CN100460421C - Immunoglobulin variants and uses thereof - Google Patents
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Abstract
Description
Translated fromChinese技术领域technical field
本发明涉及抗-CD20抗体及其在治疗B细胞相关疾病中的用途。The present invention relates to anti-CD20 antibody and its use in the treatment of B cell related diseases.
背景技术Background technique
淋巴细胞是白细胞的几个群体之一；它们特异性地识别和对外部抗原作出反应。淋巴细胞的三种主要类别是B淋巴细胞(B细胞)、T淋巴细胞(T细胞)与自然杀伤(NK)细胞。B淋巴细胞是负责抗体产生与提供体液免疫的细胞。B细胞在骨髓内部成熟并离开骨髓，在它们的细胞表面表达与抗原结合的抗体。当幼稚B细胞首次遇到其膜-结合抗体所特异的抗原时，细胞开始迅速分裂，其后代分化成记忆B细胞与被称为“浆细胞”的效应细胞。记忆B细胞具有较长寿命期限，并且继续表达与最初的母细胞具有相同特异性的膜结合抗体。浆细胞不产生膜结合抗体，但是相反产生分泌形式的抗体。分泌的抗体是体液免疫的主要效应物。Lymphocytes are one of several groups of white blood cells; they specifically recognize and respond to external antigens. The three main classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK) cells. B lymphocytes are the cells responsible for antibody production and providing humoral immunity. B cells mature inside the bone marrow and exit the bone marrow, expressing antibodies that bind to antigens on their cell surface. When a naive B cell first encounters an antigen to which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into memory B cells and effector cells called "plasma cells." Memory B cells have a longer lifespan and continue to express membrane-bound antibodies with the same specificity as the original parent cells. Plasma cells do not produce membrane-bound antibodies, but instead produce secreted forms of antibodies. Secreted antibodies are the major effectors of humoral immunity.
CD20抗原(也被称作人B淋巴细胞限制的分化抗原，Bp35)是位于前-B与成熟B淋巴细胞上的疏水跨膜蛋白质，具有分子量大约35kD。J.Biol.Chem.264(19)：11282-11287(1989)；与Einfeld et al.EMBO J.7(3)：711-717(1988))。该抗原也在超过90％的B细胞非何杰金氏淋巴瘤(NHL)上表达Blood 63(6)：1424-1433(1984))，但是在造血干细胞、原B细胞、正常浆细胞或者其它正常组织中没有找到(Tedder et al.J.Immunol.135(2)：973-979(1985))。CD20被认为调节细胞周期起始与分化的激活过程中的一个早期步骤。(Tedder et al.，supra)，并且可能作为钙离子通道发挥作用(Tedder et al.J.Cell.Biochem.14D：195(1990))。CD20 antigen (also known as human B lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein on pre-B and mature B lymphocytes with a molecular weight of approximately 35 kD. J. Biol. Chem. 264(19): 11282-11287 (1989); with Einfeld et al. EMBO J. 7(3): 711-717 (1988)). This antigen is also expressed on more than 90% of B-cell non-Hodgkin's lymphomas (NHL Blood 63(6):1424-1433 (1984)), but not on hematopoietic stem cells, primary B cells, normal plasma cells, or other Not found in normal tissues (Tedder et al. J. Immunol. 135(2):973-979(1985)). CD20 is thought to regulate an early step in the activation process of cell cycle initiation and differentiation. (Tedder et al., supra), and may function as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D: 195 (1990)).
由于CD20在B细胞淋巴瘤中的表达，这一抗原已被用作治疗该淋巴瘤的有用的治疗靶向。在美国有超过300,000人患有B细胞NHL，每年有超过56,000个新病例被诊断。例如，rituximab抗体被用来治疗具有复发或难治疗的低级或者滤泡的、CD20阳性的B细胞非何杰金氏淋巴瘤，该抗体是遗传改造的针对人CD20抗原的嵌合鼠/人单克隆抗体(可从Genentech，Inc.South San Francisco，California，U.S.购得)。Rituximab是在1998年4月7日发布的美国专利号5,736,137(Anderson et al.)与在美国专利号5,776,456中被称做“C2B8＂的抗体。体外作用机理研究显示
鼠抗体在人类治疗中的使用的一个主要限制之处在于人抗小鼠抗体(HAMA)反应(参见，例如Miller，R.A.et al.“Monoclonal antibody therapeutictrials in seven patients with T-cell lymphoma”Blood，62：988-995，1983；和Schroff，R.W.，et al.“Human anti-murine immunoglobulin response in patientsreceiving monoclonal antibody therapy”Cancer Res.，45：879-885，1985)。即使啮齿类动物抗体的可变(V)结构域与人恒定(C)区域融合的嵌合分子仍然能引发显著的免疫反应(HACA，人抗嵌合抗体)Nature(Lond.)，314：268-270，1985)。克服在单克隆抗体的临床利用中的这些限制的一个有效手段是“人源化”鼠抗体或者来自非人物种的抗体。(Jones et al.Nature(Lond)，321：522-525，1986；Riechman et al.，Nature(Lond)，332：323-327，1988)。A major limitation of the use of murine antibodies in human therapy is the human anti-mouse antibody (HAMA) response (see, e.g., Miller, R.A. et al. "Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma" Blood, 62 : 988-995, 1983; and Schroff, R.W., et al. "Human anti-murine immunoglobulin response in patients receiving monoclonal antibody therapy" Cancer Res., 45:879-885, 1985). Even a chimeric molecule in which the variable (V) domain of a rodent antibody is fused to a human constant (C) region can still elicit a significant immune response (HACA, human anti-chimeric antibody) Nature (Lond.), 314: 268 -270, 1985). An effective means of overcoming these limitations in the clinical utility of monoclonal antibodies are "humanized" murine antibodies or antibodies from non-human species. (Jones et al. Nature (Lond), 321:522-525, 1986; Riechman et al., Nature (Lond), 332:323-327, 1988).
因此，生产针对CD20抗原的治疗抗体是有利的，该抗体当给药给病人、特别是用于长期治疗时产生最低的或没有抗原性。本发明满足这一及其它需求。本发明提供克服目前治疗性组合物限制性的抗CD20抗体，以及提供额外的好处，这些好处将从下面的详细说明中加以明了。Accordingly, it would be advantageous to produce therapeutic antibodies directed against the CD20 antigen that produce minimal or no antigenicity when administered to a patient, especially for chronic treatment. The present invention fulfills this and other needs. The present invention provides anti-CD20 antibodies that overcome the limitations of current therapeutic compositions, as well as providing additional benefits that will become apparent from the detailed description below.
发明概述Summary of the invention
本发明提供CD20结合抗体或其功能性片段，以及其在治疗B细胞相关疾病中的用途。这些抗体是单克隆抗体。在特定的实施方式中，结合CD20的抗体是人源化的或者嵌合的。人源化的2H7变体包括在FR中具有氨基酸取代的变体，以及在移植的CDR中具有改变的亲和力成熟变体。在CDR或者FR中取代的氨基酸不局限于在供体或者受体抗体中存在的那些氨基酸。在其它实施方式中，本发明的抗-CD20抗体更进一步地包含Fc区域的氨基酸残基改变，这些改变导致效应子功能改善，包括增强CDC和/或ADCC功能与B细胞杀伤(在此也称为B细胞耗尽)。本发明的其它抗-CD20抗体包括那些具有能改善稳定性的改变的抗体。在一个特殊的具体实施方式中，稳定性增加的人源化2H7变体在下面实施例6中加以描述。还提供在体内具有改善的ADCC功能的岩藻糖缺陷的变体。在一个实施方式中，嵌合抗CD20抗体具有鼠可变区与人恒定区。一个像这样的特殊嵌合抗CD20抗体是(Rituximab；Genentech，Inc.)。The present invention provides a CD20-binding antibody or a functional fragment thereof, and its use in treating B cell-related diseases. These antibodies are monoclonal antibodies. In specific embodiments, antibodies that bind CD20 are humanized or chimeric. Humanized 2H7 variants included variants with amino acid substitutions in FRs, as well as affinity matured variants with altered grafted CDRs. Amino acids substituted in CDRs or FRs are not limited to those present in the donor or acceptor antibody. In other embodiments, the anti-CD20 antibodies of the present invention further comprise amino acid residue changes in the Fc region that result in improved effector functions, including enhanced CDC and/or ADCC function and B cell killing (also referred to herein as depleted of B cells). Other anti-CD20 antibodies of the invention include those with alterations that improve stability. In a specific embodiment, humanized 2H7 variants with increased stability are described in Example 6 below. Fucose-deficient variants with improved ADCC function in vivo are also provided. In one embodiment, a chimeric anti-CD20 antibody has murine variable regions and human constant regions. A specific chimeric anti-CD20 antibody like this one is (Rituximab; Genentech, Inc.).
在本发明的所有抗体组合物和使用方法的一个优选实施方式中，人源化的CD20结合抗体是2H7.v16，具有分别如图6和图7所示的SEQ ID NO.21和22的轻与重链氨基酸序列。当提及在图6、7和8中的多肽序列时，应该理解，形成分泌信号序列的前19个左右氨基酸在成熟多肽中不存在。除了在说明书中指示的氨基酸取代位置之外，基于version 16的所有其它变体的可变区将具有v16的氨基酸序列。除非另外指明，2H7变体将具有与v16相同的轻链。In a preferred embodiment of all antibody compositions and methods of use of the present invention, the humanized CD20-binding antibody is 2H7.v16, which has the light of SEQ ID NO.21 and 22 as shown in Figure 6 and Figure 7, respectively. and heavy chain amino acid sequence. When referring to the polypeptide sequences in Figures 6, 7 and 8, it is understood that the first 19 or so amino acids forming the secretion signal sequence are absent in the mature polypeptide. The variable regions of all other variants based on
本发明提供与人CD20结合的人源化抗体或者其抗原结合片段，其中抗体对体内消减灵长类动物B细胞是有效的，该抗体在H链可变区(VH)包含至少来自抗人CD20抗体的SEQ ID NO.12的CDR3序列、和人重链亚群(subgroup)III(VHIII)的基本上(substantially)的人共有框架(FR)残基。在一个实施方式中，灵长类动物B细胞来自于人和猕猴。在一个实施方式中，抗体更进一步包含SEQ ID NO.10的H链CDR1序列与SEQ ID NO.11的CDR2序列。在另一个实施方式中，前述抗体包含SEQ ID NO.4的L链CDR1序列、SEQ ID NO.5的CDR2序列、SEQ ID NO.6的CDR3序列，具有人轻链κ亚群I(VκI)的实质上人共有框架(FR)残基。在一优选实施方案中，VL的FR区域在位置46具有供体抗体残基；在一个具体的实施方式中，VL的FR2具有leuL46pro的氨基酸取代(在人κI共有序列中的Leu换成在m2H7相应位置存在的pro)。VH区域更进一步在框架的至少氨基酸位置49、71与73上包含供体抗体残基。在一个实施方式中，在VH中，人重链亚群III的以下FR位置被取代：FR2中的AlaH49Gly；FR3中的ArgH71Val与AsnH73Lys。在其它实施方式中，人源化抗体的CDR区域更进一步包含氨基酸取代，其中残基既不来自供体抗体也不来自受体抗体。The present invention provides a humanized antibody or an antigen-binding fragment thereof that binds to human CD20, wherein the antibody is effective for depleting primate B cells in vivo, the antibody comprising atleast The CDR3 sequence of SEQ ID NO. 12 of the CD20 antibody, and substantially human consensus framework (FR) residues of human heavy chain subgroup III (VH III). In one embodiment, the primate B cells are from humans and macaques. In one embodiment, the antibody further comprises the H chain CDR1 sequence of SEQ ID NO.10 and the CDR2 sequence of SEQ ID NO.11. In another embodiment, the aforementioned antibody comprises the L chain CDR1 sequence of SEQ ID NO.4, the CDR2 sequence of SEQ ID NO.5, the CDR3 sequence of SEQ ID NO.6, and has human light chain κ subgroup I (VκI) substantially human consensus framework (FR) residues. In a preferred embodiment, the FR region of theVL has a donor antibody residue at position 46; in a specific embodiment, the FR2 ofthe VL has an amino acid substitution of leuL46pro (in the human κI consensus sequence Leu is replaced by pro present at the corresponding position in m2H7). TheVH region further comprises donor antibody residues at least at
前述实施方式的抗体可以包含v16的SEQ ID NO 8的VH序列，如图1B所示。在前面的更进一步的实施方式中，抗体更进一步包含v16的SEQID NO 2的VL序列，如图1A所示。The antibody of the foregoing embodiment may comprise theVH sequence of
在其它实施方式中，人源化抗体是2H7.v31，具有SEQ ID NO.21与23的轻与重链氨基酸序列，分别如图6与图8所示；具有如图8所示SEQ ID NO.23的重链氨基酸序列的2H7.v31；具有v16的H链的D56A与N100A与L链的S92A的氨基酸取代的2H7.v96。In other embodiments, the humanized antibody is 2H7.v31, which has the light and heavy chain amino acid sequences of SEQ ID NO.21 and 23, as shown in Figure 6 and Figure 8, respectively; has the SEQ ID NO as shown in Figure 8 2H7.v31 of the heavy chain amino acid sequence of .23; 2H7.v96 with amino acid substitutions of D56A and N100A of the H chain and S92A of the L chain of v16.
在分开的实施方式中，任何前述实施方式中的抗体更进一步包含在Fc区域中的至少一个氨基酸取代，该取代与衍生它的原始或者亲本抗体相比改善了ADCC和/或CDC活性，在大多数场合下被比较的亲本抗体是v.16，在其它情况下是Rituxan。具有改善活性的一个象这样的抗体在Fc区域包含S298A/E333A/K334A的三重丙氨酸取代。具有S298A/E333A/K334A取代的一个抗体是具有SEQ ID NO.23的重链氨基酸序列的2H7.v31。同Rituxan比较起来抗体2H7.v114与2H7.v115显示ADCC活性改善至少10倍。In a separate embodiment, the antibody of any of the preceding embodiments further comprises at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity compared to the original or parental antibody from which it was derived, in large The parental antibody being compared was v.16 in most cases and Rituxan in others. One such antibody with improved activity contained a triple alanine substitution of S298A/E333A/K334A in the Fc region. One antibody with the S298A/E333A/K334A substitution was 2H7.v31 with the heavy chain amino acid sequence of SEQ ID NO.23. Antibodies 2H7.v114 and 2H7.v115 showed at least a 10-fold improvement in ADCC activity compared to Rituxan.
在另一个实施方式中，抗体更进一步包含在Fc区域的至少一个氨基酸取代，该取代同衍生它的亲本抗体比较起来CDC活性降低，衍生它的亲本抗体在大多数情况下是v16。同v16比较起来CDC活性降低的一个象这样的抗体包含在H链中的至少K322A取代。ADCC与CDC活性的比较可以如实施例中所描述的进行分析。In another embodiment, the antibody further comprises at least one amino acid substitution in the Fc region that has reduced CDC activity compared to the parent antibody from which it was derived, which in most cases is v16. One such antibody with reduced CDC activity compared to v16 contains at least a K322A substitution in the H chain. Comparison of ADCC and CDC activity can be analyzed as described in the Examples.
在一优选实施方式中，本发明的抗体是全长抗体，其中VH区域连接至人IgG重链恒定区。在优选实施方式中，IgG是人IgG1或者IgG3。In a preferred embodiment, the antibody of the invention is a full-length antibody, wherein theVH region is linked to a human IgG heavy chain constant region. In a preferred embodiment, the IgG is human IgG1 or IgG3.
在一个实施方式中，CD20结合抗体结合至细胞毒性剂。在优选实施方式中，该细胞毒性剂是毒素或者放射性同位素。In one embodiment, the CD20-binding antibody binds to a cytotoxic agent. In preferred embodiments, the cytotoxic agent is a toxin or a radioisotope.
在一个实施方式中，本发明的用于治疗或者诊断用途的抗体在CHO细胞中产生。In one embodiment, antibodies of the invention for therapeutic or diagnostic use are produced in CHO cells.
还提供一种组合物，该组合物包含前述实施方式中的任何一个的抗体，以及载体。在一个实施方式中，载体是药学上可以接受的载体。这些组合物可以以制品或者试剂盒提供。Also provided is a composition comprising the antibody of any one of the preceding embodiments, and a carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier. These compositions can be provided as articles of manufacture or kits.
本发明也提供液体制剂，包含20mg/mL抗体的人源化2H7抗体，10mM硫酸组氨酸pH5.8，60mg/mL蔗糖(6％)，0.2mg/mL聚山梨酸酯20(0.02％)。The invention also provides a liquid formulation, humanized 2H7 antibody comprising 20 mg/mL antibody, 10 mM histidine sulfate pH 5.8, 60 mg/mL sucrose (6%), 0.2 mg/mL polysorbate 20 (0.02%) .
本发明也提供分离的核酸，该核酸编码在此所披露的抗体中的任何一种，包括表达抗体的表达载体。The invention also provides isolated nucleic acids encoding any of the antibodies disclosed herein, including expression vectors for expressing the antibodies.
本发明的另一个方面是包含前述核酸的宿主细胞与产生该抗体的宿主细胞。在后者的一个优选实施方案中，宿主细胞是CHO细胞。提供了产生这些抗体的方法，该方法包含培养产生抗体的宿主细胞，并且从细胞培养物中回收抗体。Another aspect of the present invention is a host cell comprising the aforementioned nucleic acid and a host cell producing the antibody. In a preferred embodiment of the latter, the host cells are CHO cells. Methods of producing these antibodies are provided, the methods comprising culturing antibody-producing host cells, and recovering the antibodies from the cell culture.
本发明的另一方面是一种制品，包括一个容器与包含在其中的组合物，其中组合物包含前述实施方式中的任何一个的抗体。为用来治疗NHL，该制品更进一步包含包装说明书，指示该组合物被用来治疗非何杰金氏淋巴瘤。Another aspect of the invention is an article of manufacture comprising a container and a composition contained therein, wherein the composition comprises the antibody of any one of the preceding embodiments. For use in the treatment of NHL, the article of manufacture further comprises a package insert indicating that the composition is used to treat non-Hodgkin's lymphoma.
本发明的更进一步的方面是体内在B细胞中诱发程序性细胞死亡的方法，包含将B细胞与前述中的任何的抗体接触，从而杀死B细胞。A further aspect of the invention is a method of inducing apoptosis in B cells in vivo, comprising contacting the B cells with any of the aforementioned antibodies, thereby killing the B cells.
本发明也提供治疗在此所披露的疾病的方法，该方法通过将CD20结合抗体或者其功能性片段给药哺乳动物，例如患该疾病的人类病人，而治疗该疾病。在任何治疗自身免疫病或者CD20阳性癌症的方法中，在一个实施方式中，抗体是2H7.v16，分别具有SEQ ID NO.21与22的轻与重链氨基酸序列，如图6与图7所示。因此，一个实施方式是治疗CD20阳性癌症的方法，包含给予患该癌症的病人治疗有效量的本发明的人源化CD20结合抗体。在优选实施方式中，CD20阳性癌症是B细胞淋巴瘤或者白血病包括非何杰金氏淋巴瘤(NHL)或者淋巴细胞占优势的何杰金氏病(LPHD)、慢性淋巴细胞性白血病(CLL)或者SLL。在治疗B细胞淋巴瘤或者白血病的方法的实施方式中，抗体以剂量范围大约275-375mg/m2给药。在其它的实施方式中，治疗方法更进一步包含给予病人至少一种化疗剂，在其中对于非何杰金氏淋巴瘤(NHL)而言，化疗剂选自由阿霉素、环磷酰胺、长春新碱与氢化泼尼松(prednisolone)组成的组。The invention also provides methods of treating the diseases disclosed herein by administering a CD20-binding antibody or functional fragment thereof to a mammal, eg, a human patient suffering from the disease. In any method for treating autoimmune diseases or CD20 positive cancers, in one embodiment, the antibody is 2H7.v16, having the light and heavy chain amino acid sequences of SEQ ID NO.21 and 22, respectively, as shown in Figure 6 and Figure 7 Show. Accordingly, one embodiment is a method of treating a CD20-positive cancer comprising administering to a patient suffering from the cancer a therapeutically effective amount of a humanized CD20-binding antibody of the invention. In a preferred embodiment, the CD20 positive cancer is B-cell lymphoma or leukemia including non-Hodgkin's lymphoma (NHL) or lymphocyte-predominant Hodgkin's disease (LPHD), chronic lymphocytic leukemia (CLL) Or SLL. In an embodiment of the method of treating B cell lymphoma or leukemia, the antibody is administered in a dosage range of about 275-375 mg/m2 . In other embodiments, the method of treatment further comprises administering to the patient at least one chemotherapeutic agent, wherein for non-Hodgkin's lymphoma (NHL), the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine, A group consisting of base and prednisolone.
也提供治疗自身免疫病的方法，包含给予病人治疗有效量的前述权利要求中任何一个的人源化CD20结合抗体，该病人患有自身免疫病。自身免疫病选自由下列组成的组：类风湿性关节炎、青少年类风湿性关节炎、系统性红斑狼疮(SLE)、韦格内氏病(Wegener’s disease)、炎症性肠病、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、自身免疫性血小板减少、多发性硬化、牛皮癣、IgA肾病、IgM多神经病、重症肌无力、血管炎、糖尿病、雷诺氏征(Reynaud’s syndrome)、斯耶格仑氏综合症(sjorgen’ssyndrome)与肾小球肾炎。当自身免疫病是类风湿性关节炎时，抗体可以与第二治疗剂联合给药，该第二治疗剂优选地是氨甲喋呤。Also provided is a method of treating an autoimmune disease comprising administering a therapeutically effective amount of a humanized CD20-binding antibody of any one of the preceding claims to a patient suffering from an autoimmune disease. The autoimmune disease is selected from the group consisting of: rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic platelet ITP, TTP, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome syndrome), Sjogren's syndrome (sjorgen'ssyndrome) and glomerulonephritis. When the autoimmune disease is rheumatoid arthritis, the antibody may be administered in combination with a second therapeutic agent, preferably methotrexate.
在这些治疗方法中，CD20结合抗体可以单独或者与第二治疗剂结合给药，第二治疗剂例如第二抗体、或者化疗剂或免疫抑制剂。第二抗体可以是结合CD20或者不同B细胞抗原、或者NK或T细胞抗原的抗体。在一个实施方式中，第二抗体是放射性同位素标记的抗CD20抗体。在其它实施方式中，CD20结合抗体偶联至细胞毒性剂，包括毒素或者放射性同位素。In these methods of treatment, the CD20-binding antibody can be administered alone or in combination with a second therapeutic agent, such as a second antibody, or a chemotherapeutic or immunosuppressive agent. The second antibody can be an antibody that binds CD20 or a different B cell antigen, or an NK or T cell antigen. In one embodiment, the second antibody is a radiolabeled anti-CD20 antibody. In other embodiments, the CD20-binding antibody is conjugated to a cytotoxic agent, including a toxin or a radioisotope.
在另一方面，本发明提供治疗自身免疫病的方法，自身免疫病选自由皮肌炎(Dermatomyositis)、韦格内氏肉芽肿病、ANCA、再生障碍性贫血、自身免疫性溶血性贫血(AIHA)、凝血因子VIII缺乏、甲型血友病、自身免疫性中性白细胞减少、Castleman′s综合症、古德帕斯彻氏综合症(Goodpasture’s syndrome)、实体器官移植排斥、移植物抗宿主疾病(GVHD)、IgM介导的神经病、血栓性血小板减少性紫癜(TTP)、桥本氏甲状腺炎(Hashimoto’s Thyroiditis)、自身免疫性肝炎、淋巴组织间质肺炎(HIV)、闭塞性细支气管炎(非移植)对应的NSIP(bronchiolitis obliterans(non-transplant)vs.NSIP)、格-巴二氏综合症(Guillain-Barre syndrome)、大血管血管炎、巨细胞(高安氏)动脉炎(giant cell(Takayasu’s)ateritis)、中等血管血管炎(mediumvessel vasculitis)、川崎病(Kawasaki’s Disease)、结节性多动脉炎(polyateritisnodosa)，包含给予患该疾病的患者治疗有效量的CD20结合抗体。在该方法的一个实施方式中，CD20结合抗体是
本发明还提供分离的核酸，包含猕猴(Cynomolgus monkey)CD20的SEQID NO.：24的核苷酸序列(如图19所示)，或该序列的简并变体。一个实施方式是分离的核酸，包含一序列，该序列编码具有SEQ ID NO.25(如图20所示)的氨基酸序列的多肽，或具有保守性氨基酸取代的SEQ ID NO.25(图20)。另一个实施方式是包含前述核酸的载体，包括用来在宿主细胞表达的表达载体。也包括包含该载体的宿主细胞。还提供分离的多肽，该多肽包含猕猴CD20的氨基酸序列(SEQ ID NO.25；图20)。The present invention also provides an isolated nucleic acid comprising the nucleotide sequence of SEQID NO.: 24 of Cynomolgus monkey CD20 (as shown in FIG. 19 ), or a degenerate variant of the sequence. One embodiment is an isolated nucleic acid comprising a sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO. 25 (as shown in Figure 20), or SEQ ID NO. 25 (Figure 20) with conservative amino acid substitutions . Another embodiment is a vector comprising the aforementioned nucleic acid, including an expression vector for expression in a host cell. Host cells comprising the vector are also included. Also provided is an isolated polypeptide comprising the amino acid sequence of Cynomolgus CD20 (SEQ ID NO. 25; Figure 20).
附图说明Description of drawings
图1A是序列比对，比较小鼠2H7(SEQ ID NO.1)、人源化2H7.v16变体(SEQ ID NO.2)、和人κ轻链亚群I(SEQ ID NO.3)中每一个的轻链可变结构域(VL)的氨基酸序列。2H7和hu2H7.v16的VL的CDR如下：CDR1(SEQ IDNO.4)、CDR2(SEQ ID NO.5)、与CDR3(SEQ ID NO.6)。Figure 1A is a sequence alignment comparing mouse 2H7 (SEQ ID NO.1), humanized 2H7.v16 variant (SEQ ID NO.2), and human kappa light chain subgroup I (SEQ ID NO.3) The amino acid sequence of the light chain variable domain (VL ) of each of . The CDRs of theVL of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO. 4), CDR2 (SEQ ID NO. 5), and CDR3 (SEQ ID NO. 6).
图1B是序列比对，比较如下来源的VH序列：鼠2H7(SEQ ID NO.7)、人源化2H7.v16变体(SEQ ID NO.8)、和重链亚群III的人共有序列(SEQ IDNO.9)。2H7和hu2H7.v16的VH的CDR如下：CDR1(SEQ ID NO.10)、CDR2(SEQ ID NO.11)、与CDR3(SEQ ID NO.12)。Figure 1B is a sequence alignment comparingVH sequences from the following sources: murine 2H7 (SEQ ID NO. 7), humanized 2H7.v16 variant (SEQ ID NO. 8), and the human consensus of heavy chain subgroup III Sequence (SEQ ID NO. 9). The CDRs of theVH of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO. 10), CDR2 (SEQ ID NO. 11), and CDR3 (SEQ ID NO. 12).
在图1A与图1B中，每一链的CDR1、CDR2与CDR3用括号围起来，其两侧为框架区FR1-FR4，如图中所指示。2H7指鼠2H7抗体。两行序列之间的星号表示在两序列之间不同的位置。残基编号根据Kabat et al.，Sequences of Immunological Interest.5th Ed.Public Health Service，NationalInstitutes of Health，Bethesda，Md.(1991)，插入物用a、b、c、d、与e表示。In FIG. 1A and FIG. 1B , CDR1, CDR2 and CDR3 of each chain are surrounded by brackets, flanked by framework regions FR1-FR4, as indicated in the figure. 2H7 refers to the mouse 2H7 antibody. An asterisk between two sequences indicates a position that differs between the two sequences. Residue numbering is according to Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), inserts are indicated by a, b, c, d, and e.
图2显示用来构建2H7Fab质粒的噬菌粒pVX4的序列(SEQ ID NO.13)以及CDR移植的抗IFN-α人源化抗体的Fab的轻链(SEQ ID NO.14)与重链(SEQ ID NO.15)的氨基酸序列。Fig. 2 shows the light chain (SEQ ID NO.14) and the heavy chain (SEQ ID NO.14) of the Fab of the sequence (SEQ ID NO.13) that is used to construct the phagemid pVX4 of 2H7Fab plasmid and the anti-IFN-α humanized antibody of CDR transplantation The amino acid sequence of SEQ ID NO.15).
图3显示编码嵌合2H7.v6.8 Fab的表达质粒的序列(SEQ ID NO.16)。显示轻链(SEQ ID NO.17)与重链(SEQ ID NO.18)的氨基酸序列。Figure 3 shows the sequence (SEQ ID NO.16) of the expression plasmid encoding chimeric 2H7.v6.8 Fab. Amino acid sequences of light chain (SEQ ID NO.17) and heavy chain (SEQ ID NO.18) are shown.
图4显示如实施例1中所描述的用于表达免疫球蛋白轻链的质粒pDR1的序列(SEQ ID NO19；5391bp)。pDR1包含编码一无关抗体、人源化抗CD3抗体的轻链(Shalaby et al.，J.Exp.Med.175：217-225(1992))的序列，其起始和终止密码子以粗体和下划线指示。Figure 4 shows the sequence of plasmid pDR1 (SEQ ID NO 19; 5391 bp) used to express immunoglobulin light chains as described in Example 1. pDR1 contains the sequence encoding the light chain of an unrelated antibody, a humanized anti-CD3 antibody (Shalaby et al., J. Exp. Med. 175:217-225 (1992)), with start and stop codons in bold and underscored.
图5显示如实施例1中描写的用于表达免疫球蛋白重链的质粒pDR2的序列(SEQ ID NO.20；6135bp)。pDR2包含一编码无关抗体、人源化抗CD3抗体的重链的序列(Shalaby et al.，上文)，其起始和终止密码子以粗体与下划线指示。Figure 5 shows the sequence of plasmid pDR2 (SEQ ID NO. 20; 6135bp) used for expression of immunoglobulin heavy chain as described in Example 1. pDR2 contains a sequence encoding the heavy chain of an unrelated antibody, a humanized anti-CD3 antibody (Shalaby et al., supra), with start and stop codons indicated in bold and underlined.
图6显示2H7.v16完整轻链的氨基酸序列(SEQ ID NO.21)。DIQ以前的前19个氨基酸是在成熟多肽链中不存在的分泌性信号序列。Figure 6 shows the amino acid sequence of the complete light chain of 2H7.v16 (SEQ ID NO.21). The first 19 amino acids preceding DIQ are a secretory signal sequence that is absent in the mature polypeptide chain.
图7显示2H7.v16完整重链的氨基酸序列(SEQ ID NO.22)。EVQ以前的前19个氨基酸是在成熟多肽链中不存在的分泌性信号序列。将图1B的VH序列(SEQ ID NO.8)与完整重链序列比对，人γ1恒定区来自于SEQ ID NO.22中的氨基酸位置114-471。Figure 7 shows the amino acid sequence of the complete heavy chain of 2H7.v16 (SEQ ID NO. 22). The first 19 amino acids preceding EVQ are a secretory signal sequence that is absent in the mature polypeptide chain. Aligning theVH sequence (SEQ ID NO.8) in Figure 1B with the complete heavy chain sequence, the human γ1 constant region is from amino acid positions 114-471 in SEQ ID NO.22.
图8显示2H7.v31完整重链的氨基酸序列(SEQ ID NO.23)。EVQ以前的前19个氨基酸是在成熟多肽链中不存在的分泌性信号序列。轻链与2H7.v16相同(参见图6)。Figure 8 shows the amino acid sequence of the complete heavy chain of 2H7.v31 (SEQ ID NO.23). The first 19 amino acids preceding EVQ are a secretory signal sequence that is absent in the mature polypeptide chain. The light chain is identical to 2H7.v16 (see Figure 6).
图9显示如实施例中所描述的2H7.v16与2H7.v73 IgG变体的相对稳定性。分析结果校正至孵育前数值，以孵育后的剩余百分比报告。Figure 9 shows the relative stability of 2H7.v16 and 2H7.v73 IgG variants as described in the Examples. Assay results were corrected to pre-incubation values and reported as percent remaining after incubation.
图10是一流程图，总结从鼠2H7至直到v75的人源化型式的亚群的氨基酸改变。Figure 10 is a flowchart summarizing the amino acid changes from murine 2H7 to a subset of humanized versions up to v75.
图11是在所有组(结合2H7研究与Rituxan研究)中平均绝对B细胞计数[CD3-/CD40+]的总结，如实施例10中所描述。Figure 11 is a summary of mean absolute B cell counts [CD3-/CD40+] in all groups (2H7 study combined with Rituxan study), as described in Example 10.
图12显示如在实施例11中所描述的在岩藻糖缺陷的2H7变体上代表性ADCC试验的结果。Figure 12 shows the results of a representative ADCC assay as described in Example 11 on fucose-deficient 2H7 variants.
图13显示将膜联蛋白V染色以抗体浓度的函数作图的结果。在交联第二抗体存在下，将Ramos细胞用无关IgG1对照抗体(Herceptin；圆形)、Rituximab(方形)、或者rhuMAb 2H7.v16(三角形)处理，用FACS分析。图13-15在实施例13中加以描述。Figure 13 shows the results of plotting Annexin V staining as a function of antibody concentration. Ramos cells were treated with an irrelevant IgGl control antibody (Herceptin; circles), Rituximab (squares), or rhuMAb 2H7.v16 (triangles) in the presence of a cross-linked secondary antibody and analyzed by FACS. Figures 13-15 are described in Example 13.
图14显示膜联蛋白V与碘丙锭双染色作为抗体浓度的函数作图的结果。在交联第二抗体存在下，将Ramos细胞用无关IgG1对照抗体(
图15显示活的、未染色细胞计数(每10个)作为抗体浓度的函数作图。在交联第二抗体存在下，将Ramos细胞用无关IgG1对照抗体(；圆形)、Rituximab(方形)、或者rhuMAb 2H7.v16(三角形)处理，用FACS分析。Figure 15 shows viable, unstained cell counts (per 10) plotted as a function of antibody concentration. In the presence of a cross-linked secondary antibody, Ramos cells were treated with an irrelevant IgG1 control antibody ( ; circles), Rituximab (squares), or rhuMAb 2H7.v16 (triangles) treatment, analyzed by FACS.
图16、17、18显示如实施例14中所描述的在裸鼠中对Raji细胞肿瘤生长的抑制。动物用PBS(对照)或者用
图19显示如实施例15中所描述的猕猴CD20的核苷酸(SEQ ID NO.24)与氨基酸(SEQ ID NO.25)序列。Figure 19 shows the nucleotide (SEQ ID NO.24) and amino acid (SEQ ID NO.25) sequence of rhesus monkey CD20 as described in Example 15.
图20显示猕猴CD20的氨基酸序列(SEQ ID NO.25)。与人CD20不同的残基有下划线，人残基(SEQ ID NO.26)直接在猴残基下标示。猴CD20的推定胞外结构域以黑体字表示。Figure 20 shows the amino acid sequence (SEQ ID NO.25) of macaque CD20. Residues that differ from human CD20 are underlined and human residues (SEQ ID NO. 26) are indicated directly under monkey residues. The putative extracellular domain of monkey CD20 is in bold.
图21显示如在实施例15中所描述的，表达CD20的猕猴细胞与hu2H7.v16、.v31与Rituxan的结合。分析抗体结合猕猴CD20并且取代FITC偶联的鼠2H7与猕猴CD20结合的能力。Figure 21 shows the binding of CD20 expressing macaque cells to hu2H7.v16, .v31 and Rituxan as described in Example 15. Antibodies were assayed for their ability to bind macaque CD20 and displace FITC-conjugated murine 2H7 from binding macaque CD20.
图22显示类风湿性关节炎临床试验阶段I/II剂量渐增的方案。Figure 22 shows the phase I/II dose escalation scheme of the rheumatoid arthritis clinical trial.
图23显示在CHO细胞中表达2H7.v16的载体。Figure 23 shows vectors expressing 2H7.v16 in CHO cells.
优选实施方案的详细说明Detailed Description of the Preferred Embodiment
“CD20＂抗原是一非糖基化的跨膜磷蛋白，具有分子量大约35kD，在来自外周血或淋巴器官的超过90％的B细胞的表面上发现。CD20在早期前B细胞发育期间表达，并且保持直到浆细胞分化。其在人干细胞、淋巴祖先细胞或者正常浆细胞上没有发现。CD20在正常B细胞以及恶性B细胞上存在。在文献中CD20的其它名字包括“B淋巴细胞限制的分化抗原”和“Bp35＂。CD20抗原在例如Clark与Ledbetter，Adv.Can.Res.52：81-149(1989)与Valentine et al.J.Biol.Chem.264(19)：11282-112871989)中描述。The "CD20" antigen is a non-glycosylated transmembrane phosphoprotein with a molecular weight of approximately 35 kD, found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. It is not found on human stem cells, lymphoid progenitor cells, or normal plasma cells. CD20 is present on normal B cells as well as malignant B cells. Other names for CD20 in the literature include "B-lymphocyte-restricted differentiation antigen" and "Bp35". The CD20 antigen is described, eg, in Clark and Ledbetter, Adv. Can. Res. 52:81-149 (1989) and Valentine et al. J. Biol. Chem. 264(19):11282-112871989).
术语“抗体”以最宽泛的意义使用，并且特别地涵盖单克隆抗体(包括全长单克隆抗体)、多特异性抗体(例如双特异性抗体)、与抗体片段，只要它们显示出所需的生物活性或功能。The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (such as bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function.
本发明的CD20结合抗体与人源化CD20结合抗体的生物活性至少包括抗体与人CD20的结合，更优选与人及其它灵长类动物(包括猕猴、恒河猴、黑猩猩)CD20结合。抗体将以不高于1 x 10-8的Kd值结合CD20，优选地Kd值不高于大约1 x 10-9，并且能体内杀死或消减B细胞，优选地当与合适的负对照比较时杀死或者消减至少20％，所述负对照未用这样的抗体处理。B细胞消减可以是ADCC、CDC、细胞程序死亡或者其它机制中一个或多个的结果。在这里的疾病治疗的一些实施方式中，特定的效应子功能或者机制与其它相比可能更需要，并且人源化2H7的某些变体对取得生物学功能例如ADCC是优选的。The biological activity of the CD20-binding antibody and humanized CD20-binding antibody of the present invention at least includes the binding of the antibody to human CD20, more preferably binding to human and other primate (including rhesus monkey, rhesus monkey, chimpanzee) CD20. The antibody will bind CD20 with a Kd value of no greater than 1 x10-8 , preferably a Kd value of no greater than about 1 x10-9 , and is capable of killing or depleting B cells in vivo, preferably when compared to a suitable negative control When killed or reduced by at least 20%, the negative control was not treated with such antibodies. B cell depletion can be the result of one or more of ADCC, CDC, apoptosis, or other mechanisms. In some embodiments of the disease treatments herein, certain effector functions or mechanisms may be required more than others, and certain variants of humanized 2H7 are preferred for achieving biological functions such as ADCC.
“抗体片段”包含全长抗体的一部分，通常是其抗原结合或可变区。抗体片段的例子包括Fab、Fab′、F(ab′)2、与Fv片段；双抗体；线状抗体(linearantibody)；单链抗体分子；和由抗体片段形成的多特异性抗体。An "antibody fragment" comprises a portion of a full-length antibody, usually its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules;
“Fv”是包含完全抗原识别与结合部位的最小抗体片段。这个片段由一个重链与一个轻链可变区结构域以紧密、非共价联合而形成的二聚物组成。这两个结构域的折叠产生六个高变环(重链与轻链各三个环)，提供抗原结合的氨基酸残基并且赋予抗体抗原结合特异性。然而，即使单个的可变结构域(或者仅仅包含特定于抗原的三个CDR的Fv的一半)具有识别与结合抗原的能力，虽然亲合力比整个结合部位低。"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain and a light chain variable region domain in tight, non-covalent association. Folding of these two domains creates six hypervariable loops (three loops each for the heavy and light chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only the three CDRs specific to an antigen) has the ability to recognize and bind antigen, albeit with lower avidity than the entire binding site.
术语“单克隆抗体”如在这里所使用的指获得自实质上均一的抗体群体的抗体，即构成该群体的单独抗体除了那些可能少量存在的自然发生的突变之外是同一的。单克隆抗体是高度特异性的，针对单一抗原位点。此外，与常规的(多克隆)抗体制备相反——所述常规抗体一般包括针对不同决定簇(表位)的不同抗体——每个单克隆抗体针对抗原上的单一决定簇。修饰语“单克隆”指示抗体的特性为获得自实质上均一群体的抗体，不允许被解释为要求经过任何特定的方法生产抗体。例如，依照本发明使用的单克隆抗体可能是通过Kohler et al.，Nature 256：495(1975)首次描述的杂交瘤方法生产，或可通过重组DNA方法生产(参见，例如，美国专利号4,816,567)。利用例如在Clackson et al.，Nature 352：624-628(1991)与Marks et al.，J.Mol.Biol.222：581-597(1991)中描述的技术，“单克隆抗体”也可以从噬菌体抗体文库中分离。The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for those naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, in contrast to conventional (polyclonal) antibody production, which generally comprises different antibodies directed against different determinants (epitopes). The modifier "monoclonal" indicates the properties of the antibody as one obtained from a substantially homogeneous population and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567) . "Monoclonal antibodies" can also be obtained from isolated from a phage antibody library.
本发明的CD20结合抗体的“功能性片段”是这样一些片段，其保有与其所衍生的完整全长分子相比实质上相同的亲合力结合CD20的能力，并且在经过例如在这里所描述的体外或体内分析而进行的测量中显示生物活性，包括消减B细胞。"Functional fragments" of the CD20-binding antibodies of the invention are those fragments that retain substantially the same ability to bind CD20 with substantially the same avidity as the intact full-length molecule from which they are derived, and undergo in vitro assays such as those described herein. Biological activity, including depletion of B cells, is demonstrated in measurements performed by or in vivo assays.
术语“可变”指这一事实，即可变结构域的某些片段在抗体序列中存在广泛差别。V结构域介导抗原结合，并且定义特定的抗体对其特定抗原的特异性。然而，可变性在可变结构域的110个氨基酸跨度内不是均匀分布的。相反，可变区包括相对不变的几段序列称为框架区(FR)，为15-30氨基酸长，其间被极端可变的较短区域分开，称为“高变区”，每个9-12氨基酸长。天然重与轻链的可变结构域每个包含四个FR，基本上采取β片层构型，由三个高变区连接，高变区形成连接环，并且有时候形成β片层结构的一部分。每个链的高变区由FR十分接近地维系在一起，并且与另一链的高变区一起参与抗体的抗原结合部位的形成(参见Kabat et al.，Sequences of Proteinsof Immunological Interest，5th Ed.Public Health Service，National Institutes ofHealth，Bethesda，MD.1991))。恒定结构域不直接涉及抗体与抗原的结合，但是显示出各种效应子功能，例如在抗体参与抗体依赖细胞细胞毒性(ADCC)中。The term "variable" refers to the fact that certain segments of the variable domains differ widely in antibody sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable domain. In contrast, variable regions consist of several relatively invariant sequences called framework regions (FR), 15-30 amino acids long, separated by shorter regions of extreme variability, called "hypervariable regions," each 9 -12 amino acids long. The variable domains of the native heavy and light chains each contain four FRs, adopt essentially a beta-sheet configuration, and are connected by three hypervariable regions that form connecting loops and sometimes form the beta-sheet structure part. The hypervariable regions of each chain are held together in close proximity by FRs and, together with the hypervariable regions of the other chain, participate in the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, for example in the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
术语“高变区”当在这里使用时指抗体负责抗原结合的氨基酸残基。高变区通常包括来自“补体决定区”或“CDR”(例如VL中大约残基24-34(L1)、50-56(L2)与89-97(L3)周围，与VH中大约31-35B(H1)、50-65(H2)与95-102(H3)周围(Kabat et al.，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD.(1991)))，和/或来自“高变环”的残基(例如VL中残基26-32(L1)、50-52(L2)与91-96(L3)，与VH中26-32(H1)、52A-55(H2)与96-101(H3)(Chothia与Lesk，J.Mol.Biol.196：901-917(1987))。The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically include regions from "complement determining regions" or "CDRs" (e.g. around residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inVL , and about residues 89-97 (L3) inVH Around 31-35B(H1), 50-65(H2) and 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))), and/or residues from "hypervariable loops" (e.g. residues 26-32(L1), 50-52(L2) and 91-96(L3) inVL , and residues 91-96(L3) inVH , 26-32(H1), 52A-55(H2) and 96-101(H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
如在此所提到的，“共有序列”或者共有V结构域序列是人工序列，源自对已知人免疫球蛋白可变区序列的氨基酸序列的比较。基于这些比较，制备编码V结构域氨基酸的重组核酸序列，该序列对于源自人和人H链亚群III V结构域的序列是共有的。该共有V序列没有任何已知抗体结合特异性或亲合力。As referred to herein, a "consensus sequence" or consensus V domain sequence is an artificial sequence, derived from a comparison of the amino acid sequences of known human immunoglobulin variable region sequences. Based on these comparisons, recombinant nucleic acid sequences encoding V domain amino acids that are common to sequences derived from human and human H chain subgroup III V domains were prepared. This consensus V sequence does not have any known antibody binding specificity or avidity.
“嵌合抗体”(免疫球蛋白)重和/或轻链的一部分与来源于特定的物种或者属于特定的抗体种类或者亚类的抗体的相应序列相同或同源，而链的其余部分与来源于另一个物种或者属于另一个抗体种类或者亚类的抗体的相应序列相同或同源，只要它们显示出该所需生物活性(美国专利号4,816，567；与Morrison et al.，Proc.Natl.Acad.Sci.USA 81：6851-6855(1984))。在此所使用的人源化抗体是嵌合抗体的亚群。A portion of the heavy and/or light chains of a "chimeric antibody" (immunoglobulin) is identical or homologous to the corresponding sequence of an antibody derived from a specified species or belonging to a specified antibody class or subclass, while the remainder of the chain is identical to that of the source The corresponding sequences of antibodies belonging to another species or belonging to another antibody class or subclass are identical or homologous, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; with Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). As used herein, humanized antibodies are a subset of chimeric antibodies.
非人(例如，鼠的)抗体的“人源化”形式是嵌合抗体，其包含来源于非人免疫球蛋白的最小序列。在极大程度上，人源化抗体是人免疫球蛋白(受者或受体抗体)，其中受者的高变区残基被来自非人物种(供体抗体)例如小鼠、大鼠、兔或非人类灵长类动物的高变区残基替代，该高变区残基具有所需的特异性、亲合力和能力。在有些情况下，人免疫球蛋白的Fv框架区(FR)残基被相应的非人残基替代。此外，人源化抗体可以包含受者抗体或供体抗体中没有发现的残基。这些修饰被用来更进一步地改善抗体性能例如结合亲合力。一般地，人源化抗体将基本上包含所有至少一个、并且典型地两个可变结构域，其中相当于非人免疫球蛋白的高变环的所有或者基本上所有高变环、以及所有或者基本上所有FR区域是人免疫球蛋白序列的，虽然FR区域可以包括改善结合亲合力的一或多个氨基酸取代。在FR中的这些氨基酸取代的数目在重链中典型地不超过6个，在轻链中不超过3个。人源化抗体任意地也包括免疫球蛋白恒定区(Fc)的至少一部分，典型地是人免疫球蛋白恒定区的至少一部分。详细内容参见Jones et al.，Nature321：522-525(1986)；Reichmann et al.，Nature 332：323-329(1988)；与Presta，Curr.Op.Struct.Biol.2：593-596(1992)。"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or recipient antibodies) in which hypervariable region residues from the recipient have been replaced by those from a non-human species (donor antibody) such as mouse, rat, Rabbit or non-human primate hypervariable region residues are substituted that have the desired specificity, avidity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications are used to further improve antibody properties such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two variable domains, of which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all or Essentially all FR regions are of human immunoglobulin sequences, although FR regions may include one or more amino acid substitutions that improve binding affinity. The number of such amino acid substitutions in the FRs will typically not exceed 6 in the heavy chain and 3 in the light chain. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. See Jones et al., Nature 321: 522-525 (1986) for details; Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992 ).
抗体“效应子功能”指可归因于抗体Fc区域(天然序列Fc区域或者氨基酸序列变体Fc区域)的那些生物活性，并且随抗体同种型而变化。抗体效应子功能的例子包括：C1q结合与补体依赖的细胞毒；Fc受体结合；抗体依赖的细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)的下调；与B细胞活化。Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), and vary with antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Downregulation; associated with B cell activation.
“抗体依赖的细胞介导的细胞毒性”或“ADCC”指一种细胞毒性形式，其中与存在于某些细胞毒细胞(例如自然杀伤(NK)细胞、嗜中性白细胞与巨噬细胞)上的Fc受体结合的分泌Ig使得这些细胞毒效应细胞特定地与携带抗原的靶细胞结合，并且接着用细胞毒素杀死该靶细胞。抗体“武装”细胞毒细胞，并且对这样的杀伤是绝对需要的。介导ADCC的主要细胞—NK细胞—仅仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII与FcγRIII。在造血细胞上的FcR表达在Ravetch与Kinet，Annu.Rev.Immunol 9：457-92(1991)第464页表3中概括。为评估目的分子的ADCC活性，可进行体外ADCC试验，例如在美国专利号5,500,362或5,821,337中描述的。对这样的试验有用的效应细胞包括外周血单核细胞(PBMC)与自然杀伤(NK)细胞。或者或另外地，目的分子的ADCC活性可以体内评估，例如，在例如Clynes et al.PNAS(USA)95：652-656(1998)中所披露的动物模型中。"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which the The Fc receptor-bound secreted Ig allows these cytotoxic effector cells to specifically bind the antigen-bearing target cell and subsequently kill the target cell with cytotoxin. Antibodies "arm" cytotoxic cells and are absolutely required for such killing. The primary cells that mediate ADCC—NK cells—express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
“Fc受体”或者“FcR”描述与抗体Fc区域结合的受体。优选的FcR是天然序列人FcR。此外，优选的FcR是结合IgG抗体的FcR(一个γ受体)，并且包括FcγRI、FcγRII、与FcγRIII亚类的受体，包括等位基因变体和这些受体的不同拼接形式。FcγRII受体包括FcγRIIA(“激活受体”)与FcγRIIB(“抑制受体”)，其具有类似氨基酸序列，该氨基酸序列的主要不同在于其细胞质结构域。激活受体FcγRIIA在它的细胞质结构域包含免疫受体酪氨酸为基础的激活基序(ITAM)。抑制受体FcγRIIB在它的细胞质结构域包含免疫受体酪氨酸为基础的抑制基序(ITIM)。(参见综述M.in 
“人效应细胞”是表达一或多个FcR并且行使效应子功能的白细胞。优选地，细胞至少表达FcγRIII并且行使ADCC效应子功能。介导ADCC的人白细胞的例子包括外周血单核细胞(PBMC)、自然杀伤(NK)细胞、单核细胞、细胞毒性T细胞与中性粒细胞，以PBMC与自然杀伤细胞为优选。效应细胞可能从天然来源，例如从血中分离。A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMC and natural killer cells being preferred. Effector cells may be isolated from natural sources, such as blood.
“依赖于补体的细胞毒性”或“CDC”指补体存在下靶细胞的溶解。通过补体系统的第一组分(C1q)与合适亚类的抗体结合而启动经典补体途径的激活，所述抗体是与其相关抗原结合的。为评估补体激活，可进行CDC试验，例如如Gazzano-Santoro et al.，J.Immunol.Methods 202：163(1996)中描写的。"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (Clq) to an antibody of the appropriate subclass that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
在美国专利号6,194,551B1与WO99/51642中描述了多肽变体，具有改变的Fc区域氨基酸序列与增加或降低的C1q结合能力。那些专利出版物的内容在此特别地引入作为参考。也参见Idusogie et al.J.Immunol.164：4178-4184(2000)。Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in US Patent No. 6,194,551B1 and WO99/51642. The contents of those patent publications are hereby expressly incorporated by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).
IgG中N-糖基化位点在CH2结构域的Asn297。本发明也提供CD20结合的、人源化抗体组合物，该抗体具有Fc区域，其中组合物中抗体的大约80-100％(并且优选地大约90-99％)包含成熟核心糖结构，该糖结构缺乏岩藻糖，附着于糖蛋白的Fc区域。象这样的组合物在此被证明显示出在与FcγRIIIA(F158)结合上的惊人改善，该结合不如FcγRIIIA(V158)与人IgG相互作用有效。因此，在这里的组合物预计优于以前描述的抗CD20抗体组合物，特别是对于治疗表达FcγRIIIA(F158)的病人而言。在正常、健康的非洲裔美国人与白种人中，FcγRIIIA(F158)比FcγRIIIA(V158)更常见。参见Lehrnbecher et al.Blood 94：4220(1999)。本申请更进一步显示FcγRIII结合和/或ADCC功能的协同增加，该协同增加源自在此的糖基化作用变异与糖蛋白Fc区域的氨基酸序列修饰的联合。The N-glycosylation site in IgG is Asn297 in the CH2 domain. The invention also provides CD20-binding, humanized antibody compositions having an Fc region, wherein about 80-100% (and preferably about 90-99%) of the antibodies in the composition comprise a mature core sugar structure, the sugar The structure lacks fucose and is attached to the Fc region of the glycoprotein. Compositions like this are demonstrated here to show a surprising improvement in binding to FcyRIIIA(F158), which is not as efficient as FcyRIIIA(V158) interacting with human IgG. Thus, the compositions herein are expected to be superior to previously described anti-CD20 antibody compositions, especially for the treatment of patients expressing FcyRIIIA(F158). FcγRIIIA(F158) is more common than FcγRIIIA(V158) in normal, healthy African-Americans and Caucasians. See Lehrnbecher et al. Blood 94:4220 (1999). The present application further shows a synergistic increase in FcyRIII binding and/or ADCC function resulting from the combination of glycosylation variations herein and amino acid sequence modifications in the Fc region of a glycoprotein.
“分离的”抗体是已经从它的自然环境的组分中鉴别与分离和/或回收的抗体。其自然环境的污染物组分是将干扰抗体的诊断或治疗使用的物质，并且可包括酶、激素及其它蛋白的或者非蛋白的溶解物。在优选实施方式中，抗体将被纯化至(1)如经过Lowry方法确定的，按重量计算大于抗体的95％，并且最优选按重量计算超过99％，(2)足够获得通过利用转杯测序仪(spinning cup sequenator)N末端或者内部氨基酸序列的至少15个残基的程度，或者(3)在还原或者非还原条件下利用考马斯蓝或优选地银染色在SDS-PAGE下达到均一。分离的抗体包括在重组细胞内部的原位抗体，因为抗体自然环境中的至少一个组分将不存在。然而，一般地，分离的抗体将通过至少一个纯化步骤制备。An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with the diagnostic or therapeutic use of antibodies, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody will be purified to (1) greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method, (2) sufficient to obtain (spinning cup sequencer) to the extent of at least 15 residues of the N-terminal or internal amino acid sequence, or (3) achieve uniformity under SDS-PAGE using Coomassie blue or preferably silver staining under reducing or non-reducing conditions. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
“分离的”核酸分子是从至少一个污染物核酸分子中鉴别与分离的核酸分子，在抗体核酸的天然来源中其一般地与该污染物核酸分子有联系。分离的核酸分子在其形式或环境上与其在自然界中发现时不同。分离的核酸分子因此不同于其在自然细胞中存在的核酸分子。然而，分离的核酸分子包括在通常表达抗体的细胞中加以包含的核酸分子，在该细胞中例如核酸分子位于与自然细胞不同的染色体位置上。An "isolated" nucleic acid molecule is a nucleic acid molecule identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of antibody nucleic acid. An isolated nucleic acid molecule differs in form or environment from that as it is found in nature. An isolated nucleic acid molecule thus is distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express antibodies where, for example, the nucleic acid molecule is at a different chromosomal location than natural cells.
表示法“控制序列”指在一特定宿主有机体中表达可操作连接的编码序列所需的DNA序列。适于原核生物的控制序列例如包括启动子，任选操纵子序列，以及核糖体结合位点。已知真核细胞利用启动子、聚腺苷酸化信号与增强子。The notation "control sequences" refers to DNA sequences required for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
当核酸被置于与另一核酸序列的功能性关系时其是“可操作连接的”。例如，如果前序列或分泌性前导序列的DNA作为参与多肽分泌的前蛋白质加以表达，其与多肽的DNA是可操作连接的。如果启动子或者增强子影响序列的转录，其与编码序列是可操作地连接的。或如果核糖体结合位点被置于促进翻译的位置，则其与编码序列是可操作地连接的。一般地，“可操作地连接”指被连接的DNA序列是邻近的，并且在分泌性前导序列的情况下，是邻近的和在阅读状态下。然而，增强子不必是邻近的。连接通过在适宜限制性位点的连接而完成。如果这样的位点不存在，根据通常的做法使用合成寡核苷酸连接物或接头。A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a pre-sequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a pre-protein involved in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Or a ribosome binding site is operably linked to the coding sequence if it is placed in a position to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading condition. However, enhancers need not be contiguous. Linking is accomplished by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adapters are used according to common practice.
“载体”包括穿梭与表达载体。典型地，质粒构建物包括复制起始点(例如ColE1复制起始点)与选择性标记(例如氨苄青霉素或四环素抗性)，分别用于质粒在细菌中的复制与选择。“表达载体”指一种载体，其包含在细菌或真核细胞中表达本发明的抗体、包括抗体片段所需的控制序列或调控元件。合适的载体在下面披露。"Vector" includes both shuttle and expression vectors. Typically, the plasmid construct includes an origin of replication (eg ColE1 origin of replication) and a selectable marker (eg ampicillin or tetracycline resistance) for replication and selection of the plasmid in bacteria, respectively. An "expression vector" refers to a vector comprising the control sequences or regulatory elements required to express the antibodies of the invention, including antibody fragments, in bacterial or eukaryotic cells. Suitable carriers are disclosed below.
产生本发明的人源化CD20结合抗体的细胞包括细菌与真核宿主细胞，其中已经引入编码抗体的核酸。合适的宿主细胞在下面披露。Cells that produce the humanized CD20-binding antibodies of the invention include bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Suitable host cells are disclosed below.
用词“标记”当在这里使用时指可检测的化合物或组合物，其直接或间接地与抗体接合。标记可本身是可检测的(例如放射性同位素标记或荧光的标记)或者，在酶促标记的情况下，可以催化可检测的底物化合物或组合物的化学改变。The word "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label may itself be detectable (eg, a radioisotopic label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a detectable substrate compound or composition.
“自身免疫病”在此是来自并针对个体自身(自己的)抗原和/或组织的非恶性疾病或紊乱。An "autoimmune disease" herein is a non-malignant disease or disorder arising from and directed against an individual's own (own) antigens and/or tissues.
如同在这里使用的，＂B细胞消减＂指在药物或者抗体处理之后同像这样的处理以前的水平比较起来在动物或者人中B细胞水平的减少。利用众所周知的试验，例如在实验实施例中描述的试验，测量B细胞水平。B细胞消减可以完全的或者部分的。在一个实施方式中，表达CD20的B细胞消减至少25％。不受任何机制的限制，B细胞消减的可能机制包括ADCC、CDC、细胞程序死亡、钙流的调制或者在前机制中两个或更多的组合。As used herein, "B cell depletion" refers to the reduction in the level of B cells in an animal or human after drug or antibody treatment compared to the level before such treatment. B cell levels are measured using well known assays, such as those described in the Experimental Examples. B cell depletion can be complete or partial. In one embodiment, CD20 expressing B cells are depleted by at least 25%. Without being limited by any mechanism, possible mechanisms of B cell depletion include ADCC, CDC, apoptosis, modulation of calcium flux, or a combination of two or more of the preceding mechanisms.
术语“细胞毒性剂”如同在此使用的指一种物质，该物质抑制或防碍细胞功能和/或导致细胞破坏。术语意图包括放射性同位素(例如I131、I125、Y90与Re186)、化疗剂和毒素例如细菌、真菌、植物或动物来源的酶促活性毒素或其片段。The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to include radioisotopes (egI131 ,I125 ,Y90 andRe186 ), chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof.
“化疗剂”是在癌症治疗中有用的化合物。化疗剂实例包括烷化剂，如噻替哌(thiotepa)；环膦酰胺(cyclosphamide)(CYTOXANTM)；烷基磺酸酯如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶如苄替哌(benzodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和methylamelamine包括六甲蜜胺(altretamine)，三乙撑蜜胺(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin，洋红霉素，嗜癌素(carzinophilin)，色霉素(chromomycin)，放线菌素D，道诺红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin，Adriamycin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin，嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶呤，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，丁蝶翼素(pteropterin)，三甲曲沙(trimetrexate)；嘌呤类似物氟达拉滨(fludarabine)，6-巯基嘌呤，硫咪嘌呤，硫鸟嘌呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，多西氟尿啶，依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮，丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，邻氯苯对氯苯二氯乙烷(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如frolinic acid；醋葡内酯；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；磷氨氮芥(defofamine)；秋水仙胺；地吖醌(diaziquone)；依洛尼塞(elfornithine)；醋酸羟吡咔唑(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；二胺硝吖啶(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；；雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺(spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2′，2＂-三氯三乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；溴丙哌嗪(pipobroman)；gacytosine；阿拉伯糖苷(“Ara-C”)；塞替哌(thiotepa)；紫杉烷(taxoid)，如紫杉醇(
“治疗”或“处理”或“缓和”既指治疗性处理也指预防性或者预防性措施，其中目的是防止或减慢(减弱)所针对的病理情况或紊乱。如果个体根据本发明的方法接受治疗量的本发明的CD20结合抗体后，显示该特定疾病的一或多个体征和症状的可观测的和/或可度量的减少，该个体被成功地“治疗”CD20阳性癌症或自身免疫病。例如，对于癌症来说，癌细胞数目减少或者癌细胞消失；肿瘤体积减小；抑制(即在某种程度上减慢并且优选地停止)肿瘤转移；在某种程度上抑制肿瘤生长；延长症状缓解的时间，和/或在某种程度上减轻与该特定的癌症有联系的一个或多个症状；减低发病率与死亡率，与生命质量的改进等事宜。病人也可以感到疾病征候或症状的减低。治疗可以取得完全响应，定义为所有癌症迹象的消失，或者部分响应，其中肿瘤尺寸减小，优选地超过50％，更优选地超过75％。如果病人病情平稳，也被认为是已治疗的。在一优选实施方式中，在一年之后癌症病人的癌症仍然没有进展，优选地在15个月之后。评估疾病成功治疗与改善的这些参数通过为本领域医师所熟悉的常规程序是容易度量的。"Treatment" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down (attenuate) the targeted pathological condition or disorder. An individual is successfully "treated" if the individual exhibits an observable and/or measurable reduction in one or more signs and symptoms of the specified disease after receiving a therapeutic amount of a CD20-binding antibody of the invention according to the methods of the invention. "CD20 positive cancer or autoimmune disease. For example, in the case of cancer, reduction in number of cancer cells or disappearance of cancer cells; reduction in tumor volume; inhibition (i.e. slowing and preferably stopping to some extent) of tumor metastasis; inhibition of tumor growth to some extent; prolongation of symptoms Duration of remission, and/or reduction in some degree of one or more symptoms associated with that particular cancer; reduction in morbidity and mortality, and improvement in quality of life, among other matters. The patient may also experience a reduction in disease signs or symptoms. Treatment may result in a complete response, defined as disappearance of all signs of cancer, or a partial response, wherein tumor size is reduced, preferably greater than 50%, more preferably greater than 75%. Patients were also considered treated if their condition was stable. In a preferred embodiment, the cancer patient is still cancer-free after one year, preferably after 15 months. These parameters for assessing successful treatment and improvement of disease are readily measurable by routine procedures familiar to physicians in the art.
“治疗有效量”指在个体中有效“治疗”疾病或紊乱的抗体或药物的量。对于癌症，药物治疗有效量可减少癌细胞的数量；减小肿瘤体积；抑制(即在一定程度上减慢并优选停止)癌细胞对周围器官的浸润；抑制(即在一定程度上减慢到并优选停止)肿瘤转移；在一定程度上抑制肿瘤生长；和/或在一定程度上减轻与癌症相关的一种或多种症状。参见前述对“治疗”的定义。A "therapeutically effective amount" refers to an amount of an antibody or drug effective to "treat" a disease or disorder in an individual. For cancer, a therapeutically effective amount of the drug reduces the number of cancer cells; reduces tumor volume; inhibits (i.e. slows and preferably stops) the invasion of surrounding organs by cancer cells; inhibits (i.e. slows to some extent to and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate to some extent one or more symptoms associated with cancer. See definition of "treatment" above.
“慢性”给药指以与急性方式相反的连续方式给药所述药剂，从而在更长的时间内维持最初的治疗效果(活性)。“间歇性”给药是并非不间断地连续进行、而实际上是周期性的治疗。"Chronic" administration refers to administration of the agent in a continuous manner as opposed to an acute manner, so that the initial therapeutic effect (activity) is maintained over a longer period of time. "Intermittent" administration is treatment that is not continuous without interruption, but is, in fact, periodic.
本发明的方法与组合物Methods and compositions of the invention
本发明提供结合人CD20、并且优选地也结合其它灵长类动物CD20的人源化抗体，包含一个重链，该重链包含非人物种抗人CD20抗体(供体抗体)的至少一个、优选地两个或者所有H链CDR，和作为受者抗体的人共有抗体的基本上所有框架残基。供体抗体可以来自于各种的非人物种，包括小鼠、大鼠、豚鼠、山羊、兔、马、灵长类动物，但是最经常地是小鼠抗体。在这里“基本上所有的”指人源化抗体的受者FR区域可以包括在人共有FR序列中原来不存在的一或多个氨基酸取代。这些FR改变可以包含在受者或供体抗体中未找到的残基。The present invention provides humanized antibodies that bind human CD20, and preferably also other primate CD20, comprising a heavy chain comprising at least one, preferably Two or all of the H chain CDRs, and substantially all framework residues of the human consensus antibody as the recipient antibody. Donor antibodies can be from various non-human species, including mouse, rat, guinea pig, goat, rabbit, horse, primate, but most often mouse antibodies. "Substantially all" herein means that the recipient FR region of the humanized antibody may include one or more amino acid substitutions that are not originally present in the human consensus FR sequence. These FR changes may contain residues not found in either the recipient or the donor antibody.
在一个实施方式中，供体抗体是鼠抗体，可变区包括图1A与1B所示的H与L链的每一个的CDR与FR序列。在一个特定的实施方式中，人Fab框架的残基相应于人Vκ亚群I和VH亚群III的共有序列，这些共有序列分别地如图1A与图1B所示。本发明的人源化2H7抗体具有小鼠供体抗体的H链的至少一个CDR。在一个实施方式中，结合人CD20的人源化2H7抗体包含供体抗体H与L链两者的CDR。In one embodiment, the donor antibody is a murine antibody, and the variable regions include the CDR and FR sequences of each of the H and L chains shown in Figures 1A and 1B. In a specific embodiment, the residues of the human Fab framework correspond to the consensus sequences of human VK subgroup I andVH subgroup III, which are shown in Figure 1A and Figure 1B, respectively. The humanized 2H7 antibody of the present invention has at least one CDR of the H chain of the mouse donor antibody. In one embodiment, the humanized 2H7 antibody that binds human CD20 comprises the CDRs of both the H and L chains of the donor antibody.
在一个全长抗体中，本发明的人源化CD20结合抗体将包含人源化的可变结构域，该结构域与人免疫球蛋白的恒定结构域连接。在一优选实施方式中，重链恒定区来自人IgG，优选地IgG1或者IgG3。轻链恒定结构域优选地来自人κ链。In a full-length antibody, the humanized CD20-binding antibodies of the invention will comprise humanized variable domains linked to human immunoglobulin constant domains. In a preferred embodiment, the heavy chain constant region is from human IgG, preferably IgG1 or IgG3. The light chain constant domain is preferably from a human kappa chain.
除非另外指示，除了在下面的实验实施例中指示的氨基酸取代或改变位置之外，在这里的人源化2H7抗体型式将具有2H7.v16轻链(图6，SEQ IDNO.21)与H链(图7，SEQ ID NO.22)的V与C结构域序列。Unless otherwise indicated, the humanized 2H7 antibody versions herein will have a 2H7.v16 light chain (Figure 6, SEQ ID NO. (Fig. 7, SEQ ID NO.22) V and C domain sequence.
该人源化CD20结合抗体将至少结合人CD20，并且优选地结合其它灵长类动物CD20，例如猿猴(monkey)包括猕猴和恒河猴，与黑猩猩的CD20。猕猴CD20的序列在实施例15与图19中披露。The humanized CD20 binding antibody will bind at least human CD20, and preferably binds other primate CD20, for example CD20 of monkeys including macaques and rhesus monkeys, and chimpanzees. The sequence of macaque CD20 is disclosed in Example 15 and Figure 19 .
本发明的CD20结合抗体与人源化CD20结合抗体的生物活性包括该抗体与人CD20的结合，更优选与人和灵长类动物(包括猕猴、恒河猴、黑猩猩)CD20结合，其Kd值不高于1 x 10-8，优选地Kd值不高于大约1 x 10-9，甚至更优选地Kd值不高于大约1 x 10-10，并且能体外或体内杀死或者消减B细胞，优选地当与基线水平或未用这样的抗体处理过的恰当的负对照相比杀死或者消减至少20％。The biological activity of the CD20-binding antibody of the present invention and the humanized CD20-binding antibody includes the binding of the antibody to human CD20, more preferably the binding to CD20 of humans and primates (including rhesus monkeys, rhesus monkeys, and chimpanzees), and its Kd value No higher than 1 x 10-8 , preferably a Kd value no higher than about 1 x 10-9 , even more preferably a Kd value no higher than about 1 x 10-10 , and capable of killing or depleting B cells in vitro or in vivo , preferably by at least 20% when compared to baseline levels or an appropriate negative control not treated with such antibodies.
B细胞消减的所需水平将取决于疾病。对于治疗CD20阳性癌症来说，也许需要最大限度消减作为本发明的抗CD20抗体目标的B细胞。因此，对于治疗CD20阳性B细胞肿瘤而言，最好对B细胞的消减是足够的，以至少防止疾病的进展，而疾病的进展是本领域熟练医师可以评估的，例如通过监测肿瘤生长(大小)、癌细胞类型的增殖、转移、及该特定癌症的其它体征和症状。优选地，B细胞消减足以防止疾病的进展至少2个月，更优选3个月，甚至更优选4个月，更优选5个月，甚至更优选6或更多个月。在甚至更优选的实施方式中，B细胞消减足以提高症状缓解时间至少6个月，更优选9个月，更优选一年，更优选2年，更优选3年，甚至更优选5年或更多年。在一个最优选实施方式中，B细胞消减足以治疗疾病。在优选实施方案中，在癌症病人中的B细胞消减是治疗以前基线水平的至少大约75和优选地80％、85％、90％、95％、99％并且甚至100％。The desired level of B cell depletion will depend on the disease. For the treatment of CD20 positive cancers, it may be desirable to minimize the depletion of B cells that are targeted by the anti-CD20 antibodies of the invention. Thus, for the treatment of CD20-positive B cell tumors, depletion of B cells is preferably sufficient to at least prevent disease progression, which can be assessed by a physician skilled in the art, for example by monitoring tumor growth (size ), proliferation of cancer cell types, metastasis, and other signs and symptoms of that particular cancer. Preferably, the B cell depletion is sufficient to prevent progression of the disease for at least 2 months, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 or more months. In an even more preferred embodiment, the B cell depletion is sufficient to increase the time to remission for at least 6 months, more preferably 9 months, more preferably one year, more preferably 2 years, more preferably 3 years, even more preferably 5 years or more for many years. In a most preferred embodiment, B cell depletion is sufficient to treat the disease. In preferred embodiments, B cell depletion in cancer patients is at least about 75% and preferably 80%, 85%, 90%, 95%, 99% and even 100% of pre-treatment baseline levels.
对于治疗自身免疫病，也许需要依赖个体病人的疾病和/或病症的严重程度，通过调整CD20结合抗体的剂量，调制B细胞消减的程度。因此，可以消减B细胞、但不必是完全的。或者，在起始治疗中可能需要总B细胞消减，但在随后治疗中，可能调整剂量以达到仅部分消减。在一个实施方式中，与治疗前的基线水平相比，B细胞消减至少20％，即余留80％或者更少的CD20阳性B细胞。在其它实施方式中，B细胞消减25％、30％、40％、50％、60％、70％或者更多。优选地，B细胞消减足以使疾病进展停顿，更优选缓和在治疗下的特定疾病的体征和症状，甚至更优选治愈疾病。For the treatment of autoimmune diseases, it may be necessary to modulate the degree of B cell depletion by adjusting the dose of CD20-binding antibody, depending on the severity of the disease and/or condition of the individual patient. Thus, B cells can be depleted, but need not be complete. Alternatively, total B-cell depletion may be required in initial therapy, but in subsequent treatments, the dose may be adjusted to achieve only partial depletion. In one embodiment, the B cell depletion is at least 20%, ie, 80% or less CD20 positive B cells remain, compared to baseline levels prior to treatment. In other embodiments, the B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% or more. Preferably, the B cell depletion is sufficient to arrest disease progression, more preferably alleviate the signs and symptoms of the particular disease under treatment, and even more preferably cure the disease.
本发明还提供双特异性CD20结合抗体，其中抗体的一个臂具有本发明的人源化CD20结合抗体的人源化重与轻链，并且另一个臂具有针对第二抗原的结合特异性的可变区。在特定的实施方式中，第二抗原选自由CD3、CD64、CD32A、CD16、NKG2D或者其它NK激活配体组成的组。The invention also provides a bispecific CD20-binding antibody, wherein one arm of the antibody has the humanized heavy and light chains of a humanized CD20-binding antibody of the invention, and the other arm has a variable binding specificity for a second antigen. Variable area. In specific embodiments, the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.
同Rituxan(rituximab)比较起来)，v16显示出ADCC效力增加大约2至5倍，CDC比Rituxan降低3-4倍。Compared to Rituxan (rituximab), v16 showed approximately 2-5 fold increase in ADCC potency and 3-4 fold decrease in CDC compared to Rituxan.
抗体产生antibody production
单克隆抗体Monoclonal antibodies
单克隆抗体可用Kohler et al，Nature，256：495(1975)首先描述的杂交瘤法制备或通过重组DNA法(美国专利4,816,567)制备。Monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975) or by recombinant DNA methods (US Patent 4,816,567).
在杂交瘤方法中，如上述免疫小鼠或其它适宜的宿主动物，例如仓鼠，来激发产生或能产生特异结合免疫所用蛋白的抗体的淋巴细胞。可选地，所述淋巴细胞可被体外免疫。免疫后，淋巴细胞可被分离并随后使用适宜的融合剂例如聚乙二醇与骨髓瘤细胞系融合，以形成杂交瘤细胞(Goding，单克隆抗体：Principles and Practice，59-103页(Academic Press，1986))。In the hybridoma approach, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. Following immunization, lymphocytes can be isolated and subsequently fused with a myeloma cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press , 1986)).
由此制备的杂交瘤细胞被接种于适宜的培养基并在其中生长，该培养基优选包含一种或多种抑制未融合亲代骨髓瘤细胞(也称为融合伴侣)的生长或存活的物质。例如，如果亲代骨髓瘤细胞缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，杂交瘤的选择培养基通常包括次黄嘌呤，氨基蝶呤，和胸腺嘧啶(“HAT培养基”)这些抑制HGPRT-缺陷细胞生长的物质。The hybridoma cells thus prepared are seeded and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also known as fusion partners). For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the selection medium for hybridomas typically includes hypoxanthine, aminopterin, and thymine ("HAT medium"). Substances that inhibit the growth of HGPRT-deficient cells.
优选融合伴侣骨髓瘤细胞为那些能有效融合、支持所选抗体生成细胞以稳定的高水平产生抗体、并对选择未融合亲代的选择培养基敏感的那些融合伴侣骨髓瘤细胞系。优选的骨髓瘤细胞系是鼠骨髓瘤系，如由SalkInstitute Cell Distribution Center，San Diego，California USA提供的MOPC-21和MPC-11小鼠肿瘤，和由美国典型培养物保藏中心，Rockville，MarylandUSA提供的SP-2或其衍生物，如X63-Ag8-653细胞。也描述了人骨髓瘤和小鼠-人异源骨髓瘤细胞系可用于产生人单克隆抗体[Kozbor，J.Immunol.，133：3001(1984)；和Brodeur等，单克隆抗体的制备技术和应用(MarcelDekker，Inc.，New York纽约，(1987))51-63页)。Preferred fusion partner myeloma cells are those fusion partner myeloma cell lines that fuse efficiently, support stable high levels of antibody production by the selected antibody-producing cells, and are sensitive to selection media that select for unfused parents. Preferred myeloma cell lines are murine myeloma lines such as the MOPC-21 and MPC-11 mouse tumors provided by the Salk Institute Cell Distribution Center, San Diego, California USA, and the American Type Culture Collection, Rockville, Maryland USA. SP-2 or its derivatives, such as X63-Ag8-653 cells. It has also been described that human myeloma and mouse-human heteromyeloma cell lines can be used to produce human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Techniques for the Preparation of Monoclonal Antibodies and Applications (Marcel Dekker, Inc., New York, (1987)) pp. 51-63).
可在含有生长的杂交瘤细胞的培养基中分析抗所述抗原的单克隆抗体的产生。优选地，杂交瘤细胞所产生的单克隆抗体的结合特异性通过免疫沉淀或通过体外结合试验，如放射免疫分析(RIA)或酶联免疫吸附试验(ELISA)来分析。The production of monoclonal antibodies directed against the antigens can be assayed in the medium containing the growing hybridoma cells. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is analyzed by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
单克隆抗体的结合亲合力可通过，例如Muson等，Anal.Biochem.，107：220(1980)所述Scatchard分析来测定。The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described by Muson et al., Anal. Biochem., 107:220 (1980).
一旦鉴定出产生具有所需特异性、亲合力和/或活性的抗体的杂交瘤细胞，这些细胞克隆可通过有限稀释进行亚克隆，并通过标准方法(Goding，单克隆抗体：Principles and Practice，59-103页(Academic Press，1986))使其生长。适合此目的的培养基包括例如D-MEM或RPMI-1640培养基。此外，通过例如将细胞腹膜内注射入小鼠中，可以使杂交瘤细胞以动物腹水肿瘤的形式在体内生长。Once hybridoma cells producing antibodies with the desired specificity, avidity and/or activity have been identified, these cell clones can be subcloned by limiting dilution and processed by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, 59 -103 pages (Academic Press, 1986)) to make it grow. Media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals by, for example, injecting the cells intraperitoneally into mice.
上述亚克隆所分泌的单克隆抗体可用经典抗体纯化方法如，例如，亲和层析(例如使用蛋白A或蛋白G琼脂糖)或离子交换层析，羟基磷灰石层析，凝胶电泳，透析等从培养基，腹水或血清中分离。Monoclonal antibodies secreted by the above subclones can be purified by classical antibody purification methods such as, for example, affinity chromatography (e.g. using protein A or protein G agarose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, Separation from culture medium, ascitic fluid or serum by dialysis etc.
使用传统方法(例如通过使用能与编码鼠抗体重链和轻链的基因特异结合的寡核苷酸探针)可轻易分离并测序编码单克隆抗体的DNA。杂交瘤细胞是此DNA的优选来源。一旦分离出该DNA，可以将它置入表达载体中，该载体随后被转染至宿主细胞中例如大肠杆菌细胞，猴COS细胞，中国仓鼠卵巢(CHO)细胞，或不另外生成抗体蛋白的骨髓瘤细胞，从而在重组宿主细胞中合成单克隆抗体。关于在细菌中重组表达编码抗体的DNA的综述文字包括Skerra et al.，Curr.Opinion in Immunol.，5：256-262(1993)和Plückthun，Immunol.Revs.，130：151-188(1992)。DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes that bind specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are a preferred source of this DNA. Once the DNA is isolated, it can be placed into an expression vector that is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or bone marrow that do not otherwise produce the antibody protein Tumor cells to synthesize monoclonal antibodies in recombinant host cells. Review texts on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992) .
在进一步的实施方案中，可从使用McCafferty等，Nature，348：552-554(1990)描述的技术产生的抗体噬菌体文库中分离单克隆抗体或抗体片段。Clackson等，Nature，352：624-628(1991)和Marks等，J.Mol.Biol.，222：581-597(1991)分别描述了使用噬菌体文库分离鼠和人抗体。随后的文章描述了通过链改组产生高亲和性(纳米级)人抗体(Marks等，Bio/Technology，10：779-783(1992))，以及将组合感染和体内重组作为策略构建非常大的噬菌体文库(Waterhouse等，Nuc.Acids.Res.，21：2265-2266(1993))。因此，这些技术是用于分离单克隆抗体的传统单克隆抗体杂交瘤技术的可行(viable)选择。In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent papers described the generation of high-affinity (nanoscale) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), and combined infection and in vivo recombination as strategies for constructing very large Phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.
也可采用以下方法对编码抗体的DNA进行修饰以产生嵌合或融合抗体多肽：例如，以人重链和轻链恒定区(CH和CL)的序列代替同源小鼠序列(美国专利4,816,567；和Morrison等，Proc.Natl Acad.Sci.USA，81：6851(1984))，或将免疫球蛋白编码序列与非免疫球蛋白多肽(异源多肽)的完整或部分编码序列融合。非免疫球蛋白多肽序列可取代抗体的恒定区，或它们可取代抗体的一个抗原结合位点的可变区以产生嵌合二价抗体，该抗体包含针对一个抗原的一个抗原结合位点，和具有对不同抗原的特异性的另一抗原结合位点。Antibody-encoding DNA can also be modified to generate chimeric or fusion antibody polypeptides by, for example, replacing the homologous mouse sequences with sequences from human heavy and light chain constant regions (CH andCL ) (U.S. Pat. 4,816,567; and Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or fusion of an immunoglobulin coding sequence to the complete or partial coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may replace the constant region of the antibody, or they may replace the variable region of an antigen-binding site of the antibody to generate a chimeric bivalent antibody comprising an antigen-binding site directed against an antigen, and Another antigen binding site with specificity for a different antigen.
人源化抗体Humanized Antibody
使非人抗体人源化的方法已在本领域中描述。优选地，人源化抗体具有一或多个从非人来源引入它的氨基酸残基。这些非人氨基酸残基常称为“引进的”残基，它们通常来自“引进的”可变区。人源化过程基本是按照Winter及其同事(Jones等，Nature，321：522-525(1986)；Riechmann等，Nature，332：323-327(1988)；Verhoeyen等，Science，239：1534-1536(1988))所述，通过高度可变区序列替换人抗体的对应序列来进行。因此，这样的“人源化”抗体是嵌合抗体(美国专利4,816,567)，其中完整人类可变区的很少一部分被非人物种的相应序列取代。实践中，人源化抗体通常是人的抗体，其中有些高变区残基且可能有些FR残基被啮齿动物抗体中类似位点的残基取代。Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues, which are usually from an "imported" variable region. The humanization process is essentially in accordance with Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting the hypervariable region sequence for the corresponding sequence of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (US Patent 4,816,567) in which a small portion of an intact human variable region has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous positions in rodent antibodies.
当抗体用于人类治疗用途时，选择人源化抗体制备所用的人轻链和重链可变区，对于降低抗原性和HAMA反应(人抗鼠抗体)非常重要。根据所谓的“最适”方法，针对已知人可变区序列的整个文库筛选啮齿类抗体的可变区序列。鉴定与啮齿类V区序列最接近的人V区序列，并且其中的人框架区(FR)作为人源化抗体被接受(Sims et al.，J.Immunol.，151：2296(1993)；Chothia et al.，J.Mol.Biol.，196：901(1987))。另一种方法使用从具有轻链或重链特定亚群的所有人抗体的共有序列衍生的特定框架区。同样的框架区可被用于几种不同的人源化抗体(Carter等，Proc.Natl.Acad.Sci.USA，89：4285(1992)；Presta等，J.Immunol.，151：2623(1993))。Selection of human light and heavy chain variable regions for humanized antibody preparation is important to reduce antigenicity and HAMA reactivity (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called "best fit" method, the variable region sequences of rodent antibodies are screened against the entire library of known human variable region sequences. The human V region sequence closest to the rodent V region sequence was identified, and the human framework regions (FR) therein were accepted as humanized antibodies (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies with specific subgroups of light or heavy chains. The same framework regions can be used in several different humanized antibodies (Carter et al., Proc. )).
重要的是，将抗体人源化后保留了对抗原的高结合亲和力和其它有利的生物特性。为达到此目的，在一种优选方法中，通过用亲本序列和人源化序列的三维模型分析亲本序列和各种概念性人源化产物的方法来制备人源化抗体。免疫球蛋白三维模型已有商品，是本领域技术人员所熟悉的。还有用于描述和展示所选候选免疫球蛋白序列可能的三维构象结构的计算机程序。通过检查这些展示结果可分析残基在候选免疫球蛋白序列的功能中可能发挥的作用，即分析能影响候选免疫球蛋白与其抗原结合的能力的残基。通过这种方法，可从受体序列和引进序列中选出FR残基并组合，从而得到所需抗体性质，如对靶抗原的亲和力增加。总之，高变区残基直接并且最主要涉及对抗原结合的影响。Importantly, the high binding affinity for the antigen and other favorable biological properties are retained after humanization of the antibody. To this end, in a preferred method, humanized antibodies are prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are commercially available and are familiar to those skilled in the art. There are also computer programs for describing and displaying probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows for analysis of the likely role that residues play in the function of the candidate immunoglobulin sequence, ie, for residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody property, such as increased affinity for the target antigen(s), is obtained. In conclusion, the hypervariable region residues are directly and primarily involved in influencing antigen binding.
人源化抗体可以是抗体片段，例如Fab，其可选地与一种或多种细胞毒性制剂偶联以产生免疫偶联物。可选地，人源化抗体可以是全长的抗体，例如全长IgG1抗体。Humanized antibodies can be antibody fragments, such as Fab, optionally conjugated to one or more cytotoxic agents to generate immunoconjugates. Alternatively, a humanized antibody may be a full-length antibody, such as a full-length IgG1 antibody.
人抗体和噬菌体展示方法Human Antibodies and Phage Display Methods
作为人源化的备选，可制备人抗体。例如，现在可以制备转基因动物(如小鼠)，它经过免疫能在缺乏内源性免疫球蛋白生成的情况下产生全套人抗体。例如，已指出在嵌合和胚系(germ-line)突变小鼠中，抗体重链连接区(JH)基因的纯合缺失导致内源性抗体生成的完全抑制。将人胚系免疫球蛋白基因阵列转移到此胚系突变小鼠中，将导致在抗原攻击的情况下产生人抗体。见例如，Jakobovits等Proc.Natl.Acad.Sci.USA，90：2551(1993)；Jakobovits等，Nature，362：255-258(1993)；Bruggemann等，Year in Immuno.，7：33(1993)；美国专利5,545,806，5,569,825，5,591,669(所有均授予GenPharm)；5,545,807；和WO 97/17852.As an alternative to humanization, human antibodies can be prepared. For example, transgenic animals (eg, mice) can now be prepared that, upon immunization, produce a repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been shown that homozygous deletion of the antibody heavy chain joining region (JH ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into this germline mutant mouse will result in the production of human antibodies in response to antigenic challenge. See eg, Jakobovits et al. Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993) ; U.S. Patents 5,545,806, 5,569,825, 5,591,669 (all to GenPharm); 5,545,807; and WO 97/17852.
或者，可用噬菌体展示技术(McCafferty等，自然348：552-553(1990))从未免疫供体的免疫球蛋白可变(V)区基因库体外产生人类抗体和抗体片段。依据此技术，抗体V区基因在框架内(in-frame)克隆入丝状噬菌体(如M13或fd)主要或次要衣壳蛋白基因，并在噬菌体颗粒的表面展示为功能性抗体片段。因为丝状颗粒包含噬菌体基因组的单链DNA拷贝，根据抗体的功能特点进行的选择也导致对显示这些性质的抗体的编码基因进行选择。因此，噬菌体模仿了B细胞的部分特点。噬菌体展示可以以多种形式进行；这些综述见例如Johnson，Kevin S.和Chiswell，David J.，Current Opinion inStructural Biology 3,564-571(1993)。可使用V基因节段的多个来源进行噬菌体展示。Clackson等，Nature 352，624-628(1991)从免疫小鼠脾脏来源的V基因小随机组合文库中分离出一批多样的抗-噁唑酮抗体。可基本如Marks等，J.Mol.Biol.222：581-597(1991)，或Griffith等，EMBO J.12：725-734(1993)所述，构建未免疫的人类供体的V基因库，并分离针对多种多样抗原(包括自身抗原)的抗体。也见美国专利5,565,332和5,573,905。Alternatively, human antibodies and antibody fragments can be produced in vitro using phage display technology (McCafferty et al., Nature 348:552-553 (1990)) from immunoglobulin variable (V) region gene repertoires from unimmunized donors. According to this technique, antibody V region genes are cloned in-frame into filamentous phage (such as M13 or fd) major or minor capsid protein genes and displayed as functional antibody fragments on the surface of phage particles. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection for the gene encoding the antibody exhibiting these properties. Thus, phages mimic some of the features of B cells. Phage display can be performed in a variety of formats; for a review of these see, eg, Johnson, Kevin S. and Chiswell, David J., Current Opinion in
如上讨论，人抗体也可通过体外活化的B细胞产生(见美国专利5,567,610和5,229,275)。As discussed above, human antibodies can also be produced by activated B cells in vitro (see US Patent Nos. 5,567,610 and 5,229,275).
抗体片段antibody fragment
在特定的情况下，使用抗体片段比整个抗体更有优势。片段的较小体积允许快速清除，并可导致更好地接近实体肿瘤。In certain situations, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows rapid clearance and may lead to better access to solid tumors.
已开发了多种技术用于生产抗体片段。传统上，这些片段通过对完整抗体进行蛋白水解消化来衍生(见例如，Morimoto等，J.Biochem.Biojphys.Methods 24：107-117(1992)和Brennan等，Science 229：81(1985))。然而，这些片段现在可通过重组宿主细胞直接生产。Fab，Fv和ScFv抗体片段均可在大肠杆菌中表达并从中分泌，从而使这些片段的大量生产变容易。也可自上述的抗体噬菌体文库分离抗体片段。可选地，Fab’-SH片段可自大肠杆菌直接回收并利用化学方法偶联形成F(ab’)2片段(Carter等，Bio/Technology 10：163-167(1992))。根据另一方法，F(ab’)2片段可直接从重组宿主细胞培养物中分离。包括补救受体结合表位残基并具有延长的体内半寿期的Fab和F(ab′)2片段在美国专利5,869,046中描述。熟练技术人员掌握其它产生抗体片段的技术。其它实施方案中，选择的抗体是单链Fv片段(scFv)。见WO93/16185；美国专利5,571,894；和美国专利5,587,458。Fv和sFv是具有完整结合位点的种类中唯一缺乏恒定区的；因此，其适于在体内应用期间降低非特异结合。可以构建sFv融合蛋白，从而在sFv的氨基或羧基末端融合效应蛋白。见上文的Antibody Engineering，Borrebaeck编辑。抗体片段也可以是“线性抗体”，例如在美国专利5,641,870中描述。此种线型抗体片段可以是单特异或双特异的。Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, eg, Morimoto et al., J. Biochem. Biojphys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, which facilitates the mass production of these fragments. Antibody fragments can also be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments that include salvage receptor binding epitope residues and have extended in vivo half-lives are described in US Patent 5,869,046. Other techniques for generating antibody fragments are within the skill of the artisan. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93/16185; US Patent 5,571,894; and US Patent 5,587,458. Fv and sFv are the only species with intact binding sites that lack a constant region; thus, they are suitable for reducing non-specific binding during in vivo applications. sFv fusion proteins can be constructed such that an effector protein is fused to the amino or carboxy terminus of the sFv. See Antibody Engineering, edited by Borrebaeck above. Antibody fragments can also be "linear antibodies," as described, for example, in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
双特异抗体bispecific antibody
双特异抗体是对至少两种不同表位具有结合特异性的抗体。典型的双特异抗体可结合CD20蛋白质的两种不同表位。其它此种抗体可将CD20结合位点与针对另一蛋白质的结合位点组合。或者，抗CD20臂可以与结合白细胞上触发分子的臂结合，触发分子例如T细胞受体分子(例如CD3)或IgG的Fc受体(FcγR)例如FcγRI(CD64)、FcγRII(CD32)和FcγR(CD16)，或NKG2D或其它NK细胞激活配体，以将细胞防御机制针对和定位在CD20表达细胞上。双特异抗体也可用来将细胞毒制剂定位在表达CD20的细胞上。这些抗体具CD20臂和结合细胞毒性制剂的臂(例如皂草素(saporin)，抗α-干扰素，长春花生物碱，蓖麻毒蛋白A链，氨甲蝶呤或放射性同位素半抗原)。可以将双特异抗体制备成全长抗体或抗体片段(例如F(ab′)2双特异抗体)。Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. A typical bispecific antibody binds to two different epitopes of the CD20 protein. Other such antibodies may combine a CD20 binding site with a binding site for another protein. Alternatively, the anti-CD20 arm can be combined with an arm that binds a triggering molecule on a leukocyte, such as a T cell receptor molecule (e.g. CD3) or an Fc receptor for IgG (FcγR) such as FcγRI (CD64), FcγRII (CD32) and FcγR ( CD16), or NKG2D or other NK cell activating ligands to target and localize cellular defense mechanisms on CD20 expressing cells. Bispecific antibodies can also be used to target cytotoxic agents to CD20-expressing cells. These antibodies have a CD20 arm and an arm that binds a cytotoxic agent (eg saporin, anti-alpha-interferon, vinca alkaloids, ricin A chain, methotrexate or radioisotope haptens). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).
WO96/16673描述了双特异性抗ErbB2/抗FcγRIII抗体，美国专利5,837，234披露了双特异性抗ErbB2/抗FcγRI抗体。在WO98/02463中显示双特异性抗ErbB2/Fcα抗体。美国专利5,821,337教导了双特异性抗ErbB2/抗CD3抗体。WO96/16673 describes bispecific anti-ErbB2/anti-FcyRIII antibodies and US Patent 5,837,234 discloses bispecific anti-ErbB2/anti-FcyRI antibodies. Bispecific anti-ErbB2/Fca antibodies are shown in WO98/02463. US Patent 5,821,337 teaches bispecific anti-ErbB2/anti-CD3 antibodies.
制备双特异性抗体的方法是本领域已知的。全长双特异性抗体的传统制备是基于两个免疫球蛋白重链-轻链对的共表达，其中这两条链具有不同特异性(Millstein et al，Nature，305：537-539(1983))。由于免疫球蛋白重链轻链随机分配，这些杂交瘤(“四链瘤”(quadroma))产生10种不同抗体分子的可能混合物，其中只有一种具有正确的双特异性结构。对所述正确分子的纯化(通常通过亲和层析步骤来进行)非常复杂，且产量很低。类似的方法在WO93/08829和Traunecker等，EMBO J，10：3655-3659(1991)中披露。Methods of making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983) ). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas ("quadromas") produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually by an affinity chromatography step, is very complex and yields very low. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBO J, 10:3655-3659 (1991).
依据另一不同方法，可将具有所需结合特异性的抗体可变区(抗体-抗原结合位点)与免疫球蛋白恒定区序列融合。该融合优选与包含铰链区、CH2及CH3区的至少一部分的免疫球蛋白重链恒定区融合。优选使含有轻链结合所需位点的第一重链恒定区(CH1)出现在至少在一种融合中。可将编码免疫球蛋白重链融合体，以及必要时，编码免疫球蛋白轻链的DNA插入不同表达载体，共转染至适当宿主细胞。在使用非等比的三种多肽链进行构建以获得所需双特异性抗体最佳产量的实施方案中，这提供调整三种多肽片段的相互比例上的较大灵活性。但也可在至少两种多肽链以等比例表达而获得高产时或所述比例对所需链组合的产量无特别意义时，将两种或所有三种多肽链的编码序列插入同一表达载体。According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant region sequences. The fusion is preferably to an immunoglobulin heavy chain constant region comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site required for light chain binding present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain can be inserted into different expression vectors and co-transfected into appropriate host cells. In embodiments where unequal ratios of the three polypeptide chains are used for construction to obtain optimal yields of the desired bispecific antibody, this provides greater flexibility in adjusting the relative ratios of the three polypeptide fragments. However, it is also possible to insert the coding sequences for two or all three polypeptide chains into the same expression vector when at least two polypeptide chains are expressed in equal ratios to obtain high yields or when said ratios have no particular significance for the yield of the desired chain combination.
在该方法的一个优选实施方案中，所述双特异性抗体由一条臂上的具有第一结合特异性的杂合免疫球蛋白重链和另一条臂上的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)构成。已发现这种不对称结构有利于从不想要的免疫球蛋白链的混合中分离出所需双特异性化合物，因为只有该双特异性分子的一半存在免疫球蛋白轻链，这使得分离更加容易。此方法公开于1994年3月3日公开的WO94/04690中。制备双特异性抗体的进一步细节可以参见，例如Suresh等，Methods in Enzymology，121：210(1986)。In a preferred embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with the first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain on the other arm. Strand pair (providing the second binding specificity) constitutes. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from a mixture of unwanted immunoglobulin chains, as only half of the bispecific molecule is present with immunoglobulin light chains, which makes separation easier . This method is disclosed in WO94/04690 published March 3,1994. Further details of making bispecific antibodies can be found in, eg, Suresh et al., Methods in Enzymology, 121:210 (1986).
根据美国专利5,731,168所述的另一种方法，可改造一对抗体分子之间的界面，使得从重组细胞培养中获得的异二聚体的百分比最大。优选的界面包括CH3结构域的至少一部分。在该方法中，源于第一抗体分子界面上的一条或多条氨基酸小侧链被较大侧链(如酪氨酸或色氨酸)取代。与所述大侧链大小相同或相近的互补“沟”可通过将氨基酸大侧链用小侧链(如丙氨酸或苏氨酸)取代而在第二抗体分子的界面上形成。这提供了一种机制，其使异二聚体的产量比其它不想要的终产物如同型二聚体高。According to another approach described in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers obtained from recombinant cell culture. A preferred interface includes at least a portion of the CH3 domain. In this method, one or more small amino acid side chains originating from the interface of the first antibody molecule are replaced with larger side chains such as tyrosine or tryptophan. Complementary "grooves" of the same or similar size as the large side chains can be formed at the interface of the second antibody molecule by replacing large amino acid side chains with smaller side chains such as alanine or threonine. This provides a mechanism that results in higher yields of heterodimers than other unwanted end products such as isodimers.
双特异性抗体包括交联抗体或“异源偶联的”抗体。例如，可使异源偶联物中的抗体之一与抗生物素蛋白偶联，使另一抗体与生物素偶联。有观点认为，这类抗体可用于将免疫系统细胞导向不想要的细胞(美国专利4,676,980)，也可用于治疗HIV感染(WO91/00360，WO92/200373，EP03089)。异源偶联抗体可通过任何适当的交联方法制备。适当的交联制剂和多种交联技术为本领域已知，在美国专利4,676,980号中获得。Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. It has been suggested that such antibodies could be used to direct immune system cells to unwanted cells (US Patent 4,676,980) and to treat HIV infection (WO91/00360, WO92/200373, EP03089). Heteroconjugate antibodies can be prepared by any suitable cross-linking method. Suitable crosslinking formulations and various crosslinking techniques are known in the art and are found in US Patent No. 4,676,980.
从抗体片段制备双特异性抗体的技术已有文献。例如，双特异性抗体可利用化学连接制备。Brennan等，科学229：81(1985)中描述了将完整抗体经蛋白水解切割制备F(ab′)2片段的方法。这些片段在二巯基复合剂亚砷酸钠存在时被还原，从而稳定相邻的巯基，并阻止分子间二硫键的形成。生成的Fab′片段被转化为硫硝基苯甲酸盐(TNB)衍生物。其中一种Fab′-TNB衍生物经巯基乙胺还原再转化成Fab′-硫醇，再与等摩尔量的其它Fab′-TNB衍生物混合形成双特异性抗体。如此产生的双特异性抗体可作为酶的选择性固相化中所用的试剂。Techniques for preparing bispecific antibodies from antibody fragments are documented. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe the proteolytic cleavage of intact antibodies to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite, which stabilizes adjacent thiols and prevents the formation of intermolecular disulfide bonds. The resulting Fab' fragments are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is reduced by mercaptoethylamine and converted into Fab'-thiol, and then mixed with other Fab'-TNB derivatives in equimolar amounts to form a bispecific antibody. The bispecific antibodies thus produced can be used as reagents for the selective immobilization of enzymes.
近期的进展促进了Fab′-SH片段从大肠杆菌的直接回收，该片段可经化学偶联形成双特异性抗体。Shalaby等，J.Exp.Med.，175：217-225(1992)描述了完全人源化双特异抗体F(ab′)2分子的产生。每一Fab′片段分别从大肠杆菌中分泌出来，体外直接化学偶联形成双特异性抗体。如此制备的双特异性抗体能与过表达ErbB2受体的细胞和正常人T细胞结合，还能引发人类细胞毒淋巴细胞对人乳腺肿瘤靶向的裂解活性。Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is separately secreted from Escherichia coli, and directly chemically coupled in vitro to form a bispecific antibody. The bispecific antibody prepared in this way can bind to cells overexpressing ErbB2 receptors and normal human T cells, and can also trigger the lytic activity of human cytotoxic lymphocytes targeting human breast tumors.
直接从重组细胞培养中制备并分离双特异性抗体片段的多种技术也已有描述。例如，可用亮氨酸拉链制备双特异性抗体。Kostelny等，J.Immunol.，148(5)：1547-1553(1992)。将来自Fos和Jun蛋白的亮氨酸拉链肽与两种不同抗体的Fab′部分通过基因融合而连接。使抗体的同二聚体在铰链区被还原成单体，然后被再氧化形成抗体的异二聚体。该方法也可用于制备抗体同二聚体。Hollinger等，Proc.Natl.Acad.Sci.USA，90：6444-6448(1993)所述的“二价抗体”技术提供了另一种制备双特异性抗体片段的方法。所述片段中含有VH，其通过接头与VL相连，该接头非常短，使得同一链的两个结构域之间无法配对。因此，同一片段上的VH和VL结构域被迫与另一片段上的互补VL和VH结构域配对，从而形成两个抗原结合位点。此外还报道了另一种用单链Fv(sFv)二聚体来制备双特异性抗体的策略。见Gruber等，J.Immunol.，152：5368(1994)。Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies can be prepared using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked by gene fusion to the Fab' portions of two different antibodies. The homodimer of the antibody is reduced to a monomer at the hinge region, and then reoxidized to form a heterodimer of the antibody. This method can also be used to prepare antibody homodimers. The "bivalent antibody" technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provides another method for preparing bispecific antibody fragments. The fragment contains aVH connected to aVL by a linker which is so short that pairing between the two domains of the same chain is impossible. Thus, theVH andVL domains on the same fragment are forced to pair with the complementaryVL andVH domains on another fragment, thereby forming two antigen-binding sites. In addition, another strategy for preparing bispecific antibodies using single-chain Fv (sFv) dimers has been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
本发明还涉及二价以上的抗体。例如可制备三特异抗体。Tutt等J.Immunol.147：60(1991)。The present invention also relates to antibodies that are more than bivalent. For example trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).
多价抗体multivalent antibody
多价抗体比二价抗体更容易被表达与该抗体结合的抗原的细胞内化(和/或异化)。本发明的抗体可以是具有三个或更多个抗原结合位点的多价抗体(例如四价抗体)(它们不是IgM类)，通过使编码所述抗体多肽链的核酸重组表达可轻易制备该抗体。多价抗体可以包含二聚体化结构域和三个或更多个抗原结合位点。优选的二聚体化结构域包括Fc区或铰链区，或由其组成。在本文中，抗体包含一个Fc区和三个或更多个位于Fc区氨基端的抗原结合位点。优选的多价抗体在此包括三到约八个抗原结合位点，或由它们组成，但优选四个抗原结合位点。多价抗体包括至少一条多肽链(并优选两条多肽链)，其中所述多肽链包含两个或多个可变区。例如，多肽链可包含VD1-(X1)n-VD2-(X2)n-Fc，其中VD1是第一可变区，VD2是第二可变区，Fc是Fc区的一条多肽链，X1和X2代表氨基酸或多肽，n是0或1。例如，多肽链可包括VH-CH1-柔韧接头(flexible linker)-VH-CH1-Fc区链；或VH-CH1-VH-CH1-Fc区链。本文的多价抗体优选进一步包含至少两个(优选四个)轻链可变区多肽。例如本文的多价抗体可包含从约两个到约八个轻链可变区多肽。本文的轻链可变区多肽包含轻链可变区以及可选地进一步包含CL结构域。Multivalent antibodies are more readily internalized (and/or catabolic) by cells expressing an antigen to which the antibody binds than bivalent antibodies. Antibodies of the invention may be multivalent antibodies (e.g., tetravalent antibodies) (which are not of the IgM class) having three or more antigen-binding sites, which can be readily prepared by recombinant expression of a nucleic acid encoding the polypeptide chain of said antibody. Antibody. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise or consist of an Fc region or a hinge region. Herein, an antibody comprises an Fc region and three or more antigen-binding sites located amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise or consist of three to about eight antigen binding sites, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chains comprise two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1)n -VD2-(X2)n -Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represents amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide chain may comprise a VH-CH1 -flexible linker-VH -CH1-Fc region chain; or a VH- CH1-VH -CH1-Fc region chain. The multivalent antibodies herein preferably further comprise at least two (preferably four) light chain variable region polypeptides. For example, a multivalent antibody herein may comprise from about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides herein comprise a light chain variable region and optionally further comprise a CL domain.
其它氨基酸修饰Other Amino Acid Modifications
本发明还涉及对本文所述CD20结合抗体的氨基酸序列修饰。例如，可能希望改进抗体的结合亲和力和/或其它生物学特性。抗CD20抗体的氨基酸序列变体可通过在抗CD20抗体核酸中导入适当的核苷酸改变，或通过肽合成法来制备。所述修饰包括，如该抗CD20抗体的氨基酸序列中的残基缺失，和/或插入和/或取代。可对缺失、插入、和取代进行任意组合以获得最终构建体，只要该最终构建体具有所需特性。氨基酸的变化还可改变抗CD20抗体的翻译后加工，如改变糖基化位点的数目或位置。The invention also relates to amino acid sequence modifications to the CD20-binding antibodies described herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of anti-CD20 antibodies can be prepared by introducing appropriate nucleotide changes into the anti-CD20 antibody nucleic acid, or by peptide synthesis. The modification includes, for example, deletion, and/or insertion and/or substitution of residues in the amino acid sequence of the anti-CD20 antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired properties. Amino acid changes can also alter the post-translational processing of the anti-CD20 antibody, such as altering the number or location of glycosylation sites.
一种鉴别抗CD20抗体中处于诱变优选位置的特定残基或区域的有效方法是Cunningham和Wells，科学244：1081-1085(1989)所述的“丙氨酸扫描诱变”。这里，鉴定一个残基或一组靶残基(例如，带电的残基如精氨酸、天冬氨酸、组氨酸、赖氨酸和谷氨酸)并用中性或带负电的氨基酸(最优选丙氨酸或多聚丙氨酸)取代，以便影响氨基酸与CD20抗原的相互作用。那些证实对取代具有功能敏感性的氨基酸位置通过在取代点引入进一步的或其它的变体而改进。故，尽管引入氨基酸序列变异的位点是预先决定的，但突变本身不必是预定的。例如，为分析在指定位点处突变的作用，在所述靶密码子或区域实行丙氨酸扫描或随机诱变，并筛选所表达的具有预期活性的抗CD20抗体变体。A useful method for identifying specific residues or regions in anti-CD20 antibodies that are preferred positions for mutagenesis is "alanine scanning mutagenesis" as described by Cunningham and Wells, Science 244: 1081-1085 (1989). Here, a residue or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and neutral or negatively charged amino acids ( Alanine or polyalanine) substitutions are most preferred in order to affect the interaction of the amino acid with the CD20 antigen. Those amino acid positions that demonstrate functional sensitivity to substitution are improved by introducing further or other variants at the point of substitution. Thus, although the sites to introduce amino acid sequence variations are predetermined, the mutations themselves need not be predetermined. For example, to analyze the effect of mutations at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed anti-CD20 antibody variants are screened for the expected activity.
氨基酸序列插入包括氨基-和/或羧基-端的融合(其长度从一个残基至包括100个或更多残基的多肽)，以及序列内单个或多个氨基酸残基的插入。末端插入的例子包括带有N-末端甲硫氨酰残基的抗CD20抗体或与细胞毒性多肽融合的抗体。抗CD20抗体分子的其它插入变体包括使该抗CD20抗体的N或C末端与酶(例如ADEPT)或增加该抗体血清半寿期的多肽融合形成的融合体。Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides comprising 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-CD20 antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-CD20 antibody molecule include fusions of the N- or C-terminus of the anti-CD20 antibody to an enzyme (eg, ADEPT) or a polypeptide that increases the serum half-life of the antibody.
另一类变体是氨基酸取代变体。这些变体使抗CD20抗体分子中至少一个氨基酸残基被不同残基取代。最有兴趣进行取代诱变的位点包括高变区，也可以改变FR。保守取代见表1的“优先取代”栏。如果这些取代引起生物学活性的改变，则可引入表1中“取代举例”栏的更实质性改变，或进一步在下文的氨基酸分类中所述的更实质性改变，并筛选产物。Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-CD20 antibody molecule substituted by a different residue. The sites of most interest for substitution mutagenesis include hypervariable regions, which can also alter FRs. For conservative substitutions, see the "Preferred Substitutions" column in Table 1. If these substitutions lead to changes in biological activity, more substantial changes in the "Example of Substitution" column in Table 1, or further substantive changes described in the amino acid classification below, can be introduced, and the products can be screened.
氨基酸取代表amino acid substitution table
对抗体的生物学特性的实质性修改可通过选择取代来完成，所述取代的效应在维持(a)取代区多肽框架的结构，例如片层结构或螺旋构象，(b)该分子靶位点的电荷或疏水性，(c)侧链的大小这几方面有显著差异。天然残基根据共有的侧链特性可分为：Substantial modification of the biological properties of an antibody can be accomplished by selecting substitutions that have the effect of maintaining (a) the structure of the polypeptide framework, such as a sheet or helical conformation, at the region of substitution, (b) the molecular target site There are significant differences in the charge or hydrophobicity of (c) the size of the side chain. Natural residues can be grouped according to shared side chain properties:
(1)疏水性：正亮氨酸，蛋氨酸，丙氨酸，缬氨酸，亮氨酸，异亮氨酸(1) Hydrophobic: norleucine, methionine, alanine, valine, leucine, isoleucine
(2)中性亲水：半胱氨酸，丝氨酸，苏氨酸(2) Neutral hydrophilic: cysteine, serine, threonine
(3)酸性：天冬氨酸，谷氨酸(3) Acidity: aspartic acid, glutamic acid
(4)碱性：天冬酰胺，谷氨酰胺，组氨酸，赖氨酸，精氨酸(4) Alkaline: asparagine, glutamine, histidine, lysine, arginine
(5)影响侧链定向的残基：甘氨酸，脯氨酸，和(5) Residues affecting side chain orientation: glycine, proline, and
(6)芳香族：色氨酸，酪氨酸，苯丙氨酸。(6) Aromatic: tryptophan, tyrosine, phenylalanine.
非保守取代将限定上述某一类的成员被另一类取代。Non-conservative substitutions will restrict the substitution of a member of one of the aforementioned classes by another class.
任何不参与保持抗CD20抗体正确构型的半胱氨酸残基可被替代，通常由丝氨酸替代，来提高分子的氧化稳定性并防止错误交联。反之，可在抗体中添加半胱氨酸连接以提高其稳定性(特别当抗体为抗体片段如FV片段时)。Any cysteine residues not involved in maintaining the correct conformation of the anti-CD20 antibody can be substituted, usually by serine, to increase the oxidative stability of the molecule and prevent mis-crosslinking. Conversely, cysteine linkages can be added to the antibody to increase its stability (particularly when the antibody is an antibody fragment such as a FV fragment).
取代变体的特别优选类型包括取代亲本抗体(例如人源化抗体或人抗体高变区的一或多个残基。通常，所选用于进一步开发的所得变体相对于其所来源的亲本抗体应具有改进的生物学活性。产生这种取代变体的一个方便方法是利用了噬菌体展示的亲和力成熟。简单地说，使高变区的几个位点(如6-7个位点)突变以便在每一位点产生所有可能的氨基酸取代。这样产生的抗体变体以单价形式展示在丝状噬菌体颗粒上，其为与每个颗粒内包装的M13基因III产物的融合体。然后筛选噬菌体展示的变体是否具有本文所述生物学活性(如结合亲和力)。为了鉴定备选的高变区修饰位点，可通过丙氨酸扫描诱变来鉴定对抗原结合作出主要贡献的高变区残基。或者或此外，分析抗原-抗体复合物的晶体结构以确定CD20与抗体之间的接触点也较有利。这些接触残基及邻近残基是根据本文所述技术进行取代的候选位点。一旦产生这样的变体，如本文所述对它们全部进行筛选，选出在一或多个相关实验中具有良好特性的抗体以便进一步开发。A particularly preferred type of substitutional variant involves substituting one or more residues of a hypervariable region of a parental antibody (e.g., a humanized antibody or a human antibody. Typically, the resulting variant selected for further development is relative to the parental antibody from which it was derived. Should have improved biological activity. A convenient method to generate such substitution variants is to utilize affinity maturation of phage display. Simply put, several sites (such as 6-7 sites) of the hypervariable region are mutated In order to generate all possible amino acid substitutions at each position. The antibody variants thus produced are displayed in monovalent form on filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage are then screened Whether the displayed variant has the biological activity (such as binding affinity) described herein. To identify alternative hypervariable region modification sites, alanine scanning mutagenesis can be used to identify hypervariable regions that make a major contribution to antigen binding Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to determine the contact points between CD20 and the antibody. These contact residues and neighboring residues are candidates for substitution according to the techniques described herein Once such variants are generated, they are all screened as described herein, and antibodies with favorable properties in one or more relevant experiments are selected for further development.
抗体的另一种氨基酸变体改变了该抗体原来的糖基化模式。所谓改变就是去掉抗体中的一或多个糖组分，和/或添加一或多个原本不存在于该抗体中的糖基化位点。Another amino acid variant of an antibody alters the antibody's original glycosylation pattern. The so-called modification is to remove one or more sugar components in the antibody, and/or add one or more glycosylation sites that do not originally exist in the antibody.
抗体的糖基化通常为N-连接或O-连接。N-连接指将糖组分与天冬酰胺残基的侧链相连。三肽序列天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸(其中X是除脯氨酸以外的任何氨基酸)是使糖组分与天冬酰胺进行侧链酶促相连的识别序列。因此，多肽中存在上述任一种三肽序列都可产生潜在的糖基化位点。O-连接糖基化指将N-乙酰半乳糖胺、半乳糖、或木糖附着于羟基氨基酸，主要是丝氨酸、苏氨酸，但也可用5-羟脯氨酸和5-羟赖氨酸。Glycosylation of antibodies is typically either N-linked or O-linked. N-linking refers to the attachment of a sugar component to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are the recognition for the enzymatic attachment of the sugar component to the side chain of asparagine sequence. Therefore, the presence of any of the above-mentioned tripeptide sequences in a polypeptide can generate potential glycosylation sites. O-linked glycosylation refers to the attachment of N-acetylgalactosamine, galactose, or xylose to hydroxyamino acids, primarily serine, threonine, but also 5-hydroxyproline and 5-hydroxylysine .
在抗体分子中添加糖基化位点可通过改变氨基酸序列，使其包含一或多个上述三肽序列(在添加N-连接糖基化位点的情况下)而实现。这种改变也可通过在原始抗体序列中添加或取代一或多个丝氨酸或苏氨酸残基来实现(在添加O-连接的情况下)。Addition of glycosylation sites to antibody molecules can be accomplished by altering the amino acid sequence to contain one or more of the tripeptide sequences described above (in the case of addition of N-linked glycosylation sites). Such alterations may also be accomplished by the addition or substitution of one or more serine or threonine residues in the original antibody sequence (in the case of addition of an O-linkage).
编码抗CD20抗体的氨基酸序列变体的核酸分子由本领域已知的各种方法制备。这些方法包括但不限于从天然来源分离(在天然氨基酸序列变体的情况下)，或通过对早期制备的变体或未变异的抗CD20抗体进行寡核苷酸介导的(或定点)诱变，PCR诱变和盒式诱变来制备。Nucleic acid molecules encoding amino acid sequence variants of anti-CD20 antibodies are prepared by various methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of natural amino acid sequence variants), or by oligonucleotide-mediated (or site-directed) induction of variant or unvaried anti-CD20 antibodies produced earlier. mutagenesis, PCR mutagenesis and cassette mutagenesis.
也许希望在效应子功能方面修饰本发明的抗体，例如以增强抗体的抗原依赖的细胞介导的细胞毒性(ADCC)和/或依赖于补体的细胞毒性(CDC)。这可通过在抗体Fc区域引入一或多个氨基酸取代而达到。二者择一地或者另外，半胱氨酸残基可被引入Fc区域，从而允许在该区内形成链间二硫键。如此生产的同型二聚体抗体可具有改善的内在化能力，和/或增加补体介导的细胞杀伤与抗体依赖的细胞的细胞毒性(ADCC)。见Caron et al.，J.ExpMed.176：1191-1195(1992)与Shopes，B.J.Immunol.148：2918-2922(1992)。也可以利用如Wolff et al.Cancer Research 53：2560-2565(1993)描述的异双功能交联剂制备具有增强的抗肿瘤活性的同型二聚体抗体。或者，抗体可以被改造使其具有两个Fc区域，并且可以由此具有增强的补体介导的溶解与ADCC能力。见Stevenson et al.Anti-Cancer Drug Design 3：219-230(1989)。It may be desirable to modify the antibodies of the invention with respect to effector function, eg, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or in addition, cysteine residues may be introduced into the Fc region, allowing interchain disulfide bond formation within this region. The homodimeric antibodies thus produced may have improved internalization capabilities, and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. ExpMed. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers as described by Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, antibodies can be engineered to have two Fc regions and can thus have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
为延长抗体的血清半衰期，可以将补救受体结合表位加入抗体(特别是抗体片段)，例如在美国专利5,739,277中描述的。如同在这里所使用的，术语“补救受体结合表位”指IgG分子(例如IgG1，IgG2，IgG3，或IgG4)Fc区域的表位，其负责延长IgG分子的体内血清半衰期。To prolong the serum half-life of antibodies, salvage receptor binding epitopes can be added to antibodies (particularly antibody fragments), as described, for example, in US Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (eg, IgGl, IgG2, IgG3, or IgG4) that is responsible for extending the serum half-life of the IgG molecule in vivo.
其它抗体修饰Other Antibody Modifications
在此也涉及抗体的其它修饰。例如，抗体可以与各种非蛋白聚合物中的一种连接，例如聚乙二醇，聚丙二醇，聚氧化烯，或聚乙二醇与聚丙二醇的共聚物。抗体也可以在胶体药物递送系统(例如脂质体、白蛋白微球粒、微乳剂、纳米颗粒与纳米胶囊)中，或者在粗乳状液中捕捉在微胶囊中，所述微胶囊例如通过凝聚技术或界面聚合作用制备，例如，分别是羟甲基纤维素或明胶微胶囊与聚异丁烯酸甲酯微胶囊。这一技术在Remington′sPharmaceutical Sciences，16th edition，Oslo，A.，Ed.，(1980)中披露。Other modifications of antibodies are also contemplated herein. For example, the antibody can be linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be entrapped in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, such as by coacervation technology or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively. This technique is disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
筛选具有所需特性的抗体Screen for antibodies with desired properties
可如实验实施例中描述的选择具有某些生物学特性的抗体。Antibodies with certain biological properties can be selected as described in the experimental examples.
本发明的抗CD20抗体的生长抑制效果可以通过本领域已知的方法评估，例如利用内源或者在用CD20基因转染之后表达CD20的细胞。例如，肿瘤细胞系与CD20转染的细胞可以用本发明的抗CD20单克隆抗体在各种浓度处理几天(例如，2-7天)，用结晶紫或MTT染色，或用其它比色定量进行分析。另一个测量增殖的方法是比较在用本发明的抗CD20抗体存在或不存在下处理，细胞3H-胸腺嘧啶核苷的摄取。在抗体处理之后，收获细胞，并且在闪烁计数器上测定掺入DNA的放射性的量。合适的正对照包括用已知抑制该细胞系生长的生长抑制性抗体处理选定的细胞系。The growth inhibitory effect of the anti-CD20 antibodies of the present invention can be assessed by methods known in the art, such as using cells expressing CD20 endogenously or after transfection with the CD20 gene. For example, cells transfected with a tumor cell line and CD20 can be treated with anti-CD20 monoclonal antibodies of the invention at various concentrations for several days (e.g., 2-7 days), stained with crystal violet or MTT, or quantified with other colorimetric methods. for analysis. Another way to measure proliferation is to compare the uptakeof3H -thymidine by cells treated with or without an anti-CD20 antibody of the invention. Following antibody treatment, cells were harvested and the amount of radioactivity incorporated into the DNA was determined on a scintillation counter. Suitable positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit the growth of that cell line.
为选择诱导细胞死亡的抗体，可相对于对照评估膜完整性的丧失，膜完整性的丧失用例如碘化丙锭(PI)、锥虫蓝或7AAD摄取指示。PI摄取试验可以在缺乏补体与免疫效应细胞下进行。表达CD20的肿瘤细胞单独与培养基孵育，或者与包含浓度例如大约10μg/ml的合适的单克隆抗体的培养基一起孵育。细胞孵育3天。每次处理后，洗细胞，等分入35毫米带过滤器帽的12x75管(每管1ml，每试验组3管)，以除去细胞团块。样品可用FACSCANTM流式细胞仪与FACSCONVERTTMCellQuest软件(BectonDickinson)分析。用PI摄取确定诱导统计上显著意义的细胞死亡水平的抗体可被选为诱导细胞死亡的抗体。To select antibodies that induce cell death, loss of membrane integrity as indicated by, for example, propidium iodide (PI), trypan blue or 7AAD uptake can be assessed relative to controls. PI uptake assays can be performed in the absence of complement and immune effector cells. Tumor cells expressing CD20 are incubated with culture medium alone or with medium containing an appropriate monoclonal antibody at a concentration, eg, about 10 μg/ml. Cells were incubated for 3 days. After each treatment, cells were washed and aliquoted into 35mm filter-capped 12x75 tubes (1ml per tube, 3 tubes per test group) to remove cell clumps. Samples can be analyzed using a FACSCAN(TM) flow cytometer with FACSCONVERT(TM) CellQuest software (Becton Dickinson). Antibodies that induce statistically significant levels of cell death as determined by PI uptake can be selected as cell death-inducing antibodies.
为了筛选与被目的抗体结合的CD20上的表位结合的抗体，可以进行常规杂交阻滞试验，例如在antibodies，aLaboratory Manual，Cold SpringHarbor Laboratory，Ed Harlow and David Lane(1988)中所描述的。这一试验可用于判断受试抗体是否与本发明的抗CD20抗体结合在相同的位点或表位上。或者或另外，可以用本领域已知的方法进行表位作图。例如，抗体序列可以例如通过丙氨酸扫描进行诱变处理，以鉴别接触残基。一开始测试突变抗体对多克隆抗体的结合以保证适当的折叠。在一不同方法中，相应于CD20不同区域的肽可用在与测试抗体或者与测试抗体和具有已表征或已知表位的抗体的竞争试验中。To screen for antibodies that bind to epitopes on CD20 bound by the antibody of interest, conventional hybridization retardation assays, such as those described in Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether the test antibody binds to the same site or epitope as the anti-CD20 antibody of the present invention. Alternatively or additionally, epitope mapping can be performed using methods known in the art. For example, antibody sequences can be mutagenized, eg, by alanine scanning, to identify contact residues. Mutant antibodies were initially tested for binding against polyclonal antibodies to ensure proper folding. In a different approach, peptides corresponding to different regions of CD20 can be used in competition assays with test antibodies or with test antibodies and antibodies with characterized or known epitopes.
载体，宿主细胞和重组方法Vectors, Host Cells and Recombination Methods
本发明还提供编码人源化CD20结合抗体的分离的核酸，包含该核酸的载体和宿主，以及制备该抗体的重组技术。The present invention also provides an isolated nucleic acid encoding a humanized CD20-binding antibody, vectors and hosts comprising the nucleic acid, and recombinant techniques for producing the antibody.
为了重组制备所述抗体，分离其编码核酸并插入复制型载体中以便进一步克隆(扩增DNA)或表达。编码所述抗体的DNA可以用常规方法很容易地分离并测序(例如使用能够特异性结合编码抗体重链和轻链的基因的寡核苷酸探针)。有多种载体可以利用。载体组分通常包括但不限于一或多个下列组分：信号序列，复制起点，一或多个标记基因，增强子元件，启动子，和转录终止序列。For recombinant production of the antibodies, their encoding nucleic acids are isolated and inserted into replicating vectors for further cloning (amplification of DNA) or expression. DNA encoding the antibodies can be readily isolated and sequenced by conventional methods (eg, using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). There are various vectors available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
(i)信号序列组分(i) Signal sequence components
本发明的CD20结合抗体不仅可以直接重组产生，还可作为与异源多肽融合的融合多肽来重组产生，所述异源多肽优选信号序列或其它在成熟蛋白或多肽的N末端上具有特异性裂解位点的多肽。所选异源信号序列优选是被宿主细胞识别并加工(即被信号肽酶裂解)的序列。对于不能识别和加工天然CD20结合抗体信号序列的原核宿主细胞，信号序列被选自，例如，碱性磷酸酶，青霉素酶，1pp或热稳定肠毒素II前导序列的原核生物信号序列取代。为了进行酵母分泌，可以将天然信号序列取代为，例如，酵母转化酶前导序列，α因子前导序列(包括糖酵母(Saccharomyces)α因子前导序列和克鲁维酵母(Kluyveromyces)α因子前导序列)，或酸性磷酸酶前导序列，白色念珠菌葡萄糖淀粉酶前导序列，或WO 90/13646描述的信号。在哺乳动物细胞表达中，哺乳动物信号序列以及病毒的分泌前导序列，如单纯疱疹病毒gD信号都可以采用。The CD20-binding antibody of the present invention can not only be directly recombinantly produced, but can also be recombinantly produced as a fusion polypeptide fused with a heterologous polypeptide, preferably a signal sequence or other specific cleavage at the N-terminus of a mature protein or polypeptide. site of the polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that are unable to recognize and process the native CD20-binding antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, 1pp or the thermostable enterotoxin II leader sequence. For yeast secretion, the native signal sequence can be replaced by, for example, the yeast invertase leader, the alpha factor leader (including the Saccharomyces alpha factor leader and the Kluyveromyces alpha factor leader), Or the acid phosphatase leader sequence, the C. albicans glucoamylase leader sequence, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex virus gD signal, can be used.
这些前体区的DNA与编码CD20结合抗体的DNA连接在阅读框内。The DNA of these precursor regions was ligated in-frame with the DNA encoding the CD20-binding antibody.
(ii)复制起点(ii) origin of replication
表达载体和克隆载体都包含能使该载体在一或多种选定的宿主细胞中复制的核酸序列。一般情况下，在克隆载体中，这种序列是能使该载体独立于宿主染色体DNA而复制的序列，包括复制起点或自主复制序列。这样的序列在各种细菌、酵母和病毒中都是众所周知的。质粒pBR322的复制起点适合大多数革兰氏阴性细菌，2μ质粒起点适合酵母菌，多种病毒起点(SV40，多瘤病毒(Polyoma)，腺病毒，VSV或BPV)可用于哺乳动物细胞中的克隆载体。复制起点组分一般不是哺乳动物表达载体所必需的(SV40起点的使用通常仅仅是由于其包含早期启动子)。Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, such sequences are those that enable the vector to replicate independently of the host chromosomal DNA, including origins of replication or autonomously replicating sequences. Such sequences are well known in various bacteria, yeast and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and a variety of viral origins (SV40, Polyoma, adenovirus, VSV or BPV) can be used for cloning in mammalian cells carrier. An origin of replication component is generally not required for mammalian expression vectors (the SV40 origin is often used only because it contains an early promoter).
(iii)选择基因组分(iii) Selection of genetic components
表达载体和克隆载体可以包含选择基因，也称选择标记。典型的选择基因编码具有以下性质的蛋白：(a)赋予对抗生素或其它毒素(如氨苄青霉素，新霉素，氨甲蝶呤或四环素)的抗性，(b)弥补营养缺陷，或(c)提供复合培养基不能供给的关键营养物，例如编码芽孢杆菌(Bacilli)D-丙氨酸消旋酶的基因。Expression and cloning vectors may contain a selectable gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate, or tetracycline), (b) compensate for auxotrophs, or (c ) provide key nutrients that complex media cannot supply, such as the gene encoding Bacilli D-alanine racemase.
选择方案的一个实例是利用药物限制(arrest)宿主细胞的生长。那些被异源基因成功转化的细胞产生赋予药物抗性的蛋白，从而在该选择环境中存活。这种显性选择的实例采用药物新霉素，霉酚酸和潮霉素。An example of a selection strategy is the use of drugs to arrest the growth of host cells. Those cells successfully transformed with the heterologous gene produce the protein conferring drug resistance and thus survive the selection environment. Examples of such dominant selection employ the drugs neomycin, mycophenolic acid and hygromycin.
适合于哺乳动物细胞的另一例选择标记是允许鉴定能摄取所述抗体核酸的细胞的那些，如DHFR，胸苷激酶，金属硫蛋白-I型和-II型，优选灵长类金属硫蛋白基因，腺苷脱氨酶，鸟氨酸脱羧酶等。Another example of selectable markers suitable for mammalian cells are those that allow the identification of cells capable of taking up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-type I and -II, preferably the primate metallothionein gene , adenosine deaminase, ornithine decarboxylase, etc.
例如，细胞被DHFR选择基因转化后，先将所有转化体培养在包含氨甲蝶呤(Mtx，为DHFR的一种竞争型拮抗剂)的培养基中来鉴定细胞。当采用野生型DHFR时，合适的宿主细胞是DHFR活性有缺陷的中国仓鼠卵巢(CHO)细胞系。For example, after cells are transformed with a DHFR selection gene, all transformants are cultured in a medium containing methotrexate (Mtx, a competitive antagonist of DHFR) to identify the cells. When using wild-type DHFR, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
或者，宿主细胞(尤其包含内源DHFR的野生型宿主)被编码CD20结合抗体的DNA序列，野生型DHFR蛋白，以及另一种选择标记如氨基糖苷3’-磷酸转移酶(APH)转化或共转化以后，可以通过在含有针对该选择标记的选择试剂如氨基糖苷类抗生素(如卡那霉素，新霉素或G418)的培养基中培养细胞来进行选择。参见美国专利4,965,199。Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) are transformed or co-transformed with a DNA sequence encoding a CD20-binding antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH). After transformation, selection can be performed by culturing the cells in a medium containing a selection agent for the selection marker, such as an aminoglycoside antibiotic (eg kanamycin, neomycin or G418). See US Patent 4,965,199.
适用于酵母的合适选择基因是存在于酵母质粒YRp7中的trp1基因(Stinchcomb等，Nature，282：39(1979))。trp1基因为不能在色氨酸中生长的酵母突变株(例如ATCC 44076或PEP4-1)提供了选择标记(Jones，Genetics，85：12(1977))。此后，酵母宿主细胞基因组中trp1损伤的存在提供了通过在缺乏色氨酸的条件中生长而检测转化的有效环境。类似地，Leu2-缺陷型酵母菌株(ATCC 20,622或38,626)可以由携带Leu2基因的已知质粒来互补。A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene provides a selectable marker for mutant strains of yeast that cannot grow in tryptophan (eg, ATCC 44076 or PEP4-1) (Jones, Genetics, 85:12 (1977)). Thereafter, the presence of trp1 lesions in the yeast host cell genome provides an efficient environment for detection of transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) can be complemented by known plasmids carrying the Leu2 gene.
此外，源自1.6μm环状质粒pKD1的载体可以用于转化克鲁维酵母(Kluyveromyces)。备选地，Van den Berg，Bio/Technology，8：135(1990)报道了一种用于在乳克鲁维酵母(K.lactis)中大规模制备重组小牛凝乳酶的表达系统。还有人公开了用于通过克鲁维酵母工业菌株分泌成熟重组人血清白蛋白的稳定、多拷贝表达载体。Fleer等，Bio/Technology，9：968-975(1991)。In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces. Alternatively, Van den Berg, Bio/Technology, 8:135 (1990) reported an expression system for the large-scale production of recombinant calf chymosin in K. lactis. A stable, multi-copy expression vector for the secretion of mature recombinant human serum albumin by an industrial strain of Kluyveromyces has also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
(iv)启动子组分(iv) Promoter components
表达载体和克隆载体通常包含能被宿主生物识别的启动子，它与所述编码CD20结合抗体的核酸可操作相连。适用于原核宿主的启动子包括phoA启动子，β-内酰胺酶和乳糖启动子系统，碱性磷酸酶，色氨酸(trp)启动子系统，和杂合(hybrid)启动子如tac启动子。但其它已知的细菌启动子也是合适的。适用于细菌系统的启动子还可以包含与编码CD20结合抗体的DNA可操作相连的Shine-Dalgarno(S.D.)序列。Expression and cloning vectors typically contain a promoter recognized by the host organism operably linked to the nucleic acid encoding the CD20-binding antibody. Promoters suitable for prokaryotic hosts include the phoA promoter, the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter . However, other known bacterial promoters are also suitable. Promoters suitable for use in bacterial systems may also comprise a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the CD20-binding antibody.
真核生物的启动子序列是已知的。几乎所有的真核基因在转录起始点上游约25-30个碱基处具有AT-富集区。很多基因在其转录起始点上游70-80个碱基处有另一种序列：CNCAAT区，其中N可以是任何核苷酸。大多数真核基因的3’端是AATAAA序列，它可以作为一种信号用于将poly-A尾添加到编码序列3’端。所有这些序列都适合于插入真核表达载体中。Eukaryotic promoter sequences are known. Almost all eukaryotic genes have an AT-rich region approximately 25-30 bases upstream of the transcription start point. Many genes have another sequence 70-80 bases upstream of their transcription start point: CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which serves as a signal for adding a poly-A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.
适用于酵母宿主的启动子序列的实例包括：3-磷酸甘油酸激酶或其它糖酵解酶的启动子，所述其它糖酵解酶如烯醇化酶，甘油醛-3-磷酸脱氢酶，己糖激酶，丙酮酸脱羧酶，磷酸果糖激酶，葡萄糖-6磷酸异构酶，3-磷酸甘油变位酶，丙酮酸激酶，磷酸丙糖异构酶，磷酸葡萄糖异构酶和葡萄糖激酶。Examples of promoter sequences suitable for use in yeast hosts include: promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, Hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 phosphate isomerase, glycerol 3-phosphate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.
其它的酵母启动子，即那些另具有由生长条件控制转录的优点的诱导型启动子，是下述基因的启动子区：醇脱氢酶2、异细胞色素C(isocytochromeC)、酸性磷酸酶、与氮代谢相关的降解酶、金属硫蛋白、甘油醛-3-磷酸脱氢酶，以及负责麦芽糖和半乳糖利用的酶。在EP 73,657中进一步描述了适用于酵母表达的载体和启动子。酵母增强子与酵母启动子联合使用也是有利的。Other yeast promoters, i.e. those inducible promoters which additionally have the advantage of controlling transcription by growth conditions, are the promoter regions of the following genes:
在哺乳动物宿主细胞中，从载体转录CD20结合抗体是受，例如启动子调控，所述启动子来自病毒基因组，如多形瘤病毒、鸡痘病毒(fowlpoxvirus)、腺病毒(如腺病毒2)、牛乳头瘤病毒、禽肉瘤病毒、巨细胞病毒、逆转录病毒、乙型肝炎病毒，最优选猴病毒40(SV40)的启动子，来自异源哺乳动物的启动子，如肌动蛋白启动子或免疫球蛋白启动子等，来自热休克启动子，前提是这些启动子与宿主细胞系统相容。In mammalian host cells, transcription of a CD20-binding antibody from a vector is regulated by, for example, a promoter from a viral genome, such as polyoma virus, fowlpox virus, adenovirus (e.g. adenovirus 2) , bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, most preferably the promoter of Simian Virus 40 (SV40), a promoter from a heterologous mammal such as the actin promoter or immunoglobulin promoters, etc., from heat shock promoters, provided these are compatible with the host cell system.
SV40病毒的早期和晚期启动子可以作为还包含SV40病毒复制起点的SV40限制性片段而方便地获得。人巨细胞病毒的立即早期启动子可以作为Hind III E限制性片段方便地获得。美国专利4,419,446中公开了在哺乳动物宿主中用牛乳头瘤病毒作为载体来表达DNA的系统。美国专利4,601,978中叙述了对这个系统改进。另见Reyes等，Nature，297：598-601(1982)关于在单纯疱疹病毒胸苷激酶启动子控制下在小鼠细胞中表达人β干扰素cDNA。备选地，可以将劳氏肉瘤病毒长末端重复序列用作启动子。The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a Hind III E restriction fragment. US Patent No. 4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. Improvements to this system are described in US Patent 4,601,978. See also Reyes et al., Nature, 297:598-601 (1982) on the expression of human beta interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.
(v)增强子元件组分(v) Enhancer element components
编码本发明CD20结合抗体的DNA在高等真核生物中的转录常常通过将增强子序列插入载体中来增加。目前已知很多哺乳动物基因(球蛋白、弹性蛋白酶、白蛋白、甲胎蛋白和胰岛素)的增强子序列。但通常使用真核细胞病毒的增强子。实例包括在复制起始点晚期侧的SV40增强子(bp100-270)，巨细胞病毒早期启动子增强子，在复制起始点晚期侧的多形瘤增强子，和腺病毒增强子。也可参见Yaniv，Nature，297：17-18(1982)所述用于活化真核启动子的增强元件。增强子可以剪接插入载体中抗体编码序列的5’或3’侧，但优选位于启动子的5’侧。Transcription in higher eukaryotes of DNA encoding a CD20-binding antibody of the invention is often increased by inserting enhancer sequences into the vector. Enhancer sequences are known for many mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Usually, however, enhancers of eukaryotic viruses are used. Examples include the SV40 enhancer (bp100-270) on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer. See also Yaniv, Nature, 297: 17-18 (1982) for enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector on the 5' or 3' side of the antibody coding sequence, but is preferably located on the 5' side of the promoter.
(vi)转录终止组分(vi) Transcription termination component
用于真核宿主细胞(酵母、真菌、昆虫、植物、动物、人或来自其它多细胞生物的有核细胞)的表达载体，还可以包括对转录终止和mRNA稳定所必需的序列。这些序列通常来自真核细胞或病毒的DNA或cDNA的5’(偶尔为3’)非翻译区。这些区域包含转录为编码多价抗体的mRNA的非翻译区中聚腺苷酸化片段的核苷酸片段。一种有效的转录终止组分是牛生长激素聚腺苷酸化区。参见WO94/11026以及本文公开的表达载体。Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) may also include sequences necessary for transcription termination and mRNA stabilization. These sequences are usually derived from the 5' (occasionally 3') untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated region of the mRNA encoding the multivalent antibody. One potent transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vectors disclosed therein.
(vii)选择并转化宿主细胞(vii) Selection and transformation of host cells
克隆或表达本文所述载体中的DNA的适宜宿主细胞，包括上述原核生物、酵母或高等真核细胞。适于此目的的原核生物包括真细菌，如革兰氏阴性或革兰氏阳性细菌，例如肠杆菌科(Enterobacteriaceae)，如埃希氏菌属(Escherichia)，例如，大肠杆菌(E.coli)，肠杆菌属(Enterobacter)，欧文菌属(Erwinia)，克雷白菌属(Klebsiella)，变形菌杆属(Proteus)，沙门菌属(Salmonella)(如鼠伤寒沙门菌(Salmonella typhimurium))，沙雷菌属(Serratia)(如粘质沙雷菌(Serratia marcescans))和志贺菌属(Shigella)等，以及芽孢杆菌属(Bacilli)如枯草芽孢杆菌(B.subtilis)和地衣芽孢杆菌(B.licheniformis)(例如1989年4月12日出版的DD 266，710中所述地衣芽孢杆菌41P)等，假单胞菌属(Pseudomonas)如铜绿菌假单胞菌(P.aeruginosa)，及链霉菌(Streptomyces)。优选的大肠杆菌克隆宿主是大肠杆菌294(ATCC 31，446)，但其它菌株，如大肠杆菌B，大肠杆菌X1776(ATCC 31，537)和大肠杆菌W3110(ATCC 27，325)也是合适的。这些实例是用于说明，并非限制。Suitable host cells for cloning or expressing the DNA in the vectors described herein include the aforementioned prokaryotes, yeast or higher eukaryotic cells. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria, for example Enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia), for example, Escherichia coli (E.coli) , Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (such as Salmonella typhimurium), Serratia (such as Serratia marcescans) and Shigella (Shigella), etc., and Bacilli (Bacilli) such as Bacillus subtilis (B.subtilis) and Bacillus licheniformis (B. .licheniformis) (such as Bacillus licheniformis 41P described in DD 266,710 published on April 12, 1989), etc., Pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (P.aeruginosa), and chain Mold (Streptomyces). A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are for illustration, not limitation.
全长抗体、抗体片段与抗体融合蛋白质可以在细菌中产生，特别是当不需要糖基化作用与Fc效应子功能时，例如当治疗抗体偶联至细胞毒药剂(例如毒素)并且免疫偶联物自身显示在破坏肿瘤细胞上的效力时。全长抗体在循环中具有更长的半衰期。在大肠杆菌中生产是更快和更具有成本效率的。对于抗体片段与多肽在细菌中的表达，见例如美国专利5,648,237(Carter et.al.)，美国专利5,789,199(Joly et al.)，与美国专利5,840,523(Simmons et al.)，其中描绘了用于最优化表达与分泌的翻译起始区域(TIR)和信号顺序，这些专利在此引入作为参考。在表达之后，抗体从大肠杆菌细胞糊状混合物中在可溶组分中分离，并且取决于同种型，可以通过例如蛋白A或者G柱纯化。最后纯化可以以类似于纯化在CHO细胞中表达的抗体的程序进行。Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, such as when therapeutic antibodies are conjugated to cytotoxic agents (such as toxins) and immunoconjugated The drug itself shows efficacy in destroying tumor cells. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent 5,648,237 (Carter et. al.), U.S. Patent 5,789,199 (Joly et al.), and U.S. Patent 5,840,523 (Simmons et al.), which describe methods for Translation initiation region (TIR) and signal sequences optimized for expression and secretion, these patents are hereby incorporated by reference. After expression, antibodies are separated in the soluble fraction from the E. coli cell paste mixture and, depending on the isotype, can be purified, for example, by protein A or G columns. Final purification can be performed in a procedure similar to the purification of antibodies expressed in CHO cells.
除了原核生物，真核微生物如丝状真菌或酵母也是适合于编码CD20结合抗体的载体的克隆或表达宿主。酿酒酵母(Saccharomyces cerevisiae)或常见的面包酵母是最常用的低等真核宿主微生物。还有多个其它属、种和株已有商品供应，并且可以用于本发明，例如粟酒裂殖酵母(Schizosaccharomyces pombe)；克鲁维酵母属(Kluyveromyces)宿主，例如乳克鲁维酵母(K.lactis)、脆壁克鲁维酵母(K.fragilis)(ATCC 12，424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16，045)、威克曼氏克鲁维酵母(K.wickeramii)(ATCC 24，178)、K.waltii(ATCC 56，500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC 36，906)、耐热克鲁维酵母(K.thermotolerans)和马克斯克鲁维氏酵母(K.marxianus)等；西洋蓍霉(yarrowia)(EP 402，226)；巴斯德毕赤酵母(pichia pastoris)(EP 183，070)；念珠菌属(Candida)；Trichodermareesia(EP 244，234)；粗糙链孢霉(Neurospora crassa)；许旺氏酵母属(schwanniomyces)如西方许旺氏酵母(schwanniomyces occidentalis)等；和丝状真菌，例如链孢霉属(Neurospora)、青霉属(Penicillium)、Tolypocladium以及曲霉属宿主如构巢曲霉(A.nidulans)和黑曲霉(A.niger)等。In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding CD20-binding antibodies. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. There are also a number of other genera, species and strains that are commercially available and can be used in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as Kluyveromyces lactis ( K.lactis), Kluyveromyces fragilis (K.fragilis) (ATCC 12,424), Kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), Kluyveromyces wickmannii (K .wickeramii) (ATCC 24,178), K.waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906), K.thermotolerans and K. marxianus, etc.; yarrowia (EP 402, 226); Pichia pastoris (EP 183, 070); Candida (Candida); Trichodermareesia (EP 244, 234); Neurospora crassa; Schwanniomyces such as schwanniomyces occidentalis etc.; and filamentous fungi such as Neurospora , Penicillium (Penicillium), Tolypocladium and Aspergillus hosts such as Aspergillus nidulans (A.nidulans) and Aspergillus niger (A.niger).
适合于表达糖基化CD20结合抗体的宿主细胞来自多细胞生物。无脊椎动物细胞的实例包括植物和昆虫细胞。目前已经从下述宿主中鉴定了大量的杆状病毒株和变体以及相应的容许型昆虫宿主细胞：草地夜蛾(Spodoptera Frugiperda，毛虫)、埃及伊蚊(Aedes aegypti，蚊子)、白纹伊蚊(Aedes albopictus，蚊子)、Drosophila melanogaster(果蝇)和家蚕蛾(Bombyxmori)等。用于转染的各种病毒株可以公开地获得，例如加利福尼亚Y级夜蛾(Autographa california)NPV的L-1变体和家蚕蛾NPV的Bm-5株，并且这些病毒可以在此用作本发明的病毒，尤其是用于转染草地夜蛾细胞。Suitable host cells for expression of glycosylated CD20-binding antibodies are from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A large number of baculovirus strains and variants and corresponding permissive insect host cells have been identified from the following hosts: Spodoptera Frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes aegypti (mosquito), Mosquito (Aedes albopictus, mosquito), Drosophila melanogaster (fruit fly), silkworm moth (Bombyxmori), etc. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa california NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used here as present Invented virus, especially for transfection of Spodoptera frugiperda cells.
棉花(cotton)、玉米(corn)、土豆(potato)、大豆(soybean)、矮牵牛(petunia)、西红柿(tomato)和烟草(tobacco)的植物细胞培养物也可以用作宿主。Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.
然而，关注最多的是脊椎动物细胞，而且在培养(组织培养)中繁殖脊椎动物细胞已经成为常规方法。有效哺乳动物宿主细胞系的实例是用SV40转化的猴肾CV1细胞系(COS-7，ATCC CRL 1651)；人胚肾细胞系(293细胞或经过再克隆后能在悬浮培养物中生长的293细胞，Graham等，J.Gen Virol.36：59(1977))；仓鼠幼鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub等，Proc.Natl.Acad.Sci.U.S.A.77：4216(1980))；小鼠足细胞(TM4，Mather，Biol.Reprod.23：243-251(1980))；猴肾细胞(CV1 ATCCCCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK ATCC CCL 34)；布法罗(buffalo)大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝细胞(Hep G2，HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL 51)；TRI细胞(Mather等，Annals N.Y.Acad.Sci.383：44-68(1982))；MRC 5细胞；FS4细胞；和人肝细胞癌细胞系(Hep G2)。However, vertebrate cells have received the most attention, and propagating vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 cells or 293 Cells, Graham et al., J.Gen Virol.36:59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.Natl.Acad. Sci.U.S.A.77:4216 (1980)); Mouse podocyte (TM4, Mather, Biol.Reprod.23:243-251 (1980)); Monkey kidney cell (CV1 ATCCCCL 70); African green monkey kidney cell (VERO -76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N.Y.Acad.Sci.383:44 -68(1982));
宿主细胞用上述用于制备CD20结合抗体的表达或克隆载体转化，并在改进型传统营养培养基上培养，所述培养基经改进已适于诱导启动子、筛选转化体、或扩增编码所需序列的基因。Host cells are transformed with the expression or cloning vectors described above for the production of CD20-binding antibodies and cultured on conventional nutrient media modified to induce promoters, select transformants, or amplify genes encoding the The gene to be sequenced.
(viii)培养宿主细胞(viii) Culturing host cells
用于产生本发明的CD20结合抗体的宿主细胞可以在各种培养基中培养。市售培养基如Ham’s F10(Sigma)，极限必需培养基((MEM)，Sigma)，RPMI-1640(Sigma)，和Dulbecco改良Eagle培养基((DMEM)，Sigma)都适于培养所述宿主细胞。此外，Ham et al.，Meth.Enz.58：44(1979)，Barnes et al.，Anal.Biochem.102：255(1980)，U.S.Pat.Nos.4,767,704；4,657,866；4,927,762；4,560,655；or 5,122,469；WO 90/03430；WO 87/00195；或U.S.Patent Re.30，985所述的任一种培养基也可以用作宿主细胞培养基。任何上述培养基可以根据需要添加激素和/或其它生长因子(如胰岛素，运铁蛋白，或表皮生长因子)，盐(如氯化钠，钙，镁，和磷酸盐)，缓冲液(如HEPES)，核苷酸(如腺苷和胸苷)，抗生素(如GentamycinTM)，痕量元素(定义为通常以微摩尔水平的终浓度出现的无机化合物)，和葡萄糖或等效能源。还可以包括本领域技术人员已知的适当浓度的任何其它必需添加物。培养条件，如温度，pH等，都是在选定的表达宿主上已用到的那些，并且对本领域技术人员而言也是显而易见的。Host cells for producing the CD20-binding antibody of the present invention can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing the host cell. Additionally, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 90/03430; WO 87/00195; or any of the media described in US Patent Re. 30,985 can also be used as a host cell culture medium. Any of the above media can be supplemented with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES ), nucleotides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ ), trace elements (defined as inorganic compounds usually present at final concentrations on the micromolar level), and glucose or an equivalent energy source. Any other necessary additives may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those that have been used on the chosen expression host and will be apparent to those skilled in the art.
(ix)抗体纯化(ix) Antibody purification
使用重组技术时，所述抗体可产生在细胞内，在周质空间中，或直接分泌到培养基中。如果所述抗体是产生在细胞内，首先要通过例如离心或超滤作用除去颗粒状残渣(为宿主细胞或裂解的片段)。Carter et al.，Bio/Technology 10：163-167(1992)描述了一种将已分泌到大肠杆菌周质空间中的抗体分离的方法。简言之，在有乙酸钠(pH 3.5)，EDTA，和苯甲基磺酰氟(PMSF)存在的情况下将细胞团解冻约30分钟。细胞碎片可以通过离心除去。在抗体分泌至培养基的情况中，通常首先使用诸如Amicon或MilliporePellicon超滤单元等市售蛋白浓缩滤器浓缩这类表达系统的上清。上述任一步骤中可以包括PMSF等蛋白酶抑制剂，以便抑制蛋白水解，还可以包括抗生素，以便防止外来污染物(adventitious contaminant)的生长。Using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris (either host cells or lysed fragments) is first removed, eg, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for isolating antibodies that have been secreted into the periplasmic space of E. coli. Briefly, cell pellets were thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. In the case of antibody secretion into culture medium, supernatants from such expression systems are usually first concentrated using commercially available protein concentration filters such as Amicon or MilliporePellicon ultrafiltration units. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can also be included to prevent the growth of adventitious contaminants.
从所述细胞制备的抗体组合物可以利用，例如，羟基磷灰石层析、凝胶电泳、透析、和亲和层析等方法纯化，优选亲和层析纯化技术。蛋白A作为亲和配体的适宜性有赖于所述抗体中存在的任何免疫球蛋白Fc区的种类和同种型。蛋白A可以用来纯化基于人γ1，γ2或γ4重链的抗体(Lindmarket al.，J.Immunol.Meth.62：1-13(1983))。蛋白G被建议用于所有小鼠同种型以及用于人γ3(Guss et al.，EMBO J.5：15671575(1986))。亲和配体附着的基质最常是琼脂糖，也可以采用其它基质。机械稳定的基质例如控制孔径的玻璃(controlled pore glass)或聚(苯二乙烯)苯(poly(styrenedivinyl)benzene)与琼脂糖相比，其流速更快且处理时间更短。当抗体包含CH3区时，可以用Bakerbond ABXTM树脂(J.T.Baker，Phillipsburg，NJ)纯化。其它蛋白纯化技术，例如离子交换柱分级分离，乙醇沉淀，反相HPLC，硅层析，肝素SEPHAROSETM层析，阳离子或阴离子交换树脂(例如聚天冬氨酸柱)层析，层析聚焦，SDS-PAGE，和硫酸铵沉淀也可以采用，这取决于所回收的抗体。The antibody composition prepared from the cells can be purified by, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and the affinity chromatography purification technique is preferred. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region present in the antibody. Protein A can be used to purify antibodies based on
在任何初步提纯步骤之后，在pH大约2.5-4.5之间利用洗脱缓冲液对包含目的抗体与污染物的混合物进行低pH疏水相互作用层析，优选地在低盐浓度下进行(例如大约0-0.25M盐)。After any preliminary purification steps, the mixture comprising the antibody of interest and contaminants is subjected to low pH hydrophobic interaction chromatography using an elution buffer between pH about 2.5-4.5, preferably at a low salt concentration (e.g. about 0 -0.25M salt).
抗体偶联物antibody conjugate
抗体可被偶联至细胞毒性剂例如毒素或放射性同位素。在某些实施方式中，毒素是加利车霉素、maytansinoid、多拉司他汀、auristatin E与其类似物或衍生物是优选的。Antibodies can be conjugated to cytotoxic agents such as toxins or radioisotopes. In certain embodiments, it is preferred that the toxin is calicheamicin, maytansinoid, dolastatin, auristatin E, and analogs or derivatives thereof.
优选的药物/毒素包括DNA损伤剂、微管聚合或解聚抑制剂与抗代谢物。细胞毒药剂的优选类别包括，例如，酶抑制剂例如二氢叶酸还原酶抑制剂，与胸苷酸合酶抑制剂，DNA嵌入剂，DNA裂解剂、拓扑异构酶抑制剂、蒽环类抗生族药物、长春花属药物、丝裂霉素素、博来霉素、细胞毒核苷、喋啶族药物、diynenes、鬼臼霉素与分化诱导物。Preferred drugs/toxins include DNA damaging agents, inhibitors of microtubule polymerization or depolymerization, and antimetabolites. Preferred classes of cytotoxic agents include, for example, enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, anthracycline antibiotics family of drugs, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, pteridine family of drugs, diynenes, podophyllotoxin and differentiation inducers.
这些类别的特别有用的成员包括，例如，氨甲喋呤，甲氨喋呤，二氯氨甲喋呤，5-氟尿嘧啶，6-巯基嘌呤，阿糖胞苷，美法仑，环氧长春碱，洛诺西丁、放线菌素、道诺红菌素、阿霉素、N-(5，5-二醋氧戊基)阿霉素、吗啉代阿霉素、1-(2-氯乙基)-1，2-二精脒甲磺酰酰肼、N8-乙酰基精脒、氨基蝶呤甲氨喋呤、esperamicin、丝裂霉素C、丝裂霉素A、放线菌素、博来霉素、洋红霉素、氨基蝶呤、太利苏霉素、鬼臼霉素与鬼臼霉素衍生物例如鬼臼亚乙苷或鬼臼亚乙苷磷酸盐、长春碱、长春新碱、去乙酰长春酰胺、紫杉酚、泰索帝、视黄酸、丁酸、N8-乙酰基精脒、喜树碱、加利车霉素、苔藓抑素、cephalostatins、美登木素、布拉地新钠、maytansinoids例如DM-1、美登素、美登醇、N-去甲基-4，5-去环氧基美登醇、C-19-去氯美登醇、C-20-羟美登醇、C-20-去甲氧基美登醇、C-9-SH美登醇、C-14-烷氧基甲基美登醇、C-14-羟基或乙酰氧甲基美登醇、C-15-羟基/乙酰氧美登醇、C-15-甲氧基美登醇、C-18-N-去甲基美登醇与4，5-脱氧美登醇、auristatins例如auristatinE、M、PHE与PE；dolostatins例如dolostatin A、dolostatin B、dolostatin C、dolostatin D、dolostatin E、(20-epi与11-epi)、dolostatin G、dolostatin H、dolostatin I、dolostatin 1、dolostatin 2、dolostatin 3、dolostatin 4、dolostatin 5、dolostatin 6、dolostatin 7、dolostatin 8、dolostatin 9、dolostatin 10、deo-dolostatin10、dolostatin 11、dolostatin 12、dolostatin 13、dolostatin 14、dolostatin 15、dolostatin 16、dolostatin 17、与dolostatin 18；cephalostatins例如cephalostatin1、cephalostatin 2、cephalostatin 3、cephalostatin 4、cephalostatin 5、cephalostatin6、cephalostatin 7、25′-epi-cephalostatin 7、20-epi-cephalostatin 7、cephalostatin8、cephalostatin 9、cephalostatin 10、cephalostatin 11、cephalostatin 12、cephalostatin 13、cephalostatin 14、cephalostatin 15、cephalostatin 16、cephalostatin 17、cephalostatin 18、与cephalostatin 19。Particularly useful members of these classes include, for example, methotrexate, methotrexate, methotrexate, 5-fluorouracil, 6-mercaptopurine, cytarabine, melphalan, epoxyvinblastine, lonoxetine, Cactomycin, daunorubicin, doxorubicin, N-(5,5-diecetoxypentyl) doxorubicin, morpholino doxorubicin, 1-(2-chloroethyl)-1, 2-ditomycin methanesulfonyl hydrazide, N8-acetyl spermidine, aminopterin methotrexate, esperamicin, mitomycin C, mitomycin A, actinomycin, bleomycin, Carmine, aminopterin, telizumycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, deacetylvinblastine Amide, taxol, taxotere, retinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, bryostatin, cephalostatins, maytansinoid, bradixin sodium , maytansinoids such as DM-1, maytansine, maytansinol, N-desmethyl-4,5-deepoxy maytansinol, C-19-desclomaytansinol, C-20-hydroxymaytansinol Alcohol, C-20-desmethoxy maytansinol, C-9-SH maytansinol, C-14-alkoxymethyl maytansinol, C-14-hydroxy or acetoxymethyl maytansinol, C-15-Hydroxy/Acetoxymaytansinol, C-15-Methoxymaytansinol, C-18-N-desmethylmaytansinol and 4,5-deoxymaytansinol, auristatins such as auristatinE, M , PHE and PE; dolostatins such as dolostatin A, dolostatin B, dolostatin C, dolostatin D, dolostatin E, (20-epi and 11-epi), dolostatin G, dolostatin H, dolostatin I, dolostatin 1, dolostatin 2, dolostatin 3, dolostatin 4, dolostatin 5, dolostatin 6, dolostatin 7, dolostatin 8, dolostatin 9, dolostatin 10, deo-dolostatin 10, dolostatin 11, dolostatin 12, dolostatin 13, dolostatin 14, dolostatin 15, dolostatin 16, dolostatin 17, and dolostatin 18; Cephalostatins such as cephalostatin 1, cephalostatin 2, cephalostatin 3, cephalostatin 4, cephalostat in 5, cephalostatin 6, cephalostatin 7, 25′-epi-cephalostatin 7, 20-epi-cephalostatin 7, cephalostatin 8, cephalostatin 9, cephalostatin 10, cephalostatin 11, cephalostatin 12, cephalostatin 13, cephalostatin phal, cephalostatin 14, cephalostatin 1 17, cephalostatin 18, and cephalostatin 19.
Maytansinoids是有丝分裂抑制剂，通过抑制微管蛋白聚合而起作用。美登素最初从东非灌木齿叶美登木分离出来(美国专利3,896,111)。随后，人们发现某些微生物也产生maytansinoids，例如美登醇与C-3美登醇酯(美国专利4,151,042)。在美国专利4,137,230；4,248,870；4,256,746；4,260,608；4,265,814；4,294,757；4,307,016；4,308,268；4,308,269；4,309,428；4,313,946；4,315,929；4317,821；4,322,348；4,331,598；4,361,650；4,364,866；4,424,219；4,450,254；4,362,663；与4,371,533中披露了合成美登醇与其衍生物和类似物，在此特别地引入作为参考。Maytansinoids are mitosis inhibitors that work by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub maytansine (US Patent 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinoid esters (US Patent 4,151,042).在美国专利4,137,230；4,248,870；4,256,746；4,260,608；4,265,814；4,294,757；4,307,016；4,308,268；4,308,269；4,309,428；4,313,946；4,315,929；4317,821；4,322,348；4,331,598；4,361,650；4,364,866；4,424,219；4,450,254；4,362,663；与4,371,533中披露了Synthesis of maytansinol and its derivatives and analogs are expressly incorporated herein by reference.
美登素与maytansinoids已与特定地与肿瘤细胞抗原结合的抗体偶联。包含maytansinoids的免疫偶联物及其治疗用途在例如美国专利5,208,020，5,416,064与欧洲专利EP 0425235B1中披露，其公开在此特别地引入作为参考。Liu et al.，Proc.Natl.Acad.Sci.USA 93：8618-8623(1996)描述了包含指定为DM1的maytansinoid，该maytansinoid与针对人结肠直肠癌症的单克隆抗体C242连接。偶联物被发现对培养的结肠癌细胞具有高度细胞毒性，并且在体内肿瘤生长试验中显示抗肿瘤活性。Chari et al.Cancer Research52：127-131(1992)描述了免疫偶联物，其中maytansinoid通过二硫键接头与鼠抗体A7偶联，该鼠抗体A7与人结肠癌细胞系上的抗原结合，或者maytansinoid偶联至另一鼠单克隆抗体TA.1，该抗体与HER-2/neu癌基因结合。Maytansinoids and maytansinoids have been conjugated to antibodies that specifically bind tumor cell antigens. Immunoconjugates comprising maytansinoids and their therapeutic uses are disclosed, for example, in US Pat. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describe a maytansinoid comprising a designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and showed antitumor activity in an in vivo tumor growth assay. Chari et al. Cancer Research 52: 127-131 (1992) describe immunoconjugates in which maytansinoid is coupled via a disulfide linker to a murine antibody A7 that binds to an antigen on a human colon cancer cell line, or maytansinoid is conjugated to another murine monoclonal antibody, TA.1, which binds to the HER-2/neu oncogene.
本领域已知许多连接基团用于制备抗体maytansinoid偶联物，包括，例如，在美国专利5,208,020或EP专利0425235B1，与Chari et al.CancerResearch 52：127-131(1992)中所披露的。连接基团包括如上述专利中所披露的二硫化物基团、硫醚基团、对酸敏感的基团、对光敏感的基团、对肽酶敏感的基团、或对酯酶敏感的基团，优选二硫化物与硫醚基团。A number of linking groups are known in the art for preparing antibody maytansinoid conjugates, including, for example, those disclosed in US Patent 5,208,020 or EP Patent 0425235B1, and Chari et al. Cancer Research 52:127-131 (1992). Linking groups include disulfide groups, thioether groups, acid-sensitive groups, light-sensitive groups, peptidase-sensitive groups, or esterase-sensitive groups as disclosed in the above patents. groups, preferably disulfide and thioether groups.
可以使用各种双功能蛋白偶联剂制备抗体与maytansinoid的偶联物，例如N-琥珀酰亚胺基-3-(2-吡啶基二巯基)丙酸盐(SPDP)、琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸盐、iminothiolane(IT)、亚氨酸酯的双功能衍生物(例如二甲基己二酰亚氨酯HCL)，活性酯(例如二琥珀酰亚胺基辛二酸酯)、醛(例如戊二醛)、二叠氮化物(例如二-(p-叠氮苯甲酰基)已二胺)、二重氮衍生物(例如二-(p-重氮苯甲酰基)-乙二胺)、二异氰酸盐(例如甲苯2，6-二异氰酸盐)、与二-活性氟化合物(例如1，5-二氟-2，4-二硝基苯)。特别优选的偶联剂包括N-琥珀酰亚胺基-3-(2-吡啶基二巯基)丙酸盐(SPDP)(Carlsson et al.，Biochem.J.173：723-737[1978])与N-琥珀酰亚胺基-4-(2-吡啶基巯基)戊酸盐(SPP)，以提供二硫键。Antibody-maytansinoid conjugates can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl -4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidate (e.g. dimethyladipimide HCL ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazides (such as bis-(p-azidobenzoyl)hexamethylenediamine), double Nitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as
取决于连接类型，接头可以附着于maytansinoid分子的各个位置。例如，利用常规的偶联技术，用羟基反应可以形成酯键。反应可以发生在有羟基的C-3位置，C-14位置用羟甲基修饰，C-15位置用羟基修饰，C-20位置具有羟基。在优选实施方式中，在美登醇或美登醇类似物的C-3位置形成连接Depending on the type of linkage, linkers can be attached at various positions on the maytansinoid molecule. For example, an ester bond can be formed by reacting a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position with a hydroxyl group, the C-14 position is modified with a hydroxymethyl group, the C-15 position is modified with a hydroxyl group, and the C-20 position has a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or maytansinol analog
加利车霉素Calicheamicin
另一个感兴趣的免疫偶联物包含偶联至一或多个加利车霉素分子的CD20结合抗体。加利车霉素族抗生素能够在皮摩尔浓度以下产生双链DNA断裂。制备加利车霉素族的偶联物参见美国专利5,712,374，5,714,586，5,739,116，5,767285，5,770,701，5,770,710，5,773,001，5,877,296(均属于American Cyanamid Company)。可被使用的加利车霉素的结构类似物包括，但不局限于，γ1I，α2I，α3I，N-乙酰基γ1I，PSAG与2I1(Hinman et al.CancerResearch 53：3336-3342(1993)，Lode et al.Cancer Research 58：2925-2928(1998)与前面提到过的属于American Cyanamid的美国专利)。另一个可以偶联至抗体的抗肿瘤药物是QFA，其为抗叶酸物。加利车霉素与QFA均具有胞内作用位点，并且不容易越过质膜。因此，通过抗体介导的内在化所导致的这些药剂的细胞摄取大大增强他们的细胞毒效果。Another immunoconjugate of interest comprises a CD20 binding antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-strand DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, refer to US Patents 5,712,374, 5,714,586, 5,739,116, 5,767285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all belonging to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, γ1I , α2I , α3I , N-acetyl γ1I , PSAG and 2I 1 (Hinman et al. Cancer Research 53:3336 -3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998) and the aforementioned US patent belonging to American Cyanamid). Another antineoplastic drug that can be conjugated to an antibody is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not easily cross the plasma membrane. Thus, cellular uptake of these agents through antibody-mediated internalization greatly enhances their cytotoxic effects.
放射性同位素radioisotope
抗体可以包含高度放射性原子以选择性破坏肿瘤。已有各种放射性同位素用于生产放射性偶联的抗CD20抗体。实例包括At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、p32、Pb212与Lu的放射性同位素。当偶联物被用于诊断时，其可包含放射性原子用于闪烁法研究，例如tc99m或I123，或包含自旋标记物用于核磁共振(NMR)成像(又名磁共振成象，mri)，例如还是碘-123，碘-131，铟-111，氟-19，碳-13，氮-15，氧-17，钆，锰或铁。Antibodies can contain highly radioactive atoms to selectively destroy tumors. Various radioisotopes have been used for the production of radioconjugated anti-CD20 antibodies. Examples include At211 , I131 , I125 , Y90 , Re186 , Re188 , Sm153 , Bi212 , p32 ,Pb212 and radioactive isotopes of Lu. When the conjugate is used diagnostically, it may contain a radioactive atom for scintigraphic studies, such as tc99m or I123 , or a spin label for nuclear magnetic resonance (NMR) imaging (aka magnetic resonance imaging, mri), for example also iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
放射性或其它标记可以已知方式掺入偶联物。例如，肽可以生物合成或可以利用合适的氨基酸前体经由化学氨基酸合成来合成，化学氨基酸合成涉及例如用氟-19代替氢。标记例如tc99m或I123、Re186、Re188与In111可以通过肽中的半胱氨酸残基附着。钇-90可以通过赖氨酸残基附着。IODOGEN方法(Fraker et al(1978)Biochem.Biophys.Res.Commun.80：49-57)可用于掺入碘-123。“Monoclonal Antibodies in Immunoscintigraphy”(Chatal，CRC Press1989)详细描述了其它方法。Radioactive or other labels can be incorporated into the conjugates in known manner. For example, peptides can be biosynthesized or can be synthesized via chemical amino acid synthesis involving, for example, the substitution of fluorine-19 for hydrogen, using suitable amino acid precursors. Labels such as tc99m or I123 , Re186 , Re188 and In111 can be attached via cysteine residues in the peptide. Yttrium-90 can be attached via lysine residues. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
可以使用各种双功能蛋白偶联剂制备抗体与细胞毒性剂的偶联物，例如N-琥珀酰亚胺基-3-(2-吡啶基二巯基)丙酸盐(SPDP)、琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸盐、iminothiolane(IT)、亚氨酸酯的双功能衍生物(例如二甲基己二酰亚氨酯HCL)，活性酯(例如二琥珀酰亚胺基辛二酸酯)、醛(例如戊二醛)、二叠氮化物(例如二-(p-叠氮苯甲酰基)已二胺)、二重氮衍生物(例如二-(p-重氮苯甲酰基)-乙二胺)、二异氰酸盐(例如甲苯2，6-二异氰酸盐)、与二-活性氟化合物(例如1，5-二氟-2，4-二硝基苯)。例如，可以如Vitetta et al.Science 238：1098(1987)描述制备蓖麻毒免疫毒素。碳-14-标记的1-异硫氰酸苄基-3-甲基二乙烯-三胺五乙酸(MX-DTPA)是示例性的螯合剂，用来将放射性核苷酸结合至抗体。见WO94/11026。接头可以是“可断裂的接头”，以促进细胞毒类药物在细胞中的释放。例如，可使用酸不稳定的接头、肽酶敏感的接头、光敏感的接头、二甲基接头或包含二硫化物的接头(Chari et al.Cancer Research 52：127-131(1992)；美国专利5,208,020)。Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimide Amino-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidate (e.g. dimethyladipimide ester HCL), active esters (e.g. disuccinimidyl suberate), aldehydes (e.g. glutaraldehyde), diazides (e.g. di-(p-azidobenzoyl)hexamethylenediamine), Diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as
CD20结合抗体的治疗用途Therapeutic uses of CD20 binding antibodies
本发明的CD20结合抗体可用于治疗多种恶性与非恶性疾病，包括自身免疫病与相关病症，与CD20阳性癌症，包括B细胞淋巴瘤与白血病。骨髓中的干细胞(B细胞祖先)缺乏CD20抗原，允许健康B细胞在治疗后再生，并在几个月之内回复至正常水平。The CD20-binding antibodies of the invention are useful in the treatment of a variety of malignant and non-malignant diseases, including autoimmune diseases and related disorders, and CD20-positive cancers, including B-cell lymphomas and leukemias. Stem cells (B cell progenitors) in the bone marrow lack the CD20 antigen, allowing healthy B cells to regenerate after treatment and return to normal levels within a few months.
自身免疫病或自身免疫相关病症包括关节炎(类风湿性关节炎，青年类风湿性关节炎，骨关节炎，牛皮癣关节炎)，牛皮癣，皮炎包括异位性皮炎；慢性自身免疫荨麻疹，多肌炎/皮肌炎，中毒性表皮坏死松解，全身性硬皮病/硬化症，与炎性肠疾病(inflammatory bowel disease(IBD))有关的应答(Crobn’s病，溃疡性结肠炎)，呼吸窘迫综合症，成人呼吸窘迫综合症，脑膜炎，过敏性鼻炎，脑炎，葡萄膜炎，结肠炎，肾小球肾炎，过敏性病症，湿疹，哮喘，T细胞浸润有关的病症和慢性炎症反应，动脉粥样硬化，自身免疫性心肌炎，白细胞粘附缺陷，系统性红斑狼疮(SLE)，狼疮(包括肾炎型，非肾型，盘型，脱发型)，少年发作的糖尿病(juvenile onset diabetes)，多发性硬化，变应性脑脊髓炎，细胞因子与T淋巴细胞介导的与急性和迟发性过敏反应有关的免疫反应，肺结核，结节病，肉芽肿病包括韦格内氏肉芽肿病，粒细胞缺乏症，血管炎，再生障碍性贫血，库姆斯氏阳性贫血，戴布二氏贫血，免疫性溶血性贫血包括自身免疫性溶血性贫血(AIHA)，恶性贫血，纯红细胞发育不全(PRCA)，凝血因子VIII缺乏，甲型血友病，自身免疫性中性白细胞减少，全血细胞减少症，白血球减少症，涉及白细胞血细胞渗出的疾病，CNS炎性紊乱，多器官损伤综合症，重症肌无力，抗原抗体复合物介导的疾病，抗肾小球基底膜疾病，抗磷脂抗体综合症，过敏性神经炎，Bechet病，Castleman综合症，古德帕斯彻氏综合症，兰-伊二氏类重症肌无力综合征，雷诺氏综合症，斯耶格仑氏综合症，斯-约二氏综合症，实体器官移植排斥(包括对高位反应性抗体滴度的预处理，IgA在组织沉积，等等)，移植物抗宿主病(GVHD)，天疱疮样大疱，天疱疮(所有的，包括寻常的、叶状的)，自身免疫性多内分泌腺病，赖特尔氏病(Reiter′sdisease)，僵人综合症，巨细胞性动脉炎，免疫复合物性肾炎，IgA肾病，IgM多神经病或IgM介导的神经病，特发性血小板减少性紫癜(ITP)，血栓性血小板减少性紫癜(TTP)，自身免疫性血小板减少，睾丸与卵巢的自身免疫病包括自身免疫性睾丸炎与卵巢炎，原发性甲状腺功能减退，自身免疫性内分泌疾病包括自身免疫性甲状腺炎，慢性甲状腺炎(桥本氏甲状腺炎)，亚急性甲状腺炎，自发性甲状腺机能减退，阿狄森氏病，格雷夫斯氏病、自身免疫的多腺综合症(或多腺内分泌病综合症)，I型糖尿病也称为胰岛素依赖性糖尿病(IDDM)与席汉氏综合症，自身免疫性肝炎，淋巴结间质肺炎(HIV)，闭塞性细支气管炎(非移植)与NSIP，格-巴二氏综合症，大血管血管炎(包括风湿性多肌痛与巨细胞(高安氏)动脉炎)，中等血管血管炎(包括川崎病与结节性多动脉炎)，强直性脊柱炎，贝尔格尔氏病(IgA肾病)，快速进展的肾小球肾炎，原发性胆汁性肝硬变，Celiac sprue(粘胶质肠病)，冷球蛋白血症，ALS，冠状动脉病。Autoimmune or autoimmune-related conditions including arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis including atopic dermatitis; chronic autoimmune urticaria, multiple Myositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma/sclerosis, responses related to inflammatory bowel disease (IBD) (Crobn's disease, ulcerative colitis), respiratory Distress syndrome, adult respiratory distress syndrome, meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, T cell infiltration related conditions and chronic inflammatory response , atherosclerosis, autoimmune myocarditis, leukocyte adhesion defect, systemic lupus erythematosus (SLE), lupus (including nephritic type, non-renal type, disc type, alopecia type), juvenile onset diabetes (juvenile onset diabetes) , multiple sclerosis, allergic encephalomyelitis, cytokine and T lymphocyte mediated immune responses associated with acute and delayed hypersensitivity reactions, tuberculosis, sarcoidosis, granulomatous diseases including Wegener's granulomatosis disease, agranulocytosis, vasculitis, aplastic anemia, Coombs' positive anemia, Debbie's anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red blood cell development Insufficiency (PRCA), Factor VIII Deficiency, Hemophilia A, Autoimmune Neutropenia, Pancytopenia, Leukopenia, Diseases Involving Leukocyte Hematopoiesis, CNS Inflammatory Disorders, Multiple Organ Injury Syndrome myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman syndrome, Goodpasture syndrome, Myasthenia gravis syndrome, Raynaud's syndrome, Sjögren's syndrome, Stein-Joe's syndrome, solid organ transplant rejection (including pretreatment for high reactive antibody titers, IgA in tissue deposition, etc.), graft-versus-host disease (GVHD ), pemphigoid bullae, pemphigus (all, including vulgaris, phyllodes), autoimmune polyendocrine disease , Reiter's disease, stiff man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP ), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune diseases of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine diseases including autoimmune Chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndrome Syndrome (or polyglandular endocrine disease syndrome), type 1 diabetes also known as insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome, autoimmune hepatitis, lymph node interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplantation) and NSIP, Guillain-Barr syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (high Angle) arteritis), medium vessel vasculitis (including Kawasaki disease and polynodular nodular arteritis), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, Celiac sprue (mucosal enteropathy), cryoglobulinemia syndrome, ALS, coronary artery disease.
CD20阳性癌症是那些包含在细胞表面表达CD20的细胞的异常增殖的癌症。CD20阳性B细胞肿瘤包括CD20阳性何杰金氏病，包括淋巴细胞占优势的何杰金氏病(LPHD)；非何杰金氏淋巴瘤(NHL)；滤泡中心细胞(FCC)淋巴瘤；急性淋巴细胞性白血病(ALL)；慢性淋巴细胞性白血病(CLL)；毛细胞白血病。非何杰金氏淋巴瘤包括低级/滤泡的非何杰金淋巴瘤(NHL)，小淋巴细胞性淋巴瘤(SLL)，中等/滤泡NHL，中等扩散NHL，高级成免疫细胞的(high grade immuoblastic)NHL，高级成淋巴细胞的NHL，高级小非核裂细胞(high grade small non-cleaved cell)NHL，bulky disease NHL，类浆细胞淋巴细胞性淋巴瘤，套细胞淋巴瘤，AIDS相关的淋巴瘤与瓦尔登斯特伦氏巨球蛋白血症。也涉及这些癌症复发的治疗。LPHD是一类何杰金氏病，尽管用放射或化学疗法治疗仍倾向经常复发，其特征在于CD20阳性的恶性细胞。CLL是四种主要类型的白血病之一。作为称为淋巴细胞的成熟B细胞的癌症，CLL表现为细胞在血液、骨髓与淋巴组织中的进行性堆积。CD20 positive cancers are those comprising abnormal proliferation of cells expressing CD20 on the cell surface. CD20-positive B-cell neoplasms include CD20-positive Hodgkin's disease, including lymphocyte-predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicle center cell (FCC) lymphoma; Acute lymphocytic leukemia (ALL); Chronic lymphocytic leukemia (CLL); Hairy cell leukemia. Non-Hodgkin's lymphomas include low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), intermediate/follicular NHL, intermediate-diffusion NHL, high-grade immunoblastic (high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, plasmacytic lymphocytic lymphoma, mantle cell lymphoma, AIDS-related lymphoma tumor and Waldenstrom's macroglobulinemia. Treatment of recurrence of these cancers is also involved. LPHD is a type of Hodgkin's disease that tends to recur frequently despite treatment with radiation or chemotherapy, and is characterized by CD20-positive malignant cells. CLL is one of four main types of leukemia. A cancer of mature B cells called lymphocytes, CLL appears as a progressive buildup of cells in the blood, bone marrow, and lymphoid tissues.
在具体的实施方式中，人源化CD20结合抗体与其功能性片段被用于治疗非何杰金氏淋巴瘤(NHL)，淋巴细胞占优势的何杰金氏病(LPHD)，小淋巴细胞性淋巴瘤(SLL)，慢性淋巴细胞性白血病，类风湿性关节炎与青年类风湿性关节炎，系统性红斑狼疮(SLE)包括狼疮肾炎，韦格内氏病，炎症性肠病，特发性血小板减少性紫癜(ITP)，血栓性血小板减少性紫癜(TTP)，自身免疫性血小板减少，多发性硬化，牛皮癣，IgA肾病，IgM多神经病，重症肌无力，血管炎，糖尿病，雷诺氏症，斯耶格仑氏综合症与肾小球肾炎。In specific embodiments, humanized CD20-binding antibodies and functional fragments thereof are used to treat non-Hodgkin's lymphoma (NHL), lymphocyte-predominant Hodgkin's disease (LPHD), small lymphocytic Lymphoma (SLL), chronic lymphocytic leukemia, rheumatoid arthritis and juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE) including lupus nephritis, Wegener's disease, inflammatory bowel disease, idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Autoimmune Thrombocytopenia, Multiple Sclerosis, Psoriasis, IgA Nephropathy, IgM Polyneuropathy, Myasthenia Gravis, Vasculitis, Diabetes, Raynaud's, Sjogren's syndrome and glomerulonephritis.
人源化CD20结合抗体或其功能性片段可用在对，例如，复发或难治的低级或滤泡CD20阳性B细胞NHL的单一药剂治疗中，或者可以在多种药物疗法中与其它药物联合对病人给药。Humanized CD20-binding antibodies or functional fragments thereof can be used, for example, in single-agent therapy for relapsed or refractory low-grade or follicular CD20-positive B-cell NHL, or can be combined with other drugs in multi-drug therapy The patient is given the drug.
无痛淋巴瘤是一缓慢增长、不可治愈的疾病，其中一般的病人存活六到十年之间，经历多个症状缓解与复发周期。在一个实施方式中，人源化CD20结合抗体或其功能性片段被用来治疗无痛NHL。Painless lymphoma is a slow-growing, incurable disease in which the average patient survives between six and ten years through multiple cycles of remission and relapse. In one embodiment, a humanized CD20-binding antibody or a functional fragment thereof is used to treat indolent NHL.
评估肿瘤治疗效力或成绩的参数对于对该恰当疾病熟悉的医师来说是已知的。通常，熟练医师将试图减少该特定疾病的体征和症状。参数可以包括疾病进行的中值时间、症状缓解时间及病情平稳。The parameters for assessing the efficacy or success of tumor therapy are known to a physician familiar with the appropriate disease. Typically, a skilled physician will attempt to reduce the signs and symptoms of that particular disease. Parameters may include median time to disease onset, time to remission of symptoms, and stable disease.
以下参考文献描述了淋巴瘤与CLL，其诊断、治疗和用于测量治疗效力的标准医学程序。Canellos GP，Lister，TA，Sklar JL：The Lymphomas.W.B.Saunders Company，Philadelphia，1998；van Besien K and Cabanillas，F：Clinical Manifestations，Staging and Treatment of Non-Hodgkin′s Lymphoma，第70章，pp 1293-1338，在Hematology，Basic Principles and Practice，3rd ed.Hoffman et al.(editors).Churchill Livingstone，Philadelphia，2000中；和Rai，Kand Patel，D：Chronic Lymphocytic Leukemia，第72章，pp 1350-1362，在Hematology，Basic Principles and Practice，3rd ed.Hoffman et al.(editors).Churchill Livingstone，Philadelphia，2000.The following references describe lymphoma and CLL, their diagnosis, treatment and standard medical procedures used to measure the efficacy of treatment. Canellos GP, Lister, TA, Sklar JL: The Lymphomas. W.B. Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, F: Clinical Manifestations, Staging and Treatment of Non-Hodgkin's Lymphoma,
评估治疗自身免疫或自身免疫相关疾病效力或成绩的参数对于在该恰当疾病领域熟练的医师来说是已知的。通常，熟练医师将试图减少该特定疾病的体征和症状。以下举例说明。The parameters for assessing the efficacy or success of treating an autoimmune or autoimmune-related disease are known to a physician skilled in the appropriate disease area. Typically, a skilled physician will attempt to reduce the signs and symptoms of that particular disease. The following example illustrates.
在一个实施方式中，本发明的抗体用于治疗类风湿性关节炎。类风湿性关节炎的特征在于多关节炎症、软骨损耗与骨侵蚀，导致关节破坏，并且最终减低关节功能。另外，由于RA是全身疾病，其可对其它组织例如肺、眼与骨髓产生作用。在患RA超过10年的病人中不到50％能继续进行日常工作或生活。In one embodiment, the antibodies of the invention are used to treat rheumatoid arthritis. Rheumatoid arthritis is characterized by polyarticular inflammation, cartilage loss and bone erosion, leading to joint destruction and ultimately reduced joint function. Additionally, since RA is a systemic disease, it can have effects on other tissues such as the lungs, eyes, and bone marrow. Fewer than 50% of patients with RA for more than 10 years can continue to carry out daily work or life.
抗体可以在早期RA(即未用氨甲喋呤(MTX)治疗的)病人中作为第一线治疗使用，可作为单一治疗药物使用，或者与例如MTX或环磷酰胺联合使用。或者，抗体可以在治疗DMARD和/或MTX难治的病人中作为第二线治疗药物使用，可作为单一治疗药物使用，或者与例如MTX联合使用。人源化CD20结合抗体在预防和控制关节损害、延缓结构破坏、降低与RA中炎症相关的疼痛中有用，并且通常在中度至严重RA中减少体征和症状。可以在用其它用于治疗RA的药物治疗之前、之后或过程中用人源化CD20抗体治疗RA病人(见下面的联合治疗)。在一个实施方式中，先前曾用具有减轻疾病效果的抗风湿药治疗失败和/或对仅用氨甲喋呤治疗反应不足的病人用本发明的人源化CD20结合抗体治疗。在这一治疗的实施方式中，对病人实施17天的治疗方案，其中：仅接受人源化CD20结合抗体(在第1与第15天，1g iv输注)；CD20结合抗体加环磷酰胺(在第3与第17天，750mg iv输注)；或者CD20结合抗体加氨甲喋呤。Antibodies can be used as first-line therapy in patients with early RA (ie, not treated with methotrexate (MTX)), as monotherapy, or in combination with, for example, MTX or cyclophosphamide. Alternatively, antibodies may be used as second-line therapy in patients refractory to DMARDs and/or MTX, as monotherapy, or in combination with, for example, MTX. Humanized CD20 binding antibodies are useful in preventing and managing joint damage, delaying structural destruction, reducing pain associated with inflammation in RA, and generally reducing signs and symptoms in moderate to severe RA. RA patients can be treated with the humanized CD20 antibody before, after or during treatment with other drugs used to treat RA (see Combination Therapy below). In one embodiment, patients who have previously failed treatment with disease-modifying antirheumatic drugs and/or have had an inadequate response to treatment with methotrexate alone are treated with a humanized CD20-binding antibody of the invention. In this embodiment of treatment, patients are administered a 17-day regimen in which: humanized CD20-binding antibody alone (1 g iv infusion on
评价RA治疗效力的一个方法是以美国风湿病学学院(ACR)标准为基础，该标准测量在关节触痛与肿胀上改善的百分比，同时测量其它内容。与未用抗体治疗(例如治疗前基线)或用安慰剂治疗相比较，RA病人可被评分为，例如ACR20(改善20％)。评价抗体治疗效力的其它途径包括X线评分，例如Sharp X线评分，该评分被用来对结构破坏例如骨侵蚀与关节间隙变窄评分。在治疗期间或之后，也可以健康评估调查表[HAQ]评分、AIMS评分、SF36为基础，评价病人残疾的预防或改善。ACR20标准可以包括在触痛(疼痛)关节计数与肿胀关节计数上改善20％，加上在如下5个附加测量标准中至少3个改善20％：One method of assessing the efficacy of RA treatment is based on the American College of Rheumatology (ACR) criteria, which measure percent improvement in joint tenderness and swelling, among other measures. RA patients can be scored, for example, as ACR20 (20% improvement) compared to no antibody treatment (eg, pre-treatment baseline) or treatment with placebo. Other ways of assessing the efficacy of antibody therapy include radiographic scoring, such as the Sharp radiographic score, which is used to score structural damage such as bone erosion and joint space narrowing. During or after treatment, the prevention or improvement of the patient's disability can also be evaluated based on the Health Assessment Questionnaire [HAQ] score, AIMS score, and SF36. ACR20 criteria may include a 20% improvement in tender (painful) joint count and swollen joint count, plus a 20% improvement in at least 3 of the following 5 additional measures:
用视觉模拟尺度(VAS)评估病人疼痛，Assess patient pain with a visual analog scale (VAS),
病人对疾病活动性的综合评估(VAS)，Patient's Global Assessment of Disease Activity (VAS),
医师对疾病活动性的综合评估(VAS)Physician's Comprehensive Assessment of Disease Activity (VAS)
病人通过健康评估调查表对残疾的自我评估，和Patient self-assessment of disability through the Health Assessment Questionnaire, and
急性期反应物，CRP或ESR。Acute phase reactants, CRP or ESR.
ACR50与70类似地进行定义。优选地，给予病人有效量的本发明的CD20结合抗体，以达到评分为至少ACR20，优选地至少ACR30，更优选地至少ACR50，甚至更优选地至少ACR70，优选地至少ACR75和更高。ACR50 is defined similarly to 70. Preferably, the patient is administered an effective amount of a CD20 binding antibody of the invention to achieve a score of at least ACR20, preferably at least ACR30, more preferably at least ACR50, even more preferably at least ACR70, preferably at least ACR75 and higher.
牛皮癣关节炎具有独一无二和特殊的放射线照相特征。对于牛皮癣关节炎而言，关节侵蚀与关节间隙变窄也可以用Sharp记分评估。本发明的人源化CD20结合抗体可用于防止关节损害和减少该紊乱的疾病体征和症状。Psoriatic arthritis has unique and specific radiographic features. For psoriatic arthritis, joint erosion and joint space narrowing can also be assessed using the Sharp score. The humanized CD20 binding antibodies of the invention are useful for preventing joint damage and reducing disease signs and symptoms of the disorder.
本发明的另一方面为通过给予患SLE的患者治疗有效量的本发明的人源化CD20结合抗体，治疗狼疮或SLE的方法。SLEDAI评分提供疾病活动性的数值定量。SLEDAI是24个已知与疾病活动性相关的临床与实验室参数的加权指数，数值范围为0-103。见Bryan Gescuk & John Davis，“Noveltherapeutic agent for systemic lupus erythematosus”在Current Opinion inRheumatology 2002，14：515-521中。针对双链DNA的抗体被认为导致肾性潮红(renal flares)及其它狼疮表现。可以对进行抗体治疗的病人随时间监测肾性潮红，其被定义为血清肌酸酐、尿蛋白质或血尿的显著的、可再现的增加。或者或另外，可以监测病人抗核抗体与抗双链DNA抗体的水平。对于SLE的治疗包括高剂量皮质类固醇和/或环磷酰胺(HDCC)。Another aspect of the invention is a method of treating lupus or SLE by administering to a patient suffering from SLE a therapeutically effective amount of a humanized CD20 binding antibody of the invention. The SLEDAI score provides a numerical quantification of disease activity. SLEDAI is a weighted index of 24 clinical and laboratory parameters known to be associated with disease activity, with values ranging from 0-103. See Bryan Gescuk & John Davis, "Noveltherapeutic agent for systemic lupus erythematosus" in Current Opinion in Rheumatology 2002, 14:515-521. Antibodies against double-stranded DNA are thought to cause renal flares and other lupus manifestations. Patients on antibody therapy can be monitored over time for renal flushing, which is defined as a marked, reproducible increase in serum creatinine, urine protein, or hematuria. Alternatively or additionally, the patient's levels of anti-nuclear antibodies and anti-double stranded DNA antibodies can be monitored. Treatment for SLE includes high dose corticosteroids and/or cyclophosphamide (HDCC).
脊椎关节病是关节的一组疾病，包括强直性脊柱炎、牛皮癣关节炎与克朗氏病(crohn′s disease)。治疗成果可以用经验证实的病人与医师综合评估测量工具来判定。Spondyloarthropathy is a group of diseases of the joints, including ankylosing spondylitis, psoriatic arthritis and Crohn's disease. Treatment outcomes can be judged using empirically validated integrated patient and physician assessment measures.
各种药物被用来治疗牛皮癣，治疗与疾病严重程度直接有关。具有较轻型牛皮癣的病人一般用局部治疗，例如局部类固醇、蒽林、卡泊三烯、氯氟美松、与他佐罗汀，以控制病症，而具有中度与重度牛皮癣的病人则更可能使用系统的(氨甲喋呤、类视黄醇、环孢菌素、PUVA与UVB)疗法。也使用焦油(Tars)。这些疗法的缺点在于安全性考虑、耗时或治疗过程不便利。此外，有些要求昂贵的设备与在办公室环境下设定专用空间。系统药物可产生严重的副作用，包括高血压、高脂血症、骨髓抑制、肝脏疾病、肾病与胃肠不适。并且，使用光照疗法可以增加皮肤癌的发病率。除与使用局部治疗相关的不便与不适之外，光照疗法和系统治疗要求不断地使病人在治疗与停止治疗之间反复，并由于其副作用需要监测终生照射量。Various medications are used to treat psoriasis, and treatment is directly related to the severity of the disease. Patients with milder forms of psoriasis generally use topical treatments such as topical steroids, anthralin, calcipotriene, clomethasone, and tazarotene to control the condition, while patients with moderate and severe psoriasis are more likely Use systemic (methotrexate, retinoids, cyclosporine, PUVA and UVB) therapy. Tars are also used. Disadvantages of these therapies are safety concerns, time-consuming, or inconvenient treatment procedures. In addition, some require expensive equipment and a dedicated space in an office environment. Systemic drugs can produce serious side effects, including hypertension, hyperlipidemia, bone marrow suppression, liver disease, kidney disease, and gastrointestinal distress. Also, the use of light therapy can increase the incidence of skin cancer. In addition to the inconvenience and discomfort associated with the use of topical treatments, phototherapy and systemic treatments require constant toggling of the patient between treatment and cessation of treatment, and lifetime monitoring of exposure due to their side effects.
牛皮癣治疗效力通过监测与基础状态相比较疾病临床征象与症状的改变而评估，包括医师综合评估(PGA)改变与牛皮癣面积和严重性指数(PASI)评分、牛皮癣症状评估(PSA)。可在整个治疗过程重周期性地测量病人的视觉模拟等级(Visual analog scale)，视觉模拟等级被用来表示特定时间点所感受到的痒感程度。Psoriasis treatment efficacy was assessed by monitoring changes in clinical signs and symptoms of disease compared with baseline status, including changes in Physician's Global Assessment (PGA) and Psoriasis Area and Severity Index (PASI) scores, Psoriasis Symptom Assessment (PSA). The patient's visual analog scale (Visual analog scale) can be measured periodically throughout the treatment process, and the visual analog scale is used to indicate the degree of itch felt at a specific time point.
病人在首次输注治疗性抗体时可发生输注反应或输注相关症状。这些症状在严重程度方面有差异，并且通常在药物介入后是可逆的。这些症状包括但不局限于流感样发热、寒战/强直、恶心、荨麻疹、头痛、支气管痉挛、血管性水肿。对本发明的疾病治疗方法来说，可能需要使输注反应达到最小。因此，本发明的另一方面是通过给予人源化CD20结合抗体而披露的治疗疾病的方法，其中抗体同用
剂量dose
取决于所要治疗的适应症以及本领域熟练医师所熟悉的与规定剂量有关的因素，本发明的抗体将以有效治疗适应症并使毒性与副作用减至最小的剂量被给药。对于治疗CD20阳性癌症或自身免疫病来说，治疗有效的剂量在大约250mg/m2至大约400mg/m2或500mg/m2范围内，优选地大约250-375mg/m2。在一个实施方式中，剂量范围是275-375mg/m2。在治疗CD20阳性B细胞肿瘤的一个实施方式中，抗体以300-375mg/m2的范围给药。对于治疗患B细胞淋巴瘤例如非何杰金氏淋巴瘤的病人而言，在特定的实施方式中，本发明的抗CD20抗体与人源化抗CD20抗体将以10mg/kg或375mg/m2剂量给予人类患者。对于治疗NHL来说，一个给药法是在治疗第一周以10mg/kg剂量给予一次剂量的抗体组合物，继之以二周间隔，然后给予第二次剂量的相同量的抗体。通常，NHL病人在一年内接受一次象这样的治疗，但是一旦淋巴瘤复发，象这样的治疗可以被重复。在另一个给药法中，治疗低级NHL的病人接受四周一种型式的人源化2H7，优选地v16(375mg/m2每周)，随后在第五周进行三个附加疗程的抗体加标准CHOP(环磷酰胺、阿霉素、长春新碱和强的松)或CVP(环磷酰胺、长春新碱、强的松)化疗，其每三周给药，共三个周期。Depending on the indication being treated and factors related to prescribed dosage which are familiar to physicians skilled in the art, the antibodies of the invention will be administered in a dose effective to treat the indication while minimizing toxicity and side effects. For the treatment of CD20 positive cancers or autoimmune diseases, therapeutically effective doses range from about 250 mg/m2 to about 400 mg/m2 or 500 mg/m2 , preferably about 250-375 mg/m2 . In one embodiment, the dosage range is 275-375 mg/m2 . In one embodiment for treating CD20 positive B cell tumors, the antibody is administered in the range of 300-375 mg/m2 . For the treatment of patients suffering from B-cell lymphoma such as non-Hodgkin's lymphoma, in a specific embodiment, the anti-CD20 antibody of the present invention and the humanized anti-CD20 antibody will be administered at 10 mg/kg or 375 mg/m2 Doses are administered to human patients. For the treatment of NHL, one dosing regimen is to administer one dose of the antibody composition at a dose of 10 mg/kg during the first week of treatment, followed by a two-week interval, followed by a second dose of the same amount of the antibody. Typically, NHL patients receive a treatment like this once within a year, but it can be repeated if the lymphoma recurs. In another dosing regimen, patients treated for low-grade NHL received four weeks of one form of humanized 2H7, preferably v16 (375 mg/m2 weekly), followed by three additional courses of the antibody plus standard CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CVP (cyclophosphamide, vincristine, prednisone) chemotherapy, given every three weeks for three cycles.
对于治疗类风湿性关节炎而言，在一个实施方式中，人源化抗体的剂量范围是125mg/m2(相当于大约200mg/每剂)至600mg/m2，分成两剂，例如第一剂200mg在第一天给药，继之以在第15天给予第二剂量200mg。在不同实施方式中，剂量是250mg/每剂、275mg、300mg、325mg、350mg、375mg、400mg、425mg、450mg、475mg、500mg、525mg、550mg、575mg、600mg。For the treatment of rheumatoid arthritis, in one embodiment, the dose of the humanized antibody ranges from 125 mg/m2 (equivalent to about 200 mg/dose) to 600 mg/m2 divided into two doses, e.g. the first A dose of 200 mg was given on the first day, followed by a second dose of 200 mg on day 15. In various embodiments, the dose is 250 mg per dose, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg.
在治疗疾病时，如在该疾病领域熟练的医师所决定的，本发明的CD20结合抗体可以长期或者间歇地给予病人。In treating a disease, the CD20-binding antibody of the present invention may be administered to the patient chronically or intermittently, as determined by a physician skilled in the disease field.
静脉内输注或皮下给药可使病人感觉到不利反应例如发热、寒战、烧灼感、无力和头痛。为缓和或使这样的不利反应达到最小，病人可接受抗体的初始调制剂量(initial conditioning dose)，继之以治疗剂量。调制剂量可以比治疗剂量低，以调节病人使其耐受较高剂量。Intravenous infusion or subcutaneous administration can cause patients to experience adverse reactions such as fever, chills, burning sensation, weakness and headache. To moderate or minimize such adverse reactions, patients receive an initial conditioning dose of the antibody, followed by a therapeutic dose. The modulating dose may be lower than the therapeutic dose in order to condition the patient to tolerate the higher dose.
给药途径Route of administration
根据已知方法将CD20结合抗体给予人类病人，例如通过静脉内给药，例如以丸剂(bolus)或通过在一段时期内的连续输注，经过皮下、肌肉、腹膜内、脑脊膜内、关节内、滑膜内、鞘内、或吸入途径，通常经过静脉内或皮下给药。The CD20-binding antibody is administered to a human patient according to known methods, for example by intravenous administration, for example as a bolus or by continuous infusion over a period of time, subcutaneously, intramuscularly, intraperitoneally, intrameningally, intraarticularly Intrasynovial, intrathecal, or inhalation routes, usually intravenous or subcutaneous.
在一个实施方式中，人源化2H7抗体经过静脉内输注给药，以0.9％氯化钠溶液作为输注载体。In one embodiment, the humanized 2H7 antibody is administered by intravenous infusion with 0.9% sodium chloride solution as the infusion vehicle.
联合治疗combination therapy
在治疗上面描述的B细胞肿瘤时，病人可以用本发明的CD20结合抗体与一或多种治疗剂例如化疗剂一起用多药物疗法加以治疗。CD20结合抗体可以与化疗剂同时、序贯地或者交替地给药，或在用其它治疗不反应之后进行联合治疗。淋巴瘤治疗的标准化学疗法包括环磷酰胺、阿糖胞苷、美法仑、与米托蒽醌加美法仑。CHOP是治疗非何杰金氏淋巴瘤最常见的化学疗法。以下是用于CHOP疗法的药物：环磷酰胺(商品名cytoxan，neosar)；阿霉素(亚德里亚霉素/羟基亚德里亚霉素)；长春新碱(Oncovin)；与氢化泼尼松(有时称为Deltasone或Orasone)。在一个具体的实施方式中，CD20结合抗体与下列化疗剂中的一个或多个联合给药至需要其的病人：阿霉素、环磷酰胺、长春新碱与氢化泼尼松。在特定的实施方式中，患淋巴瘤(例如非何杰金氏淋巴瘤)的病人用本发明的抗CD20抗体与CHOP(环磷酰胺、阿霉素、长春新碱和强的松)疗法联合治疗。在另一个实施方式中，癌症病人可以用本发明的人源化CD20结合抗体与CVP(环磷酰胺、长春新碱与强的松)化疗联合治疗。在特定的实施方式中，患CD20阳性NHL的病人用人源化2H7.v16与CVP结合进行治疗。在治疗CLL的特定实施方式中，CD20结合抗体与用氟达拉滨与环磷酰胺中的一个或两个进行的化学疗法联合给药。In treating the B cell tumors described above, patients may be treated with multidrug therapy with a CD20-binding antibody of the invention together with one or more therapeutic agents, such as chemotherapeutic agents. CD20-binding antibodies can be administered simultaneously, sequentially, or alternately with chemotherapeutic agents, or in combination therapy after failure to respond to other treatments. Standard chemotherapy for lymphoma treatment includes cyclophosphamide, cytarabine, melphalan, and mitoxantrone plus melphalan. CHOP is the most common chemotherapy treatment for non-Hodgkin's lymphoma. The following drugs are used in CHOP therapy: cyclophosphamide (trade names cytoxan, neosar); doxorubicin (doxorubicin/hydroxydriamycin); vincristine (Oncovin); and prednisone (sometimes called Deltasone or Orasone). In a specific embodiment, a CD20-binding antibody is administered to a patient in need thereof in combination with one or more of the following chemotherapeutic agents: doxorubicin, cyclophosphamide, vincristine, and prednisone. In a specific embodiment, patients with lymphoma (eg, non-Hodgkin's lymphoma) are treated with an anti-CD20 antibody of the invention in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy treat. In another embodiment, cancer patients can be treated with the humanized CD20-binding antibody of the present invention in combination with CVP (cyclophosphamide, vincristine and prednisone) chemotherapy. In a specific embodiment, patients with CD20 positive NHL are treated with humanized 2H7.v16 in combination with CVP. In a specific embodiment for the treatment of CLL, the CD20 binding antibody is administered in combination with chemotherapy with one or both of fludarabine and cyclophosphamide.
在治疗上面描述的自身免疫病或自身免疫相关病症中，病人可以用本发明的CD20结合抗体与第二治疗剂，例如免疫抑制剂，例如在多药物疗法中联合治疗。CD20结合抗体可以与免疫抑制剂同时、序贯地或者交替地给药，或在不反应时与其它疗法联用。免疫抑制剂可以以现有技术中所披露的相同或较少剂量给药。优选的附加免疫抑制剂将取决于许多因素，包括所治疗的疾病种类以及病人的病史。In the treatment of an autoimmune disease or an autoimmune-related disorder as described above, a patient may be treated with a CD20-binding antibody of the invention in combination with a second therapeutic agent, eg, an immunosuppressant, eg, in multidrug therapy. CD20 binding antibodies can be administered simultaneously, sequentially or alternately with immunosuppressants, or in combination with other therapies in the absence of response. Immunosuppressants can be administered in the same or lesser doses than those disclosed in the prior art. The preferred additional immunosuppressant will depend on many factors including the disease being treated and the patient's medical history.
在此用于附加治疗的“免疫抑制剂”指抑制或掩蔽病人的免疫系统的物质。象这样的药剂将包括抑制细胞因子生产、下调或抑制自身抗原表达、或掩蔽MHC抗原的物质。象这样的药剂的例子包括类固醇例如糖皮质激素，例如强的松、甲基强的松龙、与地塞米松、2-氨基-6-芳基-5-取代的嘧啶(见美国专利4,665,077)、咪唑硫嘌呤(或环磷酰胺，如果有对咪唑硫嘌呤的副反应)；溴隐亭；戊二醛(掩蔽MHC抗原，如美国专利4,120,649所描述)；针对MHC抗原与MHC片段的抗特应抗体；环孢菌素A；细胞因子或细胞因子受体拮抗剂包括抗干扰素γ，β，或α抗体；抗肿瘤坏死因子α抗体；抗肿瘤坏死因子β抗体；抗白介素-2抗体与抗IL-2受体抗体；抗L3T4抗体；异源的抗淋巴细胞球蛋白；泛T抗体，优选地抗CD3或抗CD4/CD4a抗体；包含LFA-3结合结构域的可溶性肽(WO90/08187，公开日7/26/90)；链激酶；TGF-β；链道酶；来自宿主的RNA或DNA；FK506；RS-61443；脱氧精胍菌素；雷帕霉素；T细胞受体(美国专利5,114,721)；T细胞受体片段(Offner et al.，Science 251：430-432(1991)；WO 90/11294；与WO91/01133)；与T细胞受体抗体(EP 340,109)例如T10B9。"Immunosuppressant" as used herein for adjunctive therapy refers to substances that suppress or mask a patient's immune system. Such agents would include substances that inhibit cytokine production, downregulate or inhibit expression of self-antigens, or mask MHC antigens. Examples of such agents include steroids such as glucocorticoids, such as prednisone, methylprednisolone, and dexamethasone, 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077) , azathioprine (or cyclophosphamide, if there is a side reaction to azathioprine); bromocriptine; glutaraldehyde (masking of MHC antigens, as described in US Patent 4,120,649); anti-specific antibodies against MHC antigens and MHC fragments Cytokines or cytokine receptor antagonists including anti-interferon gamma, beta, or alpha antibodies; anti-tumor necrosis factor alpha antibodies; anti-tumor necrosis factor beta antibodies; anti-interleukin-2 antibodies and Anti-IL-2 receptor antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; pan-T antibody, preferably anti-CD3 or anti-CD4/CD4a antibody; soluble peptide containing LFA-3 binding domain (WO90/08187 , published date 7/26/90); streptokinase; TGF-β; streptase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; US Patent 5,114,721); T cell receptor fragments (Offner et al., Science 251:430-432 (1991); WO 90/11294; and WO91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.
为了治疗类风湿性关节炎，病人可以用本发明的CD20抗体结合下述药物中的任何一个或更多进行治疗:DMARDS(减轻病症的抗风湿病药物(例如氨甲喋呤)，NSAI或NSAID(非类固醇类抗炎药物)，HUMIRATM(adalimumab；Abbott Laboratories)，
对于治疗强直性脊柱炎、牛皮癣关节炎与克朗氏病而言，病人可以用本发明的CD20结合抗体与例如
对于SLE的治疗包括高剂量皮质类固醇和/或环磷酰胺(HDCC)。Treatment for SLE includes high dose corticosteroids and/or cyclophosphamide (HDCC).
对于治疗牛皮癣而言，可以给予病人CD20结合抗体结合局部治疗，例如局部类固醇、蒽林、卡泊三烯、氯氟美松与他佐罗汀，或结合氨甲喋呤、类视黄醇、环孢菌素、PUVA与UVB治疗。在一个实施方式中，牛皮癣病人用CD20结合抗体顺序处理或者同时用环孢菌素处理。For psoriasis, patients can be given CD20-binding antibodies in combination with topical treatments such as topical steroids, anthralin, calcipotriene, clomethasone, and tazarotene, or in combination with methotrexate, retinoids, cyclosporine Toxin, PUVA and UVB treatment. In one embodiment, psoriasis patients are treated sequentially with CD20 binding antibodies or concurrently with cyclosporine.
药物制剂pharmaceutical preparations
根据本发明使用的CD20结合抗体的治疗制剂通过以冻干制剂或水溶液的形式混合具有所需纯度的抗体和任选药学上可接受的载体、赋形剂或稳定剂(Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980))用于储存而制备。可接受的载体、赋形剂、稳定剂在所用剂量及浓度对受体无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐，及其它有机酸；抗氧化剂包括抗坏血酸和甲硫氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；苯扎氯铵，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它糖包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、海藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)和/或非离子表面活性剂如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。Therapeutic formulations of CD20-binding antibodies used in accordance with the present invention are prepared by mixing antibodies of the desired purity and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th) in the form of lyophilized formulations or aqueous solutions. edition, Osol, A.Ed. (1980)) prepared for storage. Acceptable carriers, excipients, stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives agents (such as octadecyldimethylbenzyl ammonium chloride; hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as Methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (fewer than about 10 residues); Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes) and/or nonionic surfactants such as TWEEN™ , PLURONICS™ or polyethylene glycol (PEG).
示例性的抗CD20抗体制剂在WO98/56418中描述，在此特别地引入作为参考。另一种制剂是液体多剂量制剂，包含40mg/mL抗CD20抗体、25mM醋酸盐、150mM海藻糖、0.9％苯甲醇、0.02％ pH 5.0的聚山梨酸酯20，在2-8℃最短保存期限两年。另一感兴趣的抗CD20制剂包含溶解在9.0mg/mL氯化钠、7.35mg/mL柠檬酸钠二水合物、0.7mg/mL聚山梨酸酯80与灭菌注射水中的10mg/mL抗体，pH6.5。另一水性药物制剂包含大约pH 4.8至大约pH5.5、优选地pH5.5的10-30mM乙酸钠、含量大约0.01-0.1％v/v的聚山梨酸酯作为表面活性剂、含量大约2-10％w/v的海藻糖与作为防腐剂的苯甲醇(U.S.6,171,586)。适宜于皮下给药的冻干制剂在WO97/0480l中描述。象这样的冻干制剂可以用合适的稀释剂重新配制成高蛋白浓度溶液，并且重新配制的制剂可皮下给药至在此将要被治疗的哺乳动物。Exemplary anti-CD20 antibody formulations are described in WO98/56418, which is hereby expressly incorporated by reference. The other formulation is a liquid multi-dose formulation containing 40 mg/mL anti-CD20 antibody, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02
人源化2H7变体的一个制剂是12-14mg/mL抗体，溶解在10mM组氨酸、6％蔗糖、0.02％聚山梨酸酯20中，pH5.8。One formulation of the humanized 2H7 variant was 12-14 mg/mL antibody dissolved in 10 mM histidine, 6% sucrose, 0.02
在一个具体的实施方式中，2H7变体并且特别地是2H7.v16，以20mg/mL抗体溶解在10mM组氨酸硫酸酯、60mg/mL蔗糖、0.2mg/mL聚山梨酸酯20与灭菌注射水中，pH5.8的形式配制。In a specific embodiment, 2H7 variants, and specifically 2H7.v16, are dissolved at 20 mg/mL antibody in 10 mM histidine sulfate, 60 mg/mL sucrose, 0.2 mg/
制剂在此也可包含特定适应症所需的多于一种活性化合物，优选地是那些具有不对彼此产生不利影响的互补活性的化合物。例如，可能需要进一步提供细胞毒药剂、化疗剂、细胞因子或免疫抑制剂(例如作用于T细胞的药剂例如环孢菌素，或作用于结合T细胞的抗体的药剂例如结合LFA-1的药剂)。象这样的其它药剂的有效量取决于制剂中存在的抗体量、疾病或紊乱或治疗的种类、以及上面所讨论的其它因素。这些通常以相同剂量、和经由在此所描述的给药途径、或在此以前所使用的剂量的大约1-99％而使用。The formulations herein may also contain more than one active compound as required for a particular indication, preferably those compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide cytotoxic agents, chemotherapeutic agents, cytokines, or immunosuppressants (e.g., agents that act on T cells, such as cyclosporine, or agents that act on antibodies that bind T cells, such as agents that bind LFA-1 ). Effective amounts of such other agents depend on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used at the same dosage, and via the routes of administration described herein, or about 1-99% of the dosage used heretofore.
活性成分也可以在胶体药物递送系统(例如脂质体、白蛋白微球粒、微乳剂、纳米颗粒与纳米胶囊)中，或者在粗乳状液中捕捉在微胶囊中，所述微胶囊例如通过凝聚技术或界面聚合作用制备，例如，分别是羟甲基纤维素或明胶微胶囊与聚异丁烯酸甲酯微胶囊。这一技术在Remington′sPharmaceutical Sciences，16th edition，Oslo，A.，Ed.，(1980)中披露。The active ingredient can also be entrapped in microcapsules in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions, e.g. by Prepared by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively. This technique is disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
可以制备缓释制剂。合适的缓释制剂的例子包括含拮抗剂的固态疏水聚合物的半渗透性基质，其中基质是以成形物品的形式，例如薄膜或微囊剂。缓释基质的例子包括聚酯、水凝胶(例如聚(2-羟乙基-异丁烯酸)、或聚(乙烯醇))、聚交酯(美国专利3,773,919)、L-谷氨酸与乙基-L-谷氨酸的共聚物、非可降解乙烯-醋酸乙烯酯、可降解的乳酸-羟基乙酸共聚物例如LUPRONDEPOTTM(由乳酸-羟基乙酸共聚物与醋酸亮丙瑞林组成的可注射微球粒)、与聚-D-(-)-3-羟丁酸。Sustained release formulations can be prepared. Examples of suitable sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, wherein the matrix is in the form of a shaped article, such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (US Patent 3,773,919), L-glutamic acid and ethyl L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOTTM (an injectable drug composed of lactic acid-glycolic acid copolymer and leuprolide acetate microspheres), and poly-D-(-)-3-hydroxybutyric acid.
用于体内给药的制剂必须是无菌的。这通过经由无菌过滤膜过滤而很容易地完成。Preparations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
制品和试剂盒Products and Kits
本发明的另一实施方式是一种制品，包含对治疗自身免疫病与相关病症和CD20阳性癌症例如非何杰金氏淋巴瘤有用的物质。制品包含容器和标签或包装说明书，该标签或包装说明书在容器上或与容器相关。合适的容器包括，例如，瓶子、小瓶、注射器等等。容器可能是由各种材料形成，例如玻璃或塑料。容器容纳对治疗该病症有效的组合物，并且可具有无菌进入孔(例如容器可以是具有可被皮下注射针穿透的塞子的静脉内溶液袋或小瓶)。组合物中至少一种活化剂是本发明的CD20结合抗体。标签或包装说明书指示该组合物用于治疗该特定病症。标签或包装说明书将进一步包含将抗体组合物给药病人的指示。Another embodiment of the invention is an article of manufacture comprising materials useful for the treatment of autoimmune diseases and related disorders and CD20 positive cancers such as non-Hodgkin's lymphoma. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from various materials such as glass or plastic. The container contains a composition effective for treating the condition, and may have a sterile access port (eg, the container may be an intravenous solution bag or vial with a stopper penetrated by a hypodermic needle). At least one activator in the composition is a CD20-binding antibody of the invention. The label or package insert indicates that the composition is used to treat that particular condition. The label or package insert will further contain instructions for administering the antibody composition to a patient.
包装说明书指通常包括在治疗产品商业包装中的指示，该指示包含有关适应症、用法、剂量、给药、禁忌症和/或关于使用象这样的治疗产品的警告的信息。在一个实施方式中，包装说明书指示该组合物用于治疗非何杰金氏淋巴瘤。Package insert refers to the directions usually included in commercial packages of therapeutic products that contain information on the indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. In one embodiment, the package insert indicates that the composition is for the treatment of non-Hodgkin's lymphoma.
另外，该制品可进一步包含第二容器，该容器包含药学上可接受的缓冲液，例如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏液与右旋糖溶液。其可进一步包括从商业与用户角度需要的其它材料，包括其它缓冲液、稀释剂、滤器、针头与注射器。Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials, including other buffers, diluents, filters, needles and syringes, as may be desired from a commercial and user standpoint.
也提供用于各种用途的试剂盒，例如用于B细胞杀伤试验、作为细胞程序死亡试验的正对照、用于从细胞纯化或免疫沉淀CD20。为了分离和纯化CD20，试剂盒可包含偶联至珠(例如琼脂糖珠)的抗CD20抗体。可提供试剂盒，该试剂盒包含用于体外，例如在ELISA或者蛋白质印迹中检测与定量CD20的抗体。如同制品一样，试剂盒包含容器，和在容器上或与容器相关的标签或包装说明书。容器容纳包含本发明的至少一个抗CD20抗体的组合物。可包括额外的容器，该容器包含例如稀释剂和缓冲液、对照抗体。标签或包装说明书可提供对组合物的描述，以及对预期体外或诊断用途的指示。Kits are also available for various uses, eg, for B cell killing assays, as a positive control for apoptosis assays, for purification or immunoprecipitation of CD20 from cells. For the isolation and purification of CD20, the kit may comprise an anti-CD20 antibody coupled to beads (eg, sepharose beads). Kits may be provided comprising antibodies for the detection and quantification of CD20 in vitro, eg, in ELISA or Western blot. As with an article of manufacture, a kit comprises a container, and a label or package insert on or associated with the container. The container contains a composition comprising at least one anti-CD20 antibody of the invention. Additional containers may be included containing, eg, diluents and buffers, control antibodies. A label or package insert can provide a description of the composition, as well as an indication of intended in vitro or diagnostic use.
猕猴CD20macaque CD20
本发明还提供分离的核酸，该核酸包含如图19所示的猕猴CD20的核苷酸序列SEQ ID NO.：24。在一个实施方式中，核酸是cDNA。在一个实施方式中，编码猴CD20的核酸位于用于在宿主细胞中表达的表达载体中。表达载体中的SEQ ID NO.：24的核苷酸序列可操作地连接至表达控制序列，例如启动子、或启动子与增强子。表达控制序列可以是通常与猕猴CD20基因相关的天然序列，或者是对该基因异源的序列。还提供分离的多肽，该多肽包含猕猴CD20的氨基酸顺序[SEQ ID NO.25；图19和20]，以及包含猕猴CD20核酸的宿主细胞。在一个方面，宿主细胞是真核细胞，例如CHO细胞。也考虑包含猕猴CD20氨基酸序列或序列片段的融合蛋白。The present invention also provides an isolated nucleic acid, which comprises the nucleotide sequence SEQ ID NO.: 24 of Cynomolgus monkey CD20 as shown in FIG. 19 . In one embodiment, the nucleic acid is cDNA. In one embodiment, the nucleic acid encoding monkey CD20 is in an expression vector for expression in a host cell. The nucleotide sequence of SEQ ID NO.: 24 in the expression vector is operably linked to an expression control sequence, such as a promoter, or a promoter and an enhancer. The expression control sequence may be a native sequence normally associated with the macaque CD20 gene, or a sequence heterologous to the gene. Also provided are isolated polypeptides comprising the amino acid sequence of Cynomolgus CD20 [SEQ ID NO. 25; Figures 19 and 20], and host cells comprising the Cynomolgus CD20 nucleic acid. In one aspect, the host cell is a eukaryotic cell, such as a CHO cell. Fusion proteins comprising the cynomolgus CD20 amino acid sequence or sequence fragments are also contemplated.
实验实施例Experimental example
实施例1Example 1
2H7抗CD20鼠单克隆抗体的人源化Humanization of 2H7 anti-CD20 mouse monoclonal antibody
鼠抗人CD20抗体2H7(在这里也称为m2H7，m指鼠的)的人源化在一系列定点诱变步骤中进行。鼠2H7抗体可变区序列和小鼠V与人C的嵌合2H7已被描述，见例如美国专利5,846,818与6,204,023。通过将鼠2H7可变区的氨基酸序列(披露在U.S.5,846,818中)与已知抗体的序列(Kabat etal.，sequences of proteins of immunological interest，Ed.5.Public Health Service，National Institutes of Health，Bethesda，MD(1991))进行比较，而鉴别2H7的CDR残基。基于序列高变性(Kabat et al.，上文)定义轻与重链的CDR区域，并分别显示于图1A与图1B。利用合成寡核苷酸(表1)，用定点诱变(Kunkel，Proc.Natl.Acad.Sci.82：488-492(1985))将所有六个鼠2H7CDR区域引入包含在质粒pVX4上包含的相应于共有序列VκI、VHIII(VLκ亚群I、VH亚群III)的完整人Fab框架(图2)。Humanization of the murine anti-human CD20 antibody 2H7 (also referred to herein as m2H7, m for murine) was performed in a series of site-directed mutagenesis steps. Murine 2H7 antibody variable region sequences and chimeric 2H7 of mouse V and human C have been described, see eg US Patent Nos. 5,846,818 and 6,204,023. By combining the amino acid sequence of the mouse 2H7 variable region (disclosed in US5,846,818) with the sequence of known antibodies (Kabat et al., sequences of proteins of immunological interest, Ed.5. Public Health Service, National Institutes of Health, Bethesda , MD (1991)) for comparison to identify the CDR residues of 2H7. The CDR regions of the light and heavy chains were defined based on sequence hypervariability (Kabat et al., supra) and are shown in Figures 1A and 1B, respectively. Using synthetic oligonucleotides (Table 1), all six murine 2H7 CDR regions were introduced into the region contained on plasmid pVX4 using site-directed mutagenesis (Kunkel, Proc. Complete human Fab framework corresponding to consensus sequences VKI,VHIII (VLK subgroup I,VH subgroup III) (Figure 2).
噬菌粒pVX4(图2)被用于诱变以及用于F(ab)s在大肠杆菌中的表达。pVX4以pb0475(Cunningham et al.，Science 243：1330-1336(1989))的衍生物、噬菌粒pb0720为基础，包含一DNA片段，该片段编码人源化的共有κ亚群I轻链(VLκI-CL)和人源化的共有亚群III重链VHIII-CH1抗IFN-α(干扰素-α)抗体。pVX4还具有碱性磷酸酶启动子和Shine-Dalgamo序列，两者均来源于另一以前描述过的以pUC119为基础的质粒pAK2(Carter et al.，Proc.Natl.Acad.Sci.USA 89：4285(1992))。在编码F(ab)轻与重链DNA之间引入唯一的Spel限制性位点。抗IFN-α重与轻链中的前23个氨基酸是StII分泌信号序列(Chang et al.，Gene 55：189-196(1987))。Phagemid pVX4 (Figure 2) was used for mutagenesis and for expression of F(ab)s in E. coli. pVX4 is based on phagemid pb0720, a derivative of pb0475 (Cunningham et al., Science 243: 1330-1336 (1989)), and contains a DNA fragment encoding a humanized consensus kappa subgroup I light chain (VLκI -CL ) and humanized consensus subgroup III heavy chainVHIII -CH1 anti-IFN-α (interferon-α) antibody. pVX4 also has an alkaline phosphatase promoter and a Shine-Dalgamo sequence, both derived from another previously described pUC119-based plasmid pAK2 (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)). A unique Spel restriction site was introduced between the DNA encoding the F(ab) light and heavy chains. The first 23 amino acids in the anti-IFN-[alpha] heavy and light chains are the StII secretion signal sequence (Chang et al., Gene 55:189-196 (1987)).
为了构建2H7的CDR交换型式(CDR-swap version)(2H7.v2)，在包含脱氧尿苷的pVX4模板上进行定点诱变；抗IFN-α的所有六个CDR都换成小鼠2H7CDR。所得到的分子称为人源化的2H72型(2H7.v2)或2H7的“CDR交换型式”；其具有图1A与1B所示的带有共有人FR残基的m2H7 CDR残基。人源化的2H7.v2被用于进一步人源化。To construct a CDR-swap version of 2H7 (2H7.v2), site-directed mutagenesis was performed on a pVX4 template containing deoxyuridine; all six CDRs of anti-IFN-α were exchanged for mouse 2H7 CDRs. The resulting molecule is referred to as humanized 2H72 type (2H7.v2) or "CDR-swapped version" of 2H7; it has m2H7 CDR residues with consensus human FR residues as shown in Figures 1A and 1B. Humanized 2H7.v2 was used for further humanization.
表1显示被用来生成重链与轻链中每一鼠2H7(m2H7)CDR的寡核苷酸序列。例如，CDR-H1寡核苷酸被用来再生成m2H7重链CDR1。CDR-H1、CDR-H2与CDR-H3分别指重链CDR1、CDR2、与CDR3；类似地，CDR-L1、CDR-L2与CDR-L3指轻链CDR中的每一个。CDR-H2中的取代用两个寡核苷酸，CDR-H2A与CDR-H2B，在两个步骤中进行。Table 1 shows the oligonucleotide sequences used to generate each of the murine 2H7 (m2H7) CDRs in the heavy and light chains. For example, CDR-H1 oligonucleotides were used to regenerate m2H7 heavy chain CDR1. CDR-H1 , CDR-H2 and CDR-H3 refer to the heavy chain CDR1 , CDR2 and CDR3 respectively; similarly, CDR-L1 , CDR-L2 and CDR-L3 refer to each of the light chain CDRs. Substitutions in CDR-H2 were performed in two steps with two oligonucleotides, CDR-H2A and CDR-H2B.
表1用于将鼠2H7 CDR的CDR交换构建入pVX4中的人框架的寡核苷酸。在被每一寡核苷酸改变的残基下划线。Table 1 Oligonucleotides used to construct the CDR swap of the murine 2H7 CDRs into the human framework in pVX4. Residues altered by each oligonucleotide are underlined.
为与人源化的构建物进行比较，使用合成寡核苷酸、经由定点诱变(Kunkel，上文)构建表达嵌合2H7 Fab(包含鼠VL和VH结构域，和人CL与CH1结构域)的质粒，以将鼠框架残基引入2H7.v2。所得到的质粒构建物序列示于图3，该构建物用于表达称为2H7.v6.8的嵌合Fab。Fab的每一编码链具有如上面为pVX4(图2)所描述的23个氨基酸的StII分泌信号序列。For comparison with humanized constructs, synthetic oligonucleotides were used to construct expressing chimeric 2H7 Fab (comprising murineVL andVH domains, and humanCL andCH1 domain) to introduce murine framework residues into 2H7.v2. The sequence of the resulting plasmid construct, which was used to express a chimeric Fab designated 2H7.v6.8, is shown in Figure 3. Each coding strand of the Fab has a 23 amino acid Stll secretion signal sequence as described above for pVX4 (Figure 2).
基于鼠2H7框架残基与人VκI，VHIII共有框架(图1A与1B)和先前人源化的抗体(Carter et al.，Proc.Natl.Acad.Sci.USA 89：4285-4289(1992))进行的序列比较，通过定点诱变将几个框架突变引入2H7.v2 Fab构建物。这些突变导致某些人共有框架残基改变成在鼠2H7框架中发现的那些残基，所述改变在那些可能影响CDR构型或抗原接触的位点发生。型式3包含VH(R71V，N73K)，型式4包含VH(R71V)，型式5包含VH(R71V，N73K)和VL(L46P)，型式6包含VH(R71V，N73K)和VL(L46P，L47W)。Based on murine 2H7 framework residues shared with human VκI,VHIII framework (Figures 1A and 1B) and previously humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992 )), several framework mutations were introduced into the 2H7.v2 Fab construct by site-directed mutagenesis. These mutations result in changes in certain human consensus framework residues to those found in the murine 2H7 framework at positions that may affect CDR configuration or antigen contact.
如下述在大肠杆菌中表达与纯化m2H7抗体的人源化与嵌合Fab型式。质粒被转化入大肠杆菌株XL-1 Blue(Stratagene，San Diego，CA)用于制备双链DNA与单链DNA。对于每一变体，使用双脱氧核苷酸法(Sequenase，，U.S.Biochemical Corp.)对轻与重链进行完全测序。质粒被转化入MM294的衍生物、大肠杆菌株16C9，铺于含5μg/ml羧苄青霉素的LB板上，选择用于蛋白表达的单一菌落。单个菌落生长在5ml LB-100μg/ml羧苄青霉素上，37℃5-8小时培养。5ml培养物被加至500ml AP5-100μg/ml羧苄青霉素中，并允许在4L带障板的摇瓶中在37C生长16小时。AP5培养基包括：1.5g葡萄糖，11.0 Hycase SF，0.6g酵母抽提物(经过检验的)，0.19g无水MgSO4，1.07gNH4Cl，3.73gKCl，1.2gNaCl，120ml 1M三乙醇胺，pH 7.4，加水至1L，然后通过0.1μMSealkeen过滤器无菌过滤。Humanized and chimeric Fab versions of the m2H7 antibody were expressed and purified in E. coli as described below. Plasmids were transformed into E. coli strain XL-1 Blue (Stratagene, San Diego, CA) for preparation of double- and single-stranded DNA. For each variant, the light and heavy chains were fully sequenced using the dideoxynucleotide method (Sequenase, US Biochemical Corp.). The plasmid was transformed into a derivative of MM294, E. coli strain 16C9, plated on LB plates containing 5 μg/ml carbenicillin, and single colonies were selected for protein expression. A single colony was grown on 5ml LB-100μg/ml carbenicillin and cultured at 37°C for 5-8 hours. 5ml of the culture was added to 500ml of AP5-100[mu]g/ml carbenicillin and allowed to grow for 16 hours at 37C in a 4L baffled shake flask. AP5 medium consists of: 1.5g glucose, 11.0 Hycase SF, 0.6g yeast extract (tested), 0.19g anhydrousMgSO4 , 1.07gNH4Cl, 3.73gKCl, 1.2gNaCl, 120ml 1M triethanolamine, pH 7.4, water to 1 L, then sterile filter through a 0.1 μM Sealkeen filter.
通过在1L离心瓶(Nalgene)中在3000xg离心收获细胞，并且除去上清液。冰冻1小时后，沉淀小丸被再悬浮在25ml冷的10mM MES-10mMEDTA，pH 5.0(缓冲液A)。加入250μl0.1M PMSF(Sigma)以抑制蛋白质水解，并且加入3.5ml 10mg/ml鸡蛋白溶菌酶(hen egg white lysozyme)(Sigma)储存液，以帮助细菌细胞壁溶解。冰上轻轻摇动1小时后，样品在40,000xg离心15分钟。上清液用缓冲液A加至体积50ml，并且加样至用缓冲液A平衡的2ml DEAE柱上。然后将流出物(flow-through)上样至用缓冲液A平衡的G蛋白-琼脂糖CL-4B(Pharmacia)柱(0.5ml柱床体积)。柱子用10ml缓冲液A洗，用3ml 0.3M甘氨酸pH 3.0洗脱入1.25ml 1MTris，pH 8.0中。然后用Centricon-30(Amicon)将F(ab)缓冲液更换入PBS，并且浓缩至终容积0.5ml。所有F(ab)的SDS-PAGE凝胶均进行电泳以保证纯度，每个变体的分子量用电喷射质谱法证实。Cells were harvested by centrifugation at 3000 xg in 1 L centrifuge bottles (Nalgene) and the supernatant removed. After freezing for 1 hour, the pellet was resuspended in 25 ml of cold 10mM MES-10mM EDTA, pH 5.0 (buffer A). 250 μl 0.1 M PMSF (Sigma) was added to inhibit proteolysis and 3.5
在以细胞为基础的ELISA结合试验中(如下所述)，包括嵌合2H7 Fab在内的Fab与CD20的结合难以检测。因此，2H7 Fab型式被重新格式化(reformatted)成全长IgG1抗体，以进行试验和进一步诱变。Binding of Fabs, including the chimeric 2H7 Fab, to CD20 is difficult to detect in cell-based ELISA binding assays (described below). Therefore, the 2H7 Fab format was reformatted into a full length IgGl antibody for testing and further mutagenesis.
通过将嵌合2H7(v6.8)Fab的VL与VH结构域和人源化Fab型式2至型式6亚克隆至先前描述过的用于哺乳动物细胞表达的pRK载体(Gorman etal.，DNA Prot.Eng.Tech.2：3(1990))，构建表达全长IgG的质粒。简要地说，每个Fab构建物用EcoRV与BlpI消化以切除VL片段，该片段被克隆入质粒pDR1的EcoRV/BlpI位点(图4)用于完整轻链(VL-CL结构域)的表达。另外，每个Fab构建物用PvuII与ApaI消化以切除VH片段，该片段被克隆入质粒pDR2的PvuII/ApaI位点(图5)用于完整重链(VH-CH1-铰链-CH2-CH3结构域)的表达。对于每个IgG变体，通过将表达轻链的质粒与表达重链的质粒共转染入用腺病毒转化的人胚肾细胞系293(Graham et al.，J.Gen.Virol.，36：59-74，(1977))，进行瞬时转染。简要地说，在转染的前一天将293细胞分瓶，并且铺于含血清的培养基上。第二天，加入以磷酸钙沉淀制备的双链DNA，继之以pAdVAntageTMDNA(Promega，Madison，WI)，细胞在37℃孵育过夜。细胞在无血清培养基中培养，在4天之后收获。用蛋白质A-琼脂糖CL-4B从培养物上清液纯化抗体，然后缓冲液更换入10mM琥珀酸钠、140mM NaCl，pH6.0，并用Centricon-10(Amicon)浓缩。用定量的氨基酸分析测定蛋白质浓度。By subcloning the VL andVH domains of the chimeric 2H7(v6.8) Fab andthe humanized
为了测量对CD20抗原的相对结合亲合力，发展出以细胞为基础的ELISA试验。将人B类淋巴母WIL2-S细胞(ATCC CRL 8885，American TypeCulture Collection，Rockville，MD)在补充有2mM L-谷氨酰胺、20mMHEPES，pH 7.2与10％热灭活的胎牛血清的RPMI 1640中、在湿润的5％CO2培育箱中生长。细胞用含1％FBS的PBS(分析缓冲液)洗，以250-300,000细胞/孔接种于96孔圆底平板上(Nunc，Roskilde，Denmark)。向板中加入溶于分析缓冲液的两倍连续稀释的标准液(15.6-1000ng/ml的2H7v6.8嵌合的IgG)与三倍连续稀释的样品(2.7-2000ng/ml)。将平板埋在冰中，孵育45分钟。为了除去未结合抗体，向孔中加入0.1mL分析缓冲液。将平板离心，除去上清。细胞再用0.2mL分析缓冲液洗两次。通过向平板中加入过氧化物酶偶联的山羊抗人Fc抗体(Jackson ImmunoResearch，Grove，PA)，检测与平板结合的抗体。孵育45分钟后，如以前所述洗细胞。板中加入TMB底物(3，3′，5，5′-四甲基联苯胺；Kirkegaard & Perry Laboratories，Gaithersburg，MD)。加入1M磷酸以停止反应。用四参数非线性回归曲线拟合程序(KaleidaGraph，Synergy software，Reading，PA)拟合滴定曲线。测定滴定曲线中点的吸收率(中点-OD)确定标准物的相应浓度。然后测定每个变体在这一中点OD所对应的浓度，标准物浓度被每个变体的浓度除。因此所得数值是每个变体的结合相对于标准的比率。相对亲合力(当量浓度)在实验间的标准偏差通常为+/-10％。To measure relative binding affinities to the CD20 antigen, a cell-based ELISA assay was developed. Human B lymphoid parent WIL2-S cells (ATCC CRL 8885, American TypeCulture Collection, Rockville, MD) were incubated in RPMI 1640 supplemented with 2mM L-glutamine, 20mM HEPES, pH 7.2 and 10% heat-inactivated fetal calf serum. grown in a humidified 5% CO2 incubator. Cells were washed with PBS (assay buffer) containing 1% FBS and seeded in 96-well round bottom plates (Nunc, Roskilde, Denmark) at 250-300,000 cells/well. Two-fold serial dilutions of standards (15.6-1000 ng/ml 2H7v6.8 chimeric IgG) and three-fold serial dilutions of samples (2.7-2000 ng/ml) in assay buffer were added to the plate. The plates were buried in ice and incubated for 45 minutes. To remove unbound antibody, 0.1 mL of assay buffer was added to the wells. The plates were centrifuged and the supernatant removed. Cells were then washed twice with 0.2 mL assay buffer. Antibody bound to the plate was detected by adding peroxidase-conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, Grove, PA) to the plate. After 45 min of incubation, cells were washed as previously described. TMB substrate (3,3',5,5'-tetramethylbenzidine; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the plate. 1M phosphoric acid was added to stop the reaction. Titration curves were fitted using a four-parameter nonlinear regression curve fitting program (KaleidaGraph, Synergy software, Reading, PA). The absorbance at the midpoint of the titration curve (midpoint-OD) was determined to determine the corresponding concentration of the standard. The concentration of each variant at this midpoint OD was then determined and the standard concentration divided by the concentration of each variant. The resulting value is therefore the ratio of binding of each variant relative to the standard. The standard deviation between experiments for relative affinities (normalities) was typically +/- 10%.
如表2所示，与嵌合2H7(v.6.8)相比，CDR交换变体(CDR-swapvariant)(v.2)的结合被极度降低。然而，型式3至6显示结合改善。为了测定将结合亲合力恢复至嵌合2H7的结合亲合力所需突变的最小数目，通过定点诱变构建额外的突变与突变组合，以生成变体7至17，如表2中所指示。特别地，这些包括VH突变A49G、F67A、I69L、N73K和L78A；与VL突变M4L、M33I和F71Y。型式16和17显示最好的相对结合亲合力，在嵌合型式的2倍范围内，型式16和17之间没有显著差异(s.d.＝+/-10％)。为了使突变数目最少，因此选择型式16作为人源化形式用于额外表征，该型式仅具有4个由人框架残基变为鼠框架残基的突变(表3)。As shown in Table 2, binding of the CDR-swap variant (v.2) was extremely reduced compared to the chimeric 2H7 (v.6.8). However,
表2.使用以细胞为基础的ELISA，将人源化的2H7 IgG变体和CD20的相对结合亲合力与嵌合2H7和CD20的相对结合亲合力进行比较。相对结合用嵌合2H7的浓度比等量结合所需变体浓度来表示；因此比值<1指示对变体亲合力较弱。相对亲合力测定中的标准偏差平均为+/-10％。可变结构域中的框架取代系根据Kabat的编号系统(Kabat et al.，上文)相对于CDR-交换型式。Table 2. Comparison of the relative binding affinities of humanized 2H7 IgG variants and CD20 to that of chimeric 2H7 and CD20 using a cell-based ELISA. Relative binding is expressed as the concentration of chimeric 2H7 to the concentration of the variant required for equivalent binding; thus a ratio <1 indicates weaker affinity for the variant. The standard deviation in relative affinity determinations averaged +/- 10%. Framework substitutions in the variable domains are relative to CDR-swapped patterns according to Kabat's numbering system (Kabat et al., supra).
表3用来在人源化2H7型式16(2H7.v16)中构建突变VH(A49G，R71V，N73K)与VL(L46P)的寡核苷酸序列。带下划线的密码子编码所指示的氨基酸取代。对于VH(R71V，N73K)与VL(L46P)，寡核苷酸以有意义链显示，因为这些被用于在Fab模板上诱变，而对于VH(A49G)，寡核苷酸以反义链显示，因为该链被与pRK(IgG重链)模板一起使用。型式16的蛋白质序列示于图6和图7。Table 3 Oligonucleotide sequences used to construct mutantVH (A49G, R71V, N73K) and VL (L46P ) in humanized 2H7 version 16 (2H7.v16). Underlined codons code for the indicated amino acid substitutions. ForVH (R71V, N73K) and VL (L46P ), oligonucleotides are shown as sense strands, since these were used for mutagenesis on the Fab template, while forVH (A49G), oligonucleotides are shown as The antisense strand is shown because this strand was used with the pRK (IgG heavy chain) template. The protein sequence of
实施例2Example 2
2H7的抗原结合决定簇(互补位)Antigen-binding determinant (paratope) of 2H7
在2H7.v16或2H7.v17中进行丙氨酸取代(Cunningham & Wells，Science244：1081-1085(1989)以检验抗体的个别侧链在与CD20结合中的作用。在293细胞中从pDR1与pDR2载体表达IgG变体，纯化，并如上述分析相对结合亲合力。几处丙氨酸取代导致WIL-2S细胞上与CD20相对结合的显著降低(表4)。Alanine substitutions (Cunningham & Wells, Science 244:1081-1085 (1989)) were performed in 2H7.v16 or 2H7.v17 to examine the role of individual side chains of the antibody in binding to CD20. In 293 cells from pDR1 and pDR2 The IgG variants were expressed by the vector, purified, and analyzed for relative binding affinities as above.Several alanine substitutions resulted in a significant decrease in relative binding to CD20 on WIL-2S cells (Table 4).
表4利用以细胞为基础的ELISA(WIL2-S细胞)测量得到的丙氨酸取代对人源化2H7.v16的CDR区域的效应。相对结合用2H7.v16亲本的浓度比等量结合所需的变体浓度来表示，因此比值<1指示变体具有较弱亲合力；比值>1指示变体具有较高亲合力。相对亲合力测定中的标准偏差平均为+/-10％。可变结构域中的框架取代系根据Kabat的编号系统(Kabat et al.，上文)相对于2H7.v16。NBD的意思是没有可检测的结合。型式45中的两个数字来自于各自独立的实验。Table 4 Effects of alanine substitutions on the CDR regions of humanized 2H7.v16 measured by cell-based ELISA (WIL2-S cells). Relative binding is expressed as the concentration of the 2H7.v16 parent to the concentration of the variant required for equivalent binding, so a ratio <1 indicates a variant with weaker affinity; a ratio >1 indicates a variant with higher affinity. The standard deviation in relative affinity determinations averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.v16 according to Kabat's numbering system (Kabat et al., supra). NBD means no detectable binding. The two numbers in
实施例3Example 3
2H7 CDR区域内部的附加突变Additional mutations within 2H7 CDR regions
也测试了额外残基的取代以及通过丙氨酸扫描被鉴定为是重要的CDR位置的取代的结合。几种变体组合，特别是v.96，似乎比v.16结合更紧密。The combination of substitutions of additional residues as well as substitutions at CDR positions identified as important by alanine scanning was also tested. Several variant combinations, notably v.96, seem to be more tightly bound than v.16.
表5使用以细胞为基础的ELISA(WIL2-S细胞)测量在人源化2H7.v16的CDR区域中的突变组合与非丙氨酸取代的效应。对CD20的相对结合用2H7.v16亲本的浓度比等量结合所需的变体浓度来表示，因此比值<1指示变体具有较弱亲合力；比值>1指示变体具有较高亲合力。相对亲合力测定中的标准偏差平均为+/-10％。可变结构域中的框架取代系根据Kabat的编号系统(Kabat et al.，上文)相对于2H7.v16。Table 5 The effect of mutation combinations and non-alanine substitutions in the CDR regions of humanized 2H7.v16 was measured using a cell-based ELISA (WIL2-S cells). Relative binding to CD20 is expressed as the concentration of the 2H7.v16 parent versus the concentration of the variant required for equivalent binding, so a ratio <1 indicates a variant with weaker affinity; a ratio >1 indicates a variant with higher affinity. The standard deviation in relative affinity determinations averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.v16 according to Kabat's numbering system (Kabat et al., supra).
实施例4Example 4
在框架人源化取代位点的突变Mutations at framework humanization substitution sites
在人源化期间发生改变的框架位置中附加残基的取代也在2H7.v16背景中被测试。特别地，在VL(P46)与VH(G49，V71，与K73)中进行既不在鼠2H7亲本中也不在人共有框架中存在的可替换的框架取代。Substitutions of additional residues in framework positions that were altered during humanization were also tested in the 2H7.v16 background. In particular, alternative framework substitutions that were present neither in the murine 2H7 parent nor in the human consensus framework were made in VL (P46 ) andVH (G49, V71, and K73).
这些取代通常导致相对结合的极子的改变(表6)，指示在这些位置的框架残基中存在一些灵活性。These substitutions generally resulted in changes in the relative binding poles (Table 6), indicating that there is some flexibility in the framework residues at these positions.
表6在框架取代的以细胞为基础的(WIL2-S)试验中的相对结合。用相对于2H7.v16背景的突变显示IgG变体。相对结合用2H7.v6.8嵌合体的浓度比等量结合所需的变体浓度来表示，因此比值<1指示变体具有较弱亲合力；比值>1指示变体具有较高亲合力。相对亲合力测定中的标准偏差平均为+/-10％。可变结构域中的框架取代系根据Kabat的编号系统(Kabat et al.，上文)相对于2H7.v16的。Table 6 Relative binding in a framework-substituted cell-based (WIL2-S) assay. IgG variants are shown with mutations relative to the 2H7.v16 background. Relative binding is expressed as the concentration of the 2H7.v6.8 chimera compared to the concentration of the variant required for equivalent binding, so a ratio <1 indicates a variant with weaker affinity; a ratio >1 indicates a variant with higher affinity. The standard deviation in relative affinity determinations averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.v16 according to Kabat's numbering system (Kabat et al., supra).
(*)用2H7.v16作为标准比较物分析的变体；相对值校正至嵌合体的值。(* ) Variants analyzed with 2H7.v16 as standard comparator; relative values corrected to chimera values.
(*)用2H7.v16作为标准比较物分析的变体；相对值校正至嵌合体的值。(* ) Variants analyzed with 2H7.v16 as standard comparator; relative values corrected to chimera values.
实施例5Example 5
具有增强的效应子功能的人源化2H7变体Humanized 2H7 variants with enhanced effector function
由于2H7可通过补体依赖的细胞毒性(CDC)和抗体依赖的细胞的细胞毒性(ADCC)介导B细胞溶解，我们设法产生具有改善的CDC与ADCC活性的人源化2H7.v16的变体。其它抗体的Fc区域内的某些残基突变也已经被描述(Idusogie et al.，J.Immunol.166：2571-2575(2001))，用于通过增强与补体成分C1q的结合而改善CDC。用于改善ADCC的某些突变也已被描述(Shields et al.，J.Biol.Chem.276：6591-6604(2001)；Presta et al.，Biochem.Soc.Trans.30：487-490(2002))，所述改善是通过增强IgG与激动性Fcγ受体的结合、并降低IgG与抑制性Fcγ受体的结合而达到的。特别地，三种突变已被鉴别具有改善的CDC与ADCC活性：如所描述的(Idusogie et al.，上文(2001)；Shields et al.，上文)，S298A/E333A/K334A(在这里也称为三重丙氨酸突变体或变体；Fc区域的编号方式根据EU编号系统；Kabat et al.，上文)。Since 2H7 can mediate B cell lysis through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), we sought to generate variants of humanized 2H7.v16 with improved CDC and ADCC activities. Mutations of certain residues within the Fc region of other antibodies have also been described (Idusogie et al., J. Immunol. 166:2571-2575 (2001)) for improving CDC by enhancing binding to the complement component C1q. Certain mutations for improving ADCC have also been described (Shields et al., J.Biol.Chem.276:6591-6604 (2001); Presta et al., Biochem.Soc.Trans.30:487-490( 2002)), the improvement is achieved by enhancing IgG binding to agonistic Fcγ receptors and reducing IgG binding to inhibitory Fcγ receptors. In particular, three mutations have been identified with improved CDC and ADCC activity: S298A/E333A/K334A (here Also known as triple alanine mutant or variant; numbering of the Fc region is according to the EU numbering system; Kabat et al., supra).
为了增强2H7的CDC与ADCC活性，构建2H7 Fc的三重丙氨酸突变体。用突变S298A/E333A/K334A也已经产生抗HER2抗体4d5的人源化变体，称为4D5Fc110(即抗p185HER2IgG1(S298A/E333A/K334A)；Shields et al.，上文)。编码抗体4D5Fc110的质粒p4D5Fc110(Shields et al.，上文)用ApaI与HindIII消化，Fc片段(包含突变S298A/E333A/K334A)连接入2H7重链载体pDR2-v16的ApaI/HindIII位点，产生pDR2-v31。31型完整重链的氨基酸序列示于图8。轻链与v16的轻链相同。In order to enhance the CDC and ADCC activities of 2H7, a triple alanine mutant of 2H7 Fc was constructed. A humanized variant of the anti-HER2 antibody 4d5, termed 4D5Fc110, has also been generated with the mutation S298A/E333A/K334A (i.e., anti-p185HER2 IgG1 (S298A/E333A/K334A); Shields et al., supra). Plasmid p4D5Fc110 (Shields et al., supra) encoding antibody 4D5Fc110 was digested with ApaI and HindIII, and the Fc fragment (containing the mutations S298A/E333A/K334A) was ligated into the ApaI/HindIII site of the 2H7 heavy chain vector pDR2-v16 to generate pDR2 - The amino acid sequence of the complete heavy chain of type v31.31 is shown in FIG. 8 . The light chain is identical to that of v16.
虽然IgG1抗体的Fc区域恒定区在一特定物种内相对保守，仍存在等位基因变异(综述见Lefranc与Lefranc，在“The human IgG subclasses：molecular analysis of structure，function，and regulation”中，pp.43-78，F.Shakib(ed.)，Pergammon Press，Oxford(1990))。Although the Fc region constant region of IgG1 antibodies is relatively conserved within a given species, allelic variation exists (reviewed in Lefranc and Lefranc, in "The human IgG subclasses: molecular analysis of structure, function, and regulation", pp. 43-78, F. Shakib (ed.), Pergammon Press, Oxford (1990)).
表7Fc区域的取代对CD20结合的影响。与CD20的相对结合在对框架取代所进行的以细胞为基础的(WIL2-S)试验中加以测量。Fc突变(*)用EU编号方式(Kabat，上文)加以指示，并相对于2H7.v16亲本。v.31的Fc区域中三个丙氨酸改变的组合被称作“Fc110”。用相对于2H7.v16背景的突变显示IgG变体。相对结合用2H7.v6.8嵌合体的浓度比等量结合所需的变体浓度来表示；因此比值<1指示变体的亲合力较弱。相对亲合力测定中的标准偏差平均为+/-10％。Table 7 Effects of substitutions in the Fc region on CD20 binding. Relative binding to CD20 was measured in a cell-based (WIL2-S) assay for framework substitutions. Fc mutations (* ) are indicated with EU numbering (Kabat, supra) and relative to the 2H7.v16 parent. The combination of three alanine changes in the Fc region of v.31 was designated "
实施例6Example 6
稳定性增强的人源化2H7变体Humanized 2H7 variants with enhanced stability
为了开发出治疗性蛋白质，需要选择在适当制剂缓冲液中，在氧化、脱胺或其它可影响产品质量的过程中保持稳定的变体。在2H7.v16中，几个残基被鉴定为不稳定性的可能来源：VL(M32)与VH(M34，N100)。因此，突变被引入这些位点，用于与v16进行比较。In order to develop therapeutic proteins, it is necessary to select variants that are stable during oxidation, deamination, or other processes that can affect product quality in appropriate formulation buffers. In 2H7.v16, several residues were identified as possible sources of instability:VL (M32) andVH (M34, N100). Therefore, mutations were introduced at these sites for comparison with v16.
表8.被设计具有增强的稳定性和/或效应子功能的2H7变体在以细胞为基础的(WIL2-S)试验中对CD20的相对结合。用相对于2H7.v16背景的突变显示IgG变体。相对结合用2H7.v6.8嵌合体的浓度比等量结合所需的变体浓度来表示；因此比值<1指示变体的亲合力较弱。相对亲合力测定中的标准偏差平均为+/-10％。可变结构域框架取代相对于2H7.v16，并根据Kabat的编号系统，Fc突变(*)用EU编号方式(Kabat et al.，上文)指示。(**)用2H7.v16测量的变体，作为标准比较物；相对值校正至嵌合体的值。Table 8. Relative binding to CD20 of 2H7 variants designed with enhanced stability and/or effector function in a cell-based (WIL2-S) assay. IgG variants are shown with mutations relative to the 2H7.v16 background. Relative binding is expressed as the concentration of the 2H7.v6.8 chimera compared to the concentration of the variant required for equivalent binding; thus a ratio of <1 indicates a weaker affinity for the variant. The standard deviation in relative affinity determinations averaged +/- 10%. Variable domain framework substitutions are relative to 2H7.v16, and according to Kabat's numbering system, Fc mutations (* ) are indicated with EU numbering (Kabat et al., supra). (** ) Variants measured with 2H7.v16 as standard comparator; relative values corrected to chimera values.
基于先前报导过的突变(Idusogie et al.(2000)；Idusogie et al.(2001)；Shields et al.(2001))，附加的Fc突变与稳定性或亲合力增强的突变结合起来，以改变或增强效应子功能。这些改变包括实施例中所描述的S298、E333A、K334A；K322A，以降低CDC活性；D265A，以降低ADCC活性；K326A或K326W，以增强CDC活性；和E356D/M358L，以测试Fc区域异型改变(allotypic change)的效果。这些突变均不导致CD20结合亲合力的显著变化。Based on previously reported mutations (Idusogie et al. (2000); Idusogie et al. (2001); Shields et al. (2001)), additional Fc mutations were combined with stability or affinity enhancing mutations to alter Or enhance effector function. These changes include S298, E333A, K334A described in the Examples; K322A, to reduce CDC activity; D265A, to reduce ADCC activity; K326A or K326W, to enhance CDC activity; allotypic change) effect. None of these mutations resulted in significant changes in CD20 binding affinity.
(**)用2H7.v16测量作为比较物的变体相对结合值校正至嵌合体的值(** ) Variant relative binding values measured with 2H7.v16 as comparator corrected to chimera values
为了测试稳定性突变对蛋白质降解率的影响，2H7.v16与2H7.v73以12-14mg/mL配制在10mM组氨酸、6％蔗糖、0.02％聚山梨酸酯20，pH5.8中，在40℃孵育16天。所孵育的样品然后用离子交换层析分析电荷变体的改变、用大小排阻层析分析聚合与断裂、通过测试以细胞为基础的(WIL2-S)试验分析相对结合。To test the effect of stability mutations on protein degradation rates, 2H7.v16 and 2H7.v73 were formulated at 12-14 mg/mL in 10 mM histidine, 6% sucrose, 0.02
结果(图9)显示，与2H7v.16相比，2H7v.73具有较高稳定性，这是通过在增加稳定性的条件下离子交换层析中主峰级分的丢失而检测的。在聚合、断裂或结合亲合力上未见明显差别。The results ( FIG. 9 ) showed that 2H7v.73 had a higher stability compared to 2H7v.16, as detected by the loss of the main peak fraction in ion exchange chromatography under conditions of increased stability. No significant differences were seen in aggregation, fragmentation or binding affinities.
实施例7Example 7
在WIL2-S细胞上抗体与CD20结合的Scatchard分析Scatchard analysis of antibody binding to CD20 on WIL2-S cells
使用放射性同位素标记的2H7 IgG测定2H7 IgG变体与WIL2-S细胞结合的平衡离解常数(Kd)。IgG变体在CHO细胞中产生。
表9从Scatchard分析得到的2H7变体的平衡结合亲合力Table 9 Equilibrium binding affinities of 2H7 variants from Scatchard analysis
实施例8Example 8
补体依赖的细胞毒性(CDC)分析Complement-Dependent Cytotoxicity (CDC) Assay
基本上如所描述的(Idusogie et al.，J.Immunol.164：4178-4184(2000)；Idusogie et al.，J.Immunol.166：2571-2575(001))，分析2H7 IgG变体介导WIL2-S细胞补体依赖的溶解的能力，该细胞是表达CD20的成淋巴细胞样(limphoblastoid)的B细胞系。抗体从0.1mg/mL贮存液中1:3连续稀释。向包含0.05mL正常人补体(Quidel，San Diego，CA)溶液的96孔组织培养板中加入每一稀释的0.05mL等分试样。向这一混合物中加入0.05mL体积的50,000 WIL2-S细胞。37℃孵育2小时后，加入0.05mL Alamar blue溶液(Accumed International，Westlake，OH)，在37℃再继续孵育18小时。将盖子从板上拿开，板子在定轨摇床上于室温下摇动15分钟。使用530nm激发滤光器和590nm发射滤光器读取相对荧光单位(RFU)。使用KaleidaGraph软件，通过将RFU拟合为每一抗体的浓度的函数计算EC50。2H7 IgG variants were analyzed essentially as described (Idusogie et al., J. Immunol. 164:4178-4184 (2000); Idusogie et al., J. Immunol. 166:2571-2575 (001)). Ability to induce complement-dependent lysis of WIL2-S cells, a CD20-expressing limphoblastoid B-cell line. Antibodies were serially diluted 1:3 from a 0.1 mg/mL stock. A 0.05 mL aliquot of each dilution was added to a 96-well tissue culture plate containing 0.05 mL of normal human complement (Quidel, San Diego, CA) solution. To this mixture was added 50,000 WIL2-S cells in a volume of 0.05 mL. After incubation at 37°C for 2 hours, 0.05 mL of Alamar blue solution (Accumed International, Westlake, OH) was added, and incubation was continued at 37°C for another 18 hours. The lid was removed from the plate and the plate was shaken on an orbital shaker at room temperature for 15 minutes. Relative fluorescence units (RFU) were read using a 530nm excitation filter and a 590nm emission filter.EC50 was calculated by fitting the RFU as a function of the concentration of each antibody using KaleidaGraph software.
结果(表10)显示人源化2H7抗体导致CDC令人惊奇的改善，v.73的相对效能类似于
表10与Rituxan相比2H7抗体的CDC活性。数字>1表示CDC活性不如
实施例9Example 9
抗体依赖的细胞毒性(ADCC)分析Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay
基本上如所描述的(Shields et al.，J.Biol.Chem.276：6591-6604(2001))，利用乳酸脱氢酶(LDH)读数，分析2H7 IgG变体介导自然杀伤细胞(NK细胞)溶解WIL2-S细胞的能力，该细胞是表达CD20的成淋巴细胞样的B细胞系。NK细胞制备自用100mL PBS稀释的100mL肝素化的血液，该血液获得自已经为FcγRIII(又名CD16)(Koene et al.，blood 90：1109-111409-1114(1997))同种型的(isotyped)正常人供体。在这一实验中，NK细胞来自于对CD16杂合的人供体(F158/V158)。稀释的血液在15mL淋巴细胞分离介质(ICNBiochemical，Aurora，Ohio)上进行分层，在2000 RPM离心20分钟。将层间分界面上的白细胞分配于4个干净的50mL管中，管里充满含15％胎牛血清的RPMI培养基。管子在1400 RPM离心5分钟，弃去上清液。细胞小团重悬浮于MACS缓冲液(0.5％BSA，2mM EDTA)中，NK细胞用小珠(NK细胞分离试剂盒，130-046-502)根据厂家的操作程序(Miltenyi Biotech，)纯化。NK细胞在MACS缓冲液中稀释至2 x 106细胞/毫升。2H7 IgG variants were analyzed to mediate natural killer (NK) cells using lactate dehydrogenase (LDH) readout essentially as described (Shields et al., J. Biol. Chem. 276:6591-6604 (2001)). cells) ability to lyse WIL2-S cells, a CD20-expressing lymphoblastoid B cell line. NK cells were prepared from 100 mL of heparinized blood diluted with 100 mL of PBS obtained from (isotyped) FcγRIII (aka CD16) (Koene et al., blood 90: 1109-111409-1114 (1997)) isotype ) normal human donor. In this experiment, NK cells were derived from a human donor heterozygous for CD16 (F158/V158). Diluted blood was layered on 15 mL of lymphocyte separation medium (ICNBiochemical, Aurora, Ohio) and centrifuged at 2000 RPM for 20 minutes. Distribute the leukocytes at the interlayer interface into 4 clean 50mL tubes filled with RPMI medium containing 15% fetal bovine serum. Centrifuge the tubes at 1400 RPM for 5 minutes and discard the supernatant. The cell pellet was resuspended in MACS buffer (0.5% BSA, 2 mM EDTA), and NK cells were purified using beads (NK Cell Isolation Kit, 130-046-502) according to the manufacturer's procedure (Miltenyi Biotech, ). NK cells were diluted to 2 x106 cells/ml in MACS buffer.
将分析培养基(F12/DMEM 50:50无甘氨酸，1mM HEPES缓冲液pH 7.2，青霉素/链霉素(100单位/mL；Gibco)，谷氨酰胺，与1％热灭活的胎牛血清)中的抗体系列稀释加至96孔圆底组织培养板中。WIL2-S细胞在分析缓冲液中稀释至浓度4 x 105/mL。WIL2-S细胞(0.05mL每孔)在96孔板中与稀释抗体混合，在室温下孵育30分钟，让抗体与CD20结合(调理作用)。Assay medium (F12/DMEM 50:50 without glycine, 1 mM HEPES buffer pH 7.2, penicillin/streptomycin (100 units/mL; Gibco), glutamine, with 1% heat-inactivated fetal calf serum) Antibody serial dilutions in 96-well round-bottom tissue culture plates. WIL2-S cells were diluted to a concentration of 4 x 105 /mL in assay buffer. WIL2-S cells (0.05 mL per well) were mixed with diluted antibody in a 96-well plate and incubated for 30 minutes at room temperature to allow antibody binding to CD20 (opsonization).
通过向每孔中加入0.1mLNK细胞起动ADCC反应。在对照孔中，加入2％Triton X-100。板子在37℃孵育4小时。释放的乳酸脱氢酶水平用细胞毒性(LDH)检测试剂盒(试剂盒#1644793，Roche Diagnostics，Indianapolis，Indiana)按照厂家指示进行测量。每孔加入0.1mL LDH显色剂，随后混合10秒。板子然后用铝箔覆盖，在黑暗中室温下孵育15分钟。然后读取490nm光密度，并通过被对照细胞中测得的总LDH除，来计算溶解百分比。溶解被绘制为抗体浓度的函数，四参数曲线拟合(KaleidaGraph)被用于判定EC50浓度。ADCC reactions were initiated by adding 0.1 mL NK cells to each well. In control wells, 2% Triton X-100 was added. Plates were incubated at 37°C for 4 hours. Released lactate dehydrogenase levels were measured using a Cytotoxicity (LDH) Assay Kit (Kit #1644793, Roche Diagnostics, Indianapolis, Indiana) according to the manufacturer's instructions. 0.1 mL of LDH chromogenic reagent was added per well followed by mixing for 10 seconds. Plates were then covered with aluminum foil and incubated for 15 minutes at room temperature in the dark. The optical density at 490 nm was then read and the percent lysis was calculated by dividing by the total LDH measured in control cells. Dissolution was plotted as a function of antibody concentration and a four-parameter curve fit (KaleidaGraph) was used to determineEC50 concentrations.
结果表明，人源化2H7抗体在ADCC方面活跃，v.31与v.75的相对功效比
表11基于n实验，与2H7.v16相比，2H7抗体对WIL2-S细胞的ADCC活性。(数值>1表示比2H7.v16效力较低，数值<1表示效力较强。)Table 11 ADCC activity of 2H7 antibody on WIL2-S cells compared to 2H7.v16 based on n experiments. (A value > 1 means less potency than 2H7.v16, and a value < 1 means stronger potency.)
进行额外的ADCC分析，以将2H7组合变体与
表12基于n实验，与
实施例10Example 10
猕猴pilot研究中2H7变体的体内效应In vivo effects of 2H7 variants in macaque pilot studies
在正常雄性猕猴(Macaca fascicularis)中测试瞬时转染CHO细胞所产生的2H7变体，以评定其体内活性。其它抗CD20抗体，例如C2B8(
用FACS方法测定外周B细胞浓度，该方法计数CD3-/CD40+细胞。通过如下门化(gating)策略在猴样品中获得CD3-CD40+B细胞在总淋巴细胞中的百分率。在前向散射/侧向散射点图上标记淋巴细胞群体以定义区域1(R1)。使用R1中的事件，显示CD40和CD3标志物的荧光强度点图。荧光标记的同种型(isotype)对照被用来判定CD40和CD3阳性各自的截止点。Peripheral B cell concentrations were determined by the FACS method, which counts CD3-/CD40+ cells. The percentage of CD3-CD40+ B cells in total lymphocytes was obtained in monkey samples by gating strategy as follows. Lymphocyte populations were marked on the forward scatter/side scatter dot plot to define region 1 (R1). Fluorescence intensity dot plots of CD40 and CD3 markers are shown using events in R1. Fluorescently labeled isotype controls were used to determine the respective cutoff points for CD40 and CD3 positivity.
结果指示2H7.v16和2H7.v31能在10mg/kg剂量产生完全外周B细胞削减、并且在0.05mg/kg剂量产生部分外周B细胞削减(图11)。时间进程与在给药的第一个72小时期间测量得到的B细胞削减的程度在两种抗体间类似。对恢复动物的随后分析表明，同用2H7.v16给药的动物比较起来，那些用2H7.v31处理的动物显示B细胞削减的持续时间较长。特别地，在用10mg/kg2H7.v16处理的恢复动物中，B细胞在第10天与第36天取样之间的某个时间显示实质性(substantial)的B细胞恢复。然而，对于用10mg/kg2H7.v31处理的恢复动物中，B细胞直到第36天与第67天之间的某个时间才显示恢复(图11)。这暗示，与2H7.v16相比，2H7.v31完全削减的持续时间长出大约一个月。The results indicated that 2H7.v16 and 2H7.v31 were able to produce complete peripheral B cell depletion at the 10 mg/kg dose and partial peripheral B cell depletion at the 0.05 mg/kg dose (Figure 11). The time course and extent of B cell depletion measured during the first 72 hours of dosing was similar between the two antibodies. Subsequent analysis of recovered animals showed that those treated with 2H7.v31 showed a longer duration of B cell depletion compared to animals dosed with 2H7.v16. In particular, in recovery animals treated with 10 mg/kg 2H7.v16, B cells showed substantial B cell recovery sometime between
在猕猴研究中，低或高剂量未观察到毒性，总病理学(gross pathology)也是正常的。在其它研究中，在这些猴子中相隔两周i.v.给药两次后，v16直到所评估的最高剂量(100mg/kgx2＝1200mg/m2x2)仍被良好耐受。In macaque studies, no toxicity was observed at low or high doses and gross pathology was normal. In other studies, v16 was well tolerated up to the highest dose evaluated (100 mg/kgx2 = 1200 mg/m2x2 ) after two iv doses two weeks apart in these monkeys.
在猕猴中用2H7.v16与
实施例11Example 11
具有增强的效应子功能的岩藻糖缺陷的2H7变体抗体Fucose-deficient 2H7 variant antibody with enhanced effector function
正常CHO与HEK293细胞将岩藻糖加至IgG寡糖至高程度(97-98％)。来自血清的IgG也是高度岩藻糖基化的。Normal CHO and HEK293 cells add fucose to IgG oligosaccharides to a high extent (97-98%). IgG from serum is also highly fucosylated.
DP12和Lec13被用来产生本研究的抗体，DP12是具有岩藻糖基化能力的无二氢叶酸还原酶缺陷的(DHFR-)CHO细胞系，Lec13是蛋白质岩藻糖基化缺陷的细胞系。CHO细胞系Pro-Lec13.6a(Lec13)获得自犹太高等学校大学阿尔伯特爱因斯坦医学院Pamela Stanley教授。亲本系是Pro-(脯氨酸营养缺陷型)和Gat-(甘氨酸、腺苷、胸苷营养缺陷型)。CHO-DP12细胞系是CHO-K1细胞系的衍生物(ATCC#CCL-61)，后者是二氢叶酸还原酶缺陷的，并且对胰岛素的需求降低。使用Superfect方法(Qiagen，Valencia，CA)用cDNA转染细胞系。使用嘌呤霉素二盐酸化物(Calbiochem，San Diego，CA)在10μg/ml浓度在生长培养基中选择表达转染的抗体的Lec13细胞，所述生长培养基含有：具有L-谷氨酰胺、核糖核苷与脱氧核糖核苷酸的MEM α培养基(GIBCO-BRL，Gaithersburg，MD)、补充有10％灭活FBS(GIBCO)、10mMHEPES、与1X青霉素/链霉素(GIBCO)。在无GHT的包含Ham′s F12生长培养基中类似地选择CHO细胞：无甘氨酸、有NaHCO3、补充有5％FBS的低葡萄糖DMEM(GIBCO)、10mM HEPES、2mM L-谷氨酰胺、1X GHT(甘氨酸、次黄嘌呤、胸苷)与1X青霉素/链霉素。DP12 and Lec13 were used to generate the antibodies in this study, DP12 is a dihydrofolate reductase-deficient (DHFR-)CHO cell line capable of fucosylation, and Lec13 is a protein fucosylation-deficient cell line . The CHO cell line Pro-Lec13.6a (Lec13) was obtained from Professor Pamela Stanley, Albert Einstein College of Medicine, University of Jewish Higher Education. The parental lines are Pro- (proline auxotroph) and Gat- (glycine, adenosine, thymidine auxotroph). The CHO-DP12 cell line is a derivative of the CHO-K1 cell line (ATCC #CCL-61 ), which is deficient in dihydrofolate reductase and has a reduced insulin requirement. Cell lines were transfected with cDNA using the Superfect method (Qiagen, Valencia, CA). Lec13 cells expressing the transfected antibody were selected using puromycin dihydrochloride (Calbiochem, San Diego, CA) at a concentration of 10 μg/ml in a growth medium containing: with L-glutamine, ribose Nucleosides and deoxyribonucleotides in MEM alpha medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% inactivated FBS (GIBCO), 10 mM HEPES, and 1X penicillin/streptomycin (GIBCO). CHO cells were similarly selected in GHT-free growth medium containing Ham's F12: no glycine, with NaHCO3 , low glucose DMEM (GIBCO) supplemented with 5% FBS, 10 mM HEPES, 2 mM L-glutamine, 1X GHT (Glycine, Hypoxanthine, Thymidine) with 1X Penicillin/Streptomycin.
在两至三周之内形成菌落，混合起来用于进行扩增和蛋白质表达。细胞集合体最初以3 x 106细胞/10厘米板的浓度接种，用于小批量蛋白质表达。一旦细胞生长至90-95％满板，便转移至无血清培养基中，并且在3-5天之后收集细胞上清液，在Fc IgG与完整IgG ELISA中测试以估计蛋白质表达水平。Lec13与CHO细胞在转移至PS24生产培养基之前一天以大约8 x 106细胞/15厘米板接种，所述PS24生产培养基补充有10mg/L重组人胰岛素与1mg/L微量元素。Colonies form within two to three weeks and are pooled for amplification and protein expression. Cell aggregates were initially seeded at a concentration of 3 x106 cells/10 cm plate for small batches of protein expression. Once the cells had grown to 90-95% confluence, they were transferred to serum-free medium, and cell supernatants were collected after 3-5 days and tested in Fc IgG and intact IgG ELISAs to estimate protein expression levels. Lecl3 and CHO cells were seeded at approximately 8 x106 cells/15 cm plate one day before transfer to PS24 production medium supplemented with 10 mg/L recombinant human insulin and 1 mg/L trace elements.
Lec13细胞与DP12细胞在无血清生产培养基中保持3-5天。收集上清液，在150ml锥形管中通过离心作用使之澄清以除去细胞和碎片。加入蛋白酶抑制剂PMSF与抑肽酶(Sigma，St.Louis，MO)，使用MWCO30过滤器(Amicon，Beverly，MA)在搅动的细胞上浓缩上清液5倍，然后使用G蛋白层析法(Amersham Pharmacia Biotech，Piscataway，NJ))立即纯化上清液。所有蛋白质均利用Centripriep-30浓缩器(Amicon)缓冲液更换入磷酸盐缓冲盐水(PBS)，并用SDS-聚丙烯酰胺凝胶电泳分析。利用A280测定蛋白质浓度，并利用氨基酸组成分析验证。Lec13 cells were maintained with DP12 cells in serum-free production medium for 3-5 days. The supernatant was collected and clarified by centrifugation in a 150ml conical tube to remove cells and debris. The protease inhibitors PMSF and aprotinin (Sigma, St.Louis, MO) were added, and the supernatant was concentrated 5-fold on stirred cells using a MWCO30 filter (Amicon, Beverly, MA), followed by protein G chromatography ( Amersham Pharmacia Biotech, Piscataway, NJ)) immediately purified the supernatant. All proteins were buffer exchanged into phosphate buffered saline (PBS) using a Centripriep-30 concentrator (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentration was determined using A280 and verified using amino acid composition analysis.
使用表达人源化2H7v16、2H7v.31的载体转染CHO细胞，并如所描述的进行选择。2H7v.16抗体保留有野生型Fc区域，而v.31(见上面实施例5，表7)的Fc区域中3个氨基酸发生改变(S298A、E333A、K334A)，导致对FcγRIIIa受体的高亲合力(Shields et al.J.Biol.Chem.276(9)：6591-6604(2001))。转染和选择后，分离出细胞的单个集落，并评估蛋白质表达水平，产量最高的细胞进行氨甲喋呤选择以选择那些质粒拷贝数被扩增并且因此生产较高水平抗体的细胞。使细胞生长，转移至无血清培养基中7天，然后收集培养基，装载至A蛋白柱上，用标准技术洗脱抗体。使用测定完整抗体的Elisa测定抗体的最后浓度。使用Centripriep-30浓缩器(Amicon)将所有蛋白质缓冲液更换入磷酸盐缓冲盐水(PBS)并用SDS-聚丙烯酰胺凝胶电泳分析。CHO cells were transfected with vectors expressing humanized 2H7v16, 2H7v.31 and selected as described. The 2H7v.16 antibody retained the wild-type Fc region, whereas v.31 (see Example 5, Table 7 above) had three amino acid changes (S298A, E333A, K334A) in the Fc region, resulting in a high affinity for the FcγRIIIa receptor. Synergy (Shields et al. J. Biol. Chem. 276(9):6591-6604(2001)). After transfection and selection, single colonies of cells are isolated and protein expression levels are assessed, with the highest producing cells subjected to methotrexate selection to select for those cells whose plasmid copy number is amplified and thus produce higher levels of antibody. Cells were grown and transferred to serum-free medium for 7 days, after which the medium was harvested, loaded onto a protein A column, and antibodies eluted using standard techniques. The final concentration of antibody was determined using an Elisa that measures intact antibody. All proteins were buffer exchanged into phosphate buffered saline (PBS) using Centripriep-30 concentrators (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis.
天冬酰胺连接的寡糖的基质辅助的激光解吸/电离飞行时间(MALDI-TOF)质谱分析：使用Papac et al.，Glycobiology 8，445-454(1998)的程序从重组糖蛋白上释放N-连接的寡糖。简要地说，96孔带有PVDF衬里的微滴定板(Millipore，Bedford，MA)用100μl甲醇调理，通过在Millipore多屏幕真空歧管上施加真空使该甲醇透过PDVF膜。调理过的PVDF膜用3X250μl水洗。在所有洗涤步骤之间，通过向歧管轻轻施加真空使孔完全排干。用还原和羧甲基化缓冲液(RCM)洗膜，所述缓冲液包含6M盐酸胍、360mM Tris、2mM EDTA、pH 8.6。将糖蛋白样品(50μg)加至各个孔中，再次轻轻施加真空使其透过PVDF膜，孔用2 X 50μl的RCM缓冲液洗。通过向每孔中加入50μl 0.1M二硫苏糖醇(DTT)溶液并将微滴定板在37℃孵育1小时来还原被固定化的样品。用真空除去DTT，孔用4 x 250μl水洗。通过添加50μl的0.1M碘乙酸(IAA)溶液来羧甲基化半胱氨酸残基，所述碘乙酸溶液在1MNaOH中新鲜配制，并用RCM缓冲液稀释至0.1M。通过在室温下在黑暗中孵育30分钟完成羧甲基化。向板子施加真空以除去IAA溶液，孔用4 x 250μl净化水洗。通过添加100μl的1％PVP360(聚乙烯吡咯烷360，000MW)(Sigma)溶液并在室温下孵育1小时封闭PVDF膜。通过轻轻施加真空除去PVP-360溶液，孔用4 x 250μl水洗。PNGase F(New England Biolabs，Beverly，MA)消化溶液(在10mM Tris醋酸盐中的25μl的25单位/ml溶液，pH 8.4)被加入每孔中，消化在37℃进行3小时。消化之后，样品转移至500μl微量离心管中，每个样品中加入2.5μl的1.5M乙酸溶液。酸化样品在室温孵育3小时以将寡糖从葡基胺转换成羟基形式。在MALDI-TOF质谱分析之前，所释放的寡糖用匀浆填充入压紧的反应管(US Biochemical，Cleveland，OH)的0.7ml床的阳离子交换树脂(以氢形式的AG50W-X8树脂)(Bio-Rad，Hercules，CA)脱盐。Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of asparagine-linked oligosaccharides: release of N- linked oligosaccharides. Briefly, 96-well PVDF-lined microtiter plates (Millipore, Bedford, MA) were conditioned with 100 [mu]l methanol which was permeated through the PDVF membrane by applying vacuum on the Millipore multi-screen vacuum manifold. The conditioned PVDF membrane was washed with 3X250 μl of water. Between all wash steps, the wells were completely drained by gently applying vacuum to the manifold. Membranes were washed with reducing and carboxymethylation buffer (RCM) containing 6M guanidine hydrochloride, 360 mM Tris, 2 mM EDTA, pH 8.6. Glycoprotein samples (50 μg) were added to each well, vacuum was gently applied again to permeate the PVDF membrane, and the wells were washed with 2×50 μl of RCM buffer. Immobilized samples were reduced by adding 50 μl of 0.1 M dithiothreitol (DTT) solution to each well and incubating the microtiter plate at 37° C. for 1 hour. DTT was removed by vacuum and the wells were washed with 4 x 250 μl of water. Cysteine residues were carboxymethylated by adding 50 μl of a 0.1 M iodoacetic acid (IAA) solution freshly prepared in 1 M NaOH and diluted to 0.1 M with RCM buffer. Carboxymethylation was accomplished by incubation at room temperature in the dark for 30 min. Vacuum was applied to the plate to remove the IAA solution and the wells were washed with 4 x 250 μl of purified water. The PVDF membrane was blocked by adding 100 μl of a 1% PVP360 (polyvinylpyrrolidine 360, 000 MW) (Sigma) solution and incubating at room temperature for 1 hour. The PVP-360 solution was removed by gently applying vacuum, and the wells were washed with 4 x 250 μl of water. PNGase F (New England Biolabs, Beverly, MA) digestion solution (25 μl of a 25 units/ml solution in 10 mM Tris acetate, pH 8.4) was added to each well, and digestion was performed at 37° C. for 3 hours. After digestion, samples were transferred to 500 μl microcentrifuge tubes, and 2.5 μl of 1.5M acetic acid solution was added to each sample. Acidified samples were incubated at room temperature for 3 hours to convert the oligosaccharides from the glucosylamine to the hydroxyl form. Prior to MALDI-TOF mass spectrometry analysis, the released oligosaccharides were filled with a homogenate into a 0.7 ml bed of cation exchange resin (AG50W-X8 resin in the hydrogen form) of a compacted reaction tube (US Biochemical, Cleveland, OH) ( Bio-Rad, Hercules, CA) for desalting.
为了以阳性方式(positive mode)进行样品的MALDI-TOF质谱分析，将脱盐的寡糖(0.5μl等分试样)用0.5μl的2，5二羟苯甲酸基质(sDHB)上样至不锈钢靶(stainless target)，所述2，5二羟苯甲酸基质通过将2mg 2，5二羟苯甲酸用溶解在1ml乙醇/1mM氯化钠1:1(v/v)中的0.1mg的5-methoxyslicylicacid溶解而制备。样品/基质混合物用真空干燥。为了以负方式(negative mode)进行分析，将脱盐的N-连接的寡糖(0.5μl等分试样)与0.5μl 2’，4’，6’-三羟基乙酰苯基质(THAP)一起上样至不锈钢靶，所述2’，4’，6’-三羟基乙酰苯在1:3(v/v)乙腈/13.3mM柠檬酸铵缓冲液中制备。真空干燥样品/基质混合物，然后在分析之前让其吸收空气中的水分。所释放的寡糖在PerSeptiveBioSystems Voyager-质谱仪上用MALDI-TOF进行分析。质谱仪在20kV以正性或负性方式用线状排列(linear configuration)并利用延迟提取(delayedextraction)进行操作。使用1300的激光功率并以数据总和方式(240扫描)获得数据以改善信噪比。仪器用标准寡糖的混合物校准，并在质量分配前用19点Savitsky-Golay算法将数据变平滑(smoothed)。利用Caesar 7.0数据分析软件包(SciBridge software)得到质谱数据的整合。For MALDI-TOF mass spectrometry analysis of samples in positive mode, desalted oligosaccharides (0.5 μl aliquots) were loaded onto stainless steel targets with 0.5 μl of 2,5 dihydroxybenzoic acid matrix (sDHB) (stainless target), the 2,5 dihydroxybenzoic acid matrix was prepared by dissolving 2mg of 2,5 dihydroxybenzoic acid with 0.1mg of 5- Prepared by dissolving methoxyslicylacid. The sample/matrix mixture was vacuum dried. For analysis in negative mode, desalted N-linked oligosaccharides (0.5 µl aliquots) were loaded with 0.5 µl 2',4',6'-trihydroxyacetophenone (THAP) The 2',4',6'-trihydroxyacetophenone was prepared in 1:3 (v/v) acetonitrile/13.3 mM ammonium citrate buffer to a stainless steel target. Vacuum dry the sample/matrix mixture and allow it to absorb moisture from the air prior to analysis. The released oligosaccharides were analyzed by MALDI-TOF on a PerSeptiveBioSystems Voyager-Mass Spectrometer. The mass spectrometer operates at 20 kV in a positive or negative mode with a linear configuration and with delayed extraction. Data were acquired in a data summation mode (240 scans) using a laser power of 1300 to improve the signal-to-noise ratio. The instrument was calibrated with a mixture of standard oligosaccharides and the data were smoothed with a 19 point Savitsky-Golay algorithm prior to mass assignment. Mass spectrometry data were integrated using Caesar 7.0 data analysis software package (SciBridge software).
自然杀伤(NK)细胞抗体依赖的细胞毒性试验Natural Killer (NK) Cell Antibody-Dependent Cytotoxicity Assay
如实施例9所描述进行ADCC试验。NK细胞与靶细胞(WIL2-S)的比例为4:1，试验进行4小时，如前面使用乳糖脱氢酶试验那样测量毒性。在添加NK细胞之前用所指示的抗体浓缩物调理靶细胞30分钟。所使用的
结果表明，低岩藻糖基化的抗体比完全岩藻糖化的抗体更有效地介导NK细胞靶细胞杀伤。低岩藻糖基化抗体2H7v.31在介导靶细胞杀伤上最有效。这一抗体在较低浓度下有效，并能在较高浓度下比其它抗体介导更高比率的靶细胞杀伤。抗体活性如下：Lec13衍生的2H7v31>Lec13衍生的2H7v16>Dp12衍生的2H7v31>Dp12衍生的2H7v16>或＝Rituxan。附加蛋白质与糖的改变。比较Lec13产生的与CHO产生的IgG中天然IgG上的糖，显示在半乳糖苷化程度上没有可明显察觉的差异，由此该结果可以完全归因于岩藻糖的存在或缺乏。The results showed that hypofucosylated antibodies mediate NK cell target cell killing more efficiently than fully fucosylated antibodies. The hypofucosylated antibody 2H7v.31 was the most effective at mediating target cell killing. This antibody is effective at lower concentrations and mediates a higher rate of target cell killing than other antibodies at higher concentrations. Antibody activity was as follows: Lec13-derived 2H7v31 > Lec13-derived 2H7v16 > Dp12-derived 2H7v31 > Dp12-derived 2H7v16 > or = Rituxan. Additional protein and sugar changes. Comparing the sugars on native IgG in Lecl3-generated versus CHO-generated IgG revealed no appreciable difference in the degree of galactosidation, so this result could be entirely attributed to the presence or absence of fucose.
实施例12Example 12
具有体内增强的ADCC的岩藻糖缺陷2H7变体抗体Fucose-deficient 2H7 variant antibodies with enhanced ADCC in vivo
本实施例描述了在表达人CD16[FcR III]和人CD20的小鼠中，Lec13产生的岩藻糖缺陷的人源化2H7变体包括v.16与v.31与在DP12中产生的正常的岩藻糖基化的同类抗体相比，体内ADCC活性。This example describes the fucose-deficient humanized 2H7 variants produced by Lec13 including v. In vivo ADCC activity compared to a fucosylated antibody of the same class.
huCD20Tg+ huCD16Tg+ mCD16-/-小鼠的产生Generation of huCD20Tg+ huCD16Tg+ mCD16−/− mice
人CD20转基因小鼠从人CD20 BAC DNA(Invitrogen，Carlsbad，CA)产生。基于人CD20表达的FACS分析筛选小鼠。HuCD20Tg+小鼠然后与huCD16Tg+mCD16-/-小鼠杂交以生成huCD20Tg+huCD16Tg+mCD16-/-小鼠。Human CD20 transgenic mice were generated from human CD20 BAC DNA (Invitrogen, Carlsbad, CA). Mice were screened based on FACS analysis of human CD20 expression. HuCD20Tg+ mice were then crossed with huCD16Tg+ mCD16−/− mice to generate huCD20Tg+ huCD16Tg+ mCD16−/− mice.
体内治疗In vivo treatment
经腹膜内注射将10至100μg的每一2H7变体或
小鼠淋巴细胞制备Mouse lymphocyte preparation
来自全血、脾、淋巴结、与骨髓的小鼠淋巴细胞根据在“Current Protocolsin Immunology，John Coligan、Ada Kruisbeek、David Margulies编辑，EthanShevach and Warren Strober，1994”中所描述的标准程序制备。Mouse lymphocytes from whole blood, spleen, lymph nodes, and bone marrow were prepared according to standard procedures described in "Current Protocols in Immunology, edited by John Coligan, Ada Kruisbeek, David Margulies, Ethan Shevach and Warren Strober, 1994".
FACS分析FACS analysis
洗50万个细胞，重悬在100μl的FACS缓冲液中，该缓冲液是含1％BSA的磷酸盐缓冲盐水，包含5μl的染色或对照抗体。所有染色抗体，包括同种型对照，均获自PharMingen，San Diego，CA。通过用
在注射后第一周每天、并在此后每周通过分析外周B细胞计数和通过对脾、淋巴结和骨髓进行FACS分析hCD20+B细胞，来评估B细胞削减与恢复。监测所注射的2H7变体抗体的血清水平。B cell depletion and recovery were assessed daily for the first week after injection and weekly thereafter by analysis of peripheral B cell counts and hCD20+ B cells by FACS analysis of spleen, lymph nodes and bone marrow. Serum levels of injected 2H7 variant antibodies are monitored.
这一体内试验的结果进一步证实了体外研究结果，即岩藻糖缺陷的2H7变体与野生型(在岩藻糖基化方面)糖基化同类抗体相比具有较高ADCC活性和较多的B细胞削减。The results of this in vivo assay further confirmed the in vitro findings that the fucose-deficient 2H7 variant had higher ADCC activity and more B cell reduction.
实施例13Example 13
细胞程序性死亡活性apoptosis activity
包括
利用第二交联抗体在体外细胞程序死亡试验中比较RhuMAb2H7.v16[人源化2H7v16；RhuMAb表示重组人单克隆抗体]与
当与抗人Fc交联和同无关IgG1对照抗体比较时，rhuMAb 2H7.v16和Rituximab两者都诱导Ramos细胞的细胞程序死亡(图13-15)。rhuMAb 2H7的细胞程序死亡活性稍低于Rituximab。在交联rhuMAb 2H7、Rituximab和对照IgG1抗体的浓度为10nM时，膜联蛋白V染色的细胞的比例分别是18.5％、16.5％、2.5％，双标记细胞的比例为29％、38％与16％，并且每10秒计数的活细胞数目为5200、3100与8600。Both rhuMAb 2H7.v16 and Rituximab induced apoptosis in Ramos cells when compared to anti-human Fc cross-linked and to an irrelevant IgGl control antibody (Figures 13-15). The apoptosis activity of rhuMAb 2H7 was slightly lower than that of Rituximab. When the concentration of cross-linked rhuMAb 2H7, Rituximab and control IgG1 antibody was 10 nM, the proportion of Annexin V-stained cells was 18.5%, 16.5%, 2.5%, and the proportion of double-labeled cells was 29%, 38% and 16%, respectively. %, and the number of viable cells counted per 10 seconds was 5200, 3100 and 8600.
这些体外数据显示，细胞程序死亡是体内B细胞削减的一个可能的机制。与细胞表面CD20结合的rhuMAb 2H7或Rituximab的体内交联可能通过免疫效应细胞表面上的FcγR发生。These in vitro data suggest that apoptosis is a possible mechanism of B cell depletion in vivo. In vivo crosslinking of rhuMAb 2H7 or Rituximab bound to cell surface CD20 likely occurs via FcγRs on the surface of immune effector cells.
实施例14Example 14
肿瘤生长的体内抑制In vivo inhibition of tumor growth
在Balb/c裸(无胸腺)小鼠中评估rhuMAb 2H7.v16抑制淋巴瘤细胞系Raji人B细胞(ATCC CCL86)生长的能力。Raji细胞表达CD20，并已报道可在裸鼠中生长，产生转移性疾病；肿瘤生长被
这一研究的结果表明rhuMAb 2H7.v16与
实施例15Example 15
猕猴CD20的克隆与抗体结合Cloning and antibody binding of macaque CD20
从猕猴脾cDNA文库分离编码CD20的cDNA之后，测定猕猴(Macacafascicularis)CD20 DNA序列。cDNA合成与质粒克隆的SUPERSCRIPTTM质粒系统(目录号18248-013，Invitrogen，Carlsbad，CA)稍加改变后被用于构建文库。使用限制性位点XhoI和NotI将cDNA文库连接入pRK5E载体。从脾组织分离mRNA((California Regional Research Primate Center，Davis，CA)。基于人CD20的非编码序列设计用于扩增编码CD20的cDNA的引物。N末端区域引物5′-AGTTTTGAGAGCAATG-3′(SEQ ID NO.37)与C末端区域引物5′-AAGCTATGAACACTAATG-3′(SEQ ID NO.38)被用来通过聚合酶链式反应(PCR)克隆编码猕猴CD20的cDNA。使用Platinum Taq DNAPolymerase High Fidelity根据厂家建议(Gibco，Rockville，MD)进行PCR反应。PCR产物亚克隆入
猕猴CD20的氨基酸序列示于图19。图20显示猕猴与人CD20的比较。猕猴CD20与人CD20具有97.3％的相似性，有8处差异。细胞外结构域在V157A包含一个改变，而其余7个残基可在细胞质或跨膜区域找到。The amino acid sequence of macaque CD20 is shown in FIG. 19 . Figure 20 shows a comparison of macaque and human CD20. Cynomolgus CD20 has 97.3% similarity with human CD20, with 8 differences. The extracellular domain contains one change at V157A, while the remaining seven residues can be found in the cytoplasmic or transmembrane regions.
分析针对抗人CD20的抗体结合并替代FITC偶联的鼠2H7结合表达CD20的猕猴细胞的能力。从两只猕猴(California Regional Research PrimateCenter，Davis，CA)取血20毫升加入肝素钠，并直接运至健泰科公司。在同一天，汇集血样，通过添加40ml的磷酸盐缓冲盐溶液(PBS)1:1稀释。20ml的稀释血液分层铺于50ml锥形管(目录号352098，Falcon，Franklin Lakes，NJ)中的4 x 20ml Ficoll-PaqueTMPlus(Amersham Biosciences，Uppsala，Sweden)，在Sorval 7离心机(Dupont，Newtown，CT)上以1300rpm室温离心30分钟。分离出PBMC层，在PBS中洗。红细胞在0.2％NaCl溶液中裂解，用等体积的1.6％NaCl溶液恢复至等渗，以1000RPM离心10分钟。PBMC小丸再悬浮在包含5％胎牛血清(FBS)的RPMI1640(Gibco，Rockville，MD)中，在37℃分散入10cm组织培养皿1小时。非粘附的B和T细胞群体用吸出除去，离心并计数。回收得到总共2.4 x 107细胞。重新悬浮的PBMC分配入20个12 x 75mm培养管(目录号352053，Falcon)，每管包含1 x 106细胞，体积0.25ml。管子被分成四组，每组五管。每组加入培养基(RPMI1640，5％FBS)、滴定量的对照人IgG1抗体、
图21中的数据显示，2H7 v.16与2H7 v.31竞争性替代FITC-鼠2H7与猕猴细胞的结合。此外，
实施例16Example 16
rhuMAb 2H7(2H7.v16)在中度至重度类风湿性关节炎中的I/II期研究A Phase I/II Study of rhuMAb 2H7 (2H7.v16) in Moderate to Severe Rheumatoid Arthritis
过程提要process summary
在患中度至重度类风湿性关节炎的个体中，所述个体同时接受稳定剂量的氨甲喋呤，对逐渐升高剂量的PRO70769(rhuMAb 2H7)的安全性进行随机的、安慰剂对照的、多中心(multicenter)、盲的I/II期研究。A randomized, placebo-controlled, multicenter study of the safety of escalating doses of PRO70769 (rhuMAb 2H7) in individuals with moderate to severe rheumatoid arthritis receiving a stable dose of methotrexate. (multicenter), blinded Phase I/II study.
目的Purpose
本研究的主要目的是评定在患中度至重度类风湿性关节炎(RA)个体中逐渐增高的静脉内(IV)剂量的PRO70769(rhuMAb 2H7)的安全性与耐受性。The primary objective of this study was to assess the safety and tolerability of escalating intravenous (IV) doses of PRO70769 (rhuMAb 2H7) in individuals with moderate to severe rheumatoid arthritis (RA).
研究设计Research design
这是一项随机的、安慰剂对照的、多中心的、盲的、I/II期、研究者与受试者双盲研究，研究逐渐增高剂量的PRO70769与MTX联合在中度至重度RA个体中的安全性。研究包括剂量逐渐增高期和第二期，其中第二期中有更多数量的个体加入。主办者对治疗方案保持不参与。This is a randomized, placebo-controlled, multicenter, blinded, phase I/II, investigator-subject double-blind study of escalating doses of PRO70769 in combination with MTX in individuals with moderate to severe RA security in . The study consisted of a dose escalation phase and a second phase in which a larger number of subjects were enrolled. The sponsor remained out of the treatment protocol.
可以加入治疗的受试者：患中度至重度RA，遭受一种至五种具有减轻症状作用的抗风湿药或生物制剂治疗失败，目前用MTX治疗的临床反应不佳。Subjects eligible for treatment: Moderate to severe RA, failure of one to five symptom-modifying antirheumatic drugs or biologics, current poor clinical response to MTX therapy.
在研究开始之前，受试者要每周接受10-25mg范围内的MTX至少12周，并接受稳定剂量至少4周，然后接受其首次量的研究药物(PRO70769或安慰剂)。受试者也可以接受稳定剂量的口服皮质激素(最多达每天10mg或等量强的松)和稳定剂量的非类固醇类抗炎药(NSAID)。根据下面的剂量渐增计划(参见图22)，受试者将在第1与第15天以指定剂量接受PRO70769或安慰剂的两次静脉内输注。Subjects received MTX in the range of 10-25mg weekly for at least 12 weeks prior to study initiation, and received a stable dose for at least 4 weeks before receiving their first dose of study drug (PRO70769 or placebo). Subjects also received stable doses of oral corticosteroids (up to 10 mg per day or equivalent prednisone) and stable doses of nonsteroidal anti-inflammatory drugs (NSAIDs). Subjects will receive two intravenous infusions of PRO70769 or placebo at the indicated doses on
根据特定标准，并且在由内部安全数据检查委员会对安全性数据进行检查之后，并且在每一组的最后一个受试者接受第二次输注后72小时对急性毒性进行了评估之后，进行剂量升级。剂量升级期后，另外40个受试者(32个接受药物的和8个接受安慰剂的)将被随机分配入以下剂量水平的每一剂量：2 x 50mg、2 x 200mg、2 x 500mg、和2 x 1000mg，如果该剂量水平在剂量升级期内已经被证实是可耐受的。大约205个受试者将加入该研究。Dosing was administered according to specified criteria and after review of safety data by the internal Safety Data Review Committee and after assessment of acute toxicity 72 hours after the last subject in each arm received the second infusion upgrade. Following the dose escalation period, an additional 40 subjects (32 drug-receiving and 8 placebo-receiving) will be randomly assigned to each of the following dose levels: 2 x 50mg, 2 x 200mg, 2 x 500mg, and 2 x 1000 mg, if this dose level has been shown to be tolerated during the dose escalation period. Approximately 205 subjects will join the study.
获得B细胞计数并登记。在为时6个月的效力估价期之外的48周追踪期内，利用流式细胞计评价B细胞计数。B细胞削减将不会被认为是剂量限制的毒性(dose-limiting toxicity，DLC)，而是PRO70769治疗的预期药效学结果。Obtain B cell counts and register. B cell counts were assessed by flow cytometry during a 48-week follow-up period beyond the 6-month efficacy assessment period. B-cell depletion will not be considered a dose-limiting toxicity (DLC), but rather an expected pharmacodynamic consequence of PRO70769 treatment.
在一可选的亚研究中，将在各个时间点从受试者获得血清血和RNA分析、以及尿样。这些样品可被用来鉴定生物标志物，这些生物标志物可能对患中度至重度RA的受试者对PRO70769治疗响应性有预示作用。In an optional substudy, serum blood and RNA analysis, and urine samples will be obtained from subjects at various time points. These samples can be used to identify biomarkers that may be predictive of responsiveness to PRO70769 treatment in subjects with moderate to severe RA.
结果的测量measurement of results
本研究的主要结果测量是患中度至重度RA的受试者中PRO70769的安全性与耐受性。The primary outcome measure of this study was the safety and tolerability of PRO70769 in subjects with moderate-to-severe RA.
研究治疗study treatment
受试者群体将根据以下升级计划在第1与第15天以指定剂量接受PRO70769或等量安慰剂的两次静脉内输注：Subject cohorts will receive two intravenous infusions of PRO70769 or an equivalent amount of placebo on
-10mg PRO70769或等量安慰剂：4个受试者接受活性药物，1个接受对照- 10mg PRO70769 or equivalent placebo: 4 subjects received active drug and 1 received control
-50mg PRO70769或等量安慰剂：8个受试者接受活性药物，2个接受对照- 50mg PRO70769 or equivalent placebo: 8 subjects received active drug and 2 received control
-200mg PRO70769或等量安慰剂：8个受试者接受活性药物，2个接受对照- 200mg PRO70769 or equivalent placebo: 8 subjects received active drug and 2 received control
-500mg PRO70769或等量安慰剂：8个受试者接受活性药物，2个接受对照- 500mg PRO70769 or equivalent placebo: 8 subjects received active drug and 2 received control
-1000mg PRO70769或等量安慰剂：8个受试者接受活性药物，2个接受对照- 1000mg PRO70769 or equivalent placebo: 8 subjects received active drug and 2 received control
效力effectiveness
PRO70769的效力将通过ACR反应测量。取得ACR20、ACR50、和ACR70反应的受试者百分率将通过处理组汇总，每一组生成95％置信区间。这些反应的组分以及其从基线的变化将通过治疗与访问加以汇总。The potency of PRO70769 will be measured by ACR response. The percentages of subjects achieving ACR20, ACR50, and ACR70 responses will be pooled by treatment group, generating 95% confidence intervals for each group. Components of these responses and their changes from baseline will be summarized by treatment and visit.
结论in conclusion
上面的数据证明制备人源化CD20结合抗体，特别是人源化2H7抗体变体的成功，所述抗体保持、甚至增强其生物学特性。本发明的人源化2H7抗体以与小鼠供体和嵌合2H7抗体类似的亲合力与CD20结合，在灵长类动物的B细胞杀伤中是有效的，导致B细胞削减。某些变体与目前用于治疗NHL的嵌合抗CD20抗体相比显示出增强的ADCC，有利于在患者中使用较低剂量的治疗抗体。此外，尽管对带有鼠FR残基的嵌合抗体而言，需要以足以取得完全B细胞削减的剂量给药以消除针对其的抗体反应，本发明人源化抗体可以以取得部分或完全B细胞削减的剂量给药，并且依该特定疾病和病人所需按不同持续时间给药。此外，这些抗体显示出在溶液中的稳定性。人源化2H7抗体的这些性质使得它们在CD20阳性癌症和自身免疫疾病中可用作理想的免疫治疗药剂；在人类患者中这些抗体预期是不具免疫原性的，或将至少比完全的鼠或嵌合抗CD20抗体免疫原性小。The above data demonstrate the success of generating humanized CD20-binding antibodies, in particular humanized 2H7 antibody variants, which maintain, or even enhance, their biological properties. The humanized 2H7 antibody of the invention binds to CD20 with similar affinity to the mouse donor and the chimeric 2H7 antibody and is effective in B cell killing in primates, resulting in B cell depletion. Certain variants showed enhanced ADCC compared to chimeric anti-CD20 antibodies currently used to treat NHL, facilitating the use of lower doses of therapeutic antibody in patients. In addition, although chimeric antibodies with murine FR residues need to be administered at doses sufficient to achieve complete B cell depletion to eliminate antibody responses against them, the humanized antibodies of the present invention can be obtained in a manner that achieves partial or complete B cell depletion. Cell-reduced doses are administered and are administered for varying durations as required by the particular disease and patient. Furthermore, these antibodies showed stability in solution. These properties of humanized 2H7 antibodies make them ideal immunotherapeutic agents in CD20-positive cancers and autoimmune diseases; these antibodies are expected to be non-immunogenic in human patients, or to be at least less immunogenic than fully murine or The chimeric anti-CD20 antibody is less immunogenic.
参考文献references
在本申请中所引用的参考文献，包括专利、公开的申请及其它出版物，在此引入作为参考。References cited throughout this application, including patents, published applications, and other publications, are hereby incorporated by reference.
除非另外指明，本发明的实施将使用本领域范围内的分子生物学的传统方法等等。这样的技术在文献中充分加以解释。参见，例如：The practice of the present invention will employ, unless otherwise indicated, conventional methods of molecular biology and the like, within the skill of the art. Such techniques are explained fully in the literature. See, for example:
Molecular Cloning：A Laboratory Manual，(J.Sambrook et al.，Cold SpringHarbor Laboratory，Cold Spring Harbor，N.Y.，1989)；Current Protocols inMolecular Biology(F.Ausubel et al.，eds.，1987 updated)；Essential MolecularBiology(T.Brown ed.，IRL Press 1991)；Gene Expression Technology(Goeddeled.，Academic Press 1991)；Methods for Cloning and Analysis of EukaryoticGenes(A.Bothwell et al.eds.，Bartlett Publ.1990)；Gene Transfer andExpression(M.Kriegler，Stockton Press 1990)；Recombinant DNA MethodologyII(R.Wu et al.eds.，Academic Press 1995)；PCR：A Practical Approach(M.McPherson et al.，IRL Press at Oxford University Press 1991)；OligonucleotideSynthesis(M.Gait ed.，1984)；Cell Culture for Biochemists(R.Adams ed.，Elsevier Science Publishers 1990)；Gene Transfer Vectors for Mammalian Cells(J.Miller & M.Calos eds.，1987)；Mammalian Cell Biotechnology(M.Butlered.，1991)；Animal Cell Culture(J.Pollard et al.eds.，Humana Press 1990)；Culture of Animal Cells，2nd Ed.(R.Freshney et al.eds.，Alan R.Liss 1987)；Flow Cytometry and Sorting(M.Melamed et al.eds.，Wiley-Liss 1990)；theseries Methods in Enzymology(Academic Press，Inc.)；Wirth M.and Hauser H.(1993)；Immunochemistry in Practice，3rd edition，A.Johnstone & R.Thorpe，Blackwell Science，Cambridge，MA，1996；Techniaues in Immunocytochemistry，(G.Bullock & P.Petrusz eds.，Academic Press 1982，1983，1985，1989)；Handbook of Experimental Imrmunology，(D.Weir & C.Blackwell，eds.)；CurrentProtocols in Immunology(J.Coligan et al.eds.1991)；Immunoassay(E.P.Diamandis & T.K.Christopoulos，eds.，Academic Press，Inc.，1996)；Goding(1986)Monoclonal Antibodies：Principles and Practice(2d ed)Academic Press，New York；Ed Harlow and David Lane，Antibodies A laboratory Manual，ColdSpring Harbor Laboratory，Cold Spring Harbor，New York，1988；AntibodyEngineering，2nd edition(C.Borrebaeck，ed.，Oxford University Press，1995)；和the series Annual Review of Immunology；the series Advances in Immunology。Molecular Cloning: A Laboratory Manual , (J. Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989);Current Protocols inMolecular Biology (F. Ausubel et al., eds., 1987 updated);Essential MolecularBiology (T. Brown ed., IRL Press 1991);Gene Expression Technology (Goeddeled., Academic Press 1991);Methods for Cloning and Analysis of EukaryoticGenes (A. Bothwell et al. eds., Bartlett Publ. 1990);Gene Transfer andExpression (M.Kriegler, Stockton Press 1990);Recombinant DNA MethodologyII (R.Wu et al.eds., Academic Press 1995); PCR:A Practical Approach (M.McPherson et al., IRL Press at Oxford University Press 1991);OligonucleotideSynthesis (M.Gait ed., 1984);Cell Culture for Biochemists (R.Adams ed., Elsevier Science Publishers 1990);Gene Transfer Vectors for Mammalian Cells (J.Miller & M.Calos eds., 1987);Mammalian Cell Biotechnology (M.Butlered., 1991);Animal Cell Culture (J.Pollard et al.eds., Humana Press 1990);Culture of Animal Cells ,2nd Ed. (R.Freshney et al.eds ., Alan R. .Liss 1987);Flow Cytometry and Sorting (M.Melamed et al.eds., Wiley-Liss 1990); theseseriesMethods in Enzymology (Academic Press, Inc.); Wirth M.and Hauser H.(1993);Immunochemistry in Practice , 3rd edition, A.Johnstone & R.Thorpe, Blackwell Science, Cambridge, MA, 1996;Techniaues in Immunocytochemistry , (G.Bullock & P.Petrusz eds., Academic Press 1982, 1983, 1985, 1989); Handbook of Experimental Imrmunology, (D. Weir & C. Blackwell, eds.);CurrentProtocols in Immunology (J. Coligan et al. eds. 1991);Immunoassay (EP Diamandis & TK Christopoulos, eds., Academic Press, Inc., 1996); Goding(1986)Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; Ed Harlow and David Lane,Antibodies A laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988;AntibodyEngineering , 2nd edition (C. Borrebaeck, ed., Oxford University Press, 1995); and the series Annual Review of Immunology; the series Advances in Immunology.
序列表sequence listing
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<213>人工序列<213> Artificial sequence
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<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
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<211>213<211>213
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>45<400>45
<210>46<210>46
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<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>46<400>46
<210>47<210>47
<211>213<211>213
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>47<400>47
<210>48<210>48
<211>452<211>452
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>48<400>48
<210>49<210>49
<211>452<211>452
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>49<400>49
<210>50<210>50
<211>452<211>452
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>50<400>50
<210>51<210>51
<211>122<211>122
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>51<400>51
<210>52<210>52
<211>107<211>107
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>52<400>52
<210>53<210>53
<211>107<211>107
<212>PRT<212>PRT
<213>人工序列<213> Artificial sequence
<220><220>
<223>该序列是人工合成的<223> The sequence is artificially synthesized
<400>53<400>53
Claims (67)
Translated fromChinesePriority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410056459.3ACN103833854B (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and application thereof |
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| US43411502P | 2002-12-16 | 2002-12-16 | |
| US60/434,115 | 2002-12-16 | ||
| US60/526,163 | 2003-12-01 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2008101748156ADivisionCN101418043A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CN200810174816.0ADivisionCN101418044B (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CN201410056459.3ADivisionCN103833854B (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and application thereof |
| CNA2008101748175ADivisionCN101418045A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
Publications (2)
| Publication Number | Publication Date |
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| CN1747969A CN1747969A (en) | 2006-03-15 |
| CN100460421Ctrue CN100460421C (en) | 2009-02-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003801063031APendingCN1751236A (en) | 2002-12-16 | 2003-12-11 | Transgenic mice expressing human CD20 |
| CNA2003801096904APendingCN1748143A (en) | 2002-12-16 | 2003-12-11 | Transgenic mice expressing human CD20 and/or CD16 |
| CNA2008101748156APendingCN101418043A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CNA2008101748175APendingCN101418045A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CN200810174816.0AExpired - LifetimeCN101418044B (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CNB200380109682XAExpired - LifetimeCN100460421C (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
Family Applications Before (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003801063031APendingCN1751236A (en) | 2002-12-16 | 2003-12-11 | Transgenic mice expressing human CD20 |
| CNA2003801096904APendingCN1748143A (en) | 2002-12-16 | 2003-12-11 | Transgenic mice expressing human CD20 and/or CD16 |
| CNA2008101748156APendingCN101418043A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CNA2008101748175APendingCN101418045A (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
| CN200810174816.0AExpired - LifetimeCN101418044B (en) | 2002-12-16 | 2003-12-16 | Immunoglobulin variants and uses thereof |
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| CN (6) | CN1751236A (en) |
| CA (1) | CA2835591A1 (en) |
| CL (1) | CL2003002639A1 (en) |
| NO (1) | NO338313B1 (en) |
| TN (1) | TNSN06133A1 (en) |
| ZA (1) | ZA200504221B (en) |
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| CN103936858A (en)* | 2014-03-27 | 2014-07-23 | 安徽大学 | Antibody L5H6 for resisting CD20 antigen and application thereof |
| CN103936855A (en)* | 2014-03-27 | 2014-07-23 | 合肥泰瑞生物技术有限公司 | Antibody L4H5 with CD20-resistant antigen and application thereof |
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| NO346624B1 (en) | 2004-12-28 | 2022-11-07 | Univ Di Genova | Monoclonal antibody against NKG2A |
| US8206709B2 (en) | 2006-06-30 | 2012-06-26 | Novo Nordisk A/S | Anti-NKG2A antibodies and uses thereof |
| WO2009092805A1 (en)* | 2008-01-24 | 2009-07-30 | Novo Nordisk A/S | Humanized anti-human nkg2a monoclonal antibody |
| JP2012517806A (en)* | 2009-02-16 | 2012-08-09 | バイオレックス・セラピューティクス インコーポレイテッド | Humanized anti-CD20 antibodies and methods of use |
| EP4502152A3 (en) | 2009-10-06 | 2025-04-30 | Regeneron Pharmaceuticals, Inc. | Genetically modified mice and engraftment |
| CN102190728B (en) | 2010-03-17 | 2014-07-02 | 永卓博济(上海)生物医药技术有限公司 | Novel humanized anti-CD20 monoclonal antibody |
| PT4233537T (en)* | 2011-02-15 | 2025-09-08 | Regeneron Pharmaceuticals Inc | Humanized m-csf mice and uses thereof |
| WO2012172102A1 (en) | 2011-06-17 | 2012-12-20 | Novo Nordisk A/S | Selective elimination of erosive cells |
| JP6475017B2 (en)* | 2011-10-27 | 2019-02-27 | ゲンマブ エー／エス | Production of heterodimeric protein |
| WO2013085893A1 (en) | 2011-12-05 | 2013-06-13 | Immunomedics, Inc. | Therapeutic use of anti-cd22 antibodies for inducing trogocytosis |
| US8883979B2 (en)* | 2012-08-31 | 2014-11-11 | Bayer Healthcare Llc | Anti-prolactin receptor antibody formulations |
| CN103880958B (en)* | 2014-03-27 | 2016-01-20 | 安徽大学 | Antibody L4H6 for resisting CD20 antigen and application thereof |
| CN103880957B (en)* | 2014-03-27 | 2016-01-20 | 安徽大学 | Antibody L1H1 for resisting CD20 antigen and application thereof |
| CN103897059B (en)* | 2014-03-27 | 2016-03-23 | 中国人民解放军军事医学科学院生物工程研究所 | Anti-CD20 Antigen Antibody L5H7 and Its Application |
| US10123518B2 (en) | 2015-04-13 | 2018-11-13 | Regeneron Pharmaceuticals, Inc | Genetically modified non-human animals and methods of use thereof |
| US20190183101A1 (en)* | 2016-08-11 | 2019-06-20 | The Jackson Laboratory | Methods and compositions relating to improved human red blood cell survival in genetically modified immunodeficient non-human animals |
| CN106872713A (en)* | 2016-12-27 | 2017-06-20 | 许洋 | A kind of immunomic mass spectrometry kit and preparation method of trace protein in situ detection |
| CN108531561A (en)* | 2017-03-01 | 2018-09-14 | 云南序源生物技术开发有限公司 | A kind of quick detection is used to identify the kit and method in the SNP features site of Y chromosome haplotype pedigree |
| CN111705084B (en)* | 2020-08-18 | 2020-12-08 | 江苏集萃药康生物科技有限公司 | Construction method of mouse B cell lymphoma cell line stably expressing luciferase and knocking out mouse CD20 from human CD20 |
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| CN118222630B (en)* | 2024-05-23 | 2024-08-27 | 江西赛基生物技术有限公司 | Construction method and application of cell line for stably expressing CD16 transmembrane protein |
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- 2003-12-16CACA2835591Apatent/CA2835591A1/ennot_activeAbandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103936858A (en)* | 2014-03-27 | 2014-07-23 | 安徽大学 | Antibody L5H6 for resisting CD20 antigen and application thereof |
| CN103936855A (en)* | 2014-03-27 | 2014-07-23 | 合肥泰瑞生物技术有限公司 | Antibody L4H5 with CD20-resistant antigen and application thereof |
| CN103936858B (en)* | 2014-03-27 | 2016-05-04 | 安徽大学 | Antibody L5H6 for resisting CD20 antigen and application thereof |
| CN103936855B (en)* | 2014-03-27 | 2016-05-04 | 合肥泰瑞生物技术有限公司 | Antibody L4H5 and the application thereof of anti-CD20 antigen |
Also Published As
| Publication number | Publication date |
|---|---|
| NO20150245L (en) | 2005-08-31 |
| TNSN06133A1 (en) | 2007-11-15 |
| CN1751236A (en) | 2006-03-22 |
| CN101418044A (en) | 2009-04-29 |
| CN101418043A (en) | 2009-04-29 |
| CN1748143A (en) | 2006-03-15 |
| NO338313B1 (en) | 2016-08-08 |
| CN101418045A (en) | 2009-04-29 |
| CN101418044B (en) | 2014-03-12 |
| CA2835591A1 (en) | 2004-07-08 |
| CL2003002639A1 (en) | 2005-04-08 |
| CN1747969A (en) | 2006-03-15 |
| ZA200504221B (en) | 2006-08-30 |
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