Anti-Nectin-4 antibody and multispecific antibody comprising same

Abstract

The present invention relates to isolated antibodies or antigen binding fragments that specifically bind to Nectin-4 protein and to multispecific antibodies comprising isolated antibodies or antigen binding fragments that specifically bind to Nectin-4 protein. The invention also relates to an isolated antibody or antigen binding fragment for coding the specific binding Nectin-4 protein, a nucleic acid of the multispecific antibody, and a host cell comprising the isolated antibody or antigen binding fragment for specifically binding Nectin-4 protein and the multispecific antibody, and a method for preparing the isolated antibody or antigen binding fragment that specifically binds to the Nectin-4 protein, and the multispecific antibody. Furthermore, the present invention relates to said isolated antibodies or antigen-binding fragments that specifically bind to the Nectin-4 protein, the diagnostic, prophylactic and / or therapeutic use of said multispecific antibodies.

Description

Anti-nectin-4 antibodies and multispecific antibodies containing the same Technical Field

[0001] The present invention generally relates to antibodies and their uses. More specifically, the present invention relates to isolated antibodies or antigen-binding fragments that specifically bind to nectin-4, and multispecific antibodies comprising the same. The present invention also relates to diagnostic, prophylactic, and / or therapeutic uses of isolated antibodies or antigen-binding fragments that specifically bind to nectin-4, and multispecific antibodies. Background Art

[0002] The first generation of immune checkpoint inhibitors (ICIs), represented by PD-1 monoclonal antibodies and / or PD-L1 monoclonal antibodies, has a limited response rate. Only a small number (10% to 30%) of cancer patients have a therapeutic effect after administration of PD-1 monoclonal antibodies and / or PD-L1 monoclonal antibodies (Lawrence P. Andrews et al. (2020) Sci Immunol. 5(49):eabc2728). Therefore, in recent years, how to improve the efficacy of ICIs through drug combinations and solve the problem of immunotherapy resistance has become a research hotspot in academia and the pharmaceutical industry.

[0003] Nectin-4 is a 66kDa type I transmembrane glycoprotein and a member of the nectin family of immunoglobulin-like adhesion molecules, which mediate cell-cell adhesion, recruit cadherins, and regulate cytoskeletal rearrangements. Nectin-4 is highly expressed in bladder cancer, breast cancer, lung cancer, pancreatic cancer, and ovarian cancer, while its expression in normal tissues is very low, making it an ideal tumor-associated antigen target (Pia M. Challita-Eid et al. (2016) Cancer Res. 76(10):3003-13.).

[0004] In 2020, J Immunother Cancer reported that Nectin-4 is a ligand of the immune checkpoint TIGIT, and Nectin-4 antibodies block the binding of Nectin-4 to TIGIT, thereby exerting the tumor-killing effect of Nectin-4 antibodies (Adi Reches et al. (2020) J Immunother Cancer. 8(1): e000266.).

[0005] PADCEV targeting nectin-4 TM(Also known as enfortumab vedotin-ejfv; Chinese name: Vitin-Enfortumab) was approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma, proving that nectin-4 is a good tumor-associated antigen. TM It is an ADC drug that targets Nectin-4 protein, a cell surface protein that is highly expressed in urothelial carcinoma. The ADC drug is composed of enfortumab, a human IgG1 monoclonal antibody targeting Nectin-4 protein, coupled with MMAE (monomethyl auristatin E, a microtubule disrupting agent) (Pia M. Challita-Eid et al. (2016) Cancer Res. 76 (10): 3003-13). But PADCEV TM Can cause serious skin adverse reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) (2019 FDA announcement).

[0006] 4-1BB, also known as CD137 or TNFRSF9, is a member of the tumor necrosis factor receptor family. Its most important feature is that it is a co-stimulatory molecule that regulates TCR-induced T cell activation on the surface of T cells. After 4-1BB binds to 4-1BBL (the 4-1BBL is the main natural ligand of 4-1BB expressed on activated antigen-presenting cells), it upregulates anti-apoptotic molecules and promotes the production of cytokines and effector functions. 4-1BB is also present on dendritic cells, activated monocytes, NK cells, neutrophils, eosinophils and mast cells, and it is reported that 4-1BB signaling stimulates IFN-γ secretion and promotes NK cell proliferation and dendritic cell activation (Cariad Chester et al. (2018) Blood 131(1): 49-57).

[0007] Preclinical results of agonistic monoclonal antibodies against 4-1BB in multiple induced and spontaneous tumor models have shown that targeting 4-1BB with agonistic antibodies can lead to tumor clearance and durable anti-tumor immune effects.

[0008] Clinical trials of two agonist antibodies, urelumab and utomilumab, have shown preliminary signs of efficacy, but inflammatory hepatotoxicity at doses >1 mg / kg has hindered the clinical development of urelumab. Utomilumab has a better safety profile but has lower 4-1BB agonist efficacy than urelumab (Cariad Chester et al. (2018) Blood 131(1):49-57).

[0009] To optimally utilize 4-1BB-mediated immune activation, the next generation of 4-1BB targeting strategies aims to enhance anti-tumor efficacy while minimizing hepatotoxicity. Attempts have been made to restrict the effects of 4-1BB agonists to the tumor microenvironment to reduce immune-related adverse events. 4-1BB is a potent target for tumor immunotherapy, showing great promise for robust immune activation (Cariad Chester et al. (2018) Blood 131(1):49-57).

[0010] Therefore, there is still a need in the art to develop novel antibodies that strike an optimal balance between safety and agonism. The present invention meets this need by providing anti-nectin-4 antibodies that bind to nectin-4 with high target specificity and high affinity, as well as bispecific antibodies comprising the anti-nectin-4 and an anti-4-1BB that binds to 4-1BB with low affinity.

[0011] Summary of the Invention

[0012] Through research, the present inventors have developed a novel group of isolated anti-nectin-4 antibodies that bind to nectin-4 with high affinity. The isolated antibodies or antigen-binding fragments of the present invention that specifically bind to nectin-4 have one or more of the following characteristics:

[0013] (a) having the ability to bind to human nectin-4 molecules expressed on cells and cynomolgus monkey nectin-4 molecules expressed on cells as measured by flow cytometry assay; and

[0014] (b) having the ability to specifically bind only to nectin-4 and not to nectin-1, nectin-2, and nectin-3, members of the nectin family, as measured by an ELISA assay; and / or

[0015] (c) less than about 10 nM, e.g., about 1 nM, about 10 nM, or less than about 10 nM, as measured in biofilm interferometry. -1 nM, about 10 -2 nM, about 10 -3 The binding and dissociation equilibrium constant K in nM D Bind to human Nectin-4 protein; and / or at a concentration of less than about 100 nM, such as about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 The binding and dissociation equilibrium constant K in nM D Binds to cynomolgus monkey nectin-4 protein.

[0016] Therefore, in a first aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein, comprising

[0017] (a) three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 1 and three CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or variants having a single or multiple CDRs with no more than three amino acid changes in each CDR region;

[0018] For example, the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 18 and the three CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NOs: 20-24; or

[0019] The three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 19 and the three CDRs in the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs: 20-24;

[0020] (b) three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 3 and three CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 4; or variants having a single or multiple CDRs with no more than three amino acid changes in each CDR region; or

[0021] (c) 3 CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5 and 3 CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 6; or a variant having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region.

[0022] In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to a nectin-4 protein, comprising a nucleotide sequence according to Kabat numbering.

[0023] (a) HCDR1 set forth in SEQ ID NO:40, or a variant of HCDR1 set forth in SEQ ID NO:40 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:41, or a variant of HCDR2 set forth in SEQ ID NO:41 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:42, or a variant of HCDR3 set forth in SEQ ID NO:42 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:43, or a variant of LCDR1 set forth in SEQ ID NO:43 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:44, or a variant of LCDR2 set forth in SEQ ID NO:44 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:45, or a variant of LCDR3 set forth in SEQ ID NO:45 with no more than two amino acid changes;

[0024] (b) HCDR1 set forth in SEQ ID NO:46, or a variant of HCDR1 set forth in SEQ ID NO:46 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:47, or a variant of HCDR2 set forth in SEQ ID NO:47 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:48, or a variant of HCDR3 set forth in SEQ ID NO:48 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:49, or a variant of LCDR1 set forth in SEQ ID NO:49 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:50, or a variant of LCDR2 set forth in SEQ ID NO:50 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:51, or a variant of LCDR3 set forth in SEQ ID NO:51 with no more than two amino acid changes; or

[0025] (c) HCDR1 set forth in SEQ ID NO:52, or a variant of HCDR1 set forth in SEQ ID NO:52 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:53, or a variant of HCDR2 set forth in SEQ ID NO:53 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:54, or a variant of HCDR3 set forth in SEQ ID NO:54 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:55, or a variant of LCDR1 set forth in SEQ ID NO:55 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:56, or a variant of LCDR2 set forth in SEQ ID NO:56 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:57, or a variant of LCDR3 set forth in SEQ ID NO:57 with no more than two amino acid changes.

[0026] Preferably, the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein comprises a

[0027] (a) HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45;

[0028] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:45;

[0029] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45;

[0030] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:62, and LCDR3 shown in SEQ ID NO:45;

[0031] HCDR1 set forth in SEQ ID NO:40, HCDR2 set forth in SEQ ID NO:58, and HCDR3 set forth in SEQ ID NO:42; LCDR1 set forth in SEQ ID NO:60, LCDR2 set forth in SEQ ID NO:62, and LCDR3 set forth in SEQ ID NO:45;

[0032] (b) HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:47, and HCDR3 shown in SEQ ID NO:48; LCDR1 shown in SEQ ID NO:49, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:51; or

[0033] (c) HCDR1 shown in SEQ ID NO: 52, HCDR2 shown in SEQ ID NO: 53, and HCDR3 shown in SEQ ID NO: 54; LCDR1 shown in SEQ ID NO: 55, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 57.

[0034] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein of the present invention comprises

[0035] (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 1, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 2, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0036] For example, the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2;

[0037] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 20;

[0038] The heavy chain variable region set forth in SEQ ID NO:18 and the light chain variable region set forth in SEQ ID NO:21;

[0039] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 22;

[0040] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 23;

[0041] The heavy chain variable region set forth in SEQ ID NO:18 and the light chain variable region set forth in SEQ ID NO:24;

[0042] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 20;

[0043] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 21;

[0044] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 22;

[0045] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 23;

[0046] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 24;

[0047] (b) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0048] For example, the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 4; or

[0049] (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:5, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:6, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0050] For example, the heavy chain variable region shown in SEQ ID NO: 5 and the light chain variable region shown in SEQ ID NO: 6.

[0051] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein of the present invention comprises

[0052] (a) SEQ ID NO: 11, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO: 12, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0053] For example, the heavy chain sequence shown in SEQ ID NO: 11, and the light chain sequence shown in SEQ ID NO: 12;

[0054] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:27;

[0055] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:28;

[0056] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:29;

[0057] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:30;

[0058] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:31;

[0059] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:27;

[0060] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:28;

[0061] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:29;

[0062] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:30;

[0063] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:31;

[0064] (b) SEQ ID NO: 13, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO: 14, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0065] (c) SEQ ID NO: 15, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO: 16, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0066] In some embodiments, the antigen-binding fragment of the isolated anti-nectin-4 antibody of the present invention is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab.

[0067] In some embodiments, the isolated antibody or antigen-binding fragment of the present invention that specifically binds to a Nectin-4 protein is a fully human antibody.

[0068] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the present invention is an IgG antibody; preferably, it is a human IgG antibody; more preferably, it is a human IgG1 or human IgG4 antibody.

[0069] In a second aspect, the present invention provides a multispecific antibody comprising an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein, for example, comprising an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein according to the first aspect of the present invention. Preferably, the multispecific antibody is a trispecific antibody or a bispecific antibody.

[0070] In some embodiments, the present invention provides a bispecific antibody comprising an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein and an isolated antibody or antigen-binding fragment that specifically binds to a T cell co-stimulatory receptor (e.g., ICOS, 4-1BB, CD28, or CD86). For example, the bispecific antibody comprises an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein according to the first aspect of the present invention and an isolated antibody or antigen-binding fragment that specifically binds to a T cell co-stimulatory receptor (e.g., ICOS, 4-1BB, CD28, or CD86).

[0071] In some embodiments, the present invention provides a bispecific antibody comprising, from amino terminus to carboxyl terminus:

[0072] (a) a first part, which is an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, for example, an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein according to the first aspect of the present invention, and

[0073] (b) a second portion, which is an isolated antibody or antigen-binding fragment that specifically binds to the T cell co-stimulatory receptor 4-1BB;

[0074] The antigen-binding fragments in the first part and the second part are Fab, Fab', F(ab')2, Fv, single-chain Fv, and single-chain Fab.

[0075] In some embodiments, the present invention provides a bispecific antibody, which is an anti-Nectin-4 / 4-1BB bispecific antibody, comprising:

[0076] (a) a first portion, which is an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein, and

[0077] (b) a second portion, which is an isolated antibody or antigen-binding fragment that specifically binds to the T cell co-stimulatory receptor 4-1BB; and

[0078] The bispecific antibody has high affinity for Nectin-4 and low affinity for 4-1BB, and targets the K D The K value of the target human 4-1BB protein is DThe value is two orders of magnitude higher, and its high affinity for human Nectin-4 can effectively reduce the side effects of its targeting of human 4-1BB. This anti-Nectin-4 / 4-1BB bispecific antibody, by targeting Nectin-4 and the co-stimulatory receptor 4-1BB, can not only block the binding of Nectin-4 to TIGIT, but also promote effective tumor-directed immune T cell activation and killing by binding to the T cell co-stimulatory receptor 4-1BB.

[0079] In some embodiments, the antigen-binding fragment of the isolated antibody of the present invention that specifically binds to 4-1BB is Fab, Fab', F(ab')2, Fv, single-chain Fv, or single-chain Fab.

[0080] In a third aspect, the present invention provides nucleic acids encoding the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein described in the first aspect, or encoding the multispecific antibody described in the second aspect, vectors (preferably, expression vectors) comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. In some embodiments, the host cells are prokaryotic or eukaryotic, for example, selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies, antigen-binding fragments, or multispecific antibodies. In some embodiments, the host cells are HEK 293 cells or CHO cells.

[0081] In a fourth aspect, the present invention provides a method for preparing the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein as described in the first aspect, or the multispecific antibody as described in the second aspect, the method comprising culturing the host cell of the present invention under conditions suitable for expressing a nucleic acid encoding the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein as described in the first aspect, or encoding the multispecific antibody as described in the second aspect, and optionally recovering the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein as described in the first aspect, or the multispecific antibody as described in the second aspect from the host cell or from the culture medium.

[0082] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein as described in the first aspect, or the multispecific antibody as described in the second aspect, and a pharmaceutically acceptable carrier.

[0083] In some embodiments, the pharmaceutical composition of the present invention comprises the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein as described in the first aspect above, or the multispecific antibody as described in the second aspect above, and comprises other therapeutic agents selected from oncolytic drugs, cytotoxic agents, cytokines, inhibitors of other immune checkpoint molecules (e.g., PD-1), and a pharmaceutically acceptable carrier.

[0084] In a sixth aspect, the present invention provides the use of the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein described in the first aspect, or the multispecific antibody described in the second aspect, for preparing a medicament for diagnosing, preventing, and / or treating a disease associated with overexpression of nectin-4, for example, cancer. In some embodiments, the cancer is a solid tumor or a hematological cancer, for example, selected from colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial cancer, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, and prostate cancer.

[0085] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective amount of an isolated antibody or antigen-binding fragment of the present invention that specifically binds to a nectin-4 protein, or a multispecific antibody of the present invention, or a nucleic acid of the present invention, or a vector of the present invention, or a host cell of the present invention, wherein the subject is a mammal; preferably, the subject is a human; wherein the disease is a disease associated with high expression of nectin-4, for example, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematological cancer, for example, the cancer is selected from colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial cancer, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, prostate cancer.

[0086] In an eighth aspect, the present invention provides a method for detecting the presence or level of Nectin-4 protein in a sample, comprising contacting the sample with an isolated antibody or antigen-binding fragment of the present invention that specifically binds to Nectin-4 protein under conditions that allow formation of a complex between the antibody or antigen-binding fragment and the Nectin-4 protein, and detecting the formation of the complex, for example, the detection method is a detection method for non-diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0088] FIG1A shows a graph showing the binding ability of murine chimeric anti-nectin-4 antibodies (ie, antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) to human nectin-4-expressing tumor cells HT-1376 at the cellular level.

[0089] FIG1B shows a graph showing the binding ability of murine chimeric anti-nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) to CHO-K1 / cynoNectin4 cells expressing cynomolgus monkey nectin-4 protein at the cellular level.

[0090] Figures 2A and 2B respectively show the binding ability detection graphs of murine chimeric anti-nectin-4 antibodies 23-H4F3 and 6-B9B6 to nectin family proteins Nectin-1, Nectin-2, Nectin-3 and Nectin-4.

[0091] Figures 3A and 3B respectively show the binding ability of humanized anti-nectin-4 antibodies (i.e., humanized antibodies huH4F3-1 to huH4F3-10) to tumor cells HT-1376 expressing human nectin-4 and to cells CHO-K1 / cynoNectin4 expressing cynomolgus monkey nectin-4 protein at the cellular level.

[0092] Figure 4A shows the activation of the NF-κB-Luc / 4-1BB reporter cell line by the anti-Nectin-4 × 4-1BB bispecific antibody in the presence of nectin-4-expressing tumor cells HT-1376. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the relative light unit (RLU) value.

[0093] Figure 4B shows the activation of the NF-κB-Luc / 4-1BB reporter cell line by the anti-Nectin-4 × 4-1BB bispecific antibody in the presence of 293T cells that do not express nectin-4. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the RLU value.

[0094] Figure 5 shows the changes in tumor volume after administration of the anti-Nectin-4×4-1BB bispecific antibody to tumor-bearing mice. The horizontal axis in the figure represents the number of days after the tumor-bearing mice were grouped.

[0095] Figure 6 shows the changes in body weight of tumor-bearing mice after administration of the anti-Nectin-4×4-1BB bispecific antibody. The horizontal axis in the figure represents the number of days after the tumor-bearing mice were grouped. DETAILED DESCRIPTION

[0096] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0097] I. Definition

[0098] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0099] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0100] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0101] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0102] The term "antibody" is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as long as they show the desired antigen-binding activity. Antibodies can be complete antibodies (e.g., having two full-length light chains and two full-length heavy chains) of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2). The monomer of a complete antibody is a tetrapeptide chain molecule formed by two full-length light chains and two full-length heavy chains connected by disulfide bonds, also referred to as a monomer of an Ig molecule. Antibody monomers are the basic structures that constitute antibodies.

[0103] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody or antigen-binding fragment thereof that specifically binds to a nectin-4 protein is substantially free of Abs that specifically bind to antigens other than the nectin-4 protein). In certain embodiments, the antibody is purified to a purity greater than 80% or 90%, preferably greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0104] "Epitope" or "antigenic determinant" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, called a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. An epitope may be formed by contiguous amino acids or by discontinuous amino acids joined by the tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding generally disappear upon treatment with denaturing solvents. An epitope typically comprises at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0105] The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than the intact or complete antibody, which is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), single-chain Fab, diabodies, single-domain antibodies (sdAb, nanobodies), camelid Ig, Ig NAR, F(ab)'3 fragment, bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, and disulfide-stabilized Fv proteins ("dsFv"). The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heterojunction antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. For more detailed descriptions, see also: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Journal of Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0106] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins comprising α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM), and disulfide bonds (between chains) are formed in the hinge. The heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increasing flexibility; the heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains," respectively. Each arm of the Y includes the variable region and the first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0107] The light chain variable region and the heavy chain variable region each comprise a "framework" region interspersed with three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" (used interchangeably herein with hypervariable regions "HVRs") are regions in the antibody variable domain that are highly variable in sequence and form structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR numbering systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (world wide web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0108] However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different numbering systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different numbering systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but due to the application of different schemes (e.g., different numbering system rules or combinations), the claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention.

[0109] The CDRs of the antibodies of the present invention can be manually assessed to determine their boundaries according to any protocol or combination thereof in the art. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0110] The sequences of the framework regions of different light or heavy chains are relatively conserved within species (e.g., humans). The framework region of an antibody (which is the combined framework region of the component light and heavy chains) is used to locate and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to the epitope of the antigen. Antibodies with different specificities (i.e., having different combination sites for different antigens) have different CDRs. Although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specificity determining residues (SDRs).

[0111] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes for a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0112] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, the two-chain Fv species is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight non-covalent association. In the single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can associate in a "dimer" structure similar to the two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen binding specificity to the antibody. Unless otherwise noted, as used herein, scFv can have a VL variable region and a VH variable region in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0113] Fab fragments contain the heavy and light chain variable domains and also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0114] The term "chimeric antibody" refers to (a) changing, replacing or exchanging the constant region or a portion thereof so that the antigen binding site is connected to a constant region of a different or changed class and / or species, etc.; or (b) changing, replacing or exchanging the variable region or a portion thereof with a variable region having a different or changed antigenic specificity. In some embodiments of the present invention, the constant region of a murine antibody is replaced with a constant region from a human immunoglobulin for modification. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original murine antibody.

[0115] "Humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in the humanized antibody correspond to the CDRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. Humanized antibodies optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to a humanized antibody. In some embodiments, the amino acid residues of the CDR of the humanized antibody are not precisely corresponding to the amino acid residues of the mouse CDR as a parent, for example, mutations are made at specific positions of the CDR to contact with a target antigen with high affinity.

[0116] "Fully human antibody" or "human antibody" refers to any antibody whose variable and constant region sequences are all human sequences, without any non-human antibody sequences. The term also includes antibodies produced recombinantly in non-human cells, which may have additional glycosylation not typical of human cells.

[0117] The term "multispecific antibody" refers to an antibody that has binding specificities for multiple different epitopes. The epitopes of a multispecific antibody can be derived from the same antigen or from multiple different antigens. The term "bispecific antibody" refers to an antibody that has binding specificities for two different epitopes. The epitopes of a bispecific antibody can be derived from the same antigen or from two different antigens.

[0118] The term "tumor antigen" encompasses tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). The term "tumor-associated antigen" (TAA) refers to proteins that are present on tumor cells, as well as proteins that are present on normal cells in selected organs during the fetal period (formerly embryonic antigens) and after birth, but at concentrations much lower than those in tumor cells. Tumor-associated antigens may also be present in the stroma near tumor cells, but are expressed in lower amounts in other stroma in the body. The term "tumor-specific antigen" (TSA) refers to antigens that are primarily present on tumor cells in mammalian subjects but are not typically found on normal cells in mammalian subjects.

[0119] The terms "nectin family" and "nectin family" are used interchangeably and are cell adhesion molecules that form physical connections between adjacent cells to enable intercellular communication, migration, and other important cellular processes. The nectin family includes at least four human nectin proteins: nectin-1, nectin-2, nectin-3, and nectin-4. Nectin-4 expression in healthy humans is primarily restricted to placental and embryonic tissues. Compared to healthy adult tissues, many types of tumor cells have high expression of nectin-4. Nectin-4 is a tumor-associated antigen.

[0120] The term "specific binding" or "binding" used when referring to an antigen and an antibody means that the antibody forms a complex with the antigen which is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2): p.270-278), ForteBio affinity assays (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8), etc.

[0121] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the association dissociation equilibrium constant (K D Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0122] As used herein, the term "variant" refers to a heavy chain variable region or light chain variable region that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions, wherein the modified antigen-binding protein comprising the heavy chain or light chain variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant heavy chain variable region or light chain variable region sequence retains 60%, 70%, 80%, 90%, or 100% of the biological characteristics of the antigen-binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. The antigen-binding proteins of the present disclosure comprise heavy chain variable region amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequences described herein. The antigen-binding proteins of the present disclosure include light chain variable region amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The percentage of homology can be over the entire heavy chain variable region and / or the entire light chain variable region, or the percentage homology can be limited to the framework region, and the sequence corresponding to the CDR has 100% identity with the CDR disclosed herein within the heavy chain variable region and / or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, such as 1, 2, or 3 amino acid substitutions, deletions, or additions, wherein the modified antigen-binding protein comprising the CDR variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, or 100% of the biological characteristics of the antigen-binding protein before modification. It will be understood that each CDR that can be modified can be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, particularly a conservative substitution.

[0123] "Polynucleotide" or "nucleic acid," used interchangeably herein, refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into a chain by DNA or RNA polymerase.

[0124] Calculation of sequence identity between sequences is performed as follows.

[0125] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0126] Mathematical algorithms can be utilized to realize the sequence comparison between two sequences and the calculation of percent identity. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (available at http: / / www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package is used, using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0127] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0128] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0129] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0130] The term "immune checkpoint" refers to a class of inhibitory signaling molecules present in the immune system that prevent tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). Studies have found that one of the reasons why tumor cells can evade the immune system in the body and proliferate uncontrollably is that they utilize the inhibitory signaling pathway of immune checkpoints, thereby inhibiting the activity of T lymphocytes, making it impossible for T lymphocytes to effectively exert their killing effect on tumors (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146). Immune checkpoint molecules include but are not limited to programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, TIM-3, and proteins with T cell immunoglobulin and ITIM domains (TIGIT).

[0131] The term "costimulatory molecules" and "costimulatory receptors" are used interchangeably, and refer to the corresponding binding partners on lymphocytes that specifically bind to costimulatory ligands to mediate the costimulatory response (such as but not limited to proliferation) of lymphocytes.Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens.Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activation of NK cell receptors, inducible co-stimulators (inducible co-stimulator, ICOS) (i.e. CD278), OX40, CD40, GITR, 4-1BB (i.e. CD137), CD27, CD28 and CD86. In some embodiments, "costimulatory molecules" are ICOS, 4-1BB, CD28 and / or CD86.

[0132] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0133] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably herein and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cell screened or selected in the initial transformed cell are included herein.

[0134] The present invention also relates to a method for producing a monoclonal antibody, which comprises culturing the host cell of the present invention to thereby produce the monoclonal antibody of the present invention.

[0135] As used herein, "subject," "individual," or "subject" refers to an animal, preferably a mammal, and more preferably a human, in need of alleviation, prevention, and / or treatment of a disease or condition, such as a viral infection. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects who have a disease or are at risk of having a disease. In the present invention, administering an antibody or pharmaceutical composition or preparation of the present invention to a subject in need thereof means administering an effective amount of the antibody, pharmaceutical composition, or preparation, etc.

[0136] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0137] II. Isolated anti-nectin-4 antibodies of the present invention

[0138] The present invention provides isolated antibodies against nectin-4 and antigen-binding fragments thereof. The protein or fragment thereof used as the sensitizing antigen for obtaining the isolated antibodies against nectin-4 of the present invention is preferably derived from a human nectin-4 protein or an extracellular portion thereof. The base sequence and amino acid sequence of human nectin-4 and its homologs can be obtained, for example, by accessing GenBank (NCBI, USA) (e.g., NCBI Accession No.: NP_112178.2). In one embodiment, the extracellular region of the human nectin-4 protein has the amino acid sequence shown in SEQ ID NO:39.

[0139] The terms "antibody that binds to Nectin-4 protein," "antibody that binds to Nectin-4," "anti-Nectin-4 protein antibody," "anti-Nectin-4 antibody," "isolated antibody that binds to Nectin-4 protein," and "Nectin-4 protein antibody" are used interchangeably herein to refer to antibodies of the present invention that are capable of binding to Nectin-4 protein with sufficient affinity such that the antibodies can be used as diagnostic, prophylactic, and / or therapeutic agents targeting Nectin-4 protein.

[0140] The isolated anti-nectin-4 antibodies and antigen-binding fragments of the present invention specifically bind to nectin-4 protein with high affinity, comprising

[0141] (a) three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 1 and three CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or variants having a single or multiple CDRs with no more than three amino acid changes in each CDR region;

[0142] For example, the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 18 and the three CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NOs: 20-24; or

[0143] The three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 19 and the three CDRs in the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs: 20-24;

[0144] (b) three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 3 and three CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 4; or variants having a single or multiple CDRs with no more than three amino acid changes in each CDR region; or

[0145] (c) three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5 and three CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO: 6; or variants having a single or multiple CDRs with no more than three amino acid changes in each CDR region;

[0146] The amino acid change is an addition, deletion or substitution of an amino acid, for example, the amino acid change is a conservative amino acid substitution.

[0147] There are several methods known in the art for determining the CDR sequences of a given antibody molecule. The CDR sequences from the antibody heavy and light chain variable regions can be determined according to any method known in the art, including but not limited to Kabat, Chothia, and IMGT numbering schemes or combinations thereof.

[0148] In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to a nectin-4 protein, comprising a nucleotide sequence according to Kabat numbering.

[0149] (a) HCDR1 set forth in SEQ ID NO:40, or a variant of HCDR1 set forth in SEQ ID NO:40 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:41, or a variant of HCDR2 set forth in SEQ ID NO:41 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:42, or a variant of HCDR3 set forth in SEQ ID NO:42 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:43, or a variant of LCDR1 set forth in SEQ ID NO:43 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:44, or a variant of LCDR2 set forth in SEQ ID NO:44 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:45, or a variant of LCDR3 set forth in SEQ ID NO:45 with no more than two amino acid changes;

[0150] (b) HCDR1 set forth in SEQ ID NO:46, or a variant of HCDR1 set forth in SEQ ID NO:46 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:47, or a variant of HCDR2 set forth in SEQ ID NO:47 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:48, or a variant of HCDR3 set forth in SEQ ID NO:48 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:49, or a variant of LCDR1 set forth in SEQ ID NO:49 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:50, or a variant of LCDR2 set forth in SEQ ID NO:50 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:51, or a variant of LCDR3 set forth in SEQ ID NO:51 with no more than two amino acid changes; or

[0151] (c) HCDR1 set forth in SEQ ID NO:52, or a variant of HCDR1 set forth in SEQ ID NO:52 with no more than two amino acid changes, HCDR2 set forth in SEQ ID NO:53, or a variant of HCDR2 set forth in SEQ ID NO:53 with no more than three amino acid changes, and HCDR3 set forth in SEQ ID NO:54, or a variant of HCDR3 set forth in SEQ ID NO:54 with no more than two amino acid changes; LCDR1 set forth in SEQ ID NO:55, or a variant of LCDR1 set forth in SEQ ID NO:55 with no more than three amino acid changes, LCDR2 set forth in SEQ ID NO:56, or a variant of LCDR2 set forth in SEQ ID NO:56 with no more than three amino acid changes, and LCDR3 set forth in SEQ ID NO:57, or a variant of LCDR3 set forth in SEQ ID NO:57 with no more than two amino acid changes.

[0152] Preferably, the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein comprises a

[0153] (a) HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45;

[0154] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:45;

[0155] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45;

[0156] HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:62, and LCDR3 shown in SEQ ID NO:45;

[0157] HCDR1 set forth in SEQ ID NO:40, HCDR2 set forth in SEQ ID NO:58, and HCDR3 set forth in SEQ ID NO:42; LCDR1 set forth in SEQ ID NO:60, LCDR2 set forth in SEQ ID NO:62, and LCDR3 set forth in SEQ ID NO:45;

[0158] (b) HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:47, and HCDR3 shown in SEQ ID NO:48; LCDR1 shown in SEQ ID NO:49, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:51; or

[0159] (c) HCDR1 shown in SEQ ID NO: 52, HCDR2 shown in SEQ ID NO: 53, and HCDR3 shown in SEQ ID NO: 54; LCDR1 shown in SEQ ID NO: 55, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 57.

[0160] In some embodiments, the isolated anti-nectin-4 protein antibody of the present invention binds to mammalian nectin-4 protein, such as human nectin-4 protein or monkey nectin-4 protein.

[0161] In some embodiments, the isolated anti-Nectin-4 protein antibodies of the present invention have one or more of the following characteristics:

[0162] (a) having the ability to bind to human nectin-4 molecules expressed on cells and cynomolgus monkey nectin-4 molecules expressed on cells as measured by flow cytometry assay; and

[0163] (b) having the ability to specifically bind only to Nectin-4 and not to Nectin 1, Nectin 2, and Nectin 3, members of the Nectin family, as measured by an ELISA assay; and / or

[0164] (c) less than about 10 nM, e.g., about 1 nM, about 10 nM, or less than about 10 nM, as measured in biofilm interferometry. -1 nM, about 10 -2 nM, about 10 -3 The binding and dissociation equilibrium constant K in nM D Bind to human Nectin-4 protein; and / or at a concentration of less than about 100 nM, such as about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 The binding and dissociation equilibrium constant K in nM D Binds to cynomolgus monkey nectin-4 protein.

[0165] In some embodiments, the isolated anti-nectin-4 protein antibody of the present invention specifically binds to the nectin-4 protein, thereby preventing the binding of the nectin-4 protein to the immune checkpoint molecule TIGIT, thereby inhibiting the signal transduction pathway of the immune checkpoint molecule TIGIT. In addition, the nectin-4 protein antibody of the present invention also has a direct effect on target cells expressing the nectin-4 protein. Therefore, the nectin-4 protein antibody of the present invention can enhance anti-tumor immunity by reversing the suppression of the immune system by TIGIT without being coupled to any toxin or anti-tumor agent. Therefore, the nectin-4 protein antibody of the present invention can block the interaction between nectin-4 on cancer cells and TIGIT on immune cells such as T and NK cells in cancer immunotherapy, thereby restoring the activity of immune cells such as T and NK cells.

[0166] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

[0167] (a) the heavy chain variable region comprises a sequence of SEQ ID NO: 1, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 2, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0168] For example, the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2;

[0169] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 20;

[0170] The heavy chain variable region set forth in SEQ ID NO:18 and the light chain variable region set forth in SEQ ID NO:21;

[0171] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 22;

[0172] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 23;

[0173] The heavy chain variable region set forth in SEQ ID NO:18 and the light chain variable region set forth in SEQ ID NO:24;

[0174] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 20;

[0175] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 21;

[0176] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 22;

[0177] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 23;

[0178] The heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 24;

[0179] (b) the heavy chain variable region comprises a sequence of SEQ ID NO: 3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0180] For example, the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 4; or

[0181] (c) the heavy chain variable region comprises a sequence of SEQ ID NO:5, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:6, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0182] For example, the heavy chain variable region shown in SEQ ID NO: 5 and the light chain variable region shown in SEQ ID NO: 6.

[0183] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention comprises

[0184] (a) SEQ ID NO: 11, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO: 12, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0185] For example, the heavy chain sequence shown in SEQ ID NO: 11, and the light chain sequence shown in SEQ ID NO: 12;

[0186] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:27;

[0187] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:28;

[0188] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:29;

[0189] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:30;

[0190] The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:31;

[0191] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:27;

[0192] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:28;

[0193] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:29;

[0194] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:30;

[0195] The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:31;

[0196] (b) SEQ ID NO: 13, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO: 14, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0197] (c) SEQ ID NO: 15, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO: 16, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0198] In some embodiments, the isolated anti-nectin-4 protein antibody of the present invention comprises an Fc region, and the Fc region is derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region is derived from human IgG1 or human IgG4.

[0199] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention comprises a constant region sequence selected from the group consisting of mouse IgG, human IgG, eg, human IgG1 or human IgG4.

[0200] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention is a chimeric antibody comprising a murine antibody variable region and a human antibody constant region.

[0201] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention is a humanized antibody.

[0202] In some embodiments, the amino acid changes present in the isolated anti-nectin-4 protein antibodies of the present invention include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another amino acid within the same class, for example, the substitution of an acidic amino acid with another acidic amino acid, the substitution of a basic amino acid with another basic amino acid, or the substitution of a neutral amino acid with another neutral amino acid.

[0203] In preferred embodiments, the amino acid changes described herein may also occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in the Fc region. In some embodiments, anti-nectin-4 protein antibodies are provided that include an Fc domain containing one or more mutations that enhance or diminish binding of the antibody to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention includes a CDR in the Fc domain. H 2 or C HAn anti-nectin-4 protein antibody comprising a mutation in region 3, wherein the one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modification at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or modification at position 250 and / or position 428; or modification at positions 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modification includes 428L (e.g., M428L) and 434S (e.g., N434S) modification; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modification; 433K (e.g., H433K) and 434 (e.g., 434Y) modification; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; 250Q and 428L modification (e.g., T250Q and M428L); and 307 and / or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification includes 265A (e.g., D265A) and / or 297A (e.g., N297A) modification.

[0204] For example, the present invention includes an anti-nectin-4 protein antibody comprising an Fc domain comprising one or more pairs (groups) of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). Any possible combination of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein is within the scope of the present invention.

[0205] In some embodiments, the isolated anti-nectin-4 protein antibodies provided herein are modified to increase or decrease their degree of glycosylation. Addition or deletion of glycosylation sites to the nectin-4 protein antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the nectin-4 protein antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the nectin-4 protein antibodies provided herein to generate Fc region variants, thereby enhancing the effectiveness of the isolated anti-nectin-4 protein antibodies of the present invention in diagnosing, preventing, and / or treating diseases.

[0206] In some embodiments, the isolated anti-nectin-4 protein antibody or antigen-binding fragment of the present invention can be used to conjugate with a drug to form an antibody-drug conjugate (ADC). The drug used for conjugation includes but is not limited to a chemotherapeutic agent, an immunomodulator (e.g., an immunopotentiator), an oligonucleotide, a toxin, a radiotoxin, a cytokine, an apoptotic agent, an anti-angiogenic agent, or a lymphokine.

[0207] In some embodiments, the drug conjugated to the isolated anti-nectin-4 protein antibody or antigen-binding fragment of the present invention is a cytotoxin.

[0208] In some embodiments, the isolated anti-nectin-4 protein antibodies or antigen-binding fragments of the present invention are conjugated to diagnostic or detectable agents. Such immunoconjugates can be used to monitor the progression, severity, and / or prognosis of a disease. Diagnosis and detection can be accomplished by coupling the antibody to a detectable substance, including but not limited to various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and positron-generating metals and non-radioactive paramagnetic metal ions using various positron emission tomography methods.

[0209] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention can be used as part of a multispecific antibody to prepare a multispecific antibody.

[0210] III. Multispecific Antibodies of the Present Invention

[0211] Although monoclonal antibodies have been established as anti-tumor therapeutics over the past two decades, their ability to mobilize immune cells, such as T cells and NK cells, at tumor sites and to enable immune cells to effectively exert cytotoxic activity is limited.

[0212] Because multispecific antibodies (e.g., bispecific antibodies) can specifically bind to different antigens, when one antigen is located on a specific immune cell and the other antigen is located on a disease cell, multispecific antibodies (e.g., bispecific antibodies) can redirect the specific immune cell to the disease cell, thereby enhancing the immune cell's ability to kill the disease cell. In addition, multispecific antibodies (e.g., bispecific antibodies) can also be designed to act simultaneously on the signal transduction pathways of two or more different mediators.

[0213] A large number of imaginative multispecific antibody (e.g., bispecific antibody) formats have been developed through antibody engineering and their applicability in disease applications has been investigated (Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9(2): 182-212).

[0214] Multispecific antibodies (e.g., bispecific antibodies) can be classified into many categories based on their components and construction methods. For example, based on the basic symmetry of the left and right sides of the multispecific antibody structure, they can be divided into symmetrical and asymmetrical structures; based on the presence or absence of the IgG Fc region, they can be divided into antibody formats with and without Fc regions; and based on the number of antigen-binding sites in the multispecific antibody, they can be divided into bivalent, trivalent, tetravalent, or higher-valent antibodies.

[0215] The present invention provides a multispecific antibody capable of simultaneously binding to molecules on immune cells and Nectin-4 proteins on tumor cells.

[0216] In some embodiments, the multispecific antibodies of the present invention include antibodies (e.g., anti-nectin-4 protein antibodies of the present invention) or antigen-binding fragments that specifically bind to nectin-4 protein and antibodies or antigen-binding fragments that bind to costimulatory molecules on immune cells. The costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and NK cell receptors. Examples of such molecules include, but are not limited to, CD27, CD28, 4-1BB (CD137), CD86, OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83.

[0217] In some exemplary embodiments of the present invention, the present invention provides bispecific antibodies comprising an antibody or antigen-binding fragment that specifically binds to a nectin-4 protein (e.g., an anti-nectin-4 protein antibody of the present invention) and an antibody or antigen-binding fragment that binds to a co-stimulatory molecule on an immune cell (e.g., an antibody or antigen-binding fragment that binds to a T cell co-stimulatory receptor such as ICOS, 4-1BB, CD28, or CD86).

[0218] The isolated anti-nectin-4 protein antibody or antigen-binding fragment of the present invention contained in the bispecific antibody of the present invention comprises at least a nectin-4 protein binding domain, i.e., at least a heavy chain variable domain and a light chain variable domain that specifically bind to the nectin-4 protein. The antibody or antigen-binding fragment that binds to a costimulatory molecule on an immune cell contained in the bispecific antibody of the present invention comprises at least a costimulatory molecule binding domain on an immune cell, i.e., at least a heavy chain variable domain and a light chain variable domain that specifically bind to a costimulatory molecule on an immune cell.

[0219] The Nectin-4 protein binding domain and the costimulatory molecule binding domain on the immune cell can be directly or indirectly connected to each other to form the bispecific antibody molecule of the present invention. Alternatively, the Nectin-4 protein binding domain and the costimulatory molecule binding domain on the immune cell can each be connected to a respective multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the Nectin-4 protein binding domain and the costimulatory molecule binding domain on the immune cell, thereby forming a bispecific antibody molecule. The multimerization domain can be a domain comprising immunoglobulin C.H 3 domain polypeptides, for example, the Fc portion of an immunoglobulin (including C H 2-C H 3 domains), e.g., an IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and an Fc domain of any isotype within each isotype group.

[0220] Any bispecific antibody format or technology can be used to prepare the bispecific antibodies of the present invention. Specific exemplary bispecific formats that can be used in the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusion, dual variable domains (OVO)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs, etc.), CrossMab, CrossFab, (SEEO) body, leucine zipper, Ouobody, IgG1 / lgG2, dual-action Fab (OAF)-lgG, and Mab 2 Bispecific formats (see, eg, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0221] In some embodiments, the bispecific antibodies of the invention comprise, from amino terminus to carboxyl terminus:

[0222] (a) a first part, which is an antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein, and

[0223] (b) a second part, which is an anti-4-1BB scFv,

[0224] For example, the anti-4-1BB scFv comprises 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 32 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 33; or a variant having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region with the above 6 CDR regions.

[0225] In some embodiments, the bispecific antibodies of the invention comprise, from amino terminus to carboxyl terminus:

[0226] (a) a first part, which is an antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein, and

[0227] (b) a second portion, which is an anti-4-1BB scFv, comprising HCDR1 shown in SEQ ID NO: 63, HCDR2 shown in SEQ ID NO: 64, and HCDR3 shown in SEQ ID NO: 65 according to Kabat numbering; LCDR1 shown in SEQ ID NO: 66, LCDR2 shown in SEQ ID NO: 67, and LCDR3 shown in SEQ ID NO: 68;

[0228] In some embodiments, the bispecific antibodies of the invention comprise, from amino terminus to carboxyl terminus:

[0229] (a) a first part, which is an antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein, and

[0230] (b) a second portion, which is an anti-4-1BB scFv, comprising a heavy chain variable region set forth in SEQ ID NO: 32, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region set forth in SEQ ID NO: 33, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0231] wherein the carboxyl terminus of the heavy chain of the first portion is covalently linked to the amino terminus of the second portion.

[0232] In some embodiments, the bispecific antibodies of the present invention comprise two heavy chains and two light chains, wherein each heavy chain comprises, from amino terminus to carboxyl terminus, a heavy chain of a first portion, a connecting peptide, and a second portion, and each light chain is a light chain of the first portion, preferably, each heavy chain comprises the amino acid sequence of SEQ ID NO: 35, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereof; and each light chain comprises the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereof;

[0233] each heavy chain comprises the amino acid sequence of SEQ ID NO:36, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; and each light chain comprises the amino acid sequence of SEQ ID NO:16, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; or

[0234] Each heavy chain comprises the amino acid sequence of SEQ ID NO:37, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; and each light chain comprises the amino acid sequence of SEQ ID NO:31, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0235] The type of the connecting peptide is not particularly limited. In an embodiment, the connecting peptide is a peptide having an amino acid sequence of 1 to 100, particularly 1 to 50, more particularly 1 to 20 amino acids in length. In some embodiments, the peptide connecting peptide is (GxS)n or (GxS)nGm, wherein G=glycine, S=serine and x=any integer in 1-4, n=any integer in 1-7, and m=any integer in 0-3. In a specific embodiment, the connecting peptide is (G4S)3 (SEQ ID NO: 69).

[0236] In some embodiments, the anti-nectin-4×4-1BB bispecific antibodies of the present invention have one or more of the following properties:

[0237] (a) measured in biofilm interferometry at a concentration of less than about 10 nM, e.g., about 1 nM, about 10 -1 nM, about 10 -2 nM, about 10 -3 The binding and dissociation equilibrium constant K in nM D Binds to human nectin-4 protein, and at a concentration of less than about 1000 nM, such as about 100 nM, about 10 nM, about 1 nM, about 10 -1 The binding and dissociation equilibrium constant K in nM D Bind to human 4-1BB protein; or at a concentration of less than about 100 nM, such as about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 The binding and dissociation equilibrium constant K in nMD Binds to cynomolgus monkey nectin-4 protein, and has a binding dissociation equilibrium constant K of less than about 150 nM, such as about 15 nM, about 1.5 nM, about 0.15 nM, about 0.015 nM D Binds to cynomolgus monkey 4-1BB protein;

[0238] (b) as measured in an NF-κB-Luc / 4-1BB reporter gene cell line, the bispecific antibody can activate the human 4-1BB signaling pathway only in the presence of Nectin-4 protein; and the bispecific antibody cannot activate the human 4-1BB signaling pathway in the absence of Nectin 4 protein; and / or

[0239] (c) having anti-tumor efficacy in vivo, and the bispecific antibody is safe in test animals.

[0240] In the bispecific antibodies of the invention, the Fc domain may comprise one or more amino acid changes (eg, insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of the Fc domain.

[0241] In some embodiments, the present invention provides bispecific antibodies comprising an Fc domain comprising one or more mutations that enhance or diminish binding of the bispecific antibody to an FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention includes a C-terminal region of the Fc domain comprising one or more mutations that enhance or diminish binding of the bispecific antibody to an FcRn receptor, for example, at acidic pH compared to neutral pH. H 2 or C HA bispecific antibody comprising a mutation in region 3, wherein the one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH in the range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modification at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or modification at position 250 and / or position 428; or modification at positions 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modification includes 428L (e.g., M428L) and 434S (e.g., N434S) modification; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modification; 433K (e.g., H433K) and 434 (e.g., 434Y) modification; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; 250Q and 428L modification (e.g., T250Q and M428L); and 307 and / or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification includes 265A (e.g., D265A) and / or 297A (e.g., N297A) modification.

[0242] For example, the present invention includes bispecific antibodies comprising an Fc domain comprising one or more pairs (sets) of mutations selected from: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). Any possible combination of the aforementioned Fc domain mutations and other mutations within the antibody variable domain disclosed herein is within the scope of the present invention.

[0243] In some embodiments, the bispecific antibodies provided herein are modified to increase or decrease the degree of their glycosylation. Addition or deletion of the glycosylation sites of the bispecific antibodies can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the bispecific antibody comprises an Fc region, the carbohydrate connected to the Fc region can be changed. In some applications, it can be useful to remove the modification of unwanted glycosylation sites, for example, to remove the fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosidation can be performed to regulate complement-dependent cytotoxicity (CDC).

[0244] IV. Nucleic acids of the present invention and host cells containing the same

[0245] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned Nectin-4 protein antibodies or antigen-binding fragments thereof or any one chain thereof.

[0246] In yet another aspect, the present invention provides a nucleic acid encoding any of the above multispecific antibodies of the present invention or any one chain thereof.

[0247] In one embodiment, a vector comprising the nucleic acid described above is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or HEK 293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0248] The present invention also encompasses nucleic acids that hybridize to the above-described nucleic acids under stringent conditions or nucleic acids encoding polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions, or insertions compared to the above-described nucleic acids.

[0249] In one embodiment, one or more vectors comprising the nucleic acid described above are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0250] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in culture medium and screened for suitable activity. Methods and conditions for cultivating the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, according to the specific expression vector and mammalian host cell used, change or optimize.

[0251] In addition, can allow to select one or more markers of transfected host cell by introducing, select the cell that stablizes DNA and is incorporated into its chromosome.Marker can for example provide prototrophy, biocidal resistance (for example, antibiotic) or heavy metal (such as copper) resistance etc. to auxotrophic host.Selectable marker gene can be directly connected with dna sequence dna to be expressed or be introduced into identical cell by cotransformation.Also may need extra element so that best synthetic mRNA.These elements can comprise splicing signal, and transcription promoter, enhancer and termination signal.

[0252] In one embodiment, a host cell comprising a polynucleotide of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NSO, P3X63 and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a HEK 293 cell or a CHO cell, for example, a HEK 293-6E cell.

[0253] V. Production and purification of isolated anti-nectin-4 protein antibodies and multispecific antibodies containing the same of the present invention

[0254] In one embodiment, the present invention provides a method for preparing an isolated anti-Nectin-4 protein antibody or a multispecific antibody comprising the same, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the isolated anti-Nectin-4 protein antibody or the multispecific antibody, or an expression vector comprising the nucleic acid, under conditions suitable for expressing the nucleic acid encoding the anti-Nectin-4 protein antibody or the multispecific antibody, and optionally isolating the anti-Nectin-4 protein antibody or the multispecific antibody. In certain embodiments, the method further comprises recovering the anti-Nectin-4 protein antibody or the multispecific antibody from the host cell (or host cell culture medium).

[0255] To recombinantly produce the isolated anti-nectin-4 protein antibody or multispecific antibody of the present invention, nucleic acid encoding the nectin-4 protein antibody or multispecific antibody of the present invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that specifically bind to the nucleic acid encoding the nectin-4 protein antibody or multispecific antibody of the present invention.

[0256] The antibodies or multispecific antibodies of the present invention prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, as will be apparent to those skilled in the art. The purity of the antibodies or multispecific antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0257] VI. Activity Assays for the Isolated Anti-Nectin-4 Protein Antibodies or Antigen-Binding Fragments thereof or Multispecific Antibodies Comprising the Same of the Present Invention

[0258] Nectin-4 protein antibodies or multispecific antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0259] In one aspect, the nectin-4 protein antibodies or multispecific antibodies of the present invention are tested for antigen-binding activity, for example, by known methods such as ELISA. Binding to each antigen directed by the nectin-4 protein and / or multispecific antibody can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, binding of the nectin-4 protein antibodies or multispecific antibodies of the present invention to the nectin-4 protein is determined using SPR or biofilm interferometry.

[0260] The present invention also provides an assay for identifying multispecific antibodies with biological activity. The biological activity can include, for example, measuring activation of the 4-1BB signaling pathway by the anti-nectin-4×4-1BB bispecific antibody using a HEK293 / NF-κB-Luc / 4-1BB reporter cell line.

[0261] VII. Pharmaceutical Compositions and Pharmaceutical Formulations

[0262] In some embodiments, the present invention provides compositions comprising any of the isolated anti-nectin-4 protein antibodies or multispecific antibodies described herein, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a combination of the nectin-4 protein antibody or multispecific antibody of the present invention and one or more other therapeutic agents (e.g., a chemotherapeutic drug, a tumor vaccine, an antibody that binds to a specific antigen on a tumor cell, an antibody that depletes a tumor cell).

[0263] In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the present invention comprises suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0264] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical formulations comprising the isolated anti-nectin-4 protein antibody or multispecific antibody described herein can be prepared by mixing the isolated anti-nectin-4 protein antibody or multispecific antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution.

[0265] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, as required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. When used to treat cancer, such active ingredients include, but are not limited to, anticancer agents and chemotherapeutic agents; when used to treat infectious diseases, such active ingredients include, but are not limited to, antiviral agents and antibiotics. The active ingredients are suitably combined in amounts effective for the intended use.

[0266] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the Nectin-4 protein antibody or multispecific antibody of the invention, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0267] VIII. Combination Products or Kits

[0268] In some embodiments, the present invention also provides a combination product comprising at least one isolated anti-nectin-4 protein antibody or multispecific antibody of the present invention, or further comprising one or more other anti-tumor agents.

[0269] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately, or simultaneously.

[0270] In some embodiments, the present invention also provides a kit comprising the isolated anti-nectin-4 protein antibody, multispecific antibody, pharmaceutical composition or combination product of the present invention, and optionally a package insert for instructions for administration.

[0271] In some embodiments, the present invention also provides a pharmaceutical product comprising the isolated anti-Nectin-4 protein antibody, multispecific antibody, pharmaceutical composition, or combination product of the present invention, optionally further comprising a package insert for guiding administration.

[0272] IX. Uses of the Isolated Anti-Nectin-4 Protein Antibody and Multispecific Antibody Containing the Same of the Present Invention

[0273] The isolated anti-nectin-4 protein antibodies or multispecific antibodies comprising the same disclosed herein have in vitro and in vivo diagnostic uses as well as therapeutic and prophylactic uses. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, e.g., a human subject, to treat, prevent, and / or diagnose a disease associated with the nectin-4 protein antigen, such as cancer.

[0274] In one aspect, the present invention provides a diagnostic method for detecting the presence of a relevant nectin-4 protein antigen in a biological sample, such as serum, semen, or urine, or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally, a control sample) with an antibody or multispecific antibody to a nectin-4 protein as described herein, or administering the antibody or multispecific antibody to a subject, under conditions permissive for interaction; and (ii) detecting the formation of a complex between the antibody or multispecific antibody to a nectin-4 protein and the sample (and optionally, a control sample). The formation of a complex indicates the presence of the relevant antigen and can indicate the suitability or need for a treatment and / or prevention as described herein.

[0275] In some embodiments, the relevant antigen is detected before treatment, for example, before the initiation of treatment or before a treatment after a treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (e.g., RT-PCR), or in vivo imaging techniques. Generally, the nectin-4 protein antibodies or multispecific antibodies used in in vivo and in vitro detection methods are directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound conjugates. Suitable detectable substances include a variety of biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (e.g., nuclear magnetic resonance active) substances, and radioactive substances.

[0276] In some embodiments, the level and / or distribution of the relevant antigen is determined in vivo, for example, non-invasively (e.g., by detecting a detectably labeled nectin-4 protein antibody or multispecific antibody of the invention using a suitable imaging technique (e.g., positron emission tomography (PET) scanning). In one embodiment, for example, by detecting a nectin-4 protein antibody or multispecific antibody of the invention using a PET agent (e.g., 18 The level and / or distribution of the relevant antigen can be determined in vivo using an antibody or multispecific antibody of the present invention that is detectably labeled with F-fluorodeoxyglucose (FDG).

[0277] In one embodiment, the present invention provides a diagnostic kit comprising the Nectin-4 protein antibody or multispecific antibody described herein and instructions for use.

[0278] In another aspect, the present invention relates to the use of the nectin-4 protein antibody or multispecific antibody of the present invention for treating or preventing cancer in vivo and modulating the immune response in a subject, thereby inhibiting or reducing the occurrence or recurrence of related diseases, such as cancer. The nectin-4 protein antibody or multispecific antibody of the present invention can be used alone. Alternatively, the nectin-4 protein antibody or multispecific antibody can be administered in combination with other cancer therapeutic / preventive agents. When the nectin-4 protein antibody or multispecific antibody of the present invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.

[0279] Therefore, in one embodiment, the present invention provides a method for treating or preventing cancer and regulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or multispecific antibody against a nectin-4 protein as described herein. In another embodiment, the present invention provides a method for preventing the occurrence or recurrence of cancer in a subject, comprising administering to the subject a prophylactically effective amount of an antibody or multispecific antibody against a nectin-4 protein as described herein.

[0280] In some embodiments, cancers treated and / or prevented using antibodies or multispecific antibodies to the Nectin-4 protein include, but are not limited to, solid tumors expressing the Nectin-4 protein, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma). In some embodiments, cancers treated and / or prevented using antibodies or multispecific antibodies to the Nectin-4 protein include, but are not limited to, carcinoma in situ and metastatic cancer expressing the Nectin-4 protein.

[0281] In one embodiment, the cancer is a solid tumor that expresses the Nectin-4 protein. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas of multiple organ systems, such as those affecting the esophagus, lung, breast, ovary, lymphoid, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma), and pancreas, as well as adenocarcinomas, including malignancies. The cancer may be in the early, middle, or late stages or be metastatic.

[0282] In some embodiments, the cancer is selected from colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial cancer, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, prostate cancer.

[0283] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0284] Example

[0285] Example 1. Generation of anti-human nectin-4 antibodies

[0286] The anti-human nectin-4 antibody hybridoma of the present invention can be prepared by the method described below. However, the method for preparing the anti-human nectin-4 antibody of the present invention is not limited to this method and can also be prepared by other methods known in the art.

[0287] 1.1 Animal immunization

[0288] Human Nectin-4-his protein (ACRO Biosystems, catalog number: NE4-H52H3) was used as an immunogen to immunize 5 BALB / c mice. The BALB / c mice were immunized once every two weeks, for a total of four times (Table 1). Venous blood was collected from the mice 7 days after the second immunization and 7 days after the third immunization, and the antibody titer in the serum of the immunized animals was detected by ELISA using human Nectin-4-his protein. After the antibody titer was observed to have risen sufficiently, a final booster immunization was performed. Finally, two immunized mice were selected to obtain antibody-producing cells. Four days after the last booster immunization, the spleens of the immunized mice were taken, and the lymphocytes isolated from the spleens were fused with myeloma cells to produce hybridomas.

[0289] Table 1. Immunization schedule for BALB / c mice

[0290] 1.2 Screening of target hybridomas

[0291] Cell culture supernatants from 4,800 hybridomas from Example 1.1 were screened using an ELISA assay for human nectin-4-his protein. Following the ELISA assay, 198 positive supernatants were selected for flow cytometry analysis against the MCF-7 cell line (a human breast cancer cell line). Hybridoma clones corresponding to positive supernatants that bound to MCF-7 cells were confirmed by a subsequent ELISA assay. Finally, 21 hybridoma clones were selected for the next subcloning stage.

[0292] 1.3 Subcloning of parental hybridomas

[0293] The selected hybridoma cell line (parent clone) was subcloned to ensure monoclonality. Subcloning was performed by re-plating the parent clone using a single-step cloning system. The 21 subclones from Example 1.2 were transferred to a 96-well cell culture plate. The subclones were screened by ELISA using human nectin-4 (ACRO, Catalog No.: NE4-H52H3) and cynomolgus monkey nectin-4 (KACTUS, Catalog No.: NEC-CM104) as antigens. Hybridomas that produced antibodies that bound to human nectin-4 and cynomolgus monkey nectin-4 were obtained. The antibodies produced were designated as: mouse antibody 23-H4F3, mouse antibody 6-B9B6, and mouse antibody 32-A12C7, respectively.

[0294] 1.4 Sequencing of anti-nectin-4 antibodies produced by hybridomas

[0295] Antibody sequencing was completed by GenScript NGS sequencing. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) are shown in Table 2.

[0296] Table 2: Amino acid sequences of heavy chain variable regions and light chain variable regions of anti-nectin-4 antibodies

[0297] Example 2: Characterization of anti-Nectin-4 chimeric antibodies

[0298] 2.1 Synthesis and expression of anti-nectin-4 chimeric antibody

[0299] The DNA sequences of the murine antibodies listed in Table 2 were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The genes encoding the VH regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 7) to obtain plasmids encoding the heavy chain of each anti-nectin-4 antibody. The genes encoding the VL regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human κ light chain constant region (SEQ ID NO: 8) to obtain plasmids encoding the light chain of each anti-nectin-4 antibody.

[0300] Plasmids encoding the heavy and light chains of each anti-nectin-4 antibody were co-transfected into ExpiCHO-S cells to express the murine chimeric antibodies shown in Table 3. Enfortuzumab (a fully human monoclonal antibody against nectin-4) (heavy chain amino acid sequence: SEQ ID NO: 9, light chain amino acid sequence: SEQ ID NO: 10) was used as a control antibody (PCT Publication No. WO2021030240A1). Similarly, the control antibody, Enfortuzumab, was expressed. The antibodies were purified accordingly.

[0301] Specifically, use ExpiCHO according to the manufacturer's product manual. TM ExpiCHO-S cells were co-transfected with the plasmids encoding the heavy and light chains of the anti-nectin-4 antibodies listed in Table 3 using the Expression System (ThermoFisher, Cat# A29133) to express the anti-nectin-4 chimeric antibodies. Similarly, a control antibody, Enfortumab, was expressed. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the expressed and secreted antibodies in the supernatant were purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibodies were concentrated and sterile filtered. The purity of the antibody protein was determined by SDS-PAGE and size exclusion. The results showed that the antibody purity was >90%, indicating that it was suitable for use in further experiments.

[0302] Table 3: Chimeric antibody names and corresponding heavy chain and light chain combinations

[0303] 2.2 Anti-Nectin-4 Chimeric Antibody Binds to HT-1376 or Cynomolgus Monkey Nectin-4 Engineered Cells

[0304] Cell binding experiments were performed to determine whether the anti-nectin-4 chimeric antibody of the present invention could bind to human nectin-4 protein expressed by tumor cells (eg, human bladder cancer cell line HT-1376) or engineered cells expressing cynomolgus monkey nectin-4 protein.

[0305] The extracellular and transmembrane regions encoding cynomolgus monkey nectin-4 (XP_005541277.1) (SEQ ID NO: 17) were cloned into the PiggyBac Dual promoter (SBI, Catalog No.: PB513-B1) expression vector and transfected into CHO-K1 cells (ATCC, Catalog No.: CCL-61) by electroporation. TM ), and then screened with 8 μg / mL puromycin (Gibco, catalog number: A1113802) to obtain CHO-K1 cells that highly express cynomolgus monkey nectin-4 (hereinafter also referred to as: CHO-K1 / cynoNectin4).

[0306] Adherent HT-1376 (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60310) or CHO-K1 / cynoNectin4 cell lines were enzymatically digested to obtain single-cell suspensions of both cells. The suspensions were centrifuged at 300 × g for 4 min at room temperature, and the culture medium was discarded. The resulting cell pellets were washed once with PBS. The cells were resuspended in a serial dilution of the mouse chimeric anti-nectin-4 antibody obtained in Example 2.1 (initial concentration: 200 nM, 4-fold serial dilutions, a total of 8 concentrations) and incubated at 4°C for 30 min. The cells were washed once with PBS, and then a 1:200 dilution of the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson ImmunoResearch, Cat# 109-116-098) was added. The cells were incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS and resuspended. Finally, the fluorescence signal in two channels was detected by flow cytometry. The results are shown in Figure 1A and Figure 1B.

[0307] Figure 1A shows that murine chimeric anti-nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) can all bind to HT-1376 cells expressing human nectin-4, with the EC50 of antibody 23-H4F3 being 0.06405 nM; the EC50 of antibody 6-B9B6 being 0.2489 nM; and the EC50 of antibody 32-A12C7 being 0.2998 nM. Figure 1B shows that murine chimeric anti-nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) can also bind to cells expressing cynomolgus monkey nectin-4 protein. The EC50 for antibody 23-H4F3 is 0.4113 nM; the EC50 for antibody 6-B9B6 is 0.7186 nM; and the EC50 for antibody 32-A12C7 is 1.053 nM. The chimeric antibody 23-H4F3 has superior binding to each cell type compared to antibodies 6-B9B6 and 32-A12C7.

[0308] 2.3 Binding of anti-nectin-4 antibodies to the nectin family

[0309] The ELISA method was used to detect the binding properties of the anti-nectin-4 antibody obtained in Example 2.1 to nectin family members.

[0310] Human nectin-1 protein (ACRO Biosystems, Catalog No. PV1-H5223), human nectin-2 protein (Sino Biological, Catalog No. 10005-H08H), human nectin-3 protein (Sino Biological, Catalog No. 10852-H08H), and human nectin-4 protein (Sino Biological, Catalog No. HPLC-19771-H08H) were immobilized onto 96-well plates by incubation overnight at 4°C. The 96-well plates were then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, the 96-well plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-nectin-4 chimeric antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6) were prepared in binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with each nectin family protein immobilized on a 96-well plate for 1 hour at 37° C. After incubation, the 96-well plate was washed three times with PBST, incubated with peroxidase-labeled goat anti-human Fc IgG (Jackson Immuno Research, Cat. No. 109-035-098) diluted 1 / 25,000 in binding buffer at 37° C. for 1 hour, washed again, developed with TMB, and stopped using 1 M H 2 SO 4 .

[0311] Representative binding curves of antibody 23-H4F3 and antibody 6-B9B6 binding to human nectin family proteins are shown in Figures 2A and 2B, respectively.

[0312] As can be seen from Figures 2A and 2B, antibody 23-H4F3 and antibody 6-B9B6 specifically bind only to human nectin-4, and do not bind to nectin-1, nectin-2, and nectin-3.

[0313] Example 3: Humanization and characterization of murine anti-nectin-4 antibodies

[0314] 3.1 Humanization of mouse anti-nectin-4 antibody

[0315] Antibody 23-H4F3 obtained in Example 2.1 was humanized. Specifically, the sequence of antibody 23-H4F3 was searched and aligned in the IMGT database. The human germline gene sequence IGHV1-3*01, which showed high homology to the heavy chain variable region of antibody 23-H4F3, was obtained as the humanized framework for the heavy chain variable region. The human germline gene sequences IGKV1-6*01 and IGKV4-1*01, which showed high homology to the light chain variable region, were used as the humanized framework for the light chain variable region. The CDRs in the heavy and light chain variable regions of antibody 23-H4F3 were transplanted into the corresponding humanized frameworks to form the humanized variable regions of antibody 23-H4F3. To maintain the affinity of the humanized antibody for nectin-4, the obtained humanized antibody variable regions were backmutated.

[0316] The sequences of the humanized heavy chain variable region and humanized light chain variable region were obtained. The variable region amino acid sequences were sent to Universal Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis. The gene encoding the humanized VH region was inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 7) to obtain a plasmid encoding the full-length heavy chain of the anti-nectin-4 humanized antibody; the gene encoding the humanized VL region was inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human kappa light chain constant region (SEQ ID NO: 8) to obtain a plasmid encoding the full-length light chain of the anti-nectin-4 humanized antibody. Table 4 lists the names and heavy chain variable region sequences of each heavy chain, as well as the names and light chain variable region sequences of each light chain after humanization of the murine antibody 23-H4F3.

[0317] Table 4. Names of heavy and light chains and variable region sequences of humanized antibodies

[0318] 3.2 Expression and purification of humanized anti-nectin-4 antibody

[0319] Similar to Example 2.1, plasmids encoding the full-length heavy chain of the anti-nectin-4 humanized antibody and plasmids encoding the full-length light chain of the anti-nectin-4 humanized antibody were combined (Table 5) and co-transfected into ExpiCHO-S cells to express each anti-nectin-4 humanized antibody. Purification was then performed as described in Example 2.1. The purified anti-nectin-4 humanized antibody was concentrated and sterile filtered, and the purity of the anti-nectin-4 humanized antibody was determined by SDS-PAGE and size exclusion chromatography (SEC).

[0320] Table 5: Humanized antibody expression combination

[0321] 3.3 Physicochemical Analysis of Humanized Anti-Nectin-4 Antibodies

[0322] For the obtained humanized anti-nectin-4 antibodies, the purity of each antibody in Table 5 was confirmed by size exclusion chromatography. Specifically, 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) was used as the running buffer, and 20 μg of each humanized anti-nectin-4 antibody sample in Table 5 was injected into the TSK G3000SWXL column. Run for 30 minutes. The collected effluent was measured using Agilent 1220HPLC, and the data was analyzed using OpenLAB software. The results showed that the purity of each humanized anti-nectin-4 antibody in Table 5 was >90%, and it could be used for the next experiment.

[0323] 3.4 Binding of humanized anti-nectin-4 antibodies to tumor cell lines and engineered cells expressing cynomolgus monkey nectin-4

[0324] Cell binding assays were performed to determine whether the humanized anti-nectin-4 antibodies of the present invention could bind to stably expressed human or cynomolgus macaque nectin-4 proteins on the cell surface. The specific assay method was as described in Example 2.2, except that the antibodies used were the humanized anti-nectin-4 antibodies listed in Table 5. The results are shown in Figures 3A and 3B.

[0325] Figures 3A and 3B respectively show that each anti-nectin-4 humanized antibody in Table 5 can bind to HT-1376 cells and cells expressing cynomolgus monkey nectin-4 protein, and the binding ability of each anti-nectin-4 humanized antibody is comparable to that of the control antibody Enfortuzumab.

[0326] Example 4 Preparation and Affinity Detection of Anti-Nectin-4×4-1BB Bispecific Antibody

[0327] 4.1 Bispecific Antibody Construction

[0328] This example constructs an anti-Nectin-4×4-1BB bispecific antibody, wherein the anti-Nectin-4 portion is derived from the chimeric antibodies 23-H4F3, 32-A12C7 of Example 2.1 or the humanized antibody huH4F3-5 of Example 3.2, and the anti-4-1BB portion has the heavy chain variable region shown in SEQ ID NO: 32 and the light chain variable region shown in SEQ ID NO: 33. This application uses standard construction methods to obtain the anti-Nectin-4×4-1BB bispecific antibody, the amino acid sequence of which is listed in the sequence listing, which, from N-terminus to C-terminus, comprises a sequence derived from an anti-Nectin-4 antibody and a single-chain antibody portion of anti-4-1BB connected to the C-terminus of the Fc region of the anti-Nectin-4 antibody.

[0329] Specifically, an anti-nectin-4×4-1BB bispecific antibody molecule was constructed as follows.

[0330] Anti-Nectin-4×4-1BB bispecific antibody heavy chain: The nucleotide sequence encoding the anti-Nectin-4 antibody heavy chain variable region was amplified and cloned into the pcDNA3.1(+) vector containing the human IgG1 mutant heavy chain constant region (SEQ ID NO: 34); using antibody P4B-3 (see CN114555638A) as a template, anti-4-1BB VH and VL were amplified, and the nucleotide sequence encoding the anti-4-1BB ScFv (i.e., VH-(G4S)3 linker-VL) was linked to the C-terminus of the heavy chain constant region of the pcDNA3.1(+) vector containing the nucleotide sequence encoding the anti-Nectin-4 antibody heavy chain variable region and the human IgG1 mutant heavy chain constant region (SEQ ID NO: 34), and the terminal lysine of Fc was replaced with alanine to prevent breakage substitution. An expression vector for the anti-nectin-4×4-1BB heavy chain was obtained, wherein the anti-nectin-4×4-1BB heavy chain includes, from N-terminus to C-terminus, an anti-nectin-4 heavy chain (with the terminal K mutated to A) and an anti-4-1BB ScFv. The heavy chains of the chimeric bispecific antibodies H4F3-41BB heavy chain (SEQ ID NO: 35), A12C7-41BB heavy chain (SEQ ID NO: 36), and the humanized bispecific antibody huH4F3-5-41BB heavy chain (SEQ ID NO: 37) were obtained.

[0331] Bispecific antibody light chain: The corresponding light chain of the bispecific antibody is shown in Table 6

[0332] Table 6. Chimeric / humanized bispecific antibody expression

[0333] The obtained heavy chain and light chain expression vectors (plasmids containing H4F3-41BB / A12C7-41BB / huH4F3-5-41BB heavy chain encoding nucleic acids and plasmids containing 23-H4F3-L / A12C7-L / huH4F3L-B light chain encoding nucleic acids, respectively) were co-transfected into ExpiCHO-S cells, and the bispecific antibody protein was expressed under appropriate conditions. The expression, purification, and preliminary analysis steps were the same as in Example 2.1.

[0334] 4.2 Affinity Detection of Bispecific Antibodies to Human / Cynomolgus Monkey Nectin-4 and 4-1BB

[0335] In this experiment, the binding affinity of the huH4F3-5-41BB bispecific antibody to human Nectin-4 protein (ACRO Biosystems, Catalog No.: NE4-H52H3), cynomolgus macaque Nectin-4 protein (KACTUS, Catalog No.: NEC-CM104), human 4-1BB protein (Sino Biological, Catalog No.: 10041-H08H), or cynomolgus macaque 4-1BB protein (Sino Biological, Catalog No.: 90847-K08H) was detected using the ForteBio Octet RED96e according to the manufacturer's instructions.

[0336] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1X PBS XiGene, Catalog No. XG3650, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0337] a) Equilibrate the baseline with running buffer for 180 s;

[0338] b) adding each bispecific antibody diluted in running buffer to a final concentration of 5 μg / mL and solidifying for 200 s;

[0339] c) Equilibrate the baseline with running buffer for 300 s;

[0340] d1) Add the following concentrations of human nectin-4 protein or cynomolgus monkey nectin-4 protein diluted in running buffer to each well: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. Bind for 200 s and dissociate for 600 s. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software; or

[0341] d2) Add the following concentrations of human 4-1BB protein or cynomolgus monkey 4-1BB protein diluted in running buffer to each well: 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. Bind for 240 s and dissociate for 400 s. Bind for 240 s and dissociate for 600 s. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0342] Table 7 summarizes the binding affinity of the huH4F3-5-41BB bispecific antibody to human Nectin-4 protein, cynomolgus monkey Nectin-4 protein, human 4-1BB protein, or cynomolgus monkey 4-1BB protein.

[0343] Table 7 Binding affinity of huH4F3-5-41BB bispecific antibody to Nectin-4 or 4-1BB protein

[0344] Table 7 shows that the bispecific antibody of the present invention can specifically bind to human Nectin-4 protein, cynomolgus monkey Nectin-4 protein, human 4-1BB protein or cynomolgus monkey 4-1BB protein. D The K value is higher than that of human 4-1BB protein. D The anti-nectin-4×4-1BB bispecific antibody targets human nectin-4 with high affinity, which can effectively reduce the side effects of targeting human 4-1BB.

[0345] Example 5 Activation of the 4-1BB Signaling Pathway by Humanized Bispecific Antibodies

[0346] HEK293 cell line (ATCC, catalog number: CRL-1573) was maintained in DMEM medium containing 10% FBS and placed in a humidified incubator at 37°C and 5% CO2. TM The plasmid encoding the nucleic acid sequence of human 4-1BB (NP_001552.2) and pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, catalog number: E849A) were co-transfected into HEK293 cells using 2000 transfection reagent (Invitrogen, catalog number: 11668019), and after selection with puromycin (Gibco, catalog number: A1113802) and hygromycin (Gibco, catalog number: 10687010), clones stably expressing human 4-1BB and NF-κB-Luc were obtained by limiting dilution and designated HEK293 / NF-κB-Luc / 4-1BB cells.

[0347] HEK293 / NF-κB-Luc / 4-1BB cells were seeded into 384-well plates (20,000 cells / well, 20 μL). HT-1376 cells or 293T cells (not expressing Nectin-4) (ATCC, catalog number: CRL-1573) were then seeded into 384-well plates (10,000 cells / well, 20 μL). 5X gradient dilutions of antibodies (final concentration starting from 10 nM, 5-fold dilution, 9 points) were prepared with DMEM culture medium containing 10% FBS, and 10 μL of antibody was added to each well. The culture plate was incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, 30 μL of One-GloTM Reagent (Promega, catalog number: E6130) was added to the wells of the assay plate and luminescence was measured using a luminescence microplate reader (Tecan F200 Pro). The results are shown in Figures 4A and 4B.

[0348] As can be seen from Figures 4A and 4B, only in the presence of Nectin-4 protein, the huH4F3-5-41BB bispecific antibody and the H4F3-41BB bispecific antibody can activate the human 4-1BB signaling pathway and further activate NF-κB, and show a dose-dependent trend in the reporter gene system; in the absence of Nectin-4 protein, neither the huH4F3-5-41BB bispecific antibody nor the H4F3-41BB bispecific antibody can activate the human 4-1BB signaling pathway.

[0349] Example 6 In vivo efficacy of anti-Nectin-4×4-1BB bispecific antibody in MC38-hNectin4 tumor model

[0350] The in vivo antitumor effect of the anti-Nectin-4×4-1BB bispecific antibody was investigated in the MC38-hNectin4 tumor model.

[0351] Humanized mouse C57BL / 6-Tnfrsf9tm1 (TNFRSF9) / Bcgen expressing the extracellular portion of human 4-1BB was purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0352] Mouse colon cancer MC38 cells were purchased from Sunran Shanghai Biotechnology Co., Ltd. Biocytogen (Beijing) Pharmaceuticals Co., Ltd. genetically modified MC38 cells to overexpress human nectin-4, and these cells were designated MC38-hNectin4. Cells were cultured in a 37°C, 5% CO2 incubator in DMEM supplemented with 10% inactivated fetal bovine serum.

[0353] Thirty-two C57BL / 6-Tnfrsf9tm1(TNFRSF9) / Bcgen mice were subcutaneously implanted with 5×10 5 MC38-hNectin4 cells / 0.1 mL PBS / mouse. When the average tumor volume reached 100 mm 3At the time of the experiment, 20 suitable mice were selected according to the tumor volume and body weight of the mice and randomly assigned to 4 experimental groups, with 5 mice in each group. On days 0, 3, 7, 10, 14, 17, 21 and 24 after grouping, the mice were intraperitoneally administered with blank control (PBS) (G1 group), A12C7-41BB bispecific antibody (5 mg / kg) (G2 group), H4F3-41BB bispecific antibody (5 mg / kg) (G3 group), and equimolar amounts of fusion protein Fc-41BB (SEQ ID NO: 38) (2.6 mg / kg) (G4 group). The fusion protein Fc-41BB is a double-chain form and is prepared in the same manner as in Example 2.1. The maximum long axis (L) and maximum wide axis (W) of the tumor in the tumor-bearing mice were measured twice a week with a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2.

[0354] Treatment with the anti-Nectin-4×4-1BB bispecific antibodies A12C7-41BB and H4F3-41BB, as well as the fusion protein Fc-41BB, resulted in significant tumor growth inhibition compared to the PBS group. The A12C7-41BB and H4F3-41BB groups showed a higher tumor growth inhibition rate (TGI rate) than the Fc-41BB group (97.5% and 102.8%, respectively, relative to 81.8% in the Fc-41BB control group). At the end of the experiment, three mice survived in the A12C7-41BB bispecific antibody group and two survived in the H4F3-41BB bispecific antibody group. The results are shown in Figure 5 and Table 8 below.

[0355] Table 8. In vivo growth inhibition of MC38-hNectin4 tumors Note: a. Mean ± standard error;

[0356] b. TGI = [1-(tumor volume change in the treatment group / tumor volume change in the control group)] × 100%;

[0357] c. Statistical analysis (One-way ANOVA) of the tumor volumes of the drug-treated groups and the PBS control group on day 28 of group administration was performed. ****p<0.0001.

[0358] Throughout the experiment, the animals were active and ate well during the dosing period. Animals in groups G1-G4 all showed some weight gain, indicating good tolerance to the test product. Weight changes for all animals are shown in Figure 6 and Table 9.

[0359] Table 9. Effects of the test articles on the body weight of mice Note: a. Mean ± standard error; b. Body weights of the drug-treated group and the PBS control group were statistically analyzed on the 28th day after group administration (One-way ANOVA).

[0360] Exemplary sequences

[0361] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Claims

1. An isolated antibody or an antigen-binding fragment of the isolated antibody that specifically binds to a nectin-4 protein, comprising (a) 3 CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO:1 and 3 CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO:2; or variants having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region; For example, the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 18 and the three CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NOs: 20-24; or The three CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO:19 and the three CDRs in the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs:20-24; (b) 3 CDRs in the heavy chain variable region amino acid sequence set forth in SEQ ID NO:3 and 3 CDRs in the light chain variable region amino acid sequence set forth in SEQ ID NO:4; or a variant having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region; or (c) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:5 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:6; or a variant having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region.

2. An isolated antibody or an antigen-binding fragment of the isolated antibody that specifically binds to the nectin-4 protein, comprising (a) HCDR1 shown in SEQ ID NO:40 or a variant of HCDR1 shown in SEQ ID NO:40 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:41 or a variant of HCDR2 shown in SEQ ID NO:41 with no more than 3 amino acid changes, and HCDR3 shown in SEQ ID NO:42 or a variant of HCDR3 shown in SEQ ID NO:42 with no more than 2 amino acid changes; LCDR1 shown in SEQ ID NO:43 or a variant of LCDR1 shown in SEQ ID NO:43 with no more than 3 amino acid changes, LCDR2 shown in SEQ ID NO:44 or a variant of LCDR2 shown in SEQ ID NO:44 with no more than 3 amino acid changes, and LCDR3 shown in SEQ ID NO:45 or a variant of LCDR3 shown in SEQ ID NO:45 with no more than 2 amino acid changes; (b) HCDR1 shown in SEQ ID NO:46 or a variant of HCDR1 shown in SEQ ID NO:46 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:47 or a variant of HCDR2 shown in SEQ ID NO:47 with no more than 3 amino acid changes, and HCDR3 shown in SEQ ID NO:48 or a variant of HCDR3 shown in SEQ ID NO:48 with no more than 2 amino acid changes; LCDR1 shown in SEQ ID NO:49 or a variant of LCDR1 shown in SEQ ID NO:49 with no more than 3 amino acid changes, LCDR2 shown in SEQ ID NO:50 or a variant of LCDR2 shown in SEQ ID NO:50 with no more than 3 amino acid changes, and LCDR3 shown in SEQ ID NO:51 or a variant of LCDR3 shown in SEQ ID NO:51 with no more than 2 amino acid changes; or (c) HCDR1 shown in SEQ ID NO:52 or a variant of HCDR1 shown in SEQ ID NO:52 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:53 or a variant of HCDR2 shown in SEQ ID NO:53 with no more than 3 amino acid changes, and HCDR3 shown in SEQ ID NO:54 or a variant of HCDR3 shown in SEQ ID NO:54 with no more than 2 amino acid changes; LCDR1 shown in SEQ ID NO:55 or a variant of LCDR1 shown in SEQ ID NO:55 with no more than 3 amino acid changes, LCDR2 shown in SEQ ID NO:56 or a variant of LCDR2 shown in SEQ ID NO:56 with no more than 3 amino acid changes, and LCDR3 shown in SEQ ID NO:57 or a variant of LCDR3 shown in SEQ ID NO:57 with no more than 2 amino acid changes; Preferably, the isolated antibody or antigen-binding fragment that specifically binds to the nectin-4 protein comprises a (a) HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45; HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:45; HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45; HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:62, and LCDR3 shown in SEQ ID NO:45; HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:58, and HCDR3 shown in SEQ ID NO:42; LCDR1 shown in SEQ ID NO:60, LCDR2 shown in SEQ ID NO:62, and LCDR3 shown in SEQ ID NO:45; (b) HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:47, and HCDR3 shown in SEQ ID NO:48; LCDR1 shown in SEQ ID NO:49, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:51; or (c) HCDR1 shown in SEQ ID NO:52, HCDR2 shown in SEQ ID NO:53, and HCDR3 shown in SEQ ID NO:54; LCDR1 shown in SEQ ID NO:55, LCDR2 shown in SEQ ID NO:56, and LCDR3 shown in SEQ ID NO:

57.

3. The isolated antibody or antigen-binding fragment of the isolated antibody according to claim 1 or 2, comprising (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 1, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 2, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; For example, the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2; The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:20; The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:21; The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:22; The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:23; The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:24; The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:20; The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:21; The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:22; The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:23; The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:24; (b) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; For example, the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 4; or (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:5, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:6, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; For example, the heavy chain variable region shown in SEQ ID NO:5 and the light chain variable region shown in SEQ ID NO:6; Preferably, the isolated antibody comprises (a) SEQ ID NO:11, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:12, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; For example, the heavy chain sequence shown in SEQ ID NO: 11, and the light chain sequence shown in SEQ ID NO: 12; The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:27; The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:28; The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:29; The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:30; The heavy chain sequence shown in SEQ ID NO:25, and the light chain sequence shown in SEQ ID NO:31; The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:27; The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:28; The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:29; The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:30; The heavy chain sequence shown in SEQ ID NO:26, and the light chain sequence shown in SEQ ID NO:31; (b) SEQ ID NO:13, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO:14, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (c) SEQ ID NO:15, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:16, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Preferably, the isolated antibody or antigen-binding fragment is a fully human antibody.

4. The isolated antibody or antigen-binding fragment of the isolated antibody according to any one of claims 1 to 3, which is an IgG antibody; preferably, it is a human IgG antibody; more preferably, it is a human IgG1 or human IgG4 antibody; wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab.

5. The isolated antibody or antigen-binding fragment of the isolated antibody according to any one of claims 1 to 4, having one or more of the following properties: (a) having the ability to bind to human nectin-4 molecules expressed on cells and cynomolgus monkey nectin-4 molecules expressed on cells as measured by flow cytometry assay; (b) having the ability to specifically bind only to Nectin-4 and not to Nectin-1, Nectin-2 and Nectin-3, members of the Nectin family, as measured by an ELISA assay; and / or (c) less than about 10 nM, such as about 1 nM, about 10 nM, or about 10 nM, as measured in biofilm interferometry. -1 nM, about 10 -2 nM, about 10 -3 nM binding dissociation equilibrium constant K D Bind to human nectin-4 protein; and / or at less than about 100 nM, such as about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 nM binding dissociation equilibrium constant K D Binds to cynomolgus monkey Nectin-4 protein; Preferably, the isolated antibody or antigen-binding fragment is used to prepare a multispecific antibody or an antibody-drug conjugate (ADC).

6. A multispecific antibody comprising an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein, for example, comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 5 that specifically binds to a Nectin-4 protein, preferably, the multispecific antibody is a trispecific antibody or a bispecific antibody, For example, the multispecific antibody is a bispecific antibody, which comprises an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein and an isolated antibody or antigen-binding fragment that specifically binds to a T cell co-stimulatory receptor such as ICOS, 4-1BB, CD28 or CD86. For example, the bispecific antibody comprises an isolated antibody or antigen-binding fragment of any one of claims 1 to 5 that specifically binds to a Nectin-4 protein and an isolated antibody or antigen-binding fragment that specifically binds to a T cell co-stimulatory receptor such as ICOS, 4-1BB, CD28 or CD86.

7. The multispecific antibody according to claim 6, which is a bispecific antibody, comprising from the amino terminus to the carboxyl terminus: (a) a first part, which is an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, for example, an isolated antibody or antigen-binding fragment of any one of claims 1 to 5 that specifically binds to Nectin-4 protein, and (b) a second portion, which is an isolated antibody or antigen-binding fragment that specifically binds to the T cell co-stimulatory receptor 4-1BB; The antigen-binding fragments in the first part and the second part are Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab; For example, the bispecific antibody comprises, from the amino terminus to the carboxyl terminus: (a) a first part, which is an isolated antibody or antigen-binding fragment according to any one of claims 1 to 5 that specifically binds to the nectin-4 protein, and (b) a second part, which is an anti-4-1BB scFv, For example, the anti-4-1BB scFv comprises 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 32 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 33; or a variant having a single or multiple CDRs with no more than 3 amino acid changes in each CDR region with the above 6 CDR regions; For example, the anti-4-1BB scFv comprises HCDR1 shown in SEQ ID NO:63 according to Kabat numbering, or a variant thereof with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:64, or a variant thereof with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:65, or a variant thereof with no more than 2 amino acid changes; LCDR1 shown in SEQ ID NO:66, or a variant thereof with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:67, or a variant thereof with no more than 2 amino acid changes. or a variant thereof with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:68 or a variant thereof with no more than 2 amino acid changes; for example, the anti-4-1BB scFv comprises HCDR1 shown in SEQ ID NO:63, HCDR2 shown in SEQ ID NO:64, and HCDR3 shown in SEQ ID NO:65 according to Kabat numbering; LCDR1 shown in SEQ ID NO:66, LCDR2 shown in SEQ ID NO:67, and LCDR3 shown in SEQ ID NO:68; For example, the anti-4-1BB scFv comprises a heavy chain variable region as set forth in SEQ ID NO: 32, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region as set forth in SEQ ID NO: 33, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; The carboxyl terminus of the heavy chain of the first part is connected to the amino terminus of the second part via a linker or directly connected without a linker, for example, the linker is a connecting peptide shown in SEQ ID NO:69 or SEQ ID NO:

70.

8. The multispecific antibody according to claim 7, wherein the bispecific antibody comprises two heavy chains and two light chains, wherein each heavy chain comprises, from amino terminus to carboxyl terminus, a heavy chain of a first portion, a connecting peptide and a second portion, and each light chain is a light chain of the first portion, preferably, each heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereof; and each light chain comprises the amino acid sequence shown in SEQ ID NO: 12, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereof; each heavy chain comprises the amino acid sequence of SEQ ID NO:36, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; and each light chain comprises the amino acid sequence of SEQ ID NO:16, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; or Each heavy chain comprises the amino acid sequence of SEQ ID NO:37, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; and each light chain comprises the amino acid sequence of SEQ ID NO:31, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto.

9. The multispecific antibody according to claim 7 or 8, wherein the bispecific antibody has one or more of the following properties: (a) measured in biofilm interferometry at less than about 10 nM, such as about 1 nM, about 10 -1 nM, about 10 -2 nM, about 10 -3 nM binding dissociation equilibrium constant K D binds to human nectin-4 protein, and at less than about 1000 nM, For example, about 100 nM, about 10 nM, about 1 nM, about 10 -1 nM binding dissociation equilibrium constant K D Bind to human 4-1BB protein; or at less than about 100 nM, such as about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 nM binding dissociation equilibrium constant K D Binds to cynomolgus monkey nectin-4 protein, and has a binding dissociation equilibrium constant K of less than about 150 nM, such as about 15 nM, about 1.5 nM, about 0.15 nM, about 0.015 nM D Binds to cynomolgus monkey 4-1BB protein; (b) when measured in a NF-κB-Luc / 4-1BB reporter gene cell line, the bispecific antibody can activate the human 4-1BB signaling pathway only in the presence of Nectin-4 protein; in the absence of Nectin-4 protein, the bispecific antibody cannot activate the human 4-1BB signaling pathway; and / or (c) having anti-tumor efficacy in vivo, and the bispecific antibody is safe to test animals.

10. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment of any one of claims 1 to 5 or encoding the multispecific antibody of any one of claims 6 to 9.

11. A vector comprising the nucleic acid of claim 10, preferably said vector is an expression vector.

12. A host cell comprising the nucleic acid of claim 10 or the vector of claim 11, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments, multispecific antibodies, most preferably, the host cell is a HEK293 cell or a CHO cell.

13. A method for preparing the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or the multispecific antibody of any one of claims 6 to 9, the method comprising culturing the host cell of claim 12 under conditions suitable for expression of a nucleic acid encoding the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or encoding the multispecific antibody of any one of claims 6 to 9, and optionally recovering the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or the multispecific antibody of any one of claims 6 to 9 from the host cell or from the culture medium.

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or the multispecific antibody of any one of claims 6 to 9, and a pharmaceutically acceptable carrier; or A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or the multispecific antibody of any one of claims 6 to 9, and other therapeutic agents selected from oncolytic drugs, cytotoxic agents, cytokines, inhibitors of other immune checkpoint molecules (e.g., PD-1), and a pharmaceutically acceptable carrier.

15. Use of the isolated antibody or antigen-binding fragment according to any one of claims 1 to 5, or the multispecific antibody according to any one of claims 7 to 10, for preparing a medicament for diagnosing, preventing and / or treating a disease associated with high expression of nectin-4, for example, the disease is cancer, for example, the cancer is a solid tumor or a hematological cancer, for example, the cancer is colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial carcinoma, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, prostate cancer.

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