Treatment of cancer

Abstract

The present invention relates to the treatment of cancer. The present invention provides antigen binding proteins capable of binding to Nectin-4 polypeptides conjugated to chemotherapeutic agents for use in increasing the sensitivity of tumours to said chemotherapeutic agents for use in the treatment of cancers characterised by Nectin-4 expressing tumour cells. In one embodiment, the present invention provides conjugates of an anti-Nectin-4 antibody conjugated to a camptothecin analogue such as exatecan or SN-38 via a cleavable linker.

Description

[0001] This application is a divisional application. The original application, with application number 202180007603.2 and filing date of January 28, 2021, is entitled "Treatment of Cancer," the entirety of which is incorporated herein by reference.

[0002] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 968,175, filed January 31, 2020; which is incorporated herein by reference in its entirety; and contains any accompanying drawings.

[0003] Sequence List Reference This application is submitted together with an electronic sequence list. The sequence list is provided as a 29 KB file named "Nectin-4-1_ST25" created on January 25, 2021. Information from the electronic version of the sequence list is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention provides an antigen-binding protein capable of binding to a Nectin-4 polypeptide conjugated to camptothecin, for the treatment of cancers characterized by Nectin-4 expressing tumor cells, specifically comprising different levels of Nectin-4 expression in the tumor, including low and heterogeneous expression. Background Technology

[0005] In the United States, an estimated 70,000 new cases of bladder cancer were diagnosed in 2016, with approximately 16,000 deaths. Urothelial carcinoma encompasses bladder cancer, ureteral cancer, and renal pelvis cancer, with an incidence rate of 50:3:1, respectively. Urothelial carcinoma is a multifocal process. Patients with upper urinary tract cancer have a 30% to 50% chance of developing bladder cancer at some stage in their lives. Bladder cancer occurs when cells in the bladder begin to grow abnormally or uncontrollably. The most common type of bladder cancer is called urothelial carcinoma (UC). In UC, abnormal growth occurs in the lining of the bladder (urothelium). As the disease progresses, it can spread. It can spread to areas surrounding the bladder or other parts of the body (metastasis). This is called advanced urothelial carcinoma.

[0006] Urothelial carcinoma (UC) is characterized by increased expression of a range of different cell surface antigens, thus providing an opportunity for targeted therapy using antibody-drug conjugates (ADCs). Among these surface antigens, several have proven suitable for ADC development, including TROP-2 (human trophoblast cell surface antigen), SLITRK family proteins (e.g., SLITRK6), EpCAM, HER2, TF-Ag (Thomsen-Friedrich antigen), FGF1V, Fn14 (FGF-inducible 14), PSMA (prostate-specific membrane antigen), and Nectin-4 (poliovirus receptor-associated protein 4, also known as PVRL4).

[0007] Nectin-4 was initially cloned from the human trachea by the Lopez group in 2001 (see Reymond et al. (2001) J. Biol. Chem. 276(46):43205-15). Increased copy number of the Nectin-4 gene has been reported as a common event in carcinogenesis, and it can promote epithelial-mesenchymal transition, invasion, and metastasis. While expression of nectin-4 protein is restricted in healthy tissues, its expression levels are significantly elevated in several tumors, and are particularly overexpressed in breast cancer, including triple-negative breast cancer (TNBC) (see M-Rabet et al. Ann Oncol. 2017 Apr 1; ​​28(4):769-776), pancreatic cancer, and UC. However, nectin-4 is also expressed in tumor samples from non-small cell lung cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer. Challita-Eid et al. (2016) in Cancer Research 76(10): 3003-3013 reported moderate to strong staining by immunohistochemistry (H score ≥100) in bladder (60%) and breast (53%) tumor tissues. Zeindler et al. (2019) in Frontiers in Medicine 6:200 reported high expression of Nectin-4 in 86 (58%) of 148 cases of TNBC.

[0008] Challita-Eid et al. (2016), ibid., developed an anti-Nectin-4 antibody conjugated to MMAE, a highly effective microtubule disruptor based on antibody AGS-22. This work led to the ADC candidate enfortumab vedotin (see U.S. Patent No. 8,637,642 and PCT Publication No. WO2012 / 047724, Agensys Inc.), which showed promising results in human clinical trials for patients with locally advanced or metastatic urothelial carcinoma who had previously received platinum-based chemotherapy and PD-1 / PD-L1 checkpoint inhibitors in neoadjuvant / adjuvant, locally advanced, or metastatic settings. Several other groups have also proposed anti-Nectin-4 agents that can be conjugated to a variety of cytotoxic agents. PCT patent application WO2018 / 158398 (INSERM) reports several anti-Nectin-4 antibodies and proposes potential conjugations to a range of cytotoxic agents. Similarly, U.S. Patent No. 8,637,642 (Agensys) also discloses an anti-Nectin-4 antibody and proposes potential conjugation with a range of cytotoxic agents. Furthermore, Bicycle Therapeutics reported the development of an anti-Nectin-4 target comprising a Nectin-4 binding protein conjugated to a cytotoxic aurestatin (MMAE) payload via a cleavable valine-citrulline dipeptide.

[0009] Reportedly, the ADCs being developed for UC include sacituzumabgovitecan (IMMU-132, anti-TROP2) for advanced UC, vetin-entaftuzumab (ASG-22ME, anti-Nectin-4), sirtratumab verdotin (ASG-15ME, anti-SLITRK6) for UC, and oportuzumab monatox (VB4-845, anti-EpCAM) for non-muscle-invasive bladder cancer.

[0010] Unfortunately, while ADCs have shown promising results, many targeted therapies fail to provide adequate and / or durable antitumor responses in patients. For example, although vitin-enterutumab (an anti-Nectin-4 ADC) demonstrated impressive treatment responses in the EV-201 phase 2 study (2019), with an ORR (objective response rate) of 44% and a CR (complete response rate) of 12% in UC, approximately half of the patients discontinued treatment. Most discontinuation was due to disease progression, as assessed by RECIST (48%) or clinical symptoms (5%). Furthermore, 18% of patients who discontinued treatment experienced adverse events, particularly neuropathy. Therefore, Nectin-4-targeted ADCs have limitations, and there is a need in the field for improved benefits for patients with UC and other cancers.

[0011] In urothelial carcinoma (UC), when the cancer has not yet spread to distant parts of the body or when it has already spread, individuals are typically treated first with a cisplatin-based regimen (with or without radiation). Camptothecin compounds are not typically used to treat UC. de Jonge et al., 2004, *Invest New Drugs* 22: 329-333, investigated the effect of the camptothecin analog RFS2000 in patients with advanced or metastatic urothelial carcinoma. De Jonge et al. (2004) concluded that the camptothecin analog had no significant activity in patients with advanced / metastatic urothelial carcinoma who had failed prior chemotherapy, and that the results of this study did not recommend further investigation of RFS2000. Numerous camptothecin analogs have been manufactured over the past few decades, including exatecan. Abou-Alfa et al., 2006 Journal of Clinical Oncology 24(27): 4441-4447 evaluated the efficacy of eczema in treating pancreatic cancer in a phase 3 clinical trial involving 349 patients and found that, apart from gemcitabine, eczema was not more effective than gemcitabine alone. Summary of the Invention

[0012] In one aspect, this disclosure provides a Nectin-4 conjugate conjugated to an eczema molecule, and its use in the treatment of Nectin-4 expressing cancers, particularly UC, breast cancer (e.g., TNBC), non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In another aspect, this disclosure provides a highly efficient linker comprising an intracellularly cleavable dipeptide, a self-eliminating spacer, and a camptothecin analogue. The highly efficient linker can be conjugated to an antibody that binds a tumor antigen. Therefore, antibodies and antibody compositions conjugated to such linkers (e.g., wherein the antibody binds a tumor antigen, optionally, Nectin-4) are also provided. In another aspect, this disclosure provides a treatment method that can be used in individuals with Nectin-4 expressing cancers, regardless of the level of Nectin-4 expression on tumor cells.

[0013] In one respect, this disclosure provides a treatment method that can be advantageously used in individuals whose tumor cells express P-glycoprotein (Pgp).

[0014] In one respect, this disclosure provides a treatment method that can be advantageously used for individuals who have previously received treatment with chemotherapeutic agents (e.g., P-glycoprotein (Pgp) transporters), platinum-based agents (e.g., oxaliplatin, cisplatin, carboplatin, nedaplatin, phenanthreneplatin, pyridineplatin, saxaplatin), or taxanes (e.g., paclitaxel and docetaxel).

[0015] In one aspect, this disclosure provides treatment methods that can be advantageously used for individuals whose tumors or cancers are resistant, i.e., unresponsive to or progressing after treatment with a composition comprising an anti-HER2 antibody (e.g., trastuzumab, an ADC comprising heavy and light variable regions, CDRs, or polypeptide chains of trastuzumab).

[0016] In one aspect, this disclosure provides treatment methods that can be used to mediate antitumor effects in individuals at doses lower than those used with conventional anti-Nectin-4 ADCs, for example, less than 5 mg / kg body weight, less than 3 mg / kg body weight, less than 1.25 mg / kg body weight, less than 1 mg / kg body weight, or a fixed dose of less than 125 mg.

[0017] In one respect, this disclosure provides treatment methods that can be used for individuals suffering from pre-existing neuropathy, diabetes or hyperglycemia, heart failure, or eye disease.

[0018] In one aspect, this disclosure provides a treatment method that can be used in individuals with Nectin-4 expressing cancer, characterized by low or intermediate levels of Nectin-4 peptide expression in tumor cells (e.g., expression of Nectin-4 peptide at the tumor cell membrane).

[0019] One treatment is provided which involves administering a Nectin-4 conjugate conjugated to a camptothecin analog or derivative (e.g., 5- or 6-cyclic camptothecin, eczema, or the SN-38 molecule). Nectin-4 is normally expressed on tumor cells; however, the level of Nectin-4 expression can affect the therapeutic efficacy of anti-Nectin-4 agents such as ADCs. While Nectin-4 expression in tumor cells may be very high in some individuals (e.g., as assessed by immunohistochemistry), others will have disease in which tumor cells express Nectin-4 at low, moderate, or below levels considered high, and these individuals may consequently respond poorly or not at all to agents such as vitin-enterotoxin. The ability to treat individuals with Nectin-4-expressing cancers characterized by non-high levels of Nectin-4 expression on tumor cells (e.g., low or intermediate levels) offers an advantage for use in a broader population, not limited to those with high Nectin-4 expression levels, such as treating subjects characterized by varying levels of Nectin-4 expression on tumor cells (e.g., low or intermediate Nectin-4 subjects and high Nectin-4 subjects). This advantage is of interest to Nectin-4-expressing cancers known to frequently (but not always) exhibit very high tumor Nectin-4 expression (such as UC), and is likely of even greater interest to Nectin-4-expressing cancers in which there are significant differences in Nectin-4 expression levels on tumor cells, such as breast cancer (e.g., TNBC, Her2+ breast cancer), non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In one embodiment, the Nectin-4-expressing cancer is further characterized by the expression of the Her2 peptide (e.g., Her2 overexpression or high expression cancer, Her2 low expression cancer).

[0020] The treatment disclosed herein can, for example, be advantageously used for cancer types known to be characterized by tumors within an individual, wherein tumor cells exhibit high heterogeneity in the levels of tumor antigens (e.g., Nectin-4) on their surface.

[0021] In one aspect, this disclosure provides a method for treating an individual with Nectin-4 expressing cancer, characterized by low or intermediate levels of Nectin-4 expression in tumor cells. The patient may have advanced or refractory cancer, or may have early-stage cancer in which tumor Nectin-4 expression remains low or intermediate. The treatment comprises administering a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or an SN-38 molecule. In one embodiment, the tumor is characterized by low or intermediate Nectin-4 expression as determined by immunohistochemical scoring, for example, an H score not greater than or less than 290, 250, 200, 180, 170, 160, 150, 140, 130, 120, or 100. In one embodiment, the individual has urothelial carcinoma or breast cancer. In one embodiment, the individual has non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In one embodiment, the cancer or tumor is advanced recurrent or metastatic cancer, optionally advanced recurrent or metastatic urothelial carcinoma. In one aspect, this disclosure provides a method for treating a population of individuals with Nectin-4 expressing cancer, wherein the population comprises individuals with cancer or tumors characterized by low or intermediate Nectin-4 expression as determined by immunohistochemical scoring, for example, an H score not greater than or less than 290, 250, 200, 180, 170, 160, 150, 140, 130, 120, or 100.

[0022] In one aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a camptothecin analog via an intracellularly cleavable (e.g., protease-cleavable) oligopeptide (e.g., dipeptide, tripeptide, tetrapeptide, or pentapeptide). In another aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a camptothecin analog via an intracellularly cleavable (e.g., protease-cleavable) dipeptide, tripeptide, tetrapeptide, or pentapeptide and a self-eliminating spacer (e.g., a self-eliminating spacer positioned between the intracellularly cleavable peptide and the camptothecin). In another aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a camptothecin analog via an intracellularly cleavable (e.g., protease-cleavable) dipeptide or tripeptide and a self-eliminating spacer. In yet another aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a camptothecin analog via an intracellularly cleavable (e.g., protease-cleavable) tetrapeptide or pentapeptide and a self-eliminating or non-self-eliminating spacer.

[0023] Optionally, the antibody is functionalized with a linker-toxin of any one of Formulas I to XI.

[0024] On one hand, the camptothecin analogue is eczema or SN-38 molecule.

[0025] In one aspect, this disclosure provides a Nectin-4 binding protein, antibody, or antibody fragment conjugated (e.g., covalently bound) to camptothecin, such as camptothecin analogs, eczema or eczema derivatives, or SN-38 molecules.

[0026] In one aspect, this disclosure provides a method for treating an individual with cancer, the method comprising treating the individual with a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or an SN-38 molecule. In one embodiment, the individual has urothelial carcinoma or breast cancer.

[0027] In one aspect, this disclosure provides a method for treating an individual without prior steps of determining individual suitability for treatment based on tumor cell expression levels of Nectin-4, the method comprising treating the individual with a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or SN-38. In one embodiment, the individual has urothelial carcinoma or breast cancer.

[0028] In one aspect, this disclosure provides a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or SN-38, for the treatment of cancer in an individual population comprising individuals with tumors characterized by high levels of Nectin-4 on tumor cells and individuals with tumors characterized by low levels of Nectin-4 on tumor cells. In one embodiment, the individuals have urothelial carcinoma or breast cancer.

[0029] In one aspect, this disclosure provides a method for treating an individual with cancer (e.g., Nectin-4 expressing cancer), wherein the individual has cancer that is resistant, unresponsive, recurrent, and / or progressive despite treatment with an antibody or antigen-binding molecule conjugated to aurestatin or MMAE molecules (e.g., a Nectin-4 antibody or antigen-binding molecule) (e.g., Vitin-Enfertuzumab), the method comprising treating the individual with a Nectin-4 conjugate conjugated to a camptothecin analog or derivative, such as eczema or an SN-38 molecule. In one embodiment, the individual has urothelial carcinoma, breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In one embodiment, the cancer or tumor is advanced recurrent or metastatic cancer, optionally advanced recurrent or metastatic urothelial carcinoma.

[0030] In one aspect, this disclosure provides a method for treating an individual with cancer (e.g., Nectin-4 expressing cancer), wherein the individual has locally advanced or metastatic urothelial carcinoma and has previously received treatment with an antibody or antigen-binding agent (e.g., Vittin-Entorutumab) conjugated with an auristatin or MMAE molecule, the method comprising treating the individual with a Nectin-4 conjugate conjugated with a camptothecin analog or derivative, such as eczema or an SN-38 molecule.

[0031] In one aspect, this disclosure provides a method for reducing or preventing drug resistance in individuals with cancer (e.g., Nectin-4 expressing cancer), the method comprising treating the individual with a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or SN-38 molecules.

[0032] In any embodiment of this document, the individual may be defined as having resistance, non-response, relapse, and / or progression following prior treatment with an antibody conjugated to an auristatin or MMAE molecule (e.g., Vitin-Enflutumab).

[0033] In any embodiment herein, the individual may be designated as having a HER2-positive tumor or cancer (e.g., Nectin-4 positive, HER2-positive tumor or cancer), optionally a HER2-overexpressing or HER2-high-expressing tumor or cancer, optionally a HER2-low-expressing tumor or cancer. In other embodiments herein, the individual may be designated as having a HER2-negative tumor or cancer. Optionally, the tumor or cancer is triple-negative breast cancer (TNBC).

[0034] In one aspect, this disclosure provides a method for treating an individual with cancer (e.g., Nectin-4 expressing cancer), said cancer being characterized by tumor cells expressing a Her2 polypeptide (e.g., tumor cells expressing both Nectin-4 and Her2 on their surface), said method comprising treating said individual with a Nectin-4 binder conjugated to a camptothecin analog or derivative, such as eczema or an SN-38 molecule (e.g., via an intracellular cleavable linker). In one embodiment, said cancer is characterized by tumor cells overexpressing or expressing high levels of Her2 on their surface. In another embodiment, said cancer is characterized by tumor cells expressing low levels of Her2 on their surface. In one embodiment, the method further includes treating the individual (e.g., administering a combination of the treatment and administration of an antibody binding to a Her2 peptide, such as trastuzumab or pertuzumab); optionally, the Her2-binding antibody is an ADC; optionally, the Her2-binding antibody is conjugated to a cytotoxic agent, optionally olipattin, maytansine (e.g., DM1), or a camptothecin analog or derivative (e.g., eczema or a derivative thereof, SN-38); optionally, the Her2-binding antibody is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a). In one embodiment, the individual has breast cancer. In one embodiment, the individual has gastric cancer. In one embodiment, the individual has colorectal cancer. In one embodiment, the individual has pancreatic cancer. In one embodiment, the individual has bladder cancer. In one embodiment, the individual has head and neck cancer.

[0035] Nectin-4 conjugates, which are conjugated with camptothecin analogues or derivatives, can be advantageously applied 1 to 4 times per month, for example, once every two weeks, once every three weeks, or once every four weeks.

[0036] Nectin-4 conjugates conjugated with camptothecin analogues or derivatives, such as eczema or SN-38 molecules, can be advantageously administered 1-4 times per month at a dose of 0.1-10 or 1-10 mg / kg body weight, for example, once every two weeks, once every three weeks, or once every four weeks.

[0037] In one aspect, this disclosure provides a Nectin-4 binder that is conjugated (e.g., covalently bound) to a camptothecin analog or derivative, such as eczetidine or an eczetidine derivative or an SN-38 molecule.

[0038] In any embodiment herein, a Nectin-4 binder conjugated (e.g., covalently bound) to a camptothecin analog or derivative, such as eczema or SN-38, can be characterized as comprising an antigen-binding protein (e.g., antibody, non-antibody peptide, or protein scaffold) specifically binding to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine, lysine, glutamine residues, or non-natural amino acid residues), said one or more amino acid residues being functionalized by a linker with a molecule comprising a structure of compound 1 or 2, or with a linker-camptothecin molecule of formula I or II. In any embodiment herein, an anti-Nectin-4 antibody or antibody fragment can be characterized as functionalized with a linker-camptothecin molecule having a structure of formula III, IV, V, VI, VII, VII, IX, X, or XI, or functionalized with any one of compounds 3 to 17.

[0039] In one embodiment, the anti-Nectin-4 antibody or antibody fragment conjugated with a camptothecin analog is a Nectin-4 binding antibody or antibody fragment conjugated with an eczema molecule, such as a molecule having the structure of compound 1 (1a or 1b). In one embodiment, the anti-Nectin-4 binding antibody or antibody fragment conjugated with a camptothecin analog is a Nectin-4 binding antibody or antibody fragment conjugated with an SN-38 molecule, such as a molecule having the structure of compound 2. In one embodiment, the Nectin-4 binding antibody or antibody fragment may be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine, lysine, glutamine, or non-natural amino acid residues), said one or more amino acid residues being functionalized by a molecule having the following structure via a linker (e.g., a cleavable linker molecule having or not having an additional spacer, such as the spacer (Y') described herein): Compound 1a or Compound 1b or Compound 2.

[0040] In one embodiment, the Nectin-4 binding protein, antibody, or antibody fragment conjugated to a cytotoxic agent may be designated as an immunoconjugate represented by formula (I): Ab–X–Z formula (I) in, Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to human Nectin-4 polypeptide. X is a linker molecule connecting Ab and Z (e.g., covalently bound to each of Ab and Z), wherein X comprises, for example, a cleavable moiety under physiological conditions, optionally under intracellular conditions, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, optionally wherein X further comprises a self-eliminating or non-self-eliminating spacer subsystem (Y') located between the cleavable moiety and Z, optionally wherein X further comprises a spacer (Y) located between Ab and the cleavable moiety; and Z is a camptothecin analogue, optionally an eczetidine molecule or an SN-38 molecule.

[0041] In one embodiment, this document provides cleavable peptide-containing linkers (e.g., oligopeptide linkers, or linkers containing dipeptides, tripeptides, tetrapeptides, or pentapeptides) and linker-toxin molecules that can be conjugated to antibody or other antigen-binding peptides or proteins, such as antibody- or non-antibody peptide or protein scaffolds that bind to tumor antigens (e.g., Nectin-4 or another suitable tumor antigen), as well as antibodies and antibody compositions conjugated to such linkers, and methods of use thereof in the treatment of cancer. In one embodiment, the dipeptide-containing linker-toxin molecule comprises: a structure (XZ), wherein X is a linker molecule including a reactive group, said reactive group optionally protected to react with a complementary reactive group on an antigen-binding protein; a spacer portion (Y) positioned between said reactive group and said cleavable dipeptide; a cleavable dipeptide selected from valine-citrulline, valine-alanine, or phenylalanine-lysine; a self-eliminating spacer subsystem (Y') positioned between said cleavable dipeptide and Z, and wherein Z is a camptothecin analog, optionally an eczema molecule or an SN-38 molecule. Furthermore, a method is provided for conjugating this linker containing a cleavable peptide to an antigen-binding protein.

[0042] In one embodiment, the antibody conjugated with a camptothecin analogue can be designated as an immunoconjugate represented by formula (I): Ab–X–Z formula (I) in, Ab is an antigen-binding protein (such as an antibody) that specifically binds to human tumor antigens. X is a linker molecule connecting Ab and Z (e.g., covalently bonded to each of Ab and Z), wherein X comprises a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide, wherein X further comprises a self-eliminating or non-self-eliminating spacer subsystem (Y') positioned between the cleavable moiety and Z, and wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety; and Z is a camptothecin analogue, optionally an eczetidine molecule or an SN-38 molecule.

[0043] In one embodiment, a method is provided for delivering or targeting a camptothecin analogue to a tumor or for releasing a camptothecin analogue in a tumor (e.g., in a subject with cancer), the method comprising administering an immunoconjugate represented by formula (I) to a subject with cancer: Ab–X–Z formula (I) in, Ab is an antigen-binding protein (such as an antibody) that specifically binds to human tumor antigens. X is a linker molecule connecting Ab and Z (e.g., covalently bonded to each of Ab and Z), wherein X comprises a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide, wherein X further comprises a self-eliminating or non-self-eliminating spacer subsystem (Y') positioned between the cleavable moiety and Z, and wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety; and Z is a camptothecin analogue, optionally an eczetidine molecule or an SN-38 molecule.

[0044] In one embodiment, the antibody or Nectin-4 conjugate conjugated with a camptothecin analogue can be designated as an immunoconjugate represented by formula (II): Ab–(X–(Z) n ) m Equation (II) in, Ab is an antigen-binding protein (e.g., antibody) that specifically binds to human Nectin-4 peptide or other tumor antigens. X is a linker molecule connecting Ab and Z, wherein X includes, for example, a cleavable moiety under physiological conditions, optionally under intracellular conditions, optionally a dipeptide, tripeptide, tetrapeptide or pentapeptide that is cleavable by a protease, optionally wherein X further includes a self-eliminating or non-self-eliminating spacer subsystem (Y') located between the cleavable moiety and Z, optionally wherein X further includes a spacer (Y) located between Ab and the cleavable moiety. Z is a camptothecin analogue, and optionally Z is a molecule that includes an eczema molecule or an SN-38 molecule, such as a molecule having the structure of compound 1 or 2; n is 1; and m is 4 to 8, or optionally m is an integer selected from 4, 5, 6, 7 or 8.

[0045] In one embodiment, Nectin-4 conjugated with a camptothecin analogue can be characterized as a composition of an immunoconjugate represented by formula (II): Ab–(X–(Z) n ) m Equation (II) in, Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to human Nectin-4 polypeptide. X is a molecule that connects Ab and Z, wherein X includes, for example, a cleavable moiety under physiological conditions, optionally under intracellular conditions, optionally a dipeptide, tripeptide, tetrapeptide or pentapeptide that is cleavable by a protease, optionally wherein X further includes a self-eliminating or non-self-eliminating spacer subsystem (Y') located between the cleavable moiety and Z, optionally wherein X further includes a spacer (Y) located between Ab and the cleavable moiety. Z is a camptothecin analogue, and optionally Z is a molecule including eczema or SN-38 molecules; Where n is 1, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the composition have m (the number of XZ portions) between 2 and 4, between 4 and 8, and optionally between 6 and 8. Optionally, the m of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the composition is, or at least 4, 6, 7, or 8.

[0046] In formula I or II, the (XZ) portion may optionally be characterized as having the structure of any one of formulas III to XI or any one of compounds 3-17.

[0047] In Formula I or II, molecule X or spacer Y may optionally be specified as comprising a reactive group (R) or a residue of the reactive group (R) reacting with an amino acid of an antigen-binding protein (e.g., an antibody) or reacting with a complementary reactive group (R') attached to an amino acid of an antigen-binding protein (e.g., an antibody).

[0048] In any embodiment herein, the eciletcan molecule may be designated to bind to the linker (X) via the amine at position 1 of the eciletcan (NH replaces NH2 at position 1 when the eciletcan molecule is incorporated into the linker). In any embodiment herein, the SN-38 molecule may be designated to bind to the linker (X) via the OH at position 9 (O replaces OH at position 9 when the SN-38 molecule shown in Compound 2 is incorporated into the linker).

[0049] In one embodiment, the Nectin-4 conjugate with eczetidine can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues), said one or more amino acid residues being functionalized via a spacer (Y) with an eczetidine molecule comprising the following structure: .

[0050] In one embodiment, the Nectin-4 conjugate with eczetidine can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues), said one or more amino acid residues being functionalized by a linker-eczetidine comprising a spacer (Y) with the following structure: .

[0051] In one embodiment, the Nectin-4 conjugate with eczetidine can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues), said one or more amino acid residues being functionalized via a spacer (Y) with an eczetidine molecule comprising the following structure: .

[0052] In one embodiment, the Nectin-4 conjugate with eczetidine can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues), said one or more amino acid residues being functionalized via a spacer (Y) with an eczetidine molecule comprising the following structure: .

[0053] The spacer (Y) may be specified as or comprise a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-100 atoms or 2-40 atoms, optionally 2-30, 2-20, 4-40, 4-30, or 4-20 atoms, optionally one or more of which may not be carbon, but may be oxygen, sulfur, nitrogen, or other atoms, optionally any carbon atom of the chain is substituted with an alkoxy group, hydroxyl group, alkyl carbonyl group, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide. For example, Y may comprise one or more ethylene oxide monomers, optionally Y comprises a polyethylene oxide moiety, optionally Y comprises the structure -(CH2CH2O). x - where x is 1 to 12, arbitrarily 1 to 8, and arbitrarily 1 to 6.

[0054] In one aspect, this disclosure provides a treatment that exhibits improved (lower) resistance compared to existing anti-Nectin-4 ADC therapies (e.g., anti-Nectin-4 antibodies conjugated with oliquistatin; vitin-entaftuzumab). In another aspect, a method is provided for treating and / or preventing cancer and / or killing tumor cells in an individual in need, wherein said treatment comprises administering 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Nectin-4 conjugates conjugated with a camptothecin analogue (e.g., eczema or SN-38 molecule) at a frequency of 1 to 2 times per month (e.g., once every two weeks, once every three weeks, or once every four weeks).

[0055] In one aspect, this disclosure provides a method for treating and / or preventing cancer and / or killing tumor cells in an individual in need, or a method for delivering and / or releasing camptothecin molecules in an individual's tumor, the method comprising treating the individual with a Nectin-4 binding protein (e.g., an antibody or antibody fragment) conjugated to a camptothecin molecule such as a camptothecin analogue (e.g., an anti-Nectin-4 antibody or antibody fragment conjugated to one or more camptothecin portions), optionally wherein the camptothecin is eczema or SN-38. In one embodiment, the individual has a Nectin-4 expressing tumor, optionally wherein the tumor is a HER2-expressing tumor or a HER-negative tumor, such as urothelial carcinoma, breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In one embodiment, the cancer or tumor is an advanced recurrent or metastatic cancer, optionally an advanced recurrent or metastatic urothelial carcinoma. In one embodiment, the cancer or tumor is triple-negative breast cancer (TNBC).

[0056] In any aspect of any embodiment herein, the individual has received prior treatment such as radiotherapy, surgery, chemotherapy, and / or treatment with biological agents.

[0057] Compositions of the immunoconjugates disclosed herein are also provided. Pharmaceutically acceptable compositions and kits comprising the immunoconjugates disclosed herein, and one or more additional components (e.g., various carriers) that may generally be active or inactive components that promote the formulation, delivery, stability, or other properties of the composition. Methods for screening, testing, and preparing immunoconjugates and ADCs are also provided.

[0058] These aspects will be described more fully in the description of the invention provided herein, and other aspects, features and advantages will be apparent. Attached Figure Description

[0059] Figure 1The expression levels of HER2 and Nectin-4 peptides on the surface of SUM190 human breast cancer tumor cells are shown, as determined by FACS (MFI: mean fluorescence intensity). SUM190 tumor cells express HER2 at low to moderate levels (median fluorescence unit 1777) and Nectin-4 at low levels (median fluorescence unit 991).

[0060] Figure 2 The expression levels of HER2 and Nectin-4 peptides on the surface of SUM185 human breast cancer tumor cells are shown, as determined by FACS (MFI: mean fluorescence intensity). SUM185 cells expressed HER2 at moderate to high levels (median fluorescence unit 2880) and Nectin-4 at high levels (median fluorescence unit 4326).

[0061] Figure 3 The right-hand image illustrates the efficacy of N4 ADC1 (anti-Nectin-4) in inducing death in HER-2 and Nectin-4 expressing SUM185 and SUM190 tumor cells. The two left-hand images show the efficacy of HER2 ADC1 (anti-Her2) in the same corresponding cells. N4 ADC1 exhibits a 20-fold increase in efficacy compared to HER2 ADC1 in SUM190 cells, and also demonstrates higher comparative efficacy even in SUM185 cells, which are characterized by lower surface Nectin-4 expression (approximately 1 / 4 the surface Nectin-4 in SUM185 compared to SUM190).

[0062] Figure 4 The internalization of human SUM185 breast cancer cells against the antibodies entorumab (mAbA) and N41 mAbC, as well as the isotype control antibody (IC), is shown. The luminescence on the Y-axis (indicating cell viability) is compared with the concentration of anti-Nectin-4 antibody on the X-axis.

[0063] Figure 5 The killing effect of the anti-Ig-like V domain antibody entferotumab (mAbA) and the isotype control (IC) on human SUM185 breast cancer cells is shown. Cell viability on the Y-axis is compared with the concentration of anti-Nectin-4 antibody on the X-axis. The antibody and the isotype control are conjugated to eczema via a linker (VC-eczema) having a first spacer, a Val-Cit cleavable portion and a PAB self-eliminating spacer.

[0064] Figure 6The killing effect of the anti-Ig-like V domain antibody entorumab (mAbA) and the isotype control (IC) on human SUM190 breast cancer cells was shown. Cell viability on the Y-axis was compared with the concentration of anti-Nectin-4 antibody on the X-axis. The antibody and the isotype control were conjugated to ecilecan via the VC-ecilecan linker or to camptothecin DxD via the GGFG cleavable linker (GGFG-DxD).

[0065] Figure 7 The killing effect of the anti-Ig-like V domain antibody entferotumab (mAbA) and the isotype control (IC) on human SUM190 breast cancer cells is shown. Cell viability on the Y-axis is compared with the concentration of anti-Nectin-4 antibody on the X-axis. The antibody and the isotype control are conjugated to ecilecan via a VC-ecilecan linker, either through a linker having a first spacer, a Val-Ala cleavable portion and a PAB self-eliminating spacer (VC-ecilecan), or through a VC-ecilecan linker in which the first spacer includes a PEG8 portion (8 PEG units).

[0066] Figure 8 The in vivo efficacy of a single 5 mg / kg camptothecin ADC based on entroxytocin (mAbA) is demonstrated, wherein the entroxytocin is conjugated to a GGFG-DxD linker, a VC-ecinotecan linker, a VA-ecinotecan linker, or a PEG8-VA-ecinotecan linker at an equivalent drug-to-antibody ratio (DAR=8).

[0067] Figure 9 The in vivo efficacy of a single 5 mg / kg camptothecin ADC against two different anti-Nectin-4 antibodies was demonstrated. Enfretumab (mAbA) or an alternative anti-Nectin-4 specific antibody (mAbB) was conjugated to the PEG8-VA-ecithecan linker at an equivalent drug-to-antibody ratio (DAR=8). Enfretumab (mAbA) conjugated to the GGFG-DxD linker and an isotype control were also tested for comparison. Detailed Implementation

[0068] definition As used in the specification, "a" or "an" may mean one or more. As used in one or more claims, when combined with the word "comprising," the word "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more.

[0069] When using “includes”, this can optionally be replaced by “consistent with” or “comprises with”.

[0070] “Nectin-4” and “Nectin-4 polypeptide” refer to proteins or polypeptides encoded by the NECTIN4 gene (see Uniprot accession number Q96NY8) or cDNA prepared from such genes. Any naturally occurring isotype, allele, or variant is encompassed under the term Nectin-4 polypeptide (e.g., Nectin-4 polypeptide having 95%, 98%, or 99% identity with SEQ ID NO: 1 or with a sequential sequence of at least 100, 200, 300, 400, or 500 amino acid residues). A typical human Nectin-4 (isomer 1) sequence of 510 amino acid residues contains a 31-amino acid signal peptide, as shown below: MPLSLGAEMW GPEAWLLLLL LLASFTGRCP AGELETSDVV TVVLGQDAKL PCFYRGDSGE QVGQVAWARV DAGEGAQELA LLHSKYGLHV SPAYEGRVEQ PPPPRNPLDG SVLLRNAVQA DEGEYECRVS TFPAGSFQAR LRLRVLVPPL PSLNPGPALE EGQGLTLAAS CTAEGSPAPS VTWDTEVKGT TSSRSFKHSR SAAVTSEFHL VPSRSMNGQP LTCVVSHPGL LQDQRITHIL HVSFLAEASV RGLEDQNLWH IGREGAMLKC LSEGQPPPSY NWTRLDGPLP SGVRVDGDTL GFPPLTTEHS GIYVCHVSNE FSSRDSQVTV DVLDPQEDSG KQVDLVSASV VVVGVIAALL FCLLVVVVVL MSRYHRRKAQ QMTQKYEEEL TLTRENSIRR LHSHHTDPRS QPEESVGLRA EGHPDSLKDN SSCSVMSEEP EGRSYSTLTT VREIETQTEL LSPGSGRAEE EEDQDEGIKQ AMNHFVQENG TLRAKPTGNG IYINGRGHLV (SEQ ID NO: 1).

[0071] SEQ ID NO: 1 corresponds to UniProt KB identifier Q96NY8-1, the contents of which are incorporated herein by reference.

[0072] Certain aspects of this disclosure provide anti-Nectin-4 antibodies that bind to human Nectin-4 or its homologs, said homologs including, but not limited to, mammalian Nectin-4 protein and orthologs of Nectin-4 from other species, such as non-human primates, cynomolgus monkeys.

[0073] The term "HER2" (also known as HER2 / neu and ErbB-2) stands for "human epidermal growth factor receptor 2". It includes variants and isotypes of HER2.

[0074] The term "tumor antigen," used interchangeably with "cancer antigen," refers to antigens differentially expressed by cancer cells, or antigens expressed by non-tumor cells (e.g., immune cells) in tumors or adjacent tissues, which have pro-tumorigenic effects (e.g., immunosuppressive effects) and can therefore be used to target cancer. Tumor antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, although they are not necessarily expressed by said normal cells, or are expressed at low levels or frequencies. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens expressed only at certain stages of differentiation, and temporarily expressed antigens, such as embryonic and fetal antigens. Other tumor antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogenes), repressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Other tumor antigens can also be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Other tumor antigens can also be expressed on immune cells that contribute to or mediate pro-tumorigenic effects, such as cells that facilitate immune evasion, monocytes, or macrophages, optionally suppressor T cells, regulatory T cells, or myeloid-derived suppressor cells. Tumor antigens are typically normal cell surface antigens that are either overexpressed, expressed at abnormal times, or expressed by the target cell population. Ideally, target antigens are expressed only on proliferating cells (e.g., tumor cells) or pro-tumorigenic cells present in the tumor or adjacent tissues (e.g., immunosuppressive immune cells), but this is rarely observed in practice. As a result, in many cases, target antigens are selected based on differential expression between proliferating / disease-prone tissues and healthy tissues. Examples of tumor antigens include: Nectin-4, receptor tyrosine kinase-like orphan receptor 1 (ROR1), Crypto, CD4, CD20, CD30, CD19, CD38, CD47, glycoprotein NMB, CanAg, Siglec family members such as CD22 (Siglec2) or CD33 (Siglec3), CD79, CD123, CD138, CD171, PSCA, L1-CAM, PSMA (prostate-specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, black transferrin, Mud 6, and TMEFF2. Examples of tumor antigens also include the immunoglobulin superfamily (IgSF), such as cytokine receptors, cytokine Ig-like receptors, and CD28 family proteins, such as cytokine Ig-like receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1, and IL-6 receptors.Examples also include MAGE, MART-1 / Melan-A, gp100, major histocompatibility complex class I related chain A and B peptides (MICA and MICB), or optionally antigens other than MICA and / or MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, protein tyrosine kinase 7 (PTK7), receptor protein tyrosine kinase 3 (TYRO-3), conjugates (e.g., nectin-4), UL16-binding protein (ULBP) family, retinoic acid early transcript-1 (RAET1) family proteins, prostate-specific antigen (PSA), B cell maturation antigen (BCMA), anti-Müllerian hormone type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as receptor tyrosine kinase-like orphan receptor 1 or NTRKR1 (EC 2.7.10.1), TROP2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptor, angiopoietin-2, PDGF, TGF-α, EGF, EGF receptor, members of the human EGF-like receptor family, such as HER-2, HER-3, HER-4 or heterodimer receptors including at least one HER subunit, gastrin-releasing peptide receptor antigen, Muc-1, CA125, integrin receptor, αvβ3 integrin, α5β1 integrin, αIIbβ3-integrin, PDGF β-receptor, SVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, CSF1R (tumor-associated monocytes and macrophages), α-fetoprotein, E-cadherin, α-catenin, β-catenin, p120ctn, PRAME, NY-ESO-1, gp75, GM2, and GD2 gangliosides, although this is not exhaustive. On the one hand, antigens of interest are those capable of undergoing intracellular internalization (such as any of the antigens listed above), for example, when bound by human antibodies.

[0075] The term "immunoconjugate" refers to an antigen-binding agent (e.g., an antibody-binding peptide or antibody) conjugated to another molecule (e.g., a camptothecin analog, eczemab molecule, or SN-38 molecule). When an immunoconjugate includes an antibody conjugated to a therapeutic agent (e.g., a camptothecin analog, eczemab molecule, or SN-38 molecule), the immunoconjugate may also be referred to as an "antibody-drug conjugate" or "ADC".

[0076] As used herein, “treatment (and treating, etc.)” generally means achieving a desired pharmacological and physiological effect. The effect may be preventative in relation to the prevention or partial prevention of a disease, its symptoms, or condition, and / or therapeutic in relation to the partial or complete cure of the disease, condition, symptom, or side effects attributable to the disease. As used herein, the term “treatment” covers any treatment of a disease in mammals (specifically, humans) and includes: (a) preventing the disease from occurring in subjects who may be susceptible to the disease but have not yet been diagnosed with it, such as preventative early asymptomatic intervention; (b) suppressing the disease, such as halting its development; or alleviating the disease, such as causing the resolution of the disease and / or its symptoms or condition, such as improvement or remedy of damage, for example, in subjects who have been diagnosed with the disease. Optionally, treatment may cause (e.g., may be characterized as a method of causing) a reduction in tumor burden, a reduction in the size and / or number of lesions, a reduction or delay in cancer progression (e.g., an increase in progression-free survival), a delay or prevention of cancer metastasis, and / or an increase in survival. Optionally, the treatment may induce or provide (e.g., may be characterized as a method of inducing or providing) a stable disease, partial response, or complete response in the subject, for example, according to standardized criteria, optionally the RECIST criteria.

[0077] Whenever Nectin-4 binding agents (such as antibodies or antibody fragments) are mentioned in the context of "cancer treatment," it includes: (a) A method of treating cancer, the method comprising the steps of administering a Nectin-4 binder (for at least one treatment) to an individual, mammal, particularly a human, in need of such treatment at a dose (therapeutic effective amount) that allows for the treatment of cancer (therapeutic effective amount). (b) Use of Nectin-4 binders in the treatment of cancer; (c) Nectin-4 binders, which are used to treat cancer (especially in humans); (d) Use of Nectin-4 binders in the preparation of pharmaceutical formulations for the treatment of cancer; (e) A method for preparing a pharmaceutical formulation for treating cancer using a Nectin-4 binder, the method comprising mixing the Nectin-4 binder with a pharmaceutically acceptable carrier; (f) A pharmaceutical preparation comprising an effective dose of a Nectin-4 binder suitable for treating cancer; (g) Any combination of (a), (b), (c), (d), (e) and (f) conforms to the subject matter that is patentable in the country where this application is filed.

[0078] As used herein, the term "biopsy" is defined as the removal of tissue for examination purposes, such as to establish a diagnosis. Examples of biopsy types include removal by aspiration, such as by a needle attached to a syringe; removal of tissue fragments by instruments; removal by endoscopy with appropriate instruments; surgical excision, such as the entire lesion; and so on.

[0079] As used herein, the term "antibody" refers to both polyclonal and monoclonal antibodies. Antibodies are designated as one of five major classes based on the type of constant domain in the heavy chain: IgA, IgD, IgE, IgG, and IgM. Several of these are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50–70 kDa). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term variable light chain (V... L ) and variable heavy chain (V H The terms "α", "δ", "ε", "γ", and "μ" refer to these light and heavy chains, respectively. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are referred to as "α", "δ", "ε", "γ", and "μ", respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. IgG is the exemplary antibody class used herein because it is the most common antibody in physiological conditions and because it is the easiest to prepare in a laboratory setting. Optionally, an antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human, or other suitable human antibodies. "Antibody" includes full-length antibodies as well as any fragment or derivative of any antibody described herein.

[0080] The amino acid residues in an antibody responsible for antigen binding can also be called hypervariable regions. Hypervariable regions typically include amino acid residues from the complementarity-determining region or the CDR (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and / or residues from the hypervariable ring (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, Journal of Molecular Biology 1987; 196:901-917), or similar systems used to determine the essential amino acids responsible for antigen binding. Typically, the amino acid residues in this region are numbered using Kabat et al., as described above. Phrases such as “Kabat position,” “variable domain residue numbering as in Kabat,” and “according to Kabat” refer herein to this numbering system used for heavy chain or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to a shortening or insertion of the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion following residue 52 of CDR H2 (residue 52a according to Kabat) and inserted residues following heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat residue numbering of a given antibody can be determined by comparing the homologous region of the antibody sequence with a “standard” Kabat numbered sequence.

[0081] The term "specific binding" means that an antibody can preferably bind to a binding partner, such as Nectin-4, in a competitive binding assay, as assessed using a protein, a recombinant form of an epitope therein, or a native protein present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected by radioactive labeling, physical methods (such as mass spectrometry), or direct or indirect fluorescent labeling using methods such as cellular fluorescence analysis (e.g., FACScan). A binding amount higher than that seen with a control nonspecific agent indicates that the agent binds to the target.

[0082] When an antibody is described as “competing” with a specific monoclonal antibody, it means that the antibody uses a recombinant molecule (e.g., Nectin-4) or a surface-expressed molecule (e.g., Nectin-4) in a binding assay to compete with the monoclonal antibody. For example, if an antibody is tested in a binding assay to reduce the binding of an antibody having a heavy chain variable region of any one of SEQ ID NO: 3, 7, or 9 and a corresponding light chain variable region of SEQ ID NO: 4, 8, or 10 to a Nectin-4 peptide or Nectin-4-expressing cells, then the antibody is described as “competing” with such an antibody, respectively.

[0083] The term "internalization," used interchangeably with "intracellular internalization," refers to the molecular, biochemical, and cellular events associated with the process of transferring molecules from the extracellular surface of a cell to its intracellular surface. The processes responsible for intracellular molecular internalization are well-known and particularly likely involve the internalization of: extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-associated molecules (such as cell surface receptors); and complexes of membrane-associated molecules that bind to extracellular molecules (e.g., ligands binding to transmembrane receptors or antibodies binding to membrane-associated molecules). Therefore, "inducing and / or increasing internalization" includes events that trigger and / or increase the rate and / or extent of intracellular internalization.

[0084] As used herein, the term "affinity" refers to the strength of antibody-epitaxygesis. Antibody affinity is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. Affinity constant K aDefined by 1 / Kd. Methods for determining the affinity of monoclonal antibodies can be found in the following references: Harlow et al., *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Coligan et al. (eds.), *Current Protocols in Immunology*, Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993); and Muller, *Meth. Enzymol.* 92:589-601 (1983), all of which are incorporated herein by reference in their entirety. A standard method well-known in the art for determining the affinity of monoclonal antibodies is the use of surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore™ SPR analyzer).

[0085] In the context of this article, "determinant cluster" refers to a site that interacts with or binds to a polypeptide.

[0086] The term "epitope" refers to an antigenic determinant and is a region or area on an antigen that an antibody binds to. Protein epitopes can include amino acid residues that directly participate in binding, as well as amino acid residues that are effectively blocked by specific antigen-binding antibodies or peptides, i.e., amino acid residues within the antibody's "footprint." It is the simplest form or smallest structural region on a complex antigen molecule that can combine with, for example, an antibody or receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope consisting of consecutive amino acid residues in a linear sequence (primary structure). The term "conformational or structural epitope" is defined as an epitope consisting of incompletely consecutive amino acid residues, and therefore represents separated portions of the linear sequence of amino acids that are close to each other through folding of the molecule (secondary, tertiary, and / or quaternary structures). Conformational epitopes depend on the 3D structure. Therefore, the terms 'conformation' and 'structure' are often used interchangeably.

[0087] The term "pharmaceutical" is used herein to refer to a mixture of chemical compounds, biological macromolecules, or extracts made from biological materials. The term "therapeutic agent" refers to a pharmaceutical agent that is biologically active.

[0088] The terms “Fc domain,” “Fc moiety,” and “Fc region” refer to the C-terminal segment of an antibody heavy chain, such as approximately amino acids (aa) 230 to approximately aa 450 from the human γ (γ) heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or its naturally occurring allotypes. Unless otherwise stated, the generally accepted Kabat amino acid numbers for immunoglobulins are used throughout this disclosure (see Kabat et al. (1991), Sequences of Protein of Immunological Interest), 5th edition, United States Public Health Service, National Institute of Health, Bethesda, MD).

[0089] As used herein, “framework” or “FR” residues refer to regions of the antibody variable domain other than those defined as CDRs. Each antibody intermediate variable domain frame can be further subdivided into consecutive regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0090] The terms "isolated," "purified," or "biologically pure" refer to materials that are substantially or essentially free of the components typically found accompanying them in their natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. Proteins, as the main species present in the formulation, are substantially purified.

[0091] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The terminology applies to amino acid polymers, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, wherein one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids.

[0092] When used in relation to, for example, cells or nucleic acids, proteins, or vectors, the term "recombinant" means that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the cell is derived from such a modified cell. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0093] In the context of this article, the term “binding” antibody to a polypeptide or epitope means an antibody that binds to the determinant cluster with specificity and / or affinity.

[0094] When used in relation to the sequences of two or more polypeptides, the term "identity" or "identity" refers to the degree of sequence correlation between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are resolved by a specific mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated using known methods. Such methods include, but are not limited to, those described in the following literature: *Computational Molecular Biology*, Lesk, AM (ed.), Oxford University Press, New York, 1988; *Biocomputing: Informatics and Genome Projects*, Smith, DW (ed.), Academic Press, New York, 1993; *Computer Analysis of Sequence Data*, Part 1, Griffin, AM and Griffin, HG (ed.), Humana Press, New Jersey, 1994; *Sequence Analysis in Molecular Biology*, von Heinje, G., Academic Press, 1987; *Sequence Analysis Primer*, Gribskov, M. and Devereux, J. (ed.), M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0095] Methods for determining identity are designed to give the maximum match between test sequences. Methods for determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG package, which includes GAP (Devereux et al., Nucleic Acid Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX procedure is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, Md. 20894; Altschul et al., ibid.). The well-known Smith-Waterman algorithm can also be used to determine consistency.

[0096] As used herein, “alkyl” refers to a straight-chain or branched hydrocarbon chain comprising fully saturated (without double or triple bonds) hydrocarbon groups. An alkyl group can have, for example, 1 to 20 carbon atoms (wherever it appears herein, numerical ranges such as “1 to 20” refer to every integer within a given range; for example, “1 to 20 carbon atoms” means that an alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also covers the occurrence of the term “alkyl” where no numerical range is specified). The alkyl group of a compound can be named “C1-C4 alkyl” or similar names. By way of example only, “C1-C4 alkyl” indicates the presence of one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. Alkyl groups can be substituted or unsubstituted.

[0097] As used herein, the term "heteroalkyl" refers to a straight-chain or branched alkyl group containing one or more heteroatoms, i.e., an element other than carbon (including but not limited to oxygen, sulfur, nitrogen, and phosphorus) replacing one or more carbon atoms.

[0098] Whenever a group is described as “substituted,” the group is substituted by one or more specified substituents. If no substituent is specified, it means that the specified “substituted” group may be substituted individually and independently by one or more groups selected from the following: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heterocycloalkyl, aralkyl, heteroarylalkyl, (heterocycloalkyl)alkyl, hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamoyl, thiocarbamoyl, amide, sulfonamide, sulfonamide, carboxyl, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, sulfinyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, monosubstituted amino and disubstituted amino, and their protected derivatives.

[0099] When the number of substituents (e.g., haloalkyl) is not specified, one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may contain one or more of the same or different alkoxy groups containing one, two, or three atoms.

[0100] Unless the context clearly indicates otherwise, references to a "compound" or "formula" with a specific number (e.g., "compound 1", "compound 2", "formula I" or "formula II") indicate all compounds derived from the compound or formula with that specific number. Compound 1, for example, includes references to compounds 1a and 1b.

[0101] Nectin-4 binder The Nectin-4 binding domain used to prepare the Nectin-4 binding ADC compositions of this disclosure can be readily derived from any of a variety of immunoglobulin or non-immunoglobulin scaffolds, such as affinity variants based on the Z domain of staphylococcal protein A, engineered Kunitz domains, monomers or adnectin based on the 10th extracellular domain of human fibronectin III, anticarrier proteins derived from lipid carriers, DARPins (designed ankyrin repeat domains, polymerized LDLR-A modules, high-affinity polymers, or... Cysteine-rich desmin peptides. Hypervariable regions, heavy and light chain CDRs, heavy and light chain variable regions, and antibodies including them (e.g., full-length antibodies or antibody fragments) bind to human Nectin-4 expressed on the surface of cells (e.g., tumor cells). When used in therapies to eliminate Nectin-4-expressing tumor cells, the Nectin-4 binder, when conjugated to a cytotoxic molecule disclosed herein, will be able to induce death of Nectin-4-expressing tumor cells, as determined in assays in which the Nectin-4-binding ADC comes into contact with tumor cells in the absence of immune effectors and / or cells other than tumor cells.

[0102] In one embodiment, an anti-Nectin-4 antigen-binding protein or antibody binds to a mature Nectin-4 polypeptide (e.g., a polypeptide having the amino acid sequence of residues 32-510 of SEQ ID NO: 1).

[0103] In one embodiment, an anti-Nectin-4 antigen-binding protein or antibody binds to the Ig-like V-type domain of the Nectin-4 polypeptide. For example, the antigen-binding protein or antibody may be characterized as being able to bind to a domain of Nectin-4 having the amino acid sequence of residues 32-144 of SEQ ID NO: 1 (also shown as SEQ ID NO: 2 below) (or bind to an epitope within or at least partially within said domain).

[0104] geletsdvvtvvlgqdaklpcfyrgdsgeqvgqvawarvdagegaqelallhskyglhv spayegrveqpppprnpldgsvllrnavqadegeeyecrvstfpagsfqarlrlr (SEQ ID NO: 2) In one embodiment, the antigen-binding protein or antibody includes a hypervariable region (e.g., heavy and light chain CDR1, 2, and 3, according to Kabat numbering) of any known anti-Nectin-4 antibody. In one embodiment, the antigen-binding protein or antibody competitively binds to the Nectin-4 polypeptide with any one or more known anti-Nectin-4 antibodies (such as antibodies ASG-22ME, 14A5.2, or N41). In one embodiment, the antigen-binding protein or antibody recognizes an epitope or "epitope site" on the Nectin-4 polypeptide that is substantially or substantially the same as, binds to, or has immune specificity to, any of the antibodies ASG-22ME, 14A5.2, or N41.

[0105] In some embodiments, an anti-Nectin-4 antibody may be selected to exhibit significantly lower binding to mutant human Nectin-4 peptide compared to wild-type Nectin-4 peptide, wherein one, two, three, four or more residues within the binding site or epitope on Nectin-4 of any of the antibodies ASG-22ME, 14A5.2 or N41 are substituted with different amino acids.

[0106] In some embodiments, anti-Nectin-4 antibodies may be characterized as exhibiting little or no binding to mutant human Nectin-4 peptides lacking an Ig-like V-domain (e.g., the domain is missing) compared to wild-type Nectin-4 peptides. In some embodiments, anti-Nectin-4 antibodies may be selected to exhibit significantly lower binding to mutant human Nectin-4 peptides compared to wild-type Nectin-4 peptides, wherein one, two, three, four, or more residues within the Ig-like V-domain are substituted with different amino acids.

[0107] In some embodiments, anti-Nectin-4 antibodies may be selected or characterized to exhibit significantly lower binding to mutant human Nectin-4 peptides compared to wild-type Nectin-4 peptides, wherein the Ig-like V-domain (or a portion thereof) is absent or substituted by amino acid sequences from different peptides or different domains (e.g., non-human or non-primate Nectin-4 peptides, non-Nectin-4 peptides, non-Ig-like V-domains).

[0108] The binding of anti-Nectin-4 antibodies to cells transfected with a Nectin-4 mutant can be measured and compared to the ability of anti-Nectin-4 agents to bind to wild-type Nectin-4 peptides (e.g., SEQ ID NO: 1). Decreased binding between anti-Nectin-4 agents and mutant Nectin-4 peptides implies reduced binding affinity (e.g., as measured by known methods, such as FACS assays of cells expressing the specific mutant, or by a Biacore assay for binding to the mutant peptide) and / or reduced overall binding capacity of the anti-Nectin-4 agent (e.g., as demonstrated by a decrease in Bmax in a plot comparing anti-Nectin-4 agent concentration to peptide concentration). A significant decrease in binding indicates that, when the anti-Nectin-4 agent binds to Nectin-4, the mutated residues are directly involved in the binding to the anti-Nectin-4 agent or are very close to the binding protein.

[0109] In some embodiments, a significant reduction in binding means a decrease in binding affinity and / or capacity between the anti-Nectin-4 antibody and the mutant Nectin-4 peptide greater than 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% relative to the binding between the antibody and the wild-type Nectin-4 peptide. In some embodiments, the binding is reduced to below the detectable limit. In some embodiments, a significant reduction in binding is demonstrated when the binding of the anti-Nectin-4 antibody to the mutant Nectin-4 peptide is less than 50% of the binding observed between the anti-Nectin-4 antibody and the wild-type Nectin-4 peptide (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%).

[0110] In some embodiments, the anti-Nectin-4 antibody exhibits significantly lower binding to the mutant Nectin-4 peptide (e.g., showing loss of binding), wherein residues in the region corresponding to residues 32-144 (or a subsequence thereof, optionally at least 4, 5, 6, 10, 20, or 40 residues) of the wild-type Nectin-4 peptide (e.g., including the sequence of SEQ ID NO: 1) are missing (e.g., deleted or substituted with different amino acids).

[0111] Antibodies can be produced using a variety of techniques known in the art. Typically, they are produced by immunizing a non-human animal (preferably a mouse) with an immunogen comprising a Nectin-4 polypeptide (preferably a human Nectin-4 polypeptide). The Nectin-4 polypeptide may comprise the full-length sequence of the human Nectin-4 polypeptide or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of a polypeptide comprising an epitope (e.g., an epitope recognized by an ASG-22ME, 14A5.2, or N41 antibody) exposed on the surface of a cell expressing the Nectin-4 polypeptide. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, and more preferably at least about 10 consecutive amino acids. The fragment is typically substantially derived from the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human Nectin-4 polypeptide in a lipid membrane, typically located on the cell surface. In a specific embodiment, the immunogen comprises intact cells, particularly intact human cells, optionally treated or lysed. In another preferred embodiment, the polypeptide is a recombinant Nectin-4 polypeptide. In a specific embodiment, the immunogen comprises intact Nectin-4 expressing cells.

[0112] The step of immunizing a non-human mammal with an antigen can be performed in any manner known in the art to stimulate antibody production in mice (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), the entire contents of which are incorporated herein by reference).

[0113] Antibodies can also be generated by selecting a combination library of immunoglobulins, as disclosed, for example, in (Ward et al., Nature, 341 (1989), p. 544, the full contents of which are incorporated herein by reference).

[0114] Using any of a variety of immune screening assays that can assess antibody competition, the identification of one or more antibodies that compete with monoclonal antibodies ASG-22ME, 14A5.2, or N41 for binding to Nectin-4 can be readily determined. Many of these assays are routine practice and are well known in the art (see, for example, U.S. Patent No. 5,660,827, which is incorporated herein by reference).

[0115] For example, when the test antibodies to be examined are obtained from different animal sources, or even different Ig isotypes, a simple competitive assay can be used, in which a control (e.g., ASG-22ME, 14A5.2, or N41) and the test antibody are mixed (or pre-adsorbed) and applied to a sample containing the Nectin-4 peptide. Protocols based on Western blotting and using surface plasmon resonance (e.g., Biacore™) analysis are suitable for such competitive studies.

[0116] In some embodiments, a control antibody (e.g., ASG-22ME, 14A5.2, or N41) is premixed with varying amounts of the test antibody (e.g., approximately 1:10 or approximately 1:100) for a period of time before application to a Nectin-4 antigen sample. In other embodiments, the control and varying amounts of the test antibody can be simply mixed during exposure to the Nectin-4 antigen sample. Whether the test antibody reduces the binding of ASG-22ME, 14A5.2, or N41 to the antigen can be determined as long as the bound antibody can be distinguished from the free antibody (e.g., by using separation or washing techniques to remove unbound antibodies) and ASG-22ME, 14A5.2, or N41 can be distinguished from the test antibody (e.g., by using species-specific or isotype-specific secondary antibodies or by specifically labeling ASG-22ME, 14A5.2, or N41 with a detectable label). In the absence of completely unrelated antibodies, the binding of the (labeled) control antibody can serve as a high control value. Low control values ​​can be obtained by incubating a labeled antibody (ASG-22ME, 14A5.2, or N41) together with an unlabeled antibody of the exact same type (ASG-22ME, 14A5.2, or N41), where competition occurs and reduces the binding of the labeled antibody. The test antibody can, for example, reduce the binding of ASG-22ME, 14A5.2, or N41 to the Nectin-4 antigen by at least about 50%, such as at least about 60%, or more preferably at least about 80% or 90% (e.g., about 65-100%), wherein any ratio of ASG-22ME, 14A5.2, or N41 to the test antibody is between about 1:10 and about 1:100. Optionally, such test antibodies reduce the binding of ASG-22ME, 14A5.2, or N41 to the Nectin-4 antigen by at least about 90% (e.g., about 95%).

[0117] Competition can also be assessed using, for example, flow cytometry assays. In such assays, cells carrying a given Nectin-4 peptide can be incubated first with, for example, ASG-22ME, 14A5.2, or N41, and then with a test antibody labeled with a fluorescent dye or biotin. If the binding obtained when pre-incubated with a saturated amount of ASG-22ME, 14A5.2, or N41 is about 80%, preferably about 50%, about 40%, or lower (e.g., about 30%, 20%, or 10%), of the binding obtained with an antibody not pre-incubated with ASG-22ME, 14A5.2, or N41, as measured by the average fluorescence, then the antibody is said to compete with ASG-22ME, 14A5.2, or N41. Alternatively, if the binding obtained with the labeled ASG-22ME, 14A5.2, or N41 antibody (by fluorescent dye or biotin) on cells pre-incubated with a saturated amount of the test antibody is about 80%, preferably about 50%, about 40%, or less (e.g., about 30%, 20%, or 10%), the antibody is said to compete with ASG-22ME, 14A5.2, or N41.

[0118] A simple competitive assay can also be used, in which the test antibody is pre-adsorbed and applied at a saturation concentration to a surface on which Nectin-4 antigen is immobilized. The surface in the simple competitive assay is preferably a Biacore™ chip (or other media suitable for surface plasmon resonance analysis). A control antibody (e.g., ASG-22ME, 14A5.2, or N41) is then contacted with the surface at a saturation concentration of Nectin-4, and the binding of the control antibody to Nectin-4 and the surface is measured. This binding of the control antibody is compared to the binding of the control antibody to a surface containing Nectin-4 in the absence of the test antibody. In the test assay, the binding of the control antibody to a surface containing Nectin-4 is significantly reduced in the presence of the test antibody, indicating that the test antibody recognizes substantially the same region on Nectin-4 as the control antibody, resulting in a “cross-reaction” between the test antibody and the control antibody. The test antibody can, for example, reduce the binding of the control antibody (such as ASG-22ME, 14A5.2, or N41) to the Nectin-4 antigen by at least about 30% or more, preferably about 40%. Optionally, such test antibodies will reduce the binding of control antibodies (e.g., ASG-22ME, 14A5.2, or N41) to the Nectin-4 antigen by at least about 50% (e.g., at least about 60%, at least about 70%, or more). It should be understood that the order of the control and test antibodies can be reversed: that is, the control antibody can be bound to the surface first, and then the test antibody can be contacted with the surface in a competitive assay. Preferably, the antibody with a higher affinity for the Nectin-4 antigen binds to the surface first, as a larger order of magnitude of the reduction in binding seen with the second antibody is expected (assuming antibody cross-reactivity). Further examples of such assays are provided in, for example, Saunal (1995) *Journal of Immunol. Methods* 183: 33-41, the disclosure of which is incorporated herein by reference.

[0119] The antibody will bind to Nectin-4-expressing tumor cells from one or more individuals with cancer characterized by Nectin-4-positive tumor cells (i.e., candidate individuals treated with an anti-Nectin-4 antibody via one of the methods described herein). Therefore, once an antibody that specifically recognizes Nectin-4 on cells is obtained, its ability to bind to Nectin-4-positive cells (e.g., cancer cells) can be optionally tested. Its ability to bind to tumor cells expressing a high level of Nectin-4 peptide on their surface and / or tumor cells expressing a low level of Nectin-4 peptide on their surface can be optionally tested. Specifically, prior to treating a patient with one of the antibodies of the present invention, the ability of the antibody to bind to malignant cells taken from the patient (e.g., in a blood sample or tumor biopsy) can be optionally tested to maximize the likelihood that the therapy will benefit the patient.

[0120] In one embodiment, antibodies are validated in an immunoassay to test their ability to bind to Nectin-4-expressing cells (e.g., malignant cells). For example, a blood sample or tumor biopsy is performed, and tumor cells are collected. The ability of a given antibody to bind to cells is then assessed using standard methods well known to those skilled in the art. To assess antibody- / cell binding, antibodies can be labeled directly or indirectly. When indirectly labeled, a secondary labeled antibody is typically added.

[0121] Determining whether an antibody binds within an epitope region can be done in a manner known to those skilled in the art. As an example of such mapping / characterization methods, the epitope region of an anti-Nectin-4 antibody can be determined using epitope “foot-printing”, which involves the chemical modification of exposed amine / carboxyl groups in the Nectin-4 protein. A specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen / deuterium exchange, binding, and reverse exchange occur between the amide protons of the receptor and ligand proteins, where the backbone amide group involved in protein binding is protected from reverse exchange and thus remains deuterated. The relevant region can then be identified by proteolytic digestion, rapid microporous high-performance liquid chromatography, and / or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267(2), pp. 252-259, (1999); Engen, JR and Smith, DL (2001), Analytical Chemistry, 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), in which the positions of signals in the two-dimensional NMR spectra of a free antigen and an antigen complexed with an antigen-binding peptide (such as an antibody) are typically compared. Antigens are usually selectively labeled with a 15N isotope such that only the signal corresponding to the antigen is seen in the NMR spectrum, and no signal from the antigen-binding peptide is observed. Compared to the spectrum of the free antigen, the antigen signal derived from the amino acid involved in the interaction with the antigen-binding peptide will typically be shifted in the spectrum of the complex, and the amino acid involved in the binding can be identified in this manner. See, for example, Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huang et al., Journal of Molecular Biology, Vol. 281 (1), pp. 61-67 (1998); and Saito and Patterson, Methods. June 1996; 9 (3): 516-24.

[0122] Mass spectrometry can also be used for epitope mapping / characterization. See, for example, Downard, *Journal of Mass Spectrometry*, April 2000; 35(4): 493-503 and Kiselaar and Downard, *Analytical Chemistry*, May 1, 1999; 71(9): 1792-1801. Protease digestion techniques can also be used for epitope mapping and identification. Antigenic determinant-related regions / sequences can be determined by protease digestion, for example by digestion with Nectin-4 at a ratio of approximately 1:50 or at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by anti-Nectin-4 binding agents can then be identified by comparing samples that have undergone trypsin digestion with those that have been incubated with antibodies and then digested with trypsin, for example (thus revealing the binder footprint). Other enzymes, such as chymotrypsin and pepsin, can or are alternatively used in similar epitope characterization methods. Furthermore, enzymatic digestion can provide a rapid method for analyzing whether potential antigenic determinant sequences lie in unexposed regions of the Nectin-4 peptide, and therefore is least likely irrelevant in terms of immunogenicity / antigenicity.

[0123] Site-directed mutagenesis is another technique that can be used to elucidate binding epitopes. For example, in an alanine scan, each residue within a protein segment is replaced with an alanine residue, and the binding affinity is measured. If the mutation results in a significant decrease in binding affinity, it is likely involved in binding. Monoclonal antibodies specific to structural epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to verify that alanine substitution does not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995; 267:383-386; and Wells, Proceedings of the National Academy of Sciences of the United States of America (ProcNatl Acad Sci USA) 1996; 93:1–6.

[0124] Electron microscopy can also be used for epitope “footprinting”. For example, Wang et al., Nature 1992; 355:275-278 used a coordinated application of cryo-electron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of the Fab fragment on the capsid surface of the native cowpea mosaic virus.

[0125] Other forms of “label-free” assays for epitope assessment include surface plasmon resonance (SPR, BIACORE™) and reflectance interference spectroscopy (RifS). See, for example, Fägerstam et al., Journal of Molecular Recognition 1990; 3:208-14; Nice et al., Journal of Chromatography 1993; 646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308–3311; Kröger et al., Biosensors and Bioelectronics 2002; 17:937-944.

[0126] It should also be noted that antibodies binding to the same or substantially the same epitopes as the antibody can be identified in one or more of the exemplary competitive assays described herein.

[0127] When immunizing and generating antibodies in vertebrates or cells, or when generating candidate antibody or amino acid sequence libraries (e.g., via phage display technology), specific selection steps can be performed to isolate antibodies or non-antibody peptides or protein scaffolds. In this regard, in a specific embodiment, a method for generating such antibodies is provided, the method comprising: (a) immunizing a non-human mammal with an immunogen comprising a Nectin-4 polypeptide or preparing an antibody or polypeptide sequence library; and (b) preparing an antibody from said immunized animal or from said antibody or sequence library; and (c) selecting from step (b) an antibody capable of binding Nectin-4, optionally selecting from step (b) an antibody capable of binding to the Ig-like V set domain of Nectin-4.

[0128] On the one hand, the average dissociation constant (Ki) of the antibody relative to human Nectin-4 D It can not exceed 1 x 10 -8 M, optionally less than 1 x 10 -9 M, as determined by, for example, surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore™ SPR analysis device). In a more specific exemplary aspect, an anti-Nectin-4 antibody is provided having a KD of approximately 1 x 10⁻⁶ for Nectin-4. -8 M to approximately 1 x 10 -10 M, or approximately 1 x 10 -9 M to approximately 1 x 10 -11 M.

[0129] In any one aspect of the embodiments, the antibody prepared according to the method of the present invention is a monoclonal antibody. In another aspect, the non-human animal used to produce the antibody is a mammal, such as a rodent, cattle, pig, poultry, horse, rabbit, goat, or sheep. The antibodies of the present invention may optionally be specified as antibodies other than any one of ASG-22ME, 14A5.2, or N41, or derivatives thereof, for example, said antibodies comprising all or part of their respective heavy and light chain CDRs or antigen-binding regions.

[0130] DNA encoding an antibody that binds to an epitope present on the Nectin-4 polypeptide is isolated from a hybridoma and placed in an expression vector, which is then transfected into host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins, to obtain monoclonal antibodies synthesized in recombinant host cells. As described elsewhere in this specification, such DNA sequences can be modified for any of a variety of purposes, such as for humanizing antibodies, producing fragments or derivatives, or for modifying antibody sequences, for example, at antigen-binding sites, to optimize antibody binding specificity. In one embodiment, an isolated nucleic acid sequence encoding the light and / or heavy chains of an antibody is provided, along with a recombinant host cell comprising (e.g., in its genome) such nucleic acid.

[0131] In any embodiment, the ability of an anti-Nectin-4 binding protein (e.g., an antibody or antibody fragment) to induce intracellular internalization of Nectin-4 expressed in tumor cells can be evaluated. For example, the Fab-Zap assay described in the examples herein can be used as a convenient method to assess the internalization of Nectin-4-expressing cells (e.g., tumor cells). In any embodiment herein, the anti-Nectin-4 binding protein, antibody, or antibody fragment, or antibody-drug conjugate comprising such an antibody or fragment, is characterized by its ability to undergo intracellular internalization upon binding to Nectin-4 on the surface of tumor cells.

[0132] In one respect, the anti-Nectin-4 antibody is an antibody that is a functionally conserved variant of any of the exemplary antibodies described herein, such as a functionally conserved variant of an antibody having the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4 (ASG-22ME), a functionally conserved variant of an antibody having the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8 (14A5.2), or a functionally conserved variant of an antibody having the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 10 (N41). A “functionally conserved variant” is a variant in which a given amino acid residue in a protein (e.g., an antibody or antibody fragment) has been altered without changing the overall conformation and function of the polypeptide, including but not limited to the substitution of amino acids with amino acids having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conserved may differ in the protein, such that the percentage of protein or amino acid sequence similarity between any two proteins with similar functions can vary and can be, for example, 70% to 99% as determined by an alignment scheme, such as by a clustering method, where the similarity is based on the MEGALIGN algorithm. "Functionally conserved variants" also include polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, as determined by BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein to which they are compared.

[0133] Exemplary anti-Nectin-4 VH and VL pairs, which can be used to prepare ADCs for use according to this disclosure, can be derived from the antibody enterotoxin antibody (or ASG-22ME) using the VH and VL (or hypervariable amino acid residues) of the antibody component ASG-22ME used in Vittin-Enterotoxin, the amino acid sequence of which is listed below (SEQ ID NO: 3), and the amino acid sequence of which is listed below (SEQ ID NO: 4). CDRs according to Kabat numbers are underlined in SEQ ID NO: 3 and 4. See also U.S. Patent No. 8,637,642 and PCT Publication No. WO2012 / 047724, the disclosure of which is incorporated herein by reference. Optionally, VH and VL comprise (e.g., modified to incorporate) a human receptor framework. In one embodiment, the anti-Nectin-4 antibody used according to this disclosure comprises VH CDR1, CDR2 and / or CDR3 of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 (e.g., according to Kabat number). In one embodiment, the anti-Nectin-4 antibody comprises VL CDR1, CDR2 and / or CDR3 of the light chain variable region having the amino acid sequence of SEQ ID NO: 4 (e.g., according to Kabat number).

[0134] ASG-22ME (Enterotolumab) VH: EVQLVESGGGLVQPGGSLRL SCAASGFTFS SYNMN WVRQA PGKGLEWVS Y ISSSSSTIYY ADSVKG RFTI SRDNAKNSLS LQMNSLRDED TAVYYCAR AY YYGMDV WGQG TTVTVSS (SEQ ID NO: 3).

[0135] ASG-22ME (Entertorumab) VL: DIQMTQSPSS VSASVGDRVT ITC RASQGIS GWLA WYQQKP GKAPKFLIY A ASTLQS GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ ANSFPPT FGG GTKVEIKRTV A (SEQ ID NO: 4).

[0136] The complete heavy and light chains of entferutuzumab or ASG-22CE / ASG-22ME are shown in SEQ ID NO: 5 and 6. In one embodiment, the anti-Nectin-4 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 5 and a light chain having the amino acid sequence shown in SEQ ID NO: 6.

[0137] ASG-22CE / ASG-22ME (Enterotolumab) complete heavy chain: EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYNMNWVRQA PGKGLEWVSY ISSSSSTIYY ADSVKGRFTI SRDNAKNSLS LQMNSLRDED TAVYYCARAY YYGMDVWGQG TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 5).

[0138] ASG-22CE / ASG-22ME (Enterotolumab) complete light chain: DIQMTQSPSS VSASVGDRVT ITCRASQGIS GWLAWYQQKP GKAPKFLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ ANSFPPTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 6).

[0139] Another exemplary anti-Nectin-4 VH and VL pair, which can be used to prepare an ADC for use according to this disclosure, is derived from antibody 14A5.2, the amino acid sequence of its heavy chain variable region (SEQ ID NO: 7) and the amino acid sequence of its light chain variable region (SEQ ID NO: 8) are listed below. CDRs according to Kabat numbers are underlined in SEQ ID NO: 7 and 8. The antibody sequences are also disclosed in PCT Publication No. WO2018 / 158398, the contents of which are incorporated herein by reference. Optionally, VH and VL comprise (e.g., modified to incorporate) a human receptor framework. In one embodiment, the anti-Nectin-4 antibody according to this disclosure comprises VH CDR1, CDR2 and / or CDR3 (e.g., according to Kabat numbers) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In one embodiment, the anti-Nectin-4 antibody comprises VL CDR1, CDR2 and / or CDR3 (e.g., according to Kabat numbers) of the light chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0140] 14A5.2 VH: EVLLQQSGPELVKPGASVKIPCKASGYTFT DYTMD WVKQSHGKSLEWIG DINPNNDVTMY NEKFKG RATLTVDKSSSTAYMEVRSLTSEDTAVYYCVR GRGFAY WGQGTLVTVSA (SEQ ID NO: 7).

[0141] 14A5.2 VL: DIVLTQSPASLIVSLGQRATISC RASQSVSTSSNSYMH WYQQKPGQPPKLLIR FASNLES GVPARFSGSGSGTYFTLNIHPVEEEDSATYYC QHSWEIPYT FGGGTKLEIK (SEQ ID NO: 8).

[0142] Another exemplary anti-Nectin-4 VH and VL pair, available from antibody N41, can be used to prepare an ADC for use according to this disclosure. The amino acid sequence of its heavy chain variable region (SEQ ID NO: 9) and the amino acid sequence of its light chain variable region (SEQ ID NO: 10) are listed below. CDRs according to Kabat numbers are underlined in SEQ ID NO: 9 and 10. The antibody VH, VL, and corresponding CDR sequences are also disclosed in PCT Publication No. WO2017 / 042210, the contents of which are incorporated herein by reference. Optionally, VH and VL include (e.g., modified to incorporate) a human receptor framework. In one embodiment, the anti-Nectin-4 antibody used according to this disclosure includes VH CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbers) having the amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-Nectin-4 antibody comprises VL CDR1, CDR2 and / or CDR3 of the light chain variable region having the amino acid sequence of SEQ ID NO: 10 (e.g., according to the Kabat number).

[0143] N41 VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGSTDYN AAFIS RLSISKDTSKSQVFFKMNSLQADDTAIYYCAR ELIHAMDN WGQGTSVTVSS (SEQ ID NO: 9).

[0144] N41 VL: DIQMTQSPASLSVSVGETVTITC RASENIYSNLA WYQQKQGNSPQLLVF AATNLAD GVPS RFSGSGSGTQYSLKINSLQSEDFGTYYC QHFWGTPT FGGGTKLEIK (SEQ ID NO: 10).

[0145] Fragments and derivatives of antibodies (which are covered by the term "antibody" or "antibodies" as used herein, unless otherwise stated or the context clearly contradicts) can be produced by techniques known in the art. A "fragment" includes a portion of a complete antibody, typically an antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; biantibodies; any antibody fragment that is a polypeptide having a primary structure consisting of a single, uninterrupted sequence of amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv molecules; (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing three CDRs of a light chain variable domain without an associated heavy chain molecule; and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing three CDRs of a heavy chain variable region without an associated light chain molecule; and multispecific antibodies formed from antibody fragments. In particular, this includes nanobodies, domain antibodies, single-domain antibodies, or "dAbs".

[0146] In one embodiment, the antibody is humanized. A “humanized” form of antibody is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from mouse immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the receptor's complementarity-determining region (CDR) are replaced by residues from the CDR of the original antibody (donor antibody), while maintaining the specificity, affinity, and potency required of the original antibody.

[0147] In some cases, the Fv framework residues of human immunoglobulins can be replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the receptor antibody or the introduced CDR or framework sequence. These modifications are made to further improve and optimize antibody performance. Typically, humanized antibodies will include substantially all at least one, and usually two, variable domains, wherein all or substantially all CDR regions correspond to those of the original antibody, and all or substantially all FR regions are those in the common sequence of human immunoglobulins. Humanized antibodies will also preferably include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., Nature, 321, p. 522 (1986); Reichmann et al., Nature, 332, p. 323 (1988); Presta, Curr. Op. Struct. Biol., 2, p. 593 (1992); Verhoeyen et al., Science, 239, p. 1534; and U.S. Patent No. 4,816,567, the entire disclosure of which is incorporated herein by reference.

[0148] The choice of either the light or heavy chain for the human variable domain used in the preparation of humanized antibodies is crucial for reducing antigenicity. According to the so-called "best fit" method, sequences of the variable domains of antibodies are screened against an entire library of known human variable domain sequences. The human frame (FR) of the humanized antibody is then accepted as the sequence closest to the mouse sequence (Sims et al., *Journal of Immunology* 151, p. 2296 (1993); Chothia and Lesk, *Journal of Molecular Sciences* 196, 1987, p. 901). Another approach uses a specific frame from a common sequence of all human antibodies derived from a specific light or heavy chain subgroup. The same frame can be used for several different humanized antibodies (Carter et al., *Proceedings of the National Academy of Sciences* 89, p. 4285 (1992); Presta et al., *Journal of Immunology* 151, p. 2623 (1993)).

[0149] More importantly, the antibody is humanized, retaining its high affinity for Nectin-4 and other favorable biological properties. To achieve this, humanized antibodies are prepared according to a preferred method, involving the analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that display and show the possible three-dimensional structures of selected candidate immunoglobulin sequences. Examining these displays allows for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequences, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from shared and input sequences to achieve desired antibody properties, such as increased affinity for the target antigen. Typically, CDR residues are directly and most substantially involved in influencing antigen binding. In one embodiment, the FR of the humanized antibody chain is derived from a human variable region having at least about 60% overall sequence identity with a variable region of a non-human donor (e.g., ASG-22ME, 14A5.2, or N41 antibody), and preferably at least about 70%, 75%, or 80% overall sequence identity. Optionally, the humanized heavy chain and / or light chain variable regions share at least about 60%, 70%, or 80% overall sequence identity with the corresponding heavy chain and / or light chain variable regions of the non-human donor (e.g., ASG-22ME, 14A5.2, or N41 antibody). Another method for preparing “humanized” monoclonal antibodies is to use a transgenic mouse (XenoMouse) (Abgenix, Fremont, CA) as the mouse for immunization. XenoMouse is a mouse host whose immunoglobulin genes have been replaced with functional human immunoglobulin genes. Therefore, antibodies produced from hybridomas prepared from such mice or from B cells of such mice have been humanized. Transgenic mice are described in U.S. Patent No. 6,162,963, which is incorporated herein by reference in its entirety. Human antibodies can also be generated according to a variety of other techniques, such as by using other transgenic animals engineered to express a human antibody library for immunization (Jakobovitz et al., Nature 362 (1993) 255), or by selecting from an antibody library using a phage display method. Such techniques are known to those skilled in the art and can be implemented starting with the monoclonal antibodies disclosed in this application.

[0150] Advantageously, the linker containing a camptothecin analog of this disclosure can be used in the preparation of conjugated antigen-binding agents (e.g., peptides, polypeptides, antibodies, or antibody fragments) to obtain antigen-binding agent-drug conjugates, such as antibody-drug conjugates (ADCs). In one embodiment, the process for preparing an antigen-binding agent-conjugate includes conjugating a camptothecin analog (Z) to an antigen-binding agent. In one embodiment, the camptothecin analog (Z) can be specified as being conjugated to the antigen-binding agent via a linker (X). X is a linker connecting an antigen-binding agent such as an antibody (Ab) and the camptothecin analog (Z), for example, in conjugation, X is a residue of the linker covalently linked to one or both of Ab and Z.

[0151] In the embodiments herein, the process for preparing the antibody-conjugate includes the step of contacting and / or reacting an antigen-binding agent such as an antibody (Ab) with a camptothecin analog (Z). The contact may be performed under suitable conditions to form or obtain an antigen-binding pharmaceutical conjugate of one aspect of this disclosure. Z may, for example, be included in a compound comprising a camptothecin analog (Z) and a linker (X) or a portion thereof, such that the step comprises contacting the antigen-binding agent with a compound comprising a camptothecin analog (Z) and a linker (X) or a portion thereof. The method may optionally specify the steps of: isolating or recovering the formed antigen-binding pharmaceutical conjugate, and optionally further processing the composition for use as a drug, and optionally formulating the antigen-binding conjugate (e.g., together with a pharmaceutical excipient) for administration to a human subject.

[0152] Optionally, the method for preparing an ADC includes conjugating an antibody (Ab) to 2, 3, 4, 5, 6, 7, or 8 camptothecin analog molecules. Optionally, the resulting composition is characterized by a DAR between 2 and 4, between 4 and 6, or between 6 and 8. Optionally, the method includes conjugating the antibody to 4 camptothecin analog molecules. Optionally, the method further includes evaluating the DAR, and if the DAR corresponds to a predetermined specification (e.g., the DAR or DAR range disclosed herein, DAR approximately 2, 4, 6, or 8, etc.), further processing the composition for use as a drug, and optionally formulating the antibody (e.g., together with a pharmaceutical excipient) for administration to a human subject.

[0153] In some embodiments, the connector (X)-(Z) element is prepared and separated before contacting (and reacting) the compound comprising (X) and (Z) with (Ab) to form the drug conjugate.

[0154] In some embodiments, the method includes: (a) Contacting and / or reacting the connector (X) or a portion thereof with (Ab) to form an Ab-X conjugate, and (b) Contacting and / or reacting the Ab-X from step (a) with a camptothecin analog (Z) or a compound comprising a second part of the linker (X) and (Z) to form the antibody-drug conjugate.

[0155] X can, for example, represent a molecule comprising a cleavable portion, for example, under physiological conditions, and optionally under intracellular conditions. In one embodiment, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable portion, and (iii) an optional self-eliminating or non-self-eliminating spacer subsystem (Y'). The cleavable portion can, for example, be an oligopeptide (e.g., a dipeptide, tripeptide, tetrapeptide, or pentapeptide). The spacer Y can be located between Ab and the cleavable portion, and the spacer subsystem (Y') can be located between the cleavable portion and Z.

[0156] In some embodiments, the linker X or spacer Y may optionally be designated to include a reactive group (R) capable of reacting with an amino acid of the antibody or a complementary reactive group (R') to an amino acid attached to the antibody (e.g., optionally after deprotection under suitable conditions). Optionally, R is a group reactive with a free amino, hydroxyl, thiol, or carboxyl group on the antibody.

[0157] In some embodiments, the linker X or spacer Y may optionally be specified as a residue comprising a reactive group R that reacts with an amino acid of the antibody or with a complementary reactive group (R') of an amino acid attached to the antibody. Optionally, R is a residue that reacts with a group reactive with a free amino, hydroxyl, thiol, or carboxyl group on the antibody.

[0158] In any embodiment, prior to the step of contacting and / or reacting the antibody or antibody fragment with a compound (e.g., a linker and / or a camptothecin analogue), the method includes the steps of preparing, selecting, or providing the antibody or antibody fragment. In one embodiment, the steps include preparing, selecting, or providing an anti-Nectin-4 antibody or antibody fragment, and determining or testing whether the antibody or antibody fragment has the characteristics of the anti-Nectin-4 antibody or antibody fragment disclosed herein.

[0159] For example, the ability of an anti-Nectin-4 antibody or antibody fragment to bind to Nectin-4 or to the V domain of Nectin-4 can be tested. The antibody or antibody fragment determined to bind to Nectin-4 (or to the V domain) is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a camptothecin analog (Z)). For example, the ability of an anti-Nectin-4 antibody or antibody fragment to bind to a mutant Nectin-4 peptide (e.g., a mutant Nectin-4 peptide lacking an Ig-like V set domain) can be tested. The antibody fragment determined to have reduced or lost binding to the mutant Nectin-4 peptide (e.g., compared to binding to a wild-type Nectin-4 peptide) is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a camptothecin analog (Z)).

[0160] As further described herein, some well-known methods for conjugating cytotoxic agents with antibodies involve multiple reaction steps, wherein the antibody is first modified with a linker or a portion thereof, and then a reaction is carried out to conjugate the cytotoxic agent with the antibody-linker composition.

[0161] In one embodiment, a process for preparing an antigen-binding drug conjugate is provided, the process comprising: (i) Contacting an antigen-binding agent (e.g., a peptide, polypeptide, antibody, or antibody fragment (e.g., its binding to Nectin-4) with a compound (L) comprising (a) a first reactive group capable of reacting with an amino acid of the agent (e.g., a side chain or glycan of the amino acid, or a group attached to the amino acid or the glycan of said amino acid) and (b) a second reactive group (R') to obtain a modified agent comprising one or more amino acids functionalized with compound (L); and (ii) Reacting the modified agent of step (i) with a compound comprising: (a) a reactive group (R) complementary to the reactive group (R'); (b) an amino acid unit (e.g., dipeptide, tripeptide, tetrapeptide, or pentapeptide) cleaved by an intracellular peptidase or protease; (c) an optional non-suicidal or suicidal spacer (Y'); and (d) a cytotoxic agent (Z). Optionally, the compound of step (ii) further comprises a spacer (Y) positioned between R and the amino acid unit.

[0162] In one embodiment, R and R' are capable of click reaction or cycloaddition, optionally wherein R comprises or is an alkyne moiety and R' comprises or is an azide moiety, or wherein R' comprises or is an alkyne moiety and R comprises or is an azide moiety, and wherein the reaction in step (ii) is a 1,3-dipolar cycloaddition.

[0163] In one embodiment, the reaction in step (i) is carried out in the presence of a catalyst, optionally an enzyme (e.g., transglutaminase).

[0164] In one embodiment, step (i) includes modifying the anti-Nectin-4 antibody or antibody fragment before contacting the compound (L). For example, the antibody or antibody fragment can be modified by reacting or contacting it with an enzyme capable of modifying antibody glycosylation (e.g., at Kabat residue N297). In one example, the modification includes deglycosylation of an antibody glycan having a core N-acetylglucosamine in the presence of an endoglucosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Examples of endoglucosidases include EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH and / or combinations thereof.

[0165] Antigen-binding protein (e.g., antibody) molecules and camptothecin analog molecules are linked by a linker. In such embodiments, the immunoconjugate can be represented, for example, by formula (II): Ab–(X–(Z) n ) m Equation (II) in, Ab is an anti-Nectin4 antigen-binding protein (e.g., an antibody). X is a linker that connects Ab and Z, for example, a linker residue that is covalently linked to one or both of Ab and Z; Z is a camptothecin analogue, and optionally Z includes the structure of compound 1 or 2 (eixotecan or SN-38 molecule). n is 1 or 2; and When n is 1, m is 1 to 8, or optionally m is an integer selected from 1 to 8 or 1 to 6, optionally m is an integer selected from 1 to 4, optionally m is 2 or 4; optionally, m is 2, 3, 4, 5, 6, 7 or 8; and when n is 2, m is 1 to 4, or optionally m is an integer selected from 1 to 4 or 1 to 3, optionally m is an integer selected from 1 to 4, optionally m is 2 or 4; optionally, m is 1, 2, 4 or 4. Optionally, "n" can be specified to represent the degree of branching or polymerization. "n" and "m" can be specified to represent the average value in a composition comprising multiple antibodies.

[0166] In one embodiment, X represents a molecule comprising, for example, a cleavable portion under physiological conditions, optionally under intracellular conditions. In one embodiment, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable portion, and (iii) an optional self-eliminating or non-self-eliminating spacer subsystem (Y'). The spacer Y may be located between Ab and the cleavable portion, and the spacer subsystem (Y') may be located between the cleavable portion and Z. The molecule X or the spacer Y may optionally be specified as comprising a reactive group (R) or a residue of the reactive group R that reacts with an amino acid of the antibody or with a complementary reactive group (R') of an amino acid attached to the antibody.

[0167] The variable m represents the –X–(Z) of each antibody molecule in the immunoconjugate. n The number of portions. In compositions comprising multiple anti-Nectin-4 ADCs, the number "m" of the -X-Z portions of each antibody molecule can vary. Thus, in an exemplary composition comprising multiple immunoconjugates of the formula herein, m is the –X-(Z) portion of each Ab. n The average quantity of the fraction, in this case, m can also be referred to as the average drug load or drug:antibody ratio (DAR). The average drug load or DAR can advantageously be 1 to about 8 per antibody (–X–(Z)). n Within the range of part X. The number "n" of Z parts attached to part X can be, for example, 1 or 2. Typically, n is 1. In some embodiments, n is 1, and m represents the average drug loading, m being between 2 and 8. In some embodiments, n is 1, and m represents the average drug loading, m being between 2 and 6. In some embodiments, n is 1, and m represents the average drug loading, m being between 4 and 8. In some embodiments, n is 1, and m represents the average drug loading, m being between 6 and 8, optionally about 6, 7, or 8. In some embodiments, n is 1, and m represents the average drug loading, m being between 4 and 6, optionally about 4, 5, or 6.

[0168] The quantity of the (–X–Z) fraction of each Ab can be characterized by conventional methods such as mass spectrometry, ELISA, and HPLC. The quantitative distribution of the immunoconjugate with respect to m can also be determined. In some cases, homogeneous immunoconjugates with specific m values ​​can be separated, purified, and characterized by techniques such as reversed-phase HPLC or electrophoresis to distinguish them from immunoconjugates with other drug loadings.

[0169] In one embodiment, the anti-Nectin-4 composition for the treatment methods of this disclosure is characterized by comprising a plurality of immunoconjugates represented by formula (I): Ab–(X–(Z) n ) mEquation (II) in, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment). X is a molecule that connects Ab and Z, for example, a linker residue that is covalently linked to one or both of Ab and Z; Z is a camptothecin analogue, which includes molecules of eczema or SN-38, such as molecules that include the structure of compound 1 or 2; n is 1 or 2; and The number of m (XZ portions) of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the antibody sample is 2 or 4, at least 2, between 2 and 4, at least 4, between 4 and 6, or between 4 and 8, optionally where n is 1, and the number of m (XZ portions) of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the antibody sample is 2 or 4, at least 2, between 2 and 4, at least 4, between 4 and 6, or between 4 and 8.

[0170] In one embodiment, the anti-Nectin-4 composition for the treatment methods of this disclosure is characterized by comprising a plurality of immunoconjugates represented by formula (I): Ab–(X–(Z) n ) m Equation (II) in, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment). X is a molecule that connects Ab and Z, for example, a linker residue that is covalently linked to one or both of Ab and Z; Z is a camptothecin analogue, which includes molecules of eczema or SN-38, such as molecules that include the structure of compound 1 or 2; n is 1; and The number of the XZ portion of the immunoconjugate in the antibody sample is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and m (the number of XZ portions) is 6, at least 6, between 6 and 8, or 8.

[0171] In one embodiment, the anti-Nectin-4 composition for the treatment methods of this disclosure is characterized by comprising a plurality of immunoconjugates represented by formula (I): Ab–(X–(Z) n ) m Equation (II) in, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment). X is a molecule that connects Ab and Z, for example, a linker residue that is covalently linked to one or both of Ab and Z; Z is a camptothecin analogue, which includes molecules of eczema or SN-38, such as molecules that include the structure of compound 1 or 2; n is 1; and The antibody sample contains at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of immunoconjugates with a m (number of XZ portions) of 8.

[0172] Various methods can be used to covalently link linkers, including cytotoxic agents, to antibodies or antigen-binding proteins, either nonspecifically or specifically, to specific amino acid residues. The linker (X) may include, for example, a portion that is cleavable under physiological conditions, optionally as shown in the examples, under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent in the intracellular environment. The linker may bind to chemically reactive groups on the antibody molecule, such as free amino, imino, hydroxyl, thiol, or carboxyl groups (e.g., bound to the N-terminus or C-terminus, to an ε-amino group bound to one or more lysine residues, a free carboxylic acid group bound to one or more glutamic acid or aspartic acid residues, or a thiol group bound to one or more cysteine ​​residues), to carbohydrates, or generally to any reactive group introduced or engineered into the antibody. The binding site for the adapter can be a natural residue in the amino acid sequence of the antibody molecule, or it can be introduced into the antibody molecule, for example by DNA recombination technology (e.g., by introducing cysteine ​​or protease cleavage sites into the amino acid sequence, by introducing non-natural amino acid residues) or by protein biochemistry (e.g., reduction, pH regulation or proteolysis, by glycoengineering, enzymatic modification of amino acid-binding glycans).

[0173] In some embodiments, an intermediate serving as a precursor to the linker (X) reacts with a cytotoxic agent (Z) under appropriate conditions. In some embodiments, a reactive group is used on the cytotoxic agent and / or the intermediate. In some embodiments, the reaction product between the cytotoxic agent and the intermediate or a derived cytotoxic agent is subsequently reacted with an antibody molecule under appropriate conditions. In other embodiments, the precursor to the linker (X) is first reacted with an antibody molecule under appropriate conditions to generate an antibody that binds to the precursor of the linker (X), which is then reacted with a molecule comprising the cytotoxic agent (Z).

[0174] In some embodiments, the linker (X) can be cleaved by a cleaving agent present in the intracellular environment (e.g., in lysosomes, endosomes, or pits). The linker may comprise, for example, a peptide linker or amino acid unit cleaved by intracellular peptidases or proteases (including, but not limited to, lysosomal or endosomal proteases). In some embodiments, the peptide linker portion is at least two amino acids long or at least three amino acids long. The cleaving agent may comprise cathepsins B and D, as well as plasmin, all known to hydrolyze dipeptide drug derivatives, thereby releasing the active drug within the target cell. Most typically, the peptide linker is cleavable by enzymes present in the cell. In specific embodiments, the peptide linker cleavable by intracellular proteases is a Val-Cit linker or a phenylalanine-lysine (Phe-Lys) linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker). The valine-citrulline (Val-Cit) element may have the following structure: .

[0175] In another specific embodiment, the peptide linker that can be cleaved by intracellular proteases is a valine-alanine (Val-Ala) linker. The val-ala element can have the following structure: .

[0176] In another specific embodiment, the peptide linker that can be cleaved by intracellular proteases is a glycine-containing oligopeptide linker, such as an oligopeptide linker containing glycine and phenylalanine, optionally a GGFG, GGFGG, or GGFGGG linker (see, for example, U.S. Patent No. 6,835,807, the disclosure of which is incorporated herein by reference).

[0177] In some embodiments, and optionally in addition to being cleavable by intracellular proteases, the linker may also function as a spacer or extension to keep the antibody at a distance from Z to avoid interfering with the antibody's ability to bind Nectin-4 and / or inhibit Nectin-4-mediated intercellular interactions. The linker may include a spacer unit (Y) and / or a spacer or spacer system (Y'). Thus, the spacer Y may be positioned between the Ab and the cleavable portion. The spacer system (Y') may be positioned between the cleavable portion and Z, or the spacer Y (or the linker X including it) may optionally be specified as including a reactive group (R) or residues of the reactive group R that react with an amino acid of the antibody or a complementary reactive group (R') of an amino acid attached to the antibody. The spacer Y can be, for example, a molecule that forms a bond (e.g., through its reactive group R) with an amino acid of the antibody (e.g., the sulfur atom, primary or secondary amino group, or carbohydrate group of the antibody), and the spacer or extension (Y) links the antibody to a cytotoxic agent (Z) or a cleavable amino acid unit (e.g., a peptide linker, a cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide), the cleavable amino acid unit optionally further having a self-eliminating and / or non-self-eliminating spacer (Y'), which in turn is linked to Z. Therefore, when one end of the spacer (Y) is linked to an amino acid unit (e.g., a cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide), the cleavable amino acid unit can then be directly linked to Z or may include additional spacers (Y'), such as non-suicidal or suicidal spacers linking the amino acid unit and Z.

[0178] The spacer (Y) may optionally be specified as or comprise a substituted or unsubstituted alkyl or heteroalkyl chain, wherein the chain length of Y is 2-100 atoms, optionally 2-40, 2-30, 2-20, 4-40, 4-30 or 4-20 atoms, wherein one or more atoms may not be carbon, but may be oxygen, sulfur, nitrogen or other atoms, wherein any carbon atom of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0179] The spacer (Y) may optionally be specified to include a stability-enhancing portion. For example, the spacer Y may be an orthogonal polyethylene glycol (PEG) portion or a polysarcosine (poly-N-methylglycine or PSAR) portion in the joint design (see, for example, WO2019 / 081455, WO2015 / 057699 and WO2016 / 059377, the disclosures of which are incorporated herein by reference).

[0180] In some specific embodiments, the spacer (Y) may include one or more ethylene oxide monomers, optionally Y includes a polyethylene oxide moiety, optionally Y includes 1 to 24, optionally 1 to 12, optionally 1 to 8, optionally 1 to 6 polyethylene oxide moieties, optionally Y includes the structure -(CH2CH2O). x - where x is 1 to 12, arbitrarily 1 to 8, and arbitrarily 1 to 6.

[0181] Examples of suitable stability-enhancing portions, spacer subchain Y, may include the stability-enhancing portions disclosed in PCT Publications WO2015 / 057699 or WO2019 / 081455. For example, spacer subchain Y may include an orthogonal linker portion and a stability-enhancing portion. The stability-enhancing portion may be a PEG homopolymer, or typically any single molecular weight homopolymer (e.g., PEG or polysarcosine homopolymer) bound to the orthogonal linker portion. The homopolymer may have, for example, 1-4, 1-6, 1-8, 1-10, 1-12, at least 6, 8, or 10, or 6-12, 6-24, or 6-72 PEG units or other monomers. The term orthogonal linker refers to a branched linker unit assembly that connects a linker portion (e.g., a chain of spacer Y) to a homopolymer unit and a cytotoxic agent (Z) via a linker (e.g., a cleavable oligopeptide (Pep) and spacer Y') such that the homopolymer unit is in a parallel configuration relative to the cytotoxic agent (opposite to a tandem configuration) (the homopolymer is parallel to the Pep-Y'-Z portion). The orthogonal linker portion can be, for example, one or more natural or non-natural amino acids, optionally selected from glutamic acid, lysine, and glycine. Optionally, the amino acid orthogonal linker portion is positioned at the end of the spacer chain Y such that the amino acid residue of the orthogonal linker portion is linked to the amino acid residue of the peptide linker (e.g., (Pep) in formula V or VI) via a peptide bond between the α-carboxyl group of one amino acid and the α-amino group of another amino acid. Y can, for example, include the result of a partial reaction of the orthogonal linker portion with a portion of formula D: Formula D R1 and R2 are different, and One of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group. The group is reactive to covalently bind to the bindable group of the orthogonal linker portion. Under such reaction conditions where the inert group is nonreactive, Z1 and Z2, whether the same or different, are optional spacers, and n is 1 or greater and k is 2 or greater.

[0182] In another instance, the spacer Y includes a group disclosed in U.S. Patent Publication No. US2017 / 0072068A1 (the disclosure of which is incorporated herein by reference), such as a group according to formula (E) or a salt thereof: Formula E in a is 0 or 1; and R 1 Choose from the following groups: hydrogen, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C24 (hetero)aryl, C3-C24 alkyl (hetero)aryl and C3-C 24 (Hetero)arylalkyl, the C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C24 (hetero)aryl, C3-C24 alkyl (hetero)aryl and C3-C 24 (Hetero)arylalkyl groups are optionally selected from O, S, and NR. 3 One or more heteroatoms are substituted and optionally interrupted by said one or more heteroatoms, wherein R 3 The group is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; and the group according to formula E or its salt is located between the first and second ends of the spacer chain Y.

[0183] The spacer or spacer system (Y') placed between the amino acid unit (e.g., a cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide) and Z can be self-eliminating or non-self-eliminating. For example, spacer Y' can comprise a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-30 atoms, optionally 2-20, 4-20, 2-10, or 4-20 atoms, optionally one or more of which may not be carbon, such as oxygen, sulfur, nitrogen, or other atoms, optionally wherein any carbon atom of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide. In one embodiment, Y' comprises a p-aminobenzyloxycarbonyl group. In one embodiment, Y' is a non-self-eliminating spacer and includes a (CH2–C(=O)) group, such as Y' being or including –O–CH2–C(=O)–, HO–O–CH2–C(=O)–, –CH2CH2–C(=O)–, –CH2CH2CH2–C(=O)–, –CH2–O–CH2–C(=O)– or –CH2CH2–O–CH2–C(=O)– groups.

[0184] "Self-eliminating" spacer units allow for the release of drug moieties without a separate hydrolysis step. When using a self-eliminating spacer, after the cleavage or conversion of an amino acid unit, the side of the spacer connected to the amino acid unit becomes unclosed, resulting in the eventual release of one or more moieties Z. Self-eliminating spacer systems can be, for example, those described in WO02 / 083180 and WO2004 / 043493 (the disclosures of which are incorporated herein by reference in their entirety), as well as other self-eliminating spacers known to those skilled in the art. In some embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methyl carbamate, or carbonate is prepared between benzyl alcohol and the cytotoxic agent. In one embodiment, the spacer unit is a p-aminobenzyloxycarbonyl (PAB). Examples of self-eliminating spacer units further include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, for example, US 2005 / 0256030 A1), such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999), Bioorg. Med. Chem. Lett. 9:2237) and o-aminobenzyl acetals or p-aminobenzyl acetals. Cyclization can be achieved using the spacer upon hydrolysis of the amide bond, as in substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223) and 2-aminophenylpropionic acid amides (Amsberry et al., Journal of Organic Chemistry, 1990, 55, 5867). Elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., Journal of Medicinal Chemistry). (Chem.) , 1984, 27, 1447) is also an example of a suicide spacer. p-aminobenzyl self-eliminating spacers (e.g., PAB) are particularly suitable for use with Phe-Lys, Val-Ala or Val-Cit cleavable dipeptide units (PAB is positioned between the dipeptide and the camptothecin analog (Z)).

[0185] A “non-self-eliminating” spacer unit is a spacer unit in which part or all of the spacer unit remains bound to part Z upon enzymatic (e.g., proteolytic) cleavage of the antibody-part-of-interest conjugate. Examples of non-self-eliminating spacers suitable for use as spacers between Gly-Gly-Phe-Gly amino acid units and ixotecan molecules include, but are not limited to, –O–CH2–C(=O)–, HO–O–CH2–C(=O)–, –CH2CH2–C(=O)–, –CH2CH2CH2–C(=O)–, –CH2–O–CH2–C(=O)–, and –CH2CH2–O–CH2–C(=O)– (e.g., to form a GGFG–CH2CH2–O–CH2–C(=O)–ixotecan unit). Use of such a spacer between the GGFG amino acid unit and ixotecan results in the release of a molecule containing ixotecan having the structure of compound 3. Other examples of non-self-eliminating spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other known combinations of peptide spacers sensitive to sequence-specific enzymatic cleavage can be used in a similar manner. For example, enzymatic cleavage of an antibody-part-of-interest conjugate containing a glycine-glycine spacer unit by a tumor cell-associated protease will result in the release of the glycine-glycine-drug moiety from the remainder of the antibody-part-of-interest conjugate. In one such embodiment, the glycine-glycine-drug moiety subsequently undergoes a separate hydrolysis step in tumor cells, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[0186] An exemplary linker-camptothecin moiety (X–Z) may include any structure shown in Formulas III and IV below, where z is a camptothecin analogue and Y and Y' are spacers. Formula IIIa Formula IIIb Formula IIIc Formula IVa Formula IVb Formula IVc.

[0187] Spacers (Y) and (Y') can optionally be specified as independently selected from the group consisting of straight or branched C1-C. 20 Alkylene, C2-C 20 alkenyl, C2-C20 Ethyne group, C3-C 20 Cycloalkylene, C5-C 20 Cycloalkylene, C8-C 20 cycloacetylenic, C7-C 20 Alkyl arylene, C7-C 20 Arylalkylene, C8-C 20 Aryleneide and C9-C 20 Arylynylene, alkylene, alkenylene, ynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylynylene are optionally selected from O, S, and NR. 1 One or more heteroatoms are substituted and optionally interrupted by said one or more heteroatoms, wherein R 1 Independently select from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 Cycloalkyl, wherein the alkyl, alkenyl, ynyl and cycloalkyl groups are optionally substituted.

[0188] Spacers (Y) and (Y') may optionally be specified as or include C1-C 10 Alkylene-, -C1-C 10 Heteroalkyl-, -C3-C8 carbocyclic-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbide ring)-, -(C3-C8 carbide ring)- C1-C 10 Alkylene, -C3-C8 heterocyclic, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Alkylene -C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocyclic-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene -(C3-C8 carbide ring)-C(=O)-, -(C3-C8 carbide ring)-C1-C 10Alkylene -C(=O)-, -C3-C8 heterocyclic -C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkyl-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbide ring)-NH-, -(C3-C8 carbide ring)-C1-C 10 Alkylene -NH-, -C3-C8 heterocyclic -NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkyl-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbide ring)-S-, -(C3-C8 carbide ring)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(=O)-, -C3-C8 carbocyclic-OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene-OC(=O)-, -C1-C 10 Alkylene-arylene-OC(=O)-, -arylene-C1-C 10 Alkylene -OC(=O)-, -C1-C 10 Alkylene -(C3-C8 carbide ring)-OC(=O)-, -(C3-C8 carbide ring)-C1-C 10 Alkylene -OC(=O)-, -C3-C8 heterocyclic -OC(=O)-, -C1-C 10Alkylene-(C3-C8 heterocyclic)-OC(=O)-, -(C3-C8 heterocyclic)-C1-C 10 The alkylene group -OC(=O)- is optionally substituted with one or more substituents selected from the following groups: -X, -R', -O, -OR', =O, -SR', -S - -NR'2, -NR'3 + ,=NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3, -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2, and C(=NR')NR'2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R' is independently -H, -Cl-C 20 Alkyl, -C6-C 20 Aryl or -C3-C 14 Heterocyclic rings.

[0189] The spacer (Y) may optionally be specified to include, for example, at one end of the chain: a reactive group (R) that is reactive to a free amino, hydroxyl, thiol, or carboxyl group or carbohydrate on the antibody, or to a complementary reactive group (R') of an amino acid attached to the antibody (e.g., via a free amino, hydroxyl, thiol, or carboxyl group or carbohydrate); or, when conjugated with an anti-Nectin-4 antibody, a residue in which the reactive group (R) reacts to a free amino, hydroxyl, thiol, or carboxyl group on the antibody or to a complementary reactive group (R') of an amino acid attached to the antibody. Examples of reactive groups for R and R' include a series of groups capable of undergoing bisorthogonal reactions, preferably cycloadditions, such as Diels-Alder reactions or 1,3-dipolar cycloadditions, such as between azides and cyclooctyne (copper-free click chemistry), between nitroketones and cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, and tetrazine linkages (see also WO2013 / 092983 or US2017 / 0072068A1, the disclosures of which are incorporated herein by reference). For example, R may be an alkyne and R' may be an azide, or R may be an azide and R' may be an alkyne. The resulting linker and functionalized antibody or its Y element may therefore include, in any embodiment, a group (RR') generated by the reaction of R and R', for example, RR' may be or include a triazole generated by the reaction of an alkyne and an azide.

[0190] In one embodiment, the reactive groups R and R' are complementary reagents that together enable a "click" reaction (i.e., clicking the chemical reagent or the reactive group). For example, 1,3-dipolar functional compounds can react with alkynes in cyclization reactions to form heterocyclic compounds, preferably in the absence of substantially added catalysts (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipolar group attached thereto (having a triatomic π-electron system containing four electrons delocalized on three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipolar groups include, but are not limited to, azides, nitrile oxides, nitrones, azooxy groups, and acyldiazo groups.

[0191] Examples include o-phosphonoaramid esters, azides, fulminates, alkynes (including any strained cyclic alkynes), cyanides, anthracene, 1,2,4,5-tetraazine, or norbornene (or other strained cyclic alkenes).

[0192] In one embodiment, R is a portion having a terminal alkyne or azide; such portions are disclosed, for example, in U.S. Patent No. 7,763,736, the disclosure of which is incorporated herein by reference. Suitable reaction conditions for using copper (and other metal salts) as a click reaction catalyst between the terminal alkyne and the azide are disclosed in U.S. Patent No. 7,763,736.

[0193] In one embodiment, R is a substituted or unsubstituted cycloalkyne. Cycloalkynes containing specific compounds are described, for example, in U.S. Patent No. 7,807,619, the disclosure of which is incorporated herein by reference.

[0194] In some embodiments, the cycloalkyne may be a compound of formula A: Formula A in: R 1 Selected from carbonyl, alkyl ester, aryl ester, substituted aryl ester, aldehyde, amide, arylamide, alkyl halide, thioester, sulfonyl ester, alkyl ketone, aryl ketone, substituted aryl ketone and halosulfonyl group; R 1 It can be at any position on the cyclooctyne group, rather than on the two carbons connected by a triple bond.

[0195] In some embodiments, the modified cycloalkyne has formula A, wherein one or more carbon atoms in the cyclooctyne ring, except for the two carbon atoms connected by a triple bond, are substituted with one or more electron-withdrawing groups, such as halogens (bromine, chlorine, fluorine, iodine), nitro, cyano, sulfone, or sulfonic acid groups. Thus, for example, in some embodiments, the subject-modified cycloalkyne has formula B: Formula B in: R 2 and R 3 Each of these is independently: (a) H; (b) a halogen atom (e.g., bromine, chlorine, fluorine, iodine); (c) -W-(CH2). n -Z (where: n is an integer from 1 to 4 (e.g., n = 1, 2, 3, or 4); W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or halogen); (d)-(CH2) n -W-(CH2) m -R 4 (Where: n and m are each independently 1 or 2; W is O, N, S or sulfonyl; if W is O, N or S, then R) 4 It can be nitro, cyano, or halogen; and if W is sulfonyl, then R 4 (e)-(CH2); or (e)-(CH2) n - R 4 (Where: n is an integer from 1 to 4 (e.g., n = 1, 2, 3, or 4); and R 4 (It can be nitro, cyano, sulfonic acid, or halogen); and R 1Selected from carbonyl, alkyl esters, aryl esters, substituted aryl esters, aldehydes, amides, arylamides, alkyl halides, thioesters, sulfonyl esters, alkyl ketones, aryl ketones, substituted aryl ketones, and halosulfonyl groups. R 1 It can be at any position on the cyclooctyne group, rather than on the two carbons connected by a triple bond.

[0196] In one embodiment, R is a substituted or unsubstituted heterocyclic strained alkyne. Cycloalkynes comprising specific compounds are described, for example, in U.S. Patent No. 8,133,515, the disclosure of which is incorporated herein by reference. In one embodiment, the alkyne has the formula C: Formula C in: Each R 1 Independently selected from the group consisting of: hydrogen, halogens, hydroxyl groups, alkoxy groups, nitrates, nitrites, sulfates, and C1-C. 10 Alkyl or heteroalkyl; Each R 2 Independently selected from the group consisting of: hydrogen, halogens, hydroxyl groups, alkoxy groups, nitrates, nitrites, sulfates, and C1-C. 10 Organic group; X represents NR 3 R 4 NH-R 4 CH-N-OR 4 CN-NR 3 R 4 CHOR4 or CHNHR4; each R 3 Represents hydrogen or an organic group, and R 4 The connector portion C is indicated. In one embodiment, R or R' is the following DBCO (dibenzylcyclooctyl) group: DBCO The alkynes described above can react with at least one 1,3-dipolar functional compound in cyclization reactions to form heterocyclic compounds, preferably in the absence of substantially any added catalyst (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipolar group attached thereto (having a triatomic π-electron system containing four electrons delocalized on three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipolar groups include, but are not limited to, azides, nitrile oxides, nitrones, azooxy groups, and acyldiazo groups.

[0197] In the chemical formula herein, Y' may optionally be absent or may be a spacer, optionally a self-eliminating spacer, for example including a p-aminobenzyl unit, or a non-self-eliminating spacer. Optionally, Y' is or includes a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-40 atoms, optionally 2-30, 2-20, 4-40, 4-30, or 4-20 atoms, optionally one or more of which may not be carbon, for example oxygen, sulfur, nitrogen, or other atoms, optionally wherein any carbon atom of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide.

[0198] An exemplary linker that can conjugate with anti-Nectin-4 binding proteins—camptothecin molecule (e.g., the XZ portion of formulas I to XI)—can optionally be represented by formula V: (R)–(Y) – (Pep) – (Y') – (Z) Formula (V) in, R is a group that is reactive to a free amino, hydroxyl, thiol, or carboxyl group on the antibody or to a complementary reactive group (R') of an amino acid attached to the antibody, or, when conjugated with anti-Nectin-4 binding protein, R is a residue in which the reactive group (R) reacts with a free amino, hydroxyl, thiol, or carboxyl group on the antibody or to a complementary reactive group (R') of an amino acid attached to the antibody. Y can be either absent or a spacer; Pep is or includes a peptide linker that is cleaved by intracellular peptidases or proteases, such as valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide. Y' is optionally absent or a spacer, optionally a self-eliminating spacer or a non-self-eliminating spacer; and Z is a camptothecin analog or derivative, optionally eczema or SN-38 molecule.

[0199] The Nectin-4 binding immunoconjugate obtained according to the present invention can be represented, for example, by formula (VI): Ab – (Y) – (Pep) – (Y') – (Z) Formula (VI) in, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody). Y is optionally absent or a spacer. Optionally, Formula VI includes residues between (Ab) and (Y) that react with the side chain of a reactive group (e.g., maleimide, primary amine) and a carbohydrate of an amino acid of the anti-Nectin-4 antigen-binding protein (Ab). Optionally, residues that react with the side chain of a reactive group (e.g., maleimide, primary amine) and a amino acid of the anti-Nectin-4 antigen-binding protein (Ab) may be specified to be included in Y; Pep is or includes an amino acid unit (e.g., a peptide linker) that is cleaved by an intracellular peptidase or protease (e.g., (Pep) is a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, such as a valine-citrulline, valine-alanine, or phenylalanine-lysine unit). Y' is optionally absent or a spacer, optionally a self-eliminating spacer or a non-self-eliminating spacer; and Z is a camptothecin analog or derivative, optionally eczema or SN-38 molecule.

[0200] Optionally, the formula may be specified as (e.g., between the ends of (Ab) and Y (or (Pep or X, if Y is not present)) including a reactive group (R) that reacts with a residue (RR') that reacts with a free amino, hydroxyl, thiol, or carboxyl group on the antibody or with a complementary reactive group (R') of an amino acid attached to the antibody.

[0201] In one example, where (RR') is a residue (e.g., R' attached to the side chain of an amino acid or a glycan of the antibody) that reacts with a reactive group (R) to a complementary reactive group (R') attached to the antibody, the Nectin-4 binding immunoconjugate according to the invention can be, for example, from formula (VI) bis )express: Ab – (RR') – (Y) – (Pep) – (Y’) – (Z) Formula (VI bis ) Wherein, Ab, Y, Pep, Y', and Z are as defined in Formula VI, and RR' is the result of a biorthogonal reaction (preferably a cycloaddition, such as a Diels-Alder reaction or a 1,3-dipolar cycloaddition). In one embodiment, RR' has a structure selected from the group consisting of: (RR') a (RR') b (RR') c (RR') d ) (RR') e (RR')f (RR') g (RR') h ) Where X 8 For O or NH, X 9 Selected from H, methyl, and pyridyl, and in structures (RR'c) and (RR'd), ---- A key indicates a single or double bond.

[0202] In any embodiment, the ixotecan molecule (or other 6-cyclic camptothecin) may be specified to bind to Y' (or (Pep, if Y' is not present) via the amine at position 1 of ixotecan.

[0203] In any embodiment, the SN-38 molecule (or other 5-cyclic camptothecin) may be specified as being bound to Y' (or (Pep, if Y' is not present) via the amine at position 9 of SN-38.

[0204] Camptothecin is well known, as are a wide range of camptothecin derivatives and analogs, which share a core ring system with various substitutions, but preferably have modifications or substitutions in rings A and / or B compared to the following basic camptothecin structure: Rings (A)(B)(C)(D)(E) Numerous camptothecin analogues have been reported, including topotecan, inirotecan, ixenotecan, DXd, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin, gimatecan, belotecan, and rubitecan. Other camptothecin analogues are disclosed in the following literature: Li et al., *ACS Med. Chem. Lett.* 2019, 10, 10, 1386–1392; *Japanese Journal of Cancer Research* 86: 776-782; and Takiguchi et al. 1997, *Japanese Journal of Cancer Research* 88: 760-769. The contents of these references are incorporated herein by reference. Four analogues, topotecan, irinotecan, belotecone, and DXd (as part of delutec-trastuzumab), have been approved by the FDA. In one embodiment, the camptothecin analogue is a pentacyclic compound (e.g., camptothecin lacks the F ring). In one embodiment, the camptothecin analogue is a hexacyclic compound, e.g., including the F ring.

[0205] Some examples, such as the basic camptothecin structure, the SN-38 molecule (7-ethyl-10-hydroxycamptothecin; the active metabolite of irinotecan), and the camptothecin analogues disclosed by Li et al., ACS Medicinal Chemistry Letters 2019, 10, 10, 1386–139, have five rings (rings A, B, C, D, and E) and can be attached to a linker (e.g., spacer Y or Y', or linker X) for example, via a substituent on ring B.

[0206] SN-38: Li et al., compounds in *ACS Medicinal Chemistry Letters*, 2019, 10, 1386–1392: Optionally, camptothecin analogues are hexacyclic compounds (with an additional F ring) in which the compound is attached to a linker via a substituent on such an F ring. Examples of such hexacyclic compounds include, but are not limited to, DXd and eczetidine.

[0207] Therefore, camptothecin analogues include any of the following molecules: eczetidine, SN-38, and a series of molecules including such a moiety, such as eczetidine which may be unsubstituted or substituted at the amine at position 1, for example, wherein the substituent is or includes –O–CH2–C(=O)–, HO–O–CH2–C(=O)–, –CH2CH2–C(=O)–, –CH2CH2CH2–C(=O)–, –CH2–O–CH2–C(=O)–, –CH2CH2–O–CH2–C(=O)– groups or other groups as shown in U.S. Patent No. 6,835,807, the disclosure of which is incorporated herein by reference.

[0208] In one embodiment, the antibody of this disclosure releases eczemac molecules having the structure of compound 1 in vivo or in vitro in the presence of Nectin-4 expressing tumor cells (e.g., during enzymatic cleavage of the cleavable portion followed by self-elimination of spacer Y').

[0209] Camptothecin analogues or analogues such as eciletcan are described in the following references: Mitsui et al. 1995, *Japanese Journal of Cancer Research* 86: 776-782, and Takiguchi et al. 1997, *Japanese Journal of Cancer Research* 88: 760-769, the contents of which are incorporated herein by reference. The structure of eciletcan is shown in the following compound 1a: Compound 1a.

[0210] Ecinotecan can be coupled to the linker via the nitrogen atom of the amino group at position 1, such that when the ecilenotecan moiety is bound to the linker or exists within the linker-ecilenotecan molecule ((XZ) molecule), for example when conjugated with an antibody, ecilenotecan will have the structure of compound 1b: Compound 1b.

[0211] Therefore, it should be understood that when eczetidine of compound 1a is attached to a linker via an amine at position 1 (and, for example, when the linker is further attached to an antibody), eczetidine will be understood as a modified group at position 1 (i.e., the NH2 group at position 1 is replaced by NH, or alternatively by an OH or O group). For example, eczetidine can be conjugated to an antibody via a linker comprising a cleavable oligopeptide. Examples include dipeptides, tripeptides, tetrapeptides, and pentapeptides, such as the glycine and phenylalanine peptides shown in U.S. Patent No. 6,835,807, or dipeptides valine-citrulline or valine-alanine attached to a PAB molecule, or the disclosure of said U.S. Patent, which is incorporated herein by reference. Various suitable linker-Z structures are known to release active eczetidine or eczetidine derivatives at the amino group at position 1. For example, eciletecan can be linked to a cleavable oligopeptide via a (CH2–C(=O)) group attached to an amine at position 1, as shown in Formula III and Compound 13, resulting in the release of eciletecan-containing Compound 13. Examples of substituents at the NH2 position 1 of eciletecan in Compound 1a include (CH2–C(=O)), including groups such as –O–CH2–C(=O)–, HO–O–CH2–C(=O)–, –CH2CH2–C(=O)–, –CH2CH2CH2–C(=O)–, –CH2–O–CH2–C(=O)–, and –CH2CH2–O–CH2–C(=O)–. Ecinotecan can be designated as an eciletecan derivative wherein the NH2 position 1 is substituted with, for example, an NH group, or an OH–CH2–C(=O)-NH group. In one embodiment, the substituted eczetidine or eczetidine derivative (e.g., derived at position 1) has the structure of compound 13.

[0212] In one embodiment, the linker portion (X–Z) is or includes the structure shown in Formula VII, wherein (Y) is a spacer (e.g., at its end) comprising a reactive group (R) with an amino acid residue, such as a free amino, hydroxyl, thiol, or carboxyl residue on an antibody (e.g., an ε-amino group of one or more lysine residues, a free carboxyl group of one or more glutamic acid or aspartic acid residues, or an S atom of one or more cysteine ​​residues). Anti-Nectin-4 binding proteins functionalized with a linker comprising the structure of Formula VII or Compound 3 or 4 will release or produce (e.g., intracellularly, in the presence of Nectin-4 expressing tumor cells) a compound having the structure of Compound 1a. Equation VII An exemplary linker having maleimide as the R group can have the structure of compound 3 below. After reducing the interchain disulfide bonds with a reducing agent (e.g., tris(2-carboxyethyl)phosphonic acid hydrochloride), such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 3 The resulting antibody-drug conjugate will include an antibody comprising one or more cysteine ​​residues functionalized with a compound having the structure of compound 3 (wherein compound 3 is bound via the S atom of the cysteine ​​residue).

[0213] In another embodiment, the linker may have a primary amine as the R group, and when reacted with an antibody in the presence of transglutaminase, an antibody comprising one or more receptor glutamine residues functionalized with the linker may be produced. For example, the linker (X–Z) or the antibody functionalized with said linker has or comprises a structure as shown in Compound 4 below: Compound 4.

[0214] In one embodiment, the linker portion (X–Z) is or includes the structure shown in Formula VIII, wherein (Y) is a spacer (e.g., at its end) comprising a reactive group (R) with an amino acid residue, such as a residue reacting with a free amino, hydroxyl, thiol, or carboxyl group on an antibody (e.g., an ε-amino group of one or more lysine residues, a free carboxyl group of one or more glutamic or aspartic acid residues, or an S atom of one or more cysteine ​​residues), or a glycan structure such as glycosylated amino acid residues (e.g., a native, truncated, or otherwise modified N-glycan that binds to the Kabat residue N297 of the antibody). The anti-Nectin-4 binding protein functionalized with a linker comprising the structure of Formula VIII or compounds 5, 6, or 7 will release or produce (e.g., intracellularly, in the presence of Nectin-4 expressing tumor cells) a compound having the structure of compound Ia. Formula VIII.

[0215] Exemplary linkers having maleimide as the R group can have the structure of compounds 5, 6, or 7. After reducing the interchain disulfide bonds with a reducing agent, such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 5 (mal-Val-Cit-PAB-icienta). Compound 6 (mal-PEG8-Val-Cit-PAB-Ecinotecan). Compound 7 (Mal-Glu-(Val-Cit-PAB-Ecinotecan)-PEG8).

[0216] In another embodiment, the linker may have a primary amine as the R group, and when reacted with an antibody in the presence of transglutaminase, an antibody comprising one or more receptor glutamine residues functionalized with the linker may be produced. For example, the linker (X–Z) or the antibody functionalized with said linker has or comprises a structure as shown in Compound 8 below: Compound 8.

[0217] In one embodiment, the linker portion (X–Z) is or includes a structure as shown in Formula IX, wherein (Y) is a spacer (e.g., at its end) comprising a reactive group (R) with an amino acid residue, such as a residue reacted with a free amino, hydroxyl, thiol, or carboxyl group on an antibody (e.g., an ε-amino group of one or more lysine residues, a free carboxyl group of one or more glutamic acid or aspartic acid residues, or an S atom of one or more cysteine ​​residues), or a glycan structure such as glycosylated amino acid residues (e.g., a native, truncated, or otherwise modified N-glycan that binds to the Kabat residue N297 of an antibody). The anti-Nectin-4 binding protein functionalized with a linker comprising Formula IX will release (e.g., intracellularly, in the presence of Nectin-4 expressing tumor cells) a compound having the structure of compound Ia.

[0218] Formula IX.

[0219] Exemplary linkers having maleimide as the R group can have the structures of compounds 9a, 9b, 9c, and 9d. After reducing the interchain disulfide bonds with a reducing agent, such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 9a. Compound 9b. Compound 9c. Compound 9d In another embodiment, the linker may have a primary amine as the R group, and when reacted with an antibody in the presence of transglutaminase, an antibody comprising one or more receptor glutamine residues functionalized with the linker may be produced. For example, the linker (X–Z) or the antibody functionalized with said linker has or comprises a structure as shown in compound 10 below: Compound 10.

[0220] In one embodiment, the linker portion (X–Z) is or includes a structure as shown in Formula X, where (Y) is a spacer (e.g., at its end) comprising a reactive group (R) with an amino acid residue, such as a residue reacted with a free amino, hydroxyl, thiol, or carboxyl group on an antibody (e.g., an ε-amino group of one or more lysine residues, a free carboxyl group of one or more glutamic acid or aspartic acid residues, or an S atom of one or more cysteine ​​residues), or a glycan structure such as glycosylated amino acid residues (e.g., a native, truncated, or otherwise modified N-glycan bound to the Kabat residue N297 of an antibody). Formula X.

[0221] An exemplary linker having maleimide as the R group can have the structure of compound 11. After reducing the interchain disulfide bonds with a reducing agent (e.g., tris(2-carboxyethyl)phosphonic acid hydrochloride), such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 11.

[0222] In one embodiment, the linker portion (X–Z) is or includes a structure as shown in Formula XI, wherein (Y) is a spacer (e.g., at its end) comprising a reactive group (R) with an amino acid residue, such as a residue reacted with a free amino, hydroxyl, thiol, or carboxyl group on an antibody (e.g., an ε-amino group of one or more lysine residues, a free carboxyl group of one or more glutamic acid or aspartic acid residues, or an S atom of one or more cysteine ​​residues), or a glycan structure such as glycosylated amino acid residues (e.g., a native, truncated, or otherwise modified N-glycan bound to the Kabat residue N297 of an antibody). Formula XI.

[0223] An exemplary linker having maleimide as the R group can have the structure of compound 12. After reducing the interchain disulfide bonds with a reducing agent, such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 12.

[0224] In another embodiment, the linker may have a primary amine as the R group, and when reacted with an antibody in the presence of transglutaminase, an antibody comprising one or more receptor glutamine residues functionalized with the linker may be produced. For example, the linker (X–Z) or the antibody functionalized with said linker has or comprises a structure as shown in compound 13 below: Compound 13.

[0225] The release (e.g., intracellularly, in the presence of Nectin-4-expressing tumor cells) of the oligopeptide linker-functionalized anti-Nectin-4 binding protein of formula XI or compounds 12 and 13, of the substituted eczema, having an OH–CH2–C(=O) substituent at the amine at position 1, as shown in the structure of eczema below: Compound 14.

[0226] Further examples of suitable connectors shown in compounds 15 and 16 below have a maleimide as an R group and a phenylalanine-containing peptide attached to the PAB molecule. Compound 15. Compound 16.

[0227] An exemplary linker having maleimide as an R group and orthogonal polysarcosine moiety is shown, which can have the structure of compound 17. After reducing the interchain disulfide bonds with a reducing agent, such linkers can be conjugated to antibodies via cysteine ​​residues in the antibody. Compound 17 (mal-PSAR16-ecciotecan).

[0228] In any exemplary linker, when bound to an antibody, the reactive group at the end may be specified to be replaced by a residue that reacts with an amino acid residue on the antibody (e.g., a free amino, hydroxyl, thiol, or carboxyl group of an amino acid).

[0229] When prepared as a structure having a primary amine, exemplary linkers of formulas III, IV, V, VI, VII, VIII, IX, X, or XI can react with the antibody in the presence of a transglutaminase (e.g., bacterial transglutaminase, BTG), such that the transglutaminase catalyzes the conjugation of the linker to a receptor glutamine residue within the antibody's primary structure (e.g., within an immunoglobulin constant domain or within a TGase recognition tag inserted into or attached to (e.g., fused to) a constant region). Methods and linkers for BTG-mediated antibody conjugation are described in PCT Publication WO2014 / 202773, the disclosure of which is incorporated herein by reference. BTG-catalyzed conjugation allows for precise control of the average drug:antibody ratio in the composition. The term "transglutaminase," used interchangeably with "TGase" or "TG," refers to an enzyme capable of cross-linking proteins to form ε-(γ-glutamyl)-lysine isopeptide bonds via an acyl transfer reaction between the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of a lysine or a structure-related primary amine (such as an aminopentyl group, e.g., peptide-bound lysine). TGase particularly includes bacterial transglutaminases (BTG), such as enzymes having EC reference EC 2.3.2.13 (protein-glutamine-γ-glutamyltransferase). The term "receptor glutamine" residue, when referring to an antibody's glutamine residue, refers to a glutamine residue recognized by TGase and capable of being cross-linked by TGase via a reaction between glutamine and a lysine or a structure-related primary amine (such as an aminopentyl group). Preferably, the receptor glutamine residue is a surface-exposed glutamine residue. The term "TGase recognition tag" refers to an amino acid sequence comprising receptor glutamine residues, which, when incorporated (e.g., attached to) a polypeptide sequence under suitable conditions, is recognized by TGase and results in TGase crosslinking through a reaction between the amino acid side chains within the amino acid sequence and the reaction partner. The recognition tag can be a peptide sequence not naturally present in the polypeptide comprising the enzyme recognition tag. Examples of TGase recognition tags include the amino acid sequences: LLQ, LLQG, LSQG, GLLQ, SLLQG, GGGQGGL, LLQGG, LLQGA, LLQGG, and LLQGA, or EQKLISEEDL, or variants having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) sequence modifications.

[0230] As illustrated in WO2013 / 092983 and WO2020 / 188061 (the disclosures of which are incorporated herein by reference), the linker-camptothecin analog moiety (XZ) can be conjugated to a glutamine residue (receptor glutamine) in an antibody in a two-step process comprising: a first step in which the moiety comprising a primary amine and a first reactive group (R) is conjugated to the antibody in the presence of BTG; and a subsequent step of reacting the antibody-linker conjugate with a molecule comprising: (i) a second reactive group (R') reactive to the first reactive group; and (ii) a camptothecin analog (Z). Examples of reactive groups for R and R' include a range of groups capable of bisorthogonal reactions, such as 1,3-dipolar cycloaddition between azide and cyclooctyne (copper-free click chemistry), 1,3-dipolar cycloaddition between nitroketone and cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, and tetrazine linkages (see also WO2013 / 092983). The resulting linkers and functionalized antibodies, or their Y-elements, can therefore include RR' groups, such as triazoles, generated from the reactions of R and R'.

[0231] The anti-Nectin-4 immunoconjugate may be incorporated into the pharmaceutical formulation at a concentration of 1 mg / ml to 500 mg / ml, wherein the pH of the formulation is 2.0 to 10.0. The formulation may further include a buffer system, one or more preservatives, one or more tension agents, one or more chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation comprising water. Such formulations are typically solutions or suspensions. In another embodiment, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0232] In another embodiment, the drug formulation is a lyophilized formulation, to which a physician or patient adds solvents and / or diluents prior to use.

[0233] In another embodiment, the pharmaceutical formulation is a dry formulation (e.g., freeze-dried or spray-dried) that can be used without any pre-dissolution.

[0234] In another aspect, the drug formulation comprises an aqueous solution and a buffer solution of such antibodies, wherein the antibodies are present at a concentration of 1 mg / ml or higher, and wherein the pH of said formulation is from about 2.0 to about 10.0.

[0235] In another embodiment, the pH of the formulation is within a range selected from the following list: about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

[0236] In another embodiment, the buffer is selected from the group consisting of: sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)aminomethane, dihydroxyethylglycine, trimethylglycine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

[0237] In other embodiments, the formulation further comprises a pharmaceutically acceptable preservative. In other embodiments, the formulation further comprises an isotonic agent. In other embodiments, the formulation further comprises a chelating agent. In other embodiments of the invention, the formulation further comprises a stabilizer. In other embodiments, the formulation further comprises a surfactant. For convenience, refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0238] Other components may be present in the pharmaceutical formulations disclosed herein. These additional components may include wetting agents, emulsifiers, antioxidants, fillers, tension modifiers, chelating agents, metal ions, oily mediators, proteins (e.g., human serum albumin, gelatin, or other proteins), and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, and histidine). Of course, these additional components should not adversely affect the overall stability of the pharmaceutical formulations disclosed herein.

[0239] The pharmaceutical composition according to the invention can be administered via several routes, such as intravenous administration. Suitable antibody formulations can also be determined by examining experience with other developed therapeutic ADCs.

[0240] In any embodiment, the composition may be characterized as comprising a plurality of the Nectin-4 binding immunoconjugates disclosed herein, wherein at least 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the sample have at least 4, 6, or 8 amino acid residues per antibody, said amino acid residues being functionalized with the linker disclosed herein. In any embodiment, the composition may be characterized as comprising a plurality of the Nectin-4 binding immunoconjugates disclosed herein, wherein at least 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the sample have at least 2, 4, 6, or 8 amino acid residues per antibody, said amino acid residues being functionalized with a linker-camptothecin moiety, such as the (X–Z) unit or (–(Y)–(Pep)–(Y')–(Z)) unit of the chemical formula herein. In any embodiment, the composition may be characterized as comprising a plurality of Nectin-4 binding immunoconjugates of the present disclosure, wherein at least 70%, 80%, 90%, 95%, 98%, or 99% of the immunoconjugates in the sample have the same number of functionalized amino acids for each antibody, optionally wherein said number is 4, 6, or 8.

[0241] Diagnosis, prognosis and treatment of malignant tumors In some aspects, methods for diagnosing, prognosing, monitoring, and treating cancers characterized by tumor cells expressing Nectin-4 on their surface are described, along with antigen-binding proteins (e.g., antibodies, antibody fragments) and immunoconjugates. In therapeutic use, the treatment comprises administering an antibody-camptothecin analog of the present disclosure to a human subject or individual. Examples are methods for diagnosing, prognosing, monitoring, and treating Nectin-4-expressing cancers, wherein a Nectin-4 binder is conjugated to a camptothecin analog or derivative molecule, such as eczema or SN-38. Suitable Nectin-4 binders are typically conjugated to multiple molecules of camptothecin analogs. Camptothecin analogs can be conjugated to antibodies via adapters including protease-cleavable oligopeptide adapters. Exemplary pharmaceutical compositions may comprise an average of 1 to 8 camptothecin analog molecules per antibody molecule, optionally 2-8, 4-8, 6-8 camptothecin analog molecules per antibody molecule, or, for example, about 4, 5, 6, 7, or 8 camptothecin analog molecules per antibody molecule.

[0242] Nectin-4 conjugates (e.g., anti-Nectin-4 antibodies or antibody fragments) conjugated with camptothecin analogues can be advantageously used to treat individuals with Nectin-4-expressing cancers characterized by tumor cells expressing Nectin-4 (e.g., at the tumor cell membrane or cell surface). Examples of such cancers include urothelial carcinoma, breast cancer (e.g., triple-negative breast cancer; HER2-positive breast cancer), non-small cell lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, colorectal cancer (e.g., colon cancer), head and neck squamous cell carcinoma, and esophageal cancer.

[0243] Nectin-4 conjugates, which are conjugated with camptothecin analogues, can be used in tumors that highly express Nectin-4.

[0244] Nectin-4 conjugates conjugated with camptothecin analogues can also be used for heterogeneous and / or low-Nectin-4-expressing tumors. In such tumors, the immunoconjugates of this disclosure can provide advantageous efficacy, optionally by avoiding MDR1-mediated resistance and / or bystander antitumor effects.

[0245] Nectin-4 conjugates conjugated with camptothecin analogues can be advantageously used to treat individuals regardless of their Nectin-4 expression levels, the heterogeneity of Nectin-4 expression levels on tumor cells within the individual, and / or whether the individual has previously been treated with vitin-enterotolumab. For example, Nectin-4 conjugates conjugated with camptothecin analogue molecules can be advantageously used to treat individuals who have previously been treated with vitin-enterotolumab. Such individuals may optionally have cancer characterized by heterogeneous and / or low Nectin4 expression tumors following vitin-enterotolumab treatment. An individual may have cancer that, despite treatment with an antibody conjugated to an auristatin or MMAE molecule (e.g., vitin-entaftuzumab), remains (e.g., during or after) resistant, unresponsive, recurrent, and / or progressive. For example, an individual may have locally advanced or metastatic urothelial carcinoma and has previously received treatment with an antibody conjugated to an auristatin or MMAE molecule (e.g., vitin-entaftuzumab).

[0246] In advanced recurrent or metastatic urothelial carcinoma, a significant proportion of individuals express high levels of Nectin-4 on tumor cells, with an H score of at least 290 (see Nectin-4 expression in cohort 1 of the EV-201 clinical trial). However, a subset of patients have an H score less than 250, and some less than 200. A minority of patients have an H score less than 150, and some have an H score less than 100. In triple-negative breast cancer (TNBC), 62% of patients have been reported to have high Nectin-4 expression on tumor cells, and 38% have low Nectin-4 expression (Rabat et al., 2017 Annals Onc. 28: 769-776). In other cancer types, the median H score for Nectin-4 expression is generally lower than that observed in UC, particularly in non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer.

[0247] In one embodiment, the individual treated according to this disclosure has advanced recurrent or metastatic cancer, optionally advanced recurrent or metastatic urothelial carcinoma.

[0248] In one embodiment, an individual treated according to this disclosure has breast cancer that is positive for estrogen receptor and / or progesterone receptor tests but negative for epidermal growth factor receptor 2 (HER2) or excess HER2 protein tests, optionally the cancer being positive for HER2 but HER2 being expressed at low levels.

[0249] In one embodiment, an individual treated according to this disclosure has triple-negative breast cancer (TNBC), such as breast cancer that is negative for estrogen receptor, progesterone receptor and excess HER2 protein tests.

[0250] In one embodiment, the individual treated according to this disclosure has breast cancer that tests positive for HER2 protein, optionally wherein the cancer expresses an excess of HER2 protein (HER2 overexpression), or optionally the cancer expresses a low level of HER2 protein (below the level of HER2 overexpression). In one embodiment, the individual is treated with a combination of an anti-Nectin-4 ADC according to this disclosure and an agent that binds a HER2-binding peptide (e.g., an antibody, such as trastuzumab, pertuzumab); optionally wherein the HER2-binding antibody is an ADC; optionally wherein the HER2-binding antibody is conjugated to a cytotoxic agent, optionally olistatin, maytansine (e.g., DM1), or camptothecin analogue (e.g., compound 1, 2, or 13); optionally wherein the HER2-binding antibody is emtansine-trastuzumab or delutec-trastuzumab (DS-8201a).

[0251] In one embodiment, the individual treated according to this disclosure has non-small cell lung cancer, optionally with lung adenocarcinoma.

[0252] In one embodiment, the individual treated according to this disclosure has pancreatic cancer.

[0253] In one embodiment, the individual treated according to this disclosure has ovarian cancer.

[0254] In one embodiment, the individual treated according to this disclosure has squamous cell carcinoma of the head and neck.

[0255] In one embodiment, the individual treated according to this disclosure has esophageal cancer.

[0256] In one embodiment, the individual treated according to this disclosure has colorectal cancer. As used herein, colorectal cancer (CRC) refers to colon cancer, rectal cancer, and colorectal cancer (cancer of both the colonic region and the rectal region).

[0257] In one embodiment, the individual treated according to this disclosure has NSCLC or lung adenocarcinoma, gastric cancer, colorectal cancer, pancreatic cancer, urothelial carcinoma, or bladder cancer that tests positive for HER2 protein, optionally wherein said cancer expresses an excess of HER2 protein (HER2 overexpression), optionally said cancer expresses a low level of HER2 protein (below the level of HER2 overexpression). In one embodiment, the individual is treated in combination with an anti-Nectin-4 ADC according to this disclosure and an agent that binds a Her2 peptide (e.g., an antibody, such as an antibody comprising the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab); optionally said Her2-binding antibody is an ADC; optionally said Her2-binding antibody is conjugated to a cytotoxic agent, optionally olistatin, maytansine (e.g., DM1), or camptothecin analogue (e.g., compound 1, 2, or 14); optionally said Her2-binding antibody is emtansine-trastuzumab or delutec-trastuzumab (DS-8201a).

[0258] In one respect, the treatment method disclosed herein does not depend on the assessment or detection of Nectin-4 expression in tumor tissue and / or on the expression level of Nectin-4 on tumor cells and / or the frequency or number of Nectin-4-expressing tumor cells in tissue samples from said individuals.

[0259] In one aspect, this disclosure provides methods for treating cancer and / or inducing an antitumor immune response in individuals in need, wherein the individuals have advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC), wherein the methods do not require prior determination of whether the individuals have tumor tissue comprising cells expressing Nectin-4 (e.g., tumor cells).

[0260] In one aspect, this disclosure provides a method for treating cancer and / or killing tumor cells in an individual in need, wherein the individual has advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC), wherein the method does not require prior determination of whether the individual has tumor tissue comprising cells expressing high levels of Nectin-4 (e.g., tumor cells), as defined by immunohistochemical assessment (e.g., H score or other appropriate IHC scoring method).

[0261] On the one hand, methods for treating cancer and / or killing tumor cells in individuals do not require prior determination of the Nectin-4 expression level of tumor cells.

[0262] On the one hand, methods for treating cancer in individuals, optionally advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC, HER2-positive cancer), include treating individuals with cancer characterized by Nectin-4 expression and an H score of no greater than or less than 290, 250, 200, 150, or 100.

[0263] In any embodiment for treating or preventing cancer in an individual, the method may be specified to include the steps of: (i) identifying an individual whose tumor cells express Nectin-4 (e.g., determined by immunohistochemistry); and (ii) administering to the individual an effective amount of the disclosed anti-Nectin-4 antibody camptothecin analogue drug conjugate.

[0264] In any embodiment used for treating or preventing cancer in an individual, the method may be specified to include the following steps: (i) identifying an individual whose tumor cells express (a) Nectin-4 (e.g., determined by immunohistochemistry) and (b) HER2, optionally wherein said tumor cells express low levels of HER2 (e.g., determined by immunohistochemistry; determined by Herceptest™); and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody conjugated to a camptothecin analog or derivative molecule, optionally in combination with an agent that binds a Her2 peptide (e.g., an antibody, such as trastuzumab, pertuzumab); optionally wherein said Her2-binding antibody is an ADC; optionally wherein said Her2-binding antibody is conjugated to a cytotoxic agent, optionally olistatin, maytansine (e.g., DM1), or a camptothecin analog (e.g., compound 1, 2, or 14); optionally wherein said Her2-binding antibody is emtansine-trastuzumab or delutec-trastuzumab (DS-8201a).

[0265] In any embodiment used for treating or preventing cancer in an individual, the method may be specified to include the following steps: (i) identifying an individual whose tumor cells have low or intermediate levels of Nectin-4 expression (e.g., determined by immunohistochemistry); and (ii) administering an effective amount of an anti-Nectin-4 antibody conjugated to a camptothecin analog or derivative molecule to the individual identified in step (i).

[0266] In any embodiment used for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method may be specified to include the following steps: (i) identifying an individual whose cancer is characterized by low levels of Nectin-4 expression (e.g., determined by immunohistochemistry); and (ii) administering an effective amount of the disclosed anti-Nectin-4 antibody-camptothecin analogue drug conjugate to the individual identified in step (i). In one embodiment, the individual has cancer characterized by an H score of no greater than or less than 150 or 100 for Nectin-4 expression.

[0267] In any embodiment for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method may be specified to include the steps of: (i) identifying an individual whose cancer is characterized by intermediate levels of tumor Nectin-4 expression (e.g., determined by immunohistochemistry); and (ii) administering to the individual an effective amount of the disclosed anti-Nectin-4 antibody-camptothecin analogue drug conjugate. In one embodiment, the individual has cancer characterized by an H score of Nectin-4 expression no greater than or less than 290, 250, 200, or 150, optionally further wherein the cancer is characterized by an H score of at least 100 for Nectin-4 expression.

[0268] In yet another embodiment, a method is provided for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method comprising: (i) identifying an individual whose cancer is characterized by an H score of no greater than or less than 290, 250, 200, 150, 120, or 100 for tumor Nectin-4 expression; and (ii) administering to the individual an effective amount of the disclosed anti-Nectin-4 antibody-camptothecin analogue drug conjugate. Optionally, step (i) may be specified as including a step of assessing Nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0269] In yet another embodiment, a method is provided for treating or preventing cancer (e.g., Nectin-4 positive cancer; breast cancer) in an individual, the method comprising: (i) identifying an individual whose cancer is characterized by a QS score of no greater than or less than 200, 150, 120, or 100 for tumor Nectin-4 expression; and (ii) administering to the individual an effective amount of the disclosed anti-Nectin-4 antibody-camptothecin analogue drug conjugate. Optionally, step (i) may be specified as including a step of assessing Nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0270] Biological samples from individuals, such as those from biopsies, can be acquired and evaluated. Optionally, samples can be preserved as formaldehyde (e.g., formalin)-fixed paraffin-embedded (FFPE) samples. After dewaxing, the slides are suitable for methods that detect Nectin-4 (and / or HER2) expression.

[0271] The expression of Nectin-4 and / or HER2 in tumor cells can be determined by any method known in the art. In some embodiments, the assay includes immunohistochemical (IHC) assays, fluorescence activated cell sorting (FACS) assays, such as quantitative FACS, ELISA, immunoblotting (e.g., Western blotting, dot blot, or intracellular Western blotting), and other immunoassays.

[0272] IHC staining of tissue sections has proven to be a reliable method for assessing or detecting the presence of proteins in a sample. Immunohistochemistry utilizes antibodies to detect and visualize cellular antigens in situ, typically via colorimetric or fluorescent methods. Therefore, antibodies or antisera (in some embodiments, polyclonal antisera, and in some embodiments, monoclonal antibodies specific to each marker) are used to detect expression. Antibodies can be detected by directly labeling the antibody itself, for example, with radiolabeling, fluorescent labeling, hapten labeling (such as biotin), or enzyme labeling (such as horseradish peroxidase or alkaline phosphatase). Alternatively, unlabeled primary antibodies can be used in combination with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific to the primary antibody. Immunohistochemical protocols and kits are well known in the art and are commercially available.

[0273] In some embodiments, IHC assays are direct assays, where the binding of an antibody to a target antigen is directly determined. This direct assay uses labeled reagents, such as fluorescent tags or enzyme-labeled primary antibodies, which can be observed without additional antibody interactions. In some embodiments, IHC assays are indirect assays. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, followed by a labeled secondary antibody binding to the primary antibody. When the secondary antibody binds to an enzyme label, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. Primary and / or secondary antibodies used in immunohistochemistry are typically labeled with detectable molecules. A variety of tags are available, including radioisotopes, colloidal gold particles, fluorescent tags, and enzyme substrate tags.

[0274] Strong staining, moderate staining, and weak staining are descriptions well known to those skilled in the art. In some aspects, strong staining, moderate staining, and weak staining are calibrated staining levels, where a range is established and staining intensity is graded within that range. In some embodiments, strong staining is staining above the 75th percentile of the intensity range, moderate staining is staining between the 25th and 75th percentiles of the intensity range, and weak staining is staining below the 25th percentile of the intensity range. In some aspects, those skilled in the art, particularly those skilled in a particular staining technique, adjust the bin size and define the staining categories.

[0275] (For example, when stained with anti-Nectin-4 antibody) Control cell lines with different staining intensities (e.g., centrifuged into clumps, formalin fixed and paraffin embedded, prepared as tissue microarrays, and stained with anti-Nectin-4 antibody, for example) can be used as controls for IHC analysis. Those skilled in the art will understand that other control cell clumps with negative, weak, moderate, and high c-met staining intensities can be readily identified using the teachings and methods known in the art and disclosed herein.

[0276] In some embodiments, a cancer or tumor is considered Nectin-4 expressing when it is (e.g., determined using an IHC assay) Nectin-4 positive. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 5% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 10% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 20% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 30% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 40% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein expressed at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 50% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein expressed at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 60% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein expressed at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 70% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein expressed at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 80% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein expressed at any intensity). In some embodiments, an individual’s cancer or tumor is Nectin-4 positive when 90% or more of the tumor cells in a sample express Nectin-4 protein (e.g., Nectin-4 protein is expressed at any intensity).

[0277] In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 5% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 10% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 20% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 30% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 40% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 50% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 60% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 70% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 80% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 90% or more of the tumor cells in a sample express Nectin-4 protein with moderate and / or strong staining intensity.

[0278] Evaluating immunohistochemical assays to determine whether an individual’s cancer or tumor is characterized by high Nectin-4 expression (e.g., low or moderate Nectin-4 expression) will typically involve applying known scoring methods.

[0279] Low, intermediate, and high tumor Nectin-4 expression can be determined based on the “H score” described in U.S. Patent No. 2013 / 0005678. The H score is obtained by the following formula: (3 × percentage of strongly stained cells) + (2 × percentage of moderately stained cells) + (percentage of weakly stained cells), resulting in a range of 0 to 300. The H score is specifically used in ulcerative colitis (UC).

[0280] In some embodiments of any of the methods described herein, low or moderate Nectin-4 expression (e.g., tumors or tumor cells with low or moderate Nectin-4 expression levels) corresponds to an H score of about 250 or less, about 220 or less, about 200 or less, about 180 or less, about 160 or less, about 150 or less, about 140 or less, about 130 or less, about 120 or less, about 110 or less, or about 100 or less.

[0281] In some embodiments of any of the methods described herein, low Nectin-4 expression (e.g., tumors or tumor cells with low Nectin-4 expression levels) corresponds to an H score of 200 or lower, about 180 or lower, about 160 or lower, about 150 or lower, about 140 or lower, about 130 or lower, about 120 or lower, about 110 or lower, or about 100 or lower.

[0282] In some embodiments of any of the methods described herein, high Nectin-4 expression (e.g., tumors or tumor cells with high Nectin-4 expression levels) corresponds to an H score of approximately 290 or higher.

[0283] In another instance, Nectin-4 staining can be scored using the Quick score (QS) formula: QS = P (percentage of positive cells) × I (intensity), with a maximum score of 300. QS has been used, for example, in breast cancer. For instance, in TNBC, some research groups have defined a low Nectin-4 expression group as having a QS = or < 100. In some embodiments of any of the methods described herein, low or moderate Nectin-4 expression (e.g., tumors or tumor cells with low or moderate Nectin-4 expression levels) corresponds to a QS score of approximately 200 or lower, approximately 180 or lower, approximately 160 or lower, approximately 150 or lower, approximately 140 or lower, approximately 130 or lower, approximately 120 or lower, approximately 110 or lower, or approximately 100 or lower.

[0284] Assays for assessing HER2 expression in tumor cells are well-known in the field. For example, assays such as the FDA-approved SPoT-Light HER2 CISH can be used to detect HER2 overexpression. Chromogenic in situ hybridization (CISH) detects HER2 gene amplification. This technique, also known as subtraction probe technology chromogenic in situ hybridization, is a test used to detect whether breast cancer cells overexpress the HER2 receptor protein on their cell surface.

[0285] Another widely used HER2 assay is HercepTest™ (Dako NorthAmerica, Inc.), a semi-quantitative immunohistochemical assay used to determine HER2 protein overexpression in formalin-fixed, paraffin-embedded cancer tissue. For example, HercepTest™ can be used to identify tumors expressing low levels of HER2 with a score of +1 to +2.

[0286] In one aspect, the treatment is intended for individuals with pre-existing neuropathy, diabetes or hyperglycemia, heart failure, or eye disease. Such conditions may make an individual unsuitable for treatment with anti-Nectin-4 ADCs (such as Vitin-Emfretuzumab) that have higher toxicity or a narrower therapeutic window than the anti-Nectin-4 antibody-drug conjugates disclosed herein.

[0287] In any embodiment, the treatment method may optionally include the following steps: (a) assessing the individual’s cancer stage and / or disease progression; and (b) if the individual has recurrent, metastatic, and / or progressive cancer, administering to the individual an effective amount of an anti-Nectin-4 antibody conjugated to a camptothecin analog or derivative molecule.

[0288] In some embodiments, the present invention includes a method of treating a tumor in an individual with urothelial carcinoma, the method comprising: (a) assessing the individual’s cancer stage and / or disease progression; and (b) if the individual has recurrent, metastatic, and / or progressive cancer, administering to the individual an effective amount of an anti-Nectin-4 antibody conjugated to an eczema molecule.

[0289] Optionally, the individual may have cancer that is resistant, unresponsive, recurrent, and / or progressive despite treatment with surgery and / or therapeutic agents (e.g., during or after treatment with chemotherapy agents, antibodies, ADCs, or radiotherapy) (e.g., urothelial carcinoma, breast cancer (e.g., triple-negative breast cancer; HER2-positive cancer), non-small cell lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma, or esophageal cancer).

[0290] In any of the embodiments described herein, treatment response may be defined and / or assessed according to well-known criteria, such as the Solid Tumor Response Evaluation Criteria (RECIST), version 1.1, see Eisenhauer et al. (2009), European Journal of Cancer 45:228-247, or the Immune-Related Response Criteria (irRC), see Wolchock et al. (2009), Clinical Cancer Research 15:7412-7420.

[0291] Optionally, individuals treated with the anti-Nectin-4 antibody-drug conjugates of this disclosure have tumors or cancers that have shown resistance, are unresponsive to, or progressed after treatment with chemotherapeutic agents known to be transported by P-glycoproteins (Pgp), such as anthracyclines (doxorubicin, daunorubicin, taxanes (paclitaxel, docetaxel), vinblastine alkaloids (vincristine, vinblastine, vindesine), and etoposide. Compounds recognized by Pgp are generally characterized as moderately hydrophobic (octanol partition coefficient to water, logP>1), typically contain titratable protons with a net cationic charge under physiological conditions, and are primarily “natural products” with an aromatic moiety.

[0292] In some embodiments, an ADC comprising an anti-Nectin-4 antibody or an antibody fragment is used or administered without the combination of chemotherapeutic agents. Optionally, the individual may be characterized as having cancer that has progressed, relapsed, or is unresponsive to prior treatment with a prior therapy, optionally further wherein the prior therapy comprises administration of vetin-entaftuzumab and / or administration of a PD-1 neutralizer (e.g., pembrolizumab, atezolizumab, nivolumab), optionally wherein the prior therapy is a chemotherapeutic agent.

[0293] Optionally, in any embodiment, the individual may be characterized as unsuitable for treatment with vitin-entaftuzumab and / or suffering from cancer unsuitable or inappropriate for vitin-entaftuzumab treatment.

[0294] An exemplary treatment regimen for treating a person with an anti-Nectin-4 antibody conjugated to a camptothecin analog molecule includes, for example, administering to a patient an effective amount of the anti-Nectin-4 antibody conjugated to a camptothecin analog molecule, wherein the method includes at least one administration cycle in which at least one dose of the anti-Nectin-4 antibody conjugated to a camptothecin analog molecule is administered at doses of 0.1-10 mg / kg body weight, 0.1-5 mg / kg body weight, 0.1-1 mg / kg body weight, 1-10 mg / kg body weight, or 1-5 mg / kg body weight is administered. In one embodiment, multiple doses are administered, such as at least 2, 3, 4, 5, 6, 8, or 10 doses. In one embodiment, the doses are administered at intervals of at least 2, 3, or 4 weeks. In one embodiment, the administration cycle is between 2 and 8 weeks, or at least 4, 6, 8, or 16 weeks.

[0295] In one embodiment, the anti-Nectin-4 antibody conjugated to a camptothecin analog molecule is administered via intravenous administration.

[0296] Example Example 1: Human tumor cells co-expressing Her2 and Nectin-4.

[0297] Based on multidimensional maps of key genomic changes in different types of cancer, the expression of HER2 and Nectin-4 genes was investigated using the Cancer Genome Atlas (a collaboration between the National Cancer Institute and the National Human Genome Institute). Significant correlations between HER2 and Nectin-4 expression were observed, particularly in samples from pancreatic cancer, lung adenocarcinoma, breast cancer, and bladder cancer patients. The highest observed correlation was in pancreatic cancer, with correlation values ​​of Spearman 0.71 and Pearson 0.78.

[0298] HER2 and Nectin-4 expression in the SUM185 and SUM190 human breast cancer cell lines (Biovit, Inc.) were determined by flow cytometry. SUM185 was derived from pleural effusion in ER-negative patients. It represents PR-negative and Her2-positive anaplastic breast cancer, with overexpression of Her2. SUM190 was derived from primary tumors in ER-negative patients. It represents PR-negative and Her2-positive (amplified) breast cancer. Tumor cells were stained with anti-Nectin-4 antibody (ASG-22ME, modified to contain the N297Q mutation in the human IgG1 isotype with reduced Fcγ receptor binding) or anti-Her2 antibody (trastuzumab, modified to contain the N297Q mutation in the human IgG1 isotype with reduced Fcγ receptor binding) and an isotype control at a concentration of 10 µg / m² (at 4°C for 15 minutes), followed by staining with a 1:200 dilution of PE-conjugated polyclonal goat anti-human antibody. Samples were analyzed using Canto II (HTS) via cellular fluorescence analysis.

[0299] Figure 1 Representative results from SUM190 human breast cancer tumor cells were presented, and Figure 2 Representative results for SUM185 human breast cancer tumor cells are shown. MFI: mean fluorescence intensity. SUM190 tumor cells expressed HER2 at low to moderate levels (median fluorescence unit 1777) and Nectin-4 at low levels (median fluorescence unit 991). SUM185 cells expressed HER2 at moderate to high levels (median fluorescence unit 2880) and Nectin-4 at high levels (median fluorescence unit 4326). Example 2: Efficacy of anti-Nectin-4 ADC functionalized with camptothecin analogs against HER2+ Nectin4+ human tumor cells Anti-Nectin-4 antibody-drug conjugates were prepared and their efficacy against HER2+Nectin4+ human tumor cells was compared with that of trastuzumab antibody-drug conjugates. VH and VL, having SEQ ID NO: 9 and 10, were prepared as anti-Nectin-4 antibody-drug conjugates of the human IgG1 isotype. Anti-Her2 antibody-drug conjugates containing the heavy and light chains of trastuzumab (human IgG1 isotype) were prepared. After partially reducing the interchain disulfide bonds, both the anti-Nectin-4 antibody and the anti-Her antibody were randomly conjugated to a linker-camptothecin analog via cysteine ​​residues in the antibody. The disulfide was reduced by incubating 2-10 molar equivalents of the reducing agent tris(2-carboxyethyl)phosphonic acid hydrochloride with the antibody (3 mg / mL) at stirring (350-400 rpm, +37°C) for 2 hours. The linker-toxin conjugation was performed by adding a molar excess (9.2 or 12 molar equivalents) of the linker-toxin, which was incubated overnight at +37°C on a stir wheel. The average drug loading (drug:antibody ratio) of the resulting ADC was approximately 8. In another example, the anti-Nectin-4 antibody was also conjugated to a second camptothecin (SN-38) containing a linker using the same method. The ADC used in this example is as follows.

[0300] N4 ADC1: Anti-Nectin-4 coupled with a connector having the following structure: Her2 ADC1: Her2-resistant connector with a connector having the following structure: N4 ADC2: Anti-Nectin-4 coupled with a connector having the following structure: .

[0301] The ability of the obtained ADCs to induce cell death in Nectin-4 / Her2-expressing SUM190 and SUM185 tumor cells of Example 1 was tested. Briefly, cells were seeded in 96-well plates (V=80 µl). N4 ADC1 and Her2 ADC1 or human IgG1-isotype control (IC)-adaptor-toxin or culture medium (concentration 5x) were tested against N4 ADC1 and the isotype control in 1:2 serial dilutions (from 530 nM to 30 nM) and 1:5 serial dilutions (7 nM to 7 x 10⁻² nM). N4 ADC2 and the isotype control were tested in 1:10 serial dilutions (from 530 nM to 5.3 x 10⁻²). The ability of the ADCs to induce cell death was determined by confluence assay using an Incucyte S3-2 device; cell viability was determined on day 6 post-treatment using the Cell TiterGlo™ (CTG) assay with an Enspire2 device. The IC50 value for each ADC was determined using GraphPad Prism8 with day 6 luminescent cell viability data. The experiment was repeated twice.

[0302] Representative results are shown in Figure 3 middle. Figure 3 The two right-hand images show that N4 ADC1 (anti-Nectin-4) effectively induces tumor cell death, with N4 ADC1 shown as a solid line with squares and the isotype control shown as a dashed line. Figure 3 The two left-hand images show the efficacy of Her2 ADC1 (anti-Her2) in the same corresponding cells, where HER2ADC1 is shown as a dotted solid line and the isotype control is shown as a dashed line. IC 50 The values ​​are summarized in the table below. N4 ADC1 was particularly effective even in SUM190 cells, which are characterized by much lower surface Nectin-4 expression (surface Nectin-4 in SUM190 is about 1 / 4 that in SUM185). N4 ADC2 (anti-Nectin-4) with the camptothecin analog SN38 also showed good potency (see IC50 below). 50 surface).

[0303] Table: IC50 values ​​of ADCs The results showed that in SUM185 expressing Nectin-4, the anti-Nectin-4 ADC (N4 ADC1) was much more effective than the anti-Her2 ADC (Her2 ADC1), with the Nectin-4 ADC having a lower IC50 value. 5040-fold lower. Notably, SUM185 cells express Nectin-4 at relatively high levels, approximately twice the level of Her2 expression in these cells (see Example 1). However, interestingly, when tested in SUM190 cells, where Nectin-4 surface expression levels are much lower, the anti-Nectin-4 ADC (N4 ADC1) remains highly effective. In these SUM190 cells, Nectin-4 surface expression is only half that of Her2 surface expression, yet the anti-Nectin-4 camptothecin ADC remains at least as effective as, or possibly more effective than, the anti-Her2 ADC, with an IC50 of 40 times higher than that of the anti-Nectin-4 ADC. 50 It is an IC that resists Her2 ADCs. 50 1 / 6 of.

[0304] Therefore, the combination of intracellular cleavable peptide linkers with camptothecin analogues may represent an effective means of eliminating Nectin-4-expressing tumor cells, including those characterized by low Nectin-4 expression levels, without increased off-target toxicity compared to the most widely used cytotoxic agents such as pyrrolobenzodiazepines and olistatin. Anti-Nectin-4 ADCs may also offer a valuable approach for treating HER2-positive cancers, including but not limited to those with low and / or moderate HER2 expression.

[0305] Example 3: The efficacy of combining an anti-Nectin-4 camptothecin analog ADC with an anti-HER2 ADC Anti-Nectin-4 ADC (N4 ADC1) and anti-Her2 ADC (Her2 ADC1) were tested to evaluate their ability to induce tumor cell death when used in combination.

[0306] The anti-Nectin-4 camptothecin analog ADC used was N4 ADC1, as shown in Example 2. The anti-Her2 ADC was also one of those used in Example 2 (Her2 ADC1). As described in Example 2, the ability of the ADCs to induce cell death was determined by assessing the confluence and viability of SUM190 cells on day 6. The results showed that the combination of the anti-Nectin-4 ADC and the anti-Her2 ADC had enhanced potency (lower IC50) in inducing death in Nectin-4+ Her2+ tumor cells compared to using either ADC alone.

[0307] Example 4: Intracellular internalization The ability of anti-nectin-4 antibodies to induce nectin-4 internalization was evaluated in the SUM190 cell line expressing low levels of nectin-4 and the SUM185 cell line expressing high levels of nectin-4. The antibodies tested were emfretuzumab (VH and VL of SEQ ID NO: 3 and 4 as human IgG1 isotypes) and N41 (VH and VL of SEQ ID NO: 9 and 10 as human IgG1 isotypes). Internalization was indirectly determined using the Fab-ZAP Human Internalization Kit (Advanced Targeting System) to allow anti-Nectin-4 antibodies to target and eliminate nectin-4-expressing cells, and cell viability was measured using CTG substrate (CellTiter-Glo® Luminescent Cell Viability Assay (Promega)). Fab-ZAP is a chemical conjugate of a goat anti-human monovalent antibody and the ribosomal inactivating protein saponin. The antibodies used were affinity-purified polyclonal antibodies against both the heavy and light chains of human IgG. This secondary antibody was used to evaluate the potential for internalization by the primary antibody.

[0308] In short, a specific concentration of antibody or control is incubated on top of SUM190 or SUM185 cells. After incubation, unbound antibody is washed away, and Fab-ZAP is added to the top of the cells. The newly formed complex (anti-nectin-4 antibody + Fab-ZAP) is internalized into the saponin-releasing cells and stops protein synthesis, leading to cell death. The internalization ability of the antibody is indirectly determined by cell viability: the higher the cell-killing efficiency, the better the antibody's internalization ability.

[0309] A graph was plotted showing the contrast between the luminescence of each antibody and the concentration of the anti-Nectin-4 antibody. The results are shown in... Figure 4 Both entaftumab and N41 exhibit strong ability to induce internalization in SUM185 and SUM190 cells.

[0310] Example 5: Binding to an anchored Nectin-4 domain-deficient protein The ability of the internalizing antibody entaftozumab and N41 to bind to different domains on human Nectin-4 was evaluated.

[0311] The wild-type huNectin4 protein consists of three extracellular Ig-like domains (V, C1, and C2) summarized in Table 1 below.

[0312] Table 1 The binding of antibodies to the Nectin-4 domains was characterized by flow cytometry using cells prepared to express wild-type human Nectin-4 protein and cells prepared to express modified Nectin-4, wherein the modified Nectin-4 has Ig-like C21 and Ig-like C22 domains and lacks the Ig-like V domain (C1C2 construct). The latter protein also carries a V5 tag for flow cytometry cell sorting, and the cells were used for Nectin4-C1C2-V5 sorting.

[0313] Wild-type Nectin-4 (Cl.3C8 cell line), the C2 construct (containing an Ig-like C2 type 2 domain but lacking the V and C1 domains), and the C1C2 construct allowed for the determination of whether the test antibody binds to the V domain or a different C domain. Briefly, nucleic acid sequences encoding different human Nectin-4 domains were amplified by PCR. The PCR products were inserted into expression vectors at appropriate restriction sites. A leader peptide was added, and for C1C2 and C2, an N-terminal V5 tag with the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO: 13) was added, and expression on the cell surface was confirmed by flow cytometry. The amino acid sequences of the resulting proteins containing different human Nectin-4 domain fragments are shown below (V5 tag underlined). The vectors were then transfected into CHO cell lines to obtain stable clones expressing proteins with different Nectin-4 domains on the cell surface.

[0314] The amino acid sequence of Nectin-4 in the huNectin4 Cl.3C8 cell line (wild-type Nectin-4): GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 14).

[0315] Amino acid sequence of Nectin-4 in the huNectin4-C1C2-V5 cell line (lacking the V domain): PPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 15).

[0316] The Nectin-4 amino acid sequence in the huNectin4-C2-V5 cell line (lacking the V and C1 domains): ASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQ QMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 16).

[0317] Both the internalizing antibody N41 and emfretuzumab bind to the entire Nectin-4 protein and lose binding to the C1C2 construct lacking the V domain. Therefore, N41 and emfretuzumab bind to Nectin-4 within the V domain.

[0318] Example 6: In vitro cytotoxicity of ixenolide ADC against breast cancer cells (Nectin-4 high / SUM185 model) We evaluated the killing effect of an Ig-like V-domain antibody, entaftumab (mAbA), on SUM185 cells, having VH and VL of SEQ ID NO: 3 and 4 as human IgG1 isotypes conjugated with eczemab. In this experiment, the entaftumab antibody and a control antibody conjugated with the same toxin at an equivalent drug-to-antibody ratio were tested. ADCs were prepared in which the antibody was conjugated with eczemab via a linker comprising a spacer, an intracellularly cleavable dipeptide valine-citrulline (VC), and a PAB self-eliminating spacer; each antibody contained 8 toxins (DAR=8). (VC-Ecitabine).

[0319] For each ADC, a specific concentration range of ADC was incubated on top of nectin-4 expressing cells. After incubation, CTG substrate was added at a 1:1 ratio, and the luminescent signal was read using an Enspire plate reader to quantify the presence of ATP (an indicator of metabolically active cells) in proportion to cell viability.

[0320] The results are shown in Figure 5In comparison with the non-targeted isotype control ADC (IC-VC-Ecinotecan), mAbA-VC-Ecinotecan was more effective at killing SUM185.

[0321] Example 7: In vitro cytotoxicity of ixotecan and Dxd ADC on breast cancer cells (Nectin-4 low / SUM190 model) The anti-Ig-like V-domain antibody entorumab (mAbA) is conjugated to eciletidine via a VC-eciletidine linker having the structure shown in Example 6, or to camptothecin analogue DxD via an intracellularly cleavable tetrapeptide linker called GGFG-DxD. Each linker is also conjugated to an allotype control (IC) antibody. Each linker is conjugated to the antibody with 8 toxins per antibody (DAR=8).

[0322] The structure of the GGFG-DxD connector (GGFG-DxD) is as follows: (GGFG-DxD).

[0323] In vitro cytotoxicity was evaluated as described in Example 6. Results are shown in... Figure 6 Compared with the mAbA-GGFG-DxD immunoconjugate, the mAbA-VC-ecithecan immunoconjugate was more effective in killing SUM190 cells with low Nectin-4 expression.

[0324] Example 8: In vitro cytotoxicity of eczemab ADCs with different cleavable connectors against breast cancer cells (Nectin-4 low / SUM190 model) We evaluated the killing effect of an Ig-like V-domain antibody, entaftumab (mAbA), on SUM190 cells having VH and VL as human IgG1 isotypes conjugated to ezetidine (via valine-citrulline-PAB linkers, valine-alanine-PAB linkers, or PEG8-valine-alanine-PAB linkers) of SEQ ID NO: 3 and 4. In each case, entaftumab and isotype control (IC) antibodies conjugated to the same toxin at an equivalent drug-to-antibody ratio were tested. ADCs were prepared in which antibodies were conjugated to ezetidine at 8 toxins per antibody via linkers (DAR=8).

[0325] The structure of the valine-citrulline-PAB linker (VC-ecithecan) is shown in Example 6. The structure of the PEG8-valine-citrulline-PAB linker (PEG8-VC-ecithecan) is shown herein as compound 6.

[0326] The structure of the valine-alanine-PAB linker (VA-eczetidine) is as follows: The structure of the PEG8-valine-alanine-PAB linker (PEG8-VA-ethicane) is as follows: In vitro cytotoxicity was evaluated as described in Example 6. Results are shown in... Figure 7 In the middle. When conjugated with mAbA, each eczetine linker allows for efficient killing of SUM190 cells with low Nectin-4 expression.

[0327] Example 9: In vivo efficacy of ADCs in a human breast cancer mouse model (Nectin-4 low / SUM190 model) We compared the in vivo efficacy of enflutuzumab (mAbA) isotype control (IC) antibodies, each conjugated to ixenotec via a valine-citrulline-PAB linker (VC-ixenotec), a valine-alanine-PAB linker (VA-ixenotec), or a PEG8-valine-alanine-PAB linker (PEG8-VA-ixenotec). Hexane-camptothecin analogues DxD conjugated to antibodies via an intracellularly cleavable tetrapeptide linker (GGFG-DxD) were also tested. Each linker and toxin was conjugated at an equivalent drug-to-antibody ratio of 8 toxins per antibody (DAR=8).

[0328] SUM190 cells were subcutaneously injected into CB17-SCID immunodeficient mice at a dose of 100 µl of Matrigel containing 500,000 cells, with growth factors diluted ½ in PBS. When the tumor reached a volume between 195 and 250 mm³, mice were randomly assigned to receive a single intravenous injection of 5 mg / kg camptothecin ADC. Tumor growth was monitored twice weekly. Kaplan-Meier survival curves were constructed using GraphPad Prism V7 software based on the following criteria: when the tumor volume reached 1500 mm³. 3 When the tumor showed signs of necrosis, the mouse was euthanized and considered dead on the same day.

[0329] The results showed that at a dose of 5 mg / kg, VC-eixotecan, VA-eixotecan, and PEG8-VA-eixotecan were equally effective in preventing tumor volume increase, and were all more effective than GGFG-DxD in preventing tumor volume increase. The results indicated that... Figure 8 middle.

[0330] Example 10: Effects of different antibodies on the efficacy of ADC in a human breast cancer mouse model (Nectin-4 low / SUM190 model) We compared the in vivo efficacy of entaftoxin (mAbA) and another Nectin-4 binding antibody when each antibody was conjugated to eczema via the same PEG8-valine-alanine-PAB linker (PEG8-VA-eczema), which at least partially binds to the IgV domain of Nectin-4 (mAbB). An isotype control (IC) antibody conjugated to PEG8-VA-eczema, and a hexane camptothecin analogue DxD conjugated via an intracellularly cleavable tetrapeptide linker (GGFG-DxD) were also tested. Each linker and toxin was conjugated at an equivalent drug-to-antibody ratio of 8 toxins per antibody (DAR=8).

[0331] SUM190 cells were subcutaneously injected into CB17-SCID immunodeficient mice at a dose of 100 µl of Matrigel containing 500,000 cells, with growth factors diluted ½ in PBS. When the tumor reached a volume between 195 and 250 mm³, mice were randomly assigned to receive a single intravenous injection of 5 mg / kg camptothecin ADC. Tumor growth was monitored twice weekly. Kaplan-Meier survival curves were constructed using GraphPad Prism V7 software based on the following criteria: when the tumor volume reached 1500 mm³. 3 When the tumor showed signs of necrosis, the mouse was euthanized and considered dead on the same day.

[0332] The results showed that at a dose of 5 mg / kg, mAbA-PEG8-VA-ecithecan and mAbB-PEG8-VA-ecithecan were equally effective in preventing tumor volume increase. Both were more effective than mAbA-GGFG-DxD in preventing tumor volume increase. The results indicated that... Figure 9 middle.

[0333] All references cited in this document, including publications, patent applications and patents, are hereby incorporated in their entirety by reference, to the extent that each reference is individually and specifically indicated to be incorporated by reference and set forth in its entirety in this document (to the maximum extent permitted by law), regardless of whether the specific document referenced elsewhere in this document is separately provided for incorporation.

[0334] Unless otherwise stated, all precise values ​​provided herein represent corresponding approximations (e.g., all exact exemplary values ​​provided with respect to a particular factor or measurement can be considered as also providing a corresponding approximate measurement, modified by “about” where appropriate). When “about” is used in conjunction with a number, this can be specified as including a value corresponding to + / - 10% of the specified number.

[0335] The description of any aspect or embodiment of this invention using terms such as “comprising,” “having,” “including,” or “containing” is intended to provide support for similar aspects or embodiments of the invention that are “composed of the one or more specific elements,” “substantially composed of the one or more specific elements,” or “substantially comprise the one or more specific elements,” unless otherwise stated or explicitly contradicted by the context (e.g., a composition comprising a specific element described herein should be understood to also describe a composition composed of said element, unless otherwise stated or explicitly contradicted by the context).

[0336] Unless otherwise stated, the use of any and all instances or exemplary language (e.g., "such") provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating any unclaimed element as necessary for practicing the invention.

Claims

1. A method of treating cancer and / or killing tumor cells in an individual, the method comprising administering to the individual a therapeutically effective amount of a Nectin-4 binding protein conjugated with a camptothecin analogue, wherein the Nectin-4 binding protein conjugated with a camptothecin analogue is an immunoconjugate represented by formula (I): Equation (I) Ab–(X–(Z)) in, Ab is an antigen-binding protein that specifically binds to human Nectin-4 polypeptide; X is a linker molecule connecting Ab and Z, wherein X includes a cleavable portion under physiological conditions, optionally under intracellular conditions, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, optionally wherein X further includes a self-eliminating spacer positioned between the protease-cleavable peptide and Z; and Z is the molecule of ixotecan.

2. The method of claim 1, wherein the individual suffers from cancer, the cancer being characterized by low or moderate Nectin-4 expression on tumor cells as determined by immunohistochemistry.

3. A method for treating cancer and / or killing tumor cells in an individual in need, wherein the individual suffers from Nectin-4 expressing cancer, the cancer being characterized by low or moderate Nectin-4 expression on tumor cells, the method comprising administering to the individual a therapeutically effective amount of Nectin-4 binding protein conjugated to a camptothecin analog via a linker.

4. The method according to any one of the preceding claims, wherein the individual relapses and / or progresses after treatment with a Nectin-4 binding agent conjugated with olistatin, optionally wherein the Nectin-4 binding protein conjugated with olistatin is vitin-enterotoxin.

5. A method for treating cancer and / or killing tumor cells in an individual with cancer that is unresponsive, relapsed, and / or progressed after treatment with a Nectin-4 binding agent conjugated to olistatin, optionally divestin-entaftuzumab, the method comprising administering to the individual a therapeutically effective amount of Nectin-4 binding protein conjugated to a camptothecin analog via a linker.

6. The method according to any one of claims 1 to 5, wherein the individual has urothelial carcinoma, optionally advanced recurrent or metastatic urothelial carcinoma.

7. The method according to any one of claims 3 to 5, wherein the Nectin-4 binding protein conjugated to a camptothecin analog via a linker is an immunoconjugate represented by formula (I): Ab–X–Z formula (I) in, Ab is an antibody that specifically binds to human Nectin-4 polypeptide, wherein the antibody is capable of inducing intracellular internalization of Nectin-4 in tumor cells that express Nectin-4 on their surface, and wherein the antibody comprises a light chain variable domain and a heavy chain variable domain. X is a linker molecule connecting Ab and Z, wherein X includes a cleavable portion under intracellular conditions, optionally wherein X further includes a self-eliminating or non-self-eliminating spacer subsystem (Y') positioned between the cleavable portion and Z, optionally wherein X further includes a spacer (Y) positioned between the Ab and the cleavable portion; and Z is a camptothecin analogue.

8. The method according to claim 7, wherein the camptothecin analog molecule shares a core ring system with camptothecin and has modifications or substitutions in rings A and / or B compared to the following basic camptothecin structures: 。 9. The method according to any one of claims 1 to 5, wherein the individual has breast cancer.

10. The method according to any one of claims 1 to 5, wherein the individual has non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer.

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