（y-軸）對時間（x_軸）的圖。在實例中 描述其細節。 圖10B敘述在利用根據本發明之肽體2xC〇n4_c，或 2xCon4-C與5-FU的組合，或以磷酸緩衝之生理鹽水（pBS) ，或以PBS加5-FU處理的帶有C〇1〇205異種移植腫瘤的小 鼠中，腫瘤體積（y-轴）對時間（X-軸）的圖。在實例中描述其 細節。 圖11A敘述在佐劑-引起之關節炎模式中，在以根據本發 明之肽體2xCon4-C，或填酸緩衝之生理鹽水（pbs)，或以 對照組肽體，或正常或關節炎對照組處理的大鼠中，腳掌 腫脹程度（AUC±SE)的圖。在實例中描述其細節。 圖11B敘述在佐劑-引起之關節炎模式中，在以根據本發 明之肽體2xCon4-C，或磷酸緩衝之生理鹽水（pbS)，或以 對照組肽體，或正常或關節炎對照組處理的大鼠中，腳掌 骨礦物質密度（BMD)的圖。在實例中描述其細節。 O:\121\121929.DOC -195- 200808833 圖lie敘述在佐劑-引起之關節炎模式中，在以根據本發 明之肽體2xCon4-C，或攝酸緩衝之生理鹽水（pBS)，或以 對照組肽體，或正常或關節炎對照組處理的大鼠中，體重 變化的圖。在實例中描述其細節。 圖12敘述兩個在大鼠中，敘述抑制VEGF_引起之角膜 血管生成作用的圖。第一個圖敘述在以牛血清白蛋白 (BSA)、VEGF加磷酸緩衝之生理鹽水（PBS)，或…郎加本 發明之肤體C 〇n4 - C處理的大鼠中，測量到的血管數目。第 二個圖敘述在以BSA、VEGF加磷酸緩衝之生理鹽水（pBS) ，或VEGF加本發明之肽體Con4_c處理之大鼠中的血管面 積（平方毫米）。在實例中描述其細節。 圖13A、13B和13C敘述全長的人類Ang_2 (hAng-2)，分 別對hAng-2的N-終端，以及對hAng-2的C-終端，根據本發 明之肽體TN8-Con4-C、L1-7-N和12-9-3-C，以及對照組肽 體Tie2-Fc、C2B8或5B12的抗原決定位作圖資料（〇d.37〇) 。在實例中描述其細節。 圖14敘述根據本發明之2xC〇n_4-C肽體的結合親和力 (KD)，使用Sapidyne KinExA測定。在實例中描述其細節。Figure 2 depicts the peptidic concentration in wild-type mice treated with a 50 microgram dose of 2xCon4-C, L1-7-N or L1-21-NO:\121\121929.DOC-193-200808833 skin (y_) Axis) A graph of the time after administration (X-axis). The details are described in the examples. Figure 3 depicts tumor volume (y_) in mice bearing A431 tumors treated with peptidom 2xc〇n4-C according to the invention, or acid-buffered saline (PBS), or control peptibody. Axis) A graph of time (x-axis). The details are described in the examples. Figure 4 depicts a graph showing growth in vitro of cultured A43 1 cells treated with peptidom body con4_c, control peptone, or untreated according to the present invention. The details are described in the examples. Figure 5 illustrates the use of a peptone C0n4-c, a peptibody L1-7-N, a peptibody L1-21-N, or a peptibody 2xCon4_C according to the present invention, or an acid-buffered physiological saline (PBS), anti- Tumor volume (y-axis) versus time (x-axis) in Ang-2 antibody (Ab53 6), or Fc-treated C〇1〇205 tumor cells. The details are described in the examples. Figure 6 depicts a mouse with a C〇i〇2〇5 xenograft tumor treated with various doses of the peptibody 2xC〇n4-C ' or phosphate buffered saline (PBS) or Fc according to the present invention. , a plot of tumor volume (y_axis) versus time (χ_axis). The details are described in the examples. Figure 7 depicts tumor volume (y-axis) vs. time in mice bearing C〇1〇205 xenograft tumors treated with peptibody 2xC〇n4-C according to the invention or treated with control peptibodies ( Diagram of the X-axis). Figure 7 also depicts a plot of CD31 stained area/total tumor area of these peptibodies. The details are described in the examples. Figure 8 depicts c带有l〇2〇5 O:\121\ treated with peptidom 2xc〇n4-C according to the invention, or physiologically buffered saline (PBS), or control peptibody. 121929JDOC -194- 200808833 A plot of tumor volume (y_axis) versus time (χ_axis) in mice xenografted. The details are described in the examples. The figure shows that the anti-Ang_2 peptibosome can inhibit C〇1〇205 tumor growth without considering when to start drug administration. Figure 9 is a summary of the complete reaction (CR) rate obtained in antibody nude mice using either antibody Ab536 or peptibody 2xC〇n4_cm in both A431*c〇i〇_2〇5 xenograft models. The details are described in the examples. Figure 10A depicts a c〇 with a combination of the peptibody 2xC〇n4_c, or 2xCon4-C according to the present invention, or a phosphate buffered saline (PBS), or a PBS plus cancer. Tumor volume (y-axis) vs. time (x-axis) in 1〇2〇5 xenografted tumor mice. The details are described in the examples. Figure 10B depicts a C with a peptibody 2xC〇n4_c according to the invention, or a combination of 2xCon4-C and 5-FU, or phosphate buffered saline (pBS), or PBS plus 5-FU. Tumor volume (y-axis) vs. time (X-axis) plot of 1 〇 205 xenografted tumor mice. The details are described in the examples. Figure 11A depicts in the adjuvant-induced arthritis mode, in the peptidom 2xCon4-C according to the invention, or in acid-buffered saline (pbs), or in the control peptibody, or normal or arthritic control A graph of the degree of swelling of the foot (AUC ± SE) in the rats treated in the group. The details are described in the examples. Figure 11B depicts in the adjuvant-induced arthritis model, in the peptidom 2xCon4-C, or phosphate buffered saline (pbS) according to the present invention, or in the control peptibody, or normal or arthritic control group. A map of the mineral density (BMD) of the metacarpal bone in treated rats. The details are described in the examples. O:\121\121929.DOC-195-200808833 Figure lie is described in the adjuvant-induced arthritis mode, in the peptidom 2xCon4-C according to the invention, or in acid buffered saline (pBS), or A graph of changes in body weight in rats treated with control peptibodies, or normal or arthritic controls. The details are described in the examples. Fig. 12 is a graph showing the effects of inhibition of VEGF_induced corneal angiogenesis in two rats. The first figure depicts the blood vessels measured in rats treated with bovine serum albumin (BSA), VEGF plus phosphate buffered saline (PBS), or ... Lang plus the body of the invention C 〇 n4 - C number. The second panel depicts the vascular area (square millimeters) in rats treated with BSA, VEGF plus phosphate buffered saline (pBS), or VEGF plus the peptibody Con4_c of the invention. The details are described in the examples. Figures 13A, 13B and 13C depict full-length human Ang_2 (hAng-2), the N-terminus of hAng-2, and the C-terminus of hAng-2, respectively, according to the peptibody TN8-Con4-C, L1 of the present invention -7-N and 12-9-3-C, and epitope mapping data of the control peptide Tie2-Fc, C2B8 or 5B12 (〇d.37〇). The details are described in the examples. Figure 14 depicts the binding affinity (KD) of the 2xC〇n_4-C peptibody according to the present invention, as determined using Sapidyne KinExA. The details are described in the examples.
O:\121\121929.DOC 196- 200808833 序列一覽表 <110> AMGEN INC. < 120>人類血管生成素-2的專一結合劑O:\121\121929.DOC 196- 200808833 Sequence Listing <110> AMGEN INC. <120> Specific Binding Agent for Human Angiopoietin-2
<130> A-810A <140>尚未指派 <141> 2002-10-11 <150>US (尚未指派） <151> 2002-09-27 <150> US 60/328,624 <151> 2001-10-11 <160> 359 <170>PatentIN 版本 3.1 <210> 1 <211> 14 <212〉蛋白質 <213> Ait Η <220> <223> Ang-2結合肽<130> A-810A <140> has not been assigned <141> 2002-10-11 <150>US (not yet assigned) <151> 2002-09-27 <150> US 60/328,624 <151> 2001-10-11 <160> 359 <170> PatentIN Version 3.1 <210> 1 <211> 14 <212>Protein<213> Ait Η <220><223> Ang- Binding peptide
O:\121\121929.DOC 200808833 <400> 1O:\121\121929.DOC 200808833 <400> 1
Lys Arg Pro Cys Glu Glu Met Trp Gly Gly Cys Asn Tyr Asp 1 5 10 <210> 2 <211> 14 ·· <212> 蛋白質 <213> 人造的序列 <220> <223> Ang-2結合肽 <400> 2 His Gin lie Cys Lys Trp Asp Pro Trp Thr Cys Lys His Trp 1 5 10 <210> 3 <211〉 14 <212> 蛋白質 <213> 人造的序列 <220〉 <223> Ang-2結合多肽 <400> 3 Lys Arg Pro Cys Glu Glu lie Phe Gly Gly Cys Thr Tyr Gin 1 5 10 <210> 4 <211〉 14 <212> 蛋白質 <213> 人造的序列Lys Arg Pro Cys Glu Glu Met Trp Gly Gly Cys Asn Tyr Asp 1 5 10 <210> 2 <211> 14 ·· <212> Protein <213> Artificial Sequence <220><223> Ang -2 binding peptide <400> 2 His Gin lie Cys Lys Trp Asp Pro Trp Thr Cys Lys His Trp 1 5 10 <210> 3 <211> 14 <212> Protein <213> Artificial sequence< 220> <223> Ang-2 binding polypeptide <400> 3 Lys Arg Pro Cys Glu Glu lie Phe Gly Gly Cys Thr Tyr Gin 1 5 10 <210> 4 <211> 14 <212> Protein <213> artificial sequence
O:\121\121929.DOC 2- 200808833 <220> <223> Ang-2結合多肽 <400> 4O:\121\121929.DOC 2- 200808833 <220><223> Ang-2 binding polypeptide <400> 4
Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met 15 10 <210> 5 <211> 18 <212>蛋白質 <213>人造的序列 <220> <223> Ang-2結合多肽 <400> 5Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met 15 10 <210> 5 <211> 18 <212>Protein <213> Artificial Sequence <220><223> Ang-2 Combination Peptide <400> 5
Phe Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys Asp 1 5 10 15Phe Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys Asp 1 5 10 15
Asn His <210〉 6 <211> 20 <212>蛋白質 <213> 人造的序列 <220> <223> Ang-2結合多肽 <400> 6Asn His <210> 6 <211> 20 <212> Protein <213> Artificial sequence <220><223> Ang-2 binding polypeptide <400>
Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe 15 10 15Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe 15 10 15
Thr Phe Gin Gin 20Thr Phe Gin Gin 20
O:\121\121929.DOC 200808833 <210> 7 <211> 14 <212>蛋白質 <213>人造的序列 <220> <223> Ang-2結合肽 <400> 7 Gin Tyr Gly Cys Asp Gly Phe Leu Tyr Gly Cys Met lie Asn 1 5 <210> 8 <211> 6Ί <212> DNA <213> 人造的序列 <220> <223> 寡核荅酸 <400〉 8 acaaacaaac atatgggtgc acagaaagcg gccgcaaaaa aactcgaggg tggaggcggt ggggaca <210> 9 <211> 20 <212> DNA <213> 人造的序列 <220> <223> 寡核甞酸 60 67O:\121\121929.DOC 200808833 <210> 7 <211> 14 <212> Protein <213> Artificial sequence <220><223> Ang-2 binding peptide <400> 7 Gin Tyr Gly Cys Asp Gly Phe Leu Tyr Gly Cys Met lie Asn 1 5 <210> 8 <211> 6Ί <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide < 400> 8 acaaacaaac atatgggtgc acagaaagcg gccgcaaaaa aactcgaggg tggaggcggt ggggaca <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide 60 67
O:\121\121929.DOC -4- 200808833 <400> 9 ggtcattact ggaccggatc 20 <210〉 10 <211> 25 <212〉 DNA <213> 人造的序列 <220> <223> 寡核甞酸 <400> 10 cgtacaggtt tacgcaagaa aatgg 25 <210> 11 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 寡核甞酸 <400> 11 tttgttggat ccattactcg agtttttttg cggccgcttt ctgtgcacca ccacctccac 60 ctttac 66 <210〉 12 <211> 32 <212> 蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體O:\121\121929.DOC -4- 200808833 <400> 9 ggtcattact ggaccggatc 20 <210> 10 <211> 25 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 10 cgtacaggtt tacgcaagaa aatgg 25 <210> 11 <211> 66 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 11 tttgttggat ccattactcg agtttttttg cggccgcttt ctgtgcacca ccacctccac 60 ctttac 66 <210> 12 <211> 32 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2
O:\121\121929.DOC 200808833 <220> <221> misc_特徵 <222> (32)..(32) <223> Xaa = Fc <400> 12O:\121\121929.DOC 200808833 <220><221> misc_features <222> (32)..(32) <223> Xaa = Fc <400> 12
Met Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu 15 10 15Met Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu 15 10 15
Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 30 <210> 13 <211> 29 <212>蛋白質 <213>人造的序列 <220〉 <223>能夠與Ang-2結合的肽體 <220> <221> misc _特徵 <222> (29).· .(29) <223> Xaa = Fc <400> 13Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 30 <210> 13 <211> 29 <212>Protein<213> Man-made sequence <220><223> Peptide bound to Ang-2 <220><221> misc _ feature <222> (29). (29) <223> Xaa = Fc <400>
Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin 15 10 15Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin 15 10 15
Phe Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 14Phe Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 14
O:\121\121929.DOC 200808833 <211〉 51 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222〉 (51)..(51) <223> Xaa = Fc <400〉 14O:\121\121929.DOC 200808833 <211> 51 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i>; misc_ feature <222> (51)..(51) <223> Xaa = Fc <400> 14
Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr· Glu Gin 15 10 15Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr· Glu Gin 15 10 15
Phe Thr Phe Gin Gin Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser 20 25 30Phe Thr Phe Gin Gin Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser 20 25 30
Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Leu Glu Gly Gly Gly 35 40 45Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Leu Glu Gly Gly Gly 35 40 45
Gly Gly Xaa 50 <210> 15 <211> 60 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽雅 <220>Gly Gly Xaa 50 <210> 15 <211> 60 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220>
O:\121\121929.DOC 200808833 <221> misc_特徵 <222> (60) . . (60) <223> Xaa = Fc <400> 15O:\121\121929.DOC 200808833 <221> misc_features <222> (60) . . . (60) <223> Xaa = Fc <400>
Met Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu 15 10 15Met Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu 15 10 15
Tyr Glu Gin Phe Thr Phe Gin Gin Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30Tyr Glu Gin Ghe Ghe Gly Gly Gly Gly Gly Gly 20 25 30
Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe 35 40 45Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe 35 40 45
Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 60 <210> 16 <211> 56 <212〉 蛋白質 <213〉 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (56) . . (56) <223> Xaa = Fc <400> 16Thr Phe Gin Gin Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 60 <210> 16 <211> 56 <212> Protein <213> Artificial Sequence <220><223><220> Peptide bound to Ang-2 <221> misc_ feature <222> (56) . (56) <223> Xaa = Fc <400>
Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin 15 10 15Met Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin 15 10 15
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Phe Thr Phe Gin Gin Gly Gly Gly Gly Gly Gly Gly Lys Phe Asn Pro 20 25 30Phe Thr Phe Gin Gin Gly Gly Gly Gly Gly Gly Gly Lys Phe Asn Pro 20 25 30
Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin 35 40 45Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin 35 40 45
Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 <210〉 17 <211> 26 <212> 蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc_ 特徵 <222> (26)..(26) <223> Xaa = Fc <400> 17Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 <210> 17 <211> 26 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <;220><221> misc_ feature <222> (26)..(26) <223> Xaa = Fc <400> 17
Met Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu 15 10 15Met Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu 15 10 15
His Met Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 18 <211> 45 <212>蛋白質 <213>人造的序列His Met Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 18 <211> 45 <212> Protein <213> Artificial Sequence
O:\121\121929.DOC 200808833 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 (45)..(45) <223> Xaa = Fc <400> 18O:\121\121929.DOC 200808833 <220> <223> Peptide capable of binding to Ang-2 <220><22i> misc-characteristic <222> (45)..(45) <;223> Xaa = Fc <400> 18
Met Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly 15 10 15Met Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly 15 10 15
Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser 20 25 30Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser 20 25 30
Gly Ser Ala Thr His Leu Glu Gly Gly Gly Gly Gly Xaa 35 40 45 <210> 19 <211〉 62 <212> 蛋白質 <213〉 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc__特徵 <222> (62) . . (62) <223> Xaa = Fc <400〉 19Gly Ser Ala Thr His Leu Glu Gly Gly Gly Gly Gly Xaa 35 40 45 <210> 19 <211> 62 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc__ feature <222> (62) . . . (62) <223> Xaa = Fc <400> 19
Met Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu 15 10 15 -10·Met Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu 15 10 15 -10·
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
His Met Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser 20 25 30His Met Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser 20 25 30
Ser Gly Ser Gly Ser Ala Thr His Gin Glu Glu Cys Glu Trp Asp Pro 35 -4 0 45Ser Gly Ser Gly Ser Ala Thr His Gin Glu Glu Cys Glu Trp Asp Pro 35 -4 0 45
Trp Thr Cys Glu His Met Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 60 <210> 20 <211〉 31 <212>蛋白質 ei3>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc_ _特徵 <222〉 (2) · · (2) <223> Xaa = Fc <400> 20Trp Thr Cys Glu His Met Leu Glu Gly Gly Gly Gly Gly Xaa 50 55 60 <210> 20 <211> 31 <212> Protein ei3> Man-made sequence <220><223> can be combined with Ang-2 Bound Peptide <220><221> misc_ _Features <222> (2) · · (2) <223> Xaa = Fc <400> 20
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu 15 10 15
Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu 20 25 30 <210> 21 <211> 53 <212> 蛋白質 <213>人造的序列Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu 20 25 30 <210> 21 <211> 53 <212> Protein <213> Artificial sequence
O:\121\121929.DOC -11 - 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> ^ <22i> misc 一特徵 <222> (2)..(2) <223> Xaa = Fc <400> 21O:\121\121929.DOC -11 - 200808833 <220><223> Peptide capable of binding to Ang-2 <220> ^ <22i> misc a feature <222> (2).. (2) <223> Xaa = Fc <400> 21
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 15 10 15
Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Lys 20 25 30Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Lys 20 25 30
Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr 35 40 45Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr 35 40 45
Phe Gin Gin Leu Glu 50 <210〉 22 <211> 59 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc一特徵 <222〉 （2)..(2) <223> Xaa = Fc -12-Phe Gin Gin Leu Glu 50 <210> 22 <211> 59 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220>;22i>misc-feature<222> (2)..(2) <223> Xaa = Fc -12-
O:\121\121929.DOC 200808833 <400> 22O:\121\121929.DOC 200808833 <400> 22
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Phe Asn Pro Leu Asp Glu 15 10 15
Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Gly·Gly Gly 20 25 30Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Gly Gly Gly 20 25 30
Gly Gly Gly Gly Gly Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr 35 40 45Gly Gly Gly Gly Gly Lys Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr 35 40 45
Leu Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu 50 55 <210> 23 <211> 25 <212> 蛋白質 <213> 人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220〉 <221〉 misc_特徵 <222> (2) .. (2) <223> Xaa = Fc <400> 23Leu Tyr Glu Gin Phe Thr Phe Gin Gin Leu Glu 50 55 <210> 23 <211> 25 <212> Protein <213> Artificial sequence <220><223> capable of binding to Ang-2 Peptide <220> <221> misc_feature<222> (2) .. (2) <223> Xaa = Fc <400> 23
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp 1 5 10 15
Pro Trp Thr Cys Glu His Met Leu Glu 20 25 <210〉 24 13-Pro Trp Thr Cys Glu His Met Leu Glu 20 25 <210〉 24 13-
0:\121\121929.D0C 200808833 <211〉 4 7 <212> 蛋白質 <213> 人造的序列 <220> <223> <220〉 能夠與Ang-2結合的肽體 <221〉 misc_特徵 <222〉 (2) · . (2) <223> Xaa = Fc <400> 240: \121\121929.D0C 200808833 <211> 4 7 <212> Protein <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 < 221> misc_features<222> (2) · . (2) <223> Xaa = Fc <400> 24
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 15 10 15
Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Gin 20 25 30Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Gin 20 25 30
Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Leu Glu 35 40 45 <210> 25 <211> 61 <212> 蛋白質 <213> 人造的序列 <220> <223> <220〉 能夠與Ang-2結合的肽體 <221> misc^_特徵 <222> (2) .. (2) -14-Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Leu Glu 35 40 45 <210> 25 <211> 61 <212> Protein <213> Artificial Sequence <220><223>< 220> Peptide capable of binding to Ang-2 <221> misc^_Features <222> (2) .. (2) -14-
O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 25O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 25
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Glu Glu Cys Glu Trp Asp 1 5 10 15
Pro Trp Thr Cys Glu His Met Gly Ser Gly Ser Ala Thr Gly Gly Ser 20 25 30Pro Trp Thr Cys Glu His Met Gly Ser Gly Ser Ala Thr Gly Gly Ser 20 25 30
Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Gin Glu Glu 35 40 45Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Gin Glu Glu 35 40 45
Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Leu Glu 50 55 60 <210〉 26 <211> 75 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc 一特徵 <222〉 (75)..(75) <223> Xaa = Fc <400> 26Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Leu Glu 50 55 60 <210> 26 <211> 75 <212>Protein<213> Artificial sequence <220><223> 2-bound peptide body <220><221> misc-characteristic <222> (75)..(75) <223> Xaa = Fc <400>
Met Gly Ala Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15Met Gly Ala Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15
Met Gly Gly Gly Gly Gly Gly Gly Gly Lys Phe Asn Pro Leu Asp Glu 2〇 25 30 -15-Met Gly Gly Gly Gly Gly Gly Gly Gly Lys Phe Asn Pro Leu Asp Glu 2〇 25 30 -15-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Gly Ser Gly 35 40 45Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr Phe Gin Gin Gly Ser Gly 35 40 45
Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser 50 55 60Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser 50 55 60
Ala Thr His Leu Glu Gly Gly Gly Gly Gly Xaa 65 70 75 <210> 27 <211〉 72 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220〉 <221〉misc 一特徵 <222〉 (2)..(2) <223> Xaa = Fc <400> 2Ί Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 1 5 10 15 Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Lys 20 25 30 Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr 35 40 45 Phe Gin Gin Gly Gly Gly Gly Gly Gin Glu Glu Cys Glu Trp Asp Pro 50 55 60 Trp Thr Cys Glu His' Met Leu Glu 65 70 -16-Ala Thr His Leu Glu Gly Gly Gly Gly Gly Xaa 65 70 75 <210> 27 <211> 72 <212>Protein<213> Artificial sequence <220><223> can be combined with Ang-2 Peptide <220> <221>misc a feature <222> (2)..(2) <223> Xaa = Fc <400> 2Ί Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Ser Gly Ser Ala Thr Gly 1 5 10 15 Gly Ser Gly Ser Thr Ala Ser Ser Gly Ser Gly Ser Ala Thr His Lys 20 25 30 Phe Asn Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Glu Gin Phe Thr 35 40 45 Phe Gin Gin Gly Gly Gly Gly Gly Gin Glu Glu Cys Glu Trp Asp Pro 50 55 60 Trp Thr Cys Glu His' Met Leu Glu 65 70 -16-
O:\121\121929.DOC 200808833 <210> 28 <211> <212> <213> 30 蛋白質 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (30)..(30) <22 3 > Xaa = Fc <400> 28O:\121\121929.DOC 200808833 <210> 28 <211><212><213> 30 Protein artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> (30)..(30) <22 3 > Xaa = Fc <400> 28
Met Gly Ala Gin Phe Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr 1 5 10 15Met Gly Ala Gin Phe Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr 1 5 10 15
Phe Gly Cys Asp Asn His Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 30 <210> 29 <211〉 26 <212>蛋白質 <213> 人造的序列 <220> <223〉 能夠與Ang-2結合的肽雜 <220> <22i> misc 特徵 — <222> (26)..(26)Phe Gly Cys Asp Asn His Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 30 <210> 29 <211> 26 <212> Protein <213> Artificial Sequence <220><223> -2 bound peptide heterozygous <220><22i> misc characteristics - <222> (26)..(26)
O:\121\121929.DOC •17· 200808833 <223> Xaa = Fc <400> 29O:\121\121929.DOC •17· 200808833 <223> Xaa = Fc <400> 29
Met Gly Ala Gin Gin Tyr Gly Cys Asp Gly Phe Leu Tyr Gly Cys Met 1.5 10 、15 lie Asn Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210〉 30 <211> 26 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc^特徵 <222> (26) . . (26) <223> Xaa = Fc <400> 30Met Gly Ala Gin Gin Tyr Gly Cys Asp Gly Phe Leu Tyr Gly Cys Met 1.5 10, 15 lie Asn Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 30 <211> 26 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc^ feature <222> (26) . . . (26) <223> Xaa = Fc <400> 30
Met Gly Ala Gin Lys Arg Pro Cys Glu Glu Met Trp Gly Gly Cys Asn 15 10 15Met Gly Ala Gin Lys Arg Pro Cys Glu Glu Met Trp Gly Gly Cys Asn 15 10 15
Tyr Asp Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 31 <211> 26 <212>蛋白質 <213>人造的序列Tyr Asp Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 31 <211> 26 <212> Protein <213> Artificial Sequence
O:\121\121929.DOC -18- 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> niisc _特徵 <222> (26)..(26) <223> Xaa = Fc <400> 31O:\121\121929.DOC -18- 200808833 <220><223> Peptide capable of binding to Ang-2 <220><22i> niisc _feature <222> (26)..( 26) <223> Xaa = Fc <400> 31
Met Gly Ala Gin His Gin lie Cys Lys Trp Asp Pro Trp Thr Cys Lys 15 10 15Met Gly Ala Gin His Gin lie Cys Lys Trp Asp Pro Trp Thr Cys Lys 15 10 15
His Trp Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210〉 32 <211> 26 <212〉 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc_特徵 <222> (26)··(26) <223> Xaa = Fc <400> 32His Trp Leu Glu Gly Gly Gly Gly Gly Xaa 20 25 <210> 32 <211> 26 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 Body <220><221>misc_feature<222> (26)·(26) <223> Xaa = Fc <400> 32
Met Gly Ala Gin Lys Arg Pro Cys Glu Glu lie Phe Gly Gly Cys Thr 1 5 10 15 -19-Met Gly Ala Gin Lys Arg Pro Cys Glu Glu lie Phe Gly Gly Cys Thr 1 5 10 15 -19-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Tyr Gin Leu Glu Gly Gly Gly Gly Gly Xaa <210〉 33 <211> 784Tyr Gin Leu Glu Gly Gly Gly Gly Gly Xaa <210> 33 <211> 784
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA <400> 33 atgggtgcac agaaattcaa cccgctggac gaactggaag aaactctgta cgaacagttc 60 actttccagc agctcgaggg tggaggcggt ggggacaaaa ctcacacatg tccaccttgc 120 ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac 180 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 240 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 300 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 360 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 420 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 480 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 540 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 600 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 660 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 720 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaataatgg 780 atcc 784 <210> 34 <211> 768≪ 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 > 33 atgggtgcac agaaattcaa cccgctggac gaactggaag aaactctgta cgaacagttc 60 actttccagc agctcgaggg tggaggcggt ggggacaaaa ctcacacatg tccaccttgc 120 ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac 180 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 240 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 300 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 360 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 420 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 480 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 540 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 600 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 660 ctcaccgtgg acaagagcag Gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 720 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaataatgg 780 atcc 784 <210> 34 <211> 768
<212> DNA <213>人造的序列<212> DNA <213> artificial sequence
O:\121\121929.DOC -20- 60200808833 <220> <223>編碼能夠與Ang-2結合之肚體的DNA <400> 34 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cagtttccag cagctcgagg gtggaggcgg tggggacaaa actcacacat gtccaccttg cccagcacct gaactcctgg ggggaccgtc agttttcctc ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaataa <210> 35 <211> 834 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽體的DNA <400> 35 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cactttccag cagggatccg gttctgctac tggtggttcc ggctccaccg caagctctgg ttcaggcagt gcgactcatc tcgagggtgg aggcggtggg gacaaaactc acacatgtcc accttgccca gcacctgaac tcctgggggg accgtcagtt ttcctcttcc ccccaaaacc caaggacacc 120 180 240 300 360 420 480 540 600 660 720 768 60 120 180 240O:\121\121929.DOC -20- 60200808833 <220><223> DNA encoding the body capable of binding to Ang-2 <400> 34 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cagtttccag cagctcgagg gtggaggcgg tggggacaaa actcacacat gtccaccttg cccagcacct gaactcctgg ggggaccgtc agttttcctc ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc Tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaataa <210> 35 <211> 834 <212&g t; DNA <213> artificial sequence <220><223> DNA encoding a peptibosome capable of binding to Ang-2 <400> 35 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cactttccag cagggatccg gttctgctac tggtggttcc ggctccaccg caagctctgg ttcaggcagt gcgactcatc tcgagggtgg aggcggtggg Galacaaactc acacatgtcc accttgccca gcacctgaac tcctgggggg accgtcagtt ttcctcttcc ccccaaaacc caaggacacc 120 180 240 300 360 420 480 540 600 660 720 768 60 120 180 240
0:\121\121929.D0C -21 - 300200808833 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ataa <210〉 36 <211> 861 <212〉 DNA <213>人造的序列 <220><223> 編碼能夠與Ang-2結合之肽體的DNA 一 <40〇> 36 atgggtgcac agaaattcaa cccgctggac gaactggaag aaactctgta cgaacagttc actttccagc agggtggtgg tggtggtggc ggtggtaagt tcaacccact ggatgagctg gaagagactc tgtatgaaca gttcactttc cagcaactcg agggtggagg cggtggggac aaaactcaca catgtccacc ttgcccagca cctgaactcc tggggggacc gtcagttttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 360 420 480 540 600 660 720 780 834 60 120 180 240 300 360 420 480 5400: \ 121 \ 121929.D0C -21 - 300200808833 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ataa < 210> 36 < 211 > 861 < 212> DNA < 213 > artificial sequence < 220 > <223> DNA encoding a peptibosome capable of binding to Ang-2 <40〇> 36 atgggtgcac agaaattcaa cccgctggac gaactggaag aaactctgta cgaacagttc actttccagc agggtggtgg tggtggtggc ggtggtaagt tcaacccact ggatgagctg gaagagactc tgtatgaa ca gttcactttc cagcaactcg agggtggagg cggtggggac aaaactcaca catgtccacc ttgcccagca cctgaactcc tggggggacc gtcagttttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 360 420 480 540 600 660 720 780 834 60 120 180 240 300 360 420 480 540
O:\121\121929.DOC -22- 600 200808833 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 660 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 720 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 780 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 840 tccctgtctc cgggtaaata a 861 <210〉 37 <211> 849O: \ 121 \ 121929.DOC -22- 600 200808833 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 660 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 720 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 780 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 840 tccctgtctc cgggtaaata a 861 < 210> 37 <211> 849
<212> DNA <213>人造的序列 <220〉<212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA <400> 37 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cactttccag 60 cagggtggtg gtggtggcgg tggtaagttc aacccactgg atgagctgga agagactctg 120 tatgaacagt tcactttcca gcaactcgag ggtggaggcg gtggggacaa aactcacaca 180 tgtccacctt gcccagcacc tgaactcctg gggggaccgt cagttttcct cttcccccca 240 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 300 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 360 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 420 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 480 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 540 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 600 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 660 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 720 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 780 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 840 ggtaaataa 849 -23-240 aaacccaagg 37 atgaaattca acccgctgga cgaactggaa gaaactctgt acgaacagtt cactttccag 60 cagggtggtg gtggtggcgg tggtaagttc aacccactgg atgagctgga agagactctg 120 tatgaacagt tcactttcca gcaactcgag ggtggaggcg gtggggacaa aactcacaca 180 tgtccacctt gcccagcacc tgaactcctg gggggaccgt cagttttcct cttcccccca; < 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 & gt acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 300 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 360 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 420 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 480 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 540 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 600 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 660 cagccggaga acaactacaa Gaccacgcct cccgtgctgg actccgacgg ctccttcttc 720 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 780 tccgtgatgc atgaggctct g Cacaaccac tacacgcaga agagcctctc cctgtctccg 840 ggtaaataa 849 -23-
O:\121\121929.DOC 200808833 <210> 38 <211> 759O:\121\121929.DOC 200808833 <210> 38 <211> 759
<212> DNA <213> 人造的序列 <220〉<212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA <400> 38 atgggtgcac agcaggaaga atgcgaatgg gacccatgga cttgcgaaca catgctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 39 <211〉 816 <212> DNA <213> 人造的序列 <220>≪ 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 > 38 atgggtgcac agcaggaaga atgcgaatgg gacccatgga cttgcgaaca catgctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt Cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 39 <211〉 816 <212> DNA <213> artificial sequence <220>
O:\121\121929.DOC -24 - 60 200808833O:\121\121929.DOC -24 - 60 200808833
<223>編碼能夠與Ang-2結合之肽體的DNA <400> 39 atgcaggaag aatgcgaatg ggacccatgg acttgcgaac acatgggatc cggttctgct actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca tctcg^agggt ggaggcggtg gggacaaaac tcacacatgt ccaccttgcc cagcacctga actcctgggg ggaccgtcag ttttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt aaataa <210> 40 <211> 867catgcgtggt ggtggacgtg 39 atgcaggaag aatgcgaatg ggacccatgg acttgcgaac acatgggatc cggttctgct actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca tctcg ^ agggt ggaggcggtg gggacaaaac tcacacatgt ccaccttgcc cagcacctga actcctgggg ggaccgtcag ttttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc cctgaggtca; < 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 & gt agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca Caaccactac acgcagaaga gcctctccct gtctccgggt aaataa <210> 40 <211> 867
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA <400〉 40 atgggtgcac agcaggaaga atgcgaatgg gacccatgga cttgcgaaca catgggatcc ggttctgcta ctggtggttc cggctccacc gcaagctctg gttcaggcag tgcgactcat caggaagaat gcgaatggga cccatggact tgcgaacaca tgctcgaggg tggaggcggt ggggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc≪ 223 > coding capable of binding to Ang-2 of peptibodies DNA < 400> 40 atgggtgcac agcaggaaga atgcgaatgg gacccatgga cttgcgaaca catgggatcc ggttctgcta ctggtggttc cggctccacc gcaagctctg gttcaggcag tgcgactcat caggaagaat gcgaatggga cccatggact tgcgaacaca tgctcgaggg tggaggcggt ggggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac Ccctgaggtc
O:\121\121929.DOC -25- 120 180 240 300 360 420 480 540 600 660 720 780 816 60 120 180 240 300 360 200808833 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaataa » <210> 41 <211〉 774O: \ 121 \ 121929.DOC -25- 120 180 240 300 360 420 480 540 600 660 720 780 816 60 120 180 240 300 360 200808833 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cacgcagaag agcctctccc tgtctccggg taaataa cgtgatgcat gaggctctgc acaaccacta »< 210 > 41 < 211> 774
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽體的DNA <400> 41 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg # gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag≪ 223 > coding capable of binding to Ang-2 of peptibodies DNA < 400 > 41 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg # gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag
O:\121\121929.DOC 26- 420 480 540 600 660 720 780 840 867 60 120 180 240 300 360 420 480 540 600 200808833 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagaaatt caacccgctg 720 gacgagctgg aagagactct gtacgaacag tttacttttc aacagctcga gtaa 774 <210> 42 ’ <211〉 840O: \ 121 \ 121929.DOC 26- 420 480 540 600 660 720 780 840 867 60 120 180 240 300 360 420 480 540 600 200808833 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagaaatt caacccgctg 720 gacgagctgg aagagactct gtacgaacag tttacttttc aacagctcga gtaa 774 <210> 42 ' <211> 840
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽tt的DNA <400> 42 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca 60 gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 240 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagggatc cggttctgct 720 actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca taaattcaac 780 ccgctggacg aactggaaga aactctgtac gaacagttca ctttccagca actcgagtaa 840 <210> 43 <211> 858 27-≪ 223 > coding capable of binding to Ang-2 of the peptide tt of DNA < 400 > 42 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca 60 gttttcctct acccaaggac accctcatga tctcccggac ccctgaggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 240 taccgtgtgg tccccccaaa tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg Taaaggtgga ggtggtggtg cacagggatc cggttctgct 720 actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca taaattcaac 780 ccgctggacg Aactggaaga aactctgtac gaacagttca ctttccagca actcgagtaa 840 <210> 43 <211> 858 27-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽體的DNA <400> 43 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg ggga ccgtca 60 gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccct ^aggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caac agcacg 240 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caag gagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctcc aaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc ccftg ctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gt ggcagcag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagaaatt ca ac<ccgctg 720 gacgaactgg aagaaactct gtacgaacag ttcactttcc agcagggtgg tggt^gtggt 780 ggcggtggta agttcaaccc actggatgag ctggaagaga ctctgtatga acagttcact 840 ttccagcaac tcgagtaa 858 <210> 44 <211> 756≪ 223 > coding capable of binding to Ang-2 of peptibodies DNA < 400 > 43 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg ggga ccgtca 60 gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccct ^ aggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caac agcacg 240 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caag gagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctcc aaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc ccftg ctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gt ggcagcag 600 gggaacgtct Tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagaaatt ca ac<ccgctg 720 gacgaactgg aagaaactct gtacgaacag ttcactttcc agcagggtgg tggt^gtggt 7 80 ggcggtggta agttcaaccc actggatgag ctggaagaga ctctgtatga acagttcact 840 ttccagcaac tcgagtaa 858 <210> 44 <211> 756
<212> DNA <213>人造的序列 <220〉<212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA -28 -<223> DNA encoding a peptibosome capable of binding to Ang-2 -
O:\121\121929.DOC 200808833 <4〇0> 44 atggacaaaa gttttcctct acatgcgtgg gacggcgtgg taccgtgtgg aagtgcaagg aaagggcagc aagaaccagg gagtgggaga tccgacggct gggaacgtct agcctctccc tgggacccat ctcacacatg tccccccaaa tggtggacgt aggtgcataa tcagcgtcct tctccaacaa cccgagaacc tcagcctgac gcaatgggca ccttcttcct tctcatgctc tgtctccggg ggacttgcga tccaccttgc acccaaggac gagccacgaa tgccaagaca caccgtcctg agccctccca acaggtgtac ctgcctggtc gccggagaac ctacagcaag cgtgatgcat taaaggtgga acacatgctc ccagcacctg accctcatga gaccctgagg aagccgcggg caccaggact gcccccatcg accctgcccc aaaggcttct aactacaaga ctcaccgtgg gaggctctgc ggtggtggtg gagtaa aactcctggg tctcccggac tcaagttcaa aggagcagta ggctgaatgg agaaaaccat catcccggga atcccagcga ccacgcctcc acaagagcag acaaccacta cacagcagga gggaccgtca ccctgaggtc ctggtacgtg caacagcacg caaggagtac ctccaaagcc tgagctgacc catcgccgtg cgtgctggac gtggcagcag cacgcagaag agaatgcgaa 60 120 180 240 300 360 420 480 540 600 660 720 756 <210> 45 <211> 822O: \ 121 \ 121929.DOC 200808833 < 4〇0 > 44 atggacaaaa gttttcctct acatgcgtgg gacggcgtgg taccgtgtgg aagtgcaagg aaagggcagc aagaaccagg gagtgggaga tccgacggct gggaacgtct agcctctccc tgggacccat ctcacacatg tccccccaaa tggtggacgt aggtgcataa tcagcgtcct tctccaacaa cccgagaacc tcagcctgac gcaatgggca ccttcttcct tctcatgctc tgtctccggg ggacttgcga tccaccttgc acccaaggac gagccacgaa tgccaagaca caccgtcctg agccctccca acaggtgtac ctgcctggtc gccggagaac ctacagcaag cgtgatgcat taaaggtgga acacatgctc ccagcacctg accctcatga gaccctgagg aagccgcggg caccaggact gcccccatcg accctgcccc aaaggcttct aactacaaga ctcaccgtgg gaggctctgc ggtggtggtg gagtaa aactcctggg tctcccggac tcaagttcaa aggagcagta ggctgaatgg agaaaaccat catcccggga atcccagcga ccacgcctcc acaagagcag acaaccacta cacagcagga gggaccgtca ccctgaggtc ctggtacgtg caacagcacg caaggagtac ctccaaagcc tgagctgacc catcgccgtg cgtgctggac gtggcagcag cacgcagaag agaatgcgaa 60 120 180 240 300 360 420 480 540 600 660 720 756 <210> 45 <211> 822
<212> DNA <213>人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽體的DNA <400> 45 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc -29- 60 120 180 240 300 360≪ 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 > 45 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa aggtgcataa ctggtacgtg gacggcgtgg taccgtgtgg tcagcgtcct caccgtcctg tgccaagaca aagccgcggg aggagcagta caacagcacg caccaggact ggctgaatgg Caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc -29- 60 120 180 240 300 360
O:\121\121929.DOC 420 480200808833 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagggatc cggttctgct actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca tcaggaagaa tgcgaatggg acccatggac ttgcgaacac atgctcgagt aa <210> 46 <211> 864 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽嫌的DNA <400> 46 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagcagga agaatgcgaa tgggacccat ggacttgcga acacatggga tccggttctg ctactggtgg ttccggctcc 540 600 660 720 780 822 60 120 180 240 300 360 420 480 540 600 660 720 780O: \ 121 \ 121929.DOC 420 480200808833 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagggatc cggttctgct actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca tcaggaagaa tgcgaatggg acccatggac ttgcgaacac atgctcgagt Aa <210> 46 <211> 864 <212> DNA <213> Artificial sequence <220><223> A DNA encoding a peptide capable of binding to Ang-2 <400> 46 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccc catcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagcagga agaatgcgaa tgggacccat ggacttgcga acacatggga tccggttctg ctactggtgg ttccggctcc 540 600 660 720 780 822 60 120 180 240 300 360 420 480 540 600 660 720 780
O:\121\121929.DOC 30- 200808833 accgcaagct ctggttcagg cagcgcgact catcaggaag aatgcgaatg ggacccatgg 840 acttgcgaac acatgctcga gtaa 864 <210〉 47 <211> 906O:\121\121929.DOC 30- 200808833 accgcaagct ctggttcagg cagcgcgact catcaggaag aatgcgaatg ggacccatgg 840 acttgcgaac acatgctcga gtaa 864 <210> 47 <211> 906
<212〉 DNA <213>人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽殖的DNA <400> 47 atgggtgcac aggaagaatg cgaatgggac ccatggactt gcgaacacat gggtggtggt 60 ggtggtggcg gtggtaaatt caacccgctg gacgaactgg aagaaactct gtacgaacag 120 ttcactttcc agcagggatc cggttctgct actggtggtt ccggctccac cgcaagctct 180 ggttcaggca gtgcgactca tctcgagggt ggaggcggtg gggacaaaac tcacacatgt 240 ccaccttgcc cagcacctga actcctgggg ggaccgtcag ttttcctctt ccccccaaaa 300 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 360 agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 420 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 480 accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 540 gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 600 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 660 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 720 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 780 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 840 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 900 aaataa 906 <210> 48 -31 -≪ 223 > coding capable of Ang-2 peptide colonization of the binding of the DNA < 400 > 47 atgggtgcac aggaagaatg cgaatgggac ccatggactt gcgaacacat gggtggtggt 60 ggtggtggcg gtggtaaatt caacccgctg gacgaactgg aagaaactct gtacgaacag 120 ttcactttcc agcagggatc cggttctgct actggtggtt ccggctccac cgcaagctct 180 ggttcaggca gtgcgactca tctcgagggt ggaggcggtg gggacaaaac tcacacatgt 240 ccaccttgcc cagcacctga actcctgggg ggaccgtcag ttttcctctt ccccccaaaa 300 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 360 agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 420 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 480 accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 540 gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 600 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 660 tgcctggtca aaggcttcta Tcccagcgac atcgccgtgg agtgggagag caatgggcag 720 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 780 tacagcaagc tcaccgtgga c Aagagcagg tggcagcagg ggaacgtctt ctcatgctcc 840 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 900 aaataa 906 <210> 48 -31 -
O:\121\121929.DOC 200808833 <211> 897 <212> DNA <213> 人造的序列 <220〉O:\121\121929.DOC 200808833 <211> 897 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽體的DNA <400> 48 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca 60 gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 240 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg taaaggtgga ggtggtggtg cacagggatc cggttctgct 720 actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca taaattcaac 780 ccgctggacg aactggaaga aactctgtac gaacagttca ctttccagca gggtggtggc 840 ggtggtcagg aagaatgcga atgggaccca tggacttgcg aacacatgct cgagtaa 897 <210> 49 <211> 771 <212> DNA <213> 人造的序列 <220> -32 -240 taccgtgtgg 48 atggacaaaa ctcacacatg tccaccttgc ccagcacctg aactcctggg gggaccgtca 60 gttttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg; < 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 & gt tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctccggg Taaaggtgga ggtggtggtg cacagggatc cggttctgct 720 actggtggtt ccggctccac cgcaagctct ggttcaggca gtgcgactca taaattcaac 780 ccgctggac g aactggaaga aactctgtac gaacagttca ctttccagca gggtggtggc 840 ggtggtcagg aagaatgcga atgggaccca tggacttgcg aacacatgct cgagtaa 897 <210> 49 <211> 771 <212> DNA <213> Artificial sequence <220> -32 -
O:\121\121929.DOC 60200808833 <223>編碼能夠與Ang-2結合之肽體的DNA <400> 49 atgggtgcac agttcgacta ctgcgaaggt gttgaagacc cgttcacttt cggttgcgac aaccacctcg agggtggagg cggtggggac aaaactcaca catgtccacc ttgcccagca cctgaactcc tggggggacc gtcagttttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaata a <210> 50 <211> 759 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽體的DNA <400> 50 atgggtgcac agcagtacgg ttgcgacggt tttctgtacg gttgcatgat caacctcgag ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 120 180 240 300 360 420 480 540 600 660 720 771 60 120 180 240 300O: \ 121 \ 121929.DOC 60200808833 < 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 > 49 atgggtgcac agttcgacta ctgcgaaggt gttgaagacc cgttcacttt cggttgcgac aaccacctcg agggtggagg cggtggggac aaaactcaca catgtccacc ttgcccagca cctgaactcc tggggggacc gtcagttttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat Gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaata a <210> 50 <211> 759 <212> DNA <21 3 > artificial sequence < 220 > < 223 > coding capable of Ang-2 peptibody binding of the DNA < 400 > 50 atgggtgcac agcagtacgg ttgcgacggt tttctgtacg gttgcatgat caacctcgag ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt Ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 120 180 240 300 360 420 480 540 600 660 720 771 60 120 180 240 300
O:\121\121929.DOC 33- 360 200808833 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tg^agtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 51 <211> 759O: \ 121 \ 121929.DOC 33- 360 200808833 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tg ^ agtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc Aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 51 <211> 759
<212> DNA <213>人造的序列 <220〉<212> DNA <213> artificial sequence <220>
<223〉 編碼能夠與Ang-2結合之肽體的DNA <400> 51 atgggtgcac agaaacgccc atgcgaagaa atgtggggtg gttgcaacta cgacctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 34-aactggtacg 51 atgggtgcac agaaacgccc atgcgaagaa atgtggggtg gttgcaacta cgacctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240; < 223> peptide precursor coding capable of binding of Ang-2 DNA < 400 & gt tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt Cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 34-
O:\121\121929.DOC 200808833 <210> 52 <211> 759 <212> DNA 、 <n3>人造的序列 <220>O:\121\121929.DOC 200808833 <210> 52 <211> 759 <212> DNA, <n3> artificial sequence <220>
<223>編瑪能夠與Ang-2結合之肽體的DNA <400> 52 atgggtgcac agcaccagat ctgcaaatgg gacccgtgga cctgcaaaca ctggctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 53 <211〉 759≪ 223 > eds Mary capable of binding to Ang-2 of peptibodies DNA < 400 > 52 atgggtgcac agcaccagat ctgcaaatgg gacccgtgga cctgcaaaca ctggctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cagttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc Aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 53 <211> 759
<212> DNA <213>人造的序列 <220> -35-<212> DNA <213> artificial sequence <220> -35-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<223> 編碼能夠與Ang-2結合之肽體的DNA <400> 53 atgggtgcac agaaacgtcc atgcgaagaa atcttcggtg gttgcaccta ccagctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cacfttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 54 <211> 25ggtcaagttc 240 aactggtacg 53 atgggtgcac agaaacgtcc atgcgaagaa atcttcggtg gttgcaccta ccagctcgag 60 ggtggaggcg gtggggacaa aactcacaca tgtccacctt gcccagcacc tgaactcctg 120 gggggaccgt cacfttttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga; < 223 > peptibody encoding capable of binding of Ang-2 DNA < 400 & gt tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660 aggtggcagc aggggaacgt Cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720 tacacgcaga agagcctctc cctgtctccg ggtaaataa 759 <210> 54 <211> 25
<212> DNA <213>人造的序列 <220> <223> 寡核芬酸 <400> 54 cggcgcaact atcggtatca agctg 25 <210> 55<212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 54 cggcgcaact atcggtatca agctg 25 <210> 55
<211〉 26 <212> DNA <213>人造的序列 36·<211> 26 <212> DNA <213> artificial sequence 36·
O:\121\121929.DOC 200808833 <220> <223>寡核甞酸 <400> 55 catgtaccgt aacactgagt ttcgtc <210> 56 <211> 14 <212〉蛋白質 <213>人造的序列 <220> <223〉從TN8-IX庫中產製的一致主題 <220> <22 1> misc—特徵 <222>(7, 12和）"(14) <223> Xaa意指任何天然存在的胺基酸。 <400> 56O:\121\121929.DOC 200808833 <220><223>oligonucleotide<400> 55 catgtaccgt aacactgagt ttcgtc <210> 56 <211> 14 <212>protein <213> Sequence <220><223> Consistent theme produced from the TN8-IX library <220><221> misc - feature <222> (7, 12 &) "(14) <223> Xaa means any naturally occurring amino acid. <400> 56
Lys Arg Pro Cys Glu Glu Xaa Trp Gly Gly Cys Xaa Tyr Xaa 1 5 10 <210> 57 <211> 14 <212>蛋白質 <213>人造的序列 <220> <223>從TN8-IX庫中產製的一致主題 <220> -37-Lys Arg Pro Cys Glu Glu Xaa Trp Gly Gly Cys Xaa Tyr Xaa 1 5 10 <210> 57 <211> 14 <212> Protein <213> Artificial Sequence <220><223> From TN8- Consistent theme produced in the IX library <220> -37-
O:\121\121929.DOC 200808833 <221> misc__特徵 <222> (7, 12和）,·（14) <223> Xaa意指任何天然存在的胺基酸。 <400> 57O:\121\121929.DOC 200808833 <221> misc__features <222> (7, 12 and), (14) <223> Xaa means any naturally occurring amino acid. <400> 57
Lys Arg Pro Cys Glu Glu Xaa Phe Gly Gly Cys Xaa Tyr Xaa 丄 5 i〇 <210> 58 <211〉 14 <212〉蛋白質 <213>人造的序列 <220> <223>從TN8-IX庫中產製的一致主題 <220> <221> misc_特徵 <222> (1，2, 3, 5和）..（13) <223> Xaa意指任何天然存在的胺基酸。 <400> 58Lys Arg Pro Cys Glu Glu Xaa Phe Gly Gly Cys Xaa Tyr Xaa 丄5 i〇<210> 58 <211> 14 <212>protein <213> artificial sequence <220><223> from TN8 Consistent theme produced in the -IX library <220><221> misc_features <222> (1,2, 3, 5 and )..(13) <223> Xaa means any naturally occurring amine Base acid. <400> 58
Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Glu Xaa Met 1 5 10 <210> 59 <211> 18 <212>蛋白質 <213>人造的序列 <220> -38-Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Glu Xaa Met 1 5 10 <210> 59 <211> 18 <212> Protein <213> Artificial Sequence <220> -38-
O:\121\121929.DOC 200808833 <223>從TN12-I庫中產製的一致主題 <220> <221〉misc—特徵 <222> (3, 8, 10-14和）··（18) <223> Xaa意指任何天然存在的胺基酸。 、 <400> 59O:\121\121929.DOC 200808833 <223> Consistent theme <220><221>misc-characteristic<222> (3, 8, 10-14 and) from the TN12-I library (18) <223> Xaa means any naturally occurring amino acid. , <400> 59
Trp Ser Xaa Cys Ala Trp Phe Xaa Gly Xaa Xaa Xaa Xaa Xaa Cys Arg <210> 60 <211> 227 <212>蛋白質 <213>人造的序列 <220> <223> 人類 Fc IgGl <400> 60Trp Ser Xaa Cys Ala Trp Phe Xaa Gly Xaa Xaa Xaa Xaa Xaa Cyas Arg <210> 60 <211> 227 <212> Protein <213> Artificial Sequence <220><223> Human Fc IgGl <;400> 60
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 15 10 15Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 15 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 > 55 60Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 > 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75 80 39·His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75 80 39·
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 85 90 95Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 100 105 110Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 100 105 110
Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 115 120 125Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 130 135 140Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 145 150 155 160Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 145 150 155 160
Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190
Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Lea Ser 210 215 220His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Lea Ser 210 215 220
Pro Gly Lys 225 <210> 61 <211> 14 <212>蛋白質 <213>人造的序列 <220> <223>從TN8-IX庫中產製的一致主題 40-Pro Gly Lys 225 <210> 61 <211> 14 <212> Protein <213> Artificial Sequence <220><223> Consistent Theme Produced From TN8-IX Library 40-
O:\121\121929.DOC 200808833 <220> <221> misc_特徵 <222>(1-3, 5, 7, 12, 13 和）..（14) <223> Xaa意指任何天然存在的胺基酸。 <400〉61O:\121\121929.DOC 200808833 <220><221>misc_features<222>(1-3, 5, 7, 12, 13 and )..(14) <223> Xaa means Any naturally occurring amino acid. <400>61
Xaa Xaa Xaa Cys Xaa Asp Xaa Tyr Trp Tyr Cys Xaa Xaa Xaa 15 10 <210> 62 <211> 14 <212〉蛋白質 <213>人造的序列 <220> <223>從TN8-IX庫中產製的一致主題 <220〉 <221> misc_特徵 <222> (1-3, 5, 7, 12, 13和）..（14) <223> Xaa意指任何天然存在的胺基酸。 <400> 62Xaa Xaa Xaa Cys Xaa Asp Xaa Tyr Trp Tyr Cys Xaa Xaa Xaa 15 10 <210> 62 <211> 14 <212>Protein <213> Artificial Sequence <220><223> From TN8-IX Consistent theme in the library <220> <221>misc_features<222> (1-3, 5, 7, 12, 13 and)..(14) <223> Xaa means any natural presence Amino acid. <400> 62
Xaa Xaa Xaa Cys Xaa Asp Xaa Tyr Thr Tyr Cys Xaa Xaa Xaa 15 10 <210> 63 <211〉 14 <212>蛋白質 <213>人造的序列 O:\121\121929.DOC -41 - 200808833 <220> <223〉從TN8-IX庫中產製的一致主題 <220> <221〉misc一特徵 <222〉（1-3, 5, 7, 12, 13和）··（14) <223〉Xaa意指任何天然存在的胺基酸。 <400> 63Xaa Xaa Xaa Cys Xaa Asp Xaa Tyr Thr Tyr Cys Xaa Xaa Xaa 15 10 <210> 63 <211> 14 <212>Protein <213> Artificial sequence O:\121\121929.DOC -41 - 200808833 <220><223> Consistent theme <220><221>misc-feature<222> (1-3, 5, 7, 12, 13 and) from the TN8-IX library 14) <223> Xaa means any naturally occurring amino acid. <400> 63
Xaa Xaa Xaa Cys Xaa Asp Xaa Phe Trp Tyr Cys Xaa Xaa Xaa 1 5 l〇 <210> 64 <211> 14 <212〉蛋白質 <213〉人造的序列 <220> <223>從了\8-1乂庫中產製的一致主題 <220> <221〉misc_特徵 <222>(1-3, 5, 7, 12, 13 和）··（14) <223〉Xaa意指任何天然存在的胺基酸。 <400> 64Xaa Xaa Xaa Cys Xaa Asp Xaa Phe Trp Tyr Cys Xaa Xaa Xaa 1 5 l〇<210> 64 <211> 14 <212>protein <213>artificial sequence <220><223> \8-1 Consistent theme in the library system <220><221>misc_features<222>(1-3, 5, 7, 12, 13 and) (14) <223>Xaa Means any naturally occurring amino acid. <400> 64
Xaa Xaa Xaa Cys Xaa Asp Xaa Phe Thr Tyr Cys Xaa Xaa Xaa 1 5 10 <210〉65 <211> 5 O:\121\121929.DOC -42- 200808833Xaa Xaa Xaa Cys Xaa Asp Xaa Phe Thr Tyr Cys Xaa Xaa Xaa 1 5 10 <210>65 <211> 5 O:\121\121929.DOC -42- 200808833
<212>蛋白質 <213>人造的序歹丨J <220> <223>能夠與Ang-2結合的多肽 <400> 65<212> Protein <213> Artificial sequence J <220><223> A polypeptide capable of binding to Ang-2 <400>
Trp Asp Pro Trp Thr 1 5 <210> 66 <211> 6 <：212>蛋白質 <21 3>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 66Trp Asp Pro Trp Thr 1 5 <210> 66 <211> 6 <:212> Protein <21 3> Artificial Sequence <220><223> A polypeptide capable of binding to Ang-2 <400>; 66
Trp Asp Pro Trp Thr Cys 1 5 <210> 67 <211> 7 <212>蛋白質 <21 3>人造的序列 <220〉 <223>能夠與Ang-2結合的多肽 <220> <22 1> misc^特徵 <222> (2)..(2)Trp Asp Pro Trp Thr Cys 1 5 <210> 67 <211> 7 <212> Protein <21 3> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <220>;<221> misc^ feature <222> (2)..(2)
O:\121\121929.DOC •43 - 200808833 <223>Xaa是酸性或中性極性的胺基酸殘基 <400〉67O:\121\121929.DOC •43 - 200808833 <223>Xaa is an acidic or neutral polar amino acid residue <400>67
Cys Xaa Trp Asp Pro Trp Thr 1 ♦ 5 <210> 68 <211〉 8 <212〉蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <220> <22 1> misc_特徵 <222> (2)..(2) <223> Xaa是酸性或中性極性的胺基酸殘基 <400> 68Cys Xaa Trp Asp Pro Trp Thr 1 ♦ 5 <210> 68 <211>8 <212>Protein<213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <220><221> misc_characteristic <222> (2)..(2) <223> Xaa is an acidic or neutral polar amino acid residue <400>
Cys Xaa Trp Asp Pro Trp Thr Cys 1 5 <210> 69 <211> 14 <212>蛋白質 <213〉人造的序列 <220> <223〉能夠與Ang-2結合的多肽 <220> O:\121\121929.DOC -44- 200808833 <221> misc_特徵 <222> (1，2和）..(3) <223>Xaa分別為胺基酸殘基。 <220> <221> misc_特徵 <222> (5)..(5) <223〉Xaa為胺基酸殘基〇 <220〉 <221> misc_特徵 <222> (12)..(12) <223> Xaa缺少或為胺基酸殘基。 <220> <221> misc j争徵 <222> (13)..(13) <223> Xaa缺少或為中性疏水性、中性極性的、或鹼性的胺基酸 殘基。 <220> <221〉misc_特徵 <222> (14)..(14) <223> Xaa為中性疏水性或中性極性的胺基酸殘基。 <400> 69Cys Xaa Trp Asp Pro Trp Thr Cys 1 5 <210> 69 <211> 14 <212> Protein < 213 > artificial sequence <220><223> 223 > polypeptide capable of binding to Ang-2 <220>O:\121\121929.DOC-44-200808833<221>misc_features<222> (1,2 and)..(3) <223> Xaa are each an amino acid residue. <220><221> misc_characteristic <222> (5)..(5) <223>Xaa is an amino acid residue 〇<220> <221> misc_features <222> (12)..(12) <223> Xaa lacks or is an amino acid residue. <220><221> misc j contending <222> (13)..(13) <223> Xaa lacks or is neutral, neutral, or basic amino acid residue base. <220><221>misc_ feature <222> (14).. (14) <223> Xaa is a neutral hydrophobic or neutral polar amino acid residue. <400> 69
Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Xaa Xaa Xaa 1 5 l〇 O:\121\121929.DOC -45- 200808833 <210> 70 <211> 20 <212〉蛋白質 <213〉人造的序列 <220> <223>能夠與Ang-2結合的多肽 <220> <221> misc_特徵 <222>(1 和）"（15) <223> Xaa缺少或為胺基酸殘基。 <220> <221>misc_ 特徵 <222> (2和）"（16) <223> Xaa缺少或為中性疏水性、中性極性的，或鹼性的胺基酸殘 基0 <220> <221> misc_特徵 <222>(3-6, 18, 19 和）··(20) <223> Xaa分別缺少或為胺基酸殘基。 <220> <221>misc—特徵 <222> (8)"(8) <223> Xaa為胺基酸殘基。 O:\121\121929.DOC -46- 200808833 <220> <221>misc_ 特徵 <222> (17)..(17) <223> Xaa缺少或為中性疏水性、中性極性的胺基酸殘基。 <400> 70Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Xaa Xaa Xaa 1 5 l〇O:\121\121929.DOC -45- 200808833 <210> 70 <211> 20 <212>Protein<213> Sequence <220><223> polypeptide capable of binding to Ang-2 <220><221> misc_features <222>(1 and)"(15) <223> Xaa is missing or Amino acid residue. <220><221>misc_features<222> (2 and)"(16) <223> Xaa lacks or is neutral hydrophobic, neutral polar, or basic amino acid residue 0 <220><221> misc_characteristic <222> (3-6, 18, 19 and) (20) <223> Xaa lacks or is an amino acid residue, respectively. <220><221>misc-characteristics<222> (8) "(8) <223> Xaa is an amino acid residue. O:\121\121929.DOC -46- 200808833 <220><221>misc_feature<222> (17)..(17) <223> Xaa lacks or is neutral hydrophobic, neutral polarity Amino acid residue. <400> 70
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Xaa Xaa 15 10 15Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Trp Asp Pro Trp Thr Cys Xaa Xaa 15 10 15
Xaa Xaa Xaa Xaa 20 <210> 71 <211> 10 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <200> <221〉misc_特徵 <222> (2)..(2) <223> Xaa為中性疏水性的胺基酸殘基。 <220> <221〉misc_特徵 <222> (4)..(4) <223>Xaa 為 a，A，D，或E。 O:\121\121929.DOC •47· 200808833 <220> <221〉misc j争徵 <222> (6)..(6) <223〉Xaa為酸性胺基酸殘基。 <220> <221> misc_特徵 <222> (7)··(7) <223〉Xaa為胺基酸殘基。 <220> <221> misc_特徵 <222> (8)..(8) <223> Xaa為中性疏水性、中性極性或鹼性的胺基酸殘基。 <400> 71Xaa Xaa Xaa Xaa 20 <210> 71 <211> 10 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <200><221〉misc_Characteristics<222> (2)..(2) <223> Xaa is a neutral hydrophobic amino acid residue. <220><221>misc_ feature <222> (4)..(4) <223> Xaa is a, A, D, or E. O:\121\121929.DOC •47· 200808833 <220><221>misc j contending <222> (6).. (6) <223> Xaa is an acidic amino acid residue. <220><221> misc_characteristic <222> (7) (7) <223> Xaa is an amino acid residue. <220><221> misc_characteristic <222> (8).. (8) <223> Xaa is a neutral hydrophobic, neutral polar or basic amino acid residue. <400> 71
Pro Xaa Asp Xaa Leu Xaa Xaa Xaa Leu Tyr 1 5 l〇 <210> 72 <211> 22 <212〉蛋白質 <213〉人造的序列 <220> <223〉能夠與Ang-2結合的多肽 <220> <221> misc 特徵 O:\121\121929.DOC -48- 200808833 <222> (1，4和）··(20) <223> Xaa缺少或為胺基酸殘基。 <220> <221>111丨8(：_特徵 ’ <222> (2, 15, 16和）··（21) <223> Xaa缺少或為中性極性的、酸性或鹼性的胺基酸殘基。 <220> <221> misc_特徵 <222> (3, 17和）..（18) <223> Xaa缺少，或為中性疏水性、或中性極性的胺基酸殘基 <220> <221> misc_特徵 <222> (6)..(6) <223> Xaa為中性疏水性的胺基酸殘基。 <220> <221〉misc_特徵 <222> (8)..(8) <223> Xaa為 a A，D，或 E 0 <220> <221> misc_特徵 <222〉（10)..(10) O:\121\121929.DOC -49- 200808833 <223> Xaa為酸性的胺基酸殘基。 <220> <221>misc_ 特徵 <222> (11)..(11) <223> Xaa為胺基酸殘基。 <220> <22 1> misc_特徵 <222> (12)..(12) <223〉Xaa為中性疏水性、中性極性或鹼性的胺基酸殘基。 <220> <221〉misc—特徵 <222> (19和）"（22) <223> Xaa缺少或中性疏水性、中性極性或鹼性的胺基酸殘基。 <400> 72Pro Xaa Asp Xaa Leu Xaa Xaa Xaa Leu Tyr 1 5 l〇<210> 72 <211> 22 <212>Protein<213>Artificial Sequence<220><223> can be combined with Ang-2 Peptide <220><221> misc characteristic O:\121\121929.DOC -48- 200808833 <222> (1,4 and) (20) <223> Xaa is absent or is amino acid Residues. <220><221>111丨8(:_Features<222> (2, 15, 16 and) (21) <223> Xaa lacks or is neutral, acidic or alkaline Amino acid residue. <220><221>misc_feature<222> (3, 17 and)..(18) <223> Xaa is absent, or is neutral hydrophobic, or neutral Polar amino acid residue <220><221> misc_characteristic <222> (6)..(6) <223> Xaa is a neutral hydrophobic amino acid residue. <220>;<221>misc_feature<222> (8)..(8) <223> Xaa is a A, D, or E 0 <220><221> misc_features <222> 10)..(10) O:\121\121929.DOC -49- 200808833 <223> Xaa is an acidic amino acid residue. <220><221>misc_feature<222> (11) (11) <223> Xaa is an amino acid residue. <220><221> misc_characteristic <222> (12)..(12) <223>Xaa is neutral hydrophobic Sexual, neutral polar or basic amino acid residue. <220><221>misc-characteristic<222> (19 and)"(22) <223> Xaa lack or neutral hydrophobicity , Neutral polar or basic amino acid residues <. 400 > 72
Xaa Xaa Xaa Xaa Pro Xaa Asp Xaa Leu Xaa Xaa Xaa Leu Tyr Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Pro Xaa Asp Xaa Leu Xaa Xaa Xaa Leu Tyr Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 73 <211> 8 <212>蛋白質 <213>人造的序列 O:\121\121929.DOC -50- 200808833 <220> <223>能夠與Ang-2結合的多肽 <220> <221> misc—特徵 <222> (3)..(3) <223> Xaa為中性極性的胺基酸殘基。 <220> <221> misc_特徵 <222> (4)..(4) <223> Xaa為酸性胺基酸殘基。 <220> <221〉misc_特徵 <222> (5)..(5) <223> Xaa為中性極性或酸性的胺基酸殘基。 <220> <221〉misc_特徵 <222> (6和）··（7) <223> Xaa為中性疏水性的胺基酸殘基。 <400> 73Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 73 <211> 8 <212> Protein <213> Artificial sequence O: \121\121929.DOC -50- 200808833 <220><223> Polypeptide bound to Ang-2 <220><221> misc-characteristic <222> (3)..(3) <223> Xaa is a neutral polar amino acid residue. <220><221> misc_characteristic <222> (4).. (4) <223> Xaa is an acidic amino acid residue. <220><221>misc_ feature <222> (5).. (5) <223> Xaa is a neutral polar or acidic amino acid residue. <220><221>misc_ feature <222> (6 and) (7) <223> Xaa is a neutral hydrophobic amino acid residue. <400> 73
Arg Pro Xaa Xaa Xaa Xaa Xaa Gly 1 5 <210> 74 <211> 20 <212〉蛋白質 51-Arg Pro Xaa Xaa Xaa Xaa Xaa Gly 1 5 <210> 74 <211> 20 <212>protein 51-
O:\121\121929.DOC 200808833 <213>人造的序列 <220> <223〉能夠與Ang-2結合的多肽 <220> <221> misc_特徵 <222>(1，2, 4, 13, 14, 19 和）··(20) <223> Xaa為中性疏水性或中性極性的胺基酸殘基。 <220> <221〉misc_特徵 <222〉（3, 9和）·_(17) <223〉Xaa為中性極性或酸性的胺基酸殘基。 <220> <221> misc_^特徵 <222>(7, 15 和）··（16) <223> Xaa為中性極性的胺基酸殘基。 <220> <221>misc—特徵 <222> (8)..(8) <223>Xaa為酸性的胺基酸殘基。 <220> <221〉misc_特徵 <222>(10 和）..（11) 52·O:\121\121929.DOC 200808833 <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <220><221> misc_feature <222> 2, 4, 13, 14, 19 and (20) <223> Xaa is a neutral hydrophobic or neutral polar amino acid residue. <220><221>misc_ feature <222>(3, 9 and)·(17) <223> Xaa is a neutral polar or acidic amino acid residue. <220><221> misc_^Characteristics <222> (7, 15 and) (16) <223> Xaa is a neutral polar amino acid residue. <220><221>misc-characteristics<222> (8).. (8) <223> Xaa is an acidic amino acid residue. <220><221>misc_features<222>(10 and)..(11) 52·
O:\121\121929.DOC 200808833 <223>Xaa為中性疏水性的胺基酸殘基。 <220> <221> misc」争徵 <222> (18)..(18) <223> Xaa為中性疏水性或鹼性的胺基酸殘基。 <400> 74O:\121\121929.DOC 200808833 <223> Xaa is a neutral hydrophobic amino acid residue. <220><221> misc" contending <222> (18).. (18) <223> Xaa is a neutral hydrophobic or basic amino acid residue. <400> 74
Xaa Xaa Xaa Xaa Arg Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Arg Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa 20 <210> 75 <211> 13 <212>蛋白質 <213>人造的序列 <220> <223>施夠與Ang-2結合的多肽 <220> <221> misc__特徵 <222> (2)..(2) <223〉Xaa為酸性的胺基酸殘基e <220> <22 1> misc—特徵 <222〉（4)..(4) <223> Xaa為中性疏水性的胺基酸殘基。 O:\121\121929.DOC -53- 200808833 <220> <221> misc_特徵 <222> (5)..(5) <223>Xaa為 E，D，或 Q ° <220> <221> misc_特徵 <222> (10)..(10) <223> Xaa為中性疏水性或中性極性的胺基酸殘基。 <220> <22 1> misc_特徵 <222> (13)..(13) <223〉Xaa為酸性的殘基。 <400> 75<210>< 221>misc__Characteristics<222> (2)..(2) <223>Xaa is an acidic amino acid residue e <220><221>misc-characteristics<222> (4) <223> Xaa is a neutral hydrophobic amino acid residue. O:\121\121929.DOC -53- 200808833 <220><221>misc_features<222> (5)..(5) <223> Xaa is E, D, or Q ° <220><221> misc_characteristic <222> (10).. (10) <223> Xaa is a neutral hydrophobic or neutral polar amino acid residue. <220><221> misc_ feature <222> (13).. (13) <223> Xaa is an acidic residue. <400> 75
Cys Xaa Gly Xaa Xaa Asp Pro Phe Thr Xaa Gly Cys Xaa 1 5 l〇 <210> 76 <211> 20 <212〉蛋白質 <213>人造的序列 <220> <223〉能夠與Ang-2結合的多肽 <400> 76Cys Xaa Gly Xaa Xaa Asp Pro Phe Thr Xaa Gly Cys Xaa 1 5 l〇<210> 76 <211> 20 <212>protein <213> artificial sequence <220><223> -2 bound polypeptide <400> 76
Pro lie Arg Gin Glu Glu Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15 O:\121\121929.DOC -54- 200808833Pro lie Arg Gin Glu Glu Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15 O:\121\121929.DOC -54- 200808833
Met Trp Glu Val 20 <210> 77 <211〉 20 * <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 77Met Trp Glu Val 20 <210> 77 <211> 20 * <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 77
Thr Asn lie Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Asp His 15 10 15Thr Asn lie Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Asp His 15 10 15
Met Pro Gly Lys 20 <210> 7 8 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 78Met Pro Gly Lys 20 <210> 7 8 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Trp Tyr Glu Gin Asp Ala Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Trp Tyr Glu Gin Asp Ala Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Ala Glu Val 20 <210> 79 55-Met Ala Glu Val 20 <210> 79 55-
O:\121\121929.DOC 200808833 <211> 20 <212>蛋白質 <213>人造的序列 <220> 4 <223>能夠與Ang_2結合的多肽 <400> 79O:\121\121929.DOC 200808833 <211> 20 <212> Protein <213> Artificial sequence <220> 4 <223> A polypeptide capable of binding to Ang_2 <400>
Asn Arg Leu Gin Glu Val Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Asn Arg Leu Gin Glu Val Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Glu Asn Val 20 <210> 80 <211> 20 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang_2結合的多肽 <400> 80Met Glu Asn Val 20 <210> 80 <211> 20 <212> Protein <213> Artificial sequence <220><223> Polypeptide capable of binding to Ang_2 <400>
Ala Ala Thr Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Ala Ala Thr Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Pro Arg Ser 20 <210> 81 <211> 20 <212>蛋白質 <213> 人造的序列 -56-Met Pro Arg Ser 20 <210> 81 <211> 20 <212> Protein <213> Artificial Sequence -56-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的多肽 <400> 81O:\121\121929.DOC 200808833 <220><223> A polypeptide capable of binding to Ang-2 <400> 81
Leu Arg His Gin Glu Gly Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1..5 10 15Leu Arg His Gin Glu Gly Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1..5 10 15
Met Phe Asp Trp .20 <210> 82 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 82Met Phe Asp Trp .20 <210> 82 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Val Pro Arg Gin Lys Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Val Pro Arg Gin Lys Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Tyr Val Gly 20 <210> 83 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能狗與Ang-2結合的多肽 <400> 83<220>
Ser He Ser His Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Ser He Ser His Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
O:\121\121929.DOC -57- 200808833O:\121\121929.DOC -57- 200808833
Met Gin Val Gly 20 <210> 84 <211> 20 1 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 84Met Gin Val Gly 20 <210> 84 <211> 20 1 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Trp Ala Ala Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Trp Ala Ala Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Gly Arg Met 20 <210> 85 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223〉 能夠與Ang-2結合的多肽 <400> 85Met Gly Arg Met 20 <210> 85 <211> 20 <212> Protein <213> Artificial sequence <220><223> Polypeptide capable of binding to Ang-2 <400>
Thr Trp Pro Gin Asp Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Thr Trp Pro Gin Asp Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Gly Ser Thr 20 <210> 86 -58-Met Gly Ser Thr 20 <210> 86 -58-
O:\121\121929.DOC 200808833 <211> 20 <212〉 蛋白質 . <213> 人造的序列 ％ <220> <223> 能夠與Ang-2結合的多肽 <400> 86O:\121\121929.DOC 200808833 <211> 20 <212> Protein . <213> Artificial sequence % <220><223> A polypeptide capable of binding to Ang-2 <400>
Gly His Ser Gin Glu Glu Cys Gly Trp Asp Pro Trp Thr Cys Glu His 15 10 15Gly His Ser Gin Glu Glu Cys Gly Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Gly Thr Ser 20 <210> 87 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 87Met Gly Thr Ser 20 <210> 87 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin His Trp Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Asp His 1 5 10 . 15Gin His Trp Gin Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Asp His 1 5 10 .
Met Pro Ser Lys 20 <210〉 88 <211> 20 <212> 蛋白質 <213〉 人造的序列 59-Met Pro Ser Lys 20 <210> 88 <211> 20 <212> Protein <213> Artificial sequence 59-
O:\121\121929.DOC 200808833 <22〇> <223> 能夠與Ang-2結合的多肽 <400> 88O:\121\121929.DOC 200808833 <22〇><223> A polypeptide capable of binding to Ang-2 <400> 88
Asn Val Arg Gin Glu Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15Asn Val Arg Gin Glu Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15
Met Pro Val Arg 20 <210> 89 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 89<220>
Lys Ser Gly Gin Val Glu Cys Asn Trp Asp Pro Trp Thr Cys Glu His 15 10 15Lys Ser Gly Gin Val Glu Cys Asn Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Pro Arg Asn 20 <210> 90 <211> 20 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 90Met Pro Arg Asn 20 <210> 90 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Val Lys Thr Gin Glu His Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15 60-Val Lys Thr Gin Glu His Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15 60-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Arg Glu Trp 20 <210> 91 <211> 20 、 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 91Met Arg Glu Trp 20 <210> 91 <211> 20 , <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Ala Trp Gly Gin Glu Gly Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15Ala Trp Gly Gin Glu Gly Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Leu Pro Met 20 <210> 92 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 92Met Leu Pro Met 20 <210> 92 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Pro Val Asn Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Pro Val Asn Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Pro Pro Met 20 <21〇> 93 61-Met Pro Pro Met 20 <21〇> 93 61-
O:\121\121929.DOC 200808833 <211〉 20 <212>蛋白質 <213>人造的序列 <220〉 ' <223> 能夠與Ang-2結合的多肽 <400> 93O:\121\121929.DOC 200808833 <211> 20 <212> Protein <213> Artificial sequence <220> ' <223> A polypeptide capable of binding to Ang-2 <400>
Arg Ala Pro Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Ala His 1 5 :· 10 15Arg Ala Pro Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Ala His 1 5 :· 10 15
Met Asp lie Lys 20 <210> 94 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽________ <400> 94Met Asp lie Lys 20 <210> 94 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 ________ <400>
His Gly Gin Asn Met Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15His Gly Gin Asn Met Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Phe Arg Tyr 20 <210> 95 <211〉 20 <212>蛋白質 <213>人造的序列 62-Met Phe Arg Tyr 20 <210> 95 <211> 20 <212> Protein <213> Artificial sequence 62-
O:\121\121929.DOC 200808833 <220> -<223>能夠與Ang-2結合的多肽 <400> 95O:\121\121929.DOC 200808833 <220>-<223> A polypeptide capable of binding to Ang-2 <400> 95
Pro Arg Leu Gin Glu Glu Cys Val Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15Pro Arg Leu Gin Glu Glu Cys Val Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15
Met Pro Leu Arg 20 <210> 96 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400〉 96<220>
Arg Thr Thr Gin Glu Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15Arg Thr Thr Gin Glu Lys Cys Glu Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15
Met Glu Ser Gin 20 <210> 97 <211〉 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 97Met Glu Ser Gin 20 <210> 97 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin Thr Ser Gin Glu Asp Cys Val Trp Asp Pro Trp Thr Cys Asp His 15 10 15 -63·Gin Thr Ser Gin Glu Asp Cys Val Trp Asp Pro Trp Thr Cys Asp His 15 10 15 -63·
O:\12 l\121929.DOC 200808833O:\12 l\121929.DOC 200808833
Met Val Ser Ser 20 <210> 98 <211> 20 』· <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <40〇> 98Met Val Ser Ser 20 <210> 98 <211> 20 』·<212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <40〇>; 98
Gin Val lie Gly Arg Pro Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Gin Val lie Gly Arg Pro Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Leu Glu Gly Leu 20 <210> 99 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 99Leu Glu Gly Leu 20 <210> 99 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Trp Ala Gin Gin Glu Glu Cys Ala Trp Asp Pro Trp Thr Cys Asp His 1 5 * 10 15Trp Ala Gin Gin Glu Glu Cys Ala Trp Asp Pro Trp Thr Cys Asp His 1 5 * 10 15
Met Val Gly Leu 20 <210> 100 -64 -Met Val Gly Leu 20 <210> 100 -64 -
O:\121\121929.DOC 200808833 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223> :能夠與Ang-2結合的多狀 <400> 100O:\121\121929.DOC 200808833 <211> 20 <212> Protein <213> Artificial sequence <220><223>: Polymorphism capable of binding to Ang-2 <400>
Leu Pro Gly Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15Leu Pro Gly Gin Glu Asp Cys Glu Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Val Arg Ser 20 <210> 101 <211> 20 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 101Met Val Arg Ser 20 <210> 101 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Pro Met Asn Gin Val Glu Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15Pro Met Asn Gin Val Glu Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Pro Arg Ser 20 <210> 102 <211> 20 <212> 蛋白質 <213> 人造的序列 O:\121\121929.DOC 65- 200808833 <220> <223> 能夠與Ang-2結合的多肽 — <400> 102Met Pro Arg Ser 20 <210> 102 <211> 20 <212> Protein <213> Artificial sequence O:\121\121929.DOC 65-200808833 <220><223> 2 bound polypeptide - <400> 102
Phe Gly Trp Ser His Gly Cys Glu Trp Asp Pro Trp Thr Cy*s" Glu His 1,5 10 15Phe Gly Trp Ser His Gly Cys Glu Trp Asp Pro Trp Thr Cy*s" Glu His 1,5 10 15
Met Gly Ser Thr 20 <210〉 103 <211> 20 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 103Met Gly Ser Thr 20 <210> 103 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Lys Ser Thr Gin Asp Asp Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15Lys Ser Thr Gin Asp Asp Cys Asp Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Val Gly Pro 20 <210〉 104 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400〉 104Met Val Gly Pro 20 <210> 104 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 104
Gly Pro Arg He Ser Thr Cys Gin Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15 -66-Gly Pro Arg He Ser Thr Cys Gin Trp Asp Pro Trp Thr Cys Glu His 1 5 10 15 -66-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Asp Gin Leu 20 <210> 105 <211> 20 ^Met Asp Gin Leu 20 <210> 105 <211> 20 ^
<212>蛋白質 <213>人造的序歹1J <220> <223>能夠與Ang-2結合的多肽 <400> 105<212> Protein <213> Artificial sequence 1J <220><223> A polypeptide capable of binding to Ang-2 <400>
Ser Thr lie Gly Asp Met Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15Ser Thr lie Gly Asp Met Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15
Met Gin Val Asp 20 <210> 106 <211> 20 <212>蛋白質 <213〉人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 106<210>
Val Leu Gly Gly Gin Gly Cys Glu Trp Asp Pro Trp Thr Cys Arg Leu 15 10 15Val Leu Gly Gly Gin Gly Cys Glu Trp Asp Pro Trp Thr Cys Arg Leu 15 10 15
Leu Gin Gly Trp 20 <210〉 107 -67-Leu Gin Gly Trp 20 <210> 107 -67-
O:\121\121929.DOC 200808833 <211〉 20 <212> 蛋白質 <213> 人造的序列 <220> * <223> 能夠與Ang-2結合的多肽 <400> 107O:\121\121929.DOC 200808833 <211> 20 <212> Protein <213> Artificial sequence <220> * <223> A polypeptide capable of binding to Ang-2 <400>
Val Leu Gly Gly Gin Gly Cys Gin Trp Asp Pro Trp Thr Cys Ser His 1 5 -. 10 15Val Leu Gly Gly Gin Gly Cys Gin Trp Asp Pro Trp Thr Cys Ser His 1 5 -. 10 15
Leu Glu Asp Gly 20 <210> 108 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 108Leu Glu Asp Gly 20 <210> 108 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Thr Thr He Gly Ser Met Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15Thr Thr He Gly Ser Met Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15
Met Gin Gly Gly 20 <210〉 109 <211> 20 <212>蛋白質 <213>人造的序列 68-Met Gin Gly Gly 20 <210> 109 <211> 20 <212> Protein <213> Artificial Sequence 68-
O:\121\121929.DOC 200808833 <22〇> <223>能夠與Ang-2結合的多肽 <400> 109O:\121\121929.DOC 200808833 <22〇><223> A polypeptide capable of binding to Ang-2 <400> 109
Thr Lys Gly Lys Ser Val Cys Gin Trp Asp Pro Trp Thr Cys Ser His 1 t 5 10 、15Thr Lys Gly Lys Ser Val Cys Gin Trp Asp Pro Trp Thr Cys Ser His 1 t 5 10, 15
Met Gin Ser Gly 20 <210> 110 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 110Met Gin Ser Gly 20 <210> 110 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Thr Thr lie Gly Ser Met Cys Gin Trp Asp Pro Trp Thr Cys Ala His 15 10 15Thr Thr lie Gly Ser Met Cys Gin Trp Asp Pro Trp Thr Cys Ala His 15 10 15
Met Gin Gly Gly 20 <210> 111 <211〉 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400〉 111Met Gin Gly Gly 20 <210> 111 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 111
Trp Val Asn Glu Val Val Cys Glu Trp Asp Pro Trp Thr Cys Asn His 15 10 15 -69-Trp Val Asn Glu Val Val Cys Glu Trp Asp Pro Trp Thr Cys Asn His 15 10 15 -69-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Trp Asp Thr Pro 20 <210> 112 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 112<220>
Val Val Gin Val Gly Met Cys Gin Trp Asp Pro Trp Thr Cys Lys His 15 10 15Val Val Gin Val Gly Met Cys Gin Trp Asp Pro Trp Thr Cys Lys His 15 10 15
Met Arg Leu Gin 20 <210> 113 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 113Met Arg Leu Gin 20 <210> 113 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Ala Val Gly Ser Gin Thr Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15Ala Val Gly Ser Gin Thr Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15
Leu Val Glu Val y 20 <210> 114 ' 70-Leu Val Glu Val y 20 <210> 114 ' 70-
O:\121\121929.DOC 200808833 <211〉 20 —- <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肤 <400> 114O:\121\121929.DOC 200808833 <211> 20 —- <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin Gly Met Lys Met Phe Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15 lie Val Tyr Arg 20 <210> 115 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang·2結合的多碑 <400> 115Gin Gly Met Lys Met Phe Cys Glu Trp Asp Pro Trp Thr Cys Ala His 15 10 15 lie Val Tyr Arg 20 <210> 115 <211> 20 <212> Protein <213> Artificial Sequence <220><223> Multi-spot <400> capable of combining with Ang·2
Thr Thr He Gly Ser Met Cys Gin Trp Asp Pro Trp Thr Cys Glu His 15 10 15Thr Thr He Gly Ser Met Cys Gin Trp Asp Pro Trp Thr Cys Glu His 15 10 15
Met Gin Gly Gly 20 <210> 116 <211> 20 <212>蛋白質 <213>人造的序列 -71-Met Gin Gly Gly 20 <210> 116 <211> 20 <212> Protein <213> Artificial Sequence -71-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的多肽 <400> 116O:\121\121929.DOC 200808833 <220><223> A polypeptide capable of binding to Ang-2 <400> 116
Thr Ser Gin Arg Val Gly Cys Glu Trp Asp Pro Trp Thr Cys Gin His 1 5 10 15Thr Ser Gin Arg Val Gly Cys Glu Trp Asp Pro Trp Thr Cys Gin His 1 5 10 15
Leu Thr Tyr Thr 20 <210〉 117 <211〉 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <_> 117Leu Thr Tyr Thr 20 <210> 117 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <_>
Gin Trp Ser Trp Pro Pro Cys Glu Trp Asp Pro Trp Thr Cys Gin Thr 15 10 15Gin Trp Ser Trp Pro Pro Cys Glu Trp Asp Pro Trp Thr Cys Gin Thr 15 10 15
Val Trp Pro Ser 20 <210> 118 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 118Val Trp Pro Ser 20 <210> 118 <211> 20 <212> Protein <213> Artificial sequence <220><223> Polypeptide capable of binding to Ang-2 <400>
Gly Thr Ser Pro Ser Phe Cys Gin Trp Asp Pro Trp Thr Cys Ser His 15 10 15 •72·Gly Thr Ser Pro Ser Phe Cys Gin Trp Asp Pro Trp Thr Cys Ser His 15 10 15 •72·
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Val Gin Gly 20 <210〉 119 <211> 22 ， <212> 蛋白質 <213> 人造的序列 <220> <223〉 能夠與Ang-2結合的多肽 <400> 119Met Val Gin Gly 20 <210> 119 <211> 22 , <212> Protein <213> Artificial sequence <220><223> Polypeptide capable of binding to Ang-2 <400>
Gin Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr Leu Tyr Glu His 15 10 15Gin Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr Leu Tyr Glu His 15 10 15
Phe lie Phe His Tyr Thr 20 <210> 120 <211〉 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 120Phe lie Phe His Tyr Thr 20 <210> 120 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Leu Asn Phe Thr Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 15Leu Asn Phe Thr Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 15
Trp Thr Leu Gin Gin Ser 20 <210> 121 -73-Trp Thr Leu Gin Gin Ser 20 <210> 121 -73-
O:\121\121929.DOC 200808833 <211> 22 <212> 蛋白質 <213> 人造的序列 \ <220> <223> 能夠與Ang-2結合的多肽 <400> 121O:\121\121929.DOC 200808833 <211> 22 <212> Protein <213> Artificial sequence \ <220><223> A polypeptide capable of binding to Ang-2 <400>
Thr Lys Phe Asn Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15Thr Lys Phe Asn Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15
Trp Thr Leu Gin His Gin 20 <210> 122 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 122<212><
Val Lys Phe Lys Pro Leu Asp Ala Leu Glu Gin Thr Leu Tyr Glu His 1 5 10 15Val Lys Phe Lys Pro Leu Asp Ala Leu Glu Gin Thr Leu Tyr Glu His 1 5 10 15
Trp Met Phe Gin Gin Ala 20 <210> 123 <211> 22 <212> 蛋白質 <213> 人造的序列 -74-Trp Met Phe Gin Gin Ala 20 <210> 123 <211> 22 <212> Protein <213> Artificial sequence -74-
0:\121\121929.D0C 200808833 <220> <223> 能夠與Ang-2結合的多肽 <400> 1230:\121\121929.D0C 200808833 <220><223> A polypeptide capable of binding to Ang-2 <400> 123
Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu lie Leu Tyr Glu Gin 1 5 10 15Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu lie Leu Tyr Glu Gin 1 5 10 15
Gin Thr Phe Gin Glu Arg 20 <210> 124 <211〉 22 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的多肚 <400> 124Gin Thr Phe Gin Glu Arg 20 <210> 124 <211> 22 <212>Protein<213> Artificial sequence <220><223> can be combined with Ang-2 <400> 124
Thr Asn Phe Met Pro Met Asp Asp Leu Glu Gin Arg Leu Tyr Glu Gin 15 10 15Thr Asn Phe Met Pro Met Asp Asp Leu Glu Gin Arg Leu Tyr Glu Gin 15 10 15
Phe lie Leu Gin Gin Gly 20 <210> 125 <211> 22 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多lii <400> 125Phe lie Leu Gin Gin Gly 20 <210> 125 <211> 22 <212> Protein <213> Artificial sequence <220><223> Multiple lii <400> capable of binding to Ang-2 125
Ser Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 15 •75-Ser Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 15 •75-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Trp Thr Leu Gin His Ala 20 <210> 126 <211〉 22 4 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 126Trp Thr Leu Gin His Ala 20 <210> 126 <211> 22 4 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 126
Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15
Phe Met Leu Gin Gin Ala 20 <210> 127 <211> 22 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 127Phe Met Leu Gin Gin Ala 20 <210> 127 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin Asn Phe Lys Pro Met Asp Glu Leu Glu Asp Thr Leu Tyr Lys Gin 1 5 10 15Gin Asn Phe Lys Pro Met Asp Glu Leu Glu Asp Thr Leu Tyr Lys Gin 1 5 10 15
Phe Leu Phe Gin His Ser 20 <210> 128 -76-Phe Leu Phe Gin His Ser 20 <210> 128 -76-
O:\121\121929.DOC 200808833 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 128O:\121\121929.DOC 200808833 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Tyr Lys Phe Thr Pro Leu Asp Asp Leu Glu Gin Thr Leu Tyr Glu Gin 1 · 5 .10 15Tyr Lys Phe Thr Pro Leu Asp Asp Leu Glu Gin Thr Leu Tyr Glu Gin 1 · 5 .10 15
Trp Thr Leu Gin His Val 20 <210> 129 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 129Trp Thr Leu Gin His Val 20 <210> 129 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin Glu Tyr Glu Pro Leu Asp Glu Leu Asp Glu Thr Leu Tyr Asn Gin 15 10 15Gin Glu Tyr Glu Pro Leu Asp Glu Leu Asp Glu Thr Leu Tyr Asn Gin 15 10 15
Trp Met Phe His Gin Arg 20 <210> 130 <211> 22 <212> 蛋白質 <213> 人造的序列 77-Trp Met Phe His Gin Arg 20 <210> 130 <211> 22 <212> Protein <213> Artificial sequence 77-
O:\121\121929.DOC <220> 200808833 <223> 能夠與Ang-2結合的多肽 <400> 130O:\121\121929.DOC <220> 200808833 <223> A polypeptide capable of binding to Ang-2 <400> 130
Ser Asn Phe Met Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 ‘15Ser Asn Phe Met Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 1 5 10 ‘15
Phe Met Leu Gin His Gin 20 <210> 131 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 131<210> <
Gin Lys Tyr Gin Pro Leu Asp Glu Leu Asp Lys Thr Leu Tyr Asp Gin 15 10 15Gin Lys Tyr Gin Pro Leu Asp Glu Leu Asp Lys Thr Leu Tyr Asp Gin 15 10 15
Phe Met Leu Gin Gin Gly 20 <210> 132 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 132"health sequence <220>
Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Lys Gin 15 10 15 -78 -Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr Leu Tyr Lys Gin 15 10 15 -78 -
O:\121\121929DOC 200808833O:\121\121929DOC 200808833
Trp Thr Leu Gin Gin Arg 20 <210> 133 <211〉 22 < <212>蛋白質 <213> 人造的序列 <220〉 <；223> 能夠與Ang-2結合的多肽 <400> 133Trp Thr Leu Gin Gin Arg 20 <210> 133 <211> 22 <<212> Protein <213> Artificial sequence <220>;223> polypeptide capable of binding to Ang-2 <400> 133
Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu Trp Leu Tyr His Gin 15 10 15Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu Trp Leu Tyr His Gin 15 10 15
Phe Thr Leu His His Gin 20 <210> 134 <211> 22 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400〉 134Phe Thr Leu His His Gin 20 <210> 134 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 134
Gin Lys Phe Met Pro Leu Asp Glu Leu Asp Glu lie Leu Tyr Glu Gin 15 10 15Gin Lys Phe Met Pro Leu Asp Glu Leu Asp Glu lie Leu Tyr Glu Gin 15 10 15
Phe Met Phe Gin Gin Ser 20 <210〉 135 -79-Phe Met Phe Gin Gin Ser 20 <210> 135 -79-
O:\121\121929.DOC 200808833 <211> 22 <212> 蛋白質 <213> 人造的序列 、 <220> <223> 能夠與Ang-2結合的多肽 — <400> 135O:\121\121929.DOC 200808833 <211> 22 <212> Protein <213> Artificial sequence, <220><223> A polypeptide capable of binding to Ang-2 - <400>
Gin Thr Phe Gin Pro Leu Asp Asp Leu Glu Glu Tyr Leu Tyr Glu Gin 1 5 10 15Gin Thr Phe Gin Pro Leu Asp Asp Leu Glu Glu Tyr Leu Tyr Glu Gin 1 5 10 15
Trp lie Arg Arg Tyr His 20 <210> 136 <211> 22 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 136Trp lie Arg Arg Tyr His 20 <210> 136 <211> 22 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Glu Asp Tyr Met Pro Leu Asp Ala Leu Asp Ala Gin Leu Tyr Glu Gin 15 10 15Glu Asp Tyr Met Pro Leu Asp Ala Leu Asp Ala Gin Leu Tyr Glu Gin 15 10 15
Phe lie Leu Leu His Gly 20 <210> 137 <211> 22 <212> 蛋白質 <213> 人造的序列 -80-Phe lie Leu Leu His Gly 20 <210> 137 <211> 22 <212> Protein <213> Artificial sequence -80-
O:\121\121929.DOC 200808833 <22〇> <223> 能夠與Ang-2結合的多肽 <400> 137O:\121\121929.DOC 200808833 <22〇><223> A polypeptide capable of binding to Ang-2 <400>
His Thr Phe Gin Pro Leu Asp Glu Leu Glu Glu Th:r Leu Tyr Tyr Gin 1,5 10 15His Thr Phe Gin Pro Leu Asp Glu Leu Glu Glu Th:r Leu Tyr Tyr Gin 1,5 10 15
Trp Leu Tyr Asp Gin Leu 20 <210> 138 <211> 22 <212> 蛋白質 <213> 人造的序列 <220〉 <223> 能夠與Ang-2結合的多肽 <400> 138Trp Leu Tyr Asp Gin Leu 20 <210> 138 <211> 22 <212> Protein <213> Artificial sequence <220> <223> A polypeptide capable of binding to Ang-2 <400>
Tyr Lys Phe Asn Pro Met Asp Glu Leu Glu Gin Thjr Leu Tyr Glu Glu 15 10 15Tyr Lys Phe Asn Pro Met Asp Glu Leu Glu Gin Thjr Leu Tyr Glu Glu 15 10 15
Phe Leu Phe Gin His Ala 20 <210> 139 <211〉 22 <212〉 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 139"health sequence <220>
Thr Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr heu Tyr Glu His 15 10 15 81-Thr Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr heu Tyr Glu His 15 10 15 81-
0:\121\121929.D0C 2008088330:\121\121929.D0C 200808833
Trp lie Leu Gin His Ser 20 <210> 140 <211> 22 ^ <212>蛋白質 <213>人造的序列； <220> <223> 能夠與Ang-2結合的多肽 <_> 140Trp lie Leu Gin His Ser 20 <210> 140 <211> 22 ^ <212>Protein <213> Artificial sequence; <220><223> A polypeptide capable of binding to Ang-2 <_>; 140
Gin Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15Gin Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr Leu Tyr Glu Gin 15 10 15
Trp Thr Leu Gin Gin Arg 20 <210> 141 <211> 22 <212>蛋白質 <n3>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 141Trp Thr Leu Gin Gin Arg 20 <210> 141 <211> 22 <212> Protein <n3> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Thr Lys Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr Leu Tyr Glu Gin 15 10 15Thr Lys Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr Leu Tyr Glu Gin 15 10 15
Trp Thr Leu Gin Gin Arg 20 <210> 142 -82 -Trp Thr Leu Gin Gin Arg 20 <210> 142 -82 -
O:\121\121929.DOC 200808833 <211> 22 <212>蛋白質 <213>人造的序列 <220> * <223> 能夠與Ang-2結合的多肽 <400> 142O:\121\121929.DOC 200808833 <211> 22 <212> Protein <213> Artificial sequence <220> * <223> A polypeptide capable of binding to Ang-2 <400>
Thr Asn Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr Leu Tyr Glu Gin 1 5 10 15Thr Asn Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr Leu Tyr Glu Gin 1 5 10 15
Trp Thr Leu Gin Gin Arg 20 <210> 143 <211> 20 <212>蛋白質 <213>人造的序列 ί <220> <223>能夠與Ang-2結合的多肽 <400> 143Trp Thr Leu Gin Gin Arg 20 <210> 143 <211> 20 <212>Protein<213> Artificial sequence ί <220><223> A polypeptide capable of binding to Ang-2 <400> 143
Ala Gly Gly Met Arg Pro Tyr Asp Gly Met Leu Gly Trp Pro Asn Tyr 15 10 15Ala Gly Gly Met Arg Pro Tyr Asp Gly Met Leu Gly Trp Pro Asn Tyr 15 10 15
Asp Val Gin Ala 20 <210> 144 <211〉 20 <212>蛋白質 <213>人造的序列 -83 -Asp Val Gin Ala 20 <210> 144 <211> 20 <212> Protein <213> Artificial Sequence -83 -
O:\121\121929.DOC 200808833 <220> <223>能夠與Ang-2結合的多肽 <400> 144O:\121\121929.DOC 200808833 <220><223> A polypeptide capable of binding to Ang-2 <400>
Gin Thr Trp Asp Asp Pro Cys Met His lie Leu Gly Pro Val Thr Trp 1 ^ 5 10 15Gin Thr Trp Asp Asp Pro Cys Met His lie Leu Gly Pro Val Thr Trp 1 ^ 5 10 15
Arg Arg Cys lie 20 <210> 145 <211> 20 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 145Arg Arg Cys lie 20 <210> 145 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Ala Pro Gly Gin Arg Pro Tyr Asp Gly Met Leu Gly Trp Pro Thr Tyr 1 5 10 . 15Ala Pro Gly Gin Arg Pro Tyr Asp Gly Met Leu Gly Trp Pro Thr Tyr 1 5 10 . 15
Gin Arg lie Val 20 <210> 146 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 146Gin Arg lie Val 20 <210> 146 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Ser Gly Gin Leu Arg Pro Cys Glu Glu He Phe Gly Cys Gly Thr Gin 1 5 10 15 -84Ser Gly Gin Leu Arg Pro Cys Glu Glu He Phe Gly Cys Gly Thr Gin 1 5 10 15 -84
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Asn Leu Ala Leu 20 <210> 147 i « <211> 20 _ <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400〉 147Asn Leu Ala Leu 20 <210> 147 i « <211> 20 _ <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400> 147
Phe Gly Asp Lys Arg Pro Leu Glu Cys Met Phe Gly Gly Pro lie Gin 15 10 15Phe Gly Asp Lys Arg Pro Leu Glu Cys Met Phe Gly Gly Pro lie Gin 15 10 15
Leu Cys Pro Arg 20 <210〉 148 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 148Leu Cys Pro Arg 20 <210> 148 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gly Gin Asp Leu Arg Pro Cys Glu Asp Met Phe Gly Cys Gly Thr Lys 15 10 15Gly Gin Asp Leu Arg Pro Cys Glu Asp Met Phe Gly Cys Gly Thr Lys 15 10 15
Asp Trp Tyr Gly 20 <210〉 149 85-Asp Trp Tyr Gly 20 <210〉 149 85-
O:\121\121929.DOC 200808833 <211〉 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 149O:\121\121929.DOC 200808833 <211>20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Gly Phe Glu Tyr Cys Asp Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 .· 15Gly Phe Glu Tyr Cys Asp Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 .· 15
Asp Lys Gin Thr 20 <210> 150 <211〉 20 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 150<210>
Lys Leu Glu Tyr Cys Asp Gly Met Glu Asp Pro Phe Thr Gin Gly Cys 15 10 15Lys Leu Glu Tyr Cys Asp Gly Met Glu Asp Pro Phe Thr Gin Gly Cys 15 10 15
Asp Asn Gin Ser 20 <210> 151 <211> 20 <212>蛋白質 <213>人造的序列 86-Asp Asn Gin Ser 20 <210> 151 <211> 20 <212> Protein <213> Artificial Sequence 86-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的多肽 <400> 151O:\121\121929.DOC 200808833 <220><223> A polypeptide capable of binding to Ang-2 <400>
Leu Gin Glu Trp Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 1 , 5 10 一15Leu Gin Glu Trp Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 1 , 5 10 - 15
Glu Lys Gin Arg 20 <210> 152 <211〉 20 <212> 蛋白質 <213> 人造的序列 <220〉 <223> 能夠與Ang-2結合的多肽 <400> 152Glu Lys Gin Arg 20 <210> 152 <211> 20 <212> Protein <213> Artificial sequence <220> <223> A polypeptide capable of binding to Ang-2 <400>
Ala Gin Asp Tyr Cys Glu Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15Ala Gin Asp Tyr Cys Glu Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15
Glu Met Gin Lys 20 <210〉 153 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的多肽 <400> 153Glu Met Gin Lys 20 <210> 153 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Leu Leu Asp Tyr Cys Glu Gly Val Gin Asp Pro Phe Thr Phe Gly Cys 15 10 15 -87-Leu Leu Asp Tyr Cys Glu Gly Val Gin Asp Pro Phe Thr Phe Gly Cys 15 10 15 -87-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Glu Asn Leu Asp 20 <210> 154 <211> 20 、 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <'400> 154Glu Asn Leu Asp 20 <210> 154 <211> 20 , <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <'400>
His Gin Glu Tyr Cys Glu Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15His Gin Glu Tyr Cys Glu Gly Met Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15
Glu Tyr Gin Gly 20 <210> 155 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 155Glu Tyr Gin Gly 20 <210> 155 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Met Leu Asp Tyr Cys Glu Gly Met Asp Asp Pro Phe Thr Phe Gly Cys 1 5 10 15Met Leu Asp Tyr Cys Glu Gly Met Asp Asp Pro Phe Thr Phe Gly Cys 1 5 10 15
Asp Lys Gin Met 20 <210> 156Asp Lys Gin Met 20 <210> 156
O:\121\121929.DOC 88- 200808833 <211> 20 <212>蛋白質 <213>人造的序列 <220〉 ? <223> 能夠與Ang-2結合的多肽 <400> 156O:\121\121929.DOC 88-200808833 <211> 20 <212> Protein <213> Artificial sequence <220> ? <223> A polypeptide capable of binding to Ang-2 <400>
Leu Gin Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 1 5 10 15Leu Gin Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 1 5 10 15
Glu Asn Gin Arg 20 <210> 157 <211> 20 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400> 157Glu Asn Gin Arg 20 <210> 157 <211> 20 <212> Protein <213> Artificial sequence <220><223> A polypeptide capable of binding to Ang-2 <400>
Leu Gin Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15Leu Gin Asp Tyr Cys Glu Gly Val Glu Asp Pro Phe Thr Phe Gly Cys 15 10 15
Glu Lys Gin Arg 20 <210> 158 <211> 20 <212>蛋白質 <213>人造的序列 -89-Glu Lys Gin Arg 20 <210> 158 <211> 20 <212> Protein <213> Artificial Sequence -89-
O:\121\121929.DOC 200808833 <220> <223〉能夠與Ang-2結合的多肽 <220> <221> misc_特徵 <222> (1)··(1) % <223>Xaa缺少或是中性疏水性、中性極性或鹼性的胺基酸殘基 <220> <221>misc_ 特徵 <222> (2, 4, 14)..(19) <223> Xaa中性疏水性或中性極性的胺基酸殘基。 <220> <221> misc_特徵 <222> (3, 6)..(17) <223> Xaa為酸性的胺基酸殘基。 <220> <221> misc j争徵 <222> (8)..(8) <223〉Xaa為中性疏水性的胺基酸殘基。 <220> <221> misc__特徵 <222> (9)..(9) <223> Xaa為 E，D，或 Q。 O:\121\121929.DOC -90- 200808833 <220> <221> misc_特徵 <222> (18)..(18) <223> Xaa為中性疏水性、中性極性或鹼性的胺基酸殘基。 <220> <221> misc」争徵 <222> (20)..(20) <223> Xaa缺少或是胺基酸殘基。 <400> 158O:\121\121929.DOC 200808833 <220><223> polypeptide capable of binding to Ang-2 <220><221> misc_feature <222> (1)·(1) % < ;223> Xaa lacks or a neutral hydrophobic, neutral polar or basic amino acid residue <220><221>misc_ feature <222> (2, 4, 14).. (19) <223> Xaa neutral hydrophobic or neutral polar amino acid residue. <220><221> misc_characteristic <222> (3, 6).. (17) <223> Xaa is an acidic amino acid residue. <220><221> misc j contending <222> (8).. (8) <223> Xaa is a neutral hydrophobic amino acid residue. <220><221> misc__Features <222> (9)..(9) <223> Xaa is E, D, or Q. O:\121\121929.DOC -90- 200808833 <220><221>misc_features<222> (18)..(18) <223> Xaa is neutral hydrophobic, neutral polarity or A basic amino acid residue. <220><221> misc" contending <222> (20)..(20) <223> Xaa is absent or an amino acid residue. <400> 158
Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Xaa Asp Pro Phe Thr Xaa Gly Cys 15 10 15Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Xaa Asp Pro Phe Thr Xaa Gly Cys 15 10 15
Xaa Xaa Xaa Xaa 20 <210> 159 <211> 60 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA <400> 159 ccgatccgtc aggaagaatg cgactgggac ccgtggacct gcgaacacat gtgggaagtt<210> 159 ccgatccgtc aggaagaatg cgactgggac ccgtggacct gcgaacacat gtgggaagtt
<210> 160 <211> 60 <212> DNA O:\121\121929.DOC -91 · 200808833 <213>人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA •’ <400> 160 accaacatcc aggaagaatg cgaatgggac ccgtggacct gcgaccacat gccgggtaaa 60 <210> 161 <211> 60 <212> DNA <213>人造的序列 <220><210> 160 <211> 60 <212> DNA O: \121\121929.DOC -91 · 200808833 <213> Artificial sequence <220><223> Encoding can be combined with Ang-2 Peptide DNA • ' <400> 160 accaacatcc aggaagaatg cgaatgggac ccgtggacct gcgaccacat gccgggtaaa 60 <210> 161 <211> 60 <212> DNA <213> Artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 161 tggtacgaac aggacgcttg cgaatgggac ccgtggacct gcgaacacat ggctgaagtt 60 <210> 162 <211> 60 <212> DNA <213> 人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 161 tggtacgaac aggacgcttg cgaatgggac ccgtggacct gcgaacacat ggctgaagtt 60 <210> 162 <211> 60 <212> DNA <213> Artificial sequence <;220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400〉 162 aaccgtctgc aggaagtttg cgaatgggac ccgtggacct gcgaacacat ggaaaacgtt 60 <210〉 163<223> DNA encoding a peptide capable of binding to Ang-2 <400> 162 aaccgtctgc aggaagtttg cgaatgggac ccgtggacct gcgaacacat ggaaaacgtt 60 <210> 163
<211> 60 <212> DNA <213>人造的序列 -92-<211> 60 <212> DNA <213> artificial sequence -92-
O:\121\121929.DOC 200808833 <220〉O:\121\121929.DOC 200808833 <220〉
<223〉 編碼能夠與Ang-2結合之肽的DNA <_> 163 ^ gctgctaccc aggaagaatg cgaatgggac ccgtggacct gcgaacacat gccgcgttcc 60 <210> 164<223> DNA encoding a peptide capable of binding to Ang-2 <_> 163 ^ gctgctaccc aggaagaatg cgaatgggac ccgtggacct gcgaacacat gccgcgttcc 60 <210>
<211> 60 <212〉 DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 164 ctgcgtcacc aggaaggttg cgaatgggac ccgtggacct gcgaacacat gttcgactgg 60 <210> 165 <211> 60 <212> DNA <213>人造的序列 <220〉<223> DNA encoding a peptide capable of binding to Ang-2 <400> 164 ctgcgtcacc aggaaggttg cgaatgggac ccgtggacct gcgaacacat gttcgactgg 60 <210> 165 <211> 60 <212> DNA <213> artificial sequence <;220〉
<223>編碼能夠與Ang-2結合之肽的DNA <400> 165 gttccgcgtc agaaagactg cgaatgggac ccgtggacct gcgaacacat gtacgttggt 60 <210> 166 <211> 60 <212> DNA <213> 人造的序列 -93-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 165 gttccgcgtc agaaagactg cgaatgggac ccgtggacct gcgaacacat gtacgttggt 60 <210> 166 <211> 60 <212> DNA <213> 93-
O:\121\121929.DOC 200808833 <220>O:\121\121929.DOC 200808833 <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 166 tgggctgctc aggaagaa.tg cgaatgggat ccgtggactt gcgaacacat gggtcgtatg 60 <210> 167 ’<223> DNA encoding a peptide capable of binding to Ang-2 <400> 166 tgggctgctc aggaagaa.tg cgaatgggat ccgtggactt gcgaacacat gggtcgtatg 60 <210> 167 ’
<211> 60 <212> DNA <213> . 人造的序列 <220> <223〉 編碼能夠與Ang-2結合之肽的DNA ! <400> 167 acttggccgc aggacaaatg cgaatgggat ccgtggactt gcgaacacat gggttctact· 60 <210> 168 <211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213>. Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 ! <400> 167 acttggccgc aggacaaatg cgaatgggat ccgtggactt gcgaacacat gggttctact· 60 <210> 168 <211> 60 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 168 ggtcactccc aggaagaatg cggttgggac ccgtggacct gcgaacacat gggtacgtcc 60 <210〉 169 <211> 60 <212> DNA <213> 人造的序列 <220> -94-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 168 ggtcactccc aggaagaatg cggttgggac ccgtggacct gcgaacacat gggtacgtcc 60 <210> 169 <211> 60 <212> DNA <213> Artificial sequence<213>;220> -94-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 169 cagcactggc aggaagaatg cgaatgggac ccgtggacct gcgaccacat gccgtccaaa <210> 170<223> DNA encoding a peptide capable of binding to Ang-2 <400> 169 cagcactggc aggaagaatg cgaatgggac ccgtggacct gcgaccacat gccgtccaaa <210> 170
<211> 60 <212> DNA <213> 人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 170 aacgttcgtc aggaaaaatg cgaatgggac ccgtggacct gcgaacacat gccggttcgt <210> 171<223> A DNA encoding a peptide capable of binding to Ang-2 <400> 170 aacgttcgtc aggaaaaatg cgaatgggac ccgtggacct gcgaacacat gccggttcgt <210>
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400〉 171 aaatccggtc aggttgaatg caactgggac ccgtggacct gcgaacacat gccgcgtaac <210> 172 <211> 60<223> DNA encoding a peptide capable of binding to Ang-2 <400> 171 aaatccggtc aggttgaatg caactgggac ccgtggacct gcgaacacat gccgcgtaac <210> 172 <211> 60
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA -95-<223> DNA-95- encoding a peptide capable of binding to Ang-2
O:\121\121929.DOC 200808833 <400> 172 gttaaaaccc aggaacactg cgactgggac ccgtggacct gcgaacacat gcgtgaatgg 60 <210〉 173 <211〉 60 - <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 173 gcttggggtc aggaaggttg cgactgggac ccgtggacct gcgaacacat gctgccgatg 60 <210> 174O:\121\121929.DOC 200808833 <400> 172 gttaaaaccc aggaacactg cgactgggac ccgtggacct gcgaacacat gcgtgaatgg 60 <210> 173 <211> 60 - <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 173 gcttggggtc aggaaggttg cgactgggac ccgtggacct gcgaacacat gctgccgatg 60 <210>
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 174 ccggttaacc aggaagactg cgaatgggac ccgtggacct gcgaacacat gccgccgatg 60 <210〉 175 <211〉 60 <212〉 DNA <213> 人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 174 ccggttaacc aggaagactg cgaatgggac ccgtggacct gcgaacacat gccgccgatg 60 <210> 175 <211> 60 <212> DNA <213> Artificial sequence<213>;220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 175 cgtgctccgc aggaagactg cgaatgggac ccgtggacct gcgctcacat ggacatcaaa 60 -96-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 175 cgtgctccgc aggaagactg cgaatgggac ccgtggacct gcgctcacat ggacatcaaa 60 -96-
O:\121\121929.DOC 200808833 <210> 176O:\121\121929.DOC 200808833 <210> 176
<211> 60 <212> DNA <213>人造的序列 、 <220><211> 60 <212> DNA <213> artificial sequence, <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 176 cacggtcaga acatggaatg cgaatgggac ccgtggacct gcgaacacat gttccgttac 60 <210〉 177 <211> 60<223> DNA encoding a peptide capable of binding to Ang-2 <400> 176 cacggtcaga acatggaatg cgaatgggac ccgtggacct gcgaacacat gttccgttac 60 <210> 177 <211> 60
<212> DNA <213>人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 177 ccgcgtctgc aggaagaatg cgtttgggac ccgtggacct gcgaacacat gccgctgcgt 60 <210> 178 <211> 60 <212> DNA <213> 人造的序列 <220〉<223> DNA encoding a peptide capable of binding to Ang-2 <400> 177 ccgcgtctgc aggaagaatg cgtttgggac ccgtggacct gcgaacacat gccgctgcgt 60 <210> 178 <211> 60 <212> DNA <213>;220〉
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 178 cgtaccaccc aggaaaaatg cgaatgggac ccgtggacct gcgaacacat ggaatcccag 60 -97-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 178 cgtaccaccc aggaaaaatg cgaatgggac ccgtggacct gcgaacacat ggaatcccag 60 -97-
O:\121\121929.DOC 200808833 <210> 179O:\121\121929.DOC 200808833 <210> 179
<211> 60 <212> DNA <213> 人造的序列 β <220><211> 60 <212> DNA <213> artificial sequence β <220>
<223>編碼能夠與Ang-2結合之肽的DN A <400> 179 cagacctccc aggaagactg cgtttgggac ccgtggacct gcgaccacat ggtttcctcc 60 <210> 180 <211> 60 <212> DNA <213>人造的序列 <220><223> DN A <400> 179 cagacctccc aggaagactg cgtttgggac ccgtggacct gcgaccacat ggtttcctcc 60 <210> 180 <211> 60 <212> DNA <213> artificial sequence encoding peptide capable of binding to Ang-2 <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 180 caggttatcg gtcgtccgtg cgaatgggac ccgtggacct gcgaacacct ggaaggtctg 60 <210〉 181 <211〉 60 <212> DNA <213>人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 180 caggttatcg gtcgtccgtg cgaatgggac ccgtggacct gcgaacacct ggaaggtctg 60 <210> 181 <211> 60 <212> DNA <213> artificial sequence<213>;220>
<223> •編碼能夠與Ang-2結合之肽的DNA <400> 181 tgggctcagc aggaagaatg cgcttgggac ccgtggacct gcgaccacat ggttggtctg 60 <210〉 182 -98-<223> • DNA encoding a peptide capable of binding to Ang-2 <400> 181 tgggctcagc aggaagaatg cgcttgggac ccgtggacct gcgaccacat ggttggtctg 60 <210> 182 -98-
O:\121\121929.DOC 200808833 <211> 60O:\121\121929.DOC 200808833 <211> 60
<212> DNA <213>人造的序列<212> DNA <213> artificial sequence
<220> ' <223>編碼能結合之肽的DNA <400〉 182 ctgccgggtc aggaagactg cgaatgggac ccgtggacct gcgaacacat ggttcgttcc <210> 183<220> ' <223> DNA encoding a peptide capable of binding <400> 182 ctgccgggtc aggaagactg cgaatgggac ccgtggacct gcgaacacat ggttcgttcc <210> 183
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 183 ccgatgaacc aggttgaatg cgactgggac ccgtggacct gcgaacacat gccgcgttcc <210> 184 <211> 60<223> DNA encoding a peptide capable of binding to Ang-2 <400> 183 ccgatgaacc aggttgaatg cgactgggac ccgtggacct gcgaacacat gccgcgttcc <210> 184 <211> 60
<212> DNA <213>人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能今與Ang-2結合之肽的DNA <400> 184 ttcggttggt ctcacggttg cgaatgggat ccgtggactt gcgaacacat gggttctacc <210> 185 <211> 60<223> DNA encoding a peptide capable of binding to Ang-2 today <400> 184 ttcggttggt ctcacggttg cgaatgggat ccgtggactt gcgaacacat gggttctacc <210> 185 <211> 60
O:\121\121929.DOC -99- 200808833O:\121\121929.DOC -99- 200808833
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 185 aaatccaccc aggacgactg cgactgggac ccgtggacct gcgaacacat ggttggtccg 60<223> DNA encoding a peptide capable of binding to Ang-2 <400> 185 aaatccaccc aggacgactg cgactgggac ccgtggacct gcgaacacat ggttggtccg 60
<210> 186 <211〉 60 <212> DNA <213> 人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA _________ <_> 186 ggtccgcgta tctccacctg ccagtgggac ccgtggacct gcgaacacat ggaccagctg 60 <210〉 187<210> 186 <211> 60 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 _________ <_> 186 ggtccgcgta tctccacctg ccagtgggac Ccgtggacct gcgaacacat ggaccagctg 60 <210〉 187
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能苎声Ang-2結合之肤的DNA <400> 187 ' ------ · tccaccatcg gtgacatgtg cgaatgggac ccgtggacct gcgctcacat gcaggttgac 60<223> DNA encoding a skin capable of binding Ang-2 binding <400> 187 ' ------ · tccaccatcg gtgacatgtg cgaatgggac ccgtggacct gcgctcacat gcaggttgac 60
<210> 188 <211> 60 <212> DNA 100-<210> 188 <211> 60 <212> DNA 100-
O:\121\121929.DOC 200808833 <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的________ ， <400> 188 gttctgggtg gtcagggttg cgaatgggac ccgtggacct gccgtctgct gcagggttgg <210> 189O:\121\121929.DOC 200808833 <213> artificial sequence <220><223> coding ________ of the peptide capable of binding to Ang-2, <400> 188 gttctgggtg gtcagggttg cgaatgggac ccgtggacct gccgtctgct gcagggttgg <210> 189
<211> 60 <212> DNA <213> 人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 189 gttctgggtg gtcagggttg ccagtgggac ccgtggacct gctcccacct ggaagacggt <210> 190 <211> 60<223> A DNA encoding a peptide capable of binding to Ang-2 <400> 189 gttctgggtg gtcagggttg ccagtgggac ccgtggacct gctcccacct ggaagacggt <210> 190 <211> 60
<212> DNA <213>人造的序列 <220> <223> 編碼能夠與Ang_2結合之肽的DNA — <400> 190 accaccatcg gttccatgtg cgaatgggac ccgtggacct gcgctcacat gcagggtggt <210> 191 <211> 60 <212> DNA <213> 人造的序列 -101 -<212> DNA <213> artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang_2 - <400> 190 accaccatcg gttccatgtg cgaatgggac ccgtggacct gcgctcacat gcagggtggt <210> 191 <211> 60 <212> DNA <213> Artificial Sequence -101 -
O:\121\121929.DOC 200808833 <220>O:\121\121929.DOC 200808833 <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 191 ^ accaaaggta aatccgtttg ccagtgggac ccgtggacct gctcccacat gcagtccggt 60 <210> 192<223> DNA encoding a peptide capable of binding to Ang-2 <400> 191 ^ accaaaggta aatccgtttg ccagtgggac ccgtggacct gctcccacat gcagtccggt 60 <210> 192
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 192 "" accaccatcg gttccatgtg ccagtgggac ccgtggacct gcgctcacat gcagggtggt 60 <210〉 193 <211> 60 <212> DNA <213> 人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 192 "&acc; accaccatcg gttccatgtg ccagtgggac ccgtggacct gcgctcacat gcagggtggt 60 <210> 193 <211> 60 <212> DNA <213> Artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 193 tgggttaacg aagttgtttg cgaatgggac ccgtggacct gcaaccactg ggacaccccg 60 <210> 194 <211> 60 <212> DNA <213> 人造的序列 -102-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 193 tgggttaacg aagttgtttg cgaatgggac ccgtggacct gcaaccactg ggacaccccg 60 <210> 194 <211> 60 <212> DNA <213> 102-
O:\121\121929.DOC 200808833 <220>O:\121\121929.DOC 200808833 <220>
<223〉 編碼能夠與Ang-2結合之肽的DNA <400> 194 gttgttcagg ttggtatgtg ccagtgggac ccgtggacct gcaaacacat gcgtctgcag <210> 195 '<223> A DNA encoding a peptide capable of binding to Ang-2 <400> 194 gttgttcagg ttggtatgtg ccagtgggac ccgtggacct gcaaacacat gcgtctgcag <210> 195 '
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400〉195 — ’ — gctgttggtt cccagacctg cgaatgggac ccgtggacct gcgctcacct ggttgaagtt <210〉 196<223> DNA encoding a peptide capable of binding to Ang-2 <400>195 — ’ — gctgttggtt cccagacctg cgaatgggac ccgtggacct gcgctcacct ggttgaagtt <210> 196
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223〉 編碼能夠與Ang-2結合之肽的DNA <400> 196 cagggtatga aaatgttctg cgaatgggac ccgtggacct gcgctcacat cgtttaccgt <210> 197<223> A DNA encoding a peptide capable of binding to Ang-2 <400> 196 cagggtatga aaatgttctg cgaatgggac ccgtggacct gcgctcacat cgtttaccgt <210>
<211> 60 <212> DNA <n3>人造的序列 <220〉 · -103-<211> 60 <212> DNA <n3> artificial sequence <220> · -103-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 197 accaccatcg gttccatgtg ccagtgggac ccgtggacct gcgaacacat gcagggtggt 60 <210> 198<223> DNA <400> 197 accaccatcg gttccatgtg ccagtgggac ccgtggacct gcgaacacat gcagggtggt 60 <210> 198 encoding a peptide capable of binding to Ang-2
<211〉 60 <212> DNA <213> 人造的序列 <220〉 <223> 編碼能夠與Ang-2結合之肽的DNj____ <400> 198 acctcccagc gtgttggttg cgaatgggac ccgtggacct gccagcacct gacctacacc 60 <210> 199<211> 60 <212> DNA <213> Artificial sequence <220><223> DNj____ <400> 198 acctcccagc gtgttggttg cgaatgggac ccgtggacct gccagcacct gacctacacc 60 <210> 199
<211> 60 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA ..... <400> 199 cagtggtcct ggccgccgtg cgaatgggac ccgtggacct gccagaccgt ttggccgtcc 60<211> 60 <212> DNA <213> artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 ..... <400> 199 cagtggtcct ggccgccgtg cgaatgggac ccgtggacct Gccagaccgt ttggccgtcc 60
<210〉 200 <211> 60 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA___________ •104-<210> 200 <211> 60 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2___________ • 104-
O:\121\121929.DOC 200808833 <400〉 200 ggtacctccc cgtccttctg ccagtgggac ccgtggacct gctcccacat ggttcagggt 60 <210> 201 <211> 42 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 201 caggaagaat gcgaatggga cccatggact tgcgaacaca tg 42 <210> 202 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA ______ <400> 202 cagaactaca aaccgctgga cgaactggac gctaccctgt acgaacactt catcttccac 60 tacacc 66O:\121\121929.DOC 200808833 <400> 200 ggtacctccc cgtccttctg ccagtgggac ccgtggacct gctcccacat ggttcagggt 60 <210> 201 <211> 42 <212> DNA <213> Artificial sequence <220><223> A DNA encoding a peptide capable of binding to Ang-2 <400> 201 caggaagaat gcgaatggga cccatggact tgcgaacaca tg 42 <210> 202 <211> 66 <212> DNA <213> artificial sequence <220>;223> DNA encoding a peptide capable of binding to Ang-2 ______ <400> 202 cagaactaca aaccgctgga cgaactggac gctaccctgt acgaacactt catcttccac 60 tacacc 66
<210> 203 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA O:\121\121929.DOC -105- 60 200808833 <400〉 203 ctgaacttca ccccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag cagtcc <210> 204 <211> 66 4 <212> DNA <213> 人造的序列 <220> <223〉 編碼能夠與Ang-2結合之肽的DNA <400〉 204 accaaattca acccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag caccag <210> 205 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 205 gttaaattca aaccgctgga cgctctggaa cagaccctgt acgaacactg gatgttccag caggct <210> 206 <211〉 66 <212> DNA <213> 人造的序列 66 60 66 60 66<210> 203 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 O: \121\121929.DOC - 105- 60 200808833 <400> 203 ctgaacttca ccccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag cagtcc <210> 204 <211> 66 4 <212> DNA <213> Artificial sequence <220><223> DNA of Ang-2-bound peptide <400>204 accaaattca acccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag caccag <210> 205 <211> 66 <212> DNA <213> artificial sequence <220><223> DNA <400> 205 gttaaattca aaccgctgga cgctctggaa cagaccctgt acgaacactg gatgttccag caggct <210> 206 <211> 66 <212> DNA <213> artificial sequence 66 60 66 60 66
O:\121\121929DOC -106- 200808833 <220>O:\121\121929DOC -106- 200808833 <220>
<223>編碼能夠與Ang-2結合之肽的DNA<223> DNA encoding a peptide capable of binding to Ang-2
<400> 206 gttaaataca aaccgctgga cgaactggac gaaatcctgt acgaacagca gaccttccag 60 gaacgt * * 66 <210> 207 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 207 accaacttca tgccgatgga cgacctggaa cagcgtctgt acgaacagtt catcctgcag . 60 cagggt 66 <210> 208 <211> 66 <212> DNA <213〉 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 208 tccaaattca aaccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag 60 cacgct 66 <210> 209 <211> 66 <212> DNA O:\121\121929.DOC -107- 200808833 <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA ^ <400> 209 ' 60 66 cagaaattcc agccgctgga cgaactggaa cagaccctgt acgaacagtt catgctgcag caggct <210> 210 <211> 66 <212> DNA <213>人造的序列 <220><400> 206 gttaaataca aaccgctgga cgaactggac gaaatcctgt acgaacagca gaccttccag 60 gaacgt * * 66 <210> 207 <211> 66 <212> DNA <213> Artificial sequence <220><223> Encoding can be combined with Ang -2 binding peptide DNA <400> 207 accaacttca tgccgatgga cgacctggaa cagcgtctgt acgaacagtt catcctgcag. 60 cagggt 66 <210> 208 <211> 66 <212> DNA <213> artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 208 tccaaattca aaccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag 60 cacgct 66 <210> 209 <211> 66 <212> DNA O:\121\121929.DOC -107- 200808833 <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 ^<400> 209 '60 66 cagaaattcc agccgctgga cgaactggaa cagaccctgt acgaacagtt catgctgcag caggct <210> 210 <211> 66 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA ........ * <400> 210 60 66 cagaacttca aaccgatgga cgaattggaa gacaccctgt acaaacagtt cctgttccag cactcc <210> 211 <211> 66 <212> DNA <213〉 人造的序列 <220〉 <223> 編碼能夠與Ang-2結合之肽的DNA <400> 211 tacaaattca ccccgctgga cgacctggaa cagaccctgt acgaacagtg gaccctgcag cacgtt 60 <210 212 -108-<223> DNA encoding a peptide capable of binding to Ang-2........ * <400> 210 60 66 cagaacttca aaccgatgga cgaattggaa gacaccctgt acaaacagtt cctgttccag cactcc <210> 211 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 <400> 211 tacaaattca ccccgctgga cgacctggaa cagaccctgt acgaacagtg gaccctgcag cacgtt 60 <210 212 -108-
O:\121\121929.DOC 66 200808833 <211> 65 <212〉 DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 212 aggaatacga accgctggac gaactggacg aaaccctgta caaccagtgg atgttccacc 60 agcgt 65 <210> 213 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA _______... <400> 213 tccaacttca tgccgctgga cgaactggaa cagaccctgt acgaacagtt catgctgcag 60 caccag 66 <210〉 214 <211> 66 <212〉 DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 214 cagaaatacc agccgctgga cgaactggac aaaaccctgt acgatcagtt catgctgcag 60 cagggt 66 -109-O:\121\121929.DOC 66 200808833 <211> 65 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 212 aggaatacga accgctggac gaactggacg aaaccctgta caaccagtgg atgttccacc 60 agcgt 65 <210> 213 <211> 66 <212> DNA <213> Artificial sequence <220><223> Encoding a peptide capable of binding to Ang-2 DNA _______... <400> 213 tccaacttca tgccgctgga cgaactggaa cagaccctgt acgaacagtt catgctgcag 60 caccag 66 <210> 214 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA of a peptide capable of binding to Ang-2 <400> 214 cagaaatacc agccgctgga cgaactggac aaaaccctgt acgatcagtt catgctgcag 60 cagggt 66 -109-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<210> 215 <211〉 66 <212> DNA <213>人造的序列 、 <220> <223〉 編碼能夠與Ang-2結合之肽的DNA <400> 215 cagaaattcc agccgctgga cgaactggaa gaaaccctgt acaaacagtg gaccctgcag cagcgt <210> 216 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400〉 216 gttaaataca aaccgctgga cgaactggac gaatggctgt accaccagtt caccctgcac caccag <210〉 217 <211> 67 <212> DNA <213> 人造的序列 <220〉 <223> 編碼能夠與Ang-2結合之肽的DNA 60 66 60 66<210> 215 <211> 66 <212> DNA <213> artificial sequence, <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 215 cagaaattcc agccgctgga cgaactggaa Gaaaccctgt acaaacagtg gaccctgcag cagcgt <210> 216 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 <400> 216 Gttaaataca aaccgctgga cgaactggac gaatggctgt accaccagtt caccctgcac caccag <210> 217 <211> 67 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 60 66 60 66
O:\121\121929.DOC -110- 60200808833 <400> 217 cagaaattca tgccgctgga cgaactggac gaaatcctgt acgaacagtt catgttccag cagtccc <210> 218 <211> 66 <212> DNA <213> 人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA <400〉 218 cagaccttcc agccgctgga cgacctggaa gaatacttgt acgaacagtg gatccgtcgt taccac <210> 219 <211> 66 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肽的DNA <400〉 219 gaagactaca tgccgctgga cgctctggac gctcagctgt acgaacagtt catcctgctg 67 60 66 cacggt <210> 220 <211> 66 <212> DNA <213> 人造的序列 60 66O:\121\121929.DOC -110- 60200808833 <400> 217 cagaaattca tgccgctgga cgaactggac gaaatcctgt acgaacagtt catgttccag cagtccc <210> 218 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 218 cagaccttcc agccgctgga cgacctggaa gaatacttgt acgaacagtg gatccgtcgt taccac <210> 219 <211> 66 <212> DNA <213> artificial sequence<213>;220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 219 gaagactaca tgccgctgga cgctctggac gctcagctgt acgaacagtt catcctgctg 67 60 66 cacggt <210> 220 <211> 66 <212> DNA <213> Artificial sequence 60 66
O:\121\121929.DOC -Ill - 200808833 <220>O:\121\121929.DOC -Ill - 200808833 <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400〉 220 cacaccttcc agccgctgga cgaactggaa gaaaccctgt actaccagtg gctgtacgac 60<223> DNA encoding a peptide capable of binding to Ang-2 <400> 220 cacaccttcc agccgctgga cgaactggaa gaaaccctgt actaccagtg gctgtacgac 60
cagctg 66 <210> 221 <211> 66 <212> DNA <213> 人造的序列 <220〉 <223> 編碼能夠與Ang-2結合之肽的DNA <400> 221 tacaaattca acccgatgga cgaactggaa cagaccctgt acgaagaatt cctgttccag 60 cacgct ββ <210> 222 <211> 66 <212〉 DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 222 accaactaca aaccgctgga cgaactggac gctaccctgt acgaacactg gatcctgcag 60 cactcc 66 <210> 223 <211〉 66 <212> DNA -112-Cagctg 66 <210> 221 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 <400> 221 tacaaattca acccgatgga Cgaactggaa cagaccctgt acgaagaatt cctgttccag 60 cacgct ββ <210> 222 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 222 accaactaca aaccgctgga cgaactggac gctaccctgt acgaacactg gatcctgcag 60 cactcc 66 <210> 223 <211> 66 <212> DNA -112-
O:\121\121929.DOC 200808833 .<213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA 、 <400> 223 cagaaattca aaccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag cagcgt <210> 224 <211> 66 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA <400> 224 accaaattcc agccgctgga cgaactggac cagaccctgt acgaacagtg gaccctgcag cagcgt <210> 225 <211〉 66 <212> DNA <213>人造的序列 <220> <223>編碼能夠與Ang-2結合之肤的DNA <400> 225 accaacttcc agccgctgga cgaactggac cagaccctgt acgaacagtg gaccctgcag cagcgt <210> 226 60 66 60 66 60O:\121\121929.DOC 200808833 .<213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2, <400> 223 cagaaattca aaccgctgga cgaactggaa cagaccctgt acgaacagtg gaccctgcag cagcgt <;210> 224 <211> 66 <212> DNA <213> Artificial sequence <220><223> DNA encoding peptide capable of binding to Ang-2 <400> 224 accaaattcc agccgctgga cgaactggac cagaccctgt acgaacagtg Gaccctgcag cagcgt <210> 225 <211> 66 <212> DNA <213> artificial sequence <220><223> DNA encoding <400> 225 accaacttcc agccgctgga capable of binding to Ang-2 Cgaactggac cagaccctgt acgaacagtg gaccctgcag cagcgt <210> 226 60 66 60 66 60
O:\121\121929.DOC -113- 66 編碼能夠與Ang-2結合之肽的DNA <400> 226 aaattcaacc cgctggacga gctggaagag actctgtacg aacagtttac <210> 227 <211> 60 <212> DNA <213> 人造的序列 <220> <223〉 編碼能夠與Ang-2結合之肽的DNA <400> 227 gctggtggta tgcgtccgta cgacggtatg ctgggttggc cgaactacga <210〉 228 <211〉 60 <212> DNA <213> 人造的序列 <220> <223> 編碼能夠與Ang-2結合之肽的DNA ---- 一 <400> 228 cagacttggg acgatccgtg catgcacatt ctgggtccgg ttacttggcg <210> 229 <211> 60 200808833 <211> 60O:\121\121929.DOC-113-66 DNA encoding a peptide capable of binding to Ang-2 <400> 226 aaattcaacc cgctggacga gctggaagag actctgtacg aacagtttac <210> 227 <211> 60 <212> DNA <213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 227 gctggtggta tgcgtccgta cgacggtatg ctgggttggc cgaactacga <210> 228 <211> 60 <212> DNA <;213> Artificial sequence <220><223> DNA encoding a peptide capable of binding to Ang-2 ---- a <400> 228 cagacttggg acgatccgtg catgcacatt ctgggtccgg ttacttggcg <210> 229 <211> 60 200808833 <211> 60
<212> DNA <213>人造的序列 <220> <223> 60 60 60<212> DNA <213> artificial sequence <220><223> 60 60 60
O:\121\121929.DOC 114- 200808833O:\121\121929.DOC 114- 200808833
<212> DNA <213> 人造的序列 <220><212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 229 gctccgggtc agcgtccgta cgacggtatg ctgggttggc cgacctacca gcgtatcgtt <210〉 230 <211> 60 <212> DNA <213>人造的序列 <220><223> A DNA encoding a peptide capable of binding to Ang-2 <400> 229 gctccgggtc agcgtccgta cgacggtatg ctgggttggc cgacctacca gcgtatcgtt <210> 230 <211> 60 <212> DNA <213> artificial sequence<220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 230 tccggtcagc tgcgtccgtg cgaagaaatc ttcggttgcg gtacccagaa cctggctctg i <210> 231<223> DNA <400> 230 tccggtcagc tgcgtccgtg cgaagaaatc ttcggttgcg gtacccagaa cctggctctg i <210> 231 encoding a peptide capable of binding to Ang-2
<211> 60 <212> DNA <213> 人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223>編碼能夠與Ang-2結合之肽的DNA <400〉 231 ttcggtgaca aacgtccgct ggaatgcatg ttcggtggtc cgatccagct gtgcccgcgt<223> DNA encoding a peptide capable of binding to Ang-2 <400> 231 ttcggtgaca aacgtccgct ggaatgcatg ttcggtggtc cgatccagct gtgcccgcgt
<210> 232 <211〉 60 <212〉 DNA 115-<210> 232 <211> 60 <212> DNA 115-
O:\121\121929.DOC 200808833 <213> 人造的序列 <220>O:\121\121929.DOC 200808833 <213> Artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 232 ggtcaggacc tgcgtccgtg cgaagacatg ttcggttgcg gtaccaaaga ctggtacggt 60 <210> 233<223> DNA encoding a peptide capable of binding to Ang-2 <400> 232 ggtcaggacc tgcgtccgtg cgaagacatg ttcggttgcg gtaccaaaga ctggtacggt 60 <210> 233
<211〉 60 <212> DNA <213> 人造的序列 <220><211> 60 <212> DNA <213> Artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 233 ggtttcgaat actgcgacgg tatggaagac ccgttcacct tcggttgcga caaacagacc 60 <210> 234 ^<223> DNA encoding a peptide capable of binding to Ang-2 <400> 233 ggtttcgaat actgcgacgg tatggaagac ccgttcacct tcggttgcga caaacagacc 60 <210> 234 ^
<211> 60 <212> DNA <213>人造的序列 <220><211> 60 <212> DNA <213> artificial sequence <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 234 aaactggaat actgcgacgg tatggaagac ccgttcaccc agggttgcga caaccagtcc 60 <210> 235<223> DNA <400> 234 aaactggaat actgcgacgg tatggaagac ccgttcaccc agggttgcga caaccagtcc 60 <210> 235 encoding a peptide capable of binding to Ang-2
<211> 60 <212> DNA <213>人造的序列 -116-<211> 60 <212> DNA <213> artificial sequence -116-
O:\121\121929.DOC 200808833 <220>O:\121\121929.DOC 200808833 <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 235 ctgcaggaat ggtgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaacagcgt <210> 236 <211> 60 <212> DNA <213>人造的序列 <220><223> A DNA encoding a peptide capable of binding to Ang-2 <400> 235 ctgcaggaat ggtgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaacagcgt <210> 236 <211> 60 <212> DNA <213> artificial sequence<220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 236 gctcaggact actgcgaagg tatggaagac ccgttcacct tcggttgcga aatgcagaaa <210〉 237 <211> 60 <212> DNA <213>人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 236 gctcaggact actgcgaagg tatggaagac ccgttcacct tcggttgcga aatgcagaaa <210> 237 <211> 60 <212> DNA <213> artificial sequence<220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 237 ctgctggact actgcgaagg tgttcaggac ccgttcacct tcggttgcga aaacctggac <210> 238 <211> 60 <212> DNA <213> 人造的序列 -117-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 237 ctgctggact actgcgaagg tgttcaggac ccgttcacct tcggttgcga aaacctggac <210> 238 <211> 60 <212> DNA <213> Artificial sequence-117 -
0:\121\121929.D0C 200808833 <220>0:\121\121929.D0C 200808833 <220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 238 caccaggaat actgcgaagg tatggaagac ccgttcacct tcggttgcga ataccagggt <210> 239 <211> 60 <212> DNA <213>人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 238 caccaggaat actgcgaagg tatggaagac ccgttcacct tcggttgcga ataccagggt <210> 239 <211> 60 <212> DNA <213> artificial sequence<220>
<223>編碼能夠與Ang-2結合之肽的DNA <400> 239 atgctggact actgcgaagg tatggacgac ccgttcacct tcggttgcga caaacagatg <210> 240 <211> 60 <212> DNA <213>人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 239 atgctggact actgcgaagg tatggacgac ccgttcacct tcggttgcga caaacagatg <210> 240 <211> 60 <212> DNA <213> artificial sequence<220>
<223> 編碼能夠與Ang-2結合之肽的DNA <400> 240 ctgcaggact actgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaccagcgt <210> 241 <211> 60 <212> DNA <213> 人造的序列 <220> -118-<223> DNA encoding a peptide capable of binding to Ang-2 <400> 240 ctgcaggact actgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaccagcgt <210> 241 <211> 60 <212> DNA <213> Artificial sequence<220> -118-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
<223> .編碼能夠與Ang-2結合之肤的DNA <400> 241 ctgcaggact actgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaacagcgt 60 <210> 242 <211> 54 1 <212> DNA <213>人造的序列 <220〉<223>. DNA encoding a peptide capable of binding to Ang-2 <400> 241 ctgcaggact actgcgaagg tgttgaagac ccgttcacct tcggttgcga aaaacagcgt 60 <210> 242 <211> 54 1 <212> DNA <213> Sequence <220〉
<223>編碼能夠與Ang-2結合之肽的DNA <400> 242 ttcgactact gcgaaggtgt tgaagacccg ttcactttcg gctgtgataa ccac 54 <210> 243 <211> 250 <212>蛋白質 <213>人造的序列 <220><223> DNA encoding a peptide capable of binding to Ang-2 <400> 242 ttcgactact gcgaaggtgt tgaagacccg ttcactttcg gctgtgataa ccac 54 <210> 243 <211> 250 <212> Protein <213> Artificial sequence<213>;220>
<223> 編碼能夠與Ang-2結合之肽的DNA <_> 243<223> DNA encoding a peptide capable of binding to Ang-2 <_> 243
Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15.Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15.
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30
Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 5,5 60 -119·His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 5,5 60 -119·
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 65 70 75 80Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 85 90 95Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 115 120 125Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val 130 135 140Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val 130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val 145 150 155 160Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val 145 150 155 160
Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190
Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 195 200 205Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 210 215 220Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 210 215 220
Ser Pro Gly Lys Gly Gly Gly Gly Gly Cys Thr Ala Gly Tyr His Trp 225 230 235 240Ser Pro Gly Lys Gly Gly Gly Gly Gly Cys Thr Ala Gly Tyr His Trp 225 230 235 240
Asn Ser Asp Cys Glu Cys Cys Arg Arg Asn 245 250Asn Ser Asp Cys Glu Cys Cys Arg Arg Asn 245 250
<210> 244 <211> 29 <212> DNA <213>人造的序列<210> 244 <211> 29 <212> DNA <213> artificial sequence
O:\121\121929.DOC -120- 200808833 <220> <223> 寡核甞酸 <400> 244 caaacgaatg gatcctcatt aaagccaga <210> 245 <211> 42 <212> DNA <213> 人造的序列 <220> <223> 寡核甞酸 <400> 245 ggtggtgcgg ccgcactcga gactgttgaa agttgtttag ca <210> 246 <211> 29 <212> DNA <213> 人造的序列 <220> <223> 寡核棼酸 <400> 246 caaacgaatg gatcctcatt aaagccagaO:\121\121929.DOC -120- 200808833 <220><223> Oligonucleotide <400> 244 caaacgaatg gatcctcatt aaagccaga <210> 245 <211> 42 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 245 ggtggtgcgg ccgcactcga gactgttgaa agttgtttag ca <210> 246 <211> 29 <212> DNA <213> Artificial sequence<220><223> Oligonucleotide <400> 246 caaacgaatg gatcctcatt aaagccaga
<210> 247 <211> 43 <212> DNA <213>人造的序列 121 -<210> 247 <211> 43 <212> DNA <213> artificial sequence 121 -
O:\121\121929.DOC 200808833 <220> <223> 寡核棼酸 <400> 247 aacacaaaag tgcacagggt ggaggtggtg gtgcggccgc act <210〉 248 <211> 91 <212>蛋白質 <213> 人造的序列 <220> <223>寡核甞酸 <400> 248O:\121\121929.DOC 200808833 <220><223> Oligonucleotide <400> 247 aacacaaaag tgcacagggt ggaggtggtg gtgcggccgc act <210> 248 <211> 91 <212>Protein<213> Artificial sequence <220><223>oligonucleotide<400> 248
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15
Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn 20 25 30Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn 20 25 30
Lys Asn Asn Lys Ser Ala Arg Thr Gly Gly Gly Ala Thr Cys Cys Gly 35 40 45Lys Asn Asn Lys Ser Ala Arg Thr Gly Gly Gly Ala Thr Cys Cys Gly 35 40 45
Thr Gly Gly Ala Ser Cys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn 50 55 60Thr Gly Gly Ala Ser Cys Asn Asn Lys Asn Asn Lys Asn Asn Lys Asn 50 55 60
Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80Asn Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80
Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 90 <210> 249 <211> 91 <212>蛋白質 <213>人造的序列 122-Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 90 <210> 249 <211> 91 <212> Protein <213> Artificial Sequence 122-
O:\121\121929.DOC 200808833 <22〇> <223>寡核甞酸 <400> 249O:\121\121929.DOC 200808833 <22〇><223> Oligonucleotide <400> 249
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15
Asn Lys Asn Asn Lys Asn Asn Lys Ala Ala Lys Cys Gly Lys Cys Cys 20 25 30Asn Lys Asn Asn Lys Asn Asn Lys Ala Ala Lys Cys Gly Lys Cys Cys 20 25 30
Lys Asn Asn Lys Gly Ala Lys Gly Ala Lys Ala Thr Lys Thr Thr Lys 35 40 45Lys Asn Asn Lys Gly Ala Lys Gly Ala Lys Ala Thr Lys Thr Thr Lys 35 40 45
Gly Gly Lys Gly Gly Lys Asn Asn Lys Ala Cys Lys Thr Ala Lys Cys 50 55 60Gly Gly Lys Gly Gly Lys Asn Asn Lys Ala Cys Lys Thr Ala Lys Cys 50 55 60
Ala Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 7 0 75 80Ala Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 7 0 75 80
Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 90 <210> 250 <211> 95 <212>蛋白質 <213>人造的序列 <220> <223>寡核苷酸 <400> 250Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 90 <210> 250 <211> 95 <212> Protein <213> Artificial Sequence <220><223> Oligonucleotide <400>; 250
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr. Asn 1 5 10 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr. Asn 1 5 10 15
Asn Lys Ala Ala Lys Thr Thr Lys Ala Ala Lys Cys Cys Lys Cys Thr 20 25 30 -123-Asn Lys Ala Ala Lys Thr Thr Lys Ala Ala Lys Cys Cys Lys Cys Thr 20 25 30 -123-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Lys Gly Ala Lys Gly Ala Lys Cys Thr Lys Gly Ala Lys Gly Ala Lys 35 40 45Lys Gly Ala Lys Gly Ala Lys Cys Thr Lys Gly Ala Lys Gly Ala Lys 35 40 45
Ala Cys Lys Cys Thr Lys Thr Ala Lys Gly Ala Lys Cys Ala Lys Thr 50 55 60Ala Cys Lys Cys Thr Lys Thr Ala Lys Gly Ala Lys Cys Ala Lys Thr 50 55 60
Thr Lys Ala Cys Lys Thr Thr Lys Cys Ala Lys Cys Ala Lys Asn Asn 65 70 75 80Thr Lys Ala Cys Lys Thr Thr Lys Cys Ala Lys Cys Ala Lys Asn Asn 65 70 75 80
Lys Cys Ala Thr Thr Cys Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 90 95 <210> 251 <211> 91 <212>蛋白質 <213>人造的序列 <220> <223> 寡核替酸 <400> 251Lys Cys Ala Thr Thr Cys Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 90 95 <210> 251 <211> 91 <212> Protein <213> Artificial Sequence <220><223> Acid <400> 251
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 1 5 10 . 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 1 5 10 . 15
Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Lys Gly Ala Lys Gly Ala 20 25 · 30Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Lys Gly Ala Lys Gly Ala 20 25 · 30
Lys Thr Gly Cys Gly Ala Lys Thr Gly Lys Gly Ala Lys Cys Cys Lys 35 40 45Lys Thr Gly Cys Gly Ala Lys Thr Gly Lys Gly Ala Lys Cys Cys Lys 35 40 45
Thr Gly Lys Ala Cys Lys Thr Gly Cys Gly Ala Lys Cys Ala Lys Ala 50 55 60Thr Gly Lys Ala Cys Lys Thr Gly Cys Gly Ala Lys Cys Ala Lys Ala 50 55 60
Thr Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80Thr Lys Asn Asn Lys Asn Asn Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80
Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 . 90 124·Thr Cys Thr Cys Gly Ala Gly Ala Thr Cys Ala 85 . 90 124·
O:\121\121929.DOC 200808833 <210> 252 <211> 89 <212>蛋白質 <213>人造的序列 <220〉 <223>寡核:y：酸 <400> 252O:\121\121929.DOC 200808833 <210> 252 <211> 89 <212> Protein <213> Artificial sequence <220> <223> Oligonuclear: y: acid <400>
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 1 5 10 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 1 5 10 15
Asn Lys Thr Thr Lys Gly Ala Lys Thr Ala Lys Asn Asn Lys Gly Ala 20 25 30Asn Lys Thr Thr Lys Gly Ala Lys Thr Ala Lys Asn Asn Lys Gly Ala 20 25 30
Lys Gly Gly Lys Gly Thr Lys Gly Ala Lys Gly Ala Lys Cys Cys Lys 35 40 45Lys Gly Gly Lys Gly Thr Lys Gly Ala Lys Gly Ala Lys Cys Cys Lys 35 40 45
Thr Thr Lys Ala Cys Lys Thr Thr Lys Gly Gly Lys Asn Asn Lys Gly 50 55 60Thr Thr Lys Ala Cys Lys Thr Thr Lys Gly Gly Lys Asn Asn Lys Gly 50 55 60
Ala Lys Ala Ala Lys Cys Ala Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80 .Ala Lys Ala Ala Lys Cys Ala Lys Asn Asn Lys Cys Ala Thr Thr Cys 65 70 75 80 .
Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 <210> 253 <211> 95 <212>蛋白質 <213>人造的序列 <220> <223>寡核甞酸 <400> 253 -125-Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 <210> 253 <211> 95 <212> Protein <213> Artificial Sequence <220><223> Oligonucleotide <400> 125-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15Cys Ala Cys Ala Gly Thr Gly Cys Ala Cys Ala Gly Gly Gly Thr Asn 15 10 15
Asn Lys Ala Ala Lys Thr Thr Lys Ala Ala Lys Cys Cys Lys Cys Thr 20 25 30Asn Lys Ala Ala Lys Thr Thr Lys Ala Ala Lys Cys Cys Lys Cys Thr 20 25 30
Lys Gly Ala Lys Gly Ala Lys Cys Thr Lys Gly Ala Lys Gly Ala Lys 35 40 45Lys Gly Ala Lys Gly Ala Lys Cys Thr Lys Gly Ala Lys Gly Ala Lys 35 40 45
Ala Cys Lys Cys Thr Lys Thr Ala Lys Gly Ala Lys Cys Ala Lys Thr 50 55 60Ala Cys Lys Cys Thr Lys Thr Ala Lys Gly Ala Lys Cys Ala Lys Thr 50 55 60
Thr Lys Ala Cys Lys Thr Thr Lys Cys Ala Lys Cys Ala Lys Asn Asn 65 70 75 80Thr Lys Ala Cys Lys Thr Thr Lys Cys Ala Lys Cys Ala Lys Asn Asn 65 70 75 80
Lys Cys Ala Thr Thr Cys Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 90 95 <210〉 254 <211> 15Lys Cys Ala Thr Thr Cys Thr Cys Thr Cys Gly Ala Gly Ala Thr 85 90 95 <210> 254 <211>
<212> DNA <213>人造的序列 <220> <223> 寡核昝酸 <400> 254 cacagtgcac agggt <210> 255<212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 254 cacagtgcac agggt <210>
<211> 16 <212〉 DNA <213>人造的序列 <220> <223> 寡核甞酸 126-<211> 16 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide 126-
O:\121\121929.DOC 200808833 <400> 255 tgatctcgag agaatg 16 .<210> 256 <211> 21 ·. <212> DNA f <213> 人造的序列 <220> <223> 寡核甞酸 <400> 256 gttagctcac tcattaggca c 21 <210> 257 <211> 21 <212> DNA <213> 人造的序列 <220> <223> 寡核钵酸 <400> 257 gtaccgtaac actgagtttc g 21 <210> 258 <211> 18 <212> DNA <213> 人造的序列 <220> <223> 寡核荅酸 <400> 258 ttacacttta tgcttccg 18 O:\121\121929.DOC - 127- 200808833 <210> 259 <211> 31 <212>蛋白質 <213>人造的序列 ' <220〉 <223>能夠與Ang-2結合的肽體 <220〉 <221> misc—特徵 <222> (2) · (2) <223〉 X33· = P c <400〉 259O:\121\121929.DOC 200808833 <400> 255 tgatctcgag agaatg 16 .<210> 256 <211> 21 ·. <212> DNA f <213> Artificial sequence <220><223> Oligonucleotide <400> 256 gttagctcac tcattaggca c 21 <210> 257 <211> 21 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 257 gtaccgtaac actgagtttc g 21 <210> 258 <211> 18 <212> DNA <213> Artificial sequence <220><223> Oligonucleotide <400> 258 ttacacttta tgcttccg 18 O :\121\121929.DOC - 127- 200808833 <210> 259 <211> 31 <212> Protein <213> Artificial sequence ' <220> <223> A peptide capable of binding to Ang-2 Body <220> <221> misc - feature <222> (2) · (2) <223> X33· = P c <400> 259
Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro lie Arg Gin Glu Glu Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro lie Arg Gin Glu Glu Cys 1 5 10 15
Asp Trp Asp Pro Trp Thr Cys Glu His Met Trp Glu Val Leu Glu 20 25 30 <210> 260 <211〉 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) -128-Asp Trp Asp Pro Trp Thr Cys Glu His Met Trp Glu Val Leu Glu 20 25 30 <210> 260 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_feature <222> (2)..(2) -128-
O:\121\121929.DOC 200808833 <223> Xaa = Fc <400〉 260O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 260
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Asn lie Gin Glu Glu Cys 1 t 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Asn lie Gin Glu Glu Cys 1 t 5 10 15
Glu Trp Asp Pro Trp Thr Cys Asp His Met Pro Gly Lys Leu Glu 20 25 30 <210> 261 <211〉 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 261Glu Trp Asp Pro Trp Thr Cys Asp His Met Pro Gly Lys Leu Glu 20 25 30 <210> 261 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) . . . (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Tyr Glu Gin Asp Ala Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Tyr Glu Gin Asp Ala Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Ala Glu Val Leu Glu 20 25 30 <210> 262 <211> 31 <212> 蛋白質 <213> 人造的序列 O:\121\121929.DOC • 129- 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <221〉 misc _特徵 <222> (2) · · (2) <223> Xaa = Fc <400> 262Glu Trp Asp Pro Trp Thr Cys Glu His Met Ala Glu Val Leu Glu 20 25 30 <210> 262 <211> 31 <212> Protein <213> Artificial sequence O:\121\121929.DOC • 129 - 200808833 <220><223> Peptide capable of binding to Ang-2 <220><221> misc _ feature <222> (2) · · (2) <223> Xaa = Fc <;400> 262
Met Xaa Gly Gly Gly Gly Gly Ala Gin Asn Arg Leu Gin Glu Val Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Asn Arg Leu Gin Glu Val Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Glu Asn Val Leu Glu 20 25 30 <210〉 263 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222〉 （2)..(2) <223> Xaa = Fc <400> 263Glu Trp Asp Pro Trp Thr Cys Glu His Met Glu Asn Val Leu Glu 20 25 30 <210> 263 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Ala Thr Gin Glu Glu Cys 1 5 10 15 •130-Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Ala Thr Gin Glu Glu Cys 1 5 10 15 •130-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Ser Leu Glu 20 25 30 <210> 264 <211> 31 <212> PRT <213〉 蛋白質 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221〉 <222> misc_ 特徵 <223> Xaa = Fc <400> 264Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Ser Leu Glu 20 25 30 <210> 264 <211> 31 <212> PRT <213> Protein artificial sequence <220><223><;220> Peptide capable of binding to Ang-2 <221><222> misc_ feature <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Arg His Gin Glu Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Arg His Gin Glu Gly Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Phe Asp Trp Leu Glu 20 25 30 <210> 265 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc 特徵 -131 -Glu Trp Asp Pro Trp Thr Cys Glu His Met Phe Asp Trp Leu Glu 20 25 30 <210> 265 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc feature-131 -
O:\121\121929.DOC 200808833 <222〉 （2)··(2) <223> Xaa = Fc <400> 265O:\121\121929.DOC 200808833 <222> (2)·(2) <223> Xaa = Fc <400> 265
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Pro Arg Gin Lys Asp Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Pro Arg Gin Lys Asp Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Tyr Val Gly Leu Glu 20 25 30 <210> 266 <211〉 31 <212>蛋白質 <213>人造的序列 <220〉 <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 266Glu Trp Asp Pro Trp Thr Cys Glu His Met Tyr Val Gly Leu Glu 20 25 30 <210> 266 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser lie Ser His Glu Glu Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser lie Ser His Glu Glu Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Gin Val Gly Leu Glu 25 30 20 <210> 267 <211> 31 <212>蛋白質 • 132.Glu Trp Asp Pro Trp Thr Cys Glu His Met Gin Val Gly Leu Glu 25 30 20 <210> 267 <211> 31 <212> Protein • 132.
O:\121\121929.DOC 200808833 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)··(2) <223> Xaa = Fc <400> 267O:\121\121929.DOC 200808833 <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc_ feature <222> (2) ··(2) <223> Xaa = Fc <400> 267
Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Ala Ala Gin Glu Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Ala Ala Gin Glu Glu Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Arg Met Leu Glu 20 25 30 <210> 268 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc一特徵 <222〉 （2)..(2) <223> Xaa = Fc <400> 268Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Arg Met Leu Glu 20 25 30 <210> 268 <211> 31 <212>Protein<213> Man-made sequence <220><223> Ang-2-bound peptide body <220><22i> misc-characteristic <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Trp Pro Gin Asp Lys Cys 1 5 10 15 -133 -Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Trp Pro Gin Asp Lys Cys 1 5 10 15 -133 -
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Ser Thr Leu Glu 20 25 30 <210> 269 <211〉 31 <212>蛋白質 <213>人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 269Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Ser Thr Leu Glu 20 25 30 <210> 269 <211> 31 <212> Protein <213> Man-made Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400> 269
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly His Ser Gin Glu Giu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly His Ser Gin Glu Giu Cys 15 10 15
Gly Trp Asp Pro Trp Thr Cys Glu His Met Gly Thr Ser Leu Glu 20 25 · 30 <210> 270 <211> 31 <212>蛋白質 <n3>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> •134-Gly Trp Asp Pro Trp Thr Cys Glu His Met Gly Thr Ser Leu Glu 20 25 · 30 <210> 270 <211> 31 <212> Protein <n3> Artificial Sequence <220><223> Peptide bound to Ang-2 <220> •134-
O:\121\121929.DOC 200808833 <22i> misc__特徵 <222> (2)..(2) <223> Xaa = Fc <400> 270 ?O:\121\121929.DOC 200808833 <22i> misc__features <222> (2)..(2) <223> Xaa = Fc <400> 270 ?
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin His Trp Gin Glu Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin His Trp Gin Glu Glu Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Asp His Met Pro Ser Lys Leu Glu 20 25 30 <210> 271 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222〉 (2) . . (2) <223> Xaa = F*c <400> 271Glu Trp Asp Pro Trp Thr Cys Asp His Met Pro Ser Lys Leu Glu 20 25 30 <210> 271 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i>misc_feature<222> (2) . . . (2) <223> Xaa = F*c <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Asn Val Arg Gin Glu Lys Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Asn Val Arg Gin Glu Lys Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Val Arg Leu Glu 20 25 30 <210〉 272 <211〉 31 -135-Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Val Arg Leu Glu 20 25 30 <210> 272 <211> 31 -135-
O:\121\121929.DOC 200808833 <212>蛋白質 <213>人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2)··(2) <223> Xaa = Fc <400> 272O:\121\121929.DOC 200808833 <212>Protein<213> Artificial sequence<220><223> Peptide capable of binding to Ang-2 <220><22i>misc-feature<;222> (2)·(2) <223> Xaa = Fc <400> 272
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Ser Gly Gin Val Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Ser Gly Gin Val Glu Cys 15 10 15
Asn Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Asn Leu Glu 20 25 30 <210> 273 <211〉 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 273 -136-Asn Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Asn Leu Glu 20 25 30 <210> 273 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_feature <222> (2)..(2) <223> Xaa = Fc <400> 273 -136-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Lys Thr Gin Glu His Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Lys Thr Gin Glu His Cys 15 10 15
Asp Trp Asp Pro Trp Thr Cys Glu His Met Arg Glu Trp Leu Glu 20 25 30 <210> 274 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc—特徵 <222> (2)..(2) <223〉 Xaa = F c <_> 274Asp Trp Asp Pro Trp Thr Cys Glu His Met Arg Glu Trp Leu Glu 20 25 30 <210> 274 <211> 31 <212> Protein <213> Artificial sequence <220><223> Ang-2-bound peptide body <220><221>misc-feature<222> (2)..(2) <223> Xaa = F c <_> 274
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Trp Gly Gin Glu Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Trp Gly Gin Glu Gly Cys 15 10 15
Asp Trp Asp Pro Trp Thr Cys Glu His Met Leu Pro Met Leu Glu 20 25 30 <210> 275 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 -137-Asp Trp Asp Pro Trp Thr Cys Glu His Met Leu Pro Met Leu Glu 20 25 30 <210> 275 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide-137-
0:\121\121929.D0C 200808833 <22〇> <221> misc__特徵 <222> (2)..(2) <223> Xa3 s Fc <400> 2750:\121\121929.D0C 200808833 <22〇><221> misc__ feature <222> (2)..(2) <223> Xa3 s Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Val Asn Gin Glu Asp Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Val Asn Gin Glu Asp Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Pro Met Leu Glu 20 25 30 <210> 276 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220〉 能夠與Ang_2結合的肽體 <221> misc__特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 276Glu Trp Asp Pro Trp Thr Cys Glu His Met Pro Pro Met Leu Glu 20 25 30 <210> 276 <211> 31 <212> Protein <213> Artificial Sequence <220><223>< 220> Peptide capable of binding to Ang_2 <221> misc__ feature <222> (2) . . . (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Ala Pro Gin Glu Asp Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Ala Pro Gin Glu Asp Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Ala His Met Asp lie Lys Leu Glu 20 25 3〇 <210> 277 138-Glu Trp Asp Pro Trp Thr Cys Ala His Met Asp lie Lys Leu Glu 20 25 3〇 <210> 277 138-
O:\121\121929.DOC 200808833 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 21ΊO:\121\121929.DOC 200808833 <211> 31 <212> Protein <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 <221>;misc_Features<222> (2) . . (2) <223> Xaa = Fc <400> 21Ί
Met Xaa Gly Gly Gly Gly Gly Ala Gin His Gly Gin Asn Met Glu 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin His Gly Gin Asn Met Glu 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Phe Arg Tyr Leu Glu 20 25 30 <210> 278 <211> 31 <212〉 蛋白質 <213> 人造的序列 <220〉 <223> <220> . 能夠與Ang-2結合的肽體 <221> misc_特徵 <222〉 ⑵··（2) <223> Xaa = Fc O:\121\121929.DOC -139 - 200808833 <400> 278Glu Trp Asp Pro Trp Thr Cys Glu His Met Phe Arg Tyr Leu Glu 20 25 30 <210> 278 <211> 31 <212> Protein <213> Artificial Sequence <220> <223><220>. Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) (2) <223> Xaa = Fc O: \121\121929.DOC -139 - 200808833 <400> 278
Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Arg Leu Gin Glu Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Arg Leu Gin Glu Glu Cys 15 10 15
Val Trp Asp Pro Trp Thr Cys Glu His Met Pro Leu Arg Leu Glu 20 25 30 <210> 27 9 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) . . (2) <223〉 Xaa = Fc <400> 279Val Trp Asp Pro Trp Thr Cys Glu His Met Pro Leu Arg Leu Glu 20 25 30 <210> 27 9 <211> 31 <212> Protein <213> Artificial Sequence <220><223><;220> Peptide capable of binding to Ang-2 <221> misc_feature <222> (2) . (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Thr Thr Gin Glu Lys Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Thr Thr Gin Glu Lys Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Glu Ser Gin Leu Glu 20 25 30 <210> 280 <211〉 31 <212> 蛋白質 <213> 人造的序列 <220> O:\121\121929.DOC - 140- 200808833 <223> 能夠與Ang-2結合的肽雅 <220> <22i> misc—特徵 <222〉 （2)..(2) 〈223> Xaa = F c <400> 280Glu Trp Asp Pro Trp Thr Cys Leu Glu 20 25 30 <210> .DOC - 140- 200808833 <223> A peptide capable of binding to Ang-2 <220><22i> misc-characteristic <222> (2)..(2) <223> Xaa = F c <;400> 280
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Thr Ser Gin Glu Asp Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Thr Ser Gin Glu Asp Cys 1 5 10 15
Val Trp Asp Pro Trp Thr Cys Asp His Met Val Ser Ser Leu Glu 20 25 30 <210> 281 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)..(2) <223> Xaa = Fc <400> 281Val Trp Asp Pro Trp Thr Cys Asp His Met Val Ser Ser Leu Glu 20 25 30 <210> 281 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Val lie Gly Arg Pro Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Val lie Gly Arg Pro Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Leu Glu Gly Leu Leu Glu 20 25 30 -141-Glu Trp Asp Pro Trp Thr Cys Glu His Leu Glu Gly Leu Leu Glu 20 25 30-141-
O:\121\121929.DOC 200808833 <210> 282 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 282O:\121\121929.DOC 200808833 <210> 282 <211> 31 <212> Protein <213> Artificial sequence <220><223><220> capable of binding to Ang-2 Peptide <221>misc_feature<222> (2)..(2) <223> Xaa = Fc <400> 282
Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Ala Gin Gin Glu Glu Cys 15 10 15 .Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Ala Gin Gin Glu Glu Cys 15 10 15 .
Ala Trp Asp Pro Trp Thr Cys Asp His Met Val Gly Leu Leu Glu 20 25 30 <210> 283 <211> 31 <212> 蛋白質 <213> 人造的序列 <220〉 <223> <220〉 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2).·(2) <223〉 Xaa = Fc 142-Ala Trp Asp Pro Trp Thr Cys Asp His Met Val Gly Leu Leu Glu 20 25 30 <210> 283 <211> 31 <212> Protein <213> Artificial Sequence <220> <223>< 220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2). (2) <223> Xaa = Fc 142-
O:\121\121929.DOC 200808833 <400> 283O:\121\121929.DOC 200808833 <400> 283
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Pro Gly Gin Glu Asp Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Pro Gly Gin Glu Asp Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Val Arg S.er Leu Glu 20 25 30 <210> 284 <211> 31 <212> 蛋白質 <213> 人造的序列 <220〉 <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2)··(2) <223> Xaa = Fc <400> 284Glu Trp Asp Pro Trp Thr Cys Glu His Met Val Arg S.er Leu Glu 20 25 30 <210> 284 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Met Asn Gin Val Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Met Asn Gin Val Glu Cys 15 10 15
Asp Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Ser Leu Glu 20 25 30 <210> 285 <211> 31 <212> 蛋白質 <213> 人造的序列 143-Asp Trp Asp Pro Trp Thr Cys Glu His Met Pro Arg Ser Leu Glu 20 25 30 <210> 285 <211> 31 <212> Protein <213> Artificial Sequence 143-
O:\121\121929.DOC <220> 200808833 <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) · · (2) <223> Xaa = Fc <400> 285O:\121\121929.DOC <220> 200808833 <223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) · · (2) <;223> Xaa = Fc <400> 285
Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Trp Ser His Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Trp Ser His Gly Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Ser Thr Leu Glu 20 25 30 <210> 286 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc—特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 286Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Ser Thr Leu Glu 20 25 30 <210> 286 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc - feature <222> (2) . . . (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Ser Thr Gin Asp Asp Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Ser Thr Gin Asp Asp Cys 1 5 10 15
Asp Trp Asp Pro Trp Thr Cys Glu His Met Val Gly Pro Leu Glu 20 25 30 -144 -Asp Trp Asp Pro Trp Thr Cys Glu His Met Val Gly Pro Leu Glu 20 25 30-144 -
O:\121\121929.DOC 200808833 <210> 287 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2).. (2) <223> Xaa - Fc <400〉 287O:\121\121929.DOC 200808833 <210> 287 <211> 31 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> (2).. (2) <223> Xaa - Fc <400> 287
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Pro Arg lie Ser Thr Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Pro Arg lie Ser Thr Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Glu His Met Asp Gin Leu Leu Glu 20 25 30 <210> 288 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2)..(2) -145 -Gin Trp Asp Pro Trp Thr Cys Glu His Met Asp Gin Leu Leu Glu 20 25 30 <210> 288 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc-feature <222> (2)..(2) -145 -
O:\121\121929.DOC 200808833 <223〉 Xaa = Fc <400> 288O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 288
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser Thr lie Gly Asp Met Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser Thr lie Gly Asp Met Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Ala His Met Gin Val Asp Leu Glu 20 25 30 <210> 289 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc _特徵 <222> (2) · · (2) <223> Xaa = Fc <400> 289Glu Trp Asp Pro Trp Thr Cys Ala His Met Gin Val Asp Leu Glu 20 25 30 <210> 289 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><221> misc_characteristic <222> (2) · · (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Leu Gly Gly Gin Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Leu Gly Gly Gin Gly Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 290 <211> 31 <212>蛋白質 <213〉 人造的序列 O:\121\121929.DOC -146 - 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc__ 特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 290Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 290 <211> 31 <212>Protein<213> Artificial sequence O:\121\121929.DOC -146 - 200808833 <220><223> Peptide capable of binding to Ang-2 <220><22i> misc__ feature <222> (2) (2) <223> Xaa = Fc <400> 290
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Leu Gly Gly Gin Gly Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Leu Gly Gly Gin Gly Cys 1 5 10 15
Gin Trp Asp Pro Trp Thr Cys Ser His Leu Glu Asp Gly Leu Glu 20 25 30 <210> 291 <211> 31 <212>蛋白質 <213> 人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) <223〉 Xaa = Fc <400> 291Gin Trp Asp Pro Trp Thr Cys Ser His Leu Glu Asp Gly Leu Glu 20 25 30 <210> 291 <211> 31 <212>Protein<213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr lie Gly Ser Met Cys 1 5 10 15 147·Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr lie Gly Ser Met Cys 1 5 10 15 147·
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Glu Trp Asp Pro Trp Thr Cys Ala His Met Gin Gly Gly Leu Glu 20 25 30 <210〉 292 <211〉 31 <212〉 蛋白質 <213〉 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) · · (2) <223〉 Xaa - Fc <400> 292Glu Trp Asp Pro Trp Thr Cys Ala His Met Gin Gly Gly Leu Glu 20 25 30 <210> 292 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) · (2) <223> Xaa - Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Lys Gly Lys Ser Val Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Lys Gly Lys Ser Val Cys 1 5 10 15
Gin Trp Asp Pro Trp Thr Cys Ser His Met Gin Ser Gly Leu Glu 20 25 30 <210> 293 <211> 31 <212> 蛋白質 <213> 人造的序列 <220〉 <223〉 <220> 能夠與Ang-2結合的肽體 <221> misc__特徵 -148-Gin Trp Asp Pro Trp Thr Cys Ser His Met Gin Ser Gly Leu Glu 20 25 30 <210> 293 <211> 31 <212> Protein <213> Artificial Sequence <220><223>220> Peptide capable of binding to Ang-2 <221> misc__ feature-148-
O:\121\121929.DOC 200808833 <222> (2)..(2) <223> Xss. = Fc <400> 293O:\121\121929.DOC 200808833 <222> (2)..(2) <223> Xss. = Fc <400> 293
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr He Gly Ser Met Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr He Gly Ser Met Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Ala His Met Gin Gly Gly Leu Glu 20 25 30 <210> 294 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc^特徵 <222> (2)··(2) <223> Xsa = F c <400> 294Gin Trp Asp Pro Trp Thr Cys Ala His Met Gin Gly Gly Leu Glu 20 25 30 <210> 294 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc^ feature <222> (2)·(2) <223> Xsa = F c <400> 294
Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Val Asn Glu Val Val Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Val Asn Glu Val Val Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Asn His Trp Asp Thr Pro Leu Glu 20 25 30 <210> 295 <211> 31 <212> 蛋白質 O:\121\121929.DOC -149- 200808833 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 （2)··(2) <223> Xaa = Fc <400> 295Glu Trp Asp Pro Trp Thr Cys Asn His Trp Asp Thr Pro Leu Glu 20 25 30 <210> 295 <211> 31 <212> Protein O:\121\121929.DOC -149- 200808833 <213> Sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> (2) (2) <223> Xaa = Fc <;400> 295
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Val Gin Val Gly Met Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Val Gin Val Gly Met Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Lys His Met Arg Leu Gin Leu Glu 20 25 30 <210〉 296 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc—特徵 一 <222> (2).·⑵ <223> Xaa = Fc <400〉 296Gin Trp Asp Pro Trp Thr Cys Lys His Met Arg Leu Gin Leu Glu 20 25 30 <210> 296 <211> 31 <212>Protein<213> Artificial sequence <220><223> Ang-2-bound peptide body <220><221> misc - feature one <222> (2). (2) <223> Xaa = Fc <400> 296
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Val Gly Ser Gin Thr Cys 15 10 15 -150-Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Val Gly Ser Gin Thr Cys 15 10 15 -150-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Glu Trp Asp Pro Trp Thr Cys Ala His Leu Val Glu Val Leu Glu 20 25 30 <210> 297 <211〉 31 · <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2) · . (2) <223> Xaa = Fc <400> 297Glu Trp Asp Pro Trp Thr Cys Ala His Leu Val Glu Val Leu Glu 20 25 30 <210> 297 <211> 31 · <212> Protein <213> Artificial Sequence <220><223><;220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2) · (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Gly Met Lys Met Phe 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Gly Met Lys Met Phe 15 10 15
Glu Trp Asp Pro Trp Thr Cys Ala His lie Val Tyr Arg Leu Glu 20 25 30 <210> 298 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 -151 -Glu Trp Asp Pro Trp Thr Cys Ala His lie Val Tyr Arg Leu Glu 20 25 30 <210> 298 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2-151 -
O:\121\121929.DOC 200808833 <221> misc__ 特徵 <222> (2).. (2) <223> Xaa = Fc <400> 298 *O:\121\121929.DOC 200808833 <221> misc__ Feature <222> (2).. (2) <223> Xaa = Fc <400> 298 *
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr He Gly Ser Met Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Thr He Gly Ser Met Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Glu His Met Gin Gly Gly Leu Glu 20 25 30 <210> 299 <211〉 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220〉 <22i> misc_ 特徵 <222〉 (2)..(2) <223> Xaa = F*c <400> 299Gin Trp Asp Pro Trp Thr Cys Glu His Met Gin Gly Gly Leu Glu 20 25 30 <210> 299 <211> 31 <212>Protein<213> Artificial sequence <220><223> Ang-2-bound peptide body <220> <22i> misc_ feature <222> (2)..(2) <223> Xaa = F*c <400> 299
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Ser Gin Arg Val Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Ser Gin Arg Val Gly Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Gin His Leu Thr Tyr Thr Leu Glu 20 25 30 <210〉 300· <211> 31 -152-Glu Trp Asp Pro Trp Thr Cys Gin His Leu Thr Tyr Thr Leu Glu 20 25 30 <210> 300· <211> 31 -152-
O:\121\121929.DOC 200808833 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc 特徵 <222> (2) ·· (2) <223> Xaa = Fc <400〉 300O:\121\121929.DOC 200808833 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc characteristic <222> (2) ·· (2) <223> Xaa = Fc <400> 300
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Trp Ser Trp Pro Pro Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Trp Ser Trp Pro Pro Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Gin Thr Val Trp Pro Ser Leu Glu 20 25 30 <210> 301 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 （2) ·· (2) <223> Xaa = Fc <400〉 301 153 -Glu Trp Asp Pro Trp Thr Cys Gin Thr Val Trp Pro Ser Leu Glu 20 25 30 <210> 301 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i>misc-feature<222> (2) ·· (2) <223> Xaa = Fc <400> 301 153 -
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Thr Ser Pro Ser Phe Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Thr Ser Pro Ser Phe Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Ser His Met Val Gin Gly Leu Glu 20 25 30 <210> 302 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc^特徵 <222> (2)··(2) <223> Xas = Fc <400> 302Gin Trp Asp Pro Trp Thr Cys Ser His Met Val Gin Gly Leu Glu 20 25 30 <210> 302 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc^ feature <222> (2) (2) <223> Xas = Fc <400> 302
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Gin Gly Leu His Gin Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Gin Gly Leu His Gin Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Lys Val Leu Trp Pro Ser Leu Glu 20 25 30 <210〉 303 <211> 31 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 -154-Glu Trp Asp Pro Trp Thr Cys Lys Val Leu Trp Pro Ser Leu Glu 20 25 30 <210> 303 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide-154-
O:\121\121929.DOC <220> 200808833 <221> misc_特徵 <222> (2) . . (2) <223〉 X3d = Fc <400> 303O:\121\121929.DOC <220> 200808833 <221> misc_features <222> (2) . . . (2) <223> X3d = Fc <400> 303
Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Trp Arg Ser Gin Val Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Val Trp Arg Ser Gin Val Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Asn Leu Gly Gly Asp Trp Leu Glu 20 25 30 <210> 304 <211> 31 <212> PRT <213> 蛋白質 <220> 人造的序列 <223> <220> 能夠與Ang-2結合的肽體 <221> <222> misc__特徵 <223> Xaa = Fc <400> 304Gin Trp Asp Pro Trp Thr Cys Asn Leu Gly Gly Asp Trp Leu Glu 20 25 30 <210> 304 <211> 31 <212> PRT <213> Protein <220> Artificial Sequence <223>;220> Peptide capable of binding to Ang-2 <221><222> misc__feature <223> Xaa = Fc <400> 304
Met Xaa Gly Gly Gly Gly Gly Ala Gin Asp Lys lie Leu Glu Glu Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Asp Lys lie Leu Glu Glu Cys 1 5 10 15
Gin Trp Asp Pro Trp Thr Cys Gin Phe Phe Tyr Gly Ala Leu Glu 20 25 30 <210> 305 155-Gin Trp Asp Pro Trp Thr Cys Gin Phe Phe Tyr Gly Ala Leu Glu 20 25 30 <210> 305 155-
O:\121\121929.DOC 200808833 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220〉 <22i> misc—特徵 <222> (2)..(2) <223> Xaa = Fc <400> 305O:\121\121929.DOC 200808833 <211> 31 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i>; misc - feature <222> (2)..(2) <223> Xaa = Fc <400> 305
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Thr Phe Ala Arg Gin Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Thr Phe Ala Arg Gin Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Ala Leu Gly Gly Asn Trp Leu Glu 20 25 30 <210〉 306 <211> 31 <212>蛋白質 <213>人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220〉 <22i> misc—特徵 <222> (2)··(2) <223> Xaa = Fc •156-Gin Trp Asp Pro Trp Thr Cys Ala Leu Gly Gly Asn Trp Leu Glu 20 25 30 <210> 306 <211> 31 <212> Protein <213> Artificial Sequence <220> <223> Ang-2-bound peptide body <220> <22i> misc-characteristic <222> (2)·(2) <223> Xaa = Fc •156-
O:\121\121929.DOC 200808833 <400> 306O:\121\121929.DOC 200808833 <400> 306
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Pro Ala Gin Glu Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Pro Ala Gin Glu Glu Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Glu Pro Leu Pro Leu Met Leu Glu 20 25 30 <210> 307 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)..(2) <223> Xaa - Fc <400> 307Glu Trp Asp Pro Trp Thr Cys Glu Pro Leu Pro Leu Met Leu Glu 20 25 30 <210> 307 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa - Fc <400> 307
Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Pro Glu Asp Met Cys Ser 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Arg Pro Glu Asp Met Cys Ser 1 5 10 15
Gin Trp Asp Pro Trp Thr Trp His Leu Gin Gly Tyr Cys Leu Glu 20 25 30 <210〉 308 <211> 31 <212>蛋白質 <213>人造的序列 <220> -157-Gin Trp Asp Pro Trp Thr Trp His Leu Gin Gly Tyr Cys Leu Glu 20 25 30 <210> 308 <211> 31 <212> Protein <213> Artificial Sequence <220> -157-
O:\121\121929.DOC 200808833 <223> 能夠與Ang-2結合的肽體 <220> <221> misc j争徵 <222> (2). . (2) <223> Xaa == Fc <400> 308O:\121\121929.DOC 200808833 <223> Peptide capable of binding to Ang-2 <220><221> misc j contending <222> (2). (2) <223> Xaa == Fc <400> 308
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Trp Gin Leu Ala Val Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Trp Gin Leu Ala Val Cys 15 10 15
Gin Trp Asp Pro Gin Thr Cys Asp His Met Gly Ala Leu Leu Glu 20 25 30 <210> 309 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)..(2) <223> Xaa = Fc <400> 309Gin Trp Asp Pro Gin Thr Cys Asp His Met Gly Ala Leu Leu Glu 20 25 30 <210> 309 <211> 31 <212>Protein<213> Artificial sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Gin Leu Val Ser Leu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Gin Leu Val Ser Leu Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Asp Gly Trp Leu Glu 20 25 3〇 -158-Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Asp Gly Trp Leu Glu 20 25 3〇 -158-
O:\121\121929.DOC 200808833 <210> 310 <211> 31 <212> 蛋白質 <213> 人造的序列 <220〉 <223> <220> 能夠與Ang-2結合的肽體 <221〉 misc_特徵 <222> (2) ·· (2) <223〉 Xaa = Fc <400> 310O:\121\121929.DOC 200808833 <210> 310 <211> 31 <212> Protein <213> Artificial sequence <220> <223><220> capable of binding to Ang-2 Peptide <221> misc_feature<222> (2) ·· (2) <223> Xaa = Fc <400> 310
Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Gly Gly Ala Gly Arg Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Gly Gly Ala Gly Arg Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Gin Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 311 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2)..(2) <223> Xaa = Fc 159-Glu Trp Asp Pro Trp Thr Cys Gin Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 311 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc-characteristic <222> (2)..(2) <223> Xaa = Fc 159-
O:\121\121929.DOC 200808833 <400> 311O:\121\121929.DOC 200808833 <400> 311
Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Phe Leu Pro Asn Glu Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Phe Leu Pro Asn Glu Cys 15 10 15
Gin Trp Asp Pro Trp Thr Cys Ser Asn Leu Pro Glu Ala Leu Glu 20 25 30 <210> 312 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)..(2) <223> Xaa 二 Fc <400> 312Gin Trp Asp Pro Trp Thr Cys Ser Asn Leu Pro Glu Ala Leu Glu 20 25 30 <210> 312 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa II Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Trp Ser His Gly Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Trp Ser His Gly Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 313 <211> 31 <212>蛋白質 <213>人造的序列 160-Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 313 <211> 31 <212> Protein <213> Artificial Sequence 160-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc_特徵 <222> (2) . . (2) <223> Xaa = Fc <400> 313O:\121\121929.DOC 200808833 <220><223> Peptide capable of binding to Ang-2 <220><221> misc_feature <222> (2) . (2) <;223> Xaa = Fc <400> 313
Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Pro Gin Thr Glu Gly Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Trp Pro Gin Thr Glu Gly Cys 1 5 10 15
Gin Trp Asp Pro Trp Thr Cys Arg Leu Leu His Gly Trp Leu Glu 20 25 30 <210> 314 <211〉 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 (2). . (2) <223> Xaa = Fc <400〉 314Gin Trp Asp Pro Trp Thr Cys Arg Leu Leu His Gly Trp Leu Glu 20 25 30 <210> 314 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i>misc-feature<222> (2). (2) <223> Xaa = Fc <400> 314
Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Asp Thr Arg Gin Gly Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Pro Asp Thr Arg Gin Gly Cys 1 5 10 15
Gin Trp Asp Pro Trp Thr Cys Arg Leu Tyr Gly Met Trp Leu Glu 20 25 30 • 161 -Gin Trp Asp Pro Trp Thr Cys Arg Leu Tyr Gly Met Trp Leu Glu 20 25 30 • 161 -
O:\121\121929.DOC 200808833 <210> 315 <211〉 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 315O:\121\121929.DOC 200808833 <210> 315 <211> 31 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><221>misc_feature<222> (2)..(2) <223> Xaa = Fc <400> 315
Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Trp Pro Gin Asp Lys Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Thr Trp Pro Gin Asp Lys Cys 15 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 316 <211> 31 <212> 蛋白質 <213> 人造的序列 <220〉 <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2).·(2) -162-Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 316 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc-characteristic <222> (2). (2) -162-
O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 316O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 316
Met Xaa Gly Gly Gly Gly Gly Ala Gin Asp Lys lie Leu Glu Glu Cys 1 ? 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Asp Lys lie Leu Glu Glu Cys 1 ? 5 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 317 <211> 31 <212〉 蛋白質 <213〉 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (2).. (2) <223> Xaa = Fc <400> 317Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 317 <211> 31 <212> Protein <213> Artificial Sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> (2).. (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Ala Thr Gin Glu Glu Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Ala Thr Gin Glu Glu Cys 1 5 10 15
Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 318 <211> 34 <212> 蛋白質 <213> 人造的序列 163 -Glu Trp Asp Pro Trp Thr Cys Arg Leu Leu Gin Gly Trp Leu Glu 20 25 30 <210> 318 <211> 34 <212> Protein <213> Artificial Sequence 163 -
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (34) .. (34 ) <223> Xaa = Fc <400> 318O:\121\121929.DOC 200808833 <220><223> Peptide capable of binding to Ang-2 <220><22i> misc_ feature <222> (34) .. (34) <223> Xaa = Fc <400> 318
Met Gly Ala Gin Thr Asn Phe Met Pro Met Asp Asp Leu Glu Gin Arg 15 10 15Met Gly Ala Gin Thr Asn Phe Met Pro Met Asp Asp Leu Glu Gin Arg 15 10 15
Leu Tyr Glu Gin Phe lie Leu Gin Gin Gly Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Phe lie Leu Gin Gin Gly Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 319 <211〉 34 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc—特徵 <222> (34) ·· (34} . <223> Xaa = Fc <400> 319 -164-Gly Xaa <210> 319 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><221> - Feature <222> (34) ·· (34} . <223> Xaa = Fc <400> 319 -164-
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Gly Ala Gin Thr Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr 15 10 15Met Gly Ala Gin Thr Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr 15 10 15
Leu Tyr Glu His Trp lie Leu Gin His Ser Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu His Trp lie Leu Gin His Ser Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 320 <211〉 34 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (34)··(34) <223> X3B — Fc <400> 320Gly Xaa <210> 320 <211> 34 <212> Protein <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 <221> _Features <222> (34)·(34) <223> X3B - Fc <400> 320
Met Gly Ala Gin Gin Lys Tyr Gin Pro Leu Asp Glu Leu Asp Lys Thr 15 10 15Met Gly Ala Gin Gin Lys Tyr Gin Pro Leu Asp Glu Leu Asp Lys Thr 15 10 15
Leu Tyr Asp Gin Phe Met Leu Gin Gin Gly Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Asp Gin Phe Met Leu Gin Gin Gly Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210〉 321 <211> 34 <212>蛋白質 -165-Gly Xaa <210> 321 <211> 34 <212> Protein -165-
O:\121\121929.DOC 200808833 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (34)..(34) <223> Xaa = Fc <400〉 321O:\121\121929.DOC 200808833 <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> )..(34) <223> Xaa = Fc <400> 321
Met Gly Ala Gin Leu Asn Phe Thr Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15Met Gly Ala Gin Leu Asn Phe Thr Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin Gin Ser Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin Gin Ser Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210〉 322 <211> 34 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc一特徵 <222> (34) .. (34) <223〉 Xaa » Fc -166-Gly Xaa <210> 322 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> A feature <222> (34) .. (34) <223> Xaa » Fc -166-
O:\121\121929.DOC 200808833 <400〉 322O:\121\121929.DOC 200808833 <400> 322
Met Gly Ala Gin Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Gin Thr 1 5 10 15Met Gly Ala Gin Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Gin Thr 1 5 10 15
Leu Tyr Glu Gin Phe Met Leu Gin Gin Ala Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Phe Met Leu Gin Gin Ala Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 323 <211> 34 <212> 蛋白質 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc—特徵 <222> (34) . . (34) <223> Xaa = Fc <400> 323Gly Xaa <210> 323 <211> 34 <212> Protein <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 <221> - Feature <222> (34) . . . (34) <223> Xaa = Fc <400> 323
Met Gly Ala Gin Gin Glu Tyr Glu Pro Leu Asp Glu Leu Asp Glu Thr 15 10 15Met Gly Ala Gin Gin Glu Tyr Glu Pro Leu Asp Glu Leu Asp Glu Thr 15 10 15
Leu Tyr Asn Gin Trp Met Phe His Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Asn Gin Trp Met Phe His Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 324 <211> 34Gly Xaa <210> 324 <211> 34
O:\121\121929.DOC 200808833 <212>蛋白度 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 · <220> <22i> misc_ 特徵 <222> (34)..(34) <223> Xaa = Fc <400> 324O:\121\121929.DOC 200808833 <212>Proteinity <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 · <220><22i> misc_ characteristics <222> (34)..(34) <223> Xaa = Fc <400> 324
Met Gly Ala Gin Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu lie 1 5 10 15Met Gly Ala Gin Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu lie 1 5 10 15
Leu Tyr Glu Gin Gin Thr Phe Gin Glu Arg Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Gin Thr Phe Gin Glu Arg Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 325 <211> 34 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (34) .. (34) <223> Xaa = Fc -168-Gly Xaa <210> 325 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> _Features <222> (34) .. (34) <223> Xaa = Fc -168-
O:\121\121929.DOC 200808833 <400> 325O:\121\121929.DOC 200808833 <400> 325
Met Gly Ala Gin Thr Lys Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr 15 10 15Met Gly Ala Gin Thr Lys Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr 15 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 326 <211> 34 <212> 蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (34) .. (34) <223> Xss = Fc <400> 326Gly Xaa <210> 326 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc_ Feature <222> (34) .. (34) <223> Xss = Fc <400> 326
Met Gly Ala Gin Thr Asn Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr 1 5 10 15Met Gly Ala Gin Thr Asn Phe Gin Pro Leu Asp Glu Leu Asp Gin Thr 1 5 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 327 169-Gly Xaa <210> 327 169-
0:\121\121929.D0C 200808833 <211> 34 <212> 蛋白質 <213> 人造的序列 <220> .. <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (34) . . (34) <223〉 Xaa = Fc <220> <221〉 ，miSL特徵 <222> (34) . . (34) <223> Xaa = Fc <400> 3270: \121\121929.D0C 200808833 <211> 34 <212> Protein <213> Artificial sequence <220> .. <223><220> Peptide capable of binding to Ang-2 <;221>misc_features<222> (34) . . . (34) <223> Xaa = Fc <220><221>, miSL feature <222> (34) (34) <223> Xaa = Fc <400> 327
Met Gly Ala Gin Gin Asn Phe Lys Pro Met Asp Glu Leu Glu Asp Thr 1 5 10 15 Leu Tyr Lys Gin Phe Leu Phe Gin His Ser Leu Glu Gly Gly Gly Gly 20 25 30 Gly Xaa <210> 328 <211> 34 <212> 蛋白質 <213> 人造的序列 O:\121\121929.DOC -170- 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (34).. (34) <223> Xaa = Fc <400> 328Met Gly Ala Gin Gin Asn Phe Lys Pro Met Asp Glu Leu Glu Asp Thr 1 5 10 15 Leu Tyr Lys Gin Phe Leu Phe Gin His Ser Leu Glu Gly Gly Gly 20 25 30 Gly Xaa <210> 328 <211> 34 <212> Protein <213> Artificial sequence O: \121\121929.DOC -170- 200808833 <220><223> Peptide capable of binding to Ang-2 <220><22i> Misc - Feature <222> (34).. (34) <223> Xaa = Fc <400> 328
Met Gly Ala Gin Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu Trp 15 10 15Met Gly Ala Gin Val Lys Tyr Lys Pro Leu Asp Glu Leu Asp Glu Trp 15 10 15
Leu Tyr His Gin Phe Thr Leu His His Gin Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr His Gin Phe Thr Leu His His Gin Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 329 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc _特徵 <222> (34) · · (34) <223> Xaa = Fc <400> 329Gly Xaa <210> 329 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><221>_Features<222> (34) · · (34) <223> Xaa = Fc <400> 329
Met Gly Ala Gin Tyr Lys Phe Thr Pro Leu Asp Asp Leu Glu Gin Thr 1 5 10 15 -171 -Met Gly Ala Gin Tyr Lys Phe Thr Pro Leu Asp Asp Leu Glu Gin Thr 1 5 10 15 -171 -
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Leu Tyr Glu Gin Trp Thr Leu Gin His Val Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin His Val Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 330 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (34) .. (34) <223> Xaa = Fc <400> 330Gly Xaa <210> 330 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> - Feature <222> (34) .. (34) <223> Xaa = Fc <400> 330
Met Gly Ala Gin Gin Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr 1 5 10 15Met Gly Ala Gin Gin Asn Tyr Lys Pro Leu Asp Glu Leu Asp Ala Thr 1 5 10 15
Leu Tyr Glu His Phe lie Phe His Tyr Thr Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu His Phe lie Phe His Tyr Thr Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210〉 331 <211> 34 <212> 蛋白質 <213>人造的序列 -172-Gly Xaa <210> 331 <211> 34 <212> Protein <213> Artificial sequence -172-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc_ 特徵 <222〉 （34)..(34) <223> Xaa = Fc <400〉 331O:\121\121929.DOC 200808833 <220><223> Peptide capable of binding to Ang-2 <220><221> misc_ feature <222> (34).. (34) <223> Xaa = Fc <400> 331
Met Gly Ala Gin Val Lys Phe Lys Pro Leu Asp Ala Leu Glu Gin Thr 1 5 10 15Met Gly Ala Gin Val Lys Phe Lys Pro Leu Asp Ala Leu Glu Gin Thr 1 5 10 15
Leu Tyr Glu His Trp Met Phe Gin Gin Ala Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu His Trp Met Phe Gin Gin Ala Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210〉 332 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc—特徵 <222〉 (34) .. (34) <223> Xaa = Fc <400> 332 -173 -Gly Xaa <210> 332 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><221> - Feature <222> (34) .. (34) <223> Xaa = Fc <400> 332 -173 -
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Gly Ala Gin Glu Asp Tyr Met Pro Leu Asp Ala Leu Asp Ala Gin 15 10 15Met Gly Ala Gin Glu Asp Tyr Met Pro Leu Asp Ala Leu Asp Ala Gin 15 10 15
Leu Tyr Glu Gin Phe lie Leu Leu His Gly Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Phe lie Leu Leu His Gly Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 333 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc一特徵 <222〉 (34) .. (34) <223> Xaa = Fc <400〉 333Gly Xaa <210> 333 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> A feature <222> (34) .. (34) <223> Xaa = Fc <400> 333
Met Gly Ala Gin Tyr Lys Phe Asn Pro Met Asp Glu Leu Glu Gin Thr 15 10 15Met Gly Ala Gin Tyr Lys Phe Asn Pro Met Asp Glu Leu Glu Gin Thr 15 10 15
Leu Tyr Glu Glu Phe Leu Phe Gin His Ala Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Glu Phe Leu Phe Gin His Ala Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210〉 334 <211〉 34 <212>蛋白質 174-Gly Xaa <210> 334 <211> 34 <212> Protein 174-
O:\121\121929.DOC 200808833 <213> 人造的序列 <220> <223> <220> 能夠與Ang-2結合的肽體 <221> misc_特徵 <222> (34) · . (34) <223> Xsci ― Fc <400> 334O:\121\121929.DOC 200808833 <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc_ feature <222> ) . . . (34) <223> Xsci ― Fc <400> 334
Met Gly Ala Gin Ser Asn Phe Met Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15Met Gly Ala Gin Ser Asn Phe Met Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15
Leu Tyr Glu Gin Phe Met Leu Gin His Gin Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Phe Met Leu Gin His Gin Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 335 <211> 34 <212> 蛋白質 <213> 人造的序列 <220> <223> <220〉 能夠與Ang-2結合的肽體 <221〉 misc_特徵 <222> (34) . . (34) <223> Xse = Fc O:\121\121929.DOC - 175- 200808833 <400> 335Gly Xaa <210> 335 <211> 34 <212> Protein <213> Artificial sequence <220><223><220> Peptide capable of binding to Ang-2 <221> misc _Features <222> (34) . . . (34) <223> Xse = Fc O:\121\121929.DOC - 175- 200808833 <400> 335
Met Gly Ala Gin Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr 15 10 15Met Gly Ala Gin Gin Lys Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr 15 10 15
Leu Tyr Lys Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 2Q 25 30Leu Tyr Lys Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 2Q 25 30
Gly Xaa <210〉 336 <211> 34 <212>蛋白質 <213>人造的序列 <220〉 <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (34) .. (34) <223> Xaa = Fc <400〉 336Gly Xaa <210> 336 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> _Features <222> (34) .. (34) <223> Xaa = Fc <400> 336
Met Gly Ala Gin Gin Lys Phe Met Pro Leu Asp Glu Leu Asp Glu lie 15 10 15Met Gly Ala Gin Gin Lys Phe Met Pro Leu Asp Glu Leu Asp Glu lie 15 10 15
Leu Tyr Glu Gin Phe Met Phe Gin Gin Ser Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Phe Met Phe Gin Gin Ser Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 337 <211〉 34 -176-Gly Xaa <210> 337 <211> 34 -176-
O:\121\121929.DOC 200808833 <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220〉 <22i> misc_ 特徵 <222> (34) .· (34) <223> Xaa = Fc <400〉 337O:\121\121929.DOC 200808833 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc_ feature <222> (34) .. (34) <223> Xaa = Fc <400> 337
Met Gly Ala Gin Thr Lys Phe Asn Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15Met Gly Ala Gin Thr Lys Phe Asn Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin His Gin Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin His Gin Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 338 <211〉 34 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (34)..(34) <223> Xaa = Fc 177-Gly Xaa <210> 338 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> - Feature <222> (34)..(34) <223> Xaa = Fc 177-
O:\121\121929.DOC 200808833 <400> 338O:\121\121929.DOC 200808833 <400> 338
Met Gly Ala Gin His Thr Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr 15 10 15Met Gly Ala Gin His Thr Phe Gin Pro Leu Asp Glu Leu Glu Glu Thr 15 10 15
Leu Tyr Tyr Gin Trp Leu Tyr Asp Gin Led Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Tyr Gin Trp Leu Tyr Asp Gin Led Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 339 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220〉 <22i> misc_ 特徵 <222> (34). . (34) <223> Xaa = Fc <400> 339Gly Xaa <210> 339 <211> 34 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc_ Feature <222> (34). (34) <223> Xaa = Fc <400> 339
Met Gly Ala Gin Gin Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15Met Gly Ala Gin Gin Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr 15 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin Gin Arg Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 340 178-Gly Xaa <210> 340 178-
O:\121\121929.DOC 200808833 <211> 34 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220〉 <22i> misc 一特徵 <222> (34) .. (34)O:\121\121929.DOC 200808833 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i>; misc a feature <222> (34) .. (34)
Xs.3. == F c <400> 340Xs.3. == F c <400> 340
Met Gly Ala Gin Gin Thr Phe Gin Pro Leu Asp Asp Leu Glu Glu Tyr 15 10 15Met Gly Ala Gin Gin Thr Phe Gin Pro Leu Asp Asp Leu Glu Glu Tyr 15 10 15
Leu Tyr Glu Gin Trp lie Arg Arg Tyr His Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp lie Arg Arg Tyr His Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 341 <211> 34 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (34) . . (34) 179 -Gly Xaa <210> 341 <211> 34 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> —Features <222> (34) . . (34) 179 -
O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 341O:\121\121929.DOC 200808833 <223> Xaa = Fc <400> 341
Met Gly Ala Gin Ser Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr 1 5 10 15Met Gly Ala Gin Ser Lys Phe Lys Pro Leu Asp Glu Leu Glu Gin Thr 1 5 10 15
Leu Tyr Glu Gin Trp Thr Leu Gin His Ala Leu Glu Gly Gly Gly Gly 20 25 30Leu Tyr Glu Gin Trp Thr Leu Gin His Ala Leu Glu Gly Gly Gly Gly 20 25 30
Gly Xaa <210> 342 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2). . (2) <223> Xaa = F*c <400> 342Gly Xaa <210> 342 <211> 31 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i>_Features<222> (2). . (2) <223> Xaa = F*c <400> 342
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser Gly Gin Leu Arg Pro Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ser Gly Gin Leu Arg Pro Cys 15 10 15
Glu Glu lie Phe Gly Cys Gly Thr Gin Asn Leu Ala Leu Leu Glu 20 25 30 <210> 343 <211> 31 180-Glu Glu lie Phe Gly Cys Gly Thr Gin Asn Leu Ala Leu Leu Glu 20 25 30 <210> 343 <211> 31 180-
O:\121\121929.DOC 200808833 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2).. (2) <223> Xaa = Fc <400> 343O:\121\121929.DOC 200808833 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i>misc-feature<;222> (2).. (2) <223> Xaa = Fc <400> 343
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Gly Gly Met Arg Pro Tyr 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Gly Gly Met Arg Pro Tyr 15 10 15
Asp Gly Met Leu Gly Trp Pro Asn Tyr Asp Val Gin Ala Leu Glu 20 25 30 <210〉 344 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2)..(2) <223> Xaa = Fc <400> 344 181 -Asp Gly Met Leu Gly Trp Pro Asn Tyr Asp Val Gin Ala Leu Glu 20 25 30 <210> 344 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><22i> misc-characteristic <222> (2)..(2) <223> Xaa = Fc <400> 344 181 -
O:\121\121929.DOC 200808833O:\121\121929.DOC 200808833
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Gin Asp Leu Arg Pro Cys 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Gin Asp Leu Arg Pro Cys 15 10 15
Glu Asp Met Phe Gly Cys Gly Thr Lys Asp Trp Tyr Gly Leu Glu 20 25 30 <210> 345 <211> 31 <212>蛋白質 <213> 人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <221> misc j寺徵 <222> (2).. (2) <223> Xsa = Fc <400> 345Glu Asp Met Phe Gly Cys Gly Thr Lys Asp Trp Tyr Gly Leu Glu 20 25 30 <210> 345 <211> 31 <212>Protein<213> Artificial sequence <220><223> Ang-2-bound peptide body <220><221> misc j temple sign <222> (2).. (2) <223> Xsa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Pro Gly Gin Arg Pro Tyr 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Pro Gly Gin Arg Pro Tyr 1 5 10 15
Asp Gly Met Leu Gly Trp Pro Thr Tyr Gin Arg lie Val Leu Glu 20 25 30 <210> 346 <211> 31 <212>蛋白質 <213〉人造的序列 <220> <223>能夠與Ang-2結合的肽體 -182-Asp Gly Met Leu Gly Trp Pro Thr Tyr Gin Arg lie Val Leu Glu 20 25 30 <210> 346 <211> 31 <212>Protein <213>Artificial Sequence <220><223> Ang-2-bound peptide-182-
O:\121\121929.DOC 200808833 <220> <221> misc_特徵 <222> ⑵..（2) <223> Xaa = Fc <400> 346O:\121\121929.DOC 200808833 <220><221> misc_features <222> (2)..(2) <223> Xaa = Fc <400> 346
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Thr Trp Asp Asp Pro Cys 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gin Thr Trp Asp Asp Pro Cys 1 5 10 15
Met His lie Leu Gly Pro Val Thr Trp Arg Arg Cys He Leu Glu 20 25 30 <210> 347 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc 一特徵 <222〉 （2)..(2) <223> Xaa = Fc <400> 347Met His lie Leu Gly Pro Val Thr Trp Arg Arg Cys He Leu Glu 20 25 30 <210> 347 <211> 31 <212>Protein<213> Artificial sequence <220><223> Ang-2-bound peptide body <220><22i>misc-feature<222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Asp Lys Arg Pro Leu 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Gly Asp Lys Arg Pro Leu 15 10 15
Glu Cys Met Phe Gly Gly Pro lie Gin Leu Cys Pro Arg Leu Glu 20 25 30 <210> 348 183-Glu Cys Met Phe Gly Gly Pro lie Gin Leu Cys Pro Arg Leu Glu 20 25 30 <210> 348 183-
O:\121\121929.DOC 200808833 <211> 25 . _ ‘· . · · <212>蛋白質 <213> 人造的序列 <220> <223>能夠與Ang-2結合的肽禮 <220> <22i> misc—特徵 <222> ⑵..（2) <223> Xaa = Fc <400> 348O:\121\121929.DOC 200808833 <211> 25 . _ '···· <212> Protein <213> Artificial sequence <220><223> Peptide binding capable of binding to Ang-2 <220><22i> misc - feature <222> (2)..(2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Arg Pro Cys Glu Glu lie 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Arg Pro Cys Glu Glu lie 1 5 10 15
Phe Gly Gly Cys Thr Tyr Gin Leu Glu 20 25 <210> 349 <211〉 31 <212>蛋白質 <213> 人造的序列 <220〉 <223〉 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> (2) ·· (2) <223> Xaa = Fc 184-Phe Gly Gly Cys Thr Tyr Gin Leu Glu 20 25 <210> 349 <211> 31 <212>Protein<213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> (2) ·· (2) <223> Xaa = Fc 184-
O:\121\121929.DOC <_> 349 <_> 349200808833O:\121\121929.DOC <_> 349 <_> 349200808833
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Glu Trp Cys Glu Gly 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Glu Trp Cys Glu Gly 1 5 10 15
Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Lys Gin Arg Leu Glu 20 25 30 <210> 350 <211> 31 <212>蛋白質 <213> 人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <221> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400> 350Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Lys Gin Arg Leu Glu 20 25 30 <210> 350 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><221> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400> 350
Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Leu Asp Tyr Cys Glu Gly 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Met Leu Asp Tyr Cys Glu Gly 1 5 10 15
Met Asp Asp Pro Phe Thr Phe Gly Cys Asp Lys Gin Met Leu Glu 20 25 30 <210> 351 <211> 蛋白質 <212> 人造的序列 <213> Artificial Sequence <220>Met Asp Asp Pro Phe Thr Phe Gly Cys Asp Lys Gin Met Leu Glu 20 25 30 <210> 351 <211> Protein <212> Artificial Sequence <213> Artificial Sequence <220>
O:\121\121929.DOC -185- 200808833 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 （2)..(2) <223> Xaa = Fc <400> 351O: \121\121929.DOC -185- 200808833 <223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> (2).. (2) <223> Xaa = Fc <400> 351
Met Xaa Gly Gly Gly Gly Gly Ala Gin His Gin Glu Tyr Cys Glu Gly 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin His Gin Glu Tyr Cys Glu Gly 1 5 10 15
Met Glu Asp Pro Phe Thr Phe Gly Cys Glu Tyr Gin Gly Leu Glu 20 25 30 <210> 352 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_ 特徵 <222> (2)..(2) <223> Xaa = Fc <400〉 352Met Glu Asp Pro Phe Thr Phe Gly Cys Glu Tyr Gin Gly Leu Glu 20 25 30 <210> 352 <211> 31 <212> Protein <213> Man-made Sequence <220><223> Ang-2-bound peptide body <220><22i> misc_ feature <222> (2)..(2) <223> Xaa = Fc <400> 352
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Asp Tyr Cys Glu Gly 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Asp Tyr Cys Glu Gly 1 5 10 15
Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Asn Gin Arg Leu Glu 20 25 30 •186·Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Asn Gin Arg Leu Glu 20 25 30 •186·
O:\121\121929.DOC 200808833 <210> 353 <211> 31 - <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc_特徵 <222> (2)..(2) <223> Xaa = Fc <400〉 353O:\121\121929.DOC 200808833 <210> 353 <211> 31 - <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <;220><22i>misc_features<222> (2)..(2) <223> Xaa = Fc <400> 353
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Leu Asp Tyr Cys Glu Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Leu Asp Tyr Cys Glu Gly 15 10 15
Val Gin Asp Pro Phe Thr Phe Gly Cys Glu Asn Leu Asp Leu Glu 20 25 30 <210> 354 <211> 31 <212>蛋白質 <213>人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222〉 （2)-.(2) <223〉 Xaa - Fc -187-Val Gin Asp Pro Phe Thr Phe Gly Cys Glu Asn Leu Asp Leu Glu 20 25 30 <210> 354 <211> 31 <212>Protein<213> Man-made sequence <220><223> Ang-2-bound peptide body <220><22i> misc-characteristic <222> (2)-.(2) <223> Xaa - Fc -187-
O:\121\121929.DOC 200808833 <400〉 354O:\121\121929.DOC 200808833 <400> 354
Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Phe Glu Tyr Cys Asp Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Gly Phe Glu Tyr Cys Asp Gly 15 10 15
Met Glu Asp Pro Phe Thr Phe Gly Cys Asp Lys Gin Thr Leu Glu 20 25 30 <210〉 355 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223>能夠與Ang-2結合的肽體 <220> <221> misc· _特徵 <222> (2) · · (2) <223> Xaa = Fc <400> 355Met Glu Asp Pro Phe Thr Phe Gly Cys Asp Lys Gin Thr Leu Glu 20 25 30 <210> 355 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220><221> misc· _ feature <222> (2) · · (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Gin Asp Tyr Cys Glu Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Ala Gin Asp Tyr Cys Glu Gly 15 10 15
Met Glu Asp Pro Phe Thr Phe Gly Cys Glu Met Gin Lys Leu Glu 20 25 30 <210> 356 <211> 31 <212>蛋白質 <213>人造的序列 •188-Met Glu Asp Pro Phe Thr Phe Gly Cys Glu Met Gin Lys Leu Glu 20 25 30 <210> 356 <211> 31 <212> Protein <213> Artificial Sequence • 188-
O:\121\121929.DOC 200808833 <220> <223> 能夠與Ang-2結合的肽體 <220> <22i> misc—特徵 <222> · (2)_ · (2) <223> Xaa = Fc <400> 356O:\121\121929.DOC 200808833 <220><223> Peptide capable of binding to Ang-2 <220><22i> misc - feature <222> · (2)_ (2) <223> Xaa = Fc <400> 356
Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Asp Tyr Cys Glu Gly 1 5 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Leu Gin Asp Tyr Cys Glu Gly 1 5 10 15
Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Lys Gin Arg Leu Glu 20 25 30 <210〉 357 <211> 31 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的肽體 <220〉 <22i> misc^ 特徵 <222> (2).·(2) <223> Xaa = Fc <400> 357Val Glu Asp Pro Phe Thr Phe Gly Cys Glu Lys Gin Arg Leu Glu 20 25 30 <210> 357 <211> 31 <212> Protein <213> Artificial Sequence <220><223> Ang-2-bound peptide body <220> <22i> misc^ feature <222> (2). (2) <223> Xaa = Fc <400>
Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Leu Glu Tyr Cys Asp Gly 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Lys Leu Glu Tyr Cys Asp Gly 15 10 15
Met Glu Asp Pro Phe TJir Gin Gly Cys Asp Asn Gin Ser Leu Glu 20 25 30 189-Met Glu Asp Pro Phe TJir Gin Gly Cys Asp Asn Gin Ser Leu Glu 20 25 30 189-
O:\121\121929.DOC 200808833 <210> 358 <211〉 29 <212>蛋白質 <213>人造的序列 <220〉 <223> 能夠與Ang-2結合的肽體 <220> <221> misc_特徵 <222〉 （2) ·· (2) <223〉 Xdd. == Fc <400〉 358O:\121\121929.DOC 200808833 <210> 358 <211> 29 <212> Protein <213> Artificial sequence <220><223> Peptide capable of binding to Ang-2 <220><221>misc_feature<222> (2) ·· (2) <223> Xdd. == Fc <400> 358
Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Asp Tyr Cys Glu Gly Val 15 10 15Met Xaa Gly Gly Gly Gly Gly Ala Gin Phe Asp Tyr Cys Glu Gly Val 15 10 15
Glu Asp Pro Phe Thr Phe Gly Cys Asp Asn His Leu Glu 20 25 <210> 359 <211> 32 <212>蛋白質 <213>人造的序列 <220> <223> 能夠與Ang-2結合的多肽 <400〉 359Glu Asp Pro Phe Thr Phe Gly Cys Asp Asn His Leu Glu 20 25 <210> 359 <211> 32 <212> Protein <213> Artificial Sequence <220><223> Bound peptide <400> 359
Cys Gly Gly Gly Gly Gly Ala Gin Thr Asn Phe Met Pro Met Asp Asp 15 10 1-5Cys Gly Gly Gly Gly Gly Ala Gin Thr Asn Phe Met Pro Met Asp Asp 15 10 1-5
Leu Glu Gin Arg Leu Tyr Glu Gin Phe He Leu Gin Gin Gly Leu Glu 20 25 30 -190-Leu Glu Gin Arg Leu Tyr Glu Gin Phe He Leu Gin Gin Gly Leu Glu 20 25 30-190-
O:\121\121929.DOCO:\121\121929.DOC
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US32862401P | 2001-10-11 | 2001-10-11 | |
| US41415502P | 2002-09-27 | 2002-09-27 |
| Publication Number | Publication Date |
|---|---|
| TW200808833Atrue TW200808833A (en) | 2008-02-16 |
| TWI336333B TWI336333B (en) | 2011-01-21 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW95125493ATWI324612B (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 (5) |
| TW91123468ATWI317281B (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| TW095125478ATW200635952A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| TW96129864ATWI336333B (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 (6) |
| TW095125490ATW200635951A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| TW095125482ATW200635950A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW95125493ATWI324612B (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 (5) |
| TW91123468ATWI317281B (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| TW095125478ATW200635952A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW095125490ATW200635951A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| TW095125482ATW200635950A (en) | 2001-10-11 | 2002-10-14 | Specific binding agents of human angiopoietin-2 |
| Country | Link |
|---|---|
| TW (6) | TWI324612B (en) |
| Publication number | Publication date |
|---|---|
| TWI324612B (en) | 2010-05-11 |
| TW200635952A (en) | 2006-10-16 |
| TW200635953A (en) | 2006-10-16 |
| TW200635950A (en) | 2006-10-16 |
| TW200635951A (en) | 2006-10-16 |
| TWI317281B (en) | 2009-11-21 |
| TWI336333B (en) | 2011-01-21 |
| Publication | Publication Date | Title |
|---|---|---|
| CN101787072B (en) | Specific binding agents of human angiopoietin-2 | |
| US20030236193A1 (en) | Methods of treatment using specific binding agents of human angiopoietin-2 | |
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| TWI324612B (en) | Specific binding agents of human angiopoietin-2 (5) | |
| HK1145692B (en) | Specific binding agents of human angiopoietin-2 | |
| HK1076113B (en) | Specific binding agents of human angiopoietin-2 | |
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| AU2006200977B2 (en) | Specific binding agents of human angiopoietin-2 | |
| HK1133017B (en) | Specific binding agents of human angiopoietin-2 |
| Date | Code | Title | Description |
|---|---|---|---|
| MK4A | Expiration of patent term of an invention patent |