Anti-Nectin-4 antibody and multispecific antibody containing same

Abstract

The present invention relates to an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, and a multispecific antibody comprising the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein. The present invention further relates to the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, nucleic acids encoding the multispecific antibody, the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, host cells comprising the multispecific antibody, and methods for preparing the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein and the multispecific antibody. The present invention also relates to diagnostic, prophylactic, and / or therapeutic uses of the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein and the multispecific antibody.

Description

[Technical Field]

[0001] The present invention relates generally to antibodies and uses thereof. 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 further relates to diagnostic, prophylactic, and / or therapeutic uses of the isolated antibodies or antigen-binding fragments that specifically bind to Nectin-4, and multispecific antibodies. [Background technology]

[0002] Only a limited number of patients respond to first-generation immune checkpoint inhibitors (ICIs), typically PD-1 and / or PD-L1 monoclonal antibodies, and only a small proportion (10%-30%) of tumor patients achieve a therapeutic effect after receiving PD-1 and / or PD-L1 monoclonal antibodies (Lawrence P. Andrews et al. (2020) Sci Immunol. 5(49):eabc2728). Therefore, in recent years, how to use drug combinations to improve the efficacy of ICIs and resolve the problem of immunotherapy resistance has become a focus of research in both academia and the pharmaceutical industry.

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

[0004] According to a 2020 study in J Immunother Cancer, Nectin-4 is a ligand for the immune checkpoint TIGIT, and it has been reported that Nectin-4 antibodies block the binding of Nectin-4 to TIGIT, thereby exerting their tumor-killing effects (Adi Reches et al (2020) J Immunother Cancer.8(1):e000266.).

[0005] PADCEV targets Nectin-4 TM The FDA approval of Nectin-4 (also known as enfortumab vedotin-ejfv, Japanese name: enfortumab vedotin-ejfv) for the treatment of locally advanced or metastatic urothelial carcinoma demonstrates that Nectin-4 is a relatively good tumor-associated antigen. TM is an ADC drug that targets the cell surface-expressed Nectin-4 protein, which is highly expressed in urothelial carcinoma. This ADC drug is formed by coupling enfortumab, a human IgG1 monoclonal antibody that targets Nectin-4, with the cytotoxic agent MMAE (monomethyl auristatin E, a microtubule-disrupting agent) (Pia M. Challita-Eid et al (2016) Cancer Res. 76(10):3003-13). However, PADCEV TM causes serious skin adverse reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) (2019 FDA publication).

[0006] 4-1BB, also known as CD137 or TNFRSF9, is a member of the tumor necrosis factor receptor family. Its most prominent feature is its function as a costimulatory molecule on the T cell surface, regulating TCR-induced T cell activation. After binding to 4-1BBL (4-1BBL is the primary natural ligand of 4-1BB expressed on activated antigen-presenting cells), 4-1BB upregulates anti-apoptotic molecules and promotes cytokine production and effector function. 4-1BB is also present on dendritic cells, activated monocytes, NK cells, neutrophils, eosinophils, and mast cells. 4-1BB signaling has been reported to stimulate IFN-γ secretion, promote NK cell proliferation, and promote 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 indicate that targeting 4-1BB with agonistic antibodies can result in tumor elimination and sustained anti-tumor immune effects.

[0008] Although clinical trials of two agonist antibodies, urelumab and utomilumab, have shown early signs of therapeutic efficacy, inflammatory hepatotoxicity at doses above 1 mg / kg has hindered the clinical development of urelumab. Utomilumab has relatively good stability, but its 4-1BB agonist effect is relatively weaker than that of urelumab (Cariad Chester et al (2018) Blood 131(1):49-57).

[0009] To optimally utilize 4-1BB-mediated immune activation, next-generation 4-1BB-targeting strategies aim to improve antitumor efficacy while simultaneously reducing liver toxicity. Efforts are underway to limit the effects of 4-1BB agonists on the tumor microenvironment, thereby reducing immune-mediated adverse events. 4-1BB is a potent target for tumor immunotherapy and shows great promise for potent immune activation (Cariad Chester et al. (2018) Blood 131(1):49-57).

[0010] Therefore, there remains a need in the art for the development of novel antibodies that achieve an optimal balance between safety and agonistic activity. The present invention fulfills this need by providing an anti-Nectin-4 antibody that binds to Nectin-4 with high target specificity and high affinity, and a bispecific antibody comprising the anti-Nectin-4 and anti-4-1BB that binds to both anti-Nectin-4 and 4-1BB with low affinity. Summary of the Invention

[0011] Through research, the present inventors have developed a set of novel isolated anti-Nectin-4 antibodies that bind to Nectin-4 with high affinity. The isolated antibodies or antigen-binding fragments thereof that specifically bind to Nectin-4 of the present invention have one or more of the following properties: (a) has the ability to bind to human Nectin-4 molecules expressed on cells and to cynomolgus monkey Nectin-4 molecules expressed on cells, as measured by flow cytometry; and (b) has the ability to specifically bind only to Nectin-4, as measured by ELISA, and does not bind to any of the Nectin family members Nectin-1, Nectin-2, and Nectin-3; and / or (c) less than about 10 nM, e.g., about 1 nM, about 10 -1 nM, about 10 -2 nM, about 10 -3 nM binding-dissociation equilibrium constant K Dand binds to human Nectin-4 protein at a concentration of less than about 100 nM, e.g., about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 nM binding-dissociation equilibrium constant K D It binds to the cynomolgus monkey Nectin-4 protein.

[0012] Accordingly, in a first aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, comprising: (a) a mutant having three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; For example, three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 18 and three CDRs in the amino acid sequence of the light chain variable region shown in any one of SEQ ID NOs: 20 to 24, or Three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 19, and three CDRs in the amino acid sequence of a light chain variable region shown in any one of SEQ ID NOs: 20 to 24; (b) a variant having three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 3 and three CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO: 4, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; or (c) Three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 5 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 6, or a variant having one or more CDRs with three or fewer amino acid changes per CDR region in each of the six CDR regions.

[0013] In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, which is based on Kabat numbering (a) an HCDR1 represented by SEQ ID NO: 40 or a variant of the HCDR1 represented by SEQ ID NO: 40 with two or fewer amino acid changes, an HCDR2 represented by SEQ ID NO: 41 or a variant of the HCDR2 represented by SEQ ID NO: 41 with three or fewer amino acid changes, and an HCDR3 represented by SEQ ID NO: 42 or a variant of the HCDR3 represented by SEQ ID NO: 42 with two or fewer amino acid changes, as well as an LCDR1 represented by SEQ ID NO: 43 or a variant of the LCDR1 represented by SEQ ID NO: 43 with three or fewer amino acid changes, an LCDR2 represented by SEQ ID NO: 44 or a variant of the LCDR2 represented by SEQ ID NO: 44 with three or fewer amino acid changes, and an LCDR3 represented by SEQ ID NO: 45 or a variant of the LCDR3 represented by SEQ ID NO: 45 with two or fewer amino acid changes, (b) an HCDR1 represented by SEQ ID NO: 46 or a variant of the HCDR1 represented by SEQ ID NO: 46 with two or fewer amino acid changes, an HCDR2 represented by SEQ ID NO: 47 or a variant of the HCDR2 represented by SEQ ID NO: 47 with three or fewer amino acid changes, and an HCDR3 represented by SEQ ID NO: 48 or a variant of the HCDR3 represented by SEQ ID NO: 48 with two or fewer amino acid changes, as well as an LCDR1 represented by SEQ ID NO: 49 or a variant of the LCDR1 represented by SEQ ID NO: 49 with three or fewer amino acid changes, an LCDR2 represented by SEQ ID NO: 50 or a variant of the LCDR2 represented by SEQ ID NO: 50 with three or fewer amino acid changes, and an LCDR3 represented by SEQ ID NO: 51 or a variant of the LCDR3 represented by SEQ ID NO: 51 with two or fewer amino acid changes, or (c) HCDR1 represented by SEQ ID NO: 52 or a variant of HCDR1 represented by SEQ ID NO: 52 with two or fewer amino acid changes, HCDR2 represented by SEQ ID NO: 53 or a variant of HCDR2 represented by SEQ ID NO: 53 with three or fewer amino acid changes, and HCDR3 represented by SEQ ID NO: 54 or a variant of HCDR3 represented by SEQ ID NO: 54 with two or fewer amino acid changes, as well as LCDR1 represented by SEQ ID NO: 55 or a variant of LCDR1 represented by SEQ ID NO: 55 with three or fewer amino acid changes, LCDR2 represented by SEQ ID NO: 56 or a variant of LCDR2 represented by SEQ ID NO: 56 with three or fewer amino acid changes, and LCDR3 represented by SEQ ID NO: 57 or a variant of LCDR3 represented by SEQ ID NO: 57 with two or fewer amino acid changes.

[0014] Preferably, the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein is (a) HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 41, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45; HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 61, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 60, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, (b) an HCDR1 represented by SEQ ID NO: 46, an HCDR2 represented by SEQ ID NO: 47, and an HCDR3 represented by SEQ ID NO: 48, and an LCDR1 represented by SEQ ID NO: 49, an LCDR2 represented by SEQ ID NO: 50, and an LCDR3 represented by SEQ ID NO: 51; or (c) Contains an HCDR1 represented by SEQ ID NO: 52, an HCDR2 represented by SEQ ID NO: 53, and an HCDR3 represented by SEQ ID NO: 54, as well as an LCDR1 represented by SEQ ID NO: 55, an LCDR2 represented by SEQ ID NO: 56, and an LCDR3 represented by SEQ ID NO: 57.

[0015] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the present invention is (a) a heavy chain variable region comprising the sequence of SEQ ID NO: 1, 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 comprising the sequence of SEQ ID NO: 2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; For example, a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2, a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 20; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 21; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 22; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 23; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 24; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 20; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 21; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 22; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 23; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 24; (b) a heavy chain variable region comprising the sequence of SEQ ID NO: 3, 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 comprising the sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; For example, a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 4, or (c) a heavy chain variable region comprising the sequence of SEQ ID NO: 5, 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 comprising the sequence of SEQ ID NO: 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; For example, it includes a heavy chain variable region shown in SEQ ID NO:5 and a light chain variable region shown in SEQ ID NO:6.

[0016] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the present invention is (a) a heavy chain sequence of SEQ ID NO: 11, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain sequence of SEQ ID NO: 12, or 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, a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 27; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 28; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 29; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 30; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 31; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 27; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 28; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 29; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 30; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 31; (b) a heavy chain sequence having SEQ ID NO: 13, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain sequence having SEQ ID NO: 14, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (c) a heavy chain sequence of SEQ ID NO: 15, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain sequence of SEQ ID NO: 16, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0017] In some embodiments, the antigen-binding fragment of the isolated anti-Nectin-4 antibody of the present invention is a Fab, a Fab', a F(ab')2, an Fv, a single-chain Fv, or a single-chain Fab.

[0018] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein of the present invention is a fully human antibody.

[0019] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein of the present invention is an IgG antibody, preferably a human IgG antibody, more preferably a human IgG1 or human IgG4 antibody.

[0020] In a second aspect, the present invention provides a multispecific antibody comprising an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein, for example, it comprises an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the first aspect of the present invention, and preferably the multispecific antibody is a trispecific antibody or a bispecific antibody.

[0021] In some embodiments, the present invention provides a bispecific antibody comprising an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein, and an isolated antibody or antigen-binding fragment that specifically binds to a T cell costimulatory 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 the Nectin-4 protein of the first aspect of the present invention, and an isolated antibody or antigen-binding fragment that specifically binds to a T cell costimulatory receptor (e.g., ICOS, 4-1BB, CD28, or CD86).

[0022] In some embodiments, the present invention provides bispecific antibodies, which comprise, from the amino terminus to the carboxy terminus: (a) a first moiety which is an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein, for example, an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the first aspect of the present invention; and (b) a second portion that is an isolated antibody or antigen-binding fragment that specifically binds to T cell costimulatory receptor 4-1BB; Here, the antigen-binding fragments in the first and second portions are Fab, Fab', F(ab')2, Fv, single-chain Fv, or single-chain Fab.

[0023] In some embodiments, the present invention provides a bispecific antibody that is anti-Nectin-4 / 4-1BB, (a) a first portion that is an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein; and (b) a second portion that is an isolated antibody or antigen-binding fragment that specifically binds to T cell costimulatory receptor 4-1BB; and The bispecific antibody has high affinity for Nectin-4 and low affinity for 4-1BB, and its K targeting human Nectin-4 protein. D The K values ​​are those of the targeting human 4-1BB protein. DThe high affinity for human Nectin-4, two orders of magnitude higher than the RI value, effectively reduces the side effects of targeting human 4-1BB. This anti-Nectin-4 / 4-1BB bispecific antibody not only blocks the binding of Nectin-4 to TIGIT by targeting Nectin-4 and the costimulatory receptor 4-1BB, but also promotes the effective activation and killing of immune T cells targeting tumors by binding to the T cell costimulatory receptor 4-1BB.

[0024] In some embodiments, the antigen-binding fragment of an isolated antibody of the present invention that specifically binds to 4-1BB is a Fab, a Fab', a F(ab')2, an Fv, a single-chain Fv, or a single-chain Fab.

[0025] In a third aspect, the present invention provides a nucleic acid encoding an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein described in the first aspect above, or encoding a multispecific antibody described in the second aspect above, a vector (preferably an expression vector) comprising the nucleic acid, or a host cell comprising the nucleic acid or vector. In some embodiments, the host cell is prokaryotic or eukaryotic, and is selected from, for example, E. coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments, or multispecific antibodies. In some embodiments, the host cell is a HEK293 cell or a CHO cell.

[0026] In a fourth aspect, the present invention provides a method for preparing an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein according to the first aspect above, or a multispecific antibody according to the second aspect above, the method comprising culturing a host cell of the invention under conditions suitable for expression of nucleic acid encoding the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein according to the first aspect above, or the multispecific antibody according to the second aspect above, and optionally recovering the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein according to the first aspect above, or the multispecific antibody according to the second aspect above, from the host cell or from the culture medium.

[0027] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein according to the first aspect above, or a multispecific antibody according to the second aspect above, and a pharmaceutically acceptable vector.

[0028] In some embodiments, the pharmaceutical composition of the present invention comprises an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein as described in the first aspect above, or a multispecific antibody as described in the second aspect above, together with another therapeutic agent selected from an oncolytic drug, a cytotoxic agent, a cytokine, or an inhibitor of another immune checkpoint molecule (e.g., PD-1), and a pharmaceutically acceptable vector.

[0029] In a sixth aspect, the present invention provides use of an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein according to the first aspect, or a multispecific antibody according to the second aspect, in the preparation of a medicament for diagnosing, preventing, and / or treating a disease associated with high Nectin-4 expression, such as cancer. In some embodiments, the cancer is a solid tumor or a blood 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, and prostate cancer.

[0030] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease, the method comprising administering an effective amount of an isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein of the present invention, 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 to a subject in need thereof, wherein the subject is a mammal, preferably a human, and 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 blood 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, and prostate cancer.

[0031] 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 thereof that specifically binds to the Nectin-4 protein of the present invention under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and the Nectin-4 protein, and detecting the formation of the complex; for example, the detection method is a non-diagnostic detection method. [Brief explanation of the drawings]

[0032] Preferred embodiments of the invention, which will now be described in detail, will be better understood when read in conjunction with the following drawings: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1A] 1 shows a graph showing the detection of the binding ability of mouse chimeric anti-Nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, antibody 32-A12C7) to tumor cells HT-1376 that express human Nectin-4 at the cellular level. [Figure 1B] 1 shows a graph showing the detection of the binding ability of mouse chimeric anti-Nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, antibody 32-A12C7) to CHO-K1 / cynoNectin4 cells expressing cynomolgus monkey Nectin-4 protein at the cellular level. [Figure 2A] and [Figure 2B] 1 shows graphs detecting the binding ability of mouse chimeric anti-Nectin-4 antibodies 23-H4F3 and 6-B9B6 to Nectin family proteins Nectin-1, Nectin-2, Nectin-3, and Nectin-4, respectively. [Figure 3A] and [Figure 3B]1 shows graphs detecting the binding ability of humanized anti-Nectin-4 antibodies (i.e., humanized antibodies huH4F3-1 to huH4F3-10) to human Nectin-4 tumor cells HT-1376 and cells CHO-K1 / cynoNectin4 expressing cynomolgus monkey Nectin-4 protein at the cellular level, respectively. [Figure 4A] 1 shows a graph showing the activation effect of the anti-Nectin-4×4-1BB bispecific antibody on the NF-κB-Luc / 4-1BB reporter gene cell line in the presence of Nectin-4-expressing tumor cells HT-1376. In the graph, the horizontal axis represents antibody concentration, and the vertical axis represents relative light units (RLU). [Figure 4B] 1 shows a graph showing the activation effect of the anti-Nectin-4×4-1BB bispecific antibody on the NF-κB-Luc / 4-1BB reporter gene cell line in the presence of 293T cells, which do not express Nectin-4. In the graph, the horizontal axis represents antibody concentration, and the vertical axis represents RLU values. [Figure 5] 1 shows changes in tumor volume in tumor-bearing mice after administration of anti-Nectin-4×4-1BB bispecific antibody, with the horizontal axis representing the number of days after the tumor-bearing mice were divided into groups. [Figure 6] 1 shows changes in mouse body weight after administration of anti-Nectin-4×4-1BB bispecific antibody to tumor-bearing mice, with the horizontal axis indicating the number of days after the tumor-bearing mice were divided into groups. DETAILED DESCRIPTION OF THE INVENTION

[0033] Before describing the present invention in detail, it should be understood that this invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary, and the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0034] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.

[0035] The term "about," when used in conjunction with numerical values, is meant to cover numerical values ​​within a range having a lower limit of 10% less than the specified numerical value and an upper limit of 10% greater than the specified numerical value.

[0036] The term "and / or," when used in conjunction with two or more options, should be understood to mean any one of the options or any two or more of the options.

[0037] As used herein, the term "comprise" or "comprises" means the inclusion of the stated elements, integers, or steps, but not the exclusion of any other elements, integers, or steps. As used herein, the term "comprise" or "comprises" also covers cases consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region that "comprises" a particular specific sequence, this also intends to cover an antibody variable region consisting of that specific sequence.

[0038] The term "antibody" is used herein in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the desired antigen-binding activity. The antibody may be an intact antibody (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). An intact antibody monomer, also called an Ig molecule monomer, is a tetrapeptide chain molecule formed from two full-length light chains and two full-length heavy chains linked by disulfide bonds. The antibody monomer is the basic structure that makes up an antibody.

[0039] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies (Abs) with different antigen specificities (e.g., an isolated antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 protein is substantially free of Abs that specifically bind to antigens other than Nectin-4 protein). In some embodiments, the antibody is purified to greater than 80% or 90% purity, preferably greater than 95% or 99% purity, which can be determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0040] An "epitope" or "antigenic determinant" refers to a cluster of antigenic determinants that interact with a specific antigen-binding site, called a paratope, in the variable region of an antibody molecule. A single antigen may have one or more epitopes. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes may be formed from contiguous amino acids or discontinuous amino acids connected by tertiary folding of a protein. Epitopes formed by contiguous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. An epitope usually comprises at least three, and more usually at least five, about nine, or about eight to ten amino acids in a unique spatial conformation.

[0041] The term "antigen-binding fragment" refers to a portion or fragment of an intact or complete antibody that contains fewer than the number of amino acid residues of the intact or complete antibody and is capable of binding to an antigen or of binding to an antigen competitively with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fv, single-chain Fv (scFv), single-chain Fab, diabody, single-domain antibody (sdAb, nanobody), camelid Ig, Ig NAR, F(ab)' fragment, bispecific-scFv, (scFv), minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, and disulfide-stabilized Fv protein ("dsFv"). The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heterozygous antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. For a more detailed description, see also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois). Kuby, Journal of Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0042] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used herein interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected via 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 containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is formed by the second and third constant regions of two heavy chains (and a fourth constant region in the case of IgE and IgM), with disulfide bonds (interchain) formed within the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem Ig domains and a hinge region used to increase flexibility, while heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0043] Each light chain variable region and heavy chain variable region contains a "framework" region interposed by three hypervariable regions (also called "complementarity-determining regions" or "CDRs"). A "complementarity-determining region" or "CDR region" or "CDR" or "hypervariable region" (which may be used interchangeably herein with hypervariable region "HVR") is a region within an antibody variable domain that is hypervariable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen 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 in an antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs in an antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.For 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 many known antibody CDR numbering systems, such as the Chothia numbering system (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)) based on the three-dimensional structure of the antibody and the topology of the CDR loops; the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; the AbM (University of Bath); the Contact (University College London); and the International ImMunoGeneTics (ImmunoGeneTics). database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and a North CDR definition based on affinity propagation clustering that utilizes a large number of crystal structures.

[0044] However, it should be recognized that there may be differences in the CDR boundaries of the variable regions of the same antibody obtained based on different numbering systems. That is, there will be differences in the CDR sequences of the variable regions of the same antibody defined by different numbering systems. Therefore, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of that antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of a different method (e.g., the rules or combinations of different numbering systems).

[0045] The CDRs of the antibodies of the present invention can be manually assessed and demarcated by any method known in the art, or a combination thereof. Unless otherwise specified, the term "CDR" or "CDR sequence" as used herein covers CDR sequences determined by any one of the above methods.

[0046] The sequences of framework regions of different light or heavy chains are relatively conserved within a species (e.g., humans). The framework regions of an antibody (the combined framework regions of the constituent light and heavy chains) are used to position and align CDRs in three-dimensional space. CDRs are primarily responsible for binding to antigen epitopes. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs differ between antibodies, there are a limited number of amino acid positions in CDRs that are directly involved in binding to antigens. These positions in CDRs are called specificity-determining residues (SDRs).

[0047] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or a cell transfected with the light and heavy chain genes of a single antibody. 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 and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0048] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one example, a two-chain Fv is formed by a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv), one heavy-chain variable domain and one light-chain variable domain are covalently linked via a flexible peptide linker, thereby associating the light and heavy chains in a "dimeric" structure in the two-chain Fv. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six HVRs together confer antigen-binding specificity to the antibody. Unless otherwise specified, as used herein, an scFv may have the VL and VH variable regions in any order (e.g., relative to the N- and C-termini of the polypeptide), and may comprise a VL-linker-VH or a VH-linker-VL.

[0049] Fab fragments contain a heavy chain variable domain, a light chain variable domain, and further contain the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab', in which the cysteine ​​residues of the constant domains bear free thiol groups. F(ab')2 antibody fragments are initially produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0050] The term "chimeric antibody" refers to (a) the alteration, substitution, or replacement of the constant region or a portion thereof, such that the antigen-binding site is linked to a constant region of a different or altered type and / or species, or (b) the alteration, substitution, or replacement of the variable region or a portion thereof, with a variable region having a different or altered antigen specificity. In some embodiments of the invention, the modification is performed by replacing the constant region of a murine antibody with a constant region derived from a human immunoglobulin. Due to the change to a human constant region, the chimeric antibody retains its specificity for antigen recognition and has reduced antigenicity in humans compared to the original murine antibody.

[0051] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In some embodiments, all or nearly all of the CDRs in a humanized antibody correspond to those of a non-human antibody, and all or nearly all of the FRs correspond to those of a human antibody. A humanized antibody may optionally 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 an antibody that has been humanized. In some embodiments, the amino acid residues in the CDRs of a humanized antibody do not exactly correspond to those in the parent mouse CDRs, e.g., are mutated at specific positions in the CDRs to allow high-affinity contact with the target antigen.

[0052] A "fully human antibody" or "human antibody" refers to any antibody in which both the variable and constant region sequences are human sequences and which does not contain any non-human antibody sequences. The term also includes antibodies produced recombinantly in non-human cells, which may add glycosylation that is not typical in human cells.

[0053] The term "multispecific antibody" refers to an antibody that has binding specificities for multiple different epitopes. The epitopes of a multispecific antibody may 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 may be derived from the same antigen or from two different antigens.

[0054] The term "tumor antigen" includes tumor-associated antigens (TAA) and tumor-specific antigens (TSA). The term "tumor-associated antigen" (TAA) refers to proteins present on tumor cells, as well as on normal cells in selected organs during fetal (formerly embryonic) and postnatal life, but at concentrations much lower than those in tumor cells. Tumor-associated antigens can also be present in the stroma near tumor cells and are expressed at lower levels in other stroma in vivo. The term "tumor-specific antigen" (TSA) refers to antigens that are present primarily on tumor cells of a mammalian subject, but are not normally found on normal cells of the mammalian subject.

[0055] The term "Nectin protein family" is used interchangeably with "Nectin family" and refers to cell adhesion molecules that form physical connections between adjacent cells to facilitate cell-cell communication, migration, and other important cellular processes. The Nectin protein family includes at least four human Nectin proteins, namely Nectin-1 protein, Nectin-2 protein, Nectin-3 protein, and Nectin-4 protein, among which, expression of Nectin-4 protein in healthy humans is mainly restricted to placental and embryonic tissues. Many types of tumor cells have higher expression of Nectin-4 protein compared to healthy adult tissues. Nectin-4 protein is a tumor-associated antigen.

[0056] The terms "specifically bind" or "to bind" when used in reference to an antigen and an antibody refer to the formation of a complex between the antibody and the antigen that 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):pp.270-278), and ForteBio affinity assays (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):pp.270-278).

[0057] "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 stated, "binding affinity" as used herein refers to the binding affinity within 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 is usually measured using the dissociation equilibrium constant (K D Affinity can be measured by methods known in the art, including those known in the art and those described herein.

[0058] As used herein, the term "variant" refers to a heavy or light chain variable region that has been modified by at least one, e.g., one, two, or three amino acid substitution, deletion, or addition, wherein the modified antigen binding protein comprising the heavy or light chain variant substantially retains the biological properties of the antigen binding protein prior to modification. In one embodiment, an antigen binding protein comprising a variant heavy or light chain variable region sequence retains 60%, 70%, 80%, 90%, or 100% of the biological properties of the antigen binding protein prior to modification. It should be understood that each heavy or light chain variable region may be modified alone or in combination with another heavy 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% identical to the heavy chain variable region amino acid sequences described herein. The antigen binding proteins of the present disclosure comprise a light chain variable region amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence described herein. The percent homology may be over the entire heavy and / or light chain variable region, or the percent homology may be limited to the framework regions, where the sequences corresponding to the CDRs have 100% identity with the CDRs disclosed herein in the heavy and / or light chain variable regions. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, e.g., one, two, or three amino acid substitutions, deletions, or additions, wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological properties of the antigen binding protein prior to modification. In one embodiment, the antigen-binding protein comprising the variant CDR retains 60%, 70%, 80%, 90%, or 100% of the biological properties of the antigen-binding protein before modification. It should be understood that each CDR that can be modified may be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, particularly a conservative substitution.

[0059] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a chain of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.

[0060] Calculate sequence identity between sequences as follows:

[0061] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In one preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 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. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position.

[0062] A mathematical algorithm can be used to compare the sequences and calculate the percentage identity between two sequences. In one preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com) is used to determine the percentage identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used to determine the percentage identity between two nucleotide sequences using a NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the one that should be used unless otherwise specified) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0063] The percentage identity between two amino acid or nucleotide sequences may also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) integrated 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.

[0064] Additionally or alternatively, the nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

[0065] In the case of polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence whereby a particular amino acid is replaced 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, polymorphic variants, interspecies homologs, and alleles of the invention. Below are eight pairs of amino acids that are conservative substitutions for one another. 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" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0066] The term "immune checkpoint" refers to an inhibitory signaling molecule present in the immune system that regulates the duration and intensity of immune responses in peripheral tissues, thereby avoiding tissue damage and maintaining tolerance to its own antigen (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4):252-264). Research has shown that one of the reasons tumor cells can evade the immune system in vivo and proliferate uncontrollably is that they utilize the inhibitory signaling pathway of immune checkpoints to suppress the activity of T lymphocytes, thereby preventing T lymphocytes from effectively exerting their killing effect on tumors (Yao S, Zhu Y, & 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 cell death 1 (PD-1), PD-L1, PD-L2, cytotoxic T-lymphocyte antigen 4 (CTLA-4), LAG-3, TIM-3, and T cell immunoglobulin and ITIM domains (TIGIT).

[0067] The terms "costimulatory molecule" and "costimulatory receptor" are used interchangeably and refer to corresponding binding partners on lymphocytes that specifically bind to a costimulatory ligand and thereby mediate a costimulatory response (e.g., but not limited to, proliferation) of the lymphocyte. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an effective lymphocyte response to an antigen. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, inducible costimulatory factor (ICOS) (i.e., CD278), OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, CD28, and CD86. In some embodiments, a "costimulatory molecule" is ICOS, 4-1BB, CD28, and / or CD86.

[0068] As used herein, "vector" refers to a construct that can deliver one or more genes or sequences of interest to a host cell and, preferably, express said 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 flocculants, DNA or RNA expression vectors encapsulated in liposomes, and eukaryotic cells such as producer cells.

[0069] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Host cells encompass "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. This specification includes mutant progeny that have the same function or biological activity as the cell screened or selected for in the originally transformed cell.

[0070] The present invention further relates to a method for producing a monoclonal antibody, which comprises culturing a host cell according to the present invention to produce the monoclonal antibody according to the present invention.

[0071] As used herein, "subject" or "individual" refers to an animal, preferably a mammal, more preferably a human, in need of mitigation, prevention, and / or treatment of a disease or condition, such as a viral infection. Mammals further include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects having or at risk of a disease. In the present invention, administering an antibody according to the present invention, or a pharmaceutical composition or product according to the present invention, to a subject in need thereof refers to administering an effective amount of the antibody, pharmaceutical composition, product, etc.

[0072] As used herein, the term "effective amount" refers to an amount of a drug or agent that elicits the biological or pharmacological response in a tissue, system, animal, or human that is being sought by, for example, a researcher or clinician. The term "therapeutically effective amount" also refers to an amount that causes improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount that effectively enhances normal physiological function.

[0073] II. Isolated anti-Nectin-4 antibodies of the present invention The present invention provides isolated antibodies against Nectin-4 and antigen-binding fragments thereof. The protein or fragment thereof used to obtain the sensitizing antigen for the isolated antibodies against Nectin-4 of the present invention is preferably human-derived Nectin-4 protein or its extracellular portion. The nucleotide and amino acid sequences of human Nectin-4 and its homologs can be obtained, for example, by accessing GenBank (NCBI, USA) (e.g., NCBI accession number: NP_112178.2). In one embodiment, the extracellular domain of human Nectin-4 protein has the amino acid sequence set forth in SEQ ID NO: 39.

[0074] 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 and refer to the antibody of the present invention that is capable of binding to Nectin-4 protein with sufficient affinity and thereby can be used as a diagnostic, preventative, and / or therapeutic agent that targets Nectin-4 protein.

[0075] The isolated anti-Nectin-4 antibodies and antigen-binding fragments of the present invention specifically bind to Nectin-4 protein with high affinity, which (a) a mutant having three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; For example, three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 18 and three CDRs in the amino acid sequence of the light chain variable region shown in any one of SEQ ID NOs: 20 to 24, or Three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 19, and three CDRs in the amino acid sequence of a light chain variable region shown in any one of SEQ ID NOs: 20 to 24; (b) a variant having three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 3 and three CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO: 4, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; or (c) three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 5 and three CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO: 6, or a variant having the above six CDR regions and one or more CDRs with three or fewer amino acid changes per CDR region; Here, 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.

[0076] There are several methods known in the art for determining the CDR sequences of a given antibody molecule. The CDR sequences from antibody heavy and light chain variable regions may be determined according to any method known in the art, including, but not limited to, the Kabat, Chothia, and IMGT numbering schemes, or a combination thereof.

[0077] In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein, which is based on Kabat numbering (a) an HCDR1 represented by SEQ ID NO: 40 or a variant of the HCDR1 represented by SEQ ID NO: 40 with two or fewer amino acid changes, an HCDR2 represented by SEQ ID NO: 41 or a variant of the HCDR2 represented by SEQ ID NO: 41 with three or fewer amino acid changes, and an HCDR3 represented by SEQ ID NO: 42 or a variant of the HCDR3 represented by SEQ ID NO: 42 with two or fewer amino acid changes, as well as an LCDR1 represented by SEQ ID NO: 43 or a variant of the LCDR1 represented by SEQ ID NO: 43 with three or fewer amino acid changes, an LCDR2 represented by SEQ ID NO: 44 or a variant of the LCDR2 represented by SEQ ID NO: 44 with three or fewer amino acid changes, and an LCDR3 represented by SEQ ID NO: 45 or a variant of the LCDR3 represented by SEQ ID NO: 45 with two or fewer amino acid changes, (b) an HCDR1 represented by SEQ ID NO: 46 or a variant of the HCDR1 represented by SEQ ID NO: 46 with two or fewer amino acid changes, an HCDR2 represented by SEQ ID NO: 47 or a variant of the HCDR2 represented by SEQ ID NO: 47 with three or fewer amino acid changes, and an HCDR3 represented by SEQ ID NO: 48 or a variant of the HCDR3 represented by SEQ ID NO: 48 with two or fewer amino acid changes, as well as an LCDR1 represented by SEQ ID NO: 49 or a variant of the LCDR1 represented by SEQ ID NO: 49 with three or fewer amino acid changes, an LCDR2 represented by SEQ ID NO: 50 or a variant of the LCDR2 represented by SEQ ID NO: 50 with three or fewer amino acid changes, and an LCDR3 represented by SEQ ID NO: 51 or a variant of the LCDR3 represented by SEQ ID NO: 51 with two or fewer amino acid changes, or (c) HCDR1 represented by SEQ ID NO: 52 or a variant of HCDR1 represented by SEQ ID NO: 52 with two or fewer amino acid changes, HCDR2 represented by SEQ ID NO: 53 or a variant of HCDR2 represented by SEQ ID NO: 53 with three or fewer amino acid changes, and HCDR3 represented by SEQ ID NO: 54 or a variant of HCDR3 represented by SEQ ID NO: 54 with two or fewer amino acid changes, as well as LCDR1 represented by SEQ ID NO: 55 or a variant of LCDR1 represented by SEQ ID NO: 55 with three or fewer amino acid changes, LCDR2 represented by SEQ ID NO: 56 or a variant of LCDR2 represented by SEQ ID NO: 56 with three or fewer amino acid changes, and LCDR3 represented by SEQ ID NO: 57 or a variant of LCDR3 represented by SEQ ID NO: 57 with two or fewer amino acid changes, Includes:

[0078] Preferably, the isolated antibody or antigen-binding fragment that specifically binds to Nectin-4 protein is (a) HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 41, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45; HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 61, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 60, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, (b) an HCDR1 represented by SEQ ID NO: 46, an HCDR2 represented by SEQ ID NO: 47, and an HCDR3 represented by SEQ ID NO: 48, and an LCDR1 represented by SEQ ID NO: 49, an LCDR2 represented by SEQ ID NO: 50, and an LCDR3 represented by SEQ ID NO: 51; or (c) HCDR1 represented by SEQ ID NO: 52, HCDR2 represented by SEQ ID NO: 53, and HCDR3 represented by SEQ ID NO: 54, and LCDR1 represented by SEQ ID NO: 55, LCDR2 represented by SEQ ID NO: 56, and LCDR3 represented by SEQ ID NO: 57; Includes:

[0079] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention binds to a mammalian Nectin-4 protein, for example, a human Nectin-4 protein or a monkey Nectin-4 protein.

[0080] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention has one or more of the following properties: (a) has the ability to bind to human Nectin-4 molecules expressed on cells and to cynomolgus monkey Nectin-4 molecules expressed on cells, as measured by flow cytometry; and (b) has the ability to specifically bind only to Nectin-4, as measured by ELISA, and does not bind to any of the Nectin family members Nectin 1, Nectin 2, and Nectin 3; and / or (c) less than about 10 nM, e.g., about 1 nM, about 10 -1 nM, about 10 -2 nM, about 10 -3 nM binding-dissociation equilibrium constant K D and binds to human Nectin-4 protein at a concentration of less than about 100 nM, e.g., about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 nM binding-dissociation equilibrium constant K D It binds to the cynomolgus monkey Nectin-4 protein.

[0081] In some embodiments, the isolated anti-Nectin-4 protein antibodies of the present invention specifically bind to Nectin-4 protein, thereby blocking the binding of Nectin-4 protein to the immune checkpoint molecule TIGIT and thereby suppressing the signaling pathway of the immune checkpoint molecule TIGIT. The Nectin-4 protein antibodies of the present invention also have a direct effect on target cells expressing Nectin-4 protein. Therefore, the Nectin-4 protein antibodies of the present invention can enhance anti-tumor immunity by reversing the suppression of the immune system by TIGIT without coupling to any toxin or anti-tumor agent. Therefore, the Nectin-4 protein antibodies of the present invention can block the interaction between Nectin-4 on cancer cells and TIGIT on immune cells such as T cells and NK cells in cancer immunotherapy, thereby restoring the activity of immune cells such as T cells and NK cells.

[0082] 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, (a) the heavy chain variable region comprises the sequence of SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; For example, a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2, a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 20; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 21; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 22; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 23; a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 24; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 20; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 21; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 22; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 23; a heavy chain variable region represented by SEQ ID NO: 19 and a light chain variable region represented by SEQ ID NO: 24; (b) the heavy chain variable region comprises the sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; For example, a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 4, or (c) the heavy chain variable region comprises the sequence of SEQ ID NO: 5, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; For example, a heavy chain variable region shown in SEQ ID NO:5 and a light chain variable region shown in SEQ ID NO:6.

[0083] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention is (a) a heavy chain sequence of SEQ ID NO: 11, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain sequence of SEQ ID NO: 12, or 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, a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 27; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 28; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 29; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 30; a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 31; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 27; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 28; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 29; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 30; a heavy chain sequence shown in SEQ ID NO: 26 and a light chain sequence shown in SEQ ID NO: 31; (b) a heavy chain sequence having SEQ ID NO: 13, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain sequence having SEQ ID NO: 14, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (c) a heavy chain sequence of SEQ ID NO: 15, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain sequence of SEQ ID NO: 16, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; Includes:

[0084] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention comprises an Fc region 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.

[0085] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention comprises a constant region sequence selected from mouse IgG, human IgG, for example, human IgG1, or human IgG4.

[0086] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention is a chimeric antibody comprising a mouse antibody variable region and a human antibody constant region.

[0087] In some embodiments, the isolated anti-Nectin-4 protein antibody of the present invention is a humanized antibody.

[0088] 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. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same type, for example, the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid.

[0089] In preferred embodiments, the amino acid changes described in the present invention may occur in regions other than CDRs (e.g., FRs). More preferably, the amino acid changes described in the present invention occur in the Fc region. In some embodiments, an anti-Nectin-4 protein antibody is provided, which comprises an Fc domain containing one or more mutations that enhance or attenuate binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the present invention provides an anti-Nectin-4 protein antibody comprising an Fc domain containing one or more mutations that enhance or attenuate binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. H 2 or C HThe present invention includes anti-Nectin-4 protein antibodies containing mutations in three regions, one or more of which improve the affinity of the Fc domain for FcRn in an acidic environment (e.g., within endosomes having a pH range of about 5.5 to about 6.0). Such mutations can improve the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0090] For example, the present invention provides 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 Q311I), and 434H (e.g., P257I and Q311I). 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 above Fc domain mutations with other mutations in the antibody variable domains disclosed herein is included within the scope of the present invention.

[0091] In some embodiments, the isolated anti-Nectin-4 protein antibodies provided herein are modified to increase or decrease their glycosylation level. Addition or deletion of glycosylation sites to a Nectin-4 protein antibody can be easily achieved by modifying the amino acid sequence to generate or remove one or more glycosylation sites. When a Nectin-4 protein antibody contains an Fc region, the sugars attached to the Fc region can be modified. In some applications, modifications to remove undesired glycosylation sites, such as removing a fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function, can be useful (see Shield et al. (2002) JBC 277:26733). In other applications, galactosidation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, the effectiveness of the isolated anti-Nectin-4 protein antibodies of the present invention for disease diagnosis, prevention, and / or treatment can be enhanced by introducing one or more amino acid modifications into the Fc region of the Nectin-4 protein antibodies provided herein to generate Fc region variants.

[0092] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention can be conjugated with a drug to form an antibody-drug conjugate (ADC). The drug used for coupling can include, but is not limited to, a chemotherapeutic agent, an immunomodulator (e.g., an immunostimulant), an oligonucleotide, a toxin, a radiotoxin, a cytokine, an apoptotic agent, an anti-angiogenic agent, or a lymphokine. In some embodiments, the drug coupled to the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention is a cytotoxin.

[0093] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention is coupled to a diagnostic or detectable reagent. Such immunoconjugates can be used to monitor disease progression, severity, and / or prognosis. Diagnosis and detection can be achieved by coupling the antibody to a detectable substance, including, but not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and various positron-emitting metals and non-radioactive paramagnetic metal ions using positron emission tomography.

[0094] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention can be used to prepare a multispecific antibody as part of a multispecific antibody.

[0095] III. Multispecific Antibodies of the Invention Monoclonal antibodies have been established as antitumor therapeutic agents over the past 20 years, but they limit the ability of immune cells, such as T cells and NK cells, to recruit and effectively exert cytotoxic activity at tumor sites.

[0096] Multispecific antibodies (e.g., bispecific antibodies) can specifically bind to different antigens. Therefore, when one antigen is located on a specific immune cell and the other antigen is located on a diseased cell, the multispecific antibody (e.g., bispecific antibody) can redirect the specific immune cell to the diseased cell, thereby enhancing the immune cell's ability to kill the diseased cell. Multispecific antibodies (e.g., bispecific antibodies) can also be designed as signal transduction pathways that simultaneously act on two or more different mediators.

[0097] Antibody engineering has led to the development of many imaginative forms of multispecific antibodies (e.g., bispecific antibodies), and their applicability in disease treatment is also being investigated (Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9(2):182-212).

[0098] Multispecific antibodies (e.g., bispecific antibodies) can be divided into many types depending on their constituent parts and construction methods. For example, they can be divided into symmetric and asymmetric structures depending on the substantial left-right symmetry of the multispecific antibody structure, into antibodies with and without an Fc region depending on whether they contain an IgG Fc region, and into bivalent, trivalent, tetravalent, or higher polyvalent antibodies depending on the number of antigen-binding sites in the multispecific antibody.

[0099] The present invention provides a multispecific antibody that can simultaneously bind to a molecule on an immune cell and to the Nectin-4 protein on a tumor cell.

[0100] In some embodiments, the multispecific antibody of the present invention comprises an antibody or antigen-binding fragment that specifically binds to 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 costimulatory molecule in immune cells. The costimulatory molecule may be represented by the protein family of TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte 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.

[0101] In some exemplary embodiments of the present invention, the present invention provides a bispecific antibody comprising an antibody or antigen-binding fragment that specifically binds to 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 costimulatory molecule in immune cells (e.g., an antibody or antigen-binding fragment that binds to a T cell costimulatory receptor such as ICOS, 4-1BB, CD28, or CD86).

[0102] The isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention included in the bispecific antibody of the present invention comprises at least a Nectin-4 protein-binding domain, i.e., comprises at least a heavy chain variable domain and a light chain variable domain that specifically bind to Nectin-4 protein. The antibody or antigen-binding fragment that binds to a costimulatory molecule in immune cells included in the bispecific antibody of the present invention comprises at least a costimulatory molecule-binding domain in immune cells, i.e., comprises at least a heavy chain variable domain and a light chain variable domain that specifically bind to a costimulatory molecule in immune cells.

[0103] The Nectin-4 protein-binding domain and the costimulatory molecule-binding domain in immune cells can be linked directly or indirectly 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 in immune cells can each be linked to a separate multimerization domain. The association of one multimerization domain with the other multimerization domain promotes the association between the Nectin-4 protein-binding domain and the costimulatory molecule-binding domain in immune cells, thereby forming the bispecific antibody molecule. The multimerization domain can be linked to immunoglobulin C H It may also be a polypeptide comprising three domains, such as the Fc portion of an immunoglobulin (C H 2~C H 3 domains), for example, an IgG selected from the isotypes IgG1, IgG2, IgG3 and IgG4, and the Fc domain of any isotype within each isotype group.

[0104] Bispecific antibodies of the invention can be prepared using any bispecific antibody format or technology. Specific exemplary bispecific formats that can be used in the invention include, for example, scFv or diabody-based bispecific formats, IgG-scFv fusions, dual variable domain (OVO)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEEO)body, leucine zipper, Ouobody, IgG1 / IgG2, dual acting Fab (OAF)-IgG, and Mab. 2 These include, but are not limited to, bispecific forms (see, for example, Klein et al., 2012, mAbs 4:6, 1-11 and references cited therein).

[0105] In some embodiments, the bispecific antibodies of the invention comprise, from the amino terminus to the carboxy terminus: (a) a first portion that is a Nectin-4 protein antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein; and (b) a second portion that is an anti-4-1BB scFv; For example, the anti-4-1BB scFv includes three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 32 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 33, or a variant having one or more CDRs with three or fewer amino acid changes per CDR region among the six CDR regions.

[0106] In some embodiments, the bispecific antibodies of the invention comprise, from the amino terminus to the carboxy terminus: (a) a first portion that is a Nectin-4 protein antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein; and (b) a second portion that is an anti-4-1BB scFv comprising, based on Kabat numbering, an HCDR1 set forth in SEQ ID NO: 63, an HCDR2 set forth in SEQ ID NO: 64, and an HCDR3 set forth in SEQ ID NO: 65, and an LCDR1 set forth in SEQ ID NO: 66, an LCDR2 set forth in SEQ ID NO: 67, and an LCDR3 set forth in SEQ ID NO: 68; In some embodiments, the bispecific antibodies of the invention comprise, from the amino terminus to the carboxy terminus: (a) a first portion that is a Nectin-4 protein antibody or antigen-binding fragment of the present invention that specifically binds to the Nectin-4 protein; and (b) a second portion that 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; The carboxy terminus of the heavy chain of the first portion and the amino terminus of the second portion are covalently linked.

[0107] In some embodiments, the bispecific antibody of the invention comprises two heavy chains and two light chains, wherein each heavy chain comprises, from amino terminus to carboxy terminus, a first portion heavy chain, a connecting peptide, and a second portion, and each light chain is a first portion light chain, and preferably each heavy chain comprises the amino acid sequence set forth 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 thereto, and each light chain comprises the amino acid sequence set forth 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 thereto; each heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 36, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and each light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; or Each heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 37, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and each light chain comprises the amino acid sequence set forth in SEQ ID NO: 31, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0108] 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, and more particularly 1 to 20 amino acids in length. In some embodiments, the connecting peptide is (GxS)n or (GxS)nGm, where G is glycine, S is serine, x is an integer of 1 to 4, n is an integer of 1 to 7, and m is an integer of 0 to 3. In one specific embodiment, the connecting peptide is (G4S)3 (SEQ ID NO: 69).

[0109] In some embodiments, the anti-Nectin-4x4-1BB bispecific antibody of the present invention has one or more of the following properties: (a) less than about 10 nM, e.g., about 1 nM, about 10 -1 nM, about 10 -2 nM, about 10 -3 nM binding-dissociation equilibrium constant K D and binds to human Nectin-4 protein at a concentration of 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 or binds to human 4-1BB protein at a concentration of less than about 100 nM, e.g., about 10 nM, about 1 nM, about 10 -1 nM, about 10 -2 nM binding-dissociation equilibrium constant K D and binds to the cynomolgus monkey Nectin-4 protein at a binding dissociation equilibrium constant K of less than about 150 nM, e.g., about 15 nM, about 1.5 nM, about 0.15 nM, or about 0.015 nM. D binds to cynomolgus monkey 4-1BB protein, (b) the bispecific antibody is capable of activating the human 4-1BB signaling pathway only in the presence of Nectin-4 protein, as measured in an NF-κB-Luc / 4-1BB reporter gene cell system, and / or the bispecific antibody is unable to activate the human 4-1BB signaling pathway in the absence of Nectin-4 protein; and / or (c) The bispecific antibody has an in vivo antitumor effect and is safe for test animals.

[0110] In the bispecific antibodies of the invention, the Fc domain may comprise one or more amino acid alterations (e.g., insertions, deletions or substitutions) compared to the wild-type, naturally occurring form of the Fc domain.

[0111] In some embodiments, the present invention provides bispecific antibodies comprising an Fc domain comprising one or more mutations that enhance or reduce binding of the bispecific antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present invention provides a bispecific antibody comprising an Fc domain comprising one or more mutations that enhance or reduce binding of the bispecific antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention includes bispecific antibodies containing mutations in three regions, one or more of which improve the affinity between the Fc domain and FcRn in an acidic environment (e.g., in an endosome at a pH of about 5.5 to about 6.0). Such mutations can improve the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0112] For example, the present invention includes bispecific antibodies comprising an Fc domain, wherein the Fc domain comprises one or more pairs (sets) of mutations selected from the following: 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 above Fc domain mutations with other mutations in antibody variable domains disclosed herein is within the scope of the present invention.

[0113] In some embodiments, the bispecific antibodies provided herein are modified to increase or decrease their degree of glycosylation. Addition or deletion of glycosylation sites in bispecific antibodies can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites. When a bispecific antibody contains an Fc region, the carbohydrates linked to the Fc region can be modified. In some applications, modifications to remove undesired glycosylation sites, such as removing a fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function, can be useful (see Shield et al. (2002) JBC 277:26733). In other applications, galactosidation modifications can be performed to modulate complement-dependent cytotoxicity (CDC).

[0114] IV. Nucleic Acids of the Invention and Host Cells Containing Them 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.

[0115] In another aspect, the present invention provides a nucleic acid encoding any of the above multispecific antibodies of the invention or any one chain thereof.

[0116] In one embodiment, a vector comprising the nucleic acid 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 HEK293 cells), or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0117] The present invention also includes nucleic acids that hybridize under stringent conditions to the above-described nucleic acids or that encode polypeptide sequences that have one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions compared to the above-described nucleic acids.

[0118] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0119] 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 utilized to achieve this, including, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, particle gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are cultured in culture and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and recovering the antibody molecules produced are known to those skilled in the art and can be modified or optimized depending on the particular expression vector and mammalian host cell used, as described herein and by methods known in the art.

[0120] Alternatively, cells that have stably integrated DNA into their chromosomes can be selected by introducing one or more selectable markers into the transfected host cells. Markers can provide, for example, prototrophy to an auxotrophic host, biocide (e.g., antibiotic) resistance, or heavy metal (e.g., copper) resistance. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may be required for optimization of mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0121] In one embodiment, a host cell is provided comprising a polynucleotide of the present invention. In some embodiments, a host cell is provided comprising an expression vector of the present invention. 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 E. coli. Host cells may also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells. Vertebrate cells may also be used as hosts. For example, mammalian cell lines modified 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 (HEK293 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, and myeloma cell lines such as Y0, NS0, P3X63, and Sp2 / 0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki & Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo eds., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one preferred embodiment, the host cells are HEK293 or CHO cells, for example, HEK293-6E cells.

[0122] V. Production and purification of the isolated anti-Nectin-4 protein antibody of the present invention and multispecific antibodies containing the same 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, the method comprising culturing a host cell comprising the isolated anti-Nectin-4 protein antibody or nucleic acid encoding the multispecific antibody, or an expression vector for said nucleic acid, under conditions suitable for expression of the nucleic acid encoding the Nectin-4 protein antibody or nucleic acid encoding the multispecific antibody, and optionally isolating the Nectin-4 protein antibody or multispecific antibody. In one embodiment, the method further comprises recovering the Nectin-4 protein antibody or multispecific antibody from the host cell (or host cell culture medium).

[0123] 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 then inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid can be easily isolated and sequenced by conventional processes, for example, by using an oligonucleotide probe capable of specifically binding to the nucleic acid encoding the Nectin-4 protein antibody or multispecific antibody of the present invention.

[0124] The Nectin-4 protein antibodies or multispecific antibodies of the present invention prepared as described herein can be purified by known conventional techniques, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions for purifying a particular protein will depend on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. The purity of the Nectin-4 protein antibodies or multispecific antibodies of the present invention can be determined by any one of a variety of well-known analytical methods, including size-exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0125] VI. Method for measuring the activity of the isolated anti-Nectin-4 protein antibody or antigen-binding fragment thereof of the present invention, or a multispecific antibody comprising the same Various assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activities of the Nectin-4 protein antibodies or multispecific antibodies provided herein.

[0126] In one embodiment, the antigen-binding activity of the Nectin-4 protein antibody or multispecific antibody of the present invention is measured by a known method, such as ELISA. Binding of the Nectin-4 protein and / or multispecific antibody to each target antigen can be measured by methods known in the art, and exemplary methods are disclosed herein. In some embodiments, binding of the Nectin-4 protein antibody or multispecific antibody of the present invention to the Nectin-4 protein is measured by SPR or biolayer interferometry.

[0127] The present invention further provides an assay method for identifying a multispecific antibody having biological activity. The biological activity may include, for example, measuring the activation of the 4-1BB signaling pathway by an anti-Nectin-4×4-1BB bispecific antibody using a HEK293 / NF-κB-Luc / 4-1BB reporter gene cell system.

[0128] VII. Pharmaceutical Compositions and Formulations In some embodiments, the present invention provides a composition comprising any of the isolated anti-Nectin-4 protein antibodies or multispecific antibodies described herein, and preferably, the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical auxiliary material. In one embodiment, the composition (e.g., pharmaceutical composition) comprises a combination of a Nectin-4 protein antibody or multispecific antibody of the present invention and one or more other therapeutic agents (e.g., a chemotherapeutic agent, a tumor vaccine, an antibody that binds to a specific antigen on tumor cells, or an antibody that consumes tumor cells).

[0129] In some embodiments, the pharmaceutical compositions or formulations of the present invention comprise suitable pharmaceutical auxiliary materials, such as pharmaceutical carriers known in the art, pharmaceutical excipients including buffering agents.

[0130] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonicity agents, absorption delaying agents, and the like. Pharmaceutical carriers suitable for the present invention may be sterile liquids, such as water and oils, including peanut oil, soybean oil, mineral oil, sesame oil, and the like, of petroleum, animal, vegetable, or synthetic origin. Pharmaceutical formulations containing 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 auxiliary materials (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution.

[0131] The pharmaceutical compositions or formulations of the present invention may further comprise two or more active ingredients, as needed for the particular indication being treated, preferably 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 present in an amount effective for the intended use and in suitable combination.

[0132] 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 present invention, which matrices are presented in shaped articles, e.g., films, or in the form of microcapsules.

[0133] VIII. Combination Products or Reagent Kits In some embodiments, the present invention further provides a combination product, which comprises at least one isolated anti-Nectin-4 protein antibody or multispecific antibody of the present invention, or which further comprises one or more other anti-tumor agents.

[0134] In some embodiments, the two or more components of the combination product may be administered to a subject sequentially, separately or simultaneously.

[0135] In some embodiments, the present invention further provides a reagent kit comprising the isolated anti-Nectin-4 protein antibody, multispecific antibody, pharmaceutical composition or combination product of the present invention, and an optional packaging addendum for administration instructions.

[0136] In some embodiments, the present invention further 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 packaging insert for administration instructions.

[0137] IX. Use of the isolated anti-Nectin-4 protein antibody of the present invention and multispecific antibodies containing the same 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 in vitro or ex vivo cultured cells or to a subject, e.g., a human subject, to treat, prevent, and / or diagnose Nectin-4 protein antigen-associated diseases, such as cancer.

[0138] 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, urine, or a tissue biopsy sample (e.g., derived 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 a Nectin-4 protein antibody or multispecific antibody described herein, or administering the Nectin-4 protein antibody or multispecific antibody to a subject, under conditions that allow for interaction; and (ii) detecting the formation of a complex between the Nectin-4 protein antibody or multispecific antibody and the sample (and optionally the control sample). The formation of a complex indicates the presence of the relevant antigen and may indicate the applicability or need for the treatment and / or prevention described herein.

[0139] In some embodiments, the relevant antigen is detected before treatment, for example, before initiating treatment or before a certain treatment after a treatment interval. Available detection methods include immunohistochemistry, immunocytochemistry, FACS, ELISA assay, PCR techniques (e.g., RT-PCR), or in vivo imaging techniques. Generally, 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 the bound or unbound conjugate. Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (e.g., nuclear magnetic resonance active) materials, and radioactive materials.

[0140] In some embodiments, the level and / or distribution of the relevant antigen is determined in vivo, e.g., by a non-invasive method (e.g., a suitable imaging technique (e.g., positron emission tomography (PET) scanning) using, for example, a Nectin-4 protein antibody or multispecific antibody of the present invention labeled with a detectable substance. In one embodiment, for example, a PET reagent (e.g., 18The level and / or distribution of the relevant antigen is measured in vivo by detecting the Nectin-4 protein antibody or multispecific antibody of the present invention detectably labeled with F-fluorodeoxyglucose (FDG).

[0141] In one embodiment, the present invention provides a diagnostic reagent kit comprising a Nectin-4 protein antibody or multispecific antibody described herein and an instruction manual.

[0142] In another aspect, the present invention relates to using the Nectin-4 protein antibody or multispecific antibody of the present invention to treat or prevent cancer in vivo and to regulate 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 may 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, such combinations may be administered in any order or simultaneously.

[0143] Thus, in one embodiment, the present invention provides a method for treating or preventing cancer and modulating immune responses in a subject, the method comprising administering to the subject a therapeutically effective amount of a Nectin-4 protein antibody or multispecific antibody described herein. In another embodiment, the present invention provides a method for preventing the occurrence or recurrence of cancer in a subject, the method comprising administering to the subject a prophylactically effective amount of a Nectin-4 protein antibody or multispecific antibody described herein.

[0144] In some embodiments, cancers treated and / or prevented using a Nectin-4 protein antibody or multispecific antibody include, but are not limited to, solid tumors that express Nectin-4 protein, and blood cancers (e.g., leukemia, lymphoma, and myeloma, e.g., multiple myeloma). In some embodiments, cancers treated and / or prevented using a Nectin-4 protein antibody or multispecific antibody include, but are not limited to, intraepithelial carcinomas and metastatic cancers that express Nectin-4 protein.

[0145] In one embodiment, the cancer is a solid tumor that expresses Nectin-4 protein. Examples of solid tumors include malignant tumors, such as sarcomas and cancers of multiple organ systems, such as those affecting the esophagus, lung, breast, ovary, lymphatic, gastrointestinal tract (e.g., colon), anus, genital and urogenital tract (e.g., kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neuronal 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 malignant tumors. The cancer may be early-stage, intermediate-stage, or late-stage cancer, or may be metastatic cancer.

[0146] 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'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.

[0147] The following examples are set forth to aid in the understanding of the present invention, and are not intended, nor should they be construed, as limiting the scope of the invention in any manner whatsoever.

[0148] Example Example 1. Production of anti-human Nectin-4 antibody Hybridomas encoding the anti-human Nectin-4 antibodies of the present invention can be prepared by the method described below. However, the method for preparing the anti-human Nectin-4 antibodies of the present invention is not limited to this method, and they can also be prepared by other methods known in the art.

[0149] 1.1 Animal immunity Five BALB / c mice were immunized with human Nectin-4-his protein (ACRO Biosystems, product number: NE4-H52H3) as the immunogen. The BALB / c mice received four immunization injections, once every two weeks (Table 1). Seven days after the second and third immunizations, blood was collected from each mouse via venous route. Antibody titers in the serum of the immunized animals were detected by ELISA using human Nectin-4-his protein. After a sufficient increase in antibody titer was observed, a final booster immunization was performed. Finally, two immunized mice were selected and used to obtain antibody-producing cells. Four days after the final booster immunization, the spleens of the immunized mice were harvested, and lymphocytes isolated from the spleens were fused with myeloma cells to produce hybridomas.

[0150] [Table 1]

[0151] 1.2 Screening of target hybridomas Cell culture supernatants from 4,800 hybridomas from Example 1.1 were initially screened by ELISA using human Nectin-4-his protein. After ELISA, 198 positive supernatants were selected and subjected to flow cytometry detection against MCF-7 cell line (human breast cancer cell line). Hybridoma clones corresponding to the positive supernatants that bound to MCF-7 cells were confirmed in the next round of ELISA. Finally, 21 hybridoma clones were selected and advanced to the next subcloning stage.

[0152] 1.3 Subclones of parent hybridomas The selected hybridoma cell line (parent clone) was subcloned to ensure monoclonality. Subcloning was performed by reseeding 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, product number: NE4-H52H3) and cynomolgus monkey Nectin-4 (KACTUS, product number: NEC-CM104) as antigens. Hybridomas producing antibodies binding to human Nectin-4 and cynomolgus monkey Nectin-4 were obtained. The produced antibodies were named mouse antibody 23-H4F3, mouse antibody 6-B9B6, and mouse antibody 32-A12C7, respectively.

[0153] 1.4 Sequencing of anti-Nectin-4 antibodies produced by hybridomas Antibody sequencing was completed by GenScript NGS sequencing, and the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) are shown in Table 2.

[0154] [Table 2]

[0155] Example 2: Characterization of anti-Nectin-4 chimeric antibodies 2.1 Synthesis and expression of anti-Nectin-4 chimeric antibody The DNA sequences of the mouse antibodies in Table 2 were codon optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd. The genes encoding the VH regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing a gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 7), thereby obtaining plasmids encoding the heavy chains of each anti-Nectin-4 antibody. The genes encoding the VL regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing a gene encoding the human κ light chain constant region (SEQ ID NO: 8), thereby obtaining plasmids encoding the light chains of each anti-Nectin-4 antibody.

[0156] Plasmids encoding the heavy and light chains of each anti-Nectin-4 antibody were co-transfected into ExpiCHO-S cells, thereby expressing the mouse chimeric antibodies shown in Table 3. Enfortumab (a fully human monoclonal antibody against Nectin-4) (heavy chain amino acid sequence is SEQ ID NO: 9, and light chain amino acid sequence is SEQ ID NO: 10) was used as a control antibody, and the sequences were derived from PCT Publication No. WO2021030240A1. Similarly, the control antibody enfortumab was expressed. The antibodies were then purified accordingly.

[0157] Specifically, the anti-Nectin-4 chimeric antibody, as well as the control antibody enfortumab, were expressed in ExpiCHO cells according to the manufacturer's instructions. TM Plasmids encoding the heavy and light chains of the anti-Nectin-4 antibodies listed in Table 3 were co-transfected into ExpiCHO-S cells using an expression system (ThermoFisher, Cat. #A29133). After transfection, the cells were cultured for 10-12 days. When cell viability dropped to 60-70%, the supernatant was collected. The expressed and secreted antibodies were purified using a MabSelect Sure Protein A affinity chromatography system (GE Healthcare). The purified antibodies were concentrated, sterile filtered, and subjected to SDS-PAGE and molecular exclusion to determine the purity of the antibody protein. The results indicated that the antibody purity was greater than 90% and suitable for subsequent experiments.

[0158] [Table 3]

[0159] 2.2 Binding of anti-Nectin-4 chimeric antibodies to HT-1376 or cynomolgus monkey Nectin-4 artificial cells Cell binding experiments were performed to determine whether the anti-Nectin-4 chimeric antibody of the present invention can bind to artificial cells expressing human Nectin-4 protein expressed by tumor cells (e.g., the human bladder cancer cell line HT-1376) or cynomolgus monkey Nectin-4 protein.

[0160] The sequence of the extracellular and transmembrane domains (SEQ ID NO: 17) encoding cynomolgus monkey Nectin-4 (XP_005541277.1) was cloned into a PiggyBac Dual promoter (SBI, product number: PB513-B1) expression vector and transfected into CHO-K1 cells (ATCC, product number: CCL-61 TM ) were transfected by electroporation, and then screened using 8 μg / mL puromycin (Gibco, product number: A1113802) to obtain CHO-K1 cells that highly express cynomolgus monkey Nectin-4 (hereinafter also referred to as CHO-K1 / cynoNectin4).

[0161] Adherent HT-1376 (Nanjing Kebai Biotechnology Co., Ltd., product number CBP60310) or CHO-K1 / cynoNectin4 cell lines were enzymatically digested to obtain single-cell suspensions of the two cells. The cells were centrifuged at 300 × g for 4 minutes at room temperature, and the medium was discarded. The resulting cell pellets were washed once with PBS. They were resuspended in gradient-diluted mouse chimeric anti-Nectin-4 antibody prepared in Example 2.1 (initial concentration 200 nM, 4-fold gradient dilution, total 8 concentrations) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098) diluted 1:200 was added and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS, resuspended, and then subjected to two-channel fluorescence signal detection using a flow cytometer. The results are shown in Figure 1A and Figure 1B.

[0162] Figure 1A shows that all of the mouse chimeric anti-Nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) were able to bind to HT-1376 cells expressing human Nectin-4, with the EC50 values ​​of antibody 23-H4F3 being 0.06405 nM, antibody 6-B9B6 being 0.2489 nM, and antibody 32-A12C7 being 0.2998 nM. Figure 1B shows that all of the mouse chimeric anti-Nectin-4 antibodies (i.e., antibody 23-H4F3, antibody 6-B9B6, and antibody 32-A12C7) were able to bind to cells expressing cynomolgus monkey Nectin-4 protein, with an EC50 of 0.4113 nM for antibody 23-H4F3, 0.7186 nM for antibody 6-B9B6, and 1.053 nM for antibody 32-A12C7. The binding ability of chimeric antibody 23-H4F3 to each cell line was superior to that of antibody 6-B9B6 and antibody 32-A12C7.

[0163] 2.3 Binding of anti-Nectin-4 antibodies to the Nectin family The binding ability of the anti-Nectin-4 antibody obtained in Example 2.1 to Nectin family members was detected by ELISA.

[0164] Human Nectin-1 protein (ACRO Biosystems, product number: PV1-H5223), human Nectin-2 protein (Sino Biological, product number: 10005-H08H), human Nectin-3 protein (Sino Biological, product number: 10852-H08H), and human Nectin-4 protein (Sino Biological, product number: HPLC-19771-H08H) were immobilized on a 96-well plate by overnight incubation at 4°C. The 96-well plate was then blocked by incubating with 1% BSA in PBS at 37°C for 1 hour. After blocking, the 96-well plate was 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 and incubated for 1 hour at 37°C with peroxidase-labeled goat anti-human Fc IgG (Jackson ImmunoResearch, product number 109-035-098) diluted 1 / 25,000 in binding buffer. The plate was then washed again, developed with TMB, and the reaction was terminated with 1 M H2SO4.

[0165] Representative binding curves of antibody 23-H4F3 and antibody 6-B9B6 to human Nectin family proteins are shown in Figures 2A and 2B, respectively.

[0166] As can be seen from Figures 2A and 2B, antibody 23-H4F3 and antibody 6-B9B6 specifically bound only to human Nectin-4, but did not bind to any of Nectin-1, Nectin-2, and Nectin-3.

[0167] Example 3: Humanization of mouse anti-Nectin-4 antibody and its characterization 3.1 Humanization of mouse anti-Nectin-4 antibody The 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 to obtain the humanized heavy chain variable region framework IGHV1-3*01, which has a relatively high homology to the antibody 23-H4F3 heavy chain variable region. The humanized light chain variable region framework IGKV1-6*01 and IGKV4-1*01, which have a relatively high homology to the light chain variable region, were obtained. The CDRs of the antibody 23-H4F3 heavy chain variable region and light chain variable region were then grafted into the corresponding humanized frameworks to form the humanized antibody 23-H4F3 variable region. To maintain the affinity of the humanized antibody for Nectin-4, back mutations were required for the resulting humanized antibody variable region.

[0168] The sequences of the humanized heavy chain variable region and humanized light chain variable region were obtained, respectively. The amino acid sequences of the variable regions were sent to General 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 a 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 a gene encoding the human κ 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 shows the names and heavy chain variable region sequences of each heavy chain and each light chain after humanization of the mouse antibody 23-H4F3.

[0169] [Table 4]

[0170] 3.2 Expression and purification of humanized anti-Nectin-4 antibody As described in Example 2.1, a plasmid encoding the full-length heavy chain of an anti-Nectin-4 humanized antibody and a plasmid encoding the full-length light chain of an anti-Nectin-4 humanized antibody were combined (Table 5) and co-transfected into ExpiCHO-S cells to express each anti-Nectin-4 humanized antibody, and the corresponding purification was 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 detected by SDS-PAGE and molecular exclusion chromatography (SEC).

[0171] [Table 5]

[0172] 3.3 Physicochemical analysis of humanized anti-Nectin-4 antibody The purity of the obtained humanized anti-Nectin-4 antibodies listed in Table 5 was confirmed by molecular exclusion chromatography. Specifically, 20 μg of each humanized anti-Nectin-4 antibody sample listed in Table 5 was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM NaSO (pH 7.0) as the running buffer. Electrophoresis was performed for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. The results indicated that the purity of each humanized anti-Nectin-4 antibody listed in Table 5 exceeded 90% and was suitable for subsequent experiments.

[0173] 3.4 Binding of humanized anti-Nectin-4 antibodies to tumor cell lines and artificial cells expressing cynomolgus monkey Nectin-4 Cell binding experiments were performed to determine whether the anti-Nectin-4 humanized antibodies of the present invention can bind to human or cynomolgus monkey Nectin-4 protein stably expressed on the cell surface. The specific detection method was as described in Example 2.2, except that the humanized anti-Nectin-4 antibodies listed in Table 5 were used. The results are shown in Figures 3A and 3B.

[0174] Figures 3A and 3B show that each of the anti-Nectin-4 humanized antibodies in Table 5 can bind to HT-1376 cells and cells expressing cynomolgus monkey Nectin-4 protein, respectively, and that the binding ability of each anti-Nectin-4 humanized antibody is comparable to that of the control antibody enfortumab.

[0175] Example 4 Preparation of anti-Nectin-4x4-1BB bispecific antibody and affinity detection 4.1 Bispecific antibody construction In this example, an anti-Nectin-4 x 4-1BB bispecific antibody was constructed, in which the anti-Nectin-4 portion was derived from the chimeric antibody 23-H4F3 or 32-A12C7 of Example 2.1 or the humanized antibody huH4F3-5 of Example 3.2, and the anti-4-1BB portion had a heavy chain variable region shown in SEQ ID NO: 32 and a light chain variable region shown in SEQ ID NO: 33. The present application obtained an anti-Nectin-4 x 4-1BB bispecific antibody using standard construction methods, and its amino acid sequence is listed in the Sequence Listing, and comprises a sequence derived from the anti-Nectin-4 antibody in order from the N-terminus to the C-terminus, and a single-chain antibody portion in which anti-4-1BB is linked to the C-terminus of the anti-Nectin-4 antibody Fc region.

[0176] Specifically, an anti-Nectin-4x4-1BB bispecific antibody molecule was constructed as follows.

[0177] Anti-Nectin-4 x 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 of the human IgG1 mutant heavy chain constant region (SEQ ID NO: 34). Anti-4-1BB VH and VL were amplified using antibody P4B-3 (see CN114555638A) as a template. The nucleotide sequence encoding the anti-4-1BB ScFv (i.e., VH-(G4S)3 linker-VL) was ligated 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). The lysine at the Fc terminus was substituted with alanine to prevent cleavage and substitution. An expression vector for the anti-Nectin-4x4-1BB heavy chain was obtained, of which the anti-Nectin-4x4-1BB heavy chain comprises, from N-terminus to C-terminus, the anti-Nectin-4 heavy chain (with the terminal K mutated to A) and the 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. Bispecific antibody light chains: The corresponding light chains of the bispecific antibodies are shown in Table 6.

[0178] [Table 6] The resulting heavy chain and light chain expression vectors (a plasmid containing the H4F3-41BB / A12C7-41BB / huH4F3-5-41BB heavy chain-encoding nucleic acid, respectively, and a plasmid containing the 23-H4F3-L / A12C7-L / huH4F3L-B light chain-encoding nucleic acid, respectively) were co-transfected into ExpiCHO-S cells and expressed under appropriate conditions to obtain the bispecific antibody protein; the expression, purification, and preliminary analysis steps were the same as in Example 2.1.

[0179] 4.2 Detection of affinity of bispecific antibodies to human / cynomolgus monkey Nectin-4 and 4-1BB In this experiment, the binding affinity of the huH4F3-5-41BB bispecific antibody to human Nectin-4 protein (ACRO Biosystems, product number: NE4-H52H3), cynomolgus monkey Nectin-4 protein (KACTUS, product number: NEC-CM104), human 4-1BB protein (Sino Biological, product number: 10041-H08H), or cynomolgus monkey 4-1BB protein (Sino Biological, product number: 90847-K08H) was detected using ForteBio Octet RED96e according to the manufacturer's instructions.

[0180] Briefly, the AHC sensor (ForteBio, product number: 18-5060) was placed in electrophoresis buffer (1X PBS XiGene, product number: XG3650, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 min. The kinetic experiment was carried out in a 96-well plate according to the following steps: a) Baseline equilibration with running buffer for 180 s; b) Each bispecific antibody diluted in electrophoresis buffer was added to a final concentration of 5 μg / mL and immobilized for 200 s. c) Equilibrate the baseline with running buffer for 300 s; d1) Human Nectin-4 protein or cynomolgus monkey Nectin-4 protein diluted with running buffer was added to each well at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. The binding time was 200 s and the dissociation time was 600 s. The experimental data were fitted and calculated using a 1:1 binding model in Fortebio Data Analysis software. d2) Human 4-1BB protein or cynomolgus monkey 4-1BB protein diluted with running buffer was added to each well at concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. Binding was allowed for 240 s, followed by dissociation for 400 s. Binding was allowed for 240 s, followed by dissociation for 600 s. The experimental data were fitted and calculated using a 1:1 binding model in Fortebio Data Analysis software.

[0181] 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.

[0182] [Table 7] Table 7 shows that the bispecific antibodies 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. The anti-Nectin-4 x 4-1BB bispecific antibodies obtained in the present invention have a K D K values ​​for human 4-1BB protein D The anti-Nectin-4 × 4-1BB bispecific antibody targets human Nectin-4 with high affinity, effectively reducing the side effects of targeting human 4-1BB.

[0183] Example 5 Activation of the 4-1BB signaling pathway by humanized bispecific antibodies HEK293 cell line (ATCC, Catalog No. CRL-1573) was maintained in DMEM medium containing 10% FBS and placed in a humidified incubator at 37°C and 5% CO2. TMHEK293 cells were co-transfected with a plasmid encoding the nucleic acid sequence of human 4-1BB (NP_001552.2) and pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Catalog No.: E849A) using 2000 Transfection Reagent (Invitrogen, Catalog No.: 11668019). After screening with puromycin (Gibco, Catalog No.: A1113802) and hygromycin (Gibco, Catalog No.: 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.

[0184] HEK293 / NF-κB-Luc / 4-1BB cells were seeded into a 384-well plate (20,000 cells / well, 20 μL). Next, HT-1376 cells or 293T cells (not expressing Nectin-4) (ATCC, Catalog No. CRL-1573) were seeded into the 384-well plate (10,000 cells / well, 20 μL). A 5-fold gradient of antibody (starting at a final concentration of 10 nM, 5-fold dilutions, 9 points) was prepared in DMEM medium containing 10% FBS, and 10 μL of antibody was added to each well. The culture plate was incubated for 6 hours in an incubator at 37°C with 5% CO2. After incubation, 30 μL of One-Glo TM The reagent (Promega, Catalog No. E6130) was added to the wells of the measurement plate, and luminescence was measured using a luminescence microplate reader (Tecan F200 Pro). The results are shown in Figures 4A and 4B.

[0185] As can be seen from Figures 4A and 4B, only in the presence of Nectin-4 protein, the huH4F3-5-41BB bispecific antibody and H4F3-41BB bispecific antibody could activate the human 4-1BB signaling pathway and further activate NF-κB, and showed a dose-dependent trend in the reporter gene system. However, in the absence of Nectin-4 protein, neither the huH4F3-5-41BB bispecific antibody nor the H4F3-41BB bispecific antibody could activate the human 4-1BB signaling pathway.

[0186] Example 6 In vivo efficacy of anti-Nectin-4x4-1BB bispecific antibody in MC38-hNectin4 tumor model The in vivo antitumor activity of the anti-Nectin-4x4-1BB bispecific antibody was investigated in the MC38-hNectin4 tumor model.

[0187] Humanized mice C57BL / 6-Tnfrsf9tm1 (TNFRSF9) / Bcgen expressing the human 4-1BB extracellular portion were purchased from Biocytogen JiangSu Co., Ltd.

[0188] Mouse colon cancer MC38 cells were purchased from Shunran Shanghai Biotechnology Co., Ltd., and genetically modified by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to overexpress human Nectin-4. These cells were designated MC38-hNectin4. The cells were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% inactivated fetal bovine serum.

[0189] 5 × 10 5 MC38-hNectin4 cells were subcutaneously implanted in 0.1 mL of PBS per mouse. The average tumor volume was 100 mm 3When the tumor volume reached 1000 mg / kg, 20 suitable mice were selected based on their tumor volume and body weight and randomly assigned to four experimental groups, with five mice per group. On days 0, 3, 7, 10, 14, 17, 21, and 24 after grouping, the mice were intraperitoneally administered blank control (PBS) (Group G1), A12C7-41BB bispecific antibody (5 mg / kg) (Group G2), H4F3-41BB bispecific antibody (5 mg / kg) (Group G3), or an equimolar amount of fusion protein Fc-41BB (SEQ ID NO: 38) (2.6 mg / kg) (Group G4). The fusion protein Fc-41BB was in a two-chain form and prepared in the same manner as in Example 2.1. During the experiment, the longest axis (L) and widest axis (W) of the tumor in the tumor-bearing mice were measured twice a week using a vernier caliper, and the tumor volume was calculated as V = L × W. 2 This was calculated using the formula / 2.

[0190] Treatment with the anti-Nectin-4x4-1BB bispecific antibodies A12C7-41BB and H4F3-41BB, and treatment with the fusion protein Fc-41BB resulted in significant tumor growth inhibition compared to the PBS group, and the A12C7-41BB and H4F3-41BB groups had higher tumor growth inhibition rates (TGI rates) than the Fc-41BB group (97.5% and 102.8%, respectively, compared to 81.8% in the Fc-41BB control group). At the end of the experiment, three mice remained alive in the A12C7-41BB bispecific antibody group, and two mice remained alive in the H4F3-41BB bispecific antibody group. The results are shown in Figure 5 and Table 8 below.

[0191] [Table 8]

[0192] Throughout the entire experimental process, the experimental animals were active and had good feeding status during the administration period, and all animals in groups G1 to G4 gained some weight, indicating that the animals tolerated the test subject well. The weight changes of all animals are shown in Figure 6 and Table 9.

[0193] [Table 9]

[0194] [Table 10] JPEG2026508244000012.jpg255161JPEG2026508244000013.jpg255168JPEG2026508244000014.jpg255166JPEG2026508244000015.jpg255159 JPEG2026508244000016.jpg255170JPEG2026508244000017.jpg248170JPEG2026508244000018.jpg253170JPEG2026508244000019.jpg189170

[0195] While exemplary embodiments of the present invention have been described above, those skilled in the art should understand that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments set forth herein.

Claims

1. An isolated antibody or an antigen-binding fragment of said isolated antibody that specifically binds to Nectin-4 protein, (a) a variant having one or more CDRs, each of which has three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 1 and three CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO: 2, or three or less amino acid changes per CDR region of the six CDR regions; For example, three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 18 and three CDRs in the amino acid sequence of the light chain variable region shown in any one of SEQ ID NOs: 20 to 24, or Three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 19, and three CDRs in the amino acid sequence of a light chain variable region shown in any one of SEQ ID NOs: 20 to 24; (b) a variant having three CDRs in the amino acid sequence of a heavy chain variable region set forth in SEQ ID NO: 3 and three CDRs in the amino acid sequence of a light chain variable region set forth in SEQ ID NO: 4, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; or (c) a variant having three CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 5 and three CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO: 6, or one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; An isolated antibody or an antigen-binding fragment of the isolated antibody.

2. An isolated antibody or an antigen-binding fragment of the isolated antibody that specifically binds to Nectin-4 protein, wherein the antibody is selected from the group consisting of: (a) HCDR1 represented by SEQ ID NO: 40 or a variant of HCDR1 represented by SEQ ID NO: 40 with two or fewer amino acid changes, HCDR2 represented by SEQ ID NO: 41 or a variant of HCDR2 represented by SEQ ID NO: 41 with three or fewer amino acid changes, and HCDR3 represented by SEQ ID NO: 42 or a variant of HCDR3 represented by SEQ ID NO: 42 with two or fewer amino acid changes, as well as LCDR1 represented by SEQ ID NO: 43 or a variant of LCDR1 represented by SEQ ID NO: 43 with three or fewer amino acid changes, LCDR2 represented by SEQ ID NO: 44 or a variant of LCDR2 represented by SEQ ID NO: 44 with three or fewer amino acid changes, and LCDR3 represented by SEQ ID NO: 45 or a variant of LCDR3 represented by SEQ ID NO: 45 with two or fewer amino acid changes, (b) HCDR1 represented by SEQ ID NO: 46 or a variant of HCDR1 represented by SEQ ID NO: 46 with two or fewer amino acid changes, HCDR2 represented by SEQ ID NO: 47 or a variant of HCDR2 represented by SEQ ID NO: 47 with three or fewer amino acid changes, and HCDR3 represented by SEQ ID NO: 48 or a variant of HCDR3 represented by SEQ ID NO: 48 with two or fewer amino acid changes, and LCDR1 represented by SEQ ID NO: 49 or a variant of LCDR1 represented by SEQ ID NO: 49 with three or fewer amino acid changes, LCDR2 represented by SEQ ID NO: 50 or a variant of LCDR2 represented by SEQ ID NO: 50 with three or fewer amino acid changes, and LCDR3 represented by SEQ ID NO: 51 or a variant of LCDR3 represented by SEQ ID NO: 51 with two or fewer amino acid changes, or (c) HCDR1 represented by SEQ ID NO: 52 or a variant of the HCDR1 represented by SEQ ID NO: 52 with two or fewer amino acid changes, HCDR2 represented by SEQ ID NO: 53 or a variant of the HCDR2 represented by SEQ ID NO: 53 with three or fewer amino acid changes, and HCDR3 represented by SEQ ID NO: 54 or a variant of the HCDR3 represented by SEQ ID NO: 54 with two or fewer amino acid changes, as well as LCDR1 represented by SEQ ID NO: 55 or a variant of the LCDR1 represented by SEQ ID NO: 55 with three or fewer amino acid changes, LCDR2 represented by SEQ ID NO: 56 or a variant of the LCDR2 represented by SEQ ID NO: 56 with three or fewer amino acid changes, and LCDR3 represented by SEQ ID NO: 57 or a variant of the LCDR3 represented by SEQ ID NO: 57 with two or fewer amino acid changes, Preferably, the isolated antibody or antigen-binding fragment that specifically binds to the Nectin-4 protein is (a) HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 41, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45; HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 61, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 59, LCDR2 represented by SEQ ID NO: 44, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 43, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, HCDR1 represented by SEQ ID NO: 40, HCDR2 represented by SEQ ID NO: 58, and HCDR3 represented by SEQ ID NO: 42, and LCDR1 represented by SEQ ID NO: 60, LCDR2 represented by SEQ ID NO: 62, and LCDR3 represented by SEQ ID NO: 45, (b) HCDR1 represented by SEQ ID NO: 46, HCDR2 represented by SEQ ID NO: 47, and HCDR3 represented by SEQ ID NO: 48, and LCDR1 represented by SEQ ID NO: 49, LCDR2 represented by SEQ ID NO: 50, and LCDR3 represented by SEQ ID NO: 51; or (c) comprising an HCDR1 represented by SEQ ID NO: 52, an HCDR2 represented by SEQ ID NO: 53, and an HCDR3 represented by SEQ ID NO: 54, and an LCDR1 represented by SEQ ID NO: 55, an LCDR2 represented by SEQ ID NO: 56, and an LCDR3 represented by SEQ ID NO: 57; An isolated antibody or an antigen-binding fragment of the isolated antibody.

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

3. An isolated antibody or an antigen-binding fragment of the isolated antibody according to claim 1 or 2.

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

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

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

7. It is a bispecific antibody, and from the amino terminus to the carboxy terminus, (a) a first moiety that is an isolated antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein, e.g., the isolated antibody or antigen-binding fragment of any one of claims 1 to 5 that specifically binds to a Nectin-4 protein; and (b) a second portion that is an isolated antibody or antigen-binding fragment that specifically binds to T-cell costimulatory receptor 4-1BB; wherein the antigen-binding fragments in the first and second portions are Fab, Fab', F(ab')2, Fv, single-chain Fv, or single-chain Fab; For example, the bispecific antibody may comprise, from the amino terminus to the carboxy terminus: (a) a first portion, which is the isolated antibody or antigen-binding fragment of any one of claims 1 to 5, which specifically binds to Nectin-4 protein; and (b) a second portion that is an anti-4-1BB scFv; For example, the anti-4-1BB scFv comprises three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 32 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 33, or a variant having one or more CDRs with three or fewer amino acid changes per CDR region of the six CDR regions; For example, the anti-4-1BB scFv includes, based on Kabat numbering, HCDR1 shown in SEQ ID NO: 63 or a variant thereof with two or less amino acid changes, HCDR2 shown in SEQ ID NO: 64 or a variant thereof with two or less amino acid changes, and HCDR3 shown in SEQ ID NO: 65 or a variant thereof with two or less amino acid changes, as well as LCDR1 shown in SEQ ID NO: 66 or a variant thereof with two or less amino acid changes, LCDR2 shown in SEQ ID NO: 67 or a variant thereof with two or less amino acid changes, and LCDR3 shown in SEQ ID NO: 68 or a variant thereof with two or less amino acid changes. For example, the anti-4-1BB scFv includes, based on Kabat numbering, HCDR1 shown in SEQ ID NO: 63, HCDR2 shown in SEQ ID NO: 64, and HCDR3 shown in SEQ ID NO: 65, as well as 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 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; wherein the carboxy terminus of the heavy chain of the first portion and the amino terminus of the second portion are linked via a linker or directly without a linker, and for example, the linker is a connecting peptide represented by SEQ ID NO: 69 or SEQ ID NO:

70. The multispecific antibody of claim 6.

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

9. The bispecific antibody has one or more of the following properties: (a) less than about 10 nM, e.g., about 1 nM, about 10 －１ nM, about 10 －２ nM, about 10 －３ Binding-dissociation equilibrium constant K in nM Ｄ and binds to human Nectin-4 protein at less than about 1000 nM, e.g., about 100 nM, about 10 nM, about 1 nM, about 10 －１ Binding-dissociation equilibrium constant K in nM Ｄ or binds to human 4-1BB protein at a concentration of less than about 100 nM, e.g., about 10 nM, about 1 nM, about 10 －１ nM, about 10 －２ Binding-dissociation equilibrium constant K in nM Ｄ and binds to cynomolgus monkey Nectin-4 protein at a binding-dissociation equilibrium constant K of less than about 150 nM, e.g., about 15 nM, about 1.5 nM, about 0.15 nM, or about 0.015 nM. Ｄ binds to cynomolgus monkey 4-1BB protein, (b) the bispecific antibody is capable of activating the human 4-1BB signaling pathway only in the presence of Nectin-4 protein, as measured in an NF-κB-Luc / 4-1BB reporter gene cell line, and / or the bispecific antibody is unable to activate the human 4-1BB signaling pathway in the absence of Nectin-4 protein; and / or (c) the bispecific antibody has an in vivo antitumor effect, and is safe for test animals. A multispecific antibody according to claim 7 or 8.

10. Encoding 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. Isolated nucleic acid.

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

12. 12. A host cell comprising the nucleic acid of claim 10 or the vector of claim 11, preferably said host cell being a prokaryotic or eukaryotic cell, more preferably selected from E. coli cells, yeast cells, mammalian cells or other cells suitable for the preparation of antibodies or antigen-binding fragments, or multispecific antibodies, most preferably said host cell being a HEK 293 cell or a CHO cell. host cell.

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

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 10. A method for the treatment of a cancer, comprising administering to a patient an isolated antibody or antigen-binding fragment of any one of claims 1 to 5, or a multispecific antibody of any one of claims 6 to 9, and another therapeutic agent selected from an oncolytic drug, a cytotoxic agent, a cytokine, an inhibitor of another immune checkpoint molecule (e.g., PD-1), and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

15. 11. 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, in the preparation of a medicament for diagnosing, preventing and / or treating a disease associated with high expression of Nectin-4, for example wherein the disease is cancer, for example wherein the cancer is a solid tumor or a blood cancer, for example wherein the cancer is 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. use.

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