Nectin-4 antibody and antibody-drug conjugate

Abstract

The present disclosure provides a nectin-4 antibody-drug conjugate and a pharmaceutical composition thereof, and a method of use for treating cancer.

Description

Technology Field

[0001] Reference to the sequence list

[0002] This application is filed together with a sequence list in ST.26 XML format. The sequence list is provided as a filename "30650_WO" created on May 3, 2024, and has a size of 120 kilobytes. The full text of the sequence list information in ST.26 XML format is incorporated herein by reference. Background Technology

[0003] The present disclosure relates to the field of medicine. More particularly, the present disclosure relates to a nectin-4 antibody-drug conjugate and a pharmaceutical composition thereof, and its use in treating cancer.

[0004] Nectin-4 is a member of the nectin family of Ca2+-independent immunoglobulin-like cell adhesion molecules. Unlike other members of the nectin family, nectin-4 expression in healthy tissues is mostly placental or embryonic, but it is overexpressed in several tumor types, including urothelial carcinoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, and ovarian cancer. Studies have associated high nectin-4 expression in several cancer types with oncology.

[0005] Antibody-drug conjugates (ADCs) for use as oncological treatments contain tumor-targeting antibodies conjugated to a payload designed to induce apoptosis upon entering tumor cells. Specific nectin-4 antibodies were used to generate ADCs with an MMAE payload (WO201247724) and specific camptothecin analogs (WO2022112356 and WO2021151984).

[0006] ADCs for use in oncology are highly challenging compounds to design because multiple aspects of the molecule must be balanced, including sufficient specificity for tumor targets compared to healthy cells, acceptable toxicity while maintaining desirable activity against bystander tumor cells, and an unstable payload that allows for intracellular delivery while maintaining excellent physical and chemical stability.

[0007] There remains a need for Nectin-4 ADCs for treating cancer. In particular, Nectin-4 ADCs need to support doses high enough to effectively kill tumor cells, but also possess a sufficient therapeutic index based on better tolerability and / or better efficacy to be acceptable to patients. In particular, there remains a need for Nectin-4 ADCs containing effector antibodies and topoisomerase I payloads. In particular, there remains a need for Nectin-4 ADCs with enhanced bystander activity against Nectin-4-low tumors while allowing for lower administration. In particular, there remains a need for Nectin-4 ADCs that avoid dermatological events observed with certain Nectin-4 ADCs or enable better management thereof. In particular, there remains a need for Nectin-4 ADCs that avoid ocular and / or peripheral neuropathy signals observed with certain Nectin-4 ADCs. In particular, there remains a need for a Nectin-4 ADC having low immunogenicity, stable in vivo pharmacokinetics, and adequate chemical and physical stability. Additionally, there remains a need for a Nectin-4 ADC possessing one or more of the following features: superior antitumor activity as measured in specific tumor models, enhanced bystander activity against Nectin-4 low tumors, lower immunogenicity, immeasurable antibody effector function, and / or superior physical and chemical stability. The ADCs provided herein address one or more of these needs.

[0008] Specific nectin-4 ADCs and compositions comprising nectin-4 ADCs are provided herein. Additionally, a method of using a nectin-4 ADC or a composition comprising a nectin-4 ADC for cancer in a subject is provided herein.

[0009] In one aspect, an antibody binding to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining region (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity determining region (LCDR) LCDR1, LCDR2, and LCDR3, wherein

[0010] a) HCDR1 includes SEQ ID NO: 4, HCDR2 includes SEQ ID NO: 5, HCDR3 includes SEQ ID NO: 6, LCDR1 includes SEQ ID NO: 7, LCDR2 includes SEQ ID NO: 8, and LCDR3 includes SEQ ID NO: 9;

[0011] b) HCDR1 includes sequence identification number: 18, HCDR2 includes sequence identification number: 19, HCDR3 includes sequence identification number: 20, LCDR1 includes sequence identification number: 21, LCDR2 includes sequence identification number: 22, and LCDR3 includes sequence identification number: 23;

[0012] c) HCDR1 includes sequence identification number: 28, HCDR2 includes sequence identification number: 29, HCDR3 includes sequence identification number: 30, LCDR1 includes sequence identification number: 31, LCDR2 includes sequence identification number: 32, and LCDR3 includes sequence identification number: 33;

[0013] d) HCDR1 includes sequence identification number: 38, HCDR2 includes sequence identification number: 39, HCDR3 includes sequence identification number: 40, LCDR1 includes sequence identification number: 41, LCDR2 includes sequence identification number: 42, and LCDR3 includes sequence identification number: 43;

[0014] e) HCDR1 includes sequence identification number: 48, HCDR2 includes sequence identification number: 49, HCDR3 includes sequence identification number: 50, LCDR1 includes sequence identification number: 41, LCDR2 includes sequence identification number: 42, and LCDR3 includes sequence identification number: 51; or;

[0015] f) HCDR1 includes sequence identification number: 56, HCDR2 includes sequence identification number: 57, HCDR3 includes sequence identification number: 58, LCDR1 includes sequence identification number: 59, LCDR2 includes sequence identification number: 60, and LCDR3 includes sequence identification number: 61; or;

[0016] g) HCDR1 includes sequence identification number: 66, HCDR2 includes sequence identification number: 67, HCDR3 includes sequence identification number: 68, LCDR1 includes sequence identification number: 69, LCDR2 includes sequence identification number: 70, and LCDR3 includes sequence identification number: 71; or

[0017] h) HCDR1 includes sequence identification number: 76, HCDR2 includes sequence identification number: 77, HCDR3 includes sequence identification number: 78, LCDR1 includes sequence identification number: 69, LCDR2 includes sequence identification number: 70, and LCDR3 includes sequence identification number: 79.

[0018] In a further aspect, an antibody binding to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein

[0019] a) HCVR includes sequence identification number: 10, and LCVR includes sequence identification number: 11, or;

[0020] b) HCVR includes sequence identification number: 14, and LCVR includes sequence identification number: 15, or;

[0021] c) HCVR includes sequence identification number: 24, and LCVR includes sequence identification number: 25, or;

[0022] d) HCVR includes sequence identification number: 34, and LCVR includes sequence identification number: 35, or;

[0023] e) HCVR includes sequence identification number: 44, and LCVR includes sequence identification number: 45, or;

[0024] f) HCVR includes sequence identification number: 52, and LCVR includes sequence identification number: 53, or;

[0025] g) HCVR includes sequence identification number: 62, and LCVR includes sequence identification number: 63, or;

[0026] h) HCVR includes sequence identification number: 72, and LCVR includes sequence identification number: 73; or

[0027] i) HCVR includes sequence identification number: 80, and LCVR includes sequence identification number: 81.

[0028] In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0029] a) HC contains amino acid 2-444 of sequence identification number: 2, and LC contains amino acid 2-215 of sequence identification number: 3;

[0030] b) HC contains amino acid 2-444 of sequence identification number: 12, and LC contains amino acid 2-215 of sequence identification number: 13;

[0031] c) HC contains amino acids 2-443 of sequence identification number: 16, and LC consists of sequence identification number: 17;

[0032] d) HC contains amino acids 2-447 of sequence identification number: 26, and LC consists of sequence identification number: 27;

[0033] e) HC contains amino acids 2-446 of sequence identification number: 36, and LC consists of sequence identification number: 37;

[0034] f) HC contains amino acids 2-446 of sequence identification number: 46, and LC consists of sequence identification number: 47;

[0035] g) HC contains amino acids 2-446 of sequence identification number: 54, and LC consists of sequence identification number: 55;

[0036] h) HC contains amino acids 2-450 of sequence identification number: 64, and LC contains amino acids 2-216 of sequence identification number: 65; or

[0037] i) HC contains amino acid 2-450 of sequence identification number: 74, and LC contains amino acid 2-216 of sequence identification number: 75.

[0038] In another aspect, an antibody-drug conjugate (ADC) comprising the nectin-4 antibody disclosed herein conjugated to a cytotoxic agent either directly or through a linker is provided herein.

[0039] In a further aspect, an ADC is provided herein, wherein the cytotoxic agent is a camptothecin analog comprising the chemical formula: XY, where:

[0040] Y is the chemical formula

[0041]

[0042] has, and *-(CH2)3N(CH3)-, *-(CH2)4N(CH3)-, *-CH2N(R 1 )-, *-(CH2)2N(R 1 )-, *-(CH2)3N(R 1 )-, *-(CH2)4N(R 1 )-, *-CH2N(CH3)C(=O)CH2O-, *-CH2N(CH2R 1 )C(=O)CH2O-, *-CH2NHC(=O)CH2O-, *-CH2NHC(=O)(CH2)2O-, *-CH2NHC(=O)(CH2)3O-, *-CH2NHC(=O)(CH2)4O-, *-CH2NHC(=O)(CH2)5O-, *-CH2NHC(=O)CH2-, *-CH2NHC(=O)(CH2)2-, *-CH2NHC(=O)(CH2)3-, *-CH2NHC(=O)(CH2)4-, *-CH2NHC(=O)(CH2)5-, *-CH2SCH2-, *-CH2S(CH2)2-, *-CH2S(CH2)3-, *-CH2S(CH2)4-, or *-CH2S(CH2)5-; Here, * is the region shared and attached to Y, and R 1 It is phenyl.

[0043] In another aspect, an antibody-drug conjugate (ADC) of the following chemical formula is provided herein:

[0044]

[0045]

[0046] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0047] In another aspect, a pharmaceutical composition comprising the nectin-4 antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients is provided herein. In another aspect, a pharmaceutical composition comprising the nectin-4 ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients is provided herein.

[0048] In another aspect, a method for treating cancer is provided herein, comprising administering an effective amount of the Nectin-4 ADC disclosed herein to a patient requiring treatment for cancer. In a further aspect, a method for treating cancer is provided herein, comprising administering an effective amount of the Nectin-4 ADC disclosed herein to a patient requiring treatment for cancer, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. Specific details for implementing the invention

[0049] Nectin-4

[0050] As used herein, “human nectin-4” refers to the human nectin-4 protein or polypeptide, also known as poliovirus receptor-associated 4, Ig superfamily receptor LNIR, or poliovirus receptor-associated protein 4 (PVRL4). The amino acid sequence of human nectin-4, including the signal peptide as provided in sequence identification number: 1, can be found in NP_112178.2.

[0051] Nectin-4 antibody (also known as anti-nectin-4 antibody)

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibodies may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0053] The exemplary antibody of the present disclosure is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region, which is primarily responsible for effector function. Each heavy chain consists of a heavy chain variable region (VH, also known as HCVR) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL, also known as LCVR) and a light chain constant region. IgG isoforms may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0054] The VH and VL regions may be further subdivided into a hypervariable region called the complementarity determining region (CDR), which is interposed with a more conserved region called the framework region (FR). The CDR is exposed on the surface of the protein and is an important region of the antibody for antigen binding specificity. Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this invention, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to CDRs was performed by Kabat et al. (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917. (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256) (2011)), or IMGT (www.imgt.It may be carried out according to widely known schemes, including those described in the international ImmunoGeneTics database available at org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The CDR of the present disclosure is determined by North.

[0055] Certain antibodies described herein contain an IgG1 Fc region, or an Fc region derived from human IgG1, for example, a modified IgG1 Fc region having an altered Fc effector function. IgG1 is known to induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Some antibodies of the present disclosure have amino acid substitutions introduced into the IgG1 Fc region that alter effector function. According to some of the disclosures, mutations are introduced at positions 234 and 235 of the Fc region (according to EU index numbering). According to some of the disclosures, mutations are introduced at positions 234, 235, and 265 of the Fc region (according to EU index numbering). In some aspects, the nectin-4 antibodies of the present disclosure comprise a modified human IgG1 Fc region (according to EU index numbering, also referred to as the hIgG1 effector or hIgG1EN Fc region) comprising alanine at residues 234 and 235, and serine at position 265. In further aspects, some antibodies have additional mutations comprising glutamine, alanine, or glycine at position 297 of the Fc region, alanine or glutamine at position 322, alanine or glycine at position 329, and / or alanine or serine at position 331 (according to EU index numbering). In some aspects, these antibody mutations are alanine at position 234, glutamate at position 235, alanine at position 237, serine at position 330, and serine at position 331 (according to EU index numbering). In addition, these amino acid substitutions introduced into the IgG1 Fc region reduced or eliminated measurable antibody effector function.

[0056] In a specific aspect of the present disclosure, the nectin-4 antibody has a modified human IgG1 or human IgG4 constant domain comprising one or more engineered cysteine ​​residues. In a further aspect, the antibody comprises engineered cysteine ​​at one or more sites within the heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and / or heavy chain constant domain 3 (CH3).

[0057] Mammalian expression of antibodies typically induces glycosylation. Glycosylation of antibodies is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid. Typically, glycosylation occurs at a highly conserved N-glycosylation site in the antibody's Fc region (e.g., position 297 of IgG1 according to IMGT or EU index numbering). Glycosylation can be altered by modifying the glycosylation site (e.g., by blocking or reducing glycosylation, or by changing the amino acid sequence to generate additional or various forms of glycosylation).

[0058] Mammalian expression of antibodies from IgG subclasses may result in clipping of C-terminal amino acids from one or both heavy chains; for example, in the case of IgG1 antibodies, one or two C-terminal amino acids may be removed. In the case of IgG1 antibodies, if C-terminal lysine is present, it may be truncated or clipped from the heavy chain during expression. Additionally, the second-to-last glycine may also be truncated or clipped from the heavy chain.

[0059] Mammalian expression of antibodies can also induce modifications to N-terminal amino acids. For example, if the most N-terminal amino acid of a heavy or light chain is glutamine or glutamate, it can be modified to pyroglutamic acid. For example, if the most C-terminal amino acid of a heavy or light chain is lysine or glycine, it can be removed.

[0060] The terms “nucleic acid” or “polynucleotide” as used interchangeably herein refer to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, into which natural, modified, and / or analog nucleotides are incorporated. Polynucleotides of this disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or by a synthetic reaction.

[0061] The polynucleotide of the present disclosure may be expressed in a host cell, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are widely known in the art. For example, the expression vector may comprise a sequence encoding one or more signal peptides that facilitate the secretion of polypeptide(s) from the host cell. The signal peptide may be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. An expression vector containing the polynucleotide of interest (e.g., a polynucleotide encoding the polypeptide of an antibody) may be delivered into a host cell by a widely known method. Additionally, the expression vector may contain one or more selection markers, for example, tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired polynucleotide sequence.

[0062] The host cell comprises a cell that is stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or part of the antibody of the present disclosure. According to some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of the antibody of the present disclosure. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC and LC polypeptides of the antibody of the present disclosure. The antibody of the present disclosure may be produced in mammalian cells, such as CHO, NS0, HEK293, or COS cells, according to techniques widely known in the art.

[0063] The culture medium in which the antibody of the present disclosure is secreted may be purified by conventional techniques, such as a mixed-mode method of ion-exchange and hydrophobic interaction chromatography. For example, the culture medium may be applied to a protein A or G column using conventional methods and eluted therefrom; a mixed-mode method of ion-exchange and hydrophobic interaction chromatography may also be used. Soluble aggregates and polymers may be effectively removed by conventional techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen at -70°C, refrigerated, or freeze-dried, for example. Various protein purification methods may be used, and such methods are known in the art and are described, for example, in the literature [Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994)].

[0064] In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining region (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity determining region (LCDR) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises sequence identification number: 4, the HCDR2 comprises sequence identification number: 5, the HCDR3 comprises sequence identification number: 6, the LCDR1 comprises sequence identification number: 7, the LCDR2 comprises sequence identification number: 8, and the LCDR3 comprises sequence identification number: 9. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 18, HCDR2 comprises sequence identification number: 19, HCDR3 comprises sequence identification number: 20, LCDR1 comprises sequence identification number: 21, LCDR2 comprises sequence identification number: 22, and LCDR3 comprises sequence identification number: 23. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 28, HCDR2 comprises sequence identification number: 29, HCDR3 comprises sequence identification number: 30, LCDR1 comprises sequence identification number: 31, LCDR2 comprises sequence identification number: 32, and LCDR3 comprises sequence identification number: 33. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 38, HCDR2 comprises sequence identification number: 39, HCDR3 comprises sequence identification number: 40, LCDR1 comprises sequence identification number: 41, LCDR2 comprises sequence identification number: 42, and LCDR3 comprises sequence identification number: 43.In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 48, HCDR2 comprises sequence identification number: 49, HCDR3 comprises sequence identification number: 50, LCDR1 comprises sequence identification number: 41, LCDR2 comprises sequence identification number: 42, and LCDR3 comprises sequence identification number: 51. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 56, HCDR2 comprises sequence identification number: 57, HCDR3 comprises sequence identification number: 58, LCDR1 comprises sequence identification number: 59, LCDR2 comprises sequence identification number: 60, and LCDR3 comprises sequence identification number: 61. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 66, HCDR2 comprises sequence identification number: 67, HCDR3 comprises sequence identification number: 68, LCDR1 comprises sequence identification number: 69, LCDR2 comprises sequence identification number: 70, and LCDR3 comprises sequence identification number: 71. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein HCDR1 comprises sequence identification number: 76, HCDR2 comprises sequence identification number: 77, HCDR3 comprises sequence identification number: 78, LCDR1 comprises sequence identification number: 69, LCDR2 comprises sequence identification number: 70, and LCDR3 comprises sequence identification number: 79.

[0065] In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the antibody comprises an HCVR comprising SEQ ID NO: 10 and an LCVR comprising SEQ ID NO: 11. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises an HCVR comprising SEQ ID NO: 14 and an LCVR comprising SEQ ID NO: 15. In yet another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises an HCVR comprising SEQ ID NO: 24 and an LCVR comprising SEQ ID NO: 25. In yet another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises an HCVR comprising SEQ ID NO: 34 and an LCVR comprising SEQ ID NO: 35. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises HCVR comprising sequence identification number: 44 and LCVR comprising sequence identification number: 45. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises HCVR comprising sequence identification number: 52 and LCVR comprising sequence identification number: 53. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises HCVR comprising sequence identification number: 62 and LCVR comprising sequence identification number: 63. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises HCVR comprising sequence identification number: 72 and LCVR comprising sequence identification number: 73. In another aspect, antibodies that bind to human nectin-4 are provided herein, wherein the antibodies include HCVR comprising sequence identification number: 80 and LCVR comprising sequence identification number: 81.

[0066] In a further aspect, an antibody binding to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the antibody has a human IgG1 or IgG4 isoform. In a further aspect, the antibody has a human IgG1 isoform. In a further aspect, the antibody comprises alanine at residues 234 and 235 (according to EU index numbering). In a further aspect, the antibody further comprises serine at position 265 (according to EU index numbering). In another aspect, the antibody has a human IgG4 isoform.

[0067] In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acid 2-444 of sequence identification number: 2 and the LC comprises amino acid 2-215 of sequence identification number: 3. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acid 2-444 of sequence identification number: 12 and the LC comprises amino acid 2-215 of sequence identification number: 13. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acid 2-443 of sequence identification number: 16 and the LC is composed of sequence identification number: 17. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-447 of sequence identification number: 26 and the LC comprises sequence identification number: 27. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-446 of sequence identification number: 36 and the LC comprises sequence identification number: 37. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-446 of sequence identification number: 46 and the LC comprises sequence identification number: 47. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-446 of sequence identification number: 54 and the LC is composed of sequence identification number: 55.In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-450 of sequence identification number: 64 and the LC comprises amino acids 2-216 of sequence identification number: 65. In one aspect, an antibody that binds to human nectin-4 is provided herein, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-450 of sequence identification number: 74 and the LC comprises amino acids 2-216 of sequence identification number: 75.

[0068] In a further aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 2 and LC is composed of sequence identification number: 3. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 12 and LC is composed of sequence identification number: 13. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 16 and LC is composed of sequence identification number: 17. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 26 and LC is composed of sequence identification number: 27. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 36 and LC is composed of sequence identification number: 37. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 46 and LC is composed of sequence identification number: 47. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 54 and LC is composed of sequence identification number: 55. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 64 and LC is composed of sequence identification number: 65. In another aspect, an antibody that binds to human nectin-4 is provided herein, wherein HC is composed of sequence identification number: 74 and LC is composed of sequence identification number: 75.

[0069] In another aspect, different mammalian cells are provided herein comprising a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequences of sequence identification numbers: 2 and 3, sequence identification numbers: 12 and 13, sequence identification numbers: 16 and 17, sequence identification numbers: 26 and 27, sequence identification numbers: 36 and 37, sequence identification numbers: 46 and 47, sequence identification numbers: 54 and 55, sequence identification numbers: 64 and 65, or sequence identification numbers: 74 and 75, wherein the cells may express the nectin-4 antibody disclosed herein.

[0070] In another aspect, a mammalian cell comprising a first DNA molecule and a second DNA molecule is provided herein, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the following amino acid sequence, and the second DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the following amino acid sequence, and wherein the cell can express the nectin-4 antibody disclosed herein:

[0071] .

[0072] In another aspect, a method for producing a nectin-4 antibody is provided herein, comprising culturing one of the mammalian cells disclosed herein under conditions that cause antibody expression and recovering the expressed antibody.

[0073] In another aspect, antibodies produced by culturing mammalian cells comprising DNA molecules comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequences of sequence identification numbers: 2 and 3, sequence identification numbers: 12 and 13, sequence identification numbers: 16 and 17, sequence identification numbers: 26 and 27, sequence identification numbers: 36 and 37, sequence identification numbers: 46 and 47, sequence identification numbers: 54 and 55, sequence identification numbers: 64 and 65, or sequence identification numbers: 74 and 75, under conditions to cause antibody expression, and recovering the expressed antibodies are provided herein.

[0074] In another aspect, an antibody produced by culturing a mammalian cell comprising a first DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the following amino acid sequence and a second DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the following amino acid sequence under conditions to express the antibody, and recovering the expressed antibody is provided herein:

[0075] .

[0076] The term "enfortumab" as used herein refers to a fully human anti-nectin-4 IgG1 kappa monoclonal antibody having the sequence as disclosed in Figures 3a and 3b of WO2012047724, expressed and purified using standard conditions.

[0077] payload

[0078] The nectin-4 antibody of the present disclosure may be conjugated to various payloads (including salts that are pharmaceutically acceptable thereto) to form antibody-drug conjugates (ADCs). Moietyes suitable for conjugation to the nectin-4 antibody disclosed herein include cytotoxic agents (e.g., chemotherapy agents), prodrug-converting enzymes, radioisotopes or compounds, toxins, and other payloads known in the art.

[0079] An exemplary ADC of the present invention utilizes a camptothecin-based payload (e.g., a camptothecin analog). Camptothecin is a topoisomerase I (TOPO 1) inhibitor that has been found to possess anticancer activity. Camptothecin and its derivatives bind to the TOPO 1 / DNA complex, which prevents re-annealing and causes apoptosis due to the accumulation of partially cleaved DNA. Other topoisomerase I inhibitors, such as SN-38 and DXd, which can be used as payloads, are known in the art.

[0080] Other payloads for the ADCs described herein are metansinoids (e.g., DM1 and DM4), pyrrolobenzodiazepines (e.g., PBD dimers), auristatin peptides (e.g., MMAE and MMAF), duocarmycin, caliceamicin, DNA small groove binders (e.g., enedine and lexitropsin) and taxanes (e.g., paclitaxel and docetaxel).

[0081] In one aspect, an ADC is provided herein, wherein the camptothecin analog comprises the chemical formula: XY, where:

[0082] Y is the chemical formula I

[0083]

[0084] With, here

[0085] R 1 is F, CH3, or CF3;

[0086] R 2 is H, F, OR3 , SR 3 , S(O)R 4 , -S(O)2R 4 , C1-C6 alkyl, or C1-C6 fluoroalkyl; or R 1 and R 2 They form a methylenedioxy or difluoromethylenedioxy ring together with the carbon atom to which they are attached;

[0087] R 3 is H or C1-C6 alkyl;

[0088] R 4 is a C1-C6 alkyl;

[0089] X is the chemical formula AB, and

[0090] Here

[0091] B is absent or -(C1-C6alkylene)-, -(C1-C6alkylene)-X 1 -(C1-C6alkylene)-, -X 1 -(C1-C6alkylene)-*, or -(C1-C6alkylene)-X 1 -L 2 -* and; where * is the shared attached part to A;

[0092] X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(-O)-, -NR 5 -, -NR 5 C(-O)-, or -C(-O)NR 5 - and;

[0093] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl;

[0094] X 1 ' is -O-, -S-, -S(O)-, or -S(O)2- and;

[0095] L 2 is phenylene;

[0096] A is -H or -X 2 And;

[0097] X 2 is OR 6 , SR 6 , S(O)R 6 , S(O)2R 6 , SSR 6 or N(R 6 )2 and;

[0098] Each R 6 is independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl;

[0099] B and L 2 Each independently halogen, -CN, -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl;

[0100] Each R 7 is independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl;

[0101] Single R 1 In the case of this F, B is -(C1-C6 alkylene)-, (C1-C6 alkylene)-X 1 -(C1-C6alkylene)-, -X 1 -(C1-C6alkylene)-*, or -(C1-C6alkylene)-X 1 -L 2 -* and; where * is the shared attached part to A; A is -X 2 and; single R 1 This is F and R 2 If ga is -OMe, -BA cannot be -NH2.

[0102] In one aspect, an ADC is provided herein, wherein the camptothecin analog comprises the chemical formula: XY, where:

[0103] Y is the chemical formula I

[0104]

[0105] With, here

[0106] R 1 is F, CH3, or CF3;

[0107] R 2 is H, F, OR 3 , SR 3 , S(O)R 4 , -S(O)2R 4 , C1-C6 alkyl, or C1-C6 fluoroalkyl; or R 1 and R 2 They form a methylenedioxy or difluoromethylenedioxy ring together with the carbon atom to which they are attached;

[0108] R 3 is H or C1-C6 alkyl;

[0109] R 4 is a C1-C6 alkyl;

[0110] X는 *-CH2O-, *-(CH2)2O-, *-(CH2)3O-, *-(CH2)4O-, *-CH2NH-, *-(CH2)2NH-, *-(CH2)3NH-, *-(CH2)4NH-, *-CH2N(CH3)-, *-(CH2)2N(CH3)-, *-(CH2)3N(CH3)-, *-(CH2)4N(CH3)-, *-CH2N(R 5 )-, *-(CH2)2N(R 5 )-, *-(CH2)3N(R 5 )-, *-(CH2)4N(R 5 )-, *-CH2N(CH3)C(=O)CH2O-, *-CH2N(CH2R 5)C(=O)CH2O-, *-CH2NHC(=O)CH2O-, *-CH2NHC(=O)(CH2)2O-, *-CH2NHC(=O)(CH2)3O-, *-CH2NHC(=O)(CH2)4O-, *-CH2NHC(=O)(CH2)5O-, *-CH2NHC(=O)CH2-, *-CH2NHC(=O)(CH2)2-, *-CH2NHC(=O)(CH2)3-, *-CH2NHC(=O)(CH2)4-, *-CH2NHC(=O)(CH2)5-, *-CH2SCH2-, *-CH2S(CH2)2-, *-CH2S(CH2)3-, *-CH2S(CH2)4-, or *-CH2S(CH2)5-; Here, * is the region shared and attached to Y, and R 5 is phenyl.

[0111] In one aspect, an ADC is provided herein, wherein the camptothecin analog comprises the chemical formula: XY, where:

[0112] Y is chemical formula II

[0113]

[0114] has, and *-(CH2)3N(CH3)-, *-(CH2)4N(CH3)-, *-CH2N(R 1 )-, *-(CH2)2N(R 1 )-, *-(CH2)3N(R 1 )-, *-(CH2)4N(R 1 )-, *-CH2N(CH3)C(=O)CH2O-, *-CH2N(R 1)C(=O)CH2O-, *-CH2NHC(=O)CH2O-, *-CH2NHC(=O)(CH2)2O-, *-CH2NHC(=O)(CH2)3O-, *-CH2NHC(=O)(CH2)4O-, *-CH2NHC(=O)(CH2)5O-, *-CH2NHC(=O)CH2-, *-CH2NHC(=O)(CH2)2-, *-CH2NHC(=O)(CH2)3-, *-CH2NHC(=O)(CH2)4-, *-CH2NHC(=O)(CH2)5-, *-CH2SCH2-, *-CH2S(CH2)2-, *-CH2S(CH2)3-, *-CH2S(CH2)4-, or *-CH2S(CH2)5-; Here, * is the region shared and attached to Y, and R 1 It is benzyl.

[0115] In another aspect, an ADC is provided herein, wherein the camptothecin analog comprises any one of the following chemical formulas:

[0116] .

[0117] In a further aspect, an ADC is provided herein, wherein the camptothecin analog comprises formula III:

[0118] .

[0119] In a further aspect, an ADC is provided herein, wherein the camptothecin analog comprises formula IV:

[0120] .

[0121] In a further aspect, an ADC is provided herein, wherein the camptothecin analog comprises formula V:

[0122] .

[0123] In a further aspect, an ADC is provided herein, wherein the camptothecin analog comprises formula VI:

[0124] .

[0125] In a further aspect, an ADC is provided herein, wherein a camptothecin analog comprises formula VII:

[0126] .

[0127] Self-sacrifice unit

[0128] The self-sacrifice or self-removal of a portion of an ADC can be designed into the overall structure of the ADC. Self-sacrifice typically involves the activation of a trigger group, followed by a chemical and / or biological reaction that triggers the spontaneous removal of the group itself, the self-sacrifice unit. The self-sacrifice unit can provide positive properties to the ADC, for example, by providing a space that reduces steric hindrance of cellular proteases reaching the peptide cleavage site of the ADC.

[0129] In some aspects of the present disclosure, the ADC described herein contains a self-sacrificial unit. Where present, the self-sacrificial unit is located after the peptide unit of the linker on the ADC, and thus the trigger group is exposed after the protease cleavage of the peptide unit of the ADC. In a further aspect, the self-sacrificial unit is a -NH-CH2- group, para-aminobenzyl oxycarbonyl (PABC), ortho-aminobenzyl carbonate (OABC), or other self-sacrificial unit known in the art.

[0130] In some aspects of the present disclosure, the ADC described herein does not contain a self-sacrificial unit. In these aspects, the terminal amine from the camptothecin analog described herein is directly linked to a peptide unit.

[0131] Linker

[0132] As disclosed herein, a payload may be conjugated to a nectin-4 antibody by a method understood by a person skilled in the art to form the nectin-4 ADC described herein. One example of such conjugation may include connecting the payload described herein to the nectin-4 antibody described herein through a linker.

[0133] The linker used in ADCs is designed for stability in plasma, allowing time for the ADC to localize in target cells. Releasing the payload too quickly lowers the therapeutic index of the ADC by damaging all types of non-targeted tissues. When the ADC is internalized into target cells, the linker must provide a payload release mechanism so that the payload can function as designed.

[0134] Linkers known to a person skilled in the art include, for example, a cleavable moiety and a non-cleavable moiety. Accordingly, an ADC is provided herein, wherein a payload, for example, a camptothecin analog, is conjugated to an antibody through a linker having a cleavable moiety or to an antibody through a linker having a non-cleavable moiety.

[0135] Any suitable linker known in the relevant art may be used to manufacture the ADC of the present disclosure. In a particular aspect, the linker comprises a reactive group capable of conjugating both the antibody of the present disclosure and a drug or cytotoxic agent. Examples include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC), V-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), Bis-maleimidopolyethyleneglycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(b-maleimidopropyloxy)succinimide ester (BMPS), G-maleimidobutyric acid N-succinimidyl ester (GMBS), E-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, and N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate). m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HC1 salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), k-maleimidoundecanate N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), l,4-bis-maleimidobutane (BMB), l,4-bis-maleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate (sulfo-SMCC), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS),N-(e-maleimidocaproyloxy)sulfosucciimido ester (sulfo-EMCS), N-(K-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH), succinimidyl hydrazinium nicotinate hydrochloride (SHNH), succinimidyl-p-formyl benzoate (SFB), and succinimidyl-p-formylphenoxyacetate (SFPA), V-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), V-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and V-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) are included, but not limited thereto.

[0136] In a specific aspect of the present disclosure, the ADC comprises a cleavable linker. Different mechanisms used in the linker to release a drug or cytotoxic agent are known in the art. Among these mechanisms are (1) using a protease cleavage site in the linker to be cleaved by a cellular protease (e.g., cathepsin B or beta-glucuronidase), (2) using a lysosome at a lower pH to induce hydrolysis of an acid-unstable unit in the linker, or (3) using a higher intracellular level of glutathione to reduce a disulfide crosslink in the linker.

[0137] In some aspects of the present disclosure, the ADC comprises a linker comprising a peptide unit that provides a site for protease cleavage. In some respects, the peptide unit is -Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe, -Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu- (Sequence ID: 100), -Leu-Ala-Leu-Ala- (Sequence ID: 101), , -Gly-Phe-Leu-Gly- (Sequence ID: 103), -Gly-Leu-Phe-Gly- (Sequence ID: 104), -Val-Arg-, -Arg-Val-, -Arg-Arg-, -Ala-Ala-, -Ala-Met-, -Met-Ala-, -Thr-Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr-, -Tyr-Met-, -Ala-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Ala-Ala- (Sequence ID: 105), -Gly-Ala-Gly-Gly- (Sequence ID: 106),-Gly-Gly-Ala-Gly- (Sequence Identification No.: 107), -Gly-Val-Gly-Gly- (Sequence Identification No.: 108), -Gly-Gly-Val-Gly- (Sequence Identification No.: 109), -Gly-Phe-Gly-Gly- (Sequence Identification No.: 110), or -Gly-Gly-Phe-Gly- (Sequence Identification No.: 102). Citrulline is represented by Cit. In the aspect of the present disclosure, the peptide unit contains all natural amino acids in the form of L-amino acids. In a further aspect, the peptide unit may include all D-amino acids or L-amino acids or combinations thereof.

[0138] In some aspects, an ADC is provided herein that further comprises a linker that links the antibody to a cytotoxic agent. In further aspects, a linker comprising a peptide unit is provided herein. In further aspects, a peptide unit comprising Ala-Ala-Ala, Val-Cit, or Gly-Gly-Phe-Gly (SEQ ID: 102) is provided herein. In one aspect, the peptide unit comprises Ala-Ala-Ala. In one aspect, the peptide unit comprises Val-Cit. In one aspect, the peptide unit comprises Gly-Gly-Phe-Gly (SEQ ID: 102). In further aspects, the peptide unit comprises all natural amino acids in the form of L-amino acids.

[0139] In some aspects of the present disclosure, the ADC comprises a linker comprising a spacer unit referred to herein as spacer unit A, which links the cysteine(s) of the antibody disclosed herein to the peptide unit and / or payload described herein. Some of the chemistry used in the art for linking to cysteine ​​includes maleimide, succinimide, or bromoacetamide chemistry, which may be used in the ADC of the present disclosure. In a further aspect of the present disclosure, maleimide-type spacers, such as maleimidocaproyl (mc) or maleimidomethylcyclohexane-1-carboxylate, may be used.

[0140] In some aspects of the present disclosure, an ADC having a spacer unit A of formula VIII is provided herein:

[0141]

[0142] Here, z is 1-5.

[0143] In some aspects of the present disclosure, an ADC having a spacer unit A of formula IX is provided herein:

[0144]

[0145] Here, z is 1-5.

[0146] In one aspect of the present disclosure, an antibody-drug conjugate (ADC) is provided herein, wherein the ADC has the following chemical formula:

[0147]

[0148]

[0149] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0150] In another aspect, an ADC is provided herein, wherein the ADC has the chemical formula X:

[0151]

[0152] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0153] In another aspect, an ADC is provided herein, wherein the ADC has the chemical formula XI:

[0154]

[0155] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0156] In another aspect, an ADC is provided herein, wherein the ADC has the chemical formula XII:

[0157]

[0158] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0159] In another aspect, an ADC is provided herein, wherein the ADC has the chemical formula XIII:

[0160]

[0161] Herein: Ab is the nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0162] In an additional aspect, n is approximately 2 to approximately 12. In another aspect, n is approximately 2 to approximately 8. In another aspect, n is approximately 4 to approximately 8. In another aspect, n is approximately 8 to approximately 12. In another aspect, n is approximately 2. In another aspect, n is approximately 4. In another aspect, n is approximately 6. In another aspect, n is approximately 8. In another aspect, n is approximately 10. In another aspect, n is approximately 12.

[0163] In one aspect, an Ab is disclosed herein comprising an HC containing amino acid 2-444 of sequence identification number: 2 and an LC containing amino acid 2-215 of sequence identification number: 3, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an HC containing amino acid 2-444 of sequence identification number: 2 and an LC containing amino acid 2-215 of sequence identification number: 3, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 2 and an LC consisting of sequence identification number: 3.

[0164] In one aspect, an Ab is disclosed herein comprising an HC containing amino acids 2-444 of sequence identification number: 12 and an LC containing amino acids 2-215 of sequence identification number: 13, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an HC containing amino acids 2-444 of sequence identification number: 12 and an LC containing amino acids 2-215 of sequence identification number: 13, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 12 and an LC consisting of sequence identification number: 13.

[0165] In one aspect, an Ab is disclosed herein comprising an HC comprising amino acids 2-443 of sequence identification number: 16 and an LC comprising sequence identification number: 17, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an HC comprising amino acids 2-443 of sequence identification number: 16 and an LC comprising sequence identification number: 17, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC comprising sequence identification number: 16 and an LC comprising sequence identification number: 17.

[0166] In one aspect, an Ab is disclosed herein comprising an LC consisting of amino acids 2-447 of sequence identification number: 26 and sequence identification number: 27, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an LC consisting of amino acids 2-447 of sequence identification number: 26 and sequence identification number: 27, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 26 and an LC consisting of sequence identification number: 27.

[0167] In one aspect, an Ab is disclosed herein comprising an LC consisting of amino acids 2-446 of sequence identification number: 36 and sequence identification number: 37, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an LC consisting of amino acids 2-446 of sequence identification number: 36 and sequence identification number: 37, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 36 and an LC consisting of sequence identification number: 37.

[0168] In one aspect, an Ab comprising an LC consisting of amino acids 2-446 of sequence identification number: 46 and sequence identification number: 47, wherein n is about 8, is disclosed herein. In one aspect, an Ab comprising an LC consisting of amino acids 2-446 of sequence identification number: 46 and sequence identification number: 47, wherein n is about 4, is disclosed herein. In a further aspect, an Ab comprising an HC consisting of sequence identification number: 46 and an LC consisting of sequence identification number: 47 is disclosed herein.

[0169] In one aspect, an Ab comprising an LC consisting of amino acids 2-446 of sequence identification number: 54 and sequence identification number: 55, wherein n is about 8, is disclosed herein. In one aspect, an Ab comprising an LC consisting of amino acids 2-446 of sequence identification number: 54 and sequence identification number: 55, wherein n is about 4, is disclosed herein. In a further aspect, an Ab comprising an HC consisting of sequence identification number: 54 and an LC consisting of sequence identification number: 55 is disclosed herein.

[0170] In one aspect, an Ab is disclosed herein comprising an LC containing amino acids 2-450 of sequence identification number: 64 and amino acids 2-216 of sequence identification number: 65, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an LC containing amino acids 2-450 of sequence identification number: 64 and amino acids 2-216 of sequence identification number: 65, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 64 and an LC consisting of sequence identification number: 65.

[0171] In one aspect, an Ab is disclosed herein comprising an LC containing amino acids 2-450 of sequence identification number: 74 and amino acids 2-216 of sequence identification number: 75, wherein n is about 8. In one aspect, an Ab is disclosed herein comprising an LC containing amino acids 2-450 of sequence identification number: 74 and amino acids 2-216 of sequence identification number: 75, wherein n is about 4. In a further aspect, an Ab is disclosed herein comprising an HC consisting of sequence identification number: 74 and an LC consisting of sequence identification number: 75.

[0172] Conjugation to anti-nectin-4 antibodies

[0173] Methods for conjugating the antibody disclosed herein to the payload disclosed herein are known in the art. In some methods, the antibody is conjugated to a linker in a first reaction, and then the antibody and the linker are conjugated to the payload in a second reaction. In some methods, the antibody is conjugated to the payload or the payload / linker in a single reaction.

[0174] In some aspects of the present disclosure, the nectin-4 antibody described herein is covalently linked to the camptothecin analog described herein through the thiol groups of one or more cysteine ​​residues located on the nectin-4 antibody. In a further aspect, the cysteine ​​residue(s) used for conjugation are each interchain disulfide cysteine ​​residues. Methods for enabling conjugation to the accompanying cysteine ​​by controlling the reduction of the interchain disulfide are known in the art. In another aspect, the cysteine ​​residue or residues used for conjugation are manipulated in the antibody separately from those used for the interchain disulfide.

[0175] In another aspect of the present disclosure, the nectin-4 antibody described herein is covalently linked to the camptothecin analog described herein through the amino group of one or more lysine residues located on the nectin-4 antibody.

[0176] In one aspect, an ADC is provided herein, wherein the linkage of a cytotoxic agent, a cytotoxic agent-self-sacrificial spacer, or a cytotoxic agent-self-sacrificial spacer-linker to an antibody is made through one or more thiol groups on cysteine ​​of the antibody. In a further aspect, one or more cysteine ​​are each natural cysteine ​​within the hinge region of the antibody.

[0177] Drug-to-Antibody Ratio (DAR)

[0178] In the present disclosure, the drug loading in the formula is represented by n, which is the number of drug molecules per antibody (also known as the drug-to-antibody ratio or DAR). Depending on the context, the subscript n may represent the number of drug molecules attached to individual antibody molecules and thus be an integer value, or it may represent the average drug load and thus be an integer or non-integer value. The average drug load represents the average number of drug-linker molecules per antibody in the composition.

[0179] While a higher DAR can produce more potent ADCs, a higher DAR can also lead to destabilization, aggregation, increased off-target toxicity, and enhanced drug clearance from systemic circulation.

[0180] In the aspect of the present disclosure, when referring to a composition comprising an ADC group, the average drug load is about 1 to about 16, about 2 to about 12, or about 2 to about 10. In a further aspect, the DAR is about 2 to about 8. In a further aspect, the DAR is 4. In yet another aspect, the DAR is about 6 to about 10. In a further aspect, the DAR is 8. The DAR in the formulation may be characterized by using techniques such as mass spectrometry, HIC, ELISA assay, or HPLC by conventional means known in the relevant art.

[0181] The present disclosure provides a method for producing an ADC, said method

[0182] (a) a step of producing a reduced nectin-4 antibody by reducing the nectin-4 antibody disclosed herein with a reducing agent; and

[0183] (b) a step of producing a conjugate by contacting a reduced nectin-4 antibody with a compound of the present disclosure, wherein the compound comprises one or more of formulas I-IX. In a further aspect, the reducing agent is DTT or TCEP.

[0184] The conjugates of the present disclosure or their salts can be readily prepared by various procedures known to those skilled in the art, some of which are exemplified in the following preparation examples and examples. Those skilled in the art recognize that specific synthesis steps for each described route may be combined in different ways or with steps from different reaction schemes to prepare the conjugates of the present disclosure or their salts. The products of each step may be recovered by conventional methods widely known in the art, including extraction, evaporation, precipitation, chromatography, filtration, softening, and crystallization. Unless otherwise indicated, all substituents are as previously defined. Reagents and starting materials are readily available to those skilled in the art. The following preparation examples, examples, and tests further exemplify the present disclosure but should not be construed as limiting the scope of the present disclosure in any way.

[0185] therapeutic use

[0186] In another aspect, a method for treating cancer is provided herein, comprising administering an effective amount of the Nectin-4 ADC or pharmaceutical composition described herein to a patient requiring treatment for cancer. In a further aspect, a method for treating cancer is provided herein, comprising administering an effective amount of the ADC or pharmaceutical composition described herein to a patient requiring treatment for cancer, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In a further aspect, the ADC or pharmaceutical composition described herein is administered in a surgical, adjuvant, or neoadjuvant setting.

[0187] In a further aspect, a method for treating cancer is provided, where the cancer is bladder cancer. In a further aspect, a method for treating cancer is provided, where the cancer is urothelial carcinoma. In a further aspect, the urothelial carcinoma is metastatic urothelial carcinoma (mUC). In a further aspect, a method for treating cancer is provided, where the cancer is breast cancer. In a further aspect, a method for treating cancer is provided, where the cancer is lung cancer. In a further aspect, a method for treating cancer is provided, where the cancer is stomach cancer. In a further aspect, a method for treating cancer is provided, where the cancer is colorectal cancer. In a further aspect, a method for treating cancer is provided, where the cancer is pancreatic cancer. In a further aspect, a method for treating cancer is provided, where the cancer is head and neck cancer. In a further aspect, a method for treating cancer is provided, where the cancer is ovarian cancer. In a further aspect, a method for treating cancer is provided, where the cancer is cervical cancer. In a further aspect, a method for treating cancer is provided, where the cancer is prostate cancer.

[0188] In a further aspect, the patient has relapsed after receiving enfortumab vedotin, or the patient has become refractory to enfortumab vedotin or standard management therapy. In a further aspect, the patient to be treated with the ADC or pharmaceutical composition described herein is ineligible for treatment with enfortumab vedotin. In a further aspect, the patient to be treated with the ADC or pharmaceutical composition described herein is naive for treatment with enfortumab vedotin. In a further aspect, the patient to be treated with the ADC or pharmaceutical composition described herein has previously received programmed death receptor-1 (PD-1) or programmed death-ligand-1 (PD-L1) inhibitors and platinum-containing chemotherapy in a neoadjuvant / adjuvant, locally advanced, or metastatic setting. In a further aspect, the patient to be treated with the ADC or pharmaceutical composition described herein has become refractory to or has relapsed with enfortumab combined with pembrolizumab, nivolumab combined with ipilimumab, or atezolizumab combined with cisplatin / gemcitabine. In a further aspect, patients to be treated with the ADC or pharmaceutical composition described herein have become refractory to or relapsed to carboplatin or cisplatin combined with gemcitabine. In a further aspect, patients to be treated with the ADC or pharmaceutical composition described herein combined with a PD-1 inhibitor or PD-L1 inhibitor are ineligible for treatment with cisplatin-containing chemotherapy.

[0189] In a further aspect, a method is provided comprising administering an effective amount of the ADC or pharmaceutical composition described herein in combination with one or more antitumor agents simultaneously, individually, or sequentially. In a further aspect, a method is provided comprising administering an effective amount of the ADC or pharmaceutical composition described herein in combination with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, individually, or sequentially.

[0190] In another aspect, a method is provided herein comprising administering an effective amount of the nectin-4 ADC or pharmaceutical composition described herein in combination with an FGFR compound, either simultaneously, individually, or sequentially. In a further aspect, the cancer is a urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, putivatinib, vopatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, logaratinib, FGF401, or pemigatinib.

[0191] In another aspect, the Nectin-4 ADC or pharmaceutical composition described herein for use in therapy is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein. In a further aspect, the cancer is urothelial carcinoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In a further aspect, the ADC or pharmaceutical composition described herein is intended for use in perioperative, adjuvant, or neoadjuvant settings.

[0192] In a further aspect, the Nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of bladder cancer is provided herein. In a further aspect, the Nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of urothelial carcinoma is provided herein. In a further aspect, urothelial carcinoma is metastatic urothelial carcinoma (mUC). In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of breast cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of lung cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of gastric cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of colorectal cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of pancreatic cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of head and neck cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of ovarian cancer is provided herein. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of cervical cancer is provided herein. In addition, the ADC or pharmaceutical composition described herein for use in the treatment of prostate cancer is provided herein.

[0193] In a further aspect, the nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein, wherein the cancer has relapsed after treatment with enfortumab vedotin or the cancer has become refractory to enfortumab vedotin. In a further aspect, the nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein, wherein the cancer has relapsed after treatment with enfortumab vedotin or the cancer has become refractory to standard management care. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein, wherein prior use of enfortumab vedotin is contraindicated. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein, wherein prior use of enfortumab vedotin has not occurred. In a further aspect, the ADC or pharmaceutical composition described herein for use in the treatment of cancer is provided herein, wherein prior use consisted of a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy in a neoadjuvant / adjuvant, locally advanced or metastatic setting, or wherein prior use consisted of enfortumab in combination with pembrolizumab, or nivolumab in combination with ipilimumab, or atezolizumab in combination with cisplatin / gemcitabine. In a further aspect, the ADC or pharmaceutical composition described herein in combination with a PD-1 inhibitor or PD-L1 inhibitor, either concurrently, individually, or sequentially, for use in the treatment of cancer is provided herein, wherein the cancer cannot be treated with cisplatin-containing chemotherapy.

[0194] In a further aspect, the ADC or pharmaceutical composition described herein is provided in combination, individually or sequentially with one or more antitumor agents for use in the treatment of cancer. In a further aspect, the antitumor agent is a PD-1 inhibitor or a PD-L1 inhibitor.

[0195] In another aspect, the present invention provides a Nectin-4 ADC or pharmaceutical composition described herein in combination with an FGFR compound, either simultaneously, individually, or sequentially, for use in the treatment of cancer. In a further aspect, the cancer is a urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, putivatinib, vopatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, logaratinib, FGF401, or pemigatinib.

[0196] In another aspect, the use of the Nectin-4 ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein. In a further aspect, the use of the ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer.

[0197] In a further aspect, the use of the Nectin-4 ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein the cancer has relapsed after treatment with enfortumab vedotin, or the cancer has become refractory to enfortumab vedotin or standard management therapy. In a further aspect, the use of the ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein prior use of enfortumab vedotin was contraindicated. In a further aspect, the use of the ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein there was no prior use of enfortumab vedotin. In a further aspect, the use of the ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein there was prior use of a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy in a neoadjuvant / adjuvant, locally advanced, or metastatic setting. In a further aspect, the use of the ADC or pharmaceutical composition described herein for the manufacture of a medicine for the treatment of cancer is provided herein, wherein the cancer cannot be treated with cisplatin-containing chemotherapy, and said medicine is administered simultaneously, individually, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0198] In a further aspect, the use of the ADC or pharmaceutical composition described herein in the manufacture of a drug for the treatment of cancer is provided herein, said drug is administered simultaneously, individually, or sequentially with one or more antitumor agents. In a further aspect, the use of the ADC or pharmaceutical composition described herein in the manufacture of a drug for the treatment of cancer is provided herein, said drug is administered simultaneously, individually, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0199] In another aspect, the use of the Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicine for the treatment of cancer is provided herein, said medicine being administered simultaneously, individually, or sequentially with an FGFR compound. In a further aspect, the cancer is a urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, putivatinib, vopatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, logaratinib, FGF401, or pemigatinib.

[0200] In additional aspects, bladder cancer is urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma. In additional aspects, bladder cancer is non-invasive, non-muscle-invasive, or muscle-invasive. In additional aspects, bladder cancer is muscle-invasive bladder cancer and is post-cystectomy. In additional aspects, bladder cancer is cancer of the bladder, renal pelvis, ureter, or urethra. In additional aspects, breast cancer is HR-positive, HER2-negative breast cancer, or triple-negative breast cancer (TNBC). In additional aspects, breast cancer is ductal breast cancer or lobular breast cancer. In additional aspects, lung cancer is squamous non-small cell lung cancer (NSCLC) or non-squamous NSCLC. In additional aspects, lung cancer is squamous, adenocarcinoma, or small cell carcinoma. In additional aspects, prostate cancer is metastatic castration-resistant prostate cancer. In additional aspects, gastric cancer is esophageal cancer or gastroesophageal junction cancer. In additional aspects, ovarian cancer is serous or mucinous. In addition, ovarian cancer is fallopian tube cancer or peritoneal cancer.

[0201] In additional aspects, the antitumor agent may be a chemotherapy agent including platinum-containing chemotherapy, or may include cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, nabelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRIES (leucovorin, fluorouracil and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, methotrexate, vinblastine and cetuximab. In additional aspects, the chemotherapy agent for bladder cancer, including one, is a combination of cisplatin (or carboplatin) and gemcitabine. In addition, the antitumor agent may be an immuno-oncology agent selected from the group consisting of nivolumab, ipilimumab, fidilizumab, pembrolizumab, tremelimumab, urelumab, rililumab, atezolizumab, epacadostat, and durvalumab.

[0202] Pharmaceutical composition and method of administration

[0203] The antibodies or ADCs described herein may be formulated as pharmaceutical compositions administered via any route that makes the antibodies or ADCs bioavailable, e.g., oral, topical, or subcutaneous administration.

[0204] In addition, pharmaceutical compositions are provided herein comprising the antibody or ADC provided herein, and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients, and adjuvants.

[0205] The antibody or ADC of the present disclosure, or a pharmaceutical composition comprising the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous). The antibody or ADC of the present disclosure may be administered to a patient in single or multiple doses together with pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition described herein may be prepared by methods widely known in the art (e.g., the literature [Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press]) and comprises the antibody or ADC as disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0206] In one aspect, a pharmaceutical composition comprising the antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients is disclosed herein. In one aspect, a pharmaceutical composition comprising the ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients is disclosed herein.

[0207] definition

[0208] As used herein, singular and similar terms used in the context of this disclosure (particularly in the context of the claims) should be interpreted to encompass both singular and plural forms unless otherwise indicated herein or clearly contradicted by the context.

[0209] The terms “to combine” and “to combine” as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, as determined by ordinary methods known in the relevant art, unless otherwise indicated.

[0210] As used herein, the term "effective dose" refers to the amount (per duration and method of administration) required to achieve the desired therapeutic result. The effective dose of a protein or conjugate may vary depending on factors such as the individual's disease state, age, sex, and body weight, and the ability of the protein or conjugate to elicit the desired response in the individual. The effective dose is also an amount in which the therapeutically beneficial effect outweighs any toxic or harmful effect of the protein or conjugate.

[0211] As used herein, the terms “treating,” “treatment,” or “treating” refer to any process capable of slowing, controlling, delaying, or stopping the progression of the disorder or disease disclosed herein, or improving the symptoms of the disorder or disease, but do not necessarily imply the complete elimination of all symptoms of the disorder or disease.

[0212] The term "patient" as used in this document refers to a human patient.

[0213] Specific abbreviations are defined as follows: "ACN" refers to acetonitrile; "DCM" refers to dichloromethane; "DIPEA" refers to N,N-diisopropylethylamine; "DBU" refers to 1,8-diazabicyclo[5.4.0]undes-7-ene; "DMTMM" refers to (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride); "DMAC" refers to dimethylacetamide; "DMF" refers to N,N-dimethylformamide; "DTT" refers to dithiothreitol; "EtOAc" refers to ethyl acetate; "EDTA" refers to ethylenediaminetetraacetic acid, and "FA" refers to formic acid; "HMPA" refers to hexamethylphosphoramide; "h" refers to time; "HEPES" refers to (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); "NMM" refers to N-methylmorpholin; "NMP" refers to (N-methyl-2-pyrrolidone); and "Su" refers to succinimide "PPTS" refers to pyridinium p-toluenesulfonate; "THF" refers to tetrahydrofuran; "TsOH" refers to p-toluenesulfonic acid; and "TCEP" refers to (tris(2-carboxyethyl)phosphine).

[0214] Examples

[0215] Example 1: Production of Nectin-4 Antibody

[0216] The CDR, variable region, complete heavy chain and light chain amino acid sequences of antibodies 1-8, and the nucleotide sequences encoding them are listed in the section titled "Amino Acid and Nucleotide Sequences" below. Additionally, the sequence identification numbers for the CDR, light chain, heavy chain, light chain variable region, and heavy chain variable region of antibodies 1-8 are presented in Tables 1 and 2.

[0217] The anti-nectin-4 antibodies of the present disclosure, including but not limited to Ab 1-8, can essentially be expressed and purified as follows. The antibodies are expressed in suitable host cells, such as HEK293 or CHO, transiently or stably transfected with an expression system for antibody secretion using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC. The expression plasmid contains cDNA versions of the LC and HC genes for the antibody (e.g., as presented in Table 3); these are expressed from constructs commonly used and suitable for this purpose, such as those based on the human cytomegalovirus major ultra-early promoter.

[0218] The culture medium in which the antibody of the present disclosure is secreted can be purified by conventional techniques, such as a mixed-mode method of ion-exchange and hydrophobic interaction chromatography. For example, the culture medium can be applied to a protein A or G column using conventional methods and eluted therefrom; a mixed-mode method of ion-exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and polymers can be effectively removed by conventional techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, refrigerated, or freeze-dried, for example, at -70°C. Various protein purification methods may be used, and such methods are known in the art and are described, for example, in the literature [30 Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994)]. The antibody can be immediately frozen at -70°C, stored for several months at 2-8°C, freeze-dried, or preserved at 4°C for immediate use.

[0219] Table 1: Sequence identification numbers for the CDR amino acid sequences of the exemplified human nectin-4 antibody

[0220]

[0221] Table 2: Sequence identification numbers for the exemplified human nectin-4 antibodies

[0222]

[0223] Table 3: Sequence identification numbers for DNA sequences of the heavy and light chains of the exemplified human nectin-4 antibody

[0224]

[0225] Example 2: Generation of Nectin-4 ADC

[0226] Synthesis of Camptothecin Analogues

[0227] Essentially, as prepared in literature [ACS Med. Chem. Lett. 2019, 10, 1386-1392] and WO2020219287, the camptothecin analog of the present disclosure, e.g. A1, can be synthesized as follows:

[0228]

[0229] Step 1: 1 M BCl3 (9.95 mL, 9.95 mmol) in DCM was added to a flask containing anhydrous 1,2-dichloroethane (50 mL), and then cooled to 0°C using an ice bath. 3-fluoro-4-methylaniline 1 (1.56 g, 12.4 mmol) was added in several portions and stirred at 0°C for 10 minutes, then 5-bromovaleronitrile 2 (1.72 mL, 14.9 mmol) was added, followed by AlCl3 (2.16 g, 16.2 mmol). The ice bath was removed, and the reaction solution was slowly heated to room temperature. After stirring at room temperature for 10 minutes, the mixture was heated under reflux for 39 hours. The solution was cooled to room temperature, cold H2O (25 mL) was slowly added, followed by the addition of a 5% aqueous HCl solution. After 30 minutes, the solution was diluted with DCM (50 mL). The organic layer was washed with H2O and brine, dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase chromatography under the following conditions: column: C18 (100 g); eluted with 25% ACN in H2O for 5 minutes, followed by a gradient of 25% to 95% ACN in H2O for 15 minutes, and then 95% ACN in H2O for 5 minutes to obtain Compound 3 as a grayish-white solid (1.42 g, 40%).

[0230] Step 2: Compound 3 (3.15 g, 15.64 mmol), Compound 4 (3.92 g, 14.89 mmol), and PPTS (0.037 g, 0.15 mmol) were suspended in toluene (200 mL) in a 50 mL flask equipped with a reflux condenser containing anhydrous toluene (10 mL). The reaction mixture was heated under reflux for 40 hours overnight under an argon atmosphere with magnetic stirring, and then allowed to cool to room temperature. The mixture was filtered, and the solid was washed with toluene (5 mL) to obtain 5 (4.74 g, 74%).

[0231] Step 3: A solution of Compound 5 (0.860 g, 1.67 mmol) in HMPA (5 mL) and deionized water (0.9 mL) was heated at 101°C for 18 hours. Upon cooling to room temperature, the solution was loaded onto a C18 cartridge and purified by reverse-phase chromatography under the following conditions: column: C18 (30 g); eluted with 25% ACN in H2O, followed by a gradient of 25% to 95% ACN in H2O for 15 minutes, followed by 95% ACN in H2O for 5 minutes, to obtain a mixture. The mixture was further purified by silica gel chromatography by eluted with a linear gradient of 0 to 20% MeOH in CH2Cl2 over 15 minutes to obtain Compound A1 as a grayish-white solid (0.392 g, 51% yield).

[0232] Synthetic reaction formula of Linker + Self-Sacrifice Unit + Camptothecin Analogue

[0233] Essentially, as prepared in the literature [ACS Med. Chem. Lett. 2019, 10, 1386-1392] and WO2020219287, the camptothecin analog of the present disclosure can be conjugated to a linker (having maleimide chemistry) and a self-sacrificial unit as follows:

[0234]

[0235] Step 4: A mixture of Fmoc-GGFG (1 g, 1.59 mmol, 1.5 equivalents), A1 (0.48 mg, 1.06 mmol, 1 equivalent), and 4 Å molecular sieve (3 g, 3X wt of A1) in NMP was treated with HCl (4 M, 4.5 equivalents) in 1,4-dioxane at 20°C. The reaction mixture was quenched by the addition of H2O. The solid was removed by filtration, and the filtrate was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal chromatography (0-10% MeOH in SiO2, DCM) to obtain Fmoc-GGFG-A1 (0.75 g, 69% yield).

[0236] Step 5: Fmoc-GGFG-A1 (0.75 g, 0.73 mmol, 1 equivalent) in DMF was treated with DBU (0.11 mL, 0.77 mmol, 1.05 equivalents) at 0°C. The reaction mixture was quenched by the addition of TsOH (0.25 g, 1.47 mmol, 2 equivalents). The resulting mixture was stirred at 0°C for 4 hours and then used directly in the subsequent step without further purification.

[0237] Step 6: A crude mixture of GGFG-A1 (0.73 mmol, 1 equivalent) and TsOH in DMF was basicized to pH 7 using NMM at 0°C, followed by the addition of NMM (0.16 mL, 1.47 mmol, 2 equivalents) and Compound 6 (0.283 g, 0.917 mmol, 1.25 equivalents). The resulting mixture was stirred at 0°C for 4-6 hours, then quenched and purified by normal chromatography (0-10% MeOH in SiO2, DCM) to obtain mc-GGFG-A1 (0.51 g, 70% yield).

[0238] The camptothecin analog of the present disclosure can be conjugated to a linker (having bromoacetamide chemistry) and a self-sacrificial unit as follows:

[0239]

[0240] Step 1: Et2NH (0.025 g, 0.34 mmol) was added to a solution of Fmoc-GGFG-A1 (0.5 g, 0.489 mmol) in DMF (5 ml). The mixture was stirred at 15-25°C for 1 hour. Subsequently, the reaction mixture was quenched by the addition of THF and then concentrated under reduced pressure. The residue was purified by reverse-phase chromatography under the following conditions: column: C18, 150*30 mm*5 μm; by eluting with 22-45% ACN in H2O (0.225% FA) to obtain GGFG-A1 as an FA salt (0.196 g, 47% yield).

[0241] Step 2: Compound 8 (2.9 g, 12.29 mmol) was added to a solution of Compound 7 (2.0 g, 12.26 mmol) in 20 ml of THF at 0°C. The reaction mixture was heated to 20°C. Stirring was continued at 20°C for 18 hours, followed by the addition of Compound 8 (0.5 g, 3.06 mmol). After stirring for an additional 4 hours at 20°C, the reaction mixture was concentrated under reduced pressure. The residue was purified by reverse-phase chromatography; column: C18 column, 150*30mm*5 μm, by elution with 22-45% ACN in H2O (0.225% FA) to obtain Compound 9 (1.15 g, 33% yield).

[0242] Step 3: A mixture of Compound 9 (430 mg, 1.51 mmol) and DMTMMT (340 mg, 1.04 mmol) in DMAC (11 ml) was cooled to 0°C. A mixture of GGFG-A1 (0.500 g, 0.63 mmol) and DIPEA (0.090 g, 0.70 mmol) in DMAC (4 ml) was added dropwise. Stirring was continued at 0°C for 0.5 hours, followed by the addition of DCM (300 mL). The mixture was washed with aqueous 10% NaBr (3 x 50 ml). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure at 0–10°C. The residue was analyzed by reverse-phase chromatography; column: C18 column, 150 × 30 mm × 5 μm; Br-GGFG-A1 (0.23 g, 34.7% yield) was obtained by purification while eluting with 15-45% ACN in H2O (0.225% FA).

[0243] Part I. Fabrication of ADCs with Maleimide Linker-Payloads

[0244] To prepare an antibody-drug conjugate having 8 drugs per antibody, the IgG1 antibody is completely reduced at 37°C for 2 hours using a reducing agent, such as DTT or TCEP, in an amount of 6 to 8 molar equivalents. Then, the reduced antibody is buffered with 50 mM HEPES containing 2 mM EDTA at pH 7.0 using a PD-10 desalting column, and the eluent is adjusted to a protein concentration of 5-10 mg / ml with HEPES buffer. The conjugation reaction can be stopped by adding an excess linker-payload, e.g., 10 molar equivalents, for 1 hour, and by adding a substantial excess of L-cysteine, e.g., 6 molar equivalents. The mixture of the generated ADCs can be purified on a PD-10 desalting column equilibrated in 25 mM histidine and 9% sucrose at pH 5.5, followed by three spin cycles using a 30 kDa MWCO centrifuge unit to remove any unreacted linker-payload related species. Finally, the generated ADCs can be sterile filtered through a 0.2 μM filter and stored at 4°C or -80°C for future use.

[0245] Part II. Preparation of ADCs with Bromoacetyl Linker-Payload

[0246] To prepare an antibody-drug conjugate having 8 drugs per antibody, the IgG1 antibody is completely reduced at 37°C for 2 hours using a reducing agent, such as DTT or TCEP, in an amount of 6 to 8 molar equivalents. Then, the reduced antibody is buffered with 50 mM HEPES containing 2 mM EDTA at pH 7.4 using a PD-10 desalting column, and the eluent is adjusted to a protein concentration of 5-10 mg / ml with HEPES buffer. The conjugation reaction can be stopped by adding an excess linker-payload, such as 12 molar equivalents, over 2-3 hours, and by adding a substantial excess of L-cysteine, such as 10 molar equivalents. The mixture of the generated ADCs can be purified on a PD-10 desalting column equilibrated in 25 mM histidine and 9% sucrose at pH 5.5, followed by three spin cycles using a 30 kDa MWCO centrifuge unit to remove any unreacted linker-payload related species. Finally, the generated ADCs can be sterile filtered through a 0.2 μM filter and stored at 4°C or -80°C for future use.

[0247] Example 3: Antibody binding affinity, cross-reactivity, and selectivity

[0248] Characterization of human and cross-species coupling of Nectin-4 ADCs by surface plasmon resonance

[0249] Using a ViaCore 8K+ instrument (Cytiva, Marlboro, Massachusetts), kinetics and affinity parameters for binding interactions between Nectin-4 ADC (each Ab1-8 conjugated as in Formula XI and DAR 8 as exemplified herein) and recombinant HIS-tagged human (Acro Biosystems, catalog number NE4-H52H3, Newark, Delaware), cino (Acro Biosystems, catalog number NE4-C52H4), and rat (R&D Systems, Minneapolis, Minnesota, catalog number 9997-N4-050) were determined.

[0250] Anti-human Fc sensor surfaces were prepared by amine-coupled goat anti-human IgG Fc (Southern Biotech Catalog No. 2014-01, Birmingham, Alabama) to the surface of a Viacor Series S CM4 (Sitiba Catalog No. BR-100534) sensor at 25°C. For immobilization, a driving buffer of 10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4 was used. Flow cells 1 & 2, comprising all 8 channels, were activated for 7 minutes at a flow rate of 10 µL / min with a 1:1 (v / v) mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 nM N-hydroxysuccinimide (NHS). Next, the anti-human IgG Fc capture reagent was coupled to the sensor surface (diluted to 50 µg / mL in 10 mM acetate pH 4.5 buffer) by injecting it into all flow cells and channels at a flow rate of 10 µL / min for 7 minutes. The remaining active group was blocked by injecting 100 mM ethylenediamine (in 200 mM borate buffer, pH 8.5) into all flow cells and channels at a flow rate of 10 µL / min for 7 minutes. Subsequently, all channels and flow cells were preconditioned using three consecutive 1-minute injections of 75 mM phosphate at a flow rate of 10 µL / min.

[0251] For kinetics / affinity analysis, the drive and sample dilution buffer was 10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween-20, pH 7.4, 1 mg / mL bovine serum albumin (BSA), and the analysis temperature was 37℃.

[0252] In each analysis cycle, different ADCs were captured on flow cell 2 of each channel by injecting at 5 µg / mL and 10 µL / min. After capture, the same analytes were injected into flow cell 1 and 2 of all 8 channels at 30 µL / min for 2 minutes, and dissociation was monitored for 10 minutes. After dissociation, all surfaces were regenerated by three 1-minute injections of 75 mM phosphate at 10 µL / min. The ADC capture and analyte cycles were repeated to obtain analyte binding of each ADC at concentrations of 0, 2.5, 7.4, 22, 67, 200, and 600 nM Nectin-4.

[0253] The sensogram data were fitted overall using the default 1:1 coupled model in VIACORE Insight evaluation software v3.0.12.15655. The kinetics and affinity parameters for the ADC are presented in Table 4.

[0254] Table 4: Kinetic and affinity parameters for ADC / nectin-4 interaction at 37°C.

[0255]

[0256]

[0257]

[0258] Although coupling is clearly observed; the data exhibits dynamic heterogeneity that causes poor fitting to the 1:1 coupled model.

[0259] k a is the association rate constant, and k d is the dissociation rate constant, and K D is the equilibrium dissociation constant (K D = k d / k a (calculated using ) and n is the number of iterations. If n = 1 or 2, the iteration value is given. If n = 3, the mean ± standard deviation is given.

[0260] Cell surface binding of nectin-4 antibodies on cell lines expressing the nectin-4 receptor

[0261] A panel of nine Nectin-4 antibodies was tested for cell surface binding to two Nectin-4 expressing tumor cell lines. Cell lines exhibiting high and low receptor densities were selected; SUM190PT tumor cells showed high endogenous expression, while NCI-H1781 tumor cells showed lower endogenous expression. SUM190PT and NCI-H1781 cells were determined to have antibody binding capacities of 108,000 and 20,000, respectively (using the MESF quantification kit, Bangs Laboratories). T24 parent cells were selected as the Nectin-4 negative cell line. Antibody binding was quantified via flow cytometry, and the EC of the binding curve was calculated. 50 and the maximum binding MFI for each antibody were both recorded.

[0262] Cells were dissociated using a non-enzymatic dissociation buffer at 37°C for 5 minutes. Cells were counted, and 10 were placed in a V-bottom polypropylene 96-well plate. 5Cells were aliquoted into wells. Cells were centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded. A series of dilutions of the 11-point antibody were prepared by down-diluting 1:4 starting at 300 nM in assay buffer (1X PBS containing 1% BSA and 0.09% sodium azide). The series of dilutions was added to the cells at 100 µL / well and mixed by pipetting. Several untreated control wells / cell lines were prepared in assay buffer alone. Cells and antibodies were incubated on an orbital shaker at 4°C for 1 hour. After incubation, the assay plates were centrifuged and washed twice with 300 µL / well of assay buffer. Subsequently, the cell pellet was stained at 100 µL / well with a 1:500 dilution of the Alexa647-conjugated mouse anti-human IgG secondary antibody in assay buffer. The assay plates were incubated in a dark room at 4°C for 1 hour with shaking. After incubation, the plates were centrifuged, and the cells were washed twice with 300 µL / well assay buffer. 100 µL / well of a 1:5000 solution of Zombie Green viability marker (BioLegend) in 1X PBS was added to the cells, and the plates were incubated in a dark room for 10 minutes with shaking at 4°C. Subsequently, the cell pellet was washed once with assay buffer and fixed in 200 µL / well of 4% paraformaldehyde in 1X PBS in a dark room at room temperature for 15 minutes. After centrifuging and washing the cells once, they were resuspended in 65 µL assay buffer for acquisition on a Sartorius iQue HTFC cytometer.

[0263] Cells were acquired on a Sartorius iQue HTFC cytometer, and an FCS file was generated using Foresight Standard Edit (v. 6.2.6652). Subsequently, the FCS file was analyzed on Flowzo (v10.8.1). Fragments were excluded from analysis by Forward Scattering (FSC) vs. Side Scattering (SSC) gating, and single cells were selected via Forward Scattering Area (FSC-A) vs. Height (FSC-H) gating. Finally, apoptotic cells positive for Zombie Green staining were excluded, and the MFI of living Alexa647-positive cells was quantified. The mean unstained cell autofluorescence was subtracted from all samples. The data were graphed and analyzed on GraphPad Prism (v9.5.1). agonist vs. response-variable slope (4-parameter) curve fitting, and EC through the % of maximum enfortumab binding calculated by setting the mean of the maximum enfortumab MFI for each cell line to 100% 50 Decided.

[0264] Nine types of nectin-4 antibodies did not bind to nectin-4 negative T24 parent cell lines. As shown in Table 5, the antibodies exhibited excellent binding to both high and low nectin-4 expressing tumor cells.

[0265] Table 5: Cell surface binding of nectin-4 antibodies to nectin-4 expressing cell lines

[0266]

[0267] MFI = Central Fluorescence Intensity

[0268] Characterization of Nectin-4 antibody and ADC binding to normal human epidermal keratinocytes using flow cytometry

[0269] To characterize the binding of the Nectin-4 antibody and ADC of the present disclosure to normal human epidermal keratinocytes, HEKα cells were plated at 1 million per well in complete culture medium (dermal cell basal medium + keratinocyte growth kit, ATCC) in 150 x 25 mm tissue culture dishes (Corning Inc.) and achieved total growth within 24 hours. The differentiation process was carried out for an additional 5–10 days. After induction of differentiation, cells were dissociated at 37°C with 2 mg / mL type XI collagenase from Clostridium histolyticum (Sigma-Aldrich). Cells were counted and plated in V-bottom polypropylene 96-well plates (Thermo Scientific Nunc) 10 5Cells were aliquoted into wells. Antibodies and ADCs were added to the cells in serial dilutions of 1:4, starting at 300 nM in assay buffer (1X DPBS containing 2% FBS, Gibbco). Cells were incubated at 4°C on a microplate shaker and protected from light for 1 hour. After incubation, cells were washed twice with 200 µL / well assay buffer and stained with Alexa Fluorine 647-conjugated Affinipure F(ab')2 fragment goat anti-human IgG (H+L) (Jackson ImmunoResearch) at a dilution of 1:1,000. After incubation at 4°C for 1 hour, cells were washed twice with 200 µL / well assay buffer and stained with Zombie Green fixable viability dye (BioLegend) at a dilution of 1:2,000. Cells were incubated on a microplate shaker at 4°C for 20 minutes. Cells were washed with assay buffer and fixed in 200 µL / well BD Cytopix fixation buffer at 4°C for 20 minutes. Cells were resuspended in assay buffer for acquisition on an Attune Cytopix flow cytometer (Thermo Fisher Scientific). Cells were determined to have an antibody binding capacity of 17,000 (using MESF quantification kit, Vance Laboratories).

[0270] As presented in Table 6, the tested Nectin-4 antibody and ADC (conjugated as in Formula XI, DAR 8) have ECs similar to or higher than those of enfortumab and enfortumab ADC (each Ab conjugated as in Formula XI, DAR 8 as exemplified herein). 50 It binds to normal human epidermal keratinocytes. Ab1 has a lower affinity for HEKα cells than enfortumab.

[0271] Table 6: Cell surface binding of Nectin-4 lead antibody and CAMP98 ADC to normal human epidermal keratinocytes

[0272]

[0273] EC 50 = Half-maximum effective concentration

[0274] Example 4: ADC Coupling and Internalization

[0275] Characterization of the internalization ability of nectin-4 antibodies and ADCs in human nectin-4 positive cells using fluorescence imaging

[0276] T24 cell lines were engineered to express human nectin-4-eGFP, and clones were selected for high expression. T24 human nectin-4-eGFP clone 3 was determined to have an antibody binding capacity of 379,000 (using the MESF quantification kit, Vance Laboratories). T24 human nectin-4-eGFP clone 3 cells were plated at 12,000 cells per well in complete culture medium (McCoy 5A, modified, + 10% FBS + Glutamax + 400 µg / mL G418 + Pen / Strep) in black clear-bottom CellCarrier Ultra 384-well microplates (Perkin Elmer). Plates were covered with AeraSeal™ sealing film and incubated overnight at 37°C and 5% CO2. The following day, antibodies and ADCs were added to the cells at a 1:3 dilution, starting at 300 nM. The plates were covered with Aerasil™ and placed in an incubator for imaging on a PerkinElmer Opera Fenix ​​Screening System over a 24-hour period. Data were processed and analyzed using Harmony and Microsoft Excel, and graphed using GraphPad Prism.

[0277] As shown in Table 7, the nectin-4 antibody and nectin-4 ADC (each exemplified herein as being conjugated to Ab1-8 as in Formula XI and having a DAR of 8) induce degradation of the nectin-4-eGFP signal. The tested nectin-4 ADC, as shown in Table 7, had an internalization potential equivalent to or greater than that of the enfortumab antibody in the same ADC format as Ab1-8.

[0278] Table 7: Percent activity and EC of Nectin-4 antibodies and ADCs 50 (nM)

[0279]

[0280] % activity = Maximum GFP signal loss compared to enfortumab mAb (100%)

[0281] Example 5: ADC Cytotoxicity and Bystander Activity

[0282] Characterization of the cytotoxicity of nectin-4 ADCs in low and high nectin-4 expression cell lines

[0283] Cells of the low-expression cell line NCI-H1781 were seeded in culture medium (RPMI 1640 + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate) in white, clear-bottom 96-well tissue culture plates. Cells were incubated overnight at 37°C under 5% CO2. The following day, ADCs were added to the culture medium at final working concentrations serially diluted 1:3 from 100 nM. The plates were covered with Breathe-Easy® sealing membranes and incubated at 37°C under 5% CO2. After 5 days of treatment, the plates were read using the Celtiter-Glo luminescence cell viability assay. 100 µl / well Celtiter-Glo reagent was incubated in the plates at room temperature for 10 minutes. Luminescence was read on a Spectramax M5e. RLU (Relative Light Unit) was obtained using Softmax Pro 5.4. The percentage of apoptosis was calculated against the untreated group as 0%. The data were graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 It was determined through log(inhibitor) vs. response - variable slope (4 parameters) curve fitting.

[0284] T24 cell lines were engineered to express human nectin-4, and clones were selected. T24-human nectin-4 clone 108, a high-expression clonal cell line, was seeded in culture medium (McCoy 5A + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 400 µg / ml G418). ADCs were added to the culture medium for 5 days at final working concentrations serially diluted from 200 nM to 1:4.

[0285] T24-human nectin-4 clone 108 and NCI H1781 cells were determined to have antibody binding capacities of 56,000 and 20,000, respectively (using MESF quantification kit, Vance Laboratories).

[0286] As presented in Table 8, the selected exemplary Nectin-4 ADCs (each Ab1-8 conjugated as in Formula XI and DAR 8 as exemplified herein) exhibited robust maximal apoptosis in Nectin-4 cell lines with differential expression levels similar to that of an enfortumab antibody in the same ADC format as Ab1-8 (enfortumab conjugated to Formula XI with an effector mutation, DAR 8). IC 50 The values ​​suggest similar efficacy across the tested ADCs in T24-human nectin-4 clone 108 cells and variable efficacy among the tested ADCs in NCI H1781 cells. No non-specific cytotoxicity was observed for the tested T24 nectin-4 negative cells.

[0287] Table 8: Cytotoxicity of nectin-4 conjugates on high and low-expression nectin-4 cell lines

[0288]

[0289] Characterization of the cytotoxicity of Nectin-4 ADCs in MMAE-resistant cell lines

[0290] In MMAE-resistant T24 Nectin-4 cells, the two Nectin-4 ADCs described herein and enfortumab vedotin were tested for activity. T24-h Nectin-4 clone 14 cells were seeded at a rate of 500 cells per well in 100 µl of culture medium in white clear-bottom 96-well tissue culture plates and incubated overnight at 37°C under 5% CO2. The next day, each ADC was serially diluted 1:4 in the culture medium, with a starting concentration of 400 nM. 100 µl of each ADC dilution was added per well, the plate was covered with a Breate-Easy® sealing membrane, and incubated at 37°C under 5% CO2. After 5 days of treatment, the plates were removed from the incubator, left at room temperature for 15 minutes, and 100 µl of medium per well was removed. 100 µl of Celtiter-Glo reagent was added per well, and the plates were incubated at room temperature for 10 minutes. RLU (Relative Light Units) were obtained using a Softmax Pro 5.4 with a Spectramax M5e. The percentage of apoptosis was calculated as 0% for DMSO alone (for free payloads) or untreated (for ADCs). Data were graphed and analyzed using GraphPad Prism version 9.5.1. IC50 was determined via log(inhibitor) vs. response – variable slope (4 parameters) curve fitting.

[0291] ADCs prepared with Ab1 and Ab2 (conjugated with formula XI and DAR 8) retained activity against MMAE-resistant cells, whereas enfortumab vedotin lost efficacy.

[0292] Bystander activity of enfortumab with the exemplified linker / payload compared to enfortumab vedotin

[0293] UMUC3 cell lines were engineered to express human nectin-4, and two clonal populations with different expression levels were selected. In flat white clear-bottom 96-well plates, UMUC3-h-nectin-4 clone F7 / UMUC3-Luc-GFP cells were mixed and seeded in assay medium (MEM + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate) at a ratio of 4:1 with a total of 1,500 cells / well / 100 µl. UMUC3-h-nectin-4 clone E3 / UMUC3-Luc-GFP cells were mixed and seeded in assay medium at a ratio of 6:1 with a total of 2,100 cells / well / 100 µl. UMUC3-Luc-GFP cells were negative for nectin-4 expression. The plates were incubated overnight at 37°C under 5% CO2. The following day, ADCs were added to the assay medium at final working concentrations serially diluted 1:3 from 100 nM. To evaluate the cell line's susceptibility to the free payload, the free payload was added to the assay medium at final working concentrations serially diluted 1:3 from 200 nM. The plates were covered with Breate-Easy® sealing membranes and incubated at 37°C under 5% CO2. The plates were read using the ONE-Glo™ Luciferase assay system after 5 days of treatment. 100 µl / well of ONE-Glo™ assay reagent was incubated in the plates at room temperature for 10 minutes. Luminescence was read on a Spectramax M5e. RLU (Relative Luminous Units) were obtained using a Softmax Pro 5.4. The percentage of UMUC3-Luc-GFP death was calculated relative to the untreated group as 0%. Data were graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 Bystander activity was determined through log(inhibitor) vs. response (3 parameters) curve fitting, and free payload cytotoxicity was determined through log(inhibitor) vs. response (4 parameters).

[0294] UMUC3 nectin-4 clone F7 and UMUC3 nectin-4 clone E3 cells were determined to have antibody binding capacities of 113,000 and 567,000, respectively (using MESF quantification kit, Bangs Laboratories).

[0295] As shown in Table 9, enfortumab conjugated as in Formula XI with a DAR of 8 exhibited a higher efficacy of bystander effect on UMUC3-Luc-GFP cells compared to enfortumab vedotin. In particular, Table 10 shows that the UMUC3-Luc-GFP cell line was similarly susceptible to the free payloads Formula XI and MMAE. Higher expression on positive cell lines resulted in higher bystander activity by enfortumab vedotin, but its efficacy remained lower than that of enfortumab conjugated as in Formula XI.

[0296] Table 9: IC50 of bystander effects by enfortumab and enfortumab vedotin conjugated with Formula XI in UMUC3-Luc-GFP nectin-4 negative cells

[0297]

[0298] Table 10: IC50 of cytotoxicity by Chemical Formula XI payload and MMAE payload against UMUC3-Luc-GFP nectin-4 negative cells

[0299]

[0300] In an experimental run essentially as described herein, the bystander effects of the four Nectin-4 ADCs and enfortumab vedotin described herein were evaluated using UMUC3-h Nectin-4 clone F7 / UMUC3-luciferase-GFP cell pairs. Cells were plated in a 4:1 ratio and treated with serial dilutions of each ADC for 5 days. To determine the bystander effects of the ADCs, luciferase luminescence was monitored using the ONE-GLO™ Luciferase assay system. ADCs prepared with Ab 1, 1a, 2, and 7 (conjugated with Formula XI and DAR 8) demonstrated potent bystander effects, whereas enfortumab vedotin exhibited a much less potent bystander effect.

[0301] Characterization of the bystander effect of nectin-4 ADCs in T24 nectin-4 negative cell lines

[0302] T24 cell lines were engineered to express mScarlet, and clones were selected and tested in a bystander assay. T24-mScarlet clone 5 and T24-human nectin-4 clone 108 were mixed and seeded in flat-clear-bottom 96-well plates in culture medium (McCoy 5A + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 400 µg / ml G418) at a total of 1,000 cells / well / 100 µl in a 9:1 ratio. T24 mScarlet clone 5 cells were negative for nectin-4 expression. The plates were incubated overnight at 37°C under 5% CO2. The following day, ADCs were added to the culture medium at final working concentrations serially diluted 1:4 from 50 nM. The plates were covered with Breate-Easy® sealing membranes, placed in BioSpar, and scanned daily at Citation 5 for 5 days.

[0303] Images of T24-mScarlet cells were captured and analyzed using Gen5 Image Prime 3.11 via Citation 5. The percentage of T24-mScarlet cell death was calculated using reduced integral intensity (area x mean intensity) and normalized for 0 hours and untreated. Data were graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 It was determined through log(inhibitor) vs. response - variable slope (4 parameters) curve fitting.

[0304] As shown in Table 11, specific nectin-4 ADCs of the present disclosure (each Ab1-8 conjugated as in Formula XI and DAR 8) exhibited bystander effects of similar potency and magnitude to enfortumab antibodies of the same ADC format as Ab1-8 (enfortumab conjugated to Formula XI, DAR 8) against T24 mScarlet clone 5 nectin-4 negative cells.

[0305] Table 11: IC50 of bystander effect by nectin-4 ADC conjugate in T24 mScarlet clone 5 nectin-4 negative cells

[0306]

[0307] Characterization of the activity of nectin-4 ADCs with different payloads and linker chemistry in low and high nectin-4 expression cell lines

[0308] NCI-H1781 cells were seeded in culture medium (RPMI 1640 + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate) in white clear-bottom 96-well tissue culture plates. Cells were incubated overnight at 37°C under 5% CO2. The next day, ADCs were added to the culture medium at final working concentrations serially diluted 1:4 from 100 nM. The plates were covered with Breate-Easy® sealing membranes and incubated at 37°C under 5% CO2. After 5 days of treatment, the plates were read using the Celtiter-Glo luminescence cell viability assay. 100 µl / well Celtiter-Glo reagent was incubated in the plates at room temperature for 10 minutes. Luminescence was read on a Spectramax M5e. RLU (Relative Luminescence Units) were obtained with a Softmax Pro 5.4. The percentage of apoptosis was calculated for the untreated group as 0%. The data were graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 It was determined through log(inhibitor) vs. response - variable slope (4 parameters) curve fitting.

[0309] UMUC3 cell lines were engineered to express human nectin-4, and clones were selected for high expression. The UMUC3-human nectin-4 clone F7 engineered cell lines were seeded in culture medium (MEM + 1x Glutamax + 10% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate + 500 µg / ml G418). ADCs were added to the culture medium for 6 days at final working concentrations serially diluted from 100 nM to 1:4.

[0310] NCI H1781 and UMUC3-hnectin4 clone F7 and cells were determined to have antibody binding capacities of 20,000 and 113,000, respectively (using MESF quantification kit, Bangs Laboratories).

[0311] As shown in Table 12, both the Ab2 ADC conjugated as in Formula XI and having a DAR of 8, and the PEG8-VA-Exatecan, the Ab2 ADC in the DAR 8 format, exhibit potent cytotoxic effects on NCI-H1781 and UMUC3-human Nectin-4 clone F7 cell lines.

[0312] Table 12: Cytotoxicity of Nectin-4 ADCs with Different Payloads and Linker Chemistries on Low- and High-Expression Nectin-4 Cell Lines

[0313]

[0314] Example 6: ADCC, ADCP, and / or CDC testing

[0315] In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assay for Nectin-4 antibody

[0316] T24 cell lines were engineered to express human Nectin-4, and clones were selected for high expression. T24-human Nectin-4 clone 147 was determined to have an antibody binding capacity of 411,000 (using the MESF quantification kit, Vance Laboratories). Target cells, T24-human Nectin-4 clone 147, were added to test medium (IMDM + 1x Glutamax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100 U / ml-100 µg / ml) in clear tissue culture 96-well plates and incubated overnight at 37°C under 5% CO2. The following day, 40 µl / well antibody was added to the test medium at a final working concentration serially diluted 1:4 from 200 nM. The antibody was incubated for 1 hour at 37°C under 5% CO2. Next, effector cells, Jurkat-Lucia NFAT-CD16, were added at 200k / 80 µl / well and incubated at 37°C under 5% CO2. After 23 hours, 20 µl of supernatant and 50 µl of pre-prepared QUANTI-Luc / well were mixed on a white opaque plate. Luminescence was read on a SpectraMax M5e. RLU (Relative Luminous Units) were obtained with SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0317] Unlike enfortumab antibodies containing wild-type IgG1 Fc, Ab1-8 is an effector antibody and did not induce antibody-dependent cytotoxicity potential.

[0318] In vitro antibody-dependent phagocytosis (ADCP) assay against Nectin-4 antibody

[0319] T24 cell lines were engineered to express high levels of human nectin4-eGFP, and clones were selected. Target cells, T24-human nectin4-eGFP clone 3, were seeded in test medium (IMDM + 1x Glutamax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100 U / ml-100 µg / ml) in clear tissue culture 96-well plates and incubated overnight at 37°C under 5% CO2. The next day, 40 µl / well mAb was added at a final working concentration serially diluted 1:4 from 200 nM in the test medium, and incubated for 1 hour at 37°C under 5% CO2. Subsequently, effector cells, Jurkat-Lucia NFAT-CD32, were added at 200k / 80 µl / well, and incubated at 37°C under 5% CO2. After 23 hours, 20 µl of supernatant and 50 µl of pre-prepared QUANTI-Luc / well were mixed on a white opaque plate. Luminescence was read on a SpectraMax M5e. RLU (Relative Luminous Units) were obtained with SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0320] Unlike enfortumab antibodies containing wild-type IgG1 Fc, Ab1-8 is an effector antibody and did not induce antibody-dependent phagocytic potential.

[0321] In vitro complement-dependent cytotoxicity (CDC) assay for nectin-4 lead antibodies

[0322] T24 cell lines were engineered to express high levels of human nectin-4, and clones were selected. T24-human nectin-4 clone 147 cells were added to assay medium (McCoy 5A + 1x Glutamax + 10% heat-inactivated fetal bovine serum) in white clear-bottom 96-well tissue culture plates and incubated overnight at 37°C under 5% CO2 in an incubator. The next day, 50 µl / well antibody was added to the assay medium at a final working concentration serially diluted 1:3 from 200 nM, and the plates were incubated for 1 hour at 37°C under 5% CO2. Subsequently, diluted human serum complement (1:3) was added to the assay medium at 50 µl / well, and the plates were incubated for 3 hours at 37°C under 5% CO2. The plates were read using the Celltiter-Glo luminescence cell viability assay. 100 µl / well Seltite-Glo reagent was incubated in plates at room temperature for 10 minutes. Luminescence was read on a SpectraMax M5e. RLU (Relative Luminous Units) were obtained using a Softmax Pro 5.4. The percentage of apoptosis was calculated relative to the untreated group. Data were graphed and analyzed using GraphPad Prism version 9.5.1.

[0323] As a positive control, Jeko-1 cells were treated with an anti-CD20 antibody in assay medium (RPMI1640 + 1x Glutamax + 10% heat-inactivated fetal bovine serum) as described above.

[0324] Like enfortumab, Ab1-8 did not exhibit CDC potential in nectin-4 expressing T24 cell lines. The anti-CD20 control antibody induced CDC activity and was used as a positive control for CD20-expressing Jeko-1 cells.

[0325] Example 7: Efficacy of Nectin-4 ADC in a Tumor Xenograft Model

[0326] To test the efficacy of the nectin-4 ADC of the present disclosure having an effector mutation in the Fc region, and to compare it with an enfortumab antibody as an ADC having wild-type Fc and the same payload background as that of the present disclosure, two tumor xenograft models having high or intermediate nectin-4 expression were tested as described. UM-UC-3 nectin-4 clone F7 cells with high nectin-4 expression were unilaterally transplanted into the right flank of 5-8 week old, immunocompromised female mice (nu / nu) weighing 18-20 g. The tumor was approximately 150-250 mm 3 When [date] was reached; animals were matched into treatment or control groups based on tumor volume, and administration was initiated (Day 0, n=8 per group). The test product was provided as a single dose (2 mg / kg) formulated with 5% dextrose. Tumors were measured every other week until Day 40.

[0327] In a separate study, MDA-MB-468 cells with moderate nectin-4 expression were injected subcutaneously into 5- to 6-week-old female NSG mice. The tumors were approximately 150-250 mm 3 When [date] was reached; animals were matched into treatment or control groups based on tumor volume, and administration was initiated (Day 0, n=8 per group). The test product was provided as a single dose (2 mg / kg) formulated with 5% dextrose. Tumors were measured every other week until Day 71.

[0328] In both models, a single dose treatment of 2 mg / kg of either the nectin-4 ADC (each Ab1-8 conjugated as in Formula XI and DAR 8 as exemplified herein) or the benchmark enfortumab ADC (enfortumab conjugated to Formula XI, DAR 8) was used. Tumor growth was measured on day 40 for the UMUC3 nectin-4 clone F7 and on day 71 for the MDAMB468 xenograft. As shown in Table 13, treatment with the nectin-4 ADC containing the effector Ab1-8 resulted in tumor growth inhibition equivalent to, or superior to, that of the benchmark enfortumab ADC with the wild-type Fc region in the case of, the specific ADC.

[0329] Table 13: Tumor measurements after treatment with Nectin-4 ADC (mean tumor volume ± SEM)

[0330]

[0331] SEM = Standard error of the mean

[0332] In a xenograft study performed in a manner similar to that described using UM-UC-3 Nectin-4 clone F7 cells, the Nectin-4 ADC described herein was administered in combination with cisplatin and gemcitabine at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg. An increase in antitumor activity was observed at low doses of 0.5 and 1 mg / kg when the SOC agonist and Nectin-4 ADC were combined.

[0333] Amino acid and nucleotide sequences

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

Claims

Claim 1 An antibody-drug conjugate (ADC) comprising an antibody conjugated to a cytotoxic agent, wherein the cytotoxic agent comprises the following chemical formula: ;Here, the antibody binds to human nectin-4 and comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein HCVR comprises heavy chain complementarity determining region (HCDR) HCDR1, HCDR2, and HCDR3, and LCVR comprises light chain complementarity determining region (LCDR) LCDR1, LCDR2, and LCDR3, where: a) HCDR1 comprises sequence identification number: 4, HCDR2 comprises sequence identification number: 5, HCDR3 comprises sequence identification number: 6, LCDR1 comprises sequence identification number: 7, LCDR2 comprises sequence identification number: 8, and LCDR3 comprises sequence identification number: 9, or; or b) an ADC in which HCDR1 includes sequence identification number: 18, HCDR2 includes sequence identification number: 19, HCDR3 includes sequence identification number: 20, LCDR1 includes sequence identification number: 21, LCDR2 includes sequence identification number: 22, and LCDR3 includes sequence identification number:

23. Claim 2 An ADC according to claim 1, wherein a) HCVR comprises sequence identification number: 10 and LCVR comprises sequence identification number: 11; or b) HCVR comprises sequence identification number: 24 and LCVR comprises sequence identification number:

25. Claim 3 An ADC according to claim 1, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein: a) the HC comprises amino acid 2-444 of sequence identification number: 2 and the LC comprises amino acid 2-215 of sequence identification number: 3; or b) the HC comprises amino acid 2-443 of sequence identification number: 16 and the LC comprises sequence identification number:

17. Claim 4 In paragraph 3, an ADC in which a) HC is composed of sequence identification number: 2 and LC is composed of sequence identification number: 3; or b) HC is composed of sequence identification number: 16 and LC is composed of sequence identification number:

17. Claim 5 An ADC according to claim 1, further comprising a linker connecting the antibody to a cytotoxic agent, wherein the linker comprises a peptide unit of Ala-Ala-Ala or Gly-Gly-Phe-Gly (Sequence Identification No.: 102). Claim 6 In claim 5, the linker further comprises a spacer unit A between the antibody and the peptide unit, wherein the spacer unit A has the following chemical formula: ,where z is an ADC of 1 to 5. Claim 7 In claim 1, having the following chemical formula: ,where: Ab is an antibody, and n is an ADC ranging from 1 to 16. Claim 8 In claim 7, an ADC having the following chemical formula: . Claim 9 In claim 7, an ADC having the following chemical formula: . Claim 10 In paragraph 7, an ADC where n is 4. Claim 11 In paragraph 7, an ADC where n is 8. Claim 12 An ADC according to claim 7, wherein the linkage to the antibody is formed through one or more thiol groups on the cysteine ​​of the antibody. Claim 13 In paragraph 12, an ADC in which one or more cystes are each natural cystes within the hinge region of the antibody. Claim 14 In claim 7, Ab comprises a.) an HC containing amino acid 2-444 of sequence identification number: 2 and an LC containing amino acid 2-215 of sequence identification number: 3, or b.) an HC containing amino acid 2-443 of sequence identification number: 16 and an LC consisting of sequence identification number:

17. Claim 15 A pharmaceutical composition for use in treating cancer, comprising an effective amount of the ADC of claim 1. Claim 16 A composition according to claim 15, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. Claim 17 A composition according to claim 15, administered simultaneously, individually, or in sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor. Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete Claim 98 delete Claim 99 delete Claim 100 delete Claim 101 delete Claim 102 delete Claim 103 delete Claim 104 delete Claim 105 delete Claim 106 delete Claim 107 delete Claim 108 delete Claim 109 delete Claim 110 delete Claim 111 delete

Images