Treatment of cancer

Abstract

The present invention provides antigen-binding proteins capable of binding to Nectin-4 polypeptides conjugated to chemotherapeutic agents for use in treating cancers characterized by Nectin-4-expressing tumor cells, for use in increasing tumor sensitivity to chemotherapeutic agents. In one embodiment, the present invention provides conjugates of anti-Nectin-4 antibodies to camptothecin analogs such as exatecan or SN-38 via cleavable linkers.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of the specification of U.S. Provisional Patent Application No. 62 / 968,175, filed on January 31, 2020, which is incorporated herein by reference in its entirety, including any drawings.

[0002] Reference to Sequence Listing This application has been filed with an electronic sequence listing. The sequence listing is provided as a file named "Nectin - 4 - 1_ST25", created on January 25, 2021, and having a size of 29 KB. The electronic information of the sequence listing is incorporated herein by reference in its entirety.

[0003] The present invention provides an antigen - binding protein capable of binding to a nectin - 4 polypeptide conjugated to camptothecin for use in the treatment of cancer characterized by nectin - 4 - expressing tumor cells, including different levels of nectin - 4 expression, including low heterogeneous expression, particularly in tumors.

Background Art

[0004] In the United States, it is estimated that more than 70,000 people were newly diagnosed with bladder cancer and approximately 16,000 people died in 2016. Urothelial carcinoma includes cancers of the bladder, ureter, and renal pelvis, which occur at a ratio of 50:3:1, respectively. Urothelial carcinoma is a multi - step process. Patients with upper urinary tract cancer have a 30% - 50% chance of developing bladder cancer at some point 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 inner lining (urothelium) of the bladder. This can spread as the disease progresses. It can spread to the surrounding bladder or other parts of the body (metastasis). This is called advanced urothelial carcinoma.

[0005] Urothelial carcinoma (UC) is characterized by increased expression of a wide range of different cell surface antigens, providing opportunities for specific therapeutic targeting using antibody-drug conjugates (ADCs). Among the surface antigens, several antigens 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 (also known as poliovirus receptor-related protein 4, PVRL4) have been shown to be suitable for ADC development.

[0006] Nectin-4 was first cloned from human trachea by the Lopez group in 2001 (see Non-Patent Document 1). An increase in the copy number of the nectin-4 gene is a frequent event in carcinogenesis and has been reported to promote epithelial-to-mesenchymal transition, invasion, and metastasis. The expression of nectin-4 protein is limited in normal tissues but is expressed at significantly high levels in several tumors, particularly breast cancer, pancreatic cancer, and UC, including triple-negative breast cancer (TNBC) (see Non-Patent Document 2). However, nectin-4 is also expressed in tumor specimens of non-small cell lung cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer. Non-Patent Document 3 reported moderate to strong staining by immunohistochemistry (H score ≥ 100) in tumor tissues of the bladder (60%) and breast (53%). Non-Patent Document 4 reported that high expression of nectin-4 was present in 86 (58%) of 148 TNBC cases.

[0007] Non-Patent Document 3 developed an anti-nectin-4 antibody conjugated with a very potent microtubule disruptor, MMAE, based on the antibody AGS-22. This research led to the development of the ADC drug candidate enfortumab vedotin, which showed promising results in a human clinical trial for the treatment of patients with locally advanced or metastatic urothelial carcinoma who had previously received platinum-containing chemotherapeutic agents and PD-1 / PD-L1 checkpoint inhibitors in the neoadjuvant / adjuvant, locally advanced, or metastatic setting (see Patent Document 1 and Patent Document 2, Agensys Inc.). Several other groups have also proposed anti-nectin-4 agents conjugated to various toxic agents. Patent Document 3 (INSERM) reported several anti-nectin-4 antibodies and proposed potential conjugation with a wide range of cytotoxic agents. Similarly, Patent Document 1 (Agensys Inc.) also provided anti-nectin-4 antibodies and proposed potential conjugation with a wide range of cytotoxic agents. Furthermore, Bicycle Therapeutics reported the development of an anti-nectin-4 targeting agent composed of a nectin-4 binding protein conjugated to a cytotoxic Auristatin (MMAE) payload.

[0008] ADCs reported for the development of UC include sacituzumab govitecan (IMMU-132, anti-TROP2), enfortumab vedotin (ASG-22ME, anti-nectin-4), siltuximab vedotin (ASG-15ME, anti-SLITRK6) for advanced UC, and oportuzumab monatox (VB4-845, anti-EpCAM) for non-muscle-invasive bladder cancer.

[0009] Unfortunately, although ADCs have shown promising results, many targeted therapies do not provide sufficient and / or durable antitumor responses to patients. For example, enfortumab vedotin (anti-nectin-4 ADC) showed impressive therapeutic efficacy with an ORR (objective response rate) of 44% and a CR (complete response rate) of 12% in UC in the EV-201 phase 2 trial, but approximately half of the patients discontinued treatment (2019). The majority of the discontinuations were due to progressive disease as evaluated by RECIST (48%) or clinical symptoms (5%). In addition, 18% of the patients who discontinued experienced adverse events, especially neuropathy. Therefore, there are limitations to ADCs targeting nectin-4, and there is a need in the art to improve the benefits to patients with UC and other cancers.

[0010] In UC, generally, an individual is first treated with a cisplatin-based regimen (with or without radiation), both when the cancer has not spread to distant parts of the body or when the cancer has spread. Camptothecin compounds are generally not used in the treatment of UC. Non-Patent Document 5 studied the camptothecin analog RFS2000 in patients with progressive or metastatic urothelial tumors. Non-Patent Document 5 concluded that it did not exhibit significant activity in patients with progressive / metastatic urothelial tumors who had failed prior chemotherapy and that the results of this study did not suggest further investigation of RFS2000. Over the past several decades, a very large number of camptothecin analogs have been prepared, among which is exatecan. Non-Patent Document 6 evaluated exatecan in the treatment of pancreatic cancer in a phase 3 trial of 349 patients and found that exatecan in addition to gemcitabine was not superior in efficacy to gemcitabine alone.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0013] In certain aspects, the present disclosure provides a nectin-4 binder conjugated to an exatecan 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 certain aspects, the present disclosure provides a very potent linker comprising an intracellularly cleavable dipeptide, a self-eliminating spacer and a camptothecin analog. The very potent linker can be conjugated to an antibody that binds to a tumor antigen. Accordingly, antibodies and antibody compositions conjugated to such linkers are also provided (e.g., when the antibody binds to a tumor antigen, optionally nectin-4). In certain aspects, the present disclosure provides a method of treatment that can be used in an individual having a nectin-4 expressing cancer, regardless of the level of nectin-4 expression on the tumor cells.

[0014] In certain aspects, the present disclosure provides a method of treatment that can be advantageously used in an individual in which the tumor cells express P-glycoprotein (Pgp).

[0015] In certain aspects, the present disclosure provides a method of treatment that can be advantageously used in an individual who has previously been treated with a chemotherapeutic agent (e.g., a chemotherapeutic agent transported by P-glycoprotein (Pgp), a platinum agent (e.g., oxaliplatin, cisplatin, carboplatin, nedaplatin, phenanthriplatin, picoplatin, satraplatin), a taxane (e.g., paclitaxel (Taxol) and docetaxel (Taxotere))).

[0016] In certain aspects, the present disclosure provides a method of treatment that can be advantageously used in an individual in which a tumor or cancer is resistant and does not respond or is progressing after treatment with a composition comprising an anti-HER2 antibody (e.g., trastuzumab, an ADC, CDR or polypeptide chain trastuzumab comprising a heavy chain variable region and a light chain variable region).

[0017] In one aspect, the present disclosure provides a treatment method that can be used to mediate an anti-tumor effect in an individual at a low dose or a dose lower than the dose used in conventional anti-nectin-4 ADCs, for example, at a dose of 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.

[0018] In one aspect, the present disclosure provides a treatment method that can be used in individuals having existing neuropathy, diabetes or hyperglycemia, heart failure, or ophthalmopathy.

[0019] In one aspect, the present disclosure provides a treatment method that can be used in individuals having nectin-4-expressing cancer characterized by low-level or moderate-level tumor cell expression of the nectin-4 polypeptide (e.g., expression of the nectin-4 polypeptide on the tumor cell membrane).

[0020] Provided are treatments comprising administration of a nectin-4 binder conjugated to a camptothecin analog or derivative (e.g., a 5-ring or 6-ring camptothecin, exatecan, or SN-38 molecule). Nectin-4 is generally expressed in tumor cells, but the expression level of nectin-4 can affect the therapeutic efficacy of anti-nectin-4 agents such as ADCs. The expression of nectin-4 in tumor cells can be very high in some individuals (e.g., as evaluated by immunohistochemistry), while other individuals have diseases in which tumor cell nectin-4 expression is low or moderate or below the level considered high expression, and thus may be less responsive or non-responsive to agents such as enfortumab vedotin. The ability to treat individuals having nectin-4 expressing cancers characterized by non-high levels of tumor cell nectin-4 expression (e.g., low or medium levels) provides an advantage in treating a broader population of subjects, e.g., a population of subjects characterized by having different levels of nectin-4 expression on their tumor cells (e.g., low or medium nectin-4 subjects and high nectin-4 subjects), without being limited to high nectin-4 expression levels. This advantage is noted in nectin-4 expressing cancers that are often (but not always) known to exhibit very high tumor nectin-4 expression such as UC, and may be of even greater interest in nectin-4 expressing cancers having a wide diversity in the level of tumor cell nectin-4 expression, 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 certain embodiments, the nectin-4 expressing cancer is further characterized by the expression of the Her2 polypeptide (e.g., Her2 overexpressing or highly expressing cancer, Her2 low expressing cancer).

[0021] The treatments of the present disclosure can advantageously be used, for example, in cancer types known to be characterized by tumors in individuals in which the tumor cells have high heterogeneity in the level of tumor antigen (e.g., nectin-4) on their surface.

[0022] In one aspect, the present disclosure provides a method of treating an individual having nectin-4 expressing cancer characterized by low or moderate levels of tumor cell nectin-4 expression. The patient may have progressive or refractory cancer, or may have cancer at an early stage where tumor nectin-4 expression remains low or moderate. The treatment comprises administration of a camptothecin analogue or derivative, such as exatecan or an SN-38 molecule conjugated to a nectin-4 binding agent. In certain embodiments, the tumor is characterized by low or moderate nectin-4 expression as determined by an immunohistochemical score, such as an H score of 290, 250, 200, 180, 170, 160, 150, 140, 130, 120 or 100 or less or less than. In certain embodiments, the individual has urothelial cancer or breast cancer. In certain embodiments, the individual has non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma or esophageal cancer. In certain embodiments, the cancer or tumor is an advanced recurrent or metastatic cancer, optionally an advanced recurrent or metastatic urothelial cancer. In one aspect, the present disclosure provides a method of treating a population of individuals having nectin-4 expressing cancer, wherein the population comprises individuals having cancer or tumors characterized by low or moderate nectin-4 expression as determined by an immunohistochemical score, such as an H score of 290, 250, 200, 180, 170, 160, 150, 140, 130, 120 or 100 or less or less than.

[0023] In certain embodiments, 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., di-, tri-, tetra- or pentapeptide). In certain embodiments, the anti-nectin-4 antibody or antibody fragment is conjugated to a camptothecin analog via an intracellularly cleavable (e.g., protease-cleavable) di-, tri-, tetra- or pentapeptide and a self-eliminating spacer (e.g., a self-eliminating spacer positioned between the intracellularly cleavable peptide and camptothecin). In certain embodiments, 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 certain embodiments, 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- or non-self-eliminating spacer.

[0024] Optionally, the antibody is functionalized with a linker toxin of any one of formulas I-XI.

[0025] In certain embodiments, the camptothecin analog is exatecan or an SN-38 molecule.

[0026] In certain embodiments, the present disclosure provides a nectin-4 binding protein, antibody or antibody fragment conjugated (e.g., covalently) to a camptothecin, such as a camptothecin analog, exatecan or an exatecan derivative or an SN-38 molecule.

[0027] In certain embodiments, the present disclosure provides a method of treating an individual having cancer, the method comprising treating the individual with a nectin-4 binder conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule. In certain embodiments, the individual has urothelial cancer or breast cancer.

[0028] In one aspect, the present disclosure provides a method of treating an individual without a pre-step of determining whether the individual is suitable for treatment based on the tumor cell expression level of nectin-4, the method comprising treating the individual with a nectin-4 binding agent conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule. In certain embodiments, the individual has urothelial cancer or breast cancer.

[0029] In one aspect, the present disclosure provides a nectin-4 binding agent conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule, for use in the treatment of cancer in a population of individuals comprising both individuals having a tumor characterized by high levels of nectin-4 on the tumor cells and individuals having a tumor characterized by low levels of nectin-4 on the tumor cells. In certain embodiments, the individual has urothelial cancer or breast cancer.

[0030] In one aspect, the present disclosure provides a method of treating an individual having cancer (e.g., nectin-4 expressing cancer), wherein the individual Auristatin or is resistant, non-responsive, has recurrent and / or progressive cancer despite treatment (e.g., during or after treatment) with an antibody or antigen-binding molecule (e.g., a nectin-4 antibody or antigen-binding molecule) conjugated to an MMAE molecule (e.g., enfortumab vedotin), the method comprising treating the individual with a nectin-4 binding agent conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule. In certain embodiments, the individual has urothelial cancer, breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer. In certain embodiments, the cancer or tumor is an advanced recurrent or metastatic cancer, optionally an advanced recurrent or metastatic urothelial cancer.

[0031] In one aspect, the present disclosure provides a method of treating an individual having cancer (e.g., nectin-4 expressing cancer), wherein the individual has locally advanced or metastatic urothelial cancer, Auristatinor has previously been treated with an antibody or antigen-binding agent conjugated to an MMAE molecule (e.g., enfortumab vedotin), and the method comprises treating the individual with a camptothecin analog or derivative, e.g., an exatecan or SN-38 molecule conjugated to a nectin-4 binding agent.

[0032] In certain embodiments, the disclosure provides a method of reducing or preventing drug resistance in an individual having cancer (e.g., nectin-4 expressing cancer), the method comprising treating the individual with a camptothecin analog or derivative, e.g., an exatecan or SN-38 molecule conjugated to a nectin-4 binding agent.

[0033] In any of the embodiments herein, the individual Auristatin can be identified as resistant, non-responsive, recurrent and / or progressive after pretreatment with an antibody (e.g., enfortumab vedotin) conjugated to an MMAE molecule.

[0034] In any of the embodiments herein, the individual can be identified as having a HER2 positive tumor or cancer (e.g., a nectin-4 positive, HER2 positive tumor or cancer), optionally a tumor or cancer with HER2 overexpression or HER2 high expression, optionally a tumor or cancer with HER2 low expression. In other embodiments herein, the individual can be identified as having a Her2 negative tumor or cancer. Optionally, the tumor or cancer is triple negative breast cancer (TNBC).

[0035] In one aspect, the present disclosure provides a method of treating an individual having a cancer (e.g., nectin-4 expressing cancer) characterized by tumor cells that express a Her2 polypeptide (e.g., tumor cells that express both nectin-4 and Her2 on their surface), the method comprising treating the individual with a nectin-4 binder conjugated (e.g., via a linker cleavable intracellularly) to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule. In certain embodiments, the cancer is characterized by tumor cells that overexpress Her2 or express it at high levels on their surface. In certain embodiments, the cancer is characterized by tumor cells that express low levels of Her2 on their surface. In certain embodiments, the method further comprises treating the individual with an antibody that binds to a Her2 polypeptide (e.g., trastuzumab, pertuzumab) (e.g., administering to the individual in combination); optionally, the antibody that binds to Her2 is an ADC; optionally, the antibody that binds to Her2 is conjugated to a cytotoxic agent, optionally Auristatin , a maytansinoid (e.g., DM1) or a camptothecin analog or derivative (e.g., exatecan or a derivative thereof, SN-38); optionally, the antibody that binds to Her2 is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a). In certain embodiments, the individual has breast cancer. In certain embodiments, the individual has gastric cancer. In certain embodiments, the individual has colorectal cancer. In certain embodiments, the individual has pancreatic cancer. In certain embodiments, the individual has bladder cancer. In certain embodiments, the individual has head and neck cancer.

[0036] The nectin-4 binder conjugated to the camptothecin analog or derivative can be advantageously administered once a month to four times a month, e.g., once every two weeks, once every three weeks, or once every four weeks.

[0037] A nectin-4 binder conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule, can be advantageously administered at 0.1-10 or 1-10 mg / kg body weight, once to four times a month, such as once every two weeks, once every three weeks, or once every four weeks.

[0038] In certain embodiments, the disclosure provides a nectin-4 binder (e.g., covalently) conjugated to a camptothecin analog or derivative, such as exatecan or an exatecan derivative or an SN-38 molecule.

[0039] In any of the embodiments herein, a nectin-4 binder (e.g., covalently) conjugated to a camptothecin analog or derivative, such as exatecan or an SN-38 molecule, can be characterized as an antigen-binding protein (e.g., an antibody, non-antibody peptide, or protein scaffold) that specifically binds to a human nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine, lysine, glutamine residues, non-natural amino acid residues) that are functionalized via a linker with a molecule comprising the structure of Compound 1 or 2 or a linker-camptothecin molecule of Formula I or II. In any of the embodiments herein, an anti-nectin-4 antibody or antibody fragment can be characterized as being functionalized with a linker-camptothecin molecule having the structure of Formula III, IV, V, VI, VII, VIII, IX, X, or XI or any of Compounds 3-17.

[0040] In certain embodiments, the anti-nectin-4 antibody or antibody fragment conjugated to a camptothecin analog is a nectin-4 binding antibody or antibody fragment conjugated to an exatecan molecule, e.g., a molecule having the structure of compound 1 (1a or 1b). In certain embodiments, the nectin-4 binding antibody or antibody fragment conjugated to a camptothecin analog is a nectin-4 binding antibody or antibody fragment conjugated to an SN-38 molecule, e.g., a molecule having the structure of compound 2. In certain embodiments, the nectin-4 binding antibody or antibody fragment is 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 unnatural amino acid residues) that are functionalized with a molecule having the following structure via a linker (e.g., a cleavable linker molecule having or not having an additional spacer, e.g., spacer (Y’) described herein). [Chemical formula]

[0041] In certain embodiments, the nectin-4 binding protein, antibody or antibody fragment conjugated to a cytotoxic agent has the formula (I): Ab-X-Z Formula (I) (wherein Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to a human nectin-4 polypeptide; X is a linker molecule that connects Ab and Z (e.g., covalently bound to each of Ab and Z), X comprises, e.g., a moiety cleavable under physiological conditions, optionally under intracellular conditions, optionally a di-, tri-, tetra- or pentapeptide cleavable by a protease, optionally X further comprises an auto-detaching or non-auto-detaching spacer system (Y’) located between the cleavable moiety and Z, and optionally X further comprises a spacer (Y) disposed between Ab and the cleavable moiety; and Z is a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule) It can be identified as an immune complex represented by

[0042] In certain embodiments, provided herein are cleavable peptide-containing linkers (e.g., oligopeptides or di-, tri-, tetra- or pentapeptides containing a linker) that can be conjugated to an antibody or other antigen-binding peptide or protein, such as a tumor antigen (e.g., nectin-4 or another suitable tumor antigen), an antibody or non-antibody peptide or protein scaffold that binds to the tumor antigen, and linker-toxin molecules, and antibodies and antibody compositions conjugated to such linkers, and methods of using them in the treatment of cancer. In certain embodiments, the dipeptide-containing linker-toxin molecule comprises a structure (X-Z), where X is a linker molecule containing a reactive group that is suitable for reacting with a complementary reactive group on the antigen-binding protein, optionally protected, the spacer portion (Y) is located between the reactive group and the cleavable dipeptide, the cleavable dipeptide is selected from valine-citrulline, valine-alanine or phenylalanine-lysine, and the self-cleaving or non-self-cleaving spacer system (Y’) is located between the cleavable moiety and Z, and Z is a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule. Further provided is a method of conjugating such a cleavable peptide-containing linker to an antigen-binding protein.

[0043] In certain embodiments, the antibody conjugated to the camptothecin analog has the formula (I): Ab-X-Z Formula (I) (wherein, Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to a tumor antigen; X is a linker molecule that links Ab and Z (e.g., covalently bound to each of Ab and Z), X comprises a valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide, X further comprises a self-cleaving or non-self-cleaving spacer system (Y’) located between the cleavable moiety and Z, and X further comprises a spacer (Y) disposed between Ab and the cleavable moiety; and Z is a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule) which can be identified as an immune complex represented by

[0044] In certain embodiments, provided are methods of delivering or targeting a camptothecin analog to a tumor or releasing a camptothecin analog in a tumor (e.g., a subject having cancer), the method comprising administering to a subject having cancer an immune complex of formula (I): Ab-X-Z Formula (I) (wherein Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to a tumor antigen; X is a linker molecule that links Ab and Z (e.g., covalently bound to each of Ab and Z), X comprises a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide, X further comprises an auto-cleavable or non-auto-cleavable spacer system (Y') located between the cleavable moiety and Z, and X further comprises a spacer (Y) disposed between Ab and the cleavable moiety; and Z is a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule) comprising administering an immune complex represented by

[0045] In certain embodiments, an antibody or nectin-4 binder conjugated to a camptothecin analog has the formula (II): Ab-(X-(Z) n ) m Formula (II) (wherein Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to a human nectin-4 polypeptide or other tumor antigen; X is a linker molecule that connects Ab and Z, and X includes, for example, a moiety cleavable under physiological conditions, optionally under intracellular conditions, and optionally a di-, tri-, tetra- or pentapeptide cleavable by protease. Optionally, X further includes a self-detaching or non-self-detaching spacer system (Y') located between the cleavable moiety and Z. Optionally, X further includes a spacer (Y) disposed between Ab and the cleavable moiety; Z is a camptothecin analog, and optionally, Z is a molecule including an exatecan molecule or an SN-38 molecule, such as a molecule having the structure of Compound 1 or 2; n is 1; and m is from 4 to 8, or optionally, m is an integer selected from 4, 5, 6, 7 or 8) and can be identified as an immune complex represented by.

[0046] In certain embodiments, the nectin-4 binder conjugated to the camptothecin analog is of formula (II): Ab-(X-(Z) n ) m Formula (II) (wherein Ab is an antigen-binding protein (e.g., an antibody) that specifically binds to the human nectin-4 polypeptide; X is a molecule that connects Ab and Z, and X includes, for example, a moiety cleavable under physiological conditions, optionally under intracellular conditions, and optionally a di-, tri-, tetra- or pentapeptide cleavable by protease. Optionally, X further includes a self-detaching or non-self-detaching spacer system (Y') located between the cleavable moiety and Z. Optionally, X further includes a spacer (Y) disposed between Ab and the cleavable moiety; Z is a camptothecin analog, and optionally, Z is a molecule including an exatecan molecule or an SN-38 molecule; n is 1, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the composition have m (the number of X-Z moieties) of 2 to 4, 4 to 8, optionally 6 to 8) It can be characterized as a composition of immune complexes represented by. Optionally, at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the composition are 4, 6, 7 or 8, or have m of at least 4, 6, 7 or 8

[0047] In Formula I or II, the (X-Z) moiety can optionally be characterized as having the structure of any of Formulas III to XI or any of Compounds 3 to 17

[0048] In Formula I or II, the molecule X or the spacer Y can optionally be identified as including the residue of a reactant of a reactive group (R) or a reactive group (R) and a complementary reactive group (R') bound to an amino acid of an antigen-binding protein (e.g., an antibody) or an antigen-binding protein (e.g., an antibody)

[0049] In any embodiment herein, the exatecan molecule can be identified as being bound to the linker (X) via the amine at the 1-position of exatecan (when the exatecan molecule is incorporated into the linker, NH replaces NH at the 1-position) 2 In any embodiment herein, the SN-38 molecule can be identified as being bound to the linker (X) via the OH at the 9-position (O replaces the OH at the 9-position when the SN-38 molecule shown in Compound 2 is incorporated into the linker)

[0050] In one embodiment, the nectin-4 binder conjugated to exatecan is characterized as including an antibody that specifically binds to a human nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine residue, glutamine residue) that are functionalized via a spacer (Y) with a linker-exatecan molecule having the following structure [Chemical]

[0051] In certain embodiments, the nectin-4 binder conjugated to exatecan 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 residue, glutamine residue) that are functionalized via a spacer (Y) with a linker-exatecan comprising the following structure. [Chemical]

[0052] In certain embodiments, the nectin-4 binder conjugated to exatecan 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 residue, glutamine residue) that are functionalized via a spacer (Y) with a linker-exatecan molecule comprising the following structure. [Chemical]

[0053] In certain embodiments, the nectin-4 binder conjugated to exatecan 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 residue, glutamine residue) that are functionalized via a spacer (Y) with a linker-exatecan molecule comprising the following structure. [Chemical]

[0054] The spacer (Y) can be identified as or include a substituted or unsubstituted alkyl or heteroalkyl chain. Optionally, Y has a chain length of 2 to 100 atoms or 2 to 40 atoms, optionally 2 to 30, 2 to 20, 4 to 40, 4 to 30 or 4 to 20 atoms. Optionally, one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms. Optionally, any carbon of the chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide. For example, Y can include one or more ethylene oxide monomers. Optionally, Y includes a polyethylene oxide moiety. Optionally, Y has the structure -(CH 2 CH 2 O) x -, where x is 1 to 12, optionally 1 to 8, optionally 1 to 6.

[0055] In certain embodiments, the present disclosure provides a treatment that exhibits improved (lower) drug resistance compared to existing anti-nectin-4 ADC therapies (e.g., Auristatin anti-nectin-4 antibody conjugated to; enfortumab vedotin). In certain embodiments, provided is a method of treating and / or preventing cancer and / or killing tumor cells in an individual in need thereof, wherein the treatment comprises administration of a nectin-4 binder conjugated to a camptothecin analog (e.g., exatecan 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), 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more.

[0056] In one aspect, the present disclosure provides a method of treating and / or preventing cancer and / or killing tumor cells in an individual in need thereof, or delivering and / or releasing a camptothecin molecule to a tumor of an individual, 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 analog (e.g., an anti-nectin-4 antibody or antibody fragment conjugated to one or more camptothecin moieties), wherein optionally, the camptothecin is exatecan or SN-38. In certain embodiments, the individual has a nectin-4 expressing tumor, and optionally, the tumor is a HER2 expressing tumor or a HER negative tumor, such as urothelial cancer, breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma or esophageal cancer. In certain embodiments, the cancer or tumor is an advanced recurrent or metastatic cancer, and optionally, an advanced recurrent or metastatic urothelial cancer. In certain embodiments, the cancer or tumor is triple negative breast cancer (TNBC).

[0057] In one aspect of any of the embodiments herein, the individual has been pre-treated by treatment with radiotherapy, surgery, chemotherapy and / or biologics.

[0058] Also provided are compositions of the immunoconjugates of the present disclosure. Also provided are pharmaceutically acceptable compositions and kits comprising the immunoconjugates of the present disclosure and one or more additional components (e.g., various carriers) that can typically be active ingredients or inactive ingredients that facilitate the formulation, delivery, stability or other characteristics of the composition. Also provided are methods of screening, testing and making immunoconjugates and ADCs.

[0059] These aspects will be described in more detail and further aspects, features and advantages will become apparent from the description of the invention provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0060]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

[0061] Definitions As used herein, "a" or "an" can mean one or more. When used in a claim, the terms "a" or "an" when used in combination with the term "comprising" can mean one or two or more. As used herein, "another" can mean at least a second or more.

[0062] When "comprising" is used, this can optionally be replaced by "consisting essentially of" or "consisting of".

[0063] "Nectin-4" and "Nectin-4 polypeptide" refer to a protein or polypeptide encoded by the Nectin 4 gene (see Uniprot accession number Q96NY8) or cDNA prepared from such a gene. The term Nectin-4 polypeptide includes any naturally occurring isoform, allele or variant (e.g., a Nectin-4 polypeptide that is 95%, 98% or 99% identical to the Nectin-4 polypeptide of SEQ ID NO: 1 or a continuous sequence of at least 100, 200, 300, 400 or 500 amino acid residues thereof). The 510 amino acid residue sequence of standard human Nectin-4 (isoform 1) (including a 31 amino acid signal peptide) is shown as follows.

Chemical formula

[0064] SEQ ID NO: 1 corresponds to the UniProt KB identifier Q96NY8-1, the disclosure of which is incorporated herein by reference.

[0065] Certain aspects of the present disclosure provide anti-Nectin-4 antibodies that bind to human Nectin-4 or homologs thereof, including, but not limited to, mammalian Nectin-4 protein and Nectin-4 orthologs of other species, such as non-human primates, cynomolgus monkeys.

[0066] The term "HER2" (also known as HER2 / neu and ErbB-2) refers to "human epidermal growth factor receptor 2", which includes variants and isoforms of HER2.

[0067] The term "tumor antigen", which can be used interchangeably with "cancer antigen", refers to an antigen differentially expressed by cancer cells or expressed by non-tumor cells (e.g., immune cells) of a tumor or tumor-adjacent tissue having a tumor-promoting effect (e.g., an immunosuppressive effect), and which can thereby be utilized to target cancer. Tumor antigens are antigens that can potentially stimulate a clearly tumor-specific immune response. Some of these antigens are encoded by normal cells but are not necessarily expressed, or are expressed at lower levels or frequencies. These antigens can be characterized as those that are silent (i.e., not expressed) in normal cells, those expressed only at specific differentiation stages, and those expressed transiently such as embryonic and fetal antigens. Other tumor antigens are encoded by mutant cellular genes such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other tumor antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. Still other tumor antigens can be expressed by immune cells that contribute to or mediate a tumor-promoting effect, e.g., cells contributing to immune evasion, monocytes or macrophages, optionally suppressor T cells, regulatory T cells, or bone marrow-derived suppressor cells. Tumor antigens are often overexpressed, or expressed at abnormal times, or are normal cell surface antigens expressed by a target cell population. Ideally, the target antigen is expressed only by proliferating cells (e.g., tumor cells) or tumor-promoting cells (e.g., immune cells having an immunosuppressive effect) present in a tumor or tumor-adjacent tissue, but in practice this is rarely observed. As a result, target antigens are often selected based on differential expression between proliferating / diseased tissue and healthy tissue.Examples of tumor antigens include nectin-4, receptor tyrosine kinase-like orphan receptor 1 (ROR1), cryptin, 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, melanotransferrin, Mud6 and TMEFF2. Examples of tumor antigens also include cytokine receptors, killer Ig-like receptors, CD28 family proteins, etc., the immunoglobulin superfamily (IgSF) such as killer Ig-like receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1, IL-6 receptor.Examples include MAGE, MART-1 / Melan-A, gp100, major histocompatibility complex class I-related chain A and B polypeptides (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), nectin (e.g., nectin-4), UL16-binding protein (ULBP) family of proteins, retinoic acid early transcript-1 (RAET1) family of proteins, prostate-specific antigen (PSA), B cell maturation antigen (BCMA), anti-Müllerian hormone type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (receptor tyrosine kinase-like orphan receptor 1 or NTRKR1 (EC 2.7.10.1) also known as), TROP2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptor, angiopoietin-2, PDGF, TGF alpha, EGF, EGF receptor, members of the human EGF-like receptor family, e.g., HER-2, HER-3, HER-4 or heterodimeric receptors composed of at least one HER subunit, gastrin-releasing peptide receptor antigen, Muc-1, CA125, integrin receptor, alpha v beta 3 integrin, alpha 5 beta 1 integrin, alpha IIb beta 3 integrin, PDGF beta receptor, SVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, CSF1R (tumor-associated monocytes and macrophages), alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, p120ctn, PRAME, NY-ESO-1, gp75, GM2 and GD2 gangliosides, although this is not intended to be exhaustive. In one aspect, the antigen of interest is, for example, an antigen capable of causing intracellular internalization when bound by a human antibody (e.g., any one of the antigens listed above).

[0068] The term "immunocomplex" refers to an antigen-binding agent (e.g., an antibody-binding polypeptide or an antibody) complexed with another molecule (e.g., a camptothecin analog, an exatecan molecule, an SN-38 molecule). When the immunocomplex contains an antibody complexed with a therapeutic agent (e.g., a camptothecin analog, an exatecan molecule, an SN-38 molecule), the immunocomplex may also be referred to as an "antibody-drug conjugate" or "ADC".

[0069] As used herein, "treating" and "treatment" and the like generally mean obtaining a desired pharmacological and physiological effect. The effect can be prophylactic in terms of preventing or partially preventing a disease, its symptoms or condition, and / or can be therapeutic in terms of partially or completely curing a disease, condition, symptom or adverse effect caused by a disease. As used herein, the term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease from occurring in a subject who may have a predisposition to the disease but has not yet been diagnosed as having it, such as prophylactic early asymptomatic intervention; (b) inhibiting a disease in a subject diagnosed as having the disease, such as preventing its onset; or alleviating a disease, such as causing regression of the disease and / or its symptoms or condition, such as improvement or repair of damage. Optionally, treatment can cause or provide (e.g., can be characterized as a method of causing or providing) a decrease in tumor burden, a decrease 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 rate. Optionally, treatment can cause or provide (e.g., can be characterized as a method of causing or providing) stable disease, partial response or complete response in a subject, e.g., according to standard criteria, optionally RECIST criteria.

[0070] Whenever reference is made to "treatment of cancer" and the like with respect to a nectin-4 binding agent (e.g., an antibody or antibody fragment), (a) A method for treating cancer, comprising administering a nectin-4 binder to an individual, mammal, particularly a human, in need of such treatment (for at least one treatment) in a dose (therapeutically effective amount) that enables the treatment of cancer, optionally in a dose (amount) specified herein; (b) Use of a nectin-4 binder for the treatment of cancer; (c) A nectin-4 binder for use in the treatment of cancer (particularly in humans); (d) Use of a nectin-4 binder for the manufacture of a pharmaceutical preparation for the treatment of cancer; (e) A method of using a nectin-4 binder for the manufacture of a pharmaceutical preparation for the treatment of cancer, 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 the treatment of cancer; (g) Depending on the subject matter for which a patent can be obtained in the country where this application is filed, any combination of (a), (b), (c), (d), (e) and (f) is included.

[0071] As used herein, the term "biopsy" is defined as the removal of tissue for examination purposes, such as to establish a diagnosis. Examples of types of biopsies include those by application of aspiration using a needle attached to a syringe, etc.; those by instrumental removal of tissue fragments; those by removal with a suitable instrument via an endoscope; those by surgical excision of an entire lesion, etc.

[0072] As used herein, the term "antibody" refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these are further classified into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Representative immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The N-terminus of each chain defines a variable region of about 100 - 110 or more amino acids that is mainly involved in antigen recognition. The terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively. The heavy chain constant domains corresponding to different classes of immunoglobulins are called "alpha", "delta", "epsilon", "gamma", and "mu", respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG is the most common antibody in physiological situations and is the most easily produced in the laboratory, and thus is a representative class of antibodies used herein. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human, or otherwise human-appropriate antibodies. "Antibody" includes full-length antibodies and any fragments or derivatives of the antibodies described herein.

[0073] The amino acid residues of an antibody involved in antigen binding may also be referred to as the "hypervariable region". The hypervariable region generally includes "complementary determining regions" or "CDRs" (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 "hypervariable loops" (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, J. Mol. Biol 1987; 196: 901-917) or amino acid residues from a similar system for determining essential amino acids involved in antigen binding. Generally, the numbering of amino acid residues in this region is performed by the method described by Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat", and "by Kabat" refer to this numbering system for the heavy chain variable domain or the light chain variable domain in this specification. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain a few or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may include one amino acid insertion (residue 52a according to Kabat) after residue 52 of CDR H2 and insertions of residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after residue 82 of the heavy chain FR. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of sequence homology of the antibody in the "standard" Kabat numbering sequence.

[0074] The term "specifically binds" means that when evaluated using a protein present on the surface of an isolated target cell, an epitope therein, or any recombinant form of the native protein, the antibody is preferably capable of binding to a binding partner, such as nectin-4, in a competitive binding assay. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected by physical methods such as radiolabeling, mass spectrometry, or by direct or indirect fluorescent labeling detected using, for example, cell fluorescence analysis (e.g., FACScan). Binding in excess of that seen with a control non-specific agent indicates that the agent binds to the target.

[0075] When an antibody is said to "compete" with a particular monoclonal antibody, this means that the antibody competes with the monoclonal antibody in a binding assay using either a recombinant molecule (e.g., nectin-4) or a surface-expressed molecule (e.g., nectin-4). For example, if a test antibody reduces the binding of an antibody having any of the heavy chain variable regions of SEQ ID NO: 3, 7, or 9 and the respective light chain variable regions of SEQ ID NO: 4, 8, or 10 to a nectin-4 polypeptide or nectin-4-expressing cell in a binding assay, the antibody is said to "compete" with such an antibody, respectively.

[0076] The term "internalization" is used interchangeably with "intracellular internalization" and refers to molecular, biochemical, and cellular events associated with the process of moving a molecule from the extracellular surface of a cell to the intracellular surface of the cell. Processes involved in the intracellular internalization of molecules are well known and can involve, inter alia, extracellular molecules (hormones, antibodies, small organic molecules, etc.); membrane-associated molecules (cell surface receptors, etc.); complexes of membrane-bound molecules bound to extracellular molecules (e.g., a ligand bound to a transmembrane receptor or an antibody bound to a membrane-bound molecule). Thus, "induction and / or increase of internalization" includes events in which intracellular internalization is initiated and / or the rate and / or extent of intracellular internalization increases.

[0077] As used herein, the term "affinity" means the strength of binding of an antibody to an epitope. The affinity of an antibody 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. The affinity constant K a is defined as 1 / Kd. Methods for determining the affinity of monoclonal antibodies can be found in Harlow, et al., Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds, Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), which references are hereby incorporated 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 (such as analysis with a BIAcore™ SPR analyzer).

[0078] As related herein, "determinant" refers to a site of interaction or binding on a polypeptide.

[0079] The term "epitope" refers to an antigenic determinant, an area or region on an antigen to which an antibody binds. A protein epitope can include the amino acid residues involved in direct binding and those amino acid residues effectively blocked by a specific antigen-binding antibody or peptide, i.e., the amino acid residues within the "footprint" of the antibody. This is, for example, the simplest form or the smallest structural region on a complex antigen molecule that can combine with an antibody or a receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope composed of adjacent amino acid residues on the linear sequence (primary structure) of amino acids. The term "conformational or structural epitope" is defined as an epitope composed of amino acid residues representing separated portions of the linear sequence of amino acids that are not all adjacent and thus come into proximity to each other by folding of the molecule (secondary, tertiary, and / or quaternary structure). Conformational epitopes are dependent on the three-dimensional structure. Thus, the term "conformational" is often used interchangeably with "structural".

[0080] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biopolymer, or an extract made from a biological substance. The term "therapeutic agent" refers to an agent having biological activity.

[0081] The terms "Fc domain", "Fc portion", and "Fc region" refer to, for example, the C-terminal fragment of an antibody heavy chain from about amino acid (aa) 230 to about aa 450 of a human γ (gamma) heavy chain or its corresponding sequence or its natural allotype in other types of antibody heavy chains (e.g., α, δ, ε, and μ in the case of human antibodies). Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et. al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0082] The term "framework" or "FR" residue, as used herein, means the region of an antibody variable domain excluding the regions defined as CDRs. Each antibody variable domain framework can be further divided into contiguous regions (FR1, FR2, FR3, and FR4) separated by the CDRs.

[0083] The terms "isolated," "purified," or "biologically pure" refer to a substance that is substantially or essentially free from the components that are normally associated with it as found in its native state. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the major species present in a preparation is substantially purified.

[0084] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as to natural amino acid polymers and non-natural amino acid polymers.

[0085] The term "recombinant," when used, for example, with respect to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found within the native (non-recombinant) form of the cell, or alternatively expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all.

[0086] As used herein in connection with a polypeptide or epitope, the term antibody that "binds" to the polypeptide or epitope refers to an antibody that binds to the determinant with specificity and / or affinity.

[0087] The terms "identity" or "identical," when used in the context of the relationship between the sequences of two or more polypeptides, refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between two or more series of amino acid residues. "Identity" assesses the percentage of exact matches between the smaller of two or more sequences, using gap alignment (if any) handled by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0088] The method for determining identity is designed to maximize the match between the sequences being tested. The method for determining identity is described in published computer programs. Examples of computer program methods for determining identity between two sequences include the GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)), and the GCG program package. The BLASTX program 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., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0089] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain containing a fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group can have, for example, from 1 to 20 carbon atoms (whenever a numerical range such as "1 to 20" appears herein, it refers to each integer within the given range; for example, "from 1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to a maximum of 20 carbon atoms, but this definition also covers occurrences of the term "alkyl" where no numerical range is specified). The alkyl group of a compound can be designated as "C 1 ~C 4 alkyl" or a similar name. As a mere example, "C 1 ~C 4"Alkyl" means that the alkyl chain has 1 to 4 carbon atoms, that is, the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl. The alkyl group may or may not be substituted.

[0090] As used herein, the term "heteroalkyl" refers to a straight or branched alkyl group containing one or more heteroatoms, i.e., elements other than carbon (including, but not limited to, oxygen, sulfur, nitrogen, phosphorus) in place of one or more carbon atoms.

[0091] Whenever a group is described as "substituted", the group is always substituted with one or more of the indicated substituents. When no substituents are indicated, the "substituted" group indicated is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroaricyclic, aralkyl, heteroaralkyl, (heteroaricyclic)alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamyl, thiocarbamyl, amide, sulfonamide, sulfonamide, carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, mono-substituted amino group and di-substituted amino group and one or more groups independently selected from their protected derivatives.

[0092] When the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens. As another example, "C 1 ~C 3"Alkoxyphenyl" may contain one or more identical or different alkoxy groups containing one, two or three atoms.

[0093] References to "compounds" or "formulas" having a specific number (e.g., "Compound 1", "Compound 2", "Formula I" or "Formula II") designate all compounds derived from the compound or formula having the specific number, unless the context clearly indicates otherwise. For example, Compound 1 includes references to Compounds 1a and 1b.

[0094] Nectin-4 binder The Nectin-4 binding domains used to prepare the Nectin-4 binding ADC compositions of the present disclosure can be readily obtained from various immunoglobulin or non-immunoglobulin scaffolds, such as affibodies based on the Z domain of staphylococcal protein A, engineered Kunitz domains, monobodies oradnectins based on the 10th extracellular domain of human fibronectin type III, anticalins derived from lipocalins, DARPins (engineered ankyrin repeat domains, multimerized LDLR-A modules, avimers or cysteine-rich knottin peptides). Hypervariable regions, heavy and light chain CDRs, heavy and light chain variable regions and antibodies containing them, such as full-length antibodies or antibody fragments, bind to human Nectin-4 expressed on the surface of cells, such as tumor cells. When used in a treatment for eliminating Nectin-4-expressing tumor cells, the Nectin-4 binder, when conjugated to a cytotoxic molecule disclosed herein, can cause the death of Nectin-4-expressing tumor cells, as determined in an assay in which Nectin-4 binding ADCs are contacted with tumor cells, for example, in the absence of immune effector and / or non-tumor cells.

[0095] In one embodiment, the anti-Nectin-4 antigen-binding protein or antibody binds to a mature Nectin-4 polypeptide, such as a polypeptide having the amino acid sequence of residues 32-510 of SEQ ID NO: 1.

[0096] In certain embodiments, the 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 can be characterized as being capable of binding to a domain of nectin-4 having the amino acid sequence of residues 32 to 144 of SEQ ID NO: 1 (also shown below as SEQ ID NO: 2) (or binding to an epitope within or at least partially within that domain). GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLR (SEQ ID NO: 2)

[0097] In certain embodiments, the antigen-binding protein or antibody comprises the hypervariable regions of any known anti-nectin-4 antibody (e.g., heavy and light chain CDR1, 2, and 3 according to Kabat numbering). In certain embodiments, the antigen-binding protein or antibody competes for binding to the nectin-4 polypeptide with any one or more known anti-nectin-4 antibodies such as antibody ASG-22ME, 14A5.2, or N41. In one embodiment, the antigen-binding protein or antibody has immunospecificity for an epitope or "epitope site" that recognizes, binds to, or is substantially or essentially the same as, or the same as, the nectin-4 polypeptide as any of antibody ASG-22ME, 14A5.2, or N41.

[0098] In some embodiments, the anti-nectin-4 antibody can be selected to exhibit significantly lower binding to a mutant human nectin-4 polypeptide in which 1, 2, 3, 4, or more residues within the binding site or epitope on nectin-4 of any of antibody ASG-22ME, 14A5.2, or N41 are substituted with different amino acids as compared to the wild-type nectin-4 polypeptide.

[0099] In some embodiments, the anti-nectin-4 antibody may be characterized by lacking binding or showing significantly reduced binding to a mutant human nectin-4 polypeptide that lacks an Ig-like V-type domain (e.g., the domain is deleted) compared to the wild-type nectin-4 polypeptide. In some embodiments, the anti-nectin-4 antibody can be selected to show significantly reduced binding to a mutant human nectin-4 polypeptide in which 1, 2, 3, 4 or more residues within the Ig-like V-type domain are substituted with different amino acids compared to the wild-type nectin-4 polypeptide.

[0100] In some embodiments, the anti-nectin-4 antibody can be selected or characterized as showing significantly reduced binding to a mutant human nectin-4 polypeptide in which the Ig-like V-type domain (or a portion thereof) is absent or substituted with an amino acid sequence from a different polypeptide or a different domain (e.g., a non-human or non-primate nectin-4 polypeptide, a non-nectin-4 polypeptide, a non-Ig-like V-type domain) compared to the wild-type nectin-4 polypeptide.

[0101] The binding of the anti-nectin-4 antibody to cells transfected with the nectin-4 mutant can be measured and compared to the ability of the anti-nectin-4 agent to bind to the wild-type nectin-4 polypeptide (e.g., SEQ ID NO: 1). A decrease in the binding between the anti-nectin-4 agent and the mutant nectin-4 polypeptide implies a decrease in binding affinity (e.g., measured by known methods such as FACS assays of cells expressing a particular mutant or Biacore assays of binding to the mutant polypeptide) and / or a decrease in the total binding capacity of the anti-nectin-4 agent (e.g., demonstrated by a decrease in Bmax in a plot of anti-nectin-4 agent concentration versus polypeptide concentration). A significant decrease in binding indicates that when the anti-nectin-4 agent binds to nectin-4, the mutated residue is directly involved in the binding to the anti-nectin-4 agent or is in proximity to the binding protein.

[0102] In some embodiments, a significant decrease in binding means that the binding affinity and / or ability between the anti-nectin-4 antibody and the mutant nectin-4 polypeptide is more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95% decreased compared to the binding between the antibody and the wild-type nectin-4 polypeptide. In certain embodiments, the binding is decreased below the limit of detection. In some embodiments, the binding of the anti-nectin-4 antibody to the mutant nectin-4 polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observed between the anti-nectin-4 antibody and the wild-type nectin-4 polypeptide, a significant decrease in binding is demonstrated.

[0103] In some embodiments, the anti-nectin-4 antibody exhibits significantly lower binding (e.g., loss of binding) to a mutant nectin-4 polypeptide in which residues (or a subsequence thereof, optionally a subsequence of at least 4, 5, 6, 10, 20 or 40 residues) corresponding to residues 32-144 in the wild-type nectin-4 polypeptide (e.g., comprising the sequence of SEQ ID NO: 1) are missing (e.g., deleted or substituted with another amino acid).

[0104] Antibodies can be produced by a variety of techniques known in the art. Generally, these 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 can comprise a full-length sequence of a human nectin-4 polypeptide or a fragment or derivative thereof, generally an immunogenic fragment, i.e., an epitope exposed on the surface of cells expressing the nectin-4 polypeptide, such as an epitope recognized by the ASG-22ME, 14A5.2 or N41 antibodies. Such fragments generally contain at least about 7 contiguous amino acids of the mature polypeptide sequence, more preferably at least about 10 contiguous amino acids thereof. The fragment generally derives essentially from the extracellular domain of the receptor. In certain embodiments, the immunogen comprises a wild-type human nectin-4 polypeptide in a lipid membrane, generally on the surface of a cell. In a specific embodiment, the immunogen comprises intact cells, particularly intact human cells, which are optionally treated or lysed. In another preferred embodiment, the polypeptide is a recombinant nectin-4 polypeptide. In certain embodiments, the immunogen comprises intact nectin-4 expressing cells.

[0105] The step of immunizing a non-human mammal with an antigen can be performed in any manner known in the art for stimulating antibody production in a mouse (see, e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is incorporated herein by reference).

[0106] Antibodies can also be produced by selection of a combinatorial library of immunoglobulins as disclosed, for example, in Ward et al. Nature, 341 (1989) p. 544 (the entire disclosure of which is incorporated herein by reference).

[0107] 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 using any one of a variety of immunological screening assays by which antibody competition can be evaluated. Many such assays are routinely performed and are well known in the art (see, e.g., U.S. Patent No. 5,660,827, which is incorporated herein by reference).

[0108] For example, a simple competition assay can be used in which the test antibody to be examined, which can be obtained from animals of different sources or of different Ig isotypes, is mixed (or pre-adsorbed) with a control (e.g., ASG-22ME, 14A5.2, or N41) and applied to a sample containing the nectin-4 polypeptide. Protocols based on the use of Western blotting and surface plasmon resonance (e.g., Biacore™) analysis are suitable for use in such competition experiments.

[0109] In certain embodiments, a control antibody (e.g., ASG-22ME, 14A5.2 or N41) is pre-mixed with various amounts of a test antibody (e.g., about 1:10 or about 1:100) a certain time before application to the nectin-4 antigen sample. In other embodiments, the control and various amounts of the test antibody can simply be mixed during exposure to the nectin-4 antigen sample. The bound antibody can be distinguished from the free antibody (e.g., by using separation or washing techniques to remove unbound antibody), and ASG-22ME, 14A5.2 or N41 can be distinguished from the test antibody (e.g., by using a species-specific or isotype-specific secondary antibody, or by specifically labeling ASG-22ME, 14A5.2 or N41 with a detectable label), as long as it is possible to determine whether the test antibody reduces the binding of ASG-22ME, 14A5.2 or N41 to the antigen. The binding of the (labeled) control antibody in the complete absence of an unrelated antibody can be a control high value. The control low value can be obtained by incubating the labeled (ASG-22ME, 14A5.2 or N41) antibody with the same type of unlabeled (ASG-22ME, 14A5.2 or N41) antibody, where competition occurs and reduces the binding of the labeled antibody. The test antibody can reduce the binding of ASG-22ME, 14A5.2 or N41 to the nectin-4 antigen by at least about 50%, e.g., at least about 60% or more preferably at least about 80% or 90% (e.g., about 65 - 100%) at any ratio of ASG-22ME, 14A5.2 or N41:test antibody, e.g., about 1:10 to about 1:100. Optionally, such a test antibody can reduce the binding of ASG-22ME, 14A5.2 or N41 to the nectin-4 antigen by at least about 90% (e.g., about 95%).

[0110] For example, competition can also be evaluated by a flow cytometry assay. In such an assay, cells bearing nectin-4 polypeptide can first be incubated, for example, with ASG-22ME, 14A5.2 or N41, and then with a test antibody labeled with a fluorescent dye or biotin. The antibody is said to compete with ASG-22ME, 14A5.2 or N41 if the binding obtained upon pre-incubation with a saturating amount of ASG-22ME, 14A5.2 or N41 is about 80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained with the antibody without pre-incubation with ASG-22ME, 14A5.2 or N41 (when measured by fluorescence means). Alternatively, the antibody is said to compete with ASG-22ME, 14A5.2 or N41 if the binding obtained with labeled ASG-22ME, 14A5.2 or N41 antibody (by a fluorescent dye or biotin) together with the antibody on cells pre-incubated with a saturating amount of the test antibody is about 80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained without pre-incubation with the test antibody.

[0111] A simple competitive assay can also be used in which the test antibody is pre-adsorbed and applied at a saturating concentration to a surface on which the nectin-4 antigen is immobilized. The surface in the simple competitive assay is preferably a Biacore™ chip (or other medium 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 nectin-4 saturating concentration and the surface binding of nectin-4 and the control antibody is measured. This binding of the control antibody is compared to the binding of the control antibody to the nectin-4-containing surface in the absence of the test antibody. In the test assay, a significant decrease in the binding of the nectin-4-containing surface by the control antibody in the presence of the test antibody indicates that the test antibody recognizes a region on nectin-4 substantially the same as that of the control antibody and that the test antibody becomes capable of “cross-reacting” with the control antibody. The test antibody can reduce the binding of a control (e.g., ASG-22ME, 14A5.2 or N41, etc.) antibody to the nectin-4 antigen by at least about 30% or more, preferably by about 40%. Optionally, such a test antibody reduces the binding of a control antibody (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). Of course, the order of the control and test antibodies can be reversed, i.e., the control antibody can be first bound to the surface and the test antibody can then be contacted with the surface in the competitive assay. Preferably, the antibody having a higher affinity for the nectin-4 antigen is first bound to the surface because it is expected that the decrease in binding seen for the second antibody (where cross-reaction of the antibodies is suspected) will be on a larger scale. Further examples of such assays are provided, for example, in Saunal (1995) J. Immunol. Methods 183:33-41, the disclosure of which is incorporated herein by reference.

[0112] The antibody binds to nectin-4-expressing tumor cells derived from an individual having cancer characterized by nectin-4 positive tumor cells, i.e., an individual who is a candidate for treatment by one of the methods described herein using an anti-nectin-4 antibody. Thus, when an antibody that specifically recognizes nectin-4 on the cell is obtained, it can optionally be tested for its ability to bind to nectin-4 positive cells (e.g., cancer cells). This can optionally be tested for its ability to bind to tumor cells that express high levels of the nectin-4 polypeptide on their surface and / or tumor cells that express low levels of the nectin-4 polypeptide on their surface. In particular, prior to treating a patient with one of the present antibodies, the ability of the antibody to bind to malignant cells collected from the patient, e.g., in a blood sample or a tumor biopsy, can optionally be tested to maximize the likelihood that the treatment will be beneficial to the patient.

[0113] In one embodiment, the antibody is validated in an immunoassay that tests its ability to bind to nectin-4 expressing cells, e.g., malignant cells. For example, a blood sample or a tumor biopsy is performed and tumor cells are collected. The ability of a given antibody to bind to the cells is then evaluated using standard methods well known to those of skill in the art. To evaluate antibody binding to the cells, the antibody can be labeled directly or indirectly. When labeled indirectly, typically a secondary labeled antibody is added.

[0114] Determination of whether an antibody binds within an epitope region can be performed as known to those skilled in the art. As an example of such a mapping / characterization method, the epitope region for an anti-nectin-4 antibody can be determined by epitope “footprinting” using chemical modification of exposed amines / carboxyls in the nectin-4 protein. A specific example of such footprinting technology is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), where hydrogen / deuterium exchange, binding, and back-exchange of the receptor and ligand protein amide protons occur, and the backbone amide groups involved in protein binding are protected from back-exchange and thus remain deuterated. The relevant regions can be identified at this point by proteolysis with pepsin, fast microbore high performance liquid chromatography separation, and / or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267(2) pp. 252-259(1999); Engen, J.R. and Smith, D.L. (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), which generally compares the positions of signals in the two-dimensional NMR spectra of an antigen that forms a complex with an antigen-binding peptide such as a free antigen and an antibody. The antigen is generally selectively isotope-labeled with 15N so that only signals corresponding to the antigen are seen in the NMR-spectrum and no signals from the antigen-binding peptide are seen. Antigen signals derived from amino acids involved in the interaction with the antigen-binding peptide generally shift in position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in binding can be identified as such. 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. 1996 Jun;9(3):516-24.

[0115] Epitope mapping / characterization can also be performed using mass spectrometry methods. See, for example, Downard, J Mass Spectrom. 2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May 1;71(9):1792-1801. Protease digestion techniques can also be useful in the context of epitope mapping and identification. Antigenic determinant-related regions / sequences can be determined by protease digestion, for example, by using trypsin at a ratio of about 1:50 to nectin-4, by using o / n digestion at pH 7-8, and subsequently performing mass spectrometry (MS) for peptide identification. Subsequently, peptides protected from trypsin cleavage by anti-nectin-4 conjugates can be identified by comparison of samples subjected to trypsin digestion and samples incubated with the antibody and subsequently subjected to digestion, for example, with trypsin (thereby revealing the footprint for the conjugate). Other enzymes such as chymotrypsin, pepsin, etc. can also be used or alternatively in a similar epitope characterization method. Furthermore, enzymatic digestion can provide a rapid method for analyzing whether potentially antigenic determinant sequences are within regions of the nectin-4 polypeptide that are not surface-exposed and thus presumably not relevant for immunogenicity / antigenicity.

[0116] Site-directed mutagenesis is another technique useful for revealing binding epitopes. For example, in "alanine scanning", each residue within a protein segment is replaced with an alanine residue and the results on binding affinity are measured. If the mutation leads to a significant decrease in binding affinity, it is likely to be involved in binding. Monoclonal antibodies specific for conformational epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to confirm that the alanine substitutions do not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995;267:383-386; and Wells, Proc Natl Acad Sci USA 1996;93:1-6.

[0117] An electron microscope can also be used for epitope "footprinting". For example, Wang et al., Nature 1992; 355: 275-278 used the simultaneous application of cryo-electron microscopy, three-dimensional image reconstruction, and X crystallography to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0118] Other forms of "label-free" assays for epitope evaluation include surface plasmon resonance (SPR, BIACORE™) and reflectometric interference spectroscopy (RifS). See, for example, Faegerstam et al., Journal Of Molecular Recognition 1990; 3: 208-14; Nice et al., J. Chromatogr. 1993; 646: 159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37: 3308-3311; Kroeger et al., Biosensors and Bioelectronics 2002; 17: 937-944.

[0119] It should also be noted that antibody binding to an epitope that is the same as or substantially the same as the antibody can be identified in one or more of the representative competitive assays described herein.

[0120] During immunization and production of antibodies in vertebrates or cells, or during generation of a library of candidate antibodies or amino acid sequences (e.g., by phage display technology), specific selection steps can be carried out to isolate antibody or non-antibody peptide or protein scaffolds. In this regard, in certain embodiments, (a) immunizing a non-human mammal with an immunogen comprising a nectin-4 polypeptide or preparing a library of antibodies or polypeptide sequences; and (b) preparing an antibody from said immunized animal or said library of antibodies or sequences; and (c) selecting an antibody from step (b) that can bind to nectin-4, optionally selecting an antibody from step (b) that can bind to the Ig-like V-set domain of nectin-4, a method for producing such an antibody is provided.

[0121] In one aspect, the antibody has an average dissociation constant (K -8 d) of 1×10 -9 M or less, optionally less than 1×10 D M, with respect to human nectin-4, as determined, for example, by surface plasmon resonance (SPR) screening (such as analysis by a BIAcore™ SPR analyzer). In a more specific exemplary aspect, anti-nectin-4 antibodies are provided that have a KD of about 1×10 -8 M to about 1×10 -10 M or about 1×10 -9 M to about 1×10 -11 M for nectin-4.

[0122] In certain aspects of any of the embodiments, the antibody prepared according to the method is a monoclonal antibody. In another aspect, the non-human animal used to produce the antibody is a mammal such as a rodent, cow, pig, poultry, horse, rabbit, goat or sheep. The antibodies of the present invention can optionally be identified as antibodies other than any of antibody ASG-22ME, 14A5.2 or N41 or derivatives thereof, for example antibodies that include all or part of their respective heavy and light chain CDRs or antigen-binding regions.

[0123] DNA encoding an antibody that binds to an epitope present on the nectin-4 polypeptide is isolated from a hybridoma, placed in an expression vector, and then transfected into a host cell such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce an immunoglobulin protein as such. As described elsewhere herein, such a DNA sequence can be modified for any of a number of purposes, such as to create a fragment or derivative that humanizes the antibody, or to modify the sequence of the antibody at the antigen-binding site to optimize, for example, the binding specificity of the antibody. In certain embodiments, an isolated nucleic acid sequence encoding the light chain and / or heavy chain of the antibody, and a recombinant host cell containing such a nucleic acid (e.g., in its genome) are provided.

[0124] In any embodiment, an anti-nectin-4 binding protein (e.g., an antibody or antibody fragment) can be evaluated for its ability to induce intracellular internalization of nectin-4 expressed by tumor cells. For example, the Fab-Zap assay described in the examples herein can be used as a convenient method to evaluate internalization in nectin-4 expressing cells (e.g., tumor cells). In any embodiment herein, an anti-nectin-4 binding protein, an antibody or antibody fragment, or an antibody-drug conjugate comprising such an antibody or fragment is characterized as being able to cause intracellular internalization when it binds to nectin-4 on the surface of a tumor cell.

[0125] In one aspect, the anti-nectin-4 antibody is an antibody that is a functionally conserved variant of any of the exemplary antibodies described herein, for example, 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 polypeptide that has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, substitution of an amino acid residue in a protein (e.g., an antibody or antibody fragment) with one having similar properties (e.g., polarity, hydrogen bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those shown to be conserved can vary within the protein, and the percent similarity of the protein or amino acid sequences between any two proteins having similar functions can vary, for example, from 70% to 99% when determined according to an alignment scheme such as the Clustal method (similarity is based on the MEGALIGN algorithm). "Functionally conserved variants" also include polypeptides having at least 60% amino acid identity as determined by the BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still more preferably at least 90%, and even more preferably at least 95%, which have the same or substantially similar properties or functions as the native, reference, or parental protein to which it is compared.

[0126] Exemplary anti-Nectin-4 VH and VL pairs that can be used to prepare an ADC for use according to the present disclosure can be obtained from the enfortumab vedotin antibody (or ASG-22ME) by using the VH and VL (or amino acid residues of the hypervariable regions) of ASG-22ME, which is the antibody component used in enfortumab vedotin, the amino acid sequence of the heavy chain variable region (SEQ ID NO: 3) listed below, and the amino acid sequence of the light chain variable region (SEQ ID NO: 4) listed below. The CDRs according to Kabat numbering are underlined in SEQ ID NOs: 3 and 4. See also U.S. Patent No. 8,637,642 and International Publication No. WO 2012 / 047724. These disclosures are incorporated herein by reference. Optionally, the VH and VL include (e.g., are modified to incorporate) a human acceptor framework. In certain embodiments, an anti-Nectin-4 antibody according to the present disclosure includes 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 numbering). In certain embodiments, the anti-Nectin-4 antibody includes 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 numbering).

[0127] ASG-22ME (enfortumab) VH:

Chemical formula

[0128] ASG-22ME (enfortumab) VL:

Chemical formula

[0129] The full-length heavy and light chains of enfortumab or ASG-22CE / ASG-22ME are shown in SEQ ID NOs: 5 and 6. In certain embodiments, an anti-Nectin-4 antibody includes 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.

[0130] Full-length heavy chain of ASG-22CE / ASG-22ME (enfortumab):

Chemical formula

[0131] Full-length light chain of ASG-22CE / ASG-22ME (enfortumab):

Chemical formula

[0132] Another exemplary anti-nectin-4 VH and VL pair that can be used to prepare an ADC for use according to the present disclosure can be derived from antibody 14A5.2, the amino acid sequence of the heavy chain variable region (SEQ ID NO: 7) listed below, and the amino acid sequence of the light chain variable region (SEQ ID NO: 8) listed below. The CDRs according to Kabat numbering are underlined in SEQ ID NOs: 7 and 8. The antibody sequences are also disclosed in WO 2018 / 158398, the disclosure of which is incorporated herein by reference. Optionally, VH and VL include (e.g., are modified to incorporate) a human acceptor framework. In certain embodiments, the anti-nectin-4 antibody according to the present disclosure includes VH CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the anti-nectin-4 antibody includes VL CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0133] 14A5.2 VH:

Chemical formula

[0134] 14A5.2 VL:

Chemical formula

[0135] Another exemplary anti - nectin - 4 VH and VL pair that can be used to prepare an ADC for use according to the present disclosure can be obtained from antibody N41, the amino acid sequence of the heavy - chain variable region (SEQ ID NO: 9) listed below, and the amino acid sequence of the light - chain variable region (SEQ ID NO: 10) listed below. The CDRs according to Kabat numbering are underlined in SEQ ID NOs: 9 and 10. The VH, VL, and their respective CDR sequences of the antibody are also disclosed in WO 2017 / 042210 pamphlet, the disclosure of which is incorporated herein by reference. Optionally, VH and VL include (e.g., are modified to incorporate) a human acceptor framework. In certain embodiments, an anti - nectin - 4 antibody for use according to the present disclosure includes VH CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbering) of the heavy - chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti - nectin - 4 antibody includes VL CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbering) of the light - chain variable region having the amino acid sequence of SEQ ID NO: 10.

[0136] N41 VH:

Chemical formula

[0137] N41 VL:

Chemical formula

[0138] Antibody fragments and derivatives (including, unless otherwise specified or clearly inconsistent with the context, one or more of the terms "antibody" as used in this application) can be prepared by techniques known in the art. A "fragment" includes a portion of an intact antibody, generally an antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments; diabodies; (1) single-chain Fv molecules, (2) single-chain polypeptides that do not have associated heavy-chain molecules and contain only one light-chain variable domain or fragments thereof containing three CDRs of the light-chain variable domain, and (3) single-chain polypeptides that do not have associated light-chain molecules and contain only one heavy-chain variable region or fragments thereof containing three CDRs of the heavy-chain variable region, including but not limited to, polypeptides having a primary structure consisting of a contiguous sequence of one set of neighboring amino acid residues (referred to herein as "single-chain antibody fragments" or "single-chain polypeptides") of any antibody fragment; and multispecific antibodies formed from antibody fragments. In particular, nanobodies, domain antibodies, single-domain antibodies or "dAbs" are included.

[0139] In certain embodiments, the antibody is humanized. A "humanized" form of an antibody is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of an antibody, etc.) that contains minimal sequences derived from mouse immunoglobulins. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of the original antibody (donor antibody), while maintaining the desired specificity, affinity, and capacity of the original antibody.

[0140] In some instances, Fv framework residues of human immunoglobulins can be replaced by corresponding non-human residues. Further, a humanized antibody can contain residues not found in either the recipient antibody or the transferred CDR or framework sequences. These modifications are made to further enhance and optimize antibody performance. Generally, a humanized antibody contains substantially all of at least one, and generally two, variable domains, all or substantially all of the CDR regions corresponding to those of the original antibody, and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. A humanized antibody optimally also contains at least a portion of an immunoglobulin constant region (Fc), which is usually that of a human immunoglobulin. For further details, see Jones et al., Nature, 321, pp. 522 (1986); Reichmann et al., Nature, 332, pp. 323 (1988); Presta, Curr. Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et al., Science, 239, pp. 1534; and U.S. Patent No. 4,816,567, the entire disclosures of which are incorporated herein by reference).

[0141] The selection of human variable domains, both light and heavy chains, to be used in making a humanized antibody is very important for reducing antigenicity. Following the so-called "best-fit" method, the sequence of the variable domain of the antibody is screened against the entire library of known human variable domain sequences. Next, the human sequence that is closest to that of the mouse is accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol. 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, 1987, pp. 901). Another method uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta et al., J. Immunol., 151, p. 2623 (1993)).

[0142] It is even more important that the antibody be humanized while retaining high affinity for nectin-4 and other advantageous biological properties. To achieve this goal, humanized antibodies are prepared by a process of analyzing the parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences, according to a preferred method. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional structures of selected candidate immunoglobulin sequences. By examining these displays, it becomes possible to analyze the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues are selected and combined from consensus and import sequences such that desired antibody properties such as improved affinity for the target antigen are achieved. Generally, CDR residues are most directly and substantially involved in affecting antigen binding. In one example, the FR of a humanized antibody chain is derived from a human variable region having at least about 60% overall sequence identity, preferably at least about 70%, 75% or 80% overall sequence identity, with the variable region of a non-human donor (e.g., ASG-22ME, 14A5.2 or N41 antibody). Optionally, the humanized heavy and / or light chain variable regions share at least about 60%, 70% or 80% overall sequence identity with the respective heavy chain and / or variable regions of a non-human donor (e.g., ASG-22ME, 14A5.2 or N41 antibody). Another method of making a "humanized" monoclonal antibody is to use a XenoMouse (Abgenix, Fremont, CA) as the mouse used for immunization. A XenoMouse is a mouse host in which its immunoglobulin genes have been replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse are already humanized. The XenoMouse is described in U.S. Patent No. 6,162,963, which is incorporated herein by reference in its entirety.Human antibodies can also be produced by a variety of other techniques, such as using other transgenic animals engineered to express a human antibody repertoire for immunization (Jakobovitz et al., Nature 362 (1993) 255) or by selection of an antibody repertoire using phage display methods. Such techniques are known to those skilled in the art and can be carried out starting from monoclonal antibodies as disclosed herein.

[0143] Advantageously, the camptothecin analog-containing linker of the present disclosure can be used in the process for preparing a conjugated antigen-binding agent (e.g., a peptide, polypeptide, antibody or antibody fragment), and an antigen-binding agent-drug conjugate, such as an antibody-drug conjugate (ADC), can be obtained. In one embodiment, the process for preparing the antigen-binding agent conjugate comprises conjugating a camptothecin analog (Z) to the antigen-binding agent. In certain embodiments, the camptothecin analog (Z) can be identified as being conjugated to the antigen-binding agent via a linker (X). X is a linker that binds an antigen-binding agent such as an antibody (Ab) and a camptothecin analog (Z). For example, upon conjugation, X is a residue of the linker that follows a covalent bond to one or both of Ab and Z.

[0144] In embodiments of the present specification, the process of preparing an antibody-drug conjugate includes the step of contacting and / or reacting an antigen-binding agent such as an antibody (Ab) with a camptothecin analog (Z). The contacting can be carried out under conditions suitable for forming or obtaining an antigen-binding agent-drug conjugate of one aspect of the present disclosure. Z can be included, for example, in a compound comprising a camptothecin analog (Z) and a linker (X) or a part of the linker (X), and the step includes contacting the antigen-binding agent with a compound comprising a camptothecin analog (Z) and a linker (X) or a part of the linker (X). The process can optionally include the step of isolating or recovering the formed antigen-binding agent-drug conjugate and optionally further processing the composition for use as a medicament, and optionally formulating the antigen-binding agent conjugate (e.g., together with a pharmaceutical excipient) for administration to a human subject.

[0145] Optionally, a method of making an ADC includes conjugating an antibody (Ab) to 2, 3, 4, 5, 6, 7, or 8 molecules of a camptothecin analog. Optionally, the resulting composition is characterized by a DAR of 2-4, 4-6, or 6-8. Optionally, the method includes conjugating the antibody to 4 molecules of a camptothecin analog. Optionally, the method includes evaluating the DAR and, if the DAR corresponds to a predetermined specification (e.g., a DAR or DAR range disclosed herein, a DAR of about 2, 4, 6, or 8, etc.), further processing the composition for use as a medicament, and optionally further formulating the antibody (e.g., together with a pharmaceutical excipient) for administration to a human subject.

[0146] In some embodiments, the linker (X)-(Z) moiety is prepared and isolated before contacting (and reacting) a compound comprising (X) and (Z) with (Ab) to thereby form a drug conjugate.

[0147] In some embodiments, the method includes (a) contacting and / or reacting a linker (X) or a part of the linker (X) with (Ab) to form an Ab-X conjugate; (b) Contacting and / or reacting the Ab-X of step (a) with a compound comprising a camptothecin analog (Z) or a linker (X) and a second moiety of (Z) to thereby form an antibody-drug conjugate comprises

[0148] X can represent, for example, a molecule comprising a moiety cleavable under physiological conditions, optionally under intracellular conditions. In certain embodiments, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable moiety, and (iii) any self-cleaving or non-self-cleaving spacer system (Y’). The cleavable moiety can be, for example, an oligopeptide (e.g., a di-, tri-, tetra- or pentapeptide). The spacer Y can be disposed between the Ab and the cleavable moiety, and the spacer system (Y’) can be disposed between the cleavable moiety and Z.

[0149] In some embodiments, the linker X or the spacer Y can be optionally specified as comprising a reactive group (R) that can react with a complementary reactive group (R’) that binds to an amino acid of the antibody or an amino acid of the antibody (e.g., under appropriate conditions, optionally after deprotection). Optionally, R is a group that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody.

[0150] In some embodiments, the linker X or the spacer Y can be optionally specified as comprising a residue of the reaction product of a reactive group R and a complementary reactive group (R’) that binds to an amino acid of the antibody or an amino acid of the antibody. Optionally, R is a group that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody and a residue of the reaction product of said free amino, hydroxyl, sulfhydryl or carboxyl group.

[0151] In any embodiment, prior to the step of contacting and / or reacting an antibody or antibody fragment with a compound (e.g., a linker and / or a camptothecin analog), the method includes the step of preparing, selecting, or providing the antibody or antibody fragment. In certain embodiments, the step includes 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 antibodies or antibody fragments disclosed herein.

[0152] For example, the anti-nectin-4 antibody or antibody fragment can be tested for its ability to bind to nectin-4 or the V domain of nectin-4. The antibody or antibody fragment that is determined to bind to nectin-4 (or 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 anti-nectin-4 antibody or antibody fragment can be tested for its ability to bind to a mutant nectin-4 polypeptide (e.g., a mutant nectin-4 polypeptide lacking the Ig-like V-set domain). The antibody or antibody fragment that is determined to have a decrease or loss of binding to the mutant nectin-4 polypeptide (e.g., as compared to binding to the wild-type nectin-4 polypeptide) is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a camptothecin analog (Z)).

[0153] As further described herein, some well-known methods for conjugating a cytotoxic agent to an antibody involve multiple reaction steps where the antibody is first modified with a linker or a portion of a linker, followed by a reaction where the cytotoxic agent is conjugated to the antibody-linker composition.

[0154] In certain embodiments, (i) contacting an antigen-binding agent (e.g., a peptide, polypeptide, antibody or antibody fragment (e.g., one that binds 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 of an amino acid or a glycan or a group attached to an amino acid or a glycan of an amino acid), and (b) a second reactive group (R') to obtain a modified agent comprising one or more amino acids functionalized with the compound (L); (ii) reacting the modifier 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., a di-, tri-, tetra- or pentapeptide) cleaved by an intracellular peptidase or protease enzyme, (c) optionally, a non-cleavable or cleavable spacer (Y'), and (d) a cytotoxic agent (Z); There is provided a process for preparing an antigen-binding agent-drug conjugate, which comprises. Optionally, the compound of step (ii) further comprises a spacer (Y) disposed between R and the amino acid unit.

[0155] In one embodiment, R and R' are capable of undergoing a click reaction or an addition cyclization. Optionally, R comprises an alkyne moiety or is an alkyne moiety, and R' comprises an azide moiety or is an azide moiety, or R' comprises an alkyne moiety or is an alkyne moiety, and R comprises an azide moiety or is an azide moiety, and the reaction of step (ii) is a 1,3-dipolar addition cyclization.

[0156] In certain embodiments, the reaction of step (i) is carried out in the presence of a catalyst, and optionally, the catalyst is an enzyme (e.g., transglutaminase).

[0157] In certain embodiments, before contacting an anti-nectin-4 antibody or antibody fragment with compound (L), step (i) comprises modifying the anti-nectin-4 antibody or antibody fragment. For example, the antibody or antibody fragment can be modified by reacting or contacting it with an enzyme that can modify the glycosylation of the antibody (e.g., at Kabat residue N297). In one example, the modification comprises deglycosylation of an antibody glycan having core N-acetylglucosamine in the presence of an endoglycosidase 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 endoglycosidases include EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH and / or combinations thereof.

[0158] An antigen-binding protein (e.g., an antibody) molecule and a camptothecin analog molecule are linked by a linker. In such embodiments, the immunocomplex is, for example, of formula (II): Ab-(X-(Z) n ) m Formula (II) (wherein, Ab is an anti-nectin 4 antigen-binding protein (e.g., an antibody); X is a linker that links Ab and Z, e.g., a residue of a linker that follows a covalent bond to one or both of Ab and Z; Z is a camptothecin analog, and optionally, Z comprises the structure of compound 1 or 2 (exatecan or SN-38 molecule); n is 1 or 2; and When n is 1, m is any one of 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; when n is 2, m is any one of 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) can be represented by. Optionally, by specifying "n", the degree of branching or polymerization can be represented. "n" and "m" can be specified to represent the average in a composition containing a plurality of antibodies.

[0159] In certain embodiments, X can represent, for example, a molecule comprising a moiety cleavable under physiological conditions, optionally intracellular conditions. In certain embodiments, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable moiety, and (iii) any self-cleaving or non-self-cleaving spacer system (Y'). The spacer Y can be disposed between the Ab and the cleavable moiety, and the spacer system (Y') can be disposed between the cleavable moiety and Z. The molecule X or the spacer Y can optionally be specified as including a residue of a reactant of a reactive group (R) or a complementary reactive group (R') that binds to an amino acid of the antibody or an amino acid of the antibody.

[0160] The variable m represents the number of -X-(Z) per one antibody molecule of the immune complex. n In a composition containing a plurality of anti-nectin-4 ADCs, the number "m" of -X-Z moieties per one antibody molecule can vary. Thus, in an exemplary composition containing a plurality of immune complexes of the formulas herein, m is the average number of -X-(Z) n moieties per one Ab, in which case m is also referred to as the average drug load or drug:antibody ratio (DAR). The average drug load or DAR is advantageously from 1 to about 8 (-X-(Z) per one Ab n) It can be within the range of the part. 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, m represents the average drug load, and m is from 2 to 8. In some embodiments, n is 1, m represents the average drug load, and m is from 2 to 6. In some embodiments, n is 1, m represents the average drug load, and m is from 4 to 8. In some embodiments, n is 1, m represents the average drug load, and m is from 6 to 8, optionally about 6, 7, or 8. In some embodiments, n is 1, m represents the average drug load, and m is from 4 to 6, optionally about 4, 5, or 6.

[0161] The number of (-X-Z) parts per Ab can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the immune complexes regarding m can also be determined. Optionally, the separation, purification, and characterization of homogeneous immune complexes where m is a specific value can be distinguished from immune complexes with other drug loads and can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0162] In certain embodiments, the anti-nectin-4 composition used in the treatment method of the present disclosure has the formula (I): Ab-(X-(Z) n ) m Formula (II) (wherein, Ab is an anti-nectin-4 antigen-binding protein (e.g., an antibody or an antibody fragment); X is a molecule that links Ab and Z, e.g., the residue of a linker following a covalent bond to one or both of Ab and Z; Z is a camptothecin analog containing an exatecan or SN-38 molecule, e.g., a molecule containing the structure of Compound 1 or 2; n is 1 or 2; At least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the antibody sample have an m (number of X-Z moieties) that is 2 or 4, at least 2, 2-4, at least 4, 4-6 or 4-8, and optionally, n is 1, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the antibody sample have an m (number of X-Z moieties) that is 2 or 4, at least 2, 2-4, at least 4, 4-6 or 4-8) characterized as comprising a plurality of immune complexes represented by

[0163] In certain embodiments, the anti-nectin-4 composition used in the treatment methods of the present disclosure has the formula (I): Ab-(X-(Z) n ) m Formula (II) (wherein, Ab is an anti-nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment); X is a molecule that links Ab and Z, e.g., the residue of a linker following a covalent bond to one or both of Ab and Z; Z is a camptothecin analog comprising an exatecan or SN-38 molecule, e.g., a molecule comprising the structure of Compound 1 or 2; n is 1; at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the antibody sample have an m (number of X-Z moieties) that is 6, at least 6, 6-8 or 8) characterized as comprising a plurality of immune complexes represented by

[0164] In certain embodiments, the anti-nectin-4 composition used in the treatment methods of the present disclosure has the formula (I): Ab-(X-(Z) n ) m Formula (II) (wherein, Ab is an anti-nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment); X is a molecule that links Ab and Z, for example, a residue of a linker following a covalent bond to one or both of Ab and Z; Z is a camptothecin analog containing an exatecan or SN-38 molecule, for example, a molecule containing the structure of compound 1 or 2; n is 1; At least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the antibody sample have an m (number of X-Z moieties) of 8) characterized as comprising a plurality of immune complexes represented by.

[0165] A variety of methods can be used to covalently attach a linker containing a cytotoxic agent to an antibody or antigen-binding protein, either non-specifically or specifically to a particular amino acid residue. The linker (X) can optionally contain a portion that is cleavable under intracellular conditions, such as physiological conditions, as shown in the examples, such that cleavage of the linker releases the cytotoxic agent in the intracellular environment. The linker can bind to a chemical reaction group on the antibody molecule, such as a free amino, imino, hydroxyl, thiol or carboxyl group (e.g., N or C terminus, epsilon amino group of one or more lysine residues, free carboxylic acid group of one or more glutamic acid or aspartic acid residues or sulfhydryl group of one or more cysteine residues), a carbohydrate or any reaction group generally introduced or engineered into the antibody. The site to which the linker binds can be a natural residue in the amino acid sequence of the antibody molecule or can be introduced into the antibody molecule by, for example, DNA recombination techniques (e.g., by introducing a cysteine or protease cleavage site into the amino acid sequence, by introducing a non-natural amino acid residue) or protein biochemistry (e.g., by glycoengineering, reduction, pH adjustment or proteolysis, enzymatic modification of amino acid-linked glycans).

[0166] In certain embodiments, an intermediate that is a precursor of linker (X) is reacted with a cytotoxic agent (Z) under suitable conditions. In certain embodiments, the reactive groups are used on the cytotoxic agent and / or the intermediate. In some embodiments, the product of the reaction between the cytotoxic agent and the intermediate or derivatized cytotoxic agent is then reacted with an antibody molecule under suitable conditions. In other embodiments, the precursor of linker (X) is first reacted with an antibody molecule under suitable conditions to produce an antibody bound to the precursor of linker (X), and then the antibody is reacted with a molecule comprising the cytotoxic agent (Z).

[0167] In some embodiments, linker (X) is cleavable by a cleaving agent present in the intracellular environment (e.g., within lysosomes or endosomes or caveolae). The linker can comprise, for example, a peptidyl linker or amino acid units that are cleaved by intracellular peptidases or protease enzymes including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptidyl linker portion is at least 2 amino acids in length or at least 3 amino acids in length. Cleaving agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug into the target cell. The most typical ones are peptidyl linkers cleavable by enzymes present within the cell. In certain embodiments, the peptidyl linker cleavable by intracellular proteases is a Val-Cit linker or a phenylalanine-lysine (Phe-Lys) linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with valine-citrulline). The valine-citrulline (Val-Cit) moiety can have the structure shown below.

Chemical formula

[0168] In another specific embodiment, the peptidyl linker cleavable by an intracellular protease is a valine-alanine (Val-Ala) linker. The val-ala element may have the structure shown below. [Chemical Formula]

[0169] In another specific embodiment, the peptidyl linker cleavable by an intracellular protease is a glycine-containing oligopeptide linker, such as a glycine and phenylalanine-containing oligopeptide linker, 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).

[0170] In some embodiments, in addition to being optionally cleavable by an intracellular protease, the linker functions as a spacer or stretcher to avoid interfering with the ability of the antibody to bind nectin-4 and / or inhibit cell-cell interactions mediated by nectin-4, so as to keep the antibody away from Z. The linker may include a spacer unit (Y) and / or a spacer or spacer system (Y’). Thus, the spacer Y can be disposed between the Ab and the cleavable moiety. The spacer system (Y’) can be disposed between the cleavable moiety and Z. Or the spacer Y (or the linker X containing it) can optionally be specified as including a residue of a reactant of a reactive group (R) or a complementary reactive group (R’) that binds to an amino acid of the antibody or an amino acid of the antibody. The spacer Y can be, for example, a molecule that forms a bond (e.g., via its reactive group R) with an amino acid of the antibody, such as a sulfur atom, a primary or secondary amino group, or a carbohydrate group of the antibody. The spacer or stretcher (Y) links the antibody to a cytotoxic agent (Z) or a cleavable amino acid unit (e.g., a peptidyl linker, a cleavable di-, tri-, tetra- or pentapeptide), and optionally further binds to a self-detaching and / or non-self-detaching spacer (Y’), and links to Z. Thus, when the spacer (Y) is linked to an amino acid unit (e.g., a cleavable di-, tri-, tetra- or pentapeptide) at one end, the cleavable amino acid unit can thus bind directly to Z or can include a further spacer (Y’), such as a non-self-destructive or self-destructive spacer that links the amino acid unit and Z.

[0171] The spacer (Y) can optionally be specified as being or including a substituted or unsubstituted alkyl or heteroalkyl chain. Optionally, Y has a chain length of 2 to 100 atoms, optionally 2 to 40, 2 to 30, 2 to 20, 4 to 40, 4 to 30 or 4 to 20 atoms. Optionally, one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms. Optionally, any carbon of the chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0172] The spacer (Y) can optionally be specified as including a stability-enhancing moiety. For example, the spacer Y can be orthogonal to a polyethylene glycol (PEG) moiety or a polysarcosine (poly-N-methylglycine or PSAR) moiety in linker design (see, for example, WO 2019 / 081455, WO 2015 / 057699 and WO 2016 / 059377, the disclosures of which are incorporated herein by reference).

[0173] In some specific embodiments, the spacer (Y) can 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 -(CH 2 CH 2 O) x -, where x is 1 to 12, optionally 1 to 8, optionally 1 to 6.

[0174] An example of a suitable stability enhancing moiety, the spacer chain Y, may comprise a stability enhancing moiety disclosed in WO 2015 / 057699 or WO 2019 / 081455. For example, the spacer chain Y may comprise an orthogonal linker moiety and a stability enhancing moiety. The stability enhancing moiety may be a PEG homopolymer or generally any single molecular weight homopolymer (e.g., PEG or polysarcosine homopolymer) attached to the orthogonal linker moiety. The homopolymer may have, for example, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, at least 6, 8 or 10 or 6 to 12, 6 to 24, 6 to 72 units of PEG or other monomer. The term orthogonal connector refers to a branched linker unit component that links a linker moiety (e.g., the chain of spacer Y) to a homopolymer unit such that the homopolymer units are arranged in parallel (not in series) in relation to the cytotoxic agent (the homopolymer is parallel to the Pep-Y’-Z moiety), and is linked to the cytotoxic agent (Z) via a linker (e.g., a cleavable oligopeptide (Pep) and spacer Y’). The orthogonal linker moiety may be, for example, one or more natural or unnatural amino acids optionally selected from glutamic acid, lysine and glycine. Optionally, the amino acid orthogonal connector moiety is arranged at the end of the spacer chain Y such that the amino acid residue of the orthogonal linker moiety is linked to the amino acid residue of the peptidyl linker (e.g., (Pep) of 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 may, for example, comprise an orthogonal connector moiety and formula D: [Chemical Formula] (wherein R 1 and R 2 are different, and R 1 and R 2 one of which is H or an inert group, and R 1 and R 2 the other of which is a functionalized reactive group, said group being reactive to covalently bond to a bondable group of the orthogonal linker moiety and non-reactive under reaction conditions such that the inert group is non-reactive, Z 1 and Z2 which are the same or different, are optional spacers, n is 1 or more, and k is 2 or more) can include the result of the reaction with the moiety of .

[0175] In another example, spacer Y includes a group disclosed in U.S. Patent Application Publication No. 2017 / 0072068A1, the disclosure of which is incorporated herein by reference, for example, formula (E):

Chemical Formula

[0176] A spacer or spacer system (Y’) disposed between an amino acid unit (e.g., a cleavable di-, tri-, tetra- or pentapeptide) and Z can be self-eliminating or non-self-eliminating. The spacer Y’ can include, for example, a substituted or unsubstituted alkyl or heteroalkyl chain. Optionally, Y has a chain length of 2 to 30 atoms, optionally 2 to 20, 4 to 20, 2 to 10 or 4 to 20 atoms, and optionally, one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms. Optionally, any carbon of the chain can be substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide. In certain embodiments, Y’ includes a p-aminobenzyloxycarbonyl group. In certain embodiments, Y’ is a non-self-eliminating spacer and includes a (CH2-C(=O)) group. For example, Y’ can be or include an -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)- group.

[0177] The "self - detaching" spacer unit enables the release of the drug moiety without another hydrolysis step. When using a self - detaching spacer, after cleavage or conversion of the amino acid unit, the side linked to the amino acid unit of the spacer is no longer blocked, and as a result, one or more moieties Z are released. Self - detaching spacer systems can be, for example, those described in WO 02 / 083180 pamphlet and WO 2004 / 043493 pamphlet, the disclosures of which are incorporated herein by reference in their entirety, and the same applies to other self - detaching spacers known to those skilled in the art. In certain embodiments, the spacer unit of the linker contains a p - aminobenzyl unit. In such an embodiment, p - aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and a carbamate, methylcarbamate or carbonate is formed between the benzyl alcohol and the cytotoxic agent. In an embodiment, the spacer unit is p - aminobenzyloxycarbonyl (PAB). Examples of self - detaching spacer units further include aromatic compounds electronically similar to p - aminobenzyl alcohol, such as 2 - aminoimidazole - 5 - methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para - aminobenzyl acetals (see, for example, US Patent Application Publication No. 2005 / 0256030A1), but are not limited thereto. The spacer can be used in mats that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4 - aminobutyric acid amide (Rodrigues et al., Chemistry Biology, 1995, 2, 223) and 2 - aminophenylpropionic acid amide (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). The elimination of amine - containing drugs substituted at the α - position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self - destructing spacer.The p-aminobenzyl self-cleaving spacer (e.g., PAB) is particularly suitable for use with Phe-Lys, Val-Ala or Val-Cit cleavable dipeptide units (PAB is placed between the dipeptide and the camptothecin analog (Z).

[0178] A "non-self-cleaving" spacer unit is one in which some or all of the spacer unit remains attached to moiety Z upon enzymatic (e.g., proteolytic) cleavage of the antibody-target moiety complex. Examples of non-self-cleaving spacer units suitable for use as a spacer between a Gly-Gly-Phe-Gly amino acid unit and an exatecan molecule include -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2 -C(=O)- and -CH 2 CH 2 -O-CH 2 -C(=O)-(e.g., to form the GGFG-CH2CH2-O-CH2-C(=O)-exatecan unit) are included, but not limited to these. Use of such a spacer between the GGFG amino acid unit and exatecan results in the release of an exatecan-containing molecule having the structure of compound 3. Other examples of non-self-cleaving spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other known combinations of peptide spacers that are susceptible to sequence-specific enzymatic cleavage can be used in a similar manner. For example, enzymatic cleavage of an antibody-target moiety complex 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-target moiety complex. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, whereby the glycine-glycine spacer unit is cleaved from the drug moiety.

[0179] Exemplary linker-camptothecin moieties (X-Z) can include either of the structures shown in Formulas III and IV below, where Z is a camptothecin analog and Y and Y’ are spacers.

Chemical formula

Chemical formula

[0180] The spacers (Y) and (Y’) can be optionally substituted by one or more heteroatoms selected from the group of O, S, and NR 1 and can be optionally interrupted, and are linear or branched C 1 ~C 20 alkylene groups, C 2 ~C 20 alkenylene groups, C 2 ~C 20 alkynylene groups, C 3 ~C 20 cycloalkylene groups, C 5 ~C 20 cycloalkenylene groups, C 8 ~C 20 cycloalkynylene groups, C 7 ~C 20 alkylarylene groups, C 7 ~C 20 arylalkylene groups, C 8 ~C 20 arylalkenylene groups, C 9 ~C 20 arylalkynylene groups, and can be specified as independently selected from the group consisting of alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups, and arylalkynylene groups, and R 1 is independently hydrogen, optionally substituted C 1 ~C 24 alkyl groups, C 2 ~C24 An alkenyl group, C 2 ~C 24 An alkynyl group and C 3 ~C 24 Is selected from the group consisting of a cycloalkyl group, an alkyl group, an alkenyl group, an alkynyl group and a cycloalkyl group.

[0181] The spacers (Y) and (Y’) are optionally C 1 ~C 10 Alkylene-, -C 1 ~C 10 Heteroalkylene-, -C 3 ~C 8 Carbocyclo-, -O-(C 1 ~C 8 Alkyl)-, -Arylene-, -C 1 ~C 10 Alkylene-arylene-, -Arylene-C 1 ~C 10 Alkylene-, -C 1 ~C 10 Alkylene-(C 3 ~C 8 Carbocyclo)-, -(C 3 ~C 8 Carbocyclo)-C 1 ~C 10 Alkylene-, -C 3 ~C 8 Heterocyclo-, -C 1 ~C 10 Alkylene-(C 3 ~C 8 Heterocyclo)-, -(C 3 ~C 8 Heterocyclo)-C 1 ~C 10 Alkylene-, -C 1 ~C 10 Alkylene-C(=O)-, -C 1 ~C 10 Heteroalkylene-C(=O)-, -C 3 ~C 8 Carbocyclo-C(=O)-, -O-(C 1 ~C 8 Alkyl)-C(=O)-, -Arylene-C(=O)-, -C 1 ~C10 alkylene-arylene-C(=O)-, -arylene-C 1 ~C 10 alkylene-C(=O)-, -C 1 ~C 10 alkylene-(C 3 ~C 8 carbocyclo)-C(=O)-, -(C 3 ~C 8 carbocyclo)-C 1 ~C 10 alkylene-C(=O)-, -C 3 ~C 8 heterocyclo-C(=O)-, -C 1 ~C 10 alkylene-(C 3 ~C 8 heterocyclo)-C(=O)-, -(C 3 ~C 8 heterocyclo)-C 1 ~C 10 alkylene-C(=O)-, -C 1 ~C 10 alkylene-NH-, -C 1 ~C 10 heteroalkylene-NH-, -C 3 ~C 8 carbocyclo-NH-, -O-(C 1 ~C 8 alkyl)-NH-, -arylene-NH-, -C 1 ~C 10 alkylene-arylene-NH-, -arylene-C 1 ~C 10 alkylene-NH-, -C 1 ~C 10 alkylene-(C 3 ~C 8 carbocyclo)-NH-, -(C 3 ~C 8 carbocyclo)-C 1 ~C 10 alkylene-NH-, -C 3 ~C 8 heterocyclo-NH-, -C 1 ~C 10 alkylene-(C 3 ~C 8 heterocyclo)-NH-, -(C3 ~C 8 heterocyclo)-C 1 ~C 10 alkylene-NH-,-C 1 ~C 10 alkylene-S-,-C 1 ~C 10 heteroalkylene-S-,-C 3 ~C 8 carbocyclo-S-,-O-(C 1 ~C 8 alkyl)-)-S-,-arylene-S-,-C 1 ~C 10 alkylene-arylene-S-,-arylene-C 1 ~C 10 alkylene-S-,-C 1 ~C 10 alkylene-(C 3 ~C 8 carbocyclo)-S-,-(C 3 ~C 8 carbocyclo)-C 1 ~C 10 alkylene-S-,-C 3 ~C 8 heterocyclo-S-,-C 1 ~C 10 alkylene-(C 3 ~C 8 heterocyclo)-S-,-(C 3 ~C 8 heterocyclo)-C 1 ~C 10 alkylene-S-,-C 1 ~C 10 alkylene-O-C(=O)-,-C 3 ~C 8 carbocyclo-O-C(=O)-,-O-(C 1 ~C 8 alkyl)-O-C(=O)-,-arylene-O-C(=O)-,-C 1 ~C 10 alkylene-arylene-O-C(=O)-,-arylene-C 1 ~C 10 alkylene-O-C(=O)-,-C 1 ~C 10 alkylene-(C 3 ~C8 Carbocyclo)-O-C(=O)-, -(C 3 ~C 8 Carbocyclo)-C 1 ~C 10 Alkylene-O-C(=O)-, -C 3 ~C 8 Heterocyclo-O-C(=O)-, -C 1 ~C 10 Alkylene-(C 3 ~C 8 Heterocyclo)-O-C(=O)-, -(C 3 ~C 8 Heterocyclo)-C 1 ~C 10 It can be specified as being or including alkylene-O-C(=O)-, and in each case, optionally, -X, -R’, -O, -OR’, =O, -SR’, -S - , -NR’ 2 , -NR’ 3 + , =NR’, -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NR’C(=O)R’, -C(=O)R’, -C(=O)NR’ 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R’, -OS(=O) 2 OR’, -S(=O) 2 NR’, -S(=O)R’, -OP(=O)(OR’) 2 , -P(=O)(OR’) 2 , -PO 3 , -PO 3 H 2 , -C(=O)X, -C(=S)R’, -CO 2 R’, -CO 2 , -C(=S)OR’, C(=O)SR’, C(=S)SR’, C(=O)NR’ 2 , C(=S)NR’ 2 and C(=NR’)NR’ 2substituted with one or more substituents selected from, and each X is, independently, a halogen: -F, -Cl, -Br or -I; each R’ is, independently, -H, -C 1 ~C 20 alkyl, -C 6 ~C 20 aryl or -C 3 ~C 14 is a heterocycle.

[0182] The spacer (Y) can optionally be specified, for example, at one end of the chain, as reacting with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or a carbohydrate, or with a complementary reactive group (R’) attached to an amino acid of the antibody (e.g., via a free amino, hydroxyl, sulfhydryl or carboxyl group or a carbohydrate), or as a residue of a reactant of a reactive group (R) with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody at the time of binding to the anti-nectin-4 antibody or as a reactive group (R) that reacts with a complementary reactive group (R’) attached to an amino acid of the antibody. Examples of the reactive group pair R and R’ include bioorthogonal reactions, preferably cycloaddition reactions, such as the Diels–Alder reaction or 1,3-dipolar cycloaddition reactions, such as between an azide and cyclooctyne (copper-free click chemistry), between a nitrone and cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, and tetrazine ligation, and a wide range of groups capable of these are included (see also WO 2013 / 092983 pamphlet or US Patent Application Publication No. 2017 / 0072068A1, the disclosures of which are incorporated herein by reference). For example, R can be an alkyne and R’ can be an azide, or R can be an azide and R’ can be an alkyne. Thus, the resulting linker and the functionalized antibody or its Y element can, in any embodiment, contain a group (RR’) resulting from the reaction of R and R’, for example, RR’ can be or can contain a triazole resulting from the reaction of an alkyne and an azide.

[0183] In certain embodiments, the reactive groups R and R’ are complementary reagents that are capable of undergoing a “click” reaction together (i.e., click chemistry reagents or reactive groups). For example, a 1,3-dipolar functional compound can react with an alkyne in a cyclization reaction to form a heterocyclic compound, preferably in the substantial absence of an added catalyst (e.g., Cu(I)). Various compounds having at least one 1,3-dipolar group (having a 3-atom pi electron system delocalized over 3 atoms and containing 4 electrons) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipolar groups include, but are not limited to, azide, nitrile oxide, nitrone, azoxy group, and acyl diazo group.

[0184] Examples include o-phosphine aromatic esters, azides, fulminates, alkynes (including strained cycloalkynes), cyanides, anthracenes, 1,2,4,5-tetrazines, or norbornenes (or other strained cycloalkenes).

[0185] In one embodiment, R is a moiety having a terminal alkyne or azide; such moieties are described, 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 catalyst for the click reaction between a terminal alkyne and an azide are provided in U.S. Patent No. 7,763,736.

[0186] In certain embodiments, R is a substituted or unsubstituted cycloalkyne. Cycloalkynes including specific compounds are described, for example, in U.S. Patent No. 7,807,619, the disclosure of which is incorporated herein by reference.

[0187] In some embodiments, the cycloalkyne has the formula A:

Chemical formula

[0188] In some embodiments, the modified cycloalkyne is of formula A, and one or more carbon atoms of the cyclooctyne ring other than the two carbon atoms bonded by a triple bond are substituted with one or more electron-withdrawing groups such as halo (bromo, chloro, fluoro, iodo), nitro group, cyano group, sulfone group or sulfonic acid group. Thus, for example, in some embodiments, the subject modified cycloalkyne is of formula B:

Chemical formula

[0189] In certain embodiments, R is a substituted or unsubstituted heterocyclic strained alkyne. Cycloalkynes containing certain compounds are described, for example, in U.S. Patent No. 8,133,515, the disclosure of which is incorporated herein by reference. In certain embodiments, the alkyne has the formula C:

Chemical formula

[0190] The alkynes as described above in this specification can preferably react with at least one 1,3-dipolar functional compound in a cyclization reaction to form a heterocyclic compound in the substantial absence of an added catalyst (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipolar group (having a 3-atom π-electron system containing 4 electrons delocalized over 3 atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipolar groups include, but are not limited to, azide, nitrile oxide, nitrone, azoxy group, and acyl diazo group.

[0191] In the formulas of this specification, Y’ can optionally be absent or be a spacer, an optionally self-eliminating spacer or non-self-eliminating spacer containing, for example, a p-aminobenzyl unit. Optionally, Y’ is or contains a substituted or unsubstituted alkyl or heteroalkyl chain, and optionally, Y’ has a chain length of 2 to 40 atoms, optionally 2 to 30, 2 to 20, 4 to 40, 4 to 30 or 4 to 20 atoms, and optionally, one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms, and optionally, any carbon of the chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0192] Exemplary linker-camptothecin molecules (e.g., the X-Z moiety of Formulas I to XI) that can complex with an anti-nectin-4 binding protein can optionally be of Formula V: (R)-(Y)-(Pep)-(Y’)-(Z) Formula (V) (wherein R is a group that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody, or reacts with a complementary reactive group (R’) bonded to an amino acid of the antibody, or upon complexation to an anti-nectin-4 binding protein, R is a residue of the reaction product of a reactive group (R) with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or a complementary reactive group (R’) bonded to an amino acid of the antibody; Y is optionally absent or is a spacer; Pep is a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, such as valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide, or includes the same; Y’ is optionally absent, or is a spacer, optionally a self-cleaving spacer or a non-self-cleaving spacer; and Z is a camptothecin analog or derivative, optionally exatecan or an SN-38 molecule) can be represented by.

[0193] The obtained nectin-4 binding immune complex according to the present invention is, for example, of formula (VI): Ab-(Y)-(Pep)-(Y’)-(Z) Formula (VI) (wherein, Ab is an anti-nectin-4 antigen-binding protein (e.g., an antibody); Y is optionally absent or is a spacer. Optionally, Formula VI includes a residue of the reaction product of a reactive group (e.g., maleimide, primary amine) with a side chain or carbohydrate of an amino acid of the anti-nectin-4 antigen-binding protein (Ab) between (Ab) and (Y). Alternatively, the residue of the reaction product of a reactive group (e.g., maleimide, primary amine) with a side chain of an amino acid of the anti-nectin-4 antigen-binding protein (Ab) can be specified as being included in Y; Pep is an amino acid unit (e.g., a peptidyl linker) that is cleaved by an intracellular peptidase or protease enzyme or contains it (e.g., (Pep) is a protease-cleavable di-, tri-, tetra- or pentapeptide, such as a valine-citrulline, valine-alanine or phenylalanine-lysine unit); Y’ is, optionally, absent or is a spacer, optionally a self-cleaving spacer or a non-self-cleaving spacer; and Z is a camptothecin analog or derivative, optionally an exatecan or SN-38 molecule) can be represented by.

[0194] Optionally, the formula can be specified as including a residue (RR’) of a reaction product between a reactive group (R) and a complementary reactive group (R’) bound to a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or an amino acid of the antibody, for example, (between (Ab) and the terminus of Y (or Pep or X if Y is absent)).

[0195] In one example, when (RR’) is a residue of a reaction product between a reactive group (R) and a complementary reactive group (R’) bound to the antibody (e.g., R’ is bound to the side chain or glycan of an amino acid of the antibody), the nectin-4 binding immune complex according to the present invention is, for example, of the formula (VI bis ): 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 bioorthogonal reaction, preferably an addition cyclization, such as a Diels-Alder reaction or a 1,3-dipolar addition cyclization) can be represented by. In certain embodiments, RR’ is

Chemical Formula

Chemical formula

[0196] In any embodiment, the exatecan molecule (or other 6-ring camptothecin) can be identified as being attached to Y’ (or Pep if Y’ is absent) via the amine at the 1-position of exatecan.

[0197] In any embodiment, the SN-38 molecule (or other 5-ring camptothecin) can be identified as being attached to Y’ (or Pep if Y’ is absent) via the amine at the 9-position of SN-38.

[0198] Camptothecin is well-known and there are a wide range of camptothecin derivatives and analogs that share a core ring system with various substitutions, but it is preferred to have modifications or substitutions in ring A and / or B compared to the following basic camptothecin structure.

Chemical formula

[0199] Numerous camptothecin analogs have been reported, including topotecan, irinotecan, exatecan, DXd, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin, gimatecan, belotecan, and rubitecan. Additional camptothecin analogs are disclosed in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392, Jpn. J. Cancer Res. 86:776-782, and Takiguchi et al. 1997 Jpn. J. Cancer Res. 88:760-769, the disclosures of which are incorporated herein by reference. Four analogs, topotecan, irinotecan, belotecan, and DXd (as part of trastuzumab deruxtecan), are approved by the FDA. In certain embodiments, the camptothecin analog is a five-ring compound (e.g., camptothecin lacks the F ring). In certain embodiments, the camptothecin analog is a six-ring compound, e.g., including the F ring.

[0200] Some examples, such as the basic camptothecin structure, the SN-38 molecule (7-ethyl-10-hydroxycamptothecin; the active metabolite of irinotecan), and the camptothecin analogs disclosed in Li et al., ACS Med. Chem. Lett. 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) via a substituent on the B ring.

[0201] SN-38:

Chem.

[0202] Compound of Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392:

Chem.

[0203] Optionally, the camptothecin analog is a hexacyclic compound (optionally with an F ring), which is attached to the linker via a substituent on such an F ring. Examples of such hexacyclic compounds include, but are not limited to, DXd and exatecan.

[0204] Thus, the camptothecin analog includes any of a wide range of molecules including exatecan, SN-38, and such moieties. For example, exatecan can be unsubstituted or substituted with an amine at the 1-position. For example, the substituent can be -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2 -C(=O)-, -CH 2 CH 2 -O-CH 2 -C(=O)- group or other groups shown in U.S. Patent No. 6,835,807 (the disclosure of which is incorporated herein by reference) or including the same.

[0205] In certain embodiments, the antibody of the disclosure releases an exatecan molecule having the structure of Compound 1 in vivo or in vitro in the presence of nectin-4 expressing tumor cells (e.g., by enzymatic cleavage of a cleavable moiety followed by self-elimination of spacer Y').

[0206] The camptothecin analog or exatecan analog is described in Mitsui et al. 1995 Jpn. J. Cancer Res. 86:776-782 and Takiguchi et al. 1997 Jpn. J. Cancer Res. 88:760-769, the disclosures of which are incorporated herein by reference. The structure of exatecan is shown in Compound 1a below.

Chemical Formula

[0207] Exatecan can be attached to a linker via the nitrogen atom of the amino group at position 1, such that the exatecan moiety, when attached to the linker or present within a linker-exatecan molecule ((X-Z) molecule), for example when conjugated to an antibody, has the structure of compound 1b.

Chemical Structure

[0208] Thus, when the exatecan of compound 1a is attached to a linker via the amine at position 1 (and for example when the linker is attached to an antibody), it is understood that the exatecan is a modified group at position 1 (i.e., the NH 2 group at position 1 is substituted with an NH or OH or O group). For example, exatecan can be attached to an antibody via a linker that includes a cleavable oligopeptide. Examples include di-, tri-, tetra- and pentapeptides such as the glycine and phenylalanine-containing peptides or the valine-citrulline or valine-alanine of a dipeptide attached to a PAB molecule shown in U.S. Patent No. 6,835,807, or the disclosure thereof is incorporated herein by reference. Various suitable linker Z structures are known that can release an exatecan or exatecan derivative active at the amino at position 1. For example, as shown in Formula III and compound 13, exatecan can be linked to a cleavable oligopeptide via a (CH2-C(=O)) group attached to the amine at position 1, thereby releasing compound 13 containing exatecan. Examples of substituents at the NH 2 at position 1 of the exatecan of compound 1a include -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2-O-CH 2 -C(=O)- and -CH 2 CH 2 -O-CH 2 -C(=O)- and the like, including groups such as (CH 2 -C(=O)) is included. Exatecan is an exatecan derivative in which the NH at the 1-position 2 is substituted, for example, by an NH group, OH-CH 2 -C(=O)-NH group. In one embodiment, the substituted exatecan or exatecan derivative (for example, derived at the 1-position) has the structure of Compound 13.

[0209] In certain embodiments, the linker moiety (X-Z) has or comprises the structure shown in Formula VII below, and (Y) is a residue of a reactant with a reactive group (R) and an amino acid residue, for example, a free amino, hydroxyl, sulfhydryl, or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteine residues) (e.g., at its terminus). An anti-nectin-4 binding protein functionalized with a linker having the structure of Formula VII or Compound 3 or 4 releases or produces a compound having the structure of Compound 1a (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells).

Chemical Structure

[0210] Exemplary linkers having maleimide as the R group can have the structure of Compound 3 below. Such linkers can be conjugated to an antibody via a cysteine residue in the antibody after the interchain disulfide bond is reduced with a reducing agent, e.g., tris(2-carboxyethyl)phosphine hydrochloride.

Chemical Structure

[0211] The resulting antibody-drug conjugate comprises an antibody comprising one or more cysteine residues functionalized with a compound having the structure of Compound 3 (wherein Compound 3 is attached via the S atom of the cysteine residue).

[0212] In another embodiment, the linker can have a primary amine as the R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can generate an antibody comprising one or more acceptor glutamine residues functionalized with the linker. For example, the linker (X-Z) or the antibody functionalized therewith has or comprises a structure represented by the following Compound 4.

Chemical formula

[0213] In certain embodiments, the linker moiety (X-Z) has or comprises a structure represented by the following Formula VIII, and (Y) is a residue of a reactant with a reactive group (R) and an amino acid residue, such as a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues or the S atom of one or more cysteine residues) or a glycan structure of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of the antibody), for example, at its terminus. An anti-nectin-4 binding protein functionalized with a linker having the structure of Formula VIII or Compounds 5, 6 or 7 releases or produces a compound having the structure of Compound Ia (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells).

Chemical formula

[0214] Exemplary linkers having maleimide as the R group may have the structures of Compounds 5, 6, or 7 below. Such linkers can bind to an antibody via a cysteine residue in the antibody after the interchain disulfide bond is reduced with a reducing agent.

Chemical formula

Chemical formula

[0215] In another embodiment, the linker can have a primary amine as the R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can generate an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (X-Z) or the antibody functionalized therewith can have or contain the structure shown as Compound 8 below.

Chemical formula

[0216] In certain embodiments, the linker moiety (X-Z) has or contains the structure shown in Formula IX below, and (Y) is a residue of a reactant with a reactive group (R) and an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl, or carboxyl group on the antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteine residues) or a glycan structure of a glycosylated amino acid residue (e.g., a native, cleaved, or otherwise modified N-glycan attached to Kabat residue N297 of the antibody), and is a spacer that contains (e.g., at its terminus) the residue of the reactant. An anti-nectin-4 binding protein functionalized with a linker containing the structure of Formula IX releases a compound having the structure of Compound Ia (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells).

Chemical formula

[0217] Exemplary linkers having maleimide as the R group can have the structures of the following compounds 9a, 9b, 9c and 9d. Such linkers can bind to an antibody via a cysteine residue in the antibody after the interchain disulfide bond is reduced with a reducing agent.

Chemical formula

Chemical formula

[0218] In another embodiment, the linker can have a primary amine as the R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can generate an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (X-Z) or the antibody functionalized therewith has or contains the structure shown as the following compound 10.

Chemical formula

[0219] In certain embodiments, the linker moiety (X-Z) has or contains the structure shown in the following formula X, and (Y) is a residue of a reactant with a reactive group (R) and an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues or the S atom of one or more cysteine residues) or a glycan structure of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of the antibody), and is a spacer that contains (e.g., at its terminus) the residue of the reactant.

Chemical formula

[0220] Exemplary linkers having maleimide as the R group may have the structure of the following Compound 11. Such linkers can bind to an antibody via a cysteine residue in the antibody after the interchain disulfide bond is reduced with a reducing agent, for example, tris(2-carboxyethyl)phosphine hydrochloride.

Chem.

[0221] In certain embodiments, the linker moiety (X-Z) has or comprises the structure shown in Formula XI below, and (Y) is a residue of a reactant with a reactive group (R) and an amino acid residue, for example, a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues or the S atom of one or more cysteine residues) or a glycan structure of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of the antibody), and is a spacer that includes (e.g., at its terminus) the residue of the reactant.

Chem.

[0222] Exemplary linkers having maleimide as the R group may have the structure of the following Compound 12. Such linkers can bind to an antibody via a cysteine residue in the antibody after the interchain disulfide bond is reduced with a reducing agent.

Chem.

[0223] In another embodiment, the linker can have a primary amine as the R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can generate an antibody comprising one or more acceptor glutamine residues functionalized with the linker. For example, the linker (X-Z) or the antibody functionalized therewith can have or comprise the structure shown as compound 13 below.

Chemical formula

[0224] The oligopeptide-containing linker-functionalized anti-nectin-4 binding protein of formula XI or compounds 12 and 13 results in the release of a substituted exatecan having an OH-CH2-C(=O) substituent present at the amine in the 1-position, as shown in the structure containing the following exatecan (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells).

Chemical formula

[0225] Further examples of suitable linkers shown in compounds 15 and 16 below have maleimide as the R group and a phenylalanine-containing peptide attached to the PAB molecule.

Chemical formula

[0226] An exemplary linker having maleimide as the R group and an orthogonal polysarcosine moiety is shown to be able to have the structure of compound 17 below. Such a linker can bind to an antibody via a cysteine residue in the antibody after the interchain disulfide bond has been reduced with a reducing agent.

Chemical formula

[0227] In any of the exemplary linkers, when binding to an antibody, the terminal reactive group can be identified as being replaced by a residue of a reaction product with an amino acid residue on the antibody, such as a free amino, hydroxyl, sulfhydryl or carboxyl group of the amino acid.

[0228] Exemplary linkers of Formulas III, IV, V, VI, VII, VIII, IX, X or XI, when prepared as structures having a primary amine, can react with an antibody in the presence of a transglutaminase enzyme (e.g., bacterial transglutaminase, BTG) such that the transglutaminase enzyme catalyzes the conjugation of the linker to an acceptor glutamine residue within the primary structure of the antibody, e.g., within an immunoglobulin constant domain or within a constant region in a TGase recognition tag inserted or added (e.g., fused). Methods and linkers for use in conjugation to an antibody via BTG are described in International Publication No. WO 2014 / 202773, the disclosure of which is incorporated by reference. Conjugation catalyzed by BTG allows for precise control of the average drug:antibody ratio in a composition. The term “transglutaminase” is used interchangeably with “TGase” or “TG” and refers to an enzyme capable of crosslinking proteins by an acyl transfer reaction between the γ-carboxamide group of a peptide-bonded glutamine and a structurally related primary amine such as a lysine or aminopentyl group, e.g., the ε-amino group of a peptide-bonded lysine, thereby resulting in an ε-(γ-glutamyl)lysine isopeptide bond. TGases include, inter alia, bacterial transglutaminase (BTG) such as an enzyme having EC reference EC2.3.2.13 (protein-glutamine-γ-glutamyltransferase). The term “acceptor glutamine” residue, when referring to a glutamine residue of an antibody, means a glutamine residue recognized by TGase and can be crosslinked by TGase via a reaction between glutamine and a structurally related primary amine such as a lysine or aminopentyl group. Preferably, the acceptor glutamine residue is a surface-exposed glutamine residue. The term “TGase recognition tag” refers to an amino acid sequence that contains an acceptor glutamine residue and, when incorporated (e.g., added) into a polypeptide sequence under appropriate conditions, is recognized by TGase and results in crosslinking by TGase via a reaction between an amino acid side chain within the amino acid sequence and a reaction partner. The recognition tag can be a peptide sequence that does not naturally occur in a polypeptide containing 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.

[0229] As exemplified in International Publication No. WO 2013 / 092983 pamphlet and International Publication No. WO 2020 / 188061 pamphlet (the disclosures of which are incorporated herein by reference), the linker-camptothecin analog moiety (X-Z) can bind to the glutamine residue of an antibody (acceptor glutamine) in a two-step process that includes a first step in which a moiety containing a primary amine and a first reactive group (R) is conjugated to the antibody in the presence of BTG, followed by reacting the antibody-linker complex with a molecule containing (i) a second reactive group (R') that reacts with the first reactive group and (ii) a camptothecin analog (Z). Examples of reactive group pairs R and R' include bioorthogonal reactions, such as 1,3-dipolar cycloadditions, such as between an azide and a cyclooctyne (copper-free click chemistry), between a nitrone and a cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, and tetrazine ligation, and a wide range of groups capable of such reactions (see also International Publication No. WO 2013 / 092983 pamphlet). Thus, the resulting linker and functionalized antibody or its Y element can contain an RR' group, such as a triazole, resulting from the reaction of R and R'.

[0230] The anti-nectin-4 immune complex can be incorporated into a pharmaceutical formulation at a concentration of 1 mg / ml to 500 mg / ml, and the formulation has a pH of 2.0 to 10.0. The formulation may further contain a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, and a surfactant. In certain embodiments, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation containing water. Such formulations are generally solutions or suspensions. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0231] In another embodiment, the pharmaceutical formulation is a lyophilized formulation, and a solvent and / or diluent is added thereto by a physician or patient prior to use.

[0232] In another embodiment, the pharmaceutical formulation is a ready-to-use dry formulation (e.g., lyophilized or spray-dried) that does not need to be pre-dissolved.

[0233] In a further aspect, the pharmaceutical formulation contains an aqueous solution of such an antibody and a buffer, the antibody is present at a concentration of 1 mg / mL or more, and the pH of the formulation is from about 2.0 to about 10.0.

[0234] In another embodiment, the pH of the formulation is in a range selected from the list consisting of 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.

[0235] In a further 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, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the present invention.

[0236] In a further embodiment, the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment, the formulation further comprises an isotonic agent. In a further embodiment, the formulation also comprises a chelating agent. In a further embodiment of the present invention, the formulation further comprises a stabilizer. In a further embodiment, the formulation further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0237] There may be a possibility that other components are present in the pharmaceutical formulations of the present disclosure. Such additional components may include wetting agents, emulsifying agents, antioxidants, fillers, isotonicity regulators, chelating agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatin, or protein), and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, and histidine). Such additional components should not, of course, have an adverse effect on the overall stability of the pharmaceutical formulations of the present disclosure.

[0238] Administration of the pharmaceutical composition according to the present invention can be by several routes of administration, such as intravenous. Suitable antibody formulations can also be determined by examining the experience with other already developed therapeutic ADCs.

[0239] In any embodiment, the composition can be characterized as comprising a plurality of nectin-4 binding immune complexes of the disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the sample have at least 4, 6 or 8 amino acid residues per antibody functionalized with a linker disclosed herein. In any embodiment, the composition can be characterized as comprising a plurality of nectin-4 binding immune complexes of the disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the sample have at least 2, 4, 6 or 8 amino acid residues per antibody functionalized with a linker-camptothecin moiety, such as the (X-Z) unit or the (-(Y)-(Pep)-(Y’)-(Z)) unit of the formulas herein. In any embodiment, the composition can be characterized as comprising a plurality of nectin-4 binding immune complexes of the disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the sample have the same number of functionalized amino acids per antibody, optionally 4, 6 or 8.

[0240] Diagnosis, prognosis and treatment of malignant tumors In some embodiments, methods useful for the diagnosis, prognosis, monitoring, and treatment of cancer characterized by tumor cells expressing nectin-4 on their surface, as well as antigen-binding proteins (e.g., antibodies, antibody fragments) and immune complexes are described. In therapeutic use, the treatment involves administering an antibody-camptothecin analog of the present disclosure to a human subject or individual. Embodiments are methods useful for the diagnosis, prognosis, monitoring, and treatment of nectin-4 expressing cancers by a nectin-4 binding agent conjugated to a camptothecin analog or derivative molecule, e.g., an exatecan or SN-38 molecule. Suitable nectin-4 binding agents are typically conjugated to multiple molecules of the camptothecin analog. The camptothecin analog can be conjugated to an antibody via a linker that includes a protease-cleavable oligopeptide linker. Exemplary pharmaceutical compositions can include, on average, from 1 to 8 camptothecin analog molecules per antibody molecule, optionally, from 2 to 8, 4 to 8, 6 to 8 camptothecin analog molecules per antibody molecule or, e.g., about 4, 5, 6, 7, or 8 camptothecin analog molecules per antibody molecule.

[0241] A nectin-4 binding agent (e.g., an anti-nectin-4 antibody or antibody fragment) conjugated to a camptothecin analog can be advantageously used to treat an individual having a nectin-4 expressing cancer characterized by tumor cells expressing nectin-4 (e.g., on the tumor cell membrane or cell surface). Examples of such cancers are urothelial cancer, 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.

[0242] A nectin-4 binding agent conjugated to a camptothecin analog can be used in nectin-4 overexpressing tumors.

[0243] The nectin-4 binder conjugated to a camptothecin analog can also be used for heterogeneous and / or low nectin-4 expressing tumors. In such tumors, the immune complexes of the present disclosure can advantageously provide efficacy, optionally via MDR1-mediated resistance and / or avoidance of the bystander anti-tumor effect.

[0244] The nectin-4 binder conjugated to a camptothecin analog can be advantageously used to treat an individual, regardless of the expression level of nectin-4, regardless of the heterogeneity of the nectin-4 expression level in tumor cells within the individual, and / or regardless of whether the individual has been previously treated with enfortumab vedotin. For example, the nectin-4 binder conjugated to a camptothecin analog molecule can be advantageously used to treat an individual who has been previously treated with enfortumab vedotin. Such an individual may optionally have cancer, optionally characterized by heterogeneous and / or low nectin 4-expressing tumors after enfortumab vedotin treatment. The individual may Auristatin also have resistant, non-responsive, recurrent, and / or progressive cancer, despite treatment (e.g., during or after treatment) with an antibody conjugated to an MMAE molecule (e.g., enfortumab vedotin). For example, the individual may have locally advanced or metastatic urothelial cancer, Auristatin and may have previously received treatment with an antibody conjugated to an MMAE molecule (e.g., enfortumab vedotin).

[0245] In advanced recurrent or metastatic urothelial carcinoma, a significant proportion of individuals express high levels of nectin-4 in tumor cells, for example, with an H-score of at least 290 (see nectin-4 expression in cohort 1 of the EV-201 clinical trial). However, the H-score of some patients is less than 250, and some patients have an H-score of less than 200. A small number of patients have an H-score of less than 150, and there are also patients with an H-score of less than 100. In triple-negative breast cancer (TNBC), it has been reported that 62% of patients have high expression of nectin-4 in tumor cells and 38% of patients have low expression of nectin-4 in tumor cells (Rabat et al., 2017 Annals Onc. 28:769-776). In other types of cancer, the median H-score of nectin-4 expression is typically lower than the values observed in UC, and is particularly prominent in non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer.

[0246] In certain embodiments, the individual to be treated according to the present disclosure has advanced recurrent or metastatic cancer, optionally advanced recurrent or metastatic urothelial carcinoma.

[0247] In certain embodiments, the individual to be treated according to the present disclosure has breast cancer that tests positive for estrogen receptor and / or progesterone receptor and tests negative for epidermal growth factor receptor 2 (HER2) or overexpressed HER2 protein, and optionally, the cancer tests positive for HER2 but HER2 is expressed at low levels.

[0248] In certain embodiments, the individual to be treated according to the present disclosure has triple-negative breast cancer (TNBC), for example, breast cancer that tests negative for estrogen receptor, progesterone receptor, and overexpressed HER2 protein.

[0249] In certain embodiments, the individual being treated in accordance with the present disclosure has breast cancer that is positive for the HER2 protein, and optionally, the cancer expresses excessive HER2 protein (HER2 overexpression), and optionally, the cancer expresses low levels of HER2 protein (lower than excessive HER2 expression). In certain embodiments, the individual is treated with an anti-nectin-4 ADC according to the present disclosure in combination with an agent (e.g., an antibody) that binds to the HER2 polypeptide (e.g., trastuzumab, pertuzumab), and optionally, the antibody that binds to HER2 is an ADC; optionally, the antibody that binds to HER2 is a cytotoxic agent, and optionally Auristatin , is conjugated to a maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compound 1, 2, or 13); optionally, the antibody that binds to HER2 is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a).

[0250] In certain embodiments, the individual being treated in accordance with the present disclosure has non-small cell lung cancer, and optionally, has lung adenocarcinoma.

[0251] In certain embodiments, the individual being treated in accordance with the present disclosure has pancreatic cancer.

[0252] In certain embodiments, the individual being treated in accordance with the present disclosure has ovarian cancer.

[0253] In certain embodiments, the individual being treated in accordance with the present disclosure has head and neck squamous cell carcinoma.

[0254] In certain embodiments, the individual being treated in accordance with the present disclosure has esophageal cancer.

[0255] In certain embodiments, the individual being treated in accordance with the present disclosure has colorectal cancer. As used herein, colorectal cancer (CRC) refers to colon cancer, rectal cancer, and colorectal cancer (cancer of both the colon and rectal regions).

[0256] In certain embodiments, the individual to be treated according to the present disclosure has NSCLC or lung adenocarcinoma, gastric cancer, colorectal cancer, pancreatic cancer, urothelial cancer or bladder cancer that is positive for the HER2 protein, and optionally, the cancer expresses an excessive amount of HER2 protein (HER2 overexpression), and optionally, the cancer expresses a low level of HER2 protein (lower than excessive HER2 expression). In certain embodiments, the individual is treated with an anti-nectin-4 ADC according to the present disclosure in combination with an agent (e.g., an antibody) that binds to a Her2 polypeptide (e.g., an antibody comprising the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab), and optionally, the antibody that binds to Her2 is an ADC; optionally, the antibody that binds to Her2 is a cytotoxic agent, optionally Auristatin , conjugated to a maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compound 1, 2 or 14); optionally, the antibody that binds to Her2 is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a).

[0257] In certain aspects, the treatment method of the present disclosure does not depend on the evaluation or detection of nectin-4 expression in tumor tissue, and does not depend on the expression level of nectin-4 on tumor cells and / or the frequency or number of nectin-4-expressing tumor cells in a tissue sample from the individual.

[0258] In certain aspects, the present disclosure provides a method for treating cancer and / or inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has advanced recurrent or metastatic urothelial cancer or breast cancer (e.g., TNBC), and the method does not require a prior determination of whether the individual has a tumor tissue containing cells (e.g., tumor cells) that express nectin-4.

[0259] In one aspect, the present disclosure provides a method of treating cancer and / or killing tumor cells in an individual in need thereof, wherein the individual has advanced recurrent or metastatic urothelial cancer or breast cancer (e.g., TNBC), and the method does not require a pre-determination of whether the individual has tumor tissue comprising cells (e.g., tumor cells) that express a high level of nectin-4 as defined, for example, by immunohistochemical evaluation (e.g., H-score or other suitable IHC scoring method).

[0260] In one aspect, a method of treating an individual's cancer and / or killing tumor cells does not require a pre-measurement of the nectin-4 expression level of the tumor cells.

[0261] In one aspect, a method of treating an individual's cancer, optionally advanced recurrent or metastatic urothelial cancer or breast cancer (e.g., TNBC, HER2-positive cancer), comprises treating an individual having cancer characterized by an H-score of nectin-4 expression of 290, 250, 200, 150 or 100 or less or less than.

[0262] In any embodiment for treating or preventing an individual's cancer, the method can be specified as comprising (i) a step of identifying an individual whose tumor cells express nectin-4 (e.g., determined by immunohistochemistry), and (ii) a step of administering to the individual an effective amount of the anti-nectin-4 antibody camptothecin analog drug conjugate of the present disclosure.

[0263] In any embodiment for treating or preventing cancer in an individual, the method can be specified as including: (i) identifying an individual whose tumor cells express (a) nectin-4 (e.g., as determined by immunohistochemistry) and (b) HER2 (optionally, the tumor cells express low levels of HER2) (e.g., as determined by immunohistochemistry; as 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 (e.g., an antibody) that binds to a Her2 polypeptide (e.g., trastuzumab, pertuzumab); optionally, the antibody that binds to Her2 is an ADC; optionally, the antibody that binds to Her2 is conjugated to a cytotoxic agent, optionally Auristatin , a maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compound 1, 2, or 14); optionally, the antibody that binds to Her2 is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a).

[0264] In any embodiment for treating or preventing cancer in an individual, the method can be specified as including: (i) identifying an individual whose tumor cells have low or medium levels of nectin-4 expression (e.g., as determined by immunohistochemistry), and (ii) administering to the individual identified in step (i) an effective amount of an anti-nectin-4 antibody conjugated to a camptothecin analog or derivative molecule.

[0265] In any embodiment for treating or preventing cancer in an individual (e.g., nectin-4 positive cancer), the method can be identified as including: (i) a step of identifying an individual whose cancer is characterized by low-level nectin-4 expression (e.g., determined by immunohistochemistry), and (ii) a step of administering to the individual identified in step (i) an effective amount of the anti-nectin-4 antibody camptothecin analog drug conjugate of the present disclosure. In certain embodiments, the individual has cancer characterized by an H score of nectin-4 expression of 150 or less or less than 150 or 100 or less or less than 100.

[0266] In any embodiment for treating or preventing cancer in an individual (e.g., nectin-4 positive cancer), the method can be identified as including: (i) a step of identifying an individual whose cancer is characterized by medium-level tumor nectin-4 expression (e.g., determined by immunohistochemistry), and (ii) a step of administering to the individual an effective amount of the anti-nectin-4 antibody camptothecin analog drug conjugate of the present disclosure. In certain embodiments, the individual has cancer characterized by an H score of nectin-4 expression of 290, 250, 200, 150 or less or less than, and optionally, the cancer is characterized by an H score of nectin-4 expression of at least 100.

[0267] In yet a further embodiment, there is provided a method for treating or preventing cancer (e.g., nectin-4 positive cancer) in an individual, comprising: (i) identifying an individual whose cancer is characterized by an H score of tumor nectin-4 expression of 290, 250, 200, 150, 120 or 100 or less or less than, and (ii) administering to the individual an effective amount of the anti-nectin-4 antibody camptothecin analog drug conjugate of the present disclosure. Optionally, step (i) can be identified as including a step of evaluating nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0268] In yet a further embodiment, provided is a method for treating or preventing cancer (e.g., nectin-4 positive cancer; breast cancer) in an individual, comprising: (i) identifying an individual characterized by a QS score of tumor nectin-4 expression of 200, 150, 120 or less or less than 100, and (ii) administering to the individual an effective amount of an anti-nectin-4 antibody camptothecin analog drug conjugate of the present disclosure. Optionally, step (i) can be identified as including the step of evaluating nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0269] Biological samples, e.g., from a biopsy, can be obtained from an individual and evaluated. Optionally, the sample is stored as a formaldehyde (e.g., formalin) fixed paraffin embedded (FFPE) sample. After deparaffinization, the slides are suitable for methods of detecting the expression of nectin-4 (and / or HER2).

[0270] The expression of nectin-4 and / or HER2 in tumor cells can be determined by any method known in the art. In certain embodiments, the assays include immunohistochemistry (IHC) assays, fluorescence activated cell sorting (FACS) assays, e.g., quantitative FACS, ELISA, immunoblotting (e.g., western blotting, dot blotting or in-cell western blotting) and other immunoassays.

[0271] IHC staining of tissue sections has been shown to be a reliable method for assessing or detecting the presence of proteins in a sample. Immunohistochemistry techniques utilize antibodies to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. Thus, antibodies or antisera, polyclonal antisera in some embodiments and monoclonal antibodies specific to each marker in some embodiments, are used to detect expression. Antibodies can be detected, for example, by directly labeling the antibody itself with a radiolabel, a fluorescent label, a hapten label such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody containing an antiserum, polyclonal antiserum or monoclonal antibody specific to the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

[0272] In some embodiments, the IHC assay is a direct assay in which the binding of an antibody to a target antigen is determined directly. In this direct assay, a labeled reagent such as a fluorescent tag or an enzyme-labeled primary antibody is used and visualization can occur without further antibody interactions. In some embodiments, the IHC assay is an indirect assay. In a typical indirect assay, an uncomplexed primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is complexed to an enzyme label, a chromogenic or fluorescent substrate is added to visualize the antigen. Signal amplification occurs because some secondary antibodies can react with different epitopes of the primary antibody. The primary and / or secondary antibodies used in immunohistochemistry are typically labeled with a detectable molecule. A number of labels are available, including radioisotopes, gold colloidal particles, fluorescent labels and enzyme substrate labels.

[0273] Strong staining, moderate staining, and weak staining are descriptions well-known to those skilled in the art. In some embodiments, strong staining, moderate staining, and weak staining are calibrated staining levels, ranges are established, and the intensity of the staining is binned within the range. In some embodiments, strong staining is staining above the 75th percentile of the intensity range, moderate staining is staining from the 25th percentile to the 75th percentile of the intensity range, and low staining is staining below the 25th percentile of the intensity range. In some embodiments, those skilled in the art are proficient in specific staining techniques and adjust the size of the bins and define the staining categories.

[0274] Control cell lines with various staining intensities (e.g., when stained with an anti-nectin-4 antibody) (e.g., centrifuged into pellets, formalin-fixed and paraffin-embedded, prepared as a tissue microarray, for example, and stained with an anti-nectin-4 antibody) can be used as controls for IHC analysis. Those skilled in the art will understand that other control cell pellets with negative, weak, moderate, and high c-met staining intensities are known in the teachings of the present application and in the art and can be readily identified using the methods disclosed herein.

[0275] In some embodiments, a cancer or tumor is considered a nectin-4-expressing carcinoma if it is nectin-4 positive (e.g., as determined using an IHC assay). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 5% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 10% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 20% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 30% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 40% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 50% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 60% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 70% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity). In some embodiments, a cancer or tumor of an individual is nectin-4 positive if 80% or more of the tumor cells in a sample express the nectin-4 protein (e.g., express the nectin-4 protein at any intensity).In some embodiments, if more than 90% of the tumor cells in the sample express nectin-4 protein (e.g., express nectin-4 protein at any intensity), the cancer or tumor of the individual is nectin-4 positive.

[0276] In some embodiments, if 5% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 10% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 20% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 30% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 40% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 50% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 60% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 70% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 80% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive. In some embodiments, if 90% or more of the tumor cells in the sample express nectin-4 protein with moderate and / or strong staining intensity, the cancer or tumor of the individual is nectin-4 positive.

[0277] The evaluation of immunohistochemical assays for determining whether an individual's cancer or tumor is characterized by high nectin-4 expression (e.g., low or moderate nectin-4 expression) typically involves the application of known scoring methods.

[0278] Low, medium, and high tumor nectin-4 expression can be determined based on the "H-score" as described in U.S. Patent Application Publication No. 2013 / 0005678. The H-score is obtained by the formula: (3 × percentage of strongly stained cells) + (2 × percentage of moderately stained cells) + (percentage of weakly stained cells), giving a range of 0 to 300. The H-score is particularly used in UC.

[0279] In some embodiments of any of the methods herein, low or medium nectin-4 expression (e.g., tumors or tumor cells having low or medium levels of nectin-4 expression) 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.

[0280] In some embodiments of any of the methods herein, low nectin-4 expression (e.g., tumors or tumor cells having low levels of nectin-4 expression) corresponds to an H-score of 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 herein, high nectin-4 expression (e.g., tumors or tumor cells having high levels of nectin-4 expression) corresponds to an H-score of about 290 or more.

[0282] In another example, nectin-4 staining can be scored according to a Quick Score (QS) using the following formula: QS = P (percentage of positive cells) × I (intensity), with a maximum score of 300. QS is used, for example, in breast cancer. For example, in TNBC, some research groups have defined a low nectin-4 expression group as QS = or <100. In some embodiments of any of the methods herein, low or medium nectin-4 expression (e.g., tumors or tumor cells having low or medium levels of nectin-4 expression) corresponds to a QS score of 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.

[0283] Assays for evaluating tumor cell expression of HER2 are well known in the art. 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 amplification of the HER2 gene. This technique, also called Subtraction Probe Technology Chromogenic In Situ Hybridization, is a test used to confirm whether breast cancer cells are overexpressing the HER2 receptor protein on the cell surface.

[0284] Another widely used assay for HER2 is HercepTest™ (Dako North America, Inc.), which is a semi-quantitative immunohistochemical assay used to determine the overexpression of HER2 protein in formalin-fixed paraffin-embedded cancer tissue. For example, tumors expressing low levels of HER2 can be identified by a score of +1 to +2 by HercepTest™.

[0285] In one aspect, the treatment is used in individuals having existing neuropathy, diabetes or hyperglycemia, heart failure, or ophthalmopathy. Such conditions may render the individual unsuitable for treatment with an anti-Nectin-4 ADC such as enfortumab vedotin, which has higher toxicity or a narrower therapeutic window than the anti-Nectin-4 antibody-drug conjugate of the present disclosure.

[0286] In any embodiment, the treatment method may optionally include (a) a step of evaluating the cancer stage and / or disease progression of the individual; and (b) a step of administering to the individual an effective amount of an anti-Nectin-4 antibody conjugated to a camptothecin analog or derivative molecule if the individual has recurrent, metastatic, and / or progressive cancer.

[0287] In some embodiments, the present invention includes a method of treating a tumor in an individual having urothelial cancer, including (a) evaluating the cancer stage and / or disease progression of the individual; and (b) administering to the individual an effective amount of an anti-Nectin-4 antibody conjugated to an exatecan molecule if the individual has recurrent, metastatic, and / or progressive cancer.

[0288] Optionally, the individual may have resistant, non-responsive, or recurrent and / or progressive cancer (e.g., urothelial cancer, 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) despite treatment with surgery and / or therapeutic agents such as chemotherapeutic agents, antibodies, ADCs, or radiation therapy (e.g., during or after).

[0289] In any embodiment of the present specification, the treatment response can be defined and / or evaluated according to well-known criteria, such as the Response Evaluation Criteria in Solid Tumors (RECIST), e.g., version 1.1 (see Eisenhauer et al. (2009) Eur. J. Cancer 45:228-247) or the immune-related Response Criteria (irRC) (see Wolchock et al. (2009) Clinical Cancer Research 15:7412-7420).

[0290] Optionally, an individual treated with an anti-nectin-4 antibody-drug conjugate of the present disclosure has a tumor or cancer that is resistant to or progresses after treatment with a chemotherapeutic agent (e.g., a chemotherapeutic agent known to be transported by P-glycoprotein (Pgp), such as anthracyclines (doxorubicin, daunorubicin), taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vindesine), and etoposide). Compounds recognized by Pgp are typically characterized by moderate hydrophobicity (octanol-water partition coefficient, logP>1) and often contain titratable protons with a net positive charge under physiological conditions, and are "natural products" having mainly aromatic moieties.

[0291] In some embodiments, an ADC comprising an anti-nectin-4 antibody, antibody fragment is used or administered without combination administration of a chemotherapeutic agent. Optionally, an individual can be characterized as having cancer that has progressed, recurred, or was non-responsive to previous treatment with a previous therapeutic agent, and optionally, further, the previous treatment includes administration of enfortumab vedotin and / or a PD-1 neutralizing agent (e.g., pembrolizumab, atezolizumab, nivolumab), and optionally, the previous therapeutic agent is a chemotherapeutic agent.

[0292] Optionally, in any embodiment, an individual can be characterized as ineligible for treatment with enfortumab vedotin and / or having a cancer that is not suitable or not indicated for treatment with enfortumab vedotin.

[0293] Exemplary treatment protocols for treating a human with an anti-nectin-4 antibody conjugated to a camptothecin analog molecule include, for example, administering to the patient an effective amount of the anti-nectin-4 antibody conjugated to the camptothecin analog molecule, and the method includes at least one dosing cycle in which at least one dose of the anti-nectin-4 antibody conjugated to the camptothecin analog molecule is administered at a dose 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. In certain embodiments, multiple doses, at least 2, 3, 4, 5, 6, 8, 10 doses are administered. In certain embodiments, the doses are administered at intervals of at least 2, 3, or 4 weeks. In certain embodiments, the dosing cycle is 2 weeks to 8 weeks or at least 4, 6, 8, or 16 weeks.

[0294] In one embodiment, the anti-nectin-4 antibody conjugated to the camptothecin analog molecule is administered by intravenous infusion.

Example

[0295] Example 1: Human tumor cells co-expressing Her2 and nectin-4. The HER2 study and the nectin-4 gene expression study were performed using The Cancer Genome Atlas (a collaborative study between the National Cancer Institute and the National Human Genome Research Institute) based on a multidimensional map of major genomic changes in different types of cancer. A significant correlation between HER2 and nectin-4 expression was observed, particularly in samples from pancreatic cancer patients, lung adenocarcinoma patients, breast cancer, and bladder cancer. The highest correlation observed was in pancreatic cancer, with correlation values of Spearman 0.71 and Pearson 0.78.

[0296] HER2 and nectin-4 expression in SUM185 and SUM190 human breast cancer tumor cell lines (Biovit inc.) was determined by flow cytometry. SUM185 is derived from the pleural effusion of ER-negative patients. Undifferentiated breast cancer that is PR-negative and Her2-positive. The cell line overexpresses Her2. SUM190 is derived from the primary tumor of ER-negative patients. PR-negative and Her2-positive (amplified) breast cancer. Tumor cells and isotype controls were stained with anti-nectin-4 antibody (ASG-22ME modified as a human IgG1 isotype containing the N297Q mutation with reduced Fc gamma receptor binding) or anti-Her2 antibody (trastuzumab modified as a human IgG1 isotype containing the N297Q mutation with reduced Fc gamma receptor binding) at 10 μg / ml (for 15 minutes, 4 °C), and then stained with PE-conjugated polyclonal goat anti-human antibody at a dilution of 1:200. Samples were analyzed by cytofluorometry using Canto II (HTS).

[0297] Representative results are shown in Figure 1 for SUM190 human breast cancer tumor cells and in Figure 2 for SUM185 human breast cancer tumor cells. MFI: mean fluorescence intensity. SUM190 tumor cells expressed HER2 at low to medium levels (mean fluorescence units 1777) and nectin-4 at lower levels (mean fluorescence units 991). SUM185 cells expressed HER2 at medium to high levels (mean fluorescence units 2880) and nectin-4 at higher levels (mean fluorescence units 4326).

[0298]

Table 1

[0299] Example 2: Efficacy of anti-nectin-4 ADCs functionalized with camptothecin analogs against HER2+ nectin4+ human tumor cells An anti-nectin-4 antibody-drug conjugate was prepared and compared with trastuzumab antibody-drug conjugate for efficacy against HER2+ nectin4+ human tumor cells. An anti-nectin-4 antibody-drug conjugate having VH and VL of SEQ ID NOs: 9 and 10 as human IgG1 isotype was prepared. An anti-Her2 antibody-drug conjugate having the heavy and light chains of trastuzumab (human IgG1 isotype) was prepared. Both the anti-nectin-4 antibody and the anti-Her antibody were stochastically conjugated to the linker-camptothecin analog via the cysteine residues of the antibody after partial reduction of the interchain disulfide. The reducing agent tris(2-carboxyethyl)phosphine hydrochloride in the range of 2 to 10 molar equivalents was incubated with the antibody (3 mg / mL) for 2 hours while stirring (350-400 rpm, +37°C) to reduce the disulfide. Conjugation of the linker toxin was performed by adding 9.2 or 12 molar equivalents of excess molar linker toxin and incubating overnight on a stirring wheel at 37°C. The average drug loading (drug:antibody ratio) of the resulting ADC was about 8. In a further example, the anti-nectin-4 antibody was also conjugated to a second camptothecin (SN-38) containing a linker using the same method. The ADCs used in this example are as follows.

[0300] N4 ADC1: Anti-nectin-4 conjugated to a linker having the following structure.

Chemical formula

[0301] Her2 ADC1: Anti-Her2 conjugated to a linker having the following structure.

Chemical formula

[0302] N4 ADC2: Anti-nectin-4 conjugated to a linker having the following structure.

Chemical formula

[0303] The resulting ADCs were tested for their ability to induce the death of nectin-4 / Her2 expressed in SUM190 and SUM185 tumor cells of Example 1. Briefly, cells were seeded in 96-well plates (V = 80 μl). N4 ADC1 and Her2 ADC1 or human IgG1-isotype control (IC)-linker-toxin or medium (concentration 5x) were tested for N4 ADC1 and the isotype control at 1:2 serial dilutions starting from (530 nM to 30 nM) and 1:5 serial dilutions (from 7 nM to 7 x 10-2 nM). N4 ADC2 and the isotype control were tested at 1:10 serial dilutions starting from (530 nM to 5.3 x 10-2). The ability of the ADCs to cause cell death was determined by assessing confluence using an Incucyte S3-2 device; viability on day 6 after treatment was determined using a Cell Titer Glo™ (CTG) assay using an Enspire2 device. The IC50 value of each ADC was determined using data on luminescent cell viability on day 6 by GraphPad Prism8. The experiment was repeated twice.

[0304] Representative results are shown in Figure 3. The two right panels in Figure 3 show that N4 ADC1 (anti-nectin-4) is effective in causing the death of tumor cells, where N4 ADC1 is shown by the solid line with squares and the isotype control is shown by the dashed line. The two left panels in Figure 3 show the efficacy of Her2 ADC1 (anti-Her2) in the same respective cells, where HER2 ADC1 is shown by the solid line with dots and the isotype control is shown by the dashed line. IC 50 values are summarized in the following table. N4 ADC1 was particularly potent even in SUM190 cells characterized by much lower nectin-4 surface expression (about 4-fold lower surface nectin-4 in SUM190 than SUM185). N4 ADC2 (anti-nectin-4) with the camptothecin analog SN38 also showed excellent potency (see the following IC 50 table).

[0305] [Table 2]

[0306] The results show that in SUM185 cells expressing nectin-4, the anti-nectin-4 ADC (N4 ADC1) is considerably more potent than the anti-Her2 ADC (Her2 ADC1), and the IC 50 is 1 / 40 of that of the nectin-4 ADC. Notably, SUM185 cells express nectin-4 at a relatively high level, and the nectin-4 expression level is approximately twice that of Her2 in these cells (see Example 1). However, interestingly, when tested in SUM190 cells with a much lower level of nectin-4 surface expression, the anti-nectin-4 ADC (N4 ADC1) remained very potent. In these SUM190 cells, the surface expression of nectin 4 was only half that of Her2, but the anti-nectin 4 camptothecin ADC was at least equivalent to or in some cases more potent than the anti-Her2 ADC, and the IC 50 of the anti-nectin-4 ADC was 6-fold lower than that of the anti-Her2 ADC.

[0307] Therefore, the combination of an intracellularly cleavable peptide linker and a camptothecin analog compound can be an effective means for eliminating nectin-4-expressing tumor cells, characterized by lower nectin-4 expression levels with unimproved off-target toxicity compared to the most widely used cytotoxic agents such as pyrrolobenzodiazepines and Auristatin etc. The anti-nectin-4 ADC can also provide a useful approach for the treatment of HER2-positive cancers including, but not limited to, HER2-low and / or HER2-medium expressing cancers.

[0308] Example 3: Efficacy of Anti-Her2 ADC in Combination with Anti-Nectin-4 Camptothecin Analog ADC An anti-nectin-4 ADC (N4 ADC1) and an anti-Her2 ADC (Her2 ADC1) were tested to evaluate their ability to cause tumor cell death when used in combination.

[0309] The anti-nectin-4 camptothecin analog ADC used was N4 ADC1 as shown in Example 2. The anti-Her2 ADC was also the one used in Example 2 (Her2 ADC1). The ability of the ADC to cause cell death was determined by evaluating the confluence of SUM190 cells and determining viability on day 6 as described in Example 2. The results showed that the combination of anti-nectin-4 ADC and anti-Her2 ADC improved the efficacy (lower IC50) of causing death of nectin-4+Her2+ tumor cells compared to either ADC alone.

[0310] Example 4: Intracellular Internalization The ability of anti-nectin-4 antibodies to induce internalization of nectin-4 was evaluated in SUM190 cell lines expressing lower levels of nectin-4 and SUM185 cells expressing higher levels of nectin-4. The antibodies tested were enfortumab (VH and VL of SEQ ID NOs: 3 and 4 as human IgG1 isotype) and N41 (VH and VL of SEQ ID NOs: 9 and 10 as human IgG1 isotype). Internalization was indirectly determined using the Fab-ZAP human internalization kit (Advanced Targeting Systems) to enable the anti-nectin-4 antibody to target and eliminate nectin-4 expressing cells by measuring cell viability using the CTG substrate (CellTiter-Glo® Luminescent Cell Viability Assay (Promega)). Fab-ZAP is a chemical conjugate of a goat anti-human monovalent antibody and saporin, a ribosome-inactivating protein. The antibodies used were polyclonal antibodies affinity purified against both the heavy and light chains of human IgG. This secondary conjugate is used to evaluate the potential of the primary antibody to internalize.

[0311] Briefly, antibodies or control items in a certain concentration range were incubated on SUM190 or SUM185 cells. After incubation, unbound antibodies were washed away, and Fab-ZAP was added onto the cells. The newly formed complex (anti-nectin 4 antibody + Fab-ZAP) was internalized into cells where saporin was released, stopping protein synthesis and resulting in cell death. The internalization ability of the antibody was indirectly determined by cell viability, and cell killing was more efficient, and the internalization ability of the antibody was better.

[0312] For each antibody, the luminescence against the nectin-4 antibody concentration was plotted on a graph. The results are shown in Figure 4. Both enfortumab and N41 were very potent in their ability to induce internalization in both SUM185 cells and SUM190 cells.

[0313] Example 5: Binding to the anchor nectin-4 domain deletion protein The internalizing antibodies enfortumab and N41 were evaluated for their ability to bind to different domains of human nectin-4.

[0314] The wild-type hu nectin 4 protein is composed of three extracellular Ig-like domains (V, C1, and C2) summarized in Table 1 below.

[0315]

Table 3

[0316] Characterization of antibody binding on the nectin-4 domain was performed by flow cytometry using cells engineered to express the wild-type human nectin-4 protein and cells engineered to express a modified nectin-4 (C1C2 construct) that has Ig-like C2 type 1 and Ig-like C2 type 2 domains and lacks the Ig-like V domain. The latter protein further has a V5 tag for flow cytometry cell sorting, and the cells were used as nectin 4-C1C2-V5 sorted cells.

[0317] The wild-type nectin-4 (Cl.3C8 cell line), C2 construct (including the Ig-like C2 type 2 domain and lacking both the V domain and the C1 domain), and C1C2 construct enable the determination of whether the test antibody binds to the V or different C domains. Briefly, nucleic acid sequences encoding different human nectin-4 domains were amplified by PCR. The PCR products were inserted into the expression vector at appropriate restriction sites. An N-terminal V5 tag with the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO: 13) was added to the leader peptide and for C1C2 and C2, and the expression on the cell surface was confirmed by flow cytometry. The amino acid sequences of the proteins containing the obtained different human nectin-4 domain fragments are shown below (the V5 tag is underlined). Then, the vector was transfected into the CHO cell line to obtain stable clones expressing different nectin-4 domain proteins on the cell surface.

[0318] Nectin-4 amino acid sequence of hu nectin4 Cl.3C8 cell line (wild-type nectin-4):

Chem.

[0319] Nectin-4 amino acid sequence of hu nectin4-C1C2-V5 cell line (V domain deleted):

Chem.

[0320] Nectin-4 amino acid sequence of hu nectin4-C2-V5 cell line (V and C1 domains deleted):

Chem.

[0321] Both the internalizing antibody N41 and enfortumab bound to the whole nectin-4 protein and lost binding to the C1C2 construct lacking the V domain. Therefore, N41 and enfortumab bind to nectin-4 within the V domain.

[0322] Example 6: In Vitro Cytotoxicity of Exatecan ADC against Breast Cancer Cells (Nectin-4 High / SUM185 Model) The inventors evaluated the killing of SUM185 cells by anti-Ig-like V domain antibody Enfortumab (mAbA) having VH and VL of SEQ ID NOs: 3 and 4 as a human IgG1 isotype conjugated to exatecan. In this experiment, the Enfortumab antibody was tested together with a control antibody conjugated with the same toxin at an equal drug-to-antibody ratio. The ADCs were prepared with 8 toxins per antibody (DAR = 8) with the antibody conjugated to exatecan via the linker shown below, which includes a spacer, the dipeptide valine-citrulline (VC) cleavable intracellularly, and a PAB self-eliminating spacer.

Chemical Structure

[0323] For each ADC, a range of concentrations of the ADC was incubated on nectin-4 expressing cells. After incubation, the CTG substrate was added at a 1 / 1 ratio and the luminescence signal was read with a plate reader (Enspire), enabling quantification of the presence of ATP (an indicator of metabolically active cells) proportional to cell viability.

[0324] The results are shown in Figure 5. mAbA-VC-exatecan was able to kill SUM185 more efficiently than the non-target isotype control ADC (IC-VC-exatecan).

[0325] Example 7: In Vitro Cytotoxicity of Exatecan and Dxd ADC against Breast Cancer Cells (Low Nectin-4 / SUM190 Model) The anti-Ig-like V domain antibody enfortumab (mAbA) was conjugated to exatecan via a VC-exatecan linker having the structure shown in Example 6, or to the camptothecin analog DxD via an intracellularly cleavable tetrapeptide linker called GGFG-D×D. Isotype control (IC) antibodies were also conjugated to each linker. Each linker was conjugated to the antibody with 8 toxins per antibody (DAR = 8).

[0326] The structure of the GGFG-DxD linker (GGFG-DxD) was as follows.

Chemical formula

[0327] In vitro cytotoxicity was evaluated as in Example 6. The results are shown in Figure 6. The mAbA-VC-exatecan immunoconjugate was able to kill nectin-4 low-expressing SUM190 cells more efficiently than the mAbA-GGFG-DxD immunoconjugate.

[0328] Example 8: In vitro cytotoxicity of exatecan ADCs with different cleavable linkers against breast cancer cells (nectin-4 low / SUM190 model) The inventors evaluated the killing of SUM190 cells by the anti-Ig-like V domain antibody enfortumab (mAbA) having VH and VL of SEQ ID NOs: 3 and 4 as a human IgG1 isotype conjugated to exatecan via any of a valine-citrulline-PAB linker, a valine-alanine-PAB linker, or a PEG8-valine-alanine-PAB linker. In each case, enfortumab was tested with an isotype control (IC) antibody conjugated with the same toxin at an equal drug-to-antibody ratio. An ADC was prepared in which the antibody was conjugated to exatecan via a linker with 8 toxins per antibody (DAR = 8).

[0329] The structure of the valine-citrulline-PAB linker (VC-exatecan) was as shown in Example 6. The structure of the PEG8-valine-citrulline-PAB linker (PEG8-VC-exatecan) is shown herein as Compound 6.

[0330] The structure of the valine-alanine-PAB linker (VA-exatecan) was as follows. [Chemical formula]

[0331] The structure of the PEG8-valine-alanine-PAB linker (PEG8-VA-exatecan) was as follows. [Chemical formula]

[0332] In vitro cytotoxicity was evaluated as in Example 6. The results are shown in Figure 7. When conjugated to mAbA, each exatecan linker enabled efficient killing of nectin-4 low-expressing SUM190 cells.

[0333] Example 9: In Vivo Efficacy of ADCs in a Mouse Model of Human Breast Cancer (Nectin-4 Low / SUM190 Model) The inventors compared the in vivo efficacy of an isotype control (IC) antibody of enfortumab (mAbA) conjugated to exatecan via either the valine-citrulline-PAB linker (VC-exatecan), the valine-alanine-PAB linker (VA-exatecan), or the PEG8-valine-alanine-PAB linker (PEG8-VA-exatecan). Also tested was the hexacyclic camptothecin analog DxD conjugated to the antibody via an intracellularly cleavable tetrapeptide linker (GGFG-DxD). Each linker and toxin was conjugated at a drug-to-antibody equivalence ratio of toxin 8 (DAR = 8) per antibody.

[0334] SUM190 cells were subcutaneously injected into CB17-SCID immunodeficient mice at a dose of 500,000 cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 with PBS. When the tumors reached a volume of 195 - 250 mm3, the mice were randomly grouped for intravenous treatment by a single injection of 5 mg / kg of camptothecin ADC. Tumor growth was followed twice a week. Kaplan-Meier survival curves were generated using GraphPad Prism V7 software according to the following criteria. When the tumor volume reached 1500 mm 3 3, the mice were euthanized and considered dead on the sacrifice day. When the tumors showed signs of necrosis, the mice were euthanized and considered dead on the same day.

[0335] The results showed that at a dose of 5 mg / kg, VC-exatecan, VA-exatecan, and PEG8-VA-exatecan were all similarly effective in preventing the increase in tumor volume and were all more effective than GGFG-DxD in preventing the increase in tumor volume. The results are shown in Figure 8.

[0336] Example 10: Effects of various antibodies on the efficacy of ADCs in a mouse model of human breast cancer (nectin-4 low / SUM190 model) The inventors compared the in vivo efficacy of enfortumab (mAbA) and another nectin-4 binding antibody (mAbB) that binds at least partially to the IgV domain of nectin-4 when each is conjugated to exatecan via the same PEG8-valine-alanine-PAB linker (PEG8-VA-exatecan). Also tested were an isotype control (IC) antibody conjugated to PEG8-VA-exatecan and a hexacyclic camptothecin analog DxD conjugated via a cleavable tetrapeptide linker (GGFG-DxD) intracellularly. Each linker and toxin was conjugated at a drug-to-antibody equivalent ratio of toxin 8 (DAR = 8) per antibody.

[0337] SUM190 cells were subcutaneously injected into CB17-SCID immunodeficient mice at a dose of 500,000 cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 with PBS. When the tumors reached a volume of 195 - 250 mm3, the mice were randomly grouped for intravenous treatment by a single injection of 5 mg / kg camptothecin ADC. Tumor growth was followed twice a week. Kaplan-Meier survival curves were generated using GraphPad Prism V7 software according to the following criteria. When the tumor volume reached 1500 mm 3 3, the mice were euthanized and considered dead on the day of sacrifice. When the tumors showed signs of necrosis, the mice were euthanized and considered dead on the same day.

[0338] The results showed that at a dose of 5 mg / kg, both mAbA-PEG8-VA-exatecan and mAbB-PEG8-VA-exatecan were equally effective in preventing the increase in tumor volume. Both were more effective than mAbA-GGFG-DxD in preventing the increase in tumor volume. The results are shown in Figure 9.

[0339] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety (to the maximum extent permitted by law), whether individually or specifically provided as an incorporation of any particular document elsewhere in this specification, and are identified and incorporated herein by reference in their entirety as if fully set forth herein.

[0340] Unless otherwise specified, all exact values provided herein are representative of the corresponding approximate values (e.g., all representative exact values or measurements provided for a particular factor may be considered to also provide the corresponding approximate measurements modified by "about" as necessary). When "about" is used in connection with a numerical value, this can be specified to include values corresponding to + / - 10% of the specified numerical value.

[0341] The description in this specification of any aspect or embodiment of the invention using terms such as "comprising", "having", "including", or "containing" with respect to one or more elements shall, unless otherwise specified or clearly inconsistent with the context, provide support for a similar aspect or embodiment of the invention "consisting of", "consisting essentially of", or "substantially comprising" that particular one or more elements (e.g., a composition described in this specification that includes a particular element shall also be understood to describe a composition consisting of that element, unless otherwise specified or clearly inconsistent with the context).

[0342] The use of any example or exemplary language provided herein (e.g., "such as") is intended merely to clarify the invention better and does not raise a limitation in the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any element not recited in any claim is essential for the practice of the invention. The present disclosure provides, for example, the following embodiments. [1] A method of treating cancer and / or killing tumor cells in an individual, comprising administering to the individual a therapeutically effective amount of a nectin-4 binding protein conjugated to a camptothecin analog, wherein the nectin-4 binding protein conjugated to the camptothecin analog has the formula (I): Ab-(X-(Z)) Formula (I) (wherein Ab is an antigen-binding protein that specifically binds to a human nectin-4 polypeptide; X is a linker molecule that links Ab and Z, and X includes a moiety cleavable under physiological conditions, optionally under intracellular conditions, a di-, tri-, tetra- or pentapeptide cleavable by a protease, and optionally, X further includes a self-cleaving spacer located between the protease-cleavable peptide and Z; and Z is an exatecan molecule) and is an immune complex represented by, a method. [2] The method according to claim 1, wherein the individual has a cancer characterized by low or moderate nectin-4 expression on tumor cells, as determined by immunohistochemistry. [3] A method of treating cancer and / or killing tumor cells in an individual in need thereof, wherein the individual has a nectin-4-expressing cancer characterized by low or moderate nectin-4 expression on tumor cells, and the method comprises administering to the individual a therapeutically effective amount of a nectin-4-binding protein conjugated to a camptothecin analog via a linker. [4] The method according to any one of claims 1 to 3, wherein the individual has relapsed and / or progressed after treatment with a nectin-4 binder conjugated to auristatin, and optionally, the nectin-4-binding protein conjugated to auristatin is enfortumab vedotin. [5] A method of treating cancer and / or killing tumor cells in an individual having a cancer that does not respond, relapses, and / or progresses after treatment with a nectin-4 binder conjugated to auristatin, optionally enfortumab vedotin, the method comprising administering to the individual a therapeutically effective amount of a 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 cancer, optionally advanced recurrent or metastatic urothelial cancer. [7] The method according to any one of claims 1 to 5, wherein the individual has breast cancer. [8] The method according to any one of claims 1 to 5, wherein the individual has TNBC. [9] The method according to any one of claims 1 to 5, wherein the individual has breast cancer that tests positive for estrogen receptor and progesterone receptor and negative for overexpressed HER2 protein.

[10] The method according to any one of items 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.

[11] The method according to any one of items 1 to 7 or 10, wherein the cancer is HER2-positive.

[12] The method according to any one of items 1 to 11, which does not depend on the evaluation or detection of the level of nectin-4 expression in the tumor.

[13] The method according to any one of items 1 to 12, including a preliminary step of determining whether the individual has tumor cells expressing nectin-4.

[14] The method according to any one of items 1 to 13, wherein the antibody that specifically binds to nectin-4 binds to the Ig-like V-set domain of the human nectin-4 polypeptide.

[15] The method according to any one of items 1 to 14, wherein the linker comprises a protease-cleavable di-, tri-, tetra- or pentapeptide.

[16] The method according to item 15, wherein the protease-cleavable di-, tri-, tetra- or pentapeptide is valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide.

[17] The method according to item 15, wherein the protease-cleavable di-, tri-, tetra- or pentapeptide comprises glycine-glycine-phenylalanine-glycine.

[18] The method according to any one of items 1 to 17, wherein the nectin-4 binding protein complexed with exatecan releases or delivers the molecules of compound 1 in vivo, optionally in the tumor and optionally in tumor cells.

[19] The method according to any one of items 1 to 18, wherein the nectin-4 binding protein competes with an antibody having the heavy and light chain variable regions of antibody ASG-22ME, 14A5.2 or N41 for binding to an epitope on the human nectin-4 polypeptide.

[20] The method according to any one of items 1 to 19, wherein the nectin-4 binding protein is a functionally conserved mutant of antibody ASG-22ME, 14A5.2 or N4.

[21] The method according to any one of items 1 to 20, wherein the nectin-4 binding protein binds to an epitope on the nectin-4 Ig-like V-set domain having the amino acid sequence of residues 32 to 144 of SEQ ID NO: 1.

[22] A composition comprising an antibody conjugated to a camptothecin analog, wherein the antibody conjugated to the camptothecin analog has the formula (X): Ab-(Y)-(X)-(Y’)-(Z) Formula (X) (wherein, Ab is an antibody; Y is optionally absent or is a spacer, an optionally substituted or unsubstituted alkyl or heteroalkyl chain, wherein optionally one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms, a substituted or unsubstituted alkyl or heteroalkyl chain, and optionally any carbon of said chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide, Y has a chain length of 2 to 100 atoms, and optionally, Y further comprises a residue of a reactant of a reactive group with the side chain of an amino acid of said antibody; X is a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme or comprises the same; Y’ is optionally absent or is a spacer optionally comprising a self-cleaving spacer or a non-self-cleaving spacer; and Z is an exatecan molecule) A composition which is an immune complex represented by.

[23] The composition according to item 22, wherein X comprises a val-cit, val-ala or val-phe dipeptide, and Y’ is a self-cleaving spacer, and optionally, Y’ comprises a p-aminobenzyl unit.

[24] The composition according to item 22 or 23, wherein the antibody conjugated to exatecan delivers or releases the molecule of compound 1 or 2 in vivo, optionally in the tumor, and optionally in the tumor cells.

[25] At least 70%, 80%, 90%, 95%, 98% or 99% of the immune complexes in the sample are functionalized with the (-(Y)-(X)-(Y’)-(Z)) unit and have at least 2, 4, 6 or 8 amino acid residues per antibody, and the composition according to any one of items 22 to 24.

[26] The composition according to any one of items 22 to 25, wherein the antibody binds to the human nectin-4 polypeptide.

[27] A linker-exatecan molecule of formula V: (R)-(Y)-(Pep)-(Y’)-(Z) Formula (V) (wherein, R is a group that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or with a complementary reactive group (R’) bound to an amino acid of an antigen-binding protein or antibody; Y is optionally absent or is a spacer; Pep is a peptidyl linker cleaved by an intracellular peptidase or protease enzyme, optionally valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide or includes it; Y’ is optionally absent, or is a spacer, optionally a self-cleaving spacer or a non-self-cleaving spacer; and Z is an exatecan molecule) A linker-exatecan molecule represented by

[28] A linker-exatecan composition having the structure of any one of formulas VIII to X, or having the structure of any one of compounds 5 to 11 or 15 to 17.

[29] A method for producing an immune complex, optionally, said immune complex is for use in the treatment of cancer, said method comprising conjugating an exatecan molecule to an antigen-binding protein via a linker (X), optionally, said linker-exatecan molecule has the structure described in item 27 or 28, method.

[30] Providing a tumor antigen, an antigen-binding protein or antibody that specifically binds to nectin-4 optionally, and contacting and / or reacting said antigen-binding protein or antibody with a molecule comprising an exatecan molecule under conditions suitable for forming an antibody-drug conjugate, and separating said antibody-drug conjugate, the method according to item 29.

[31] Said antigen-binding protein or antibody is conjugated to a camptothecin analog or said exatecan molecule via a linker (X) that links said antigen-binding protein or antibody (Ab) and the camptothecin analog or said exatecan molecule, the method according to item 29 or 30.

[32] Providing a tumor antigen, an antigen-binding protein or antibody that specifically binds to nectin-4 optionally, and said antigen-binding protein or antibody to formula V: (R)-(Y)-(Pep)-(Y’)-(Z) Formula (V) (wherein R is a group that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on said antibody or reacts with a complementary reactive group (R’) attached to an amino acid of said antigen-binding protein or antibody; Y is optionally absent or is a spacer; Pep is a peptidyl linker cleaved by an intracellular peptidase or protease enzyme, optionally valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide or comprises it; Y’ is optionally absent or is a spacer, optionally a self-cleaving spacer or a non-self-cleaving spacer; and Z is an exatecan molecule) The method according to any one of items 29 to 31, comprising contacting and / or reacting with a linker-exatecan molecule represented by

[33] The antibody conjugated to the camptothecin analog is of formula (X): Ab-(Y)-(X)-(Y’)-(Z) Formula (X) (wherein, Ab is an antibody; Y is optionally absent or is a spacer, an optionally substituted or unsubstituted alkyl or heteroalkyl chain, wherein optionally one or more atoms can be other than carbon, such as oxygen, sulfur, nitrogen or other atoms, a substituted or unsubstituted alkyl or heteroalkyl chain, and optionally any carbon of said chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide, Y has a chain length of 2 to 100 atoms, and optionally, Y further comprises a residue of a reactant of a reactive group with a side chain of an amino acid of said antibody; X is a peptidyl linker cleavable by an intracellular peptidase or protease enzyme or comprises the same; Y’ is optionally absent or is a spacer optionally comprising a self-cleaving spacer or a non-self-cleaving spacer; and Z is an exatecan molecule) The immunocomplex represented by, the method according to any one of items 29 to 32.

[34] The antigen-binding protein or antibody can induce intracellular internalization of nectin-4 in tumor cells expressing nectin-4 on their surface, the method or composition according to any one of items 1 to 26 or 28 to 33.

[35] The antigen-binding protein or antibody is conjugated to a linker-exatecan moiety comprising Val-Cit-PAB-exatecan, Val-Ala-PAB-exatecan, Phe-Lys-PAB-exatecan, (PEG) n -Val-Cit-PAB-exatecan, (PEG) n -Val-Ala-PAB-exatecan or (PEG) n -Phe-Lys-PAB-exatecan, and n is an integer from 1 to 16, optionally an integer from 1 to 8, optionally 8, according to any one of items 1 to 26 or 28 to 34 of the method or composition described.

[36] An immune complex produced by the method according to any one of items 29 to 35.

Claims

1. A pharmaceutical composition comprising a nectin-4 binding protein conjugated to a camptothecin analog for use in the treatment or prevention of cancer in an individual, wherein the nectin-4 binding protein conjugated to the camptothecin analog is of formula (I): Ab-(X-(Z)) Formula (I) (wherein Ab is an antibody that specifically binds to the human nectin-4 polypeptide; X is a linker molecule that links Ab and Z, and X comprises a moiety that is cleavable under physiological conditions, optionally under intracellular conditions, optionally a di-, tri-, tetra- or pentapeptide that is cleavable by a protease, and optionally X further comprises a self-cleaving spacer located between the protease-cleavable peptide and Z; and Z is an exatecan molecule) is an immune complex represented by, and the individual has relapsed and / or progressed after treatment with a nectin-4 binder conjugated to an auristatin, the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the individual has relapsed and / or progressed after treatment with enfortumab vedotin.

3. The pharmaceutical composition according to claim 1 or 2, wherein the individual has a cancer characterized by low or moderate nectin-4 expression on tumor cells as determined by immunohistochemistry.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the treatment or prevention of cancer in an individual comprises administering to the individual a therapeutically effective amount of the nectin-4 binding protein conjugated to a camptothecin analog.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the individual has urothelial cancer, optionally advanced recurrent or metastatic urothelial cancer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cancer is HER2 positive.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the treatment or prevention of cancer in an individual does not depend on the evaluation or detection of the level of nectin-4 expression in the tumor.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the treatment or prevention of cancer in an individual comprises a preliminary step of determining whether the individual has tumor cells that express nectin-4.

9. The nectin-4 binding protein conjugated to a camptothecin analog is of formula (X): Ab-(Y)-(X)-(Y')-(Z) Formula (X) (wherein Ab is an antibody; Y is a spacer; X is a peptidyl linker cleaved by an intracellular peptidase or protease enzyme, optionally a valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide or comprising the same; Y' is a spacer, optionally a spacer comprising a self-cleaving spacer; and Z is an exatecan molecule) The pharmaceutical composition according to any one of claims 1 to 8, which is an immune complex represented by

10. The pharmaceutical composition according to claim 9, wherein the peptidyl linker is a valine-citrulline, valine-alanine or phenylalanine-lysine dipeptide.

11. The pharmaceutical composition according to claim 9 or 10, wherein the self-cleaving spacer is p-aminobenzyloxycarbonyl (PAB).

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the nectin-4 binding protein conjugated to exatecan releases the molecule of compound 1a in vivo within tumor cells.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the antigen-binding protein can induce intracellular internalization of nectin-4 in tumor cells expressing nectin-4 on their surface.

14. The pharmaceutical composition according to any one of claims 9 to 13, wherein the antigen-binding protein is conjugated to a linker-exatecan moiety comprising Val-Cit-PAB-exatecan, Val-Ala-PAB-exatecan, Phe-Lys-PAB-exatecan.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the nectin-4 binding protein conjugated to a camptothecin analog comprises an immune complex having an exatecan molecule Z to Ab ratio of 8, wherein the exatecan molecule Z to linker X ratio is 1.

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