本揭示內容是關於治療疾病的領域。更具體來說，本揭示內容是關於一種新穎的抗體及其於治療癌症的用途。The present disclosure relates to the field of treating diseases. More specifically, the present disclosure relates to a novel antibody and its use in treating cancer.
癌症是全球主要死亡原因之一。依據國際癌症研究機構(International Agency for Research on Cancer, IARC)的統計資料，2020年全球估計約有1,000萬人死於癌症，並新增約1,930萬癌症病例。儘管全球致力於研發新的癌症療法並改善現有治療手段的功效，癌症的死亡率仍相當高。雖然目前已有諸如化學治療、手術、放射治療、荷爾蒙治療、生物治療、標靶治療及最新的免疫療法與細胞治療等不同類型的治療方法，然而癌症仍然帶來重大的健康風險並造成經濟負擔。Cancer is one of the leading causes of death worldwide. According to statistics from the International Agency for Research on Cancer (IARC), an estimated 10 million people will die from cancer in 2020, with approximately 19.3 million new cancer cases. Despite global efforts to develop new cancer treatments and improve the efficacy of existing treatments, the mortality rate from cancer remains high. Although there are currently different types of treatments such as chemotherapy, surgery, radiotherapy, hormone therapy, biological therapy, targeted therapy, and the latest immunotherapy and cell therapy, cancer still poses a significant health risk and causes an economic burden.
腫瘤生長仰賴於其周遭複雜的組織環境，該環境除了維持腫瘤細胞的生長之外，亦透過血管新生提供腫瘤細胞充足的血液供應。另一方面，腫瘤的生長及轉移也取決於其逃避宿主免疫監控及克服宿主防禦的能力。已知多數腫瘤皆會表現抗原(亦稱為「腫瘤相關抗原」(tumor-associated antigen, TAA))，基於免疫原性較弱，宿主免疫系統可不同程度地辨識該些TAA。然而，在大多數的情況下，由於癌細胞衍生的脫逃機制阻斷了宿主免疫系統的作用，腫瘤引發的免疫反應往往不足以抑制腫瘤生長，其中所述逃脫機制包含增加程式性細胞死亡配體-1 (programmed cell death ligand-1, PD-L1)的表現以調節免疫系統、釋放免疫抑制因子，以及吸引免疫抑制細胞至腫瘤環境。Tumor growth depends on the complex tissue environment around it, which not only maintains the growth of tumor cells, but also provides tumor cells with sufficient blood supply through angiogenesis. On the other hand, the growth and metastasis of tumors also depend on their ability to evade host immune surveillance and overcome host defense. It is known that most tumors express antigens (also known as tumor-associated antigens (TAAs)). Due to their weak immunogenicity, the host immune system can recognize these TAAs to varying degrees. However, in most cases, the tumor-induced immune response is often insufficient to inhibit tumor growth due to cancer cell-derived escape mechanisms that block the action of the host immune system, including increasing the expression of programmed cell death ligand-1 (PD-L1) to modulate the immune system, releasing immunosuppressive factors, and attracting immunosuppressive cells to the tumor environment.
B7-H3亦稱為CD276，是一種由胞外域、單一跨膜域及短片段胞內域所組成的第I型跨膜蛋白。B7-H3屬於B7家族中的一員，其中B7家族為具有類免疫球蛋白V域及類免疫球蛋白C域(例如，IgV-IgC)的免疫球蛋白(immunoglobulin, Ig)超家族。大多數的人類B7-H3含有兩個胞外串聯IgV-IgC域(4Ig-B7-H3)。小鼠及大鼠的B7-H3目前僅知具有2個Ig域(IgV-IgC；2Ig-B7-H3)，並具有與人類4Ig-B7-H3相似的功能。B7-H3, also known as CD276, is a type I transmembrane protein composed of an extracellular domain, a single transmembrane domain, and a short intracellular domain. B7-H3 is a member of the B7 family, which is an immunoglobulin (Ig) superfamily with immunoglobulin V-like domains and immunoglobulin C-like domains (e.g., IgV-IgC). Most human B7-H3 contains two extracellular tandem IgV-IgC domains (4Ig-B7-H3). Mouse and rat B7-H3 is currently known to have only two Ig domains (IgV-IgC; 2Ig-B7-H3) and has similar functions to human 4Ig-B7-H3.
已知B7-H3會過量表現於不同類型的癌症，包含非小細胞肺癌、咽下癌、口腔癌、腎臟癌、尿路上皮癌、結腸直腸癌、前列腺癌、多形性神經膠質母細胞瘤、卵巢癌及胰臟癌。特別是，在前列腺癌中，B7-H3的表現量會與癌症的惡性程度與進展級數呈正相關性。相似地，頭頸部癌的病患若具有高表現量的B7-H3，其存活率通常較低；而在胰臟癌及卵巢癌中，B7-H3的表現量則與淋巴結轉移及病理進展級數相關。此外，在B7-H3陽性的癌細胞株中，投予抑制B7-H3的小干擾RNA (small interfering RNA, siRNA)可降低癌細胞的轉移及侵犯能力。因此，B7-H3可作用一種潛力型藥物標的，據以研發治療癌症的治療型抗體或抗體藥物偶聯物(antibody drug conjugate, ADC)。B7-H3 is known to be overexpressed in different types of cancer, including non-small cell lung cancer, hypopharyngeal cancer, oral cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. In particular, in prostate cancer, the expression of B7-H3 is positively correlated with the malignancy and progression of the cancer. Similarly, patients with head and neck cancer who have high expression of B7-H3 generally have a lower survival rate, while in pancreatic cancer and ovarian cancer, the expression of B7-H3 is associated with lymph node metastasis and the progression of pathology. In addition, in B7-H3-positive cancer cell lines, administration of small interfering RNA (siRNA) that inhibits B7-H3 can reduce the metastasis and invasion ability of cancer cells. Therefore, B7-H3 can act as a potential drug target for the development of therapeutic antibodies or antibody drug conjugates (ADCs) for the treatment of cancer.
醫藥領域已研發數種抗-B7-H3抗體；然而，該些抗體對病患的安全性及有效性仍有待臨床試驗確認。此外，多數抗-B7-H3抗體為小鼠抗體或人源化抗體，通常具有高免疫原性而會對人類個體產生不利的影響。有鑑於此，相關領域亟需一種對B7-H3具有結合專一性的新穎抗體，以更有效且安全地治療癌症。Several anti-B7-H3 antibodies have been developed in the pharmaceutical field; however, the safety and efficacy of these antibodies for patients still need to be confirmed in clinical trials. In addition, most anti-B7-H3 antibodies are mouse antibodies or humanized antibodies, which are usually highly immunogenic and can have adverse effects on human individuals. In view of this, the relevant field is in urgent need of a novel antibody with binding specificity to B7-H3 to treat cancer more effectively and safely.
發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件或界定本發明的範圍。The content of the invention is intended to provide a simplified summary of the present disclosure so that readers can have a basic understanding of the present disclosure. This content of the invention is not a complete overview of the present disclosure, and it is not intended to point out the important/key elements of the embodiments of the present invention or to define the scope of the present invention.
本揭示內容的第一態樣是關於一種B7-H3靶向(B7-H3 targeting)的重組抗體(即，一種對B7-H3具有結合親和力及專一性的重組抗體，可靶向B7-H3)或是其片段(例如，單鏈變異片段(single-chain variable fragment, scFv))。結構上，本發明B7-H3靶向的重組抗體或抗體片段包含重鏈變異(variable heavy chain, VH)域及輕鏈變異(light chain variable, VL)域，其中VH域包含第一重鏈互補決定區(complementarity determining region, CDR) (CDR-H1)、第二重鏈CDR (CDR-H2)及第三重鏈CDR (CDR-H3)，且VL域包含第一輕鏈CDR (CDR-L1)、第二輕鏈CDR (CDR-L2)及第三輕鏈CDR (CDR-L3)。The first aspect of the present disclosure is about a B7-H3 targeting recombinant antibody (i.e., a recombinant antibody having binding affinity and specificity for B7-H3, which can target B7-H3) or a fragment thereof (e.g., a single-chain variable fragment (scFv)). Structurally, the B7-H3-targeted recombinant antibody or antibody fragment of the present invention comprises a variable heavy chain (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a first heavy chain complementarity determining region (CDR) (CDR-H1), a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3), and the VL domain comprises a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2) and a third light chain CDR (CDR-L3).
依據本揭示內容某些實施方式，所述CDR-H1、CDR-H2及CDR-H3分別包含序列編號：1、2及3的胺基酸序列，且所述CDR-L1、CDR-L2及CDR-L3分別包含序列編號：4、5及6的胺基酸序列。According to certain embodiments of the present disclosure, the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of sequence numbers: 1, 2 and 3, respectively, and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of sequence numbers: 4, 5 and 6, respectively.
依據本揭示內容某些實施方式，B7-H3靶向之重組抗體或抗體片段的VH及VL域分別包含與序列編號：7及8具有至少85%序列相似度的胺基酸序列；較佳地，分別包含與序列編號：7及8具有至少90%序列相似度的胺基酸序列；更佳地，分別包含與序列編號：7及8具有至少95%序列相似度的胺基酸序列。在本揭示內容一例示性實施方式中，B7-H3靶向之重組抗體或其片段的VH及VL域分別包含序列編號：7及8的胺基酸序列(即，分別包含與序列編號：7及8具有100%序列相似度的胺基酸序列)。According to certain embodiments of the present disclosure, the VH and VL domains of the B7-H3-targeted recombinant antibody or antibody fragment respectively comprise an amino acid sequence having at least 85% sequence similarity to sequence numbers: 7 and 8; preferably, they respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 7 and 8; more preferably, they respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 7 and 8. In an exemplary embodiment of the present disclosure, the VH and VL domains of the B7-H3-targeted recombinant antibody or fragment thereof respectively comprise the amino acid sequences of sequence numbers: 7 and 8 (i.e., they respectively comprise amino acid sequences having 100% sequence similarity to sequence numbers: 7 and 8).
本揭示內容的第二態樣是關於本發明B7-H3靶向的重組抗體或其片段於製備免疫偶聯物以治療個體之癌症的用途。依據本揭示內容的實施方式，所述免疫偶聯物包含本發明B7-H3靶向的重組抗體或抗體片段、治療劑，以及用以將治療劑連接至B7-H3靶向的重組抗體或抗體片段的連接子。依據實施目的之不同，治療劑可以是細胞毒性藥物(cytotoxic drug)、放射性核種(radioactive nuclide)、細胞激素(cytokine)、荷爾蒙藥物(hormone drug)、免疫刺激劑(immune stimulating agent)或免疫治療藥物(immunotherapeutic drug)。The second aspect of the present disclosure is about the use of the B7-H3-targeted recombinant antibody or its fragment in the preparation of an immunoconjugate for treating cancer in an individual. According to the implementation mode of the present disclosure, the immunoconjugate comprises the B7-H3-targeted recombinant antibody or antibody fragment of the present invention, a therapeutic agent, and a linker for linking the therapeutic agent to the B7-H3-targeted recombinant antibody or antibody fragment. Depending on the purpose of implementation, the therapeutic agent can be a cytotoxic drug, a radioactive nuclide, a cytokine, a hormone drug, an immune stimulating agent, or an immunotherapeutic drug.
依據本揭示內容某些較佳的實施方式，治療劑是細胞毒性藥物，舉例來說，澳瑞他汀(auristatin)或其衍生物。在一例示性實施方式中，治療劑是單甲基澳瑞他汀E (monomethyl auristatin E, MMAE)。在另一實施方式中，治療劑是單甲基澳瑞他汀F (monomethyl auristatin F, MMAF)。According to certain preferred embodiments of the present disclosure, the therapeutic agent is a cytotoxic drug, for example, auristatin or a derivative thereof. In one exemplary embodiment, the therapeutic agent is monomethyl auristatin E (MMAE). In another embodiment, the therapeutic agent is monomethyl auristatin F (MMAF).
本揭示內容亦提供一種用以治療癌症的藥學組合物。所述藥學組合物包含本發明免疫偶聯物，以及非必要地，一種藥學上可接受的載體。The present disclosure also provides a pharmaceutical composition for treating cancer, which comprises the immunoconjugate of the present invention and, optionally, a pharmaceutically acceptable carrier.
本揭示內容的另一態樣是關於一種用以治療個體之癌症的方法。本發明方法包含對個體投予一有效量之本揭示內容的免疫偶聯物或藥學組合物。Another aspect of the present disclosure is a method for treating cancer in an individual. The method of the present invention comprises administering an effective amount of the immunoconjugate or pharmaceutical composition of the present disclosure to the individual.
例示性之可接受本發明方法治療的癌症包含，但不限於，乳癌、胃癌、結腸直腸癌、膽囊癌、前列腺癌、子宮頸癌、卵巢癌、慢性或急性淋巴性白血病、膀胱癌、腎臟癌、肝癌、頭頸部鱗狀細胞癌、膠質母細胞瘤、食道癌、胰臟癌、口腔癌、肺癌、黑色素瘤及淋巴瘤。Exemplary cancers that can be treated with the methods of the present invention include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, kidney cancer, liver cancer, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, and lymphoma.
所述個體是哺乳動物；較佳為人類。The individual is a mammal; preferably a human.
在參閱下文實施方式後，本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的，以及本發明所採用之技術手段與實施態樣。After reading the following implementation methods, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation modes adopted by the present invention.
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.
I.I.定義Definition
除非本說明書另有定義，此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。此外，在不和上下文衝突的情形下，本說明書所用的單數名詞涵蓋該名詞的複數型；而所用的複數名詞時亦涵蓋該名詞的單數型。Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as those understood and used by persons of ordinary skill in the art to which the present invention belongs. In addition, singular terms used in this specification include plural forms of the terms, and plural terms also include singular forms of the terms, unless otherwise conflicting with the context.
雖然用以界定本發明較廣範圍的數值範圍與參數皆是約略的數值，此處已盡可能精確地呈現具體實施例中的相關數值。然而，任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處，「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是，「約」一詞代表實際數值落在平均值的可接受標準誤差之內，視本發明所屬技術領域中具有通常知識者的考量而定。除了實驗例之外，或除非另有明確的說明，當可理解此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此，除非另有相反的說明，本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值，且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。在此處，將數值範圍表示成由一端點至另一段點或介於二端點之間；除非另有說明，此處所述的數值範圍皆包含端點。Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person of ordinary skill in the art to which the present invention belongs. Except for experimental examples, or unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used herein (for example, to describe material usage, time duration, temperature, operating conditions, quantity ratios and the like) are modified by "about". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the attached patent application are approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated significant digits and the values obtained by applying the general rounding method. Herein, the numerical range is expressed from one end point to another or between two end points; unless otherwise stated, the numerical range described herein includes the end points.
「抗體」(antibody)一詞在本揭示內容具有最廣泛的意涵，具體包含單株抗體(monoclonal antibody, mAb；包含全長單株抗體)、多株抗體(polyclonal antibody)、多效抗體(multi-specific antibody；例如雙效抗體)、嵌合型抗體、人源化抗體或能產生特定生物活性的抗體片段。「抗體片段」(antibody fragment)一詞在本揭示內容中包含全長抗體的一部分，該部分通常為抗體中與抗原結合的位置或變異區域(例如VL及VH域)。例示性的抗體片段包含抗原結合片段(fragment antigen-binding, Fab)、Fab’、F(ab’)2、單鏈變異片段(single-chain variable fragment, scFv)、雙鏈抗體(diabody)、線性抗體(linear antibody)、單鏈抗體分子(single-chain antibody molecule)及由抗體片段形成的多效抗體。依據抗體重鏈之恆定域(constant domain)的胺基酸序列，免疫球蛋白可分為免疫球蛋白G (IgG)、免疫球蛋白A (IgA)、免疫球蛋白M (IgM)、免疫球蛋白D (IgD)及免疫球蛋白E (IgE)等類型，其中各類型可進一步分為不同亞型(同型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。習知的免疫球蛋白包含α(alpha)、γ (gamma)、δ (delta)、ε (epsilon)或μ (mu)重鏈恆定域。本發明所屬技術中具有通常知識者皆知不同類型之免疫球蛋白的次單位結構及三維構型，舉例來說，可參見Abbas等人「Cellular and Molecular Immunology」第四版(2000)。抗體可為一較大融合分子(由抗體與一或多其他蛋白或勝肽共價或非共價結合所形成)的一部分。The term "antibody" in this disclosure has the broadest meaning, specifically including monoclonal antibodies (mAbs; including full-length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies), multi-specific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, or antibody fragments capable of producing specific biological activities. The term "antibody fragment" in this disclosure includes a portion of a full-length antibody, which is usually the antigen-binding site or variable region (e.g., VL and VH domains) of the antibody. Exemplary antibody fragments include antigen-binding fragments (Fab), Fab', F(ab')2, single-chain variable fragments (scFv), bi-chain antibodies (diabody), linear antibodies (linear antibody), single-chain antibody molecules (single-chain antibody molecules) and polyvalent antibodies formed by antibody fragments. According to the amino acid sequence of the constant domain of the antibody heavy chain, immunoglobulins can be divided into types such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE), wherein each type can be further divided into different subtypes (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Known immunoglobulins contain alpha, gamma, delta, epsilon or mu heavy chain constant domains. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known to those skilled in the art, for example, see Abbas et al., "Cellular and Molecular Immunology", 4th edition (2000). Antibodies may be part of a larger fusion molecule (formed by covalent or non-covalent binding of an antibody to one or more other proteins or peptides).
在本揭示內容中，「單株抗體」(monoclonal antibody或mAb)是指由基本上均質之抗體群所得到的抗體。相較於多株抗體，其包含可靶向不同表位(epitope)的不同抗體，單株抗體僅會標的至抗原的單一抗原決定位(即，表位)。可將壽命有限之產生抗體的B細胞與快速生長的細胞(例如，永生細胞)融合以製備單株抗體。產生的融合細胞(或稱為融合瘤)可迅速增殖，以得到能產生大量抗體的選殖株。或者是，可利用重組DNA方法來製備單株抗體或保有特定生物活性的抗體片段，其中重鏈及/或輕鏈的一部分與源自一物種之抗體或屬於一抗體類型或亞型的對應序列相同或同源，而其餘部分則與源自另一物種之抗體或另一抗體類型或亞型的對應序列相同或同源。In the present disclosure, "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous group of antibodies. In contrast to polyclonal antibodies, which include different antibodies that target different epitopes, monoclonal antibodies target only a single antigenic determinant (i.e., epitope) of an antigen. Monoclonal antibodies can be prepared by fusing antibody-producing B cells with limited lifespans with rapidly growing cells (e.g., immortal cells). The resulting fused cells (or hybridomas) can be rapidly proliferated to obtain clones that can produce large amounts of antibodies. Alternatively, recombinant DNA methods can be used to prepare single antibodies or antibody fragments retaining specific biological activity, wherein a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from one species or belonging to one antibody type or subtype, and the remaining portion is identical or homologous to the corresponding sequence of an antibody derived from another species or another antibody type or subtype.
在本揭示內容中，「重組抗體」(recombinant antibody)一詞是指由細胞或細胞株所表現及分離的抗體，其中所述細胞或細胞株經包含抗體之編碼序列的表現載體(一或多個表現載體，通常為二個表現載體)所轉染，且所述編碼序列與細胞並非天然相關。As used herein, the term "recombinant antibody" refers to an antibody expressed and isolated by a cell or cell line that has been transfected with an expression vector (one or more expression vectors, usually two expression vectors) comprising a coding sequence for the antibody that is not naturally associated with the cell.
「互補性決定區域」(complementarity determining region, CDR)在本說明書是指一抗體分子的高變異區域，其可與結合抗原之三維立體表面形成互補表面。由N端到C端，各抗體的重鏈及輕鏈分別包含三個CDR (即，CDR-1、CDR-2及CDR-3)。因此，一抗原結合位點共包含6個CDR，其分別為位於重鏈變異區域的3個CDR (即CDR-H1、CDR-H2及CDR-H3)，以及位於輕鏈變異區域的3個CDR (即CDR-L1、CDR-L2及CDR-L3)。"Complementarity determining region" (CDR) in this specification refers to a highly variable region of an antibody molecule that can form a complementary surface with the three-dimensional surface of the antigen-binding molecule. From the N-terminus to the C-terminus, the heavy chain and light chain of each antibody respectively contain three CDRs (i.e., CDR-1, CDR-2, and CDR-3). Therefore, an antigen-binding site contains a total of 6 CDRs, which are 3 CDRs located in the heavy chain variable region (i.e., CDR-H1, CDR-H2, and CDR-H3), and 3 CDRs located in the light chain variable region (i.e., CDR-L1, CDR-L2, and CDR-L3).
一抗體的「變異域」(variable domain)係指該抗體重鏈或輕鏈的胺基末端域。該些位置為抗體最易變動的部分，且包含抗原結合位點。「變異」(variable)一詞係指變異域中某些部分在抗體間序列差異廣泛且用於每種特定抗體對其特定抗原的結合及專一性的實情。然而，變異性並非均勻分佈於抗體的整個變異域。其主要集中於輕鏈及重鏈變異域中3個CDR或高度變異區域。變異域中更加高度保守的部分稱作框架區域(framework region, FR)。天然重鏈和輕鏈的變異域各自包含四個FR，它們大多採取β-折疊片構形，通過形成環狀連接且在有些情況中形成β-折疊片結構一部分的三個CDR連接。每條抗體鏈中的CDR通過FR非常接近地連接在一起，並與另一條鏈的CDR一起促成抗體之抗原結合位點的形成(請見Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991))。The "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain of that antibody. These locations are the most variable parts of the antibody and contain the antigen binding site. The word "variable" refers to the fact that certain parts of the variable domain vary widely in sequence between antibodies and are used in the binding and specificity of each particular antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable domain of an antibody. It is mainly concentrated in the three CDRs or highly variable regions in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called the framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, which mostly adopt a β-sheet configuration, connected by three CDRs that form loops connecting and in some cases forming part of the β-sheet structure. The CDRs in each antibody chain are linked together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
本揭示內容及請求保護之發明概念亦包含抗體之胺基酸序列的微小變異(特別是位於抗體之FR序列的微小變異)，只要該胺基酸序列的變異維持至少85%序列相似度，例如至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列相似度。可藉由特定修飾來改變本揭示內容抗體的特性，而不影響其生理活性。舉例來說，可改變及/或刪除某些位於抗體之框架區域的胺基酸殘基而不影響本發明抗體的生理活性。特別是，保留性胺基酸取代亦包含於其中。保留性取代為具有相似/相關側鏈之胺基酸間的相互取代。一般來說，由基因編碼的胺基酸可分為四大類：(1) 酸性胺基酸，即天門冬胺酸(aspartate)、麩胺酸(glutamate)；(2)鹼性胺基酸，即離胺酸(lysine)、精胺酸(arginine)、組胺酸(histidine)；(3)非極性胺基酸，即丙胺酸(alanine)、纈胺酸(valine)、白胺酸(leucine)、異白胺酸(isoleucine)、脯胺酸(proline)、苯丙胺酸(phenylalanine)、甲硫胺酸(methionine)、色胺酸(tryptophan)；以及(4)非帶電極性胺基酸，即甘胺酸(glycine)、天門冬醯胺(asparagine)、麩醯胺酸(glutamine)、半胱胺酸(cysteine)、絲胺酸(serine)、蘇胺酸(threonine)、酪胺酸(tyrosine)。較佳的分類是：絲胺酸及蘇胺酸係屬脂肪羥基(aliphatic-hydroxy)類；天冬醯胺酸及麩醯胺係屬含醯胺(amide-containing)類；丙胺酸、纈胺酸、白胺酸及異白胺酸係屬脂肪類；而苯丙胺酸、色胺酸及酪胺酸則屬芳香(aromatic)類。舉例來說，當可想見若以異白胺酸或纈胺酸取代白胺酸、以麩胺酸取代天門冬胺酸、以絲胺酸取代蘇胺酸，或是以一結構相似的胺基酸取代另一胺基酸時，並不會造成分子結合或蛋白特性的顯著改變，特別是當該取代位置不是位於抗原結合位點(例如，CDR)時，胺基酸之間的取代更不會影響上述特性。可藉由檢測抗體衍生物之特定活性來瞭解一胺基酸的改變是否可形成一具功能性的抗體。可利用本發明所屬技術領域具有通常知識者所知的方法來製備抗體片段或類似物。片段或類似物之較佳的胺基及羧基末端是鄰近功能域的邊界。The disclosed content and the claimed inventive concept also include minor variations in the amino acid sequence of the antibody (particularly minor variations in the FR sequence of the antibody), as long as the variation in the amino acid sequence maintains at least 85% sequence similarity, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity. The characteristics of the disclosed antibody can be changed by specific modifications without affecting its physiological activity. For example, certain amino acid residues located in the framework region of the antibody can be changed and/or deleted without affecting the physiological activity of the antibody of the present invention. In particular, conservative amino acid substitutions are also included therein. Conservative substitutions are mutual substitutions between amino acids with similar/related side chains. Generally speaking, the amino acids encoded by genes can be divided into four major categories: (1) Acidic amino acids, namely aspartate and glutamate; (2) basic amino acids, namely lysine, arginine, and histidine; (3) non-polar amino acids, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) non-charged polar amino acids, namely glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. A better classification is that serine and threonine are aliphatic-hydroxy, aspartate and glutamine are amide-containing, alanine, valine, leucine, and isoleucine are aliphatic, and phenylalanine, tryptophan, and tyrosine are aromatic. For example, it is conceivable that substitution of isoleucine or valine for leucine, glutamine for aspartate, serine for threonine, or one structurally similar amino acid for another will not result in a significant change in molecular binding or protein properties, especially when the substitution is not at an antigen binding site (e.g., CDR), and substitutions between amino acids will not affect the above properties. Whether a change in an amino acid can form a functional antibody can be understood by detecting the specific activity of the antibody derivative. Antibody fragments or analogs can be prepared using methods known to those skilled in the art. The amino and carboxyl termini of the fragments or analogs are preferably adjacent to the boundaries of the functional domain.
本揭示內容針對胺基酸序列所述的「序列相似度百分比」(Percent (%) sequence identity)係指一候選序列的胺基酸殘基與一參考序列之胺基酸殘基完全相同的百分比。於進行上述比對時，可將所述候選序列與所述參考序列並排，並於必要時引入間隙，以使二序列形成最高的序列相似度。在計算相似度時，保守性置換之胺基酸殘基視為不同的殘基。相關領域已有多種方法可供進行上述並排，譬如可公開取得的軟體如BLAST、BLAST-2、ALIGN或Megalign (DNASTAR)等。本發明所屬技術領域中具有通常知識者在進行並排時，可選擇適當的參數與計算方式，以得到最佳的排列方式。在本說明書中，二胺基酸序列之間的序列比對是採用美國國家生物科技資訊中心(Nation Center for Biotechnology Information，簡稱NCBI)所提供的蛋白質-蛋白質BLAST分析資料庫Blastp來進行。一候選序列A相較於一參考序列B的胺基酸相似度(在本說明書中亦稱之為序列A與序列B具有特定百分比(%)的胺基酸相似度)的計算方式如下：% 其中X是利用BLAST分析資料庫對序列A、B進行排列後所得到的相同胺基酸殘基數目(identical matches)，而Y是A、B二序列中較短者的胺基酸殘基總數。The "percentage of sequence similarity" (Percent (%) sequence identity) described in this disclosure for amino acid sequences refers to the percentage of amino acid residues of a candidate sequence that are exactly the same as the amino acid residues of a reference sequence. When performing the above-mentioned comparison, the candidate sequence and the reference sequence can be placed side by side, and a gap can be introduced when necessary so that the two sequences form the highest sequence similarity. When calculating the similarity, the amino acid residues of conservative substitutions are regarded as different residues. There are many methods in the relevant field for performing the above-mentioned side-by-side arrangement, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR), etc. Those with ordinary knowledge in the technical field to which the present invention belongs can select appropriate parameters and calculation methods when performing side-by-side arrangement to obtain the best arrangement. In this specification, the sequence alignment between diamino acid sequences is performed using the protein-protein BLAST analysis database Blastp provided by the National Center for Biotechnology Information (NCBI). The amino acid similarity between a candidate sequence A and a reference sequence B (also referred to as the amino acid similarity between sequence A and sequence B with a specific percentage (%)) is calculated as follows: % Where X is the number of identical amino acid residues obtained after aligning sequences A and B using the BLAST analysis database, and Y is the total number of amino acid residues in the shorter of the two sequences A and B.
在本揭示內容中，「連接」(link)、「偶聯」(conjugate)及「連結」(connecting)為可互換的詞彙，用以指二元件之間的連結關係，其中二元件可以為直接連結或間接連結。In the present disclosure, “link”, “conjugate” and “connecting” are interchangeable terms used to refer to a connection relationship between two elements, wherein the two elements may be directly connected or indirectly connected.
在本揭示內容中，「治療」(treat)一詞包含部份或完全預防、改善、減輕及/或處理癌症之相關病徵(symptom)、次要病徵(secondary disorder)或症狀(condition)。「治療」(treat)一詞於本揭示內容亦指對一個體應用或投予一或多次本發明抗體或免疫偶聯物，其中該個體患有癌症之相關病徵、次要病徵或症狀，以達到部份或完全減輕、減緩、治癒疾病、延遲發病、抑制病程發展、降低疾病嚴重性，及/或降低一或多種與癌症相關之病徵、症狀、或次要病徵的發生。與癌症相關之病徵、次要病徵及/或症狀包含，但不限於，噁心、嘔吐、食慾不振、排便習慣改變、便秘、疲勞、肌肉無力、骨折、腫脹或腫塊、出血、咳嗽、發燒、神經問題(例如，癲癇、視力改變、聽力改變或面部下垂)、體重改變(即，體重增加或減少)、昏迷及疼痛。在此「治療」(treat)亦可指投予至患有早期該些病徵或症狀之個體，以降低該個體發展與癌症相關之病徵、次要病徵及/或症狀的風險。當一治療可減少一或多種病症或臨床標記時，則該治療為「有效的」(effective)。或者是，當一治療可降低、減緩或終止疾病病程、病徵或症狀的發展時，則該治療為「有效的」(effective)。In the present disclosure, the term "treat" includes partial or complete prevention, improvement, alleviation and/or treatment of cancer-related symptoms, secondary disorders or conditions. The term "treat" in the present disclosure also refers to the application or administration of one or more antibodies or immunoconjugates of the present invention to an individual, wherein the individual suffers from cancer-related symptoms, secondary disorders or symptoms, in order to partially or completely alleviate, slow down, cure the disease, delay the onset of the disease, inhibit the progression of the disease, reduce the severity of the disease, and/or reduce the occurrence of one or more cancer-related symptoms, symptoms, or secondary symptoms. Signs, side effects and/or symptoms associated with cancer include, but are not limited to, nausea, vomiting, loss of appetite, changes in bowel habits, constipation, fatigue, muscle weakness, fractures, swelling or lumps, bleeding, cough, fever, neurological problems (e.g., seizures, vision changes, hearing changes, or facial droop), weight changes (i.e., weight gain or loss), coma, and pain. Here, "treat" may also refer to administration to an individual with these early signs or symptoms to reduce the individual's risk of developing signs, side effects and/or symptoms associated with cancer. A treatment is "effective" when it reduces one or more symptoms or clinical markers. Alternatively, a treatment is "effective" if it reduces, slows, or stops the progression of a disease, or the development of signs or symptoms.
「有效量」(effective amount) 在此處係指一成分的用量足以產生欲求的療效反應。有效量亦指一種成分之治療利益效果超越其毒性或有害影響。一藥物的有效量不必然可治癒一疾病或病徵，而是對該疾病或病徵提供治療，延緩、抑制或預防該疾病或病徵的發作，或是改善該疾病或病徵的相關徵狀。有效量可以適當的形式分成一劑、二劑或多劑，據以在特定時間內對個體進行一次、二次或多次的投予。具體的有效量取決於多種因素，如欲治療的特定狀況、患者的生理條件(如，患者體重、年齡或性別)、接受治療的哺乳動物或動物的類型、治療持續時間、目前療法(如果有的話)的本質以及所用的具體配方和化合物或其衍生物的結構。舉例來說，可將有效量表示成藥物的總重量(譬如以公克、毫克或微克為單位)，或是藥物重量與體重之比例(其單位為毫克/公斤(mg/Kg))。或者是，可將有效量表示為活性成分(例如，本揭示內容的免疫偶聯物)的濃度，例如，莫耳濃度、重量濃度、體積濃度、重量莫耳濃度、莫耳分率、重量分率及混合比。習知技藝人士可依據動物模式的劑量來計算藥物(例如，本發明免疫偶聯物) 的人體等效劑量(human equivalent dose, HED)。舉例來說，習知技藝人士可依據美國食品藥物管理局(US Food and Drug Administration, FDA)所公告之「估算成人健康志願者在初始臨床治療測式之最大安全起始劑量」(Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers)來估算人體使用之最高安全劑量。"Effective amount" as used herein refers to an amount of an ingredient sufficient to produce the desired therapeutic response. An effective amount also means that the therapeutic benefits of an ingredient outweigh its toxic or harmful effects. An effective amount of a drug does not necessarily cure a disease or symptom, but rather provides treatment for the disease or symptom, delays, inhibits or prevents the onset of the disease or symptom, or improves the symptoms associated with the disease or symptom. An effective amount can be divided into one, two or more doses in an appropriate form, and administered to an individual once, twice or multiple times within a specific period of time. The specific effective amount depends on a variety of factors, such as the specific condition to be treated, the physiological condition of the patient (e.g., the patient's weight, age or sex), the type of mammal or animal being treated, the duration of treatment, the nature of the current treatment (if any), and the specific formulation used and the structure of the compound or its derivative. For example, the effective amount can be expressed as the total weight of the drug (e.g., in grams, milligrams or micrograms), or the ratio of the weight of the drug to the body weight (in milligrams/kilograms (mg/kg)). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (e.g., the immunoconjugate of the present disclosure), such as molar concentration, weight concentration, volume concentration, weight molar concentration, molar fraction, weight fraction and mixing ratio. A person skilled in the art can calculate the human equivalent dose (HED) of a drug (e.g., the immunoconjugate of the present invention) based on the dose in the animal model. For example, a person skilled in the art can estimate the highest safe dose for human use based on the "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the US Food and Drug Administration (FDA).
「個體」(subject)一詞是指包含人類的動物，其可接受本發明重組抗體、抗體片段、免疫偶聯物及/或方法的治療。除非特定指出，否則「個體」(subject)一詞同時意指男性及女性。The term "subject" refers to animals, including humans, that can be treated with the recombinant antibodies, antibody fragments, immunoconjugates and/or methods of the present invention. Unless otherwise specified, the term "subject" refers to both males and females.
II.II.發明詳細說明Invention Details
(i)(i)重組抗體Recombinant Antibodies
本揭示內容的第一態樣提供一種命名為「91H06」的抗-B7-H3單株抗體(monoclonal antibody, mAb)。依據本揭示內容的實施方式，本發明mAb的技術特徵在於對人類B7-H3蛋白具有結合親和力及專一性，可有效引發表面B7-H3蛋白的細胞內化反應(internalization)。The first aspect of the present disclosure provides an anti-B7-H3 monoclonal antibody (mAb) named "91H06". According to the implementation of the present disclosure, the technical characteristics of the mAb of the present invention are that it has binding affinity and specificity to human B7-H3 protein and can effectively induce the cell internalization reaction of surface B7-H3 protein.
依據本揭示內容某些實施方式，本發明mAb 91H06是利用噬菌體表現之scFv抗體庫製備而得。當可想見，也可藉由傳統免疫化方法(即，以特定胜肽免疫化動物使其產生抗原特異性的抗體)或重組DNA技術(亦稱為「DNA選殖技術」；即，建構用以編碼特定抗體之重組DNA後，將其轉導至宿主細胞以產生抗體)來製備本發明mAb 91H06。According to certain embodiments of the present disclosure, mAb 91H06 of the present invention is prepared using a scFv antibody library expressed by phage. It is conceivable that mAb 91H06 of the present invention can also be prepared by traditional immunization methods (i.e., immunizing animals with specific peptides to produce antigen-specific antibodies) or recombinant DNA technology (also known as "DNA cloning technology"; i.e., constructing recombinant DNA encoding specific antibodies and then transducing them into host cells to produce antibodies).
結構上，mAb 91H06包含三個位於其VH域的CDR (即，CDR-H1、CDR-H2及CDR-H3)，以及三個位於其VL域的CDR (即，CDR-L1、CDR-L2及CDR-L3)。Structurally, mAb 91H06 comprises three CDRs located in its VH domain (i.e., CDR-H1, CDR-H2, and CDR-H3), and three CDRs located in its VL domain (i.e., CDR-L1, CDR-L2, and CDR-L3).
依據本揭示內容某些實施方式，mAb 91H06的CDR-H1、CDR-H2及CDR-H3分別包含「GFTFSDYGMG」(序列編號：1)、「SISWDSSSKEYADSVKG」(序列編號：2)及「AWIAIIGGGAHFDY」(序列編號：3)的胺基酸序列，且mAb 91H06的CDR-L1、CDR-L2及CDR-L3分別包含「RASQSVSSHLA」(序列編號：4)、「LTSSLQS」(序列編號：5)及「MQSKSLPFT」 (序列編號：6)的胺基酸序列。According to certain embodiments of the present disclosure, CDR-H1, CDR-H2 and CDR-H3 of mAb 91H06 comprise the amino acid sequences of "GFTFSDYGMG" (SEQ ID NO: 1), "SISWDSSSKEYADSVKG" (SEQ ID NO: 2) and "AWIAIIGGGAHFDY" (SEQ ID NO: 3), respectively, and CDR-L1, CDR-L2 and CDR-L3 of mAb 91H06 comprise the amino acid sequences of "RASQSVSSHLA" (SEQ ID NO: 4), "LTSSLQS" (SEQ ID NO: 5) and "MQSKSLPFT" (SEQ ID NO: 6), respectively.
以下述序列編號：7及8例示性說明mAb 91H06之VH及VL域的胺基酸序列，其中以粗體依序標示各CDR (即，VH域的CDR-H1、CDR-H2及CDR-H3，以及VL域的CDR-L1、CDR-L2及CDR-L3)。The amino acid sequences of the VH and VL domains of mAb 91H06 are exemplified by the following sequence numbers: 7 and 8, wherein each CDR is marked in bold in sequence (i.e., CDR-H1, CDR-H2 and CDR-H3 of the VH domain, and CDR-L1, CDR-L2 and CDR-L3 of the VL domain).
序列編號：7 (mAb 91H06的VH域) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMGWVRQAPGKGLEWVSSISWDSSSKEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAWIAIIGGGAHFDYWGQGTLVTVSSSequence number: 7 (VH domain of mAb 91H06) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMG WVRQAPGKGLEWVSSISWDSSSKEYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAWIAIIGGGAHFDY WGQGTLVTVSS
序列編號：8 (mAb 91H06的VL域) DIQMTQSPSSLSASVGDRVTITCRASQSVSSHLAWYQQKPGKAPKLLIYLTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSKSLPFTFGQGTKVEIKRSEQ ID NO: 8 (VL domain of mAb 91H06) DIQMTQSPSSLSASVGDRVTITCRASQSVSSHLA WYQQKPGKAPKLLIYLTSSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSKSLPFT FGQGTKVEIKR
基於抗體的結合親和力及專一性主要取決於其CDR序列，當可想見，VL及VH域之FR序列可有所變異(例如，以保留性或非保留性胺基酸殘基進行置換)，而不會影響本發明抗體的結合親和力及/或專一性。較佳地，以一或多具有相似特性的胺基酸殘基來保留性置換FR序列；舉例來說，以異白胺酸、丙胺酸、纈胺酸、脯胺酸、苯丙胺酸或色胺酸(一非極性胺基酸殘基)來取代白胺酸(另一非極性胺基酸殘基)；以麩胺酸(一酸性胺基酸殘基)取代天門冬胺酸(另一酸性胺基酸殘基)；或是以精胺酸或組胺酸(一鹼性胺基酸殘基)來取代離胺酸(另一鹼性胺基酸殘基)。Since the binding affinity and specificity of an antibody is primarily determined by its CDR sequence, it is conceivable that the FR sequences of the VL and VH domains may be varied (e.g., by substitution with conservative or non-conservative amino acid residues) without affecting the binding affinity and/or specificity of the antibodies of the present invention. Preferably, the FR sequence is conservatively replaced with one or more amino acid residues having similar properties; for example, leucine (another non-polar amino acid residue) is replaced with isoleucine, alanine, valine, proline, phenylalanine or tryptophan (a non-polar amino acid residue); aspartic acid (another acidic amino acid residue) is replaced with glutamine (an acidic amino acid residue); or lysine (another basic amino acid residue) is replaced with arginine or histidine (a basic amino acid residue).
據此，習知技藝人士可置換一或多個位於mAb 91H06之VH及VL域中FR序列的胺基酸，而不會影響其活性及/或作用(即，與B7-H3結合及/或引發B7-H3的內化反應)。因此，本揭示內容的保護範疇亦涵蓋於VH及VL之FR序列中具有置換胺基酸的抗體。依據某些實施方式，mAb 91H06的VH域包含與序列編號：7具有至少85% (即，85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)序列相似度的胺基酸序列，且mAb 91H06的VL域包含與序列編號：8具有至少85% (即，85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)序列相似度的的胺基酸序列。依據某些較佳的實施方式，mAb 91H06的VH及VL域分別包含與序列編號：7及8具有至少90%序列相似度的胺基酸序列。更佳地，mAb 91H06的VH及VL域分別包含與序列編號：7及8具有至少95%序列相似度的胺基酸序列。Accordingly, those skilled in the art can replace one or more amino acids in the FR sequence of the VH and VL domains of mAb 91H06 without affecting its activity and/or function (i.e., binding to B7-H3 and/or inducing internalization reaction of B7-H3). Therefore, the protection scope of the present disclosure also covers antibodies with replaced amino acids in the FR sequences of VH and VL. According to certain embodiments, the VH domain of mAb 91H06 comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NO: 7, and the VL domain of mAb 91H06 comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NO: 8. According to certain preferred embodiments, the VH and VL domains of mAb 91H06 comprise amino acid sequences having at least 90% sequence similarity to SEQ ID NOs: 7 and 8, respectively. More preferably, the VH and VL domains of mAb 91H06 comprise amino acid sequences having at least 95% sequence similarity to SEQ ID NOs: 7 and 8, respectively.
依據實施目的不同，本發明mAb 91H06可以製備為IgG、IgA、IgM、IgD或IgE的形式。依據某些例示性的實施方式，本發明mAb是製備為IgG的形式，其包含一對Fc區，以及一對分別連接於該對Fc區之N端的Fab區。According to different implementation purposes, mAb 91H06 of the present invention can be prepared in the form of IgG, IgA, IgM, IgD or IgE. According to certain exemplary embodiments, mAb of the present invention is prepared in the form of IgG, which comprises a pair of Fc regions and a pair of Fab regions respectively connected to the N-termini of the pair of Fc regions.
依據本揭示內容某些實施方式，mAb 91H06是全人類抗-B7-H3抗體，而不含有小鼠抗體部分，因此對人類個體具有低免疫原性。According to certain embodiments of the present disclosure, mAb 91H06 is a fully human anti-B7-H3 antibody and does not contain mouse antibody portions, and therefore has low immunogenicity to human subjects.
本揭示內容亦關於本發明mAb的片段，包含scFv、Fab、Fab’、F(ab’)2及雙鏈抗體。The present disclosure also relates to fragments of the mAb of the present invention, including scFv, Fab, Fab', F(ab')2 and bispecific antibodies.
依據某些實施方式，本發明抗體(包含mAb 91H06及其片段)對B7-H3具有結合專一性，而不會與其他B7家族成員產生交叉反應。在一實施方式中，本發明抗體會辨識並結合至人類B7-H3 (序列編號：9)。在一實施方式中，本發明抗體會辨識並結合至猴子B7-H3 (序列編號：10)。在另一實施方式中，本發明抗體會辨識並結合至大鼠B7-H3 (序列編號：11)。在另一實施方式中，本發明抗體會辨識並結合至小鼠B7-H3 (序列編號：12)。依據一特定實施方式，本發明抗體會結合至人類B7-H3 IgV2及/或IgC2域，其中IgV2及IgC2域包含序列編號：13的胺基酸序列。According to certain embodiments, the antibodies of the present invention (including mAb 91H06 and fragments thereof) have binding specificity to B7-H3 without cross-reacting with other B7 family members. In one embodiment, the antibodies of the present invention recognize and bind to human B7-H3 (SEQ ID NO: 9). In one embodiment, the antibodies of the present invention recognize and bind to monkey B7-H3 (SEQ ID NO: 10). In another embodiment, the antibodies of the present invention recognize and bind to rat B7-H3 (SEQ ID NO: 11). In another embodiment, the antibodies of the present invention recognize and bind to mouse B7-H3 (SEQ ID NO: 12). According to a specific embodiment, the antibody of the present invention binds to the human B7-H3 IgV2 and/or IgC2 domains, wherein the IgV2 and IgC2 domains comprise the amino acid sequence of SEQ ID NO: 13.
(ii)(ii)包含本發明重組抗體的免疫偶聯物Immunoconjugates comprising the recombinant antibodies of the present invention
依據本揭示內容某些實施方式，本發明抗體對表現於癌細胞表面的B7-H3分子具有結合親和力，且可有效引發細胞表面B7-H3的內化反應。基於標的靶向及內化反應的特性，本發明抗體可作為一種靶向模組，將與其連接之治療劑遞送至個體的癌細胞。According to certain embodiments of the present disclosure, the antibodies of the present invention have binding affinity to B7-H3 molecules expressed on the surface of cancer cells and can effectively induce internalization reaction of B7-H3 on the cell surface. Based on the characteristics of target targeting and internalization reaction, the antibodies of the present invention can be used as a targeting module to deliver the therapeutic agent linked thereto to the cancer cells of an individual.
因此，本揭示內容的第二態樣是關於一種免疫偶聯物。結構上，本發明免疫偶聯物包含mAb 91H06或其片段(例如，scFv)、治療劑，以及用以將治療劑連接至mAb/抗體片段的連接子。Therefore, the second aspect of the present disclosure is about an immunoconjugate. Structurally, the immunoconjugate of the present invention comprises mAb 91H06 or a fragment thereof (e.g., scFv), a therapeutic agent, and a linker for linking the therapeutic agent to the mAb/antibody fragment.
依據實施目的不同，所述治療劑可以是細胞毒性藥物、放射性核種、細胞激素、荷爾蒙藥物免疫刺激劑或免疫治療藥物。Depending on the purpose of implementation, the therapeutic agent can be a cytotoxic drug, a radionuclide, a cytokine, a hormonal drug, an immunostimulant or an immunotherapeutic drug.
例示性之適合用以製備本發明免疫偶聯物的細胞毒性藥物包含，但不限於，紫杉烷(taxanes；例如，太平洋紫杉醇(paclitaxel)、多烯紫杉醇(docetaxel)及卡巴他賽(cabazitaxel))；烷化劑(例如，二氯甲二乙胺(mechlorethamine)、環磷醯胺(cyclophosphamide)、美法侖(melphalan)、苯丁酸氮芥(chlorambucil)、異環磷醯胺(ifosfamide)、白消安(busulfan)、N-亞硝基-N-甲基脲(N-Nitroso-N-methylurea, MNU)、卡莫司汀(carmustine, BCNU)、洛莫司汀(lomustine, CCNU)、司莫司汀(semustine, MeCCNU)、福莫司汀(fotemustine)、鏈佐黴素(streptozotocin)、達卡巴嗪(dacarbazine)、米托唑胺(mitozolomide)、替莫唑胺(temozolomide)、噻替派(thiotepa)、絲裂黴素(mytomycin)、二嗪醌(diaziquone, AZQ)、順鉑(cisplatin)、卡鉑(carboplatin)、奧沙利鉑(oxaliplatin)、丙卡巴肼(procarbazine)及六甲基三聚氰胺(hexamethylmelamine))；抗代謝物(例如，甲胺喋呤(methotrexate)、培美曲塞(pemetrexed)、氟尿嘧啶(fluorouracil)、卡培他濱(capecitabine)、阿糖胞苷(cytarabine)、吉西他濱(gemcitabine)、地西他濱(decitabine)、阿紮胞苷(azacitidine)、氟達拉濱(fludarabine)、奈拉濱(nelarabine)、克拉屈濱(cladribine)、氯法拉濱(clofarabine)、噴司他汀(pentostatin)、硫鳥嘌呤(thioguanine)及硫醇嘌呤(mercaptopurine))；抗微管劑(例如，微管蛋白抑制劑(tubulysin)、澳瑞他汀或其衍生物、類美登素(maytansinoid)、長春新鹼(vincristine)、長春鹼(vinblastine)、長春瑞賓(vinorelbine)、長春地辛(vindesine)、長春氟寧(vinflunine)、依託泊苷(etoposide)及替尼泊甙(teniposide))；拓樸異構酶抑制劑(例如，伊立替康(irinotecan)、拓樸替康(topotecan)、依託泊苷(etoposide)、艾黴素(doxorubicin)、米托蒽醌(mitoxantrone)、替尼泊甙(teniposide)、新生黴素(novobiocin)、默巴龍(merbarone)及阿克拉黴素(aclarubicin))；細胞毒性抗生素(例如，卡奇黴素(calicheamicin)、比咯苯偶氮駢(pyrrolobenzodiazepine)、倍癌黴素(duocarmycin)、艾黴素(doxorubicin)、道諾黴素(daunorubicin)、表柔比星(epirubicin)、艾達黴素(idarubicin)、吡柔比星(pirarubicin)、阿克拉黴素(aclarubicin)、米托蒽醌(mitoxantrone)、博萊黴素(bleomycin)、絲裂黴素C (mitomycin C)及放線菌素(actinomycin))；植物毒素(例如，蓖麻毒素(ricin)、相思子毒素(abrin)、皂草素(saporin)、蒴蓮根毒素(volkensin)、莫迪素(modeccin)、白樹素(gelonin)及槲寄生素(viscumin))；外毒素(例如，綠膿桿菌外毒素(Pseudomonas exotoxin))；肉毒桿菌毒素(botulinum toxin；例如，肉毒桿菌毒素A、肉毒桿菌毒素B、肉毒桿菌毒素C、肉毒桿菌毒素D、肉毒桿菌毒素E及肉毒桿菌毒素F)；以及內毒素(例如，白喉毒素(diphtheria toxin))。依據本揭示內容一例示性實施方式，細胞毒性藥物是澳瑞他汀或其衍生物，例如，MMAE或MMAF。Exemplary cytotoxic drugs suitable for preparing the immunoconjugates of the present invention include, but are not limited to, taxanes (e.g., paclitaxel, docetaxel, and cabazitaxel); alkylating agents (e.g., mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (Serpentine); MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mytomycin, diaziquone, AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, and hexamethylmelamine); anti-metabolites (e.g., methotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine, azathioprine, tadalafil, oxadiazine, sirolimus, tadalafil, sirolimus ... azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, and mercaptopurine); antimicrotubule agents (e.g., tubulysin, auristatin or its derivatives, maytansinoids, vincristine, vinblastine, vinorelbine); vinorelbine, vindesine, vinflunine, etoposide, and teniposide); topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclamycin); arubicin); cytotoxic antibiotics (e.g., calicheamicin, pyrrolobenzodiazepine, duocarmycin, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, bleomycin, mitomycin C (mitomycin C and actinomycin); plant toxins (e.g., ricin, abrin, saporin, volkensin, modeccin, gelonin, and viscumin); exotoxins (e.g., Pseudomonas exotoxin); botulinum toxins (e.g., botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, and botulinum toxin F); and endotoxins (e.g., diphtheria toxin). According to an exemplary embodiment of the present disclosure, the cytotoxic drug is auristatin or a derivative thereof, for example, MMAE or MMAF.
放射性核種亦稱為放射性核素(radionuclide)或放射性同位素(radioisotope或radioactive isotope)，其可以是釔-90 (yttrium-90, 90Y)、銦-111 (indium-111, 111In)、碘-131 (iodine-131, 131I)、釤-153 (samarium-153, 153Sm)、鑥-177 (lutetium-177, 177Lu)、砈-211 (astatine-211, 211At)、鉍-212 (bismuth-212, 212Bi)、錒-225 (actinium-225, 225Ac)、鐳-223 (radium-223, 223R)或釷-227 (thorium-227, 227Th)。較佳地，放射性核種是與一螯合物複合，例如乙二胺四亞甲基膦酸(ethylenediaminetetramethylenephosphonic acid, EDTMP)、1,4,7,10-四氮雜環十二烷四亞甲基膦酸(1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid, DOTMP)、1,4,7,10-四氮雜環十二烷-1,4,7,10-四乙酸(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA)、1,4,7-三氮雜環壬烷-1,4,7-三乙酸(1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA)、1,4,7-三氮雜環壬烷-1,4-二乙酸(1,4,7-triazacyclononane-1,4-diacetic acid, NODA)，或是二乙烯三胺五乙酸(diethylenetriaminepentaacetic acid, DTPA)。Radionuclides are also called radionuclides or radioisotopes. They can be yttrium-90 (90Y), indium-111 (111In), iodine-131 (131I), samarium-153 (153Sm), lutetium-177 (177Lu), astatine-211 (211At), bismuth-212 (212Bi), actinium-225 (225Ac), radium-223 (223R), or thorium-227. Preferably, the radionuclide is complexed with a chelate, such as ethylenediaminetetramethylenephosphonic acid (EDTMP), 1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (DOTA), or 1,4,7-triazacyclononane-1,4,7-triacetic acid (DOTA). NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), or diethylenetriaminepentaacetic acid (DTPA).
細胞激素可以是任何已知用以刺激免疫反應的細胞激素，舉例來說，介白素(interleukin, IL)-2、IL-10、IL-12、IL-15、IL-21、腫瘤壞死因子(tumor necrosis factor, TNF)-α、干擾素(interferon, IFN)-α、IFN-γ或顆粒細胞巨噬細胞群落刺激因子(granulocyte macrophage colony-stimulating factor, GM-CSF)。The cytokine can be any cytokine known to stimulate an immune response, for example, interleukin (IL)-2, IL-10, IL-12, IL-15, IL-21, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ, or granulocyte macrophage colony-stimulating factor (GM-CSF).
例示性之適合用以製備本發明免疫偶聯物的荷爾蒙藥物包含，但不限於，芳香酶抑制劑(aromatase inhibitor；例如，阿那曲唑(anastrozole)、依西美坦(exemestane)及來曲唑(letrozole))；選擇性雌激素受體調節劑(selective estrogen receptor modulator, SERM；例如，他莫昔芬(tamoxifen)及雷洛昔芬(raloxifene))；雌激素受體拮抗劑(例如，氟維司群(fulvestrant)及托瑞米芬(toremifene))；促黃體激素釋放激素(luteinizing hormone-releasing hormone, LHRH)促效劑(例如，戈舍瑞林(goserelin)、亮丙瑞林(leuprolide)及曲普瑞林(triptorelin))；抗雄性素(例如，阿帕魯胺(apalutamide)、恩雜魯胺(enzalutamide)、達洛魯胺(darolutamide)、比卡魯胺(bicalutamide)、氟塔醯胺(flutamide)及尼魯米特(nilutamide))；CYP17抑制劑(例如，阿比特龍(abiraterone)及克康那唑(ketoconazole))；黃體素(progestin；例如，甲羥黃體素乙酸酯(medroxyprogesterone acetate)及美皆斯妥乙酸酯(megestrol acetate))；以及解腎上腺素劑(adrenolytic；例如，米托坦(mitotane))。Exemplary hormonal drugs suitable for preparing the immunoconjugates of the present invention include, but are not limited to, aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole); selective estrogen receptor modulators (SERMs; e.g., tamoxifen and raloxifene); estrogen receptor antagonists (e.g., fulvestrant and toremifene); luteinizing hormone-releasing hormone (LH-releasing hormone); =The preferred agents include leuprolide, triptorelin, and seratinib; agonists (e.g., goserelin, leuprolide, and triptorelin); antiandrogens (e.g., apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide, and nilutamide); CYP17 inhibitors (e.g., abiraterone and ketoconazole); progestins (e.g., medroxyprogesterone acetate and megestrol acetate); and adrenolytics (e.g., mitotane).
例示性之免疫刺激劑包含，但不限於，乙醯甘露聚醣(acemannan)、臭匹立明(bropirimine)、牛蒡(burdock)、去氧膽酸(deoxycholic acid, DCA)、紫錐花(Echinacea)、PEG化重組腺苷脫氨酶(elapegademase)、類黃酮(flavonoid；例如，蘆丁(rutin)、異甘草素(isoliquiritigenin)及甘草素(liquiritigenin))、醋酸格拉替雷(glatiramer acetate)、奧普瑞介白素(oprelvekin)、腺苷脫氨酶(pegademase bovine)、普樂沙福(plerixafor)、泌乳素(prolactin)、曲拉西利(trilaciclib)、萜類(terpenes；例如，三萜類(triterpenoid))、TLR7/TLR8促效劑、TLR9促效劑、Sting促效劑及NLRP促效劑。Exemplary immunostimulants include, but are not limited to, acemannan, bropirimine, burdock, deoxycholic acid (DCA), Echinacea, PEGylated recombinant adenosine deaminase (elapegademase), flavonoids (e.g., rutin, isoliquiritigenin, and liquiritigenin), glatiramer acetate, oprelvekin, pegademe bovine), plerixafor, prolactin, trilaciclib, terpenes (e.g., triterpenoids), TLR7/TLR8 agonists, TLR9 agonists, Sting agonists, and NLRP agonists.
至於免疫治療藥物，其可以是免疫檢查點抑制劑(例如，細胞毒性T淋巴球抗原-4 (cytotoxic T-lymphocyte antigen-4, CTLA-4)、程式性細胞死亡-1 (programmed cell death-1, PD-1)或PD-L1的抑制劑)，或是免疫調節劑(例如，沙利竇邁(thalidomide)及來那度胺(lenalidomide))。As for the immunotherapy drug, it can be an immune checkpoint inhibitor (e.g., inhibitors of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death-1 (PD-1) or PD-L1), or an immunomodulator (e.g., thalidomide and lenalidomide).
用以連接本發明mAb/抗體片段及治療劑的連接子可以是可裂解型(cleavable)連接子或非裂解型(non-cleavable)連接子。例示性之可裂解型連接子包含，但不限於，蛋白酶敏感型連接子(例如，纈胺酸-瓜胺酸(valine-citrulline, VC)雙肽、纈胺酸-丙胺酸(valine-alanine, VA)雙肽、纈胺酸-離胺酸(valine-lysine, VL)雙肽、纈胺酸-精胺酸(valine-arginine, VR)雙肽及麩胺酸-纈胺酸-瓜胺酸(glutamate-valine-citrulline, EVC)三肽)、pH敏感型連接子(例如，腙(hydrazine)連接子、酯連接子及醯胺連接子)，以及麩胺基硫敏感型連接子(例如，N-琥珀醯亞胺基4-(2-吡啶二硫代)丁酸酯(N-Succinimidyl 4-(2-pyridyldithio) butanoate, SPDB)及N-琥珀醯亞胺基-4-(2-吡啶二硫代)戊酸酯(N-succinimidyl-4-(2-pyridyldithio) pentanoate, SPP))。例示性之非裂解型連接子包含，但不限於，馬來醯亞胺己醯基(maleimidocaproyl, MC)、馬來醯亞胺甲基環己烷-1-羧酸酯(maleimidomethyl cyclohexane-1-carboxylate, MCC)及4-[N-馬來醯亞胺甲基]環己烷-1-甲酸琥珀醯亞胺酯(succinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate, SMCC)。或者是，連接子可以是任何已知用以連接免疫偶聯物中二功能模體(例如，ADC之抗體及負載藥物)的連接子。習知技藝人士可依據實施目的選擇適當之用以製備本發明免疫偶聯物的連接子。依據一例示性的實施方式，用以連接本發明mAb/抗體片段及治療劑的連接子包含聚乙二醇(polyethylene glycol, PEG)鏈及與PEG鏈連接的蛋白酶敏感型連接子；較佳地，所述PEG鏈具有1到10個EG重複單元。依據本揭示內容一實施例，連接子包含PEG鏈及與PEG鏈連接的VC雙肽；在該實施例中，免疫偶聯物是製備為「mAb/抗體片段-PEG鏈-VC雙肽-治療劑」的形式。在本揭示內容另一實施例中，連接子包含PEG鏈及與PEG鏈連接的EVC三肽；在該實施例中，免疫偶聯物是製備為「mAb/抗體片段-PEG鏈-EVC三肽-治療劑」的形式。The linker used to link the mAb/antibody fragment of the present invention and the therapeutic agent can be a cleavable linker or a non-cleavable linker. Exemplary cleavable linkers include, but are not limited to, protease-sensitive linkers (e.g., valine-citrulline (VC) dipeptide, valine-alanine (VA) dipeptide, valine-lysine (VL) dipeptide, valine-arginine (VR) dipeptide, and glutamate-valine-citrulline (EVC) tripeptide), pH-sensitive linkers (e.g., hydrazine linkers, ester linkers, and amide linkers), and glutamate-sulfur-sensitive linkers (e.g., N-succinimidyl 4-(2-pyridyldithio)butyrate (N-Succinimidyl The cleavable linkers include, but are not limited to, maleimidocaproyl (MC), maleimidomethyl cyclohexane-1-carboxylate (MCC), and succinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (SMCC). Alternatively, the linker can be any known linker for linking two functional motifs in an immunoconjugate (e.g., the antibody and the drug carrier of an ADC). Those skilled in the art can select an appropriate linker for preparing the immunoconjugate of the present invention according to the purpose of implementation. According to an exemplary embodiment, the linker used to link the mAb/antibody fragment and the therapeutic agent of the present invention comprises a polyethylene glycol (PEG) chain and a protease-sensitive linker connected to the PEG chain; preferably, the PEG chain has 1 to 10 EG repeating units. According to one embodiment of the present disclosure, the linker comprises a PEG chain and a VC dipeptide connected to the PEG chain; in this embodiment, the immunoconjugate is prepared in the form of "mAb/antibody fragment-PEG chain-VC dipeptide-therapeutic agent". In another embodiment of the present disclosure, the linker comprises a PEG chain and an EVC tripeptide linked to the PEG chain; in this embodiment, the immunoconjugate is prepared in the form of "mAb/antibody fragment-PEG chain-EVC tripeptide-therapeutic agent".
如上所述，本發明mAb可依據使用目的製備為IgG、IgA、IgM、IgD或IgE的形式。依據本揭示內容某些較佳的實施方式，本發明mAb是製備為IgG的形式。在該些實施方式中，治療劑是連接於mAb的Fc區。As described above, the mAb of the present invention can be prepared in the form of IgG, IgA, IgM, IgD or IgE according to the intended use. According to certain preferred embodiments of the present disclosure, the mAb of the present invention is prepared in the form of IgG. In these embodiments, the therapeutic agent is linked to the Fc region of the mAb.
依據本揭示內容某些實施方式，本發明免疫偶聯物是利用三甘露醣(trimannosyl) ADC技術所製備，其中所述三甘露醣ADC技術是一種將負載藥物以位點專一性方式連接至靶向抗體的平台(舉例來說，請參見WO 2018/126092 A1)。在該些實施方式中，首先修飾重組抗體或抗體片段，使其與一或多個(例如，1、2、3、4、5、6、7、8或更多個)疊氮基(azide group)偶聯；經疊氮基修飾的抗體可藉由無銅點擊反應(copper-free click reaction)與一或多個(例如，1、2、3、4、5、6、7、8或更多個)具有二苯并環辛炔基(dibenzocyclooctyne group, DBCO)偶聯之連接子-負載藥物(其個別包含連接子、連結至該連接子之一末端的DBCO基團及連結至該連接子之另一末端的治療劑)連接，其中所述無銅點擊反應是發生於疊氮基與DBCO之間。當可想見，可以其他適用於點擊化學反應的基團來取代所述疊氮基或DBCO，舉例來說，炔基(alkyne group)、四嗪基(tetrazine group)或反式環辛炔(trans-cyclooctyne, TCO)。依據實施目的不同，可以其他用以合成ADC的方法來製備本發明免疫偶聯物，舉例來說，半胱胺酸接合(cysteine conjugation)、離胺酸接合(lysine conjugation)及二硫鍵橋接(disulfide re-bridging)等。本發明所屬技術領域中具有通常知識者皆知用以合成ADC的方法；為求潔簡，在此不再贅述。According to certain embodiments of the present disclosure, the immunoconjugates of the present invention are prepared using trimannosyl ADC technology, wherein the trimannosyl ADC technology is a platform for linking drug payloads to targeting antibodies in a site-specific manner (for example, see WO 2018/126092 A1). In these embodiments, a recombinant antibody or antibody fragment is first modified to be coupled with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) azide groups; the azide-modified antibody can be coupled with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) dibenzocyclooctyne (DBCO)-coupled linkers-drug-carrying agents (each comprising a linker, a DBCO group linked to one end of the linker, and a therapeutic agent linked to the other end of the linker) by a copper-free click reaction, wherein the copper-free click reaction occurs between the azide group and the DBCO. It is conceivable that the azide group or DBCO may be replaced by other groups suitable for click chemistry, for example, an alkyne group, a tetrazine group or a trans-cyclooctyne (TCO). Depending on the purpose of implementation, the immunoconjugate of the present invention may be prepared by other methods for synthesizing ADC, for example, cysteine conjugation, lysine conjugation and disulfide re-bridging. The methods for synthesizing ADC are well known to those skilled in the art to which the present invention belongs; for the sake of brevity, they will not be described in detail here.
依據本揭示內容某些例示性的實施方式，重組抗體/抗體片段具有四個疊氮基，因此重組抗體/抗體片段可與四個治療劑(例如，四個MMAE或MMAF分子)連接。在該些實施方式中，得到之免疫偶聯物的藥物與抗體比(drug-to-antibody ratio, DAR)約為4。According to certain exemplary embodiments of the present disclosure, the recombinant antibody/antibody fragment has four azide groups, and thus the recombinant antibody/antibody fragment can be linked to four therapeutic agents (e.g., four MMAE or MMAF molecules). In these embodiments, the drug-to-antibody ratio (DAR) of the resulting immunoconjugate is about 4.
(iii)(iii)包含本發明免疫偶聯物的藥學組合物Pharmaceutical compositions comprising the immunoconjugates of the present invention
本揭示內容的第三態樣是關於一種用以治療癌症的藥學組合物。依據某些實施方式，所述藥學組合物包含本發明免疫偶聯物，以及非必要地，一種藥學上可接受的載體。The third aspect of the present disclosure is a pharmaceutical composition for treating cancer. According to certain embodiments, the pharmaceutical composition comprises the immunoconjugate of the present invention and, optionally, a pharmaceutically acceptable carrier.
一般來說，本發明免疫偶聯物的重量約佔藥學組合物總重的0.1%到99%。在某些實施方式中，本發明免疫偶聯物的重量至少佔藥學組合物總重的1%。在某些實施方式中，本發明免疫偶聯物的重量至少佔藥學組合物總重的5%。在某些實施方式中，本發明免疫偶聯物的重量至少佔藥學組合物總重的10%。在其他實施方式中，本發明免疫偶聯物的重量至少佔藥學組合物總重的25%。Generally, the weight of the immunoconjugate of the present invention is about 0.1% to 99% of the total weight of the pharmaceutical composition. In certain embodiments, the weight of the immunoconjugate of the present invention is at least 1% of the total weight of the pharmaceutical composition. In certain embodiments, the weight of the immunoconjugate of the present invention is at least 5% of the total weight of the pharmaceutical composition. In certain embodiments, the weight of the immunoconjugate of the present invention is at least 10% of the total weight of the pharmaceutical composition. In other embodiments, the weight of the immunoconjugate of the present invention is at least 25% of the total weight of the pharmaceutical composition.
藥學上可接受的載體可以是任何藥學上可接受的材料或媒介物，例如液體或固體填充劑、稀釋劑、賦形劑、溶劑或包覆材料，用以攜帶活性成分(例如，本發明mAb或免疫偶聯物)，或是將活性成分(例如，本發明mAb或免疫偶聯物)由身體的一器官或部位運送到身體的另一器官或部位。載體必須是「可接受的」(acceptable)，即與配方中其他成分相容；同時使活性成分的降解最小化，並將個體產生的副作用降到最低。依據實施目的不同，本揭示內容的藥學組合物可更包含一或多種藥學上可接受的添加物，包含介面活性劑、緩衝劑、稀釋劑、穩定劑、乳化劑、分散劑、懸浮劑及防腐劑等。A pharmaceutically acceptable carrier can be any pharmaceutically acceptable material or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or coating material, used to carry an active ingredient (e.g., mAb or immunoconjugate of the present invention), or to transport an active ingredient (e.g., mAb or immunoconjugate of the present invention) from one organ or part of the body to another organ or part of the body. The carrier must be "acceptable", that is, compatible with other ingredients in the formulation; at the same time, it minimizes the degradation of the active ingredient and minimizes the side effects produced by the individual. Depending on the implementation purpose, the pharmaceutical composition of the present disclosure may further include one or more pharmaceutically acceptable additives, including surfactants, buffers, diluents, stabilizers, emulsifiers, dispersants, suspending agents and preservatives.
可依據藥學組合物的投予路徑來選擇適合與本發明免疫偶聯物合併使用之藥學上可接受的載體。本發明藥學組合物可藉由皮下、靜脈內、動脈內、腫瘤內、腹腔內或肌肉內注射投予至個體體內。The pharmaceutically acceptable carrier suitable for use in combination with the immunoconjugate of the present invention can be selected according to the administration route of the pharmaceutical composition. The pharmaceutical composition of the present invention can be administered to an individual by subcutaneous, intravenous, intraarterial, intratumoral, intraperitoneal or intramuscular injection.
用以注射投予的藥學組合物可以製備為無菌水溶性、無菌非水溶液、懸浮液或乳液。例示性之非水溶液包含，但不限於，丙二醇、聚乙二醇、橄欖油等植物油，以及油酸乙酯等注射用有機酯。例示性之水溶液包含水、乳液及懸浮液，例如生理食鹽水或緩衝介質。常見之適用於非腸胃投予的藥物載體包含，氯化鈉溶液、林格氏葡萄糖、葡萄糖、乳酸林格氏注射液或固定油，而適用於靜脈投予的藥物載體通常包含液體、營養補充物及電解質(例如，與林格氏葡萄糖相關的電解質)等。Pharmaceutical compositions for injection can be prepared as sterile aqueous solutions, sterile non-aqueous solutions, suspensions or emulsions. Exemplary non-aqueous solutions include, but are not limited to, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable organic esters such as ethyl oleate. Exemplary aqueous solutions include water, emulsions and suspensions, such as physiological saline or buffered media. Common drug carriers suitable for parenteral administration include sodium chloride solution, Ringer's dextrose, glucose, lactated Ringer's injection or fixed oils, while drug carriers suitable for intravenous administration generally include liquids, nutritional supplements and electrolytes (e.g., electrolytes associated with Ringer's dextrose), etc.
(iv)(iv)本發明重組抗體、免疫偶聯物及藥學組合物的用途Uses of the recombinant antibodies, immunoconjugates and pharmaceutical compositions of the present invention
本揭示內容的第四態樣提供一種用以治療一個體之癌症的方法。本發明方法包含對個該體投予一有效量之本揭示內容的免疫偶聯物或藥學組合物。A fourth aspect of the present disclosure provides a method for treating cancer in an individual. The method of the present invention comprises administering an effective amount of the immunoconjugate or pharmaceutical composition of the present disclosure to the individual.
依據某些實施方式，本發明方法包含將本揭示內容之免疫偶聯物投予至個體。在該些實施方式中，所述個體是小鼠，其中是將每公斤約0.01到100毫克(例如每公斤約0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100毫克)之本發明免疫偶聯物投予至個體體內。較佳地，將每公斤約0.1到50毫克之本發明免疫偶聯物投予至個體體內。更佳地，將每公斤約1到10毫克之本發明免疫偶聯物投予至個體體內。依據一實施例，將每公斤約5毫克之本發明免疫偶聯物投予至個體體內。在一實施例中，投予每公斤約1.5毫克之本發明免疫偶聯物即足以於個體產生治療功效(即，抑制腫瘤生長)。According to certain embodiments, the methods of the invention comprise administering an immunoconjugate of the disclosure to a subject. In these embodiments, the subject is a mouse, wherein about 0.01 to 100 mg/kg (e.g., about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg) of the immunoconjugate of the present invention is administered into a subject. Preferably, about 0.1 to 50 mg/kg of the immunoconjugate of the present invention is administered to an individual. More preferably, about 1 to 10 mg/kg of the immunoconjugate of the present invention is administered to an individual. According to one embodiment, about 5 mg/kg of the immunoconjugate of the present invention is administered to an individual. In one embodiment, administration of about 1.5 mg/kg of the immunoconjugate of the present invention is sufficient to produce a therapeutic effect (i.e., inhibit tumor growth) in an individual.
習知技藝人士可基於本揭示內容實施例之動物實驗所決定的投予劑量來決定本發明免疫偶聯物的人體等效劑量(human equivalent dose, HED)。據此，本發明免疫偶聯物適用於人類個體的有效劑量可以是每公斤人類個體體重1微克到每公斤人類個體體重10毫克；舉例來說，每公斤1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980或990微克，或是每公斤1、2、3、4、5、6、7、8、9或10毫克。投予劑量可單次投予，或是分多次等量投予。習知技藝人士或臨床醫療人員可依據病患的身體狀況或疾病的嚴重程度來調整投予劑量或療程。A person skilled in the art can determine the human equivalent dose (HED) of the immunoconjugate of the present invention based on the dosage determined in the animal experiments of the embodiments of the present disclosure. Accordingly, the effective dose of the immunoconjugate of the present invention for human subjects can be 1 microgram per kilogram of human subject body weight to 10 milligrams per kilogram of human subject body weight; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700 0, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 76 0, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980 or 990 micrograms, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 milligrams per kilogram. The dosage may be administered as a single dose or in multiple equal doses. A skilled person or clinical medical staff may adjust the dosage or course of treatment according to the patient's physical condition or the severity of the disease.
依據欲求目的不同，可對個體每週投予1或2次、每二週投予1次、每三週投予1次、每四週投予1次、每五週投予1次、每六週投予1次、每七週投予1次、每8週投予1次、或更久投予1次本發明免疫偶聯物及/或藥學組合物。可以習知技術及方法來偵測治療的進程。當可想見，投予療程可隨著時間而有所改變。依據本揭示內容某些例示性的實施方式，每週對個體投予1次本發明免疫偶聯物。依據另一例示性的實施方式，投予單一劑量之本發明免疫偶聯物即足以於個體產生治療功效(即，抑制腫瘤生長)。Depending on the desired purpose, the immunoconjugate and/or pharmaceutical composition of the present invention may be administered to an individual once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, or once more frequently. The progress of treatment may be detected using known techniques and methods. It is contemplated that the course of administration may vary over time. According to certain exemplary embodiments of the present disclosure, the immunoconjugate of the present invention is administered to an individual once a week. According to another exemplary embodiment, administration of a single dose of the immunoconjugate of the present invention is sufficient to produce a therapeutic effect (i.e., inhibit tumor growth) in a subject.
可藉由適當路徑對個體投予本發明免疫偶聯物或藥學組合物，其中所述路徑是選自由鼻腔、局部、黏膜及非腸胃道投予(例如，皮下、腫瘤內、肌肉內、靜脈內、動脈內或腹腔內注射)所組成的群組。依據本揭示內容某些例示的實施方式，將本發明免疫偶聯物或藥學組合物靜脈內投予至個體體內。The immunoconjugate or pharmaceutical composition of the present invention can be administered to an individual by an appropriate route, wherein the route is selected from the group consisting of nasal, topical, mucosal and parenteral administration (e.g., subcutaneous, intratumoral, intramuscular, intravenous, intraarterial or intraperitoneal injection). According to certain exemplary embodiments of the present disclosure, the immunoconjugate or pharmaceutical composition of the present invention is administered intravenously to an individual.
當可想見，本發明方法可單獨投予至個體，或是與其他有益於癌症預防或治療的額外療法(舉例來說，手術、化學治療及/或放射治療)合併投予至個體。依據所需/治療目的不同，可於投予額外治療之前、同時或之後對個體施予本發明方法。It is contemplated that the methods of the present invention may be administered to an individual alone or in combination with other additional treatments that are beneficial for cancer prevention or treatment (e.g., surgery, chemotherapy, and/or radiation therapy). Depending on the needs/therapeutic objectives, the methods of the present invention may be administered to an individual before, simultaneously with, or after the administration of the additional treatment.
可接受本發明方法治療的癌症可以是任何B7-H3陽性癌症(即，相較於正常細胞，表現或過量表現B7-H3的癌症)，例如，乳癌、胃癌、結腸直腸癌、膽囊癌、前列腺癌、子宮頸癌、卵巢癌、慢性或急性淋巴性白血病、膀胱癌、腎臟癌、肝癌、頭頸部鱗狀細胞癌、膠質母細胞瘤、食道癌、胰臟癌、口腔癌、肺癌、黑色素瘤或淋巴瘤。Cancers that can be treated with the methods of the present invention can be any B7-H3-positive cancer (i.e., a cancer that expresses or overexpresses B7-H3 relative to normal cells), for example, breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, kidney cancer, liver cancer, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma or lymphoma.
基本上，可接受本發明方法治療的個體為一哺乳動物，舉例來說，人類、小鼠、大鼠、天竺鼠、倉鼠、猴子、豬、狗、貓、馬、綿羊、山羊、牛及兔子。較佳地，個體是人類。Basically, the subject that can be treated by the method of the present invention is a mammal, for example, humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cows and rabbits. Preferably, the subject is a human.
基於對B7-H3蛋白的結合親和力及/或專一性，本發明mAb或抗體片段可作為一種偵測抗體，用以偵測表現或過量表現B7-H3的癌症/癌化細胞。因此，本揭示內容的第五態樣是關於一種利用本發明mAb來診斷一個體之癌症的方法。在此態樣中，本發明mAb較佳是與一報導分子連接，舉例來說，螢光團或酵素。本發明方法包含：(a)由個體分離一生物檢體；(b)將生物檢體與本發明mAb混合；(c)偵測報導分子的表現；以及(d)基於步驟(c)的結果來診斷個體是否罹患癌症，其中當步驟(c)偵測到報導分子時，代表個體患有癌症。Based on the binding affinity and/or specificity to B7-H3 protein, the mAb or antibody fragment of the present invention can be used as a detection antibody to detect cancer/cancerous cells that express or overexpress B7-H3. Therefore, the fifth aspect of the present disclosure is a method of using the mAb of the present invention to diagnose cancer in an individual. In this aspect, the mAb of the present invention is preferably linked to a reporter molecule, for example, a fluorophore or an enzyme. The method of the present invention comprises: (a) isolating a biological sample from an individual; (b) mixing the biological sample with the mAb of the present invention; (c) detecting the expression of a reporter molecule; and (d) diagnosing whether the individual has cancer based on the result of step (c), wherein when the reporter molecule is detected in step (c), it means that the individual has cancer.
或者是，本發明mAb可作為一種捕捉抗體，用以捕捉表現或過量表現B7-H3的癌症/癌化細胞，之後再利用與報導分子偶聯之二級抗體(即，偵測抗體)進行偵測，其中所述二級抗體可辨識本發明mAb (例如，mAb的Fc區)或癌化細胞。Alternatively, the mAb of the present invention can be used as a capture antibody to capture cancer/cancerous cells that express or overexpress B7-H3, and then detected using a secondary antibody (i.e., a detection antibody) coupled to a reporter molecule, wherein the secondary antibody can recognize the mAb of the present invention (e.g., the Fc region of the mAb) or the cancerous cells.
依據實施目的不同，本發明mAb可用於不同偵測試驗，舉例來說，免疫組織化學(immunohistochemistry, IHC)、免疫螢光(immunofluorescence)、ELISA)、流式細胞儀及西方墨點法。Depending on the purpose of implementation, the mAb of the present invention can be used in different detection tests, for example, immunohistochemistry (IHC), immunofluorescence (ELISA), flow cytometry and Western blot.
下文提出多個實驗例來說明本發明的某些態樣，以利本發明所屬技術領域中具有通常知識者實作本發明，且不應將這些實驗例視為對本發明範圍的限制。據信習知技藝者在閱讀了此處提出的說明後，可在不需過度解讀的情形下，完整利用並實踐本發明。此處所引用的所有公開文獻，其全文皆視為本說明書的一部分。實施例The following are several experimental examples to illustrate certain aspects of the present invention, so as to facilitate those with ordinary knowledge in the technical field to which the present invention belongs to implement the present invention, and these experimental examples should not be regarded as limiting thescope of the present invention. It is believed that after reading the description provided here, the skilled person can fully utilize and practice the present invention without over-interpretation. All public documents cited here are regarded as part of this specification in their entirety.
材料及方法Materials and Methods
製備PreparationB7-H3B7-H3蛋白protein
將用以編碼人類(胺基酸殘基29-466)或是小鼠或大鼠(胺基酸殘基29-244) B7-H3胞外域的核苷酸序列與小鼠Fc域融合，之後建構至pcDNA3.4載體。以FreeStyleTM293細胞分別表現及純化重組人類B7-H3-mFc、大鼠B7-H3-mFc及小鼠B7-H3-mFc蛋白。由R&D systems®購買重組石蟹獼猴(cynomolgus monkey) B7-H3蛋白。Nucleotide sequences encoding the human (amino acid residues 29-466) or mouse or rat (amino acid residues 29-244) B7-H3 extracellular domain were fused to the mouse Fc domain and then constructed into the pcDNA3.4 vector. Recombinant human B7-H3-mFc, rat B7-H3-mFc, and mouse B7-H3-mFc proteins were expressed and purified using FreeStyle™ 293 cells. Recombinant cynomolgus monkey B7-H3 protein was purchased from R&D systems® .
由合成的人類抗體庫篩選Screening by synthetic human antibody libraryB7-H3 mAbB7-H3 mAb
(A)(A)製備生物淘選的Preparation of BiopanningscFvscFv噬菌體Bacteriophage
將噬菌體表現之scFv抗體庫(包含約1011個抗體變異體)與TG1細胞培養於含有每毫升100微克胺苄青黴素(ampicillin)及2%葡萄糖的2x酵母菌胰化蛋白(yeast tryptone, YT)培養液中，之後置於37°C振盪培養，直到600奈米的OD達到0.5。加入輔助噬菌體後，於37°C水浴槽繼續培養30分鐘。以4,000 rpm的轉速離心感染細胞15分鐘後，將細胞沉澱物輕輕懸浮於2x YTAK培養液(含有每毫升100微克胺苄青黴素及每毫升25微克康黴素(kanamycin)的YT培養液)中，接著於37°C振盪培養至隔日。以10,000 rpm的轉速離心產物20分鐘後，將1/5體積的PEG/NaCl (20%聚乙二醇8000，2.5 M NaCl)加至上清液，並置於4°C培養至少一小時。以10,000 rpm的轉速離心20分鐘後，利用無菌水洗滌沉澱物以移除細菌碎片。將1/5體積的PEG/NaCl加至上清液後，置於4°C培養至少一小時。以磷酸鹽緩衝液(phosphate-buffered saline, PBS)洗滌沉澱物以移除細菌碎片。The scFv antibody library (containing approximately 1011 antibody variants) expressed by the phage was incubated with TG1 cells in 2x yeast tryptone (YT) medium containing 100 μg of ampicillin per ml and 2% glucose, and then incubated at 37°C with shaking until the OD at 600 nm reached 0.5. After adding the helper phage, the cells were incubated in a 37°C water bath for 30 minutes. After centrifugation of infected cells at 4,000 rpm for 15 minutes, the cell pellet was gently suspended in 2x YTAK medium (YT medium containing 100 μg of ampicillin and 25 μg of kanamycin per ml) and incubated at 37°C with shaking until the next day. After centrifugation of the product at 10,000 rpm for 20 minutes, 1/5 volume of PEG/NaCl (20% polyethylene glycol 8000, 2.5 M NaCl) was added to the supernatant and incubated at 4°C for at least one hour. After centrifugation at 10,000 rpm for 20 minutes, the pellet was washed with sterile water to remove bacterial debris. After adding 1/5 volume of PEG/NaCl to the supernatant, incubate at 4°C for at least one hour. Wash the precipitate with phosphate-buffered saline (PBS) to remove bacterial debris.
(B)(B)篩選噬菌體Phage screening
以人類B7-H3-mFc蛋白塗覆ELISA盤後，於4°C反應至隔日。加入300微升之5%脫脂牛奶(稀釋於PBS中)，並置於37°C反應90分鐘。以PBS洗滌三次後，將100微升之稀釋於5% MPBS (含有2%脫脂牛奶的PBS)的噬菌體(約1011到1012個噬菌體)加入ELISA盤，並於37°C培養90分鐘。以PBST (含有Tween®20的PBS)洗滌ELISA盤三次後，以PBS洗滌一次。將100微升之100 mM三甲胺(trimethylamine, TEA)加至ELISA盤以沖提噬菌體，之後加入1 M Tris (pH7.4)中和沖提產物。將得到的噬菌體與TG1細胞混合後，置於37°C反應30分鐘。以4,000 rpm的轉速離心15分鐘後，將TG1細胞懸浮於2x TY培養液中，之後接種於2x YTAG (含有胺苄青黴素及葡萄糖的YT培養液)培養盤以擴增感染的細胞。After coating the ELISA plate with human B7-H3-mFc protein, incubate at 4°C until the next day. Add 300 μL of 5% skim milk (diluted in PBS) and incubate at 37°C for 90 minutes. After washing three times with PBS, add 100 μL of phage (about 1011 to 1012 phage) diluted in 5% MPBS (PBS containing 2% skim milk) to the ELISA plate and incubate at 37°C for 90 minutes. Wash the ELISA plate three times with PBST (PBS containing Tween® 20) and once with PBS. 100 μl of 100 mM trimethylamine (TEA) was added to the ELISA plate to extract the phage, and then 1 M Tris (pH 7.4) was added to neutralize the extracted product. The obtained phage was mixed with TG1 cells and incubated at 37°C for 30 minutes. After centrifugation at 4,000 rpm for 15 minutes, the TG1 cells were suspended in 2x TY medium and then inoculated into 2x YTAG (YT medium containing ampicillin and glucose) plates to expand the infected cells.
(C)(C)製備下一輪的噬菌體Prepare the next round of phage
將5-6毫升之含有15%甘油的2x YT培養液加至細菌盤中，使細菌由盤子上刮取下來。將50-100微升之刮取的細菌培養於100毫升之2x YTAG中，於37°C振盪培養，直到600奈米的OD達到0.5。加入輔助噬菌體後，於37°C水浴槽培養30分鐘。以4,000 rpm的轉速離心感染細胞15分鐘後，將細胞沉澱物輕輕懸浮於2x YTAK培養液中，接著於37°C振盪培養至隔日。以10,000 rpm的轉速離心產物20分鐘後，將1/5體積(8毫升)的PEG/NaCl加至上清液，並置於4°C培養至少一小時。以10,000 rpm的轉速離心20分鐘後，利用PBS洗滌沉澱物以移除細菌碎片。將噬菌體抗體庫注入以人類B7-H3-mFc蛋白塗覆的多孔洞微盤。利用ELISA篩選法來確認會表現抗-B7-H3 scFv的噬菌體殖株。Add 5-6 ml of 2x YTAK medium containing 15% glycerol to the bacterial plate and scrape the bacteria from the plate. Add 50-100 μl of the scraped bacteria to 100 ml of 2x YTAK and culture at 37°C with shaking until the OD at 600 nm reaches 0.5. After adding helper phage, culture in a 37°C water bath for 30 minutes. Centrifuge the infected cells at 4,000 rpm for 15 minutes, gently resuspend the cell pellet in 2x YTAK medium, and then culture at 37°C with shaking until the next day. After centrifugation at 10,000 rpm for 20 minutes, 1/5 volume (8 ml) of PEG/NaCl was added to the supernatant and incubated at 4°C for at least one hour. After centrifugation at 10,000 rpm for 20 minutes, the precipitate was washed with PBS to remove bacterial debris. The phage antibody library was injected into a multi-well microplate coated with human B7-H3-mFc protein. Phage clones expressing anti-B7-H3 scFv were confirmed by ELISA screening.
(D)(D)利用useELISAELISA來篩選To filterB7-H3B7-H3陽性噬菌體Positive phage
將步驟(C)確認的噬菌體殖株培養於200毫升之2x YTAG培養液中，並於37°C反應至隔日。將50微升的培養產物轉移至每孔洞含有200微升之2x YTAG的96孔洞盤。將培養盤至於37°C振盪培養2小時。加入109pfu的輔助噬菌體，之後於37°C培養90分鐘。以4,000 rpm的轉速離心30分鐘後，將沉澱物懸浮於300微升的2x YTAG培養液中，並於30°C反應至隔日。以4,000 rpm的轉速離心細胞30分鐘，取100微升的上清液至ELISA盤(以人類B7-H3-mFc蛋白塗覆，並經300微升之2% MPBS預處理)中。90分鐘後，以PBST洗滌ELISA盤三次。將適當稀釋之與HRP鍵結的抗-M13抗體(稀釋於2% MPBS)加至ELISA盤，之後加入TMB受質溶液(3,3’,5,5’-四甲基聯苯胺(3,3’,5,5’-Tetramethylbenzidine))進行呈色反應。加入50微升之1 M硫酸中止反應。測量450奈米及650奈米的OD，由OD450測量的數值減去OD650測量的數值。The phage strain confirmed in step (C) was cultured in 200 ml of 2x YTAG medium and incubated at 37°C until the next day. 50 μl of the culture product was transferred to a 96-well plate containing 200 μl of 2x YTAG in each well. The culture plate was shaken and incubated at 37°C for 2 hours. 109 pfu of helper phage was added, followed by incubation at 37°C for 90 minutes. After centrifugation at 4,000 rpm for 30 minutes, the precipitate was suspended in 300 μl of 2x YTAG medium and incubated at 30°C until the next day. Centrifuge the cells at 4,000 rpm for 30 minutes and take 100 μl of the supernatant to an ELISA plate (coated with human B7-H3-mFc protein and pretreated with 300 μl of 2% MPBS). After 90 minutes, wash the ELISA plate three times with PBST. Add the appropriately diluted anti-M13 antibody conjugated to HRP (diluted in 2% MPBS) to the ELISA plate, and then add TMB substrate solution (3,3',5,5'-Tetramethylbenzidine) for color development. Add 50 μl of 1 M sulfuric acid to stop the reaction. Measure the OD at 450 nm and 650 nm, and subtract the value measured at OD650 from the value measured at OD450 .
由噬菌體表現之抗體庫進行三到四輪生物淘選後，篩選得到60個具有顯著B7-H3親和力的scFv噬菌體殖株，之後將其VH及VL鏈分別插入至含有CH及CL鏈的表現載體，以建構編碼序列。將構建載體轉染至FreeStyleTM293細胞。以蛋白A Sepharose®Fast Flow純化抗體。純化後，測量OD280奈米以定量抗體，並以還原及非還原性聚丙烯醯胺凝膠電泳(polyacrylamide gel electrophoresis, PAGE)進行分析。After three to four rounds of bio-panning from the phage-expressed antibody library, 60 scFv phage strains with significant B7-H3 affinity were screened, and their VH and VL chains were then inserted into expression vectors containing CH and CL chains, respectively, to construct the coding sequence. The constructed vectors were transfected into FreeStyleTM 293 cells. Antibodies were purified using Protein A Sepharose® Fast Flow. After purification, OD280 nm was measured to quantify the antibodies, and reduced and non-reduced polyacrylamide gel electrophoresis (PAGE) was used for analysis.
ELISAELISA結合試驗Binding test
以ELISA評估全長抗體對人類、猴子、小鼠或大鼠B7-H3的結合親和力。簡單來說，以溶於塗覆緩衝液的B7-H3蛋白(即，人類B7-H3-mFc蛋白、石蟹獼猴B7-H3蛋白、小鼠B7-H3 Fc嵌合蛋白或大鼠B7-H3 Fc嵌合蛋白)塗覆96孔洞盤後，於4°C放置至隔日。以PBS洗滌3次後，將96孔洞盤倒置並於乾淨的吸水紙上輕敲以去除多餘的液體。加入阻斷緩衝液(含有1%牛血清白蛋白(bovine serum albumin, BSA)的PBS)，之後置於37°C振盪培養1小時。以PBS洗滌3次後，加入連續稀釋的抗-B7-H3抗體(溶於含有1% BSA的PBS)。將ELISA盤至於37°C振盪培養1小時。以PBS洗滌3次後，加入二級抗體(與過氧化酶鍵結之AffiniPureTMF(ab’)2片段山羊抗-人類IgG (H＋L))，並於37°C振盪培養1小時。以PBS洗滌3次後，加入TMB受質，置於室溫(避免光照)反應5分鐘。加入1 N HCl中止溶液，接著測量450/650奈米的吸光值。利用軟體決定抗體的結合親和力(EC50)。The binding affinity of full-length antibodies to human, monkey, mouse, or rat B7-H3 was assessed by ELISA. Briefly, a 96-well plate was coated with B7-H3 protein (i.e., human B7-H3-mFc protein, stone crab macaque B7-H3 protein, mouse B7-H3 Fc chimeric protein, or rat B7-H3 Fc chimeric protein) dissolved in coating buffer and placed at 4°C until the next day. After washing three times with PBS, the 96-well plate was inverted and tapped on clean absorbent paper to remove excess liquid. Blocking buffer (PBS containing 1% bovine serum albumin (BSA)) was added and then incubated at 37°C with shaking for 1 hour. After washing 3 times with PBS, add serially diluted anti-B7-H3 antibody (dissolved in PBS containing 1% BSA). Incubate the ELISA plate at 37°C with shaking for 1 hour. After washing 3 times with PBS, add secondary antibody (AffiniPureTM F(ab')2 fragment goat anti-human IgG (H+L) conjugated with peroxidase) and incubate at 37°C with shaking for 1 hour. After washing 3 times with PBS, add TMB substrate and react at room temperature (protected from light) for 5 minutes. Add 1 N HCl to stop the solution, and then measure the absorbance at 450/650 nm. Use software to determine the binding affinity (EC50) of the antibody.
表面電漿共振Surface Plasmon Resonance(surface plasmon resonance, SPR)(surface plasmon resonance, SPR)
利用BiacoreTM試驗決定本發明抗體與人類B7-H3的結合動力學。依據使用者操作手冊活化羧甲基化葡聚醣生物感測器晶片(CM5)。利用固定化緩衝液稀釋抗-人類IgG (Fc)後，以每分鐘10微升的流速注入晶片，以產生約10,000反應單位(response unit, RU)的偶聯蛋白，接著注入1 M乙醇胺阻斷未反應的基團。將人類抗-B7-H3抗體捕捉於抗-人類IgG晶片。為測量結合動力學，將二倍連續稀釋的人類B7-H3-mFc蛋白(由40 nM稀釋至0.3125 nM)注入製造商提供的HBS-EP+BiacoreTM運行緩衝液，流速為每分鐘30微升。藉由參照扣除來補償結果以決定本發明抗體及人類B7-H3-mFc蛋白的結合親和力。The binding kinetics of the antibodies of the present invention to human B7-H3 were determined using a BiacoreTM assay. The carboxymethylated dextran biosensor chip (CM5) was activated according to the user manual. Anti-human IgG (Fc) was diluted with immobilization buffer and injected into the chip at a flow rate of 10 μl/min to generate approximately 10,000 response units (RU) of coupled protein, followed by injection of 1 M ethanolamine to block unreacted groups. Human anti-B7-H3 antibodies were captured on the anti-human IgG chip. To measure binding kinetics, two-fold serial dilutions of human B7-H3-mFc protein (from 40 nM to 0.3125 nM) were injected into the HBS-EP+ BiacoreTM running buffer provided by the manufacturer at a flow rate of 30 μl per minute. The results were compensated by reference subtraction to determine the binding affinity of the antibody of the present invention and human B7-H3-mFc protein.
流式細胞儀Flow cytometer
以間接免疫螢光染色來決定本發明抗體與B7-H3表現細胞的結合親和力。簡單來說，將十萬個頭頸部鱗狀細胞癌(head and neck squamous cell carcinoma, HNSCC)、前列線癌、非小細胞癌(non-small cell lung cancer, NSCLC)或肝癌(hepatocellular carcinoma, HCC)細胞懸浮於FACS緩衝液(含有0.5-1% BSA或5-10% FBS，以及0.1% NaN3的PBS)中，並與本發明抗體(每孔洞1微克)混合於96孔洞之U型底微盤中，之後於4°C反應60分鐘。以PBS洗滌後，加入二級抗體(溶於FACS緩衝液之與Alexa Fluor®647鍵結的抗-人類IgG)，並於4°C反應60分鐘。利用流式細胞儀測量細胞的螢光強度，之後以軟體分析結果。Indirect immunofluorescence staining was used to determine the binding affinity of the antibodies of the present invention to B7-H3 expressing cells. Briefly, 100,000 head and neck squamous cell carcinoma (HNSCC), prostate cancer, non-small cell lung cancer (NSCLC) or hepatocellular carcinoma (HCC) cells were suspended in FACS buffer (PBS containing 0.5-1% BSA or 5-10% FBS and 0.1% NaN3) and mixed with the antibodies of the present invention (1 μg per well) in a 96-well U-bottom microplate and then reacted at 4°C for 60 minutes. After washing with PBS, the secondary antibody (anti-human IgG conjugated to Alexa Fluor® 647 in FACS buffer) was added and reacted at 4°C for 60 minutes. The fluorescence intensity of the cells was measured using a flow cytometer, and the results were analyzed using software.
以間接免疫螢光染色來決定本發明抗體的內化反應。簡單來說，將癌細胞(6x105，包含HNSCC細胞及前列腺癌細胞)懸浮於FACS緩衝液，並與本發明抗體(每孔洞6微克)混合於96孔洞之U型底微盤中，之後於4°C反應60分鐘。利用PBS洗滌以移除過多的抗體後，將1.5x105細胞分為4等分，並於37°C培養30、60、120或180分鐘。接著，將細胞置於冰上，之後以二級抗體進行染色。以PBS洗滌後，加入二級抗體(溶於FACS緩衝液之與Alexa Fluor®647鍵結的抗-人類IgG)，並於4°C反應60分鐘。利用流式細胞儀測量細胞的螢光強度，之後以軟體分析結果。Indirect immunofluorescence staining was used to determine the internalization of the antibodies of the present invention. Briefly, cancer cells (6x105 , including HNSCC cells and prostate cancer cells) were suspended in FACS buffer and mixed with the antibodies of the present invention (6 μg per well) in a 96-well U-bottom microplate, followed by reaction at 4°C for 60 minutes. After washing with PBS to remove excess antibodies, 1.5x105 cells were divided into 4 equal parts and incubated at 37°C for 30, 60, 120 or 180 minutes. Then, the cells were placed on ice and stained with secondary antibodies. After washing with PBS, the secondary antibody (anti-human IgG conjugated to Alexa Fluor® 647 in FACS buffer) was added and reacted at 4°C for 60 minutes. The fluorescence intensity of the cells was measured using a flow cytometer, and the results were analyzed using software.
細胞培養Cell culture
將Detroit 562細胞(人類咽喉鱗狀細胞癌細胞株)培養於含有0.1 mM非必需胺基酸、1.0 mM丙酮酸鈉、0.1%乳清蛋白水解物及10%胎牛血清(fetal bovine serum, FBS)的最低必需培養液(minimum essential medium, MEM)中。Detroit 562 cells (a human pharyngeal squamous cell carcinoma cell line) were cultured in minimum essential medium (MEM) containing 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 0.1% whey protein hydrolysate, and 10% fetal bovine serum (FBS).
將FaDu細胞(人類HNSCC細胞株)、DU145細胞(源自前列腺癌腦轉移的人類細胞株)及HepG2細胞(人類HCC細胞株)培養於含有0.1 mM非必需胺基酸、1.0 mM丙酮酸鈉及10% FBS的MEM中。利用CRISPR/Casp9技術製備B7-H3敲除FaDu (FaDu/B7-H3 KO)細胞。FaDu cells (human HNSCC cell line), DU145 cells (human cell line derived from prostate cancer brain metastasis), and HepG2 cells (human HCC cell line) were cultured in MEM containing 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. B7-H3 knockout FaDu (FaDu/B7-H3 KO) cells were prepared using CRISPR/Casp9 technology.
將CAL 27細胞(人類口腔腺鱗狀細胞癌細胞株)、Huh7細胞(人類HCC細胞株)及A549細胞(人類NSCLC細胞株)培養於含有10%FBS的DMEM (Dulbecco’s Modified Eagle Medium)中。CAL 27 cells (human oral adenosquamous cell carcinoma cell line), Huh7 cells (human HCC cell line), and A549 cells (human NSCLC cell line) were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS.
將OECM-1細胞(人類口腔鱗狀細胞癌細胞株)、LNCaP細胞(源自轉移性淋巴結病變的人類前列腺癌細胞株)、H1299細胞(人類NSCLC細胞株)及NCI-H520細胞(人類肺鱗狀細胞癌細胞株)培養於含有10% FBS的RPMI中。OECM-1 cells (human oral squamous cell carcinoma cell line), LNCaP cells (human prostate cancer cell line derived from metastatic lymphadenopathy), H1299 cells (human NSCLC cell line), and NCI-H520 cells (human lung squamous cell carcinoma cell line) were cultured in RPMI containing 10% FBS.
將PC-3細胞(人類前列腺癌細胞株)培養於含有2 mM L-麩醯胺酸、每公升1.5公克碳酸氫鈉及7% FBS的Ham’s F12K培養液中。PC-3 cells (a human prostate cancer cell line) were cultured in Ham’s F12K medium containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 7% FBS.
將HA59T細胞(人類HCC細胞株)培養於含有4 mM L-麩醯胺酸、每公升4.5公克葡萄糖、0.1 mM非必需胺基酸及10% FBS的DMEM中。HA59T cells (a human HCC cell line) were cultured in DMEM containing 4 mM L-glutamine, 4.5 g glucose/liter, 0.1 mM non-essential amino acids, and 10% FBS.
將HuT78細胞(人類皮膚T淋巴球細胞株)培養於含有20% FBS的IMDM (Iscove’s Modified Dulbecco’s Medium)中。HuT78 cells (a human dermal T lymphocyte cell line) were cultured in IMDM (Iscove’s Modified Dulbecco’s Medium) containing 20% FBS.
將Ba/F3細胞(小鼠介白素-3相關前-B細胞株)培養於含有10% FBS及每毫升10奈克之小鼠IL-3的RPMI中。Ba/F3 cells (a mouse interleukin-3-associated pro-B cell line) were cultured in RPMI containing 10% FBS and 10 ng/mL mouse IL-3.
將EMT6細胞(小鼠乳癌細胞株)培養於含有2 mM L-麩醯胺酸及15% FBS的Waymouth’s培養液(MB 752/1)中。EMT6 cells (mouse breast cancer cell line) were cultured in Waymouth’s medium (MB 752/1) containing 2 mM L-glutamine and 15% FBS.
將ID8細胞(小鼠卵巢表層上皮細胞株)培養於含有4% FBS及1X ITS (重組人類胰島素、人類運鐵蛋白及亞硒酸鈉之混合物)的DMEM中。ID8 cells (a mouse ovarian epithelial cell line) were cultured in DMEM containing 4% FBS and 1X ITS (a mixture of recombinant human insulin, human transferrin, and sodium selenite).
抗體antibody--藥物偶聯物Drug conjugates(antibody-drug conjugate, ADC)(antibody-drug conjugate, ADC)
利用三甘露醣抗體藥物偶聯平台來製備本發明ADC。具體來說，將0.04毫升之連接子-負載藥物(10 mM，溶於二甲硫(dimethyl sulfide, DMSO)或二甲基乙醯胺(dimethylacetamide, DMA))與0.12毫升之DMSO或DMA緩慢加入含有mAb-4 疊氮基(Azide, Az) (0.4毫升，每毫升5毫克)的MES緩衝液(pH6.5)中。於37°C攪拌反應混合物18小時。將得到的抗體-藥物偶聯體於MES緩衝液(pH6.5)以標稱分子量極限(nominal molecular weight limit, NMWL)為30 kDa的離心過濾器去除鹽類並濃縮，以分別得到91H06-MMAE及91H06-MMAF。在本發明試驗中，用以連接mAb 91H06及負載藥物(MMAE或MMAF)的連接子包含一PEG連接子及一與PEG連接子連結的蛋白酶敏感型連接子(纈胺酸-瓜胺酸雙肽，或麩胺酸-纈胺酸-瓜胺酸三肽)。利用液相層析質譜法測量ADC的藥物與抗體比(drug-to-antibody ratio, DAR)。91H06-MMAE與91H06-MMAF的DAR約介於3.5到3.9之間。The ADC of the present invention was prepared using the trimannose antibody-drug conjugation platform. Specifically, 0.04 ml of linker-carrier drug (10 mM, dissolved in dimethyl sulfide (DMSO) or dimethylacetamide (DMA)) and 0.12 ml of DMSO or DMA were slowly added to MES buffer (pH 6.5) containing mAb-4 azide (Az) (0.4 ml, 5 mg per ml). The reaction mixture was stirred at 37°C for 18 hours. The obtained antibody-drug conjugate was desalted and concentrated in MES buffer (pH 6.5) using a centrifugal filter with a nominal molecular weight limit (NMWL) of 30 kDa to obtain 91H06-MMAE and 91H06-MMAF, respectively. In the present invention, the linker used to link mAb 91H06 and the loaded drug (MMAE or MMAF) comprises a PEG linker and a protease-sensitive linker (valine-citrulline dipeptide or glutamine-valine-citrulline tripeptide) linked to the PEG linker. The drug-to-antibody ratio (DAR) of the ADC was measured by liquid chromatography-mass spectrometry. The DAR of 91H06-MMAE and 91H06-MMAF is between 3.5 and 3.9.
細胞毒殺試驗Cytotoxicity assay
將細胞以三重複方式種植於96孔洞盤，並放置至隔日。將連續稀釋之本發明ADC (最終濃度：500 nM到0.025 nM)加至各孔洞。於37°C培養120小時後，依據使用者操作手冊於避光環境中配製CellTiter-Glo®試劑，並加至各孔洞。將培養盤置於振盪器振盪2分鐘，以混合溶液並誘導細胞裂解。於室溫反應10分鐘後，記錄發光值，並利用軟體計算結果。Cells were seeded in triplicate in a 96-well plate and left to stand the next day. Serially diluted ADCs of the present invention (final concentration: 500 nM to 0.025 nM) were added to each well. After 120 hours of incubation at 37°C, CellTiter-Glo® reagent was prepared according to the user manual in a dark environment and added to each well. The culture plate was placed on a shaker for 2 minutes to mix the solution and induce cell lysis. After 10 minutes of reaction at room temperature, the luminescence value was recorded and the results were calculated using the software.
動物試驗Animal testing
於NOD SCID小鼠建立Detroit 562異種移植模式及FaDu異種移植模式，以評估91H06-MMAE的活體內抗腫瘤功效。本研究使用6-8週大的NOD SCID小鼠。每籠飼養5隻小鼠。所有的動物皆飼養於19-25°C之12小時光照/12小時黑暗的環境中。動物可隨意取得囓齒動物顆粒食物及飲用水。Detroit 562 xenograft model and FaDu xenograft model were established in NOD SCID mice to evaluate the in vivo antitumor efficacy of 91H06-MMAE. NOD SCID mice aged 6-8 weeks were used in this study. Five mice were housed per cage. All animals were housed at 19-25°C in a 12-hour light/12-hour dark environment. Animals had ad libitum access to rodent pellet food and drinking water.
將Detroit 562腫瘤細胞(3×106細胞)或FaDu腫瘤細胞(1×106細胞)與Matrigel®(1:1, v/v)混合，劑量體積為0.1毫升，之後將混合物皮下(subcutaneously, SC)注射至小鼠的右前側。當平均腫瘤體積到達約160立方毫米(mm3；於Detroit 562腫瘤模式)或約100立方毫米(於FaDu腫瘤模式)時，將帶有腫瘤的小鼠隨機分為3組，其中各組是由5隻小鼠所組成。將91H06-MMAE或載體溶液(25 mM MES緩衝液, pH6.5)靜脈內投予至帶有腫瘤的小鼠。起始投予之日註記為第0天。接受載體溶液之動物在本研究作為疾病(載體)對照組，以計算腫瘤生長抑制(tumor growth inhibition, TGI)率。在Detroit 562腫瘤模式中，對實驗組動物投予每公斤1.5毫克(1.5 mpk)或每公斤5毫克(5 mpk)的91H06-MMAE，每週投予一次，連續投予三週；在FaDu腫瘤模式中，對實驗組動物投予單一劑量之每公斤1.5毫克(1.5 mpk)或每公斤5毫克(5 mpk)的91H06-MMAE。Detroit 562 tumor cells (3×106 cells) or FaDu tumor cells (1×106 cells) were mixed with Matrigel® (1:1, v/v) in a dose volume of 0.1 ml and then injected subcutaneously (SC) into the right anterior flank of mice. When the average tumor volume reached approximately 160 cubic millimeters (mm3 ; in the Detroit 562 tumor model) or approximately 100 cubic millimeters (in the FaDu tumor model), the tumor-bearing mice were randomly divided into 3 groups, each consisting of 5 mice. 91H06-MMAE or vehicle solution (25 mM MES buffer, pH 6.5) was intravenously administered to tumor-bearing mice. The day of the initial administration was noted as day 0. Animals receiving the vehicle solution served as the disease (vehicle) control group in this study to calculate the tumor growth inhibition (TGI) rate. In the Detroit 562 tumor model, animals in the experimental group were administered 1.5 mg/kg (1.5 mpk) or 5 mg/kg (5 mpk) of 91H06-MMAE once a week for three consecutive weeks; in the FaDu tumor model, animals in the experimental group were administered a single dose of 1.5 mg/kg (1.5 mpk) or 5 mg/kg (5 mpk) of 91H06-MMAE.
在評估91H06-MMAE之抗腫瘤功效的實驗中，將Detroit 562腫瘤細胞(3×106細胞)與Matrigel®(1:1, v/v)混合，劑量體積為0.1毫升，之後將混合物皮下注射至小鼠的右前側。當平均腫瘤體積到達約1,500立方毫米時，對帶有腫瘤之小鼠靜脈內投予每公斤5毫克(5 mpk)的91H06-MMAE，每週投予一次，連續投予三週。In the experiment to evaluate the anti-tumor efficacy of 91H06-MMAE, Detroit 562 tumor cells (3×106 cells) were mixed with Matrigel® (1:1, v/v) in a dose volume of 0.1 ml and then injected subcutaneously into the right front flank of mice. When the average tumor volume reached approximately 1,500 cubic millimeters, 5 mg/kg (5 mpk) of 91H06-MMAE was intravenously administered to tumor-bearing mice once a week for three consecutive weeks.
每週監測並記錄腫瘤體積、體重、死亡率及明顯的毒性徵象三次。使用數位游標尺測量並依據公式：TV = (W2×L)/2計算腫瘤體積(立方毫米)，其中W為腫瘤的寬度(毫米)，而L則為腫瘤的直徑長度(毫米)。以公式：GI=[1-(Tx-T0/Cx-C0)]×100%計算TGI百分比，其中Tx及Cx分別表示治療組及對照組在第X天的平均腫瘤體積。Tumor volume, body weight, mortality, and overt toxicity signs were monitored and recorded three times a week. Tumor volume (cubic millimeters) was measured using a digital vernier caliper and calculated according to the formula: TV = (W2 ×L)/2, where W is the width of the tumor (mm) and L is the diameter of the tumor (mm). TGI percentage was calculated using the formula: GI=[1-(Tx-T0/Cx-C0)]×100%, where Tx and Cx represent the average tumor volume on day X in the treatment group and control group, respectively.
實施例Embodiment11確認ConfirmmAbmAbs91H0691H06
1.11.1蛋白結合親和力Protein binding affinity
本實施例將評估mAb 91H06或伊非那他單抗(一種B7-H3靶向的抗體，作為正對照組)對人類、猴子、小鼠或大鼠之B7-H3的結合活性。如本揭示內容之「材料及方法」所述，將mAb 91H06加至以人類B7-H3-mFc、石蟹獼猴B7-H3、小鼠B7-H3 mFc或大鼠B7-H3 mFc嵌合蛋白塗覆的培養盤中，之後利用ELISA決定二者之間的結合親活性。This example will evaluate the binding activity of mAb 91H06 or ifenatumomab (a B7-H3-targeted antibody, as a positive control group) to human, monkey, mouse or rat B7-H3. As described in the "Materials and Methods" of this disclosure, mAb 91H06 was added to a culture plate coated with human B7-H3-mFc, stone macaque B7-H3, mouse B7-H3 mFc or rat B7-H3 mFc chimeric protein, and then the binding affinity between the two was determined by ELISA.
結果指出，本發明mAb 91H06對人類、猴子、小鼠或大鼠B7-H3具有EC50＜10-10的結合親和力 (表1)。相較之下，伊非那他單抗僅能辨識人類及猴子的B7-H3 (表1)。The results indicated that mAb 91H06 of the present invention had a binding affinity of EC50 <10-10 for human, monkey, mouse or rat B7-H3 (Table 1). In contrast, ifenatumomab could only recognize human and monkey B7-H3 (Table 1).
表1 伊非那他單抗或mAb 91H06對特定B7-H3蛋白的結合親和力
進一步BiacoreTM試驗分析mAb 91H06的結合動力學。BiacoreTM試驗的結果確認了mAb 91H06對人類、猴子、小鼠或大鼠之B7-H3的交叉反應性(表2)。The binding kinetics of mAb 91H06 were further analyzed by Biacore™ assay. The results of Biacore™ assay confirmed the cross-reactivity of mAb 91H06 to B7-H3 of human, monkey, mouse or rat (Table 2).
表2 mAb 91H06對特定B7-H3蛋白的結合親和力
已知人類B7-H3與B7-H4 (31%)及B7家族的其他成員(24%至31%) 具有序列同源性。因此，本實施例亦評估本發明mAb是否會與其他B7蛋白產生交叉反應。結果指出，本發明mAb 91H06會選擇性地與B7-H3結合，而不會結合至B7家族的其他成員(第1圖)。It is known that human B7-H3 has sequence homology with B7-H4 (31%) and other members of the B7 family (24% to 31%). Therefore, this example also evaluated whether the mAb of the present invention would cross-react with other B7 proteins. The results indicated that the mAb 91H06 of the present invention selectively binds to B7-H3 and does not bind to other members of the B7 family (Figure 1).
1.21.2細胞結合親和力Cell binding affinity
本實施例將檢測本發明mAb對不同人類癌細胞的結合活性。結果指出，mAb 91H06可辨識並結合至各種HNSCC細胞株(包含Detroit 562、CAL 27、FaDu及OECM-1細胞)，以及各種前列腺癌細胞株(包含DU 145、LNCaP及PC-3細胞) (表3)。除了HNSCC及HCC細胞株之外，mAb 91H06亦對不同HCC 細胞株(包含HepG2、HA59T及Huh7細胞)，以及不同的NSCLC細胞株(包含A549、H1299及NCI-H520細胞)具有結合親和力(表3)。值得注意的是，mAb 91H06對測試癌細胞的結合活性與對照抗體(伊非那他單抗)相當，證實mAb 91H06可有效靶向表現B7-H3的癌細胞。This example will detect the binding activity of the mAb of the present invention to different human cancer cells. The results indicate that mAb 91H06 can recognize and bind to various HNSCC cell lines (including Detroit 562, CAL 27, FaDu and OECM-1 cells), and various prostate cancer cell lines (including DU 145, LNCaP and PC-3 cells) (Table 3). In addition to HNSCC and HCC cell lines, mAb 91H06 also has binding affinity to different HCC cell lines (including HepG2, HA59T and Huh7 cells), and different NSCLC cell lines (including A549, H1299 and NCI-H520 cells) (Table 3). Notably, the binding activity of mAb 91H06 to the tested cancer cells was comparable to that of the control antibody (ifenatumomab), demonstrating that mAb 91H06 can effectively target cancer cells expressing B7-H3.
表3 mAb 91H06對特定癌細胞的細胞結合活性
此外，本實施例也評估mAb 91H06對B7-H3低表現量之癌細胞株(即，HuT78細胞)及B7-H3敲除癌細胞株(即，B7-H3敲除FaDu細胞)的結合活性。結果指出，mAb 91H06不會結合至B7-H3陰性癌細胞(表3)，證明mAb 91H06對B7-H3高表現量之癌細胞的結合專一性。In addition, the present example also evaluates the binding activity of mAb 91H06 to cancer cell lines with low B7-H3 expression (i.e., HuT78 cells) and B7-H3 knockout cancer cell lines (i.e., B7-H3 knockout FaDu cells). The results indicate that mAb 91H06 does not bind to B7-H3 negative cancer cells (Table 3), demonstrating the binding specificity of mAb 91H06 to cancer cells with high B7-H3 expression.
為確認本發明mAb的跨物種反應性，本實施例進一步分析mAb 91H06對囓齒動物細胞的結合活性。依據流式細胞儀的分析結果，相較於基準抗體伊非那他單抗，其對囓齒動物細胞不具結合親和力(第2圖之小圖(A)及(C))，mAb 91H06可辨識並結合至表現B7-H3的囓齒動物細胞株(包含EMT6及ID8細胞；第2圖之小圖(B)及(D))，而不會結合至B7-H3陰性的Ba/F3細胞(結果未顯示)。該些數據證實本發明mAb 91H06對B7-H3陽性囓齒動物細胞具有跨物種反應性。To confirm the cross-species reactivity of the mAb of the present invention, the binding activity of mAb 91H06 to rodent cells was further analyzed in this example. According to the results of flow cytometric analysis, compared with the benchmark antibody ifenatumab, which has no binding affinity to rodent cells (panels (A) and (C) of Figure 2), mAb 91H06 can recognize and bind to rodent cell lines expressing B7-H3 (including EMT6 and ID8 cells; panels (B) and (D) of Figure 2), but not to Ba/F3 cells that are negative for B7-H3 (results not shown). These data demonstrate that mAb 91H06 of the present invention has cross-species reactivity to B7-H3-positive rodent cells.
1.31.3競爭型結合試驗Competitive Binding Test
已知結合至相同標的之不同表位的抗體可能產生不同生物活性(例如，結合導介之內化反應)。因此，利用競爭型結合試驗來決定本發明mAb 91H06及伊非那他單抗於B7-H3蛋白的相對表位位置。表4的結果指出，伊非那他單抗(每毫升4微克)的存在會改變伊非那他單抗-生物素(biotin)對B7-H3蛋白的結合親和力；相較之下，本發明mAb 91H06則不會影響伊非那他單抗-生物素與B7-H3蛋白的結合。結果指出，mAb 91H06不會與伊非那他單抗競爭結合至B7-H3蛋白，且mAb 91H06辨識的B7-H3表位與伊非那他單抗辨識的B7-H3表位不同。It is known that antibodies binding to different epitopes of the same target may produce different biological activities (e.g., binding-mediated internalization reactions). Therefore, a competitive binding assay was used to determine the relative epitope locations of mAb 91H06 and ifenatumab on the B7-H3 protein. The results in Table 4 indicate that the presence of ifenatumab (4 μg/mL) changes the binding affinity of ifenatumab-biotin to the B7-H3 protein; in contrast, mAb 91H06 of the present invention does not affect the binding of ifenatumab-biotin to the B7-H3 protein. The results indicated that mAb 91H06 did not compete with ifinatumomab for binding to the B7-H3 protein and that the B7-H3 epitope recognized by mAb 91H06 was different from the B7-H3 epitope recognized by ifinatumomab.
表4 在伊非那他單抗或mAb 91H06存在或不存在時，伊非那他單抗-生物素對B7-H3蛋白的結合親和力
1.41.4細胞內化反應Cell internalization reaction
ADC是一種具潛力的癌症治療手段，其中是將負載藥物(例如，細胞毒性藥物)與抗體(例如，抗-H7-H3 mAb)偶聯，藉由抗體的結合特性將負載藥物靶向並運送到表現對應抗原(例如，B7-H3)的癌細胞，據以使個體體內的脫靶毒性及/或副作用最小化。ADC的關鍵在於抗體能夠與癌細胞上的抗原結合並被內化。被內化的ADC在溶酶體(lysosome)或晚期內體(endosome)中反應，進而在癌細胞中釋放出活性細胞毒素。基於細胞內化是ADC功能的先決條件，相關研究應廣泛評估抗體引發的內化反應，以確保其適當的亞細胞功能。整體來說， 選擇對標的抗原具有高親和力及專一性的抗體在製備ADC時相當重要。此外，標的抗原必須主要表現於標的細胞(例如，癌細胞)的表面，而於健康細胞上具有最少的表現量。再著，一個理想的ADC應快速被內化，以驅動細胞毒性負載藥物於癌細胞中的遞送及釋放。因此，本實施例將評估本發明mAb是否可誘發細胞內化反應。ADC is a potential cancer treatment in which a drug payload (e.g., a cytotoxic drug) is coupled to an antibody (e.g., anti-H7-H3 mAb) and the binding properties of the antibody are used to target and deliver the drug payload to cancer cells expressing the corresponding antigen (e.g., B7-H3), thereby minimizing off-target toxicity and/or side effects in the individual. The key to ADC is that the antibody can bind to the antigen on the cancer cell and be internalized. The internalized ADC reacts in the lysosome or late endosome, thereby releasing active cytotoxins in the cancer cell. Since cellular internalization is a prerequisite for ADC function, related studies should extensively evaluate the internalization reaction triggered by the antibody to ensure its appropriate subcellular function. In general, it is important to select antibodies with high affinity and specificity for the target antigen when preparing ADCs. In addition, the target antigen must be primarily expressed on the surface of the target cells (e.g., cancer cells) and have minimal expression on healthy cells. Furthermore, an ideal ADC should be quickly internalized to drive the delivery and release of cytotoxic cargo drugs in cancer cells. Therefore, this example will evaluate whether the mAbs of the present invention can induce a cell internalization reaction.
投予本發明mAb 91H06可誘發Detroit 562細胞表面B7-H3的內化反應，其細胞內化速度顯著快於對照抗體伊非那他單抗的細胞內化速度(表5)。相似的結果亦可見於CAL 27及FaDu細胞(表6及表7)。Administration of mAb 91H06 of the present invention can induce internalization of B7-H3 on the surface of Detroit 562 cells, and its cell internalization rate is significantly faster than that of the control antibody ifenatumomab (Table 5). Similar results can also be seen in CAL 27 and FaDu cells (Tables 6 and 7).
表5 mAb 91H06於Detroit 562細胞的內化反應
表6 mAb 91H06於Cal27細胞的內化反應
表7 mAb 91H06於FaDu細胞的內化反應
此外，投予本發明mAb 91H06亦可誘導DU 145及LNCaP細胞表面B7-H3的內化反應(表8及表9)，其造成的內化程度明顯優於對照抗體伊非那他單抗造成的內化程度。In addition, administration of mAb 91H06 of the present invention can also induce internalization of B7-H3 on the surface of DU 145 and LNCaP cells (Tables 8 and 9), and the degree of internalization caused by it is significantly better than that caused by the control antibody ifenatumomab.
表8 mAb 91H06於DU145細胞的內化反應
表9 mAb 91H06於LNCaP細胞的內化反應
該些結果暗示本發明mAb 91H06可有效誘發癌細胞中抗原的內化反應，因此可作為一種靶向模組，據以建構治療癌症的ADC。These results suggest that mAb 91H06 of the present invention can effectively induce antigen internalization reaction in cancer cells and can therefore be used as a targeting module to construct ADC for treating cancer.
實施例Embodiment22確認本發明Confirmation of the inventionADCADC
依據本揭示內容之「材料及方法」所述之流程將mAb連接至MMAE。依據分析結果，得到的91H06-MMAE具有高純度(＞95%)，且一致的DAR (DAR約為4) (結果未顯示)。The mAb was conjugated to MMAE according to the procedures described in the "Materials and Methods" of this disclosure. According to the analysis results, the obtained 91H06-MMAE had high purity (>95%) and consistent DAR (DAR was about 4) (results not shown).
SPR的數據指出，mAb 91H06及91H06-MMAE皆對B7-H3蛋白具有高結合親和力，其中mAb 91H06的結合速率常數(association rate constant, ka)、解離速率常數(dissociation rate constant, kd)及結合動力學解離常數(binding kinetics dissociation constant, KD)分別為6.86E+5、1.56E-4及2.27E-10，而91H06-MMAE的ka、kd及KD則分別為6.43E+5、1.55E-4及2.4E-10。流式細胞儀的數據進一步證實本發明91H06-MMAE維持對CAL 27細胞及Detroit 562細胞的高細胞結合活性及專一性(結果未顯示)。SPR data indicated that both mAb 91H06 and 91H06-MMAE had high binding affinity to B7-H3 protein. The association rate constant (ka ), dissociation rate constant (kd) and binding kinetics dissociation constant (KD ) of mAb 91H06 were 6.86E+5, 1.56E-4 and 2.27E-10, respectively, while theka ,kd and KD of 91H06-MMAE were 6.43E+5, 1.55E-4 and 2.4E-10, respectively. Flow cytometric data further confirmed that 91H06-MMAE of the present invention maintained high cell binding activity and specificity for CAL 27 cells and Detroit 562 cells (results not shown).
接著分析本發明ADC (即，91H06-MMAE)的細胞毒殺能力。如表10的結果所述，91H06-MMAE對B7-H3高表現量之癌細胞(包含口腔腺鱗狀細胞癌CAL 27細胞、咽喉鱗狀細胞癌Detroit 562細胞、咽下癌FaDu細胞及前列腺癌LNCaP細胞)具有細胞毒殺活性；而對B7-H3低表現量之癌細胞(即，HuT78細胞)與B7-H3敲除癌細胞(即，FaDu/B7-H3 KO細胞)則不具明顯的細胞毒性。細胞毒殺試驗的結果與實施例1.2測量之癌細胞株表面B7-H3的表現量一致。Then, the cytotoxicity of the ADC of the present invention (i.e., 91H06-MMAE) was analyzed. As shown in the results of Table 10, 91H06-MMAE has cytotoxic activity against cancer cells with high B7-H3 expression (including oral squamous cell carcinoma CAL 27 cells, laryngeal squamous cell carcinoma Detroit 562 cells, hypopharyngeal cancer FaDu cells, and prostate cancer LNCaP cells); while it has no obvious cytotoxicity against cancer cells with low B7-H3 expression (i.e., HuT78 cells) and B7-H3 knockout cancer cells (i.e., FaDu/B7-H3 KO cells). The results of the cytotoxicity assay were consistent with the expression level of B7-H3 on the surface of cancer cell lines measured in Example 1.2.
表10 91H06-MMAE對B7-H3陽性癌細胞株的細胞毒殺活性(IC50)
除了MMAE之外，本發明mAb也與不同的細胞毒性負載藥物(即，具有較MMAE更高之細胞毒性的MMAF)連接。如表11的結果所示，投予測試ADC (即，91H06-MMAF)亦可顯著造成口腔腺鱗狀細胞癌CAL 27細胞、咽喉鱗狀細胞癌Detroit 562細胞、咽下癌FaDu細胞及前列腺癌LNCaP細胞死亡，而不會對HuT78及FaDu/B7-H3 KO細胞產生明確的細胞毒性。此外，91H06-MMAF的細胞毒性更高於91H06-MMAE的細胞毒性。該些結果不僅與細胞表面B7-H3的表現量一致，也與不同毒性負載藥物的效力一致。整體來說，含有91H06之ADC的高效力及選擇性細胞毒殺能力暗示本發明91H06可作為ADC藥物研發時理想的單株抗體，據以治療B7-H3陽性癌症。In addition to MMAE, the mAb of the present invention was also linked to a different cytotoxic drug load (i.e., MMAF, which has higher cytotoxicity than MMAE). As shown in the results of Table 11, administration of the test ADC (i.e., 91H06-MMAF) can also significantly cause cell death of oral adenocarcinoma CAL 27 cells, laryngeal squamous cell carcinoma Detroit 562 cells, hypopharyngeal carcinoma FaDu cells, and prostate cancer LNCaP cells, but does not produce clear cytotoxicity to HuT78 and FaDu/B7-H3 KO cells. In addition, the cytotoxicity of 91H06-MMAF is higher than that of 91H06-MMAE. These results are consistent not only with the expression of B7-H3 on the cell surface, but also with the efficacy of different toxic loading drugs. Overall, the high potency and selective cytotoxicity of ADC containing 91H06 suggest that 91H06 of the present invention can be used as an ideal monoclonal antibody in the development of ADC drugs to treat B7-H3 positive cancers.
表11 91H06-MMAF到B7-H3陽性癌細胞株的細胞毒殺活性(IC50)
上述結果證實mAb 91H06可與不同毒性負載藥物連接，並有效毒殺不同類型的癌細胞。The above results confirm that mAb 91H06 can be linked to different toxic drug loads and effectively kill different types of cancer cells.
實施例Embodiment3391H06-MMAE91H06-MMAE於小鼠異種移植模式的活體內功效In vivo efficacy in a mouse xenograft model
本實施例將分析本發明ADC於動物模式的抗腫瘤功效。第3圖及第4圖的結果指出，相較於對照組，投予1.5 mpk及5 mpk的91H06-MMAE會顯著抑制Detroit 562異種移植模式(第3圖)及FaDu異種移植模式(第4圖)中腫瘤的生長。投予1.5 mpk及5 mpk劑量之本發明91H06-MMAE造成的腫瘤生長抑制(tumor growth inhibition, TGI)率於Detroit 562異種移植模式中分別高於80%及100% (表12)，而於FaDu異種移植模式中則高於100% (表13)。在接受91H06-MMAE治療的動物中未觀察到體重下降或異常狀況(結果未顯示)。This example analyzes the anti-tumor efficacy of the ADC of the present invention in animal models. The results of Figures 3 and 4 indicate that, compared with the control group, administration of 1.5 mpk and 5 mpk of 91H06-MMAE significantly inhibited the growth of tumors in the Detroit 562 xenograft model (Figure 3) and the FaDu xenograft model (Figure 4). The tumor growth inhibition (TGI) rate caused by administration of 1.5 mpk and 5 mpk doses of 91H06-MMAE of the present invention was higher than 80% and 100% in the Detroit 562 xenograft model, respectively (Table 12), and higher than 100% in the FaDu xenograft model (Table 13). No weight loss or abnormalities were observed in animals treated with 91H06-MMAE (results not shown).
表12 特定治療於Detroit 562異種移植模式中的抗腫瘤活性
表13 特定治療於FaDu異種移植模式中的抗腫瘤活性
值得注意的是，在帶有平均腫瘤大小超過1500立方毫米之大腫瘤的小鼠中，投予每公斤5毫克之91H06-MMAE可顯著抑制腫瘤生長(第5圖)。試驗期間並未觀察到動物之體重下降或異常狀況(結果未顯示)。It is noteworthy that in mice with large tumors with an average tumor size of more than 1500 cubic millimeters, administration of 5 mg/kg of 91H06-MMAE significantly inhibited tumor growth (Figure 5). No weight loss or abnormal conditions were observed in the animals during the experiment (results not shown).
總結上述，本揭示內容提供了一種名為mAb 91H06的新穎抗體。依據本揭示內容的實施例，mAb 91H06對B7-H3具有高結合親和力及專一性，可用以製備治療B7-H3陽性癌症(即使於大腫瘤情況)的免疫偶聯物(即，ADC)。In summary, the present disclosure provides a novel antibody named mAb 91H06. According to the embodiments of the present disclosure, mAb 91H06 has high binding affinity and specificity for B7-H3 and can be used to prepare immunoconjugates (i.e., ADCs) for treating B7-H3-positive cancers (even in the case of large tumors).
當可理解，上述實施方式僅為例示性的的闡述，習知技藝人士可進行不同修飾。上述說明書、實施例及數據提供了本發明例示性實施方式完整的說明。雖然上文實施方式中揭露了本發明的具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍當以附隨申請專利範圍所界定者為準。It should be understood that the above embodiments are merely exemplary descriptions, and those skilled in the art may make various modifications. The above description, embodiments, and data provide a complete description of the exemplary embodiments of the present invention. Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope defined in the attached patent application.
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為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之說明如下：In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the attached drawings are described as follows:
第1圖是依據本揭示內容實施例1.1所繪示關於酵素結合免疫吸附法(enzyme-linked immunosorbent assay, ELISA)的分析結果，用以闡述抗體91H06對特定蛋白的結合活性，其中抗體91H06對B7-H3具有結合專一性，而不會與其他B7家族蛋白產生交叉反應；FIG. 1 is an analysis result of an enzyme-linked immunosorbent assay (ELISA) according to Example 1.1 of the present disclosure, which is used to illustrate the binding activity of antibody 91H06 to a specific protein, wherein antibody 91H06 has binding specificity to B7-H3 and does not cross-react with other B7 family proteins;
第2圖是依據本揭示內容實施例1.2所繪示關於流式細胞儀的分析結果，用以闡述伊非那他單抗(Ifinatamab)及抗體91H06對B7-H3陽性細胞的結合親和力；小圖(A)：伊非那他單抗對EMT6細胞的結合親和力；小圖(B)：抗體91H06對EMT6細胞的結合親和力；小圖(C)：伊非那他單抗對ID8細胞的結合親和力；小圖(D)：抗體91H06對ID8細胞的結合親和力；以流式細胞儀來分析測試抗體的細胞結合活性(填充峰)；以同型(isotype)對照抗體(未填充峰)來證實染色的專一性；FIG. 2 is a flow cytometer analysis result according to Example 1.2 of the present disclosure, which is used to illustrate the binding affinity of Ifinatamab and antibody 91H06 to B7-H3 positive cells; panel (A): the binding affinity of Ifinatamab to EMT6 cells; panel (B): the binding affinity of antibody 91H06 to EMT6 cells; panel (C): the binding affinity of Ifinatamab to ID8 cells; panel (D): the binding affinity of antibody 91H06 to ID8 cells; the cell binding activity of the test antibody (filled peak) was analyzed by flow cytometer; the specificity of staining was confirmed by isotype control antibody (unfilled peak);
第3圖是依據本揭示內容實施例3所繪示的線性圖，用以闡述帶有Detroit 562腫瘤之小鼠於接受特定治療後腫瘤的體積；載體：每週對帶有腫瘤之小鼠投予一次2-(N-嗎啉代)乙磺酸(2-(N-morpholino) ethanesulfonic acid, MES)，連接投予三週；1.5 mpk：每週對帶有腫瘤之小鼠投予一次91H06-MMAE (每公斤1.5毫克)，連接投予三週；5 mpk：每週對帶有腫瘤之小鼠投予一次91H06-MMAE (每公斤5毫克)，連接投予三週；FIG. 3 is a line graph drawn according to Example 3 of the present disclosure, illustrating the tumor volume of mice bearing Detroit 562 tumors after receiving specific treatments; Vehicle: 2-(N-morpholino) ethanesulfonic acid (MES) was administered to tumor-bearing mice once a week for three consecutive weeks; 1.5 mpk: 91H06-MMAE (1.5 mg/kg) was administered to tumor-bearing mice once a week for three consecutive weeks; 5 mpk: 91H06-MMAE (5 mg/kg) was administered to tumor-bearing mice once a week for three consecutive weeks;
第4圖是依據本揭示內容實施例3所繪示的線性圖，用以闡述帶有FaDu腫瘤之小鼠於接受特定治療後腫瘤的體積；載體：對帶有腫瘤之小鼠投予單一劑量的MES緩衝液；1.5 mpk：對帶有腫瘤之小鼠投予單一劑量的91H06-MMAE (每公斤1.5毫克)；5 mpk：對帶有腫瘤之小鼠投予單一劑量的91H06-MMAE (每公斤5毫克)；以及FIG. 4 is a line graph drawn according to Example 3 of the present disclosure, illustrating the tumor volume of mice bearing FaDu tumors after receiving specific treatments; Vehicle: a single dose of MES buffer was administered to mice bearing tumors; 1.5 mpk: a single dose of 91H06-MMAE (1.5 mg/kg) was administered to mice bearing tumors; 5 mpk: a single dose of 91H06-MMAE (5 mg/kg) was administered to mice bearing tumors; and
第5圖是依據本揭示內容實施例3所繪示的線性圖，用以闡述對帶有Detroit 562腫瘤之小鼠每週投予一次91H06-MMAE (每公斤5毫克)，連續投予三週後腫瘤的體積。FIG. 5 is a line graph drawn according to Example 3 of the present disclosure, illustrating the tumor volume after once-weekly administration of 91H06-MMAE (5 mg/kg) to mice bearing Detroit 562 tumors for three consecutive weeks.
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