TW202504637A - Methods and compositions related to antibodies and antibody drug conjugates (adcs) that bind nectin-4 proteins - Google Patents

Abstract

Antibody drug conjugates (ADCs) that bind to NECTIN-4 protein(s) and variants thereof are described herein. NECTIN-4 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, the ADCs of the invention provide a therapeutic composition for the treatment of cancer.

Description

Translated fromChinese

與結合至NECTIN-4蛋白之抗體及抗體藥物結合物(ADC)相關之方法及組合物Methods and compositions related to antibodies and antibody-drug conjugates (ADCs) that bind to NECTIN-4 proteins

本文所描述之本發明係關於結合NECTIN-4蛋白之抗體、其抗原結合片段及抗體藥物結合物(ADC)。本發明進一步係關於適用於治療癌症與其他免疫及神經病症之預後、預防及治療方法，及組合物。The invention described herein relates to antibodies, antigen-binding fragments thereof, and antibody-drug conjugates (ADCs) that bind to NECTIN-4 protein. The invention further relates to prognostic, preventive and therapeutic methods and compositions suitable for treating cancer and other immune and neurological disorders.

癌症為全世界僅次於冠狀動脈疾病(coronary disease)之第二大死亡原因。Cancer is the second leading cause of death worldwide after coronary disease.

儘管癌症療法在過去數十年內已改良且生存率已提高，但癌症之異質性仍需要利用複數個治療模態的新穎治療策略。此尤其適用於治療解剖學關鍵位點處之實體腫瘤(例如神經膠質母細胞瘤、頭頸部鱗狀癌瘤及肺腺癌)，其有時限於標準放射治療及/或化學療法。然而，此等療法之不利效應為化療及放射抗性，其加劇局部區域復發、遠端轉移及次級原發性腫瘤，以及降低患者之生活品質的嚴重副作用。Although cancer treatments have improved and survival rates have increased over the past few decades, the heterogeneity of cancer still requires novel treatment strategies utilizing multiple therapeutic modalities. This is particularly true for the treatment of solid tumors at anatomically critical sites (e.g., glioblastoma, head and neck squamous carcinoma, and lung adenocarcinoma), which are sometimes limited to standard radiation therapy and/or chemotherapy. However, adverse effects of these therapies are chemo- and radioresistance, which exacerbate locoregional recurrence, distant metastases, and secondary primary tumors, as well as severe side effects that reduce the patient's quality of life.

在對抗癌症及其他醫學病況中，單株抗體(mAb) 之治療效用 (G. KOHLER及C. MILSTEIN, Nature 256:495-497 (1975))得到認識。一般而言，抗體藉由許多機制起作用，大部分機制與免疫系統之其他臂有關。The therapeutic utility of monoclonal antibodies (mAbs) has been recognized in the fight against cancer and other medical conditions (G. KOHLER and C. MILSTEIN, Nature 256:495-497 (1975)). In general, antibodies work by many mechanisms, most of which are related to other arms of the immune system.

抗體-藥物結合物(ADC)為新出現的一類靶向治療劑，具有優於傳統化學療法之治療指數。除(單株)抗體(mAb)及目標選擇之外，藥物及連接基團已為ADC開發之焦點。然而，近來，已探究結合物均質性之重要性。已報導，ADC之藥理學概況可藉由應用位點特異性結合技術改良，該等技術利用經工程改造至隨後與連接基團藥物結合之抗體中的表面暴露之半胱胺酸殘基，從而產生具有所界定藥物-抗體比率(DAR)的經位點特異性結合之ADC。Antibody-drug conjugates (ADCs) are an emerging class of targeted therapeutics with superior therapeutic indices over conventional chemotherapy. In addition to (monoclonal) antibodies (mAbs) and target selection, the drug and linker group have been the focus of ADC development. However, recently, the importance of conjugate homogeneity has been explored. It has been reported that the pharmacological profile of ADCs can be improved by applying site-specific conjugation technologies that utilize surface-exposed cysteine residues engineered into antibodies that are subsequently conjugated to linker drugs, thereby generating site-specifically conjugated ADCs with defined drug-antibody ratios (DARs).

先前技術揭示獲得ADC之若干方法。參見例如WO2006/034488 (Genentech)、SUTHERLAND等人, Blood 122(8):1455-1463 (2013)、WO2014/124316 (Novartis)、US2017/0080103 (Synthon Biopharmaceuticals)、US11,559,582 (Agensys, Inc.)及WO2019/183438 (Seattle Genetics, Inc.)等。Prior art discloses several methods for obtaining ADCs. See, for example, WO2006/034488 (Genentech), SUTHERLAND et al., Blood 122(8):1455-1463 (2013), WO2014/124316 (Novartis), US2017/0080103 (Synthon Biopharmaceuticals), US11,559,582 (Agensys, Inc.), and WO2019/183438 (Seattle Genetics, Inc.).

迄今所揭示之所有先前技術方法，均側重於在表面/溶劑暴露位置處、在展示高硫醇反應性之位置處及在單株抗體之特定恆定區中之位置處的位點結合連接基團藥物，其目的在於改良均質性及藥物動力學性質。All prior art approaches disclosed to date have focused on site-binding of the linker group drug at surface/solvent exposed positions, at positions exhibiting high thiol reactivity, and at positions in specific constant regions of monoclonal antibodies with the goal of improving homogeneity and pharmacokinetic properties.

儘管以上所描述之習知離胺酸及半胱胺酸結合方法已產生經FDA批准通過之抗體-藥物結合物且其正用於構築當前臨床前及臨床試驗中之大量ADC的大部分，但仍需要新穎結合策略，目的在於(進一步)改良ADC之物理化學、藥物動力學、藥理學及/或毒理學性質，以獲得具有可接受之抗原結合性質、活體內功效、治療指數及/或穩定性之ADC。Although the known lysine and cysteine conjugation methods described above have resulted in FDA-approved antibody-drug conjugates and are being used to construct a large portion of the large number of ADCs currently in preclinical and clinical trials, there is still a need for novel conjugation strategies aimed at (further) improving the physicochemical, pharmacokinetic, pharmacological and/or toxicological properties of ADCs in order to obtain ADCs with acceptable antigen binding properties, in vivo efficacy, therapeutic index and/or stability.

自前述，熟習此項技術者將顯而易見，需要新穎治療範式來治療癌症及免疫疾病。藉由使用現代抗體工程改造技術以及新穎結合方法，可以更有效治療、減少副作用及更低生產成本之總體目的達成一類新穎抗體。From the foregoing, it will be apparent to those familiar with this technology that new therapeutic paradigms are needed to treat cancer and immune diseases. By using modern antibody engineering techniques and novel conjugation methods, a new class of antibodies can be achieved with the overall goal of more effective treatment, reduced side effects and lower production costs.

鑒於此項技術中已知之當前不足，本發明之一目標為提供新穎且改良之抗體及結合配體與利用抗體及ADC治療癌症、免疫病症及其他疾病之方法。In view of the current deficiencies known in this art, one object of the present invention is to provide novel and improved antibodies and binding ligands and methods of using antibodies and ADCs to treat cancer, immune disorders and other diseases.

本發明提供結合至NECTIN-4蛋白及NECTIN-4蛋白之多肽片段的抗體、抗原結合片段、抗體藥物結合物(ADC)、抗體免疫調節結合物、抗體融合蛋白及抗體片段融合蛋白。在一些實施例中，本發明包含與治療劑結合之全人類抗體。The present invention provides antibodies, antigen binding fragments, antibody drug conjugates (ADCs), antibody immunomodulatory conjugates, antibody fusion proteins, and antibody fragment fusion proteins that bind to NECTIN-4 proteins and polypeptide fragments of NECTIN-4 proteins. In some embodiments, the present invention comprises fully human antibodies bound to therapeutic agents.

在某些實施例中，限制條件為不編碼表IV之整個核酸序列及/或不製備表V之整個胺基酸序列。在某些實施例中，表IV之整個核酸序列經編碼及/或表V之整個胺基酸序列經製備，其中之任一者呈各別人類單位劑型。In certain embodiments, the restriction is that the entire nucleic acid sequence of Table IV is not encoded and/or the entire amino acid sequence of Table V is not prepared. In certain embodiments, the entire nucleic acid sequence of Table IV is encoded and/or the entire amino acid sequence of Table V is prepared, either of which is in the form of a respective human unit dosage.

本發明進一步提供各種免疫原性或治療組合物，諸如抗體、抗體藥物結合物，及用於治療表現NECTIN-4之癌症，諸如表I中所列之彼等癌症的策略。The present invention further provides various immunogenic or therapeutic compositions, such as antibodies, antibody-drug conjugates, and strategies for treating cancers expressing NECTIN-4, such as those listed in Table I.

在另一實施例中，本發明教示標示為CL.Z之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.Z.

在另一實施例中，本發明教示標示為CL.X之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.X.

在另一實施例中，本發明教示標示為CL.N之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.N.

在另一實施例中，本發明教示標示為CL.I之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.I.

在另一實施例中，本發明教示標示為CL.B之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.B.

在另一實施例中，本發明教示標示為CL.D之抗體組合物。In another embodiment, the present invention teaches an antibody composition labeled CL.D.

在另一實施例中，本發明教示合成抗體之方法。In another embodiment, the present invention teaches a method of synthesizing antibodies.

在另一實施例中，本發明教示合成抗體及使藥物部分結合至抗體以形成ADC之方法。In another embodiment, the present invention teaches methods of synthesizing antibodies and conjugating drug moieties to the antibodies to form ADCs.

在另一實施例中，本發明教示治療人類之癌症之方法。In another embodiment, the present invention teaches methods of treating cancer in humans.

在另一實施例中，本發明教示治療人類之免疫或神經病症之方法。In another embodiment, the present invention teaches methods of treating immune or neurological disorders in humans.

在另一實施例中，本發明教示本文中之一或多種組合物在製造用於治療人類之癌症、免疫及/或神經病症之藥劑中的用途。In another embodiment, the present invention teaches the use of one or more compositions herein in the manufacture of a medicament for treating cancer, immune and/or neurological disorders in a human.

相關申請案之交叉參考本申請案主張2023年6月13日申請之美國臨時專利申請案第63/628,028號及2024年4月05日申請之美國臨時專利申請案第63/575,054號之優先權，其內容以引用之方式完全併入本文中。CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63/628,028 filed on June 13, 2023 and U.S. Provisional Patent Application No. 63/575,054 filed on April 5, 2024, the contents of which are incorporated herein by reference in their entirety.

序列表XML檔案(「序列表XML」)之提交以下提交內容以全文引用之方式完全併入本文中：呈XML檔案形式之序列表之電腦可讀形式(CRF)的內容(檔案名稱：9300-20000.40 - SEQ LIST - XML - 07-June-2024，錄入日期：2024年6月06日，大小：136 KB)。Submission ofSequence ListingXMLFile("Sequence ListingXML") The following submission is fully incorporated herein by reference: the contents of the Computer Readable Form (CRF) of the Sequence Listing in the form of an XML file (file name: 9300-20000.40-SEQ LIST-XML-07-June-2024, accession date: June 06, 2024, size: 136 KB).

在聯邦贊助研究下作出之發明的權利聲明不適用。Claims to inventions made under federally sponsored research do not apply.

章節概述Chapter OverviewI.)I.)定義DefinitionII.)II.)抗體antibodyIII.)III.)抗體antibody--藥物Drugs--結合物ConjugateIV.)IV.)連接基團單元Linking group unitV.)V.)延伸基團單元Extended group unitVI.)VI.)胺基酸單元Amino acid unitVII.)VII.)間隔基團單元Spacer group unitVIII.)VIII.)藥物單元Drug unitIX.)IX.)藥物負載Drug loadX.)X.)測定ADCDetermine ADC之細胞毒性效應的方法Methods for the cytotoxic effects ofXI.)XI.)表現NECTIN-4Expression of NECTIN-4之癌症的治療Cancer TreatmentXII.)     NECTIN-4 ADCXII.) NECTIN-4 ADC混合物mixtureXIII.)XIII.)組合療法Combination therapyXIV.)XIV.)套組/Set/製品Products

I.)定義：除非另外定義，否則除非上下文另外明確指示，否則本文中所使用之所有技術術語、標記及其他科學術語意欲具有熟習本發明涉及之技術者通常所理解的含義。在一些情況下，出於清楚起見及/或出於方便參考，在本文中定義具有通常所理解含義之術語，且本文中包括此類定義不應必然解釋為表示與此項技術中一般所理解存在實質性差異。按需要，除非另外指出，否則涉及可商購套組及試劑之使用的程序通常根據製造商所定義之方案及/或參數進行。I.)Definitions: Unless otherwise defined, all technical terms, indicia, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention relates, unless the context clearly indicates otherwise. In some cases, for clarity and/or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from what is generally understood in the art. As required, procedures involving the use of commercially available kits and reagents are generally performed according to protocols and/or parameters defined by the manufacturer, unless otherwise indicated.

除非上下文另外指明，否則當本文中使用商標時，提及該商標亦指商標產品的產品調配物、通用藥物及活性醫藥成分。Unless the context indicates otherwise, when a trademark is used herein, reference to the trademark also refers to the product formulation, generic drug, and active pharmaceutical ingredient of the trademarked product.

術語「晚期癌症」、「局部晚期癌症」、「晚期疾病」及「局部晚期疾病」意謂已擴展穿過相關組織囊的癌症且意謂包括在美國泌尿協會(American Urological Association；AUA)系統下之C期疾病，在惠特莫耳-朱厄特(Whitmore-Jewett)系統下之C1-C2期疾病及在TNM (腫瘤、結節、轉移)系統下之T3-T4及N+期疾病。一般而言，不建議手術用於患有局部晚期疾病之患者，且此等患者相較於患有臨床局部化(器官限制性)癌症之患者具有實質上較差的結果。The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" mean cancer that has extended through the corpuscle of associated tissue and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and such patients have substantially worse outcomes than patients with clinically localized (organ-confined) cancer.

術語「經取代」意謂指定基團或部分攜有一或多個取代基。術語「未經取代」意謂指定基團不攜有取代基。術語「視情況經取代」意謂指定基團未經取代或經一或多個取代基取代。當術語「經取代」用於描述結構系統時，取代意謂發生在系統上之任何價數允許的位置。The term "substituted" means that the specified group or moiety bears one or more substituents. The term "unsubstituted" means that the specified group bears no substituents. The term "optionally substituted" means that the specified group is unsubstituted or substituted with one or more substituents. When the term "substituted" is used to describe a structural system, substitution is intended to occur at any valence-permitted position on the system.

術語「類似物」係指結構上類似或與另一分子(例如，NECTIN-4有關蛋白)共有類似或對應屬性的分子。例如，NECTIN-4蛋白之類似物可特異性結合至NECTIN-4之抗體或T細胞。The term "analog" refers to a molecule that is structurally similar or shares similar or corresponding properties with another molecule (e.g., a protein related to NECTIN-4). For example, an analog of NECTIN-4 protein can specifically bind to an antibody or T cell against NECTIN-4.

除非另外明確指示，否則術語「抗體」沿最廣泛意義使用。因此，「抗體」可為天然存在的或合成的，諸如藉由習知融合瘤或轉殖基因小鼠技術產生之單株抗體。NECTIN-4抗體包含單株及多株抗體以及含有此等抗體之抗原結合域及/或一或多個互補決定區的片段。如本文中所使用，術語「抗體」係指特異性結合NECTIN-4及/或展現所需生物活性的任何形式之抗體或其片段，且特定言之覆蓋單株抗體(包括全長單株抗體)、多株抗體、多特異性抗體(例如雙特異性抗體)及抗體片段，只要其特異性結合NECTIN-4及/或展現所需生物活性即可。任何特異性抗體均可用於本文中所提供之方法及組合物中。因此，在一個實施例中，術語「抗體」涵蓋包含至少一個來自輕鏈免疫球蛋白分子之可變區及至少一個來自重鏈分子之可變區的分子，該等可變區組合形成針對目標抗原之特異性結合位點。在一個實施例中，抗體未IgG抗體。例如，抗體為IgG1、IgG2、IgG3或IgG4抗體。適用於本發明方法及組合物中之抗體可在細胞培養物中、在噬菌體中、在酵母中或在各種動物中生成，該等動物包括但不限於牛、兔、山羊、小鼠、大鼠、倉鼠、天竺鼠、綿羊、狗、貓、猴、黑猩猩及猿。因此，在一個實施例中，本發明之抗體為哺乳動物抗體。噬菌體技術可用於分離初始抗體或用於生成具有改變之特異性或親合力特徵的變異體。此類技術為常規的且為此項技術中熟知的。在一個實施例中，藉由此項技術中已知之重組手段產生抗體。例如，重組抗體可藉由用包含編碼該抗體之DNA序列的載體轉染宿主細胞產生。一或多種載體可用於轉染表現宿主細胞中之至少一個VL及至少一個VH區的DNA序列。抗體生成及產生之重組手段的例示性描述包括Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997)；SHEPARD等人, MONOCLONAL ANTIBODIES (Oxford University Press, 2000)；GODING, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993)及CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons,最近版本)。本發明之抗體可藉由重組手段來修飾以提高抗體介導所需功能之功效。因此，在本發明之範圍內，抗體可藉由使用重組手段進行取代來修飾。典型地，取代將為保守取代。例如，抗體恆定區中之至少一個胺基酸可經不同殘基置換。參見例如美國專利第5,624,821號、美國專利第6,194,551號、申請案第WO 9958572號；及ANGAL等人, Mol. Immunol. 30: 105-08 (1993)。胺基酸中之修飾包括胺基酸之缺失、添加及取代。在一些情況下，進行該等變化係為了降低非所需活性，例如補體依賴性細胞毒性。通常，抗體係藉由以共價或非共價方式接合提供可偵測信號之物質來標記。多種多樣的標記及結合技術為已知的且廣泛報導於科學文獻及專利文獻中。此等抗體可針對與正常或缺陷NECTIN-4的結合進行篩選。參見例如ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996)。具有所需生物活性之適合抗體可使用以下活體外分析，包括但不限於增殖、遷移、黏著、軟瓊脂生長、血管生成、細胞-細胞通信、細胞凋亡、運輸、信號轉導及以下活體內分析，諸如腫瘤生長之抑制來鑑別。本文中所提供之抗體亦可適用於診斷應用。作為捕捉抗體或非中和抗體，其可經篩選以能夠在不抑制抗原之受體結合或生物活性下與特定抗原結合。作為中和抗體，抗體可適用於競爭性結合分析。其亦可用於定量NECTIN-4及/或其受體。Unless otherwise expressly indicated, the term "antibody" is used in the broadest sense. Therefore, an "antibody" may be naturally occurring or synthetic, such as a monoclonal antibody produced by known fusion tumor or transgenic mouse technology. NECTIN-4 antibodies include monoclonal and polyclonal antibodies and fragments containing the antigen binding domain and/or one or more complementary determining regions of such antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to NECTIN-4 and/or exhibits the desired biological activity, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they specifically bind to NECTIN-4 and/or exhibit the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Therefore, in one embodiment, the term "antibody" encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule, which variable regions combine to form a specific binding site for a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. Antibodies suitable for use in the methods and compositions of the present invention can be produced in cell culture, in phage, in yeast, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, the antibody of the present invention is a mammalian antibody. Phage technology can be used to isolate the original antibody or to generate variants with altered specificity or affinity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL and at least one VH region in a host cell. Exemplary descriptions of recombinant means for antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); SHEPARD et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); GODING, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993) and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). The antibodies of the present invention can be modified by recombinant means to improve the efficacy of the antibody in mediating the desired function. Therefore, within the scope of the present invention, the antibody can be modified by substitution using recombinant means. Typically, the substitution will be a conservative substitution. For example, at least one amino acid in the constant region of the antibody can be replaced by a different residue. See, e.g., U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and ANGAL et al., Mol. Immunol. 30: 105-08 (1993). Modifications in amino acids include deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Typically, antibodies are labeled by covalently or non-covalently conjugating a substance that provides a detectable signal. A variety of labeling and binding techniques are known and widely reported in scientific and patent literature. These antibodies can be screened for binding to normal or defective NECTIN-4. See, e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biological activity can be identified using in vitro assays including, but not limited to, proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as inhibition of tumor growth. The antibodies provided herein can also be used in diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened for the ability to bind to a specific antigen without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be used in competitive binding assays. They can also be used to quantify NECTIN-4 and/or its receptors.

如本文中所使用，術語抗體之「抗原結合片段」或「抗體片段」 (或簡稱「抗體部分」)係指保持特異性結合至抗原(例如NECTIN-4及/或其變異體)之能力的NECTIN-4抗體的一或多個片段。已顯示抗體之抗原結合功能可由全長抗體之片段來實現。涵蓋於術語抗體之「抗原結合片段」內之結合片段的實例包括(i) Fab片段，一由V_L、V_H、C_L及C_H1域組成之單價片段；(ii) F(ab′)₂片段，一包含兩個藉由鉸鏈區處之二硫橋鍵鍵聯之Fab片段的二價片段；(iii)Fd片段，由V_H及C_H1域組成；(iv) Fv片段，由抗體之單臂之V_L及V_H域組成；(v) dAb片段(WARD等人, (1989) Nature 341:544-546)，其由V_H域組成；及(vi)分離之互補決定區(CDR)。此外，儘管Fv片段之兩個域V_L及V_H由獨立基因編碼，但其可使用重組方法、藉由使其能夠製備為單一蛋白鏈之合成連接基團接合，其中V_L及V_H區配對形成單價分子(稱為單鏈Fv (scFv)；參見例如BIRD等人, (1988) Science 242:423-426；及HUSTON等人(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883)。該等單鏈抗體亦意欲涵蓋於術語抗體之「抗原結合片段」內。此等抗體片段係使用熟習此項技術者已知之習知技術獲得，且以與完整抗體相同之方式針對效用來篩選片段。As used herein, the term "antigen-binding fragment" or "antibody fragment" (or simply "antibody portion") of an antibody refers to one or more fragments of a NECTIN-4 antibody that retains the ability to specifically bind to an antigen (e.g., NECTIN-4 and/or its variants). It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the_VL ,_VH ,_CL and_CH1 domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment, consisting of the_VH and_CH1 domains; (iv) a Fv fragment, consisting of the_VL and_VH domains of a single arm of an antibody; (v) a dAb fragment (WARD et al., (1989) Nature 341:544-546), which consists of a_VH domain; and (vi) isolated complementary determining regions (CDRs). In addition, although the two domains of the Fv fragment_{, VL} and_VH, are encoded by independent genes, they can be joined using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain, in which the_VL and_VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., BIRD et al., (1988) Science 242:423-426; and HUSTON et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

如本文中所使用，術語「Fc」係指包含鉸鏈區、CH₂及/或CH₃域的區域。As used herein, the term "Fc" refers to the region including the hinge region,_CH2 and/or_CH3 domains.

如本文中所使用，任何形式之「抗原」可用於生成對本發明之NECTIN-4具有特異性的抗體。因此，引發抗原可為單獨的單一抗原決定基、多個抗原決定基或整個蛋白或與此項技術中已知的一或多種免疫原性增強劑組合。引發抗原可為經分離之全長蛋白、細胞表面蛋白(例如用抗原之至少一部分轉染之細胞來免疫接種)或可溶性蛋白(例如僅用蛋白之細胞外域部分來免疫接種)。可在經遺傳修飾之細胞中產生抗原。編碼抗原之DNA可為基因體或非基因體的(例如cDNA)且編碼細胞外域之至少一部分。如本文中所使用，術語「部分」在抗原之情況下係指按需要以構成所關注抗原之免疫原性抗原決定基的最小數目個胺基酸或核酸。可採用適用於轉化所關注細胞之任何基因載體，包括但不限於腺病毒載體、質體及非病毒載體，諸如陽離子脂質。在一個實施例中，本文中之方法及組合物的抗體特異性結合所關注目標之細胞外域之至少一部分。As used herein, any form of "antigen" can be used to generate antibodies specific for NECTIN-4 of the present invention. Thus, the eliciting antigen can be a single antigenic determinant alone, multiple antigenic determinants, or the entire protein or in combination with one or more immunogenicity enhancers known in the art. The eliciting antigen can be an isolated full-length protein, a cell surface protein (e.g., immunization with cells transfected with at least a portion of the antigen) or a soluble protein (e.g., immunization with only the extracellular domain portion of the protein). The antigen can be produced in genetically modified cells. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" in the context of an antigen refers to the minimum number of amino acids or nucleic acids required to constitute the immunogenic antigenic determinant of the antigen of interest. Any gene vector suitable for transforming cells of interest may be used, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids. In one embodiment, the antibodies of the methods and compositions herein specifically bind to at least a portion of the extracellular domain of the target of interest.

本文中所提供之抗體或其抗原結合片段可構成或為「生物活性劑」之一部分。如本文中所使用，術語「生物活性劑」係指任何合成或天然存在之化合物，其結合抗原及/或增強或調節所需生物效應以增強細胞殺死毒素。在一個實施例中，適用於本發明之結合片段為具有生物活性的片段。如本文中所使用，術語「具有生物活性的」係指能夠結合所需抗原性抗原決定基且直接或間接發揮生物效應的抗體或抗體片段。直接效應包括(但不限於)生長信號之調節、刺激及/或抑制；抗細胞凋亡信號之調節、刺激及/或抑制；細胞凋亡或壞死信號之調節、刺激及/或抑制；調節、刺激及/或抑制ADCC級聯；及調節、刺激及/或抑制CDC級聯及/或Fc緘默。The antibodies or antigen-binding fragments thereof provided herein may constitute or be part of a "biologically active agent". As used herein, the term "biologically active agent" refers to any synthetic or naturally occurring compound that binds to an antigen and/or enhances or modulates a desired biological effect to enhance a cytolytic toxin. In one embodiment, the binding fragments suitable for use in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that is capable of binding to a desired antigenic epitope and exerting a biological effect directly or indirectly. Direct effects include, but are not limited to, modulation, stimulation and/or inhibition of growth signals; modulation, stimulation and/or inhibition of anti-apoptotic signals; modulation, stimulation and/or inhibition of apoptotic or necrosis signals; modulation, stimulation and/or inhibition of the ADCC cascade; and modulation, stimulation and/or inhibition of the CDC cascade and/or Fc silencing.

如本文中所使用之與抗原結合有關的術語「特異性結合」蛋白意謂抗原結合蛋白結合至目標以及目標內之離散域或離散胺基酸序列，而不結合或不顯著結合至其他(例如不相關)蛋白。然而，此術語不排除抗體或其結合片段亦可與緊密相關之分子交叉反應的事實。本文所描述之抗體及其片段以及包含其之抗體藥物結合物可以比其結合至緊密相關之分子大至少2、5、10、50、100或1000倍的親和力特異性結合至本文所揭示之NECTIN-4。As used herein, the term "specific binding" protein in relation to antigen binding means that the antigen binding protein binds to the target and discrete domains or discrete amino acid sequences within the target, but does not bind or does not significantly bind to other (e.g., unrelated) proteins. However, this term does not exclude the fact that the antibody or its binding fragment can also cross-react with closely related molecules. The antibodies and fragments thereof described herein, as well as antibody-drug conjugates comprising the same, can specifically bind to NECTIN-4 disclosed herein with an affinity that is at least 2, 5, 10, 50, 100, or 1000 times greater than their binding to closely related molecules.

「雙特異性」抗體亦可用於本發明方法及組合物中。如本文中所使用，術語「雙特異性抗體」係指對至少兩種不同抗原性抗原決定基具有結合特異性之抗體，通常單株抗體。在一個實施例中，抗原決定基來自相同抗原。在另一個實施例中，抗原決定基來自兩種不同抗原。用於製備雙特異性抗體之方法係此項技術中已知的。例如，可利用兩種免疫球蛋白重鏈/輕鏈對之共表現，以重組方式產生雙特異性抗體。參見例如MILSTEIN等人, Nature 305:537-39 (1983)。或者，可利用化學鍵聯製備雙特異性抗體。參見例如BRENNAN等人, Science 229:81 (1985)。雙特異性抗體包括雙特異性抗體片段。參見例如HOLLINGER等人, Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993)；GRUBER等人, J. Immunol. 152:5368 (1994)。"Bispecific" antibodies may also be used in the methods and compositions of the present invention. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, that has binding specificity for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly by co-expression of two immunoglobulin heavy chain/light chain pairs. See, for example, MILSTEIN et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared by chemical bonding. See, e.g., BRENNAN et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., HOLLINGER et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993); GRUBER et al., J. Immunol. 152:5368 (1994).

本文所描述之單株抗體具體包括「嵌合」抗體，其中重鏈及/或輕鏈之一部分與來源於特定物種或屬於特定抗體類別或子類別之抗體中之相應序列一致或同源，而鏈之剩餘部分與來源於另一物種或屬於另一抗體類別或子類別之抗體中之相應序列一致或同源；以及此類抗體之片段，只要其特異性結合目標抗原及/或展現所需生物活性即可 (美國專利第4,816,567號；及MORRISON等人, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984))。The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; as well as fragments of such antibodies, as long as they specifically bind to the target antigen and/or exhibit the desired biological activity (U.S. Patent No. 4,816,567; and MORRISON et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

如本文中所使用，術語「癌症」、「贅瘤」及「腫瘤」可互換使用且以單數或複數形式使用，係指已經歷惡性轉化使其對於宿主有機體為病理的細胞。原發癌細胞(亦即獲自惡性轉化位點附近之細胞)可藉由公認技術，尤其組織學檢查容易地與非癌變細胞區分開。如本文中所使用，癌細胞之定義不僅包括原發癌細胞，而且包括來源於癌細胞祖先之任何細胞。此包括已轉移癌細胞及來源於癌細胞之活體外培養物及細胞株。當提及通常表現為實體腫瘤之類型的癌症時，「臨床上可偵測」腫瘤為可基於腫瘤塊偵測之腫瘤；例如藉由諸如CAT掃描、MR成像、X射線、超音波或觸診之程序，及/或由於可獲自患者之樣本中一或多種癌症特異性抗原之表現而偵測到的腫瘤。腫瘤可為造血腫瘤，例如血球之腫瘤或類似腫瘤，意謂液體腫瘤。基於此類腫瘤之臨床病況的特定實例包括白血病，諸如慢性骨髓細胞性白血病或急性骨髓細胞性白血病；骨髓瘤，諸如多發性骨髓瘤；淋巴瘤及類似腫瘤。As used herein, the terms "cancer," "tumor," and "neoplasm" are used interchangeably and in the singular or plural and refer to cells that have undergone malignant transformation that renders them pathological to the host organism. Primary cancer cells (i.e., cells obtained near the site of malignant transformation) can be readily distinguished from non-cancerous cells by recognized techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cell that is derived from a cancer cell ancestor. This includes cancer cells that have metastasized and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that typically manifests as a solid tumor, a "clinically detectable" tumor is one that can be detected based on a tumor mass; for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound or palpation, and/or due to the expression of one or more cancer-specific antigens in a sample that can be obtained from the patient. The tumor can be a hematopoietic tumor, such as a tumor of blood cells or a tumor-like tumor, meaning a fluid tumor. Specific examples of such tumor-based clinical conditions include leukemias, such as chronic myeloid leukemia or acute myeloid leukemia; myelomas, such as multiple myeloma; lymphomas and similar tumors.

術語「治療劑」係指提供治療效益及/或如本文所定義為治療上有效的所有藥劑。治療劑可例如逆轉、改善、緩解、抑制或限制疾病、病症或病況之進展，或減輕疾病、病症或病況之嚴重程度，影響、好轉或改善疾病，諸如癌症之一或多種症狀。此類藥劑可為細胞毒性劑或細胞生長抑制劑。該術語包括但不限於化學治療劑、抗贅生劑及如本文所定義之「藥物單元」藥劑。The term "therapeutic agent" refers to all agents that provide a therapeutic benefit and/or are therapeutically effective as defined herein. Therapeutic agents can, for example, reverse, ameliorate, alleviate, inhibit or limit the progression of a disease, disorder or condition, or reduce the severity of a disease, disorder or condition, affect, ameliorate or improve one or more symptoms of a disease, such as cancer. Such agents can be cytotoxic agents or cytostatic agents. The term includes, but is not limited to, chemotherapeutic agents, anti-bacterial agents, and "drug unit" agents as defined herein.

術語「抗贅生劑」係指提供治療效益及/或治療上有效(如本文所定義)用於治療贅瘤或癌症的所有藥劑。The term "antibiotic" refers to all agents that provide a therapeutic benefit and/or are therapeutically effective (as defined herein) for the treatment of tumors or cancer.

術語「化學治療劑」係指有效抑制腫瘤生長之所有化學化合物。化學治療劑之非限制性實例包括烷化劑，例如氮芥、伸乙基亞胺化合物及烷基磺酸酯；抗代謝物，例如葉酸、嘌呤或嘧啶拮抗劑；有絲分裂抑制劑，例如抗微管蛋白劑，諸如長春花生物鹼、奧瑞他汀(auristatins)及鬼臼毒素之衍生物；細胞毒性抗生素；損害或干擾DNA表現或複製之化合物，例如DNA小溝黏合劑；及生長因子受體拮抗劑。另外，化學治療劑包括細胞毒性劑(如本文所定義)、抗體、生物分子及小分子。The term "chemotherapeutic agent" refers to all chemical compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethylenimine compounds, and alkyl sulfonates; anti-metabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin agents, such as vinca alkaloids, auristatins, and derivatives of podophyllotoxin; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove adhesives; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.

術語「互補決定區」及「CDR」在此項技術中已知，係指抗體可變區內賦予抗原特異性及結合親和力的胺基酸之非連續序列。一般而言，各重鏈可變區中存在三(3)個CDR (CDR-H1、CDR-H2、CDR-H3)，且各輕鏈可變區中存在三(3)個CDR (CDR-L1、CDR-L2、CDR-L3)。The terms "complementary determining region" and "CDR" are known in the art and refer to non-contiguous sequences of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. Generally, there are three (3) CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).

所載CDR的確切胺基酸序列邊界可使用許多熟知方案中之任一者容易地測定，包括由以下描述之彼等方案：Kabat等人(1991),「Sequences of Proteins of Immunological Interest」,第5版,國立衛生研究院公共衛生處(Public Health Service, National Institutes of Health), Bethesda, Md. (「Kabat」編號方案)；AL-LAZIKANI等人, (1997) JMB 273, 927-948 (「Chothia」編號方案)；MACCALLUM等人,J. Mol. Biol.262:732-745 (1996), 「Antibody-antigen interactions: Contact analysis and binding site topography」,J. Mol. Biol.262, 732-745. (「Contact」編號方案)；LEFRANC M. P.等人, 「IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains」,Dev Comp Immunol,2003年1月; 27(1):55-77 (「IMGT」編號方案)；及HONEGGER A.及PLICKTHUN A., 「Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool」,J Mol Biol,2001年6月8日; 309(3):657-70, (AHo編號方案)。The exact amino acid sequence boundaries of the contained CDRs can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); AL-LAZIKANI et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MACCALLUM et al.,J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography",J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); LEFRANC MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains”,Dev Comp Immunol, 2003 Jan; 27(1):55-77 (“IMGT” numbering scheme); and HONEGGER A. and PLICKTHUN A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”,J Mol Biol, 2001 Jun 8; 309(3):657-70, (AHo numbering scheme).

所載CDR之邊界可視用於鑑別之方案而變化。例如，Kabat方案係基於結構比對，而Chothia方案係基於結構資訊。Kabat及Chothia方案之編號均基於最常見抗體域序列長度，其中一些抗體中出現藉由插入字母調節之插入，例如「30a」及缺失。該兩個方案將某些插入及缺失(「插入缺失」)置於各種位置，從而產生差異編號。Contact方案係基於對複雜晶體結構之分析，且在多個方面與Chothia編號方案類似。The boundaries of the CDRs contained may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody domain sequence lengths, some of which have insertions adjusted by inserted letters, such as "30a", and deletions. The two schemes place certain insertions and deletions ("indels") in various positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many aspects.

因此，除非另外指明，否則術語既定抗體之「CDR」及「互補決定區」或其區域，諸如可變區，以及抗體之個別CDR (例如CDR-H1、CDR-H2)或其區域，應理解為涵蓋如由以上本文所描述之已知方案中之任一者定義的互補決定區。在一些情況下，指定用於鑑別一或多個特定CDR之方案，諸如藉由Kabat、Chothia或Contact方法所定義之CDR。Therefore, unless otherwise indicated, the terms "CDR" and "complementary determining region" of a given antibody or a region thereof, such as a variable region, and individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or a region thereof, should be understood to encompass complementary determining regions as defined by any of the known schemes described herein above. In some cases, schemes for identifying one or more specific CDRs are specified, such as CDRs defined by the Kabat, Chothia or Contact methods.

如本文中所使用，術語「保守取代」係指熟習此項技術者已知的胺基酸及/或胺基酸序列之取代，且可一般在不改變所得分子之生物活性的情況下進行。熟習此項技術者認識到，一般而言，多肽之非必需區中之單個胺基酸取代不實質上改變生物活性(參見例如WATSON等人, MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co.,第224頁(第4版, 1987))。此類例示性取代較佳根據表II及表III中所闡述之彼等者進行。例如，此類變化包括用異白胺酸(I)、纈胺酸(V)及白胺酸(L)中之任一者取代此等疏水性胺基酸中之任何其他胺基酸；天冬胺酸(D)取代麩胺酸(E)且反之亦然；麩醯胺酸(Q)取代天冬醯胺(N)且反之亦然；及絲胺酸(S)取代蘇胺酸(T)且反之亦然。視特定胺基酸之環境及其在蛋白三維結構中之作用而定，其他取代亦可視為保守的。例如，甘胺酸(G)與丙胺酸(A)時常為可互換，與丙胺酸(A)與纈胺酸(V)可互換一樣。相對呈疏水性之甲硫胺酸(M)常常可與白胺酸及異白胺酸互換，且有時可與纈胺酸互換。離胺酸(K)及精胺酸(R)在胺基酸殘基之顯著特徵為其電荷且此等兩個胺基酸殘基之pK差異不顯著的位置處時常可互換。其他改變在特定環境中仍可被視為「保守的」 (參見例如本文中之表III；第13-15頁「Biochemistry」,第2版, Lubert Stryer編(史丹福大學)；HENIKOFF等人, PNAS 1992,第89卷, 10915-10919；LEI等人, J Biol Chem 1995年5月19日; 270(20):11882-6)。其他取代亦可容許且可憑經驗或根據已知保守取代確定。As used herein, the term "conservative substitution" refers to the substitution of amino acids and/or amino acid sequences known to those skilled in the art, and can generally be performed without changing the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in the non-essential regions of polypeptides do not substantially change biological activity (see, for example, WATSON et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co., page 224 (4th edition, 1987)). Such exemplary substitutions are preferably performed according to those described in Table II and Table III. For example, such changes include substitution of any of isoleucine (I), valine (V), and leucine (L) for any of these hydrophobic amino acids; substitution of aspartic acid (D) for glutamine (E) and vice versa; substitution of glutamine (Q) for asparagine (N) and vice versa; and substitution of serine (S) for threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) is often interchangeable with alanine (A), as is alanine (A) with valine (V). The relatively hydrophobic methionine (M) is often interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the significant characteristic of the amino acid residue is its charge and the pK difference of these two amino acid residues is not significant. Other changes can still be considered "conservative" in a particular context (see, for example, Table III herein; pp. 13-15 "Biochemistry", 2nd edition, Lubert Stryer, ed. (Stanford University); HENIKOFF et al., PNAS 1992, Vol. 89, 10915-10919; LEI et al., J Biol Chem 1995 May 19; 270(20): 11882-6). Other substitutions are also permissible and can be determined empirically or based on known conservative substitutions.

術語「細胞毒性劑」係指抑制或阻止細胞之表現活性、細胞之功能及/或引起細胞破壞的物質。該術語意欲包括放射性同位素、化學治療劑，及毒素，諸如小分子毒素或源自細菌、真菌、植物或動物之酶活性毒素，包括其片段及/或變異體。細胞毒性劑之實例包括但不限於奧瑞他汀、奧瑞他汀衍生物、金黴素(auromycins)、喜樹鹼(拓樸異構酶1抑制劑)、類美登素、蓖麻毒素、蓖麻毒素A鏈、康普瑞汀(combrestatin)、倍癌黴素(duocarmycins)、尾海兔素(dolastatins)、多柔比星(doxorubicin)、佐柔比星(daunorubicin)、紫杉醇、順鉑(cisplatin)、cc1065、溴化乙錠、絲裂黴素、依託泊苷(etoposide)、替尼泊苷(tenoposide)、長春新鹼、長春鹼、秋水仙鹼、二羥基炭疽菌素二酮(dihydroxy anthracin dione)、放線菌素、白喉毒素、綠膿桿菌外毒素(PE) A (Pseudomonasexotoxin (PE) A)、PE40、相思子毒素(abrin)、相思子毒素A鏈、莫迪素A鏈(modeccin A chain)、α-帚麴菌素(alpha-sarcin)、白樹素(gelonin)、有絲分裂素(mitogellin)、侷限麴菌素(retstrictocin)、酚黴素(phenomycin)、伊諾黴素(enomycin)、麻瘋樹毒蛋白(curicin)、巴豆毒素(crotin)、卡奇黴素(calicheamicin)、肥皂草(Sapaonaria officinalis)抑制劑，及糖皮質激素與其他化學治療劑，以及放射性同位素諸如At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²或²¹³、P³²以及Lu之放射性同位素，包括Lu¹⁷⁷。The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression and activity of cells, the function of cells and/or causes cell destruction. The term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins, auristatin derivatives, auromycins, camptothecins (topoisomerase 1 inhibitors), maytansinoids, ricin, ricin A chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, and succinate. dione), actinomycin, diphtheria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,Sapaonaria officinalis inhibitors, and glucocorticoids and other chemotherapeutic agents, as well as radioactive isotopes such as At²¹¹ , I¹³¹ , I¹²⁵ , Y⁹⁰ , Re¹⁸⁶ , Re¹⁸⁸ , Sm¹⁵³ , Bi²¹² or²¹³ , P³² and radioactive isotopes of Lu including Lu¹⁷⁷ .

抗體，包括本發明之抗體，亦可與前述細胞毒性劑中之任一者結合且亦與能夠將前驅藥轉化為其活性形式之抗癌前驅藥活化酶結合。Antibodies, including those of the present invention, may also be bound to any of the aforementioned cytotoxic agents and also to an anticancer prodrug activating enzyme capable of converting the prodrug to its active form.

如本文中所使用，術語「雙功能抗體」係指具有兩個抗原結合位點之小抗體片段，該等片段包含連接至同一多肽鏈(V_H-V_L)中之輕鏈可變域(V_L)的重鏈可變域(V_H)。藉由使用過短而使得同一鏈上之兩個域之間不能配對的連接基團，迫使該等域與另一條鏈之互補域配對且產生兩個抗原結合位點。雙功能抗體更充分地描述於例如EP 404,097；WO 93/11161；及HOLLINGER等人, Proc. Natl. Acad. Sci. USA 90:6444-48 (1993)中。As used herein, the term "bifunctional antibodies" refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (_VL ) in the same polypeptide chain (_VH -_VL ). By using a linker that is too short to allow pairing between the two domains on the same chain,_the domains are forced to pair with complementary domains of another chain and create two antigen binding sites. Bifunctional antibodies are more fully described in, e.g., EP 404,097; WO 93/11161; and HOLLINGER et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

術語「同源物」係指因例如在相應位置處具有相同或類似之化學殘基序列而與另一分子展現同源性的分子。The term "homolog" refers to a molecule that exhibits homology to another molecule, for example by having the same or similar sequence of chemical residues at corresponding positions.

術語「一致的」或「序列一致性」指示在與恰當插入或缺失最佳比對及比較時兩個核酸或兩個胺基酸序列之間的一致性程度。The term "identical" or "sequence identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

在考慮到為了兩個序列之最佳比對而需要引入之空位數目及各空位之長度的情況下，兩個序列之間的「百分比一致性」為該等序列共有之一致位置之數目的函數(亦即，%一致性=一致位置之數目/位置之總數目乘以100)。序列之比較及兩個序列之間的百分比一致性測定可使用數學演算法實現，如下文所描述。兩個核苷酸序列之間的百分比一致性可使用GCG軟體包中之GAP程式，使用NWS gap dna CMP矩陣及40、50、60、70或80之空位權重及1、2、3、4、5或6之長度權重來測定。兩個核苷酸或胺基酸序列之間的百分比一致性亦可使用已併入至ALIGN程式(第2.0版)中之Meyers等人, Comput. Appi. Biosci., 4:11-17 (1988)中的演算法，使用PAM120權重殘基表、空位長度罰分為12及空位罰分為4測定。另外，兩個胺基酸序列之間的百分比一致性可使用已併入至GCG軟體包中之GAP程式中的NEEDLEMAN等人, J. Mol. Biol. 48:444-453 (1970)演算法，使用Blossum 62矩陣或PAM250矩陣及16、14、12、10、8、6或4之空位權重及1、2、3、4、5或6之長度權重測定。The "percent identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % identity = number of identical positions/total number of positions multiplied by 100). Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWS gap dna CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of Meyers et al., Comput. Appi. Biosci., 4:11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the NEEDLEMAN et al., J. Mol. Biol. 48:444-453 (1970) algorithm, which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

舉例而言，聚核苷酸序列可與參考聚核苷酸序列一致，亦即與參考序列100%一致，或其可包括與參考序列相比多達某一整數數目個核苷酸改變，諸如至少50%、60%、70%、75%、80%、85%、90%、95%、98%或99%一致。此類改變係選自至少一個核苷酸缺失、取代(包括轉移及顛換)或插入，且其中該等改變可發生於參考核苷酸序列之5'或3'末端位置或彼等末端位置之間的任何位置，個別地散置於參考序列中之核苷酸之間或參考序列內之一或多個鄰接基團中。核苷酸改變之數目藉由將如本文所描述之參考聚核苷酸序列中之核苷酸總數目乘以各別百分比一致性之數值百分比(除以100)且從參考聚核苷酸序列中之核苷酸總數目中減去該乘積來測定，或：n_n≤x_n-(x_ny)，其中n_n為核苷酸改變之數目，x_n為如本文所描述之參考聚核苷酸序列中的核苷酸總數目(關於例示性參考聚核苷酸序列，參見「序列表」中之核酸序列)，且y對於50%為0.50、對於60%為0.60、對於70%為0.70、對於75%為0.75、對於80%為0.80、對於85%為0.85、對於90%為0.90、對於95%為0.95、對於98%為0.98、對於99%為0.99或對於100%為1.00，為乘法算子之符號，且其中x_n及y之任何非整數乘積向下捨入為最接近之整數，之後將其自x_n中減去。類似地，多肽序列可與如本文所描述之多肽參考序列一致，亦即100%一致，或其可包括與參考序列相比多達某一整數數目個胺基酸改變，使得%一致性小於100%，諸如至少50%、60%、70%、75%、80%、85%、90%、95%、98%或99%一致。此類改變係選自由至少一個胺基酸缺失、取代(包括保守及非保守取代)或插入組成之群，且其中該等改變可發生於參考多肽序列之胺基端或羧基端位置或彼等末端位置之間的任何位置，個別地散置於參考序列中之胺基酸中或參考序列內之一或多個鄰接基團中。給定%一致性之胺基酸改變數目藉由將由多肽參考序列編碼之多肽序列中的胺基酸總數目乘以各別百分比一致性之數值百分比(除以100)且隨後從如本文所描述之多肽參考序列中之該胺基酸總數目中減去該乘積來測定，或：n_a≤x_a-(x_ay)，其中n_a為胺基酸改變之數目，x_a為如本文所描述之參考多肽序列中的胺基酸總數目，且y對於50%為0.50、對於60%為0.60、對於70%為0.70、對於75%為0.75、對於80%為0.80、對於85%為0.85、對於90%為0.90、對於95%為0.95、對於98%為0.98、對於99%為0.99或對於100%為1.00，為乘法算子之符號，且其中x_a及y之任何非整數乘積向下捨入為最接近之整數，之後將其自x_a中減去。百分比一致性可跨越整個序列長度測定。如本文中所定義，術語「超過75%相同」包括超過75%、80%、85%、95%及99%一致性以及此範圍內的所有離散值及離散子範圍。For example, a polynucleotide sequence may be identical to a reference polynucleotide sequence, i.e., 100% identical to the reference sequence, or it may include up to a certain integer number of nucleotide changes compared to the reference sequence, such as at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical. Such changes are selected from at least one nucleotide deletion, substitution (including transfer and exchange) or insertion, and wherein the changes may occur at the 5' or 3' terminal position of the reference nucleotide sequence or anywhere between those terminal positions, individually interspersed between nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in a reference polynucleotide sequence as described herein by the numerical percentage of the respective percent identity (divided by 100) and subtracting the product from the total number of nucleotides in the reference polynucleotide sequence, or: n_n ≤_{x n} -(x_ny ), wherein n_n is the number of nucleotide changes, x n is the number of nucleotide changes,_n is the total number of nucleotides in a reference polynucleotide sequence as described herein (see the nucleic acid sequences in the Sequence Listing for exemplary reference polynucleotide sequences), and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, is the sign of the multiplication operator, and wherein any non-integer product of_xn and y is rounded down to the nearest integer and then subtracted from_xn . Similarly, a polypeptide sequence may be identical to a polypeptide reference sequence as described herein, i.e., 100% identical, or it may include up to a certain integer number of amino acid changes compared to the reference sequence, such that the % identity is less than 100%, such as at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical. Such changes are selected from the group consisting of at least one amino acid deletion, substitution (including conservative and non-conservative substitutions) or insertion, and wherein the changes may occur at the amino-terminal or carboxyl-terminal position of the reference polypeptide sequence or anywhere between those terminal positions, individually interspersed in the amino acids in the reference sequence or in one or more adjacent groups within the reference sequence. The number of amino acid changes for a given % identity is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by the polypeptide reference sequence by the numerical percentage of the respective percent identity (divided by 100) and then subtracting the product from the total number of amino acids in the polypeptide reference sequence as described herein, or:_na ≤_xa -(_xay ), wherein_na is the number of amino acid changes, x is the number of amino acids in the polypeptide reference sequence._a is the total number of amino acids in a reference polypeptide sequence as described herein, and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, is the sign of the multiplication operator, and wherein any non-integer product of_xa and_y is rounded down to the nearest integer before being subtracted from xa. The percent identity can be determined across the entire length of the sequence. As defined herein, the term "greater than 75% identical" includes greater than 75%, 80%, 85%, 95% and 99% identity and all discrete values and subranges therein.

在一個實施例中，本文所提供之抗體為「人類抗體」。如本文中所使用，術語「人類抗體」係指基本上輕鏈及重鏈序列之全部序列，包括互補決定區(CDR)係來自人類基因之抗體。在一個實施例中，人類單株抗體係藉由三源融合瘤(trioma)技術、人類B細胞技術(參見例如KOZBOR等人, Immunol. Today 4: 72 (1983))、EBV轉形技術(參見例如COLE等人, MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985))或使用酵母或噬菌體顯示(參見例如MARKS等人, J. Mol. Biol. 222:581 (1991))來製備。在一特定實施例中，人類抗體生成於轉殖基因小鼠中。用於製備此類部分至全人類抗體之技術為此項技術中已知的且可使用任何此類技術。根據一個尤其較佳實施例，在經工程改造以表現人類重鏈及輕鏈抗體基因之轉殖基因小鼠中製備全人類抗體序列。製備產生人類抗體之轉殖基因小鼠的例示性說明見於申請案第WO 02/43478及美國專利第6,657,103號(Abgenix)及其子系。隨後可使來自產生所需抗體之轉殖基因小鼠的B細胞融合以製備用於連續產生抗體的融合瘤細胞株。參見例如美國專利第5,569,825號；第5,625,126號；第5,633,425號；第5,661,016號及第5,545,806號；以及JAKOBOVITS, Adv. Drug Del. Rev. 31:33-42 (1998)；GREEN等人, J. Exp. Med. 188:483-95 (1998)。In one embodiment, the antibodies provided herein are "human antibodies". As used herein, the term "human antibody" refers to antibodies in which substantially all sequences of light and heavy chain sequences, including complementary determining regions (CDRs), are derived from human genes. In one embodiment, human monoclonal antibodies are prepared by trioma technology, human B cell technology (see, e.g., KOZBOR et al., Immunol. Today 4: 72 (1983)), EBV transformation technology (see, e.g., COLE et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)) or using yeast or phage display (see, e.g., MARKS et al., J. Mol. Biol. 222: 581 (1991)). In a specific embodiment, human antibodies are produced in transgenic mice. Techniques for preparing such partially to fully human antibodies are known in the art and any such techniques may be used. According to a particularly preferred embodiment, fully human antibody sequences are prepared in transgenic mice engineered to express human heavy and light chain antibody genes. Exemplary descriptions of preparing transgenic mice that produce human antibodies are found in application WO 02/43478 and U.S. Patent No. 6,657,103 (Abgenix) and its sub-lines. B cells from transgenic mice that produce the desired antibodies can then be fused to prepare a fusion tumor cell line for continuous production of antibodies. See, e.g., U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016 and 5,545,806; and JAKOBOVITS, Adv. Drug Del. Rev. 31:33-42 (1998); GREEN et al., J. Exp. Med. 188:483-95 (1998).

如本文中所使用，術語「人源化抗體」係指含有來自非人類(例如鼠類)抗體以及人類抗體之序列的抗體形式。此類抗體係含有來源於非人類免疫球蛋白之最小序列的嵌合抗體。一般而言，人源化抗體將包含至少一個且通常兩個可變域中之基本上全部可變域，其中全部或基本上全部高變環對應於非人類免疫球蛋白之高變環且全部或基本上全部FR區為人類免疫球蛋白序列之FR區。人源化抗體視情況亦將包含免疫球蛋白恆定區(Fc)之至少一部分，通常，人類免疫球蛋白之恆定區的至少一部分。參見例如CABILLY,美國專利第4,816,567號；QUEEN等人, (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033；及ANTIBODY ENGINEERING: A PRACTICAL APPROACH (牛津大學出版社, 1996)。As used herein, the term "humanized antibody" refers to an antibody form containing sequences from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. A humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. See, e.g., CABILLY, U.S. Patent No. 4,816,567; QUEEN et al., (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996).

如本文中所使用，術語「抑制」或「……之抑制」意謂降低可量測之量或完全防止。As used herein, the term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent entirely.

術語「哺乳動物」係指歸類為哺乳動物之任何有機體，包括小鼠、大鼠、兔、狗、貓、牛、馬及人類。在本發明之一個實施例中，哺乳動物為小鼠。在本發明之另一實施例中，哺乳動物為人類。The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the present invention, the mammal is a mouse. In another embodiment of the present invention, the mammal is a human.

術語「轉移癌」及「轉移性疾病」意謂已擴散至局部淋巴結或遠端位點且意欲包括在AUA系統下之D期疾病及TNM系統下之T×N×M+期的癌症。The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or distant sites and are intended to include stage D disease under the AUA system and stage TxNxM+ under the TNM system.

如本文中所使用之術語「經修飾之」係指天然胺基酸、非天然胺基酸、天然胺基酸多肽或非天然胺基酸多肽存在變化。此類變化或修飾可藉由以下方式獲得：天然胺基酸、非天然胺基酸、天然胺基酸多肽或非天然胺基酸多肽之合成後修飾，或藉由共轉譯，或藉由天然胺基酸、非天然胺基酸、天然胺基酸多肽或非天然胺基酸多肽之轉譯後修飾。As used herein, the term "modified" refers to changes in natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. Such changes or modifications can be obtained by post-synthetic modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides, or by co-translation, or by post-translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides.

「分子識別」意謂其中主體分子能夠與第二分子(亦即客體)形成複合物的化學事件。此製程經由非共價化學鍵進行，包括但不限於氫鍵結、疏水相互作用、離子相互作用。"Molecular recognition" means a chemical event in which a host molecule is able to form a complex with a second molecule (i.e., the guest). This process occurs via non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

如本文中所使用，術語「單株抗體」係指自實質上均質抗體之群體獲得之抗體，亦即除可少量存在的可能天然存在之突變外，構成該群體之個別抗體為相同的。單株抗體具有針對單一抗原性抗原決定基的高度特異性。相比之下，習知(多株)抗體製劑典型地包括針對不同抗原決定基(或對其具有特異性)之多種抗體。在一個實施例中，多株抗體含有複數個在含有多個抗原性抗原決定基之單一抗原內具有不同抗原決定基特異性、親和力或親合力之單株抗體。修飾語「單株」指示抗體之特徵為自實質上均質之抗體群體獲得，且不應理解為需要藉由任何特定方法產生該抗體。例如，根據本發明使用之單株抗體可藉由首先由KOHLER等人, Nature 256: 495 (1975)描述之融合瘤方法製備，或可藉由重組DNA方法(參見例如美國專利第4,816,567號)製備。亦可使用例如CLACKSON等人, Nature 352: 624-628 (1991)及MARKS等人, J. Mol. Biol. 222: 581-597 (1991)中所描述之技術自噬菌體抗體庫分離出「單株抗體」。此等單株抗體通常將以至少約1 μM、更通常至少約300 nM、通常至少約30 nM、較佳至少約10 nM、更佳至少約3 nM或更佳之Kd結合，通常藉由ELISA測定。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies have a high degree of specificity for a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of antibodies that are directed to (or specific for) different antigenic epitopes. In one embodiment, a polyclonal antibody contains a plurality of monoclonal antibodies with different antigenic determinant specificities, affinities, or avidities within a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by the fusion tumor method first described by KOHLER et al., Nature 256: 495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phagosome antibody libraries using techniques described, for example, by CLACKSON et al., Nature 352: 624-628 (1991) and MARKS et al., J. Mol. Biol. 222: 581-597 (1991). Such monoclonal antibodies will typically bind with a Kd of at least about 1 μM, more typically at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, typically as determined by ELISA.

「非天然胺基酸(non-natural amino acid)」或另外書寫為「nnAA」係指並非二十(20)種常見胺基酸或焦離胺酸或硒半胱胺酸中之一者的胺基酸。可與術語「nnAA」同義使用之其他術語為「非天然編碼之胺基酸(non-natural encoded amino acid)」、「非天然胺基酸(unnatural amino acid)」、「非天然存在之胺基酸(non-natural occuring amino acid)」。另外，術語nnAA包括但不限於並非天然存在之胺基酸且可以合成方式獲得或可藉由修飾非天然胺基酸獲得。"Non-natural amino acid" or otherwise written as "nnAA" refers to an amino acid that is not one of the twenty (20) common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "nnAA" are "non-natural encoded amino acid," "unnatural amino acid," "non-natural occurring amino acid." Additionally, the term nnAA includes but is not limited to amino acids that are not naturally occurring and may be obtained synthetically or may be obtained by modifying non-natural amino acids.

「醫藥賦形劑」包含諸如佐劑、載劑、pH調節劑及緩衝劑、張力調節劑、濕潤劑、防腐劑及其類似物之材料。"Drug formulations" include materials such as adjuvants, carriers, pH adjusters and buffers, tonicity adjusters, wetting agents, preservatives and the like.

「醫藥學上可接受」係指在生理學上與人類或其他哺乳動物相容之無毒、惰性及/或組合物。"Pharmaceutically acceptable" means non-toxic, inert and/or compositions that are physiologically compatible with humans or other mammals.

術語「多肽」意謂具有至少約4、5、6、7或8個胺基酸之聚合物。在整個說明書中，使用標準三個字母(參見表II)或單個字母名稱表示胺基酸。在此項技術中，此術語通常可與「肽」或「蛋白」互換使用。The term "polypeptide" means a polymer having at least about 4, 5, 6, 7 or 8 amino acids. Throughout the specification, the amino acids are represented by the standard three-letter (see Table II) or single-letter designations. In this art, this term is often used interchangeably with "peptide" or "protein".

如本文中所使用，術語「單鏈Fv」或「scFv」或「單鏈」抗體係指包含抗體之V_H及V_L域之抗體片段，其中此等域存在於單一多肽鏈中。一般而言，Fv多肽進一步包含在V_H與V_L域之間的多肽連接基團，其使得sFv能夠形成用於抗原結合之所需結構。關於sFv之綜述，參見PLUCKTHUN, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES,第113卷, Rosenburg and Moore編, Springer-Verlag,紐約,第269-315頁(1994)。As used herein, the term "single-chain Fv" or "scFv" or "single-chain" antibody refers to an antibody fragment comprising the_VH and_VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linking group between the_VH and_VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see PLUCKTHUN, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

如本文中所使用，術語「特異性的(specific)」、「特異性結合(specifically binds)」及「特異性結合(binds specifically)」係指抗體與目標抗原的抗原決定基之選擇性結合。可藉由在一組給定條件下比較同適當抗原之結合與同不相關抗原或抗原混合物之結合對抗體進行結合特異性測試。若抗體與適當抗原的結合比與不相關抗原或抗原混合物多至少2、5、7且較佳10倍，則將其視為特異性的。在一個實施例中，特異性抗體為僅結合NECTIN-4抗原但不結合至任何其他不相關抗原之抗體。在另一實施例中，特異性抗體為結合人類NECTIN-4抗原但不結合與該NECTIN-4抗原具有70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高之胺基酸同源性之非人類NECTIN-4抗原的抗體。在另一實施例中，特異性抗體為結合人類NECTIN-4抗原但不結合與該NECTIN-4抗原之胺基酸序列具有70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高之百分比一致性之非人類NECTIN-4抗原的抗體。在另一實施例中，特異性抗體為結合人類NECTIN-4抗原且結合鼠類NECTIN-4抗原，但與人類抗原之結合程度更高的抗體。在另一實施例中，特異性抗體為結合人類NECTIN-4抗原且結合靈長類動物NECTIN4抗原，但與人類抗原之結合程度更高的抗體。在另一實施例中，特異性抗體結合至人類NECTIN-4抗原及任何非人類NECTIN-4抗原，但與人類抗原或其任何組合之結合程度更高。As used herein, the terms "specific", "specifically binds", and "binds specifically" refer to the selective binding of an antibody to an antigenic determinant of a target antigen. Antibodies can be tested for binding specificity by comparing binding to an appropriate antigen with binding to an unrelated antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to an unrelated antigen or antigen mixture. In one embodiment, a specific antibody is an antibody that binds only to the NECTIN-4 antigen but not to any other unrelated antigen. In another embodiment, the specific antibody is an antibody that binds to a human NECTIN-4 antigen but does not bind to a non-human NECTIN-4 antigen having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid homology with the NECTIN-4 antigen. In another embodiment, the specific antibody is an antibody that binds to a human NECTIN-4 antigen but does not bind to a non-human NECTIN-4 antigen having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity with the amino acid sequence of the NECTIN-4 antigen. In another embodiment, the specific antibody is an antibody that binds to human NECTIN-4 antigen and to mouse NECTIN-4 antigen, but binds to human antigen to a higher degree. In another embodiment, the specific antibody is an antibody that binds to human NECTIN-4 antigen and to primate NECTIN-4 antigen, but binds to human antigen to a higher degree. In another embodiment, the specific antibody binds to human NECTIN-4 antigen and any non-human NECTIN-4 antigen, but binds to human antigen or any combination thereof to a higher degree.

如本文中所使用之「以治療」或「治療的」及語法相關之術語，係指任何疾病後果之改善，諸如長期生存、較低發病率及/或副作用減輕，此等作為替代治療模態之副產物；如本領域中容易瞭解，完全根除疾病為較佳的，但儘管不為對治療行為之要求。As used herein, the terms "treat" or "therapeutic" and grammatically related terms refer to any improvement in disease outcomes, such as long-term survival, lower morbidity and/or reduced side effects, as a byproduct of alternative treatment modalities; as is well understood in the art, complete eradication of the disease is preferred, although not a requirement for therapeutic action.

術語「變異體」係指展現自所描述類型或標準之變化的分子，諸如在特定描述之蛋白(例如，如表IV中所示之NECTIN-4蛋白)之對應位置具有一或多個不同胺基酸殘基的蛋白。類似物為變異蛋白之一實例。剪接同功異型物及單核苷酸多型性(SNP)為變異體之其他實例。The term "variant" refers to a molecule that exhibits variation from a described type or standard, such as a protein having one or more different amino acid residues at corresponding positions in a particular described protein (e.g., NECTIN-4 protein as shown in Table IV). Analogs are one example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants.

片語「經分離之」或「生物上純的」係指一種材料，其實質上或基本上不含如在其原生狀態下所發現之通常與該材料相關的組分。因此，根據本發明之經分離之肽較佳不含有通常在其原位環境中與肽相關之材料。例如，當聚核苷酸實質上與除NECTIN-4基因以外之基因對應或互補或編碼除NECTIN-4基因產物或其片段以外之多肽的污染物聚核苷酸分離時，該聚核苷酸係稱為「經分離之」。熟習此項技術者可容易地採用核酸分離程序以獲得經分離之NECTIN-4聚核苷酸。例如，當採用物理、機械或化學方法從通常與蛋白相關之細胞成分中移除NECTIN-4蛋白時，蛋白被稱為“經分離之”。熟習此項技術者可容易地採用標準純化方法以獲得經分離之NECTIN-4蛋白。或者，經分離之蛋白可藉由化學手段製備。The phrases "isolated" or "biologically pure" refer to a material that is substantially or essentially free of components normally associated with the material as found in its native state. Thus, an isolated peptide according to the present invention is preferably free of materials normally associated with the peptide in its native environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond to or complement genes other than the NECTIN-4 gene or encode polypeptides other than the NECTIN-4 gene product or fragments thereof. One skilled in the art can readily employ nucleic acid separation procedures to obtain isolated NECTIN-4 polynucleotides. For example, a NECTIN-4 protein is said to be "isolated" when physical, mechanical or chemical methods are employed to remove the protein from cellular components normally associated with the protein. Those skilled in the art can readily employ standard purification methods to obtain isolated NECTIN-4 protein. Alternatively, the isolated protein can be prepared by chemical means.

合適之「標記」包括放射性核種、酶、基質、輔因子、抑制劑、螢光部分、化學發光部分、磁性顆粒及類似物。教示此類標記之用途的專利包括美國專利第3,817,837號；第3,850,752號；第3,939,350號；第3,996,345號；第4,277,437號；第4,275,149號；及第4,366,241號。另外，本文中所提供之抗體可用作氟抗體之抗原結合組分。(參見例如Zeytun等人,Nat. Biotechnol.21:1473-79 (2003))。Suitable "labels" include radionuclides, enzymes, matrices, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Patents Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, the antibodies provided herein can be used as antigen binding components of fluoroantibodies. (See, e.g., Zeytun et al.,Nat. Biotechnol. 21:1473-79 (2003)).

本發明之「NECTIN-4蛋白」及/或「NECTIN-4有關蛋白」包括本文中特定鑑別之蛋白(參見表IV)，以及遵循本文所概述之或可易於在此項技術中獲得之方法在無需過度實驗之情況下而分離/生成及表徵的對偶基因變異體、保守取代變異體、類似物及同源物。亦包括組合不同NECTIN-4蛋白或其片段之部分的融合蛋白，以及NECTIN-4蛋白及異源多肽之融合蛋白。此類NECTIN-4蛋白統稱為NECTIN-4有關蛋白、本發明之蛋白或NECTIN-4。術語「NECTIN-4有關蛋白」係指以下之多肽片段或NECTIN-4蛋白序列：具有4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或多於25個胺基酸；或至少30、35、40、45、50、55、60、65、70、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、225、250、275、300、325、330、335、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、515、516、517、518、519或更多個胺基酸。The "NECTIN-4 protein" and/or "NECTIN-4 related protein" of the present invention include the proteins specifically identified herein (see Table IV), as well as allele variants, conservative substitution variants, analogs and homologs that are isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. It also includes fusion proteins that combine parts of different NECTIN-4 proteins or fragments thereof, as well as fusion proteins of NECTIN-4 proteins and heterologous polypeptides. Such NECTIN-4 proteins are collectively referred to as NECTIN-4 related proteins, proteins of the present invention or NECTIN-4. The term "NECTIN-4 related protein" refers to the following polypeptide fragments or NECTIN-4 protein sequences: having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 1 500, 510, 515, 516, 517, 518, 519 or more amino acids.

II.)抗體本發明之另一態樣提供結合至如本文中所揭示之NECTIN-4的抗體。在一個實施例中，抗體結合至NECTIN-4(表IV)及其他NECTIN-4有關蛋白。II.)Antibodies Another aspect of the invention provides antibodies that bind to NECTIN-4 as disclosed herein. In one embodiment, the antibody binds to NECTIN-4 (Table IV) and other NECTIN-4 related proteins.

如此項技術中已知，本發明之NECTIN-4抗體尤其適用於癌症(參見例如表I)，用於預後分析、成像、診斷及治療方法。在一個實施例中，本文揭示用於例如在免疫分析中偵測癌症之NECTIN-4結合分析。類似地，此類NECTIN-4抗體適用於(例如，當與治療劑組合時，諸如在ADC中)癌症(例如，表I中所闡述之癌症)之治療及/或預後，NECTIN-4在此等其他癌症中亦表現或過度表現。此外，細胞內表現之抗體(例如單鏈抗體)在治療上適用於治療涉及NECTIN-4及其他目標之表現的癌症。As is known in the art, the NECTIN-4 antibodies of the present invention are particularly useful in cancer (see, e.g., Table I), for prognostic analysis, imaging, diagnosis and treatment methods. In one embodiment, NECTIN-4 binding assays for detecting cancer, e.g., in immunoassays, are disclosed herein. Similarly, such NECTIN-4 antibodies are useful (e.g., when combined with therapeutic agents, such as in ADCs) for the treatment and/or prognosis of cancer (e.g., cancers described in Table I), in which NECTIN-4 is also expressed or overexpressed. In addition, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful for treating cancers involving expression of NECTIN-4 and other targets.

用於製備抗體、尤其單株抗體之各種方法為此項技術中所熟知。例如，抗體可藉由使用呈經分離之或經免疫結合之形式的NECTIN-4有關蛋白、肽或片段對適合之哺乳動物宿主免疫接種來製備(Antibodies: A Laboratory Manual, CSH Press編, Harlow及Lane (1988)；Harlow, Antibodies, Cold Spring Harbor Press, NY (1989))。另外，亦可使用NECTIN-4之融合蛋白，諸如NECTIN-4 GST融合蛋白。在一特定實施例中，產生包含NECTIN-4之全部或大部分胺基酸序列之GST融合蛋白，且隨後用作免疫原以生成適當抗體。在另一實施例中，合成NECTIN-4有關蛋白且用作免疫原Various methods for preparing antibodies, especially monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with NECTIN-4-related proteins, peptides or fragments in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, ed., Harlow and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of NECTIN-4, such as NECTIN-4 GST fusion proteins, can also be used. In a specific embodiment, a GST fusion protein comprising all or most of the amino acid sequence of NECTIN-4 is produced and then used as an immunogen to generate appropriate antibodies. In another embodiment, a NECTIN-4-related protein is synthesized and used as an immunogen.

另外，使用此項技術中已知之裸DNA免疫接種技術(具有或不具有經純化之NECTIN-4有關蛋白或NECTIN-4表現細胞)以生成針對所編碼之免疫原的免疫反應(關於綜述，參見DONNELLY等人, 1997, Ann. Rev. Immunol. 15: 617-648)。Alternatively, naked DNA immunization techniques known in the art (with or without purified NECTIN-4-related protein or NECTIN-4-expressing cells) are used to generate an immune response to the encoded immunogen (for a review, see DONNELLY et al., 1997, Ann. Rev. Immunol. 15: 617-648).

藉助於本文中所提供之實例進一步說明用於生成NECTIN-4抗體之較佳方法。用於製備用作免疫原之蛋白或多肽的方法為此項技術中所熟知。此項技術中亦熟知用於製備蛋白與載體，諸如BSA、KLH或另一載體蛋白之免疫原性結合物的方法。在一些情況下，使用例如碳化二亞胺試劑直接結合；在其他情況下，連接試劑，諸如由Pierce Chemical Co., Rockford, Ill.供應之彼等連接試劑為有效的。如此項技術中所理解，NECTIN-4免疫原之投與通常藉由在適合時間段內注射及使用適合佐劑進行。在免疫接種時程期間，可採用抗體之力價以判定抗體形成之適合性。The preferred methods for generating NECTIN-4 antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins and carriers, such as BSA, KLH or another carrier protein, are also well known in the art. In some cases, direct conjugation is performed using, for example, carbodiimide reagents; in other cases, linking reagents, such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. As understood in the art, administration of the NECTIN-4 immunogen is typically performed by injection over an appropriate time period and using a suitable adjuvant. During the immunization schedule, the titer of the antibody can be used to determine the suitability of the antibody formation.

NECTIN-4單株抗體可藉由此項技術中熟知之各種方式產生。例如，使用Kohler及Milstein之標準融合瘤技術或使產生抗體之B細胞永生化之修飾製備分泌所需單株抗體之永生化細胞株，如通常已知。藉由免疫分析篩選分泌所需抗體之永生化細胞株，其中抗原為NECTIN-4有關蛋白。當鑑別適當永生化細胞培養物時，細胞可擴增，且抗體產生自活體外培養物或產生自腹水。NECTIN-4 monoclonal antibodies can be produced by various means well known in the art. For example, an immortalized cell line secreting the desired monoclonal antibody is prepared using the standard fusion tumor technology of Kohler and Milstein or a modification that immortalizes antibody-producing B cells, as is generally known. Immortalized cell lines secreting the desired antibody are screened by immunoassay, wherein the antigen is a NECTIN-4-related protein. When appropriate immortalized cell cultures are identified, the cells can be expanded and antibodies produced from in vitro cultures or from ascites.

本發明之抗體或片段亦可藉由重組手段產生。特異性結合至NECTIN-4蛋白之所需區的區亦可在多個物種來源之嵌合或互補決定區(CDR)移植抗體的情況下產生。人源化或人類NECTIN-4抗體亦可產生且較佳用於治療情境中。藉由用非人類抗體CDR中之一或多者取代相應人類抗體序列使鼠類及其他非人類抗體人源化的方法為熟知的(參見例如JONES等人, 1986, Nature 321: 522-525；RIECHMANN等人, 1988, Nature 332: 323-327；VERHOEYEN 等人, 1988, Science 239: 1534-1536)。亦參見CARTER等人, 1993, Proc. Natl. Acad. Sci. USA 89: 4285及SIMS等人, 1993, J. Immunol. 151: 2296。The antibodies or fragments of the present invention may also be produced by recombinant means. Regions that specifically bind to desired regions of the NECTIN-4 protein may also be produced with chimeric or complementary determining regions (CDR) grafted antibodies from multiple species. Humanized or human NECTIN-4 antibodies may also be produced and are preferably used in therapeutic settings. Methods for humanizing mouse and other non-human antibodies by replacing the corresponding human antibody sequence with one or more of the non-human antibody CDRs are well known (see, e.g., JONES et al., 1986, Nature 321: 522-525; RIECHMANN et al., 1988, Nature 332: 323-327; VERHOEYEN et al., 1988, Science 239: 1534-1536). See also CARTER et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and SIMS et al., 1993, J. Immunol. 151: 2296.

在一個實施例中，本發明之人類單株抗體可使用VelocImmune小鼠來製備，其中免疫球蛋白重鏈(VH、DH及JH片段)及/或κ輕鏈(VK及JK)基因座處攜帶內源性小鼠可變片段的基因體序列已全部或部分經攜帶人類免疫球蛋白重鏈(VH、DH及JH)及/或κ輕鏈(VK 及 JK)基因座之未經重排生殖系可變片段的人類基因體序列置換(Regeneron, Tarrytown, N.Y.)。參見例如美國專利第6,586,251號、第6,596,541號、第7,105,348號、第6,528,313號、第6,638,768號及第6,528,314號。In one embodiment, human monoclonal antibodies of the present invention can be prepared using VelocImmune mice, in which the genome sequences carrying endogenous mouse variable segments at the immunoglobulin heavy chain (VH, DH and JH segments) and/or kappa light chain (VK and JK) loci have been replaced in whole or in part with human genome sequences carrying unrearranged germline variable segments of the human immunoglobulin heavy chain (VH, DH and JH) and/or kappa light chain (VK and JK) loci (Regeneron, Tarrytown, N.Y.). See, e.g., U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

另外，本發明之人類抗體可使用含有編碼未經重排人類重鏈(mu及γ)及κ輕鏈免疫球蛋白序列之人類免疫球蛋白基因微型基因座以及使內源性mu及κ鏈基因座不活化之靶向突變的HuMAb小鼠(Medarex, Inc.)生成(參見例如LONBERG等人, (1994) Nature 368(6474): 856-859)。In addition, human antibodies of the present invention can be generated using the HuMAb mouse (Medarex, Inc.) that contains a human immunoglobulin gene miniloci encoding unrearranged human heavy chain (mu and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate the endogenous mu and κ chain loci (see, e.g., LONBERG et al., (1994) Nature 368(6474): 856-859).

在另一實施例中，本發明之全人類抗體可使用在轉殖基因及轉染色體上承載人類免疫球蛋白序列之小鼠，諸如承載人類重鏈轉殖基因及人類輕鏈轉染色體之小鼠來產生。此類小鼠在本文中稱為「KM小鼠」，此類小鼠描述於TOMIZUKA等人, (2000) Proc. Natl. Acad. Sci. USA 97:722-727及授予TOMIZUKA等人之PCT公開案WO 02/43478中。In another embodiment, the fully human antibodies of the present invention can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes. Such mice are referred to herein as "KM mice" and are described in TOMIZUKA et al., (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02/43478 to TOMIZUKA et al.

本發明之人類單株抗體亦可使用篩選人類免疫球蛋白基因庫之噬菌體或酵母顯示方法製備。在此項技術中已確立此類用於分離人類抗體之噬菌體顯示方法。參見例如：美國專利第5,223,409號；第5,403,484號；及授予LADNER等人之美國專利第5,571,698號；授予DOWER等人之美國專利第5,427,908號及第5,580,717號；授予MCCAFFERTY等人之美國專利第5,969,108號及第6,172,197號；及美國專利第5,885,793號；第6,521,404號；第6,544,731號；第6,555,313號；授予GRIFFITHS等人之第6,582,915號及第6,593,081號。The human monoclonal antibodies of the present invention can also be prepared using phage or yeast display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies have been established in the art. See, e.g., U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to LADNER et al.; 5,427,908 and 5,580,717 to DOWER et al.; 5,969,108 and 6,172,197 to MCCAFFERTY et al.; and 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to GRIFFITHS et al.

本發明之人類單株抗體亦可使用人類免疫細胞已重建於其中以使得免疫接種時可產生人類抗體反應之SCID小鼠來製備。此類小鼠描述於例如授予WILSON等人之美國專利第5,476,996號及第5,698,767號中。The human monoclonal antibodies of the present invention can also be prepared using SCID mice in which human immune cells have been reconstituted so that human antibody responses can be produced upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

另外地，本發明之人類抗體可通過使用轉殖基因小鼠之技術製備，該轉殖基因小鼠針對抗體產生而失活，經人類重鏈及輕鏈基因座工程改造，被稱為轉基因鼠(Amgen Fremont, Inc.，前稱Abgenix, Inc.)。製備產生人類抗體之轉殖基因小鼠的例示性描述可見於美國專利第6,657,103號。亦參見美國專利第5,569,825號；第5,625,126號；第5,633,425號；第5,661,016號；及第5,545,806號；及MENDEZ等人,Nature Genetics, 15: 146-156 (1998)；KELLERMAN, S. A. & GREEN, L. L., Curr. Opin. Biotechnol 13, 593-597 (2002)。Alternatively, the human antibodies of the present invention can be prepared by using transgenic mice that are inactivated for antibody production and engineered with human heavy and light chain loci, referred to as transgenic mice (Amgen Fremont, Inc., formerly Abgenix, Inc.). Exemplary descriptions of the preparation of transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and MENDEZ et al., Nature Genetics, 15: 146-156 (1998); KELLERMAN, S. A. & GREEN, L. L., Curr. Opin. Biotechnol 13, 593-597 (2002).

以上產生方法中之任一者得到具有結合NECTIN-4，或與NECTIN-4具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性的同源物或片段或多肽序列之一定能力的抗體。Any of the above production methods yields an antibody having a certain ability to bind to NECTIN-4, or a homologue or fragment or polypeptide sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with NECTIN-4.

針對NECTIN-4之抗體、其結合片段及包含其之抗體藥物結合物的結合親和力(K_D)可為1 mM或以下、100 nM或以下、10 nM或以下、2 nM或以下或1 nM或以下。或者，K_D可在5與10 nM之間；或在1與2 nM之間。K_D可在1微莫耳與500微莫耳之間或在500微莫耳與1 nM之間。The binding affinity (_KD ) of the antibodies, binding fragments thereof, and antibody-drug conjugates comprising the same for NECTIN-4 may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the_KD may be between 5 and 10 nM; or between 1 and 2 nM._{The KD} may be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.

藉由締合常數(Ka)及解離常數(Kd)測定抗原結合蛋白之結合親和力(KD=Kd/Ka)。結合親和力可藉由BIACORE，例如藉由將測試抗體捕捉於經蛋白A塗佈之感測器表面上且使NECTIN-4在此表面上流動量測。或者，結合親和力可藉由FORTEBIO，例如將測試抗體受體捕獲於經蛋白A塗佈之針上且使NECTIN-4在此表面上流動量測。熟習此項技術者可鑑別此項技術中已知量測結合親和力之其他適合的分析法。The binding affinity of the antigen binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd/Ka). Binding affinity can be measured by BIACORE, for example, by capturing the test antibody on a protein A coated sensor surface and flowing NECTIN-4 over the surface. Alternatively, binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a protein A coated needle and flowing NECTIN-4 over the surface. Other suitable analytical methods known in the art for measuring binding affinity can be identified by those skilled in the art.

本發明之經工程改造之抗體包括其中V_H及/或V_L內之構架殘基已經修飾(例如以改良抗體之性質)的抗體。通常，進行此類構架修飾以降低抗體之免疫原性。例如，一種方法係使一或多個構架殘基「回復突變」成相應生殖系序列。更特定言之，已進行體細胞突變之抗體可含有不同於獲得抗體之生殖系序列的構架殘基。此類殘基可藉由比較抗體構架序列與獲得抗體之生殖系序列來鑑別。為了使構架區序列恢復其生殖系組態，體細胞突變可藉由例如定點突變誘發或PCR介導之突變誘發而「回復突變」成生殖系序列(例如白胺酸「回復突變」成甲硫胺酸)。此類「回復突變」之抗體亦意欲包涵於本發明中。The engineered antibodies of the present invention include antibodies in which the framework residues within the_VH and/or_VL have been modified (e.g., to improve the properties of the antibody). Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "revert mutation" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic cell mutation may contain framework residues that are different from the germline sequence of the obtained antibody. Such residues can be identified by comparing the antibody framework sequence with the germline sequence of the obtained antibody. In order to restore the framework region sequence to its germline configuration, the somatic cell mutation can be "reverted" to the germline sequence by, for example, site-directed mutagenesis induction or PCR-mediated mutagenesis induction (e.g., leucine is "reverted" to methionine). Such "reverted mutation" antibodies are also intended to be encompassed by the present invention.

亦可對V_H及/或V_L進行工程改造以改變與抗原之結合親和力。例如，本發明亦意欲涵蓋改變構架及/或CDR區內之殘基以提高親和力或降低對nectin-4之親和力。_VH and/or_VL can also be engineered to alter the binding affinity to the antigen. For example, the present invention also contemplates altering residues within the framework and/or CDR regions to increase affinity or decrease affinity for nectin-4.

構架修飾之另一類型涉及使構架區內，或甚至一或多個CDR區內之一或多個殘基突變，以移除T細胞抗原決定基，藉此降低抗體之潛在免疫原性。此方法亦稱為「去免疫」且進一步詳細描述於CARR等人之美國專利公開案第2003/0153043號中。Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell antigenic determinants, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is further described in detail in U.S. Patent Publication No. 2003/0153043 by CARR et al.

除構架或CDR區內所作之修飾之外或可替代地，本發明之抗體可經工程改造以包括Fc區內之修飾，通常以改變抗體之一或多種功能性質，諸如血清半衰期、補體結合、Fc受體結合及/或抗原依賴性細胞毒性。此外，本發明之NECTIN-4抗體可經化學修飾(例如一或多個化學部分可附接至抗體)或經修飾以改變其醣基化，同樣改變抗體之一或多種功能性質。此等實施例中之各者進一步詳細描述於下文中。In addition to or in lieu of modifications made within the framework or CDR regions, the antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the NECTIN-4 antibodies of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, also altering one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

在一個實施例中，CH1之鉸鏈區經修飾以使得鉸鏈區中之半胱胺酸殘基之數目改變，例如增加或減少。此方法進一步描述於BODMER等人之美國專利第5,677,425號中。改變CH1鉸鏈區中之半胱胺酸殘基數目以例如促進輕鏈及重鏈組裝或提高或降低NECTIN-4抗體之穩定性。In one embodiment, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to BODMER et al. The number of cysteine residues in the hinge region of CH1 is changed to, for example, promote light and heavy chain assembly or increase or decrease the stability of the NECTIN-4 antibody.

在另一實施例中，抗體之Fc鉸鏈區經突變以縮短NECTIN-4抗體之生物半衰期。更特定言之，將一或多個胺基酸突變引入至Fc鉸鏈片段之CH2-CH3域界面區以使得抗體對葡萄球菌蛋白A (Staphylococcyl protein A；SpA)的結合相對於原生Fc鉸鏈域SpA結合減弱。此方法進一步詳細描述於WARD等人之美國專利第6,165,745號中。In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the NECTIN-4 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment to reduce the binding of the antibody to Staphylococcyl protein A (SpA) relative to native Fc hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745 to WARD et al.

在另一實施例中，NECTIN-4抗體經修飾以延長其生物半衰期。可進行多種方法。例如，可如授予Ward之美國專利第6,277,375號中所描述引入突變。或者，為延長生物半衰期，抗體可在CH1或CL區內改變以含有獲自IgG之Fc區CH2域之兩個環的救助受體結合抗原決定基，如PRESTA等人之美國專利第5,869,046號及第6,121,022號所描述。In another embodiment, the NECTIN-4 antibody is modified to extend its biological half-life. A variety of methods can be used. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to extend the biological half-life, the antibody can be altered in the CH1 or CL region to contain salvage receptor binding antigenic determinants derived from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to PRESTA et al.

在另一實施例中，NECTIN-4抗體包含表VI(A)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(A).

在另一實施例中，NECTIN-4抗體包含表VI(B)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(B).

在另一實施例中，NECTIN-4抗體包含表VI(C)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(C).

在另一實施例中，NECTIN-4抗體包含表VI(D)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(D).

在另一實施例中，NECTIN-4抗體包含表VI(E)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(E).

在另一實施例中，NECTIN-4抗體包含表VI(F)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody rechain sequence described in Table VI(F).

在另一實施例中，NECTIN-4抗體包含表VI(G)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(G).

在另一實施例中，NECTIN-4抗體包含表VI(H)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(H).

在另一實施例中，NECTIN-4抗體包含表VI(I)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(I).

在另一實施例中，NECTIN-4抗體包含表VI(J)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(J).

在另一實施例中，NECTIN-4抗體包含表VI(K)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(K).

在另一實施例中，NECTIN-4抗體包含表VI(L)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(L).

在另一實施例中，NECTIN-4抗體包含表VIII中所闡述之抗體重鏈可變區序列。In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain variable region sequence described in Table VIII.

在另一實施例中，NECTIN-4抗體包含表IX中所闡述之抗體輕鏈可變區序列。In another embodiment, the NECTIN-4 antibody comprises the antibody light chain variable region sequence described in Table IX.

在另一實施例中，NECTIN-4抗體包含表X中所闡述之抗體CDR序列。In another embodiment, the NECTIN-4 antibody comprises the antibody CDR sequences described in Table X.

在另一實施例中，NECTIN-4抗體結合至治療劑。In another embodiment, the NECTIN-4 antibody is conjugated to a therapeutic agent.

NECTIN-4抗體之反應性可藉由多種眾所周知的手段，包括西方墨點法、免疫沉澱、ELISA及FACS分析按需要使用NECTIN-4有關蛋白、NECTIN-4表現細胞或其萃取物建立。NECTIN-4抗體或其片段可經可偵測標記物標記或與第二分子結合。適合的可偵測標記物包括但不限於放射性同位素、螢光化合物、生物發光化合物、化學發光化合物、金屬螯合劑或酶。The reactivity of the NECTIN-4 antibody can be established by a variety of well-known means, including Western blot, immunoprecipitation, ELISA and FACS analysis, as desired, using NECTIN-4 related proteins, NECTIN-4 expressing cells or extracts thereof. The NECTIN-4 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators or enzymes.

III.)抗體藥物結合物在另一態樣中，本發明提供包含結合至治療劑之抗體(較佳地，本文所揭示之NECTIN-4抗體)的抗體-藥物結合物(ADC)。治療劑可為細胞毒性劑、細胞生長抑制劑、化學治療劑、藥物、生長抑制劑、毒素(例如，細菌、真菌、植物或動物來源之酶活性毒素或其片段)或放射性同位素(亦即放射性結合物)。在另一態樣中，本發明進一步提供使用ADC之方法。在一個態樣中，ADC包含共價附接或經由肟鍵附接至細胞毒性劑或可偵測藥劑的以上NECTIN-4抗體中之任一者。III.)Antibody Drug Conjugates In another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody (preferably, a NECTIN-4 antibody disclosed herein) bound to a therapeutic agent. The therapeutic agent may be a cytotoxic agent, a cell growth inhibitor, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof), or a radioactive isotope (i.e., a radioactive conjugate). In another aspect, the present invention further provides methods of using the ADC. In one aspect, the ADC comprises any of the above NECTIN-4 antibodies covalently attached or attached via an oxime bond to a cytotoxic agent or a detectable agent.

在另一實施例中，ADC包含使用自體水解順丁烯二醯亞胺用於半胱胺酸修飾之結合至治療劑的NECTIN-4抗體(參見WO 2013/173337)。In another embodiment, the ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using autolyzed cis-butylenediimide for cysteine modification (see WO 2013/173337).

在另一實施例中，ADC包含使用半胱胺酸修飾結合至治療劑之NECTIN-4抗體且進一步包含減少半胱胺酸殘基以形成硫氫基部分。In another embodiment, the ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using cysteine modification and further comprises reduction of the cysteine residue to form a sulfhydryl moiety.

在另一實施例中，ADC包含使用多肽部分及自分解型部分與治療劑結合的NECTIN-4抗體。In another embodiment, the ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using a polypeptide portion and a self-immolative portion.

在另一實施例中，ADC包含結合至治療劑之NECTIN-4抗體，其中該ADC具有高藥物抗體比率(DAR)。In another embodiment, the ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent, wherein the ADC has a high drug to antibody ratio (DAR).

藉助於先前技術，抗體-藥物結合物在治療癌症中用於局部遞送細胞毒性劑或細胞生長抑制劑的用途(Syrigos及Epenetos (1999) Anticancer Research 19:605-614；NICULESCU-DUVAZ及SPRINGER (1997) Adv. Drg. Del. Rev. 26:151-172；美國專利第4,975,278號)允許將藥物部分靶向遞送至腫瘤且在其中形成細胞內累積，其中全身投與此等未結合之藥物藥劑可對正常細胞以及設法消除之腫瘤細胞產生不可接受的毒性水平(BALDWIN等人, (1986) Lancet,頁碼(1986年3月15日):603-05；Thorpe, (1985) 「Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review」, in Monoclonal Antibodies '84: Biological and Clinical Applications, A. PINCHERA等人(編),第475-506頁)。藉此尋求毒性最小的最大功效。已報導多株抗體及單株抗體均適用於此等策略(Rowland等人, (1986) Cancer Immunol. Immunother., 21:183-87)。用於此等方法之藥物包括柔紅黴素(daunomycin)、多柔比星、胺甲喋呤及長春地辛(ROWLAND等人, (1986)，見上文)。用於抗體-毒素結合物中之毒素包括：細菌毒素，諸如白喉毒素；植物毒素，諸如蓖麻毒素；小分子毒素，諸如格爾德黴素(geldanamycin) (MANDLER等人, (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581；MANDLER等人, (2000) Bioorganic & Med. Chem. Letters 10:1025-1028；MANDLER等人, (2002) Bioconjugate Chem. 13:786-791)；類美登素(EP 1391213；LIU等人, (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623)；及卡奇黴素(LODE等人, (1998) Cancer Res. 58:2928；HINMAN等人, (1993) Cancer Res. 53:3336-3342)。毒素可藉由包括微管蛋白結合、DNA結合或拓樸異構酶抑制之機制影響其細胞毒性及細胞生長抑制效應。一些細胞毒性藥物在與大型抗體或蛋白受體配體結合時，傾向於為非活性的或弱活性的。With the help of the prior art, the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; NICULESCU-DUVAZ and SPRINGER (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) allows the targeted delivery of drug moieties to tumors and their intracellular accumulation, where systemic administration of such unbound drug agents may produce unacceptable levels of toxicity to normal cells as well as to the tumor cells being managed (BALDWIN et al., (1986) Lancet, pp. (March 15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, A. PINCHERA et al. (eds.), pp. 475-506). This is done to achieve maximum efficacy with minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be suitable for these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (ROWLAND et al., (1986), supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; plant toxins such as ricin; small molecule toxins such as geldanamycin (MANDLER et al., (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; MANDLER et al., (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; MANDLER et al., (2002) Bioconjugate Chem. 13:786-791); maytansines (EP 1391213; LIU et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); and kacinomycin (LODE et al., (1998) Cancer Res. 58:2928; HINMAN et al., (1993) Cancer Res. 53:3336-3342). Toxins may affect their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or weakly active when bound to large antibodies or protein receptor ligands.

迄今為止，FDA已審批通過十二(12)種ADC，包括吉妥珠單抗奧佐米星(gemtuzumab ozogamicin) (MYLOTARG，Wyeth Pharmaceuticals)，其為由FDA於2000年審批通過之第一ADC。(參見例如Drago等人, 2021 Nature Reviews 18, 327-344；Mckertish等人, 2021 Biomedicines 9, 872；Khongorzui等人, 2020 Molecular Cancer Res. 18:3-19；Bross等人, 2001 Clin. Cancer Res. 7, 1490-1496；Hamann等人, 2002 Bioconjug. Chem. 13, 47-58；Lamb, 2017 Drugs 77, 1603-1610.)。To date, the FDA has approved twelve (12) ADCs, including gemtuzumab ozogamicin (MYLOTARG, Wyeth Pharmaceuticals), which was the first ADC approved by the FDA in 2000. (See, e.g., Drago et al., 2021 Nature Reviews 18, 327-344; McKertish et al., 2021 Biomedicines 9, 872; Khongorzui et al., 2020 Molecular Cancer Res. 18:3-19; Bross et al., 2001 Clin. Cancer Res. 7, 1490-1496; Hamann et al., 2002 Bioconjug. Chem. 13, 47-58; Lamb, 2017 Drugs 77, 1603-1610.).

另外，商用抗體藥物結合物之實例為安適利(ADCETRIS) (維布妥昔單抗(brentuximab vedotin)，Seattle Genetics,)、ZEVALIN® (替伊莫單抗(ibritumomab tiuxetan)，Biogen/Idec)、KADCYLA® (恩美曲妥珠單抗(ado-trastuzumab emtansine)，Genentech)、BESPONSA® (奧英妥珠單抗(inotuzumab ozogamicin)，Pfizer/Wyeth)、POLIVY (維泊妥珠單抗(polatuzumab vedotin)，Genentech/Roche)、莫坎妥珠單抗(Cantuzumab mertansine) (Immunogen, Inc.)、MLN-2704 (Millennium Pharm.，BZL Biologics，Immunogen Inc.)及PADCEV (維汀-恩弗妥單抗-ejfv (enfortumab vedotin-ejfv)，Seattle Genetics/Astellas (Agensys, Inc.，Santa Monica，California)。In addition, examples of commercially available antibody-drug conjugates are ADCETRIS (brentuximab vedotin, Seattle Genetics,), ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec), KADCYLA® (ado-trastuzumab emtansine, Genentech), BESPONSA® (inotuzumab ozogamicin, Pfizer/Wyeth), POLIVY (polatuzumab vedotin, Genentech/Roche), Cantuzumab mertansine (Immunogen, Inc.), MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), and PADCEV (vetuximab-emtansine-ejfv) (Immunogen, Inc.). (enfortumab vedotin-ejfv), Seattle Genetics/Astellas (Agensys, Inc., Santa Monica, California).

此外，本文中描述包括但不限於適用於生成ADC之化學治療劑的治療劑。可使用之酶活性毒素及其片段包括白喉A鏈、白喉毒素之非結合活性片段、外毒素A鏈(來自綠膿桿菌(Pseudomonas aeruginosa))、蓖麻毒素A鏈、相思子毒素A鏈、莫迪素A鏈、α-帚麴菌素、光桐(Aleurites fordii)蛋白、香石竹(dianthin)蛋白、洋商陸(Phytolaca americana)蛋白(PAPI、PAPII及PAP-S)、苦瓜(momordica charantia)抑制劑、麻瘋樹毒蛋白、巴豆毒素、肥皂草抑制劑、白樹素、有絲分裂素、侷限麴菌素、酚黴素、伊諾黴素及單端孢黴烯(tricothecene)。參見例如1993年10月28日公開之WO 93/21232。多種放射性核種可用於產生放射性結合之抗體。實例包括¹⁷⁷Lu、⁸⁹Zr、²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y及¹⁸⁶Re。抗體與細胞毒性劑之結合物可使用多種雙官能蛋白偶合劑來製備，該等蛋白偶合劑諸如N-丁二醯亞胺基-3-(2-吡啶基二硫醇)丙酸酯(SPDP)、亞胺基硫雜環戊烷(IT)、醯亞胺酯之雙官能衍生物(諸如二亞胺代己二酸二甲酯鹽酸鹽)、活性酯(諸如辛二酸二丁二醯亞胺酯)、醛(諸如戊二醛)、雙疊氮基化合物(諸如雙(對疊氮基苯甲醯基)己二胺)、雙重氮鎓衍生物(諸如雙-(對重氮鎓苯甲醯基)-乙二胺)、二異氰酸酯(諸如2,6-二異氰酸甲苯酯)及雙活性氟化合物(諸如1,5-二氟-2,4-二硝基苯)。經碳-14標記之1-異硫氰基苯甲基-3-甲基二伸乙三胺五乙酸(MX-DTPA)為用於放射性核苷酸與抗體結合之例示性螯合劑(WO94/11026)。Additionally, described herein are therapeutic agents including, but not limited to, chemotherapeutic agents suitable for use in generating ADCs. Enzymatically active toxins and fragments thereof that may be used include diphtheria chain A, non-binding active fragments of diphtheria toxin, exotoxin chain A (fromPseudomonas aeruginosa ), ricin chain A, abrin A chain, modisin chain A, alpha-sarcosin,Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII and PAP-S),momordica charantia inhibitor, tardigrade protein, crotonin, saponin, scutellaria baicalensis inhibitor, mitogenins, fusin, phenomycin, enoxomycin and tricothecene. See, e.g., WO 93/21232 published Oct. 28, 1993. A variety of radionuclides can be used to generate radioconjugated antibodies. Examples include¹⁷⁷ Lu,⁸⁹ Zr,²¹² Bi,¹³¹ I,¹³¹ In,⁹⁰ Y, and¹⁸⁶ Re. Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters such as dimethyl diimidoadipate hydrochloride, active esters such as suberic acid, and 186 Re. The invention relates to a chelating agent for binding radionucleotides to antibodies. The chelating agent includes diisocyanate, ...

可與本發明之抗體結合之其他抗腫瘤劑包括BCNU；鏈脲黴素(streptozoicin)；長春新鹼及5-氟尿嘧啶；美國專利第5,053,394號、第5,770,710號中所描述之統稱為LL-E33288複合物的藥劑家族；以及埃斯波黴素(esperamicins) (美國專利第5,877,296號)。Other anti-tumor agents that can be combined with the antibodies of the present invention include BCNU; streptozoicin; vincristine and 5-fluorouracil; the family of agents collectively referred to as LL-E33288 complexes described in U.S. Patent Nos. 5,053,394 and 5,770,710; and esperamicins (U.S. Patent No. 5,877,296).

可使用之酶活性毒素及其片段包括白喉A鏈、白喉毒素之非結合活性片段、外毒素A鏈(來自綠膿桿菌)、蓖麻毒素A鏈、相思子毒素A鏈、莫迪素A鏈、α-帚麴菌素、光桐蛋白、香石竹蛋白、洋商陸蛋白(PAPI、PAPII及PAP-S)、苦瓜抑制劑、麻瘋樹毒蛋白、巴豆毒素、肥皂草抑制劑、白樹素、有絲分裂素、侷限麴菌素、酚黴素、伊諾黴素及單端孢黴烯。例如，蓖麻毒素免疫毒素可如Vitetta等人(1987) Science, 238:1098中所描述來製備。參見例如WO 93/21232 (1993年10月28日公開)。Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modisin A chain, α-sarcin, glaucoma proteins, caryophyllins, sphaerocephala proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, tartaric acid protein, crotonin, saponin, sphaerocephala inhibitor, mitogenins, fusin, phenomycin, inomycin and trichothecenes. For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science, 238:1098. See, for example, WO 93/21232 (published October 28, 1993).

本發明另外涵蓋形成於抗體與具有核分解活性之化合物(例如核糖核酸酶或DNA核酸內切酶，諸如去氧核糖核酸酶；DNA水解酶)之間的ADC。The invention further encompasses ADCs formed between an antibody and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; a DNA hydrolase).

為了選擇性毀壞腫瘤，抗體可包含高度放射性原子。多種放射性同位素可用於產生放射性結合之抗體。實例包括At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re⁸⁸、Sm⁵³、Bi²¹²、P³²、Pb²¹²及Lu之放射性同位素。當結合物用於偵測時，其可包含用於閃爍掃描研究之放射性原子，例如tc^99m或I¹²³，或用於核磁共振(NMR)成像(亦稱為磁共振成像，mri)之自旋標示，諸如同樣的碘-123、碘-131、銦-111、氟-19、碳-13、氮-15、氧-17、釓、錳或鐵。To selectively destroy tumors, antibodies may contain highly radioactive atoms. A variety of radioisotopes can be used to produce radioactively conjugated antibodies. Examples include At²¹¹ , I¹³¹ , I¹²⁵ , Y⁹⁰ , Re¹⁸⁶ , Re⁸⁸ , Sm⁵³ , Bi²¹² , P³² , Pb²¹² , and radioisotopes of Lu. When the conjugate is used for detection, it may contain a radioactive atom such as tc^99m or I¹²³ for scintillation scanning studies, or a spin label such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

放射性或其他標記可以已知方式併入結合物中。例如，肽可生物合成或可藉由使用涉及例如氟-19替代氫之適合胺基酸前驅物之化學胺基酸合成而合成。諸如tc^99m或I¹²³、Re¹⁸⁶、Re¹⁸⁸及In¹¹¹之標記可經由肽中之半胱胺酸殘基附接。釔-90可經由離胺酸殘基附接。IODOGEN方法(FRAKER等人, (1978) Biochem. Biophys. Res. Commun. 80: 49-57)可用於併入碘-123。「Monoclonal Antibodies in Immunoscintigraphy」(CHATAL, CRC Press 1989)詳細描述其他方法。Radioactive or other labels can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid promotors involving, for example, fluorine-19 replacing hydrogen. Labels such as tc^99m or I¹²³ , Re¹⁸⁶ , Re¹⁸⁸ and In¹¹¹ can be attached via cysteine residues in the peptide. Yttrium-90 can be attached via lysine residues. The IODOGEN method (FRAKER et al., (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (CHATAL, CRC Press 1989) describes other methods in detail.

本發明尤其提供用於靶向遞送治療劑之抗體-藥物結合物化合物。本案發明人已發現抗體-藥物結合物化合物具有針對表現NECTIN-4及其變異體之細胞的強效細胞毒性及/或細胞生長抑制活性。The present invention provides, inter alia, antibody-drug conjugate compounds for targeted delivery of therapeutic agents. The inventors of the present invention have discovered that the antibody-drug conjugate compounds have potent cytotoxic and/or cell growth inhibitory activity against cells expressing NECTIN-4 and its variants.

抗體-藥物結合物化合物包含共價連接至至少一個藥物單元之抗體單元。藥物單元可直接共價連接至抗體單元或經由連接基團單元(-LU-)連接至抗體單元。Antibody-drug conjugate compounds comprise an antibody unit covalently linked to at least one drug unit. The drug unit can be covalently linked to the antibody unit directly or via a linking group unit (-LU-).

在一些實施例中，抗體藥物結合物化合物具有下式： Ab-(LU-D)_pADC方案(I) 或其醫藥學上可接受之鹽或溶劑合物；其中： •  Ab為抗體單元，例如本發明之NECTIN-4抗體； •  (LU-D)為連接基團單元-藥物單元部分，其中： •  LU-為連接基團單元，且 •  -D為對目標細胞具有細胞生長抑制或細胞毒活性之藥物單元；且 •  p在1至20或可替代地1-50之範圍內。In some embodiments, the antibody-drug conjugate compound has the formula: Ab-(LU-D)_p ADC scheme (I) or a pharmaceutically acceptable salt or solvent conjugate thereof; wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; • (LU-D) is a linker unit-drug unit portion, wherein: • LU- is a linker unit, and • -D is a drug unit having cell growth inhibitory or cytotoxic activity on target cells; and • p is in the range of 1 to 20 or alternatively 1-50.

在一些實施例中，抗體藥物結合物化合物具有下式： Ab-(A_a-W_w—Y_y-D)_pADC方案(II) 或其醫藥學上可接受之鹽或溶劑合物，其中： •  Ab為抗體單元，例如本發明之NECTIN-4抗體；且 •  -A_a-W_w-Y_y-為連接基團單元(LU)，其中： •  -A-為延伸基團單元， •  a為0或1或2或3， •  各-W-獨立地為胺基酸單元， •  w為0至12範圍內之整數， •  -Y-為自分解型間隔基團單元， •  y為0、1或2； •  -D為對目標細胞具有細胞生長抑制或細胞毒活性之藥物單元；且 •  p為1至20或替代地1-50之整數。In some embodiments, the antibody-drug conjugate compound has the formula: Ab-(A_a -W_w —Y_y -D)_p ADC scheme (II) or a pharmaceutically acceptable salt or solvent complex thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; and • -A_a -W_w -Y_y - is a linking group unit (LU), wherein: • -A- is an extension group unit, • a is 0 or 1 or 2 or 3, • each -W- is independently an amino acid unit, • w is an integer in the range of 0 to 12, • -Y- is a self-degradable spacer group unit, • y is 0, 1 or 2; • -D is a drug unit having cell growth inhibition or cytotoxic activity on target cells; and • p is an integer from 1 to 20 or alternatively 1-50.

在一些實施例中，抗體藥物結合物化合物具有下式：ADC方案(III) 或ADC方案(IV) 或其醫藥學上可接受之鹽或溶劑合物，其中： •  Ab為抗體單元，例如本發明之NECTIN-4抗體； •  各R獨立地選自N、CH或C； •  R'為C或CH； •  W係選自：In some embodiments, the antibody drug conjugate compound has the formula: ADC solution (III) or ADC scheme (IV) or a pharmaceutically acceptable salt or solvent thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; • each R is independently selected from N, CH or C; • R' is C or CH; • W is selected from:

在一些實施例中，抗體藥物結合物化合物具有下式：ADC方案(V)ADC方案(VI) •  Ab為抗體單元，例如本發明之抗NECTIN-4抗體； •  各R獨立地選自N、CH或C； •  W係選自：•  X_b為選自由以下組成之群之間隔基團部分：烷基、雜烷基、聚乙二醇(PEG)及肽； •  b為0、1或2； •  Y_b為包含約1至約6個係天然及/或非天然胺基酸之胺基酸的多肽部分； •  Z_b為自分解型部分，包括但不限於：•  D為對目標細胞具有細胞生長抑制或細胞毒活性之藥物單元； •  p為1至20或替代地1-50之整數。In some embodiments, the antibody drug conjugate compound has the formula: ADC Solution (V) ADC Scheme (VI) • Ab is an antibody unit, such as the anti-NECTIN-4 antibody of the present invention; • Each R is independently selected from N, CH or C; • W is selected from: • X_b is a spacer moiety selected from the group consisting of: alkyl, heteroalkyl, polyethylene glycol (PEG) and peptide; • b is 0, 1 or 2; • Y_b is a polypeptide moiety comprising about 1 to about 6 amino acids that are natural and/or non-natural amino acids; • Z_b is a self-immolative moiety, including but not limited to: • D is a drug unit having cytostatic or cytotoxic activity on target cells; • p is an integer from 1 to 20 or alternatively 1-50.

在一些實施例中，抗體藥物結合物化合物具有下式：ADC方案(VII) 或其醫藥學上可接受之鹽或溶劑合物，其中： •  Ab為抗體單元，例如本發明之NECTIN-4抗體； •  R¹為，其中R²為未經取代或經取代之C₁-C₆烷基、雜烷基、環烷基或環雜烷基； •  R^x及R^y中之各者獨立地選自R及L-R^z，其限制條件為當R^x及R^y中之一者為NR^z時，其他者為R； •  R⁵為H或CR'₃，其中各R'獨立地為H或F； •  R⁶為H或CH₂CN； •  LU為連接基團單元；且 •  R為H或C₁-C₃烷基；且 •i為1至約20範圍內之整數。In some embodiments, the antibody drug conjugate compound has the formula: ADC scheme (VII) or a pharmaceutically acceptable salt or solvent thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; •^R1 is , wherein R² is unsubstituted or substituted C₁ -C₆ alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; • each of R^x and R^y is independently selected from R and LR^z , with the proviso that when one of R^x and R^y is NR^z , the other is R; • R⁵ is H or CR'₃ , wherein each R' is independently H or F; • R⁶ is H or CH₂ CN; • LU is a linking group unit; and • R is H or C₁ -C₃ alkyl; and •i is an integer in the range of 1 to about 20.

在一些實施例中，抗體藥物結合物化合物具有下式：ADC方案(VIII) 或其醫藥學上可接受之鹽或溶劑合物，其中： Ab為抗體單元，例如本發明之NECTIN-4抗體； R¹為，其中R²為未經取代或經取代之C₁-C₆烷基、雜烷基、環烷基或環雜烷基； R^x及R^y中之各者獨立地選自R及L-R^z，其限制條件為當R^x及R^y中之一者為NR^z時，其他者為R； R⁵為H或CR'₃，其中各R'獨立地為H或F； R⁶為H或CH₂CN； LU為連接基團單元；且 R為H或C₁-C₃烷基；且j為1至約20範圍內之整數。In some embodiments, the antibody drug conjugate compound has the formula: ADC solution (VIII) or a pharmaceutically acceptable salt or solvent thereof, wherein: Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention;^R1 is , wherein R² is unsubstituted or substituted C₁ -C₆ alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; each of R^x and R^y is independently selected from R and LR^z , with the proviso that when one of R^x and R^y is NR^z , the other is R; R⁵ is H or CR'₃ , wherein each R' is independently H or F; R⁶ is H or CH₂ CN; LU is a linking group unit; and R is H or C₁ -C₃ alkyl; andj is an integer in the range of 1 to about 20.

在一些實施例中，抗體藥物結合物化合物具有下式：ADC方案(IX) 或其醫藥學上可接受之鹽或溶劑合物，其中： Ab為抗體單元，例如本發明之NECTIN-4抗體； R¹為，其中R²為未經取代或經取代之C₁-C₆烷基、雜烷基、環烷基或環雜烷基； R^x及R^y中之各者獨立地選自R及L-R^z，其限制條件為當R^x及R^y中之一者為NR^z時，其他者為R； R⁵為H或CR'₃，其中各R'獨立地為H或F； R⁶為H或CH₂CN； LU為連接基團單元；且 R為H或C₁-C₃烷基；且k為1至約20範圍內之整數。In some embodiments, the antibody drug conjugate compound has the formula: ADC scheme (IX) or a pharmaceutically acceptable salt or solvent thereof, wherein: Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention;^R1 is , wherein R² is unsubstituted or substituted C₁ -C₆ alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; each of R^x and R^y is independently selected from R and LR^z , with the proviso that when one of R^x and R^y is NR^z , the other is R; R⁵ is H or CR'₃ , wherein each R' is independently H or F; R⁶ is H or CH₂ CN; LU is a linking group unit; and R is H or C₁ -C₃ alkyl; andk is an integer in the range of 1 to about 20.

在一些實施例中，抗體藥物結合物化合物具有下式：•  R¹為，其中R²為未經取代或經取代之C₁-C₆烷基、雜烷基、環烷基或環雜烷基； •  R³為H或C₁-C₃烷基； •  R⁵為H或CR'₃，其中各R'獨立地為H或F； •  R⁶為H或CH₂CN； •  Ab為抗體單元，例如本發明之抗NECTIN-4抗體； •  各R獨立地選自N、CH或C； •  J為結合部分； •  X_b為選自由以下組成之群之間隔基團部分：烷基、雜烷基、聚乙二醇(PEG)及肽； •  b為0、1或2； •  Y_b為包含約1至約6個係天然及/或非天然胺基酸之胺基酸的多肽部分； •  Z_b為自分解型部分，包括但不限於：In some embodiments, the antibody drug conjugate compound has the formula: • R¹ is , wherein R² is unsubstituted or substituted C₁ -C₆ alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; • R³ is H or C₁ -C₃ alkyl; • R⁵ is H or CR'₃ , wherein each R' is independently H or F; • R⁶ is H or CH₂ CN; • Ab is an antibody unit, such as the anti-NECTIN-4 antibody of the present invention; • each R is independently selected from N, CH or C; • J is a binding moiety; • X_b is a spacer group moiety selected from the group consisting of: alkyl, heteroalkyl, polyethylene glycol (PEG) and peptide; • b is 0, 1 or 2; • Y_b is a polypeptide portion comprising about 1 to about 6 amino acids that are natural and/or non-natural amino acids; • Z_b is a self-degradable moiety, including but not limited to:

對於包含複數個抗體之組合物，藥物負載由每一抗體之藥物分子的平均數目p表示。藥物負載可在每個抗體1至24個藥物(D)之範圍內。結合反應製備中之每一抗體的平均藥物數目可由諸如質譜法、ELISA分析及HPLC之習知手段來表徵。亦可根據p確定抗體-藥物-結合物之定量分佈。在一些情況下，當p為來自具有其他藥物負載之抗體-藥物-結合物的特定值時，均質抗體-藥物-結合物之分離、純化及表徵可藉由諸如逆相HPLC或電泳之手段來實現。在例示性實施例中，p為2至8。在一些實施例中，p為2至24。For compositions comprising a plurality of antibodies, drug loading is represented by the average number of drug molecules per antibody, p. Drug loading may range from 1 to 24 drugs (D) per antibody. The average number of drugs per antibody in the binding reaction preparation may be characterized by known means such as mass spectrometry, ELISA analysis, and HPLC. The quantitative distribution of the antibody-drug-conjugate may also be determined based on p. In some cases, when p is a specific value from an antibody-drug-conjugate with other drug loadings, separation, purification, and characterization of homogeneous antibody-drug-conjugates may be achieved by means such as reverse phase HPLC or electrophoresis. In exemplary embodiments, p is 2 to 8. In some embodiments, p is 2 to 24.

抗體-藥物結合物化合物之生成可藉由熟習此項技術者已知的技術來實現。簡言之，抗體-藥物結合物化合物包含NECTIN-4抗體。The production of antibody-drug conjugate compounds can be achieved by techniques known to those skilled in the art. Briefly, the antibody-drug conjugate compounds include NECTIN-4 antibodies.

在一個實施例中，NECTIN-4 ADC包含表VI(A)中所闡述之抗體重鏈序列。In one embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(A).

在另一實施例中，NECTIN-4 ADC包含表VI(B)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(B).

在另一實施例中，NECTIN-4 ADC包含表VI(C)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(C).

在另一實施例中，NECTIN-4 ADC包含表VI(D)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(D).

在另一實施例中，NECTIN-4 ADC包含表VI(E)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(E).

在另一實施例中，NECTIN-4 ADC包含表VI(F)中所闡述之抗體重鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody rechain sequence described in Table VI(F).

在另一實施例中，NECTIN-4 ADC包含表VI(G)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(G).

在另一實施例中，NECTIN-4 ADC包含表VI(H)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(H).

在另一實施例中，NECTIN-4 ADC包含表VI(I)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(I).

在另一實施例中，NECTIN-4 ADC包含表VI(J)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(J).

在另一實施例中，NECTIN-4 ADC包含表VI(K)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(K).

在另一實施例中，NECTIN-4 ADC包含表VI(L)中所闡述之抗體輕鏈序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(L).

在另一實施例中，NECTIN-4 ADC包含表VIII中所闡述之抗體重鏈可變區序列。In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain variable region sequence described in Table VIII.

在另一實施例中，NECTIN-4 ADC包含表IX中所闡述之抗體輕鏈可變區序列。In another embodiment, the NECTIN-4 ADC comprises the antibody light chain variable region sequence set forth in Table IX.

在另一實施例中，NECTIN-4 ADC包含表X中所闡述之抗體CDR序列。In another embodiment, the NECTIN-4 ADC comprises the antibody CDR sequences set forth in Table X.

在另一實施例中，前述NECTIN-4抗體結合至治療劑。In another embodiment, the aforementioned NECTIN-4 antibody is conjugated to a therapeutic agent.

在一個實施例中，治療劑為圖10中所闡述之藥物-連接基團(DL)酬載。In one embodiment, the therapeutic agent is a drug-linker (DL) payload as illustrated in FIG. 10 .

在一個實施例中，DL酬載闡述於圖10(A)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(A) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(B)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(B) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(C)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(C) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(D)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(D) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(E)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(E) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(F)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(F) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(G)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(G) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(H)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(H) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(I)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(I) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(J)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(J) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(K)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(K) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(L)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(L) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(M)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(M) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(N)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(N) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(O)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(O) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

在一個實施例中，DL酬載闡述於圖10(P)中且具有以下化學結構：或其醫藥學上可接受之鹽或溶劑合物形式。In one embodiment, the DL payload is illustrated in FIG. 10(P) and has the following chemical structure: or a pharmaceutically acceptable salt or solvent thereof.

多種不同反應可用於藥物及/或連接基團與結合劑之共價連接。此通常藉由例如抗體分子之結合劑之胺基酸殘基，包括離胺酸之胺基、麩胺酸及天冬胺酸之自由羧酸基、半胱胺酸之硫氫基及芳族胺基酸之多個部分的反應來實現。共價連接之最常用的非特異性方法中之一者為將化合物之羧基(或胺基)與抗體之胺基(或羧基)連接之碳化二亞胺反應。A variety of different reactions can be used for covalent attachment of the drug and/or linker group to the binding agent. This is usually achieved by reacting various moieties of the binding agent, such as amino acid residues of the antibody molecule, including the amine group of lysine, the free carboxylic acid groups of glutamine and aspartic acid, the sulfhydryl group of cysteine, and aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction that links the carboxyl (or amine) group of the compound to the amine (or carboxyl) group of the antibody.

另外，諸如二醛或醯亞胺酯之雙官能劑已經用於將化合物之胺基與抗體分子之胺基連接。希夫鹼(Schiff base)反應亦可用於將藥物與結合劑連接。此方法涉及含有二醇或羥基基團之藥物的過碘酸鹽氧化，由此形成隨後與結合劑反應的醛。連接係經由與結合劑之胺基形成希夫鹼而發生。異硫氰酸酯亦可用作用於將藥物與結合劑共價連接之偶合劑。其他技術為熟習此項技術者已知且在本發明之範圍內。Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amine groups of the compound to the amine groups of the antibody molecule. The Schiff base reaction can also be used to link the drug to the binding agent. This method involves the oxidation of the periodate salt of the drug containing a diol or hydroxyl group, thereby forming an aldehyde that is then reacted with the binding agent. The linking occurs via the formation of the Schiff base with the amine group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently link the drug to the binding agent. Other techniques are known to those skilled in the art and are within the scope of the present invention.

在某些實施例中，連接基團之前驅物的中間物在適當條件下與藥物反應。在某些實施例中，使用在藥物及/或中間物上之反應性基團。藥物與中間物之間的反應產物，或衍化藥物，隨後在適當條件下與NECTIN-4抗體反應。In some embodiments, the intermediate of the derivatizer is reacted with the drug under appropriate conditions before the linkage group. In some embodiments, reactive groups on the drug and/or the intermediate are used. The reaction product between the drug and the intermediate, or the derivatized drug, is then reacted with the NECTIN-4 antibody under appropriate conditions.

IV.)連接基團單元通常，抗體-藥物結合物化合物包含連接基團單元在藥物單元與抗體單元之間。在一些實施例中，連接基團在細胞內條件下可裂解，連接基團之裂解在細胞內環境中使藥物單元自抗體釋放。在另外其他實施例中，連接基團單元不可裂解，且藥物例如藉由抗體降解釋放。IV.)Linking Group Unit Typically, the antibody-drug conjugate compound comprises a linking group unit between the drug unit and the antibody unit. In some embodiments, the linking group is cleavable under intracellular conditions, and cleavage of the linking group releases the drug unit from the antibody in the intracellular environment. In still other embodiments, the linking group unit is not cleavable, and the drug is released, for example, by antibody degradation.

在一個較佳實施例中，連接基團結合至本文所描述之NECTIN-4抗體。In a preferred embodiment, the linker group is conjugated to the NECTIN-4 antibody described herein.

在一些實施例中，連接基團可藉由細胞內環境(例如溶酶體或核內體或胞膜窖(caveolea)內)中存在之裂解劑裂解。連接基團可為例如肽基連接基團，藉由細胞內肽酶或蛋白酶，包括(但不限於)溶酶體或胞內體蛋白酶裂解。連接基團亦可藉由存在於細胞外環境中(例如在細胞膜或組織空間附近)之裂解劑裂解。連接基團可為例如肽基連接基團，藉由細胞外肽酶或蛋白酶，包括(但不限於)組織蛋白酶家族酶或基質金屬蛋白酶裂解。In some embodiments, the linking group can be cleaved by a cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linking group can be, for example, a peptidyl linking group, which is cleaved by an intracellular peptidase or protease, including (but not limited to) a lysosomal or endosomal protease. The linking group can also be cleaved by a cleavage agent present in the extracellular environment (e.g., near the cell membrane or tissue space). The linking group can be, for example, a peptidyl linking group, which is cleaved by an extracellular peptidase or protease, including (but not limited to) a tissue protease family enzyme or a matrix metalloproteinase.

在其他實施例中，可裂解連接基團為pH敏感的，亦即在某些pH值對水解敏感。通常，pH敏感性連接基團可在酸性條件下水解。例如，可使用在溶酶體中可水解之酸不穩定連接基團(例如肟、腙、半卡腙、硫半卡腙、順式烏頭醯胺、原酸酯、縮醛、縮酮，或其類似物)。(參見例如美國專利第5,122,368號；第5,824,805號；第5,622,929號；DUBOWCHIK及WALKER,1999, Pharm. Therapeutics 83:67-123；NEVILLE等人, 1989, Biol. Chem.264:14653-14661。)In other embodiments, the cleavable linking group is pH sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linking group can be hydrolyzed under acidic conditions. For example, an acid-labile linking group that can be hydrolyzed in the lysosome (e.g., an oxime, a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-amide, an orthoester, an acetal, a ketone, or the like) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; DUBOWCHIK and WALKER, 1999, Pharm . Therapeutics 83:67-123; NEVILLE et al., 1989, Biol. Chem. 264:14653-14661.)

在另外其他實施例中，連接基團在此項技術中已知之還原條件下可裂解。(參見例如Thorpe等人, 1987, Cancer Res.47:5924-5931；WAWRZYNCZAK等人, 於Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer(C. W. VOGEL編,牛津大學出版社, 1987)。亦參見美國專利第4,880,935號)。連接基團亦可在細胞內(或細胞外)發現之還原條件下裂解。例如，在一個較佳實施例中，特異性連接基團N-O鍵可在形式上還原及斷裂使得連接基團裂解。In yet other embodiments, the linking group is cleavable under reducing conditions known in the art. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; WAWRZYNCZAK et al., inImmunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW VOGEL, ed., Oxford University Press, 1987). See also U.S. Patent No. 4,880,935). The linking group may also be cleaved under reducing conditions found inside (or outside) cells. For example, in a preferred embodiment, the NO bond of the specific linking group may be reduced and cleaved in form so that the linking group is cleaved.

在另外其他特定實施例中，連接基團為丙二酸酯連接基團(Johnson等人, 1995, Anticancer Res.15:1387-93)、順丁烯二醯亞胺基苯甲醯基連接基團(Lau等人, 1995, Bioorg-Med-Chem.3(10):1299-1304)，或3'-N-醯胺類似物(La 等人, 1995, Bioorg-Med-Chem.3(10):1305-12)。In yet other specific embodiments, the linking group is a malonate linking group (Johnson et al., 1995, Anticancer Res. 15:1387-93), a cis-butylenediimidobenzyl linking group (Lau et al., 1995, Bioorg -Med -Chem. 3(10):1299-1304), or a 3'-N-amide analog (La et al., 1995, Bioorg -Med -Chem. 3(10):1305-12).

在另外其他實施例中，連接基團單元不可裂解，且藥物藉由抗體降解釋放。(參見PCT公開案第WO2012/166560號(Ambrx, Inc.)，其以全文引用的方式且出於所有目的倂入本文中)。In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See PCT Publication No. WO2012/166560 (Ambrx, Inc.), which is incorporated herein by reference in its entirety and for all purposes).

通常，連接基團對細胞外環境基本上不敏感。如本文中所使用，在連接基團之情形下，「基本上對細胞外環境不敏感(not substantially sensitive to the extracellular environment)」意謂當抗體-藥物結合物化合物存在於細胞外環境(例如血漿中)中時，抗體-藥物結合物化合物樣本中不超過約20%，通常不超過約15%，更通常不超過約10%，且甚至更通常不超過約5%、不超過約3%或不超過約1%之連接基團裂解。連接基團對細胞外環境是否基本上不敏感可如下判定：例如使抗體-藥物結合物化合物與血漿一起培育預定時段(例如2、4、8、16或24小時)，且隨後定量血漿中存在之自由藥物的量。Typically, the linking group is substantially insensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment" in the context of a linking group means that when the antibody-drug conjugate compound is present in an extracellular environment (e.g., plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linking group is cleaved in a sample of the antibody-drug conjugate compound. Whether the linking group is substantially insensitive to the extracellular environment can be determined by, for example, incubating the antibody-drug conjugate compound with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free drug present in the plasma.

在其他非互斥實施例中，如此項技術中已知，連接基團促進細胞內化。In other non-mutually exclusive embodiments, the linking group promotes cellular internalization, as is known in the art.

可與本發明組合物及方法一起使用之多種例示性連接基團描述於WO 2004/010957、美國公開案第2006/0074008號、美國公開案第20050238649號及美國公開案第2006/0024317號中(其中之各者均以全文引用之方式且出於所有目的併入本文中)。A variety of exemplary linking groups that can be used with the compositions and methods of the present invention are described in WO 2004/010957, U.S. Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006/0024317 (each of which is incorporated herein by reference in its entirety and for all purposes).

出於本揭露之目的，「連接基團單元」 (LU)係可用於連接藥物單元與抗體單元以形成抗體-藥物結合物化合物之雙官能化合物。在一些實施例中，連接基團單元具有下式： -A_a-W_w—Y_y— o      其中：-A-為延伸基團單元， o      a為0或1， o      各-W-獨立地為胺基酸單元， o      w為0至12範圍內之整數， o      -Y-為自分解型間隔基團單元，且 o      y為0、1或2。For the purposes of the present disclosure, a "linker unit" (LU) is a bifunctional compound that can be used to link a drug unit and an antibody unit to form an antibody-drug conjugate compound. In some embodiments, the linker unit has the following formula:_-Aa -_Ww -_Yy -o wherein: -A- is a stretcher unit, o a is 0 or 1, o each -W- is independently an amino acid unit, o w is an integer in the range of 0 to 12, o -Y- is a self-immolative spacer unit, and o y is 0, 1 or 2.

在一些實施例中，a為0或1，w為0或1，且y為0、1或2。在一些實施例中，a為0或1，w為0或1，且y為0或1。在一些實施例中，當w為1至12時，y為1或2。在一些實施例中，w為2至12且y為1或2。在一些實施例中，a為1且w及y為0。In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

V.)延伸基團單元延伸基團單元(A)當存在時能夠將抗體單元連接至胺基酸單元(-W-) (若存在)至間隔基團單元(-Y-) (若存在)；或連接至藥物單元(-D)。天然或經由化學操縱可存在於NECTIN-4抗體上之適用官能基包括但不限於酮基、醛基、硫氫基、胺基、羥基、醣之變旋異構羥基及羧基。適合官能基為酮基、醛基、硫氫基及胺基。在一個實例中，酮基係在併入至本發明之抗體中的非天然胺基酸(nnAA)上。在另一實例中，醛基係在併入至本發明之抗體中的nnAA上。在另一實例中，硫氫基可藉由使NECTIN-4抗體之分子內二硫鍵還原而生成。在另一實施例中，硫氫基可藉由NECTIN-4抗體之離胺酸部分之胺基與2-亞胺基硫雜環戊烷(特勞特試劑(Traut's reagent))或其他硫氫基生成試劑的反應生成。在某些實施例中，NECTIN-4抗體為重組抗體且經工程改造以承載一或多個離胺酸。在某些其他實施例中，重組NECTIN-4抗體經工程改造以承載額外硫氫基，例如額外半胱胺酸。V.)Extender Unit The Extender Unit (A), when present, is capable of linking the Antibody Unit to the Amino Acid Unit (-W-) (if present) to the Spacer Unit (-Y-) (if present); or to the Drug Unit (-D). Suitable functional groups that may be present on the NECTIN-4 antibody naturally or through chemical manipulation include, but are not limited to, keto, aldehyde, sulfhydryl, amine, hydroxyl, isomeric hydroxyl of sugars, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl, and amine. In one example, the keto group is on the non-natural amino acid (nnAA) incorporated into the antibody of the present invention. In another example, the aldehyde group is on the nnAA incorporated into the antibody of the present invention. In another example, the sulfhydryl group can be generated by reducing the intramolecular disulfide bonds of the NECTIN-4 antibody. In another embodiment, the sulfhydryl group can be generated by reacting the amine group of the lysine moiety of the NECTIN-4 antibody with 2-iminothiolane (Traut's reagent) or other sulfhydryl generating reagents. In certain embodiments, the NECTIN-4 antibody is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant NECTIN-4 antibody is engineered to carry additional sulfhydryl groups, such as additional cysteine.

在一個實施例中，延伸基團單元與抗體單元之硫原子形成鍵。硫原子可來源於抗體之硫氫基。在某些實施例中，延伸基團單元經由抗體單元之硫原子與延伸基團單元之硫原子之間的二硫鍵連接至抗體單元。在另外其他實施例中，延伸基團含有可與抗體之一級或二級胺基形成鍵的反應性位點。此等反應性位點之實例包括但不限於活化酯，諸如丁二醯亞胺酯、4硝基苯酯、五氟苯酯、四氟苯酯、酸酐、酸氯化物、磺醯氯、異氰酸酯及異硫氰酸酯。In one embodiment, the extension group unit forms a bond with a sulfur atom of an antibody unit. The sulfur atom may be derived from a sulfhydryl group of an antibody. In certain embodiments, the extension group unit is linked to the antibody unit via a disulfide bond between a sulfur atom of an antibody unit and a sulfur atom of the extension group unit. In other embodiments, the extension group contains a reactive site that can form a bond with a primary or secondary amine group of an antibody. Examples of such reactive sites include, but are not limited to, activated esters, such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

在一些實施例中，延伸基團含有對可存在於抗體上之經修飾之醣的(-CHO)基團具有反應性的反應性位點。例如，可使用諸如高碘酸鈉之試劑使醣輕度氧化且經氧化之醣的所得(-CHO)單元可與含有諸如醯肼、肟、一級或二級胺、肼、硫半卡腙、肼羧酸酯及芳基醯肼(諸如Kaneko等人, 1991, Bioconjugate Chem.2:133-41描述之彼等芳基醯肼)之官能基的延伸基團縮合。In some embodiments, the stretching group contains a reactive site that is reactive toward a (-CHO) group of a modified carbohydrate that may be present on the antibody. For example, the carbohydrate may be mildly oxidized using reagents such as sodium periodate and the resulting (-CHO) unit of the oxidized carbohydrate may be condensed with a stretching group containing functional groups such as hydrazides, oximes, primary or secondary amines, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides (such as those described by Kaneko et al., 1991, Bioconjugate Chem. 2:133-41).

VI.)胺基酸單元胺基酸單元(-W-)當存在時：若間隔基團單元存在，則連接延伸基團單元至間隔基團單元；若間隔基團單元不存在，則連接延伸基團單元至藥物部分；且若延伸基團單元及間隔基團單元不存在，則連接抗體單元至藥物單元。VI.)Amino Acid Units The Amino Acid Unit (-W-) when present: if a Spacer Unit is present, the Extender Unit is linked to the Spacer Unit; if the Spacer Unit is not present, the Extender Unit is linked to the Drug Moiety; and if the Extender Unit and the Spacer Unit are not present, the Antibody Unit is linked to the Drug Unit.

在某些實施例中，胺基酸單元可包含天然胺基酸。在其他實施例中，胺基酸單元可包含非天然胺基酸。In certain embodiments, the amino acid unit may comprise a natural amino acid. In other embodiments, the amino acid unit may comprise a non-natural amino acid.

在一些實施例中，胺基酸單元可藉由一或多種酶，包括癌症或腫瘤相關蛋白酶以酶促方式裂解，以釋放藥物單元(-D)，在一個實施例中，藥物單元在釋放時經活體內質子化以提供藥物(D)。In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes, including cancer or tumor-associated proteases, to release the drug unit (-D). In one embodiment, the drug unit is protonated in vivo upon release to provide drug (D).

在胺基酸單元之一個態樣中，胺基酸單元為纈胺酸-瓜胺酸(vc或Val-Cit)。在另一態樣中，胺基酸單元為苯丙胺酸-離胺酸。在胺基酸單元之又另一態樣中，胺基酸單元為N-甲基纈胺酸-瓜胺酸。在又另一個態樣中，胺基酸單元為5-胺基戊酸、高苯丙胺酸離胺酸、四異喹啉羧酸酯離胺酸、環己基丙胺酸離胺酸、異哌啶酸離胺酸、β-丙胺酸離胺酸、甘胺酸絲胺酸纈胺酸麩醯胺酸及異哌啶酸。In one aspect of the amino acid unit, the amino acid unit is valine-citrulline (vc or Val-Cit). In another aspect, the amino acid unit is phenylalanine-lysine. In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinoline carboxylate lysine, cyclohexylalanine lysine, isopentipoic acid lysine, β-alanine lysine, glycine serine valerine glutamine and isopentipoic acid.

VII.)間隔基團單元當胺基酸單元存在時，間隔基團單元(-Y-)當存在時連接胺基酸單元至藥物單元。或者，當胺基酸單元不存在時，間隔基團單元連接延伸基團單元至藥物單元。當胺基酸單元及延伸基團單元均不存在時，間隔基團單元亦連接藥物單元至抗體單元。間隔基團單元具有兩種通用類型：非自分解型或自分解型。本發明之可能間隔基團的實例為此項技術中已知的。參見TOKI等人, 2002, J. Org. Chem. 67:1866-1872及Nature Biotechnology 21(7):778-784)。VII.)Spacer unit When the amino acid unit is present, the spacer unit (-Y-) connects the amino acid unit to the drug unit when present. Alternatively, when the amino acid unit is not present, the spacer unit connects the extension unit to the drug unit. When both the amino acid unit and the extension unit are not present, the spacer unit also connects the drug unit to the antibody unit. There are two general types of spacer units: non-self-degradable or self-degradable. Examples of possible spacer groups of the present invention are known in the art. See TOKI et al., 2002, J. Org. Chem. 67:1866-1872 and Nature Biotechnology 21(7):778-784).

自分解型間隔基團之其他實例包括但不限於以電子方式類似於PAB基團之芳族化合物，諸如2-胺基咪唑-5-甲醇衍生物(HAY等人, 1999, Bioorg. Med. Chem. Lett.9:2237)及鄰胺基苯甲基乙醛或對胺基苯甲基乙醛。Other examples of self-immolative spacer groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (HAY et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and 2-aminobenzyl acetaldehyde or 4-aminobenzyl acetaldehyde.

可使用在醯胺鍵水解時經歷環化之間隔基團，諸如經取代及未經取代之4-胺基丁酸醯胺(RODRIGUES等人, 1995, Chemistry Biology2:223)、經適當取代之雙環[2.2.1]及雙環[2.2.2]環系統(STORM等人, 1972, J. Amer. Chem. Soc.94:5815)及2-胺基苯基丙酸醯胺(AMSBERRY等人, 1990, J. Org. Chem.55:5867)。消除在甘胺酸之a位置處經取代的含胺藥物(KINGSBURY等人, 1984, J. Med. Chem.27:1447)亦為自分解型間隔基團之實例。Spacer groups that undergo cyclization upon hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (RODRIGUES et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (STORM et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (AMSBERRY et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the a-position of glycine (KINGSBURY et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer group.

VIII.)藥物單元藥物部分(D)可為任何細胞毒性、細胞生長抑制或免疫調節(例如免疫抑制)藥物。D為具有可與間隔基團單元、與胺基酸單元、與延伸基團單元或與抗體單元形成鍵之原子的藥物單元(部分)。在一些實施例中，藥物單元D具有可與間隔基團單元形成鍵的氮原子。如本文中所使用，術語「藥物單元」及「藥物部分」同義且可互換使用。VIII.)Drug Units The drug moiety (D) can be any cytotoxic, cytostatic or immunomodulatory (e.g., immunosuppressive) drug. D is a drug unit (moiety) having an atom that can form a bond with a Spacer unit, with an Amino Acid unit, with a Stretcher unit, or with an Antibody unit. In some embodiments, the drug unit D has a nitrogen atom that can form a bond with a Spacer unit. As used herein, the terms "drug unit" and "drug moiety" are synonymous and can be used interchangeably.

適用類別之細胞毒性劑、細胞生長抑制劑或免疫調節劑包括例如抗微管蛋白劑、DNA小溝黏合劑、DNA複製抑制劑及烷化劑。Suitable classes of cytotoxic agents, cytostatic agents or immunomodulatory agents include, for example, anti-tubulin agents, DNA groove binders, DNA replication inhibitors and alkylating agents.

在一些實施例中，藥物為奧瑞他汀，諸如奧瑞他汀E (在此項技術中亦稱為尾海兔素-10之衍生物)或其衍生物。奧瑞他汀可為例如形成於奧瑞他汀E與酮酸之間的酯。例如，奧瑞他汀E可與對乙醯基苯甲酸或苯甲醯基戊酸反應以分別產生AEB及AEVB。In some embodiments, the drug is an auristatin, such as auristatin E (also referred to in the art as a derivative of Aplysia-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.

在一些實施例中，藥物單元為卡奇黴素、喜樹鹼、類美登素或蒽環黴素(anthracycline)。在一些實施例中，藥物為紫杉烷、拓樸異構酶抑制劑、長春花生物鹼。In some embodiments, the drug unit is a kacinomycin, a camptothecin, a maytansine, or an anthracycline. In some embodiments, the drug is a taxane, a topoisomerase inhibitor, or a vinca alkaloid.

在一些典型實施例中，適合之細胞毒性劑包括例如DNA小溝黏合劑(例如烯二炔及偏端黴素(lexitropsins)，一種CBI化合物；亦參見美國專利第6,130,237號)、倍癌黴素、紫杉烷(例如太平洋紫杉醇及多西他賽(docetaxel))、嘌呤黴素(puromycins)及長春花生物鹼。其他細胞毒性劑包括例如CC-1065、SN-38、拓樸替康(topotecan)、嗎啉基-多柔比星、根瘤菌素(rhizoxin)、氰基-N-嗎啉基-多柔比星、棘黴素(echinomycin)、康普瑞汀(combretastatin)、紡錘菌素(netropsin)、埃博黴素(epothilone) A及B 、雌莫司汀(estramustine)、念珠藻素(cryptophysins)、西馬多丁(cemadotin)、類美登素、圓皮海綿內酯(discodermolide)、艾榴塞洛素(eleutherobin)及米托蒽醌(mitoxantrone)。In some typical embodiments, suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a type of CBI compound; see also U.S. Patent No. 6,130,237), duocarcinoids, taxanes (e.g., paclitaxel and docetaxel), puromycins, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyano-N-morpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansine, discodermolide, eleutherobin, and mitoxantrone.

在一些實施例中，藥物為抗微管蛋白劑。抗微管蛋白劑之實例包括奧瑞他汀、紫杉烷(例如Taxol® (太平洋紫杉醇)、Taxotere® (多西他賽))、T67 (Tularik)及長春花生物鹼(例如長春新鹼、長春鹼、長春地辛及長春瑞濱(vinorelbine))。其他抗微管蛋白劑包括例如巴卡丁衍生物、紫杉烷類似物(例如埃博黴素A及B)、諾考達唑(nocodazole)、秋水仙鹼及秋水醯胺、雌莫司汀、念珠藻素(cryptophycins)、西馬多丁、類美登素、康普瑞汀、圓皮海綿內酯及艾榴塞洛素。In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include auristatins, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), and vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colchicine, estramustine, cryptophycins, cematodin, maytansine, compretin, sphagnum linolenic acid, and elasinolate.

在某些實施例中，細胞毒性劑為類美登素(另一組抗微管蛋白劑)。例如，在特定實施例中，類美登素為美登素或DM-1 (ImmunoGen, Inc.；亦參見Chari等人, 1992, Cancer Res. 52:127-131)。In certain embodiments, the cytotoxic agent is a maytansinoid (another group of anti-tubulin agents). For example, in certain embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).

在某些實施例中，細胞毒性劑或細胞生長抑制劑為尾海兔素。在某些實施例中，細胞毒性劑或細胞生長抑制劑為奧瑞他汀類別，例如尾海兔素-10、奧瑞他汀E或奧瑞他汀PHE等。In some embodiments, the cytotoxic agent or cell growth inhibitor is Aplysia. In some embodiments, the cytotoxic agent or cell growth inhibitor is auristatin, such as Aplysia-10, Auristatin E, or Auristatin PHE.

在某些實施例中，藥物單元(D)為具有以下結構式之奧瑞他汀類似物：或其醫藥學上可接受之鹽， 其中 R¹為，其中R²為未經取代或經取代之C₁-C₆烷基、雜烷基、環烷基或環雜烷基； R^a、R^b及R^c中之各者係選自H及NR^xR^y，其限制條件為R^a、R^b及R^c中之僅一者為NR^xR^y且其他者中之各者為H； R^x及R^y中之各者獨立地選自R、R^r及L-R^z，其限制條件為當R^x及R^y中之一者為L-R^z或R^r時，其他者為R； R⁵為H或CR'₃，其中各R'獨立地為H或F； R⁶為H或CH₂CN； L為連接基團； R^r為(C=O)-O-(CH₂)_p-R^v或(C=O)-(CH₂)_q-R^v； R^v為R、OR、NHR、NR₂、芳基或胺基酸；p為0、1、2、3、4、5或6；q為0、1、2、3、4、5或6； R^z包含官能性或反應性基團；且 R為H或C₁-C₃烷基。In certain embodiments, the drug unit (D) is an auristatin analog having the following structural formula: or a pharmaceutically acceptable salt thereof, wherein R¹ is , wherein R² is unsubstituted or substituted C₁ -C₆ alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; each of Ra, R^b and R^c is selected from H and NR^x R^y , with the proviso that only one of^Ra ,^{R band} R^c is NR^x R^y and each of the others is H; each of R^x and R^y is independently selected from R, R^r and LR^z , with the proviso that when one of R^x and R^y is LR^z or R^r , the other is R; R⁵ is H or CR'₃ , wherein each R' is independently H or F; R⁶ is H or CH₂ CN; L is a linking group; R^r is (C=O)-O-(CH₂ )_p -R^v or (C=O)-(CH₂ )_q -R^v ; R^v is R, OR, NHR, NR₂ , aryl or amino acid;p is 0, 1, 2, 3, 4, 5 or 6;q is 0, 1, 2, 3, 4, 5 or 6; R^z comprises a functional or reactive group; and R is H or C₁ -C₃ alkyl.

IX.)藥物負載藥物負載由p表示且為分子中每一抗體之藥物部分的平均數目。藥物負載可在每個抗體1至24個藥物部分(D)之範圍內。本發明之ADC包括與1至24個範圍之藥物部分結合之抗體的集合。結合反應製備ADC中之每一抗體之藥物部分的平均數目可由諸如質譜法及ELISA分析之習知手段來表徵。亦可根據p確定ADC之定量分佈。在一些情況下，當p為來自具有其他藥物負載之ADC的特定值時，均質ADC之分離、純化及表徵可藉由諸如電泳之手段來實現。IX.)Drug Loading Drug loading is represented by p and is the average number of drug moieties per antibody in the molecule. Drug loading can range from 1 to 24 drug moieties (D) per antibody. The ADCs of the present invention include a collection of antibodies conjugated to a range of 1 to 24 drug moieties. The average number of drug moieties per antibody in the preparation of the ADC by the binding reaction can be characterized by known means such as mass spectrometry and ELISA analysis. The quantitative distribution of the ADC can also be determined based on p. In some cases, when p is a specific value from an ADC with other drug loading, separation, purification and characterization of homogeneous ADCs can be achieved by means such as electrophoresis.

對於一些抗體-藥物結合物，p可受抗體上之連接位點之數目限制。例如，在連接為半胱胺酸硫醇之情況下，如在以上示例性實施例中，抗體可僅僅具有一個或若干個半胱胺酸硫醇基，或可僅僅具有一個或若干個可通過其連接連接基團的充分反應性硫醇基。在某些實施例中，較高藥物負載，例如p＞5可引起某些抗體-藥物結合物之聚集、不可溶性、毒性或細胞滲透性損失。在某些實施例中，本發明之ADC之藥物負載在以下範圍內：1至約8；約2至約6；約3至約5；約3至約4；約3.1至約3.9；約3.2至約3.8；約3.2至約3.7；約3.2至約3.6；約3.3至約3.8；或約3.3至約3.7。實際上，已展示對於某些ADC，每個抗體之藥物部分的最佳比率可小於8且可為約2至約5。參見美國專利第7,498,298號(以全文引用之方式併入本文中)。For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, in the case of attachment to a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or a few cysteine thiol groups, or may have only one or a few fully reactive thiol groups through which the attachment group may be attached. In certain embodiments, higher drug loading, e.g., p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADC of the invention is in the following ranges: 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In fact, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody can be less than 8 and can be about 2 to about 5. See U.S. Patent No. 7,498,298 (incorporated herein by reference in its entirety).

在某些實施例中，在結合反應期間使少於理論最大值之藥物部分結合於抗體。抗體可含有例如不與藥物-連接基團中間物或連接基團試劑反應之離胺酸殘基，如下文所論述。一般而言，抗體不含許多可連接至藥物部分的自由及反應性半胱胺酸硫醇基。實際上，抗體中之大多數半胱胺酸硫醇基殘基以二硫橋鍵形式存在。在某些實施例中，抗體可用諸如二硫蘇糖醇(DTT)或三羰基乙基膦(TCEP)之還原劑在部分或完全還原條件下還原，以生成反應性半胱胺酸硫醇基。在某些實施例中，抗體經歷變性條件，以顯出反應性親核基團，諸如離胺酸或半胱胺酸。In certain embodiments, less than the theoretical maximum of the drug moiety is bound to the antibody during the binding reaction. Antibodies may contain, for example, lysine residues that do not react with drug-linker intermediates or linker reagents, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to the drug moiety. In practice, most cysteine thiol residues in antibodies exist in the form of disulfide bridges. In certain embodiments, antibodies can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or full reducing conditions to generate reactive cysteine thiol groups. In certain embodiments, antibodies are subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

ADC之負載(藥物/抗體比率)可以例如藉由以下之不同方式控制：(i)限制藥物-連接基團中間物或連接基團試劑相對於抗體之莫耳過量；(ii)限制結合反應時間或溫度；(iii)用於半胱胺酸硫醇修飾之部分或限制性還原條件；(iv)藉由重組技術工程改造抗體之胺基酸序列，使得半胱胺酸殘基之數目及位置經修飾以控制連接基團-藥物連接之數目及/或位置(諸如本文及WO2006/034488 (以全文引用的方式併入本文中)所揭示而製備之thioMab或thioFab)。The loading (drug/antibody ratio) of the ADC can be controlled in various ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the binding reaction time or temperature; (iii) partial or limiting reducing conditions for cysteine thiol modification; (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine residues are modified to control the number and/or position of linker-drug linkages (such as thioMabs or thioFabs prepared as disclosed herein and in WO2006/034488 (incorporated herein by reference in their entirety)).

應理解當超過一個親核性基團與藥物-連接物中間物或連接基團試劑反應，隨後與藥物部分試劑反應時，那麼所得產物為ADC化合物與連接至抗體之一或多個藥物部分之分佈的混合物。每一抗體之平均藥物數目可藉由對抗體具有特異性且對藥物具有特異性之雙重ELISA抗體分析由混合物計算。個別ADC分子可藉由質譜法在混合物中鑑別出且藉由HPLC，例如疏水相互作用層析分離(參見例如Hamblett, K. J.等人, 「Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate」,發明摘要編號624,美國癌症研究協會(American Association for Cancer Research), 2004年度會議, 2004年3月27-31日, AACR會議記錄(Proceedings of the AACR),第45卷, 2004年3月；Alley, S. C.等人, 「Controlling the location of drug attachment in antibody-drug conjugates」,發明摘要編號627,美國癌症研究協會, 2004年度會議, 2004年3月27-31日, AACR會議記錄,第45卷, 2004年3月)。在某些實施例中，具有單一負載值之均質ADC可藉由電泳或層析自結合混合物分離。It is understood that when more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent, and subsequently reacted with a drug moiety reagent, then the resulting product is a mixture of ADC compounds and distributions of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by dual ELISA antibody analysis specific for the antibody and specific for the drug. Individual ADC molecules can be identified in a mixture by mass spectrometry and separated by HPLC, such as hydrophobic interaction analysis (see, e.g., Hamblett, K. J. et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, S. C. et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). 2004, March 27-31, AACR Meeting Proceedings, Vol. 45, March 2004). In certain embodiments, a homogeneous ADC having a single loading value can be separated from the binding mixture by electrophoresis or chromatography.

X.)測定ADC之細胞毒性效應的方法測定藥物或抗體-藥物結合物是否在細胞上發揮細胞生長抑制及/或細胞毒性效應之方法為已知的。一般而言，ADC之細胞毒性或細胞生長抑制活性可藉由以下來量測：使表現抗體藥物結合物之目標蛋白的哺乳動物細胞暴露於細胞培養基中；培養細胞約6小時至約5天之時間段；及量測細胞存活率。基於細胞之活體外分析可用以量測抗體藥物結合物之存活率(增殖)、細胞毒性及細胞凋亡之誘導(凋亡蛋白酶活性)。X.) Methodsfor Determiningthe Cytotoxic Effects ofADCs Methods for determining whether a drug or antibody-drug conjugate exerts a cytostatic and/or cytotoxic effect on cells are known. Generally, the cytotoxic or cytostatic activity of an ADC can be measured by exposing mammalian cells expressing the target protein of the antibody-drug conjugate to a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (apoptotic protease activity) of antibody-drug conjugates.

為確定ADC是否發揮細胞生長抑制效應，可使用胸苷倂入分析。例如，可培養以96孔盤之5,000個細胞/孔之密度接種的表現目標抗原之癌細胞72小時之時間段且在72小時之時間段的最後8小時期間暴露於0.5 μCi之³H-胸苷。在ADC存在及不存在下量測³H-胸苷至培養物之細胞中的倂入。To determine whether an ADC exerts a cytostatic effect, a thymidine incorporation assay can be used. For example, cancer cells expressing the target antigen seeded at a density of 5,000 cells/well in a 96-well plate can be cultured for a 72-hour period and exposed to 0.5 μCi of^3H -thymidine during the last 8 hours of the 72-hour period. The incorporation of^3H -thymidine into the cells of the culture is measured in the presence and absence of the ADC.

為測定細胞毒性，可量測壞死或細胞凋亡(程式化細胞死亡)。壞死通常伴隨著質膜之滲透性增加；細胞腫脹及質膜破裂。細胞凋亡通常特徵在於膜起疱、細胞質凝聚及內源核酸內切酶活化。對癌細胞之此等效應中之任一者的測定表明，ADC適用於治療癌症。To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is usually accompanied by increased permeability of the plasma membrane; cell swelling and plasma membrane rupture. Apoptosis is usually characterized by membrane blebbing, cytoplasmic condensation and activation of endogenous endonucleases. The determination of any of these effects on cancer cells indicates that the ADC is useful in the treatment of cancer.

細胞存活率可藉由測定細胞中諸如中性紅、錐蟲藍或ALAMAR™藍之染料的攝取來量測(參見例如PAGE等人, 1993, Intl. J. Oncology3:473-476)。在此類分析中，在含有染料之培養基中培育細胞，洗滌細胞，且以分光光度法量測反映細胞對染料之攝取的剩餘染料。亦可使用蛋白結合染料磺醯羅丹明B (sulforhodamine B；SRB)來量測細胞毒性(SKEHAN等人, 1990, J. Natl. Cancer Inst.82:1107-12)。Cell viability can be measured by measuring the uptake of dyes such as neutral red, conifer blue, or ALAMAR™ blue by cells (see, e.g., PAGE et al., 1993, Intl. J. Oncology 3:473-476). In such assays, cells are incubated in a medium containing the dye, washed, and the residual dye, which reflects the uptake of the dye by the cells, is measured spectrophotometrically. Cytotoxicity can also be measured using the protein-bound dye sulforhodamine B (SRB) (SKEHAN et al., 1990, J. Natl. Cancer Inst. 82:1107-12).

或者，使用四唑鎓鹽，諸如MTT或CellTiter-Glo®藉由偵測活細胞而非死細胞來定量分析哺乳動物細胞生存及增殖(參見例如MOSMANN, 1983, J. Immunol. Methods65:55-63)。Alternatively, tetrazolium salts such as MTT or CellTiter-Glo® are used to quantify mammalian cell survival and proliferation by detecting live cells rather than dead cells (see, e.g., MOSMANN, 1983, J. Immunol. Methods 65:55-63).

細胞凋亡可藉由量測例如DNA片段化來定量。用於定量活體外測定DNA片段化之商用光學量測方法係可用的。此類分析之實例，包括TUNEL (其偵測經標記之核苷酸於片段化之DNA中的併入)及基於ELISA之分析描述於Biochemica,1999,第2期,第34-37頁(Roche Molecular Biochemicals)中。Apoptosis can be quantified by measuring, for example, DNA fragmentation. Commercial optical measurement methods for quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays are described inBiochemica, 1999, No. 2, pp. 34-37 (Roche Molecular Biochemicals).

細胞凋亡亦可由量測細胞中之形態變化來測定。例如，如同壞死一樣，質膜完整性之損失可藉由量測對某些染料(例如螢光染料，諸如吖啶橙或溴化乙錠)之攝取來測定。用於量測凋亡細胞數目之方法已由Duke及Cohen, Current Protocols in Immunology (COLIGAN等人編, 1992,第3.17.1-3.17.16頁)描述。細胞亦可用DNA染料(例如吖啶橙、溴化乙錠或碘化丙錠)標記且觀測細胞沿內部核膜之染色質凝聚及邊聚情況。可經量測以測定細胞凋亡之其他形態變化包括例如細胞質凝聚、膜起疱形成增加及細胞收縮。Apoptosis can also be determined by measuring morphological changes in cells. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells have been described by Duke and Cohen, Current Protocols in Immunology (COLIGAN et al., eds., 1992, pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and marginalization along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

可在培養物之附著及「浮動」區室中均量測凋亡細胞之存在。例如，兩個區室均可藉由以下收集：移除上清液，將連接細胞用蛋白胰酶消化，在離心洗滌步驟(例如在2000 rpm下10分鐘)之後合併製劑，及偵測細胞凋亡(例如藉由量測DNA片段化)。(參見例如PIAZZA等人, 1995, Cancer Research55:3110-16)。The presence of apoptotic cells can be measured in both the attached and "floating" compartments of the culture. For example, both compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations after a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, e.g., PIAZZA et al., 1995, Cancer Research 55:3110-16).

在活體內，可在適合之動物模型中評估NECTIN-4抗體治療組合物之效應。例如，可使用異種癌症模型，其中癌症外植體或繼代異種移植組織引入至免疫功能不全動物中，諸如裸小鼠或SCID小鼠(KLEIN等人, 1997, Nature Medicine 3: 402-408)。例如，PCT專利申請案WO98/16628及美國專利第6,107,540號描述人類前列腺癌之各種異種移植模型，其能夠再現原發性腫瘤之發展、微轉移及表徵晚期疾病之成骨性骨轉移的形成。可使用量測腫瘤形成之抑制、腫瘤消退或轉移及類似者的分析來預測功效。In vivo, the effect of NECTIN-4 antibody therapeutic compositions can be evaluated in suitable animal models. For example, xenograft cancer models can be used, in which cancer explants or subcultured xenograft tissues are introduced into immunocompromised animals, such as nude mice or SCID mice (KLEIN et al., 1997, Nature Medicine 3: 402-408). For example, PCT patent application WO98/16628 and U.S. Patent No. 6,107,540 describe various xenograft models of human prostate cancer that are able to reproduce the development of primary tumors, micrometastases, and the formation of osteoblastic bone metastases that characterize advanced disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

評估細胞凋亡促進之活體內分析適用於評估治療組合物。在一個實施例中，可檢查來自用治療組合物治療之荷瘤小鼠的異種移植物中細胞凋亡灶點之存在並與未治療之攜帶異種移植物之對照小鼠比較。在經治療小鼠之腫瘤中發現凋亡病灶之程度提供關於組合物治療功效之指示。In vivo assays to assess the promotion of apoptosis are suitable for evaluating therapeutic compositions. In one embodiment, the presence of apoptotic foci in xenografts from tumor-bearing mice treated with the therapeutic composition can be examined and compared to untreated xenograft-bearing control mice. The extent to which apoptotic foci are found in tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

可將用於實施前文方法之治療組合物調配至包含適用於所需遞送方法之載劑中的醫藥組合物中。適合之載劑包括當與治療組合物合倂時保留治療組合物之抗腫瘤功能且一般與患者之免疫系統無反應性的任何材料。實例包括但不限於多種標準醫藥載劑中之任一者，諸如無菌磷酸鹽緩衝鹽水溶液、抑菌水及其類似者(通常參見Remington's Pharmaceutical Sciences,第16版, A. Osal.編, 1980)。The therapeutic composition used to practice the above methods can be formulated into a pharmaceutical composition comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that retains the anti-tumor function of the therapeutic composition when combined with the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a variety of standard pharmaceutical carriers, such as sterile phosphate-buffered saline solutions, bacteriostatic water, and the like (see generally Remington's Pharmaceutical Sciences, 16th edition, A. Osal., ed., 1980).

治療調配物可經由能夠將治療組合物遞送至腫瘤位點之任何途徑溶解及投與。可能有效之投與途徑包括但不限於靜脈內、非經腸、腹膜內、肌內、腫瘤內、皮內、器官內、原位及其類似途徑。用於靜脈內注射之較佳調配物包含於保藏抑菌水、無菌未保藏水中及/或稀釋於聚氯乙烯或含有0.9%無菌注射用氯化鈉，USP之聚乙烯袋中的治療組合物。治療蛋白製劑可凍乾且儲存為無菌散劑，較佳在真空下，且隨後在注射之前於抑菌水(含有例如苄醇防腐劑)或於無菌水中重構。The therapeutic formulation can be dissolved and administered by any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, in situ, and the like. Preferred formulations for intravenous injection comprise the therapeutic composition in preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinyl chloride or polyethylene bags containing 0.9% Sterile Sodium Chloride for Injection, USP. The therapeutic protein preparation can be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (containing a preservative such as benzyl alcohol) or in sterile water prior to injection.

使用前述方法治療癌症之劑量及投與方案將隨方法及目標癌症而變化，且一般將視此項技術中所瞭解之多種其他因素而定。The dosage and administration regimen for treating cancer using the aforementioned methods will vary depending on the method and the target cancer, and will generally depend on a variety of other factors understood in the art.

在一個實施例中，由於NECTIN-4抗體之修飾，本發明之醫藥組合物可包含超過一種的本發明之ADC物種。例如，本發明包括包含本發明之ADC的醫藥組合物，其中NECTIN-4抗體為C端離胺酸部分移除或完全移除之抗體、具有N端轉譯後修飾之抗體、缺乏重鏈C端離胺酸且具有N端轉譯後修飾之抗體，及/或具有重鏈C端離胺酸且不具有N端轉譯後修飾之抗體。In one embodiment, due to the modification of the NECTIN-4 antibody, the pharmaceutical composition of the present invention may include more than one ADC species of the present invention. For example, the present invention includes a pharmaceutical composition comprising an ADC of the present invention, wherein the NECTIN-4 antibody is an antibody with a C-terminal lysine partially or completely removed, an antibody with an N-terminal post-translational modification, an antibody lacking a heavy chain C-terminal lysine and having an N-terminal post-translational modification, and/or an antibody with a heavy chain C-terminal lysine and no N-terminal post-translational modification.

在一較佳實施例中，NECTIN-4抗體闡述於表VI及表VII中。In a preferred embodiment, the NECTIN-4 antibodies are described in Table VI and Table VII.

XI.)表現NECTIN-4之癌症的治療將NECTIN-4鑑別為通常表現於受限的一組組織或細胞中但亦表現於諸如表I中列出之彼等癌症之癌症中的蛋白，為治療此類癌症開闢了多種治療方法。XI.)Treatment of CancersExpressingNECTIN-4 The identification of NECTIN-4 as a protein that is normally expressed in a restricted set of tissues or cells, but is also expressed in cancers such as those listed in Table I, opens up a variety of therapeutic approaches for treating these cancers.

值得注意的是，即使當靶向蛋白表現於正常組織或細胞上，甚至重要的正常器官組織上時，靶向抗腫瘤療法亦適用。重要器官為維持生命所必需之器官，諸如心臟或結腸。非重要器官為可被移除而個體仍然能夠存活的器官。非重要器官之實例為卵巢、乳房及前列腺。It is important to note that targeted anti-cancer therapy is applicable even when the targeted protein is expressed on normal tissues or cells, even on vital normal organ tissues. Vital organs are organs that are essential for maintaining life, such as the heart or colon. Non-vital organs are organs that can be removed and the individual still survive. Examples of non-vital organs are the ovaries, breasts, and prostate.

目標蛋白在正常組織、甚至重要正常組織中之表現不會損害該蛋白之靶向劑作為其中該蛋白亦過度表現之某些腫瘤之治療劑的效用。例如，重要器官中之表現可改變且自身有害的。另外，可在不影響死亡率之情況下移除視為非必需之器官，諸如前列腺及卵巢。最後，一些重要器官由於免疫豁免而不受正常器官表現影響。免疫豁免器官係藉由血液器官障壁保護免於血液影響的器官，且因此不可接受免疫療法。免疫豁免器官之實例為大腦及睪丸。Expression of a target protein in normal tissues, even critical normal tissues, does not compromise the utility of a targeting agent for that protein as a therapeutic for certain tumors in which that protein is also overexpressed. For example, expression in critical organs may be altered and itself harmful. Additionally, organs that are considered non-essential, such as the prostate and ovaries, may be removed without affecting mortality. Finally, some critical organs are protected from normal organ expression due to immunoprivilege. An immunoprivileged organ is an organ that is protected from the effects of blood by the blood-organ barrier and is therefore not amenable to immunotherapy. Examples of immunoprivileged organs are the brain and testes.

因此，抑制NECTIN-4蛋白之活性的治療方法適用於罹患表現NECTIN-4之癌症(諸如表I中所闡述之彼等癌症)的患者。此等治療方法一般屬於三類。第一類在NECTIN-4與腫瘤細胞生長相關時調節其功能，引起腫瘤細胞生長之抑制或阻滯或誘導其殺死。第二類包含用於抑制NECTIN-4蛋白與其結合搭配物或與其他蛋白結合或締合之各種方法。第三類包含用於抑制NECTIN-4基因之轉錄或NECTIN-4 mRNA之轉譯的多種方法。Therefore, therapeutic methods that inhibit the activity of NECTIN-4 protein are suitable for patients suffering from cancers that express NECTIN-4 (such as those described in Table I). These therapeutic methods generally belong to three categories. The first category modulates the function of NECTIN-4 when it is related to tumor cell growth, causing inhibition or retardation of tumor cell growth or inducing its killing. The second category includes various methods for inhibiting the binding or association of NECTIN-4 protein with its binding partner or with other proteins. The third category includes various methods for inhibiting the transcription of NECTIN-4 gene or the translation of NECTIN-4 mRNA.

因此，可評估癌症患者的NECTIN-4表現之存在及水平，較佳使用腫瘤組織之免疫組織化學評定、定量NECTIN-4成像或可靠地指示NECTIN-4表現之存在及程度的其他技術。若適用，腫瘤活體組織切片或手術試樣之免疫組織化學分析對於此目的為較佳的。用於腫瘤組織之免疫組織化學分析之方法為此項技術中所熟知。Therefore, cancer patients can be evaluated for the presence and level of NECTIN-4 expression, preferably using immunohistochemical assessment of tumor tissue, quantitative NECTIN-4 imaging, or other techniques that reliably indicate the presence and extent of NECTIN-4 expression. If applicable, immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

XII.) NECTIN-4 ADC混合物本發明之治療方法涵蓋投與單一NECTIN-4 ADC以及不同抗體(亦即，NECTIN-4抗體或結合另一蛋白之抗體)之組合物或混合物。此類抗體混合物可具有某些優勢，因為其含有靶向不同抗原決定基之抗體，利用不同效應機制，或將細胞毒性抗體與依賴於免疫效應子功能性之抗體直接組合。此類抗體之組合可展現協同治療功效。另外，NECTIN-4抗體可與其他治療模態同時投與，包括但不限於各種化學治療劑及生物製劑、雄性激素阻斷劑、免疫調節劑(例如，IL-2、GM-CSF、PD1、PD-L1)、手術或放射。XII.) NECTIN-4 ADCCocktails The therapeutic methods of the present invention encompass administration of a single NECTIN-4 ADC in combination or mixtures of different antibodies (i.e., NECTIN-4 antibodies or antibodies that bind another protein). Such antibody cocktails may have certain advantages because they contain antibodies that target different antigenic determinants, utilize different effector mechanisms, or directly combine cytotoxic antibodies with antibodies that rely on immune effector functionality. Such combinations of antibodies may exhibit synergistic therapeutic efficacy. In addition, NECTIN-4 antibodies may be administered concurrently with other therapeutic modalities, including but not limited to various chemotherapeutic agents and biologics, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF, PD1, PD-L1), surgery, or radiation.

在一較佳實施例中，NECTIN-4抗體以結合形式投與。In a preferred embodiment, the NECTIN-4 antibody is administered in a conjugated form.

在另一較佳實施例中，NECTIN-4抗體闡述於表VI及表VII中。In another preferred embodiment, the NECTIN-4 antibody is described in Table VI and Table VII.

NECTIN-4 ADC調配物經由能夠將抗體遞送至腫瘤細胞之任何途徑投與。投與途徑包括但不限於靜脈內、腹膜內、肌內、腫瘤內、皮內及其類似途徑。治療一般涉及經由可接受之投與途徑，諸如靜脈內注射(IV)反覆投與NECTIN-4 ADC製劑，通常劑量範圍包括但不限於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、15、20或25 mg/kg體重。一般而言，每週10-1000 mg MAb範圍內之劑量為有效的且良好耐受。NECTIN-4 ADC formulations are administered by any route that is capable of delivering the antibody to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and the like. Treatment generally involves repeated administration of NECTIN-4 ADC formulations by an acceptable route of administration, such as intravenous injection (IV), typically in a dosage range including, but not limited to, 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, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg MAb per week are effective and well tolerated.

基於Herceptin® (曲妥珠單抗(Trastuzumab))在治療轉移性乳癌中之臨床經驗，大致4 mg/kg患者體重IV之初始負載劑量，隨後約2 mg/kg IV之每週劑量之MAb製劑表示可接受之給藥方案。較佳地，初始負載劑量以90分鐘或更長輸注形式投與。週期維持劑量以30分鐘或更長輸注形式投與，限制條件為初始劑量經良好耐受。如熟習此項技術者所瞭解，許多因素可在特定情況下影響理想劑量方案。此類因素包括例如所用抗體之結合親和力及半衰期、患者中NECTIN-4的表現量、循環排出之NECTIN-4抗原量、所需穩態抗體濃度水平、治療頻率及與本發明之治療方法組合使用之化學治療劑或其他藥劑之影響，以及特定患者之健康狀況。Based on clinical experience with Herceptin® (Trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient weight IV, followed by weekly doses of approximately 2 mg/kg IV of the MAb formulation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as an infusion over 90 minutes or longer. Periodic maintenance doses are administered as infusions over 30 minutes or longer, provided that the initial dose is well tolerated. As will be appreciated by those skilled in the art, many factors can influence the ideal dosing regimen in a particular situation. Such factors include, for example, the binding affinity and half-life of the antibody used, the amount of NECTIN-4 expressed in the patient, the amount of NECTIN-4 antigen excreted in the circulation, the desired steady-state antibody concentration level, the frequency of treatment and the effects of chemotherapy or other agents used in combination with the treatment methods of the present invention, and the health status of the particular patient.

視情況而言，應評估患者給定樣本中之NECTIN-4水平(例如，循環NECTIN-4抗原及/或NECTIN-4表現細胞之水平)以輔助判定最有效之給藥方案等。此類評估亦用於監測整個療法中之目的且適用於與其他參數(例如膀胱癌療法中之尿液細胞學及/或免疫細胞水平，或類似地，前列腺癌療法中之血清PSA水平)之評估組合來衡量治療成功。As appropriate, NECTIN-4 levels (e.g., levels of circulating NECTIN-4 antigen and/or NECTIN-4 expressing cells) in a given sample from a patient should be assessed to help determine the most effective dosing regimen, etc. Such assessments are also used for monitoring purposes throughout therapy and are useful in combination with assessments of other parameters (e.g., urine cytology and/or immune cell levels in bladder cancer therapy, or similarly, serum PSA levels in prostate cancer therapy) to measure treatment success.

本發明之一目標為提供NECTIN-4 ADC，其抑制或延緩表現NECTIN-4之腫瘤細胞的生長。本發明之另一目標為提供使用此類NECTIN-4 ADC，且尤其使用此類NECTIN-4 ADC與其他藥物或免疫活性治療組合來抑制哺乳動物，較佳人類之血管生成及其他生物功能且藉此降低腫瘤生長的方法。One object of the present invention is to provide NECTIN-4 ADCs that inhibit or delay the growth of tumor cells expressing NECTIN-4. Another object of the present invention is to provide methods of using such NECTIN-4 ADCs, and particularly using such NECTIN-4 ADCs in combination with other drugs or immunoactive treatments, to inhibit angiogenesis and other biological functions in mammals, preferably humans, and thereby reduce tumor growth.

XIII.)組合療法在一個實施例中，當腫瘤，包括人類腫瘤用NECTIN-4 ADC結合化學治療劑或放射或其組合治療時，存在協同作用。換言之，當與化學治療劑或放射或其組合相組合時，NECTIN-4 ADC對腫瘤生長之抑制增強超過預期。協同作用可例如藉由與僅由NECTIN-4 ADC之治療所預期的或用NECTIN-4 ADC及化學治療劑或放射治療之累加效應相比，用組合治療對腫瘤生長之更大抑制來展示。較佳地，協同作用藉由癌症之緩解而證明，其中預期自NECTIN-4 ADC之治療或使用NECTIN-4 ADC與化學治療劑或放射或免疫療法，諸如CAR-T或NK細胞療法之累加組合的治療不緩解。XIII.)Combination Therapy In one embodiment, when a tumor, including a human tumor, is treated with NECTIN-4 ADC in combination with chemotherapy or radiation, or a combination thereof, a synergistic effect exists. In other words, the inhibition of tumor growth by NECTIN-4 ADC is enhanced beyond what would be expected when combined with chemotherapy or radiation, or a combination thereof. Synergistic effects can be demonstrated, for example, by greater inhibition of tumor growth with combination treatment than would be expected from treatment with NECTIN-4 ADC alone or compared to an additive effect of treatment with NECTIN-4 ADC and chemotherapy or radiation. Preferably, synergy is demonstrated by remission of a cancer where remission is not expected from treatment with NECTIN-4 ADC or treatment with an additive combination of NECTIN-4 ADC and chemotherapy or radiation or immunotherapy, such as CAR-T or NK cell therapy.

使用NECTIN-4 ADC及化學療法組合或放射或其兩者抑制腫瘤細胞生長之方法包含在開始化學療法或放射治療之前、期間或之後，以及其任何組合(亦即，在開始化學療法及/或放射治療之前及期間、之前及之後、期間及之後、或之前、期間及之後)投與NECTIN-4 ADC。例如，NECTIN-4 ADC通常在開始放射治療及/或化學療法之前1天與60天之間，較佳3天與40天之間，更佳5天與12天之間投與。然而，視治療方案及特定患者之需求而定，該方法以將提供最有效治療且最終延長患者壽命之方式進行。Methods of inhibiting tumor cell growth using a combination of NECTIN-4 ADC and chemotherapy or radiation or both include administering NECTIN-4 ADC before, during, or after initiating chemotherapy or radiation therapy, and any combination thereof (i.e., before and during, before and after, during and after, or before, during, and after initiating chemotherapy and/or radiation therapy). For example, NECTIN-4 ADC is typically administered between 1 day and 60 days, preferably between 3 days and 40 days, and more preferably between 5 days and 12 days prior to initiating radiation therapy and/or chemotherapy. However, depending on the treatment regimen and the needs of a particular patient, the method is performed in a manner that will provide the most effective treatment and ultimately prolong the life of the patient.

化學治療劑之投與可以多種方式來實現，包括藉由非經腸及經腸途徑全身性地投與。在一個實施例中，NECTIN-4 ADC及化學治療劑作為單獨分子投與。化學治療劑或化學療法之特定實例包括順鉑、達卡巴𠯤(dacarbazine) (DTIC)、放線菌素d (dactinomycin)、二氯甲基二乙胺(氮芥)、鏈脲佐菌素、環磷醯胺、卡莫司汀(carmustine) (BCNU)、洛莫司汀(lomustine) (CCNU)、多柔比星(阿黴素(adriamycin))、佐柔比星、丙卡巴肼(procarbazine)、絲裂黴素、阿糖胞苷、依託泊苷、胺甲喋呤、5-氟尿嘧啶、長春鹼、長春新鹼、博萊黴素(bleomycin)、太平洋紫杉醇(紫杉醇)、多西他賽(克癌易(taxotere))、阿地介白素(aldesleukin)、天冬醯胺酶、白消安(busulfan)、卡鉑(carboplatin)、克拉屈濱(cladribine)、達卡巴𠯤、氟尿苷、氟達拉濱(fludarabine)、羥基脲、依弗醯胺、干擾素α、亮丙立德(leuprolide)、甲地孕酮(megestrol)、美法侖(melphalan)、巰基嘌呤(mercaptopurine)、普卡黴素(plicamycin)、米托坦(mitotane)、培門冬酶(pegaspargase)、噴司他汀(pentostatin)、哌泊溴烷(pipobroman)、普卡黴素、鏈脲佐菌素、他莫昔芬(tamoxifen)、替尼泊苷(teniposide)、睪內酯、硫鳥嘌呤、噻替派(thiotepa)、尿嘧啶氮芥、長春瑞濱(vinorelbine)、吉西他濱(gemcitabine)、苯丁酸氮芥、紫杉醇及其組合。Administration of chemotherapeutic agents can be achieved in a variety of ways, including systemic administration by parenteral and enteral routes. In one embodiment, NECTIN-4 ADC and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, dichloromethyldiethylamine (nitrogen mustard), streptozotocin, cyclophosphamide, carmustine (BCNU), lomustine (lomustine) (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (paclitaxel), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, effulgamide, interferon alfa, leuprolide de), megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, paclitaxel, and combinations thereof.

與NECTIN-4 ADC組合使用之放射源可在所治療患者外部或內部。當放射源在病患外部時，療法被稱為外部束放射治療(EBRT)。當放射源在患者內部時，治療被稱為近接治療(BT)。在一個實施例中，放射治療為硼中子捕獲治療。在一個實施例中，放射為質子硼融合治療。The radiation source used in combination with NECTIN-4 ADC can be external or internal to the patient being treated. When the radiation source is external to the patient, the treatment is called external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT). In one embodiment, the radiation therapy is boron neutron capture therapy. In one embodiment, the radiation is proton boron fusion therapy.

上文所描述之治療方案可進一步與其他癌症治療劑及/或方案組合，例如額外化學療法、癌症疫苗、信號轉導抑制劑、適用於治療異常細胞生長或癌症之藥劑、抗體(例如，如WO/2005/092380 (Pfizer)中所描述之抗CTLA-4抗體)或藉由結合至IGF-1R及細胞介素抑制腫瘤生長之其他配體。The treatment regimens described above may be further combined with other cancer therapeutic agents and/or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies as described in WO/2005/092380 (Pfizer)), or other ligands that inhibit tumor growth by binding to IGF-1R and cytokines.

當哺乳動物經受額外化學療法時，可使用上文所描述之化學治療劑。另外，可使用生長因子抑制劑、生物反應調節劑、抗激素療法、選擇性雌性素受體調節劑(SERM)、血管生成抑制劑及抗雄性素。例如，可使用抗激素，例如抗雌性素，諸如諾瓦得士(Nolvadex) (他莫昔芬)或抗雄性素，諸如康士得(Casodex)(4'-氰基-3-(4-氟苯基磺醯基)-2-羥基-2-甲基-3′-(三氟甲基)丙醯苯胺)。When the mammal is undergoing additional chemotherapy, the chemotherapeutic agents described above may be used. In addition, growth factor inhibitors, biological response regulators, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens may be used. For example, antihormones such as antiestrogens such as Nolvadex (tamoxifen) or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) may be used.

以上治療方法可與廣泛多種手術、化學療法或放射治療方案中之任一者組合。本發明之治療方法可使得能夠使用減少之化學療法(或其他療法)劑量及/或較不頻繁之投與，其對於所有患者且尤其對於不良好耐受化學治療劑之毒性的彼等患者為一優勢。The above treatment methods can be combined with any of a wide variety of surgical, chemotherapy or radiation treatment regimens. The treatment methods of the present invention can enable the use of reduced chemotherapy (or other treatment) doses and/or less frequent administration, which is an advantage for all patients and especially for those patients who do not tolerate the toxicity of chemotherapeutic agents well.

XIV.)套組/製品對於用於本文中所描述之實驗室、預後、預防、診斷及治療應用，套組係在本發明之範圍內。此類套組可包含載劑、包裝或經分隔以接收一或多個容器(諸如小瓶、管及其類似物)之容器，容器中之各者包含用於該方法之獨立元件中之一者，以及包含使用說明書，諸如本文所描述之用途的標籤或插頁。例如，容器可包含本揭露之NECTIN-4抗體或若干NECTIN-4抗體(參見表VI及表VII)。套組可包含有包含藥物單元之容器。套組可包括NECTIN-4 ADC之全部或一部分及/或用於偵測癌症及/或其他免疫病症之診斷分析。XIV.)Kits/Articles of manufacture For use in the laboratory, prognostic, preventive, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits may include a carrier, packaging or container that is separated to receive one or more containers (such as vials, tubes and the like), each of which contains one of the independent elements for use in the method, and instructions for use, such as a label or insert for the use described herein. For example, the container may contain a NECTIN-4 antibody or a plurality of NECTIN-4 antibodies disclosed herein (see Table VI and Table VII). The kit may include a container containing a drug unit. The kit may include all or a portion of a NECTIN-4 ADC and/or a diagnostic assay for detecting cancer and/or other immune disorders.

本發明之套組將通常包含上文所描述之容器及一或多種與其相關之其他容器，該等其他容器包含就商業及使用者觀點而言合乎需要之材料，包括緩衝劑、稀釋劑、過濾器、針、注射器；載劑、包裝、容器、小瓶及/或列舉內含物之管標籤及/或使用說明書，及帶有使用說明書之藥品說明書。The kits of the present invention will generally include the container described above and one or more other containers associated therewith, which contain materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and/or tube labels listing the contents and/or instructions for use, and drug product instructions with instructions for use.

標籤可存在於容器上或與容器一起存在，以指示組合物用於特定療法或非治療應用，諸如預後、預防、診斷或實驗室應用，且亦可指示活體內或活體外使用之說明，諸如本文所描述之彼等說明。說明及/或其他資訊亦可包括在與套組一起或在套組上包括之插頁或標籤上。標籤可在容器上或與容器相關聯。當形成標籤之字母、數目或其他字元經模製或蝕刻於容器自身中時，標籤可位於容器上；當標籤存在於亦裝有容器之貯器或載體內時，標籤可與容器相關聯，例如呈藥品說明書形式。標記可指示組合物用於診斷、治療、預防或預後病況，諸如癌症或其他免疫病症。A label may be present on or with the container to indicate that the composition is for a specific therapeutic or non-therapeutic application, such as prognostic, preventive, diagnostic, or laboratory application, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and/or other information may also be included on an insert or label included with or on the kit. The label may be on or associated with the container. The label may be on the container when the letters, numbers, or other characters forming the label are molded or etched into the container itself; the label may be associated with the container when the label is present in a container or carrier that also contains the container, such as in the form of a drug package insert. The label may indicate that the composition is used to diagnose, treat, prevent, or prognose a condition, such as cancer or other immune disorders.

術語「套組」及「製品」可用作同義詞。The terms "set" and "product" may be used synonymously.

在本發明之另一實施例中，製品含有組合物，諸如本揭露之NECTIN-4 ADC。製品通常包含至少一個容器及至少一個標籤。適合之容器包括例如瓶、小瓶、注射器及試管。容器可由多種材料形成，諸如玻璃、金屬或塑膠。容器可容納一或若干種NECTIN-4 ADC及/或一或多種NECTIN-4 ADC之治療劑劑量。In another embodiment of the invention, an article of manufacture contains a composition, such as a NECTIN-4 ADC disclosed herein. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed of a variety of materials, such as glass, metal, or plastic. The container can hold one or more NECTIN-4 ADCs and/or a therapeutic dose of one or more NECTIN-4 ADCs.

容器可替代地容納對於治療、診斷、預後或預防病況有效之組合物且可具有無菌接取口(例如容器可為靜脈內溶液袋或具有可由皮下注射針刺穿之塞的小瓶)。組合物中之活性劑可為本發明之NECTIN-4抗體或ADC。The container may alternatively contain a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a NECTIN-4 antibody or ADC of the present invention.

製品可進一步包含第二容器，其包含醫藥學上可接受之緩衝液，諸如磷酸鹽緩衝鹽水、林格氏溶液(Ringer's solution)及/或右旋糖溶液。其可進一步包括就商業及使用者觀點而言合乎需要之其他材料，包括其他緩衝劑、稀釋劑、過濾器、攪拌器、針、注射器及/或帶有適應症及/或使用說明書之藥品說明書。The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes and/or a product leaflet with indications and/or instructions for use.

例示性實施例1) 一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 52、SEQ ID NO: 53及SEQ ID NO: 54中所闡述之序列的互補決定區(CDR)。 2) 如技術方案1之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO: 70、SEQ ID NO: 71及SEQ ID NO: 72中所闡述之序列的互補決定區(CDR)。 3) 如技術方案1之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 28中所闡述之序列。 4) 如技術方案2之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 34中所闡述之序列。 5) 如技術方案1之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 5中所闡述之序列。 6) 如技術方案2之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 17中所闡述之序列。 7) 如技術方案1之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO：28中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO：34中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 8) 如技術方案1之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 9) 如技術方案1之抗體或其抗原結合片段，其中該抗體為全人類抗體。 10)   如技術方案1之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 11)   如技術方案1之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 12)   如技術方案11之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 13)   如技術方案11之抗體或其抗原結合片段，其進一步包含延伸基團單元。 14)   如技術方案11之抗體或其抗原結合片段，其進一步包含間隔基團單元。 15)   如技術方案11之抗體或其抗原結合片段，其進一步包含胺基酸單元。 16)   一種醫藥組合物，其包含如技術方案1至15中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 17)   一種套組，其包含如技術方案1至16中任一項之抗體或其抗原結合片段。 18)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案16之醫藥組合物。 19)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案17之套組。 20)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案1至15中任一項之抗體或其抗原結合片段。 21)   如技術方案18至20中任一項之方法，其中該個體為人類個體。 22)   如技術方案21之方法，其中該癌症闡述於表I中。 23)   如技術方案22之方法，其中該方法進一步包含投與放射或化學治療劑。 24)   一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 55、SEQ ID NO: 56及SEQ ID NO: 57中所闡述之序列的互補決定區(CDR)。 25)   如技術方案24之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO: 73、SEQ ID NO: 74及SEQ ID NO: 75中所闡述之序列的互補決定區(CDR)。 26)   如技術方案24之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 29中所闡述之序列。 27)   如技術方案24之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 35中所闡述之序列。 28)   如技術方案24之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 6中所闡述之序列。 29)   如技術方案24之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 18中所闡述之序列。 30)   如技術方案24之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO：29中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO：35中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 31)   如技術方案24之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 32)   如技術方案24之抗體或其抗原結合片段，其中該抗體為全人類抗體。 33)   如技術方案24之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 34)   如技術方案24之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 35)   如請求項34之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 36)   如技術方案34之抗體或其抗原結合片段，其進一步包含延伸基團單元。 37)   如技術方案34之抗體或其抗原結合片段，其進一步包含間隔基團單元。 38)   如技術方案34之抗體或其抗原結合片段，其進一步包含胺基酸單元。 39)   一種醫藥組合物，其包含如技術方案24至38中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 40)   一種套組，其包含如技術方案24至39中任一項之抗體或其抗原結合片段。 41)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案39之醫藥組合物。 42)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案40之套組。 43)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案24至39中任一項之抗體或其抗原結合片段。 44)   如技術方案41至43中任一項之方法，其中該個體為人類個體。 45)   如請求項43之方法，其中該癌症闡述於表I中。 46)   如技術方案43之方法，其中該方法進一步包含投與放射或化學治療劑。 47)   一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 58、SEQ ID NO: 59及SEQ ID NO: 60中所闡述之序列的互補決定區(CDR)。 48)   如技術方案47之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO: 76、SEQ ID NO: 77及SEQ ID NO: 78中所闡述之序列的互補決定區(CDR)。 49)   如技術方案47之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 30中所闡述之序列。 50)   如技術方案47之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 36中所闡述之序列。 51)   如技術方案47之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 8中所闡述之序列。 52)   如技術方案47之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 20中所闡述之序列。 53)   如技術方案47之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO：30中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO：36中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 54)   如技術方案47之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 55)   如技術方案47之抗體或其抗原結合片段，其中該抗體為全人類抗體。 56)   如技術方案47之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 57)   如技術方案47之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 58)   如技術方案47之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 59)   如技術方案47之抗體或其抗原結合片段，其進一步包含延伸基團單元。 60)   如技術方案47之抗體或其抗原結合片段，其進一步包含間隔基團單元。 61)   如技術方案47之抗體或其抗原結合片段，其進一步包含胺基酸單元。 62)   一種醫藥組合物，其包含如技術方案47至61中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 63)   一種套組，其包含如技術方案47至62中任一項之抗體或其抗原結合片段。 64)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案62之醫藥組合物。 65)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案63之套組。 66)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案47至65中任一項之抗體或其抗原結合片段。 67)   如技術方案64至66中任一項之方法，其中該個體為人類個體。 68)   如技術方案66之方法，其中該癌症闡述於表I中。 69)   如技術方案66之方法，其中該方法進一步包含投與放射或化學治療劑。 70)   一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 61、SEQ ID NO: 62及SEQ ID NO: 63中所闡述之序列的互補決定區(CDR)。 71)   如技術方案70之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO: 79、SEQ ID NO: 80及SEQ ID NO: 81中所闡述之序列的互補決定區(CDR)。 72)   如技術方案70之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 31中所闡述之序列。 73)   如技術方案70之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 37中所闡述之序列。 74)   如技術方案70之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 10中所闡述之序列。 75)   如技術方案70之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 22中所闡述之序列。 76)   如技術方案70之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO：31中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO：37中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 77)   如技術方案70之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 78)   如技術方案70之抗體或其抗原結合片段，其中該抗體為全人類抗體。 79)   如技術方案70之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 80)   如技術方案70之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 81)   如請求項70之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 82)   如技術方案70之抗體或其抗原結合片段，其進一步包含延伸基團單元。 83)   如技術方案70之抗體或其抗原結合片段，其進一步包含間隔基團單元。 84)   如技術方案70之抗體或其抗原結合片段，其進一步包含胺基酸單元。 85)   一種醫藥組合物，其包含如技術方案70至84中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 86)   一種套組，其包含如技術方案70至84中任一項之抗體或其抗原結合片段。 87)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案85之醫藥組合物。 88)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案86之套組。 89)   一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案70至88中任一項之抗體或其抗原結合片段。 90)   如技術方案87至89中任一項之方法，其中該個體為人類個體。 91)   如技術方案89之方法，其中該癌症闡述於表I中。 92)   如技術方案89之方法，其中該方法進一步包含投與放射或化學治療劑。 93)   一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 64、SEQ ID NO: 65及SEQ ID NO: 66中所闡述之序列的互補決定區(CDR)。 94)   如技術方案93之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO: 82、SEQ ID NO: 83及SEQ ID NO: 84中所闡述之序列的互補決定區(CDR)。 95)   如技術方案93之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 32中所闡述之序列。 96)   如技術方案93之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 38中所闡述之序列。 97)   如技術方案93之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 12中所闡述之序列。 98)   如技術方案93之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 24中所闡述之序列。 99)   如技術方案93之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO: 32中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO: 38中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 100) 如技術方案93之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 101) 如技術方案93之抗體或其抗原結合片段，其中該抗體為全人類抗體。 102) 如技術方案93之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 103) 如技術方案93之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 104) 如技術方案93之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 105) 如技術方案93之抗體或其抗原結合片段，其進一步包含延伸基團單元。 106) 如技術方案93之抗體或其抗原結合片段，其進一步包含間隔基團單元。 107) 如技術方案93之抗體或其抗原結合片段，其進一步包含胺基酸單元。 108) 一種醫藥組合物，其包含如技術方案93至107中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 109) 一種套組，其包含如技術方案93至108中任一項之抗體或其抗原結合片段。 110) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案108之醫藥組合物。 111) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案109之套組。 112) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案93至107中任一項之抗體或其抗原結合片段。 113) 如技術方案110至112中任一項之方法，其中該個體為人類個體。 114) 如技術方案112之方法，其中該癌症闡述於表I中。 115) 如技術方案112之方法，其中該方法進一步包含投與放射或化學治療劑。 116) 一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 67、SEQ ID NO: 68及SEQ ID NO: 69中所闡述之序列的互補決定區(CDR)。 117) 如技術方案116之抗體或其抗原結合片段，其進一步包含輕鏈可變區，該輕鏈可變區包含具有SEQ ID NO:85、SEQ ID NO: 86及SEQ ID NO:87中所闡述之序列的互補決定區(CDR)。 118) 如技術方案116之抗體或其抗原結合片段，其中該重鏈可變區包含SEQ ID NO: 33中所闡述之序列。 119) 如技術方案116之抗體或其抗原結合片段，其中該輕鏈可變區包含SEQ ID NO: 39中所闡述之序列。 120) 如技術方案116之抗體或其抗原結合片段，其中該重鏈包含SEQ ID NO: 14中所闡述之序列。 121) 如技術方案116之抗體或其抗原結合片段，其中該輕鏈包含SEQ ID NO: 26中所闡述之序列。 122) 如技術方案116之抗體或其抗原結合片段，其包含：重鏈可變區，其包含與SEQ ID NO: 33中所闡述之重鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列；及輕鏈可變區，其包含與SEQ ID NO: 39中所闡述之輕鏈可變區胺基酸序列至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同源的胺基酸序列。 123) 如技術方案116之抗體或其抗原結合片段，其中其抗原結合片段為Fab、F(ab′)2、Fv或scFv。 124) 如技術方案116之抗體或其抗原結合片段，其中該抗體為全人類抗體。 125) 如技術方案116之抗體或其抗原結合片段，其中該抗體或其抗原結合片段以重組方式產生。 126) 如技術方案116之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。 127) 如技術方案116之抗體或其抗原結合片段，其中該藥物為奧瑞他汀類似物。 128) 如技術方案116之抗體或其抗原結合片段，其進一步包含延伸基團單元。 129) 如技術方案116之抗體或其抗原結合片段，其進一步包含間隔基團單元。 130) 如技術方案116之抗體或其抗原結合片段，其進一步包含胺基酸單元。 131) 一種醫藥組合物，其包含如技術方案116至130中任一項之抗體或其抗原結合片段及醫藥學上可接受之賦形劑。 132) 一種套組，其包含如技術方案116至131中任一項之抗體或其抗原結合片段。 133) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案131之醫藥組合物。 134) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案132之套組。 135) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案116至130中任一項之抗體或其抗原結合片段。 136) 如技術方案133至135中任一項之方法，其中該個體為人類個體。 137) 如技術方案135之方法，其中該癌症闡述於表I中。 138) 如技術方案135之方法，其中該方法進一步包含投與放射或化學治療劑或CAR-T療法或NK細胞療法。 139) 一種抗體藥物結合物(ADC)，其包含結合至藥物-連接基團(DL)酬載之NECTIN-4抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈CDR區，該重鏈CDR區包含SEQ ID NO: 52至SEQ IS NO: 69中之任一者中所闡述之胺基酸序列。 140) 如技術方案139之ADC，其進一步包含NECTIN-4抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含輕鏈CDR區，該輕鏈CDR區包含SEQ ID NO: 70至SEQ ID NO: 87中之任一者中所闡述之胺基酸序列。 141) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 142) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 143) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 144) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 145) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 146) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 147) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 148) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 149) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 150) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 151) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 152) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 153) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 154) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 155) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 156) 如技術方案139或140之ADC，其中該DL酬載包含以下化學結構：。 157) 一種醫藥組合物，其包含如技術方案139至156中任一項之ADC及醫藥學上可接受之賦形劑。 158) 一種套組，其包含如技術方案139至156中任一項之ADC。 159) 一種套組，其包含如技術方案157之醫藥組合物。 160) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案139至156中任一項之ADC。 161) 一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如技術方案157之醫藥組合物。 162) 如技術方案160之方法，其中該個體為人類。 163) 如技術方案161之方法，其中該個體為人類。 164) 如技術方案160之方法，其中該癌症闡述於表I中。 165) 如技術方案161之方法，其中該癌症闡述於表I中。 166) 如技術方案160之方法，其中該方法進一步包含投與放射或化學治療劑或CAR-T療法或NK細胞療法。 167) 如技術方案161之方法，其中該方法進一步包含投與放射或化學治療劑或CAR-T療法或NK細胞療法。Exemplary embodiments 1) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54. 2) The antibody or the antigen-binding fragment thereof of technical solution 1, further comprising a light chain variable region, the light chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72. 3) The antibody or the antigen-binding fragment thereof of technical solution 1, wherein the heavy chain variable region comprises a sequence as described in SEQ ID NO: 28. 4) The antibody or antigen-binding fragment thereof of technical solution 2, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 34. 5) The antibody or antigen-binding fragment thereof of technical solution 1, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 5. 6) The antibody or antigen-binding fragment thereof of technical solution 2, wherein the light chain comprises the sequence as described in SEQ ID NO: 17. 7) The antibody or antigen-binding fragment thereof of technical solution 1, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence described in SEQ ID NO: 28; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence described in SEQ ID NO: 34. 8) The antibody or antigen-binding fragment thereof of technical solution 1, wherein the antigen-binding fragment is Fab, F(ab′)2, Fv or scFv. 9) The antibody or antigen-binding fragment thereof of technical solution 1, wherein the antibody is a fully human antibody. 10) The antibody or antigen-binding fragment thereof of technical solution 1, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 11) The antibody or antigen-binding fragment thereof of technical solution 1, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 12) The antibody or antigen-binding fragment thereof of technical solution 11, wherein the drug is an auristatin analog. 13) The antibody or antigen-binding fragment thereof of technical solution 11, further comprising an extension group unit. 14) The antibody or antigen-binding fragment thereof of technical solution 11, further comprising a spacer group unit. 15) The antibody or antigen-binding fragment thereof of technical solution 11, further comprising an amino acid unit. 16) A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of technical solutions 1 to 15 and a pharmaceutically acceptable formulation. 17) A kit comprising an antibody or antigen-binding fragment thereof as described in any one of technical solutions 1 to 16. 18) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of technical solution 16. 19) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit of technical solution 17. 20) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof as described in any one of technical solutions 1 to 15. 21) The method of any one of technical solutions 18 to 20, wherein the individual is a human individual. 22) The method of technical solution 21, wherein the cancer is described in Table I. 23) The method of technical solution 22, wherein the method further comprises administering radiation or chemotherapy. 24) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having a sequence described in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57. 25) The antibody or antigen-binding fragment thereof of technical solution 24, further comprising a light chain variable region, the light chain variable region comprising a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75. 26) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the heavy chain variable region comprises the sequence as described in SEQ ID NO: 29. 27) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 35. 28) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 6. 29) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the light chain comprises the sequence as described in SEQ ID NO: 18. 30) The antibody or antigen-binding fragment thereof of technical solution 24, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence as described in SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence as described in SEQ ID NO: 35. 31) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the antigen-binding fragment thereof is Fab, F(ab′)2, Fv or scFv. 32) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the antibody is a fully human antibody. 33) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 34) The antibody or antigen-binding fragment thereof of technical solution 24, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 35) The antibody or antigen-binding fragment thereof of claim 34, wherein the drug is an auristatin analog. 36) The antibody or antigen-binding fragment thereof of technical solution 34, further comprising an extension group unit. 37) The antibody or antigen-binding fragment thereof of technical solution 34, further comprising a spacer group unit. 38) The antibody or antigen-binding fragment thereof of technical solution 34, further comprising an amino acid unit. 39) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 24 to 38 and a pharmaceutically acceptable formulation. 40) A kit comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 24 to 39. 41) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of technical solution 39. 42) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit of technical solution 40. 43) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described in any one of technical solutions 24 to 39. 44) The method as described in any one of technical solutions 41 to 43, wherein the individual is a human individual. 45) The method as described in claim 43, wherein the cancer is described in Table I. 46) The method as described in technical solution 43, wherein the method further comprises administering radiation or chemotherapy. 47) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60. 48) The antibody or antigen-binding fragment thereof of technical solution 47, further comprising a light chain variable region, the light chain variable region comprising a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78. 49) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the heavy chain variable region comprises the sequence as described in SEQ ID NO: 30. 50) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 36. 51) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 8. 52) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the light chain comprises the sequence as described in SEQ ID NO: 20. 53) The antibody or antigen-binding fragment thereof of technical solution 47, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence as described in SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence as described in SEQ ID NO: 36. 54) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the antigen-binding fragment thereof is Fab, F(ab′)2, Fv or scFv. 55) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the antibody is a fully human antibody. 56) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 57) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 58) The antibody or antigen-binding fragment thereof of technical solution 47, wherein the drug is an auristatin analog. 59) The antibody or antigen-binding fragment thereof of technical solution 47, further comprising an extension group unit. 60) The antibody or antigen-binding fragment thereof of technical solution 47, further comprising a spacer group unit. 61) The antibody or antigen-binding fragment thereof of technical solution 47, further comprising an amino acid unit. 62) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 47 to 61 and a pharmaceutically acceptable formulation. 63) A kit comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 47 to 62. 64) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of technical solution 62. 65) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit of technical solution 63. 66) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of technical solutions 47 to 65. 67) The method as described in any one of technical solutions 64 to 66, wherein the individual is a human individual. 68) The method as described in technical solution 66, wherein the cancer is described in Table I. 69) The method as described in technical solution 66, wherein the method further comprises administering a radiation or chemotherapy agent. 70) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63. 71) The antibody or antigen-binding fragment thereof of technical solution 70, further comprising a light chain variable region, the light chain variable region comprising a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81. 72) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the heavy chain variable region comprises the sequence as described in SEQ ID NO: 31. 73) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 37. 74) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 10. 75) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the light chain comprises the sequence as described in SEQ ID NO: 22. 76) The antibody or antigen-binding fragment thereof of technical solution 70, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence as described in SEQ ID NO: 31; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence as described in SEQ ID NO: 37. 77) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the antigen-binding fragment thereof is Fab, F(ab′)2, Fv or scFv. 78) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the antibody is a fully human antibody. 79) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 80) The antibody or antigen-binding fragment thereof of technical solution 70, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 81) The antibody or antigen-binding fragment thereof of claim 70, wherein the drug is an auristatin analog. 82) The antibody or antigen-binding fragment thereof of technical solution 70, further comprising an extension group unit. 83) The antibody or antigen-binding fragment thereof of technical solution 70, further comprising a spacer group unit. 84) The antibody or antigen-binding fragment thereof of technical solution 70, further comprising an amino acid unit. 85) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 70 to 84 and a pharmaceutically acceptable formulation. 86) A kit comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 70 to 84. 87) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of technical solution 85. 88) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit of technical solution 86. 89) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of technical solutions 70 to 88. 90) The method as described in any one of technical solutions 87 to 89, wherein the individual is a human individual. 91) The method as described in technical solution 89, wherein the cancer is described in Table I. 92) The method as described in technical solution 89, wherein the method further comprises administering radiation or chemotherapy. 93) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66. 94) The antibody or antigen-binding fragment thereof of technical solution 93, further comprising a light chain variable region, the light chain variable region comprising a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84. 95) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the heavy chain variable region comprises the sequence as described in SEQ ID NO: 32. 96) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 38. 97) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 12. 98) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the light chain comprises the sequence as described in SEQ ID NO: 24. 99) The antibody or antigen-binding fragment thereof of technical solution 93, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence as described in SEQ ID NO: 32; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence as described in SEQ ID NO: 38. 100) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the antigen-binding fragment thereof is Fab, F(ab′)2, Fv or scFv. 101) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the antibody is a fully human antibody. 102) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 103) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 104) The antibody or antigen-binding fragment thereof of technical solution 93, wherein the drug is an auristatin analog. 105) The antibody or antigen-binding fragment thereof of technical solution 93, further comprising an extension group unit. 106) The antibody or antigen-binding fragment thereof of technical solution 93, further comprising a spacer group unit. 107) The antibody or antigen-binding fragment thereof of technical solution 93, further comprising an amino acid unit. 108) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 93 to 107 and a pharmaceutically acceptable formulation. 109) A kit comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 93 to 108. 110) A method for treating cancer in an individual, comprising administering a therapeutically effective amount of the pharmaceutical composition of technical solution 108 to the individual. 111) A method for treating cancer in an individual, comprising administering a therapeutically effective amount of the kit of technical solution 109 to the individual. 112) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of technical solutions 93 to 107. 113) The method as described in any one of technical solutions 110 to 112, wherein the individual is a human individual. 114) The method as described in technical solution 112, wherein the cancer is described in Table I. 115) The method as described in technical solution 112, wherein the method further comprises administering a radiation or chemotherapy agent. 116) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69. 117) The antibody or antigen-binding fragment thereof of technical solution 116, further comprising a light chain variable region, the light chain variable region comprising a complementary determining region (CDR) having a sequence as described in SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87. 118) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the heavy chain variable region comprises the sequence as described in SEQ ID NO: 33. 119) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the light chain variable region comprises the sequence as described in SEQ ID NO: 39. 120) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the heavy chain comprises the sequence as described in SEQ ID NO: 14. 121) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the light chain comprises the sequence described in SEQ ID NO: 26. 122) The antibody or antigen-binding fragment thereof of technical solution 116, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence described in SEQ ID NO: 33; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence described in SEQ ID NO: 39. 123) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the antigen-binding fragment thereof is Fab, F(ab′)2, Fv or scFv. 124) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the antibody is a fully human antibody. 125) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. 126) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group. 127) The antibody or antigen-binding fragment thereof of technical solution 116, wherein the drug is an auristatin analog. 128) The antibody or antigen-binding fragment thereof of technical solution 116, further comprising an extension group unit. 129) The antibody or antigen-binding fragment thereof of technical solution 116, further comprising a spacer group unit. 130) The antibody or antigen-binding fragment thereof of technical solution 116, further comprising an amino acid unit. 131) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 116 to 130 and a pharmaceutically acceptable excipient. 132) A kit comprising the antibody or antigen-binding fragment thereof of any one of technical solutions 116 to 131. 133) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of technical solution 131. 134) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit of technical solution 132. 135) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of technical solutions 116 to 130. 136) The method of any one of technical solutions 133 to 135, wherein the individual is a human individual. 137) The method of technical solution 135, wherein the cancer is described in Table I. 138) The method of technical solution 135, wherein the method further comprises administering radiation or chemotherapy or CAR-T therapy or NK cell therapy. 139) An antibody-drug conjugate (ADC) comprising a NECTIN-4 antibody or an antigen-binding fragment thereof bound to a drug-linker (DL) payload, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR region, and the heavy chain CDR region comprises the amino acid sequence described in any one of SEQ ID NO: 52 to SEQ ID NO: 69. 140) The ADC of technical solution 139, further comprising a NECTIN-4 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a light chain CDR region, and the light chain CDR region comprises the amino acid sequence described in any one of SEQ ID NO: 70 to SEQ ID NO: 87. 141) The ADC of technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: 142) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 143) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 144) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 145) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 146) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 147) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 148) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 149) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 150) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 151) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 152) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 153) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 154) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 155) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 156) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 157) A pharmaceutical composition comprising an ADC as described in any one of technical solutions 139 to 156 and a pharmaceutically acceptable excipient. 158) A kit comprising an ADC as described in any one of technical solutions 139 to 156. 159) A kit comprising a pharmaceutical composition as described in technical solution 157. 160) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an ADC as described in any one of technical solutions 139 to 156. 161) A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition as described in technical solution 157. 162) The method as described in technical solution 160, wherein the individual is a human. 163) The method of technical solution 161, wherein the individual is a human. 164) The method of technical solution 160, wherein the cancer is described in Table 1. 165) The method of technical solution 161, wherein the cancer is described in Table 1. 166) The method of technical solution 160, wherein the method further comprises administering radiation or chemotherapy or CAR-T therapy or NK cell therapy. 167) The method of technical solution 161, wherein the method further comprises administering radiation or chemotherapy or CAR-T therapy or NK cell therapy.

實例：本發明之各種態樣藉助於以下若干實例進一步描述及說明，該等實例中無一者意欲限制本發明之範圍。Examples: Various aspects of the present invention are further described and illustrated with the help of the following several examples, none of which is intended to limit the scope of the present invention.

實例1：生成抗體之方法藉由使用酵母顯示人類Fab抗體庫重新發現活動來生成NECTIN-4抗體。在若干回合之富集繼之純系篩選之後，鑑別特異性識別表現於癌細胞上之人類NECTIN-4的純系。隨後，自分離自酵母純系之DNA對抗體之可變重鏈及輕鏈進行定序。為了以重組方式表現NECTIN-4抗體，分別在人類重鏈IgG1及人類輕鏈Igκ恆定區的上游選殖抗體可變重鏈及輕鏈序列。信號肽插入重鏈及輕鏈的上游以允許分泌抗體。在選殖載體中，在CMV啟動子/強化子的下游選殖完整抗nectin-4抗體人類重鏈匣及輕鏈匣。在MAb編碼序列的下游包括聚腺苷酸化位點。重組抗nectin-4抗體重鏈及輕鏈的表現構築體轉染至CHO細胞中。Example 1:Method for generating antibodies NECTIN-4 antibodies were generated by de novo discovery of a human Fab antibody library using yeast display. After several rounds of enrichment followed by clone screening, clones were identified that specifically recognized human NECTIN-4 expressed on cancer cells. Subsequently, the variable heavy and light chains of the antibody were sequenced from DNA isolated from the yeast clones. In order to express the NECTIN-4 antibody recombinantly, the antibody variable heavy and light chain sequences were cloned upstream of the constant regions of human heavy chain IgG1 and human light chain Igκ, respectively. Signal peptides were inserted upstream of the heavy and light chains to allow secretion of the antibody. In the cloning vector, the complete anti-nectin-4 antibody human heavy chain cassette and light chain cassette were cloned downstream of the CMV promoter/enhancer. A polyadenylation site was included downstream of the MAb coding sequence. The expression constructs of the recombinant anti-nectin-4 antibody heavy chain and light chain were transfected into CHO cells.

經穩定轉染之中國倉鼠卵巢(CHO)細胞經歷選擇及回收製程用於生成表現重組抗體及Fc變異體之穩定池。對於抗體生成，在採集培養基之前，對經穩定轉染之池使用典型培養持續時間為8至15天的饋料批式生產製程。替代地，在採集培養基之前，培養經短暫轉染之細胞3-15天之典型持續時間。隨後，針對所採集之細胞培養液進行蛋白-A親和力純化，且將經純化之物質經緩衝液交換為磷酸鹽緩衝鹽水(PBS)或其他較佳抗體調配物緩衝液。重組抗體之品質藉由尺寸排阻層析、SDS-PAGE及此項技術中已知之其他方法評估。Stably transfected Chinese hamster ovary (CHO) cells undergo a selection and recovery process for the generation of stable pools expressing recombinant antibodies and Fc variants. For antibody production, a fed-batch production process is used for the stably transfected pools with a typical culture duration of 8 to 15 days before the medium is harvested. Alternatively, cells that have been transiently transfected are cultured for a typical duration of 3-15 days before the medium is harvested. Subsequently, the harvested cell culture medium is subjected to protein-A affinity purification, and the purified material is buffer exchanged to phosphate-buffered saline (PBS) or other preferred antibody formulation buffer. The quality of the recombinant antibodies is assessed by size exclusion analysis, SDS-PAGE and other methods known in the art.

所得NECTIN-4抗體闡述於表VI、表VII中且包含(SEQ ID NO: 4)至(SEQ ID NO: 27)。The obtained NECTIN-4 antibodies are described in Table VI and Table VII and include (SEQ ID NO: 4) to (SEQ ID NO: 27).

實例2：NECTIN-4抗體之結合分析使用以下方案評估本發明之NECTIN-4抗體的結合親和力。簡言之，採集腫瘤細胞株，且將細胞再懸浮於FACS緩衝液(2% FBS + 5 mM EDTA於PBS中)中。將細胞接種至96孔圓底培養盤中且與抗體(10 µg/mL)一起在冰上培育一(1)小時。對於細胞結合特異性測定，添加10 µg/mL抗體。在培育之後，細胞藉由200×g離心5分鐘粒化，用FACS緩衝液洗兩次，且用R-PE標記之山羊抗人類Fc γ片段特異性二級抗體(Jackson Immuno Research；West Grove，PA)在冰上避光複染(counterstained)一(1)小時。隨後將經標記之細胞用FACS緩衝液洗兩次且藉由流式細胞分析技術使用Attune NxT流式細胞儀(Thermo Fisher Scientific；Carlsbad，CA)分析。Example 2 :Binding Analysis ofNECTIN-4 Antibodies The binding affinity of the NECTIN-4 antibodies of the present invention was evaluated using the following protocol. Briefly, tumor cell lines were harvested and cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated with antibodies (10 µg/mL) on ice for one (1) hour. For cell binding specificity assays, 10 µg/mL of antibodies were added. After incubation, cells were pelleted by centrifugation at 200×g for 5 minutes, washed twice with FACS buffer, and counterstained with R-PE-labeled goat anti-human Fcγ fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice in the dark for one (1) hour. Labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

結果顯示NECTIN-4抗體在多個癌細胞株(T-47D、RT4、NCI-H1781、NCI-H322、PC-3、L-540、SU-DHL-1及K562)上特異性結合至NECTIN-4。(參見圖1)。The results showed that the NECTIN-4 antibody specifically binds to NECTIN-4 on multiple cancer cell lines (T-47D, RT4, NCI-H1781, NCI-H322, PC-3, L-540, SU-DHL-1, and K562) (seeFigure1 ).

實例3：NECTIN-4抗體之結合分析在另一實例中，本發明之NECTIN-4抗體之結合親和力使用以下方案評估。簡言之，採集腫瘤細胞株，且將細胞再懸浮於FACS緩衝液(2% FBS + 5 mM EDTA於PBS中)中。將細胞接種至96孔圓底培養盤中且與抗體(10 µg/mL)一起在冰上培育一(1)小時。對於細胞結合特異性測定，添加10 µg/mL抗體之3倍稀釋。在培育之後，細胞藉由200×g離心5分鐘粒化，用FACS緩衝液洗兩次，且用R-PE標記之山羊抗人類Fc γ片段特異性二級抗體(Jackson Immuno Research；West Grove，PA)在冰上避光複染一(1)小時。隨後將經標記之細胞用FACS緩衝液洗兩次且藉由流式細胞分析技術使用Attune NxT流式細胞儀(Thermo Fisher Scientific；Carlsbad，CA)分析。Example 3 :Binding Analysis ofNECTIN-4 Antibodies In another example, the binding affinity of the NECTIN-4 antibodies of the present invention was evaluated using the following protocol. Briefly, tumor cell lines were harvested and the cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated with antibodies (10 µg/mL) on ice for one (1) hour. For cell binding specificity assays, a 3-fold dilution of 10 µg/mL antibody was added. After incubation, cells were pelleted by centrifugation at 200×g for 5 minutes, washed twice with FACS buffer, and counterstained with R-PE-labeled goat anti-human Fcγ fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice in the dark for one (1) hour. The labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

結果顯示NECTIN-4抗體特異性結合至T-47D乳癌細胞株上之NECTIN-4。(參見圖2及表XII)。The results showed that the NECTIN-4 antibody specifically binds to NECTIN-4 on the T-47D breast cancer cell line (seeFigure 2 andTableXII ).

實例4：NECTIN-4抗體之結合分析在另一實例中，本發明之NECTIN-4抗體之結合親和力使用以下方案評定。簡言之，採集腫瘤細胞株，且將細胞再懸浮於FACS緩衝液(2% FBS + 5 mM EDTA於PBS中)中。將細胞接種至96孔圓底培養盤中且與抗體(10 µg/mL)一起在冰上培育一(1)小時。對於細胞結合親和力測定，添加10 µg/mL抗體之3倍稀釋。在培育之後，藉由以200×g離心5分鐘粒化細胞，用FACS緩衝液洗滌兩次，且用R-PE標記之山羊抗人類Fc γ片段特異性二級抗體(Jackson Immuno Research；West Grove，PA)在冰上避光複染一(1)小時。隨後將經標記之細胞用FACS緩衝液洗滌兩次且藉由流式細胞分析技術使用Attune NxT流式細胞儀(Thermo Fisher Scientific；Carlsbad，CA)分析。Example 4 :Binding Analysis ofNECTIN-4 Antibodies In another example, the binding affinity of the NECTIN-4 antibodies of the present invention was assessed using the following protocol. Briefly, tumor cell lines were harvested and the cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated with antibodies (10 µg/mL) on ice for one (1) hour. For cell binding affinity determination, a 3-fold dilution of 10 µg/mL antibody was added. After incubation, cells were pelleted by centrifugation at 200×g for 5 minutes, washed twice with FACS buffer, and counterstained with R-PE-labeled goat anti-human Fcγ fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice in the dark for one (1) hour. Labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

結果展示NECTIN-4抗體在NCI-H292肺癌細胞株上特異性結合至NECTIN-4且亦以其各別ADC形式與NCI-H292相對結合。(參見圖3及表XIII)。The results showed that the NECTIN-4 antibody specifically binds to NECTIN-4 on the NCI-H292 lung cancer cell line and also binds relatively to NCI-H292 in its respective ADC form (seeFIG. 3 andTableXIII ).

實例5：NECTIN-4 ADC之活體外細胞毒性使用以下方案測定NECTIN-4 ADC之活體外細胞毒性。簡言之，採集腫瘤細胞株，接種至384孔白色平底培養盤中，且在37℃下培育的同時使其再附著2-4小時。隨後通過劑量調定(500 nM最大值及5倍稀釋)用ADC或自由酬載測試品處理細胞。在5天處理之後，基於製造商說明書(Promega；Madison，WI)藉由CellTiter Glo分析測定剩餘細胞存活率。資料相對於未經處理之對照細胞正規化且使用4參數邏輯方程式使用GraphPad Prism軟體(版本9；La Jolla，CA)擬合劑量反應曲線。Example 5 :In vitro cytotoxicity ofNECTIN-4 ADC The in vitro cytotoxicity of NECTIN-4 ADC was determined using the following protocol. Briefly, tumor cell lines were harvested, seeded into 384-well white flat-bottom culture plates, and allowed to attach for 2-4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test article by dose adjustment (500 nM maximum and 5-fold dilution). After 5 days of treatment, the remaining cell viability was determined by CellTiter Glo analysis based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells and dose-response curves were fit using a 4-parameter logic equation using GraphPad Prism software (version 9; La Jolla, CA).

結果展示NECTIN-4 ADC對多個癌細胞株、NECTIN-4陽性肺腺癌NCI-H322細胞(參見圖4(A))、NECTIN-4陽性PC3-NECTIN-4重組前列腺癌細胞(參見圖4(B))但不對NECTIN-4陰性細胞(參見圖4(C))具有在活體外細胞毒性效應。亦參見表XIV。The results show that NECTIN-4 ADC has in vitro cytotoxic effects on multiple cancer cell lines, NECTIN-4 positive lung adenocarcinoma NCI-H322 cells (seeFIG. 4 (A) ), NECTIN-4 positive PC3-NECTIN-4 recombinant prostate cancer cells (seeFIG. 4 (B) ), but not on NECTIN-4 negative cells (seeFIG. 4 (C) ).See also TableXIV .

在使用上文所闡述之方案的另一組實驗中，使用與九(9)種不同連接基團/酬載結合之相同NECTIN-4 Ab展示活體外細胞毒性效價(IC₅₀)。IC₅₀(nM)闡述於表XV中。此等結果進一步證實，利用多種連接基團/酬載之NECTIN-4 ADC對NECTIN-4陽性細胞株PC3-NECTIN-4具有活體外細胞毒性效應。In another set of experiments using the protocol described above, in vitro cytotoxic potency (_IC50 ) was demonstrated using the same NECTIN-4 Ab conjugated to nine (9) different linkers/payloads. The_IC50 (nM) are described inTableXV . These results further demonstrate that NECTIN-4 ADCs utilizing a variety of linkers/payloads have in vitro cytotoxic effects on the NECTIN-4 positive cell line PC3-NECTIN-4.

實例6：NECTIN-4 ADC酬載之活體外細胞毒性使用以下方案測定NECTIN-4 ADC酬載之活體外細胞毒性。簡言之，採集腫瘤細胞株，接種至384孔白色平底培養盤中，且在37℃下培育的同時使其再附著2-4小時。隨後通過劑量調定(500 nM最大值及5倍稀釋)用ADC或自由酬載測試品處理細胞。在5天處理之後，基於製造商說明書(Promega；Madison，WI)藉由CellTiter Glo分析測定剩餘細胞存活率。資料相對於未經處理之對照細胞正規化且使用4參數邏輯方程式使用GraphPad Prism軟體(版本9；La Jolla，CA)擬合劑量反應曲線。Example6:In vitro cytotoxicity ofNECTIN-4 ADC payloads The in vitro cytotoxicity of NECTIN-4 ADC payloads was determined using the following protocol. Briefly, tumor cell lines were harvested, seeded into 384-well white flat-bottom culture plates, and allowed to attach for 2-4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test article by dose adjustment (500 nM maximum and 5-fold dilution). After 5 days of treatment, remaining cell viability was determined by CellTiter Glo analysis based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells and dose-response curves were fit using a 4-parameter logic equation using GraphPad Prism software (version 9; La Jolla, CA).

結果展示在Nectin4-陽性乳癌細胞株Sum190PT中，呈遞三(3)種不同酬載實體(填充符號)之Nectin-4 ADC與各別同型對照ADC (空心符號)及各別自由酬載(點線)相比的活體外細胞毒性。(參見圖5)。Results show the in vitro cytotoxicity of Nectin-4 ADCs delivered with three (3) different payload entities (filled symbols) compared to respective isotype control ADCs (open symbols) and respective free payloads (dotted lines) in the Nectin4-positive breast cancer cell line Sum190PT (seeFigure 5 ).

實例7：NECTIN-4 ADC與維汀-恩弗妥單抗相比之旁觀者活性使用以下方案測定NECTIN-4 ADC與商用維汀-恩弗妥單抗(PADCEV)相比之旁觀者活性。簡言之，使用表現NECTIN-4之PC3-Nectin4重組細胞及經Cell Trace Violet (Invitrogen；Waltham，MA)標記之不表現Nectin-4之SU-DHL-1細胞的共培養模型在24孔培養盤中進行。目標陽性細胞與目標陰性細胞之比率為1:2。在用ADC測試品處理96小時之後，採集SU-DHL-1細胞且收集至96孔圓底培養盤中，且依序用可固定存活率染料(Fixable Viability Dye；FVD) eFluor780 (Invitrogen)及APC-Annexin V (BioLegend；San Diego，CA)染色，隨後藉由流式細胞分析技術分析。用ADC處理之SU-DHL-1細胞之單培養物充當對照以證明非目標特異性殺死或其不存在。早期凋亡(Annexin V單一陽性)、晚期凋亡(FVD eFluor780單一陽性)及壞死(Annexin V及FVD eFluor780雙陽性)細胞視為無活力的。Example 7: Bystander activity of NECTIN-4 ADC comparedto Vitin-Enfortumab The bystander activity of NECTIN-4 ADC compared to commercial Vitin-Enfortumab (PADCEV) was determined using the following protocol. Briefly, a co-culture model using PC3-Nectin4 recombinant cells expressing NECTIN-4 and SU-DHL-1 cells not expressing Nectin-4 labeled with Cell Trace Violet (Invitrogen; Waltham, MA) was performed in 24-well culture plates. The ratio of target positive cells to target negative cells was 1:2. After 96 hours of treatment with the ADC test articles, SU-DHL-1 cells were harvested and collected into 96-well round-bottom culture plates and stained sequentially with Fixable Viability Dye (FVD) eFluor780 (Invitrogen) and APC-Annexin V (BioLegend; San Diego, CA) and subsequently analyzed by flow cytometry. Single cultures of SU-DHL-1 cells treated with ADC served as controls to demonstrate non-target specific killing or its absence. Early apoptotic (Annexin V single positive), late apoptotic (FVD eFluor780 single positive) and necrotic (Annexin V and FVD eFluor780 double positive) cells were considered non-viable.

結果展示代表性ADC與維汀-恩弗妥單抗(實心圓)相比及與無處理(空心三角形)相比之旁觀者活性(實心方形)。旁觀者活性藉由在與表現Nectin4之細胞株共培養時繪製Nectin4陰性細胞株之細胞死亡圖來測定(圖6(A))。與目標陰性細胞單株培養中之維汀-恩弗妥單抗相比，代表性ADC之非特異性活性不存在(圖6(B))。Results show bystander activity (filled squares) of representative ADCs compared to Vitino-Enfortumab (filled circles) and compared to no treatment (open triangles). Bystander activity was determined by plotting cell death of Nectin4-negative cell lines when co-cultured with cell lines expressing Nectin4 (FIG.6 (A) ). Non-specific activity of representative ADCs was absent compared to Vitino-Enfortumab in monoculture of target-negative cell lines (FIG.6 (B) ).

實例8：使用多個酬載之NECTIN-4 ADC在HT-1376異種移植模型中的活體內功效使用以下方案進行NECTIN-4 ADC之活體內功效。簡言之，將HT-1376細胞懸浮液與基質膠(Matrigel) 1:1混合。將5,000,000個活細胞經皮下注射至雌性BALB/c裸小鼠之後側腹中。當平均腫瘤尺寸達到大致100 mm³時，將小鼠隨機分為五(5)組。測試品以5 mg/kg給藥一次。在第28天終止研究。Example 8:In vivoefficacyof NECTIN-4 ADC using multiple payloadsin the HT-1376xenograft model The in vivo efficacy of NECTIN-4 ADC was performed using the following protocol. Briefly, HT-1376 cell suspension was mixed 1:1 with Matrigel. 5,000,000 viable cells were injected subcutaneously into the flank of female BALB/c nude mice. When the mean tumor size reached approximately 100^mm3 , the mice were randomly divided into five (5) groups. The test article was dosed once at 5 mg/kg. The study was terminated on day 28.

結果展示當與對照組相比時，所有NECTIN-4 ADC均抑制腫瘤生長。(參見圖7)。The results showed that all NECTIN-4 ADCs inhibited tumor growth when compared to the control group (seeFigure7 ).

實例9：NECTIN-4 ADC在Sum190PT乳癌異種移植模型中的活體內功效另外，使用以下方案進行NECTIN-4 ADC (Ab5-ADC2)之活體內功效實驗。簡言之，將Sum190PT細胞懸浮液與Cultrex ECM以1:1混合。將3,000,000個活細胞皮下注射至雌性NSG小鼠之後側腹中。當平均腫瘤尺寸達到大致130 mm³時，將小鼠隨機分組，每組五(5)隻小鼠。測試品以10 mg/kg間隔一週給藥兩次。在第28天終止研究。Example 9: In vivo efficacy of NECTIN-4 ADCin the Sum190PTbreast cancer xenograft model In addition, an in vivo efficacy experiment of NECTIN-4 ADC (Ab5-ADC2) was performed using the following protocol. Briefly, Sum190PT cell suspension was mixed with Cultrex ECM at a 1:1 ratio. 3,000,000 viable cells were injected subcutaneously into the flank of female NSG mice. When the average tumor size reached approximately 130^mm3 , the mice were randomized into groups of five (5) mice per group. The test article was administered twice at 10 mg/kg, one week apart. The study was terminated on Day 28.

結果展示在Nectin4陽性Sum190PT乳癌異種移植模型中Nectin-4 ADC(實心圓)與各別同型對照ADC (空心方形)及PBS組(空心圓)相比之活體內功效。(參見圖8)。The results show the in vivo efficacy of Nectin-4 ADC (filled circles) compared to respective isotype control ADCs (open squares) and PBS group (open circles) in the Nectin4-positive Sum190PT breast cancer xenograft model (seeFigure 8 ).

實例10：NECTIN-4 ADC與PADCEV相比在患者源性頭頸癌模型中的活體內功效另外，使用以下方案進行NECTIN-4 ADC (Ab5-ADC2)與商用維汀-恩弗妥單抗(PADCEV)相比的活體內功效實驗。簡言之，來源於頭頸部鱗狀細胞癌患者之腫瘤在免疫功能不全小鼠中以患者源性異種移植物(PDX)形式活體內繁殖。採集來自原種小鼠(stock mice)之腫瘤片段(直徑2-3 mm)且用於皮下接種至雌性NOD/SCID小鼠中。當平均腫瘤尺寸達到大致150 mm³時，將小鼠隨機分組，每組五(5)隻小鼠。測試品以單次劑量形式或間隔兩(2)週兩次以5 mg/kg或10 mg/kg給藥。Example 10: In vivo efficacy of NECTIN-4 ADC comparedto PADCEVin a patient-derived head and neck cancer model In addition, an in vivo efficacy experiment of NECTIN-4 ADC (Ab5-ADC2) compared to the commercial Vidin-Enfortumomab (PADCEV) was performed using the following protocol. Briefly, tumors derived from head and neck squamous cell carcinoma patients were propagated in vivo as patient-derived xenografts (PDX) in immunocompromised mice. Tumor fragments (2-3 mm in diameter) from stock mice were harvested and used for subcutaneous inoculation into female NOD/SCID mice. When the average tumor size reached approximately 150^mm3 , the mice were randomized into groups of five (5) mice per group. The test article was administered as a single dose or twice at two (2) weeks intervals at 5 mg/kg or 10 mg/kg.

結果展示，在Nectin4陽性患者源性頭頸癌模型中，與維汀-恩弗妥單抗(方形)相比，Nectin-4 ADC (圓形)展現更好功效及完全腫瘤根除。(參見圖9)。The results showed that Nectin-4 ADC (circles) demonstrated better efficacy and complete tumor eradication compared to Vidin-Enfamil (squares) in a Nectin4-positive patient-derived head and neck cancer model (seeFigure9 ).

實例11：嵌合抗原受體(CAR) T細胞療法在表現NECTIN-4之癌症中的用途一般言之，T細胞幫助發現及對抗感染及疾病，諸如體內之癌症。許多癌症可避開T細胞，因此當T細胞無法「看到」癌症時，癌症可在體內生長。癌症免疫療法中之一種有前景的形式被稱為CAR-T療法。Example 11:Use ofChimeric Antigen Receptor (CAR) TCell Therapy in Cancers Expressing NECTIN-4 In general, T cells help detect and fight infections and diseases, such as cancer, in the body. Many cancers can evade T cells, so when T cells cannot "see" the cancer, the cancer can grow in the body. One promising form of cancer immunotherapy is called CAR-T therapy.

在CAR-T療法中，嵌合抗原受體(CAR)經設計以識別在癌症中表現之特異性標記物(例如NECTIN-4)。研究已展示當CAR連接至特異性抗原時，誘導免疫反應及T細胞識別癌症且可抑制癌症生長。In CAR-T therapy, chimeric antigen receptors (CARs) are designed to recognize specific markers expressed in cancer (e.g., NECTIN-4). Studies have shown that when CARs are linked to specific antigens, they induce an immune response and T cells recognize cancer and can inhibit cancer growth.

藉助於非限制性實例，自患有表現NECTIN-4之癌症的患者收集血液。分離來自患者血液之T細胞且基因工程改造以生成CAR-T細胞。使用此項技術中已知之技術培養及擴增CAR-T細胞。最後，將CAR-T細胞灌注至患者之血流中。參見JIN等人, Cancer Cell Int., 21:83 (2021)。By way of non-limiting example, blood is collected from a patient with a cancer expressing NECTIN-4. T cells from the patient's blood are isolated and genetically engineered to generate CAR-T cells. The CAR-T cells are cultured and expanded using techniques known in the art. Finally, the CAR-T cells are infused into the patient's bloodstream. See JIN et al., Cancer Cell Int., 21:83 (2021).

試驗起初證明安全性且其後證實重複劑量之功效。試驗為開放標記的，比較標準化學療法與標準療法加NECTIN-4 CAR-T細胞。如將瞭解，可與患者登記結合使用之一個非限制性準則為如藉由此項技術中已知之標準測定之腫瘤中的NECTIN-4濃度。The trial initially demonstrated safety and subsequently confirmed efficacy with repeated dosing. The trial is open label, comparing standard chemotherapy to standard therapy plus NECTIN-4 CAR-T cells. As will be appreciated, one non-limiting criterion that may be used in conjunction with patient enrollment is the NECTIN-4 concentration in the tumor as measured by standards known in the art.

實例12：自然殺手(NK)細胞療法在表現NECTIN-4之癌症中的用途類似於CAR-T療法，自然殺手(NK)細胞療法為已展示在治療癌症(例如表現NECTIN-4之癌症)中之前景的免疫療法形式。不同於T細胞，NK細胞不適合於特異性抗原。然而，雖然NK細胞可識別且侵襲癌細胞，但NK細胞不能存活足夠長時間或足夠快速增殖以完全對抗癌細胞。然而，研究已展示NK細胞可藉由用稱為細胞介素之免疫系統蛋白對其處理而增強。研究已展示用細胞介素增強NK細胞使得免疫反應更穩定。NK細胞療法之一個優點為與CAR-T療法相比缺乏副作用。在一些情況下，NK細胞亦用CAR增強以使其更適於對抗癌症。參見LU等人, Frontiers in Oncology,第11卷,論文720501 (2021年8月)。Example 12:Use ofNatural Killer (NK)Cell Therapy in Cancers Expressing NECTIN-4 Similar to CAR-T therapy, Natural Killer (NK) cell therapy is a form of immunotherapy that has shown promise in treating cancers, such as cancers expressing NECTIN-4. Unlike T cells, NK cells are not adapted to specific antigens. However, while NK cells can recognize and invade cancer cells, NK cells cannot survive long enough or proliferate quickly enough to completely fight cancer cells. However, studies have shown that NK cells can be enhanced by treating them with immune system proteins called cytokines. Studies have shown that enhancing NK cells with cytokines makes the immune response more stable. One advantage of NK cell therapy is the lack of side effects compared to CAR-T therapy. In some cases, NK cells are also enhanced with CAR to make them more suitable for fighting cancer.See LU et al., Frontiers in Oncology, Vol. 11, Paper 720501 (August 2021).

藉助於非限制性實例，可使用若干策略來增強NK細胞療法之功效。首先，NK細胞由末梢血液(PB)、臍帶血(UCB)、誘導性富潛能幹細胞(iPSC)及NK92細胞株生成。自前述來源分離之後，藉由細胞介素，諸如IL-2、IL-15及/或IL-18刺激NK細胞。此外，可離體修飾NK細胞以表現CAR，允許NK細胞識別特異性腫瘤相關抗原，諸如NECTIN-4。最後，將NK細胞灌注至患者之血流中。參見MEHTA等人, Int. J. of Hematology, 107:262-270 (2018)。By way of non-limiting examples, several strategies can be used to enhance the efficacy of NK cell therapy. First, NK cells are generated from peripheral blood (PB), umbilical cord blood (UCB), induced high potential stem cells (iPSCs) and NK92 cell lines. After separation from the aforementioned sources, NK cells are stimulated by interleukins, such as IL-2, IL-15 and/or IL-18. In addition, NK cells can be modified in vitro to express CAR, allowing NK cells to recognize specific tumor-related antigens, such as NECTIN-4. Finally, NK cells are infused into the patient's bloodstream. See MEHTA et al., Int. J. of Hematology, 107:262-270 (2018).

試驗起初證明安全性且其後證實重複劑量之功效。試驗為開放標記的，比較標準化學療法與標準療法加NECTIN-4 NK細胞。如將瞭解，可與患者登記結合使用之一個非限制性準則為如藉由此項技術中已知之標準測定之腫瘤中的NECTIN-4濃度。The trial initially demonstrated safety and subsequently confirmed efficacy with repeated doses. The trial was open label, comparing standard chemotherapy to standard therapy plus NECTIN-4 NK cells. As will be appreciated, one non-limiting criterion that can be used in conjunction with patient enrollment is the NECTIN-4 concentration in the tumor as measured by standards known in the art.

實例13：NECTIN-4抗體及NECTIN-4 ADC特徵分析本發明之Nectin-4抗體及NECTIN-4 ADC組合物使用此項技術中已知之分析進一步表徵。Example 13:Characterization ofNECTIN -4Antibodies and NECTIN-4 ADCs The Nectin-4 antibodies and NECTIN-4 ADC compositions of the present invention were further characterized using assays known in the art.

圖11及圖12中所展示之結果證實更好安全概況及更強功效，從而相對於此項技術中已知之其他NECTIN-4抗體及NECTIN-4 ADC具有改良之治療窗。The results shown inFigures11 and12 demonstrate a better safety profile and stronger efficacy, resulting in an improved therapeutic window relative to other NECTIN-4 antibodies and NECTIN-4 ADCs known in the art.

實例14：NECTIN-4 ADC跨越多種人類正常細胞初代培養物的活體外安全性評定使用以下方案測定NECTIN-4 ADC之活體外細胞毒性。簡言之，採集正常人類源性初代細胞，包括角膜上皮細胞、成人真皮纖維母細胞及成人表皮角質細胞，塗佈至384孔白色平底培養盤中，且在37℃下培育的同時使其再附著2-4小時。隨後通過劑量調定(1000 nM最大值及5倍稀釋)用ADC或自由酬載測試品處理細胞。隨後，在5或6天處理之後，基於製造商說明書(Promega；Madison，WI)藉由CellTiter Glo分析測定細胞存活率。資料相對於未經處理之對照細胞正規化且使用4參數邏輯方程式使用GraphPad Prism軟體(版本9；La Jolla，CA)擬合劑量反應曲線。Example 14: In vitro safety assessment of NECTIN-4 ADCacross multiple human normal cell primary cultures The in vitro cytotoxicity of NECTIN-4 ADC was determined using the following protocol. Briefly, normal human-derived primary cells, including corneal epithelial cells, adult dermal fibroblasts, and adult epidermal keratinocytes, were harvested, plated into 384-well white flat-bottom culture plates, and allowed to attach for an additional 2-4 hours while incubating at 37°C. The cells were then treated with ADC or free payload test article by dose adjustment (1000 nM maximum and 5-fold dilution). Subsequently, after 5 or 6 days of treatment, cell viability was determined by CellTiter Glo analysis based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells and dose-response curves were fitted using a 4-parameter logic equation using GraphPad Prism software (Version 9; La Jolla, CA).

結果展示，在諸如人類角膜上皮細胞(參見圖11(A))、成人真皮纖維母細胞(參見圖11(B))及成人表皮角質細胞(參見圖11(C))之多種正常初代細胞上，NECTIN-4 ADC Ab5-ADC2比維汀-恩弗妥單抗具有顯著較弱的活體外細胞毒性效應。亦參見表XVI，其展示在至多18 mg/kg的重複劑量下的ADC及總IgG之毒理動力學參數，且因此提供NHP中HNSTD增加之證據作為所描述之改良之活體外安全性概況的結果。The results show thatNECTIN-4 ADC Ab5-ADC2 has significantly weaker in vitro cytotoxic effects than vetin-enfototin on a variety of normal primary cells such as human corneal epithelial cells (see Figure11( A)), adult dermal fibroblasts (see Figure11 (B) ), and adult epidermal keratinocytes (seeFigure11 (C) ). See alsoTableXVI , which shows the toxicokinetic parameters of ADC and total IgG at repeated doses up to 18 mg/kg, and thus provides evidence of increased HNSTD in NHP as a result of the described improved in vitro safety profile.

在使用上文所闡述之方案的另一組實驗中，展示前述細胞之流式細胞分析技術直方圖。人類角膜上皮細胞(參見圖12(A))；成人真皮纖維母細胞(參見圖12(B))，其展示無NECTIN-4表現且表示由於非靶向ADC攝取而引起的毒性傾向；以及成人表皮角質細胞(參見圖12(C))，其表現NECTIN-4且表示NECTIN-4 ADC之中靶毒性傾向。In another set of experiments using the protocol described above, flow cytometry histograms are shown for human corneal epithelial cells (seeFIG.12(A) ); adult dermal fibroblasts (see FIG.12(B) ), which exhibit no NECTIN-4 expression and indicate a toxicity tendency due to non-targeted ADC uptake; and adult epidermal keratinocytes(seeFIG.12(C) ) , which express NECTIN-4 and indicate a tendency for on-target toxicity of the NECTIN-4 ADC.

實例15：Ab5-ADC2之細胞週期分析在此實驗中，使用以下方案確定Ab5-ADC2之細胞週期分析。簡言之，採集HT-1376腫瘤細胞且接種至6孔培養盤中且在37℃下培育隔夜。次日，用5 nM之Ab5-ADC2處理細胞72小時，且隨後用FxCycle PI/RNase Staining Solution (Invitrogen，Waltham，MA，USA)進行碘化丙錠染色。藉由流式細胞分析技術量測DNA含量，且使用Watson Pragmatic演算法與FlowJo軟體(版本10；BD Biosciences，Franklin Lakes，NJ，USA)分析細胞週期資料建模。Example 15 :Cell cycle analysis ofAb5-ADC2 In this experiment, the cell cycle analysis of Ab5-ADC2 was determined using the following protocol. Briefly, HT-1376 tumor cells were harvested and seeded into 6-well culture plates and incubated overnight at 37°C. The next day, cells were treated with 5 nM Ab5-ADC2 for 72 hours and then stained with propidium iodide using FxCycle PI/RNase Staining Solution (Invitrogen, Waltham, MA, USA). DNA content was measured by flow cytometry, and cell cycle data modeling was analyzed using Watson Pragmatic algorithm and FlowJo software (version 10; BD Biosciences, Franklin Lakes, NJ, USA).

結果展示HT-1376細胞在用Ab5-ADC2處理72小時之後合併之G2及子G1期之百分比(%)的細胞週期分析。(參見圖13(A))。圖13(B)展示經Ab5-ADC2處理之細胞的代表性流式細胞分析技術直方圖，相比於未經處理之對照細胞，經Ab5-ADC2處理之細胞在細胞週期之子G1及G2期均展示細胞群體增加，如針對此酬載類別所預期。Results show cell cycle analysis of HT-1376 cells in the combined G2 and sub-G1 phases after treatment with Ab5-ADC2 for 72 hours. (SeeFIG.13(A) ).FIG.13(B) shows representative flow cytometry histograms of cells treated with Ab5-ADC2, which show an increase in cell populations in both the sub-G1 and G2 phases of the cell cycle compared to untreated control cells, as expected for this payload class.

實例16：與MMAE相比之自由酬載的免疫原性細胞死亡(ICD)分析在此實驗中，使用以下方案確定與MMAE相比之自由酬載的ICD分析。簡言之，採集NCI-H292腫瘤細胞且接種至100 mm培養皿中且在37℃下培育隔夜。次日，用10 nM之自由酬載處理細胞48小時，且隨後用以下免疫原性細胞死亡(ICD)標記物進行活細胞染色：抗鈣網伴護蛋白、抗HSP70及抗HMGB1。所有標記物抗體均用AlexaFluor 488 (Novus Biologicals，Centennial，CO，USA)標記。藉由流式細胞分析技術使用Attune NxT流式細胞儀分析細胞，且相對於同型對照抗體染色測定ICD標記物陽性%。Example 16: Immunogenic Cell Death (ICD)Analysisof Free Payload Comparedto MMAE In this experiment, the ICD analysis of free payload compared to MMAE was determined using the following protocol. Briefly, NCI-H292 tumor cells were harvested and seeded into 100 mm culture dishes and incubated overnight at 37°C. The next day, cells were treated with 10 nM of free payload for 48 hours and then live cell staining was performed with the following immunogenic cell death (ICD) markers: anti-calcium chaperone, anti-HSP70, and anti-HMGB1. All marker antibodies were labeled with AlexaFluor 488 (Novus Biologicals, Centennial, CO, USA). Cells were analyzed by flow cytometry using an Attune NxT flow cytometer and the % ICD marker positivity was determined relative to isotype control antibody staining.

結果展示如對於此酬載類別所預期，兩種酬載的免疫原性細胞死亡標記物增加之程度相似。(參見圖14)。The results showed that both payloads increased immunogenic cell death markers to a similar extent, as expected for this payload class (seeFigure 14 ).

實例17：Ab5-ADC2及Ab5之補體依賴性細胞毒性(CDC)分析在此實驗中，使用以下方案確定Ab5-ADC2之CDC分析。簡言之，採集HT-1376腫瘤細胞且將其塗佈於96孔白色平底培養盤中且與指定連續稀釋之測試品一起在冰上培育一(1)小時，以允許抗體或ADC結合至細胞。隨後，在37℃下用幼兔補體對細胞調理素化1小時，且藉由CellTiter-Glo 2.0 Assay (Promega)測定由於CDC活性引起細胞溶解之後的剩餘細胞存活率。Example 17 :Complement-dependent cytotoxicity (CDC)assay ofAb5-ADC2and Ab5 In this experiment, the CDC assay of Ab5-ADC2 was determined using the following protocol. Briefly, HT-1376 tumor cells were harvested and plated in 96-well white flat-bottom culture plates and incubated with the designated serial dilutions of the test article on ice for one (1) hour to allow the antibody or ADC to bind to the cells. Subsequently, the cells were opsonized with baby rabbit complement for 1 hour at 37°C, and the remaining cell viability after cell lysis due to CDC activity was determined by CellTiter-Glo 2.0 Assay (Promega).

結果展示Ab5-ADC2以及恩弗妥單抗缺乏任何顯著的CDC活性。(參見圖15)。The results showed that Ab5-ADC2 and Enfamil lacked any significant CDC activity. (SeeFigure 15 ).

實例18：Ab5-ADC2及Ab5之抗體依賴性細胞介導之細胞毒性(ADCC)分析在此實驗中，使用以下方案確定Ab5-ADC2之ADCC分析。簡言之，採集NCI-H292腫瘤細胞且接種至96孔白色平底培養盤中且在37℃下培育隔夜。次日，將腫瘤細胞與經工程改造之效應細胞(效應細胞與目標細胞之比率為6:1)在指定抗體或ADC測試品存在下共同培育6小時，且使用商用ADCC Reporter Bioassay，V Variant套組(Promega，Madison，WI，USA)量測ADCC活性。Example 18 :Antibody-dependent cell-mediated cytotoxicity (ADCC)assay ofAb5-ADC2and Ab5 In this experiment, the ADCC assay of Ab5-ADC2 was determined using the following protocol. Briefly, NCI-H292 tumor cells were harvested and seeded into 96-well white flat-bottom culture plates and incubated overnight at 37°C. The next day, tumor cells were co-cultured with engineered effector cells (ratio of effector cells to target cells was 6:1) in the presence of the designated antibody or ADC test article for 6 hours, and ADCC activity was measured using the commercial ADCC Reporter Bioassay, V Variant kit (Promega, Madison, WI, USA).

結果展示，儘管陽性對照(Her2 Ab)展示強ADCC活性，但對於Ab5-ADC2未觀測到顯著ADCC活性。(參見圖16)。The results showed that, although the positive control (Her2 Ab) exhibited strong ADCC activity, no significant ADCC activity was observed for Ab5-ADC2 (seeFIG16 ).

實例19：Ab5-ADC2及Ab5之抗體依賴性細胞介導之吞噬作用(ADCP)分析在此實驗中，使用以下方案確定Ab5-ADC2之ADCP分析。簡言之，採集腫瘤細胞且接種於384孔白色平底培養盤中且在37℃下培育隔夜。次日，將腫瘤細胞與經工程改造之效應細胞(效應細胞與目標細胞之比率為6:1至7.5:1)在指定抗體或ADC測試品存在下共同培育6小時，且使用商用FcγRI ADCP生物分析套組(Promega)量測ADCP活性。Example 19 :Antibody-dependent cell-mediated phagocytosis (ADCP)assay ofAb5-ADC2and Ab5 In this experiment, the ADCP assay of Ab5-ADC2 was determined using the following protocol. Briefly, tumor cells were harvested and seeded in 384-well white flat-bottom culture plates and incubated overnight at 37°C. The next day, tumor cells were co-cultured with engineered effector cells (ratio of effector cells to target cells was 6:1 to 7.5:1) in the presence of the designated antibody or ADC test article for 6 hours, and ADCP activity was measured using a commercial FcγRI ADCP bioassay kit (Promega).

結果展示，與分析對照試劑(表現isoCTRL Ab之目標之RAMOS細胞中的isoCTRL Ab)相比，在表現NECTIN-4之SUM190PT及HT-1376癌細胞株中對於Ab5-ADC2觀測到弱ADCP活性。(參見圖17)。The results showed that weak ADCP activity was observed for Ab5-ADC2 in SUM190PT and HT-1376 cancer cell lines expressing NECTIN-4 compared to the assay control (isoCTRL Ab in RAMOS cells expressing the target of the isoCTRL Ab) (seeFIG17 ).

總體而言，CDC、ADCC及ADCP發現結果之缺乏排除FcR介導之效應功能活性對Ab5-ADC2之作用機制的任何顯著貢獻。Overall, the lack of CDC, ADCC, and ADCP findings excludes any significant contribution of FcR-mediated effector function activity to the mechanism of action of Ab5-ADC2.

實例20：Ab5-ADC2之ADC及自由酬載在石蟹獼猴中的藥代動力學概況在此實驗中，使用以下方案測定Ab5-ADC2及其相應自由酬載之藥物動力學概況。簡言之，以2次劑量之不同劑量水平向石蟹獼猴給與ADC測試品Ab5-ADC2。在不同時間點收集血液且將其處理成用於總抗體及ADC定量的血清與用於自由酬載分析的血漿。酶聯免疫吸附分析(ELISA)用於藉由以下定量總抗體及ADC濃度：用可溶性重組NECTIN-4捕捉及分別用生物素標記之抗人類IgG或生物素標記之抗酬載偵測，隨後為「鏈黴抗生物素蛋白-HRP」之結合。TMB用作比色基質，且藉由H2SO4溶液停止顏色反應。在讀盤儀上量測450 nm-630 nm下之吸光度。藉由針對校準樣本濃度繪製經連續稀釋之測試品之反應(各校準曲線樣本之平均吸收度減去空白之平均吸收度值)且藉由四參數邏輯模型擬合來生成校準曲線。所有資料藉由SoftMax Pro 7.0軟體分析。Example 20:Pharmacokinetic profileof ADCand free payload of Ab5-ADC2 in stone crab macaques In this experiment, the pharmacokinetic profile of Ab5-ADC2 and its corresponding free payload was determined using the following protocol. Briefly, the ADC test product Ab5-ADC2 was administered to stone crab macaques at different dose levels in 2 doses. Blood was collected at different time points and processed into serum for total antibody and ADC quantification and plasma for free payload analysis. Enzyme-linked immunosorbent assay (ELISA) was used to quantify total antibody and ADC concentrations by capturing with soluble recombinant NECTIN-4 and detecting with biotinylated anti-human IgG or biotinylated anti-payload, respectively, followed by binding of "streptavidin-HRP". TMB was used as a colorimetric matrix, and the color reaction was stopped by H2SO4 solution. The absorbance was measured at 450 nm-630 nm on a plate reader. The calibration curve was generated by plotting the response of the serially diluted test sample against the concentration of the calibration sample (the average absorbance of each calibration curve sample minus the average absorbance value of the blank) and fitting by a four-parameter logical model. All data were analyzed by SoftMax Pro 7.0 software.

對於自由酬載定量，藉由蛋白沉澱自20 μL石蟹獼猴K2EDTA血漿萃取酬載，隨後藉由AB SCIEX TRIPLE QUAD^TM6500+質譜儀使用內標偵測且定量。TK參數彙總於表XVI中。For free payload quantification, the payload was extracted from 20 μL of stone crab macaque K2 EDTA plasma by protein precipitation, followed by detection and quantification by AB SCIEX TRIPLE QUAD^™ 6500+ mass spectrometer using internal standards. TK parameters are summarized inTableXVI .

結果顯示總IgG、ADC及自由酬載之線性PK概況而無顯著累積。值得注意地，總IgG及ADC曲線在各劑量水平下及在21天給藥週期之整個時程中重疊，半衰期為約5天(表XVI)，其經由用於生成Ab5-ADC2之極穩定結合技術達成。重要地是，18 mg/kg之Ab5-ADC2的重複給藥在非人類靈長類動物中為良好耐受的，其經由結合穩定性之增加及更佳腫瘤靶向酬載累積同時保留正常組織來達成(參見圖18)。The results showed linear PK profiles for total IgG, ADC and free payload without significant accumulation. Notably, the total IgG and ADC curves overlapped at each dose level and over the entire course of the 21-day dosing cycle, with a half-life of approximately 5 days (TableXVI ), which was achieved through the extremely stable binding technology used to generate Ab5-ADC2. Importantly, repeated dosing of 18 mg/kg of Ab5-ADC2 was well tolerated in non-human primates, which was achieved through increased binding stability and better tumor-targeted payload accumulation while sparing normal tissues (seeFigure18 ).

實例21：NECTIN-4 ADC與維汀-恩弗妥單抗相比在患者源性子宮頸癌模型PDX36中的活體內功效另外，使用以下方案進行NECTIN-4 ADC (Ab5-ADC2)與商用維汀-恩弗妥單抗相比的活體內功效實驗。簡言之，在免疫功能不全小鼠中活體內繁殖來源於子宮頸癌患者之PDX (在此情況下為PDX36)。採集來自原種小鼠之腫瘤片段(直徑2-3 mm)且用於皮下接種至雌性NOD/SCID小鼠中。當平均腫瘤尺寸達到大致150 mm³時，將小鼠隨機分組，每組5隻小鼠。測試品以2.5、5或10 mg/kg給藥。Example 21 :In vivo efficacy ofNECTIN-4 ADC comparedto Vitin-Enfortumab in the patient-derived cervical cancer model PDX36In addition, an in vivo efficacy experiment of NECTIN-4 ADC (Ab5-ADC2) compared to commercial Vitin-Enfortumab was performed using the following protocol. Briefly, PDX derived from cervical cancer patients (in this case PDX36) were propagated in vivo in immunocompromised mice. Tumor fragments (2-3 mm in diameter) from stock mice were harvested and used for subcutaneous inoculation into female NOD/SCID mice. When the average tumor size reached approximately 150^mm3 , the mice were randomized into groups of 5 mice per group. The test articles were dosed at 2.5, 5, or 10 mg/kg.

結果顯示，在患者源性子宮頸癌模型中，與維汀-恩弗妥單抗(深色三角形)相比，Ab5-ADC2 (黑圈)呈現更好的功效及腫瘤抑制，此轉化為用Ab5-ADC2處理之荷瘤小鼠之更長研究中生存期。(參見圖19(A)及圖19(B))。The results showed that in the patient-derived cervical cancer model, Ab5-ADC2 (black circles) exhibited better efficacy and tumor inhibition compared to Vidin-Enfortumab (dark triangles), which translated into longer in-study survival in tumor-bearing mice treated with Ab5-ADC2. (SeeFigure 19 (A) andFigure19 (B) ).

實例22：NECTIN-4 ADC在多個患者源性子宮頸癌模型(PDX10、PDX12、PDX13、PDX16、PDX34及PDX36)中之活體內功效另外，使用以下方案在多個子宮頸癌模型中進行NECTIN-4 ADC (Ab5-ADC2)之活體內功效實驗。簡言之，在免疫功能不全小鼠中活體內繁殖來源於子宮頸癌患者之PDX (在此情況下為PDX10、PDX12、PDX13、PDX16、PDX34及PDX36)。採集來自繁殖小鼠之腫瘤片段(直徑2-3 mm)且用於皮下接種至雌性NOD/SCID小鼠中。當平均腫瘤尺寸達到大致150 mm³時，將小鼠隨機分組，每組5隻小鼠。測試品以2.5、5或10 mg/kg給藥。Example 22 :In vivo efficacy ofNECTIN-4 ADCin multiple patient-derived cervical cancer models (PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) In addition, in vivo efficacy experiments of NECTIN-4 ADC (Ab5-ADC2) were performed in multiple cervical cancer models using the following protocol. Briefly, PDXs derived from cervical cancer patients (in this case PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) were propagated in vivo in immunocompromised mice. Tumor fragments (2-3 mm in diameter) from the propagated mice were harvested and used for subcutaneous inoculation into female NOD/SCID mice. When the mean tumor size reached approximately 150^mm3 , mice were randomized into groups of 5 mice. Test articles were administered at 2.5, 5, or 10 mg/kg.

結果顯示，在多個患者源性子宮頸癌模型中，與媒劑對照相比，Ab5-ADC2 (黑圈)呈現出顯著的功效和腫瘤抑制。(參見圖20(A)至圖20(F))。The results showed that Ab5-ADC2 (black circles) exhibited significant efficacy and tumor inhibition compared to vehicle controls in multiple patient-derived cervical cancer models (seeFigures20(A) to20(F) ).

實例23：經由免疫組織化學在患者源性子宮頸癌模型中之NECTIN-4表現使用以下方案評估NECTIN-4患者源性子宮頸癌異種移植模型之表現特徵。簡言之，將組織樣本固定在10%緩衝中性福馬林中，處理且包埋於石蠟中，隨後製備為4 µm組織切片。去石蠟及再水合之後，針對抗原擷取處理切片。隨後將抗原擷取之後的組織切片與小鼠抗nectin-4初級抗體或IgG (抗體對照)一起培育。洗滌與初級抗體結合的組織切片且用經酶標記之二級抗體偵測。使用Leica Bond Refine Polymer Detection系統對結合之二級抗體進行顯色。隨後使用Aperio ScanScope CS (Aperio；Vista，CA)掃描經染色之組織切片，成像且用光學顯微鏡法評估。使用NanoZoomer-HT 2.0 Image系統使用40×放大率掃描所有經染色之切片且生成影像。Example 23: NECTIN-4Expressionin Patient-Derived Cervical Cancer Model by Immunohistochemistry The expression characteristics of NECTIN-4 in a patient-derived cervical cancer xenograft model were evaluated using the following protocol. Briefly, tissue samples were fixed in 10% buffered neutral formalin, processed and embedded in paraffin, and then prepared as 4 μm tissue sections. After deparaffinization and rehydration, the sections were processed for antigen capture. The tissue sections after antigen capture were then incubated with mouse anti-nectin-4 primary antibody or IgG (antibody control). Tissue sections bound to the primary antibody were washed and detected with an enzyme-labeled secondary antibody. Bound secondary antibodies were visualized using the Leica Bond Refine Polymer Detection system. Stained tissue sections were subsequently scanned, imaged, and evaluated by light microscopy using an Aperio ScanScope CS (Aperio; Vista, CA). All stained sections were scanned and images were generated using a NanoZoomer-HT 2.0 Image system using 40× magnification.

結果顯示使用抗Nectin-4抗體藉由IHC證實NECTIN-4之表現。PDX12 (21(A)及21(B))、PDX10 (21(C)及21(D))、PDX16 (21(E)及21(F))、PDX13 (21(G)及21(H))、PDX34 (21(I)及21(J))及PDX36 (21(K)及21(L))示於各別圖中，其中影像以高解析度(21(A)、(C)、(E)、(G)、(I)及(K))以及低解析度(21(B)、(D)、(F)、(H)、(J)及(L))示出。(參見圖21)。此等PDX模型展示一系列NECTIN-4表現模式，包括具有非常異質NECTIN-4表現之PDX模型。儘管如此，Ab5-ADC2能夠在所有模型中達成顯著腫瘤生長抑制。The results show that the expression of NECTIN-4 was confirmed by IHC using an anti-Nectin-4 antibody. PDX12 (21(A) and21(B) ), PDX10 (21(C) and21(D) ), PDX16 (21(E) and21(F) ), PDX13 (21(G) and21(H) ), PDX34 (21(I) and21(J) ) and PDX36 (21(K) and21(L) ) are shown in the respective figures, where the images are shown at high resolution (21(A) ,(C) ,(E) ,(G) ,(I) and(K) ) and low resolution (21(B) ,(D) ,(F) ,(H) ,(J) and(L) ). (SeeFIG21 ). These PDX models displayed a range of NECTIN-4 expression patterns, including PDX models with very heterogeneous NECTIN-4 expression. Despite this, Ab5-ADC2 was able to achieve significant tumor growth inhibition in all models.

實例24：經由LC-MS/MS之Ab5-ADC2或維汀-恩弗妥單抗之自由酬載濃度分析使用以下方案評估Ab5-ADC2或維汀-恩弗妥單抗在NECTIN-4陽性乳癌荷瘤小鼠之多個組織中的自由酬載濃度。簡言之，向Sum190PT炎性乳癌異種移植小鼠模型給藥Ab5-ADC2或維汀-恩弗妥單抗。收集腫瘤、正常組織及血漿且藉由LCMS/MS分析酬載濃度。Example 24: Free Payload Concentration Analysis ofAb5-ADC2or Vitin-Enfortumabby LC-MS/MS The free payload concentration of Ab5-ADC2 or Vitin-Enfortumab in multiple tissues of NECTIN-4 positive breast cancer bearing mice was evaluated using the following protocol. Briefly, Ab5-ADC2 or Vitin-Enfortumab was administered to the Sum190PT inflammatory breast cancer xenograft mouse model. Tumors, normal tissues, and plasma were collected and analyzed for payload concentration by LCMS/MS.

圖22(A)中之結果顯示相對於MMAE之自由酬載濃度。圖22(B)中之結果展示Ab5-ADC2相對於維汀-恩弗妥單抗之酬載濃度。值得注意地，Ab5-ADC2能夠將更多酬載遞送至表現NECTIN-4之腫瘤，同時減少酬載在正常組織中之暴露，此係比維汀-恩弗妥單抗更好安全性概況之基礎。The results inFigure 22(A) show the free payload concentration relative to MMAE. The results inFigure22(B) show the payload concentration of Ab5-ADC2 relative to Vitin-Enfortumab. Notably, Ab5-ADC2 was able to deliver more payload to tumors expressing NECTIN-4 while reducing exposure of the payload in normal tissues, which is the basis for a better safety profile than Vitin-Enfortumab.

實例25：Ab5-ADC2之穩定性及DAR保持分析使用以下方案進行穩定性評定及DAR保持分析。簡言之，使到達時為10-12週齡之史泊格多利大鼠(Sprague Dawley rats)適應一週，且隨後給予單次靜脈內劑量之Ab5-ADC2或維汀-恩弗妥單抗。在給藥後的不同時間點收集血液樣本：1小時、4小時、第3天、第7天、第14天及第21天。藉由在4℃下以10,000 g離心10分鐘，將收集於EDTA塗佈之管中的血液處理成血漿。將血漿等分且在-80℃下冷凍儲存直至分析。將血漿樣本稀釋於TBS中，隨後與生物素標記之捕獲抗人抗體塗層珠粒組合，隨後與鏈黴抗生物素蛋白塗佈之DynaBeads (Invitrogen)混合且培育約2小時。使珠粒上親和力捕獲之ADC經受磁體以收集複合物，隨後洗滌ADC。經親和力純化之ADC在Agilent QTOF 6550 B上在MassHunter B.07.00或等效物下運作。使用最大熵演算法對峰進行積分、提取及光譜去卷積。去卷積光譜作為CSV檔案輸出且導入DAR計算器中。測定各樣本之DAR且隨時間推移繪製。Example 25 :Stability and DARretention analysis ofAb5-ADC2 Stability assessment and DAR retention analysis were performed using the following protocol. Briefly, Sprague Dawley rats, 10-12 weeks of age upon arrival, were acclimated for one week and then given a single intravenous dose of Ab5-ADC2 or Vidin-Enfatamab. Blood samples were collected at different time points after dosing: 1 hour, 4 hours, day 3, day 7, day 14, and day 21. Blood collected in EDTA-coated tubes was processed into plasma by centrifugation at 10,000 g for 10 minutes at 4°C. The plasma was aliquoted and stored frozen at -80°C until analysis. Plasma samples were diluted in TBS and then combined with biotinylated capture anti-human antibody coated beads, followed by mixing with streptavidin coated DynaBeads (Invitrogen) and incubating for about 2 hours. The affinity captured ADC on the beads was subjected to a magnet to collect the complex, followed by washing the ADC. The affinity purified ADC was run on an Agilent QTOF 6550 B under MassHunter B.07.00 or equivalent. Peaks were integrated, extracted and spectrally deconvoluted using the maximum entropy algorithm. The deconvoluted spectra were exported as CSV files and imported into the DAR calculator. The DAR for each sample was determined and plotted over time.

圖23(A)中之結果顯示在iv注射之後，Ab5-ADC2相較於維汀-恩弗妥單抗隨時間推移之結合及藥物-抗體比率(DAR)的穩定性。圖23(B)中之結果顯示在自史泊格多利大鼠中注射1小時及21天之後，來自血漿之經親和力純化之Ab5-ADC2之重鏈及輕鏈的去卷積MS概況。綜合而言，此等結果指示Ab5-ADC2在循環中非常穩定，其允許與正常組織相比增加對腫瘤的酬載遞送，如圖22所展現。重要地是，此使得非人類靈長類動物中之HNSTD (最高非嚴重毒性劑量)劑量為18 mg/kg，如圖18中所示。The results inFIG. 23(A) show the stability of binding and drug-antibody ratio (DAR) of Ab5-ADC2 compared to Vidin-Enfortumab over time after iv injection. The results inFIG.23(B) show the deconvoluted MS profiles of the heavy and light chains of affinity-purified Ab5-ADC2 from plasma 1 hour and 21 days after injection in Sprague-Dawley rats. Taken together, these results indicate that Ab5-ADC2 is very stable in circulation, which allows for increased payload delivery to tumors compared to normal tissues, as demonstrated inFIG. 22 . Importantly, this resulted in a HNSTD (highest non-serious toxicity dose) dose of 18 mg/kg in non-human primates, as shown in FIG18 .

實例26：經由使用NECTIN-4抗體及NECTIN-4 ADC治療人類癌瘤之人類臨床試驗根據本發明合成NECTIN-4抗體及NECTIN-4 ADC，其具體地積聚於腫瘤細胞中且用於治療某些腫瘤及其他免疫病症及/或其他疾病(參見表I)。關於此等適應症中之各者，成功地實行兩種臨床方法。Example 26:Human clinical trialsusing NECTIN-4antibodies and NECTIN-4 ADCs for the treatment of human carcinomas According to the present invention, NECTIN-4 antibodies and NECTIN-4 ADCs were synthesized, which specifically accumulate in tumor cells and are used to treat certain tumors and other immune disorders and/or other diseases (see Table I). For each of these indications, two clinical approaches were successfully performed.

I.)輔助療法： 在輔助療法中，用NECTIN-4抗體及NECTIN-4 ADC以及化學治療劑或醫藥劑或生物醫藥劑或其組合治療患者。藉由添加NECTIN-4抗體及NECTIN-4 ADC，在標準方案下治療原發癌目標。方案設計解決如藉由以下實例評估之有效性，包括但不限於原發性或轉移性病灶之腫瘤塊減少、無進展生存期增加、總生存期、患者健康改善、疾病穩定以及減少標準化學療法及其他生物藥劑之常見劑量的能力。此等劑量降低藉由降低化學治療劑或生物劑之劑量相關毒性而允許額外及/或長期療法。I.)Adjuvant Therapy : In adjuvant therapy, patients are treated with NECTIN-4 antibodies and NECTIN-4 ADCs and chemotherapy or pharmaceutical agents or biopharmaceutical agents or combinations thereof. Primary cancer targets are treated under standard regimens by adding NECTIN-4 antibodies and NECTIN-4 ADCs. Regimen designs address effectiveness as assessed by the following examples, including but not limited to reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce the usual doses of standard chemotherapy and other biopharmaceuticals. These dose reductions allow for additional and/or long-term therapies by reducing the dose-related toxicity of chemotherapeutic or biologic agents.

II.)單藥療法： 結合在腫瘤之單藥療法中使用NECTIN-4抗體及NECTIN-4 ADC，在無化學治療劑或醫藥劑或生物劑的情況下向患者投與NECTIN-4抗體及NECTIN-4 ADC。在一個實施例中，在臨床上對患有廣泛轉移性疾病的末期癌症患者進行單藥療法。方案設計解決如藉由以下實例評估之有效性，包括但不限於原發性或轉移性病灶之腫瘤塊減少、無進展生存期增加、總生存期、患者健康改善、疾病穩定以及減少標準化學療法及其他生物藥劑之常見劑量的能力。II.)Monotherapy : In conjunction with the use of NECTIN-4 antibodies and NECTIN-4 ADCs in monotherapy for tumors, NECTIN-4 antibodies and NECTIN-4 ADCs are administered to patients in the absence of chemotherapy or pharmaceutical or biologic agents. In one embodiment, monotherapy is performed clinically in terminal cancer patients with extensive metastatic disease. The regimen design addresses efficacy as assessed by the following examples, including but not limited to reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce the typical dose of standard chemotherapy and other biologics.

劑量可調整劑量方案以提供最佳所需反應。例如，可投與單一NECTIN-4抗體及NECTIN-4 ADC注射，可隨時間推移投與若干分次劑量，或可如治療情形之緊急程度所指示而按比例減少或增加劑量。如本文中所使用之「劑量單位形式(Dosage Unit Form)」係指適合作為單位劑量用於待治療之哺乳動物個體的物理上不連續之單元；各單位含有與所需醫藥載劑相關聯之經計算以產生所要治療效應之預定量之活性化合物。本發明之劑量單位形式之規格由以下因素決定且直接視以下因素而定：(a) NECTIN-4抗體及NECTIN-4 ADC之獨特特徵、輻射機制(反應器)之個別機制及待達成之特定治療或預防效應，及(b)此項技術中混配此類化合物用於治療個人敏感性固有的侷限性。Dosage The dosage regimen may be adjusted to provide the optimal desired response. For example, a single NECTIN-4 antibody and NECTIN-4 ADC injection may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, "Dosage Unit Form" refers to physically discrete units suitable as unit doses for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined by and are directly dependent upon: (a) the unique characteristics of the NECTIN-4 antibodies and NECTIN-4 ADCs, the individual mechanisms of action (reactors) and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such compounds for the treatment of individual sensitivities.

臨床開發計劃(CDP)CDP遵從及開發使用本揭露之NECTIN-4抗體及NECTIN-4 ADC的癌症及/或免疫病症之治療(參見表I)。試驗起初證明安全性且其後證實重複劑量之功效。試驗為開放標記的，比較標準化學療法與標準療法加NECTIN-4抗體及NECTIN-4 ADC。如將瞭解，可與患者登記結合使用之一個非限制性準則為藉由此項技術中已知之標準偵測方法測定之腫瘤中的NECTIN-4抗體及NECTIN-4 ADC之濃度。Clinical Development Program(CDP) The CDP complies with and develops treatments for cancer and/or immune disorders using the NECTIN-4 antibodies and NECTIN-4 ADCs of the present disclosure (see Table I). The trials initially demonstrate safety and subsequently demonstrate efficacy at repeated doses. The trials are open label, comparing standard chemotherapy to standard therapy plus NECTIN-4 antibodies and NECTIN-4 ADCs. As will be appreciated, one non-limiting criterion that may be used in conjunction with patient enrollment is the concentration of NECTIN-4 antibodies and NECTIN-4 ADCs in the tumor as determined by standard detection methods known in the art.

本發明之範圍不受本文所揭示之實施例限制，該等實施例意欲作為本發明之個別態樣的單一說明，且在功能上等效之任何實施例在本發明之範圍內。熟習此項技術者根據前述說明及教示內容將顯而易知除了本文所描述之修改之外的對本發明之模型、方法及生命週期方法論之各種修改，且類似地意欲屬於本發明之範疇內。此類修改或其他實施例可在不背離本發明之真實範圍及精神的情況下實施。表I.待治療癌症之代表性清單#癌症1膀胱癌2子宮內膜癌3頭頸癌4肺癌5卵巢癌6胰臟癌7子宮頸癌8乳癌9食道癌10前列腺癌11胃癌12結腸直腸癌13腦癌14骨癌15皮膚癌16肝癌17腎癌表II.胺基酸縮寫單字母三字母全名FPhe苯丙胺酸LLeu白胺酸SSer絲胺酸YTyr酪胺酸CCys半胱胺酸WTrp色胺酸PPro脯胺酸HHis組胺酸QGln麩醯胺酸RArg精胺酸IIle異白胺酸MMet甲硫胺酸TThr蘇胺酸NAsn天冬醯胺KLys離胺酸VVal纈胺酸AAla丙胺酸DAsp天冬胺酸EGlu麩胺酸GGly甘胺酸表III.胺基酸取代矩陣The scope of the present invention is not limited by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the present invention, and any embodiments that are functionally equivalent are within the scope of the present invention. Various modifications to the models, methods, and life cycle methodology of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing descriptions and teachings, and are similarly intended to be within the scope of the present invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of the present invention.Table I.Representative List of Cancers to be Treated#cancer 1 Bladder cancer 2 Endometrial cancer 3 Head and Neck Cancer 4 Lung cancer 5 Ovarian cancer 6 Pancreatic cancer 7 Cervical cancer 8 Breast cancer 9 Esophageal cancer 10 Prostate cancer 11 Gastric cancer 12 Colorectal cancer 13 Brain cancer 14 Bone cancer 15 Skin cancer 16 Liver cancer 17 Kidney cancerTableII.Amino Acid Abbreviations Single letter Three letters Full name F Phe Phenylalanine L Leu Leucine S Ser Serine Y Tyr Tyrosine C Cys Cysteine W Trp Tryptophan P Pro Proline H His Histidine Q Gln Glutamine R Arg Arginine I Ile Isoleucine M Met Methionine T Thr Threonine N Asn Asparagine K Lys Lysine V Val Valine A Ala Alanine D Asp Aspartic acid E Glu Glutamine G Gly GlycineTableIII.Amino Acid Substitution Matrix

改編自GCG Software 9.0 BLlOSUM62胺基酸取代矩陣(嵌段取代矩陣)。值越高，在相關天然蛋白中發現取代的可能性越大。表IV.人類黏連蛋白細胞黏附分子4 (NECTIN-4)之核酸序列(SEQ ID NO: 1)及胺基酸序列(SEQ ID NO: 2)表V.人類黏連蛋白細胞黏附分子4 (NECTIN-4)之胺基酸序列(SEQ ID NO: 3)。信號肽帶下劃線。表VI(A)至VI(F). 抗體重鏈之序列表VI(A). CL.Z重鏈之cDNA序列(SEQ ID NO: 4)及胺基酸序列(SEQ ID NO: 5)。編碼可變區之核苷酸序列帶下劃線。表VI(B). CL.X重鏈之cDNA序列(SEQ ID NO: 6)及胺基酸序列(SEQ ID NO: 7)。編碼可變區之核苷酸序列帶下劃線。表VI(C). CL.N重鏈之cDNA序列(SEQ ID NO: 8)及胺基酸序列(SEQ ID NO: 9)。編碼可變區之核苷酸序列帶下劃線。表VI(D). CL.I重鏈之cDNA序列(SEQ ID NO: 10)及胺基酸序列(SEQ ID NO: 11)。編碼可變區之核苷酸序列帶下劃線。表VI(E). CL.B重鏈之cDNA序列(SEQ ID NO: 12)及胺基酸序列(SEQ ID NO: 13)。編碼可變區之核苷酸序列帶下劃線。表VI(F). CL.D重鏈之cDNA (SEQ ID NO: 14)及胺基酸序列(SEQ ID NO: 15)。編碼可變區之核苷酸序列帶下劃線。表VI(G)至VI(L). 抗體輕鏈之序列表VI(G). CL.Z輕鏈之cDNA序列(SEQ ID NO: 16)及胺基酸序列(SEQ ID NO: 17)。編碼可變區之核苷酸序列帶下劃線。表VI(H). CL.X輕鏈之cDNA序列(SEQ ID NO: 18)及胺基酸序列(SEQ ID NO: 19)。編碼可變區之核苷酸序列帶下劃線。表VI(I). CL.N輕鏈之cDNA序列(SEQ ID NO: 20)及胺基酸序列(SEQ ID NO: 21)。編碼可變區之核苷酸序列帶下劃線。表VI(J). CL.I輕鏈之cDNA序列(SEQ ID NO: 22)及胺基酸序列(SEQ ID NO: 23)。編碼可變區之核苷酸序列帶下劃線。表VI(K). CL.B輕鏈之cDNA序列(SEQ ID NO: 24)及胺基酸序列(SEQ ID NO: 25)。編碼可變區之核苷酸序列帶下劃線。表VI(L). CL.D輕鏈之cDNA序列(SEQ ID NO: 26)及胺基酸序列(SEQ ID NO: 27)。編碼可變區之核苷酸序列帶下劃線。表VII(A)至VII(F). 抗體重鏈之胺基酸序列表VII(A). CL.Z重鏈之胺基酸序列(SEQ ID NO: 28)。可變區帶下劃線，且Kabat CDR區加框。表VII(B). CL.X重鏈之胺基酸序列(SEQ ID NO: 29)。可變區帶下劃線，且Kabat CDR區加框。表VII(C). CL.N重鏈之胺基酸序列(SEQ ID NO: 30)。可變區帶下劃線，且Kabat CDR區加框。表VII(D). CL.I重鏈之胺基酸序列(SEQ ID NO: 31)。可變區帶下劃線，且Kabat CDR區加框。表VII(E). CL.B重鏈之胺基酸序列(SEQ ID NO: 32)。可變區帶下劃線，且Kabat CDR區加框。表VII(F). CL.D重鏈之胺基酸序列(SEQ ID NO: 33)。可變區帶下劃線，且Kabat CDR區加框。表VII(G)至表VII(L). 抗體輕鏈之胺基酸序列 表VII(G). CL.Z輕鏈之胺基酸序列(SEQ ID NO: 34)。可變區帶下劃線，且Kabat CDR區加框。表VII(H). CL.X輕鏈之胺基酸序列(SEQ ID NO: 35)。可變區帶下劃線，且Kabat CDR區加框。表VII(I). CL.N輕鏈之胺基酸序列(SEQ ID NO: 36)。可變區帶下劃線，且Kabat CDR區加框。表VII(J). CL.I輕鏈之胺基酸序列(SEQ ID NO: 37)。可變區帶下劃線，且Kabat CDR區加框。表VII(K). CL.B輕鏈之胺基酸序列(SEQ ID NO: 38)。可變區帶下劃線，且Kabat CDR區加框。表VII(L). CL.D輕鏈之胺基酸序列(SEQ ID NO: 39)。可變區帶下劃線，且Kabat CDR區加框。表VIII. 抗體重鏈可變區之胺基酸序列表IX.抗體輕鏈可變區之胺基酸序列表X.抗體CDR之胺基酸序列純系鏈(重鏈/輕鏈)CDR序列CL.Z重鏈AYGMH (SEQ ID NO:  52)VISYDGSYKYYADSVKG (SEQ ID NO:  53)GVGAFPWVFFDY (SEQ ID NO:  54)CL.X重鏈SYAFS (SEQ ID NO:  55)RILPPIFGTTNYAQKFQG (SEQ ID NO:  56)QAGGVLDV (SEQ ID NO:  57)CL.N重鏈GYGMH (SEQ ID NO:  58)VISFEGSNKYYADSVKG (SEQ ID NO:  59)RHYSYGFDV (SEQ ID NO:  60)CL.I重鏈NYDMH (SEQ ID NO:  61)VISFEGSNKYYADSVKG (SEQ ID NO:  62)RHYSYGFDV (SEQ ID NO:  63)CL.B重鏈SAWMS (SEQ ID NO:  64)RIKSETYGGTTDYAAPVKG (SEQ ID NO:  65)GGADHFDLYLYYGAMDV (SEQ ID NO:  66)CL.D重鏈NYAMS (SEQ ID NO:  67)TISPGGGNTDYADSVKG (SEQ ID NO:  68)GFSRFDH (SEQ ID NO:  69)CL.Z重鏈RASQSISTYLN (SEQ ID NO:  70)AASSLQS (SEQ ID NO:  71)QQGYSTPLT (SEQ ID NO:  72)CL.X輕鏈RASQSVSSNYLA (SEQ ID NO:  73)GASSRAT (SEQ ID NO:  74)QQYGTSLLT (SEQ ID NO:  75)CL.N輕鏈TGSSSNIGAGYDVH (SEQ ID NO:  76)DNRNRPS (SEQ ID NO:  77)QSYDTSLSAWV (SEQ ID NO:  78)CL.I輕鏈RASQSISRYLN (SEQ ID NO:  79)AASSLQS (SEQ ID NO:  80)QQGYSTPPT (SEQ ID NO:  81)CL.B輕鏈RASQSIYTYLN (SEQ ID NO:  82)AASSLQS (SEQ ID NO:  83)QQGYSYPPT (SEQ ID NO:  84)CL.D輕鏈RATQSVSGSYLA (SEQ ID NO:  85)GASSRAT (SEQ ID NO:  86)QQYASWPGT (SEQ ID NO:  87)表XI(A)至XI(F). 胺基酸可變重鏈區與對應生殖系序列之比對表XI(A). CL.Z重鏈可變區(SEQ ID NO: 88)之胺基酸序列與對應頂部V (SEQ ID NO: 89)、D (SEQ ID NO: 90)及J (SEQ ID NO: 91)生殖系序列之比對。Kabat CDR區加框。表XI(B). CL.X重鏈可變區(SEQ ID NO: 92)之胺基酸序列與對應頂部V (SEQ ID NO: 93)、D (SEQ ID NO: 94)及J (SEQ ID NO: 95)生殖系序列之比對。Kabat CDR區加框。表XI(C). CL.N重鏈可變區之胺基酸序列(SEQ ID NO: 96)與對應頂部V (SEQ ID NO: 97)、D (SEQ ID NO: 98)及J (SEQ ID NO: 99)生殖系序列之比對。Kabat CDR區加框。表XI(D). CL.I重鏈可變區(SEQ ID NO: 100)之胺基酸序列與對應頂部V (SEQ ID NO: 101)、D (SEQ ID NO: 102)及J (SEQ ID NO: 103)生殖系序列之比對。Kabat CDR區加框。表XI(E). CL.B重鏈可變區(SEQ ID NO: 104)之胺基酸序列與對應頂部V (SEQ ID NO: 105)、D (SEQ ID NO: 106)及J (SEQ ID NO: 107)生殖系序列之比對。Kabat CDR區加框。表XI(F). CL.D重鏈可變區之胺基酸序列(SEQ ID NO: 108)與對應頂部V (SEQ ID NO: 109)、D (SEQ ID NO: 110)及J (SEQ ID NO: 111)生殖系序列之比對。Kabat CDR區加框。表XI(G)至XI(L).胺基酸輕鏈可變區與對應生殖系序列之比對表XI(G). CL.Z輕鏈可變區(SEQ ID NO: 112)之胺基酸序列與對應頂部V (SEQ ID NO: 113)及J (SEQ ID NO: 114)生殖系序列之比對。Kabat CDR區加框。表XI(H). CL.X輕鏈可變區(SEQ ID NO: 115)之胺基酸序列與對應頂部V (SEQ ID NO: 116)及J (SEQ ID NO: 117)生殖系序列之比對。Kabat CDR區加框。表XI(I). CL.N輕鏈可變區之胺基酸序列(SEQ ID NO: 118)與對應頂部V (SEQ ID NO: 119)及J (SEQ ID NO: 120)生殖系序列之比對。Kabat CDR區加框。表XI(J). CL.I輕鏈可變區(SEQ ID NO: 121)之胺基酸序列與對應頂部V (SEQ ID NO: 122)及J (SEQ ID NO: 123)生殖系序列之比對。Kabat CDR區加框。表XI(K). CL.B輕鏈可變區之胺基酸序列(SEQ ID NO: 124)與對應頂部V (SEQ ID NO: 125)及J (SEQ ID NO: 126)生殖系序列之比對。Kabat CDR區加框。表XI(L). CL.D輕鏈可變區(SEQ ID NO: 127)之胺基酸序列與對應頂部V (SEQ ID NO: 128)及J (SEQ ID NO: 129)生殖系序列之比對。Kabat CDR區加框。表XII.彙總各種Nectin-4抗體與人類乳癌細胞株T-47D之結合的平均螢光強度比率(MFIR)值Ab純系T-47D細胞結合(MFIR)Ab1822.05Ab2814.10Ab3649.26Ab4991.03Ab5806.53Ab6745.53同型對照Ab8.54表XIII.彙總各種Nectin-4抗體及衍生之ADC與人類肺癌細胞株NCI-H292之結合參數Bmax、K_D及擬合曲線R2測試品NCI-H292細胞BmaxK_D(μg/mL)擬合曲線R2Ab1-ADC135470.095290.9972Ab1134950.069720.9816Ab2-ADC124080.094010.9857Ab2108250.14250.9806Ab3-ADC146210.081170.9929Ab3142670.047280.9959Ab4-ADC156970.080680.9938Ab4166580.096870.9945Ab5-ADC147880.13050.9949Ab5143510.10630.9966Ab6134910.15290.9903表XIV.彙總Nectin4陽性細胞株、NCI-H322及PC3-Nectin4以及Nectin4陰性細胞株PC3中之活體外細胞毒性效價(IC₅₀)樣本PC-3/Nectin4 [IC50，nM]NCI-H322 [IC50，nM]PC-3 [IC50，nM]ADC10.0851288ADC20.1549250ADC30.1654130ADC40.114880ADC50.2170130ADC60.162478同型對照ADC97157110酬載0.390.24800.41表XV.彙總含有九(9)種不同連接基團-酬載之Nectin-4 ADC在Nectin-4陽性細胞株PC3-Nectin4中的活體外細胞毒性效價(IC₅₀)測試品IC50 (nM)#個實驗之平均值Ab5-ADC10.710Ab5-ADC298.13Ab5-ADC30.315Ab5-ADC40.58Ab5-ADC5＞5002Ab5-ADC60.33Ab5-ADC7180.01Ab5-ADC828.51Ab5-ADC90.44表XVI.彙總以6、12或18 mg/kg在石蟹獼猴中靜脈內投與Ab5-ADC2之後之ADC及總IgG抗體的毒理動力學參數劑量Cmax(µg/mL)AUC 0-inf(h*µg/mL)T1/2(h)ADC6 mg/kg2102520011112 mg/kg4025020011118 mg/kg50473000130總抗體6 mg/kg2042500011312 mg/kg3915020011618 mg/kg49174400131Adapted from GCG Software 9.0 BL10 SUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the greater the probability of finding the substitution in the related natural protein.TableIV.Nucleic acid sequence(SEQ ID NO: 1)and amino acid sequence(SEQ ID NO: 2) ofhuman nectin cell adhesion molecule4 (NECTIN-4)TableV.Amino acid sequence ofhuman nectin cell adhesion molecule4 (NECTIN-4)(SEQ ID NO: 3). The signal peptide band is underlined. Tables VI(A) to VI(F). Antibody heavy chain sequencesTableVI(A).cDNAsequence(SEQ ID NO: 4)and amino acid sequence(SEQ ID NO: 5)of CL.Z heavy chain. The nucleotide sequence encoding the variable region is underlined.TableVI (B).cDNAsequence(SEQ ID NO: 6)and amino acid sequence(SEQ ID NO: 7)of CL.X recombinant. The nucleotide sequence encoding the variable region is underlined.TableVI (C).cDNAsequence(SEQ ID NO: 8)and amino acid sequence(SEQ ID NO: 9)of CL.N recombinant. The nucleotide sequence encoding the variable region is underlined.TableVI (D).cDNAsequence(SEQ ID NO: 10)and amino acid sequence(SEQ ID NO: 11)of CL.I recombinant. The nucleotide sequence encoding the variable region is underlined.TableVI (E).cDNAsequence(SEQ ID NO: 12)and amino acid sequence(SEQ ID NO: 13)of CL.B recombinant. The nucleotide sequence encoding the variable region is underlined.TableVI(F).cDNA (SEQ ID NO: 14)and amino acid sequence(SEQ ID NO: 15)of CL.D recombinant. The nucleotide sequence encoding the variable region is underlined. Tables VI(G) to VI(L). Antibody light chain sequencesTableVI(G).cDNAsequence(SEQ ID NO: 16)and amino acid sequence(SEQ ID NO: 17)of the CL.Z light chain. The nucleotide sequence encoding the variable region is underlined.TableVI(H).cDNAsequence(SEQ ID NO: 18)and amino acid sequence(SEQ ID NO: 19)ofthe light chain of CL.X. The nucleotide sequence encoding the variable region is underlined.TableVI (I).cDNAsequence(SEQ ID NO: 20)and amino acid sequence(SEQ ID NO: 21)of the CL.N light chain. The nucleotide sequence encoding the variable region is underlined.TableVI(J).cDNAsequence(SEQ ID NO: 22)and amino acid sequence(SEQ ID NO: 23)ofthe light chain of CL.I. The nucleotide sequence encoding the variable region is underlined.TableVI(K).cDNAsequence(SEQ ID NO: 24)and amino acid sequence(SEQ ID NO: 25)of the CL.B light chain. The nucleotide sequence encoding the variable region is underlined.TableVI (L).cDNAsequence(SEQ ID NO: 26)and amino acid sequence(SEQ ID NO: 27)of the CL.D light chain. The nucleotide sequence encoding the variable region is underlined. Tables VII(A) to VII(F). Amino acid sequences of antibody heavy chainsTableVII(A).Amino acid sequence of CL.Z heavy chain(SEQ ID NO: 28). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(B).Amino acid sequence of CL.X heavy chain(SEQ ID NO: 29). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(C). Amino acid sequence of CL.Nheavy chain(SEQ ID NO: 30). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(D). Amino acid sequence of CL.Iheavy chain(SEQ ID NO: 31). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(E). Amino acid sequence of CL.Bheavy chain(SEQ ID NO: 32). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(F). Amino acid sequence of CL.Dheavy chain(SEQ ID NO: 33). The variable regions are underlined and the Kabat CDR regions are boxed. Table VII(G) to Table VII(L). Amino acid sequences of antibody light chains Table VII(G). Amino acid sequence of CL.Z light chain (SEQ ID NO: 34). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(H).Amino acid sequence of the CL.X light chain(SEQ ID NO: 35). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(I).Amino acid sequence of the CL.N light chain(SEQ ID NO: 36). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(J).Amino acid sequence of the CL.I light chain(SEQ ID NO: 37). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(K).Amino acid sequence of the CL.B light chain(SEQ ID NO: 38). The variable regions are underlined and the Kabat CDR regions are boxed.TableVII(L).Amino acid sequence of the CL.D light chain(SEQ ID NO: 39). The variable regions are underlined and the Kabat CDR regions are boxed. Table VIII. Amino acid sequences of variable regions of antibody heavy chains Table IX. Amino acid sequences of variable regions of antibody light chains Table X. Amino acid sequences of antibody CDRsPureChain(heavy chain/light chain)CDRsequences CL.Z Heavy Chain AYGMH (SEQ ID NO: 52)VISYDGSYKYYADSVKG (SEQ ID NO: 53)GVGAFPWVFFDY (SEQ ID NO: 54) CL.X Heavy Chain SYAFS (SEQ ID NO: 55)RILPPIFGTTNYAQKFQG (SEQ ID NO: 56)QAGGVLDV (SEQ ID NO: 57) CL.N Heavy Chain GYGMH (SEQ ID NO: 58)VISFEGSNKYYADSVKG (SEQ ID NO: 59)RHYSYGFDV (SEQ ID NO: 60) CL.I Heavy Chain NYDMH (SEQ ID NO: 61)VISFEGSNKYYADSVKG (SEQ ID NO: 62)RHYSYGFDV (SEQ ID NO: 63) CL.B Heavy Chain SAWMS (SEQ ID NO: 64)RIKSETYGGTTDYAAPVKG (SEQ ID NO: 65)GGADHFDLYLYYGAMDV (SEQ ID NO: 66) CL.D Heavy Chain NYAMS (SEQ ID NO: 67)TISPGGGNTDYADSVKG (SEQ ID NO: 68)GFSRFDH (SEQ ID NO: 69) CL.Z Heavy Chain RASQSISTYLN (SEQ ID NO: 70)AASSLQS (SEQ ID NO: 71)QQGYSTPLT (SEQ ID NO: 72) CL.X Light chain RASQSVSSNYLA (SEQ ID NO: 73)GASSRAT (SEQ ID NO: 74)QQYGTSLLT (SEQ ID NO: 75) CL.N Light chain TGSSSNIGAGYDVH (SEQ ID NO: 76)DNRNRPS (SEQ ID NO: 77)QSYDTSLSAWV (SEQ ID NO: 78) CL.I Light chain RASQSISRYLN (SEQ ID NO: 79)AASSLQS (SEQ ID NO: 80)QQGYSTPPT (SEQ ID NO: 81) CL.B Light chain RASQSIYTYLN (SEQ ID NO: 82)AASSLQS (SEQ ID NO: 83)QQGYSYPPT (SEQ ID NO: 84) CL.D Light chain RATQSVSGSYLA (SEQ ID NO: 85)GASSRAT (SEQ ID NO: 86)QQYASWPGT (SEQ ID NO: 87) Tables XI(A) to XI(F). Alignment of amino acid variable heavy chain regions with corresponding germline sequencesTableXI(A). Alignment of amino acid sequence of CL.Zheavy chain variable region(SEQ ID NO: 88)with corresponding topV (SEQ ID NO: 89),D (SEQ ID NO: 90)andJ (SEQ ID NO: 91)germline sequences. Kabat CDR regions are boxed.TableXI(B).Alignment of the amino acid sequence of the CL.Xheavy chain variable region(SEQ ID NO: 92) with the corresponding topV (SEQ ID NO: 93),D (SEQ ID NO: 94)andJ (SEQ ID NO: 95)germline sequences. Kabat CDR regions are boxed.TableXI(C).Alignment of the amino acid sequence of the CL.N heavy chain variable region(SEQ ID NO: 96)with the corresponding topV (SEQ ID NO: 97),D (SEQ ID NO: 98)andJ (SEQ ID NO: 99)germline sequences. Kabat CDR regions are boxed.TableXI(D).Alignment of the amino acid sequence of the CL.Iheavy chain variable region(SEQ ID NO: 100) with the corresponding topV (SEQ ID NO: 101),D (SEQ ID NO: 102)andJ (SEQ ID NO: 103)germline sequences. Kabat CDR regions are boxed.TableXI(E). Alignment of the amino acid sequence of the CL.Bheavy chain variable region(SEQ ID NO: 104)with the corresponding topV (SEQ ID NO: 105),D (SEQ ID NO: 106)andJ (SEQ ID NO: 107)germline sequences. Kabat CDR regions are boxed.TableXI(F).Alignment of the amino acid sequence of the CL.D heavy chain variable region(SEQ ID NO: 108)with the corresponding topV (SEQ ID NO: 109),D (SEQ ID NO: 110)andJ (SEQ ID NO: 111)germline sequences. Kabat CDR regions are boxed. Tables XI(G) to XI(L). Alignment of amino acid light chain variable regions with corresponding germline sequencesTableXI(G). Alignment of amino acid sequence of CL.Zlight chain variable region(SEQ ID NO: 112)with corresponding topV (SEQ ID NO: 113)andJ (SEQ ID NO: 114)germline sequences. Kabat CDR regions are boxed.TableXI(H). Alignment of the amino acid sequence of the CL.Xlight chain variable region(SEQ ID NO: 115)with the corresponding topV (SEQ ID NO: 116)andJ (SEQ ID NO: 117)germline sequences. Kabat CDR regions are boxed.TableXI(I).Alignment of the amino acid sequence of the CL.N light chain variable region(SEQ ID NO: 118)with the corresponding topV (SEQ ID NO: 119)andJ (SEQ ID NO: 120)germline sequences. Kabat CDR regions are boxed.TableXI(J). Alignment of the amino acid sequence of the CL.Ilight chain variable region(SEQ ID NO: 121)with the corresponding topV (SEQ ID NO: 122)andJ (SEQ ID NO: 123)germline sequences. Kabat CDR regions are boxed.TableXI(K).Alignment of the amino acid sequence of the CL.B light chain variable region(SEQ ID NO: 124)with the corresponding topV (SEQ ID NO: 125)andJ (SEQ ID NO: 126)germline sequences. The Kabat CDR regions are boxed.TableXI(L). Alignment of the amino acid sequence of the CL.Dlight chain variable region(SEQ ID NO: 127)with the corresponding topV (SEQ ID NO: 128)andJ (SEQ ID NO: 129)germline sequences. Kabat CDR regions are boxed.TableXII. Summary of mean fluorescence intensity ratio (MFIR) values of various nectin-4 antibodies binding to human breast cancer cell line T-47D Ab pure system T-47D cell binding (MFIR) Ab1 822.05 Ab2 814.10 Ab3 649.26 Ab4 991.03 Ab5 806.53 Ab6 745.53 Isotype control Ab 8.54Table XIII. Summary of binding parameters Bmax,_KD and fitting curve R2 of various nectin-4 antibodies and derived ADCs to human lung cancer cell line NCI-H292 Test products NCI-H292 cells Bmax_KD (μg/mL) Fitting curve R2 Ab1-ADC 13547 0.09529 0.9972 Ab1 13495 0.06972 0.9816 Ab2-ADC 12408 0.09401 0.9857 Ab2 10825 0.1425 0.9806 Ab3-ADC 14621 0.08117 0.9929 Ab3 14267 0.04728 0.9959 Ab4-ADC 15697 0.08068 0.9938 Ab4 16658 0.09687 0.9945 Ab5-ADC 14788 0.1305 0.9949 Ab5 14351 0.1063 0.9966 Ab6 13491 0.1529 0.9903Table XIV. Summary of in vitro cytotoxic potency (IC₅₀ ) in Nectin4-positive cell lines, NCI-H322 and PC3-Nectin4, and Nectin4-negative cell line PC3 Sample PC-3/Nectin4 [IC50, nM] NCI-H322 [IC50, nM] PC-3 [IC50, nM] ADC1 0.085 12 88 ADC2 0.15 49 250 ADC3 0.16 54 130 ADC4 0.11 48 80 ADC5 0.21 70 130 ADC6 0.16 twenty four 78 Isotype control ADC 97 157 110 Payroll 0.39 0.2480 0.41Table XV. Summary of in vitro cytotoxic potency (IC₅₀ ) of Nectin-4 ADCs containing nine (9) different linker-payloads in the Nectin-4 positive cell line PC3-Nectin4Test productsIC50 (nM) Average of#experiments Ab5-ADC1 0.7 10 Ab5-ADC2 98.1 3 Ab5-ADC3 0.3 15 Ab5-ADC4 0.5 8 Ab5-ADC5 ＞500 2 Ab5-ADC6 0.3 3 Ab5-ADC7 180.0 1 Ab5-ADC8 28.5 1 Ab5-ADC9 0.4 4Table XVI. Summary of toxicokinetic parameters of ADC and total IgG antibodies following intravenous administration of Ab5-ADC2 at 6, 12, or 18 mg/kg in stone macaquesDosageCmax(µg/mL)AUC 0-inf(h*µg/mL)T1/2(h)ADC 6 mg/kg 210 25200 111 12 mg/kg 402 50200 111 18 mg/kg 504 73000 130Total Antibody 6 mg/kg 204 25000 113 12 mg/kg 391 50200 116 18 mg/kg 491 74400 131

圖1. Nectin-4抗體對多種細胞株之抗體結合特異性圖2. Nectin-4抗體對T-47D乳癌細胞株之抗體結合。圖2(A).展示Ab1。圖2(B).展示Ab2。圖2(C).展示Ab3。圖2(D).展示Ab4。圖2(E).展示Ab5。圖2(F).展示Ab6。圖3.Nectin-4抗體與各別ADC相比對NCI-H292肺癌細胞株之抗體親和力。圖4.Nectin4 ADC跨越多種癌細胞株之活體外細胞毒性。圖4(A).展示NCI-H322肺癌細胞株。圖4(B).展示PC3-NECTIN-4重組之表現NECTIN-4的PC3前列腺癌細胞株。圖4(C).展示NECTIN-4陰性細胞株PC3。圖5.使用多種酬載之NECTIN-4 ADC對Sum190PT乳癌細胞株的活體外細胞毒性圖6.ADC與維汀-恩弗妥單抗(enfortumab vedotin)相比之旁觀者活性。圖6(A).展示與表現NECTIN-4之細胞株共培養的NECTIN-4陰性細胞株。圖6(B).展示單株培養中之NECTIN-4陰性細胞株。圖7.使用多種酬載之NECTIN-4 ADC在HT-1376異種移植模型中的活體內功效。圖8.NECTIN-4 ADC在NECTIN-4陽性Sum190PT乳癌異種移植模型中的活體內功效。圖9.NECTIN-4 ADC在NECTIN-4陽性患者源性頭頸癌模型中與維汀-恩弗妥單抗相比的活體內功效。圖10.藥物-連接基團(DL)酬載結構。圖10(A).展示標示為DL-01之結構。圖10(B).展示標示為DL-02之結構。圖10(C).展示標示為DL-03之結構。圖10(D).展示表示為DL-04之結構。圖10(E).展示標示為DL-05之結構。圖10(F).展示標示為DL-06之結構。圖10(G).展示標示為DL-07之結構。圖10(H).展示標示為DL-08之結構。圖10(I).展示標示為DL-09之結構。圖10(J).展示標示為DL-10之結構。圖10(K).展示標示為DL-11之結構。圖10(L).展示標示為DL-12之結構。圖10(M).展示標示為DL-13之結構。圖10(N).展示標示為DL-14之結構。圖10(O).展示標示為DL-15之結構。圖10(P).展示標示為DL-16之結構。圖11.NECTIN4 ADC跨越多種正常人類細胞初代培養物的活體外細胞毒性。圖11(A).展示HCEpC細胞。圖11(B).展示HDFa細胞。圖11(C).展示HEKa細胞。圖12.NECTIN-4與恩弗妥單抗及Ab5跨越多種正常人類細胞初代培養物之流式細胞分析技術直方圖。圖12(A).展示HCEpC細胞。圖12(B).展示HDFa細胞。圖12(C).展示HEKa細胞。圖13.HT-1376細胞中之Ab5-ADC2之細胞週期分析。圖13(A).展示在用Ab5-ADC2處理72小時後HT-1376細胞的合併之G2及子G1期%。圖13(B).展示與未經處理之對照細胞相比，經Ab5-ADC2處理之細胞的代表性流式細胞分析技術直方圖。圖14.自由酬載在NCI-H292癌細胞中與MMAE相比對免疫原性細胞死亡之誘導。圖15.Ab5-ADC及Ab5在HT-1376癌細胞中與恩弗妥單抗抗體相比之補體依賴性細胞毒性分析。圖16.Ab5-ADC2及Ab5在NCI-H292癌細胞中的抗體依賴性細胞介導之細胞毒性(ADCC)活性圖17.Ab5-ADC2及Ab5跨越多種癌細胞株的抗體依賴性細胞介導之吞噬作用(ADCP)活性圖18.Ab5-ADC2之總IgG、ADC及自由酬載的藥物動力學(PK)概況。圖19.NECTIN-4 ADC Ab5-ADC2在NECTIN-4陽性患者源性子宮頸癌模型PDX36中與維汀-恩弗妥單抗相比之活體內功效。圖19(A).展示腫瘤生長動力學。圖19(B).展示卡普蘭-麥爾線圖(Kaplan-Meier plot)中之生存幾率。圖20.NECTIN-4 ADC Ab5-ADC2在六(6)個癌症模型之小鼠臨床試驗中的活體內功效。圖20(A).展示PDX10。圖20(B).展示PDX12。圖20(C).展示PDX13。圖20(D).展示PDX16。圖20(E).展示PDX34。圖20(F).展示PDX36。圖21.經由免疫組織化學在患者源性子宮頸癌異種移植模型中的NECTIN-4表現。圖21(A)、圖21(C)、圖21(E)、圖21(G)、圖21(I)及圖21(K)以高解析度展示表現。圖21(B)、圖21(D)、圖21(F)、圖21(H)、圖21(J)及圖21(L)以低解析度展示表現。圖21(A)及圖21(B). 展示PDX12。圖21(C)及圖21(D). 展示PDX10。圖21(E)及圖21(F). 展示PDX16。圖21(G)及圖21(H). 展示PDX13。圖21(I)及圖21(J). 展示PDX34。圖21(K)及圖21(L). 展示PDX36。圖22.Ab5-ADC2或維汀-恩弗妥單抗之正常組織及腫瘤組織中的自由酬載濃度。圖22(A).展示正常組織中之自由酬載濃度。圖22(B). 展示Ab5-ADC2及維汀-恩弗妥單抗之酬載濃度。圖23.Ab5-ADC2之穩定性概況。圖23(A). 展示與維汀-恩弗妥單抗相比，Ab5-ADC2之結合穩定性及初始藥物-抗體比率(DAR)之百分比。圖23(B). 展示經親和力純化之Ab5-ADC2之重鏈及輕鏈的去卷積MS概況。Figure 1. Antibody binding specificity of nectin-4 antibodies to various cell linesFigure 2. Antibody binding of nectin-4 antibodies to T-47D breast cancer cell line.Figure2 (A). Ab1 is shown.Figure2 (B). Ab2 is shown.Figure2 (C). Ab3 is shown.Figure2 (D). Ab4 is shown.Figure2 (E). Ab5 is shown.Figure2 (F). Ab6 is shown.Figure 3. Antibody affinity of nectin-4 antibodies to NCI-H292 lung cancer cell line compared to individual ADCs.Figure 4. In vitro cytotoxicity of nectin4 ADCs across various cancer cell lines.Figure4 (A). NCI-H322 lung cancer cell line is shown.Figure4 (B). PC3 prostate cancer cell line expressing nectin-4 recombinant PC3-nectin-4 is shown.Figure4(C). Shows the NECTIN-4 negative cell line PC3.Figure 5. In vitro cytotoxicity of NECTIN-4 ADCs using various payloads against Sum190PT breast cancer cell lineFigure 6. Bystander activity of ADCs compared to enfortumab vedotin.Figure6(A). Shows NECTIN-4 negative cell lines co-cultured with NECTIN-4 expressing cell lines.Figure6(B). Shows NECTIN-4 negative cell lines in monoculture.Figure 7. In vivo efficacy of NECTIN-4 ADCs using various payloads in the HT-1376 xenograft model.Figure 8. In vivo efficacy of NECTIN-4 ADCs in the NECTIN-4 positive Sum190PT breast cancer xenograft model.FIG. 9. In vivo efficacy of NECTIN-4 ADC compared to Vittin-Enfamol in a NECTIN-4 positive patient-derived head and neck cancer model.FIG. 10. Drug-linker (DL) payload structures.FIG.10(A). Structures labeled DL-01 are shown.FIG.10(B). Structures labeled DL-02 are shown.FIG.10(C). Structures labeled DL-03 are shown.FIG.10(D). Structures labeled DL-04 are shown.FIG.10(E). Structures labeled DL-05 are shown.FIG.10(F). Structures labeled DL-06 are shown.FIG.10(G). Structures labeled DL-07 are shown.FIG.10(H). Structures labeled DL-08 are shown.FIG.10(I). Structures labeled DL-09 are shown.Figure10(J). Structures labeled DL-10 are shown.Figure10(K). Structures labeled DL-11 are shown.Figure10(L). Structures labeled DL-12 are shown.Figure10(M). Structures labeled DL-13 are shown.Figure10(N). Structures labeled DL-14 are shown.Figure10(O). Structures labeled DL-15 are shown.Figure10(P). Structures labeled DL-16 are shown.Figure 11. In vitro cytotoxicity of NECTIN4 ADC across multiple normal human cell primary cultures.Figure11(A). HCEpC cells are shown.Figure11(B). HDFa cells are shown.Figure11(C). HEKa cells are shown.FIG12. Flow cytometry histograms of NECTIN-4 with Enfamil and Ab5 across various normal human cell primary cultures.FIG12(A). HCEpC cells are shown.FIG12(B). HDFa cells are shown.FIG12(C). HEKa cells are shown.FIG13. Cell cycle analysis of Ab5-ADC2 in HT-1376 cells.FIG13(A). Combined G2 and sub-G1 phase % of HT-1376 cells after 72 hours of treatment with Ab5-ADC2 are shown.FIG13(B). Representative flow cytometry histograms of cells treated with Ab5-ADC2 compared to untreated control cells are shown.Figure 14. Induction of immunogenic cell death by free payload compared to MMAE in NCI-H292 cancer cells.Figure 15. Complement-dependent cytotoxicity analysis of Ab5-ADC and Ab5 compared to Enfortumab antibody in HT-1376 cancer cells.Figure 16. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of Ab5-ADC2 and Ab5 in NCI-H292 cancer cells.Figure 17. Antibody-dependent cell-mediated phagocytosis (ADCP) activity of Ab5-ADC2 and Ab5 across multiple cancer cell lines. Figure 18. Pharmacokinetic (PK) profiles of total IgG, ADC, and free payload of Ab5-ADC2.FIG. 19. In vivo efficacy of NECTIN-4 ADC Ab5-ADC2 compared to Vittin-Enfamil in the NECTIN-4 positive patient-derived cervical cancer model PDX36.FIG. 19(A). Tumor growth kinetics are shown.FIG.19(B). Survival probability in Kaplan-Meier plot is shown.FIG. 20. In vivo efficacy of NECTIN-4 ADC Ab5-ADC2 in mouse clinical trials in six (6) cancer models.FIG.20(A). PDX10 is shown. FIG. 20(B). PDX12 is shown.FIG.20(C). PDX13 is shown.FIG . 20(D). PDX16 is shown.FIG .20(E). PDX34 is shown.FIG.20(F). PDX36 is shown.FIG21. Expression of NECTIN-4 in patient-derived cervical cancer xenograft models by immunohistochemistry.FIG21(A) ,FIG21(C) ,FIG21(E) ,FIG21(G) ,FIG21(I) , andFIG21(K) show expression at high resolution.FIG21(B) ,FIG21(D) ,FIG21(F) ,FIG21(H) ,FIG21(J) , andFIG21(L) show expression at low resolution.FIG21(A)andFIG21(B) . PDX12 is shown.FIG21(C) andFIG21(D) . PDX10 is shown.FIG21(E) andFIG21(F) . PDX16 is shown.FIG21(G) andFIG21(H) . PDX13 is shown.Figure21 (I) andFigure21 (J) . PDX34 is shown.Figure21 (K) andFigure21 (L) . PDX36 is shown.Figure 22. Free payload concentration in normal and tumor tissues of Ab5-ADC2 or Vitin-Enfortuzumab.Figure22 (A) . Free payload concentration in normal tissue is shown.Figure22 (B) . Payload concentration of Ab5-ADC2 and Vitin-Enfortuzumab is shown.Figure23. Stability profile of Ab5-ADC2.Figure23 (A) . Binding stability and percentage of initial drug-antibody ratio (DAR) of Ab5-ADC2 compared to Vitin-Enfortuzumab are shown.FIG.23(B) shows the deconvoluted MS profile of the heavy and light chains of affinity purified Ab5-ADC2.

TW202504637A_113121327_SEQL.xmlTW202504637A_113121327_SEQL.xml

Claims (36)

Translated fromChinese

一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 52、SEQ ID NO: 53及SEQ ID NO: 54中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 55、SEQ ID NO: 56及SEQ ID NO: 57中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 58、SEQ ID NO: 59及SEQ ID NO: 60中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 61、SEQ ID NO: 62及SEQ ID NO: 63中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63.一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 64、SEQ ID NO: 65及SEQ ID NO: 66中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.一種抗體或其抗原結合片段，其中該抗體或其抗原結合片段包含重鏈可變區，該重鏈可變區包含具有SEQ ID NO: 67、SEQ ID NO: 68及SEQ ID NO: 69中所述序列的互補決定區(CDR)。An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a complementary determining region (CDR) having the sequence described in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69.如請求項1至6中任一項之抗體或其抗原結合片段，其中該抗體或其抗原結合片段經由連接基團結合至藥物。The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is bound to a drug via a linker group.如請求項7之抗體或其抗原結合片段，其中該藥物為奧瑞他汀(auristatin)類似物。The antibody or antigen-binding fragment thereof of claim 7, wherein the drug is an auristatin analog.一種抗體藥物結合物(ADC)，其包含NECTIN-4抗體或其抗原結合片段結合至藥物-連接基團(DL)酬載(payload)，其中該抗體或其抗原結合片段包含如請求項1至6中任一項之抗體。An antibody-drug conjugate (ADC) comprising a NECTIN-4 antibody or an antigen-binding fragment thereof conjugated to a drug-linking group (DL) payload, wherein the antibody or the antigen-binding fragment thereof comprises the antibody of any one of claims 1 to 6.如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .如請求項9之ADC，其中該DL酬載包含以下化學結構：。The ADC of claim 9, wherein the DL payload comprises the following chemical structure: .一種醫藥組合物，其包含如請求項9至25中任一項之ADC及醫藥學上可接受之賦形劑。A pharmaceutical composition comprising the ADC of any one of claims 9 to 25 and a pharmaceutically acceptable excipient.一種套組，其包含如請求項9至25中任一項之ADC。A kit comprising an ADC as in any one of claims 9 to 25.一種套組，其包含如請求項26之醫藥組合物。A kit comprising the pharmaceutical composition of claim 26.一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如請求項9至25中任一項之ADC。A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the ADC of any one of claims 9 to 25.一種治療個體之癌症之方法，其包含向該個體投與治療有效量之如請求項26之醫藥組合物。A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of claim 26.如請求項29之方法，其中該個體為人類。The method of claim 29, wherein the individual is a human.如請求項30之方法，其中該個體為人類。The method of claim 30, wherein the individual is a human.如請求項29之方法，其中該癌症列於表I中。The method of claim 29, wherein the cancer is listed in Table I.如請求項30之方法，其中該癌症列於表I中。The method of claim 30, wherein the cancer is listed in Table I.如請求項29之方法，其中該方法進一步包含投與放射，或化學治療劑，或CAR-T療法，或NK細胞療法。The method of claim 29, wherein the method further comprises administering radiation, or chemotherapy, or CAR-T therapy, or NK cell therapy.如請求項30之方法，其中該方法進一步包含投與放射，或化學治療劑，或CAR-T療法，或NK細胞療法。The method of claim 30, wherein the method further comprises administering radiation, or chemotherapy, or CAR-T therapy, or NK cell therapy.