Nectin-4 antibody and its use

Abstract

The present disclosure provides antibodies and antibody fragments thereof that bind to Nectin-4. Such antibodies and antibody fragments are useful in the treatment of cancer, either alone or in combination with other agents.

Description

[Technical Field]

[0001] Cross-reference of related applications This international patent application claims priority to U.S. Provisional Patent Application No. 63 / 074,864, filed 4 September 2020, and U.S. Provisional Patent Application No. 63 / 166,622, filed 26 March 2021, each of which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on 3 September 2021, is named "122863_5003_WO_Sequence_Listing.TXT" and is 48 kilobytes in size.

[0003] field This disclosure relates to antibodies that bind to nectin-4 and antibody fragments thereof. This disclosure further relates to therapeutic and diagnostic compositions comprising these antibodies, as well as methods of using these compositions for the treatment and / or diagnosis of cancer. [Background technology]

[0004] The human nectin family includes nine homologs (nectin-1 to nectin-4 and nectin-like-1 to nectin-5) (Duraivelan et al., Sci Rep, 10:9434, 2020). Nectin proteins (nectin-1, nectin-2, nectin-3, and nectin-4) are calcium-independent immunoglobulin superfamily (IgSF) cell adhesion molecules that mediate cell-cell adhesion at adherent junctions in epithelial cells through homophilic or heterophilic trans-interactions. In normal epithelium, adherent junctions define cell polarity (a property often lost during tumor formation).

[0005] Nectin-1, -2, -3, and -4 are expressed as single-pass transmembrane type I glycoproteins and are characterized by a common domain configuration, consisting of an extracellular domain (ECD) with three tandem immunoglobulin-like domains / loops arranged as an N-terminal Ig-like variable domain (D1) followed by two Ig-like constant domains (D2 and D3). Nectins interact with each other via V-domain-to-V-domain binding interactions, thereby creating a trans-hetero-interaction network that supports cell-cell adhesion. Hetero-affinity interactions between nectin-3 / nectin-1, nectin-3 / nectin-2, and nectin-1 / nectin-4 have been reported (Harrison et al., Nat Struct Mol Biol, 19(9):906-915, 2012). In addition to their role in cell-cell adhesion, nectins play important roles in regulating diverse physiological cellular activities, in viral entry, and in immunomodulation.

[0006] Nectin (derived from the Latin word "necto," meaning "to connect") interacts with other nectins on cell surface molecules through their Ig-like V domains in their ECDs. Nectin functions to promote cell adhesion by first binding to form cis-dimers on the same cell, and then forming homophilic or heterophilic trans-dimers on adjacent cells with other nectins or members of the immunoglobulin superfamily (IgSF) (Miyoshi et al., Am J Nephrol, 27:590, 2007). Heterophilic trans-dimers have been reported to form stronger cell-cell interactions than homophilic trans-dimers. Binding specificity differs for each nectin (e.g., nectin-4 binds to itself as well as to nectin-1).

[0007] The ability of nectin family members to interact with additional cell surface molecules significantly expands their interaction network. Some members of the nectin family can exert immunomodulatory functions as a result of their hetero-affinity trans-interactions with other members of the IgSF. These interactions are known to influence the function of a variety of immune cell types, including natural killer (NK) cells, monocytes, dendritic cells (DCs), and T lymphocytes. Not only are some known nectin family partners IgSF members, but some nectins are known to recognize common binding partners. For example, both nectin-2 and PVR recognize CD226, TIGIT, and nectin-3 (Duraivelan et al., Sci Rep, 10:9434, 2020).

[0008] Nectin-4 has been reported to be upregulated in various epithelial cell cancers, such as breast cancer (Fabre-Lafay et al., BMC Cancer, 7:73, 2007), lung cancer (Takano et al., Cancer Res, 69(16):6694-03, 2009), ovarian cancer (Derycke et al., Am J Clin Pathol, 5:835-845, 2010), pancreatic cancer (Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015), gallbladder cancer (Zhang et al., Cancer Lett, 375:179-189, 2016), and gastric cancer (Zhang et al., Hum Pathol, 72:107-116, 2018). These cancers frequently exhibit copy number increase or local amplification of the nectin-4 locus (Pavlova et al., Elife, 2:e00358, 2013).

[0009] Recently, evidence has been accumulating suggesting that nectin contributes to the function of promoting tumorigenesis and metastasis. In particular, nectin-4 has been shown to be involved in cancer cell adhesion, migration, proliferation, and epithelial-mesenchymal transition. In breast, pancreatic, and lung cancers, overexpression of nectin-4, or detection of soluble nectin-4 in patient serum, has been reported to be associated with tumor progression and / or low survival rates (Fabre-Lafay et al., BMC Cancer, 7:73, 2007; Takano et al., Cancer Res, 69(16):6694-03, 2009; Derycke et al., Am J Clin Pathol, 5:835-845, 2010; Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015; and Lattanzio et al., Oncogenesis, 3:e118, 2014).

[0010] There is an unmet need for effective, safe, and specific anti-nectin-4 antibodies that can be used alone or in combination with other drugs in antibody-based immunotherapy. [Overview of the Initiative]

[0011] This disclosure addresses the above needs by providing anti-nectin-4 antibodies and fragments thereof that bind to nectin-4, including nectin-4 present on the surface of cancer cells, for example. These antibodies and fragments thereof are characterized by a unique set of CDR sequences, specificity to nectin-4, and are useful in cancer immunotherapy as monotherapy or in combination with other anticancer agents. More specifically, this disclosure relates to antibodies that bind to human nectin-4 and their use to modulate (e.g., antagonize) the nectin-4-mediated activity of cells localized in the tumor microenvironment.

[0012] According to some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof comprises three CDRs of heavy chain (HC) variable regions selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and three CDRs of light chain (LC) variable regions selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, or a series of six complementarity-determining region (CDR) sequences selected from the group consisting of analogs or derivatives thereof having at least 90%, 95%, or 99% sequence identity with any one of the CDRs of SEQ ID NOs: 1 to 16, provided that the antibody or fragment thereof retains binding to nectin-4.

[0013] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 17, CDR2:SEQ ID NO: 18, and CDR3:SEQ ID NO: 19; and / or a light chain variable region comprising CDR1:SEQ ID NO: 20, CDR2:SEQ ID NO: 21, and CDR3:SEQ ID NO: 22.

[0014] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 23, CDR2:SEQ ID NO: 24, and CDR3:SEQ ID NO: 25; and / or a light chain variable region comprising CDR1:SEQ ID NO: 26, CDR2:SEQ ID NO: 27, and CDR3:SEQ ID NO: 28.

[0015] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 29, CDR2:SEQ ID NO: 30, and CDR3:SEQ ID NO: 31; and / or a light chain variable region comprising CDR1:SEQ ID NO: 32, CDR2:SEQ ID NO: 33, and CDR3:SEQ ID NO: 34.

[0016] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 35, CDR2:SEQ ID NO: 36, and CDR3:SEQ ID NO: 37; and / or a light chain variable region comprising CDR1:SEQ ID NO: 38, CDR2:SEQ ID NO: 39, and CDR3:SEQ ID NO: 40.

[0017] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 41, CDR2:SEQ ID NO: 42, and CDR3:SEQ ID NO: 43; and / or a light chain variable region comprising CDR1:SEQ ID NO: 44, CDR2:SEQ ID NO: 45, and CDR3:SEQ ID NO: 46.

[0018] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 47, CDR2:SEQ ID NO: 48, and CDR3:SEQ ID NO: 49; and / or a light chain variable region comprising CDR1:SEQ ID NO: 50, CDR2:SEQ ID NO: 51, and CDR3:SEQ ID NO: 52.

[0019] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 47, CDR2:SEQ ID NO: 53, and CDR3:SEQ ID NO: 54; and / or a light chain variable region comprising CDR1:SEQ ID NO: 55, CDR2:SEQ ID NO: 56, and CDR3:SEQ ID NO: 52.

[0020] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a heavy chain variable region comprising CDR1:SEQ ID NO: 57, CDR2:SEQ ID NO: 58, and CDR3:SEQ ID NO: 59; and / or a light chain variable region comprising CDR1:SEQ ID NO: 50, CDR2:SEQ ID NO: 51, and CDR3:SEQ ID NO: 60.

[0021] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or an analog or derivative thereof having at least 90%, 95%, or 99% sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15, provided that the antibody or fragment thereof retains binding to nectin-4.

[0022] In other embodiments, the anti-nectin-4 antibody or an antibody fragment thereof comprises a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, or an analog or derivative thereof having at least 90%, 95%, or 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, provided that the antibody or the fragment thereof retains binding to nectin-4.

[0023] In other embodiments, the anti-nectin-4 antibody or an antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

[0024] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof is the following combination: (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4; (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8; (e) a variable heavy chain sequence comprising SEQ ID NO: 9 and a variable light chain sequence comprising SEQ ID NO: 10; (f) a variable heavy chain sequence comprising SEQ ID NO: 11 and a variable light chain sequence comprising SEQ ID NO: 12; (g) a variable heavy chain sequence comprising SEQ ID NO: 13 and a variable light chain sequence comprising SEQ ID NO: 14; and (h) a variable heavy chain sequence comprising SEQ ID NO: 15 and a variable light chain sequence comprising SEQ ID NO: 16 and comprises a variable heavy chain sequence and a variable light chain sequence selected therefrom.

[0025] In some embodiments, there is provided an immune complex comprising an antibody or an antibody fragment that binds to nectin-4 and is covalently attached to a cytotoxic agent, wherein the antibody or the antibody fragment is the following combination: (a) A variable heavy chain sequence containing SEQ ID NO: 1 and a variable light chain sequence containing SEQ ID NO: 2; (b) Variable heavy chain sequence containing Sequence ID 3 and variable light chain sequence containing Sequence ID 4; (c) Variable heavy chain sequence containing Sequence ID No. 5 and variable light chain sequence containing Sequence ID No. 6; (d) Variable heavy chain sequence containing SEQ ID NO: 7 and variable light chain sequence containing SEQ ID NO: 8; (e) Variable heavy chain sequence containing Sequence ID No. 9 and variable light chain sequence containing Sequence ID No. 10; (f) Variable heavy chain sequence containing sequence number 11 and variable light chain sequence containing sequence number 12; (g) Variable heavy chain sequence containing Sequence ID No. 13 and variable light chain sequence containing Sequence ID No. 14; and (h) Variable heavy chain sequence containing Sequence ID No. 15 and variable light chain sequence containing Sequence ID No. 16 It includes variable heavy chain sequences and variable light chain sequences selected from the following.

[0026] In some embodiments, an immunocomplex is provided comprising an antibody or fragment thereof that binds to nectin-4 and is covalently attached to a cytotoxic agent, in which case the antibody comprises (a) a heavy chain variable region comprising CDR1:SEQ ID NO: 17, CDR2:SEQ ID NO: 18 and CDR3:SEQ ID NO: 19; and / or a light chain variable region comprising CDR1:SEQ ID NO: 20, CDR2:SEQ ID NO: 21 and CDR3:SEQ ID NO: 22; (b) a heavy chain variable region comprising CDR1:SEQ ID NO: 23, CDR2:SEQ ID NO: 24 and CDR3:SEQ ID NO: 25; and / or (c) Light chain variable region including CDR1:SEQ ID NO: 26, CDR2:SEQ ID NO: 27, and CDR3:SEQ ID NO: 28; (c) Heavy chain variable region including CDR1:SEQ ID NO: 29, CDR2:SEQ ID NO: 30, and CDR3:SEQ ID NO: 31; and / or Light chain variable region including CDR1:SEQ ID NO: 32, CDR2:SEQ ID NO: 33, and CDR3:SEQ ID NO: 34; (d) Heavy chain variable region including CDR1:SEQ ID NO: 35, CDR2:SEQ ID NO: 36, and CDR3:SEQ ID NO: 37; and / or CDR1:SEQ ID NO: 38, CDR2:SEQ ID NO: 39 , and the light chain variable region including CDR3:SEQ ID NO: 40; and / or (e) the heavy chain variable region including CDR1:SEQ ID NO: 41, CDR2:SEQ ID NO: 42, and CDR3:SEQ ID NO: 43; and / or the light chain variable region including CDR1:SEQ ID NO: 44, CDR2:SEQ ID NO: 45, and CDR3:SEQ ID NO: 46; and / or (f) the heavy chain variable region including CDR1:SEQ ID NO: 47, CDR2:SEQ ID NO: 48, and CDR3:SEQ ID NO: 49; and / or CDR1:SEQ ID NO: 50, CDR2:SEQ ID NO: 51, and CDR 3: A light chain variable region including SEQ ID NO: 52; and / or (g) A heavy chain variable region including CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 53, and CDR3: SEQ ID NO: 54; and / or A light chain variable region including CDR1: SEQ ID NO: 55, CDR2: SEQ ID NO: 56, and CDR3: SEQ ID NO: 52, or (h) A heavy chain variable region including CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, and CDR3: SEQ ID NO: 59; and a light chain variable region including CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, and CDR3: SEQ ID NO: 60.

[0027] In some embodiments, the anti-nectin-4 antibody and its antibody fragment include one or more heavy chain variable region CDRs disclosed in Table 1 and / or one or more light chain variable region CDRs disclosed in Table 2.

[0028] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof exhibits one or more of the following structural and functional features, individually or in combination: (a) specific to human nectin-4; (b) not binding to human nectin-1, human nectin-2, or human nectin-3; (c) binding to the epitope of the N-terminal Ig-like V domain of nectin-4; (d) translocating from the surface of nectin-4-positive cells after binding to nectin-4; (e) cross-reacting with cynomolgus monkey nectin-4; (f) cross-reacting with rat and / or mouse nectin-4; (g) interfering with human nectin-4 / nectin-1 binding interactions; (h) interfering with human nectin-4 / TIGIT binding interactions; (i) reducing the level of cell surface protein expression of nectin-4 on human tumor cells; or (j) directing ADCC in human cells expressing endogenous levels of nectin-4.

[0029] In some embodiments, an anti-nectin-4 antibody or fragment thereof specifically binds to human cells expressing endogenous levels of nectin-4 and / or host cells modified to overexpress nectin-4, and does not exhibit binding to the extracellular domains of human nectin-1, nectin-2, or nectin-3 (e.g., specific binding).

[0030] In some embodiments, the nectin-4 antibody or antibody fragment binds to human nectin-4 with an affinity of less than 100 nM.

[0031] In some embodiments, the nectin-4 antibody or antibody fragment binds to the epitope of the N-terminal Ig-like V domain of nectin-4. In alternative embodiments, the nectin-4 antibody or antibody fragment binds to the epitope of the Ig-like C domain of nectin-4.

[0032] In some embodiments, the anti-nectin-4 antibody specifically binds to human nectin-4 when it is present on the surface of tumor cells, inducing the internal migration of nectin-4.

[0033] In some embodiments, the anti-nectin-4 antibody specifically binds to human nectin-4 when human nectin-4 is present on the surface of tumor cells, directing ADCC-mediated death of tumor cells.

[0034] In some embodiments, the nectin-4 antibody or antibody fragment has cross-reactive binding to cynomolgus monkey nectin-4 with an EC50 < 5 nM. In other embodiments, the nectin-4 antibody or antibody fragment binds to human and cynomolgus monkey nectin-4 and has cross-reactive binding with rat and / or mouse nectin-4 at an equivalent or lower binding affinity.

[0035] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof blocks the human nectin-4 / nectin-1 binding interaction, including by partially blocking it.

[0036] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof blocks the human nectin-4 / TIGIT (T cell immune receptor including the Ig domain and TIIM domain) binding interaction, including by partially blocking it.

[0037] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof is incorporated into an immune complex containing a conjugated anti-nectin-4 antibody or antibody fragment thereof with one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes (i.e., radiocomplexes).

[0038] In some embodiments, the nectin-4 antibody is a monoclonal antibody. This disclosure provides non-human parental (e.g., mouse) anti-nectin-4 antibodies and antibody fragments thereof, as well as methods of use thereof. Those skilled in the art will understand that the antibodies of this disclosure may be modified for intended use, such as conversion to chimeric antibodies or humanization for use as human therapeutic antibodies or fragments. In alternative embodiments, the nectin-4 antibody is a bispecific antibody.

[0039] Generally, a humanized nectin-4 antibody or fragment thereof may contain substantially all of at least one and typically two variable domains, in which case all or substantially all of the hypervariable loop corresponds to that of the mouse parental anti-nectin-4 antibody disclosed herein, and all or substantially all of the framework (FR) region is derived from a suitable human consensus immunoglobulin sequence. The humanized antibody or fragment thereof may optionally contain at least a portion of the human immunoglobulin constant region (Fc). For example, this disclosure includes humanized versions of any of the following: N4_mAb 6 antibody (including a CDR region derived from the VH sequence provided in SEQ ID NO: 11 and the VL sequence provided in SEQ ID NO: 12), N4_mAb 7 antibody (including a CDR region derived from the VH sequence provided in SEQ ID NO: 13 and the VL sequence provided in SEQ ID NO: 14), and N4_mAb 8 antibody (including a CDR region derived from the VH sequence provided in SEQ ID NO: 15 and the VL sequence provided in SEQ ID NO: 16).

[0040] In some embodiments, the nectin-4 antibody or antibody fragment is a recombinant antibody (e.g., a chimeric antibody, a humanized antibody, or a bispecific antibody) and contains six(6) CDRs, all of which are derived from the VH domain or VL domain of a single anti-nectin-4 antibody disclosed herein. For example, the binder may contain all six CDR regions of an anti-nectin-4 antibody designated “N4_mAb 1”. In a typical example, the antibody or antibody fragment thereof may contain the amino acid sequences of SEQ ID NOs: 17-19 and SEQ ID NOs: 20-22, where these sequences represent the variable heavy chain region CDR1, CDR2 and CDR3 and the variable light chain region CDR1, CDR2 and CDR3 of a mouse anti-human nectin-4 antibody also known herein as “N4_mAb 1”.

[0041] In some embodiments, the nectin-4 antibody is a full-length antibody. In some embodiments, the nectin-4 antibody is an antibody fragment. In further embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies and diabodies.

[0042] Nectin-4 antibodies and their antibody fragments can be used to treat cancer. Such methods for treating cancer may include the step of administering a composition or formulation containing a nectin-4 antibody or its antibody fragment to a target requiring it. For example, a nectin-4 antibody or its antibody fragment can be administered either alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapy. In certain embodiments, a nectin-4 antibody or its antibody fragment is used to prepare an ADC suitable for mediating the death of cancer cells expressing nectin-4.

[0043] [Brief explanation of the drawing]

[0044] The above summary of this disclosure and the following detailed description will be better understood when read in conjunction with the attached figures. For illustrative purposes, currently preferred embodiments are shown in the figures. However, please understand that this disclosure is not limited to the configurations, examples, and means shown.

[0045] [Figure 1-1] This figure shows the amino acid sequences of the VH and VL domains of a mouse anti-nectin-4 antibody, as well as their respective CDR sequences (kabat numbering). Sequence identifiers are provided, and for the CDRs, the variable domain sequences are underlined. [Figure 1-2] This figure shows the amino acid sequences of the VH and VL domains of a mouse anti-nectin-4 antibody, as well as their respective CDR sequences (kabat numbering). Sequence identifiers are provided, and for the CDRs, the variable domain sequences are underlined. [Figure 1-3] This figure shows the amino acid sequences of the VH and VL domains of a mouse anti-nectin-4 antibody, as well as their respective CDR sequences (kabat numbering). Sequence identifiers are provided, and for the CDRs, the variable domain sequences are underlined. [Figure 1-4] This figure shows the amino acid sequences of the VH and VL domains of a mouse anti-nectin-4 antibody, as well as their respective CDR sequences (kabat numbering). Sequence identifiers are provided, and for the CDRs, the variable domain sequences are underlined. [Figure 2] This figure shows the binding of chimeric nectin-4 antibody to recombinant nectin-4 (extracellular domain), as measured by ELISA. [Figure 3] [Figures 3A and 3B] These figures show the binding of the nectin-4 antibody to nectin-4 expressing cells. Figure 3A shows the binding of the chimeric nectin-4 antibody to CHO-nectin-4 cells, which ectopically express human nectin-4. Figure 3B shows the binding of the chimeric nectin-4 antibody to SKBR3 cells. SKBR3 is a human breast cancer cell line that endogenously expresses nectin-4. [Figure 4] [Figures 4A and 4B] These figures demonstrate that nectin-4 antibodies induce nectin-4-dependent antibody endocytosis. Figure 4A shows endocytosis (manifested through indirect cell death) of a chimeric nectin-4 antibody in CHO-nectin-4 cells that ectopically express human nectin-4. Figure 4B shows endocytosis of a chimeric nectin-4 antibody against SKBR3 cells. SKBR3 is a human breast cancer cell line that endogenously expresses nectin-4. [Figure 5] [Figures 5A and 5B] These figures show the internal distribution of nectin-4 antibody and the synchronous dynamics of membrane nectin-4 levels in T47D cells. T47D is a human breast cancer cell line that endogenously expresses nectin-4. Figure 5A shows the internal distribution dynamics of nectin-4 antibody. Figure 5B shows the relative levels of membrane nectin-4 protein measured at the same time point as internal distribution. [Figure 6] [Figures 6A and 6B] These figures show the ability of the nectin-4 antibody to induce antibody-dependent cytotoxicity (ADCC). Figure 6A shows the ADCC activity of the nectin-4 antibody in T47D cells, and Figure 6B shows the ADCC activity in SKBR3 cells. Both T47D and SKBR3 are human breast cancer cell lines that endogenously express nectin-4. [Modes for carrying out the invention]

[0046] To make this disclosure easier to understand, certain technical and scientific terms are defined below. Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which this disclosure belongs.

[0047] Throughout this disclosure, the following abbreviations will be used: mAb or Mab or MAb - Monoclonal antibody. CDR - Complementarity Determination Region. VH or VH - heavy chain variable region. VL or VL - Light chain variable region. FR - Antibody Framework Region.

[0048] The term “nectin-4” (N4) or “nectin-4 protein” includes human nectin-4, in particular, the native sequence polypeptide, isoforms, chimeric polypeptide, all homologs, fragments, and precursors of nectin-4. The amino acid sequences of human, cynomolgus monkey, rat, and mouse nectin-4 are provided in NCBI reference sequences: NP_112178.2 (human) (SEQ ID NO: 61), XP_005541277.1 (cynomolgus monkey) (SEQ ID NO: 62), NP_001102546.1 (rat) (SEQ ID NO: 63), and NP_082169.2 (mouse) (SEQ ID NO: 64). The orthologues of nectin-4 share >99%, approximately 94%, and approximately 92% homology with the human protein in cynomolgus monkeys, rats, and masses, respectively.

[0049] The term “nectin-1” or “nectin-1 protein” includes human nectin-1(N1), in particular the native sequence polypeptide, isoforms, chimeric polypeptide, all homologs, fragments, and precursors of nectin-1. The amino acid sequence of human nectin-1 is provided in NCBI reference sequence NP_002846.3(human)SEQ ID NO: 65.

[0050] As used herein, the term "TIGIT" refers to the "T cell immune receptor containing an Ig domain and a TIIM domain," which is a member of the PVR (poliovirus receptor) family of immunoglobulin proteins that binds to PVR / CD155, Nectin-2 / CD112, and Nectin-4 (Reches et al., J Immunotherapy Cancer, 8:e000266, 2020). TIGIT is also known as TIGIT, WUCAM, Vstm3, and Vsig9. Unless otherwise indicated or made clear from the context, references to TIGIT herein refer to human TIGIT.

[0051] The term "immunoglobulin superfamily" (IgSF) refers to a superfamily of proteins containing one or more immunoglobulin-like (Ig-like) domains. Most IgSF proteins are localized to or secreted on the cell surface and function in cell recognition, binding, or adhesion processes. There are approximately 500 non-antibody, non-T cell receptor (TCR) IgSF proteins encoded in the human genome. Most IgSF members are type I transmembrane proteins, which typically consist of an extracellular domain containing one or more Ig-like domains in either a variable (V) domain or a constant (C) domain, a single transmembrane domain, and a cytoplasmic tail.

[0052] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0053] Exemplary antibodies such as IgG contain two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0054] The hypervariable region generally encompasses amino acid residues from approximately 24–34 (LCDR1; "L" indicates the light chain), 50–56 (LCDR2), and 89–97 (LCDR3) in the light chain variable region, as well as amino acid residues around approximately 31–35B (HCDR1; "H" indicates the heavy chain), 50–65 (HCDR2), and 95–102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and / or including those residues that form the hypervariable loop (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

[0055] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for possible variant antibodies. For example, variant antibodies may contain naturally occurring mutations or arise during the manufacturing process of monoclonal antibody preparations, but such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain various antibodies directed to various determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any arbitrary method. For example, monoclonal antibodies used in accordance with this disclosure can be produced by a variety of techniques, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods for producing monoclonal antibodies and other exemplary methods are described herein.

[0056] The term "chimeric" antibody refers to recombinant antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from a different species or belonging to a different antibody class or subclass, or similarly, fragments of such antibodies, insofar as they exhibit the desired biological activity. In addition, complementarity-determining region (CDR) transplantation can be performed to alter certain properties of the antibody molecule, including affinity or specificity. Typically, the variable domain is obtained from antibodies derived from experimental animals such as rodents ("parent antibodies"), and the constant domain sequence is obtained from human antibodies, so the resulting chimeric antibodies can be directed to function as effectors in human subjects and are less likely to induce harmful immune responses than the parent (e.g., mouse) antibody from which they are derived.

[0057] The term "humanized antibody" refers to an antibody modified to include one or more human framework regions in its variable region, along with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) in the heavy and / or light chains. In certain embodiments, a humanized antibody contains a sequence that is entirely human except for the CDR region. Humanized antibodies typically have lower immunogenicity to humans than non-humanized antibodies and therefore provide therapeutic effects in certain situations. Those skilled in the art are familiar with humanized antibodies and suitable techniques for producing them. For example, each of these is incorporated herein by reference in its entirety: Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, See U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370; and Selick et al., International Publication No. 90 / 07861.

[0058] A “human antibody” is an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or is produced using any technique for producing human antibodies known to those skilled in the art. This definition of human antibodies specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including the method described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals, which are modified to produce such antibodies in response to antigen loading, and whose endogenous gene loci are rendered inactive. For example, immunized HuMab mice (for example, regarding HuMab mice, see Nils Lonberg et al., 1994, Nature). See 368:856-859, International Publication No. 98 / 24884, International Publication No. 94 / 25585, International Publication No. 93 / 1227, International Publication No. 92 / 22645, International Publication No. 92 / 03918 and International Publication No. 01 / 09187), Xenomouse (for example, with respect to XENOMOUSE® technology, see U.S. Patent No. 6,075,181 and U.S. Patent No. 6,150,584) or Trianni mouse (for example, see International Publication No. 2013 / 063391, International Publication No. 2017 / 035252 and International Publication No. 2017 / 136734).

[0059] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains corresponding to various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0060] The term “antigen-binding domain” (or simply “binding domain”) or similar terms for an antibody refer to one or more fragments of the antibody that possess the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs that can be linked as needed by a synthetic linker.

[0061] The “variable domain” (V domain) of an antibody mediates binding and confers specificity of a particular antibody to an antigen. However, the variability is not evenly distributed across the entire 110-amino acid length of the variable domain. Instead, the V domain consists of relatively invariant segments called framework regions (FRs), each consisting of 15 to 30 amino acids separated by shorter, highly variable regions called “hypervariable regions” or CDRs, each 9 to 12 amino acids long. As those skilled in the art will understand, the exact numbering and arrangement of CDRs may differ among various numbering systems. However, it should be understood that the disclosure of a variable heavy sequence and / or variable light sequence includes the disclosure of the corresponding CDR. Thus, the disclosure of each variable heavy region is a disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is a disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0062] As used herein, “complementarity-determining regions” or “CDRs” refer to short polypeptide sequences within the variable regions of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. Within each VL and each VH, there are three CDRs (referred to as CDR1, CDR2, and CDR3). Unless otherwise specified herein, CDR regions and framework regions are annotated according to the Kabat numbering scheme (Kabat EA, et al., 1991, Sequences of proteins of Immunological interest, In: NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md).

[0063] In other embodiments, the CDR of the antibody can be determined according to MacCallum RM et al, (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety, or according to the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al, (1999) Nucleic Acids Res 27: 209-212, which are each incorporated herein by reference in their entirety. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety. In other embodiments, the CDR of an antibody can be determined according to an AbM numbering scheme, which references the AbM hypervariable region, representing the compromise between Kabat's CDR and Chothia's structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0064] The term "framework," "framework region," or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0065] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in the selection of VL framework sequences or VH framework sequences of human immunoglobulins. Generally, the selection of VL or VH sequences of human immunoglobulins is derived from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al., cited above. In one embodiment, for VH, the subgroup is subgroup I, as described in Kabat et al., cited above.

[0066] The "hinge region" is generally defined as a stretching of human IgG1 from 216–238 (EU numbering) or 226–251 (Kabat numbering). The hinge can be further divided into three distinct regions: the upper, middle (e.g., core), and lower hinges.

[0067] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native Fc region and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0068] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0069] The term "effector function," derived from the interaction between the antibody Fc region and certain Fc receptors, includes, but is not limited to, FcγR-mediated effector functions such as Clq binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors. Such effector functions generally require the Fc region to be combined with an antigen-binding domain (e.g., an antibody variable domain).

[0070] An antibody that "binds to the same epitope as the reference antibody" refers to an antibody that contacts a set of amino acid residues of the antigen that overlaps with those of the reference antibody, or an antibody that blocks the binding of the reference antibody to the antigen by 50% or more in a competitive assay. The amino acid residues of the antibody that come into contact with the antigen can be determined, for example, by determining the crystal structure of the antibody conjugated with the antigen, or by performing hydrogen / deuterium exchange. In some embodiments, antibody residues that are within 5 Å of the antigen are considered to come into contact with the antigen. In some embodiments, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to the antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of its own antibody to the antigen by 50% or more in a competitive assay.

[0071] The term "antibody fragment" refers to a molecule distinct from the intact antibody, containing the portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments, but not limited to them, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; and single-chain antibody molecules (e.g., scFv). Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, a designation reflecting its ability to readily crystallize. A Fab fragment consists of the entire light (L) chain along with the variable region domain (VH) of the heavy (H) chain, and the first constant domain (CH1) of one heavy chain. Pepsin treatment of an antibody yields a single large F(ab)2 fragment, which is roughly equivalent to two disulfide-linked Fab fragments with bivalent antigen-binding activity and can still crosslink antigens. The Fab fragment differs from the Fab' fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain containing one or more cysteines from the antibody hinge region. Fab'-SH is the heretical notation for Fab', in which case the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was initially generated as a pair of Fab' fragments, having a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0072] "Fv" consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight, non-covalent association. The folding of these two domains gives rise to six hypervariable loops (three from the H chain and three from the L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0073] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing a VH antibody domain and a VL antibody domain attached to a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the sFv to form a desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0074] The term “antigen-binding domain” (or simply “binding domain”) or similar terms for an antibody refer to one or more fragments of the antibody that possess the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed within the term “antigen-binding moiety” of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs that can be linked as needed by a synthetic linker.

[0075] The term "multispecific antibody" is used most broadly to cover antibodies containing heavy chain variable domains (VH) and light chain variable domains (VL), in which case the VH-VL unit has multiple epitope specificity (e.g., it can bind to two different epitopes on one biomolecule or to each epitope on different biomolecules). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, and bispecific diabodies and triabodies. "Multiple epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0076] "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, in contrast to bispecific antibodies, dual specificity antibodies have two antigen-binding arms with identical amino acid sequences, and each Fab arm can recognize two antigens. Dual specificity allows the antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, a multispecific antibody in IgG1 form binds to each epitope with affinities of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Single specificity" refers to the ability to bind to only one epitope. Multispecific antibodies can have a structure similar to a complete immunoglobulin molecule and include an Fc region, e.g., an IgG Fc region. Such structures may include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, scFv can be attached to either the N-terminus or C-terminus of either the heavy chain or the light chain.

[0077] As used herein, the term “bispecific antibody” refers to a monoclonal antibody, often human or humanized, having binding specificity to at least two different antigens. In this disclosure, one binding specificity may be directed toward nectin-4, and the other toward any other antigen, such as a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacterial-derived protein, or bacterial surface protein.

[0078] As used herein, the term "diabody" refers to a bivalent antibody containing two polypeptide chains, in which each polypeptide chain contains a VH domain and a VL domain linked by a linker that is too short to allow intramolecular association of the VH domain and VL domain on the same peptide chain (e.g., a linker consisting of 5 amino acids). This arrangement forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimer structure. Thus, the term "tribody" refers to a trivalent antibody containing three peptide chains, each peptide chain containing one VH domain and one VL domain linked by an extremely short linker that is too short to allow intramolecular association of the VH domain and VL domain within the same peptide chain (e.g., a linker consisting of 1-2 amino acids).

[0079] The term “isolated antibody,” when used to describe the various antibodies disclosed herein, means an antibody identified, isolated, and / or recovered from cells or cell cultures on which the antibody is expressed. Isolated antibodies or antibody fragments may include variants of the antibody or antibody fragment, which have one or more cotranslational or posttranslational modifications that occur during the preparation, purification, and / or storage of the antibody or antibody fragment. Contaminations from its natural environment are substances that would normally be considered to interfere with the diagnostic or therapeutic use of the polypeptide, and contaminations may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, isolated antibodies are purified to a purity of 95% or greater than 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 2007. In preferred embodiments, the antibody is purified to a degree sufficient to obtain (1) at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining.

[0080] Regarding the binding of an antibody to a target molecule, the terms "specific binding," or "specific to" or "specific to" a particular polypeptide or epitope on a particular polypeptide target, mean a binding that is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled targets. In this case, specific binding is indicated when the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled targets. As used herein, “specific binding,” or “specific to” or “specific to” an epitope on a particular polypeptide or a particular polypeptide target, may be indicated, for example, by a molecule having a Kd of ≤10⁴M, alternatively ≤10⁵M, alternatively ≤10⁶M, alternatively ≤10⁷M, alternatively ≤10⁸M, alternatively ≤10⁹M, alternatively ≤10⁻¹M, alternatively ≤10⁻¹M, alternatively ≤10⁻¹M, alternatively ≤10⁻¹²M, or a molecule having a Kd in the range of 10⁴M to 10⁶M, or 10⁶M to 10⁻¹M, or 10⁷M to 10⁹M. As those skilled in the art will understand, affinity and KD values ​​are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term “specific binding” refers to a binding in which a molecule binds to nectin-4 or a nectin-4 epitope without substantially binding to any other polypeptide or polypeptide epitope.

[0081] As used herein, the term "specifically binds to nectin-4" means the ability of an antibody or antigen-binding fragment to recognize and bind to endogenous human nectin-4 when it is present on the surface of normal or malignant cells, but not to human nectin-1, nectin-2, or nectin-3 or other homologs of the human nectin family.

[0082] As used herein, the term "affinity" refers to the binding strength of an antibody to an epitope. Antibody affinity is given by the dissociation constant Kd, defined as [Ab] × [Ag] / [Ab-Ag] (where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody, and [Ag] is the molar concentration of the unbound antigen). The affinity constant Ka is defined as 1 / Kd. Methods for determining mAb affinity can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), and these references are incorporated herein by reference in their entirety. A well-known standard method in this field for determining mAb affinity is the use of surface plasmon resonance (SPR) screening (e.g., analysis using a BIAcore® SPR analyzer).

[0083] An "epitope" is a technical term that indicates the site(s) of interaction between an antibody and its antigen(s). As stated by Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc.): "Antibodies generally recognize only small regions on the surface of large molecules such as proteins..." [A particular epitope] is likely to consist of amino acids from different parts of the [antigen] polypeptide chain that are brought together by protein folding. This type of antigenic determinant is known as a conformational epitope or discontinuous epitope because the recognized structure consists of protein segments that are discontinuous in the amino acid sequence of the antigen but come together in the three-dimensional structure. In contrast, epitopes consisting of a single segment of a polypeptide chain are called serial epitopes or linear epitopes (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc.).

[0084] The term "KD," as used herein, refers to the equilibrium dissociation constant, derived from the ratio of kd to ka (i.e., kd / ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Preferred methods for determining the KD of an antibody include biolayer interferometry (BLI) analysis, preferably using a Fortebio Octet RED device; surface plasmon resonance, preferably using a biosensor system such as a BIACORE® surface plasmon resonance system; or flow cytometry and scatchard analysis.

[0085] "EC50" for a drug and a specific activity (e.g., cell binding, inhibition of enzyme activity, activation or inhibition of immune cells) refers to the effective concentration of the drug that produces 50% of the drug's maximum response or effect with respect to such activity. "EC100" for a drug and a specific activity refers to the effective concentration of the drug that produces substantially the maximum response with respect to such activity.

[0086] As used herein, the term “antibody-drug conjugate” (ADC) refers to an immune complex consisting of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (known as the payload) via a synthetic linker. Immune complexes (antibody-drug conjugates, ADCs) are a class of highly potent antibody-based cancer therapies. ADCs consist of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (known as the payload) via a synthetic linker. ADCs combine the specificity of monoclonal antibodies with the potency of small molecule chemotherapeutic drugs, while facilitating the direct targeted delivery of a highly cytotoxic small molecule drug moiety to tumor cells.

[0087] As used herein, the term “endocytosis” refers to the process by which eukaryotic cells move segments of the plasma membrane, cell surface receptors, and components from the extracellular fluid into the cell. Mechanisms of endocytosis include receptor-mediated endocytosis. The term “receptor-mediated endocytosis” refers to a biological mechanism in which, upon binding of a ligand to its target, causes membrane invagination and pinching, leading to internal movement and delivery to the cytosol or transfer to an appropriate intracellular compartment.

[0088] The term "bystander effect" refers to the target cell-mediated death of healthy cells adjacent to tumor cells targeted by an antibody-drug conjugate. The bystander effect is generally caused by the cellular efflux of hydrophobic cytotoxic drugs that can diffuse from antigen-positive target cells to adjacent antigen-negative healthy cells. The presence or absence of the bystander effect may be due to the linker and conjugation chemistry used to construct the immune complex.

[0089] As used herein, the terms “antibody-based immunotherapy” and “immunotherapy” are used broadly to refer to any form of therapy that mediates direct or indirect effects on nectin-4 expressing cells, depending on the targeting specificity of an anti-nectin-4 antibody, a bispecific molecule, an antigen-binding domain, or a fusion protein containing an anti-nectin-4 antibody or its antibody fragment or CDR. The terms are intended to encompass methods of treatment using naked antibodies, bispecific antibodies (including T-cell binding, NK-cell binding, and other immune cell / effector cell binding), antibody-drug conjugates, cell therapies using oncolytic viruses containing modified T cells (CAR-T) or NK cells (CAR-NK) containing a nectin-4 specific chimeric antigen receptor, and gene therapies by delivering the antigen-binding sequence of an anti-nectin-4 antibody, and intended to express the corresponding antibody fragment in vivo.

[0090] Nectin protein family Members of the nectin family are expressed as single-pass transmembrane type I glycoproteins and are characterized by a common domain configuration, consisting of three Ig-like domains in the outer domain (a distal IgV domain followed by two IgC domains), a transmembrane region, and a cytoplasmic domain that binds to the actin cytoskeleton via the adapter protein afadin (Samanta et al., Cell Mol Life Sci, 72(4):645-658, 2015).

[0091] Many viruses utilize IgSF member proteins to facilitate viral tropism, adhesion, and subsequent entry into host cells. Some members of the nectin family were identified as viral receptors before their physiological function as cell adhesion molecules was discovered. Initially, members of the nectin family were independently identified by multiple groups as viral entry receptors and named based on their observed function. Nectin-1, -2, and -3 were initially described as molecules homologous to the poliovirus receptor (PVR, necl-5, CD155), and were subsequently named poliovirus receptor-associated (PRR) proteins (nectin-1 / PRR1 / CD111, nectin-2 / PRR2 / CD112, and nectin-3 / PRR3) (Reymond et al., J Biol Chem, 276(46):43205-15, 2001), and later assigned the names CD111, CD112, and CD113, respectively. Subsequently, it was demonstrated that nectin-4 recognizes measles virus hemagglutinin (MV-H) and functions as an epithelial cell receptor against measles virus invasion (Samanta et al., Cell Mol Life Sci, 72(4):645-658, 2015).

[0092] Nectin-4 (also known as poliovirus receptor-like 4 or PVRL4) was first described in 2001 as a novel ligand for nectin-1. More specifically, it was described as an afadin-related member of the nectin family that trans-interacts with nectin-1 through V-domain interactions but does not trans-interact with nectin-2, nectin-3, or PVR (Reymond et al., J Biol Chem, 276(46):43205-15, 2001).

[0093] Nectin functions as a cell adhesion molecule by first forming homocis-dimers on the cell surface, and then trans-dimers on adjacent cells in both homophilic and heterophilic ways. The specificity of binding differs for each nectin. Nectin-4 binds to itself and to nectin-1 (Reymond et al., J Biol Chem, 276(46):43205-15, 2001; Fabre et al., J Biol Chem, 277(30):27006-27013, 2002). Intercellular contact is thought to be initiated by interactions between nectins on adjacent cells. Subsequently, cadherin-catenin complexes are recruited to nectin-based intercellular adhesion sites, and trans-interactions of cadherins occur on adjacent cells, thereby forming adhesion junctions (Boylan et al., Oncotarget, 8(6):9717-9738, 2017).

[0094] The external domains of nectin proteins share between 30 and 55% amino acid sequence identity. Nectins are linked to actin cytoskeleton afadin (F-actin-binding protein) through binding motifs in their cytoplasmic domains and are involved in the formation of epithelial and endothelial junctions. In complex interactions with other cell adhesion molecules (CAMs), signaling molecules regulate several diverse physiological cellular activities, including migration, proliferation, survival, differentiation, polarization, and viral entry.

[0095] The ability of nectin family members to interact with additional cell surface molecules in mammals significantly expands their interaction network. Nectins are known to interact cis-interact with other cell surface membrane receptors, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, vascular endothelial growth factor receptor, prolactin receptor, ErbB2, ErbB3, and ErbB4, as well as integrins such as integrin αvβ3 and integrin α6β4, and to regulate not only cell-cell adhesion but also cell migration, proliferation, differentiation, and survival (Kedashiro et al., Sci Rep, 9:18997, 2019).

[0096] Some members of the nectin family can exert immunomodulatory functions as a result of hetero-affinity trans-interactions with other members of the immunoglobulin superfamily. These interactions are known to affect the function of a variety of immune cell types, including natural killer (NK) cells, monocytes, dendritic cells (DCs), and T lymphocytes. Not only are some of the known nectin family interactors IgSF members, but some nectins are known to recognize common binding partners. For example, both nectin-2 and PVR recognize CD226, TIGIT, and nectin-3 (Duraivelan et al., Sci Rep, 10:9434, 2020).

[0097] Bioinformatics analysis using an algorithm to classify proteins into functionally related families predicted that five additional IgSF members, CD96 (TACTILE), CD226 (DNAM-1), TIGIT (WUCAM, VSTM3), CRTAM, and CD200, are functionally and evolutionarily related to nectin and nectin-like proteins and could be binding partners for members of the nectin family (Rubinstein et al., Structure, 21(5):766-776, 2013). To date, with the exception of CD200, all of these proteins have been reported to bind to members of the nectin / nectin-like family (Rubenstein, et al).

[0098] Nectin-4 Nectin-4 was initially identified through bioinformatics searches using sequences from known nectin protein extradomains to identify the relevant sequence (Reymond et al., J Biol Chem, 276(46):43205-15, 2001). Human nectin-4 was cloned from the human trachea and described as an antigen whose expression pattern is restricted in normal human tissues.

[0099] Based on their findings, Reymond and his colleagues identified nectin-4 as a novel ligand for nectin-1 (Reymond et al., J Biol Chem, 276(46):43205-15, 2001): i) the soluble chimeric recombinant nectin-4 external domain (nectin-4-Fc) interacts with cells expressing nectin-1 but not with cells expressing PVR / CD155, nectin-2, or nectin-3, while nectin-1Fc binds to cells expressing nectin-4; ii) nectin-1-Fc precipitates nectin-4 expressed in COS cells; and iii) mutual in vitro physical interactions were observed between the soluble recombinant nectin-4-Fc protein and the soluble recombinant nectin-1-Fc protein (Reymond, N et al.). Nectin-4-Fc / nectin-4-Fc interactions were also detected, indicating that nectin-4 possesses both homophilic and heterophilic properties.

[0100] The human nectin-4 gene contains nine exons that encode the nectin-4 adhesion receptor, a 55.5 kDa protein containing 510 amino acids. According to the protein knowledge database UniProtKb, nectin-4 (Q96NY8) contains an N-terminal signal peptide (1-31 amino acids), an extracellular domain (32-349 amino acids) with three immunoglobulin-like subdomains (V-1 type 32-144 amino acids, C2-1 type 148-237 amino acids, C2-2 type 248-331 amino acids), a transmembrane domain (350-370 amino acids), and a cytoplasmic domain (371-510 amino acids).

[0101] It has been reported that the V-like domain of nectin-4 is sufficient to mediate its trans-interaction with nectin-1, and that the membrane-proximal nectin-4 C-like domain contributes to improving the affinity of the trans-interaction (Fabre et al., J Biol Chem, 277(30):27006-27013, 2002). Nectin-4 and nectin-3 share a common binding region in the V-like domain of nectin-1 (Harrison et al., Nat Struct Mol Biol, 19(9):906-915, 2012).

[0102] The nectin-4 / nectin-1 trans-interaction has also been reported to be blocked by an anti-nectin-1 monoclonal antibody (R1.302) whose epitope is localized to the V-like domain of nectin-1 (Reymond et al., J Biol Chem, 276(46):43205-15, 2001). Subsequent publications have demonstrated that a monoclonal antibody specific to the Ig-like V domain of nectin-4 blocks the adhesion of ovarian cancer cell lines modified to overexpress human nectin-4 (NIH:OVCAR5) to nectin-1 (Boylan et al., Oncotarget, 8(6):9717-9738, 2017).

[0103] Targeting Nectin-4 for Cancer Immunotherapy Nectin-4 was identified as a potential ADC target using suppression-subtractive hybridization due to its high mRNA expression levels in bladder cancer (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016). Nectin-4 was initially described as a tumor-specific antigen (TSA) because early publications reported limited expression of nectin-4 by human placental endothelial cells (Reymond et al., J Biol Chem, 276(46):43205-15, 2001), lack of expression in normal adult tissues, and re-expression in various cancer tissues, including breast, ovarian, pancreatic, and lung cancers (Fabre-Lafay et al., BMC Cancer, 7:73, 2007, Takano et al., Cancer Res, 69(16):6694-03, 2009, Derycke et al., Am J Clin Pathol, 5:835-845, 2010, Pavlova et al., Elife, 2:e00358, 2013, Nishiwada et al., J Exp Clin). Cancer Res, 34(1):30, 2015, Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016).

[0104] Immunohistochemical (IHC) studies using a mouse antibody (M22-244b3) directed against the extracellular domain of human nectin-4 and a panel of normal human tissue samples (representing 36 human organs) demonstrated broader expression in normal tissues at lower or moderate levels than previously reported (Challita-Eid et al.). These findings also identify normal tissues that may be at increased risk of intentionally inducing anti-nectin-4 toxicity. Low levels of nectin-4 with weak to moderate uniform staining have been reported in human dermal keratinocytes, dermal appendages (sweat glands and hair follicles), and the epithelium of the bladder, stomach, breast, esophagus, and salivary glands (tubules) (Challita-Eid et al., Reymond et al., J Biol Chem, 276(46):43205-15, 2001, Brancati et al., Am J Hum Gen, 87:265-273, 2010). This suggests that nectin-4 is a tumor-associated antigen (TAA) rather than a tumor-like antigen (TSA).

[0105] Nectin-4 is overexpressed in several cancers, particularly urothelial carcinoma, lung cancer, pancreatic cancer, breast cancer, and ovarian cancer (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016; Fabre-Lafay et al., BMC Cancer, 7:73, 2007; Takano et al., Cancer Res, 69(16):6694-03, 2009; Derycke et al., Am J Clin Pathol, 5:835-845, 2010). Extensive immunohistochemistry of nectin-4 expression in human cancer tumor microarrays (TMAs) representing 34 tumors across seven different indications (e.g., bladder cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer) demonstrated that 69% of TMA samples were nectin-4 positive across all evaluated cancer indications. The highest overall frequencies of nectin-4 expression were observed in bladder, breast, and pancreatic tumors. The prevalence of nectin-4 positive samples with moderate to strong staining was generally lower in ovarian, lung, head and neck, and esophageal cancer samples (Chalittta-Eid et al.). The higher nectin-4 expression levels observed in cancer theoretically provide a treatment window characterized by an acceptable safety profile for anti-nectin-4 targeted ADCs and antibody-based immunotherapies (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016, and Shim et al., Biomolecules, 10(3):360, 2020).

[0106] The early stages of epithelial carcinoma progression are characterized by genetic alterations that confer the ability to survive and proliferate in the absence of extracellular matrix colonization. The ability of cancer cells to tolerate loss of colonization is crucial for cancer cell survival and for the pathological progression of tumorigenesis (e.g., invasion into the basal layer, extravasation into blood vessels, and metastatic growth as a distal site) (Pavlova et al., Elife, 2:e00358, 2013). Nectin-4 was identified in a gain-of-function screening of genes that enable matrix-independent cell proliferation in TL-HMEC (hTERT immortalized human mammary epithelial cells transduced with the SV40 large T antigen) (Pavlova et al., Elife, 2:e00358, 2013).

[0107] Pavlova et al. further reported that nectin-4 drives the rapid association of TL-HMECs into multicellular clusters in suspension, and that the observed clustering can be disrupted using antibodies directed at the extracellular domain of nectin-4. Cell clustering was completely abolished in the presence of an anti-nectin-4 antibody. Similarly, antibodies targeting the extracellular region of nectin-1 also inhibited nectin-4-induced cell clustering.

[0108] Pavlova et al. further demonstrated that nectin-4 promotes clustering of tumor cells by binding to nectin-1 receptors on adjacent cells, i.e., an interaction that triggers integrin β4 / SHP-2 / c-Src activation in a matrix adhesion-independent manner. Pavlova et al. proposed a model in which tumor-specific intercellular contact and signaling via nectin-4 / nectin-1 interactions provides an alternative to cell-matrix signaling, conferring a survival advantage that enables avoidance of annoikis (i.e., induction of apoptosis of cells based on loss of adhesion to the extracellular matrix (ECM) and adjacent cells).

[0109] Studies conducted to determine the biological significance of nectin-4 in cellular functions underlying ovarian cancer progression (i.e., cell adhesion, spheroid formation, migration, and proliferation) have reported in vitro data demonstrating that mAbs targeting the IgV-like domain of nectin-4 nearly completely blocked the adhesion of ovarian cancer cells to nectin-1 (Boylan et al., Oncotarget, 8(6):9717-9738, 2017). Boylan et al. noted that Pavlova observed in vivo disruption of tumor cell adhesion and reduced tumor growth in a mouse xenograft model of breast cancer using the same anti-nectin-4 antibody compared to tumors treated with control IgG. Based on these combined results, it can be inferred that blocking nectin-4 cell adhesion may be a key element in the therapeutic efficacy of anti-nectin-4 antibodies used in cancer immunotherapy (Boylan et al.).

[0110] Publications reporting the results of preclinical trials evaluating the use of anti-nectin-4 ADCs as monotherapy for the treatment of nectin-4 expressing tumors validated the clinical development of anti-nectin-4 antibody-based immunotherapy. For example, AGS-22M6E ADC monotherapy was reported to inhibit tumor growth in four mouse xenograft models of human bladder cancer, pancreatic cancer, breast cancer, and lung cancer. Subsequent publications by M-Rabet et al. confirmed that nectin-4 is a therapeutic target for primary and metastatic triple-negative breast cancer (TNBC). This is based on the observation that ADCs (N41mAb-vcMMAE) prepared using various anti-nectin-4 antibodies (International Publication No. 2017 / 042210) induced complete and sustained responses in vitro and in vivo against primary tumors, metastatic lesions, and local recurrences in three TNBC models developed in immunodeficient NSG mice (M-Rabet et al., Annals of Oncology, 28(4):769-776, 2017).

[0111] Nectin-4 / TIGIT TIGIT belongs to the immunoglobulin superfamily and is known to interact with members of the human nectin family, including the poliovirus receptor (PVR or CD155 or Necl-5), PVRL2 (CD112 or Nectin-2), and CD113 (Nectin-3) (Stanietsky et al., Proc. Natl. Acad. Sci. USA, 106:17585-63, 2009; Yu et al., Nat Immunol, 10:48-57, 2009; Boles et al., Eur J Immunol, 39:695-703, 2009).

[0112] TIGIT is expressed by most NK cells and several T cell subsets, including memory T cells and regulatory T cells (Yu et al. (2009)), as well as on CD8+ tumor-infiltrating lymphocytes (TILs) (Reches et al., J Immunotherapy Cancer, 8:e000266, 2020). Following interaction with PVR or nectin-2, TIGIT inhibits the activation of effector function in T cells or NK cells. TIGIT-mediated suppression of T cell activation was attributed to the generation of immunomodulatory dendritic cells (Yu, X., et al. Nat. Immunol. (10) 48-57 (2009)). PVR is a common ligand for TIGIT, TACTILE, and DNAM-1. DNAM-1 (CD226) is a costimulatory counterreceptor that competes with both TIGIT for PVR binding. However, the binding affinities of PVR / receptor interactions differ significantly, with TIGIT exhibiting greater affinity for PVR than either DNAM-1 or TACTILE in this case (Yu et al., 2009). TIGIT's superiority in PVR ligand binding is advantageous for effector cell inhibition over effector cell costimulation. Given its apparent central role in suppressing antitumor responses, TIGIT has emerged as a particularly attractive target for cancer therapy.

[0113] Nectin-4 has recently been identified as a functional ligand for TIGIT, and published data have demonstrated that the nectin-4 / TIGIT interaction inhibits natural killer cell activity (Reches et al., J Immunotherapy Cancer, 8:e000266, 2020). According to Reches et al.'s publication, antibodies capable of blocking the nectin-4 / TIGIT interaction have also been reported to enhance tumor cell death in vitro and in vivo (Reches et al., and international publication no. 2019 / 215782), suggesting that blocking the nectin-4 / TIGIT interaction using anti-nectin-4 antibodies may lead to a specific and significant induction of an immune response against tumors.

[0114] Anti-nectin-4 antibody The anti-nectin-4 antibodies (N4_mAb 1 to N4_mAb 8) of this disclosure specifically bind to human nectin-4 and disrupt the binding interactions of nectin-4 / nectin-1 and / or nectin-4 / TIGIT. The antibodies and fragments thereof of this disclosure are characterized by a unique set of CDR sequences, specificity to nectin-4, and are useful in cancer immunotherapy, either as monotherapy or in combination with other anticancer agents. More specifically, this disclosure relates to antibodies that bind to human nectin-4 and their use to modulate the nectin-4-mediated activity of cells localized in the tumor microenvironment.

[0115] In some embodiments, the antibody of this disclosure may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody that specifically binds to human nectin-4, or an antigen-binding portion thereof. In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof exhibits one or more of the following structural and functional features, individually or in combination: (a) specific to human nectin-4; (b) not binding to human nectin-1, human nectin-2, or human nectin-3; (c) binding to the epitope of the N-terminal Ig-like V domain of nectin-4; (d) translocating internally from the surface of nectin-4-positive cells after binding; (e) cross-reacting with cynomolgus monkey nectin-4; (f) cross-reacting with rat and / or mouse nectin-4; (g) interfering with human nectin-4 / nectin-1 binding interactions, including reducing them; (h) interfering with human nectin-4 / TIGIT binding interactions, including reducing them; (i) reducing the level of cell surface protein expression of nectin-4 on human tumor cells; or (j) directing ADCC in human cells expressing endogenous levels of nectin-4.

[0116] Based on in vitro evaluations of maximum binding capacity, EC50, cell surface penetration, and cytotoxicity, the anti-nectin-4 antibodies and fragments thereof of this disclosure can be evaluated for suitability for use as ADC-based targeting antibodies or antibody fragments for cancer treatment. In other embodiments, the anti-nectin-4 antibodies or fragments thereof of this disclosure can be used to induce antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or to block oncogenic receptor signaling in the nectin-4 / nectin-1 axis or nectin-4 / TIGIT axis, or to neutralize secreted nectin-4.

[0117] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof comprises a VH having the set of CDRs disclosed in Table 1 (HCDR1, HCDR2, and HCDR3). For example, an anti-nectin-4 antibody or an antibody fragment thereof may include a set of CDRs corresponding to such CDRs in one or more of the anti-nectin-4 antibodies disclosed in Table 1 (e.g., the CDR of N4_mAb 1).

[0118] In another embodiment, an anti-nectin-4 antibody or an antibody fragment thereof includes a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 2. For example, an anti-nectin-4 antibody or an antibody fragment thereof may include a set of CDRs corresponding to such CDRs in one or more of the anti-nectin-4 antibodies disclosed in Table 2 (e.g., the CDR of N4_mAb 2).

[0119] In alternative embodiments, the anti-nectin-4 antibody or its antibody fragment comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1, and a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2. In one embodiment, the antibody may be a monoclonal antibody, a chimeric antibody, a bispecific antibody, a humanized antibody, or a human antibody, or an antigen-binding portion thereof, that specifically binds to human nectin-4. In one embodiment, the anti-nectin-4 antibody or its antibody fragment comprises all six CDR regions of each antibody, N4_mAb 1, N4_mAb 2, N4_mAb 3, N4_mAb 4, N4_mAb 5, N4_mAb 6, N4_mAb 7, or N4_mAb 8, formatted as a chimeric antibody or humanized antibody.

[0120] [Table 1]

[0121] [Table 2]

[0122] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof is (i) CDR1: Sequence ID 17, CDR2: Sequence ID 18, CDR3: Sequence ID 19; (ii) CDR1: Sequence ID 23, CDR2: Sequence ID 24, CDR3: Sequence ID 25; (iii) CDR1: Sequence ID 29, CDR2: Sequence ID 30, CDR3: Sequence ID 31; (iv) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO: 37; (v)CDR1:Sequence ID 41, CDR2:Sequence ID 42, CDR3:Sequence ID 43; (vi) CDR1: Sequence ID 47, CDR2: Sequence ID 48, CDR3: Sequence ID 49; (vii) CDR1: Sequence ID 47, CDR2: Sequence ID 53, CDR3: Sequence ID 54; and (viii) CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, CDR3: SEQ ID NO: 59, The VH includes a series of complementarity determination regions (CDR1, CDR2, and CDR3) selected from the group consisting of the following.

[0123] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof is (i) CDR1: Sequence ID 20, CDR2: Sequence ID 21, CDR3: Sequence ID 22; (ii) CDR1: Sequence ID 26, CDR2: Sequence ID 27, CDR3: Sequence ID 28; (iii) CDR1: Sequence ID 32, CDR2: Sequence ID 33, CDR3: Sequence ID 34; (iv) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40; (v)CDR1:Sequence ID 44, CDR2:Sequence ID 45, CDR3:Sequence ID 46; (vi) CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 52; (vii) CDR1: Sequence ID 55, CDR2: Sequence ID 56, CDR3: Sequence ID 52; and (viii) CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 60, It includes a VL having a series of complementarity determination regions (CDR1, CDR2, and CDR3) selected from the group consisting of the following.

[0124] In another embodiment, an anti-nectin-4 antibody or an antibody fragment thereof is (a) (i) CDR1: Sequence ID 17, CDR2: Sequence ID 18, CDR3: Sequence ID 19; (ii) CDR1: Sequence ID 23, CDR2: Sequence ID 24, CDR3: Sequence ID 25; (iii) CDR1: Sequence ID 29, CDR2: Sequence ID 30, CDR3: Sequence ID 31; (iv) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO: 37; (v)CDR1:Sequence ID 41, CDR2:Sequence ID 42, CDR3:Sequence ID 43; (vi) CDR1: Sequence ID 47, CDR2: Sequence ID 48, CDR3: Sequence ID 49; (vii) CDR1: Sequence ID 47, CDR2: Sequence ID 53, CDR3: Sequence ID 54; and (viii) CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, CDR3: SEQ ID NO: 59 A VH having a series of complementarity determination regions (CDR1, CDR2, and CDR3) selected from the group consisting of, (b) (i) CDR1: Sequence ID 20, CDR2: Sequence ID 21, CDR3: Sequence ID 22; (ii) CDR1: Sequence ID 26, CDR2: Sequence ID 27, CDR3: Sequence ID 28; (iii) CDR1: Sequence ID 32, CDR2: Sequence ID 33, CDR3: Sequence ID 34; (iv) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40; (v)CDR1:Sequence ID 44, CDR2:Sequence ID 45, CDR3:Sequence ID 46; (vi) CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 52; (vii) CDR1: Sequence ID 55, CDR2: Sequence ID 56, CDR3: Sequence ID 52; and (viii) CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 60, A VL having a series of complementarity determination regions (CDR1, CDR2, and CDR3) selected from the group consisting of, Includes.

[0125] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof is (i) VH:CDR1:Sequence ID 17, CDR2:Sequence ID 18, CDR3:Sequence ID 19, VL:CDR1:Sequence ID 20, CDR2:Sequence ID 21, CDR3:Sequence ID 22; (ii) VH:CDR1:Sequence ID 23, CDR2:Sequence ID 24, CDR3:Sequence ID 25, VL:CDR1:Sequence ID 26, CDR2:Sequence ID 27, CDR3:Sequence ID 28; (iii) VH:CDR1:Sequence ID 29, CDR2:Sequence ID 30, CDR3:Sequence ID 31, VL:CDR1:Sequence ID 32, CDR2:Sequence ID 33, CDR3:Sequence ID 34; (iv) VH:CDR1:SEQ ID NO: 35, CDR2:SEQ ID NO: 36, CDR3:SEQ ID NO: 37, VL:CDR1:SEQ ID NO: 38, CDR2:SEQ ID NO: 39, CDR3:SEQ ID NO: 40; (v)VH:CDR1:Sequence ID 41, CDR2:Sequence ID 42, CDR3:Sequence ID 43, VL:CDR1:Sequence ID 44, CDR2:Sequence ID 45, CDR3:Sequence ID 46; (vi)VH:CDR1:Sequence ID 47, CDR2:Sequence ID 48, CDR3:Sequence ID 49, VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 52; (vii)VH:CDR1:Sequence ID 47, CDR2:Sequence ID 53, CDR3:Sequence ID 54, VL:CDR1:Sequence ID 55, CDR2:Sequence ID 56, CDR3:Sequence ID 52; (viii) VH:CDR1:Sequence ID 57, CDR2:Sequence ID 58, CDR3:Sequence ID 59, and VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 60 It includes combinations of VH and VL having a series of complementarity determination regions (CDR1, CDR2, and CDR3) selected from the group consisting of the following.

[0126] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof includes a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and / or a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

[0127] In one embodiment, an anti-nectin-4 antibody or an antibody fragment thereof comprises a pair of variable heavy chain sequences and variable light chain sequences, which are selected from the following combinations: a variable heavy chain sequence containing SEQ ID NO: 1 and a variable light chain sequence containing SEQ ID NO: 2; a variable heavy chain sequence containing SEQ ID NO: 3 and a variable light chain sequence containing SEQ ID NO: 4; a variable heavy chain sequence containing SEQ ID NO: 5 and a variable light chain sequence containing SEQ ID NO: 6; a variable heavy chain sequence containing SEQ ID NO: 7 and a variable light chain sequence containing SEQ ID NO: 8; a variable heavy chain sequence containing SEQ ID NO: 9 and a variable light chain sequence containing SEQ ID NO: 10; a variable heavy chain sequence containing SEQ ID NO: 11 and a variable light chain sequence containing SEQ ID NO: 12; a variable heavy chain sequence containing SEQ ID NO: 13 and a variable light chain sequence containing SEQ ID NO: 14; a variable heavy chain sequence containing SEQ ID NO: 15 and a variable light chain sequence containing SEQ ID NO: 16. Those skilled in the art will further understand that it is possible to prepare anti-nectin-4 antibodies or antibody fragments thereof comprising combinations of variable heavy chains and variable light chains that are entirely different from the pairings specified above, by independently selecting or mixing and matching variable light chains and variable heavy chains.

[0128] In an alternative embodiment, the anti-nectin-4 antibody or an antibody fragment thereof comprises a pair of variable heavy chain sequences and variable light chain sequences, which are selected from the following combinations: a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 2; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 4; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 6; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 7 and a variable light chain sequence A variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 8; a variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 9 and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 10; a variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 11 and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 12; a variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 13 and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 14; a variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 15 and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 16. Advantageously, such an antibody or fragment thereof retains binding specificity to nectin-4. Those skilled in the art will further understand that it is possible to independently select, mix, or match variable light chains and variable heavy chains to prepare anti-nectin-4 antibodies containing combinations of variable heavy and variable light chains that are entirely different from the pairings identified above.

[0129] In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment selected from the group consisting of, for example, Fab, Fab', F(ab)2, Fv, domain antibodies (dAb), and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, miniantibodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer nectin-4 specific binding thereto.

[0130] In some embodiments, the variable region domain of the anti-nectin-4 antibody disclosed herein can be covalently attached at its C-terminal amino acid to at least one other antibody domain or fragment thereof. For example, the VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, the VL domain can be linked to a CK domain or fragment thereof. Thus, for example, the antibody may be a Fab fragment, in which case the antigen-binding domain contains associated VH and VL domains, which are covalently linked at their C-terminuses to the CH1 and CK domains, respectively. The CH1 domain can be extended with further amino acids to supply, for example, a hinge region or a portion of a hinge region domain as seen in the Fab fragment, or to supply further domains such as the CH2 and CH3 domains of the antibody.

[0131] Therefore, in one embodiment, the antibody fragment comprises at least one CDR as described herein. The antibody fragment may contain at least two, three, four, five, or six CDRs as described herein. The antibody fragment may further comprise at least one variable region domain of the antibody described herein. The variable region domain may be of any size or amino acid composition, but as a whole, it will comprise at least one CDR sequence responsible for binding to human nectin-4, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 as described herein, which is adjacent to or in-frame with one or more framework sequences.

[0132] In some embodiments, the anti-nectin-4 antibody is a monoclonal antibody. In some embodiments, the anti-nectin-4 antibody is a human antibody. In alternative embodiments, the anti-nectin-4 antibody is a mouse antibody. In some embodiments, the anti-nectin-4 antibody is a chimeric antibody, a bispecific antibody, or a humanized antibody.

[0133] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof exhibits one or more of the following structural and functional features, individually or in combination: (a) specific to human nectin-4; (b) not binding to human nectin-1, human nectin-2, or human nectin-3; (c) binding to the epitope of the N-terminal Ig-like V domain of nectin-4; (d) translocating internally from the surface of nectin-4-positive cells after binding; (e) cross-reacting with cynomolgus monkey nectin-4; (f) cross-reacting with rat and / or mouse nectin-4; (g) interfering with human nectin-4 / nectin-1 binding interactions, including reducing them; (h) interfering with human nectin-4 / TIGIT binding interactions, including reducing them; (i) reducing the level of cell surface protein expression of nectin-4 on human tumor cells; or (j) directing ADCC in human cells expressing endogenous levels of nectin-4.

[0134] In some embodiments, an anti-nectin-4 antibody or an antibody fragment thereof contains one or more conserved amino acid substitutions. Those skilled in the art will understand that a conserved amino acid substitution is the substitution of one amino acid with another amino acid having similar structural or chemical properties, such as similar side chains. Exemplary conserved substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Ed. (1987).

[0135] "Conservative modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing an amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is a substitution in which an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, as has already been described regarding alanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol Scand Suppl 643: 55-67; ​​Sasaki et al. (1998) Adv Biophys 35: 1-24), any native residue in the polypeptide can also be substituted with alanine. Amino acid substitutions for the antibodies of this disclosure can be carried out by known methods, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195).

[0136] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15. In other embodiments, the anti-nectin-4 antibody or an antibody fragment thereof retains the binding activity and / or functional activity of the anti-nectin-4 antibody or an antibody fragment thereof that includes the variable heavy chain sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15. In yet another embodiment, the anti-nectin-4 antibody or an antibody fragment thereof includes the variable heavy chain sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15, and the heavy chain variable sequence has one or more conserved amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5. In yet another embodiment, one or more conservative amino acid substitutions are contained within one or more framework regions of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 (based on Kabat's numbering system).

[0137] In certain embodiments, an anti-nectin-4 antibody or an antibody fragment thereof comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the anti-nectin-4 heavy chain variable region sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15, and comprises one or more conserved amino acid substitutions (based on Kabat's numbering system) in the framework region, and retains the binding activity and / or functional activity of an anti-nectin-4 antibody or an antibody fragment thereof comprising the variable heavy chain sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15 and the variable light chain sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16.

[0138] In some embodiments, the anti-nectin-4 antibody or an antibody fragment thereof includes a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16. In other embodiments, the anti-nectin-4 antibody or an antibody fragment thereof retains the binding activity and / or functional activity of the anti-nectin-4 antibody or an antibody fragment thereof that includes the variable light chain sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16. In yet another embodiment, the anti-nectin-4 antibody or an antibody fragment thereof includes the variable light chain sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, and the light chain variable sequence has one or more conserved amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5. In yet another embodiment, one or more conserved amino acid substitutions are contained within one or more framework regions of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16 (based on Kabat's numbering system).

[0139] In certain embodiments, an anti-nectin-4 antibody or an antibody fragment thereof comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the anti-nectin-4 light chain variable region sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, comprising one or more conserved amino acid substitutions (based on Kabat's numbering system) in the framework region, and retaining the binding activity and / or functional activity of an anti-nectin-4 antibody or an antibody fragment thereof comprising the variable heavy chain sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15 and the variable light chain sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16.

[0140] Characteristics of bonding The antibodies of the present disclosure (N4_mAb 1, N4_mAb 2, N4_mAb 3, N4_mAb 4, N4_mAb 5, N4_mAb 6, N4_mAb 7 and N4_mAb 8) and antibody fragments thereof specifically bind to human nectin-4 when human nectin-4 is present on the surface of normal or malignant cells, but do not specifically bind to the extracellular domains of human nectin-1, nectin-2 or nectin-3.

[0141] Antibodies typically bind specifically to their cognate antigens with high affinity, as reflected by a dissociation constant (KD) of 10 -8 , -8 , -10 , , -10 , , -10 ,

[0142] ~10 -11 M or less. Any KD greater than about 10 [[ID=⑨]] -6 M is generally considered to indicate non-specific binding. As used herein, an antibody that "specifically binds" to an antigen binds with high affinity to the antigen and substantially identical antigens (this means having a KD of 10 -7 M or less, preferably 10 -8 M or less, more preferably 5×10 -9 M or less, most preferably between 10 -8 M and 10 -10 M or less), but does not bind with high affinity to unrelated antigens. The antibodies of the present disclosure bind to human nectin-4 ECD with high affinity, and the KD determined by SPR ranges from 1.72×10 -8 M to 3.75×10 -10 M. N4_mAb 6, N4_mAb 7 and N4_mAb 8 represent the group with the highest affinity, having a KD less than 4×10 -10 M.

[0142] As used herein, the term “cross-reactivity” refers to the ability of the anti-human nectin-4 specific antibodies described herein to bind to nectin-4 from a different species. For example, the antibodies described herein may also bind to nectin-4 from another species (e.g., cynomolgus monkey or rat or mouse nectin-4). As used herein, cross-reactivity can be measured by detecting specific reactivity with a purified antigen in a binding assay (e.g., SPR, ELISA), or by binding to cells that physiologically express nectin-4, or otherwise by functionally interacting with them. Methods for determining cross-reactivity include, for example, BIACORE® surface plasmon resonance (SPR) analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or standard binding assays described herein by flow cytometry techniques.

[0143] The human nectin-4 antibodies N4_mAb 1 to N4_mAb 8 of this disclosure all bind to cynomolgus monkey-derived nectin-4 with remarkable affinity. Their binding affinities to rat nectin-4 vary, with N4_mAb 5, N4_mAb 6, N4_mAb 7, and N4_mAb 8 being the most potent. They show weak or no binding to mouse nectin-4.

[0144] Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain types of chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody contains a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody contains its antibody fragment.

[0145] Antibodies can be prepared as chimeric antibodies or antibody fragments thereof, possessing a mouse variable region and a human constant region. The heavy chain constant region uses the consensus human IgG1 constant region sequence (Uniprot P01857), while the light chain constant region uses the consensus human kappa constant region sequence (UniProt P01834). Human IgG1 can be chosen because it is one of the most common subtypes for chimeric antibody production and can provide effector function. The human kappa constant region can be used because all mouse parent antibodies have a mouse kappa light chain.

[0146] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans, while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody contains one or more variable domains, in which case the HVR, e.g., CDR, (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may also contain at least a portion of the human constant region, as required. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore or improve, for example, antibody-binding specificity or affinity.

[0147] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) transplantation); and Padlan, Mol. Immunol. 28:489-498. Further information is available in (1991) (described on "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (described on "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (described on the "guide selection" approach to FR shuffling).

[0148] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the “best fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of specific subgroups of light chain or heavy chain variable regions of human antibodies (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684). This includes (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0149] Nectin-4 internal distribution and dose-dependent cytotoxicity The antibodies of this disclosure, which are specific to nectin-4, can mediate internal translocation of nectin-4, including induced internal translocation, thereby leading to dose-dependent cytotoxicity in the presence of an ADC-conjugated secondary antibody. In CHO cell lines overexpressing nectin-4, the observed EC50 for cell death ranged from 0.21 nM to 0.63 nM. In the cancer cell line SKBR3, the EC50 for cell death ranged from 0.61 nM to 2.14 nM.

[0150] Antibody-based immunotherapy The goal of antibody-based immunotherapy, which uses antibodies that target tumor antigens, is to eliminate cancer cells without harming normal tissue. Therefore, the efficacy and safety of antibody-based immunotherapy in oncology vary greatly depending on the intended mechanism of action, the relevant effector function of the immune system, and the nature of tumor-specific or tumor-associated target antigens.

[0151] Antibody-drug conjugates (ADCs) are a class of highly potent antibody-based cancer therapies. ADCs consist of recombinant monoclonal antibodies covalently linked to a cytotoxic agent (known as the payload) via a synthetic linker. ADCs combine the specificity of monoclonal antibodies with the efficacy of small-molecule chemotherapeutic agents, while facilitating the direct, targeted delivery of the highly cytotoxic small-molecule drug portion to tumor cells. The targeted nature of ADCs, coupled with limited systemic exposure, allows for improved drug efficacy. Furthermore, these characteristics confer desirable traits to ADCs, such as fewer side effects and a wider therapeutic window (Peters et al., Biosci Rep, 35(4):e00225, 2015).

[0152] Generally, when an ADC binds to an antigen on the surface of a cancer cell, it is transported internally and delivered along the endosomal / lysosomal pathway for degradation. In the lysosome, the payload is released either through specific cleavage of the linker by lysosomal enzymes or through general degradation of the antibody. The released cytotoxic compound then leaves the lysosome, accumulates to the required threshold level, and ultimately causes the death of the targeted cancer cell. An ideal ADC is one that retains the selectivity and killing ability of the mAb while still being able to release a cytotoxic drug in an amount large enough to kill tumor cells.

[0153] Cell surface antigens suitable for use as ADC targets are characterized by two important properties: (i) high expression levels by target cells and limited or no expression in normal tissues, and (ii) internal translocation in response to antibody binding (e.g., efficient internal translocation). Nectin-4 is overexpressed in several cancers, particularly bladder cancer, lung cancer, pancreatic cancer, head and neck cancer, esophageal cancer, breast cancer, and ovarian cancer (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016; Fabre-Lafay et al., BMC Cancer, 7:73, 2007; Takano et al., Cancer Res, 69(16):6694-03, 2009; Derycke et al., Am J Clin Pathol, 5:835-845, 2010). It is also known that monoclonal antibodies specific to nectin-4 can mediate the induced and efficient internal translocation of nectin-4 (Doronina et al., Nat Biotechnol, 21:778-784, 2003; M-Rabet et al., Annals of Oncology, 28(4):769-776, 2017; International Publication No. 2012 / 047724; U.S. Patent No. 8,637,642; International Publication No. 2004 / 016799; U.S. Patent No. 7,968,090; International Publication No. 2017 / 042210; U.S. Patent No. 10,675,048). Therefore, the anti-nectin-4 antibodies (N4_mAb 1 to N4_mAb 8) of this disclosure are suitable for use as ADC-based targeting antibodies for the development of antibody-based immunotherapies for the treatment of cancer.

[0154] The production of antibody-drug conjugates can be carried out by any technique known to those skilled in the art, using a suitable payload drug, synthetic linker, and conjugation chemistry. Those skilled in the art are familiar with ADCs and also know the following: the development of ADCs requires evaluation of several factors, including the biology of the target antigen, the specificity of the antibody, the cytotoxicity and mechanism of action of the payload drug, the stability and cleavage of the linker, the site of linker attachment, and the level of heterogeneity of the ADC produced by conjugation chemistry. Heterogeneity can lead to the production of drug products containing inactive species (no drug payload) and species with more than four drug moieties per antibody molecule (high load) (these species are cleared more rapidly and may contribute to toxicity) in terms of the number of cytotoxic molecules attached to each antibody. Furthermore, the presence of inactive species (antibodies with no cytotoxic payload at all) can lead to reduced efficacy due to competition for binding to the ADC target antigen. Therefore, it is desirable to produce an ADC drug product containing a homogeneous mixture of antibodies characterized by a consistent drug-to-antibody ratio (DAR).

[0155] Most ADC candidates currently undergoing clinical evaluation use one of the three main classes of drugs as their cytotoxic payload: maytansinoids, auristatin, and PBD dimers; however, other classes of payloads, such as calicheamycin (for gemtuzumab ozogamicin and inotuzumab ozogamicin), duocalmycin, exatecan, or SN-38, are also used (Shim et al., Biomolecules, 10(3):360, 2020). Generally speaking, cytotoxic drugs act either as tubulin inhibitors (auristatin and maytansinoids) or as DNA disruptors, which include duocalmycin (DNA alkylation), calicheamicin (DNA double-strand breaks), camptothecin analogs such as SN-38 and exatecan (topoisomerase inhibitors), or pyrrolobenzodiazepine (PBD) dimers (DNA crosslinking) (Shim et al.).

[0156] One of the key functions of linkers is to maintain the stability of ADCs in the bloodstream, while simultaneously enabling toxin release during internal translocation by target cells. Important parameters to consider in the process of identifying a suitable linker include the linker's cleavage properties and conjugate chemical details (i.e., the location and nature of the chain).

[0157] In a broad sense, linkers are classified into two broad categories: cleavageable and non-cleavageable. Cleavageable linkers utilize the difference between the normal physiological state in the bloodstream and the intracellular state present in the cytoplasm of cancer cells (Peters et al., Biosci Rep, 35(4):e00225, 2015). Changes in the microenvironment after the ADC-antigen complex has been translocated trigger linker cleavage, releasing a cytotoxic payload that effectively delivers toxicity to target cancer cells expressing the target antigen. In a broad sense, there are three types of cleavageable linkers: hydrazone, disulfide, and peptide linkers. In contrast, non-cleavageable linkers simply rely on the process of lysosomal degradation following the translocation of the ADC antigen. After the translocation of the ADC-antigen complex, protease enzymes in lysosomes degrade the antibody protein structure, yielding a single amino acid (usually cysteine ​​or lysine) attached to the linker and a cytotoxic agent released into the cytoplasm as the active drug. It is well known that linker chemistry is a crucial determinant of the specificity, potency, activity, and safety of ADCs.

[0158] Those skilled in the art will recognize that there are many techniques for chemically modifying proteins suitable for use in the conjugation of linker payloads to TSA- or TAA-specific antibodies. Those skilled in the art will also recognize that various methods of conjugation chemistry provide varying levels of control over the number and site of drug attachment, and that the pharmacokinetics, toxicity, and therapeutic window of the anti-nectin-4 ADCs produced may be affected. Antibody-drug conjugates can be prepared by conjugating drugs to antibodies according to conventional techniques.Techniques for conjugating the therapeutic portion into antibodies are well known to those skilled in the art. For example, see: Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506. (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

[0159] Those skilled in the art will understand that, in addition to conventional conjugation techniques (which include conjugation to either lysine or cysteine ​​residues exposed on the surface of the antibody as a result of its natural amino acid sequence composition), there are numerous other methods relating to site-directed drug conjugation that can be used to prepare anti-nectin-4 specific immune complexes.

[0160] Site-directed conjugation chemistry aims to produce relatively homogeneous ADC products without altering the antibody binding affinity. Generally speaking, three main strategies are used for site-directed antibody conjugation: the use of modified cysteine, the incorporation of non-natural amino acids, and enzymatic conjugation, which uses an antibody reaction site designed to react specifically with bacterial enzymes (e.g., transglutaminase, glycotransferase, saltase, or formylglycine-producing enzymes) that produce post-translational modifications of proteins in a site-directed manner. Techniques for site-specifically conjugating therapeutic portions into antibodies are well known to those skilled in the art, and include, but are not limited to, U.S. Patent Nos. 7,723,485; 8,937,161; 9,000,130; 9,884,127; 9,717,803; 10,639,291; 10,357,472; U.S. Patent Publication Nos. 2015 / 0283259; 2017 / 0362334; 2018 / 0140714; and International. The methods of disclosure are included in pamphlets No. 2013 / 092983; No. 2013 / 092998; No. 2014 / 072482; No. 2014 / 202773; No. 2014 / 202775; No. 2015 / 155753; No. 2015 / 191883; No. 2016 / 102632; No. 2017 / 059158; No. 2018 / 140590 and No. 2018 / 185526.

[0161] ADCs can be designed to kill not only target antigen-positive cells but also other nearby cells, regardless of the expression of the target antigen on their surface, through a mechanism commonly known as the "bystander effect" (Kovtun et al., Cancer Res, 66(6):3214-21, 2006). While the bystander effect weakens the concept of absolute target specificity of ADCs, it can be advantageous when treating solid tumors that lack uniform expression of the target antigen. Enfortumab vedotin is known to exert a bystander effect by releasing cell-permeable MMAEs from nectin-4-positive cells in mixed cell assays, thereby killing nectin-4-negative cancer cells (Liu et al., Abstract 5581, Poster presented at the American Association of Cancer Research Virtual Meeting II, 2020). Documentation on the bystander effect of enfortumab vedotin supports future clinical trials of nectin-4-targeted ADCs, either alone or in combination with other checkpoint inhibitors, for the treatment of tumors characterized by heterogeneous expression of nectin-4.

[0162] Enfortumab vedotin Enfortumab vedotin-ejfv (PADCEV®) is the first and only FDA-approved nectin-4 targeted ADC. Enfortumab vedotin (AGS-22M6E) consists of a fully human IgG1-kappa anti-nectin-4 antibody (AGS-22C3). The ADC is conjugated to the small molecule monomethyl auristatin E (MMAE, a microtubule disruptor) by a protease-cleaving linker (maleimidocaproylvaline-citrulline) (see, for example, International Publication No. 2012 / 047724, U.S. Patent Nos. 8,637,642, 9,078,931, and 9,962,454). The ADC binds to nectin-4 expressed on the cell surface, and the entire complex moves into the cell. MMAE is cleaved from the complex, leading to disruption of the intracellular microtubule network, resulting in cell cycle arrest and apoptosis (programmed cell death).

[0163] The parental antibody (AGS-22M6) used to generate enfortumab was produced by immunizing the XENOMOUSE® strain (Amgen / Abgenix) with the extracellular domain (ECD) of human nectin-4. AGS-22M6 binds to transfected human, monkey, and rat nectin-4 expressed on the surface of human PC3 (prostate cancer) host cells (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016). AGS-22M6 recognizes the epitope of the first Ig-like domain of nectin-4 and blocks the nectin-4 / nectin-1 trans-interaction in vitro, but has not been reported to have any effect on cell viability (Challita-Eid et al.). Notably, the parental antibody did not mediate antitumor activity in any preclinical model, and the efficacy of enfortumab vedotin correlated with the bound cytotoxic payload and nectin-4 expression.

[0164] Enfortumab vedotin was first approved by the U.S. Food and Drug Administration in December 2019 for use in patients with locally advanced and metastatic urothelial carcinoma who have received prior treatment with immune checkpoint inhibitors (i.e., programmed cell death protein 1 (PD-1) / programmed death-ligand 1 (PD-L1) therapy) and platinum-based chemotherapy, either as neoadjuvant or adjuvant treatment in a locally advanced and metastatic environment. Urothelial carcinoma, which accounts for more than 90% of bladder cancers, begins with cells covering the bladder and surrounding organs. Platinum-containing chemotherapy, PD-1 inhibitors, and PD-L1 inhibitors are standard treatments for patients with bladder cancer, the sixth most common cancer in the United States.

[0165] Published reports indicate that, following the binding of enfortumab vedotin to nectin-4 expressing cancer cells (i.e., bladder cancer cell line T24-nectin-4), the enfortumab vedotin / nectin-4 complex is translocated internally and catabolized in the intracellular lysosomal compartment (Doronina et al., Nat Biotechnol, 21:778-784, 2003). The resulting intracellular release of MMAE into the cytosol is known to induce growth arrest in the G2 / M phase, followed by apoptotic cell death (Francisco, JA et al, Blood, 102:1458 2003). As expected, higher levels of cytotoxic cell death correlated with higher levels of intracellular MMAE release. The unconjugated parental antibody (AGS-22M6) used to prepare the ADC was reported to have no cytotoxic activity.

[0166] During its preclinical development, enfortumab vedotin demonstrated antitumor activity in nectin-4 expressing cell lines and animal models. Enfortumab vedotin inhibited the growth of nectin-4 expressing tumors in mouse xenograft models of human bladder, pancreatic, breast, and lung cancer. Enfortumab vedotin treatment significantly inhibited the growth of all four tumor types and resulted in tumor regression in bladder and breast xenografts (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016). In a xenograft model of bladder cancer (T-24 cells transplanted into nude mice), the effects of enfortumab vedotin ADC-based immunotherapy have been reported to include not only targeted auristatin delivery, cell cycle arrest, and apoptosis, but also immunogenic cell death (ICD) including bystander cell death, extracellular release of adenosine triphosphate and HMGB1, immune cell recruitment, and activation of antigen-presenting cells (Liu et al., Abstract 5581, Poster presented at the American Association of Cancer Research Virtual Meeting II, 2020).

[0167] The FDA granted accelerated approval for enfortumab vedotin based on the response rate and response durability observed in the multicenter Phase II EV-201 trial (ClinicalTrials.gov identifier NCT03219333). The EV-201 trial included 125 patients with locally advanced or metastatic urothelial carcinoma who had received prior treatment with a PD-1 inhibitor or PD-L1 inhibitor and platinum-based chemotherapy. Patients received enfortumab vedotin-ejfv at 1.25 mg / kg on days 1, 8, and 15 of a 28-day cycle until disease progression or unacceptable toxicity. Reflecting the percentage of patients with a certain degree of tumor reduction, the overall response rate was 44%, with 12% achieving a complete response and 32% achieving a partial response. The median duration of response was 7.6 months (Rosenberg et al., J Clin Oncol, 37(29):2592-2600, 2019).

[0168] The EV-201 trial will be advanced to a second cohort (Cohort 2) of patients who have received prior anti-PD-1 / L1 therapy and are cisplatin-ineligible without prior platinum-based treatment, to determine if similar effects are observed. Enfortumab vedotin has also been evaluated in other solid tumors, including hormone receptor-positive / HER-negative breast cancer, triple-negative breast cancer, non-squamous non-small cell lung cancer, head and neck cancer, as well as gastric and esophageal cancer, in the Phase 2 EV-202 trial (ClinicalTrials.gov identifier NCT04225117).

[0169] In addition, a Phase III clinical trial (EV-301; ClinicalTrials.gov identifier: NCT03474107) demonstrated the survival benefit of enfortumab vedotin in patients with prior platinum-based and prior anti-PD-1 / L1 therapy by comparing enfortumab vedotin monotherapy with monotherapy in such patient populations. Enfortumab vedotin has also been tested in Phase I / II clinical trials in combination with anti-PD-1-based and / or platinum-based therapies (EV-103; ClinicalTrials.gov identifier: NCT03288545) and is being evaluated in a broader patient population of urothelial carcinoma, including as a first-line treatment.

[0170] Method for producing antibodies Anti-nectin-4 antibodies or antibody fragments thereof can be prepared by any method known in the art. For example, a recipient can be immunized with soluble recombinant nectin-4 (N4) protein or a fragment of N4 peptide conjugated with a carrier protein thereof. Any suitable immunization method can be used. Such methods include the use of adjuvants, other immunostimulants, repeated booster immunization, and one or more immune pathways.

[0171] Any suitable source of human nectin-4 can be used as an immunogen for the production of non-human or human anti-nectin-4 antibodies of the compositions and methods disclosed herein.

[0172] Various forms of nectin-4 antigens can be used to induce an immune response for the identification of a biologically active anti-nectin-4 antibody. Thus, the inducing nectin-4 antigen may be a single epitope, multiple epitopes, or an entire protein, either alone or in combination with one or more immunogenicity enhancers. In some embodiments, the inducing antigen is an isolated soluble full-length protein, or a soluble protein containing less than a full-length sequence (e.g., immunizing with a peptide containing either or both of the V-like domain or C-like domain of human nectin-4). As used herein, the term “part” refers, where necessary, to the minimum number of amino acids or nucleic acids that constitute the immunogenic epitope of the antigen of interest. Any genetic vector suitable for transforming the cells of interest may be used, including, but not limited to, adenovirus vectors, plasmids, and non-viral vectors such as cationic lipids.

[0173] It is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts such as mice, rodents, primates, and humans. Techniques for preparing such monoclonal antibodies can be found, for example, in Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and the references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; and Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells derived from animals immunized with the desired antigen are immortalized, usually by fusion with myeloma cells. See Kohler and Milstein (196) Eur. J. Immunol. 6:511-519. Alternative methods for immortalization include transformation using Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the field. See, for example, Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies arising from a single immortalized cell are screened for the production of antibodies with desired specificity and affinity for an antigen. The yield of monoclonal antibodies produced by such cells can be increased by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, monoclonal antibodies or DNA sequences encoding their antigen-binding fragments can be isolated by screening a human B-cell-derived DNA library according to a general protocol outlined, for example, Huse et al. (1989) Science 246: 1275-1281.Therefore, antibodies can be obtained by various techniques well known to researchers skilled in this field.

[0174] Other appropriate techniques involve the selection of antibody libraries in phages, yeasts, viruses, or similar vectors. See, for example, Huse et al. cited above; and Ward et al. (1989) Nature 341:544–546. The polypeptides and antibodies disclosed herein can be used with or without modification, including chimeric or humanized antibodies. Often, polypeptides and antibodies are labeled by covalently or noncovalently linking them with substances that provide a detectable signal. A wide variety of labeling and conjugation techniques are known and widely reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, and magnetic particles. Patents teaching the use of such signs include U.S. Patent Nos. 3,817,837; 3,850,752; 3,9396,345; 4,277,437; 4,275,149; and 4,366,241. Furthermore, recombinant immunoglobulins can also be produced; see Cabilly U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10023; or, they can be produced in transgenic mice; see Nils Lonberg et al., (1994), Nature 368:856-859; and Mendez et al. (1997) Nature Genetics 15: 146-156; TRANSGENIC ANIMALS AND METHODS OF USE (International Publication No. 2012 / 62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour and other techniques.

[0175] In some embodiments, the ability of the produced antibody to bind to nectin-4 and / or other relevant members of the nectin family can be evaluated using standard binding assays, such as surface plasmon resonance (SPR), FoteBio (BLI), ELISA, Western blotting, immunofluorescence, flow cytometry analysis, chemotaxis assays, and cell migration assays. In some embodiments, the produced antibody can also be evaluated for its ability to block / inhibit nectin-4 from binding to nectin-1 or TIGIT, or for its ability to efficiently translocate nectin-4 to nectin-4-expressing cells upon binding.

[0176] Antibody compositions prepared from hybridomas or host cells can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the standard purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and human gamma 3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those obtainable with agarose. If the antibody contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation with ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin SEPHAROSE®, chromatography with anion or cation exchange resins (e.g., polyaspartate columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody being recovered.

[0177] Following any preliminary purification steps, the mixture containing the antibody of interest and any contaminants can be subjected to low-pH hydrophobic interaction chromatography, typically at low salt concentrations (e.g., from about 0–0.25 M salt), but using an elution buffer at a pH between approximately 2.5 and 4.5.

[0178] The disclosure also includes nucleic acids that hybridize to all or part of a nucleotide sequence (e.g., the portion encoding the variable region) represented by an isolated polynucleotide sequence encoding an antibody or antibody fragment of the disclosure, under low, medium, and high stringency conditions as defined herein. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides long. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98%, identical to the sequence of part or all of the nucleic acid or its complementary chain encoding an anti-nectin-4 polypeptide (e.g., the heavy chain variable region or the light chain variable region). The types of hybridizing nucleic acids described herein can be used, for example, as cloning probes, primers, e.g., PCR primers, or diagnostic probes.

[0179] Polynucleotides, vectors, and host cells Other embodiments include isolated polynucleotides comprising sequences encoding an anti-nectin-4 antibody or an antibody fragment thereof, vectors comprising the polynucleotide, and host cells, as well as recombinant techniques for producing the antibody. The isolated polynucleotides can encode any desired form of anti-nectin-4 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, chimeric antibodies, humanized antibodies, bispecific antibodies, and multispecific antibodies formed from antibody fragments.

[0180] Some embodiments include isolated polynucleotides containing a sequence encoding the heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15. Some embodiments also include isolated polynucleotides containing a sequence encoding the light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16.

[0181] In one embodiment, the isolated polynucleotide sequence is (a) Variable heavy chain sequence containing Sequence ID 1 and variable light chain sequence containing Sequence ID 2; (b) Variable heavy chain sequence containing Sequence ID 3 and variable light chain sequence containing Sequence ID 4; (c) Variable heavy chain sequence containing Sequence ID No. 5 and variable light chain sequence containing Sequence ID No. 6; (d) Variable heavy chain sequence containing SEQ ID NO: 7 and variable light chain sequence containing SEQ ID NO: 8; (e) Variable heavy chain sequence containing Sequence ID No. 9 and variable light chain sequence containing Sequence ID No. 10; (f) Variable heavy chain sequence containing sequence number 11 and variable light chain sequence containing sequence number 12; (g) A variable heavy chain sequence containing SEQ ID NO: 13 and a variable light chain sequence containing SEQ ID NO: 14; or (h) Variable heavy chain sequence containing Sequence ID No. 15 and variable light chain sequence containing Sequence ID No. 16 It encodes an antibody or antibody fragment having a light chain variable region and a heavy chain variable region, containing the amino acid sequence.

[0182] In another embodiment, the isolated polynucleotide sequence is (a) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 2; (b) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 4; (c) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 6; or, (d) Variable heavy chain sequences that are 90%, 95%, or 99% identical to SEQ ID NO: 7 and variable light chain sequences that are 90%, 95%, or 99% identical to SEQ ID NO: 8 (e) Variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 9 and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 10; (f) A variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 11 and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 12; (g) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 13 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 14; or (h) Encodes an antibody or antibody fragment having a light chain variable region and a heavy chain variable region, which include an amino acid sequence of a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 15 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 16.

[0183] A polynucleotide containing a sequence encoding an anti-nectin-4 antibody or an antibody fragment thereof can be fused to one or more regulatory sequences known in the art, and this can be incorporated into a suitable expression vector or host cell known in the art. Each polynucleotide molecule encoding a heavy chain variable domain or a light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, thereby enabling the production of intact antibodies. Alternatively, polynucleotides or parts thereof can be fused to each other, resulting in templates for the production of single-chain antibodies.

[0184] In recombinant production, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (DNA amplification) or expression. Many suitable vectors for expressing recombinant antibodies are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0185] Anti-nectin-4 antibodies or their antibody fragments may also be produced as fusion polypeptides, in which case the antibody or fragment is fused with a heterologous polypeptide such as a signal sequence, or another polypeptide having a cleavage site specific to the amino terminus of a mature protein or polypeptide. The selected heterologous signal sequence is typically recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the anti-nectin-4 antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence can be, for example, alkaline phosphatase, penicillinase, lipoprotein, or a thermostable enterotoxin II leader. In yeast secretion, the native signal sequence can be replaced with, for example, a leader sequence obtained from the signals described in International Publication No. 90 / 13646, such as yeast invertase alpha factor (including the alpha factor leaders of Saccharomyces and Kluyveromyces), acid phosphatase, C. albicans glucoamylase, or other signals. In mammalian cells, mammalian signal sequences, as well as viral secretion leaders, such as the herpes simplex gD signal, can be used. The DNA of such precursor regions is ligated within the reading frame to the DNA encoding the anti-nectin-4 antibody.

[0186] Expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, these sequences within a cloning vector enable the vector to replicate independently of host chromosomal DNA, and include origins of replication or autonomous replication sequences. Such sequences are well known for various bacteria, yeasts, and viruses. Origins of replication derived from plasmid pBR322 are suitable for most Gram-negative bacteria, 2-υ plasmid origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, origins of replication components are not required for mammalian expression vectors (the SV40 origin can usually be used simply because it contains an initial promoter).

[0187] Expression vectors and cloning vectors may contain genes encoding selection markers to facilitate the identification of expression. Typical selection marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or alternatively, complementary nutritional requirement deficiencies, or, in other options, the selection marker gene supplies specific nutrients that are not present in the complex medium, for example, the gene encoding D-alanine racemase for Bacilli.

[0188] Antibody composition and treatment method This disclosure also provides compositions, including, for example, pharmaceutical compositions, that include an anti-nectin-4 antibody or an antibody fragment thereof for use in the treatment of patients having cancer, including, for example, primary or metastatic cancer of epithelial cell origin. In certain embodiments, the compositions described herein are administered to cancer patients to kill tumor cells. For example, the compositions described herein can be used to treat patients with solid tumors characterized by the presence of cancer cells expressing or overexpressing nectin-4. In some embodiments, the compositions of this disclosure can be used to treat breast cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, gallbladder cancer, or urothelial cancer.

[0189] In some embodiments, cancer treatment is an area where combination strategies are particularly desirable because the combined action of two, three, four, or more anticancer drugs / therapies often produces synergistic effects that are considerably stronger than those of monotherapy approaches. The drugs and compositions provided herein (e.g., pharmaceutical compositions) can be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, allogeneic, allogeneic, or unrelated). The drugs and compositions may also be used in combination with one or more of the following: antitumor agents, chemotherapeutic agents, proliferation inhibitors, cytotoxic agents, immune checkpoint inhibitors, costimulatory molecules, kinase inhibitors, angiogenesis inhibitors, small molecule targeted therapies, and multiepitope strategies. Thus, in another embodiment of this disclosure, cancer treatment can be effectively combined with a variety of other drugs.

[0190] In one treatment method, the pharmaceutical composition containing an anti-nectin-4 antibody may further contain a therapeutic agent or a poison, either conjugated to or unconjugated to the anti-nectin-4 antibody or antibody fragment. In a particular embodiment, an anti-nectin-4 antibody is used to deliver an ADC containing a cytotoxic payload to a target tumor that expresses and / or overexpresses nectin-4.

[0191] The Nectin-4 antibody of this disclosure can be administered either alone or in combination with other compositions useful for treating cancer. In one embodiment, the antibody of this disclosure can be administered either alone or in combination with other immunotherapies, including other antibodies useful for treating cancer. For example, in one embodiment, the other immunotherapies are antibodies against immune checkpoint molecules selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, lymphocyte activation gene 3 (LAG3), NKG2A, B7-H3, B7-H4, CTLA-4, GITR, VISTA, CD137, TIGIT, and any combination thereof. In an alternative embodiment, the second immunotherapy is an antibody against tumor-specific antigen (TSA) or tumor-associated antigen (TAA). Each combination represents a separate embodiment of this disclosure.

[0192] The therapeutic agent combinations discussed herein may be administered simultaneously as components of a bispecific or multispecific binder or fusion protein, or as single compositions contained in a pharmaceutically acceptable carrier. Alternatively, the therapeutic agent combination may be administered simultaneously as separate compositions having each agent contained in a pharmaceutically acceptable carrier. In another embodiment, the therapeutic agent combination may be administered sequentially.

[0193] Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, in accordance with conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. In some embodiments, the pharmaceutical compositions are administered to subjects to treat cancer.

[0194] As used herein, “pharmaceutically acceptable carriers” include any physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Preferably, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, active compounds, i.e., antibodies, bispecific molecules, and multispecific molecules, can be coated with a substance to protect them from the action of acids and other natural conditions that may inactivate them.

[0195] Typically, compositions for administration by injection are solutions of sterile isotonic aqueous buffer. If necessary, the pharmacopoeia may also contain a solubilizer and a local anesthetic such as lignocaine to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage forms, and supplied as lyophilized powders or water-free concentrates in sealed containers, such as ampoules or sachets indicating the amount of the active agent. When administered by infusion, the pharmacopoeia may be administered using drip bottles containing sterile pharmaceutical-grade water or saline. When administered by injection, the pharmacopoeia may be supplied in ampoules of sterile water for injection or saline so that the components can be mixed before administration.

[0196] The compositions of this disclosure can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. The active compound can be prepared with a carrier that will protect the compound from rapid release, such as a controlled-release formulation, including delivery systems by implant, transdermal patch, and microencapsulation. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0197] In alternative embodiments, nucleic acids encoding the antibodies or derivatives thereof described herein can be introduced into mammalian cells or target tissues using conventional viral and nonviral-based gene transfer methods. Such methods can be used to administer antibody-encoding nucleic acids to cells in vitro. In some embodiments, nucleic acids encoding antibodies or derivatives thereof are administered for in vivo or ex vivo gene therapy applications. In other embodiments, gene delivery techniques are used to study the activity of antibodies in cell-based or animal models. Nonviral vector delivery systems include nucleic acids complexed with delivery vehicles such as DNA plasmids, naked nucleic acids, and liposomes. Viral vector delivery systems include DNA viruses and RNA viruses that have either an episomal genome or an integrated genome after delivery to cells. Such methods are well known in the art.

[0198] Methods for nonviral delivery of nucleic acids encoding the modified polypeptides described herein include lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Publication No. 91 / 17424, and International Publication No. 91 / 16024. They can be delivered to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid conjugates, including targeted liposomes such as immunolipid conjugates, is well known to those skilled in the art.

[0199] The RNA virus-based or DNA virus-based systems for delivering antibody-encoding nucleic acids described herein take advantage of highly advanced methods for targeting viruses to specific cells in the body and delivering viral payloads to the nuclei. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro and then the modified cells can be administered to patients (ex vivo). Conventional virus-based systems for polypeptide delivery of the disclosure include retroviral, lentiviral, adenovirus, adeno-associated herpesvirus, and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method for gene transfer in target cells and tissues. Integration in the host genome is possible using retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in a wide variety of cell types and target tissues.

[0200] The dosage level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of the active ingredient that is non-toxic to the subject and effective in achieving the desired therapeutic response with respect to a particular subject, composition, and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of this disclosure to be employed, the route of administration, the time of administration, the excretion rate of the particular compound to be employed, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition to be employed, and factors well known in the medical field such as the age, sex, weight, condition, overall health, and prior medical history of the patient being treated.

[0201] The pharmaceutical compositions described herein may be administered in an effective dose. “Effective dose” means an amount that, alone or in combination with additional doses, achieves the desired response or effect. In the case of treating a particular disease or condition, the desired response is preferably related to inhibiting the course of the disease. This includes delaying the progression of the disease, and in particular interrupting or improving the progression of the disease.

[0202] Non-therapeutic use Both the soluble (sN4) and transmembrane nectin-4 isoforms have been described in rodents and humans (Reymond et al., J Biol Chem, 276(46):43205-15, 2001). Soluble nectin-4 is produced by proteolytic cleavage on the cell surface by the metalloproteinases ADAM17 / TACE Soluble N4 (Fabre-Lafay et al., J Biol Chem, 289(20):19543-19550, 2005) and ADAM10 (Buchanan et al., J Biol Chem, 292(15):6339-6351, 2017), and can be detected in the serum of breast, ovarian, and lung cancer patients (Buchanan et al., Fabre-Lafay et al., BMC Cancer, 7:73, 2007, Takano et al., Cancer Res, 69(16):6694-03, 2009).

[0203] Several clinical studies have revealed that nectin-4 functions as a tumor biomarker, and that overexpression of nectin-4 in cancer tissue is significantly associated with cancer progression and low survival rates in patients (Fabre-Lafay et al., BMC Cancer, 7:73, 2007; Siddharth et al., Int J Biochem Cell Biol, 102:151-160, 2018; Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015; Zhang et al., Oncology Lett 18:1163-1170, 2019; Derycke et al., Am J Clin Pathol, 5:835-845, 2010; Deng et al., Cancer Cell Int, 19:106, 2019).

[0204] Abnormal expression of both the membranous and soluble forms of nectin-4 has been reported in human breast cancer tissue and its serum (Fabre-Lafay et al., BMC Cancer, 7:73, 2007), and nectin-4 has been proposed as a useful histological and serological tumor-related marker and a prognostic factor for breast cancer patients (Fabre-Lafay et al., BMC Cancer, 7:73, 2007, M-Rabet et al., Annals of Oncology, 28(4):769-776, 2017, Siddharth et al., Int J Biochem Cell Biol, 102:151-160, 2018). In human pancreatic cancer, overexpression of nectin-4 significantly promotes cancer cell proliferation and contributes to intratumoral angiogenesis (Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015). Compared to normal gastric tissue, human gastric cancer tissue shows higher nectin-4 expression, and it has been demonstrated that nectin-4 expression levels are significantly associated with cancer cell differentiation, lymph node metastasis, advanced TNM stage, and poor prognosis in patients (Zhang et al., Hum Pathol, 72:107-116, 2018). Nectin-4 has also been reported to be overexpressed in esophageal cancer and colorectal cancer (Deng et al., Cancer Cell Int, 19:106, 2019, Zhang et al., Oncology Lett 18:1163-1170, 2019).

[0205] The detection of nectin-4 can be a useful prognostic factor for tumor progression. Such a method comprises the steps of contacting a biological sample from a subject with an anti-nectin-4 antibody or an antibody fragment thereof, and detecting the binding of the antibody to nectin-4. “Biological sample” refers to any biological sample obtained from individual cell lines, tissue cultures, or other sources of cells potentially expressing nectin-4. Methods for obtaining tissue biopsies for immunohistochemical analysis and bodily fluids for detection of soluble proteins in serum or plasma of human subjects are well known in the art.

[0206] Anti-nectin-4 antibodies or antibody fragments are also useful in diagnostic assays to detect and / or quantify the nectin-4 protein, for example, by detecting nectin-4 expression in specific cells, tissues, or serum. Anti-nectin-4 antibodies can be used diagnostically, for example, as part of a clinical trial procedure to monitor the onset or progression of a disease, thereby determining, for example, the effectiveness of a given treatment and / or prophylactic regimen.

[0207] In some embodiments, for example for diagnostic purposes, it may be advantageous to label the antibody at a detectable portion. A number of detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, etc. Labels can be indirectly conjugated to antibodies using various known techniques. For example, an antibody can be conjugated with biotin, and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin selectively binds to avidin, and therefore the label can be conjugated to the antibody in this indirect manner. Alternatively, to obtain indirect conjugation of a label with an antibody, the antibody can be conjugated with a small hapten (e.g., digoxin), and one of the various types of labels mentioned above can be conjugated with an anti-hapten antibody (e.g., an anti-digoxin antibody). This allows for indirect conjugation of the label with the antibody.

[0208] Exemplary radioisotope labels include 35S, 14C, 125I, 3H, and 131I. Antibodies can be labeled with radioisotopes using techniques described, for example, in *Current Protocols in Immunology*, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured, for example, by scintillation assays.

[0209] Exemplary fluorescent labels include those derived from rare earth chelators (europium chelators), or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine, phycoerythrin, and Texas Red are available. Fluorescent labels can be conjugated to antibodies via known techniques, such as those disclosed in Current Protocol in Immunology. Fluorescence can be quantified using a fluorophotometer. A variety of well-characterized enzyme substrate labels are known in the art (see, for example, U.S. Patent No. 4,275,149). Enzymes generally catalyze chemical changes in chromogenic substrates, which can be measured using various techniques. For example, the change may be a change in the hue of the substrate, which can be measured by spectrophotometry. Alternatively, enzymes can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence are described above. Chemiluminescent substrates can be electrically excited by a chemical reaction and then emit light, which can be measured, for example, using a chemiluminometer, or they can donate energy to a fluorescence receptor.

[0210] Examples of enzyme labeling include luciferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes with antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, NY, 73: 147-166.

[0211] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as the substrate, in which case the hydrogen peroxidase oxidizes a pigment precursor such as orthophenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) with para-nitrophenyl phosphate as the chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate such as p-nitrophenyl-β-D-galactosidase or a fluorescent substrate such as 4-methylumbelliferyl-β-D-galactosidase.

[0212] In another embodiment, the anti-nectin-4 antibody or an antibody fragment thereof is used unlabeled and detected with a labeled antibody that binds to the anti-nectin-4 antibody or its antibody fragment.

[0213] The antibodies and antibody fragments described herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0214] All identified patents and publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methods described in such publications that may be used in connection with this disclosure. These publications are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventors that they have no prior rights to such disclosure, whether on the grounds of prior disclosure or for any other reason. All statements regarding the dates or content of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or content of these documents.

[0215] Unless otherwise indicated, it will be understood by those skilled in the art that any of the various embodiments described and illustrated herein may be further modified to incorporate the characteristics shown in any of the other embodiments disclosed herein.

[0216] The broad scope of this disclosure will be best understood by reference to the following examples, which are not intended to limit this disclosure to any particular embodiment. The specific embodiments described herein are provided for illustrative purposes only, and this disclosure is limited by the terms of the appended claims, including the entire scope of equivalents to which such claims are entitled. [Examples]

[0217] General method Methods for protein purification, including immunoprecipitation, chromatography, and electrophoresis, are described. For example, see Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, fusion protein generation, and protein glycosylation are also described. For example, see Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra.

[0218] The supernatant of hybridomas or cell cultures containing anti-nectin-4 antibody was purified using a HiTrap Protein G column (GE, catalog no. 17040401) according to the manufacturer's procedure. Briefly, the supernatant was equilibrated with 5 CVs of DPBS (Gibco, catalog no. 14190-136) and loaded via a syringe / injection pump (Legato 200, KDS) at ambient temperature with a residence time of 3 minutes. The column was washed with 5 CVs of DPBS and eluted with 4 CVs of pH 2.8 elution buffer (Fisher Scientific, catalog no. PI21004). The elutes were fractionated, and the fractions were neutralized with 1 M Tris-HCl, pH 8.5 (Fisher Scientific, catalog no. 50-843-270) and assayed with A280 (DropSense96, Trinean). The peak fractions were pooled and the buffer was replaced with DPBS. A centrifugal filter (EMD Millipore, catalog number UFC803024) was equilibrated in DPBS at 4,000 × g for 2 minutes. The purified sample was loaded, DPBS was added, and the sample was rotated at 4,000 × g for 5-10 minutes until the total DPBS volume was ≥6 DV. The final pool was analyzed by A280.

[0219] Standard methods in molecular biology are described. For example, see Maniatis et al., (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods can also be found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), complex carbohydrate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0220] Stable cell lines expressing human (Homo sapiens) nectin-4 (NCBI accession number NM_030916.2) were generated by transfecting selected host cells (i.e., CHO-K1) with a pcDNA3.1-based plasmid expressing human nectin-4 using electroporation-based transfection. Integrated cells were selected using Geneticin. Seven to ten days after Geneticin selection, stable clones were isolated by FACS using a PE-conjugated anti-nectin-4 antibody (R&D Systems, catalog number FAB2659P). After proliferation, stable clones were further confirmed for nectin-4 expression by flow cytometry.

[0221] The sequences of the heavy and light chain variable regions of the hybridoma clone were determined as follows. Total RNA was analyzed using the RNeasy Plus Mini Kit from Qiagen (Germantown, MD, USA), at a rate of 1-2 × 10⁶. 6 cDNA was extracted from hybridoma cells. cDNA was generated by performing a 5' RACE reaction using the SMARTer RACE 5' / 3' Kit from Takara (Mountainview, CA, USA). PCR was performed using Q5 High-Fidelity DNA Polymerase from NEB (Ipswitch, MA, USA) to amplify the variable regions from the heavy and light chains using the Takara Universal Primer mix in combination with gene-specific primers for the 3' mouse constant region of appropriate immunoglobulins. The amplified variable regions for the heavy and light chains were run on a 2% agarose gel, appropriate bands were excised, and then the gel was purified using the Mini Elute Gel Extraction Kit from Qiagen. The purified PCR products were cloned using the Zero Blunt PCR Cloning Kit from Invitrogen (Carlsbad, CA, USA), transformed into Takara Stellar Competent E.Coli cells, and seeded on LB agar + 50 μg / mL kanamycin plates. Direct colony-Sanger sequencing was performed using GeneWiz (South Plainfield, NJ, USA). The resulting nucleotide sequences were analyzed using IMGT V-QUEST to identify productive rearrangements, and the translated protein sequences were analyzed. CDR determination was based on Kabat numbering.

[0222] The selected VH or VL chain was PCR amplified and cloned into a pcDNA3.4-based expression vector containing a constant region derived from human IgG1 (Uniprot P01857) or human kappa light chain (UniProt P01834). Paired heavy- and light-expression plasmids were transfected into Expi293 cells (Thermo Fisher Scientific) according to the provider's Expi293 expression system protocol. Five days after transfection, the culture supernatant was collected by centrifugation. Chimeric antibodies were purified by a one-step affinity purification using a Protein A column and buffered PBS pH 7.2.

[0223] Flow cytometry methods are available, including the Fluorescence-Activated Cell Sorting and Detection System (FACS®). See, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Suitable fluorescent reagents for modifying nucleic acids are available, including nucleic acid primers and probes, polypeptides, and antibodies for use as diagnostic reagents. See, for example, Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0224] Standard techniques are available for characterizing ligand / receptor interactions. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York. Standard methods for antibody functional characterization suitable for characterizing antibodies with specific mechanisms of action are also well known to those skilled in the art.

[0225] An in-house developed nectin-4 specific antibody based on the fully human anti-nectin-4 antibody enfortumab (AGS-22M6), referred herein as “Positive Control 1” (PC1), was prepared based on publicly available information published in International Publication No. 2012 / 047724 (VH, SEQ ID NO: 7; and VL, SEQ ID NO: 8). The PC1 antibody was used to confirm nectin-4 expression by the transfectants and tumor cell lines used in the examples, and to establish binding and functional assays used to evaluate and characterize the anti-nectin-4 specific antibodies disclosed herein. A second in-house developed nectin-4 antibody, referred herein as “Positive Control 2” (PC2), was prepared based on publicly available information published in International Publication No. 2019 / 215728 (VH, SEQ ID NO: 37; and VL, SEQ ID NO: 38). The PC2 antibody was used as a control in assays to block the interaction between TIGIT and nectin-4.

[0226] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites, and sequence alignments.

[0227] [Example 1] Production of anti-nectin-4 antibodies Mouse anti-nectin-4 antibodies were generated by immunizing Balb / c mice with recombinant human nectin-4 protein.

[0228] Immunization: Balb / c mice were immunized with recombinant human nectin-4 protein either intraperitoneally (IP) or subcutaneously (SC). The immune response was monitored by post-orbital blood sampling. Plasma was screened by ELISA, flow cytometry (FACS), or imaging (as described below), and mice with sufficient anti-nectin-4 titers were used for fusion. Mice were further immunized intraperitoneally or intravenously with immunogen, then sacrificed, and the spleen and lymph nodes were removed.

[0229] Selection of mice producing anti-nectin-4 antibodies: To select mice producing antibodies that bind to nectin-4, serum from immunized mice was screened by ELISA, FACS, or imaging for binding to recombinant nectin-4 protein or nectin-4 expressing cell lines (CHO-nectin-4) or nectin-4 expressing endogenous cell lines (T-47D, purchased from ATCC), and for non-binding to parental CHO cells that do not express nectin-4. For ELISA, briefly, ELISA plates coated with recombinant human nectin-4 were incubated with diluted serum from immunized mice at room temperature for 1 hour, the assay plates were washed, and specific antibody binding was detected using HRP-labeled anti-mouse IgG antibody. The plates were read using an ELISA reader (Biotek). For FACS, briefly, CHO-nectin-4 cells, parental CHO cells, or nectin-4 expressing endogenous cells (T-47D) were incubated with diluted serum from immunized mice. Cells were washed, and specific antibody binding was detected using Alexa 647-labeled goat anti-mouse IgG antibody (Invitrogen, catalog number: A21235, lot number: 2161043). Flow cytometry analysis was performed using flow cytometry instruments (Intellicyte, IQue plus, Sartorius). In addition, mouse serum was tested by imaging. Briefly, CHO-nectin-4 cells or T-47D cells were incubated with diluted serum from immunized mice. Cells were washed, fixed with paraformaldehyde, washed again, and specific antibody binding was detected using secondary Alexa 488 goat anti-mouse antibody and Hoechst (Invitrogen). Plates were scanned and analyzed using an imaging machine (Cytation 5, Biotek). Hybridoma supernatants were tested for nectin-4 specific binding by ELISA, imaging, and FACS as described above.

[0230] Generation of hybridomas producing mAbs against nectin-4: To generate hybridomas producing the mouse antibodies of this disclosure, splenocytes and lymph node cells were isolated from immunized mice and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas were screened for antigen-specific antibody production. For example, single-cell suspensions of splenocytes and lymph node cells from immunized mice were fused to an equal number of Sp2 / 0 mouse IgG non-secretory myeloma cells (ATCC, CRL1581) by electrofusion. The cells were seeded in flat-bottomed 96-well tissue culture plates, followed by incubation in selective medium (HAT medium) for 2 weeks, and then switched to hybridoma culture medium. Approximately 10–14 days after cell seeding, the supernatant from individual wells was screened by ELISA, imaging, or FACS as described above. Antibody-secreting hybridomas were transferred to 24-well plates and screened again. If still positive for anti-nectin-4, the positive hybridomas were subcloned by sorting using a single-cell sorter. Stable subclones were then cultured in vitro to generate small amounts of antibody for purification and characterization.

[0231] [Example 2] Binding of anti-nectin-4 specific antibody The binding specificity of the anti-nectin-4 antibody disclosed herein was evaluated by ELISA. Briefly, human recombinant nectin-4 protein was directly coated onto an ELISA plate. Purified antibody was then added to the plate, followed by detection with goat anti-mouse IgG-HRP. After adding the ABTS substrate, the ELISA plate was read using an ELISA plate reader (Bioteck). In Figure 2, the control PC1 refers to the reference antibody (known to be a nectin-4 specific recombinant antibody, manufactured by Novarock Biotherapeutics, lot number: 03042020KD); the negative control is human IgG1 (Dendritics, catalog number: DDXCH01P-100, lot number: DDXCH01-028).

[0232] Figure 2 shows the binding activity of the eight nectin-4 specific antibodies of this disclosure. N4_mAb 1 to N4_mAb 8 bind to human nectin-4 protein in a dose-dependent manner. In addition, the positive control also binds to human nectin-4 protein in a dose-dependent manner. Human IgG1 does not bind to human nectin-4 protein.

[0233] The ELISA-conjugated EC50 values ​​for eight anti-nectin-4 antibodies, N4_mAb 1 to N4_mAb 8, and a positive control antibody are listed in Table 3 below.

[0234] [Table 3]

[0235] The results in Figure 2 and Table 3 indicate that anti-human nectin-4 specific antibodies are characterized by binding to human nectin-4 at a low EC50 value according to ELISA.

[0236] The binding specificity of the anti-nectin-4 antibody described herein was also evaluated by FACS. Briefly, CHO-nectin-4 cells (vs. parental CHO cells) or an endogenous cell line expressing nectin-4 (T-47D) were incubated with a diluted purified recombinant antibody. The cells were washed, and specific antibody binding was detected using Alexa 647-labeled goat anti-mouse IgG antibody. Flow cytometry analysis was performed using flow cytometry instruments (Intellicyte, IQue plus, Sartorius).

[0237] Figures 3A and 3B show the binding activity of the nectin-4 specific antibodies of this disclosure. Figure 3A shows that the anti-nectin-4 antibodies, N4_mAb 1 to N4_mAb 8, bind to human CHO-nectin-4 cells in a dose-dependent manner; the positive control also binds to CHO-nectin-4 cells in a dose-dependent manner. The negative control human IgG1 does not bind to CHO-nectin-4 cells. Figure 3B shows that the anti-nectin-4 antibodies, N4_mAb 1 to N4_mAb 8, bind to the SKBR3 cell line, which endogenously expresses nectin-4, in a dose-dependent manner. The positive control also binds to SKBR3 cells in a dose-dependent manner.

[0238] Table 4 shows the binding EC50 values ​​of the eight Nectin-4 antibodies of this disclosure. Antibodies N4_mAb 1 to N4_mAb 8 bind to CHO-Nectin-4 cells and SKBR3 cells according to FACS.

[0239] [Table 4]

[0240] Figures 3A and 3B and Table 4 indicate that anti-human nectin-4 specific antibodies bind to CHO-nectin-4 cells and SKBR3 cells at low EC50 values ​​according to FACS.

[0241] The binding kinetics of the anti-nectin-4 specific antibodies of the present disclosure to recombinant human nectin-4 were determined by surface plasmon resonance (SPR) using a BIAcore3000 system. Briefly, a CM5 chip was immobilized via an amine coupling chemical reaction with an anti-human IgG antibody (GE, catalog number BR-1000-12, lot 10283568) according to the application wizard in flow cell 4. Flow cell 3 was left unmodified and used as a reference cell for subtracting systematic instrument noise and drift. Fc4-3 detection was performed using a double blank (Fc3 and blank analyte buffer). Antibody samples were diluted to 1 μg / mL in HBS-EP and injected at a flow rate of 10 μL / min for 1 minute. The recombinant human nectin 4-His protein to be analyzed was diluted from 10 to 0.156 nM (1:4 dilution, 5 points down to 0 nM in HBS-EP buffer), injected at 50 μL / min for 2 minutes, followed by dissociation for 6 minutes. All analyses were performed using a double blank, Fc3 and blank analyte buffer to reduce background. Data were analyzed using BIAevaluation software (version 4.1.1) with a mass transfer model with global fit for 1:1 binding to determine the apparent binding kinetics.

[0242] The binding KD values of the anti-nectin-4 antibodies of the present disclosure are provided in Table 5. As the results indicate, the anti-nectin-4 specific antibodies bind to human recombinant nectin-4 with KD values in the range of 1.72E-08 to 3.75E-10 M.

[0243] [Table 5]

[0244] [Example 3] Blocking of nectin-1 binding to nectin-4 expressing cells The ability of the nectin-4 specific antibodies of the present disclosure to block nectin-1 binding to nectin-4 expressing cells (CHO cells transfected with human nectin-4) was determined by FACS. Briefly, CHO-nectin-4 cells were incubated with dilutions of the purified recombinant antibodies of nectin-4 of the present disclosure. Then, His-tagged biotinylated human nectin-1 (ACROBiosystems, catalog number: PV1-H5223, lot: 733-38GS1-47, 0.75 ug / ml) was added to the plate. After 30 minutes of incubation, the cells were washed, followed by addition of streptavidin Alexa 647 (1:1000) to the sample. After 30 minutes of incubation, the samples were analyzed for nectin-1 blocking activity on a flow cytometry instrument (Intellicyte, IQue plus, Sartorius). The positive control antibody used in this blocking assay is a reference antibody (manufactured by Novarock Biotherapeutics, lot: 03042020KD, known as a recombinant nectin-4 specific antibody).

[0245] As shown by the data in Table 6, the anti-nectin-4 antibodies (N4_mAb 1 to N4_mAb 8) of the present disclosure block human nectin-1 binding to nectin-4-CHO cells with low EC50 values (0.12 to 130 nM). The positive control also blocked human nectin-1 binding to CHO-nectin-4 cells (EC50 0.16 nM).

[0246] [Table 6]

[0247] [Example 4] Blocking of TIGIT Binding to Nectin-4 Expressing Cells The ability of the Nectin-4 specific antibody of this disclosure to block TIGIT binding to CHO-Nectin-4 cells was evaluated by FACS. Briefly, CHO-Nectin-4 cells were incubated with a dilution of the purified recombinant antibody of this disclosure. Biotinylated human TIGIT protein (SinoBiologic, catalog: 10917-H08H-B, lot: LC13AP0902) was then added to the plate. After 30 minutes of incubation, the cells were washed, followed by the addition of streptavidin Alexa 647 (1:1000) to the sample. After 30 minutes of incubation, the sample was analyzed for TIGIT blocking activity using flow cytometry equipment (Intellicyte, IQue plus, Sartorius). The positive control antibody used in this blocking assay was PC2, a Nectin-4 antibody with known blocking activity.

[0248] The data in Table 7 shows that the anti-nectin-4 antibodies of this disclosure (N4_mAb 1 to N4_mAb 8) blocked the binding of human TIGIT to CHO-nectin-4 cells (EC50 in the range of 0.13 to 3.11 nM). The positive control also blocked the binding of human TIGIT to CHO-nectin-4 cells (EC50 0.85 nM).

[0249] [Table 7]

[0250] [Example 5] Antibody binding specificity to nectin family proteins The anti-nectin-4 antibodies (N4-mAb 1 to N4-mAb 8) disclosed herein were tested for binding to human nectin family proteins, i.e., nectin-1 (ACROBiosystems, catalog number: PV1-H5223, lot number: 733-38GS1-47), nectin-2 (ACROBiosystems, catalog number: PV2-H52E2, lot number: 1982-61MS1-FD), and nectin-3 (ACROBiosystems, catalog number: PV3-H52E4, lot number: 1984-61MS1-AC), by either ELISA or Gator binding assay. None of the nectin-4 antibodies bound to nectin-1, nectin-2, or nectin-3 (data not shown).

[0251] [Example 6] Determination of the Nectin-4 antibody-binding domain The extracellular portion of human nectin-4 (SEQ ID NO: 61) has three domains: one Ig-like V domain (amino acids 32-144) and two Ig-like C domains (amino acids 148-237 and 248-331, respectively). To determine the binding domain of the nectin-4 antibody, three pcDNA3.1-based expression plasmids were constructed: one encoding full-length human nectin-4, one encoding a variant with a deletion between amino acids 148-331 (ΔC domain), and one expressing a variant with a deletion between amino acids 32-147 (ΔV domain). The plasmids were transfected into human 293 T cells (ATCC) using TransIT-293 transfection reagent (Mirus Bio), and the binding affinity of the disclosed antibodies to the transiently expressed nectin-4 variants was measured by flow cytometry.

[0252] Table 8 shows the antibody binding strengths: "+++" indicates an EC50 of less than 5 nM, and "-" indicates that the EC50 is greater than the highest antibody concentration tested (133 nM). All antibodies except N4_mAb 4 bind to variants lacking the Ig-like C domain with similar affinity to full-length human nectin-4. However, these antibodies lost binding to variants lacking the Ig-like V domain, which suggests that the binding epitope for each antibody is located within the Ig-like V domain. N4_mAb 4 has a unique binding epitope (amino acids 148-331) within the Ig-like C domain.

[0253] [Table 8]

[0254] [Example 7] Interspecies nectin-4 binding cDNA encoding the nectin-4 protein from cynomolgus monkeys (SEQ ID NO: 62), rats (SEQ ID NO: 63), or mice (SEQ ID NO: 64) was separately cloned into pcDNA3.1-based mammalian expression plasmids. Each plasmid was transfected into human 293T cells in the same manner as described in Example 6. The binding EC50 of the human nectin-4 antibody of this disclosure to species-specific nectin-4 transiently expressed on the surface of 293T cells was measured by flow cytometry.

[0255] The binding strength is reported in Table 9 and classified into four categories. The strongest binding with an EC50 value of less than 5 nM is defined as "+++", a binding EC50 value between 5 nM and 25 nM is defined as "++", a binding EC50 value between 25 nM and 133 nM is marked as "+", and a binding EC50 of approximately 133 nM is indicated as "-". All antibodies bind to nectin-4 derived from cynomolgus monkeys and show diverse affinities to nectin-4 derived from rodent species.

[0256] [Table 9]

[0257] [Example 8] Anti-nectin-4-mediated cytotoxicity Endocytosis of the nectin 4-specific antibody of the present disclosure bound to nectin-4-positive cells was measured by a cytotoxicity-based endocytosis assay using co-internalization of the target-binding antibody together with an anti-human IgG Fc-MMAF antibody.

[0258] The CHO-nectin4 cell line and the SKBR3 cell line were cultured in growth media (F12K + 10% FBS and McCoy 5a medium + 10% FBS, respectively). Cells were harvested, resuspended in their respective growth media, and seeded onto assay plates. The cells were incubated overnight at 37°C. The anti-nectin 4 antibody was incubated with an MMAF-conjugated Fab anti-hFc fragment (Moradec, catalog number AH-202AF-50) for 30 minutes, then added to the cell plates and incubated for an additional 96 hours. CellTiter-Glo (Promega catalog number G7570) was added to evaluate the cell viability of each well. Signals were quantified using a Neo2 plate reader (BioTek).

[0259] As demonstrated in Table 10 and Figure 4A, the lead panel of anti-nectin-4 antibodies induced endocytosis-derived cytotoxicity in CHO-nectin-4 cells with EC50 values ranging from 0.21 to 0.63 nM. Similarly, as shown in Table 10 and Figure 4B, the panel of anti-nectin-4 antibodies induced endocytosis-derived cytotoxicity in SKBR3 cells with EC50 values ranging from 0.61 to 2.14 nM.

[0260] The lead panel antibodies also exhibited endocytosis-derived cytotoxicity in the breast tumor cell line T47D, which endogenously expresses nectin-4 (data not shown).

[0261]

Table 10

[0262] [Example 9] Antibody internal distribution and Nectin-4 protein level dynamics The internal distribution of selected anti-nectin-4 antibodies and the dynamics of nectin-4 protein levels were measured in T47D cells. Briefly, the antibodies shown were labeled with Alexa Flour 488 using a labeling kit (Thermo Fisher, catalog number A20181). The labeled antibodies were incubated with T47D cells at 37°C for 0, 1, 4, and 24 hours. At each time point, the cells were transferred to 4°C and an unlabeled antibody was added for binding to the membrane nectin-4 protein. The cells were then fixed and an Alexa Flour 647-labeled secondary antibody was added. The plates were then imaged by Cytation 5 (BioTek) for both dyes (green and red).

[0263] Figure 5A shows the internal migration dynamics of the selected anti-nectin-4 antibodies. All four antibodies showed time-dependent internal migration from 0 to 24 hours. At the same time point, membrane nectin-4 levels were quantified, and the results are shown in Figure 5B. Among the antibodies, N4_mAb 2 showed faster internal migration, but also caused depletion of membrane nectin-4. The other three antibodies did not show a significant effect on cell surface nectin-4 protein levels.

[0264] [Example 10] Antibody-dependent cytotoxicity (ADCC) in tumor cells endogenously expressing nectin-4. The ADCC activity of anti-nectin-4 antibodies was measured by a bioluminescence assay. Briefly, anti-nectin-4 antibodies were serially diluted with an assay buffer containing RPMI + 4% low-IgG FBS and added to a mixture of individual target cell lines (either T47D or SKBR3) and ADCC effector cells. ADCC effector cells were Jurkat cells expressing CD16a, which is activated upon recognition of the Fc portion of the bound nectin-4 antibody. Effector cell activation was detected using the Promega bioluminescence assay (Promega, catalog number E6130) according to the manufacturer's instructions.

[0265] As shown in Figure 6A, ADCC activity was observed only for N4_mAb 4 on T47D cells. None of the other antibodies, including PC1, showed ADCC activity. On SKBR3 cells, N4_mAb 4 also showed strong ADCC activity, while N4 mAb 2 showed very mild activity, and all other antibodies showed no ADCC activity (Figure 6B).

[0266] Unless otherwise indicated, all numbers used in the specification and claims to express quantities, molecular weights, and other properties of components, reaction conditions, etc., should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations, which may vary depending on the desired properties to be obtained by this disclosure. At the very least, each numerical parameter should be interpreted by considering the reported number of significant figures and applying the usual rounding techniques, not in an attempt to limit the application of the doctrine of equivalents to the claims.

[0267] While the numerical ranges and parameters representing the broad scope of this disclosure are approximations, the figures described in specific examples are reported as accurately as possible. Each figure, however, inherently contains a certain degree of error that inevitably arises from the standard deviation observed in their respective test measurements.

[0268] The terms “a,” “an,” “the,” and similar references used in the context describing this disclosure (particularly in the context of the claims below) are construed to cover both singular and plural forms unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context. The enumeration of ranges of values ​​herein is intended merely as a way of omitting individual references to each individual value that falls within the range. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein can be performed in any appropriate order unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context. Any use of any examples or exemplary phrases provided herein (e.g., “such as”) is intended merely to better illustrate this disclosure and does not impose any limitation on the scope of the disclosure unless otherwise specifically asserted. Nothing in this specification should be construed as indicating that any non-claimed element is essential for the practice of the invention.

[0269] The grouping of alternative elements or embodiments of the disclosures disclosed herein should not be construed as limiting. Each group's components may be referred to and claimed individually, or in any combination with other components of the group or other components found herein. One or more components of a group may be included in or removed from a group for convenience and / or patentability reasons. Where any such inclusion or removal occurs, this specification shall include the modified groups and thus satisfy the written specification of all Markush groups used in the appended claims.

[0270] Certain embodiments of this disclosure are described herein, including the best mode for carrying out the disclosure as known to the inventors. Of course, variations of those embodiments described herein will be apparent to those skilled in the art by reading the foregoing description. The inventors believe that those skilled in the art will adopt such variations as appropriate, and the disclosure is intended to be carried out in ways other than those specifically described herein. Accordingly, the disclosure encompasses all variations and equivalents of the subject matter enumerated in the claims appended herein, where applicable law permits. Furthermore, any combination of the above elements in all possible variations thereof is also encompassed herein, unless otherwise specifically indicated herein, or unless it is clearly inconsistent with the context.

[0271] Specific embodiments of the disclosure herein may be further limited in the claims using the phrases "consisting of" or "essentially consisting of." Where used in the claims, whether filed or added by amendment, the transitional phrase "consisting of" excludes any element, step or component not provided for in the claims. The transitional phrase "essentially consisting of" limits the scope of the claims to those which do not substantially affect the specified material or step, or the basic and novel features. Embodiments of the disclosure as claimed herein are essentially or expressly described herein and are effective herein.

[0272] Embodiments of the Disclosure disclosed herein are to be understood as illustrative examples of the principles of the Disclosure. Other modifications that may be adopted are within the scope of the Disclosure. Accordingly, alternative configurations of the Disclosure, though not limited to examples, may be used in accordance with the teachings herein. Accordingly, the Disclosure is not strictly limited to those shown and described herein.

[0273] While this disclosure has been described and illustrated herein by reference to various specific materials, procedures, and examples, it is understood that this disclosure is not limited to any particular combination of materials and procedures selected for its purposes. Numerous variations of such details may be implicitly included, as will be understood by those skilled in the art. This specification and the examples are intended to be illustrative only, and the true scope and intent of this disclosure is intended to be indicated by the following claims. All references, patents, and patent applications referenced herein are incorporated herein by reference in their entirety. The present invention includes the following embodiments. [1] An anti-nectin-4 antibody comprising a variable weight (VH) region and a variable light weight (VL) region, The VH region and the VL region are, (a) VH:CDR1:Sequence ID 17, CDR2:Sequence ID 18, CDR3:Sequence ID 19; VL:CDR1:Sequence ID 20, CDR2:Sequence ID 21, CDR3:Sequence ID 22; (b) VH: CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, CDR3: SEQ ID NO: 25; VL: CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, CDR3: SEQ ID NO: 28; (c)VH:CDR1:Sequence ID 29, CDR2:Sequence ID 30, CDR3:Sequence ID 31;VL:CDR1:Sequence ID 32, CDR2:Sequence ID 33, CDR3:Sequence ID 34; (d)VH:CDR1:SEQ ID NO: 35, CDR2:SEQ ID NO: 36, CDR3:SEQ ID NO: 37; VL:CDR1:SEQ ID NO: 38, CDR2:SEQ ID NO: 39, CDR3:SEQ ID NO: 40; (e) VH:CDR1:Sequence ID 41, CDR2:Sequence ID 42, CDR3:Sequence ID 43; VL:CDR1:Sequence ID 44, CDR2:Sequence ID 45, CDR3:Sequence ID 46; (f)VH:CDR1:Sequence ID 47, CDR2:Sequence ID 48, CDR3:Sequence ID 49;VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 52; (g) VH: CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 53, CDR3: SEQ ID NO: 54; VL: CDR1: SEQ ID NO: 55, CDR2: SEQ ID NO: 56, CDR3: SEQ ID NO: 52; or (h)VH:CDR1:SEQ ID NO: 57, CDR2:SEQ ID NO: 58, CDR3:SEQ ID NO: 59;VL:CDR1:SEQ ID NO: 50, CDR2:SEQ ID NO: 51, CDR3:SEQ ID NO: 60 A set of CD-Rs selected from, Anti-nectin-4 antibody. [2] (a) A heavy chain variable region having the sequence described in Sequence ID No. 1 and a light chain variable region having the sequence described in Sequence ID No. 2; (b) Heavy chain variable region having the sequence described in Sequence ID No. 3 and light chain variable region having the sequence described in Sequence ID No. 4; (c) Heavy chain variable region having the sequence described in Sequence ID No. 5 and light chain variable region having the sequence described in Sequence ID No. 6; (d) Heavy chain variable region having the sequence described in Sequence ID 7 and light chain variable region having the sequence described in Sequence ID 8; (e) A heavy chain variable region having the sequence described in Sequence ID No. 9 and a light chain variable region having the sequence described in Sequence ID No. 10; (f) A heavy chain variable region having the sequence described in Sequence ID No. 11 and a light chain variable region having the sequence described in Sequence ID No. 12; (g) A heavy chain variable region having the sequence described in Sequence ID No. 13 and a light chain variable region having the sequence described in Sequence ID No. 14; or (h) Heavy chain variable region having the sequence described in Sequence ID No. 15 and light chain variable region having the sequence described in Sequence ID No. 16 The anti-nectin-4 antibody described in [1] above, including the above. [3] A mouse antibody, the anti-nectin-4 antibody described in [1] above. [4] The anti-nectin-4 antibody described in [1] above, which is a chimeric antibody. [5] A humanized antibody, the anti-nectin-4 antibody described in [1] above. [6] An anti-nectin-4 antibody as described in any of [1] to [5] above, conjugated to a cytotoxic agent. [7] The anti-nectin-4 antibody described in [1] above, which is a full-length antibody. [8] The antibody fragment is the anti-nectin-4 antibody described in [1] above. [9] The antibody fragments are Fab, Fab', and F(ab'). 2 An anti-nectin-4 antibody as described in [8] above, selected from the group consisting of Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies and diabodies.

[10] An anti-nectin-4 antibody as described in [1] above, which binds to human nectin-4.

[11] A pharmaceutical composition comprising an antibody described in any of the above [1] to

[10] and a pharmaceutically acceptable carrier.

[12] The pharmaceutical composition described above

[11] for use in modulating the immune system by inhibiting the binding of nectin-4 to nectin-1.

[13] The pharmaceutical composition described above

[11] for use in modulating the immune system by inhibiting the binding of nectin-4 to TIGIT.

[14] A pharmaceutical composition as described in

[11] above, for use in treating cancer.

[15] A method for treating cancer in a subject requiring the same, comprising the step of administering the pharmaceutical composition described in

[11] above to the subject requiring the same.

[16] A method for diagnosing cancer in a subject, comprising the step of contacting a biological sample with an antibody or antibody fragment described in any of [1] to

[10] above.

[17] An isolated polynucleotide containing a sequence encoding an anti-nectin-4 antibody as described in any of the above [1] to

[10] .

[18] The isolated polynucleotide described in

[17] above, which encodes the amino acid sequence described in any one of sequence numbers 1 to 17.

[19] A vector containing the polynucleotide described above

[18] .

[20] A host cell containing the polynucleotide described in

[18] above, and / or the vector described in

[19] above.

[21] A method for producing an anti-nectin-4 antibody as described in [1] above, comprising the step of culturing the host cells described in

[20] above.

Claims

1. An anti-nectin-4 antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH region and the VL region are, (a) VH: CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, CDR3: SEQ ID NO: 59; VL: CDR1: Sequence ID 50, CDR2: Sequence ID 51, CDR3: Sequence ID 60; (b) VH: CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 48, CDR3: SEQ ID NO: 49; VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 52; or (c) VH: CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 53, CDR3: SEQ ID NO: 54; VL: CDR1: Sequence ID 55, CDR2: Sequence ID 56, CDR3: Sequence ID 52; A set of CD-Rs selected from, Anti-nectin-4 antibody.

2. (a) A heavy chain variable region having the sequence described in Sequence ID No. 15 and a light chain variable region having the sequence described in Sequence ID No. 16; (b) A heavy chain variable region having the sequence described in Sequence ID No. 11 and a light chain variable region having the sequence described in Sequence ID No. 12; or (c) Heavy chain variable region having the sequence described in Sequence ID No. 13 and light chain variable region having the sequence described in Sequence ID No. 14; The anti-nectin-4 antibody according to claim 1, comprising:

3. The anti-nectin-4 antibody according to claim 1, which is a mouse antibody.

4. The anti-nectin-4 antibody according to claim 1, which is a chimeric antibody.

5. The anti-nectin-4 antibody according to claim 1, which is a humanized antibody.

6. An anti-nectin-4 antibody according to any one of claims 1 to 5, conjugated with a cytotoxic agent.

7. The anti-nectin-4 antibody according to claim 1, which is a full-length antibody.

8. The anti-nectin-4 antibody according to claim 1, which is an antibody fragment.

9. The antibody fragments are Fab, Fab', and F(ab'). ２ The anti-nectin-4 antibody according to claim 8, selected from the group consisting of Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies and diabodies.

10. An anti-nectin-4 antibody according to claim 1, which binds to human nectin-4.

11. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, for use in regulating the immune system by inhibiting the binding of nectin-4 to nectin-1.

13. The pharmaceutical composition according to claim 11, for use in regulating the immune system by inhibiting the binding of nectin-4 to TIGIT.

14. A pharmaceutical composition according to claim 11 for use in treating cancer.

15. A composition for diagnosing cancer in a subject, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10.

16. An isolated polynucleotide comprising a sequence encoding an anti-nectin-4 antibody according to any one of claims 1 to 10.

17. The isolated polynucleotide according to claim 16, encoding the amino acid sequence described in any one of sequence numbers 11 to 16.

18. A vector comprising the polynucleotide described in claim 17.

19. A host cell comprising the polynucleotide according to claim 17 and / or the vector according to claim 18.

20. A method for producing an anti-nectin-4 antibody according to claim 1, comprising the step of culturing the host cells according to claim 19.

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