RU2805252C2 - Nectin-4 binding proteins and methods of their application - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention is related to an antibody or its antigen-binding fragment that binds to human nectin-4. A polynucleotide encoding said antibody; a vector and a cell containing said polynucleotide; a kit containing the specified antibody are also disclosed. A method for producing said antibody; a method for determining the expression of nectin-4 using said antibody; a method of assessing the susceptibility of an individual with a malignant disease to an antitumor therapeutic agent using said antibody are disclosed.

EFFECT: invention makes it possible to effectively assess the susceptibility of an individual with a malignant disease to an antitumor therapeutic agent.

30 cl, 12 ex, 23 tbl, 13 dwg

Description

1. FIELD TO WHICH THE INVENTION RELATES

[0001] Provided herein are compositions, methods and uses of antibodies that specifically bind to human nectin-4 and detect the level of expression of nectin-4.

2. CROSS-REFERENCES TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62/515,454, filed June 5, 2017, the entire contents of which are incorporated herein by reference.

3. LINK TO THE LIST OF SEQUENCES SUBMITTED IN IN ELECTRONIC FORMAT

[0003] This application includes a reference to a sequence listing in machine readable form (CRF) in ASCII text format filed with this application, designated 14369-208-228_SEQ_LISTING.txt, created on May 31, 2018 and having a size of 37280 bytes.

4. BACKGROUND OF THE ART

[0004] Nectin-4, also known as poliovirus receptor-associated protein 4 (PVRL4), is a single type I transmembrane protein of approximately 52 kDa that belongs to the nectin family of cell adhesion molecules. Nectins mediate Ca2+-independent cell-cell adhesion at adhesive junctions through homophilic interactions, in which one nectin-4 interacts with another nectin-4, and through heterophilic interactions, in which nectin-4 interacts with another nectin family protein, such as nectin-1 , nectin-2 or nectin-3. (Miyoshi J et al, 2007, Am J Nephrol 27:590-604; Takai et al, 2003, Cancer Sci 94:655-667; Reymond N. et al , 2001, Journal of Biological Chemistry , 276:43205-15) . The extracellular domain of nectin-4 has three Ig-like subdomains designated V, C1, and C2. The C1 domain of nectin-2 has been shown to be responsible for homophilic interactions, while the V domains of most nectin molecules are involved in heterophilic interactions and cell-cell adhesion. (Miyoshi J et al , 2007, Am J Nephrol 27:590-604; Takai et al, 2003, Cancer Sci 94:655-667; Reymond N. et al , 2001, Journal of Biological Chemistry , 276:43205 15).

[0005] Nectins are expressed in various tissues, including, for example, hematopoietic, neuronal, endothelial and epithelial cells. (Mendelsohn C et al, 1989, Cell 56:855-865; Lope; 72:9992-10002; Takahashi K. et al. , 1999, Journal of Cell Biology 145:539-549; Miyoshi J et al ., 2007 Am J Nephrol 27:590-604; Takai et al. , 2003, Cancer Sci 94:655-667).

[0006] In addition to mediating cell-cell adhesions through homophilic interactions or heterophilic interactions with other nectin family proteins, nectins can recruit other cell adhesion molecules, such as cadherins, or other cell surface receptors, such as the prolactin receptor. By recruiting other cell surface receptors, nectins can also play the role of a stimulatory coreceptor and, thus, perform a signaling function. For example, in the mouse mammary gland, nectin-4 interacts with the prolactin receptor through its extracellular and transmembrane domain and binds to suppressor of cytokine signaling 1 (SOCS-1), which normally inhibits the kinase activity of Janus kinase 2 (JAK2), thereby upregulating prolactin -induced JAK2 activation and signaling. (Maruoka et al ., 2017, Journal of Biological Chemistry, doi: 10.1074/jbc.M116.769091, jbc.M116.769091, Epub Ahead of Print; Kitayama et al ., 2016, Journal of Biological Chemistry 291:5817-5831 ). Nectins can also interact with cadherins to induce and then rapidly suppress Rac1 activity during the initial stages of cell-cell adhesion. (Khameeka et al. , 2011, PLoS ONE 6: e17841).

[0007] To date, all attempts to generate antibodies against nectin-4 to specifically detect and evaluate the expression of nectin-4 in various biological samples have been futile. Thus, there is a need to develop an anti-nectin-4 antibody that can specifically detect and evaluate the expression of nectin-4 in biological samples.

5. BRIEF DESCRIPTION OF THE INVENTION

[0008] Disclosed herein are proteins that bind to nectin-4 (eg, human nectin-4, SEQ ID NO:43), including binding proteins, such as antibodies, that bind to nectin-4. Such binding proteins, including antibodies, can bind to a nectin-4 polypeptide, a fragment of nectin-4, and/or an epitope of nectin-4.

[0009] Also disclosed herein are, in some embodiments, binding proteins, including antibodies or fragments thereof, that specifically bind to a cell expressing nectin-4.

[0010] Also disclosed herein are polynucleotides and vectors containing sequences encoding such antibodies, cells (eg, host cells) containing such polynucleotides or vectors, and compositions, reagents, and kits containing such antibodies. In yet another aspect, disclosed herein are methods for modulating Nectin-4 activity (eg, activation of the Nectin-4 signaling pathway) or Nectin-4 expression levels, diagnostic methods, and the use of such anti-Nectin-4 antibodies.

[0011] In some embodiments, the binding protein (eg, an anti-Nectin-4 antibody) contains six complementarity determining regions (CDRs) or fewer than six CDRs. In other embodiments, the binding protein (eg, an anti-nectin-4 antibody) comprises one, two, three, four, five, or six CDRs selected from: heavy chain variable region (VH) CDR1, VH CDR2, and VH CDR3, variable region CDR1 light chain (VL) regions, CDR2 VL and/or CDR3 VL. In some embodiments, the binding protein (e.g., anti-nectin-4 antibody) contains one, two, three, four, five, or six CDRs selected from: CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and/or CDR VL of a monoclonal antibody designated M22-321b41.1 as described herein, or a humanized variant thereof. In some embodiments, the binding protein (eg, anti-nectin-4 antibody) further comprises a backbone region or framework region (FR), including FR1 VH, FR2 VH, FR3 VH, FR4 VH, FR1 VL, FR2 VL, FR3 VL and/or FR4 VL amino acid sequence of human immunoglobulin or variant thereof.

[0012] In some embodiments, the antibody or antigen binding fragment thereof comprises a VL comprising the CDR1 VL, CDR2 VL, and CDR3 VL of antibody M22-321b41.1, as set forth in Table 1.

[0013] In other embodiments, the antibody or antigen binding fragment thereof comprises a VH comprising the CDR1 VH, CDR2 VH and CDR3 VH of antibody M22-321b41.1, as set forth in Table 1 .

[0014] In other embodiments, the antibody or antigen binding fragment thereof comprises:

(a) VL containing FR1 VL, FR2 VL, FR3 VL and FR4 VL antibodies M22-321b41.1, as indicated in Table 2 ; And

(b) VH containing FR1 VH, FR2 VH, FR3 VH and FR4 VH antibodies M22-321b41.1, as indicated in Table 2 .

[0015] In some embodiments, the CDR1 VL, CDR2 VL, and CDR3 VL of the antibody or antigen binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 7, 10, and 12, respectively, and the CDR1 VH, CDR2 VH, and CDR3 VH of the antibody or antigen binding fragment thereof comprise amino acid sequences SEQ ID NO: 15, 19 and 23, respectively.

[0016] In yet another embodiment, the antibody or antigen binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the amino acid sequence contains one or more conservative modifications.

[0017] In specific embodiments, the antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the amino acid sequence contains one or more conservative modifications.

[0018] In specific embodiments, the antibody or antigen binding fragment thereof comprises: (a) VL comprising the amino acid sequence of SEQ ID NO:3; and (b) VH containing the amino acid sequence of SEQ ID NO:4.

[0019] In some embodiments, the antibody comprises a human IgG1 Fc fragment. In other embodiments, the antibody comprises a mutant variant of the human IgG1 Fc fragment.

[0020] In certain embodiments, the antibody or antigen binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:33.

[0021] In other embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain Fc fragment comprising the amino acid sequence of SEQ ID NO:35.

[0022] In yet another embodiment, the antibody or antigen binding fragment further comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:33; and a heavy chain Fc fragment containing the amino acid sequence of SEQ ID NO:35.

[0023] In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:5.

[0024] In specific embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 31-346 within the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 31-150 within the amino acid sequence of SEQ ID NO:1. In specific embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 1-150 within the amino acid sequence of SEQ ID NO:1.

[0025] In one embodiment, the epitope of human nectin-4 is different from the ligand binding site of nectin-4.

[0026] In one aspect, the present description discloses an antibody or antigen binding fragment thereof that binds to an epitope of human nectin-4, wherein the antibody or antigen binding fragment thereof specifically binds to a cell expressing nectin-4.

[0027] In one embodiment, an antibody or antigen-binding fragment thereof that binds to an epitope of human nectin-4 is used to assess the expression of nectin-4 in a tissue sample of an individual suspected of having a malignancy, comprising: (a) subjecting said tissue sample to contact with an antibody or its antigen-binding fragment; (b) detecting the binding of said antibody or antigen-binding fragment thereof to said tissue sample; and (c) determining the expression of nectin-4 in the tissue sample, wherein the expression level of nectin-4 in the tissue sample is compared to a reference level of nectin-4 expression.

[0028] In certain embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized, human, or chimeric antibody. In yet another embodiment, the humanized antibody is a deimmunized human antibody or a composite antibody. In specific embodiments, the antibody is a humanized antibody. In specific embodiments, the antibody is a humanized antibody that specifically binds to human nectin-4.

[0029] In some embodiments, the antibody or antigen binding fragment thereof is a Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, tribody, or tetrabody. In some embodiments, the antibody or antigen-binding fragment thereof is a multispecific antibody derived from antibody fragments.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an agent. In one embodiment, the agent is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.

[0031] Also disclosed herein is a composition comprising an antibody or antigen binding fragment thereof of the present invention. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0032] Also disclosed herein is a polynucleotide comprising nucleotide sequences encoding the VH, VL, or both VH and VL regions of the antibodies of the present invention.

[0033] Also disclosed herein is, in some embodiments, a polynucleotide comprising nucleotide sequences encoding the heavy chain, the light chain, or both the heavy and light chains of the antibodies of the present invention.

[0034] In some embodiments, the polynucleotide is operably linked to a promoter.

[0035] Also disclosed herein is a population of polynucleotides comprising: (a) a first polynucleotide comprising nucleotide sequences encoding the VH or heavy chain of an antibody of the present invention, and (b) a second polynucleotide containing nucleotide sequences encoding the VL or light chain of an antibody according to the present invention. In some embodiments, the first polynucleotide is operably linked to a first promoter and the second polynucleotide is operably linked to a second promoter.

[0036] Also disclosed herein is a vector containing a polynucleotide of the present invention.

[0037] Also disclosed herein is, in some embodiments, a population of vectors comprising: (a) a first vector containing nucleotide sequences encoding the VH or heavy chain of an antibody of the present invention, and (b) a second vector containing nucleotide sequences the VL or light chain encoding antibodies of the present invention.

[0038] The present description further discloses, in some embodiments, a population of vectors comprising: (a) a first vector containing nucleotide sequences encoding the VH or heavy chain of an antibody of the present invention operably linked to a first promoter, and (b) a second a vector containing nucleotide sequences encoding the VL or light chain of an antibody of the present invention, operably linked to a second promoter.

[0039] The disclosure of this application, in some embodiments, discloses a cell containing a polynucleotide or population of polynucleotides of the present invention.

[0040] The description of the present application also discloses, in some embodiments, a cell containing a vector or population of vectors of the present invention.

[0041] The description of the present application further discloses, in some embodiments, an isolated cell producing an antibody or antigen binding fragment thereof of the present invention.

[0042] Also disclosed herein is a population of cells comprising: (a) a first host cell containing a polynucleotide containing a nucleotide sequence encoding a VH or heavy chain of an antibody of the present invention, and (b) a second host cell containing a polynucleotide containing a nucleotide sequence encoding the VL or light chain of an antibody of the present invention.

[0043] Further disclosed herein is a population of cells comprising: (a) a first host cell containing a polynucleotide comprising a nucleotide sequence encoding a VH or heavy chain of an antibody of the present invention operably linked to a first promoter, and (b) a second cell -a host comprising a polynucleotide comprising a nucleotide sequence encoding a VL or light chain of an antibody of the present invention, operably linked to a second promoter.

[0044] Also disclosed herein is a kit containing an antibody or antigen binding fragment thereof of the present invention.

[0045] Also disclosed herein is a method for producing an antibody or antigen binding fragment thereof that specifically binds to an epitope of human nectin-4. In some embodiments, the method includes culturing a cell of the present invention to express an antibody or antigen-binding fragment thereof. In other embodiments, the method comprises expressing a polynucleotide of the present invention. In one embodiment, the epitope of human nectin-4 is different from the ligand binding site of nectin-4.

6. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 shows the result of the experiment showing that all malignant cells were positively stained in the IHC staining assay of the nectin-4 mRNA-positive AG-B1 xenograft with 2.5 μg/ml M22-321b41.1.

[0047] Figure 2 shows the result of the experiment showing that all malignant cells were positively stained in the IHC staining assay of the nectin-4 mRNA-positive AG-B1 xenograft with 2.5 μg/ml M22-244b3.1.1.1.

[0048] Figure 3 shows the result of the experiment showing that all cells were not stained in the IHC staining assay of the nectin-4 mRNA-negative xenograft AG-K24 with 2.5 μg/ml M22-321b41.1.

[0049] Figure 4 shows the result of the experiment showing that the majority of cells stained positively in the IHC staining assay of the nectin-4 mRNA-negative xenograft AG-K24 with 2.5 μg/ml M22-244b3.1.1.1.

[0050] Figure 5 shows the result of the experiment showing that all cells were not stained in the IHC staining assay of nectin-4 mRNA-negative xenograft MDA-MB-231-MFP-XCL with 2.5 μg/ml M22-321b41.1 .

[0051] Figure 6 shows the result of the experiment showing that the majority of cells were positively stained in the IHC staining assay of nectin-4 mRNA-negative xenograft MDA-MB-231-MFP-XCL with 2.5 μg/ml M22-244b3.1.1 .1. [0052] Figure 7 shows the result of the experiment showing that all cells were not stained in the IHC staining assay of the nectin-4 mRNA-negative AG-B1 xenograft with 2.5 μg/ml IgG2a negative control antibody.

[0053] Figure 8 shows the result of the experiment showing that all malignant cells were positively stained in the IHC staining assay of the nectin-4 mRNA-positive AG-L16 xenograft with 2.5 μg/ml M22-321b41.1.

[0054] Figure 9 shows the result of the experiment showing that the majority of malignant cells were positively stained in the IHC staining assay of nectin-4 mRNA-positive AG-B11 xenograft with 2.5 μg/ml M22-321b41.1.

[0055] Figure 10 presents an experimental result showing strong staining of nectin-4 in the IHC staining assay of urothelial carcinoma with M22-321b41.1.

[0056] Figure 11 shows the result of an experiment showing the heterogeneity of nectin-4 staining in a breast carcinoma IHC staining assay with M22-321b41.1.

[0057] Figure 12A shows the results of a Western blot experiment showing specific detection of the nectin-4 band only in those cells and xenografts that express nectin-4.

[0058] Figure 12B shows the results of a control FACS experiment showing that Rat1(E)-nectin-4 cells that stained positively with M22-321b41.1 in Figure 12.A expressed high levels of nectin-4; and other Rat1(E) cells that stained negatively with M22-321b41.1 in Figure 12A expressed high levels of nectin-1, nectin-2, and nectin-3, indicating that M22-321b41.1 is specific in relation to nectin-4.

[0059] Figure 13 shows the results of a vestent blot experiment showing that antibody M22-321b41.1 recognizes a protein band containing the V fragment/domain (amino acid residues 1-150) of nectin-4. In the top panel, antibody M22-321b41.1 was tested in a Western blot assay against lysates of cells expressing native full-length (wt) protein, V fragment/domain (V), V and C1 fragment/domain (V/C1). , C1 and C2 fragment/domain, C2 fragment/domain, and negative control (neo). In the bottom panel, GAPDH was used as a loading control.

7. DETAILED DESCRIPTION OF THE INVENTION

[0060] Disclosed herein are binding proteins, such as antibodies, that bind to nectin-4, including human nectin-4. In one embodiment, anti-nectin-4 antibodies bind to human nectin-4. In some embodiments, the anti-nectin-4 antibody binding assay to nectin-4 is performed in vitro . In other embodiments, the anti-nectin-4 antibody binding assay to nectin-4 is performed in vivo . In some embodiments, the assays include immunohistochemical (IHC) assays, fluorescent cell sorting (FACS) assays, ELISAs, immunoblotting (eg, Western blotting, dot blotting, or cell-based Western blotting), and other immunoassays.

[0061] In particular embodiments, binding proteins, such as antibodies that bind to nectin-4 described herein, have in common the ability to compete with each other for binding to nectin-4. This competitive inhibition may indicate that each antibody binds to the same region of nectin-4 (eg, the same epitope), thereby exhibiting similar effects. In some embodiments, the anti-nectin-4 antibodies described herein include humanized anti-nectin-4 antibodies, such as antibodies derived from or based on antibody M22-321b41.1. In other embodiments, the anti-nectin-4 antibodies described herein compete for binding with an antibody derived from or based on antibody M22-321b41.1. In some embodiments, anti-nectin-4 antibodies have CDR sequences as described in Table 1. In some embodiments, anti-nectin-4 antibodies bind to a specific domain of human nectin-4 (e.g., residues 31-346, 1-150, or 31- 150 in amino acid sequence SEQ ID NO:1). In addition, such binding may be significantly associated with certain amino acid residues within this region that contain an epitope recognized by the anti-nectin-4 antibodies described herein. Together, these results described herein suggest that the effects observed for an anti-nectin-4 antibody that is derived from or based on the M22-321b41.1 antibody, including an antibody with one or more of the CDRs described in Table 1 , can be extrapolated to other anti-nectin-4 antibodies described herein having the same or similar epitope specificity (eg, the same or similar CDRs).

[0062] In some embodiments of the present invention, binding proteins, such as anti-nectin-4 antibodies, may contain immunoglobulin variable regions that contain one or more CDRs, as described in Table 1. In such binding proteins (for example, anti-nectin-4 antibodies), 4) The CDRs may be coupled to one or more backbone regions or framework regions (FR), such as those described in Table 2, that orient the one or more CDRs such that the proper antigen binding properties of the one or more CDRs are realized. Such binding proteins, including anti-nectin-4 antibodies as described herein, can bind to nectin-4 in various assays.

7.1 General techniques

[0063] The techniques and procedures described or referenced herein include techniques and procedures that are well known and/or commonly used using conventional methodology by those skilled in the art, such as, for example, the widely used methodologies described in Sambrook et al ., Molecular Cloning: A Laboratory Manual (3d ed.2001); Current Protocols in Molecular Biology (Ausubel et al . eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed.2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed.2010); and Antibody Engineering , volume 1 and 2 (Kontermann and Dübel eds., 2d ed.2010).

7.2 Terminology

[0064] Unless otherwise specified, all technical and scientific terms used herein have meanings that are well known to one skilled in the art. For the purpose of interpreting the description of this application, the following definitions of terms are used and, where necessary, terms used in the singular also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that the description of any of the listed terms conflicts with any of the documents incorporated herein by reference, then the description of the terms provided below should be considered as controlling.

[0065] The terms "nectin-4", "nectin-4 polypeptide" or "nectin-4 protein" include a polypeptide ("polypeptide" and "protein" are used interchangeably herein), including any native polypeptide from any vertebrate source including mammals, e.g., primates (e.g., human and cyno), dogs, and rodents (e.g., mouse and rat), unless otherwise noted. In some embodiments, the terms include “nectin-4-related polypeptides” including SNP variants thereof. The term "nectin-4" also includes "full-length" full-length nectin-4, as well as any form of nectin-4 that is produced after processing in the cell. In some embodiments, nectin-4 has the amino acid sequence SEQ ID NO: 1. Under GenBank™ accession number NM_030916 (mRNA), NG_028109 (genomic DNA), Gene ID 81607 (gene), NP_112178 (precursor amino acid sequences), another variant of human nectin-4 nucleic acids or amino acid sequences is registered. An example amino acid sequence of human nectin-4 is given below:

[0066] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPC FYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTS EFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSH HTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO:1).

[0067] GenBank™ accession number NM_030916 provides information on yet another human nectin-4 nucleic acid sequence variant:

[0068] atgcccctgtccctgggagccgagatgtggggggcctgaggcctggctgctgc tgctgctactgctggcatcatttacaggccggtgccccgcgggtgagctggagacctcagacgtggtaactgtggtgctgggccaggacgcaaaactgccctgcttctaccgaggggactccggcgagcaagtggggcaagtggcatgggctcgg gtggacgcgggcgaaggcgcccaggaactagcgctactgcactccaaatacgggcttcatgtgagcccggcttacgagggccgcgtggagcagccgccgcccccacgcaaccccctggacggctcagtgctcctgcgcaacgcagtgcaggcggatgagggcgagtacgagtgccgggtcagcaccttccccgcc ggcagcttccaggcgcggctgcggctccgagtgctggtgcctcccctgccctcactgaatcctggtccagcactagaagagggccagggcctgaccctggcagcctcctgcacagctgagggcagcccagcccccagcgtgacctgggacacggaggtcaaaggcacaacgtccagccgttccttcaagcactcccgctctg ctgccgtcacctcagagttccacttggtgcctagccgcagcatgaatgggcagccactgacttgtgtggtgtcccatcctggcctgctccaggaccaaaggatcacccacatcctccacgtgtccttccttgctgaggcctctgtgaggggccttgaagaccaaaatctgtggcacattggcagagaagggctatgctcaagtg cctgagtgaagggcagccccctccctcatacaactggacacggctggatgggcctctgcccagtggggtacgagtggatggggacactttgggctttcccccactgaccactgagcacagcggcatctacgtctgccatgtcagcaatgagttctcctcaagggattctcaggtcactgtggatgttcttgacccccaggaagactctgggaag caggtggacctagtgtcagcctcggtggtggtggtgggtgtgatcgccgcactcttgttctgccttctggtggtggtggtggtgctcatgtcccgataccatcggcgcaaggcccagcagatgacccagaaatatgaggagggctgaccctgaccagggagaactccatccggaggctgcattcccatcacacggaccccaggagcca gccggagaggagagtgtagggctgagagccgagggccaccctgatagtctcaaggacaacagtagctgctctgtgatgagtgaagagcccgagggccgcagttactccacgctgaccacggtgagggatagaaacacagactgaactgctgtctccaggctctgggcgggccgaggagggaagatcaggatgaaggcatcaaacaggccatgaaccatttt gttcaggagaatgggaccctacgggccaagcccacgggcaatggcatctacatcaatgggcggggacacctggtctga (SEQ ID NO:36).

[0069] Under GenBank™ accession number AF472510, NM_027893, XM_203738, NM_001122680, a variant of the nucleic acid and amino acid sequences of mouse nectin-4 is registered; GenBank™ accession numbers XM_005541220 and XM_005541223 provide example nucleic acid and amino acid sequences of monkey nectin-4.

[0070] “Nectin-4 related polypeptides” include allelic variants (eg, SNP variants); splicing variants; fragments; derivatives; variants with substitutions, deletions and insertions; fusion polypeptides; and interspecies homologs that may retain nectin-4 activity. As will be understood by those skilled in the art, an anti-nectin-4 antibody disclosed herein can bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 antigen, and/or a nectin-4 epitope. The "epitope" may be part of a larger nectin-4 antigen, which may be part of a larger nectin-4 polypeptide fragment, which in turn may be part of a larger nectin-4 polypeptide. Nectin-4 can exist in native or denatured form. The nectin-4 polypeptides described herein can be isolated from various sources, such as various types of human tissue or other sources, or produced by recombinant or synthetic methods. Orthologs of nectin-4 polypeptide are also well known in the art.

[0071] “Nectin-4 ligand” refers to a molecule that binds or otherwise interacts with nectin-4. Nectin-4 can interact homophilically with another nectin-4 molecule and heterophilically interact with other nectin family proteins such as nectin-1, nectin-2 and nectin-3. Nectin-4 may also interact with other cell surface adhesion molecules, such as cadherins, and with other cell surface receptors, such as prolactin receptors. Nectin-4 can also interact with viral particles and viral proteins on viral particles, such as measles viruses and polioviruses. Therefore, nectin-4 ligands may include nectin-1, nectin-2, nectin-3 or nectin-4 or fragments of any of the nectin family proteins. Nectin-4 ligands may also include domains or full-length molecules of other cell surface receptors, such as cadherins or fragments thereof, or prolactin receptors or fragments thereof. Nectin-4 ligands may further include viral particles, viral capsid proteins, viral surface proteins, and fragments of any viral proteins that can bind nectin-4. Non-limiting examples of nectin-4 ligand, in addition to the natural ligands described herein or known to one of ordinary skill in the art, include artificial ligands, for example, ligands improved by screening a library of peptides for ligands that can bind to nectin-4.

[0072] The terms "nectin-4 activity" and "nectin signaling pathway" mean the biological effect or biological response caused by the binding of a nectin-4 ligand and nectin-4, for example, in vivo or in vitro . The terms also refer to a similar effect or response caused by molecules that can mimic nectin-4 ligand function. Such nectin-4 ligand mimetics induce a biological response or biological effects that would otherwise result from binding of the nectin-4 ligand.

[0073] The term “binding protein” refers to a protein containing a portion (e.g., one or more binding regions, such as CDRs) that binds to nectin-4, and optionally a portion of the backbone or framework (e.g., one or more regions skeletal or scaffold) that allows the binding moiety to adopt a conformation that facilitates binding of the binding protein to a nectin-4 polypeptide, fragment, or epitope. Examples of such binding proteins include antibodies such as human antibody, humanized antibody, chimeric antibody, recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab')2 fragment, IgD antibody, IgE antibody, antibody IgM, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody and fragments thereof. The binding protein may comprise, for example, an alternative protein scaffold or an artificial scaffold grafted with CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds derived from antibodies containing mutations introduced, for example, to stabilize the three-dimensional structure of the binding protein, as well as fully synthetic scaffolds containing, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics 53(1):121–29; and Roque et al ., 2004, Biotechnol. Prog.20:639-54. Additionally, peptide mimetic antibodies (“PAMs”) can be used, as well as antibody mimetic scaffolds using fibronectin components as the scaffold. In the context of the present disclosure, the binding protein is considered to specifically bind or selectively bind to nectin-4.

[0074] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably and in its broadest sense herein and specifically includes, for example, individual anti-nectin-4 monoclonal antibodies (including full-length or intact monoclonal antibodies), anti-nectin-4 antibody compositions, Nectin-4 with polyepitope or monoepitope specificity, polyclonal or monovalent antibodies, multivalent antibodies formed from at least two intact antibodies, single-chain anti-nectin-4 antibodies and anti-nectin-4 antibody fragments, as described below. The antibody may be human, humanized, chimeric, and/or affinity matured, as well as other species, such as mouse and rabbit, and the like. The term “antibody” should be understood as a polypeptide product of B cells within the immunoglobulin class of polypeptides, which is capable of binding to a specific molecular antigen and consists of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain includes a variable region ranging from about 100 to about 130 or more amino acids in length, and the carboxy-terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed.1995); and Kuby, Immunology (3d ed.1997). In specific embodiments, a specific molecular antigen may be associated with an antibody of the present invention, including a nectin-4 polypeptide, a nectin-4 fragment, or a nectin-4 epitope. Antibodies also include, but are not limited to, synthetic antibodies, recombinant antibodies, camellized antibodies, intra-antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments, such as nectin-4-binding fragments) of any of the above variants, which refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (eg, antigen binding fragments, such as nectin-4 binding fragments) include single chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, and F(ab) fragments. ')2-fragments linked by a disulfide bond Fv (dsFv), Fd-fragments, Fv-fragments, diabody, tribody, tetrabody and minibody. In particular, the antibodies of the present invention include immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, for example, antigen binding domains or molecules that contain an antigen binding site that binds to a nectin-4 antigen (for example, one or more CDRs of an anti-nectin-4 antibody). Such antibody fragments can be found, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al ., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol.178:497-515; and Day, Advanced Immunochemistry (2d ed.1990). The antibodies of the present invention can be immunoglobulin molecules of any class (eg, IgG, IgE, IgM, IgD and IgA) or any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

[0075] "Antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. In some embodiments, the target antigen is a polypeptide.

[0076] The terms "antigen binding fragment", "antigen binding domain2", "antigen binding region" and similar terms refer to that portion of an antibody that contains amino acid residues that interact with the antigen and give the binding agent specificity and affinity for the antigen (e.g., CDR).

[0077] The terms “binds” or “linking” refer to interactions between molecules, including, for example, the formation of a complex. The interactions may be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions and/or van der Waals interactions. A complex may also involve the linking of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of a complete non-covalent interaction between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as nectin-4, is the binding of the antibody or functional fragment to that epitope. The ratio of the rate of dissociation (koff) and the rate of association (kon) of an antibody to a monovalent antigen (koff/kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KDvalue, the higher the affinity of the antibody. The value of KD varies depending on the antibody-antigen complexes and depends on both kon and koff. The dissociation constant KD of an antibody of the present invention can be determined using any method of the present invention or any other method well known to those skilled in the art. Affinity at a single binding site does not always reflect the true strength of the interaction between antibody and antigen. If complex antigens containing multiple, repetitive antigenic determinants, such as polyvalent nectin-4, come into contact with antibodies containing multiple binding sites, then the interaction of the antibody with the antigen at one site increases the likelihood of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity. The avidity of an antibody may be a better measure of its binding ability than the affinity of its individual binding sites. For example, high avidity can compensate for low affinity, as is sometimes found in pentameric IgM antibodies, which may have lower affinity than IgG, but IgM's high avidity, due to its multivalency, allows it to bind effectively to antigen.

[0078] The terms “antibodies that specifically bind to nectin-4,” “antibodies that specifically bind to an epitope of nectin-4,” and similar terms are also used interchangeably herein to refer to antibodies that specifically bind to a nectin-4 polypeptide , such as a nectin-4 antigen, or fragment, or epitope (for example, human nectin-4, such as a human nectin-4 polypeptide, antigen or epitope). An antibody that specifically binds to nectin-4 (eg, human nectin-4) may bind to the extracellular domain or a peptide derived from the extracellular domain of nectin-4. An antibody that specifically binds to a nectin-4 antigen (eg, human nectin-4) may cross-react with related antigens (eg, cynomolgus nectin-4). In some embodiments, an antibody that specifically binds to a nectin-4 antigen does not cross-react with other antigens, such as nectin-1, nectin-2, and/or nectin-3. An antibody that specifically binds to nectin-4 antigen can be identified, for example, using an immunoassay, Biacore®, or other methods known to those skilled in the art.

[0079] An antibody that “binds to an antigen of interest” (e.g., a target antigen such as nectin-4) is an antibody that binds with sufficient affinity to the antigen to be used as an agent for targeting or detecting a cell or tissue , expressing this antigen, and essentially does not cross-react with other proteins. In such embodiments, the extent of binding of the antibody to the “non-target” protein will be less than about 10% of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) or RIA analysis. With respect to the binding of an antibody to a target molecule, the term “specific binding,” “specifically binds,” or “specifically for” a particular polypeptide or epitope on a particular target polypeptide means binding that is significantly different from nonspecific binding. Specific binding can be measured, for example, by determining binding to a molecule compared to binding to a control molecule, which is typically a molecule of similar structure but lacking binding activity. For example, specific binding may be determined by competition with a control molecule that is similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated by competitive inhibition of the binding of the labeled target to the probe by an excess of labeled target. The term “anti-nectin-4 antibody” or “antibody that binds nectin-4” includes an antibody that is capable of binding to nectin-4 with sufficient affinity to permit use of the antibody, for example, as a diagnostic agent targeting nectin-4 . The term “specific binding,” “specifically binds,” or “is specific for” a particular polypeptide or epitope on a particular target polypeptide, as used herein, refers to binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide, substantially without binding to any other polypeptide or polypeptide epitope. Disclosed herein is an anti-nectin-4 antibody that specifically binds to nectin-4. Also disclosed herein is an anti-nectin-4 antibody that specifically binds to nectin-4 over nectin-1, nectin-2 and/or nectin-3.

[0080] An antibody specifically binds to a nectin-4 antigen if it binds to the nectin-4 antigen with higher affinity than any cross-reacting antigen, as determined by experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). ELISA). Typically, the specific or selective response is at least twice the background signal or noise, and can be more than 10 times the background. See, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for information on antibody binding. Alternatively, antibody specificity can be determined qualitatively, for example, in an IHC assay. In such an IHC assay, an antibody specifically binds to nectin-4 antigen or has specificity for nectin-4 antigen if the antibody binds, e.g., immunohistochemically stains, to cells expressing nectin-4 but does not bind substantially, e.g. stains cells expressing nectin-4. For example, an antibody does not bind substantially to a cell, e.g., does not stain, to a cell by IHC, if the antibody binds, e.g., to a cell, e.g., immunohistochemically stains, at a level substantially similar to the level of binding observed with immunoglobulin isotype control.

[0081] The terms “fully human antibody” or “human antibody” are used interchangeably herein and refer to an antibody that contains a human variable region and, for example, a human constant region. In specific embodiments, these terms refer to an antibody that contains a variable region and a constant region of human origin. “Fully human” anti-nectin-4 antibodies, in some embodiments, may also include antibodies that bind to nectin-4 polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of a human germline immunoglobulin nucleic acid sequence. In a specific embodiment, the anti-nectin-4 antibodies disclosed herein are fully human antibodies. The term "fully human antibody" includes antibodies with variable and constant regions corresponding to human germline immunoglobulin sequences, as described by Kabat et al . (See Kabat et al . (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Examples of methods for producing fully human antibodies are provided, for example, in the examples provided herein, but any method known in the art can be used.

[0082] The phrase "recombinant human antibody" includes human antibodies that are produced, expressed, created or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library human, antibodies isolated from an animal (eg, mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, for example, Taylor, LD et al. (1992) Nucl. Acids Res 20:6287-6295 ) or antibodies produced, expressed, generated, or isolated by any other method that involves splicing immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat et al . (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). In some embodiments, however, such recombinant human antibodies are subject to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are so arranged that when derived from the human germline and compared to human germline VH and VL sequences, may not naturally exist in the human germline antibody repertoire in vivo .

[0083] The term “competition,” when used in the context of anti-nectin-4 antibodies (e.g., antibodies and binding proteins that bind to nectin-4 and compete for the same epitope or binding site on a target), means competition that is determined in an assay in which a test antibody (or binding fragment) prevents or inhibits the specific binding of a reference molecule (eg, a reference ligand or a reference antigen-binding protein, such as a reference antibody) to a common antigen (eg, nectin-4 or a fragment thereof). Various variations of competitive binding assays can be used to determine whether a test antibody competes with a reference antibody for binding to nectin-4 (eg, human nectin-4). Examples of assays that can be used include solid-phase direct or indirect RIA, solid-phase direct or indirect enzyme-linked immunosorbent assay (EIA), competitive sandwich assay (see, for example, Stahli et al ., 1983, Methods in Enzymology 9: 242-53) , solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al ., 1986, J. Immunol.137:3614-19), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, for example, Harlow and Lane, Antibodies, A Laboratory Manual (1988)), labeled solid phase RIA using I-125 label (see, e.g., Morel et al ., 1988, Mol. Immunol.25:7-15) and direct RIA with a label (Moldenhauer et al ., 1990, Scand. J. Immunol.32:77-82). Typically, such an assay involves the use of purified antigen (eg, nectin-4, such as human nectin-4) bound to a solid surface, or cells carrying either an unlabeled test antigen-binding protein (eg, a test anti-nectin-4 antibody) or a labeled reference antigen-binding protein (eg, reference anti-nectin-4 antibody). Other examples of competitive binding assays that may be used include ELISA, FACS, cell adhesion assays (eg, as described in Humphries et al . Methods Mol Biol. 2009;522:203-10), surface plasmon resonance (SPR), or others competitive binding assays known to one skilled in the art. Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cells in the presence of the antigen binding protein of interest. Typically, the antigen binding protein being tested is present in excess. Antibodies identified by a competition assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and/or antibodies that bind to a neighboring epitope, an epitope located close enough to the epitope bound by the reference antibody to cause steric interference for antibodies. These competitive binding determination methods are described more similarly herein. Typically, if the competing antibody protein is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 30%, e.g. 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even more.

[0084] as used herein, the term “in combination” in the context of administering other types of therapeutic agents refers to the use of more than one therapeutic agent. The use of the term “in combination” does not limit the order in which therapeutic agents are prescribed to an individual with an infection. The first therapeutic agent can be administered before (eg, 1 minute before, 45 minutes before, 30 minutes before, 45 minutes before, 1 hour before, 2 hours before, 4 hours before, 6 hours before, 12 hours before, 24 hours before, before 48 hours, 72 hours before, 96 hours before, 1 week before, 2 weeks before, 3 weeks before, 4 weeks before, 5 weeks before, 6 weeks before, 8 weeks before or 12 weeks before, simultaneously or after (for example, after 1 minute, after 45 minutes, after 30 minutes, after 45 minutes, after 1 hour, after 2 hours, after 4 hours, after 6 hours, after 12 hours, after 24 hours, after 48 hours, after 72 hours, after 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) after administration of the second therapeutic agent to an individual who has suffered, is suffering from or is susceptible to a disease caused by nectin-4. Any additional therapeutic agent may be administered in any order with other additional therapeutic agents. In some embodiments, antibodies of the present invention may be administered in combination with one or more therapeutic agents (e.g., therapeutic agents that are not antibodies of the present invention that are currently used to prevent, treat, control, and/or ameliorate a disease mediated by nectin-4 Non-limiting examples of therapeutic agents that can be administered in combination with an antibody of the present invention include analgesics, anesthetics, antibiotics or immunomodulatory agents or any other agents specified in US Pharmacopoeia and/or Physician 's Desk Reference .

[0085] An "isolated" antibody is substantially free of cellular material or other contaminant proteins from the cell or tissue source and/or other contaminants from which the antibody is derived, or is substantially free of chemical precursors or other chemicals in chemical synthesis. The term “substantially free of cellular material” includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations that contain less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) heterologous protein (also called “contaminant protein” in this case). In some embodiments, if the antibody is produced recombinantly, it is substantially free of culture medium, for example, the culture medium constitutes less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In some embodiments, if the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals involved in protein synthesis. Accordingly, such antibody preparations contain less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) chemical precursors or compounds other than the antibody of interest. Contaminants may also include, but are not limited to, substances that adversely affect the therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, as determined by the Lawry method (Lowry et al ., 1951, J. Bio. Chem. 193: 265-75), such as 96%, 97%, 98% or 99%, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by a spin plate sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, the isolated antibody is obtained by at least one purification step. In specific embodiments, the antibodies of the present invention are isolated.

[0086] The 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically approximately 150,000 Daltons in size. Each L chain is linked to an H chain by a single covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. The H and L chains also each have regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each α and γ chain and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. VL is aligned with VH and CL is aligned with the first heavy chain constant domain (CH1). Certain amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of VH and VL results in a single antigen-binding site. For a description of the structure and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al . eds., 8th ed.1994).

[0087] The term "variable region", "variable domain", "V region" or "V domain" refers to the portion of the light or heavy chains of an antibody that is typically located at the amino terminus of the light or heavy chain and is about 120 in length -130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, and are used in the context of the binding and specificity of each particular antibody for its particular antigen. The heavy chain variable region may be designated "VH". The light chain variable region may be designated "VL". The term "variability" refers to the fact that certain segments of the variable regions vary greatly in sequence between different antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the 110-amino acid span of the variable regions. Instead, V regions are composed of less variable (e.g., relatively invariant) regions called framework regions (FRs) of about 15–30 amino acids in length, separated by shorter regions of greater variability (e.g., extremely variable) called "hypervariable regions" each of which is about 9-12 amino acids long. Each heavy and light chain variable region includes four FRs, generally adopting a β-sheet configuration, connected by three hypervariable regions that form loops connecting and in some cases forming part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity to each other and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen binding site of antibodies (see, for example, Kabat et al ., Sequences of Proteins of Immunological Interest (5th ed.1991)). Constant regions are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the antibody's involvement in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Variable regions vary significantly in sequence between antibodies. In specific embodiments, the variable region is a human variable region.

[0088] The term “Kabat variable region residue numbering” or “Kabat amino acid residue numbering” and variants thereof refers to the numbering system used for heavy chain variable regions or light chain variable regions in antibody formulation according to Kabat et al ., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the truncation or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may include one amino acid insertion (Kabath residue 52a) after residue 52 and three inserted residues (e.g., Kabat residues 82a, 82b and 82c, and the like) after residue 82. Kabat numbering of residues may be determined for a given antibody by alignment in regions of homology of the antibody sequence with the Kabat "standard" sequence. The Kabat numbering system is commonly used to refer to a residue in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g. Kabat et al ., supra.) "EU numbering system" or "EU index" are commonly used to refer to a residue in the constant region of an immunoglobulin heavy chain (e.g., EU index, as described in Kabat et al ., supra.) The "Kabat EU index" refers to the residue numbering of the human IgG 1 antibody EU. Other numbering systems have been described, such as AbM, Chothia, Contact, IMGT and AHon and are well known to one of ordinary skill in the art.

[0089] An "intact" antibody is an antibody containing an antigen binding site, as well as CL and at least the heavy chain constant regions, CH1, CH2 and CH3. The constant regions may comprise human constant regions or amino acid sequence variants thereof. In some embodiments, the intact antibody has one or more effector functions.

[0090] “Antibody fragments” include a portion of an intact antibody, such as an antigen binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies and di-diabodies (see, for example, Holliger et al ., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al ., 2005, J. Biol. Chem. 280:19665-72 ;Hudson et al ., 2003, Nat. Med.9:129-34; WO 93/11161; and US patent Nos. 5837242 and 6492123); single chain antibody molecules (see, for example, US Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); double variable domain antibodies (see, for example, US Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, for example, Woolven et al ., 1999, Immunogenetics 50: 98-101; and Streltsov et al ., 2004, Proc Natl Acad Sci USA.101:12444-49) ; and multispecific antibodies formed from antibody fragments.

[0091] A "functional fragment", "binding fragment" or "antigen binding fragment" of a therapeutic antibody exhibits at least one, if not all, biological functions of an intact antibody, wherein the function includes at least binding to a target antigen (e.g. nectin-4 binding fragment or a fragment that binds to nectin-4).

[0092] The term "fusion protein" as used herein refers to a polypeptide that contains the amino acid sequence of an antibody and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of an antibody (e.g., an antigen binding antibody, which does not target nectin-4).The term "fusion", as used in relation to nectin-4 or anti-nectin-4 antibody, refers to the combination of a peptide or polypeptide, or a fragment, variant and/or derivative thereof, with a heterologous peptide or polypeptide. In some embodiments, the fusion protein retains the biological activity of nectin 4 or an anti-nectin 4 antibody. In some embodiments, the fusion protein comprises a VH region, a VL region, a VH CDR (one, two, or three VH CDRs), and/or a VL CDR (one, two or three CDR VL) anti-nectin-4 antibodies, where the fusion protein binds to a nectin-4 epitope, a nectin-4 fragment and/or a nectin-4 polypeptide.

[0093] The term "heavy chain" as used in relation to an antibody refers to a polypeptide chain of about 50-70 kDa in size, in which the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids in length, and the carboxy-terminal portion includes constant region. The constant region can be one of five different types (eg, isotypes), called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The different heavy chains vary in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to five known classes (i.e., isotypes) of immunoglobulins, immunoglobulins IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. The heavy chain may be a human heavy chain.

[0094] The term "light chain" as used in relation to an antibody refers to a polypeptide chain of approximately 25 kDa in size, in which the amino-terminal portion includes a variable region of about 100 to 110 or more amino acids in length and the carboxy-terminal portion includes a constant region region. The approximate length of the light chain is 211 to 217 amino acids. There are two different types, referred to as (κ) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0095] The term “host” as used herein refers to an animal, such as a mammal (eg, a human).

[0096] The term “host cell” as used herein refers to the particular cell in question that can be transfected with a nucleic acid molecule, and the progeny or possible progeny of such cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the genome of the host cell.

[0097] The term "monoclonal antibody" as used herein refers to an antibody derived from a population of essentially homogeneous antibodies, e.g., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minute quantities, and each monoclonal antibody typically recognizes one epitope on an antigen. In specific embodiments, a “monoclonal antibody” as used herein is an antibody produced by a single hybridoma or other cells, where the antibody binds only to an epitope of nectin-4, as determined, for example, by an ELISA or other antigen-binding or competitive binding assay, well known in this field. The term "monoclonal" is not limited to any particular method of producing an antibody. For example, monoclonal antibodies that can be used as described can be produced using the hybridoma technique first described by Kohler et al ., 1975, Nature 256: 495, or can be produced by recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see ., for example, US patent 4816567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using methods described by Clackson et al ., 1991, Nature 352: 624-28 and Marks et al ., 1991, J. Mol. Biol.222:581-97, for example. Other methods for producing clonal cell lines and the monoclonal antibodies expressed by them are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al . eds., 5th ed. 2002). Examples of methods for producing monoclonal antibodies are given in the "Examples" section of this description.

[0098] The term "native", as used in connection with biological substances such as nucleic acid molecules, polypeptides, host cells and the like, refers to substances that are natural and have not been manipulated, modified and/or altered (eg , isolation, purification, selection) by man.

[0099] Antibodies of the present invention may include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain ( it) is identical or homologous to the corresponding sequences of antibodies obtained from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, if they exhibit the desired biological activity (see US patent No. 4816567; and Morrison et al ., 1984, Proc Natl Acad Sci USA 81:6851-55).

[00100] "Humanized" forms of antibodies that are not human (eg, murine) are chimeric antibodies that include human immunoglobulins (eg, receptor antibody) in which the native CDR residues are replaced with residues of the corresponding CDR from a non-human species to a human (eg, donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capabilities. In some cases, one or more residues of the FR region of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These changes are made to fine-tune the properties of the antibodies. A humanized antibody heavy or light chain may contain substantially all of at least one or more variable regions in which all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. In some embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. For more information, see Jones et al ., 1986, Nature 321: 522-25; Riechmann et al ., 1988, Nature 332: 323-29; Presta, 1992, Curr. Op. Struct. Biol.2:593-96; Carter et al ., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; US Patent No. 6800738; 6719971; 6639055; 6407213; and 6054297.

[00101] A “human antibody” is an antibody that has an amino acid sequence that corresponds to the sequence of an antibody produced by a human and/or produced using any method for producing human antibodies described herein. This definition of a human antibody excludes a humanized antibody containing non-human antigen-binding moieties. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al ., 1991, J. Mol. Biol. 222 :581) and yeast display libraries (Chao et al ., 2006, Nature Protocols 1: 755-68). Also available are methods for producing human monoclonal antibodies as described in Cole et al ., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al ., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol.5: 368-74. Human antibodies can be produced by introducing an antigen into a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation, but whose endogenous loci have been disabled, for example in mice (see, for example, Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and US Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE<TM>). See also, for example, Li et al ., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 regarding human antibodies produced by human B cell hybridoma technologies.

[00102] The term "CDR" refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework VH region of an immunoglobulin (Ig or antibody) with a β-sheet structure or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL antibody with a β-sheet structure. Accordingly, CDRs are variable region sequences arranged within framework sequences. CDR regions are well known to those skilled in the art and have been identified, for example, by Kabat as regions of greatest hypervariability within antibody variable domains (V) (Kabath et al ., 1997, J. Biol. Chem.252:6609-16; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR sequences have also been structurally defined by Chothia as sequences whose residues are not part of the conserved β-sheet structure, and thus as capable of adopting different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17) . Both terms are well known in the art. CDR sequences were also determined using AbM, Contact and IMGT. CDR positions within the canonical variable region of an antibody are determined by comparison of numerous structures (Al-Lazikani et al ., 1997, J. Mol. Biol. 273:927-48; Morea et al ., 2000, Methods 20:267-79). Because the number of residues within the hypervariable region varies among antibodies, additional residues to the canonical positions are usually numbered as a, b, c and the like next to the residue number determined by the canonical variable region numbering scheme (Al-Lazikani et al ., supra. ) Such nomenclature is also well known to those skilled in the art.

[00103] The term “hypervariable region,” “HVR,” or “HV,” as used herein, refers to variable regions of an antibody that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). The present description covers and uses a number of images of hypervariable regions. Kabat complementarity determining regions (CDRs) are based on sequence variation and are the most commonly used (see, e.g., Kabat et al ., supra.) Instead, Chothia indicates the location of structural loops (see, e.g., Chothia and Lesk, 1987 , J Mol Biol 196:901-17). The end of the Chothia CDR-H1 loop, when using Kabat numbering, changes at H32 and H34 and depending on the length of the loop (this is because the Kabat numbering scheme places insertions H35A and H35B; if neither of 35A and 35B is not present, then the loop ends on 32; if only 35A is present, then the loop ends on 33; if both 35A and 35B are present, then the loop ends on 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dübel eds., 2d ed. 2010)). “Contact” hypervariable regions are based on the analysis of existing complex crystal structures. The residues of each of these hypervariable regions or CDRs are noted below.

[00104] Recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System®, has been developed and is widely used (Lafranc et al ., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) in humans and other vertebrates. Here, CDRs are specified in terms of both amino acid sequence and location on the light or heavy chain. Because the "location" of the CDRs in the immunoglobulin variable domain structure is conserved across species and is present in structures called loops, CDRs and framework residues are easily identified by numbering systems that align variable domain sequences according to structural features. This information can be used when transplanting and replacing CDR residues of immunoglobulins of one type into an acceptor structure from, as a rule, a human antibody. An additional numbering system (AHon) was developed by Honegger and Plückthun, 2001, J. Mol. Biol.309:657-70. The correspondence between the numbering system, including, for example, Kabat numbering and the unique IMGT numbering system, is well known to one of ordinary skill in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al ., supra) .

IMGT Kabat AbM Chothia Contact CDR1 VH 27-38 31-35 26-35 26-32 30-35 CDR2 VH 56-65 50-65 50-58 53-55 47-58 CDR3 VH 105-117 95-102 95-102 96-101 93-101 V _L CDR1 27-38 24-34 24-34 26-32 30-36 CDR2 VL 56-65 50-56 50-56 50-52 46-55 CDR3 VL 105-117 89-97 89-97 91-96 89-96

[00105] Hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL , and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. In this document, the terms “HVR” and “CDR” are used interchangeably.

[00106] The term “constant region” or “constant domain” refers to the carboxy-terminal portion of the light and heavy chain that is not directly involved in antibody-antigen binding but exhibits various effector functions, such as interaction with the Fc receptor. The term refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin, that is, the variable region that contains the antigen binding site. The constant region may comprise the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.

[00107] The term "framework" or "FR" refers to the variable region residues that flank the CDR. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies and bispecific antibodies. FR residues are variable domain residues that are different from hypervariable region residues or CDR residues.

[00108] An "affinity matured" antibody is an antibody with one or more changes (e.g., amino acid sequence variants, including changes, additions, and/or deletions) in one or more HVRs that result in improved affinity of the antibody for an antigen compared to parent antibody that does not have such change(s). Affinity matured antibodies can have nanomolar or even picomolar activity against the target antigen. Affinity matured antibodies are prepared by techniques known in the art. Reviewed in Hudson and Souriau, 2003, Nature Medicine 9:129-34; Hoogenboom, 2005, Nature Biotechnol.23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51.

[00109] “Binding affinity” generally refers to the strength of the net non-covalent interaction between a single binding site of a molecule (eg, a binding protein such as an antibody) and its binding partner (eg, an antigen). Unless otherwise specified as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a binding molecule X for its binding partner Y can usually be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies typically bind to antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind to antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present description. Specific illustrative embodiments include the following. In one embodiment, the “K _D ” or “K _D value” can be measured using assays known in the art, for example, using a binding assay. KD can be measured in an RIA, for example, by performing an assay with a Fab variant of the antibody of interest and its antigen (Chen et al ., 1999, J. Mol Biol 293: 865-81). The KD or KD value can also be measured using a Biacore® surface plasmon resonance assay, using, for example, the Biacore®TM-2000 or Biacore®TM-3000, or by dowsing interferometry, using, for example, the Octet®QK384 system. The "forward reaction rate" or "association rate" or "k _on " can also be determined by the same surface plasmon resonance or biolayer interferometry techniques described above, using, for example, Biacore®TM-2000 or Biacore®TM-3000, or Octet systems ®QK384.

[00110] The phrase “substantially similar” or “substantially similar” denotes a sufficiently high degree of similarity between two numerical values or signals (e.g., IHC staining signals) of two images (e.g., one associated with an antibody of the present invention and the other associated with the reference antibody) such that one skilled in the art would consider the difference between the two values or two images to be of little or no biological and/or statistical significance in the context of the biological characteristic measured by the values (eg, KD values). For example, the difference between the two values may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% , depending on the value of the reference antibody.

[00110] The phrase "substantially increased" or "substantially decreased" as used herein denotes a sufficiently high degree of difference between two numbers or signals (e.g., IHC staining signals) of two images (e.g., one associated with an antibody of the present invention, and the other associated with the reference antibody), such that one skilled in the art would consider the difference between the two values or two images to be insignificant in the context of the biological characteristic measured by the values. For example, the difference between the two values may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, depending on the value for the reference antibodies.

[00112] "Effect functions" of an antibody refer to those biological activities that are associated with the Fc region (eg, the native Fc region sequence or the amino acid sequence of a variant Fc region) of the antibody and vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and B cell activation.

[00113] The term “effective amount” as used herein refers to an amount of an antibody or pharmaceutical composition herein that is sufficient to achieve the desired result.

[00114] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is often defined by the extent from the amino acid residue at Cys226, or Pro230, to the carboxy terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant methods based on the nucleic acid encoding the antibody heavy chain. Accordingly, an intact antibody composition may contain antibody populations with all K447 residues deleted, antibody populations with no K447 residues deleted, and antibody populations containing a mixture of antibodies with and without K447 residue.

[00115] A “functional Fc region” has the “effector function” of a native Fc region sequence. An example of “effector functions” includes C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; etc. Such effector functions typically require association of an Fc region with a binding region or binding domain (eg, a variable region or antibody domain) and can be assessed using various assays as described.

[00116] A “native Fc region sequence” includes an amino acid sequence identical to the naturally occurring Fc region amino acid sequence that has not been manipulated, modified, and/or altered (e.g., isolated, purified, selected, incorporated, or combined with other sequences such as human variable region sequences. Native human IgG1 Fc region sequences include native human IgG1 Fc region sequence (non-A and A allotypes); the native sequence of the Fc region of human IgG2; the native sequence of the Fc region of human IgG3; and the native sequence of the Fc region of human IgG4, as well as their natural variants. For example, the native amino acid sequence of the Fc region of human IgG1 is shown below (SEQ ID NO: 35).

[00117] An "Fc region variant" contains an amino acid sequence that differs from the native Fc region sequence by at least one amino acid modification (eg, substitution, addition, or deletion). In some embodiments, the Fc region variant has at least one amino acid substitution compared to the native Fc region sequence or compared to the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in the Fc region sequence or in the Fc region of the parent polypeptide. A variant Fc region of the present invention may have at least about 80% homology to the native Fc region sequence and/or the Fc region of the parent polypeptide, or at least about 90% homology, e.g., at least about 95% homology .

[00118] The term "variant" when used in relation to nectin-4 or an anti-nectin-4 antibody may refer to a peptide or polypeptide containing one or more (such as 1 to 25, 1 to 20, 1 to 15 , from 1 to 10 or from 1 to 5) substitutions, deletions and/or additions in the amino acid sequence compared to the native or unmodified sequence. For example, a nectin-4 variant may result from one or more (e.g., 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5) changes in the amino acid sequence of native nectin-4 . Also by way of example, a variant anti-nectin-4 antibody may result from one or more (such as 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5) changes in amino acid the sequence of a native or previously unmodified anti-nectin-4 antibody. Variants may be natural, such as allelic or splice variants, or may be artificially engineered. Polypeptide variants can be derived from the corresponding nucleic acid molecules encoding the variants. In specific embodiments, the nectin-4 variant or anti-nectin-4 antibody variant at least retains specific binding to nectin-4 compared to the reference antibody. In some embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes VH or VL regions or subregions, such as one or more CDRs, of nectin-4 or anti-nectin-4 antibodies.

[00119] The term "vector" refers to a substance that is used to transfer or insert a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an anti-nectin-4 antibody, as described herein, to introduce the nucleic acid sequence into a cell - owner. Vectors that can be used include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which may include selection sequences or markers functional for stable integration into the host cell chromosome. In addition, the vectors may include one or more selection marker genes and corresponding expression control sequences. Selection marker genes that may be included, for example, confer resistance to antibiotics or toxins, complement an auxotrophic deficiency, or provide critical nutrients not contained in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. If two or more nucleic acid molecules are to be expressed together (eg, both the heavy and light chains of an antibody or the VH and VL of an antibody), both nucleic acid molecules can be inserted, for example, into the same expression vector or into different expression vectors. For expression of a single vector, the encoding nucleic acids may be operably linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into a host cell can be confirmed by methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will appreciate that nucleic acid molecules are expressed in sufficient quantities to produce the desired product (e.g., anti-nectin-4 antibodies as described herein), and it will also be appreciated that expression levels can be optimized to produce expression at a sufficient level using methods well known in the art.

[00120] A nectin-4 “extracellular domain” or “ECD” polypeptide refers to a form of nectin-4 polypeptide that is substantially free of transmembrane and cytoplasmic domains. For example, in a nectin-4 ECD polypeptide, at least 1% of such transmembrane and/or cytoplasmic domains may be present and less than 0.5% of such domains may be present.

[00121] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by sequence alignment and comparison.""Percentage (%) amino acid sequence identity" compared to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and the introduction of gaps if necessary to achieve maximum percent sequence identity and without considering any conservative substitutions in the context of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN software (DNAStar, Inc. ). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[00122] A “modification” of an amino acid residue/position refers to a change in the primary amino acid sequence compared to the original amino acid sequence, the change resulting from a change in the sequence containing the specified amino acid residue/position. For example, typical modifications include replacing a residue with another amino acid (eg, a conservative or non-conservative substitution), inserting one or more (eg, typically less than 5, 4, or 3) amino acids adjacent to a specified residue/position, and/or deleting a specified residue/position.

[00123] An "epitope" is a region on the surface of an antigen molecule to which one antibody molecule binds, for example, a localized region on the surface of an antigen, such as a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide that is capable of binding to one or more antigen-binding regions of an antibody and which has antigenic or immunogenic activity in an animal, such as a mammal (eg, a human), which is capable of inducing an immune response. An immunogenic epitope is the portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is the portion of a polypeptide to which an antibody binds, as determined by any method well known in the art, including, for example, an immunoassay. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes often consist of chemically active surface groups of molecules, such as the side chains of amino acids or sugars, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are interrupted in the protein sequence, but which are combined when the protein folds into a three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of a protein is in an altered conformation, such as subsequent activation or binding of another protein or ligand. In some embodiments, a nectin-4 epitope is a three-dimensional surface feature of a nectin-4 polypeptide. In other embodiments, the Nectin-4 epitope is a linear feature of a nectin-4 polypeptide. Typically an antigen has several or many different epitopes and can interact with many different antibodies.

[00124] An antibody binds to an “epitope,” “substantially the same epitope,” or “the same epitope” as a reference antibody if the two antibodies recognize identical, overlapping, or adjacent epitopes in three-dimensional space. The most widely used and rapid methods for determining the binding of two antibodies to identical, overlapping or adjacent epitopes in three-dimensional space are competitive assays, which can be configured in a number of different formats, for example, using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on the cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent, or enzyme labels.

[00125] "Epitope mapping" is the process of identifying the binding sites or epitopes of antibodies to their target antigens. “Epitope-specific sorting” is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope-specific sorting includes methods and systems for distinguishing between the epitope recognition properties of different antibodies, using competitive assays in combination with computational processes to cluster antibodies based on their epitope recognition properties and identifying antibodies having different binding specificities.

[001] The term “carriers” as used herein includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal exposed to them at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; a low molecular weight polypeptide (eg, less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™. The term "carrier" may also refer to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient or carrier of the drug. Such carriers may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a typical carrier when the composition (eg, a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injection solutions. Suitable excipients (e.g. pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. The compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral compositions, including formulations, may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington 's Pharmaceutical Sciences (18th ed.1990). The compositions, including pharmaceutical compositions, may contain an anti-nectin-4 antibody, for example, isolated or purified, together with a suitable amount of carriers.

[00126] The term "pharmaceutically acceptable" as used herein means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia, European Pharmacopeia or other well known and accepted pharmacopoeia for use in animals and, in particular, in humans.

[00127] "Polyclonal antibodies" as used herein refers to a population of antibodies resulting from an immunogenic response to a protein with multiple epitopes, and thus includes many different antibodies directed to the same or different epitopes of the protein. Methods for producing polyclonal antibodies are known in the art (see, for example, Short Protocols in Molecular Biology (Ausubel et al. Eds., 5th ed.2002)).

[00128] An "isolated nucleic acid" is a nucleic acid, such as RNA, DNA, or mixed nucleic acid, that is substantially isolated from other DNA genomic sequences, as well as from proteins or complexes, such as ribosomes and polymerases, that are co-located with native sequence in nature. An "isolated" nucleic acid molecule is a molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. In addition, an “isolated” nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant methods, or may be substantially free of chemical precursors or other chemicals if it is synthesized chemically. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term includes nucleic acid sequences that have been extracted from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

[00129] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to nucleotide polymers of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, the term "oligonucleotide" refers to short, typically single-stranded synthetic polynucleotides, typically, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description of polynucleotides above applies equally and entirely to oligonucleotides. The cell that produces the anti-nectin-4 antibody of the present invention may be the parent cell of the hybridoma, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Suitable host cells are described below.

[00130] Unless otherwise indicated, the left end of any single-stranded polynucleotide sequence described herein is the 5'end; the direction of movement to the left along a double-stranded polynucleotide sequence is called the 5' direction. The direction from 5' to 3' when adding the resulting RNA transcripts is called the direction of transcription; regions of sequences on a DNA strand that have the same sequence as RNA and are 5'-terminal to the 5'-end of the RNA transcript are called "sequences to the left (or in the direction of the 5'-end)", and regions of sequences on a DNA strand , having the same sequence as RNA and being 3'-terminal to the 3'-end of the RNA transcript, are called "sequences to the right (or in the direction of the 3'-end)".

[00131] The term "recombinant antibody" refers to an antibody that is produced, expressed, created or isolated in a recombinant manner. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial antibody library, antibodies isolated from an animal (eg, mouse or cow) that is transgenic and/or transchromosomal for genes human immunoglobulins (see, for example, Taylor et al., 1992, Nucl. Acids Res. 20:6287-95), or antibodies produced, expressed, created or isolated by any other method that involves splicing immunoglobulin gene sequences with other sequences DNA. Such recombinant antibodies may have variable and constant regions, including those derived from human germline immunoglobulin sequences (see Kabat et al., supra). However, in some embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or, if a transgenic animal is used for human Ig sequences, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from or related to human germline VH and VL sequences that may exist naturally in the human germline antibody repertoire in vivo.

[00132] The terms "individual" and "patient" can be used interchangeably. As used herein, in certain embodiments, the individual is a mammal, such as a non-primate (eg, cow, pig, horse, cat, dog, rat, and the like) or a primate (eg, monkey and human). In some embodiments, the individual is a human. In one embodiment, the individual is a mammal, such as a human, who has been diagnosed with a condition or disorder described herein. In another embodiment, the individual is a mammal, such as a human, at risk of developing a condition or disease described herein.

[00133] "Substantially all" refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, according to at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[00134] The term "detection probe" refers to a substance that produces a detectable signal. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment and the like that provides a detectable signal through its activity.

[00135] The term “detectable agent” refers to a substance that can be used to determine the presence or presence of a desired molecule, such as an anti-nectin-4 antibody as described herein, in a sample or in an individual. The detectable agent may be a substance that can be visualized, or a substance that can be detected and/or otherwise measured (eg, by quantitation).

[00136] The term "diagnostic agent" refers to a substance administered to an individual that facilitates the diagnosis of a disease, disorder or conditions. Such substances can be used to identify, detect and/or localize the process causing the disease. In some embodiments, the diagnostic agent comprises an anti-nectin-4 antibody or fragment thereof, as described herein, which, alone or as a conjugate to a substance administered to an individual or contacted with a sample from an individual, facilitates the diagnosis of a nectin-4-mediated disease.

[00137] The term "encoding nucleic acid" or its grammatical equivalents, as used in relation to a nucleic acid molecule, refers to a nucleic acid molecule in its native state or produced by methods well known to those skilled in the art, which can be transcribed from by producing mRNA, which is then translated into a polypeptide and/or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid, and the coding sequence can be isolated from it.

[00138] The term "excipient" refers to an inert substance that is typically used as a diluent, drug carrier, preservative, binding or stabilizing agent, and includes, but is not limited to, proteins (for example, serum albumin and the like), amino acids (for example, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine and the like), fatty acids and phospholipids (for example, alkyl sulfonates, caprylate and the like), surfactants (for example, SDS, polysorbate, nonionic surfactant -active substance and the like), saccharides (for example, sucrose, maltose, trehalose and the like) and polyols (for example, mannitol, sorbitol and the like). See also Remington and Gennaro, Remington 's Pharmaceutical Sciences ( ^18th ed. 1990), which is incorporated herein by reference in its entirety.

[00139] In the context of a peptide or polypeptide, the term “fragment” as used herein refers to a peptide or polypeptide that contains a less than full-length amino acid sequence. Such a fragment may arise, for example, as a result of truncation at the amino terminus, truncation at the carboxy terminus and/or as a result of internal deletion of residue(s) of the amino acid sequence. The fragments may, for example, be generated by alternative RNA splicing or protease activity in vivo. In some embodiments, nectin-4 fragments or anti-nectin-4 antibody fragments include polypeptides comprising an amino acid sequence consisting of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least at least 20 contiguous amino acid residues, at least less than 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues at least 200 contiguous amino acid residues, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, or at least 550 contiguous amino acid residues of the amino acid sequence of a nectin-4 polypeptide or an anti-nectin-4 antibody. In a specific embodiment, the nectin-4 polypeptide or anti-nectin-4 antibody fragment retains at least 1, at least 2, at least 3 or more functions of the polypeptide or antibody.

[00140] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 6%, within 5%, within 4 %, within 3%, within 2%, within 1% or less of the specified value or range.

[00141] "Administer" or "inject" refers to the act of injecting an injection or other physical delivery of a substance, if external to the body (e.g., anti-nectin-4 antibodies as described herein), to a patient, e.g., transmucosally, intradermally , intravenous, intramuscular delivery, and/or any other method of physical delivery as described herein or known in the art.

[00142] In the context of a polypeptide, the term “analog” as used herein refers to a polypeptide that has similar or identical function to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody, but does not necessarily contain similar or identical amino acid sequence of a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide or an anti-nectin-4 antibody, or has a similar or identical structure to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide or an anti-nectin-4 antibody. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following conditions: (a) the polypeptide has an amino acid sequence that is at least 30%, at least 35%, at least by 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of nectin-4 polypeptide, fragment of nectin-4 polypeptide or antibodies against nectin-4 described herein; (b) the polypeptide is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody (or a VH or VL region thereof) as described herein, consisting of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least from 100 amino acid residues of at least 125 amino acid residues or at least 150 amino acid residues (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2001); and Maniatis et al., Molecular Cloning: A Laboratory Manual (1982)); or (c) the polypeptide is encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , is at least 95% or at least 99% identical to the nucleotide sequence encoding a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody (or a VH or VL region thereof) described herein. A polypeptide with a similar structure to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody as described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide 4 or antibodies against nectin-4 described herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

[00143] In the context of a polypeptide, the term “derivative” as used herein refers to a polypeptide that contains the amino acid sequence of a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an antibody that binds to a nectin-4 polypeptide that is altered by introducing a substitution, deletion or addition of an amino acid residue. As used herein, the term “derivative” also refers to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an antibody that binds to a nectin-4 polypeptide that is chemically modified, for example, by covalently attaching any type of molecule to the polypeptide. For example, but not limited to, a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody may be chemically modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, by introducing known protective/blocking functional groups groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, binding to a cellular ligand or other protein, and the like. Derivatives are modified so that they differ from the natural or parent peptide or polypeptides either in the type or arrangement of the molecules attached. Derivations further involve the deletion of one or more chemical groups that are naturally present in the peptide or polypeptide. In addition, a derivative of a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or an anti-nectin-4 antibody may contain one or more non-classical amino acids. The polypeptide derivative has the same or identical function as the nectin-4 polypeptide, nectin-4 polypeptide fragment, or anti-nectin-4 antibody described herein.

[00144] The term “composition” is intended to mean a product containing specified ingredients (eg, an antibody of the present invention), optionally in specified amounts.

[00145] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to completely or partially inhibiting the development, relapse, occurrence, or progression of a nectin-4-associated disease and/or associated symptom, in resulting from the administration of a therapeutic agent or combination of therapeutic agents of the present invention (eg, a combination of prophylactic or therapeutic agents, such as an antibody of the present invention).

[00146] As used herein, the term “prophylactic agent” refers to any agent that can completely or partially inhibit the development, relapse, occurrence or progression of a nectin-4-related disease, and/or an associated symptom, in an individual . In certain embodiments, the term “prophylactic agent” refers to an antibody of the present invention. In some other embodiments, the term “prophylactic agent” refers to an agent other than an antibody of the present invention. In some embodiments, the prophylactic agent is an agent that is known to be, has been, or is currently being used to prevent a nectin-4-related disease and/or symptom associated therewith, or to prevent the occurrence of, development, progression and/or increasing severity of nectin-4-related disease and/or associated symptom. In specific embodiments, the prophylactic agent is a fully human anti-nectin-4 antibody, such as a fully human anti-nectin-4 monoclonal antibody.

[00147] In certain embodiments, a “prophylactically effective serum titer” is a serum titer in an individual, such as a human, that completely or partially inhibits the development, relapse, occurrence, or progression of a disease associated with and/or nectin-4 symptom in the specified individual.

[00148] “Nectin-4-related disease” and “nectin-4-related disorder” are used interchangeably herein and refer to any disease that is wholly or partly caused by, results from, or is correlated with nectin-4. In some embodiments, nectin-4 may be abnormally (eg, highly active) expressed on the cell surface. In some embodiments, nectin-4 may be pathologically activated on a specific cell type. In other embodiments, normal, pathological, or excessive cell signaling is due to the binding of nectin-4 to a nectin-4 ligand. In some embodiments, a snectin-4-associated disease is a disease in which nectin-4 can be used as a biomarker in a patient, such as a human. In some embodiments, a nectin-4-associated disease is a disease in which the expression of nectin-4 may reflect the progression or prognosis of the disease in an individual, e.g., a human. In some embodiments, the nectin-4 ligand is, for example, a nectin-4 receptor that is expressed on the surface of a cell.

[00149] The term "serum titer" as used herein refers to the average serum titer in a population of at least 10, at least 20, at least 30, or at least 40 individuals to about 100, 1000, or more.

[00150] As used herein, the term “side effects” includes unwanted and unfavorable effects of a therapy (eg, a prophylactic or therapeutic agent). Undesired effects are not necessarily unfavorable. An adverse effect of a therapy (eg, a prophylactic or therapeutic agent) may be harmful or cause discomfort or risk of complications. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, shortness of breath, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dryness in the mouth and loss of appetite, rash or swelling at the injection site, flu-like symptoms such as fever, chills and fatigue, digestive disorders and allergic reactions. Other undesirable effects experienced by patients include many other effects and are well known in the art. Many of them are described in Physician 's Desk Reference (60 ^th end., 2006).

[00151] As used herein, the term “therapeutic agent” refers to any agent that can be used to treat, control, or reduce a nectin-4-associated disease and/or associated symptom. In some embodiments, the term “therapeutic agent” refers to an antibody of the present invention. In some other embodiments, the term “therapeutic agent” refers to an agent other than an antibody of the present invention. In some embodiments, the therapeutic agent is an agent that is known to be, has been, or is currently being used to treat, control, or reduce a nectin-4-related disease or one or more symptoms associated therewith.

[00152] A therapeutic combination (e.g., the use of prophylactic or therapeutic agents) that is more effective than the additive effects of any two or more individual therapeutic agents, e.g., the synergistic effect of a combination of prophylactic and/or therapeutic agents is more effective than a single agent, and/or allows the use of lower doses of one or more agents and/or less frequent administration of the agents to an individual suffering from a nectin-4 related disease. The ability to use lower doses of prophylactic or therapeutic agents and/or administer these drugs less frequently reduces the toxicity associated with administering the drugs to an individual without reducing the effectiveness of the drugs to prevent, treat, control or improve nectin-related disease. -4. In addition, the synergistic effect may result in increased effectiveness of therapy in preventing or treating, controlling or ameliorating nectin-4-related disease. Finally, the synergistic effect of a therapeutic combination (eg, prophylactic or therapeutic agents) may avoid or reduce adverse or undesirable side effects associated with the use of either drug alone.

[00153] In some embodiments, a “therapeutically effective serum titer” is a serum titer in an individual, such as a human, that reduces the severity, duration, and/or symptoms associated with nectinome-4-related disease in said individual.

[00154] As used herein, the term “therapy” refers to any protocol, method and/or agent that can be used to prevent, treat, control and/or improve a condition associated with nectin-4. In some embodiments, the terms “therapy” and “drugs” refer to biotherapies, supportive care, and/or other treatments that may be used to prevent, treat, control, and/or ameliorate nectin-4-associated disease known specialist in the field, for example, medical personnel.

[00155] As used herein, the terms “treat” and “treatment” refer to reducing or reducing the progression, severity and/or duration of nectinome-4 disease as a result of the administration of one or more drugs (including, but not but not limited to, administration of one or more prophylactic or therapeutic agents, such as the antibody of the present invention).

[00156] As used herein, the terms “control” and “control” refer to the beneficial effects that an individual receives from a therapy (eg, a prophylactic or therapeutic agent) that does not cure the nectin-4-related condition. In certain embodiments, one or more drugs (e.g., prophylactic or therapeutic agents, such as an antibody of the present invention) are administered to an individual to “control” a condition or disease described herein, one or more symptoms thereof, to prevent progression or deterioration of condition or disease.

[00157] As used herein, the terms “prevention” and “prevention” refer to the complete or partial inhibition of the development, relapse, occurrence or progression of a nectin-4-related disease and/or associated symptom resulting from the administration of a drug an agent or therapeutic combination described herein (eg, a combination of prophylactic or therapeutic agents, such as an antibody of the present invention).

[00158] As used herein, the term "administration" or "administer" or "administer" refers to the act of injecting or otherwise physically delivering a substance (eg, the anti-nectin-4 antibody of the present invention or an antigen-binding fragment thereof) to an individual or patient (eg, human), such as transmucosal, topical, intradermal, parenteral, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery as described herein or known in the art.

[00159] As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount of a drug (e.g., an antibody or pharmaceutical composition of the present invention) that is sufficient to reduce and/or reduce the severity and/or duration of the specified condition, disorder or disease and/or symptom associated therewith. These terms also include an amount necessary to reduce, delay, or delay the development or progression of the specified disease, reduce, delay or reduce the likelihood of relapse, progression or occurrence of the specified disease and/or to improve or enhance the preventive or therapeutic effect(s). another drug (eg, a drug other than the anti-nectin-4 antibody of the present invention). In some embodiments, "effective amount" as used herein also refers to the amount of an antibody of the present invention to achieve a specified result.

[00160] The terms "stability" and "stable", as used herein in the context of a liquid composition containing an antibody that immunospecifically binds to the nectin-4 antigen, refer to the stability of the antibody in the composition to maintain conformation when exposed to thermal and chemical influences, aggregation , degradation or fragmentation under existing conditions of production, receipt, transportation and storage. The "stable" compositions of the present invention retain biological activity equal to or greater than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under existing conditions of production, receipt, transportation and storage. Antibody stability can be assessed by the degree of aggregation, degradation, or fragmentation by methods known to those skilled in the art, including, but not limited to, capillary gel electrophoresis (rCGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and HPSEC comparison with a reference antibody. The overall stability of a composition containing an antibody that immunospecifically binds to the nectin-4 antigen can be assessed by various immunoassays, including, for example, ELISA and radioimmunoassay using a specific nectin-4 epitope. In some exemplary formulations, the anti-nectin-4 antibody may be stable for two months, four months, six months, eight months, ten months, one year, eighteen months, two years, three years, or longer.

[00161] As used herein, the term “substantially surfactant-free” refers to an antibody formulation that immunospecifically binds to the nectin-4 antigen, wherein the formulation contains less than 0.0005%, less than 0.0003%, or less than 0.0001% surfactants and/or less than 0.0005%, less than 0.0003% or less than 0.0001% surfactants.

[00162] As used herein, “substantially free of salt” refers to an antibody composition that immunospecifically binds to the nectin-4 antigen, where the composition contains less than 0.0005%, less than 0.0003%, or less than 0.0001 % inorganic salts.

[00163] As used herein, the term "inorganic salt" refers to any non-carbon containing compounds that result from the replacement of part or all of the hydrogen of an acidic group or acidic group with a metal or group acting as a metal, and which typically , are used as tonicity-regulating compounds in pharmaceutical compositions and preparations of biological substances. The most common inorganic salts are NaCl, KCl, NaH ₂ PO ₄ and the like.

[00164] As used herein, the term "surfactant" refers to organic substances having amphipathic structures; namely, they consist of groups with opposing solubility properties, typically groups with an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified based on the charge of the surfactant component into anionic, cationic and nonionic surfactants. Surfactants are often used as wetting, emulsifying, solubilizing and dispersing agents for various pharmaceutical and biological formulations.

8. Compositions and methods of their manufacture.

[00165] The present invention provides antibodies that immunospecifically bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or a nectin-4 etipop. The invention also relates to isolated nucleic acids encoding antibodies that immunospecifically bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide or a nectin-4 etipop. The invention further provides vectors and host cells containing nucleic acids encoding antibodies that immunospecifically bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or a nectin-4 epitope. Also provided are methods for producing antibodies that immunospecifically bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or a nectin-4 epitope.

[00166] In certain embodiments, the antibodies of the present invention bind to human and/or cynomolgus nectin-4. In one embodiment, anti-nectin-4 antibodies bind to human nectin-4. In one embodiment, anti-nectin-4 antibodies bind to cynomolgus nectin-4. In one embodiment, anti-nectin-4 antibodies bind to both human nectin-4 and cynomolgus nectin-4. In other embodiments, the antibodies of the present invention do not bind to rodent nectin-4.

[00167] In some embodiments, anti-nectin-4 antibodies bind to the extracellular domain (ECD) of nectin-4. In some embodiments, anti-nectin-4 antibodies bind to an epitope in the nectin-4 ECD that is different from the nectin-4 ligand binding site.

[00168] Also disclosed are antibodies that competitively block the binding of the anti-nectin-4 antibody of the present invention to a nectin-4 polypeptide.

[00169] Antibodies that compete with the anti-nectin-4 antibody of the present invention for binding to the nectin-4 polypeptide are also provided.

[00170] In some embodiments, anti-nectin-4 antibodies do not block the binding of a nectin-4 ligand to a nectin-4 polypeptide.

[00171] In some embodiments, anti-nectin-4 antibodies do not compete with the nectin-4 ligand for binding to the nectin-4 polypeptide.

[00172] In some embodiments, the binding of nectin-4 ligand to nectin-4 is not inhibited by the antibody.

[00173] The anti-nectin-4 antibodies of the present invention may also be conjugated or recombinantly fused to, for example, a diagnostic agent, a detection agent, and/or another agent. Also disclosed herein are compositions containing an anti-nectin-4 antibody.

[00174] Also provided herein are isolated nucleic acid molecules encoding the heavy chain, light chain, VH region, VL region, CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and/or CDR3 VL of anti-nectin-4 antibodies , which bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope.

[00175] Also provided herein are vectors and host cells containing nucleic acid molecules encoding anti-nectin-4 antibodies that bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope. 4. Also provided are methods for producing antibodies that bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope.

[00176] In one embodiment, the present invention provides anti-nectin-4 antibodies that may find use as diagnostic agents. Examples of antibodies include polyclonal, monoclonal, humanized, human and heteroconjugate antibodies, as well as variants thereof with improved affinity or other properties.

9. Antibodies against nectin-4

[00177] Antibodies of the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, internal antibodies, single chain Fv (scFv), camel antibodies, Fab- fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above.

[00178] In particular, the antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen-binding site that immunospecifically binds to nectin-4 antigen. The immunoglobulin molecules of the present invention can be any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In a specific embodiment, the antibody of the present invention is an IgG antibody, such as an IgG1 antibody.

[00179] Antibody variants and derivatives include antibody fragments that retain the ability to specifically bind an epitope. Examples of fragments include Fab fragments (an antibody fragment that contains an antigen binding domain and contains a light chain and a heavy chain portion linked by a disulfide bridge); Fab' (an antibody fragment containing one anti-binding domain containing a Fab and an additional heavy chain portion connected by a hinge region); F(ab') ₂ (two Fab' molecules connected by interchain disulfide bonds at the hinge regions of the heavy chains; Fab' molecules can be directed to the same or different epitopes); a single-chain Fab chain containing a variable region, also known as sFv (variable antigen-binding determinant region of one light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (variable antigen-binding determinant region of one light and heavy chain of an antibody linked together by a disulfide bond); camel VH (the variable, antigen-binding determinant region of a single heavy chain antibody in which some of the amino acids at the VH boundary are amino acids found in the heavy chain of natural camel antibodies); diabody (a dimerized sFv is formed when the VH domain of the first sFv binds to the VL domain of the second sFv, and the VL domain of the first sFv binds to the VH domain of the second sFv; the two antigen-binding regions of the diabody can be directed to the same or different epitopes); and tribody (trimerized sFv, formed in a manner similar to diabody, but in which three antigen-binding domains are assembled into one complex; the three antigen-binding domains may be directed to the same or different epitopes). Antibody derivatives also include one or more antibody combining site CDR sequences. CDR sequences may be linked together on a scaffold if two or more CDR sequences are present. In certain embodiments, the antibody of the present invention comprises a single chain Fv (“scFv”). scFvs are antibody fragments containing the VH and VL domains of the antibody, where these domains are present on the same polypeptide chain. Typically, the scFv polypeptide also contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. scFv is reviewed in Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[00180] The antibodies of the present invention can be of any animal origin, including birds and mammals (eg, human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken). In certain embodiments, the antibodies of the present invention are human or humanized monoclonal antibodies. As used in the present invention, “human” antibodies include antibodies with the amino acid sequence of human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.

[00181] In some embodiments, the antibodies are fully human antibodies, such as fully human antibodies that immunospecifically bind to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, or a nectin-4 epitope. Such fully human antibodies will have an advantage over fully murine (or other fully or partially non-human antibodies), humanized antibodies or chimeric antibodies in terms of maximizing the reduction in the development of unwanted or unnecessary side effects, such as immune responses directed at incompletely human antibodies (eg , antibodies against nectin-4 obtained from other species) when administered to an individual.

[00182] In one embodiment, an antibody of the present invention may include a bispecific antibody or antigen binding fragment thereof having two antibodies or antigen binding fragments thereof, each directed to different epitopes of nectin-4.

[00183] In some embodiments, the antibodies of the present invention are monospecific for a given nectin-4 polypeptide epitope and do not bind immunospecifically to other epitopes.

[00184] In some embodiments, the antibodies of the present invention are antibodies that bind to nectin-4, including a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope. In one embodiment, an antibody of the present invention binds to human nectin-4. In another embodiment, the antibody of the present invention binds to cynomolgus nectin-4. In another embodiment, the antibody of the present invention binds to human nectin-4 and cynomolgus nectin-4. In some embodiments, anti-nectin-4 antibodies do not block the binding of a nectin-4 ligand to a nectin-4 polypeptide. In other embodiments, anti-nectin-4 antibodies are humanized antibodies (e.g., containing human constant regions) that bind to nectin-4, including a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope .

[00185] In one embodiment, the antibodies of the antibody compositions and methods of using the antibodies of the present invention include antibody M22-321b41.1. Also disclosed herein are hybridomas that produce the antibody M22-321b41.1. In some embodiments, the M22-321b41.1 antibody is derived from the M22-321b41.1 hybridoma cell line. In some embodiments, the M22-321b41.1 antibody is of the mouse IgG2a/kappa isotype.

[00186] M22-321b41.1 is a hybridoma cell line of mouse origin. The hybridoma cell line M22-321b41.1 was deposited on June 13, 2017 (ATCC accession number PTA-124245).

[00187] In some embodiments, the anti-nectin-4 antibody comprises the VH region, VL region, CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and/or CDR3 VL of a mouse monoclonal antibody described herein, such as an amino acid sequence depicted in Table 1 . Accordingly, in some embodiments, an isolated antibody or functional fragment thereof of the present invention comprises one, two and/or three heavy chain CDRs and/or one, two and/or three light chain CDRs from the anti-nectin-4 antibody M22-321b41.1 .

Table 1: CDR sequences of antibody M22-321b411

Example Kabat Chothia Contact VL CDR VL CDR1 RSSKSLLHSNGITYLY
(SEQ ID NO:7) RSSKSLLHSNGITYLY
(SEQ ID NO:7) RSSKSLLHSNGITYLY (SEQ ID NO:7) ITYLYWY
(SEQ ID NO:8) CDR2 VL LLIYHMSNLAS
(SEQ ID NO:9) HMSNLAS
(SEQ ID NO:10) HMSNLAS
(SEQ ID NO:10) LLIYHMSNLA
(SEQ ID NO:11) CDR3 VL AQNLELPFT
(SEQ ID NO:12) AQNLELPFT
(SEQ ID NO:12) AQNLELPFT
(SEQ ID NO:12) AQNLELPF
(SEQ ID NO:13) VH CDR CDR1 VH GYTFTTYWMQ
(SEQ ID NO:14) TYWMQ
(SEQ ID NO:15) GYTFTY
(SEQ ID NO:16) TTYWMQ
(SEQ ID NO:17) CDR2 VH WIGSIYPGDGDTRYTQKFKG
(SEQ ID NO:18) SIYPGDGDTRYTQKFKG
(SEQ ID NO:19) YPGDGD
(SEQ ID NO:20) WIGSIYPGDGDTR
(SEQ ID NO:21) CDR3 VH AREYYGLDY
(SEQ ID NO:22) EYYGLDY
(SEQ ID NO:23) EYYGLDY
(SEQ ID NO:23) AREYYGLD
(SEQ ID NO:24) VL sequence:
DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYHMSNLASGVPDRFTSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPFTFGGGTKLETKRADAAPTVSIFPPSSEQLTSG (SEQ ID NO: 3) VH sequence:
QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQGLEWIGSIYPGDGDTRYTQKFKGKATLTADKSSSTAYIQLSTLASEDSAVYYCAREYYGLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTG (SEQ ID NO: 4)

[00188] In some embodiments, the antibody of the present invention contains or consists of six CDRs, for example, CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and/or CDR3 VL, listed in Table 1 . In some embodiments, an antibody of the present invention may contain less than six CDRs. In some embodiments, the antibody contains or consists of one, two, three, four, or five CDRs selected from the group consisting of CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and/or CDR3 VL specified in Table 1 . In some embodiments, the antibody contains or consists of one, two, three, four, or five CDRs selected from the group consisting of CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and/or CDR3 VL of the monoclonal antibody M22-321b41 .1.

[00189] In some embodiments, the antibodies of the present invention comprise one or more (eg, one, two, or three) VH CDRs listed in Table 1 . In other embodiments, the antibodies of the present invention comprise one or more (eg, one, two, or three) VL CDRs listed in Table 1 . In other embodiments, the antibodies of the present invention comprise one or more (eg, one, two, or three) VH CDRs listed in Table 1 and one or more VL CDRs listed in Table 1 . Accordingly, in some embodiments, the antibodies comprise a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, as an example, CDR1 VH (SEQ ID NO:14). In some embodiments, the antibodies comprise a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21 ) and, as an example, CDR2 VH (SEQ ID NO:18). In some embodiments, the antibodies comprise a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24 ) and, as an example, CDR3 VH (SEQ ID NO:22). In some embodiments, the antibodies comprise a CDR1 VH and/or a CDR2 VH and/or a CDR3 VH independently selected from one of the amino acid sequences CDR1 VH, CDR2 VH, CDR3 VH, as set forth in Table 1 . In some embodiments, the antibodies comprise a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8 ) and, as an example, CDR1 VL (SEQ ID NO:7). In yet another embodiment, the antibodies comprise a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO: 11) and, as an example, CDR2 VL (SEQ ID NO:9). In some embodiments, the antibodies comprise a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13 ) and, as an example, CDR3 VL (SEQ ID NO:12). In some embodiments, the antibodies comprise CDR1 VL and/or CDR2 VL and/or CDR3 VL independently selected from one of the amino acid sequences CDR1 VL, CDR2 VL, CDR3 VL, as shown in Table 1 .

[00190] In some embodiments, antibodies of the present invention comprise a VH region comprising: (1) a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); (2) a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, in as an example, CDR2 VH (SEQ ID NO:18); and (3) a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, as an example, CDR3 VH (SEQ ID NO:22); and a VL region comprising: (1) a VL CDR1 with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO :8) and, by way of example, CDR1 VL (SEQ ID NO:7); (2) a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, in as an example, CDR2 VL (SEQ ID NO:9); and (3) a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, as an example, CDR3 VL (SEQ ID NO:12).

[00191] In some embodiments, antibodies of the present invention comprise a VH region comprising: (1) a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); (2) a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, in as an example, CDR2 VH (SEQ ID NO:18); and (3) a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, as an example, CDR3 VH (SEQ ID NO:22).

[00192] In other embodiments, the antibodies provided herein comprise a VL region comprising: (1) CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO :7), Contact CDR1 VL (SEQ ID NO:8) and, as an example, CDR1 VL (SEQ ID NO:7); (2) a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, in as an example, CDR2 VL (SEQ ID NO:9); and (3) a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, as an example, CDR3 VL (SEQ ID NO:12).

[00193] The present invention also provides antibodies comprising one or more (eg, one, two, or three) VH CDRs and one or more (eg, one, two, or three) VL CDRs listed in Table 1 . In particular, the present invention relates to an antibody comprising a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17 ) and, as an example, CDR1 VH (SEQ ID NO:14); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In some embodiments, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21 ) and, by way of example, CDR2 VH (SEQ ID NO:18); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In one embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO: 24) and, as an example, CDR3 VH (SEQ ID NO:22); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In other embodiments, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In some embodiments, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24 ) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In some embodiments, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21 ) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In one embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In some embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17 ) and, as an example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO: 7), Contact CDR1 VL (SEQ ID NO:8) and, as an example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In some embodiments, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of . Cabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In one embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO: 24) and, as an example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In other embodiments, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In some embodiments, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24 ) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of . Cabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example , CDR1 VL (SEQ ID NO:7). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In some embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17 ) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of . Cabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9);. In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In some embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17 ) and, as an example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In one embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In other embodiments, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22);a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example , CDR2 VL (SEQ ID NO:9). In some embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17 ) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In one embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In other embodiments, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO:17) and, by way of example, CDR1 VH (SEQ ID NO:14); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In some embodiments, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO:21 ) and, by way of example, CDR2 VH (SEQ ID NO:18); CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises a CDR1 VH with an amino acid sequence selected from the group consisting of Kabat CDR1 VH (SEQ ID NO:15), Chothia CDR1 VH (SEQ ID NO:16), Contact CDR1 VH (SEQ ID NO: 17) and, as an example, CDR1 VH (SEQ ID NO:14); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In one embodiment, the antibody comprises a CDR2 VH with an amino acid sequence selected from the group consisting of Kabat CDR2 VH (SEQ ID NO:19), Chothia CDR2 VH (SEQ ID NO:20), Contact CDR2 VH (SEQ ID NO: 21) and, as an example, CDR2 VH (SEQ ID NO:18); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In other embodiments, the antibody comprises a CDR3 VH with an amino acid sequence selected from the group consisting of Kabat CDR3 VH (SEQ ID NO:23), Chothia CDR3 VH (SEQ ID NO:23), Contact CDR3 VH (SEQ ID NO:24) and, by way of example, CDR3 VH (SEQ ID NO:22); CDR1 VL with an amino acid sequence selected from the group consisting of Kabat CDR1 VL (SEQ ID NO:7), Chothia CDR1 VL (SEQ ID NO:7), Contact CDR1 VL (SEQ ID NO:8) and, by way of example, CDR1 VL (SEQ ID NO:7); CDR2 VL with an amino acid sequence selected from the group consisting of Kabat CDR2 VL (SEQ ID NO:10), Chothia CDR2 VL (SEQ ID NO:10), Contact CDR2 VL (SEQ ID NO:11) and, by way of example, CDR2 VL (SEQ ID NO:9); and a CDR3 VL with an amino acid sequence selected from the group consisting of Kabat CDR3 VL (SEQ ID NO:12), Chothia CDR3 VL (SEQ ID NO:12), Contact CDR3 VL (SEQ ID NO:13) and, by way of example , CDR3 VL (SEQ ID NO:12). In yet another embodiment, the antibody comprises any combination of CDR VH and CDR VL listed in Table 1 .

[00194] The frame regions described herein are defined by the boundaries of the CDR numbering system. In other words, if CDRs are defined, for example, by Kabat, IMGT or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3 -CDR3-FR4. For example, FR1 is defined as the amino acid residues from the N-terminus to the amino acid residues of CDR1, as defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between the amino acid residues of CDR1 and CDR2, as defined, for example , by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between amino acid residues CDR2 and CDR3, as defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues from the C-terminus to the amino acid residues of CDR3, as defined, for example, by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[00195] In some embodiments, an isolated antibody or functional fragment thereof of the present invention further comprises one, two, three and/or four light chain FRs of the M22-321b41.1 antibody, as shown in Table 2 .

Table 2. Amino acid sequences of FR VL and VH M22-321b41.1

Ab FR1 FR2 FR3 FR4 VL DIVMTQAAFSN
PVTLGTSASIS
(SEQ ID NO:25) WYLQKPGQSP
QLLIY
(SEQ ID NO:26) GVPDRFTSSGSGT
DFTLRISRVEAED
VGVYYC
(SEQ ID NO:27) FGGGTKLETKR
(SEQ ID NO:28) VH QVQLQQSGAE
LARPGASVKLS
CKASGYTFT
(SEQ ID NO:29) WVKQRPGQGL
EWIG
(SEQ ID NO:30) KATLTADKSSSTAYIQLSTLASEDSAVYYCAR
(SEQ ID NO:31) WGQGTTLTVSS
(SEQ ID NO:32)

[00196] In some embodiments, an isolated antibody or functional fragment thereof of the present invention further comprises one, two, three and/or four heavy chain FRs of the M22-321b41.1 antibody, as shown in Table 2 . In some embodiments, one or more antibody heavy chain FRs are derived from antibody M22-321b41.1.

[00197] In some embodiments, an isolated antibody or functional fragment thereof of the present invention further comprises one, two, three and/or four light chain FRs of the M22-321b41.1 antibody, as shown in Table 2 . In some embodiments, the antibody light chain FR is derived from antibody M22-321b41.1.

[00198] In certain embodiments, an antibody or fragment thereof of the present invention comprises a VH region that comprises: (1) an FR1 VH with the amino acid sequence of SEQ ID NO: 29; (2) FR2 VH with amino acid sequence SEQ ID NO: 30; (3) FR3 VH with amino acid sequence SEQ ID NO: 31; and/or (4) FR4 VH with the amino acid sequence of SEQ ID NO: 32. In particular embodiments, the antibody contains a VH region containing all four of the above FR1 VH, FR2 VH, FR3 VH and FR4 VH.

[00199] Thus, in one embodiment, the humanized antibody comprises a VH region that contains an FR1 VH with the amino acid sequence of SEQ ID NO:29. In some embodiments, the humanized antibody comprises a VH region that contains an FR2 VH with the amino acid sequence of SEQ ID NO:30. In one embodiment, the humanized antibody comprises a VH region that contains an FR3 VH with the amino acid sequence of SEQ ID NO:31. In other embodiments, the humanized antibody comprises a VH region that contains an FR4 VH with the amino acid sequence of SEQ ID NO:32.

[00200] In some embodiments, the VL region comprises: (1) VL FR1 with the amino acid sequence of SEQ ID NO:25; (2) FR2 VL with amino acid sequence SEQ ID NO:26; (3) FR3 VL with amino acid sequence SEQ ID NO:27; and/or (4) FR4 VL with the amino acid sequence of SEQ ID NO:28.

[00201] Thus, in some embodiments, the humanized antibody comprises a VL region that includes VL FR1 with the amino acid sequence of SEQ ID NO:25. In some embodiments, the humanized antibody comprises a VL region that includes FR2 VL with the amino acid sequence of SEQ ID NO:26. In one embodiment, the humanized antibody comprises a VL region that includes VL FR3 with the amino acid sequence of SEQ ID NO:27. In other embodiments, the humanized antibody comprises a VL region that includes FR4 VL with the amino acid sequence of SEQ ID NO:28.

[00202] In some embodiments, the antibody or fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) FR1 VH with the amino acid sequence of SEQ ID NO:29; (2) FR2 VH with amino acid sequence SEQ ID NO:30; (3) FR3 VH with amino acid sequence SEQ ID NO:31; and/or (4) FR4 VH with the amino acid sequence of SEQ ID NO:32; and wherein the VL region comprises: (1) FR1 VL with the amino acid sequence of SEQ ID NO:25; (2) FR2 VL with amino acid sequence SEQ ID NO:26; (3) FR3 VL with amino acid sequence SEQ ID NO:27; and/or (4) FR4 VL with the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody comprises a VH region containing all four of the above FR1 VH, FR2 VH, FR3 VH and FR4 VH. In other embodiments, the antibody comprises a VL region containing all four of the above VL FR1, VL FR2, VL FR3 and VL FR4. In other embodiments, the antibody comprises a VH region comprising all four of the above FR1 VH, FR2 VH, FR3 VH and FR4 VH and a VL region containing all four of the above VL FR1, VL FR2, VL FR3 and VL FR4.

[00203] Also disclosed herein are antibodies comprising one or more (e.g., one, two, three, or four) VH FRs and one or more (e.g., one, two, three, or four) VL FRs listed in Table 2. In particular, an antibody comprising FR1 VH (SEQ ID NO:29) and FR1 VL (SEQ ID NO:25) is disclosed herein. In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29) and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30) and FR1 VL (SEQ ID NO:25). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30) and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody contains FR2 VH (SEQ ID NO:30) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody contains FR3 VH (SEQ ID NO:31) and FR1 VL (SEQ ID NO:25). In other embodiments, the antibody comprises FR3 VH (SEQ ID NO:31) and FR2 VL (SEQ ID NO:26). In yet another embodiment, the antibody contains FR3 VH (SEQ ID NO:31) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR3 VH (SEQ ID NO:31) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody contains FR4 VH (SEQ ID NO:32) and FR1 VL (SEQ ID NO:25). In yet another embodiment, the antibody contains FR4 VH (SEQ ID NO:32) and FR2 VL (SEQ ID NO:26). In one embodiment, the antibody contains FR4 VH (SEQ ID NO:32) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR4 VH (SEQ ID NO:32) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30) and FR1 VL (SEQ ID NO:25). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), and FR2 VL (SEQ ID NO:26). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), and FR1 VL (SEQ ID NO:25). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR2 VL (SEQ ID NO:26). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR3 VL (SEQ ID NO:27). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25), and FR2 VL (SEQ ID NO:26). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VL (SEQ ID NO:27), and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), and FR2 VL (SEQ ID NO:26). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26), and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In other embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), and FR3 VL (SEQ ID NO:27). In other embodiments, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR1 VL (SEQ ID NO:25). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR2 VL (SEQ ID NO:26). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), and FR1 VL (SEQ ID NO:25). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32) and FR2 VL (SEQ ID NO:26). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), and FR1 VL (SEQ ID NO:25). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR2 VL (SEQ ID NO:26). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR1 VL (SEQ ID NO:25). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), and FR2 VL (SEQ ID NO:26). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO: 27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), and FR2 VL (SEQ ID NO:26). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), and FR2 VL (SEQ ID NO:26). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In other embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27), and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27), and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR1 VL ( SEQ ID NO:25). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR2 VL ( SEQ ID NO:26). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR2 VL ( SEQ ID NO:26). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR3 VL ( SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL ( SEQ ID NO:26). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL ( SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL ( SEQ ID NO:26). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL ( SEQ ID NO:26). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL ( SEQ ID NO:27). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL ( SEQ ID NO:28). In other embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In yet another embodiment, the antibody comprises FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL ( SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR2 VL (SEQ ID NO:26). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL ( SEQ ID NO:25) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL ( SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL ( SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL ( SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL ( SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL ( SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In some embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL ( SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR3 VL (SEQ ID NO:27). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL ( SEQ ID NO:25), FR2 VL (SEQ ID NO:26) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL ( SEQ ID NO:25), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR2 VL ( SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In yet another embodiment, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR1 VL (SEQ ID NO:25), FR2 VL ( SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR2 VH (SEQ ID NO:30), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In other embodiments, the antibody comprises FR1 VH (SEQ ID NO:29), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL (SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In one embodiment, the antibody comprises FR2 VH (SEQ ID NO:30), FR3 VH (SEQ ID NO:31), FR4 VH (SEQ ID NO:32), FR1 VL (SEQ ID NO:25), FR2 VL ( SEQ ID NO:26), FR3 VL (SEQ ID NO:27) and FR4 VL (SEQ ID NO:28). In some embodiments, the antibody comprises any combination of FR VH (SEQ ID NO:29-32) and FR VL (SEQ ID NO:25-28) listed in Table 2 .

[00204] In some embodiments, the antibodies of the present invention contain a VH region or a VH domain. In other embodiments, the antibodies of the present invention comprise a VL region or a VL domain. In some embodiments, the antibodies of the present invention comprise a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. In other embodiments, the antibodies of the present invention comprise a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region selected from the group consisting of SEQ ID NO:3 and 4, as set forth in Table 1 .

[00205] In some embodiments, the antibodies of the present invention comprise a VH region comprising: (1) a VH CDR1 with the amino acid sequence of SEQ ID NO:15; (2) CDR2 VH with amino acid sequence SEQ ID NO:19; and (3) CDR3 VH with amino acid sequence SEQ ID NO:23; and a VL region selected from the group consisting of SEQ ID NO:3, as shown in Table 1 .

[00206] In other embodiments, the antibodies of the present invention contain a VH region selected from the group consisting of SEQ ID NO:4, as set forth in Table 1 ; and a VL region comprising: (1) VL CDR1 with amino acid sequence SEQ ID NO:7; (2) CDR2 VL with amino acid sequence SEQ ID NO:10; and (3) CDR3 VL with amino acid sequence SEQ ID NO:12.

[00207] Also provided herein are isolated nucleic acid molecules encoding the heavy chain, light chain, VH region, VL region, CDR1 VH, CRD2 VH, CDR3 VH, CDR1 VL, CDR2 VL and/or CDR3 VL of anti-nectin antibody immunoglobulin 4, which binds to a nectin-4 polypeptide, a fragment of a nectin-4 polypeptide, a nectin-4 peptide, or a nectin-4 epitope. Representative nucleic acid sequences of the VL region, light chain, VH region and heavy chain of antibody M22-321b41.1 are shown in Tables 3-4 .

Table 3. VL nucleic acid sequences

Antibodies Nucleotide sequences DNA sequence M22-321b41.1,
region VL GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCATATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCACTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGC AGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGCGCTCAAAATCTAGAACTTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAACAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGA (SEQ ID NO:37) DNA sequence M22-321b41.1 light chain
(SEQ ID NO:39)

Table 4. VH nucleic acid sequences

Antibody Nucleotide sequences DNA sequence M22-321b41.1, VH region CAGGTTCAGCTCCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTATACCTTTACTACCTACTGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGTCTATTTATCCTGGAGATGGTGATACTAGGTACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATTCAACT CAGCACCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAATACTACGGTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGC (SEQ ID NO:38) DNA sequence M22-321b41.1, heavy chain CAGGTTCAGCTCCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTATACCTTTACTACCTACTGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGTCTATTTATCCTGGAGATGGTGATACTAGGTACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATTCAACT CAGCACCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAATACTACGGTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTG GTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTTACACCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAAGAT CAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGA CCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGC TGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA (SEQ ID NO:40)

[00208] In some embodiments, the antibody of the present invention has the VH and VL amino acid sequences of antibody M22-321b41.1. In some embodiments, the antibody comprises a VH having the amino acid sequence of SEQ ID NO:4 and a VL having the amino acid sequence of SEQ ID NO:3.

[00209] In some embodiments, an antibody or antigen binding fragment thereof of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the light chain contains a constant region with the amino acid sequence:

GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSSTSPIVKTFNRNEC (SEQ ID NO:33).

[00210] In some embodiments, an antibody or antigen binding fragment thereof of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the heavy chain contains a constant region with the amino acid sequence:

SSVTLGCLVKGYFPEPVTLTWNSGSLSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO:34).

[00211] In other embodiments, an antibody or antigen binding fragment thereof of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the heavy chain contains the Fc region of human IgG1 with the amino acid sequence:

PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO:35).

[00212] In some embodiments, an antibody or antigen binding fragment thereof of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the heavy chain does not contain the human IgG1 Fc region with the amino acid sequence of SEQ ID NO:35.

[00213] In yet another embodiment, an antibody or antigen binding fragment thereof of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the light chain contains a constant region with the amino acid sequence SEQ ID NO:33; and the heavy chain contains an Fc region with the amino acid sequence of SEQ ID NO:35.

[00214] In some embodiments, an antibody of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the light chain comprises the following amino acid sequence :

DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYHMSNLASGVPDRFTSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPFTFGGGTKLETKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSSTSPIVKTFNRNEC (SEQ ID NO:5).

[00215] In some embodiments, an antibody of the present invention that specifically binds to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) comprises a light chain and a heavy chain, wherein the heavy chain comprises the following amino acid sequence :

QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQGLEWIGSIYPGDGDTRYTQKFKGKATLTADKSSSTAYIQLSTLASEDSAVYYCAREYYGLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTS STWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVE WTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO:6).

[00217] In yet another aspect, antibodies are provided that compete with one of the exemplary antibodies or functional fragments for binding to nectin-4. Such antibodies may also bind to the same epitope as one of the example antibodies provided herein, or to an overlapping epitope. Antibodies and fragments that compete for binding or bind to the same epitope as the exemplified antibodies are expected to exhibit similar functional properties. Exemplary antigen binding proteins and fragments thereof include those with the VH and VL regions and CDRs set forth herein, including those listed in Table 1 . Thus, by specific example, antibodies provided include antibodies that compete with an antibody containing: (a) 1, 2, 3, 4, 5, or all 6 CDRs listed for the antibody as listed in Table 1 ; (b) VH and VL selected from the VH and VL regions listed for the antibody as indicated in Table 1; or (c) two light chains and two heavy chains containing VH and VL, as specified for the antibody in Table 1 . In some embodiments, the antibody is antibody M22-321b41.1.

[00218] In certain embodiments, an antibody described herein, or an antigen binding fragment thereof, contains amino acid sequences with a certain percentage of identity compared to the M22-321b41.1 antibody.

[00219] Determining the percentage identity of two sequences (eg, amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used to compare two sequences is that of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264 2268, modified as Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:58735877. This algorithm is included in the NBLAST and XBLAST programs Altschul et al., 1990, J. Mol. Biol. 215:403. A BLAST nucleotide search can be performed with a set of NBLAST nucleotide program parameters, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed with XBLAST program parameters set, for example, score 50, word length=3, to obtain amino acid sequences homologous to the protein molecule described herein. To align with gaps for comparison purposes, Gapped BLAST can be used, as described in Altschul et al., 1997, Nucleic Acids Res.25: 3389 3402. Alternatively, PSI BLAST can be used to perform iterative searches that detect distant connections between molecules ( Id . ). When using the BLAST, Gapped BLAST, and PSI Blast programs, you can use the default parameters of the corresponding programs (for example, XBLAST and NBLAST) (see, for example, National Center for Biotechnology Information (NCBI) on the Internet: ncbi.nlm.nih.gov ). Another preferred, non-limiting example of a mathematical algorithm used for sequence comparison is that of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, you can use the PAM120 residue significance table, a gap length penalty of 12, and a gap length penalty of 4.

[00220] The percent identity of two sequences can be determined using techniques similar to those described above, with or without regard to gaps. When calculating percent identity, only complete matches are usually taken into account.

[00221] In some embodiments, an antibody described herein, or an antigen binding fragment thereof, comprises a VL domain that is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO:3, where the antibody immunospecifically binds to nectin-4. In certain embodiments, an antibody of the present invention or an antigen binding fragment thereof comprises a VL domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:3, where the antibody immunospecifically binds to nectin-4, and where the antibody contains CDRs (eg, CDR VL 1-3) that are identical to the CDRs (eg, CDR VL 1-3) of the M22- antibody 321b41.1.

[00222] In certain embodiments, an antibody described herein, or an antigen binding fragment thereof, comprises a VL domain comprising VL framework regions that are at least 80%, at least 85%, at least 90% consistent , is at least 95% or at least 98% identical to the amino acid sequence of the framework regions of SEQ ID NO:3, where the antibody immunospecifically binds to nectin-4. In a specific embodiment, the antibody contains CDR VLs that are identical to the CDR VLs of antibody M22-321b41.1.

[00223] In certain embodiments, an antibody of the present invention or an antigen binding fragment thereof comprises a VH domain that is at least 80%, at least 85%, at least 90%, at least 95%, or is at least 98% identical to the amino acid sequence of SEQ ID NO:4, where the antibody immunospecifically binds to nectin-4. In some embodiments, an antibody of the present invention or an antigen binding fragment thereof comprises a VH domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:4, where the antibody immunospecifically binds to nectin-4, and where the antibody contains CDRs (eg, VH CDR 1-3) that are identical to the CDRs (eg, VH CDR 1-3) of the M22- antibody 321b41.1.

[00224] In certain embodiments, an antibody of the present invention, or an antigen binding fragment thereof, comprises a VH domain comprising VH framework regions that are at least 80%, at least 85%, at least 90%, is at least 95% or at least 98% identical to the amino acid sequence of the framework regions of SEQ ID NO:4, where the antibody immunospecifically binds to nectin-4. In specific embodiments, such an antibody contains CDRs (eg, VH CDR 1-3) identical to the CDRs (eg, VH CDR 1-3) of the M22-321b41.1 antibody.

[00225] In certain embodiments, an antibody of the present invention or an antigen binding fragment thereof comprises (i) a VL domain that is at least 80%, at least 85%, at least 90%, at least 95 % or at least 98% identical to the amino acid sequence of SEQ ID NO:3 and (ii) a VH domain, the sequence of which is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO:4, respectively, where the antibody immunospecifically binds to nectin-4. In specific embodiments, such an antibody or antigen binding fragment thereof comprises a CDR (e.g., CDR VL 1-3 and/or CDR VH 1-3) identical to the CDR (e.g., CDR VL 1-3 and/or CDR VH 1-3) of the antibody M22-321b41.1.

[00226] In yet another aspect, the antibodies or antigen-binding fragments thereof of the present invention bind to a region, including an epitope, of human nectin-4. For example, in some embodiments, an antibody of the present invention binds to the human nectin-4 region (SEQ ID NO:1) containing amino acid residues 31-346 (SEQ ID NO:2), the human nectin-4 region (SEQ ID NO:1 ), containing amino acid residues 1-150 (SEQ ID NO:45), or the human nectin-4 region (SEQ ID NO:1), containing amino acid residues 31-150 (SEQ ID NO:46) of human nectin-4. In yet another aspect, the antibodies of the present invention bind to a specific epitope of human nectin-4. The amino acid sequence of amino acid residues 31-346 of human nectin-4 (SEQ ID NO:1) is:

AGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQ RITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLV (SEQ ID NO:2). The amino acid sequence of amino acid residues 1-150 of human nectin-4 (SEQ ID NO:1) is: MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVL VPPL (SEQ ID NO:45). The amino acid sequence of amino acid residues 31-150 of human nectin-4 (SEQ ID NO:1) is:

AGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPL (SEQ ID NO:46).

[00227] In some embodiments, the antibodies of the present invention bind to a nectin-4 epitope that is a three-dimensional surface feature of a nectin-4 polypeptide (eg, in the multimeric form of a nectin-4 polypeptide). The region of a nectin-4 polypeptide that comprises an epitope may be contiguous amino acids of the polypeptide, or the epitope may result from the joining of two or more non-contiguous regions of the polypeptide. The nectin-4 epitope may be present in (a) a multimeric form (“nectin-4 multimeric epitope”) of nectin-4, (b) a monomeric form (“nectin-4 monomeric epitope”) of nectin-4, (c) both multimeric and and a monomeric form of nectin-4, (d) a multimeric form but not a monomeric form of nectin-4, or (e) a monomeric form but not a multimeric form of nectin-4. For example, in some embodiments, the epitope is present or only available for binding in the multimeric (native) form, but is absent or not available for binding in the monomeric (denatured) form of the anti-nectin-4 antibody. In other embodiments, the nectin-4 epitope is a linear feature of a nectin-4 polypeptide (eg, in a multimeric form or a monomeric form of a nectin-4 polypeptide). Antibodies of the present invention can immunospecifically bind to (a) an epitope of the monomeric form of nectin-4, (b) an epitope of the multimeric form of nectin-4, (c) an epitope of the monomeric but not multimeric form of nectin-4, (d) an epitope of the multimeric but not a monomeric form of nectin-4, or (f) both a monomeric form and a multimeric form of nectin-4.

[00228] Also disclosed herein are antibodies that bind to the same or overlapping nectin-4 epitope (e.g., an epitope located in the human nectin-4 ECD), as does the M22-321b41.1 antibody described herein for example, antibodies that compete (e.g., in a dose-dependent manner) for binding to nectin-4 (e.g., human ECD nectin-4) with antibody M22-321b41.1 described herein, or that competitively inhibit (e.g., in a dose-dependent manner) in a dose-dependent manner) the antibody M22-321b41.1 described herein from binding to nectin-4 (eg, an epitope located on the ECD of human nectin-4).

[00229] In a specific aspect, antibodies that bind to the same or overlapping nectin-4 epitope (eg, an epitope located in the human nectin-4 ECD) as antibody M22-321b41.1 are also disclosed herein.

[00230] Antibodies that bind to the same or overlapping epitopes of nectin-4 (for example, an epitope located in the ECD of human nectin-4) can be identified using routine techniques, such as those used in the examples presented herein . An immunoassay, for example, is used to demonstrate the ability of one antibody to block the binding of another antibody to a target antigen, i.e. Competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen, such as nectin-4. Numerous types of competitive binding assays are known, for example: direct or indirect radioimmunoassay (RIA), direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., (1983) Methods in Enzymology 9:242) ; solid phase direct biotin-avidin EIA (see Kirkland et al., (1986) J. Immunol.137: 3614); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, (1988) Antibody: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA assay using the I-125 tag (see Morel et al., (1988) Mol. Immunol.25 (1): 7); biotin-avidin solid-phase direct EIA (Cheung et al., (1990) Virology 176: 546); and direct labeled RIA assay (Moldenhauer et al., (1990) Scand J. Immunol.32:77). Typically, such an assay involves the use of purified antigen (eg, nectin-4, such as human ECD nectin-4) bound to a solid surface or cells bearing either of an unlabeled test immunoglobulin and a labeled control immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to a solid surface, or to cells, in the presence of a test antibody. Usually the immunoglobulin being tested is present in excess. Typically, when the competing antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. The competitive binding assay can be configured in a variety of different formats using either a labeled antigen or a labeled antibody. In the conventional version of this assay, the antigen is immobilized onto a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is then measured using radioactive or enzyme labels. A more detailed description is given, for example, Wagener et al., J. Immunol., 1983, 130:2308-2315; Wagener et al., J. Immunol. Methods 1984, 68:269-274; Kuroki et al., Cancer Res., 1990, 50:4872-4879; Kuroki et al., Immunol. Invest., 1992, 21:523-538; Kuroki et al., Hybridoma, 1992, 11:391-407, and Using Antibodies: A Laboratory Manual : Ed Harlow and David Lane (Clod Springs Harbor Laboratory Press, Clod Springs Harbor, NY, 1999), pp.386-389 .

[00231] In some aspects, competitive binding assays can be used to determine whether the binding of an antibody is competitively inhibited, for example, in a dose-dependent manner, by another antibody, thereby indicating that the antibodies bind to substantially the same epitope or overlapping epitopes , for example, sterically overlapping epitopes. Such competitive binding assays may include, for example, competitive ELISA assays, which can be configured in all different formats using either a labeled antigen or a labeled antibody. In a specific embodiment, the antibody may be tested in competitive binding assays with the M22-321b41.1 antibody described herein, or a chimeric variant thereof or a Fab antibody, or an antibody comprising the CDR VH and CDR VL of the M22-321b41.1 antibody .

[00232] In specific aspects, disclosed herein is an antibody that competitively blocks (e.g., in a dose-dependent manner) the binding of antibodies containing amino acid sequences described herein to specifically bind to a nectin-4 polypeptide (e.g., nectin-4 ECD , eg, human nectin-4) as determined by assays known to one of ordinary skill in the art or described herein (eg, competitive ELISA assays). In specific aspects, disclosed herein is an antibody that competes (e.g., in a dose-dependent manner) for specific binding to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4) with an antibody comprising the amino acid sequences described herein (eg, the VL and/or VH amino acid sequences of antibody M22-321b41.1), as determined using assays known to one of ordinary skill in the art or described herein (eg, competitive ELISA assays).

[00233] In specific aspects, the present invention provides an antibody that competes (e.g., in a dose-dependent manner) for specific binding to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4), antibody M22- 321b41.1.

[00234] In specific aspects, the present invention provides an antibody that competes (e.g., in a dose-dependent manner) for specific binding to a nectin-4 polypeptide (e.g., nectin-4 ECD, e.g., human nectin-4), with an antibody comprising VL having the amino acid sequence SEQ ID NO:3, and VH having the amino acid sequence SEQ ID NO:4.

[00235] In a particular aspect, antibodies (i) are also disclosed herein that compete (e.g., in a dose-dependent manner) with any M22-321b41.1 antibody for specific binding to an epitope of a nectin-4 polypeptide (e.g., an ECD epitope of nectin-4 4 humans), and (ii) which are able to specifically bind to nectin-4 in an IHC assay.

[00236] In a specific embodiment, an antibody of the present invention is an antibody that is competitively blocked (e.g., in a dose-dependent manner) by an antibody that specifically binds to nectin-4 and contains a VL with the amino acid sequence of SEQ ID NO:3 and a VH with the amino acid sequence of SEQ ID NO: 4, for specific binding to a nectin-4 polypeptide (eg, nectin-4 ECD, eg, human nectin-4).

[00237] In a specific aspect, antibodies (i) are also disclosed herein that are competitively blocked (e.g., in a dose-dependent manner) by antibody M22-321b41.1 from specifically binding to an epitope of a nectin-4 polypeptide (e.g., an ECD epitope of nectin-4 4 humans) and (ii), which are able to specifically bind to nectin-4 in the IHC assay.

[00238] In specific aspects, disclosed herein is an antibody or antigen binding fragment thereof that immunospecifically binds to the same epitope as antibody M22-321b41.1 containing the amino acid sequences described herein with respect to specific binding to a nectin polypeptide. 4 (eg ECD of nectin-4, eg human nectin-4). Assays known to one of ordinary skill in the art or described herein (eg, ELISA assays) can be used to determine whether two antibodies bind to the same epitope or not.

[00239] In a specific embodiment, the antibody described herein, or an antigen binding fragment thereof, immunospecifically binds to the same epitope as the antibody of antibody M22-321b41.1, which specifically binds nectin-4 and contains a VL with the amino acid sequence of SEQ ID NO:3, and VH with the amino acid sequence of SEQ ID NO:4.

[00240] In a specific aspect, antibodies (i) that immunospecifically bind to the same nectin-4 polypeptide epitope (eg, human nectin-4 ECD epitope) as antibody M22-321b41.0.1 are disclosed herein; and (ii) which are capable of specifically binding to nectin-4 in an IHC assay.

[00241] In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 31-346 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 31-75 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 75-125 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 100-150 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 125-175 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 150-200 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 175-225 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 200-250 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 225-275 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 250-300 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 275-325 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 300-346 in the amino acid sequence of SEQ ID NO:1.

[00242] In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 1-150 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 31-150 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 1-10 in the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 5-15 in the amino acid sequence of SEQ ID NO:1. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 10-20 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4 binds to at least one of residues 15-25 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 20-30 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 25-35 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen-binding fragment thereof, when binding to nectin-4 binds to at least one of residues 30-40 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment thereof, when binding to nectin-4, binds to at least one of residues 35- 45 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 40-50 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or an antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 45-55 in the amino acid sequence of SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 50-60 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 65-75 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 70-80 in the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4 binds to at least one of residues 75-85 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 80-90 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 85-95 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen-binding fragment thereof, when binding to nectin-4 binds to at least one of residues 90-100 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment thereof, when binding to nectin-4, binds to at least one of residues 95- 105 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 100-110 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or an antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 105-115 in the amino acid sequence of SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 110-120 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 115-125 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 120-130 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4 binds to at least one of residues 125-135 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen binding fragment thereof, when bound to nectin-4, binds to at least one of residues 130-140 in the amino acid sequence SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to nectin-4, binds to at least one of residues 135-145 in the amino acid sequence SEQ ID NO: 1. In other embodiments, the antibody or antigen-binding fragment thereof, when binding to nectin-4 binds to at least one of residues 140-150 in the amino acid sequence SEQ ID NO: 1.

[00243] Any combination of two, three, four, five, six, seven, eight, nine, ten or more of the above amino acid nectin-4 binding sites is also contemplated.

[00244] In certain embodiments, the antibodies specifically bind to an ECD epitope of human nectin-4 that is different from the nectin-4 ligand binding site. In some embodiments, nectin-4 ligand binding is not inhibited by the antibody.

[00245] In one aspect, provided herein are antibodies that specifically bind to nectin-4. In certain embodiments, provided herein are antibodies that specifically bind to human nectin-4 ECD.

[00246] In some embodiments, antibodies that specifically bind to nectin-4 bind to the human nectin-4 ECD or an epitope of the human nectin-4 ECD. In some embodiments, the antibodies specifically bind to an ECD epitope of human nectin-4 that is different from the nectin-4 ligand binding site.

[00247] Nectin-4 activity may refer to any nectin-4 activity, such as an activity known or described in the art. Non-limiting examples of nectin-4 activity include: dimerization of the nectin-4 receptor, heterodimerization of the nectin-4 receptor with other receptors, phosphorylation of the nectin-4 receptor, signaling in the nectin-4 receptor signaling pathway, cell proliferation such as ligand-nectin-4- induced enhancement of cell proliferation (eg, malignant cell proliferation) or cell survival (eg, malignant cells), nectin-4 ligand-induced anti-apoptosis. Nectin-4 activity or nectin-4 function are used interchangeably herein. In certain aspects, nectin-4 activity is induced by binding of a nectin-4 ligand to a nectin-4 receptor. In some embodiments, an increase in nectin-4 activity or signaling may occur in the absence of the nectin-4 ligand-binding nectin-4 receptor due to high (or overexpression) expression of nectin-4 receptors. High or overexpression of nectin-4 in a cell refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% greater than the expression level of a reference cell known to have normal nectin-4 expression or nectin-4 activity, or greater than the average level nectin-4 expression in a population of cells or samples known to have normal nectin-4 expression or nectin-4 activity. Nectin-4 expression levels can be assessed by methods described herein or known to one skilled in the art (eg, Western blotting, ELISA or IHC).

10. Polyclonal antibodies

[00248] The antibodies of the present invention may be polyclonal antibodies. Methods for producing polyclonal antibodies are known to one skilled in the art. Polyclonal antibodies can be obtained from a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant is administered to the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a nectin-4 polypeptide or a fusion protein thereof. It may be advantageous to conjugate the immunizing agent to a protein that is known to be immunogenic in the mammal being immunized, or to immunize the mammal with the protein and one or more adjuvants. Examples of such immunogenic proteins include, but are not limited to, snail limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Ribi, CpG, Poly 1C, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation. Blood can then be drawn from the mammal and the serum analyzed for antibody titer against nectin-4. If desired, the mammal can be stimulated until the antibody titer increases or reaches a plateau. Additionally or alternatively, lymphocytes can be obtained from the immunized animal for fusion and production of monoclonal antibodies from the hybridoma, as described below.

11. Monoclonal antibodies

[00249] Antibodies of the present invention may alternatively be monoclonal antibodies. Monoclonal antibodies can be produced using hybridoma technology, first described by Kohler et al., 1975, Nature 256:495-97, or can be produced by recombinant DNA methods (see, for example, US Pat. No. 4,816,567).

[00250] In hybridoma technology, a mouse or other suitable host animal, such as a hamster, is immunized as described above to induce the production of lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Following immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent such as polyethylene glycol to form hydridoma cells (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[00251] The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which in certain embodiments contains one or more substances that inhibit the growth or survival of parental myeloma cells that have not been fused (also referred to as fusion partners). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then selective culture medium for the hybridoma typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[00252] Typical myeloma cells that are fusion partners are cells that fuse efficiently, support stable, highly efficient antibody production by selected antibody-producing cells, and are sensitive to a selective environment that is selective for non-fused parent cells. Typical myeloma cell lines are mouse myeloma lines such as SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and cells derived from MOPC-21 and MPC mouse tumors -11, available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol.133:3001-05; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987) ).

[00253] The culture medium in which hybridoma cells grow is analyzed for the production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using immunoprecipitation or an in vitro binding assay such as RIA or ELISA. The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described in Munson et al., 1980, Anal. Biochem.107:220-39.

[00254] Once hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity have been identified, clones can be subcloned by limited dilution procedures and grown using standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal, for example, by intraperitoneal injection of cells into mice.

[00255] Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional antibody purification procedures, such as, for example, affinity chromatography (for example, using protein A or protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis and the like.

[00256] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes capable of specifically binding to genes encoding mouse antibody heavy and light chains). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, with ensuring the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., 1993, Curr. Opinion in Immunol.5:256-62 and Plückthun, 1992, Immunol. Revs.130: 151-88.

[00257] In some embodiments, an antibody that binds to a nectin-4 epitope comprises a VH domain amino acid sequence and/or a VL domain amino acid sequence encoded by a nucleotide sequence that hybridizes to (1) a complementary nucleotide sequence encoding either of the VH domains and /or VL described herein under stringent conditions (e.g., hybridization with filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2x SSC /0.1% SDS at about 50-65°C), under very stringent conditions (e.g. hybridization with filter-bound nucleic acid in 6x SSC at about 45°C followed by one or more washes in 0.1x SSC /0.2% SDS at about 68°C) or under other stringent hybridization conditions known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Vol. I, 6.3.1-6.3.6 and 2.10.3 (Ausubel et al. eds., 1989).

[00258] In some embodiments, an antibody that binds to a nectin-4 epitope comprises a CDR VH amino acid sequence or a CDR VL amino acid sequence encoded by a nucleotide sequence that hybridizes to a complementary nucleotide sequence encoding any CDR VH and/or CDR VL, as listed in Table 1, under stringent conditions (e.g., hybridization with filter-bound DNA in 6x SSC at about 45°C followed by one or more washes in 0.2x SSC/0.1% SDS at about 50-65 °C), under very stringent conditions (e.g. hybridization with filter-bound nucleic acid in 6x SSC at approximately 45°C followed by one or more washes in 0.1x SSC/0.2% SDS at approximately 68°C C) or under other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel et al., supra).

[00259] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries prepared using techniques described, for example, in Antibody Phage Display: Methods and Protocols (O'Brien and Aitken eds., 2002). In phage display methods, the functional domains of an antibody are “displayed” on the surface of phage particles that carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to produce the antibodies described herein include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J Immunol 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT application PCT/GB91/01134; International Publication WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO97/13844; and US patents 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108.

[00260] In principle, synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of the antibody variable region (Fv) fused to the phage coat protein. Such phage libraries are screened for the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and are thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and optionally further saturated with additional antigen adsorption/elution cycles.

[00261] Variable domains can be functionally "displayed" on phage either as single-chain Fv fragments (scFv), in which VH and VL are covalently linked through a short flexible peptide, or as Fab fragments, in which each is fused to a constant domain and non-covalently linked, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol.12:433-55.

[00262] The VH and VL gene repertoires can be cloned independently by PCR and randomly recombined in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., supra. Libraries from immunized sources produce high-affinity antibodies against an immunogen without the need to create hybridomas. Alternatively, a repertoire of naïve sequences can be cloned to produce a single source of human antibodies against a wide range of foreign antigens and self-antigens without immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naïve sequence libraries can also be generated synthetically by cloning segments of unranked stem cell V genes and using PCR primers containing random sequences to encode highly variable CDR3 regions and perform rearrangements in vitro, as described, for example, by Hoogenboom and Winter, J. Mol. Biol 227:381–88.

[00263] Screening of libraries can be accomplished by various techniques known in the art. For example, nectin-4 (e.g., nectin-4 polypeptide, fragment, or epitope) can be used to coat adsorption plate wells, expressed on host cells fixed to adsorption plates, or used in cell sorting, conjugated to biotin for bead capture, coated with streptavidin, or used in any other method for panning-based display libraries. Selection of antibodies with slow dissociation kinetics (eg, strong binding affinity) can be enhanced by long wash times and monovalent phage display as described by Bass et al., 1990, Proteins 8: 309-14 and WO 92/09690, and using low coverage antigen as described in Marks et al., 1992, Biotechnol.10:779-83.

[00264] Anti-nectin-4 antibodies can be generated by establishing a suitable antigenic screening procedure to select the phage clone of interest, followed by constructing a full-length anti-nectin-4 antibody clone using VH and/or VL sequences (e.g., Fv sequences) or various CDR sequences VH and VL sequences from the phage clone of interest and suitable constant region sequences (eg Fc) described by Kabat et al., supra.

[00265] The antibodies described herein may also, for example, include chimeric antibodies. A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and US Patent Nos. 5807715, 4816567, 4816397 and 6331415.

[00266] Antibodies or antigen binding fragments obtained using the techniques described herein can be isolated by standard, well known methods. For example, antibodies or antigen-binding fragments can be suitably separated, for example, from culture medium, ascites fluid, serum, cell lysate, synthesis reaction material, or the like, by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography , gel electrophoresis, dialysis or affinity chromatography. As used herein, an “isolated” or “purified” antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals from chemical synthesis.

12. Antibody fragments

[00267] The present disclosure discloses antibodies and antibody fragments that bind to nectin-4. Under certain circumstances, it is preferable to use antibody fragments rather than full-length antibodies. Smaller fragment sizes result in faster clearance and may provide improved access to cells, tissues or organs. A review of some antibody fragments is given in Hudson et al., 2003, Nature Med.9:129-34.

[00268] Various techniques have been developed to obtain antibody fragments. Typically, fragments were obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83) . However, fragments can now be produced directly using recombinant host cells. All Fab, Fv and scFv antibody fragments can be expressed and secreted from E. coli or yeast cells, making it easy to obtain large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries described above. Alternatively, Fab'-SH fragments can be directly isolated from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al., 1992, Bio/Technology 10:163-67). According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell cultures. A Fab and F(ab') 2 fragment with an increased in vivo half-life containing salvage receptor binding epitope residues is described, for example, in US Pat. No. 5,869,046. Other techniques for preparing antibody fragments will be apparent to one skilled in the art. In some embodiments, the antibody is a single chain Fv fragment (scFv) (see, for example, WO 93/16185; US Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact binding sites lacking constant regions; thus, they can be used to reduce nonspecific binding when used in vivo. ScFv fusion proteins can be designed to fusion an effector protein at the amino or carboxy terminus of the scFv (see, eg, Borrebaeck ed., supra). The antibody fragment may also be a "linear antibody", for example, as described in the links above.

[00269] Small structures derived from antibodies are individual variable domains (V domains), also called single domain antibodies (sdAbs). In some organisms, camels and cartilaginous fish, part of the immune system is represented by high-affinity V-like single-domain structures located on the equivalent Fc domain. (Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA.101:12444-49). V-like domains (called camel VhH and shark V-NAR) typically have long surface loops that allow entry into the cavities of target antigens. They also stabilize isolated VH domains by capping hydrophobic regions on the surface.

[00270] These VhH and V-NAR domains were used to create sdAbs. Human V domain variants have been constructed using selection from phage libraries and other approaches that have resulted in stable, highly efficient binding domains derived from the VL and VH domains.

[00271] Antibodies described herein include, but are not limited to, immunoglobulin molecules and active moieties of immunoglobulin molecules, for example, molecules that contain an antigen-binding site that binds to an epitope of nectin-4. The immunoglobulin molecules described herein can be any class of molecules (eg, IgG, IgE, IgM, IgD, and IgA) or any subclass of immunoglobulin molecules (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[00272] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to the nectin-4 epitope. Exemplary functional fragments include Fab fragments (eg, an antibody fragment that contains an antigen binding domain and contains a light chain and a portion of a heavy chain bridged by a disulfide bond); Fab' (eg, an antibody fragment containing a single antigen-binding domain containing a Fab and an additional heavy chain portion connected through a hinge region); F(ab')2 (eg, two Fab' molecules linked by interchain disulfide bonds at the heavy chain hinge regions; Fab' molecules may be directed to the same or different epitopes); a single chain containing a variable region, also known as a scFv (eg, the variable antigen-binding determinant region of one light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (eg, the variable antigen-binding determinant region of one light and heavy chain of an antibody linked together by a disulfide bond); camel VH (eg, the variable antigen-binding determinant region of a single heavy chain antibody, in which some of the amino acids at the VH boundary are amino acids that are found in the heavy chain of natural camel antibodies).

13. Humanized antibodies

[00273] Antibodies described herein may, for example, include humanized antibodies, such as deimmunized or compounded human antibodies.

[00274] The humanized antibody may contain human constant region sequences. In specific embodiments, the humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ . In certain embodiments, the humanized antibody may contain kappa or lambda light chain constant sequences.

[00275] Humanized antibodies can be produced using various techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO91/09967; and US Patent Nos. 5225539, 5530101 and 5585089), veining or surface modification (European patents EP 592106 and EP 519596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805- 814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (US Patent No. 5565322) and methods described, for example, in US Patent No. 6407213, US Patent No. 5766886, WO9317105, Tan et al., J. Immunol.169:111925 (2002), Caldas et al., Protein Eng.13(5):353-60 (2000), Morea et al., Methods 20(3):26779 (2000) , Baca et al., J. Biol. Chem.272(16):10678-84 (1997), Roguska et al., Protein Eng.9(10):895904 (1996), Couto et al., Cancer Res.55 (23 Supp):5973s-5977s ( 1995), Couto et al., Cancer Res.55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol.235(3):959-73 (1994). See also US Patent Publication No. US2005/0042664 A1 (Feb. 24-2005), each reference herein incorporated by reference in its entirety.

[00276] In some embodiments, the antibodies of the present invention may be humanized antibodies that bind to nectin-4, including human nectin-4. For example, the humanized antibodies of the present invention may contain one or more CDRs, as shown in Table 1 . Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from the "donor" variable domain. Humanization can be performed, for example, according to the method of Jones et al., 1986, Nature 321: 522-25; Riechmann et al., 1988, Nature 332: 323-27; and Verhoeyen et al., 1988, Science 239: 1534-36) by replacing the hypervariable region sequences with the corresponding human antibody sequences.

[00277] In some cases, humanized antibodies are constructed by CDR grafting, where the amino acid sequences of the six CDRs of a parent non-human (eg, rodent) antibody are grafted onto a human antibody scaffold. For example, Padlan et al. determined that only about a third of the residues in the CDR actually bind to the antigen and called them “specificity-determining residues” or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In the SDR grafting technique, only the remnants of the SDR are grafted onto a human antibody scaffold (see, eg, Kashmiri et al., 2005, Methods 36:25-34).

[00278] The selection of human variable domains, both light and heavy, for use in the production of humanized antibodies may be important to reduce antigenicity. For example, according to the so-called “best match” method, the variable domain sequence of a non-human (eg, rodent) antibody is screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence can be selected as the human backbone for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308; and Chothia et al., 1987, J. Mol. Biol. .196:901-17). Another method uses a specific scaffold derived from the consensus sequence of all human antibodies from a particular subset of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151: 2623-32). In some cases, the framework is derived from consensus sequences of the most common human subclasses, subgroup IV _L 6 (V _L 6I) and subgroup V _H III (V _H III). Another method uses human germline genes as a source of framework regions.

[00279] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is not important. The method consists of comparing non-human sequence with the functional repertoire of human germline genes. Genes encoding the same or closely related canonical structures to mouse sequences are then selected. Then, from the genes that share canonical structures with non-human antibodies, genes that have the highest homology in the CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, eg, Tan et al., 2002, J. Immunol.169:1119-25).

[00280] In addition, it is generally desirable that antibodies be humanized while maintaining their antigen affinity and other preferred biological properties. To achieve this goal, according to one method, humanized antibodies are obtained through a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and known to those skilled in the art. Computer programs are available that illustrate and show possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer ( Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Examination of these displays allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected from recipient and donor sequences and combined to produce a desired antibody characteristic, such as increased affinity for the target antigen(s). In general, hypervariable region residues directly and most significantly influence antigen binding.

[00281] Another method of humanizing antibodies is based on a system of indicators of the “humanization” of antibodies called Human String Content (HSC). This method compares the mouse sequence to the human germline gene repertoire and differences are scored as HSC. The target sequence is then humanized by maximizing its HSC value, without using a global identification system, to produce a variety of diverse humanized variants (Lazar et al., 2007, Mol. Immunol.44:1986-98).

[00282] In addition to the methods described above, empirical methods can be used to create and select humanized antibodies. These methods include methods that rely on generating large libraries of humanized variants and selecting the best clones using enrichment technologies or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal and yeast libraries, as well as by screening bacterial colonies (see, for example, Hoogenboom, 2005, Nat. Biotechnol.23:1105-16; Dufner et al., 2006, Trends Biotechnol.24 :523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21: 163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17: 847-60).

[00283] In the FR library approach, a collection of residue variants is introduced into specific positions in the FR, and the library is then screened to select FRs that best preserve the transplanted CDR. The residues to be replaced may include some or all of the "Vernier" residues identified as possibly improving CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99) or more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[00284] In FR shuffling, full-length FRs were combined with non-human CDRs rather than creating combinatorial libraries of selected residue variants (see, eg, Dall'Acqua et al., 2005, Methods 36:43-60). Libraries can be screened for binding in a two-step process by first humanizing VL and then VH. Alternatively, a one-step FR shuffling process can be used. This process has been shown to be more efficient than two-step screening, since the resulting antibodies exhibit improved biochemical and physicochemical properties, including increased expression, increased affinity and thermal stability (see, for example, Damschroder et al., 2007, Mol. Immunol .44:3049-60).

[00285] The humanization method is based on the experimental identification of major minimal specificity determinants (MSDs) and the sequential replacement of non-human fragments in a human FR library and binding assessment. It starts with the CDR3 regions of the non-human VH and VL chains and gradually replaces other regions of the non-human antibody with the human FR, including CDR1 and CDR2 of both VH and VL. These methods typically result in epitope conservation and identification of antibodies from multiple subclasses with different human V segment CDRs. “Humanization” allows the isolation of antibodies that are 91-96% homologous to human germline antibody genes (see, for example, Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[00286] A method of "engineering with human sequence data" involves modifying a non-human antibody or antibody fragment, such as a mouse, or a chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody to produce a modified antibody with reduced immunogenicity for humans, which still retains the desired binding properties of the original non-human antibodies. Typically, the technique involves classifying the amino acid residues of a non-human (eg, murine) antibody into “low risk,” “medium risk,” or “high risk” residues. Classification is accomplished using an overall risk/benefit calculation that evaluates the predicted benefit of making a particular substitution (eg, immunogenicity in humans) versus the risk that the substitution will affect the folding of the resulting antibody. The specific human amino acid residue to be replaced at a predetermined position (eg, low or moderate risk) of the non-human (eg, murine) antibody sequence can be selected by aligning the amino acid sequence of the variable regions of the non-human antibody and the corresponding region of the specific or human antibody consensus sequence. Amino acid residues at low or moderate risk positions in the non-human sequence can be replaced by the corresponding residues of the human antibody sequence according to this alignment. Techniques for engineering proteins from human sequence data are described in more detail in Studnicka et al., 1994, Protein Engineering 7: 805-14; US Patent No. 5766886; 5770196; 5821123; and 5869619; and in PCT publication WO93/11794.

[00287] A composite human antibody can be produced using, for example, composite human antibody technology (Composite Human Antibody™, Antitope Ltd., Cambridge, United Kingdom). To create compound human antibodies, variable region sequences are constructed from fragments of the variable region sequences of multiple human antibodies, avoiding T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody. Such antibodies may contain human constant region sequences, for example human light chain and/or heavy chain constant regions.

[00288] A deimmunized antibody is an antibody in which T cell epitopes have been removed. Methods for producing deimmunized antibodies are described. See, for example, Jones et al., Methods Mol Biol.2009;525:405-23, xiv, and De Groot et al., Cell. Immunol.244:148-153(2006)). Deimmunized antibodies contain variable regions that lack T cell epitopes and human constant regions. Briefly, VH and VL antibodies are cloned, and T cell epitopes are then identified by testing overlapping peptides derived from VH and VL antibodies in a T cell proliferation assay. T cell epitopes are identified through in silico methods to identify peptide binding to human MHC class II. Introduction of mutations in VH and VL abolishes binding to MHC class II. The mutated VH and VL are then used to create a deimmunized antibody.

14. Human antibodies

[00289] In certain embodiments, the antibody is a fully human antibody. Fully human antibodies can be produced by any method known in the art. Antibodies against nectin-4 can be constructed by combining Fv clone variable domain sequence(s) selected from human phage display libraries with known human constant domain sequence(s), as described above. Alternatively, the human monoclonal antibodies against nectin-4 of the present invention can be produced by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, in Kozbor, 1984, J. Immunol.133:3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol.147:86-95.

[00290] It is also possible to produce transgenic animals (eg, mice) that are capable of producing the full repertoire of human antibodies after immunization without producing endogenous immunoglobulin. To generate high-affinity monoclonal antibodies with human sequence against a wide range of possible therapeutic targets, transgenic mice that express human antibody repertoires are used (see, for example, Jakobovits A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; US Patent Nos. 6075181 and 6150584; and Lonberg et al., 2005, Nature Biotechnol. 23:1117-25).

[00291] Alternatively, a human antibody may be produced by immortalizing human B lymphocytes that produce antibody directed to a target antigen (e.g., such B lymphocytes may be isolated from an individual or may be immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and US Patent No. 5,750,373).

[00292] Gene shuffling can be used to produce human antibodies from non-human antibodies, such as rodent antibodies, where the human antibody has similar affinity and specificity to the original non-human antibody. According to this method, which is also called "epitope imprinting" or "guided selection", the heavy or light chain variable region of a non-human antibody fragment produced by phage display methods as described herein is replaced with a repertoire of human V domain genes , creating a population of non-human chain/human chain scFv or Fab chimeras. Antigen-assisted selection results in the isolation of a chimeric scFv or Fab with a non-human/human chain in which the human chain restores the antigen-binding site disrupted by removal of the corresponding non-human chain in the primary phage display clone (e.g., controlled selection ( epitope imprinting) of a non-human chain partner). If the process is repeated to replace the remaining non-human chain, a human antibody is obtained (see, for example, PCT WO 93/06213; and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique produces fully human antibodies that do not have non-human FR or CDR residues. Examples of "guided selection" to humanize mouse antibodies against cell surface antigens include folate binding protein found on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res. 58:991-96) and CD147, which is highly expressed on hepatocellular carcinoma cells (see, for example, Bao et al., 2005, Cancer Biol. Ther. 4:1374-80).

[00293] A possible disadvantage of the "guided selection" approach is that shuffling one antibody chain while maintaining another may result in epitope drift. To preserve an epitope recognized by non-human antibodies, CDR retention can be used (see, for example, Klimka et al., 2000, Br. J. Cancer.83:252-60; and Beiboer et al., 2000, J. Mol Biol 296:833–49). In this method, the non-human VH CDR3 is typically retained since this CDR may be located at the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. However, in some cases, CDR3 VH and CDR3 VL, as well as CDR2 VH, CDR2 VL and CDR1 VL non-human antibodies may be retained.

15. Multivalent antibodies

[00294] A multivalent antibody may be more quickly taken up (and/or catabolized) by a cell expressing the antigen to which the antibody binds than a divalent antibody. The antibodies of the present invention may be multivalent antibodies (other than IgM antibodies) with three or more antigen binding sites (eg, quadrivalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. A multivalent antibody may contain a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In such a scenario, the antibody will contain an Fc region and three or more antigen binding sites, amino-terminal to the Fc region. In specific embodiments, the multivalent antibody contains (or consists of) three to about eight antigen binding sites. In one such embodiment, the multivalent antibody contains (or consists of) four antigen binding sites. A multivalent antibody contains at least one polypeptide chain (eg, two polypeptide chains), where the polypeptide chain(s) contains two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1) _n -VD2-(X2) _n -Fc, where VD1 is the first variable domain, VD2 is the second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 is an amino acid or a polypeptide, and n is 0 or 1. For example, the polypeptide chain(s) may comprise: a VH-CH1 chain-flexible linker-VH-CH1-Fc region; or chain VH-CH1-VH-CH1-Fc region. The multivalent antibody of the present invention may further comprise at least two (eg, four) light chain variable domain polypeptides. A multivalent antibody of the present invention may, for example, contain from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides described herein comprise a light chain variable domain and optionally further comprise a CL domain.

16. Construction of Fc

[00295] It may be desirable to modify the anti-nectin-4 antibody of the present invention by engineering the Fc. In some embodiments, modification of the Fc region of an antibody results in decreased or eliminated effector function of the antibody. In some embodiments, the effector function is ADCC, ADCP, and/or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. The modification can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody.

[00296] To increase the half-life of an antibody in serum, a salvage receptor binding epitope can be included in the antibody (especially an antibody fragment), for example, as described in US Pat. No. 5,739,277. The term "salvage receptor binding epitope" refers to the Fc epitope -region of the IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

17. Alternative coupling agents

[00297] The present disclosure discloses non-immunoglobulin binding agents that specifically bind to the same epitope as the anti-nectin-4 antibody disclosed herein. In some embodiments, a non-immunoglobulin binding agent is defined as one that replaces or is replaced by the anti-nectin-4 antibody of the present invention in a competitive binding assay. These alternative coupling agents may include, for example, any engineered protein scaffolds known in the art. Such scaffolds may contain one or more CDRs, as shown in Table 1. Such scaffolds include, for example, anticalins, which are based on a lipocalin scaffold, a protein structure characterized by a rigid beta-sheet structure that supports four hypervariable loops that form the ligand-binding site. New binding specificity properties can be engineered using targeted random mutagenesis in loop regions combined with functional mapping and controlled selection (see, for example, Skerra, 2008, FEBS J.275:2677-83). Other suitable scaffolds may include, for example, adnectins or monobodies based on the tenth extracellular domain of human fibronectin III (see, for example, Koide and Koide, 2007, Methods Mol. Biol. 352: 95-109); affibodies based on the Z domain of staphylococcal protein A (see, for example, Nygren et al., 2008, FEBS J. 275:2668-76); DARPins based on ankyrin repeat proteins (see, for example, Stumpp et al., 2008, Drug. Discov. Today 13: 695-701); finomers based on the SH3 domain of the human protein kinase Fyn (see, for example, Grabulovski et al., 2007, J. Biol. Chem. 282: 3196-204); affins based on Sac7d from Sulfolobus acidolarius (see, for example, Kprebrink et al., 2008, J. Mol. Biol. 383: 1058-68); γ-B-crystallin-based affins (see, for example, Ebersbach et al., 2007, J. Mol. Biol. 372: 172-85); avimers based on the A-domain of membrane receptor proteins (see, for example, Silverman et al., 2005, Biotechnol.23: 1556-61); cysteine-rich knottin peptides (see, for example, Kolmar, 2008, FEBS J.275: 2684-90); and engineered Kunitz-type inhibitors (see, eg, Nixon and Wood, 2006, Curr. Opin. Drug. Discov. Dev. 9:261-68). For a review, see, for example, Gebauer and Skerra, 2009, Curr. Opin. Chem. Biol.13:245-55.

18. Screening for nectin-4-specific antibodies

[00298] Methods for producing antibodies have been described above. Antibodies can be further screened or selected for certain biological characteristics, such as nectin-4 binding specificity, if desired. Screening for antibodies specific to a target antigen involves two aspects. First, antibody screening to identify antibodies that do not bind or have low binding affinity to a sample that does not contain nectin-4. This aspect of the screen identifies antibodies that specifically recognize and bind to nectin-4, but do not substantially recognize or bind to molecules other than nectin-4. Second, screening antibodies to identify those that bind strongly or with high affinity to a sample that contains nectin-4. This aspect of screening identifies antibodies that have high affinity for nectin-4 and can give strong detection signals in various assays. as described below. Screening for antibodies in both aspects can be accomplished by immunoassays further described below.

[00299] In certain embodiments, antibodies are first screened using an ELISA in which microtiter plates are coated with nectin-4 protein or a fragment thereof. Antibody clones that do not bind or bind with weak affinity to immobilized nectin-4 are removed. In some embodiments, antibodies that bind to nectin-4 can be further screened for reactivity in an immunoassay, such as an ELISA-based assay or Western blot assay, against other nectin isoforms, for example, using nectin-1-coated microtiter plates , nectin-2 or nectin-3. Antibody clones that interact with another nectin isoform are removed, and antibody clones that only interact with nectin-4 can be selected for further screening.

[00300] Antibodies against nectin-4 can be further screened for their binding to negative controls that do not express or express nectin-4 at low levels. For such screening, the binding of various antibody clones and negative controls can be determined using an immunoassay, such as an IHC assay, an ELISA assay, or an immunoblotting assay. Antibody clones that interact with any negative controls are removed. Different negative controls (e.g., different tissues or cell lines negative for nectin-4 expression) may contain a different set of molecules, so multiple negative controls can be used to ensure that putative anti-nectin-4 antibodies are selected against a diverse set of molecules that are not nectin-4.

[00301] Antibodies against nectin-4 can be further screened for binding to positive controls known to express nectin-4. For such screening, the binding of various antibody clones and positive controls can be determined using an immunoassay, such as an IHC assay, an ELISA assay, or an immunoblotting assay. Antibody clones are selected that bind strongly to positive controls. An anti-nectin-4 antibody that binds strongly to the positive control and weakly to the negative control can be selected as a nectin-4-specific antibody. The difference between the amount of anti-nectin-4 antibody bound to the positive and negative controls lies in the specific binding of this anti-nectin-4 antibody to nectin-4. Specific binding can be used as one of the criteria for selecting specific antibodies against nectin-4. The specific antibody provides highly specific binding in the immunoassay, as described in detail below.

[00302] In some embodiments, the specificity of an anti-nectin-4 antibody can be determined by correlating or comparing the anti-nectin-4 antibody binding to negative and positive standards having known levels of nectin-4 expression in those standards as determined by other means. The nectin-4 reference expression can be a positive control of nectin-4 or a negative control of nectin-4, as described below. Binding of a specific anti-nectin-4 antibody to a sample or reference may correlate or be proportional to the expression of nectin-4 in the sample or reference. That is, a specific anti-nectin-4 antibody binds highly to a sample or reference that expresses nectin-4 at a high level, binds moderately if the expression level is intermediate, binds weakly if the expression level is low, and minimally binds (e.g., binds at approximately the same level at which an isotype control antibody that does not express nectin-4 would bind. Known nectin-4 expression in the negative or positive control can be determined independently using a qPCR assay as detailed below. Known nectin-4 expression can also be independently determined using any of the immunoassays described below with an anti-nectin-4 antibody that has previously been determined to be specific for nectin-4. Suitable immunoassays to confirm the expression of nectin-4 in negative and positive controls include, by way of example and without limitation, an IHC assay, an immunoblot assay, a FACS assay, or an ELISA assay, as described in detail below. In some embodiments, the nectin-4 mRNA level may differ from being zero in the negative control due to background noise of the assay or due to biologically insignificant low levels of retained nectin-4, as described below. A negative control for nectin-4 can be considered negative for nectin-4, for example, if the level of nectin-4 mRNA in the qPCR assay is substantially similar to the level of mRNA detected using a set of nonspecific primers, or if the level of nectin-4 mRNA is biologically insignificant due to the level Nectin-4 mRNA in nectin-4 positive control. Likewise, nectin-4 detected by immunoassays using another nectin-4-specific antibody in the negative control may not be null due to background noise of the assay or due to biologically insignificant low levels of retained nectin-4. Background noise may be caused by nonspecific binding of non-nectin-4 assay reagents to samples. A nectin-4 negative control can be considered negative for nectin-4, for example, if the level of nectin-4 in the negative control detected with a specific anti-nectin-4 antibody is substantially similar to the level detected with an isotype control antibody in the same analysis. In some assays, background noise may account for a significant proportion of the detected signal in the positive control.

[00303] In addition, positive and negative controls have been defined and published by those skilled in the art, including positive and/or negative tissues, cells (including, for example, cell lines), and pathological samples. Such literature can be easily found by searching databases such as Pubmed (eg, using a search term such as nectin-4, expression, positive, negative, and/or distribution) and analyzing the search results.

[00304] Accordingly, the relative amount of nectin-4-specific antibody or antigen-binding fragment thereof bound to a negative control cell not expressing nectin-4 may be about 50%, 40%, 30%, 20%, 10%. 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.03%, 0.01%, 0.001% or less compared to the amount of antibody or antigen binding fragment bound to a positive control cell expressing nectin-4.

[00305] In some embodiments of an antibody or antigen binding fragment thereof of the present invention, negative or positive expression of nectin-4 in control cells is independently determined using a qPCR assay, an IHC assay with a second antibody, an immunoblotting assay with a second antibody, a FACS assay with a second antibody or ELISA with a second antibody.

[00306] In some embodiments of an antibody or antigen binding fragment thereof of the present invention, the amount of antibody or antigen binding fragment thereof bound to cells is determined by IHC analysis, immunoblotting analysis, FACS analysis, or ELISA. 19. Antibody variants

[00307] In some embodiments, amino acid sequence modification(s) of antibodies that bind to nectin-4 or that are described herein are contemplated. For example, it may be desirable to improve binding affinity and/or other biological properties of an antibody, including, but not limited to, specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the anti-nectin-4 antibodies described herein, it is contemplated that variants of the anti-nectin-4 antibodies can be produced. For example, anti-nectin-4 antibody variants can be produced by introducing appropriate nucleotide changes into the coding DNA and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid substitutions can alter the post-translational processes of an anti-nectin-4 antibody, such as changing the number or position of glycosylation sites or changing membrane anchoring characteristics.

[00308] In some embodiments, the glutamine at the first residue may be replaced by pyroglutamate in any of the antibodies of the present invention, which also includes a polypeptide having a first glutamine in its sequence. Such substitution occurs naturally under physiological conditions, or in vitro or ex vivo in a buffer with a pH of about 7. Such substitution can be accomplished by placing an antibody that contains a first glutamine-containing polypeptide under conditions favorable to cyclization of the first glutaminyl glutamine with by forming pyroglutamate, for example, into a weak acid solution such as acetate and trifluoroacetate.

[00309] In some embodiments, the antibodies of the present invention are chemically modified, for example, by covalently linking any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, modified with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and the like. Any of a variety of chemical modifications can be accomplished by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more non-classical amino acids.

[00310] Variants may be a substitution, deletion, or insertion of one or more codons encoding an antibody or polypeptide, resulting in a change in amino acid sequence compared to the native sequence antibody or polypeptide. Amino acid substitutions may result from the replacement of one amino acid with another amino acid with similar structural and/or chemical properties, such as the replacement of leucine with serine, for example conservative amino acid substitutions. To introduce mutations into the nucleotide sequence encoding a molecule of the present invention, any standard techniques known to those skilled in the art may be used, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, resulting in amino acid substitutions. The insertions or deletions may optionally range from 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions. compared to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted nonessential amino acid residues. The tolerance can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for activity exhibited by the full-length sequence or the mature native sequence.

[00311] Amino acid sequence insertions include N- and/or C-terminal fusions ranging in length from one residue to polypeptides containing one hundred residues or more, as well as insertions of one or more amino acid residues within a sequence. Examples of terminal inserts include an antibody with an N-terminal methionine residue. Other options for inserting an antibody molecule include fusion at the N- or C-terminus of the antibody with an enzyme (eg, antibody-targeted prodrug enzyme therapy) or polypeptide, thereby increasing the serum half-life of the antibody.

[00312] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a side chain having a similar charge. In the prior art, families of amino acid residues having side chains with similar charges have been identified. These families include amino acid residues with basic side chains (e.g., lysine, arginine, histidine), with acidic side chains (e.g., aspartic acid, glutamic acid), with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine , tyrosine, cysteine), with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), with beta-branched side chains (e.g. threonine, valine, isoleucine) and with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly throughout all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[00313] Significant modifications to the biological properties of an antibody are made by selecting substitutions that vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of the substitution, such as folding or helical conformation, and (b) the charge or hydrophobicity of the molecule at the target site or (c) side chain size. Alternatively, conservative (eg, within an amino acid group with similar properties and/or side chains) substitutions may be made to maintain properties or to prevent significant changes therein. Amino acids can be grouped by similarity in the properties of their side chains (see, for example, Lehninger, Biochemistry 73-75 (2d ed.1975)): (1) non-polar: Ala (A), Val (V), Leu (L) , Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[00314] Alternatively, natural moieties can be divided into groups based on general side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[00315] Non-conservative substitutions result in replacing a member of one of these classes with a member of another class. Such substituted residues may also be introduced into conserved substitution sites or into remaining (non-conserved) sites. Accordingly, in one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 35%, at least 40%, at least 45% by 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 35% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 40% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 45% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 50% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 55% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 60% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 65% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 70% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 75% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 80% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 85% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 90% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 95% identical to the amino acid sequence of a mouse monoclonal antibody as described herein. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 99% identical to the amino acid sequence of a mouse monoclonal antibody as described herein.

[00316] In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence shown in tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 35% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 40% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 45% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 50% identical to the amino acid sequence as shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 55% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 60% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 65% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 70% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 80% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 85% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 95% identical to the amino acid sequence shown in Tables 1-2. In one embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains an amino acid sequence that is at least 99% identical to the amino acid sequence shown in Tables 1-2.

[00317] In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 35%, at least 40%, at least by 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 35% identical to the amino acid sequence of a CDR VH shown in Table 1 , and/or the VL CDR amino acid sequence shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to a nectin-4 epitope contains a VH CDR and/or VL CDR amino acid sequence that is at least 40% is identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains the amino acid sequence of the CDR VH and/or A CDR VL that is at least 45% identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin- 4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 50% identical to the CDR VH amino acid sequence shown in Table 1 and/or the CDR VL amino acid sequence shown in Table 1. In yet another embodiment, the antibody or a fragment thereof that binds to an epitope of nectin-4 contains a VH CDR and/or VL CDR amino acid sequence that is at least 55% identical to the VH CDR amino acid sequence given in Table 1 and/or the VL CDR amino acid sequence given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a VH CDR and/or VL CDR amino acid sequence that is at least 60% identical to the VH CDR amino acid sequence shown in Table 1 , and/or the CDR VL amino acid sequence shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 65% is identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains the amino acid sequence of the CDR VH and/or A CDR VL that is at least 70% identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin- 4, contains a VH CDR and/or VL CDR amino acid sequence that is at least 75% identical to the VH CDR amino acid sequence shown in Table 1 and/or the VL CDR amino acid sequence shown in Table 1. In yet another embodiment, the antibody or a fragment thereof that binds to an epitope of nectin-4 contains a VH CDR and/or VL CDR amino acid sequence that is at least 80% identical to the VH CDR amino acid sequence given in Table 1 and/or the VL CDR amino acid sequence given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 85% identical to the amino acid sequence of a CDR VH shown in Table 1 , and/or the CDR VL amino acid sequence shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 90% is identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL given in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin-4 contains the amino acid sequence of the CDR VH and/or A CDR VL that is at least 95% identical to the amino acid sequence of the CDR VH shown in Table 1 and/or the amino acid sequence of the CDR VL shown in Table 1. In yet another embodiment, an antibody or fragment thereof that binds to an epitope of nectin- 4 contains a CDR VH and/or CDR VL amino acid sequence that is at least 99% identical to the VH CDR amino acid sequence shown in Table 1 and/or the VL CDR amino acid sequence shown in Table 1.

[00318] Variations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis (see, for example, Carter, 1986, Biochem J.237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10: 6487-500), cassette mutagenesis (see, for example, , Wells et al., 1985, Gene 34: 315-23) or other known techniques can be applied to the cloned DNA to produce a DNA variant of the anti-nectin-4 antibody.

[00319] Any cysteine residue not involved in maintaining the correct conformation of the anti-nectin-4 antibody can also be replaced, for example, with another amino acid such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant cross-linking. Conversely, cysteine linkage(s) can be added to an anti-nectin-4 antibody to improve its stability (eg, if the antibody is an antibody fragment, such as an Fc fragment).

[00320] In some embodiments, the anti-nectin-4 antibody molecule of the present invention is a “de-immunized” antibody. A “deimmunized” anti-nectin-4 antibody is an antibody derived from a humanized or chimeric anti-nectin-4 antibody that has one or more changes in its amino acid sequence that results in reduced immunogenicity of the antibody compared to the corresponding parent non-immunized antibody. One procedure for generating such antibody mutants involves identifying and removing T cell epitopes of the antibody molecule. In the first step, the immunogenicity of an antibody molecule can be determined by several methods, for example, by determining T cell epitopes in vitro or predicting such epitopes in vitro, as is known in the art. Once critical residues for T cell epitope function have been identified, mutations can be made to remove immunogenicity and preserve antibody activity. A review is given, for example, in Jones et al., 2009, Methods in Molecular Biology 525: 405-23.

20. In vitro affinity maturation

[00321] In some embodiments, antibody variants having improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be produced by in vitro affinity maturation. Like its natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Antibody libraries are displayed as Fab-, scFv-fragments or V-domain fragments either on the surface of an organism (eg, phage, bacterium, yeast cell or mammalian cell) or as binding (eg, covalent or non-covalent) to their encoding mRNA or DNA. Affinity selection of display antibodies allows the isolation of organisms or complexes that carry genetic information encoding antibodies. Two or three rounds of mutation and selection using display-based methods such as phage display typically result in antibody fragments with affinities in the low nanomolar range. Affinity mature antibodies may have nanomolar or even picomolar affinity for the target antigen.

[00322] Phage display is a widely used antibody display and selection method. Antibodies are located on the surface of Fd or M13 bacteriophages as fused constructs with the bacteriophage coat protein. Selection involves exposing an antigen to allowing antibodies located on the phage to bind to their targets, a process called “panning.” The phage associated with the antigen is recovered and used to infect bacteria and produce phages for further selection cycles. For a review, see, for example, Hoogenboom, 2002, Methods. Mol. Biol.178: 1-37; and Bradbury and Marks, 2004, J. Immunol. Methods 290: 29-49.

[00323] In a yeast display system (see, for example, Boder et al., 1997, Nat. Biotech. 15: 553-57; and Chao et al., 2006, Nat. Protocols 1: 755-68), the antibody may located in the form of single-chain variable fusion constructs (scFv), in which the heavy and light chains are linked by a flexible linker. ScFv is fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall via disulfide bonds with Aga1p. Protein localization by Aga2p projects the protein away from the cell surface, minimizing possible interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with a biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Changes in antibody surface expression can be measured by immunofluorescent labeling of either hemagglutinin or the c-Myc epitope tag flanking the scFv. Expression has been shown to correlate with the stability of the surface-presented protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol. 292: 949-56). An additional advantage of yeast display is that the displayed proteins fold in the endoplasmic reticulum of eukaryotic yeast cells, taking advantage of the endoplasmic reticulum chaperones and quality control mechanism. Once maturation is complete, the antibody's affinity can be conveniently "titrated" while the antibody is on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast display is the potentially smaller working library size compared to other display methods; however, a recent approach used the yeast cell mating system to generate combinatorial diversity, the size of which was estimated to be 10 ¹⁴ (see, for example, US Patent Publication 2003/0186374; and Blaise et al., 2004, Gene 342: 211-18).

[00324] In ribosomal display, antibody-ribosome-mRNA (ARM) complexes are created for selection in a cell-free system. A DNA library encoding a particular antibody library is genetically hybridized to a spacer sequence that lacks a stop codon. This spacer sequence, if translated, remains attached to the peptidyl tRNA and occupies the ribosomal tunnel, thereby allowing the protein of interest to protrude from the ribosome and fold. The resulting complex of mRNA, ribosome and protein can bind to a surface-bound ligand, allowing simultaneous release of the antibody and its encoding mRNA through affinity capture with the ligand. The ribosome-bound mRNA is then transcribed back into cDNA, which can then be mutagenized and used in the next round of selection (see, for example, Fukuda et al., 2006, Nucleic Acids Res. 34:e127). In mRNA display, a covalent bond is established between antibody and mRNA using puromycin as an adapter molecule (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).

[00325] Because these methods are carried out entirely in vitro, they provide two major advantages over other breeding technologies. First, the diversity of the library is not limited by the efficiency of transformation of bacterial cells, but only by the number of ribosomes and various mRNA molecules present in the analytical tube. Second, random mutations can be easily introduced after each round of selection, for example using non-proofreading polymerases, since no library needs to transform after any diversification step.

[00326] In a mammalian cell-based display system (see, e.g., Bowers et al., 2011, Proc Natl Acad Sci USA.108:20455-60), a fully human IgG library is constructed based on the germline sequence of V gene segments , connected to pre-recombined D(J) regions. The full-length heavy chain and light chain V regions are assembled together with the human heavy chain and light chain constant regions and transfected into a mammalian cell line (eg, HEK293). The transfected library is expanded and subjected to several rounds of negative selection against streptavidin (SA) on magnetic beads, followed by a round of positive selection against SA on magnetic beads coated with a biotinylated target protein, peptide fragment, or epitope. Cells selected by positive selection are expanded in number and then sorted using FACS rounds to isolate single cell clones containing antibodies that specifically bind to the target protein, peptide fragment or epitope. The heavy and light chain pairs of these single-cell clones are retransfected with AID for further maturation. Multiple rounds of mammalian cell-based display combined with AID-induced somatic hypermutation produce antibodies with high specificity and high affinity.

[00327] Diversity can also be introduced into CDRs or entire V genes of antibody libraries in a targeted manner or by random introduction. This approach involves sequential high- or low-level targeted mutagenesis of all antibody CDRs or targeting somatic hypermutation hot spots (see, e.g., Ho et al., 2005, J. Biol. Chem. 280: 607-17) or on residues suspected of influencing affinity, on an experimental basis or for structural reasons. Diversity can also be obtained by replacing regions that vary in nature through DNA shuffling or similar methods (see, e.g., Lu et al., 2003, J. Biol. Chem. 278:43496-507; US Pat. No. 5,565,332 and 6989250). Alternative methods target hypervariable loops extending into framework residues (see, e.g., Bond et al., 2005, J. Mol. Biol. 348:699-709), using loop deletion and CDR insertions or hybridization-based diversification (See, for example, US Patent Publication No. 2004/0005709). Additional methods for producing differences in CDR are described, for example, in US patent No. 7985840. Other methods that can be used to create antibody libraries and/or antibody affinity maturation are disclosed, for example, in US patent Nos. 8685987 and 8603990, as well as in publications US Nos. 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855, and 2009/0075378, each incorporated herein by reference.

[00328] Screening of libraries can be accomplished by various techniques known in the art. For example, nectin-4 can be immobilized on solid supports, columns, rods or cellulose-poly(vinylidene fluoride) membranes/other filters, expressed on host cells fixed on adsorption plates, or used in cell sorting, or conjugated to biotin for uptake beads coated with streptavidin or used in any other method for panning display libraries.

[00329] A review of in vitro affinity maturation methods is presented, for example, in Hoogenboom, 2005, Nature Biotechnology 23: 1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4: 39-51; and is incorporated by reference herein.

21. Modifications of antibodies against nectin-4.

[00330] Covalent modifications of anti-nectin-4 antibodies are included within the scope of the present invention. Covalent modifications involve the interaction of target amino acid residues of the anti-nectin-4 antibody with an organic functional group-containing agent that is capable of reacting with selected side chains or N- or C-terminal residues of the anti-nectin-4 antibody. Other modifications include deamidation of glutaminyl and aspartyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the side chain α-amino groups of lysine, arginine and histidine (see, for example, Creighton, Proteins : Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[00331] Other types of covalent modification of an anti-nectin-4 antibody included within the scope of the present invention include changing the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482- 501; and Walsh, 2010, Drug Discov. Today 15:773-80) and binding the antibody to one of a variety of non-protein polymers, for example, polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes, in a manner set forth, for example, in US Pat. No. 4,640,835 ; 4496689; 4301144; 4670417; 4791192; or 4179337.

[00332] The anti-nectin-4 antibody of the present invention can also be modified to form chimeric molecules containing the anti-nectin-4 antibody fused to another, heterologous polypeptide or amino acid sequence, such as an epitope tag (see, for example, Terpe, 2003 , Appl. Microbiol. Biotechnol. 60:523-33) or the Fc region of the IgG molecule (see, for example, Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[00333] The present disclosure also discloses fusion proteins comprising an antibody of the present invention that binds a nectin-4 antigen and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused may be effective in targeting the antibody to cells with cell surface expressed nectin-4.

[00334] Also disclosed herein are panels of antibodies that bind to the nectin-4 antigen. In specific embodiments, panels of antibodies have different rates of association, different rates of dissociation, different targeting of the nectin-4 antigen, and/or different specificities for the nectin-4 antigen. In some embodiments, the panels comprise or consist of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450 , about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950 or about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96-well or 384-well plates for assays such as ELISA.

22. Obtaining antibodies against nectin-4

[00335] Antibodies against nectin-4 can be obtained by culturing cells transformed or transfected with a vector containing nucleic acids encoding antibodies against nectin-4.

The polynucleotide sequences, the polypeptide components of the antibodies of the present invention, can be obtained by standard recombination methods. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or by PCR. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. A large number of vectors that are available and known in the art can be used for the purposes of the present description. Selection of a suitable vector depends primarily on the size of the nucleic acids to be incorporated into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expression of antibodies of the present invention include prokaryotes such as Archaebacteria and Eubacteria, including gram-negative or gram-positive organisms, eukaryotic microorganisms such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells , such as mammalian host cell lines. Host cells are transformed with the expression vectors described above and cultured in conventional growth media modified, if necessary, to induce promoters, select transformants, or amplify genes encoding desired sequences. Antibodies produced by host cells are purified using standard protein purification methods known in the art.

[00336] Methods for producing antibodies, including vector construction, expression and purification, are further described in Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherischia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

[00337] It is of course contemplated that alternative methods, which are well known in the art, can be used to obtain antibodies against nectin-4. For example, a suitable amino acid sequence or parts thereof can be obtained by direct peptide synthesis using solid-phase methods (see, for example, Stewart et al., Solid-Phase Peptide Synthesis (1969); and Merrifield, 1963, J. Am Chem. Soc. 85:2149-54). In vitro protein synthesis can be accomplished using automated or non-automated methods. The various portions of the anti-nectin-4 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-nectin-4 antibody. Alternatively, antibodies can be purified from cells or biological fluids, such as milk, of a transgenic animal engineered to express the antibody, as described, for example, in US Pat. Nos. 5,545,807 and 5,827,690.

23. Immunoconjugates

[00338] Also disclosed herein are conjugates comprising any anti-nectin-4 antibody of the present invention covalently linked by a synthetic linker to one or more non-antibody agents.

[00339] In some embodiments, the antibodies of the present invention are conjugated or recombinantly fused, for example, to a diagnostic or detection molecule. Conjugated or recombinantly fused antibodies can be used, for example, to monitor or predict the onset, development, progression and/or severity of nectin-4-mediated disease.

[00340] Such diagnosis and detection can be accomplished, for example, by attaching an antibody to a detectable substance including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; to prosthetic groups such as, but not limited to, streptavidin/biotin or avidin/biotin; to fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; to luminescent substances such as, but not limited to, luminol; to bioluminescent substances such as, but not limited to, luciferase, luciferin or aequorin; to chemiluminescent substances such as, but not limited to, acridinium-based compound or HALOTAG; to radioactive compounds such as, but not limited to, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, and ¹²¹ I), carbon ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In , ¹¹³ In, ¹¹² In and ¹¹¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga and ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge , ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn or ¹¹⁷ Sn; to positron-active metals using various positron emission tomographs; and to non-radioactive paramagnetic metal ions.

[00341] Also disclosed herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently linked) to a heterologous protein or polypeptide (or fragment thereof, e.g., a polypeptide of about 10, about 20, about 30, about 40, about 50 in length , about 60, about 70, about 80, about 90, or about 100 amino acids) for the preparation of fusion proteins, as well as their use. In particular, the specification discloses fusion proteins comprising an antigen binding fragment of an antibody of the present invention (e.g., a Fab fragment, an Fc fragment, an Fv fragment, an F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, or CDR VL) and heterologous protein, polypeptide or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide to which the antibody is fused can be used to target the antibody to a specific cell type, for example, cells that express nectin-4. For example, an antibody that binds to a cell surface receptor expressed by a particular cell type may be fused or conjugated to a modified antibody of the present invention.

[00342] In addition, the antibodies of the present invention can be fused to marker or "tag" sequences, such as a peptide, to facilitate purification. In specific embodiments, the amino acid sequence of the marker or tag is a hexa-histidine peptide, such as a tag introduced into a pQE vector (see, for example, QIAGEN, Inc.), among others, most of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine provides convenient purification of the fusion protein. Other peptide tags used for purification include, but are not limited to, hemagglutinin tag (“HA”), which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78) binding peptide strepavidin/avidin and "FLAG" tag.

[00343] Methods for preparing fusion constructs or conjugating (including polypeptides) with antibodies are known (see, for example, Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy : A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al. eds., 1985); Thorpe et al., 1982, Immunol. Rev. 62:119-58; US patent Nos. 5,336,603; 5622929; 5359046; 5349053; 5447851; 5723125; 578318 1 ; 5908626; 5844095; and 5112946; EP 307344; EP 367.166; EP 394827; PCT Publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631 and WO 99/048 13; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10535-39; Traunecker et al., 1988, Nature, 331:84-86; Zheng et al., 1995, J. Immunol.154:5590-600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

[00344] Fusion proteins can be produced, for example, using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques (collectively referred to as “DNA shuffling”). DNA shuffling can be used to alter the activity of anti-nectin-4 antibodies as provided herein, including, for example, antibodies with higher affinity and lower dissociation rates (see, for example, US Pat. No. 5,605,793; 5,811,238; 5,830,721; 5,834,252 ; and 5837458; Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson et al., 1999, J. Mol. Biol .287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). Antibodies or encoded antibodies can be altered by random mutagenesis through error-based PCR, random nucleotide insertions, or other methods prior to recombination. The polynucleotide encoding an antibody of the present invention may be recombined with one or more components, motifs, sections, portions, domains, fragments, and the like, of one or more heterologous molecules.

[00345] An antibody of the present invention can also be conjugated to a second antibody to form an antibody heteroconjugate, as described, for example, in US Pat. No. 4,676,980.

[00346] Antibodies that bind to nectin-4 can also be attached to solid carriers, which are particularly useful for immunoassays or target antigen purification. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[00347] The linker may be a "cleavable linker" that facilitates the release of the conjugated agent into the cell, but the present invention also includes non-cleavable linkers. Linkers for use in the conjugates of the present invention include, but are not limited to, labile acid linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, e.g., valine and/or citrulline , such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, for example, Chari et al., 1992, Cancer Res. 52:127-31; and US patent No. 5208020), thioether linkers or hydrophilic linkers designed against the occurrence of multidrug resistance mediated by a drug transporter (see, for example, Kovtun et al., 2010, Cancer Res. 70: 2528-37).

[00348] Antibody-agent conjugates can be prepared using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo- EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB and SVSB (succinimidyl (4-vinyl sulfone) benzoate). The present description also provides that antibody-agent conjugates can be prepared using any suitable methods that are disclosed in the art (see, for example, Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).

[00349] Conventional strategies for conjugating antibodies and agents rely on random conjugation chemistry including the ε-amino group of Lys residues or the thiol group of Cys residues, resulting in heterogeneous conjugates. Recently developed methods provide site-specific conjugation to antibodies, resulting in uniform loading and avoiding subpopulations of conjugates with altered antigen-binding or pharmacokinetic activity. These include the design of “thioMabs” containing cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J . Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26: 925-32). In another method, selenocysteine is cotranslationally inserted into the antibody sequence by recoding the UGA stop codon from the end to the selenocysteine insert, which allows site-specific covalent conjugation on the nucleophilic selenol group of selenocysteine in the presence of other natural amino acids (see, for example, Hofer et al., 2008 , Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[00350] In yet another embodiment, the present invention discloses antibodies conjugated or recombinantly fused to a therapeutic molecule (or one or more therapeutic molecules). The antibody may be conjugated or recombinantly fused to a therapeutic molecule, such as a cytotoxin, eg, a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, such as an alpha emitter. A cytotoxin or cytotoxic agent includes any agent that has a negative effect on cells. Therapeutic moieties include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine); alkylating agents (eg, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum) (II); anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (eg actinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)); auristatin molecules (eg, auristatin PHE, bryostatin 1, and solastatin 10; see Woyke et al., Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother. 45:3580- 4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad et al., Int. J Oncol 15:367-72 (1999), all incorporated herein by reference); hormones (eg, glucocorticoids, progestins, androgens and estrogens), DNA repair enzyme inhibitors (eg, etoposide or topotecan), kinase inhibitors (eg, compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7) :2167-76 (2002)); cytotoxic agents (eg, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs and compounds described in US patents 6245759, 6399633, 6383790, 6335156, 6271242, 6242196, 621841 0, 6218372, 6057300, 6034053, 5985877, 5958769, 5925376, 5922844, 5911995, 5872223, 5863904, 5840745, 5728868, 5648239, 5587459); farnesyltransferase inhibitors (for example, R115777, BMS-214662 and compounds described ny, for example, in US patents: 6458935, 6451812, 6440974, 6436960, 6432959, 6420387, 6414145, 6410541, 6410539, 6403581, 6399615, 6387905, 6372747, 6369034, 6362188, 6342765, 6342487, 6300 501, 6268363, 6265422, 6248756, 6239140, 6232338, 6228865, 6228856, 6225322, 6218406, 6211193, 6187786, 6169096, 6159984, 6143766, 6133303, 6127366, 6124465, 6124295, 6103723, 6093737, 6090948, 6080870, 6077853, 6071935, 6066 738, 6063930, 6054466, 6051582, 6051574 and 6040305); topoisomerase inhibitors (eg, captothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; riazoloacridine; XR-5000; saintopine; UCE6; UCE1022; TAN -1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamcyin); bulgarein; minor groove DNA binders such as Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronini; epiberberine; coraline; beta-lapakone; VS-4-1; bisphosphonates (eg, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, pyridronate, pamidronate, zoledronate) HMG-CoA reductase inhibitors (eg, lovastavatin, cvastatatin) Lexol, Lupitor, rosuvastatin and atorvastatin ) ; antisense oligonucleotides (eg, those disclosed in US Pat. Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminase inhibitors (eg, fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) and its pharmaceutically acceptable salts, solvates, clathrates and prodrugs.

[00351] In addition, the antibody of the present invention can be conjugated or recombinantly fused to a therapeutic molecule or drug molecule that modifies a desired biological response. Therapeutic molecules or drug molecules should not be considered as limited to classical chemical therapeutics. For example, the drug molecule may be a protein, peptide, or polypeptide with a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin or diphtheria toxin; protein such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agent such as TNF-γ, TNF-γ, AIM I (see international publication WO 97/33899), AIM II (see international publication WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6: 1567-1574) and VEGF (see international publication WO 99/ 23105), an antiangiogenic agent, such as angiostatin, endostatin, or a component of the coagulation pathway (eg, tissue factor); or a biological response modifier such as, for example, a lymphokine (eg, interferon gamma, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-5 (“IL-5”) , interleukin-6 (“IL-6”), interleukin-7 (“IL-7”), interleukin 9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12 (“IL -12"), interleukin-15 ("IL-15"), interleukin-23 ("IL-23"), granulocyte macrophage colony-stimulating factor ("GM-CSF") and granulocyte colony-stimulating factor ("G-CSF")) , or a growth factor (e.g., growth hormone (“GH”)), or a coagulating agent (e.g., calcium, vitamin K, tissue factors such as, but not limited to, Hageman factor (factor XII), high molecular weight kininogen (HMWK), prekallikrein (PK), coagulation factor proteins II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid and fibrin monomer).

[00352] A therapeutic molecule or drug conjugated or recombinantly fused to an antibody of the present invention that immunospecifically binds to nectin-4 antigen should be selected to achieve the desired prophylactic or therapeutic effect(s). In certain embodiments, the antibody is a modified antibody. When deciding which therapeutic or drug molecule to select for conjugation or recombinant fusion with the antibody of the present invention, consideration should be given to the nature of the disease, the severity of the disease, and the condition of the patient.

[00353] As used herein, the term “anti-nectin-4 antibody-drug conjugate” or “anti-nectin-4 antibody ADC” refers to an anti-nectin-4 antibody or antigen-binding fragment thereof conjugated or recombinantly fused to a therapeutic molecule (or one or more therapeutic molecules) or to an anti-nectin-4 antibody or antigen-binding fragment thereof conjugated or recombinantly fused to a therapeutic molecule or drug molecule that modifies a given biological response, as described above.

[00354] Disclosed herein is an anti-nectin-4 antibody ADC, including an anti-nectin-4 antibody that specifically binds to nectin-4. Also disclosed herein are anti-nectin-4 antibody ADCs, including antibodies against nectin-4 or an antigen-binding fragment thereof, of the present invention.

24. Methods of using antibodies and compositions.

[00355] In one aspect, disclosed herein is a method of inhibiting (e.g., reducing the rate of cell doubling, slowing the rate of cell increase, or blocking or preventing cell increase) proliferation of a cell, comprising contacting the cell with an effective amount of an anti-nectin antibody ADC -4 according to the present invention.

[00356] In one aspect, disclosed herein are methods of inhibiting cell proliferation comprising contacting the cells with an anti-nectin-4 antibody ADC, including an anti-nectin-4 antibody that specifically binds to nectin-4 (e.g., nectin-4 ECD -4 human or the ECD epitope of human nectin-4) of the present invention. In certain embodiments, the cells are contacted with an effective amount of an anti-nectin-4 antibody ADC as described herein. In some embodiments, the anti-nectin-4 antibody ADC binds to the human nectin-4 ECD. In some embodiments, the anti-nectin-4 antibody ADC binds to the ECD epitope of human nectin-4. In some embodiments, the anti-nectin-4 antibody ADC specifically binds to an ECD epitope of human nectin-4 that is different from the nectin-4 ligand binding site. In some embodiments, cell proliferation is inhibited by at least about 10%. In some embodiments, cell proliferation is inhibited by at least about 15%. In some embodiments, cell proliferation is inhibited by at least about 20%. In some embodiments, cell proliferation is inhibited by at least about 25%. In some embodiments, cell proliferation is inhibited by at least about 30%. In some embodiments, cell proliferation is inhibited by at least about 35%. In some embodiments, cell proliferation is inhibited by at least about 40%. In some embodiments, cell proliferation is inhibited by at least about 45%. In some embodiments, cell proliferation is inhibited by at least about 50%. In some embodiments, cell proliferation is inhibited by at least about 55%. In some embodiments, cell proliferation is inhibited by at least about 60%. In some embodiments, cell proliferation is inhibited by at least about 65%. In some embodiments, cell proliferation is inhibited by at least about 70%. In some embodiments, cell proliferation is inhibited by at least about 75%. In some embodiments, cell proliferation is inhibited by at least about 80%. In some embodiments, cell proliferation is inhibited by at least about 85%. In some embodiments, cell proliferation is inhibited by at least about 90%. In some embodiments, cell proliferation is inhibited by at least about 95%. In some embodiments, cell proliferation is inhibited by at least about 98%. In some embodiments, cell proliferation is inhibited by at least about 99%. In some embodiments, cell proliferation is inhibited by at least about 100%. In certain embodiments, cell proliferation is inhibited by at least about 25% to about 65%. In specific embodiments, inhibition of cell proliferation is assessed by the methods described herein. In some embodiments, inhibition of cell proliferation is assessed by methods known to one of ordinary skill in the art. In some embodiments, inhibition of cell proliferation is associated with inhibition of proliferation of cells that do not contact the anti-nectin-4 antibody ADC. In some embodiments, inhibition of cell proliferation involves inhibition of proliferation of cells that contact an unrelated antibody (eg, an antibody that does not specifically bind nectin-4), an ADC of an unrelated antibody, or an unconjugated anti-nectin-4 antibody.

25. Pharmaceutical compositions

[00357] In one aspect, the present invention further provides pharmaceutical compositions comprising at least one anti-nectin-4 antibody ADC of the present invention. In some embodiments, the pharmaceutical composition comprises 1) an anti-nectin-4 antibody and 2) a pharmaceutically acceptable carrier.

[00358] Pharmaceutical compositions containing anti-nectin-4 antibody ADCs are prepared for storage by mixing the anti-nectin-4 antibody ADCs of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers (see, for example, Remington, Remington's Pharamceutical Science (18th edition 1980)) in the form of aqueous solutions or lyophilized or other dried forms.

[00359] The antibodies of the present invention can be formulated in any suitable form for delivery to the target cell/tissue, for example, in the form of microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10: 146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), as sustained release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57) or in liposomes (Maclean et al. , 1997, Int. J. Oncol. 11: 325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8: 39-45).

[00360] The anti-nectin-4 antibody ADC of the present invention can also be entrapped in a microcapsule prepared, for example, by coacervation or interfacial polymerization methods, for example, hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal systems drug delivery (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described, for example, in Remington, supra.

[00361] Various compositions and delivery systems are known and can be used with an anti-nectin-4 antibody ADC that binds to nectin-4 as described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing ADC antibodies against nectin-4, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), constructing nucleic acid as part of a retroviral or another vector, and so on. In another embodiment, the composition may be in the form of a controlled or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, for example, Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14: 201-40; Buchwald et al., 1980, Surgery 88: 507-16; and Saudek et al., 1989, N. Engl. J. Med. 321: 569-74). In yet another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (eg, anti-nectin-4 ADC antibody as described herein) or composition of the present invention (see, for example, Medical Applications of Controlled Release (Langer and Wise eds., 1974), Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984), Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23 :61-126; Levy et al., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J. Neurosurg. 71:105 -12; US patents 5679377; 5916597; 5912015; 5989463; and 5128326; PCT publication numbers WO 99/15154 and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release composition is inert, free of leachable impurities, shelf stable, sterile, and biodegradable.

[00362] In yet another embodiment, a controlled or sustained release system can be placed in close proximity to a specific target tissue, such as the nasal passages or lungs, thereby reducing the systemic dose to a fraction (see, for example, Goodson, Medical Applications of Controlled Release Vol.2, 115-38 (1984)). Controlled release systems are described, for example, in Langer, 1990, Science 249:1527-33. Any technique known to one of ordinary skill in the art can be used to prepare sustained release formulations containing one or more antibodies that bind to nectin-4 as described herein (see, for example, US Pat. No. 4,526,938, PCT Publication WO 91/05548 and WO 96/20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

[00363] Therapeutic compositions containing one or more antibodies of the present invention can be prepared for storage by mixing the anti-nectin-4 antibody ADC at the desired level of purity, together with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) )). Mack Publishing Co., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate and other organic salts; antioxidants including ascorbic acid and methionine; preservatives (eg, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, for example polyvinylpyrrolidone; amino acids, for example glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars, for example sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal-containing complexes (for example, complexes based on Zn protein); and/or nonionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00364] Antibodies of the present invention can also, for example, be formulated into liposomes. Liposomes containing the molecule of interest are prepared by methods known in the art, such as those described in Epstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030; and US Patents 4,485,045 and 4,544,545. Extended circulation time liposomes are disclosed in US Pat. No. 5,013,556.

[00365] Particularly effective immunoliposomes can be prepared by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and a PEG derivative of phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of specific pore sizes to produce liposomes with the desired diameter. Fab' fragments of the anti-nectin-4 ADC antibody of the present invention can be conjugated to liposomes as described in Martin et al. (1982) J. Biol. Chem.257:286-288 via disulfide exchange reaction. The liposome does not necessarily contain a chemotherapeutic agent (such as doxorubicin); see Gabizon et al., (1989) J. National Cancer Inst.81(19):1484.

[00366] Formulations, such as those described herein, may also contain more than one active compound as needed for a particular treatment indication. In certain embodiments, the formulations comprise an anti-nectin-4 ADC antibody of the present invention and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose. For example, the anti-nectin-4 antibody ADC herein can be combined with one or more other therapeutic agents. Such combination therapy can be administered to the patient serially, simultaneously or sequentially.

[00367] The anti-nectin-4 antibody ADC of the present invention can also be entrapped in a microcapsule prepared, for example, by coacervation or interfacial polymerization methods, for example, hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal systems drug delivery (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[00368] Formulations for in vivo use may be sterile. Sterility is easily achieved by filtration through membranes, such as sterile filtration membranes.

[00369] Sustained release formulations can also be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing anti-nectin-4 ADC antibody, the matrices being in the form of molded articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for more than 100 days, some hydrogels release proteins over shorter periods of time. When encapsulated antibodies remain in the body for long periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be developed for stabilization depending on the mechanism used. For example, if the mechanism of aggregation is found to be the formation of an intermolecular SS bond via thiodisulfide exchange, stabilization can be achieved by modification of sulfhydryl moieties, lyophilization from acidic solutions, control of moisture content, use of appropriate additives, and development of specific polymer matrix compositions.

[00370] The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more antibodies of the present invention and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be used to prevent, treat, control or alleviate a nectin-4-mediated disease or one or more symptoms thereof.

[00371] Pharmaceutical carriers suitable for administering the compounds of the present invention include any such carriers known to those skilled in the art that are suitable for the particular route of administration.

[00372] In addition, the antibodies of the present invention may be formulated as the sole pharmaceutically active ingredient of the composition or may be combined with other active ingredients (such as one or more other prophylactic or therapeutic agents).

[00373] The compositions may contain one or more antibodies of the present invention. In one embodiment, the antibodies are formulated in suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or sterile solutions or suspensions for parenteral administration, and also transdermal patch and dry powder inhalers. In one embodiment, the antibodies described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel (1985), Introduction to Pharmaceutical Dosage Forms, 4th edition, pp. 126).

[00374] In the compositions, effective concentrations of one or more antibodies or derivatives thereof are mixed with a suitable pharmaceutical carrier. The concentrations of the compounds in the compositions are effective to deliver the amount, when administered, necessary to treat, prevent, or alleviate a nectin-4 mediated disease or symptom thereof.

[00375] In one embodiment, the compositions are formulated for single administration. To prepare the composition, a weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the condition being treated is ameliorated, prevented, or one or more of its symptoms is improved.

[00376] The anti-nectin-4 antibody ADC of the present invention is included in a pharmaceutically acceptable carrier in an effective amount sufficient to provide a therapeutically beneficial effect in the absence of undesirable side effects for the patient being treated. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems using conventional methods and then extrapolating these to human doses.

[00377] The concentration of the anti-nectin-4 antibody ADC in the pharmaceutical composition depends, for example, on the physicochemical characteristics of the anti-nectin-4 antibody ADC, the dosing schedule and the number of administrations, as well as other factors known to those skilled in the art.

[00378] In one embodiment, a therapeutically effective dose results in a serum concentration of anti-nectin-4 ADC antibody from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions in another embodiment provide a dose of from about 0.001 mg to about 2000 mg of anti-nectin-4 antibody ADC per kilogram of body weight per day. Pharmaceutical unit dosage forms can be prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg and, in one embodiment, from about 10 mg to about 500 mg ADC anti-nectin-4 antibodies and/or combinations of other optional essential ingredients per unit dosage form.

[00379] The anti-nectin-4 antibody ADC may be administered all at once or may be divided into several smaller doses administered at intervals. It should be understood that the exact dose and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro testing data. Additionally, it should be noted that concentrations and dosage values may vary depending on the severity of the condition being treated. In addition, it should be understood that for any particular patient, specific dosage regimens must be adjusted over time according to individual need and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges given herein are those given by way of example only and are not intended to limit the scope or practice of the compositions claimed.

[00380] Once the anti-nectin-4 antibody ADC is mixed or added, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen vehicle. An effective concentration is sufficient to reduce the symptoms of the disease, disorder or condition being treated and can be determined empirically.

[00381] Pharmaceutical compositions are intended for administration to humans and animals in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions and oil-in-water emulsions containing suitable quantities of compounds or pharmaceutically acceptable derivatives thereof. The anti-nectin-4 antibody ADC, in one embodiment, is formulated and administered in unit dosage forms or multiple dosage forms. As used herein, unit dosage forms refer to physically discrete units suitable for use in humans and animals and individually packaged as is known in the art. Each unit dose contains a predetermined amount of an anti-nectin-4 antibody ADC sufficient to produce the desired therapeutic effect, in combination with the desired pharmaceutical carrier, excipient or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dose forms can be administered as fractions or multiples. A multiple dose form is a plurality of unit dose forms packaged in a single container for administration in a separate unit dose form. Examples of multiple dose forms include vials, tablet or capsule bottles, or pint or gallon bottles. Therefore, a multiple dose form is a plurality of unit doses that are not separated within the package.

[00382] In some embodiments, one or more anti-nectin-4 antibodies of the present invention are in a liquid pharmaceutical composition. Liquid pharmaceutically administered compositions can, for example, be prepared by dissolving, dispersing or otherwise mixing the active compound as described above and optional pharmaceutical adjuvants in a carrier such as, for example, water, saline, aqueous dextrose, glycerin, glycols, ethanol, and the like. similar, thereby forming a solution or suspension. If desired, the pharmaceutical composition for administration may also contain minor amounts of non-toxic excipients such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitol monolaurate, triethanolamine sodium acetate , triethanolamine oleate and other similar agents.

[00383] Currently used methods for preparing such dosage forms are known or obvious to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[00384] Dosage forms or compositions can be prepared containing anti-nectin-4 ADC antibodies in the range of 0.005% to 100%, equilibrated with a non-toxic carrier. Methods for preparing such compositions are known to those skilled in the art.

[00385] Oral pharmaceutical dosage forms are either solid, liquid or gel based. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed, chewable lozenges, and tablets that may be enteric-coated, sugar-coated, or film-coated. Capsules may be hard or soft gelatin capsules, and granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[00386] In certain embodiments, the formulations are solid dosage forms. In certain embodiments, the formulations are capsules or tablets. Tablets, pills, capsules, lozenges and the like may contain one or more of the following ingredients or compounds of a similar nature: a binder; lubricants; diluent; gliding substance; disintegrating agent; dye; sweetener; flavoring; wetting agent; vomit coating; and film coating. Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silica. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Dyes include, for example, any of the approved certified water-soluble dyes FD and C, mixtures thereof; and water-insoluble dyes FD and C suspended in alumina hydrate. Sweeteners include sucrose, lactose, mannitol and artificial sweeteners such as saccharin, and any number of spray-dried flavors. Flavors include natural flavors extracted from plants, such as fruits, and synthetic mixtures of compounds that cause a pleasant sensation, such as, but not limited to, peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laureate ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

[00387] Antibodies can be provided in a composition that protects them from the acidic environment of the stomach. For example, the composition may be presented as an enteric-coated mixture that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be in the form of a formulation in combination with an antacid or other similar ingredient.

[00388] If the unit dosage form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, unit dosage forms may contain various other substances that modify the physical form of the dosage unit, such as coatings of sugar and other enteric agents. The compounds may also be administered as a component of an elixir, suspension, syrup, wafer, bulk composition, chewing gum, or the like. In addition to active compounds, the syrup may contain sucrose as a sweetener and some preservatives, dyes and flavors.

[00389] Anti-nectin-4 ADCs can also be mixed with other active agents that do not impair the desired effect, or with agents that are additive to the desired effect, such as antacids, H2 blockers, and diuretics. The active ingredient is an anti-nectnia-4 antibody ADC or a pharmaceutically acceptable derivative thereof, as described herein. Higher concentrations may be included, up to about 98% by weight of the active ingredient.

[00390] In all embodiments, tablets and capsules may be coated, as known to those skilled in the art, to modify or maintain dissolution of the active ingredient. For example, they can be coated with conventional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

[00391] In some embodiments, the compositions are liquid dosage forms. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil emulsions.

[00392] Elixirs are clear, sweetened hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugar, such as sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small beads in another liquid. Pharmaceutically acceptable carriers used in emulsions include non-aqueous liquids, emulsifiers and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in the non-effervescent granules for reconstitution into a liquid oral dosage form include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in the effervescent granules for reconstitution into a liquid oral dosage form include organic acids and a source of carbon dioxide. Colors and flavors are used in all of the above dosage forms.

[00393] Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite and surfactants such as polyoxyethylene sorbitol monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, veegum and acacia. Sweeteners include sucrose, syrups, glycerin and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitol monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acids. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Dyes include any approved certified water soluble FD and C dye and mixtures thereof. Flavors include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant taste sensation.

[00394] For a solid dosage form, the solution or suspension, for example, in propylene carbonate, vegetable oils or triglycerides, in one embodiment is enclosed in a gelatin capsule. Such solutions, as well as their preparation and encapsulation, are described in US patent No. 4328245; 4409239; and 4,410,545. For a liquid dosage form, a solution, for example, in polyethylene glycol, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, for example, water, to more easily measure it upon administration.

[00395] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (eg, propylene carbonate) and other such vehicles, and encapsulating these solutions or suspensions in shells hard or soft gelatin capsules. Other effective formulations include those set forth in US Patent Nos. RE28819 and 4358603. Briefly, such formulations include, but are not limited to, formulations that contain a compound of the present invention, dialkylated mono- or polyalkylene glycol, including, but not limited to, 1 ,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, where 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and also dithiocarbamates.

[00396] Other formulations include, but are not limited to, aqueous-alcoholic solutions, including pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl)acetal of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

[00397] Parenteral administration, in one embodiment, is characterized by injection, subcutaneous, intramuscular or intravenous, which are also disclosed herein. Injectable preparations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Injectable solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other agents such as, for example, sodium acetate, sorbitol monolaurate, triethanolamine oleate and cyclodextrins.

[00398] Implantation of a slow or sustained release system such that a constant dose level is maintained (see, for example, US Pat. No. 3,710,795) is also disclosed herein. Briefly, the compound of the present invention is dispersed in a solid internal matrix, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxanes , silicone carbonate copolymers, hydrophilic polymers such as acrylic and methacrylic acid ester hydrogels, collagen, crosslinked polyvinyl alcohol and crosslinked partially hydrolyzed polyvinyl acetate, which is surrounded by an outer polymer membrane, for example polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/copolymers ethyl acrylate, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymers, vinylidene chloride, ethylene and propylene, ionomer, polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, copol ethylene/vinyl alcohol imer, ethylene terpolymer /vinyl acetate/vinyl alcohol and ethylene/vinyloxyethanol copolymer, insoluble in body fluids. The anti-nectin-4 antibody ADC diffuses across the outer polymer membrane in a rate-controlled release step. The amount of anti-nectin-4 antibody ADC contained in such parenteral compositions is strictly dependent on its specific nature, as well as the activity of the compound and the needs of the patient.

[00399] Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent immediately before use, including subcutaneous tablets, sterile suspensions, ready for injection, sterile dry insolubles products ready for use with the vehicle immediately before use and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[00400] For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[00401] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable agents.

[00402] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral products packaged in multi-dose containers that include phenols or cresols, mercuries, benzyl alcohol, chlorobutanol esters, methyl and propyl p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN 80). The metal ion sequestering or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

[00403] The concentration of the pharmaceutically active compound is adjusted such that the injection provides an effective amount to produce the desired pharmacological effect. The exact dosage will depend on the age, weight and condition of the patient or animal, as is known in the art.

[00404] Standard parenteral medications may be packaged in an ampoule, vial, or syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration must be sterile.

[00405] As an example, intravenous or intra-arterial infusion of a sterile aqueous solution containing the active compound is an effective route of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active substance administered as needed to obtain the desired pharmacological effect.

[00406] Injectable preparations are intended for local and systemic administration. In one embodiment, a therapeutically effective dose is administered to the composition such that it has a concentration of at least about 0.1% w/w. up to about 90% w/w or more, in some embodiments, more than 1% wt./mass. active compound to the tissue(s) being treated.

[00407] The anti-nectin-4 antibody ADC may be suspended in micronized or other suitable form. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen vehicle. The effective concentration is sufficient to reduce the symptoms of the condition and can be determined empirically.

[00408] In other embodiments, pharmaceutical preparations are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[00409] A lyophilized powder is prepared by dissolving the anti-nectin-4 antibody ADC of the present invention or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or reconstituted solution obtained from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as a citrate buffer, a sodium or potassium phosphate buffer, or other buffer known to those skilled in the art, in one embodiment having an approximately neutral pH value.

[00410] Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired composition. In one embodiment, the resulting solution is distributed into vials for lyophilization. Each vial contains one dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at about 4°C to room temperature. [00410] Reconstitution of the lyophilized powder with water for injection produces a preparation for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable vehicle. The exact amount depends on the selected compound. This amount can be determined empirically.

[00411] Topical mixtures are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion or the like and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigants, sprays, suppositories, dressings, skin patches or any other formulations suitable for topical use.

[00412] The antibodies of the present invention can be formulated as aerosols for topical use, such as inhalation (see, for example, US Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which disclose aerosols for the delivery of steroid drugs effective in the treatment of inflammatory diseases diseases, in particular asthma). These respiratory compositions may be in the form of an aerosol or nebulizer solution or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the composition will, in one embodiment, have a diameter of less than 50 microns, in one embodiment, less than 10 microns.

[00413] The compounds may be formulated for topical or topical use, for example, for topical use on the skin and mucous membranes such as the eyes, in the form of gels, creams and lotions and for application to the eyes or for intracisternal or intraspinal use. Topical administration is envisaged for transdermal delivery, as well as for ocular or mucosal administration or for inhalation therapy. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[00414] These solutions, especially those intended for ophthalmic use, can be formulated as 0.01-10% isotonic solutions with a pH of approximately 5-7, with appropriate salts.

[00415] Other routes of administration are also disclosed herein, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration.

[00416] Transdermal patches, including isophoretic and electrophoretic devices, are well known to those skilled in the art. For example, such patches are disclosed in US patents 6267983, 6261595, 6256533, 6167301, 6024975, 6010715, 5985317, 5983134, 5948433 and 5860957.

[00417] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic use. Rectal suppositories, as used herein, mean solid bodies for insertion into the rectum that melt or soften at body temperature, releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or carriers and melting point elevating agents. Examples of bases include cocoa butter (cocoa butter), glycerol-gelatin, carbowax (polyoxyethylene glycol) and corresponding mixtures of mono-, di- and triglycerides of fatty acids. Combinations of different bases can be used. Agents for increasing the melting point of suppositories include spermaceti and wax. Suppositories for rectal administration can be prepared by compression or molding. The weight of the rectal suppository, in one embodiment, is about 2 to 3 grams.

[00418] Tablets and capsules for rectal administration can be prepared using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.

[00419] Antibodies and other compositions of the present invention can also be formulated to target a specific tissue, receptor, or other area of the body of a patient receiving treatment. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in instant compositions. For non-limiting examples of targeting methods, see, for example, US Patent Nos. 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 60 04534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5,709,874. In some embodiments, the anti-nectin-4 antibodies of the present invention are targeted (or otherwise administered) to the colon, for example, in a patient suffering from or at risk of developing a nectin-4-mediated disease.

[00420] In one embodiment, liposome suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art. For example, liposome formulations can be prepared as described in US Pat. No. 4,522,811. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) inside a flask. A solution of the compound of the present invention in phosphate buffered saline lacking divalent cations (PBS) is added and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation and then resuspended in PBS.

26. Methods of administration and dosage

[00421] In a specific embodiment, provided herein is a composition for use in the prevention, control, treatment and/or amelioration of a nectin-4-mediated disease comprising an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in the prevention of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in the control of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in the treatment of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in ameliorating a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In certain embodiments, the patient is a patient in need of such effects. In some embodiments, the patient suffers from a nectin-4 mediated disease. In other embodiments, the patient is at risk of developing a nectin-4-mediated disease. In some embodiments, administration results in the prevention, control, treatment, or amelioration of a nectin-4-mediated disease. In one embodiment, the anti-nectin-4 antibody ADC is an ADC in which the anti-nectin-4 antibody is the M22-321b41.1 antibody.

[00422] In one embodiment, provided herein is a composition for use in the prevention, control, treatment, and/or amelioration of a nectin-4-mediated disease, comprising an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in preventing a symptom of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in controlling a symptom of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in treating a symptom of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a composition for use in improving a symptom of a nectin-4-mediated disease, wherein the composition comprises an anti-nectin-4 antibody ADC of the present invention. In certain embodiments, the patient is a patient in need of such effects. In some embodiments, the patient suffers from a nectin-4 mediated disease. In other embodiments, the patient is at risk of developing a nectin-4-mediated disease. In some embodiments, administration results in the prevention, control, treatment, or improvement of a symptom of a nectin-4-mediated disease. In one embodiment, the anti-nectin-4 antibody is M22-321b41.1 antibody.

[00423] In another embodiment, provided herein is a method of preventing, controlling, treating and/or ameliorating a nectin-4 mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of preventing a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of controlling a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of treating a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of improving a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In certain embodiments, the patient is a patient in need of such effects. In some embodiments, the patient suffers from a nectin-4 mediated disease. In other embodiments, the patient is at risk of developing a nectin-4-mediated disease. In some embodiments, administration results in the prevention, control, treatment, or amelioration of a nectin-4-mediated disease. In one embodiment, the anti-nectin-4 antibody ADC is an ADC in which the anti-nectin-4 antibody is the M22-321b41.1 antibody.

[00424] In another embodiment, provided herein is a method of preventing, controlling, treating and/or ameliorating a symptom of a nectin-4 mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of preventing a symptom of a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of controlling a symptom of a nectin-4 mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of treating a symptom of a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In one embodiment, provided herein is a method of improving a symptom of a nectin-4-mediated disease in a patient, comprising administering an effective amount of an anti-nectin-4 antibody ADC of the present invention. In certain embodiments, the patient is a patient in need of such effects. In some embodiments, the patient suffers from a nectin-4 mediated disease. In other embodiments, the patient is at risk of developing a nectin-4-mediated disease. In some embodiments, administration results in the prevention, control, treatment, or improvement of a symptom of a nectin-4-mediated disease. In one embodiment, the anti-nectin-4 antibody ADC is an ADC in which the anti-nectin-4 antibody is the M22-321b41.1 antibody.

[00425] Antibodies of the present invention can also be used, for example, to purify, detect and target nectin-4 antigens in both diagnostic and therapeutic methods in vitro and in vivo. For example, modified antibodies are used in immunoassays to qualitatively and quantitatively measure nectin-4 levels in biological samples. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988) (incorporated herein by reference in its entirety).

[00426] Also disclosed herein are methods of preventing, controlling, treating, and/or ameliorating a nectin-4-related condition by administering to a patient an effective amount of an anti-nectin-4 antibody ADC or a pharmaceutical composition comprising an anti-nectin-4 antibody ADC of the present invention. In one aspect, the anti-nectin-4 antibody ADC is substantially purified (ie, substantially free of substances that limit its action or cause unwanted side effects). In certain embodiments, the anti-nectin-4 antibody ADC is a fully human monoclonal antibody, such as a fully human monoclonal antibody. The patient being treated may be a mammal, such as a non-primate (eg, cows, pigs, horses, cats, dogs, rats and the like) or a primate (eg, a monkey such as a cynomolgus or a human). In one embodiment, the patient is a human. In another embodiment, the patient is a human with a nectin-4-mediated disease.

[00427] Various delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., the anti-nectin-4 ADC antibody of the present invention), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing ADC antibodies against nectin-4, receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of nucleic acid as part of a retroviral or other vector, etc. similar. Methods of administration of a prophylactic or therapeutic agent (e.g., the anti-nectin-4 ADC antibody of the present invention) or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal administration. membranes (eg, intranasal and oral routes). In a specific embodiment, the prophylactic or therapeutic agent (eg, the anti-nectin-4 antibody ADC of the present invention) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agents or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through an epithelial or mucosal surface (e.g., oral mucosa, oral mucosa, rectal and intestinal mucosa, and the like) and can be administered together with other biologically active substances. Administration may be systemic or local. In addition, pulmonary administration can be used, for example, using an inhaler or nebulizer and a nebulizing agent formulation. See, for example, US Pat. Nos. 6,019,688, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT publications WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety.

[00428] In a particular embodiment, it may be desirable to administer a prophylactic or therapeutic agent or pharmaceutical composition of the present invention locally to the area in need of treatment. This can be achieved, for example, without limitation, by local infusion, topical administration (eg, intranasal spray), injection, or implant, wherein said implant is a porous, non-porous or gelatinous material, including membranes such as sialastic membranes, or fibers. In some embodiments, when administering the anti-nectin-4 antibody ADC of the present invention, care must be taken when using substances that do not absorb the anti-nectin-4 antibody ADC.

[00429] In another embodiment, a prophylactic or therapeutic agent or composition of the present invention provided herein can be delivered in a vesicle, particularly in a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see .general description there).

[00430] In another embodiment, the prophylactic or therapeutic agent or composition of the present invention may be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In yet another embodiment, polymeric substances can be used to achieve a controlled or sustained release of a prophylactic or therapeutic agent (e.g., the anti-nectin-4 antibody ADC of the present invention) or composition of the present invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg.71: 105); US Patent No. 5679377; US Patent No. 5916597; US Patent No. 5912015; US Patent No. 5989463; US Patent No. 5128326; PCT Publication No. WO99/15154; and PCT publication WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release composition is inert, free of leachable impurities, shelf stable, sterile, and biodegradable. In yet another embodiment, the controlled release system can be placed in close proximity to the therapeutic target, i.e., the nasal passages or lungs, while requiring only a portion of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra , vol. 2, pp. 115-138 (1984)). Controlled release systems as reviewed by Langer (1990, Science 249:1527-1533) can be used. Any technique known to one skilled in the art can be used to prepare sustained release formulations containing one or more antibodies of the present invention. See, for example, US Pat. No. 4,526,938, PCT Publication WO 91/05548, PCT Publication WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854, and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in its entirety.

[00431] In a particular embodiment, where the composition of the present invention is a nucleic acid encoding a prophylactic or therapeutic agent (e.g., the anti-nectin-4 antibody ADC of the present invention), the nucleic acid can be administered in vivo to stimulate expression of the encoded prophylactic or therapeutic agent by constructing it as part of an appropriate nucleic acid expression vector and introducing it so that it becomes intracellular, for example, by using a retroviral vector (see US Pat. No. 4,980,286) or by direct injection, or by using microparticle bombardment (eg, gene gun; Biolistic, Dupont) or coating with lipids or cell surface receptors or transfecting agents, or by introducing together with a homeobox-like peptide known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the host cell DNA for expression by homologous recombination.

[00432] In a specific embodiment, the composition of the present invention contains one, two or more antibodies of the present invention. In another embodiment, the composition of the present invention contains one, two or more antibodies of the present invention and a prophylactic or therapeutic agent other than the anti-nectin-4 antibody ADC of the present invention. In one embodiment, the agents are agents known or currently used to prevent, control, treat, and/or ameliorate nectin-4-related conditions. In addition to prophylactic or therapeutic agents, the compositions of the present invention may also contain a carrier.

[00433] The compositions of the present invention include the base material of drug compositions used in the manufacture of pharmaceutical compositions (eg, compositions that are suitable for administration to a patient or individual) that can be used to prepare unit dosage forms. In one embodiment, the composition of the present invention is a pharmaceutical composition. Such compositions contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (eg, an antibody of the present invention, or another prophylactic or therapeutic agent) and a pharmaceutically acceptable carrier. Pharmaceutical compositions may be formulated to be suitable for the respective route of administration to a patient.

[00434] In a specific embodiment, the term “carrier” refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient or carrier with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including oil of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly in solutions for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like . The composition, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The oral formulation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions contain a prophylactically or therapeutically effective amount of an anti-nectin-4 antibody ADC, for example in purified form, together with a suitable amount of a carrier to form a form suitable for administration to a patient. The composition must correspond to the method of administration.

[00435] In one embodiment, the composition is formulated in accordance with routine procedures in the form of a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in a sterile isotonic aqueous buffer solution. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Such compositions, however, may be administered by a route other than intravenous.

[00436] Typically, the ingredients of the compositions of the present invention are supplied either individually or as a mixture in a unit dosage form, for example, as a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the amount of active agent . If the composition is to be administered by infusion, it may be placed, for example, in an infusion bottle containing sterile water or pharmaceutical grade saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

[00437] The anti-nectin-4 antibody or ADC of the present invention may be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the amount of the anti-nectin-4 antibody or ADC. In one embodiment, the anti-nectin-4 antibody or ADC is provided as a dry, sterilized, lyophilized powder or anhydrous concentrate in a sealed container and can be reconstituted, for example, in water or saline to a suitable concentration for administration to a patient. In some embodiments, the anti-nectin-4 antibody or ADC is provided as a dry, sterile lyophilized powder in a sealed container at a unit dosage of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least at least 2 mg or at least 3 mg, for example at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg. Lyophilized anti-nectin-4 ADC can be stored at 2 to 8°C in its original packaging, and anti-nectin-4 ADC can be administered within 12 hours, for example within 6 hours, within 5 hours, within 3 hours or within 1 hour after recovery. In an alternative embodiment, the anti-nectin-4 antibody or ADC is provided in liquid form in a sealed container indicating the amount and concentration of the anti-nectin-4 antibody or ADC. In some embodiments, a liquid form of the anti-nectin-4 antibody or ADC is provided in a sealed container, at least 0.1 mg/ml, at least 0.5 mg/ml, or at least 1 mg/ml and, for example , at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 40 mg/ml, according to at least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml, at least 80 mg/ml, at least 90 mg/ml or at least 100 mg/ml.

[00438] The compositions of the present invention can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as those formed with hydrochloric, phosphoric, acetic, oxalic, tartaric acid and the like, and salts formed with cations, such as salts formed with sodium, potassium, ammonium, calcium , iron hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

[00439] The amount of a prophylactic or therapeutic agent (e.g., the anti-nectin-4 antibody ADC of the present invention) or composition of the present invention that will be effective for the prevention, control, treatment and/or amelioration of a nectin-4-mediated disease can be determined by standard clinical methods.

[00440] Accordingly, a dose of an anti-nectin-4 antibody ADC or composition that produces a serum titer of from about 0.1 μg/ml to about 450 μg/ml, and in some embodiments, at least 0.1 μg/ml, at at least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, according to at least 1 µg/ml, at least 1.5 µg/ml, for example at least 2 µg/ml, at least 5 µg/ml, at least 10 µg/ml, at least 15 µg/ml , at least 20 µg/ml, at least 25 µg/ml, at least 30 µg/ml, at least 35 µg/ml, at least 40 µg/ml, at least 50 µg/ml, according at least 75 µg/ml, at least 100 µg/ml, at least 125 µg/ml, at least 150 µg/ml, at least 200 µg/ml, at least 250 µg/ml, at least 300 μg/ml, at least 350 μg/ml, at least 400 μg/ml, or at least 450 μg/ml can be administered to a human for the prevention, control, treatment and/or amelioration of nectin-4-mediated disease. Additionally, in vitro assays may optionally be used as aids in determining optimal dosage ranges. The exact dosage to be used in a formulation will also depend on the route of administration and the severity of the disease, disorder or condition, and should be selected in accordance with the judgment of the practitioner and the circumstances of each patient.

[00441] Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

[00442] For the antibodies of the present invention, the dose administered to a patient is typically from 0.1 mg/kg to 100 mg/kg of the patient's body weight. In some embodiments, the dose administered to the patient is from about 1 mg/kg to about 75 mg/kg of the patient's body weight. In some embodiments, the dose administered to the patient is from 1 mg/kg to 20 mg/kg of the patient's body weight, such as from 1 mg/kg to 5 mg/kg of the patient's body weight. In general, human antibodies have a longer half-life in humans than antibodies derived from other species, due to the immune response to foreign polypeptides. Thus, lower doses of human antibodies can be used more often and administered less frequently. In addition, the dose and frequency of administration of the antibodies of the present invention can be reduced by enhancing the uptake and penetration of the antibodies into tissues through modifications such as, for example, lipidation.

[00443] In one embodiment, about 100 mg/kg or less, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 0.1 mg/kg or less anti-nectin-4 ADC 5 times, 4 times, 3 times, 2 times or 1 time for the treatment of nectin-4-mediated disease. In some embodiments, the nectin-4 antibody ADCs of the present invention are administered about 1 to 12 times, which doses can be administered as needed, such as weekly, biweekly, monthly, bimonthly, trimonthly, and the like, as determined by a doctor. In some embodiments, a lower dose (eg, 1-15 mg/kg) may be administered more frequently (eg, 3-6 times). In other embodiments, a higher dose (eg, 25-100 mg/kg) may be administered less frequently (eg, 1-3 times). However, as will be apparent to those skilled in the art, other amounts and dosage schedules are readily ascertainable and are within the scope of the present disclosure.

[00444] In a specific embodiment, about 100 mg/kg, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg are administered /kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, about 0.1 mg/kg or less anti-nectin-4 antibody ADC in a sustained release formulation to a patient, such as a human, for prevention, control, treatment and/or amelioration of nectin-4-mediated disease. In another specific embodiment, about 100 mg/kg, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg/kg, or less than, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 0.1 mg/kg or less bolus of non-sustained release anti-nectin-4 antibody ADC to a patient, such as a human , for the prevention, control, treatment and/or amelioration of nectin-4 mediated disease, and over a period of time, approximately 100 mg/kg, approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg are administered. kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 5 mg/kg or less anti-nectin antibody ADC- 4 of the present invention as an extended release to a specified patient (eg, intranasally or intramuscularly) two, three or four times (eg, once). According to this embodiment, the specified period of time may be from 1 to 5 days, a week, two weeks or a month.

[00445] In some embodiments, a single dose of an anti-nectin-4 antibody ADC of the present invention is administered to a patient for the prevention, control, treatment, and/or amelioration of a nectin-4-mediated condition, two, three, four, five, six, seven, eight , nine, ten, eleven, twelve times, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five or twenty-six with two weeks (for example, about 14 days) at intervals throughout the year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg /kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg , about 100 mg/kg or a combination thereof (that is, each dose of the monthly dose may or may not be identical).

[00446] In another embodiment, a single dose of the anti-nectin-4 ADC of the present invention is administered to a patient for the prevention, control, treatment and/or amelioration of a nectin-4 mediated condition, two, three, four, five, six seven, eight, nine , ten, eleven or twelve times at intervals of about a month (for example, about 30 days) for a year, where the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg /kg, approximately 5 mg/kg, approximately 10 mg/kg, approximately 15 mg/kg, approximately 20 mg/kg, approximately 25 mg/kg, approximately 30 mg/kg, approximately 35 mg/kg, approximately 40 mg/kg , approximately 45 mg/kg, approximately 50 mg/kg, approximately 55 mg/kg, approximately 60 mg/kg, approximately 65 mg/kg, approximately 70 mg/kg, approximately 75 mg/kg, approximately 80 mg/kg, approximately 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof (ie, each dose of the monthly dose may or may not be identical).

[00447] In one embodiment, a single dose of the anti-nectin-4 ADC of the present invention is administered to a patient to prevent, control, treat, and/or ameliorate a nectin-4-mediated condition two, three, four, five, or six times with at intervals of about two months (eg, about 60 days) over the course of a year, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof (ie, each bimonthly dose may or may not be identical).

[00448] In some embodiments, a single dose of an anti-nectin-4 antibody ADC of the present invention is administered to a patient for the prevention, control, treatment, and/or amelioration of a nectin-4-mediated condition two, three, or four times over three weeks (e.g., about 120 days) at intervals over an annual course, where the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/ kg, about 100 mg/kg, or a combination thereof (that is, each three-month dose may or may not be identical).

[00449] In some embodiments, the route of dosing the anti-nectin-4 antibody ADC of the present invention to a patient is intranasal, intramuscular, intravenous, or a combination thereof, but other routes described herein are also suitable. Each dose may be administered by the same route of administration or by a different route. In some embodiments, the anti-nectin-4 antibody ADC of the present invention can be administered by multiple routes of administration simultaneously or sequentially at different doses of the same or another anti-nectin-4 antibody ADC of the present invention.

[00450] In certain embodiments, the anti-nectin-4 ADC antibodies of the present invention are administered prophylactically or therapeutically to a patient. The anti-nectin-4 antibody ADCs of the present invention can be prophylactically or therapeutically administered to a patient to prevent, reduce, or ameliorate a nectin-4-mediated disease or symptom thereof.

27. Gene therapy

[00451] In a specific embodiment, nucleic acids containing sequences encoding antibodies or functional derivatives thereof are administered to a patient for use in the method of the present invention, for example, for the prevention, control, treatment and/or amelioration of a nectin-4 mediated disease, disorder or conditions through gene therapy. Such therapy includes therapy provided by administering an expressed or expressible nucleic acid to a patient. In one embodiment, the nucleic acids produce an encoded antibody that can be conjugated to produce an anti-nectin-4 antibody ADC, and the anti-nectin-4 antibody or ADC provides a prophylactic or therapeutic effect.

[00452] Any of the recombinant gene expression (or gene therapy) methods available in the art can be used. Examples of methods are described below and given in the “Examples” section.

[00453] For a general review of gene therapy techniques, see Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapeutics 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol 32:573-596; Mulligan 1993 Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIBTECH 11(5):155-215.

Methods generally known in the field of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[00454] In a specific embodiment, the composition contains nucleic acids encoding an anti-nectin-4 antibody of the present invention, wherein the nucleic acids are part of an expression vector that expresses the antibody or chimeric proteins or heavy or light chains, or a therapeutic agent or drug, as appropriate owner. In particular, such nucleic acids have promoters, such as heterologous promoters, operably linked to the coding region of the anti-nectin-4 antibody, the promoter being inducible or constitutive and optionally tissue specific. In another specific embodiment, nucleic acid molecules are used in which anti-nectin-4 antibody-encoding sequences and any other desired sequences are flanked by regions that promote homologous recombination at the desired site in the genome, thereby allowing intrachromosomal expression of the anti-nectin-4 antibody-encoding nucleic acid. nectin-4 (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). In some embodiments, the expressed anti-nectin-4 antibody molecule comprises a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains or fragments thereof in the anti-nectin-4 antibody.

[00455] Delivery of nucleic acids to a patient can be either direct, in which case the patient is directly exposed to the nucleic acid or vectors carrying the nucleic acid, or indirect, in which case the cells are first transformed with the nucleic acids in vitro and then transplanted into the patient. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

[00456] In a particular embodiment, the nucleic acid sequences are administered directly in vivo, where the sequences are expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, for example, by constructing them as part of a suitable nucleic acid expression vector and introducing the vector such that the sequences become intracellular, for example, by infection using defective or attenuated retroviruses or other viral vectors (see US Pat. No. 4,980,286), either by direct injection of naked DNA, or by bombardment with microparticles (eg, gene gun; Biolistic, Dupont), or by application of lipids or cell surface receptors or transfecting agents, encapsulation in liposomes, microparticles or microcapsules or by introducing them in connection with a peptide that is known to enter the nucleus, by introducing it in connection with a ligand captured by receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem.262: 4429-4432) (which can be used to target cell types that specifically express receptors) and the like. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand contains a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for specific cellular uptake and expression by targeting a specific receptor (see, for example, PCT publications WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188 WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the host cell DNA for expression by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature 342:435–438).

[00457] In a specific embodiment, viral vectors are used that contain nucleic acid sequences encoding an anti-nectin-4 antibody. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors contain components necessary for proper packaging of the viral genome and integration into the DNA of the host cell. Nucleic acid sequences encoding an anti-nectin-4 antibody for use in gene therapy can be cloned into one or more vectors to facilitate delivery of the gene to a patient. More information on retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6: 291-302, which describes the use of a retroviral vector to deliver the mdr 1 gene into hematopoietic stem cells to increase the resistance of the stem cells to chemotherapy. Other references, examples of the use of retroviral vectors in gene therapy, are: Clowes et al., 1994, J. Clin. Invest.93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Development.3:110-114.

[00458] Adenoviruses are other viral vectors that can be used in recombinant antibody production. Adenoviruses are particularly convenient carriers for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause moderate disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells and muscle. The advantage of adenoviruses is their ability to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503 provides an overview of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrates the use of adenoviral vectors to transfer genes into the respiratory epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are given in Rosenfeld et al., 1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; Mastrangeli et al., 1993, J. Clin. Invest.91:225-234; PCT publication W094/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In a specific embodiment, adenovirus vectors are used.

[00459] Adeno-associated virus (AAV) can also be used (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; and US Pat. No. 5,436,146). In a specific embodiment, AAV vectors are used to express the anti-nectin-4 antibody ADCs of the present invention. In certain embodiments, AAV comprises a nucleic acid encoding a VH domain. In other embodiments, AAV comprises a nucleic acid encoding a VL domain. In certain embodiments, AAV comprises a nucleic acid encoding a VH domain and a VL domain. In some embodiments of the methods of the present invention, an AAV containing a nucleic acid encoding a VH domain and an AAV containing a nucleic acid encoding a VL domain is administered to a patient. In other embodiments, the patient is administered an AAV containing a nucleic acid encoding a VH domain and a VL domain. In certain embodiments, the VH and VL domains are overexpressed.

[00460] Another gene therapy approach involves transferring the gene into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method involves transferring a selectable marker into cells. The cells are then selected to isolate those cells that have taken up and express the transferred gene. These cells are then delivered to the patient.

[00461] In this embodiment, the nucleic acid is introduced into the cell prior to in vivo introduction of the resulting recombinant cell. Such administration can be accomplished by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellulose gene transfer fusion spheroplasts and the like. Numerous methods are known in the art for introducing foreign genes into cells (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644 ; Clin. Pharma. Ther. 29:69-92 (1985)) and can be used in accordance with the methods of the present invention, provided that the developmental and physiological functions of the recipient cells are not impaired. The technique must ensure stable transfer of the nucleic acid into the cell so that the nucleic acid is expressed by the cell, for example, inherited and expressed by its cellular progeny.

[00462] The resulting recombinant cells can be delivered to the patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem or progenitor cells) can be administered intravenously. The number of cells contemplated for use depends on the desired effect, the condition of the patient and the like, and can be determined by one skilled in the art.

[00463] Cells into which a nucleic acid may be introduced for gene therapy purposes include any desired cell type available and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; lymphoid and myeloid cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, for example, obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver and the like.

[00464] In a specific embodiment, the cell used for gene therapy is autologous to the patient.

[00465] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an anti-nectin-4 antibody are introduced into the cells such that they are expressed by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a particular embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells that can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the methods of the present invention (see, for example, PCT publication WO 94/08598; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A: 229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[00466] In a specific embodiment, the nucleic acid to be administered for gene therapy purposes comprises an inducible promoter operably linked to a coding region such that expression of the nucleic acid is controlled by controlling the presence or absence of a corresponding transcription inducer.

28. Polynucleotides encoding an antibody

[00467] Also disclosed are polynucleotides containing a nucleotide sequence encoding an antibody of the present invention that immunospecifically binds to an epitope of nectin-4. Also disclosed herein are polynucleotides that hybridize under high, intermediate, or lower stringency hybridization conditions, such as those defined above, with polynucleotides that encode a modified antibody of the present invention.

[00468] Polynucleotides can be prepared and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Since the amino acid sequences of certain anti-nectin-4 antibodies of the present invention are known, the nucleotide sequences encoding these antibodies and modified versions of such antibodies can be determined using methods well known in the art, that is, the nucleotide codons that, are known to encode specific amino acids so as to produce a nucleic acid that encodes an antibody. Such an antibody-encoding polynucleotide can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier et al., 1994, BioTechniques 17: 242), which briefly involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence or fragments thereof variants, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides using PCR.

[00469] Alternatively, a polynucleotide encoding an antibody of the present invention can be obtained from a nucleic acid from a suitable source (for example, hybridoma M22-321b41.1, as disclosed herein, having ATCC accession number PTA-124245, deposit date June 13 2017). If a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (for example, an antibody cDNA library or a cDNA library constructed from a nucleic acid such as poly-A+ RNA isolated from any tissue or cells expressing an antibody, such as hybridoma cells, selected to express the antibody of the present invention by PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library that encodes an antibody. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in this area.

[00470] In certain embodiments, the nucleic acid molecules of the present invention contain or consist of a nucleic acid sequence of any of SEQ ID NO: 38 (encoding VH) and/or SEQ ID NO: 37 (encoding VL), or any combination thereof (eg , in the form of a nucleotide sequence encoding an antibody of the present invention, for example, a full-length antibody, a heavy and/or light chain of an antibody, or a single chain antibody of the present invention).

[00471] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody of the present invention, or a fragment thereof (e.g., a light chain variable region and/or a heavy chain variable region) that immunospecifically binds to a nectin-4 antigen, and vectors, eg vectors, containing such polynucleotides for recombinant expression in host cells (eg, E. coli cells and mammalian cells). In certain aspects, cells (eg, host cells) are disclosed herein. Also provided herein are methods for producing the antibodies and antigen binding fragments described herein.

[00472] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody of the present invention. In certain embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding a light chain and a heavy chain of an antibody of the present invention. The polynucleotides may contain nucleotide sequences encoding a light chain containing the FR VL and CDR of the antibodies of the present invention (see, for example, tables 1-2, respectively). The polynucleotides may contain nucleotide sequences encoding a heavy chain containing the VH FR and CDR of the antibodies of the present invention (see, for example, Tables 1-2, respectively). In certain embodiments, the polynucleotide of the present invention encodes a VL chain region containing the amino acid sequence of SEQ ID NO: 37. In particular embodiments, the polynucleotide of the present invention encodes a VH chain region containing the amino acid sequence of any of SEQ ID NO: 38.

[00473] In specific embodiments, disclosed herein are polynucleotides comprising a nucleotide sequence encoding an anti-nectin-4 antibody comprising three VL CDR chains, such as those containing the VL CDR1, VL CDR2 and VL CDR3 of the M22-321b41.1 antibody (e.g. see table 1). In specific embodiments, provided herein are polynucleotides comprising three VH CDR chains, such as those comprising the CDR1 VH, CDR2 VH and CDR3 VH of antibody M22-321b41.1 (eg, see Table 1).

[00474] In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-nectin-4 antibody comprising a VL chain region, for example, comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequence, described in this document (for example, see Tables 1-2). In specific embodiments, disclosed herein are polynucleotides comprising a nucleotide sequence encoding an anti-nectin-4 antibody comprising a VH chain region, for example, comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising the amino acid sequence described herein document (for example, see tables 1-2).

[00475] In some embodiments, a polynucleotide described herein comprises a nucleotide sequence encoding an antibody of the present invention comprising a light chain variable region (VL) comprising an amino acid described herein (e.g., see Table 1), wherein the antibody immunospecifically binds to nectin-4 polypeptide.

[00476] In certain embodiments, a polynucleotide of the present invention comprises a nucleotide sequence encoding an antibody of the present invention, comprising a heavy chain variable region (VH) comprising an amino acid sequence described herein (e.g., see Table 1), wherein the antibody is immunospecific binds to nectin-4 polypeptide.

[00477] In certain aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody described herein, including a VL chain region containing one or more VL FRs having the amino acid sequence described herein (e.g., see Table 2), wherein the antibody binds immunospecifically to nectin-4 polypeptide. In certain aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody of the present invention, comprising a VH chain region comprising one or more VH FRs having the amino acid sequence described herein (e.g., see Table 2), wherein the antibody immunospecifically binds to a nectin polypeptide -4, for example, human nectin-4 polypeptide.

[00478] In specific embodiments, a polynucleotide of the present invention comprises a nucleotide sequence encoding an antibody of the present invention comprising framework regions (e.g., VL domain and VH domain framework regions) that are human framework regions where the antibody immunospecifically binds to a nectin polypeptide. 4.

[00479] In a particular embodiment, the polynucleotide of the present invention contains the nucleotide sequence described in Table 3 encoding VH or VL, respectively, of the antibody M22-321b41.1 described herein, which immunospecifically binds to nectin-4 polypeptide.

Recombinant antibody production

[00480] Recombinant expression of an antibody of the present invention (e.g., a full-length antibody, a heavy and/or light chain antibody, or a single chain antibody as described herein) that immunospecifically binds to nectin-4 antigen requires the construction of an expression vector containing polynucleotide encoding an antibody. After obtaining a polynucleotide encoding an antibody molecule, an antibody heavy or light chain, or a fragment thereof (such as, but not necessarily, a fragment containing a heavy and/or light chain variable domain) of the present invention, a vector for producing the antibody molecule can be obtained using technology recombinant DNA using methods well known in the art. Thus, methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and corresponding signals to control transcription and translation. These methods include, for example, in vitro recombinant DNA technology techniques, in vivo synthesis techniques, and genetic recombination. Also disclosed are replicable vectors containing a nucleotide sequence encoding an antibody molecule of the present invention, an antibody heavy or light chain, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, WO 86/05807 and WO 89/01036; and US Pat. No. 5,124,644), and the antibody variable domain can be cloned into such a vector to express the entire the heavy chain, the entire light chain, or both the heavy and light chains.

[00481] The expression vector is transfected into a host cell by conventional methods, and then the transfected cells are cultured by conventional methods to produce the antibody of the present invention. Thus, also disclosed herein are host cells containing a polynucleotide encoding an antibody of the present invention or fragments thereof, or a heavy or light chain or fragment thereof, or a single chain antibody of the present invention, operably linked to a heterologous promoter. In some embodiments, for the expression of double chain antibodies, vectors encoding the heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below.

[00482] Various host expression vector systems can be used to express the antibody molecules provided herein (see, for example, US Pat. No. 5,807,155). Such host expression systems provide vehicles by which coding sequences of interest can be produced and subsequently purified, but also provide cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the antibody molecule described herein in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus (eg, baculovirus) expression vectors containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with expression vectors based on a recombinant plasmid (eg, Ti plasmid) containing antibody coding sequences; or mammalian cell systems (eg, COS, CHO, BHK, 293, NS0 and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (eg, late adenovirus; vaccinia virus promoter 7.5K). Bacterial cells such as Escherichia coli or eukaryotic cells can be used to express the recombinant antibody molecule, especially to express the entire recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector, such as the core intermediate early gene promoter element from human cytomegalovirus, are an effective antibody expression system (Foecking et al., 1986, Gene 45:101 and Cockett et al., 1990, Bio/Technology 8:2). In some embodiments, the antibodies of the present invention are produced in CHO cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies of the present invention that immunospecifically bind to nectin-4 antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[00483] In bacterial systems, a number of expression vectors may advantageously be selected depending on the intended use of the antibody molecule being expressed. For example, if it is necessary to produce large quantities of such an antibody to produce a pharmaceutical composition based on an antibody molecule, vectors that control the expression of large quantities of fusion protein products that are easy to purify may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that fusion protein; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and similar pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione. The design of pGEX vectors includes the inclusion of thrombin or factor Xa protease cleavage sites, which allows the cloned target gene product to be cleaved from the GST group.

[00484] In the Autographa californica insect system, nuclear polyhedrosis virus (AcNPV) is used as a vector for the expression of foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be separately cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

[00485] A number of virus-based expression systems can be used in mammalian host cells. When using an adenovirus as an expression vector, the antibody coding sequence of interest can be ligated to the adenovirus transcription/translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome through in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg, see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Efficient translation of embedded antibody coding sequences may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must be synchronized with the reading frame of the desired coding sequence to ensure translation of the entire inserted sequence. These exogenous translation control signals and initiation codons can have different origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators and the like (see, for example, Bittner et al., 1987, Methods in Enzymol.153:51-544).

[00486] In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the desired specific manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of the expressed foreign protein. In this regard, it is possible to use eukaryotic host cells that have cellular mechanisms for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not produce endogenously any immunoglobulin chains), CRL7O3O and HsS78Bst cells. In some embodiments, the fully human anti-nectin-4 monoclonal antibodies of the present invention are produced in mammalian cells, such as CHO cells.

[00487] Stable expression can be used for long-term production of recombinant proteins in high yield. For example, cell lines can be constructed that stably express the antibody molecule. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by suitable expression regulatory elements (eg, promoter, enhancer sequences, transcription terminators, polyadenylation sites, and the like) and a selectable marker. After administration of the vector, cells can be allowed to grow for 1-2 days in enriched medium and then transferred to selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to construct cell lines that express the antibody molecule. Such engineered cell lines may be particularly useful for screening and evaluating compositions that directly or indirectly interact with the antibody molecule.

[00488] A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase genes (Lowy et al., 1980, Cell 22: 8-17) can be used in tk-, hgprt- or aprt-cells, respectively. Also, resistance to antimetabolites can be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc Natl Acad Sci USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072); neo, which confers resistance to aminoglycoside G-418 (Wu and Wu, Biotherapy, 3:87-95 (1991); Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods well known in the art of recombinant DNA technology can typically be used to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol.150:1, incorporated herein by reference in their entirety.

[00489] Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel. The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the antibody is amplifiable, increasing the level of inhibitor in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[00490] A host cell can be co-transfected with two expression vectors provided herein: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain identical selectable markers that provide equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The heavy and light chain coding sequences may comprise cDNA or genomic DNA.

[00491] Once the antibody molecule of the present invention is produced by recombinant expression, it can be purified by any method known in the art to purify an immunoglobulin molecule, for example, chromatography (e.g., ion exchange, affinity, in particular, affinity for a specific protein A antigen, and calibration chromatography), centrifugation, varying solubility, or any other standard protein purification technique. In addition, the antibodies of the present invention can be fused to heterologous polypeptide sequences described herein or other methods known in the art to facilitate purification.

29. Diagnostic tests and detection methods

[00492] In one aspect, the anti-nectin-4 antibodies and fragments thereof of the present invention can be used to detect nectin-4 in a biological sample. Such anti-nectin-4 antibodies may include antibodies that bind to human nectin-4 but do not induce nectin-4 signaling activity. The term "detection" or "detection" as used herein includes quantitative or qualitative detection and refers to the use of readings or assay results to determine the presence/absence of nectin-4 in a sample, the relative expression level of nectin-4 in the sample (e.g., relative one or more reference values, relative to other samples or relative to one or more expression scales) or nectin-4 concentration in the sample. Detected assay results may be numerical data, such as qPCR results, immunoassay results, or measured protein concentration; the detected read may be non-numeric data, such as anti-nectin-4 antibody staining of cells or tissues in microscopic images or nectin-4 staining in flow cytometry imaging; or the detected readout may be any experimental data that can be used as a proxy for nectin-4 expression as is well known to one of ordinary skill in the art, such as fluorescence intensity, luminescence intensity, colorimetric readout, and absorption/emission spectroscopy. The detected reading in the assays is referred to herein as the assay signal.

[00493] As provided herein, a biological sample is samples or substances that are of biological origin. Biological samples include a biological fluid, cell or tissue sample. In some embodiments, the cell may be a cultured cell line, engineered cells, in vitro or ex vivo cell culture obtained from an individual, such as a human, or cells obtained from an individual. In other embodiments, the tissue samples may include body fluid or body tissue obtained from an individual, including a human. In some embodiments, the tissue samples include samples from an area of a patient suspected of having the disease. Such tissue samples may be, but are not limited to, tissues, biological fluid, tissue fractions and/or cells isolated from an organism, such as a mammal, particularly a human. In a specific embodiment, tissue samples are sectioned, formalin-fixed, and paraffin-embedded (FFPE). Cells, body fluid and/or tissue can be obtained from an individual, such as a human, by methods well known to one skilled in the art, for example, biopsy (including liquid biopsy), surgical procedures, cell smears and phlebotomy.

[00494] In one aspect, a method for detecting nectin-4 in a biological sample is disclosed herein. In some embodiments, the method includes contacting a biological sample with an anti-nectin-4 antibody under conditions favorable for binding of the anti-nectin-4 antibody to nectin-4, and detecting the binding of the anti-nectin-4 antibody to nectin-4.

[00495] As is well known to one skilled in the art, the amount of antibody bound to an antigen in a biological sample correlates with the amount of antigen in the biological sample. Therefore, the amount of binding of the anti-nectin-4 antibody of the present invention to nectin-4 in a biological sample can be used to measure the amount or expression level of nectin-4 in the biological sample. In some embodiments, the amount of nectin-4 in a biological sample is determined within the linear detection range of the anti-nectin-4 antibody. “Linear detection range” refers to the range of antigen amount or concentration within which the bound antibody is linearly correlated with the amount or concentration of antigen in the biological sample. This linear range depends on factors such as the affinity of the antibody for the antigen, the concentration of the antibody used for antibody-antigen binding, and the conditions used for antibody-antigen binding.

[00496] The concentration of antibody used for antibody-antigen binding can be assessed in titration experiments. In one embodiment of such titration experiments, the antibody concentration is serially diluted two, three, five, or ten times, and each of the serially diluted antibodies is coupled to control samples with a known amount of the corresponding antigen. In one embodiment, the desired antibody concentration is the concentration at which the amount of binding antibody varies over the widest linear detection range.

[00497] Nectin-4 expression in a biological sample refers to the amount of nectin-4 in the biological sample at a given time. Measured nectin-4 expression reflects the accumulation of nectin-4 in a biological sample over a period of time and may include degradation or modification products such as full-length nectin-4, fragments of nectin-4, and naturally modified, eg, glycosylated, nectin-4. Since proteins are translated from mRNA, mRNA levels in biological samples can be used as a proxy for nectin-4 expression in biological samples.

[00498] Disclosed herein are methods for assessing the expression of nectin-4 in a tissue sample from an individual suspected of having a cancer, comprising: (a) contacting said tissue sample with an antibody or antigen-binding fragment thereof of the present invention; (b) detecting the binding of said antibody or antigen-binding fragment thereof to said tissue sample; (c) determining the expression of nectin-4 in the tissue sample, wherein the expression level of nectin-4 in the tissue sample is compared with a control level of nectin-4 expression.

[00499] Also provided herein are methods for assessing nectin-4 expression in a tissue sample of an individual suspected of having a cancer, comprising: (a) performing an IHC assay on the tissue sample with an antibody or antigen-binding fragment thereof of the present invention; (b) determining the expression of nectin-4 in the tissue sample, wherein the expression level of nectin-4 in the tissue sample is compared with the control level of nectin-4 expression.

[00500] Also disclosed herein are methods for assessing the susceptibility of a patient with a malignant disease to an anticancer therapeutic agent, said method based on the expression of nectin-4 in a tissue sample of said patient, comprising: (a) contacting said tissue sample with an antibody or an antigen binding fragment of the present invention; (b) detecting the binding of said antibody or antigen-binding fragment thereof to said tissue sample; (c) determining the expression of nectin-4 in the tissue sample, wherein the expression level of nectin-4 in the tissue sample is compared with a control level of nectin-4 expression; where the increased level of Nectin-4 expression compared to the control indicates susceptibility to the specified antitumor therapy.

[00501] Also disclosed herein are methods for assessing the susceptibility of a patient with a malignant disease to an anticancer therapeutic agent, said method based on the expression of nectin-4 in a tissue sample of said patient, comprising: (a) performing an IHC assay on the patient's tissue sample with the antibody, or an antigen binding fragment thereof according to the present invention; (b) determining the expression of nectin-4 in the tissue sample, wherein the expression level of nectin-4 in the tissue sample is compared with a control level of nectin-4 expression; wherein an increased level of nectin-4 expression in a tissue sample compared to a control indicates susceptibility to said antitumor therapy.

[00502] Disclosed herein are methods of treating a cancer in a patient, comprising (a) determining the level of expression of nectin-4 in a tissue sample of the patient using an antibody or antigen-binding fragment thereof of the present invention, wherein the level of expression of nectin-4 in the tissue sample is higher, than the control level of nectin-4 expression; (b) administering an antitumor therapeutic agent to a patient.

[00503] Disclosed herein are methods of treating cancer in an individual comprising (a) contacting a tissue sample of the individual with an antibody or antigen-binding fragment thereof of the present invention; (b) determining the expression level of nectin-4 in the patient's tissue sample using an antibody or antigen binding fragment thereof of the present invention, wherein the expression level of nectin-4 in the tissue sample is higher than the control level of nectin-4 expression; (c) administering an antitumor therapeutic agent to a patient.

[00504] Provided herein are methods of treating cancer in an individual, comprising (a) obtaining a tissue sample from the patient; (b) contacting a sample of patient tissue with an antibody or antigen binding fragment thereof of the present invention; (c) determining the expression level of nectin-4 in the patient's tissue sample using an antibody or antigen binding fragment thereof of the present invention, wherein the expression level of nectin-4 in the tissue sample is higher than the control level of nectin-4 expression; (d) administering an antitumor therapeutic agent to a patient.

[00505] The patient in any method of the present invention can be a human, and the patient can have any malignancy for which determination of nectin-4 expression can be performed and/or for which nectin-4 expression can provide diagnostic, prognostic or prognostic value . Examples of malignancies include: acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myeloblastic leukemia; acute myeloid leukemia (adults/children); adrenocortical carcinoma; AIDS-related malignancy; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bile duct cancer, extrahepatic (cholangiocarcinoma); bladder cancer; osteosarcoma of bone/malignant fibrous histiocytoma; brain cancer (adults/children); brain tumor, cerebellar astrocytoma (in adults/children); brain tumor, cerebral astrocytoma/malignant cerebral glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumors; tumor of the brain, visual pathways and hypothalamic glioma; brainstem glioma; mammary cancer; bronchial adenomas/carcinoids; bronchial tumor; Burkitt's lymphoma; malignant tumors of children; carcinoid of the gastrointestinal tract; carcinoid; adult carcinoma, unknown primary site; carcinoma with unknown primary site; embryonal tumor of the central nervous system; lymphoma of the central nervous system, primary; cervical cancer; adrenocortical carcinoma in children; malignant tumors in children; childhood cerebral astrocytoma; chordoma in children; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small cell tumor; emphysema; endometrial cancer; ependymoblastoma; ependymoma; esophageal carcinoma; Ewing's sarcoma in the Ewing tumor family; extracranial embryonal cell tumor; extragonal embryonic cell tumor; extrahepatic bile duct cancer; gallbladder cancer; stomach cancer; gastric carcinoid; gastrointestinal carcinoids; gastrointestinal stromal tumor; germ cell tumor: extracranial, extragonal or trophoblastic tumor of the ovary; gestational trophoblastic tumor, unknown primary site; glioma; brainstem glioma; glioma of the visual pathways in children and the hypothalamus; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver) cancer; Hodgkin's lymphoma; hypopharyngeal cancer; glioma of the hypothalamus and optic pathway; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; kidney cancer (renal cell carcinoma); Langerhans cell histocytosis; laryngeal cancer; cancer of the lips and oral cavity; liposarcoma; liver cancer (primary); lung cancer; lymphoma, primary central nervous system; macroglobulinemia, Waldenström; breast cancer in men; malignant fibrous bone histiocytoma/osteosarcoma; medulloblastoma; medulloepithelioma; melanoma; melanoma, intraocular (eye); Merkel cell cancer; Merkel skin carcinoma; mesothelioma; mesothelioma, a malignant tumor in adults; metastatic squamous cell carcinoma of the neck with unknown primary location; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma/plasmocyte neoplasm; mycoses fungoides, myelodysplastic syndromes; myelodysplastic/myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, acute in adults; myeloid leukemia, acute in children; multiple myeloma (bone marrow cancer); myeloproliferative disorders, chronic; cancer of the nasal cavity and paranasal sinuses; nasopharyngeal cancer; neuroblastoma, non-small cell lung cancer; non-Hodgkin's lymphoma; oligodendroglioma; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma/malignant fibrous histiocytoma of bone; ovarian cancer; epithelial ovarian cancer (superficial epithelial-stromal tumor); ovarian germ cell tumor; ovarian tumor with low malignant potential; pancreas cancer; pancreatic cancer, islet cells; papillomatosis; cancer of the paranasal sinuses and nasal cavity; parathyroid cancer; penile cancer; nasopharyngeal cancer; pheochromocytoma; pineal astrocytoma; germinoma of the pineal gland; tumors of the pineal gland parenchyma of moderate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma; pleuropulmonary blastoma; primary lymphoma of the central nervous system; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); transitional cell carcinoma of the renal pelvis and ureter; respiratory tract carcinoma involving the NUT gene on chromosome 15; retinoblastoma; rhabdomyosarcoma, in children; salivary gland cancer; sarcoma, Ewing tumor family; Sézary syndrome; skin cancer (melanoma); skin cancer (non-melanoma); small cell lung cancer; small bowel cancer; soft tissue sarcoma; soft tissue sarcoma; spinal cord tumor; squamous cell carcinoma; squamous cell carcinoma of the neck with unknown primary localization, metastatic; stomach cancer; supratentorial primitive neuroectodermal tumor; cutaneous T-cell lymphoma (fungoid granuloma and Sezary syndrome); testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, in children; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; cancer of the uterus, endometrium; urethral cancer, bladder cancer, ureteral cancer, urethral and bladder cancer, uterine sarcoma; vaginal cancer; vulvar cancer; Wilms tumor; and other cancers of epithelial origin in which nectin-4 is expressed.

[00506] In some embodiments of the methods of the present invention, a patient, such as a human, is suspected of having a malignancy selected from the group consisting of endometrial cancer, urothelial cancer, bladder cancer, ureteral cancer, urethral cancer, lung cancer, ovarian cancer, breast cancer, esophageal cancer, pancreatic cancer, head and neck cancer, prostate cancer, penile cancer, anal cancer, vulvar cancer, primary urachal cancer, and other cancers of epithelial origin in which nectin is expressed -4.

[00507] As used herein, the term "susceptibility" refers to the likelihood that a cell or patient or individual will respond or will respond to treatment with an anticancer therapeutic agent. Susceptibility may include the likelihood of a wide range of reactions to an antineoplastic therapeutic agent, such as, but not limited to, decreased proliferation or growth of malignant cells, increased death of malignant cells, decreased number of malignant cells or tumor size, decreased rate of increase in tumor size, slowed progression of stages malignancy, regression of cancer stage or tumor size, decreased expression of cancer markers, decreased cancer metastasis, and/or improved patient outcome such as patient weight restoration, patient survival at 6, 1, 2, 3, or 5 years , and/or the length of patient survival. In certain embodiments, susceptibility may be scaled as no susceptibility (ie, little likelihood of response), sensitive (response likely), and/or uncertain susceptibility. In certain embodiments, a cell or tissue, or individual, or patient is more likely to respond to an antitumor therapeutic agent if the cell, tissue, patient, or individual has significant expression of nectin-4 in the tumor or malignant cell. In some embodiments, susceptibility increases with increasing levels of nectin-4 in the tumor or malignant cells/tissues. In some embodiments, susceptibility correlates with the level of nectin-4 expression in the tumor or malignant cells/tissues. In some embodiments, susceptibility is linearly proportional to the level of nectin-4 expression in the tumor or malignant cells/tissues. In other embodiments, the susceptibility of a cell, tissue, patient, or individual to an antitumor agent may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70, 80, 90, 100 or more than for cells negative for nectin-4 expression.

[00508] As used herein, the term “susceptibility score” refers to the prediction that susceptibility will be higher in a cell, tissue, person, or individual compared to another cell, tissue, person, or individual. Such forecasts are made based on certain criteria. In some embodiments, the prediction of susceptibility is made based on the level of nectin-4 expression in a cell, tissue, tumor, human, or individual. As described above, in certain embodiments, a cell or tissue, or individual, or patient is predicted to be more likely to respond to an antitumor therapeutic agent if the cell, tissue, patient, or individual has significant expression of nectin-4 in the tumor or cancer cell. In some embodiments, susceptibility is predicted to increase with increasing levels of nectin-4 in the tumor or malignant cells/tissues. In some embodiments, susceptibility is predicted to correlate with the level of nectin-4 expression in the tumor or malignant cells/tissues. In some embodiments, susceptibility is predicted to be linearly proportional to the level of nectin-4 expression in the tumor or malignant cells/tissues. In other embodiments, the susceptibility of a cell, tissue, patient, or individual to an antitumor agent is predicted to be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times , 90, 100 or more than for cells negative for nectin-4 expression.

[00509] An antitumor therapeutic agent may be any antitumor therapeutic agent or any anticancer therapy for which nectin-4 expression may provide diagnostic, prognostic, or prognostic value. In some embodiments, the therapeutic agent is an agent that targets a biological process, such as a signaling pathway, metabolic pathway, or protein synthesis/degradation pathway in which nectin-4 plays a role. In some embodiments, the therapeutic agent is an agent that targets nectin-4 activity, nectin-4 signaling, or nectin-4-mediated diseases. In specific embodiments, the therapeutic agent is an anti-nectin-4 antibody or a conjugate of an anti-nectin-4 antibody and a drug.

[00510] To determine the expression of nectin-4 in a tissue sample, the expression level of nectin-4 in the tissue sample is compared or correlated with a control level of nectin-4 expression. As used herein, “control expression level” refers to the expression level in the control sample, which when compared with the expression level of nectin-4 in the test sample provides relative information about the expression of nectin-4 in the test sample. The control expression level may be a known expression level, for example expression in a control sample in which the amount, concentration and/or molar amount of nectin-4 is known. For example, the control level of expression may be expression of Nectin-4 in a cell line in which the cells have been transfected or otherwise engineered to express a known amount of nectin-4. In some embodiments, the control level of expression may be expression of nectin-4 in a cell in which nectin-4 expression has been quantified by methods disclosed herein or known to one of ordinary skill in the art, such as ELISA, SDS-PAGE, quantitative immunoprecipitation, and /or quantitative Western blotting. In other embodiments, the control expression level includes more than one control value, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40 45, 50, 75, 100 and more. In one embodiment, the control level has 4 values: one negative for nectin-4 expression, one for weak nectin-4 expression, one for moderate nectin-4 expression, and one for high/strong nectin-4 expression.

[00511] Alternatively, the control expression level may be a level at which the exact concentration or amount of nectin-4 is unknown, but the state, activity and/or function of the control is known. In some embodiments, the control expression levels may be the levels of a non-cancerous cell from the same patient suspected of having a malignant neoplasm. In such embodiments, comparison of nectin-4 expression in a test sample and expression in a control sample allows the expression of nectin-4 in the test sample to be determined relative to (eg, higher, lower, or substantially the same) the expression of nectin-4 in non-malignant cells. In other embodiments, the control expression levels may be those for the cancer cell, where by comparing the expression of nectin-4 in the test sample and the expression in the control sample, the expression of nectin-4 in the test sample can be determined relative to (e.g., higher, lower, or substantially the same) expression of nectin-4 in malignant cells. In other embodiments, the control expression levels may be those of a cell of a second individual, where by comparing the expression of nectin-4 in the test sample with the expression in the control sample, the expression of nectin-4 in the test sample can be determined relative to (e.g., higher, lower, or substantially the same) expression of nectin-4 in cells from the second individual. The second individual may be a normal person not suffering from a cancer, a person suffering from the same type of cancer, a patient suspected of having a cancer, or a person suffering from a cancer different from the one suspected of being present in the patient.

[00512] The control sample and control cells, as described herein, may be a cell line, in vitro or ex vivo culture of cells obtained from a patient suspected of having a malignancy, cells obtained from a patient suspected of having a malignancy, culture in vitro or ex vivo cells obtained from a second individual, cells obtained from a second individual.

[00513] Therefore, in some embodiments of the methods of the present invention, the reference level of nectin-4 expression may be the level of nectin-4 expression in cancer cells, non-cancerous cells of a specified patient, or non-cancerous cells of a second individual.

[00514] As described above, in some embodiments, the expression of nectin-4 in a sample can be determined by correlating or comparing the binding of anti-nectin-4 antibody to the sample and the binding of anti-nectin-4 antibody to a negative and positive control, where the levels of nectin expression are - 4 are known in the control. The control expression of nectin-4 can be a positive control of nectin-4 or a negative control of nectin-4. As used herein, the term “nectin-4 positive control” or “positive control” refers to a cell, tissue, tumor, human and/or individual that is known to express a significant amount of nectin-4. “Nectin-4 negative control” or “negative control” refers to cells and/or tissues that are known not to express nectin-4 or to express low levels of nectin-4, such that nectin-4 in the cells or tissues is not biologically significant. A level of nectin-4 is biologically insignificant if (1) one skilled in the art would consider the level of nectin-4 expression to be low in light of the protein product of the housekeeping gene; (2) the presence or absence of small amounts of nectin-4 makes no biological difference to the cell or tissue; and (3) if this small amount of nectin-4 is removed from cells or tissues, the cells or tissues will continue to function substantially the same as before such removal. The known expression of nectin-4 can be determined independently using qPCR analysis. Known nectin-4 expression can also be independently determined using any immunoassay described below with an anti-nectin-4 antibody that has been determined to be specific for nectin-4 (e.g., a nectin-4-specific antibody identified in specificity screening methods) as described higher). Suitable immunoassays include, by way of example and without limitation, an IHC assay, an immunoblot assay, a FACS assay, or an ELISA. In some embodiments, the nectin-4 mRNA level may differ from being zero in the negative control due to background noise of the assay or due to biologically insignificant low levels of retained nectin-4. A negative control for nectin-4 can be considered negative for nectin-4, for example, if the level of nectin-4 mRNA in the qPCR assay is substantially similar to the level of mRNA detected using a set of nonspecific primers, or if the level of nectin-4 mRNA is biologically insignificant due to the level Nectin-4 mRNA in nectin-4 positive control. Likewise, nectin-4 detected by immunoassays using another nectin-4-specific antibody in the negative control may not be null due to background noise of the assay or due to biologically insignificant low levels of retained nectin-4. Background noise may be caused by nonspecific binding of non-nectin-4 assay reagents to samples. A nectin-4 negative control can be considered negative for nectin-4, for example, if the level of nectin-4 in the negative control detected with an anti-nectin-4 antibody is substantially similar to the level detected with an isotype control antibody in the same assay . In some assays, background noise may account for a significant proportion of the detected nectin-4 expression signal.

[00515] In addition, positive and negative controls have been defined and published by those skilled in the art, including positive and/or negative tissues, cells (including, for example, cell lines), and pathological samples. Such literature can be easily found by searching databases such as Pubmed (eg, using a search term such as nectin-4, expression, positive, negative, and/or distribution) and analyzing the search results.

[00516] Accordingly, the relative amount of antibody or antigen binding fragment thereof bound to a negative control cell not expressing nectin-4 may be about 50%, 40%, 30%, 20%, 10%. 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.03%, 0.01%, 0.001% or less compared to the amount of antibody or antigen-binding fragment associated with the image of a cell expressing nectin-4.

[00517] Therefore, in some embodiments of the methods of the present invention, the control level of nectin-4 expression is the level of expression of nectin-4 in the negative control, wherein the expression of nectin-4 in the negative control is independently determined using a qPCR assay, an IHC assay with a second antibody , immunoblotting assay with a secondary antibody, FACS assay with a secondary antibody, or ELISA with a secondary antibody.

[00518] In other embodiments of the methods of the present invention, the control level of nectin-4 expression is the level of expression of nectin-4 in a positive control cell expressing nectin-4, wherein the expression of nectin-4 in the positive control is determined independently using a qPCR assay, the assay IHC with a second antibody, immunoblotting with a second antibody, FACS with a second antibody, or ELISA with a second antibody.

[00519] The level of expression of nectin-4 in the sample may differ from the level of expression in the positive control. The relative amount of antibody or antigen binding fragment thereof bound to a tissue sample may be approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120 %, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80, 90, 100, 200, 500 or greater amount of antibody or antigen binding fragment thereof bound to a positive control cell expressing nectin-4.

[00520] Many different PCR or qPCR protocols are known in the art and are exemplified below herein and can be directly used or adapted to determine the level of nectin-4 mRNA in samples or controls that can be used as a proxy for nectin-4 protein expression levels in samples or controls. Quantitative PCR (qPCR) (also called real-time PCR) is used and adapted in some embodiments because it not only provides a quantitative measurement but also reduces time and contamination. As used herein, the term "quantitative PCR" (or "qPCR") refers to directly monitoring the progress of PCR amplification as it occurs, without the need for repeated sampling of reaction products. In qPCR, reaction products can be monitored by a signaling mechanism (e.g. fluorescence) as they are generated and monitored after the signal rises above background levels but before the reaction reaches a plateau. The number of cycles required to reach a detectable or "threshold" level of fluorescence is directly dependent on the concentration of the targets being amplified at the start of the PCR process, allowing signal intensity to be measured to provide a real-time measurement of the amount of target nucleic acid in the sample. When qPCR is used to determine the level of mRNA expression, an additional step of reverse transcription of the mRNA into DNA is performed before the qPCR analysis. Examples of PCR methods can be found in the literature (Wong et al., BioTechniques 39:75-85 (2005); D'haene et al., Methods 50:262-270 (2010)), which is incorporated herein by reference in its entirety. volume. Examples of PCR assays can be found in US Pat. No. 6,927,024, which is incorporated herein by reference in its entirety. Examples of RT-PCR methods can be found in US Pat. No. 712,799, which is incorporated herein by reference in its entirety. The fluorescence in situ PCR method is described in US Pat. No. 7,186,507, which is incorporated herein by reference in its entirety.

[00521] In one specific embodiment, qPCR can be performed to detect or measure nectin-4 mRNA levels as follows. Briefly, the average Ct (cycle threshold) (or referred to interchangeably as Cq (cycle quantitation)) values of replicate qPCR reactions for nectin-4 and one or more housekeeping genes are determined. The average Ct values for nectin-4 can then be normalized to the Ct values for housekeeping genes using the following approximate formula: nectin-4 - ΔCt = (average Ct value for nectin-4 - average Ct value for housekeeping gene A). Relative nectin-4-ΔCt can then be used to determine the relative level of nectin-4 mRNA, for example, using the expression formula mRNA=2 ^-ΔCt . The sum of Ct and Cq values is given in the MIQE guideline (Bustin et al., The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55:4 (2009)).

[00522] Other conventional methods known in the art can also be used to quantify mRNA expression in a sample, including Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106: 247-283 (1999)); RNase protection assays (Hod, Biotechniques 13: 852-854 (1992)); microarrays (Hoheisel et al., Nature Reviews Genetics 7: 200-210 (2006); Jaluria et al., Microbial Cell Factories 6: 4 (2007)); and polymerase chain reaction (PCR) (Weis et al., Trends in Genetics 8: 263-264 (1992)). Alternatively, mRNA expression levels can be determined by sequencing methods. Representative methods for sequencing-based gene expression analysis include sequential analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) analysis of gene expression.

[00523] As previously stated, tissue images can be, but are not limited to, tissues, biological fluid, tissue fractions and/or cells isolated from an organism, such as a mammal, particularly a human. Therefore, tissue samples can be obtained from various organs of an individual, including humans. In some embodiments, tissue samples are obtained from organs suspected of having a disease, dysfunction, or disorder, such as cancer. In other embodiments, tissue samples are obtained from normal organs of the patient being tested or from a second individual.

[00524] In certain embodiments of the methods of the present invention, the tissue includes tissue from the bladder, ureter, breast, lung, colon, rectum, ovary, fallopian tube, esophagus, cervix, uterine endometrium, skin, larynx, bone marrow, saliva iron, kidney, prostate, brain, spinal cord, placenta, adrenal gland, pancreas, parathyroid gland, pituitary gland, testicle, thyroid gland, spleen, tonsil, thymus, heart, stomach, small intestine, liver, skeletal muscle, peripheral nerve , mesothelium or eyes.

[00525] Anti-nectin-4 antibodies associated with samples can be detected using a variety of immunoassays known in the art, including IHC analysis, immunoblotting analysis, FACS analysis and ELISA.

[00526] Nectin-4 can be detected using an anti-nectin-4 antibody in various IHC assays. IHC staining of tissue sections has been shown to be a reliable method for assessing or detecting proteins in a sample. IHC techniques use an antibody to probe and visualize cellular antigens in situ, usually by chromogenic or fluorescent methods. Primary antibodies or antisera, such as polyclonal antisera and monoclonal antibodies, that specifically target nectin-4 can be used to detect expression in an IHC assay. In some embodiments, the tissue sample is contacted with a primary antibody for a particular target for a period of time sufficient for binding of the antibody to the target to occur. As discussed in detail previously, antibodies can be detected using direct labels on the antibodies themselves, such as radioactive labels, fluorescent labels, hapten tags such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody containing an antiserum, a polyclonal antiserum, or a monoclonal antibody specific for the primary antibody. IHC protocols and kits are well known in the art and are commercially available. Automated systems for slide acquisition and IHC processing are commercially available. The Leica BOND Autostainer and Leica Bond Refine Detection are examples of such an automated system.

[00527] In some embodiments, the IHC assay is performed with an unlabeled primary antibody in combination with a labeled secondary antibody in an indirect assay. The indirect assay uses two antibodies to detect target proteins, such as nectin-4, in a tissue sample. First, an unconjugated primary antibody is applied to the tissue (first layer), which reacts with the target antigen in the tissue sample. An enzyme-labeled secondary antibody that specifically recognizes the antibody isotype of the primary antibody is then applied (second layer). The secondary antibody reacts with the primary antibody, followed by application of a chromogen substrate. The second layer antibody may be labeled with an enzyme such as peroxidase, which reacts with the chromogen 3,3-diaminobenzidine (DAB) to form a brown precipitate at the reaction site. This method is sensitive and versatile due to the strong signal amplification through the signal amplification system.

[00528] In some embodiments, a signal amplification system may be used to increase detection sensitivity. "Signal amplification system" as used herein means a system of reagents and methods that can be used to amplify the signal when detecting a bound primary or secondary antibody. The signal amplification system increases the sensitivity of target protein detection, increases the detected signal and reduces the lower limit of detection limits. There are several types of signal amplification systems, including enzyme labeling systems and macrolabeling systems. These systems/approaches are not mutually exclusive and can be used in combination for additive effect.

[00529] Macrolabeling or macrolabeling systems are arrays of tags numbering in the tens (eg, phycobiliproteins) to millions (eg, fluorescent microspheres) attached or embedded in a common scaffold. The scaffold can be coupled to a target specific affinity reagent, such as an antibody, and thus the embedded tags bind together to the target upon binding. Labels in macrotags can be any of the labels described herein, such as fluorophores, haptens, enzymes and/or radioisotopes. In one embodiment, the signal amplification system uses a labeled polymer conjugated to a second antibody. Polymer technology uses an inert dextran “needle” molecule labeled with the HRP enzyme, to which 1, 2, 3, 4, 5, 6, 7, 8, 910, 15, 20, 25, 30, 50 or more molecules can be attached a second antibody, which makes the system even more sensitive.

[00530] A signal amplification system based on an enzyme tagging system uses the catalytic activity of enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase to produce high density in situ tagging of a target protein or nucleic acid sequence. In one embodiment, tyramide can be used to increase the HRP signal. In such a system, HRP enzymatically converts the labeled tyramide derivative into highly reactive, short-lived tyramide radicals. The labeled reactive tyramide radicals are then covalently bound to residues (mainly the phenolic residue of protein tyrosine residues) in close proximity to the HRP-antibody-target interaction site, resulting in an amplification of the number of tags per site with minimal diffusion-related loss of signal localization. Therefore, the signal can be amplified by 1, 2, 3, 4, 5, 6, 7, 8, 910, 15, 20, 25, 30, 50, 75 or 100 times. As is known to one skilled in the art, labels on tyramide can be any of the labels described herein, including fluorophores, enzymes, haptens, radioisotopes and/or photophores. Other enzymatic reactions can also be used to enhance the signal. For example, enhanced enzyme-labeled fluorescence (ELF) is available for alkaline phosphatase, where alkaline phosphatase enzymatically cleaves a weakly blue fluorescent substrate (ELF 97 phosphate) and converts it into a bright yellow-green fluorescent precipitate that exhibits an unusually large Stokes shift and excellent photostability . Both the tyramide-based signal amplification system and the ELF signal amplification system are commercially available, for example, from ThermoFisher Scientific (Waltham, MA USA 02451).

[00531] Thus, in some embodiments of the methods of the present invention, the expression level of nectin-4 is determined using a signal amplification system.

[00532] In some embodiments, the sample is then contrasted to identify cellular and subcellular elements.

[00533] In some embodiments, detection of the level of nectin-4 expression can also be achieved using antibodies described herein using immunoblotting analysis. In some embodiments of immunoblotting analysis, proteins are often (but not necessarily) separated by electrophoresis and transferred to membranes (usually a nitrocellulose or PVDF membrane). Similar to IHC assays, primary antibodies or antisera, such as polyclonal antisera and monoclonal antibodies, that specifically target nectin-4 can be used to detect protein expression. In some embodiments, the membrane is contacted with a primary antibody for a particular target for a period of time sufficient for antibody-antigen binding, and the bound antibodies can be detected using direct labels on the primary antibodies themselves, e.g., radiolabeled fluorescent tags, hapten tags , such as biotin, or enzymes such as horseradish peroxidase or alkaline phosphatase. In other embodiments, an unlabeled primary antibody is used in an indirect assay as described above in combination with a labeled second antibody specific for the first antibody. As described herein, the second antibodies can be labeled, for example, with enzymes or other detectable labels, such as fluorescent labels, luminescent labels, colorimetric labels, or radioisotopes. Immunoblotting protocols and kits are well known in the art and are commercially available. Automated immunoblotting systems, such as iBind Western Systems for Western blotting (ThermoFisher, Waltham, MA USA 02451), are commercially available. Immunoblotting includes, but is not limited to, Western blot, intracellular Western blot, and dot blot. Dot blot is a simplified procedure in which protein samples are not separated by electrophoresis, but are placed directly on a membrane. Intracellular Western blotting involves seeding cells into microtiter plates, fixing/permeabilizing the cells, and then detecting with a labeled first antibody or an unlabeled first antibody followed by a labeled second antibody as described herein.

[00534] In other embodiments, nectin-4 expression levels can also be detected using the antibodies described herein in a flow cytometry assay, including a fluorescence activated cell sorting (FACS) assay. Similar to IHC or immunoblotting assays, first antibodies or antiserum, such as polyclonal antiserum and monoclonal antibodies, that specifically target nectin-4 can be used to detect protein expression in a FACS analysis. In some embodiments, cells are stained with primary antibodies against a specific target protein for a time sufficient to allow antibody-antigen binding to occur, and bound antibodies can be detected using direct labels on the primary antibodies, such as fluorescent labels or hapten labels such as biotin on first antibodies. In other embodiments, an unlabeled primary antibody is used in an indirect assay as described above in combination with a fluorescently labeled second antibody specific for the first antibody. FACS provides a method for sorting or analyzing a mixture of fluorescently tagged biological cells, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. Thus, the flow cytometer detects and reports the intensity of the fluorichrome-labeled antibody, which indicates the level of expression of the target protein. Therefore, the expression level of surface proteins (such as nectin-4) can be detected using antibodies against the target protein. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. Methods for performing staining and FACS analysis are well known to one skilled in the art and are described by Teresa S. Hawley and Robert G. Hawley in Flow Cytometry Protocols, Humana Press, 2011 (ISBN 1617379506, 9781617379505).

[00535] In other embodiments, nectin-4 expression levels can also be detected using immunoassays such as an enzyme-linked immunosorbent assay (EIA) or ELISA. EIA and ELISA assays are known in the art, for example, for the analysis of a wide range of tissues and samples, including blood, plasma, serum or bone marrow. A wide variety of ELISA assay formats are available, see, for example, US Patent Nos. 4,016,043, 4,424,279, and 4,018,653, which are incorporated herein by reference in their entirety. These include both single-site and dual-site or “sandwich” non-competitive assays as well as traditional competitive binding assays. These assays also involve direct binding of the labeled antibody to the target protein. Sandwich assays are commonly used assays. There are a number of variations of sandwich analysis. For example, in a typical direct assay, an unlabeled antibody is immobilized on a solid support and the test sample is brought into contact with the bound molecule. After a sufficient period of incubation, for a time sufficient to form an antibody-antigen complex, a second antibody specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is added and incubated for a time sufficient for the formation of another antibody-antigen complex -labeled antibody. Any unreacted material is washed away and the presence of antigen is determined by monitoring the signal with a reporter molecule. The results can be either qualitative, by simply observing the visible signal, or can be quantitatively assessed by comparison with a control sample containing known amounts of the target protein.

[00536] In some embodiments of the EIA or ELISA assays, the enzyme is conjugated to a second antibody. In other embodiments, fluorescently labeled second antibodies can be used in place of an enzyme-labeled second antibody to produce a detectable signal in an ELISA assay format. When activated by a light beam of a specific wavelength, a fluorochrome-labeled antibody absorbs light energy, causing the molecule to become excitable and then emit light with a characteristic color that is visually detectable using a light microscope. As in EIA and ELISA, the fluorescently labeled antibody can bind to the first antibody-target protein complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of the appropriate wavelength, the observed fluorescence indicating the presence of the target protein of interest. Immunofluorescence and EIA methods are well known in the art and are described herein.

[00537] For the immunoassays described herein, any enzyme or non-enzyme label can be used, provided that enzymatic activity or the non-enzyme label, respectively, can be detected. Thus, the enzyme produces a detectable signal that can be used to detect the target protein. Particularly effective detectable signals are chromogenic or fluorogenic signals. Accordingly, particularly effective enzymes for use as a tag include tags for which a chromogenic or fluorogenic substrate is available. Such chromogenic or fluorogenic substrates can be converted by an enzymatic reaction into an easily detectable chromogenic or fluorescent product that can be readily detected and/or quantified by microscopy or spectroscopy. Such enzymes are well known to those skilled in the art, including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes that have well-known chromogenic or fluorogenic substrates include various peptidases, where chromogenic or fluorogenic peptide substrates can be used to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates is also well known in the field of bacterial diagnostics, including, but not limited to, the use of α- and β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactosidase, 6-phosphogalactohydrolase, β-glucosidase, α-glucosidases, amylases, neuraminidases, esterases, lipases, and the like (Manafi et al., Microbiol. Rev. 55: 335-348 (1991)), and such enzymes with known chromogenic or fluorogenic substrates can be easily adapted for use in the methods of the present invention.

[00538] Various chromogenic or fluorogenic substrates for producing detectable signals are well known to those skilled in the art and are commercially available. Examples of substrates that can be used to produce detectable signals include, but are not limited to, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), chloronaphthol (4-CN)( 4-chloro-1-naphthol), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD) and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase; 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR/AS-MX), and p-nitrophenyl phosphate (PNPP) for alkaline phosphatase; 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase; etc. Examples of fluorogenic substrates include, but are not limited to, 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis (2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis (cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatases; QuantaBlu™ and QuantaRed™ for horseradish peroxidase; 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside) and naphthofluorescein di-(β-D-galactopyranoside) for β-galactosidase; 3-acetylumbelliferyl β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside for β-glucosidase; and 4-methylumbelliferyl-α-D-galactopyranoside for α-galactosidase. Examples of enzymes and substrates for producing a detectable signal are also described, for example, in US publication 2012/0100540. Various detectable enzyme substrates, including chromogenic or fluorogenic substrates, are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Typically, substrates are converted into products that form precipitates that are deposited at the site of the target nucleic acid. Other examples of substrates include, but are not limited to, HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black™, Warp Red™, Vulcan Fast Red and Ferangi Blue others manufacturers (Concord CA; biocare.net/products/detection/chromogens).

[00539] In some embodiments of the immunoassay, the detectable label may be directly associated with either a first antibody or a second antibody that detects an unlabeled first antibody. Examples of detectable labels are well known to those skilled in the art, including, but not limited to, chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Examples of fluorophores that can be used as labels include, but are not limited to, rhodamine derivatives such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110 and derivatives thereof such as tetramethylrhodamine-5 -(or 6), lissamine rhodamine B and the like; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalenes such as dansyl (5-dimethylaminonapthalene-1-sulfonyl); coumarin derivatives such as 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), Alexa fluorescent dyes (Molecular Probes) and the like; 4,4-difluoro-4-boron-3a,4a-diaza-s-indacene (BODIPY™) and its derivatives (leMolcular Probes; Eugene Oreg.); pyrene and sulfonated pyrene, such as Cascade Blue™ and derivatives thereof, including 8-methoxypyrene-1,3,6-trisulfonic acid, and the like; pyridyloxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Lucifer Yellow (3,6-disulfonate-4-aminonaphthalimide) and their derivatives; fluorescent dyes CyDye™ (Amersham/GE Healthcare Life Sciences; Piscataway NJ) and the like. Examples of chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatics such as nitrophenyl, diazo dyes, dabsil (4-dimethylaminoazobenzene-4'-sulfonyl) and the like.

[00540] Techniques well known to one skilled in the art, such as microscopy or spectroscopy, can be used to visualize chromogenic or fluorescent detectable signals associated with the bound first or second antibodies.

[00541] "Determination of nectin-4 expression" refers to placing the expression of nectin-4 in a test biological sample on a control system (or control scale) so that a person familiar with the control system can determine the relative expression of nectin-4 in the test sample compared to with other samples that are located in the specified system. Therefore, such determination involves comparing the expression of nectin-4 in the tissue sample under study with the control level of nectin-4 expression.

[00542] In some embodiments, the system may be a quantitative system in which the actual molar concentration or molar amount of nectin-4 in the test sample is compared to the molar concentration or molar amount of control expression levels. In some embodiments, other surrogate numerical measurements that are linearly proportional to the molar concentration or number of moles may be used. Examples of these surrogate measurements include fluorescence intensity measurements of bound antibodies, luminescence intensity measurements of bound antibodies, radioactivity measurements of bound antibodies, and/or colorimetric or chromogenic measurements of bound antibodies.

[00543] In other embodiments, said system may be a categorization system. The categorization system can have only 2 categories and as many as 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more categories. In one embodiment, the system has a positive category for samples that are positive for nectin-4 expression (positive) and a negative category for samples that are negative for nectin-4 expression (negative). In such a categorization system, determining the expression of nectin-4 in a test sample involves comparing the expression of nectin-4 in the test sample with the nectin-4 expression of a positive control sample and/or a negative control sample and placing the test sample in the positive category if the test sample expresses nectin-4 at the same or higher level than the positive control. In another embodiment, the categorization system has 3 categories, for example, negative, medium and high expression categories. In another embodiment, the categorization system has 4 categories, such as negative, low/weak, medium/moderate, and high/strong expression. In some aspects, determining nectin-4 expression in a test sample includes comparing nectin-4 expression in the test sample to one or more known category nectin-4 expression controls and placing the test sample into categories according to its relative expression relative to the control(s). .

[00544] In some embodiments, said system may be an evaluation system. The scoring system can be similar to the categorization system, except that the categories are replaced by points. For example, a system of negative and positive expression of nectin-4 in 2 categories could be a scoring system of 0 and 1, where 0 means negative and 1 means positive. In another embodiment, the 3 category system may be a 0, 1, and 2 rating system, where 0 is negative, 1 is low/weak expression, and 2 is moderate/medium and high/strong expression. In another embodiment, the 4 category system may be a 0, 1, 2, and 3 rating system, where 0 is negative, 1 is low/weak, 2 is moderate/moderate, and 3 is high/strong expression. Similar to the categorization system, determining the expression of nectin-4 in a test sample in a scoring system involves comparing the expression of nectin-4 in the test sample with one or more controls for nectin-4 expression having a known score, and assigning a score to the test sample according to its relative expression compared to the control. A scoring system, however, may have scores with discrete numbers (for example, a system of 0, 2, 4, 6, 8 and 10 or a system of 1, 4, 6, 11, 13 and 19), with fractions (for example, 1 , 1.5, 2, 2.5, 3, 3.5 and the like), negative numbers, numbers starting with any number (for example, 1000, 2000, 3000 and the like), and any set of numbers that can be used to track relative protein expression in biological samples.

[00545] In some embodiments, the system may be a percentage of cells combined with categorization or scoring systems. Combining cell percentage with a categorization or scoring system results in a more accurate grading of samples. In such a combined system, each cell of a biological sample is assigned a value or placed into a categorization or scoring system, and the approximate percentage of cells in a test sample in each category or with one score is determined. In one example, measuring the percentage of cells can be combined with a 2-category system - positive and negative nectin-4 expression, where the sample is not measured as a whole, but is broken down into the percentage of cells positive for nectin-4 expression and the percentage of cells negative for nectin-4 expression. expression of nectin-4. For example, in a 2 category system positive and negative for nectin-4 expression, a test sample may be negative for nectin-4 expression if placed in a 2 category system, but have 10% of cells positive for nectin-4 expression and 90% of cells positive for nectin-4 expression. , negative for nectin-4 expression. In one embodiment, the system includes a percentage of cells combined with a 2 category or 2 point system. In one embodiment, the system includes a percentage of cells combined with a 3 category or 3 point system. In one embodiment, the system includes a percentage of cells in combination with a 4 category or 4 point system. In one embodiment, the system includes a percentage of cells in combination with a 5 category or 5 point system. In one embodiment, the system includes a percentage of cells in combination with a 6 category or 6 point system. In one embodiment, the system includes a percentage of cells in combination with a 7 category or 7 point system. In one embodiment, the system includes a percentage of cells in combination with an 8 category or 8 point system. In one embodiment, the system includes a percentage of cells in combination with a 9 category or 9 point system. In one embodiment, the system includes a percentage of cells in combination with a 10 category or 10 point system. In one embodiment, the system includes cell percentages in combination with any categorization or scoring system. In one embodiment, the system includes a percentage of cells combined with a 4-point system consisting of 0 (nectin-4 negative), 1 (low/weak nectin-4 expression), 2 (medium/moderate nectin-4 expression), and 3 (high/strong expression of nectin-4).

[00546] In some aspects, the reference system may be the ratio of nectin-4 expression to expression of at least one control protein. In some embodiments, this ratio may be the ratio of quantitative measurements of nectin-4 expression and measurements of a control protein, where quantitative measurements include measurements described herein, such as molar concentration, number of moles, fluorescence intensity measurements of bound antibodies, luminescence intensity measurements bound antibodies, measurements of radioactivity of bound antibodies, and/or colorimetric or chromogenic measurements of bound antibodies. In other embodiments, this ratio may also be the ratio of the scores assigned to nectin-4 and control protein expression, in accordance with any scoring system presented herein. The control protein may be a protein of normal cellular function, such as actin, GAPDH, GDI, or any protein expressed by a housekeeping gene. For example, if a test sample has a score of 3 for nectin-4 expression and a score of 1 for actin expression, the ratio could be defined as 3.

[00547] In other aspects, the control system may use the results of a mathematical function using one or more categories, scores, ratios, percentages, and quantities as input. In some embodiments, a mathematical function uses addition, addition, multiplication, or a combination of these operators to combine various inputs to produce results that provide more detailed information about nectin-4 expression in a sample and/or a more precise scale gradation of nectin-4 expression. In one embodiment, the mathematical function calculates H scores based on a combination of a percentage and a four-point system. For example, H scores are calculated by summing the products of the percentage of cells (0-100) having each nectin-4 expression score (0=negative, 1=low/weak, 2=moderate/moderate, and 3=high/strong). For example: a sample with 10% cells with a score of 3, 30% of cells with a score of 2, 20% of cells with a score of 1, and 40% of cells with a score of 0 would have an H score of (3x10) + (2x30) + (1x20) ) + (0×40) = 110.

[00548] Therefore, as discussed herein, the level of nectin-4 expression can be determined using a categorization system, a scoring system, the ratio of nectin-4 expression to the expression of at least one control protein, the percentage of cells in said categorization system, or scoring, quantifying the nectin-4 staining signal, or the result of a mathematical function using one or more of said categorization, scoring, ratio, percentage, and quantifying the input data.

[00549] Most formalin-fixed tissues require an antigen retrieval step prior to immunohistochemical staining. Methylene bridges are formed during the fixation of protein cross-links and cover the epitopes of antigens. In antigen retrieval methods, these methylene bridges are broken and epitopes are exposed, allowing antibodies to bind. In some embodiments, antigens are produced by heat-induced epitope generation (HIER). In other embodiments, antigens are isolated by enzymatic retrieval (eg, proteolytic digestion). For HIER, formalin-fixed paraffin-embedded (FFPE) tissue sections can be heated at 50, 55, 60, 65, 70, 75, 80, 85°C, 90°C, 95°C, 100°C, 105°C , 110°C, 115°C, 120°C, 125°C or 130°C. FFPE tissue sections can be heated at any of these temperatures for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95. 100, 105, 110, 115, 120, 150, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, 960, 1020, 1080, 1140, 120 0.1260 1320 , 1380 and 1440 minutes. The HIER procedure at any of the above temperatures and for any of the above time periods can be performed in a solution with a pH of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8, 2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, 10, 10.2, 10.4, 10.6, 10.8, 11, 11.5, or 12. In some embodiments, HIER may be performed at a temperature selected from the group consisting of 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110° C, 115°C, for a time selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40 and at a pH selected from the group consisting of 8, 8.2, 8.4 , 8.6, 8.8, 9, 9.2, 9.4 9.6, 9.8, 10, 10.2, 10.4.

[00550] Thus, the methods of the present invention further include the step of isolating the nectin-4 epitope by heat-induced epitope release (HIER).

[00551] Unlabeled antibodies, labeled antibodies and derivatives thereof and analogs thereof that immunospecifically bind to nectin-4 antigen can be used for diagnostic purposes to detect, diagnose or monitor nectin-4-mediated disease. Thus, provided herein are methods for detecting a nectin-4-mediated disease, comprising: (a) analyzing the expression of a nectin-4 antigen in cells or a tissue sample of an individual using one or more antibodies of the present invention that immunospecifically bind to a nectin-4 antigen. 4; and (b) comparing the level of nectin-4 antigen with a control level, for example with levels in normal tissue samples (for example, in tissue samples from a patient who does not have a nectin-4-mediated disease, in tissue samples from the same patient before the onset of the disease, a normal cell in tissue samples from the same patient or normal tissue samples from a normal organ from the same patient), whereby an increase in the test level of nectin-4 antigen compared with the control level of nectin-4 antigen is indicative of nectin-4-mediated disease.

[00552] Also provided herein is a diagnostic assay for diagnosing a nectin-4-mediated disease, comprising: (a) analyzing the level of nectin-4 antigen in cells or a tissue sample of an individual using one or more antibodies of the present invention that immunospecifically bind to nectin-4 antigen; and (b) comparing the level of nectin-4 antigen with a control level, such as levels in normal tissue samples, wherein an increase in the level of the tested nectin-4 antigen compared with a control level of nectin-4 antigen is indicative of a nectin-4-mediated disease. In some embodiments, provided herein is a method of treating a nectin-4-mediated disease in a patient, comprising: (a) analyzing the level of nectin-4 antigen in cells or a tissue sample of the patient using one or more antibodies of the present invention that immunospecifically bind to nectin-4 antigen; and (b) comparing the level of nectin-4 antigen with a control level, such as levels in normal tissue samples, wherein an increase in the level of the tested nectin-4 antigen compared with a control level of nectin-4 antigen is indicative of a nectin-4-mediated disease. In some embodiments, the method further comprises (c) administering an effective amount of an antibody of the present invention to a patient identified as having a nectin-4-mediated disease. A more accurate diagnosis of nectin-4-mediated disease may allow healthcare providers to use preventive measures or aggressive treatment at an earlier stage, thereby preventing the development or further progression of nectin-4-mediated disease.

[00553] Antibodies of the present invention can be used to analyze levels of nectin-4 antigen in a biological sample using classical immunohistological methods, as described herein, or as known to those skilled in the art (for example, see Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; and Jalkanen et al., 1987, J. Cell Biol. 105: 3087-3096). Other antibody-based methods that can be used to detect protein gene expression include immunoassays such as ELISA and radioimmunoassay (RIA). Suitable labeled antibody assays are known in the art and include enzymatic tags such as glucose oxidase; radioisotopes such as iodine ( ^125I , ^121I ), carbon ( ^14C ), sulfur ( ^35S ), tritium ( ^3H ), indium ( ^121In ) and technetium ( ^99Tc ); luminescent tags such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

[00554] One aspect presented herein is the detection and diagnosis of nectin-4-mediated disease in humans. In one embodiment, the diagnosis includes: a) administering (eg, parenterally, subcutaneously, or intraperitoneally) to the patient an effective amount of a labeled antibody that immunospecifically binds to nectin-4 antigen; b) waiting for a period of time after administration to allow the labeled antibody to concentrate in areas of the patient that express nectin-4 antigen (and to allow unbound labeled molecule to disappear to background levels); c) determining the background level; and d) detecting the labeled antibody in the patient, such that detection of the labeled antibody above background levels indicates that the patient has a nectin-4-mediated disease. The background level can be determined in a variety of ways, including comparing the amount of detected labeled molecule with a standard value previously determined for a particular system.

[00555] It is known in the art that the size of the patient and the imaging system used can determine the number of imaging slices needed to obtain diagnostic images. In the case of a human radioactive isotopic fragment, the amount of radioactivity applied is typically between 5 and 20 millicurides ^{of 99} Tc. The labeled antibody will then accumulate at the site of cells that contain the specific protein. In vivo tumor imaging is described in SW Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

[00556] Depending on several variables, including the type of label used and the route of administration, the time interval after the individual and for clearance of unbound labeled antibody to background levels is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is from 5 to 20 days or from 5 to 10 days.

[00557] In one embodiment, monitoring for a nectin-4-mediated disease is performed by repeating the method for diagnosing the nectin-4-mediated disease, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, and so on. similar.

[00558] The labeled molecule can be detected in a patient using methods known in the art for in vivo scanning. These methods depend on the type of label being used.

One skilled in the art will be able to determine a suitable method for detecting a particular mark. Methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scans such as emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

[00559] In a specific embodiment, the molecule is radiolabeled and detected in the patient using a radiation sensitive surgical instrument (Thurston et al., US Pat. No. 5441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence-sensitive scanning instrument. In another embodiment, the molecule is labeled with a positron-emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI). 30. Sets

[00560] Also disclosed herein are kits containing an antibody (eg, anti-nectin-4 antibody) of the present invention or a composition thereof (eg, a pharmaceutical composition) packaged in a suitable packaging material. The kit optionally contains a label or package insert that includes a description of the components or instructions for the in vitro, in vivo or ex vivo use of the components of the kit.

[00561] The term "packaging material" refers to the physical structure in which the components of the kit are housed. The packaging material may contain the components in a sterile state and may be made from material commonly used for such purposes (eg, paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, and the like).

[00562] The kits of the present invention may contain labels or inserts. Labels or inserts include “printed materials,” such as paper or cardboard, separately or attached to a component, kit, or packaging material (e.g., box) or attached, for example, to an ampoule, tube, or vial containing a component of the kit. The labels or inserts may further include a computer-readable medium such as a disk (e.g., a hard disk, card, or memory disk), an optical disk such as a CD or DVD-ROM/RAM, DVD, MP3, magnetic tape, or electrical storage device, such as RAM and ROM, or hybrids thereof, such as magnetic/optical storage devices, flash media, or memory cards. Labels or inserts may include information about the manufacturer, lot number, manufacturer location, and date.

[00563] The kits of the present invention may further include other components. Each component of the kit can be in a separate container, and all the different containers can be in one package. Kits can also be designed for cold storage. In addition, a kit can be created containing antibodies of the present invention or cells that contain nucleic acids encoding antibodies of the present invention. The cells in the kit may be stored under appropriate storage conditions prior to use. Additional components may include, by way of example, a positive or negative control sample (eg, tissue samples from a patient known to have a nectin-4-mediated disease, tissue samples from a patient not known to have a nectin-4-mediated disease, tissue samples from the same patient before disease onset, normal cell tissue samples from a patient with nectin-4 mediated disease, or normal tissue samples from a normal organ from the same patient), buffers, wash solutions, antibody detection reagents, and/or enhancement reagents signal.

[00564] Also disclosed herein are panels of antibodies that immunospecifically bind to nectin-4 antigen. In specific embodiments, panels of antibodies disclosed herein have different association rate constants, different dissociation rate constants, different affinity for nectin-4 antigen, and/or different specificity for nectin-4 antigen. In some embodiments, the panels of the present invention comprise or consist of about 10, preferably about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950 or about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96-well or 384-well plates, such as plates for assays such as ELISA.

[00565] Unless otherwise specified, all technical and scientific terms used in this description have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[00566] All applications, publications, patents and other references, GenBank and ATCC references mentioned herein are incorporated by reference in their entirety. In case of any discrepancies, the present specification containing definitions shall be relied upon.

[00567] As used herein, singular forms include the plural unless the context clearly indicates otherwise. Thus, for example, reference to a “peptide sequence” includes a plurality of such sequences, and so on.

[00568] As used herein, numerical values are often presented in range format throughout this document. The use of the range format is merely for convenience and brevity and should not be construed as strictly limiting the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numeric values within that range, and all numeric values or numeric ranges, including integers within such ranges and fractions of values or integers within ranges, unless the context clearly indicates otherwise. This design applies regardless of the range and in all contexts of this patent document. Thus, for example, reference to the 90-100% range includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94 %, 91-93% and the like. Reference to the 90-100% range also includes 91%, 92%, 93%, 94%, 95%, 95%, 97% and the like, as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5% and the like, 92.1%, 92.2%, 92.3%, 92.4%, 92.5% and the like, and so on.

[00569] Additionally, reference to the range 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80 80- 90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200 -225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and the like. In a further example, reference to the range 25-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-25000, 25000-50000 includes any numerical value or range within or encompassing such values, for example, 25, 26, 27, 28, 29... 250, 251, 252, 253, 254... 500, 501, 502, 503, 504... and the like.

[00570] As also used herein, a number of ranges are disclosed herein. Applications of a number of ranges include combinations of upper and lower ranges to provide another range. This design applies regardless of the range and in all contexts of this patent document. Thus, for example, reference to a number of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150 includes ranges such as 5 - 20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150 and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150 and 20-40, 20-50, 20-75, 20-100, 20-150 and so on.

[00571] For the sake of brevity, certain abbreviations are used throughout this document. One example is the one-letter abbreviation for amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows:

Alanine Ala (A)

arginine Arg(R)

asparagine Asn(N)

aspartic acid Asp (D)

cysteine Cys(C)

glutamic acid Glu(E)

glutamine Gln (Q) glycine Gly (G)

histidine His (H)

Isoleucine Ile(I)

leucine Leu (L)

lysine Lys (K)

methionine Met (M)

Phenylalanine Phe (F)

proline Pro (P)

serine Ser(S)

threonine Thr (T)

tryptophan Trp (W)

tyrosine Tyr(Y)

valine Val(V)

[00572] The invention is generally described herein using affirmative expressions to describe numerous embodiments. The invention also, in particular, includes embodiments that do not relate to a specific subject, in whole or in part, such as substances or materials, process steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention as a whole is not expressed herein in terms of “the invention does not include,” aspects that are not expressly included in the invention are nevertheless disclosed herein.

[00573] A number of embodiments of the invention are described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not to limit, the scope of the invention as described in the claims. EXAMPLES

[00574] The examples in this section are offered by way of illustration and not by way of limitation.

EXAMPLE 1: Antigen Preparation

[00575] For subsequent immunization experiments, a fragment of human nectin-4, such as a fragment containing amino acid sequence 31-346 (SEQ ID NO: 2) of human nectin-4 (SEQ ID NO: 1), was created and used as an antigen.

[00576] 6xHN-labeled nectin 4 (aa 31-346) was produced using commercially available pET expression vectors. Expression was carried out in E.col BL21 (DE3) cells according to the manufacturer's instructions and purified by IMAC (immobilized metal affinity chromatography) purification. Purified nectin 4 (aa 31-346) was used as an immunogen.

[00577] Recombinant nectin-4 (aa (1-348)) for antibody screening was generated in 293FT cells using a modified pTag5/mychis expression vector according to the manufacturer's instructions. The protein was purified using standard IMAC-based purification.

EXAMPLE 2: OBTAINING ANTIBODIES

A. Obtaining and production of antibodies

[00578] Monoclonal antibody M22-321b41.1 was produced by immunizing Balb/C mice with a recombinant fragment (31-346; SEQ ID NO:2) of the extracellular domain of the human nectin-4 protein produced by bacteria using standard hybridoma methods described originally by Kohler and Milstein, Eur. J. Immunol.6, 511 (1976). Hybridoma M22-321b41 was subcloned as M22-321b41.1, which produces a murine kappa IgG2a antibody that specifically binds to the recombinant extracellular domain of the human nectin-4 protein by ELISA. In addition, purified protein A antibody M22-321b41.1 has been shown to stain specifically test tissues that express nectin-4 by IHC.

[00579] M22-321b41.1 hybridoma cells were then grown, expanded, and live cells were frozen for long-term storage in liquid nitrogen. B. Hybridoma sequencing

[00580] The M22-321b41.1 hybridoma antibody heavy and light chain genes were sequenced using RT-PCR.

EXAMPLE 3: SCREENING AND ANTIBODY SELECTION

A. Screening Assays (or Binding Assays)

[00581] Primary ELISA analysis was performed with a fragment of the extracellular domain of nectin-4 (amino acid residues 31-346) obtained as described above.

[00582] A secondary ELISA was performed with a fragment of the extracellular domain of nectin-4 (amino acid residues 31-346) produced in E. coli, a fragment of the extracellular domain of Nectin-4 (amino acid residues 1-346) produced in 293FT cells, nectin-4 -tag5 and two control proteins (one produced by pET and one produced by tag5). B. Antibody selection

[00583] Antibodies were first selected if they showed staining in 3T3-nectin-4 recombinant cells and were not stained in controls. The initially selected antibodies were then tested against a panel of positive and negative control tissues with known nectin-4 mRNA expression as determined by qPCR. Antibodies were tested at different concentrations and with different antigen retrieval protocols. Antibodies were selected if they stained specifically in nectin-4-mRNA-positive tissues, did not stain in nectin-4-mRNA-negative tissues, and were better than the control anti-nectin-4 antibodies tested in terms of staining intensity, ratio stained cells and nonspecific background staining.

EXAMPLE 4. Screening for antibodies specific to nectin-4.

[00584] Antibodies prepared as described above were screened to select antibodies specific for nectin-4. Screening was carried out, for example, using IHC analysis. Briefly, human cancer xenograft tissues from various origins, such as bladder, breast, ovary, pancreas, kidney, skin, lung, and colon, were first tested by qPCR to determine the expression level of nectin-4 in these tissues.

[00585] Quantitative real-time PCR (qPCR) analysis was used to determine the relative expression levels of nectin-4 mRNA. qPCR analysis was performed using a Bio-Rad CFX384 Real-Time PCR System with SsoFast EvaGreen Supermix (Bio-Rad) and 5 ng RNA equivalent of cDNA template. Nectin-4 mRNA expression levels were calculated relative to the expression levels of GAPDH, a housekeeping gene, using a modified delta Cq method with the following formula: 10000x2 ^{-(Nectin-4 Cq GAPDH Cq)} ; the relative expression of nectin-4 was described relative to GAPDH units.

[00586] Primers used in the nectin-4 qPCR assay include: forward primer 191P4D12.1: 5'-GGCTGGAGTTCAATGAGGTTTATTT -3' (SEQ ID NO:41); and reverse primer 191P4D12.2: 5'- TCCAGCAGATTTCAGACTAAGAAGA -3' (SEQ ID NO:42). Primers used in the GAPDH qPCR assay include: GAPDH.1 forward primer: 5'-AGAACATCATCCCTGCCTCTACTG -3' (SEQ ID NO:43); and reverse primer GAPDH.2: 5'-AAATGAGCTTGACAAAGTGGTCGT -3' (SEQ ID NO: 44).

[00587] The cDNA template for qPCR was synthesized using the Bio-Rad iScript Advanced cDNA Synthesis Kit from RNA isolated from tissue or cell line samples using the TRIzol RNA extraction method followed by RNA purification and DNase I digestion using the Qiagen RNeasy Cleanup kit .

[00588] Representative human xenograft tissues are shown in Table 5. The anti-nectin-4 antibodies generated above were then used in an IHC assay to stain tissues with known levels of nectin-4 expression. IHC assays were performed similarly to those detailed in Example 5 below. IHC staining of nectin-4 in various tissues correlated with nectin-4 mRNA levels in those tissues. An antibody was selected as a nectin-4-specific antibody when it produced an IHC staining signal that correlated with nectin-4 mRNA levels, e.g., strong staining in tissues with high levels of nectin-4 mRNA, moderate IHC staining signal in tissues with intermediate levels of nectin mRNA -4, low IHC staining signal in tissues with low levels of nectin-4 mRNA and no IHC staining signal in tissues not expressing nectin-4 mRNA. A total of at least 283 antibody clones were screened, leading to the identification of nectin-4-specific antibodies M22-321b41.1 and M22-244b3.1.1.1

EXAMPLE 5: FUNCTIONAL ANALYSIS: IMMUNOCHISTOCHEMICAL staining of xenographic TISSUE

[00589] Antibodies generated, screened, expressed and purified, for example, as described herein, were further assessed for their ability to specifically stain human xenograft tissues expressing nectin-4 in an IHC assay. For example, two mouse monoclonal antibodies against nectin-4, M22-244b3.1.1.1 and M22-321b41.1 (IgG1 and IgG2a isotypes, respectively), were tested in an IHC assay. Antibodies M22-244b3.1.1.1 and M22-321b41.1 were obtained at a concentration of 1.54 mg/ml and 1.3 mg/ml in phosphate-buffered saline, pH 7.4. As a negative control, a commercial mouse IgG2a isotype control from murine myeloma (clone: UPC10, Sigma-Aldrich, St. Louis, MO) was contacted with β-2,6-fructosan, which does not bind to human nectin-4, or other mammalian antigens were used. .

[00591] IHC staining was performed according to the following example protocols. Briefly, indirect IHC was used to detect nectin-4 in the IHC assay. Specimens were fixed in 10% buffered neutral formalin, processed and embedded in paraffin wax, and prepared as 4-μm tissue sections. After deparaffinization and rehydration, sections were processed and antigen extracted. Epitopes of the nectin-4 antigen were obtained, for example, by heating tissue sections in an aqueous environment, commonly referred to as a thermal epitope reduction (HIER) procedure. For example, antigenic epitopes of nectin-4 were obtained by applying Epitope Retrieval 2 (ER2, an EDTA-based buffer at pH 8.9-9.1) on an automated Leica platform for 20 minutes at 100°C. Tissue sections after antigen retrieval were then incubated with mouse primary antibody M22-321b41.1, M22-244b3.1.1.1, or IgG2a (control antibody). Tissue sections bound to the first antibody were washed and detected with an enzyme-labeled, eg peroxidase-labeled, second antibody. The bound second antibody produced color, for example, using the Leica Bond Refine polymer detection system, which can use the reaction between peroxidase and the chromogen 3,3'-diaminobenzidine (DAB) to produce a brown precipitate at the site of the reaction. Stained tissue sections were then scanned, imaged, and evaluated by light microscopy, such as using an Aperio ScanScope CS (Aperio, Vista, CA).

[00592] To evaluate the expression level of nectin-4 in tissue sections based on the level of antibody staining, stained tissue sections were examined, scored, classified and recorded into various categories or scores according to the intensity of IHC staining and the ratio of cancer cells. Categories include strong (“3” or interchangeable with “3+”), moderate (“2” or interchangeable with “2+”), and weak (“1” or interchangeable with “1+”). The absence of specific staining was recorded as negative (“0” or interchangeable with “−”). [00593] Human cancer xenograft tissues from various origins, such as bladder, breast, ovary, pancreas, kidney, skin, lung and colon, with known levels of nectin-4 mRNA expression were tested. Nectin-4 mRNA levels in these representative human xenograft tissues were first assessed by qPCR and are shown in Table 5.

Table 5. Tissues for antibody titration experiment

Human xenograft tissues Mouse number Nature of xenograft tissue Nectin-4 mRNA (qPCR) AG-B1 61668 Papillary urothelial bladder cancer 681 AG-B11 68618 Invasive keratinizing squamous cell carcinoma of the bladder 391 AG-Br29 71838 Infiltrating ductal carcinoma of the breast 76 AG-OV35 67431 Severe ovarian carcinoma, high differentiation 45 AG-Panc3 62103 Invasive times of the pancreas, average differentiation 12 AG-C6 61674 Rectal cancer, medium differentiation 6 AG-K24 65766 Renal cell carcinoma 0.4 AG-Mel10 70845 Malignant melanoma 0 MDA-MB-231-MFP-XCL 58624 Mammary cancer 0

[00594] In order to find the appropriate staining concentration for the antibodies, antibodies M22-321b41.1 and M22-244b3.1.1.1 were first titrated at 2.5 μg/ml, 5.0 μg/ml and 7.5 μg/ml ml using the xenograft tissue examples listed in Table 5 in the IHC staining assay as described above. The results are shown in Table 6.

Table 6. Summary of titration results for M22-321b41.1 and M22-244b3.1.1.1

M22-321b41.1 M22-244b3.1.1.1 Human xenograft tissues Mouse number Nectin-4 mRNA (qPCR) Conc. (µg/
ml) Point Conc. (µg/ml) Point AG-B1 61668 681 2.5 3+ 2.5 3+ AG-B11 68618 391 2.5 3+ 2.5 2+ AG-Br29 71838 76 2.5 2+ 2.5 3+ AG-OV35 67431 45 2.5 3+ 2.5 2+ AG-Panc3 62103 12 2.5 3+ 2.5 3+ AG-C6 61674 6 2.5 1+ 2.5 + AG-K24 65766 0.4 2.5 - 2.5 2+ MDA-MB-231-MFP-XCL 58624 0 2.5 - 2.5 1+ AG-Mel10 70845 0 2.5 - 2.5 - AG-B1 61668 681 5 3+ 5 3+ AG-B11 68618 391 5 3+ 5 2+ AG-Br29 71838 76 5 2+ 5 3+ AG-OV35 67431 45 5 3+ 5 3+ AG-Panc3 62103 12 5 3+ 5 3+ AG-C6 61674 6 5 + 5 + AG-K24 65766 0.4 5 - 5 3+ MDA-MB-231-MFP-XCL 58624 0 5 + 5 1+ AG-Mel10 70845 0 5 - 5 - AG-B1 61668 681 7.5 3+ 7.5 3+ AG-B11 68618 391 7.5 3+ 7.5 2+ AG-Br29 71838 76 7.5 2+ 7.5 3+ AG-OV35 67431 45 7.5 3+ 7.5 3+ AG-Panc3 62103 12 7.5 3+ 7.5 3+ AG-C6 61674 6 7.5 + 7.5 + AG-K24 65766 0.4 7.5 + 7.5 3+ MDA-MB-231-MFP-XCL 58624 0 7.5 + 7.5 1+ AG-Mel10 70845 0 7.5 - 7.5 -

[00595] The optimal concentration for anti-nectin-4 IHC antibody staining was defined as the concentration at which anti-nectin-4 IHC antibody staining was observed in tissues expressing nectin-4 mRNA but not in nectin-4 negative tissues. At a concentration of 2.5 μg/ml, both M22-321b41.1 and M22-244b3.1.1.1 antibodies showed positive staining in human xenograft tissues, positive for nectin-4 mRNA (Fig. 1 and Fig. 2), and showed the least background staining in negative control tissues. Table 6 and detailed staining results in Table 7 indicate that 2.5 μg/ml was the approximate optimal concentration for both antibodies.

Table 7. Detailed staining results for M22-321b41.1 and M22-244b3.1.1.1 titrations

Xeno
graft Mouse number Nectin-4 mRNA (qPCR) Primary antibody Conc.
(µm/ml) Staining results AG-B1 61668 681 M22-244b3.1.1.1 2.5 3+ AG-B11 68618 391 M22-244b3.1.1.1 2.5 2+ AG-Br29 71838 76 M22-244b3.1.1.1 2.5 3+ AG-OV35 67431 45 M22-244b3.1.1.1 2.5 2+ AG-Panc3 62103 12 M22-244b3.1.1.1 2.5 3+ AG-C6 61674 6 M22-244b3.1.1.1 2.5 + AG-K24 65766 0.4 M22-244b3.1.1.1 2.5 2+ AG-Mel10 70845 0 M22-244b3.1.1.1 2.5 - MDA-MB-231-MFP-XCL 58624 0 M22-244b3.1.1.1 2.5 + AG-B1 61668 681 M22-244b3.1.1.1 5.0 3+ AG-B11 68618 391 M22-244b3.1.1.1 5.0 2+ AG-Br29 71838 76 M22-244b3.1.1.1 5.0 3+ AG-OV35 67431 45 M22-244b3.1.1.1 5.0 3+ AG-Panc3 62103 12 M22-244b3.1.1.1 5.0 3+ AG-C6 61674 6 M22-244b3.1.1.1 5.0 + AG-K24 65766 0.4 M22-244b3.1.1.1 5.0 3+ AG-Mel10 70845 0 M22-244b3.1.1.1 5.0 - MDA-MB-231-MFP-XCL 58624 0 M22-244b3.1.1.1 5.0 + AG-B1 61668 681 M22-244b3.1.1.1 7.5 3+ AG-B11 68618 391 M22-244b3.1.1.1 7.5 2+ AG-Br29 71838 76 M22-244b3.1.1.1 7.5 3+ AG-OV35 67431 45 M22-244b3.1.1.1 7.5 3+ AG-Panc3 62103 12 M22-244b3.1.1.1 7.5 3+ AG-C6 61674 6 M22-244b3.1.1.1 7.5 + AG-K24 65766 0.4 M22-244b3.1.1.1 7.5 3+ AG-Mel10 70845 0 M22-244b3.1.1.1 7.5 - MDA-MB-231-MFP-XCL 58624 0 M22-244b3.1.1.1 7.5 + AG-B1 61668 681 M22-321b41.1 2.5 3+ AG-B11 68618 391 M22-321b41.1 2.5 3+ AG-Br29 71838 76 M22-321b41.1 2.5 2+ AG-OV35 67431 45 M22-321b41.1 2.5 3+ AG-Panc3 62103 12 M22-321b41.1 2.5 3+ AG-C6 61674 6 M22-321b41.1 2.5 + AG-K24 65766 0.4 M22-321b41.1 2.5 - AG-Mel10 70845 0 M22-321b41.1 2.5 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 2.5 - AG-B1 61668 681 M22-321b41.1 5.0 3+ AG-B11 68618 391 M22-321b41.1 5.0 3+ AG-Br29 71838 76 M22-321b41.1 5.0 2+ AG-OV35 67431 45 M22-321b41.1 5.0 3+ AG-Panc3 62103 12 M22-321b41.1 5.0 3+ AG-C6 61674 6 M22-321b41.1 5.0 + AG-K24 65766 0.4 M22-321b41.1 5.0 - AG-Mel10 70845 0 M22-321b41.1 5.0 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 5.0 + AG-B1 61668 681 M22-321b41.1 7.5 3+ AG-B11 68618 391 M22-321b41.1 7.5 3+ AG-Br29 71838 76 M22-321b41.1 7.5 2+ AG-OV35 67431 45 M22-321b41.1 7.5 3+ AG-Panc3 62103 12 M22-321b41.1 7.5 3+ AG-C6 61674 6 M22-321b41.1 7.5 + AG-K24 65766 0.4 M22-321b41.1 7.5 + AG-Mel10 70845 0 M22-321b41.1 7.5 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 7.5 +

[00596] Antibody M22-321b41.1 at a concentration of 2.5 μg/ml did not stain in tissues negative for nectin-4 mRNA AG-K24 and MDAMB-231-MFP-XCL (Fig. 3 and Fig. 5); whereas M22-244b3.1.1.1 had nonspecific staining in nectin-4 mRNA-negative AG-K24 (Fig. 4) and MDAMB-231-MFP-XCL (Fig. 6) xenograft cancer cells. No staining was observed in sections incubated with mouse IgG2a negative control antibody (Fig. 7). Therefore, M22-321b41.1 specifically stained tissues that express nectin-4 mRNA produce no staining or staining as low as the IgG isotype control for tissues that do not express nectin-4 mRNA.

[00597] The specificity of M22-321b41.1 was further tested in an expanded panel of positive and negative control tissues, as shown in Table 8. The results showed that the antibody at a concentration of 2.5 μg/ml was specific for tissues and cells expressing nectin mRNA 4, including AG-B1, AG-UTS, AG-L16 (Fig. 8), AG-Br29, AG-B11 (Fig. 9), AG-OV35, AG-Panc3, AG-C16, AG-C6, Rat1 (E)-nectin-4 and T47D. The results also showed that the antibody was negative for tissues and 231-MFPXCL (FIG 5), AG-Mel5, AG-K31, CALU-1, MDA-MB-231, UG-K3, Rat1(E)-neo, Rat1( E)-nectin-1, Rat1(E)-nectin-2, Rat1(E)-nectin-3, JMSU-1, Hep3B and ACHN. These data demonstrated that M22-321b41.1 was specific for nectin-4 in the IHC assay because, for all tissues and cells tested herein, M22-321b41.1 stained positively for tissues and cells that expressed nectin-4 mRNA. 4, and negatively stained those that did not express. (Table 8 and Table 9).

Table 8: Nectin-4 expression data in an expanded panel of FFPE samples using M22-321b41.1 at 2.5 μg/ml

Sample Number Nectin-4 mRNA (qPCR) Point
(coloring
M22-321b41.1) Rat1(E) Neo C974 negative - Rat1(E) nectin-1 C975 negative - Rat1(E) nectin-2 C976 negative - Rat1(E) nectin-3 C977 negative - Rat1(E) nectin-4 C978 Positively 3 T47D C1035 positively 3 MDA-MB-231 C1033 negative - JMSU-1 C979 negative - Hep3B C951 negative - ACHN C955 negative - AG-B1 61668 681 3 AG-B11 68618 391 3 AG-B10 68687 374 3 AG-UT5 71078 188 2 AG-L16 62092 105 3 AG-Br29 71838 76 2 AG-OV35 67431 45 3 AG-Panc3 62103 12 3 AG-C16 66746 7 1 AG-C6 61674 6 1 AG-K24 65766 0.4 - AG-Mel10 70845 0 - MDA-MB-231-MFP-XCL 58624 0 - MDA-MB-231 58480 0 - AG-Mel5 57775 0 - AG-K31 600-B681 0 -

Table 9. Staining results for M22-244b3.1.1.1 extended panel

Xenograft Number
mice mRNA
nectin 4 (qPCR) First antibody Conc.
(µg/ml) Staining results AG-B1 61668 681 M22-321b41.1 2.5 3+ AG-B11 68618 391 M22-321b41.1 2.5 3+ AG-Br29 71838 76 M22-321b41.1 2.5 2+ AG-OV35 67431 45 M22-321b41.1 2.5 3+ AG-Panc3 62103 12 M22-321b41.1 2.5 3+ AG-C6 61674 6 M22-321b41.1 2.5 + AG-K24 65766 0.4 M22-321b41.1 2.5 - AG-Mel10 70845 0 M22-321b41.1 2.5 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 2.5 - AG-B1 61668 681 M22-321b41.1 5.0 3+ AG-B11 68618 391 M22-321b41.1 5.0 3+ AG-Br29 71838 76 M22-321b41.1 5.0 2+ AG-OV35 67431 45 M22-321b41.1 5.0 3+ AG-Panc3 62103 12 M22-321b41.1 5.0 3+ AG-C6 61674 6 M22-321b41.1 5.0 + AG-K24 65766 0.4 M22-321b41.1 5.0 - AG-Mel10 70845 0 M22-321b41.1 5.0 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 5.0 + AG-B1 61668 681 M22-321b41.1 7.5 3+ AG-B11 68618 391 M22-321b41.1 7.5 3+ AG-Br29 71838 76 M22-321b41.1 7.5 2+ AG-OV35 67431 45 M22-321b41.1 7.5 3+ AG-Panc3 62103 12 M22-321b41.1 7.5 3+ AG-C6 61674 6 M22-321b41.1 7.5 + AG-K24 65766 0.4 M22-321b41.1 7.5 + AG-Mel10 70845 0 M22-321b41.1 7.5 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 7.5 + Rat1(E) Neo C974 negative M22-321b41.1 2.5 - Rat1(E) Nectin1 C975 negative M22-321b41.1 2.5 - Rat1(E) Nectin2 C976 negative M22-321b41.1 2.5 - Rat1(E)Nectin3 C977 negative M22-321b41.1 2.5 - Rat1(E) Nectin4 C978 negative M22-321b41.1 2.5 3+ T47D C1035 negative M22-321b41.1 2.5 3+ MDA-MB-231 C1033 negative M22-321b41.1 2.5 - JMSU-1 C979 negative M22-321b41.1 2.5 - Hep3B C951 negative M22-321b41.1 2.5 - ACHN C955 negative M22-321b41.1 2.5 - AG-B1 61668 681 M22-321b41.1 2.5 3+ AG-B11 68618 391 M22-321b41.1 2.5 3+ AG-B10 68687 374 M22-321b41.1 2.5 3+ AG-UT5 71078 188 M22-321b41.1 2.5 2+ AG-L16 62092 105 M22-321b41.1 2.5 3+ AG-Br29 71838 76 M22-321b41.1 2.5 2+ AG-OV35 67431 45 M22-321b41.1 2.5 3+ AG-Panc3 62103 12 M22-321b41.1 2.5 3+ AG-C16 66746 7 M22-321b41.1 2.5 + AG-C6 61674 6 M22-321b41.1 2.5 + AG-K24 65766 0.4 M22-321b41.1 2.5 - AG-Mel10 70845 0 M22-321b41.1 2.5 - MDA-MB-231-MFP-XCL 58624 0 M22-321b41.1 2.5 - MDA-MB-231 58480 0 M22-321b41.1 2.5 - AG-Mel5 57775 0 M22-321b41.1 2.5 - AG-K31 600-B681 0 M22-321b41.1 2.5 - CALU-1 72902 0 M22-321b41.1 2.5 - UG-K3 74323 0 M22-321b41.1 2.5 -

[00598] Anti-nectin-4 antibodies prepared and screened as described above were further compared with other commercial anti-nectin-4 antibodies in an IHC assay. For example, anti-nectin-4/PVRL4 antibody (referred to as “Novus PVRL4 antibody”) from Novus Biologicals (catalog number NBP1-82829), a rabbit polyclonal anti-nectin-4 antibody, was generated and tested for its ability to specifically stain nectin-4 in IHC analysis in various xenograft tissues in which nectin-4 mRNA levels were determined by qPCR. In experiments where the Novus PVRL4 antibody was titrated at 2 μg/ml, 1.5 μg/ml, 1 μg/ml, and 0.5 μg/ml, the Novus PVRL4 antibody produced weaker nectin-4 specific staining than the staining produced by M22- 244b3. 1.1.1.1 antibody and gave nonspecific staining at all concentrations in the MDA-MB-231 xenograft, which did not express nectin-4 mRNA as shown in Table 10. Thus, the Novus PVRL4 antibody was nonspecific for nectin-4.

Table 10: Novus PVRL4 Antibody Staining Results at Various Concentrations

Textile Mouse number RNA IHC results MDA-MB-231-MFP-XCL 58624 0 scattered positive cells even at 0.5 μg/ml AG-B10 P8 64069 286 Medium intensity spots at 2 µg/ml. Weakens at low concentrations AG-Mel10 P5 72088 0 Negative tumors with a weak cytoplasmic background at 1.5 μg/ml AG-OV20 P7 72260 67 Weak cytoplasmic staining with small foci of moderately positive cells AG-C18 P9 67921 3 Spots weak to negative at 2 µg/ml AG-C16 P4 68679 50 Mild to moderate, patchy, apical at 2 µg/ml

[00599] The Novus PVRL4 antibody was further tested in an IHC assay against a broader panel of tissues in which nectin-4 mRNA levels were determined by qPCR. Background cytoplasmic staining was observed in the majority of xenografts tested, regardless of Nectin-4 mRNA levels, as shown in Table 11.

Table 11: Novus PVRL4 Antibody Staining Results on a Wider Panel of Xenograft Tissues

Textile Number
mice RNA Novus PVRL4(NBP1-82829) 2 µg/ml AG-B10 P8 64069 286 bright spots on the membrane AG-Mel10 P5 72088 0 negative to weak background AG-OV20 P7 72260 67 Uneven cytoplasmic AG-Br8 P3 59161 41 Moderate to strong on the membrane on one side of the tumor AG-K24 P3 64812 1 Negative to weak background MDA-MB-231-MFP-XCL 58624 0 Low to moderate background AG-K9 P17 52376 0.1 Weak positive background AG-OV23 P3 55635 2 Heel of moderate brightness on the membrane/cytoplasm AG-C18 P9 67921 3 Weak to moderately positive, patchy AG-C16 P4 68679 50 Spots of moderate brightness AG-Br12 P2 62109 1024 Bright across the entire membrane

[00600] Because the Novus PVRL4 antibody is an exhaustible polyclonal antibody with potential batch-to-batch variability, another rabbit polyclonal anti-nectin antibody PA5-30837 from ThermoFischer Scientific (“Thermo PVRL4 antibody”) was generated and tested. and was compared in an IHC assay in three different xenograft tissues in which nectin-4 mRNA levels were determined by qPCR. The three xenograft tissues tested included AG-B1(+)PS26 with a nectin-4 mRNA level of 478, AG-OV35P3 with a nectin-4 mRNA level of 78, and AG-Mel5 P17 not expressing nectin-4 mRNA. Thermo PVRL4 antibody caused weak/moderate IHC staining in AG-B1 and negative IHC staining in AG-OV35.

[00601] Therefore, neither the Novus PVRL4 antibody nor the Thermo PVRL4 antibody was as specific for nectin-4 in the IHC assay as the M22-244b3.1.1.1.1 or M22-321b41.1 antibodies generated and selected in this case.

[00602] Two other commercial antibodies (monoclonal mouse anti-nectin-4 antibody MAB2659 and goat polyclonal anti-nectin-4 antibody AF2659), both from R&D Systems, were also produced and tested in IHC analysis in three different xenograft tissues in which mRNA levels nectin-4 was determined by qPCR. The three xenograft tissues tested included AG-B1(+)PS26 with a nectin-4 mRNA level of 478, AG-OV35P3 with a nectin-4 mRNA level of 78, and AG-Mel5 P17 without nectin-4 mRNA. Antibody MAB2659, when used at a concentration of 1 μg/ml or 5 μg/ml, gave dirty stippled/cytoplasmic staining at the tumor periphery in AG-B1 and AG-OV35. Goat anti-Nectin-4 polyclonal antibody AF2659 gave strong membrane staining with background stromal staining in AG-B1 and AG-OV35 at 5 μg/ml, but weak staining at 1 μg/ml. Therefore, antibody AF2659 was further evaluated at 2.5 μg/ml in other tissues. Antibody AF2659, however, caused background staining in MDA-MB-231 xenografts, which do not express nectin-4 mRNA. Therefore, neither antibody MAB2659 nor antibody AF2659 was as specific as the M22-321b41.1 antibody generated and screened in this case.

EXAMPLE 6: FUNCTIONAL ANALYSIS: IMMUNOCHISTOCHEMICAL STAINING OF PRIMARY TISSUE

[00603] Antibodies, such as anti-nectin-4 antibody M22-321b41.1, prepared, screened, expressed and purified, for example, as described in the examples herein, were further evaluated for the ability to specifically stain primary human tissues expressing nectin-4 , in IHC analysis.

[00604] IHC analysis was performed, for example, using an indirect IHC technique. The indirect IHC method used two antibodies to detect tissue antigens. First, the unconjugated primary antibody is applied to the tissue (first layer), which reacts with the tissue antigen. An enzyme-labeled second antibody, which is directed against the IgG of the animal species from which the antibody was obtained, was then applied (second layer). The secondary antibody reacts with the primary antibody, followed by application of a chromogen substrate. The second layer antibody can be labeled with the enzyme peroxidase, which reacts with the chromogen 3,3'-diaminobenzidine (DAB) to form a brown precipitate at the reaction site. This method is sensitive and versatile due to the strong signal amplification through reactions of multiple second antibodies with different antigenic sites of the first antibody.

[00605] As an example, a second antibody conjugated to a labeled polymer was used to further increase the sensitivity of the test. The polymer technology used an inert dextran “needle” molecule labeled with the HRP enzyme, to which up to 10 molecules of a second antibody can be attached, making the system even more sensitive.

[00606] The sample is then contrasted to identify cellular and subcellular elements.

6.A. Staining procedure

[00607] IHC analysis was performed according to the following exemplary protocol. Briefly, samples were fixed, processed and embedded in paraffin wax and prepared as tissue sections. The slides were then incubated in an oven at 60±5°C for 1-2 hours, removed from the oven and allowed to cool to room temperature (RT) before continuing the experiment. After routine deparaffinization and rehydration of the tissue samples, antigen retrieval was performed to reverse cross-link the proteins, which occurred after formaldehyde fixation. Antigen retrieval was achieved either by proteolytic digestion of tissues in protease solutions or by applying heat to tissue sections in an aqueous environment, commonly referred to as the HIER procedure. For this analysis, Epitope Retrieval 2 (ER2, EDTA-based buffer pH 8.9-9.1) was used on a Leica automated platform for 20 minutes at 100°C.

[00608] Staining was programmed to run on Bond instruments using Leica Bond autostainers at room temperature unless otherwise noted. Briefly, the following steps were programmed as a staining protocol for Leica Bond auto glass: (1) epitope retrieval 2 (ER2) procedure: 20 minutes at 100°C (Bond 2 epitope retrieval solution (Leica), part no. AR9640); (2) 3× bound complex wash: 0 min (bound complex wash solution (Leica), part no. AR9590); (3) marker: 15 minutes (staining with mouse anti-nectin-4 monoclonal antibody M22-321b41.1 or mouse IgG2a negative control (BD Biosciences), part no. 550339, in first binding antibody solution (Leica), part no. AR9352); (4) 3× bound complex wash: 0 minutes (bound complex wash solution (Leica), part no. AR9590); (5) after the first: 8 minutes (rabbit anti-mouse IgG2a); (6) 3x bound complex wash: 2 minutes (bound complex wash solution (Leica), part no. AR9590); (7) polymer: 8 minutes (anti-rabbit poly-HRP-IgG from Bond Polymer Refine Detection (Leica), part no. DS9800); (8) 2× washing of the bound complex: 0 min, 2 min; (9) 1× DI water: 0 minutes; (10) blocking with peroxide: 5 minutes (3-4% hydrogen peroxide) and rinsing the bound complex 3 times: 0 minutes; (11) 2 × DI water: 0 minutes; (12) mixed DAB cleanup: 0 minutes (66 mM 3,3-diaminobenzidine tetrahydrochloride and ≤0.1% hydrogen peroxide from Bond Polymer Refine Detection (Leica), part no. DS9800); (13) mixed DAB cleanup: 10 minutes (66 mM 3,3-diaminobenzidine tetrahydrochloride and ≤0.1% hydrogen peroxide from Bond Polymer Refine Detection (Leica), part no. DS9800); (14) 3× DI water: 0 minutes; (15) hematoxylin: 15 minutes (<0.1% hematoxylin from Bind Polymer Refine Detection (Leica), part #DS9800); (16) 1 × DI water: 0 minutes; (17) 1× wash: 0 minutes; (18) 1× DI water: 0 minutes. As specified in the procedure, a mouse IgG2a control reagent from BD Biosciences was used for each sample tested to assess the presence of nonspecific (background) staining. The presence of background staining was also a criterion for accepting/rejecting a particular assay.

6.B Fabric samples

[00609] Positive and negative controls were used to validate and control all steps of the IHC assay. For this analysis, mouse/human tumor xenografts with varying levels of nectin-4 expression were used as controls.

[00610] Formalin-fixed paraffin samples in human tissue were obtained as residual material from surgery or autopsy from Institutional Review Board-approved sources, for example government repositories (eg, Cooperative Human Tissue Network), through academic collaborations (eg, UCLA , UC Irvine), and from commercial sources, including, for example, normal urothelial cells and human transitional cell carcinoma cells (BL802) and normal (FDA999) tissue microarray (TMA), Biomax USA. Tissue samples were tested for nectin-4 expression using the protocol described in Example 6.A. Four mouse/human tumor xenografts were also tested, which included one negative (X14-126), one weakly positive (X14-33), one moderately positive (X14-139), and one strongly positive (X14-43) for nectin-4 expression .

6.C Titration of anti-nectin-4 antibody concentration for staining

[00611] The optimal titer of the first antibody was determined by performing a titration assay on xenograft tissue sections, for example, using the protocol described in Examples 4 and 5. A titration assay on paraffin-embedded samples was performed to determine the optimal antibody concentration for IHC assays. To perform the titration assay, 2-fold serial dilutions containing the optimal concentration (2.5 μg/ml) determined in Example 5 were prepared and tested with their corresponding negative control tissue sections.

[00612] Anti-nectin-4 antibody M22-321b41.1 and the corresponding negative control antibody were applied to tissue sections according to the appropriate IHC protocol for paraffin-embedded samples as described in Examples 4 and 5. The results were evaluated and compared by study pathologists using light microscopy. The study pathologists defined the optimal titer (optimal antibody concentration) as the lowest titer (lowest antibody concentration) that produced the greatest intensity of staining without significant background staining. In this example, as shown in Table 12, an antibody concentration of 2.5 μg/ml was selected and used in subsequent IHC analyses.

Table 12: Determination of optimal antibody concentration for anti-nectin-4 antibody M22-321b41.1.

Type No. Track no. Concent. antibodies ng/ml Background % pos. Target staining intensity Stromal staining Sub Morphol. Comments X14-126 0VE7 3.75 0 0 BUT. 0 BUT. A A small piece of positive tissue was found, likely contamination X14-126 0VE8 2.5 0 0 BUT. 0 BUT. A Same as above X14-126 0VE9 1.25 0 0 BUT. 0 BUT. A Same as above X14-33 0VDZ 3.75 0 25 1-3+ 0 M,C,Ap A X14-33 0VE0 2.5 0 25 1-3+ 0 M,C,Ap A X14-33 0VE1 1.25 0 25 1-3+ 0 M,C,Ap A X14-139 0VDS 3.75 0 90 1-3+ 0 M,C,Ap A X14-139 0VDT 2.5 0 90 1-3+ 0 M,C,Ap A X14-139 0VDU 1.25 0 80 1-3+ 0 M,C,Ap A X14-43 0VDK 3.75 0 100 3++ 1-3+ M, C A diffuse IC+ X14-43 0VDL 2.5 0 100 3++ 1-2+ M, C A X14-43 0VDM 1.25 0 100 3+ 1-2+ M, C A 2+F

6.D. Evaluation of nectin-4 staining in IHC analysis

[00613] IHC staining evaluation was performed by board certified pathologists. Staining intensity was scored and reported as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The approximate percentage of cells stained at each intensity level, or positively stained cells overall, was assessed and assigned a value from 0 to 100%.

[00614] To compare staining among tissues, different parameters for assessing nectin-4 staining were either compared directly, or the results of nectin-4 staining were further processed, for example, calculating both the H score, which takes into account both the intensity of staining and the percentage of cells stained in a certain range intensities. For example, the H score was calculated by summing the products of the percentage of cells stained at a given staining intensity (0–100) and the staining intensity (0–3). For example: a sample with 10% cells scoring 3+, 30% cells scoring 2+, 20% cells scoring 1+ and 40% unstained cells would have an H score of (3×10)+(2×30)+(1× 20)+(0×40)=110.

[00615] The coefficient of variation (CV) was used to estimate intra-assay variation or precision in the H score. The intra-assay precision was determined from five separate experiments using three positive xenograft samples. Qualitatively, intra-experimental precision was considered acceptable according to the conventional staining scheme for each tissue tested in three separate experiments. CV was calculated as follows:

CV=standard deviation/mean × 100

[00616] For example, a CV less than or equal to 35% was considered acceptable.

[00617] As shown in Table 13, to evaluate the intra-assay accuracy of the processes, the characteristics (3+, 2+, 1+ combinations) of staining intensities were matched for each tissue over five replicates. In addition, the CVs for all tissues were less than 35%, which is within the approximate acceptable range. Thus, M22-321b41.1 provided accurate detection of nectin-4 in the nectin-4 IHC assay.

[00618]

Table 13 Accuracy of anti-nectin-4 antibody M22-321b41.1 in IHC assays

GENERAL INFORMATION ON THE CUT TEST WITH STAINING OF A PREPARATION OF DIFFERENT CELLS OTHER Search clearance no. Slide no. Date of staining/equipment Tissue (diagnosis) Isotype control % and subcellular localization of cells stained at each intensity H-score % nectin-4 Comment. 3+% 3+
sub 2+
% 2+
sub 1+% 1+
sub 0% Textile X14-43 0VH4 30 June 2014 Bond III Xeno-transpl. 0 95 M, C 5 M, C 0 BUT. 0 295 100 Excl. necrosis tissue X14-43 0VH5 30 June 2014 Bond III Xeno-transpl. 0 95 M, C 5 M, C 0 BUT. 0 295 100 X14-43 0VH6 30 June 2014 Bond III Xeno-transpl. 0 98 M, C 2 M, C 0 BUT. 0 298 100 X14-43 0VH7 30 June 2014 Bond III Xeno-transpl. 0 98 M, C 2 M, C 0 BUT. 0 298 100 X14-43 0VH8 30 June 2014 Bond III Xeno-transpl. 0 98 M, C 2 M, C 0 BUT. 0 298 100 Average 296.8 100.0 STDEV 1.6 0.0 CV 0.6 0.0 X14-139 0VHB 30 June 2014 Bond III Xeno-transpl. 0 40 M, C 45 M, C 10 M, C 5 220 95 X14-139 0VHC 30 June 2014 Bond III Xeno-transpl. 0 40 M, C 40 M, C 15 M, C 5 215 95 X14-139 0VHD 30 June 2014 Bond III Xeno-transpl. 0 40 M, C 35 M, C 20 M, C 5 210 95 X14-139 0VHE 30 June 2014 Bond III Xeno-transpl. 0 45 M, C 40 M, C 10 M, C 5 225 95 X14-139 0VHF 30 June 2014 Bond III Xeno-transpl. 0 45 M, C 40 M, C 10 M, C 5 225 95 Average 219.0 95.0 STDEV 6.5 0.0 CV 3.0 0.0 X14-33 0VHI 30 June 2014 Bond III Xenotranspl. 0 10 M, C 15 M, C 5 M, C 70 65 thirty X14-33 0VHJ 30 June 2014 Bond III Xenotranspl. 0 10 M, C 10 M, C 10 M, C 70 60 thirty X14-33 0VHK 30 June 2014 Bond III Xenotranspl. 0 10 M, C 10 M, C 5 M, C 75 55 25 X14-33 0VHL 30 June 2014 Bond III Xenotranspl. 0 8 M, C 12 M, C 5 M, C 75 53 25 X14-33 0VHM 30 June 2014 Bond III Xenotranspl. 0 5 M, C 15 M, C 5 M, C 75 50 25 Average 56.6 27.0 STDEV 5.9 2.7 CV 10.5 10.1 GENERAL COMMENT/NOTE: ± = Equivalent results BUT. = Not detected Ca=Carcinoma NS=Not visible c/w=Agrees with

[00619] Assay reproducibility was determined in five separate experiments using the same xenograft samples as in the previous assay. Qualitatively, intra-experimental precision was considered acceptable according to the conventional staining scheme for each tissue tested in five separate experiments. As shown in Table 14, to evaluate intra-rater reproducibility, characteristics (3+, 2+, 1+ combinations) of staining intensities were matched for each tissue over three replicates. In addition, the CVs for all tissues were less than 35%, which is within the approximate acceptable range. Thus, M22-321b41.1 provided highly reproducible detection of nectin-4 in the nectin-4 IHC assay.

Table 14: Intra-assay reproducibility of anti-nectin-4 antibody M22-321b41.1 in IHC assays

GENERAL INFORMATION ON THE CUT TEST WITH STAINING OF A PREPARATION OF DIFFERENT CELLS No.
Search access Number of repetitions Test data
and platform Slide no. Tissue (diagnosis) Isotype
control % and subcellular localization of cells stained at each intensity H-score % nectin-4 Comment. 3+% 3+ sub 2+
% 2+ sub 1+% 1+ sub 0% TISSUE X14-43 #1 June 26, 2014 Bond III 0VDL Xeno-transpl. 0 100 M, C 0 BUT 0 BUT 0 300 100 X14-43 #2 June 30, 2014 Bond III 0VH4 Xeno-transpl. 0 95 M, C 5 M, C 0 BUT 0 295 100 X14-43 #3 June 30, 2014 Bond Max 0VHQ Xeno-transpl. 0 95 M, C 5 M, C 0 BUT 0 295 100 X14-43 #4 01 July 2014 Bond Max 0VJS Xeno-transpl. 0 100 M, C 0 BUT 0 BUT 0 300 100 X14-43 #5 01 July 2014 Bond III 0VJQ Xeno-transpl. 0 98 M, C 2 M, C 0 BUT. 0 298 100 Average 297.6 100.0 STDEV 2.5 0.0 CV 0.8 0.0 X14-139 #1 June 26, 2014 Bond III 0VDT Xenotranspl. 0 40 M, C 40 M, C 15 M, C 5 215 95 Apical marked.
having painted. X14-139 #2 June 30, 2014 Bond III 0VHB Xenotranspl. 0 40 M, C 45 M, C 10 M, C 5 220 95 X14-139 #3 June 30, 2014 Bond Max 0VHT Xenotranspl. 0 45 M, C 40 M, C 10 M, C 5 225 95 X14-139 #4 01 July 2014 Bond Max 0VJX Xenotranspl. 0 45 M, C 45 M, C 10 M, C 0 235 100 X14-139 #5 01 July 2014 Bond III 0VJV Xenotranspl. 0 45 M, C 40 M, C 15 M, C 0 230 100 Average 225.0 97.0 STDEV 7.9 2.7 CV 3.5 2.8 X14-33 #1 June 26, 2014 Bond III 0VED Xeno-transpl. 0 8 M, C 12 M, C 10 C 70 58 thirty Apical staining noted X14-33 #2 June 30, 2014 Bond III 0VHI Xeno-transpl. 0 10 M, C 15 M, C 5 M, C 70 65 thirty X14-33 #3 June 30, 2014
Bond Max 0VHW Xeno-transpl. 0 8 M, C 12 M, C 5 M, C 75 53 25 X14-33 #4 01 July 2014
Bond Max 0VK3 Xeno-transpl. 0 8 M, C 10 M, C 7 C 75 51 25 X14-33 #5 01 July 2014
Bond III 0VK0 Xeno-transpl. 0 8 M, C 12 M, C 5 C 75 53 25 Average 56.0 27.0 STDEV 5.7 2.7 CV 10.1 10.1 GENERAL COMMENT/NOTE: ± = equivalent.
results Ap=apical staining H=heterogeneous staining SCC* = squamous Ca cells BUT=Not detected B=basal layer staining I=inflammatory cells L1=Link.1 NS=not visible C=cytoplasm.
coloring Ca=carcinoma L2=Link.2 c/w=consistent with F=focal positive M=membrane staining N=nuclear staining

[00620] The parameters and results 6.A-6.D are summarized in the following table 15:

Table 15: Examples of parameters, results and their ranges.

Description of the event Methods and results Precision Analysis: An Example of Within-Experiment CV 0.6%, 0.3%, 10.5% Reproducibility Analysis: An Example of Within-Experiment CV 0.8%, 3.5%, 10.1% Dyeing in fabrics 0=no, 1=weak,
2=medium and 3=strong Parameters, results and their ranges H-score (0-300); 0-3+,
% stained cells

6.E Sensitivity and specificity of nectin-4 IHC staining.

[00621] A nectin-4 IHC assay was considered sensitive, for example, if it positively stained tissues that expressed nectin-4 antigen. A nectin-4 IHC assay was considered specific, for example, if differential staining was observed in different tissue types and NHTIN-4 IHC staining correlated with known nectin-4 antigen expression in the stained tissues. Because specificity and sensitivity studies were not mutually exclusive, the analysis often provided information on both sensitivity and specificity.

[00622] The sensitivity and specificity of the nectin-4 IHC assay with M22-321b41.1 was determined by TMA from 60 unique samples of bladder carcinoma and 20 normal urothelial tissues, 4 mouse human tumor xenografts, and 13 human whole tissue sections (WTS) of various carcinomas , including carcinoma of the bladder, lung and breast. A summary of the sensitivity and specificity of antibody M22-321b41.1 for nectin-4 staining in IHC assays is shown in Table 16 and Table 17 below. In these and several other example analyses, tissue samples were classified by H-score into one of four categories: high (201-300), medium (101-200), low (1-100), negative (0).

Table 16. Summarized tissue staining sensitivity data

Summarizing tissue staining sensitivity data Level
H-points Location Neoplastic tissues* Carcinoma, whole tissue High
(201-300) M, C Bladder: 6/8, Breast: 1/4, Lungs: 2/4, Xenograft: 2/4 Average
(101-200) M, C Bladder: 1/8, Breast: 2/4, Rectum: 2/3, Ovary: 1/1 Short
(1-100) BUT. Bladder: 1/8, Lungs: 1/4, Rectum: 1/3, Xenograft: 1/4; Mammary gland: 1/4 Negative
(<=0) BUT. Lung: 1/4, Rectum: 1/3, Xenograft: 1/4

Table 17. Summarized tissue staining specificity data

Summarizing tissue staining specificity data Level
H-points Location Normal tissue* High
(201-300) M, C esophagus: 1/3, cervix: 1/3, skin: 2/3 Average
(101-200) M, C mammary gland 1/3, cervix 1/3, larynx 1/3 Short
(1-100) M, C cervix 1/3, mammary gland 2/3, bone marrow 1/3, lungs 1/3, salivary gland 3/3, kidney 1/3, prostate 2/3, endometrium 2/3, larynx 1/3 Negative BUT. bone marrow 2/3, lung 2/3, esophagus 1/3, kidneys 2/3, prostate 1/3, endometrium 1/3, skin 1/3, larynx 1/3
The following results were negative in all repeats: brain (N), cerebellum (N), adrenal gland (N), ovaries, pancreas, parathyroid, pituitary, testis, thyroid, spleen, tonsils, thymus, cardiac muscle, stomach, small intestine, colon, liver, skeletal muscle, peripheral nerve, mesothelium and eye

[00623] In some example assays to evaluate the specificity and sensitivity of M22-321b41.1, H-scores for 13 and WTS carcinomas obtained by staining with either antibody X, which is known to be specific for nectin-4, or the antibody against nectin-4 M22-321b41.1 at 2.5 μg/ml, respectively. The comparison shown in Table 18 below revealed that IHC staining with M22-321b41.1 was consistent with known nectin-4 expression as shown by Antibody X staining in the vast majority of cases.

Table 18. Correlation between IHC staining with M22-321b41.1 and IHC staining with antibody X

Correlation between M22-321b41.1 and Antibody X M22-321b41.1 Antibody X Sample No. Access no. H-score, 2.5 µg/ml H-score, 2.5 µg/ml 1 H882 300 280 2 H704 290 275 3 H716 230 200 4 H785 257 275 5 H878 180 100 6 H695 170 180 7 H734 140 135 8 H700 40 38 9 H737 45 120 10 H788 56 55 eleven H838 90 125 12 H739 0 0 13 H774 0 0 14 X14-43 High 300 15 X14-139 high 215 16 X14-33 Low 58 17 X14-126 Negative 0

[0064] The results shown in Table 18 above were reclassified into positive and negative nectin-4 staining to determine the correlation of staining positivity between antibody X and anti-nectin-4 antibody M22-321b41.1. Tissue was considered positive if the H-score was ≥150. The overall results shown in Table 19 below showed a concordance rate of 89% for antibody X and anti-nectin-4 antibody M22-321b41.1 for positively stained tissues, a concordance rate of 100% for negatively stained tissues, and an overall agreement rate equal to 94%. Thus, there is a high degree of correlation between nectin-4 IHC staining with M22-321b41.1 and tissue nectin-4 expression. Therefore, IHC analysis of nectin -4 with M22-321b41.1 is sensitive and specific.

Table 19. Antibody X against nectin-4 versus M22-321b41.1 If the threshold is H-score ≥ 150

M22-321b41.1 Antibody X Put Negative Sum Put 8 0 8 Father 1 8 9 Sum 9 8 17

[00625] The sensitivity and specificity of M22-321b41.1 were further determined using a larger set of tissue samples, including TMA from 60 unique bladder carcinoma and 20 normal urothelial tissue samples, 4 murine human tumor xenografts, and 13 human whole tissue sections (WTS) of various carcinomas, including bladder, lung and breast carcinomas. A total of 94 cases were evaluated and a total of nectin-4 was detected in 95% of cases (82% in WTS and 98% in TMA) using IHC assays with antibody X. Representative IHC staining of carcinoma with M22-321b41.1 is shown in the figures 10 and 11. Subcellular localization was primarily membranous and cytoplasmic, with some apical staining thought to be membranous. The results presented in Table 20 further demonstrated the sensitivity and specificity of M22-321b41.1 nectin-4 IHC assays because the percentage of positive staining in M22-321b41.1 tumor samples was consistent with the known expression of nectin-4 in carcinomas as shown in IHC assays with antibody X. Therefore, the IHC assay of nectin -4 with M22-321b41.1 was both sensitive and specific.

Table 20 Sensitivity of anti-nectin-4 antibody M22-321b41.1 for IHC staining of various tissues.

Table 20 - Part I.

GENERAL INFORMATION ABOUT CUTS TEST WITH STAINING OF A PREPARATION OF DIFFERENT CELLS TEST WITH STAINING OF A PREPARATION FOR OTHER CELL TYPES Other Line Access Test date/
type
fabrics Test no. Iso
type.
counter. % and subcellular localization of cells stained for each intensity H
point % nectin-4 Normal Endothelium Glad.
muscles. Fib
rob
flipper Stroma Cell
playback Comm. 3+% 3+ sub 2+% 2+ sub 1+% 1+ sub 0% Controls 1 X14-126 7/7/2014 Xeno-transpl. 0VRV 0 0 BUT 0 BUT 0 BUT 100 0 0 BUT 0 N.S. 0 0 N.S. 2 X14-139 7/7/2014 Xeno-transpl. 0VRY 0 thirty M, C 45 M, C 20 M, C 5 200 95 BUT 0 N.S. 0 0 N.S. Textile 1 PTBL0
1172A 6/23/2014 Ca bladder. 0V6J 0 40 M, C 45 M, C 10 M, C 5 220 95 N.S. 0 0 0 0 2+F 2 PTBL0
0626B 6/23/2014 Ca bladder. 0V6M 0 50 M, C 15 M, C 15 M, C 20 195 80 N.S. 0 2+ 0 0 2+ 3 PTBL0
0696A 6/23/2014 Ca bladder. 0V6Q 0 thirty M, C 55 M, C 10 M, C 5 210 95 N.S. 0 1+ 0 0-1+ 2+ 4 PTBL0
1177A 6/23/2014 Ca bladder. 0V6T 0 50 M, C 45 M, C 5 C 0 245 100 N.S. 0 0 0 0 3+ 5 PTBL0
1228A 6/23/2014 Ca bladder. 0V6W 0 80 M, C 10 M, C 0 BUT 10 260 90 N.S. 0 3+ 0 1+ 3+ 6 PTBL0
1247A 6/23/2014 Ca bladder. 0V6Z 0 90 M, C 10 M, C 0 BUT 0 290 100 N.S. 0 1+ 0 1+ 3+ 7 PTLQ0
1824 6/23/2014 SCC lung* 0V72 0 70 M, C 20 M, C 10 C 0 260 100 N.S. 0 0 1+ 2+ 2+ 8 H882 7/7/2014 urinary.BL. 0VS1 0 80 M, C 20 M, C 0 BUT 0 280 100 3+ 0 2+ 0 0 2+F 9 H704 7/7/2014 milk
iron (IDC) 0VS4 0 80 M, C 15 M, C 5 C 0 275 100 3+ 0 N.S. 0 1-2+ 3+ 10 H716 7/7/2014 milk iron (IDC) 0VS7 0 50 M, C 20 M, C 10 C 20 200 80 2+ 0 N.S. 0 0 2+ eleven H785 7/7/2014 lung (SCC) 0VSA 0 80 M, C 15 M, C 5 C 0 275 100 2+ 0 N.S. 0 1-2+ 2+ 12 H878 7/7/2014 urinary. BL. 0VSD 0 20 M, C 15 M, C 10 C 55 100 45 N.S. 0 0 0 0 2+ 13 H695 7/7/2014 milk
iron (IDC) 0VSG 0 25 M, C 40 M, C 25 C 10 180 90 2+ 0 N.S. 0 0 2+F 14 H734 7/7/2014 rectum 0VSJ 0 5 M, C 45 M, C thirty C 20 135 80 2+ 0 0 0 0 0 15 H700 7/7/2014 milk
iron (IDC) 0VSM 0 10 M, C 3 M, C 2 C 85 38 15 N.S. 0 0 0 0 0 16 H737 7/7/2014 rectum 0VSQ 0 10 M 40 M, C 10 C 40 120 60 0 0 0 0 0 2+F M=almost all apical 17 H788 7/7/2014 light 0VST 0 5 C 15 C 10 C 70 55 thirty 3+ 0 0 0 0 3+ 18 H838 7/7/2014 ovaries 0VSW 0 10 M, C 35 M, C 25 M, C thirty 125 70 N.S. 0 N.S. 0 0 3+ 19 H739 7/7/2014 rectum 0VSZ 0 0 BUT 0 BUT 0 BUT 100 0 0 N.S. 0 0 0 0 0 20 H774 7/7/2014 light 0VT2 0 0 BUT 0 BUT 0 BUT 100 0 0 3+ 0 N.S. 0 0 0 21 X14-43 6/26/2014 xeno-transpl. 0VDL 0 100 M, C 0 BUT 0 BUT 0 300 100 N.S. 0 N.S. 0 0 N.S. 22 X14-139 6/26/2014 xeno-transpl. 0VDT 0 40 M, C 40 M, C 15 M, C 5 215 95 N.S. 0 N.S. 0 0 N.S. 23 X14-33 6/26/2014 xeno-transpl. 0VED 0 8 M, C 12 M, C 10 C 70 58 thirty N.S. 0 N.S. 0 0 N.S. 24 X14-126 6/26/2014 xeno-transpl. 0VE8 0 0 BUT 0 BUT 0 BUT 100 0 0 N.S. 0 N.S. 0 0 N.S. General comments/notes: BUT=not defined NC=negative control A=apical N=normal NS=not visible F=focal C=cytoplasmic staining PC=plasma cells II = invasive/
intraductal N=nuclear IC=inflammatory cells TCC=transitional cell carcinoma Ca=carcinoma HOT= normal tissue adjacent to cancer SCC* = squamous Ca cells LCC=large cell carcinoma PSC Ca=papillary serous cystadenocarcinoma IDC=infiltrating ductal carcinoma

Table 20-Part II.

GENERAL INFORMATION ABOUT CUTS TEST WITH STAINING OF A PREPARATION OF DIFFERENT CELLS TEST WITH STAINING OF A PREPARATION FOR OTHER CELL TYPES Number
access Test number Place
TMA Bladdet/level diagnostician Isotype control % and subcellular localization of cells stained for each intensity H-score %
Neck
tin-4 Nrm. En
before
thelium Ch.
musk Fib
ro
blast Stroma Cl.
playback Comment. 3+% 3+ sub 2+% 2+ sub 1+% 1+ sub 0% TISSUE BL802 0VK8 A1 TCC G1 0 85 M, C 15 M, C 0 BUT. 0 285 100 N.S. 0 0 0 0 N.S. BL802 0VK8 A2 TCC G1-G2 0 25 M, C 50 M, C 15 M, C 10 190 90 N.S. 0 0 0 0 3+ BL802 0VK8 A3 TCC G1 0 0 BUT. 5 M, C 10 M, C 85 20 15 N.S. 0 0 0 0 2+ BL802 0VK8 A4 TCC G1-G2 0 15 M, C 40 M, C 35 M, C 10 160 90 N.S. 0 N.S. 0 1+ 2+ BL802 0VK8 A5 TCC G2 0 65 M, C 20 M, C 10 M, C 5 245 95 N.S. 0 0 0 0 N.S. BL802 0VK8 A6 TCC G2-G3 0 25 M, C 55 M, C 15 M, C 5 200 95 N.S. 0 0 0 1+ 3+ BL802 0VK8 A7 TCC G2 0 25 M, C 35 M, C 25 M, C 15 170 85 N.S. 0 N.S. 0 0 3+ BL802 0VK8 A8 TCC G2 0 thirty M, C 45 M, C 15 M, C 10 195 90 N.S. 0 N.S. 0 0 3+ BL802 0VK8 A9 TCC G1 0 0 BUT. 5 M, C 15 M, C 80 25 20 N.S. 0 N.S. 0 0 N.S. BL802 0VK8 A10 TCC G2 0 80 M, C 10 M, C 10 M, C 0 270 100 N.S. 0 N.S. 0 0 3+ BL802 0VK8 B1 TCC G2 0 95 M, C 5 M, C 0 BUT. 0 295 100 N.S. 0 N.S. 0 0 3+ BL802 0VK8 B2 TCC G2 0 5 M, C 45 M, C thirty M, C 20 135 80 N.S. 0 N.S. 0 0 0 BL802 0VK8 B3 TCC G2 0 60 M, C 20 M, C 10 M, C 10 230 90 N.S. 0 N.S. 0 3+F 3+ BL802 0VK8 B4 TCC G2 0 thirty M, C 45 M, C 15 M, C 10 195 90 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 B5 TCC G3 0 70 M, C 15 M, C 5 M, C 10 245 90 N.S. 0 N.S. 0 2+F 3+ BL802 0VK8 B6 TCC G2 0 20 M, C 50 M, C 20 M, C 10 180 90 N.S. 0 N.S. 0 2+ 2+ BL802 0VK8 B7 TCC G3 0 thirty M, C 10 M, C 45 M, C 15 155 85 N.S. 0 0 0 2+ 3+ BL802 0VK8 B8 TCC G3 0 80 M, C 20 M, C 0 BUT. 0 280 100 N.S. 0 N.S. 0 0 N.S. Partial core BL802 0VK8 B9 TCC G1 0 20 M, C 40 M, C 20 M, C 20 160 80 N.S. 0 0 0 0 N.S. BL802 0VK8 B10 TCC G2 0 20 M, C thirty M, C 20 M, C thirty 140 70 N.S. 0 0 0 0 2+ BL802 0VK8 C1 TCC G1 0 90 M, C 10 M, C 0 BUT. 0 290 100 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 C2 TCC G2 0 15 M, C 65 M, C 15 M, C 5 190 95 N.S. 0 N.S. 0 0 2+ BL802 0VK8 C3 TCC (sparse) G1 0 60 M, C 20 M, C 10 M, C 10 230 90 N.S. 0 N.S. 0 2+,F 3+ BL802 0VK8 C4 TCC G2-G3 0 40 C 10 C 0 BUT. 50 140 50 N.S. 0 N.S. 0 3+ 3+ BL802 0VK8 C5 TCC G3 0 thirty M, C 20 M, C 20 M, C thirty 150 70 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 C6 TCC G2 0 0 M, C 40 M, C 40 M, C 20 120 80 N.S. 0 N.S. 0 0 2+ BL802 0VK8 C7 TCC G3 0 80 M, C 20 M, C 0 BUT. 0 280 100 N.S. 0 N.S. 0 2+,F 0 BL802 0VK8 C8 TCC G3 0 2 M, C 2 M, C 0 BUT. 96 10 4 N.S. 0 0 0 0 3+ BL802 0VK8 C9 TCC G3 0 65 M, C 20 M, C 15 C 0 250 100 N.S. 0 0 0 1+F 3+ BL802 0VK8 C10 TCC G2 0 5 M, C 60 M, C 10 M, C 25 145 75 2+ 0 0 0 0 0 BL802 0VK8 D1 TCC G2 0 25 M, C 55 M, C 20 C 0 205 100 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 D2 TCC G2 0 50 M, C 10 M, C 10 C thirty 180 70 N.S. 0 0 0 0 2+ BL802 0VK8 D3 TCC G3 0 35 M, C 45 M, C 15 C 5 210 95 N.S. 0 0 0 0 2+ BL802 0VK8 D4 TCC G2 0 thirty M, C 60 M, C 10 C 0 220 100 N.S. 0 N.S. 0 2+ 2+ BL802 0VK8 D5 TCC G3 0 40 C 50 C 5 C 5 225 95 N.S. 0 2+ 0 0 2+F Rarely M+ cells BL802 0VK8 D6 TCC G3 0 80 M, C 10 M, C 5 C 5 265 95 N.S. 0 2+ 0 2+ 2+ BL802 0VK8 D7 TCC G1-G2 0 55 C 40 C 3 C 2 248 98 N.S. 0 N.S. 0 2+ 2+ BL802 0VK8 D8 TCC G2 0 25 M, C 45 M, C 15 M, C 15 180 85 N.S. 0 2+ 0 0 3+ BL802 0VK8 D9 TCC G2 0 0 BUT. 20 C thirty C 50 70 50 N.S. 0 N.S. 0 0 0 BL802 0VK8 D10 TCC G3 0 40 M, C 40 M, C 20 C 0 220 100 N.S. 0 N.S. 0 2+ 2+ stroma
adiposis
textile BL802 0VK8 E1 TCC G2 0 90 M, C 10 M, C 0 BUT. 0 290 100 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 E2 TCC G2 0 70 C 15 C 10 C 5 250 95 N.S. 0 N.S. 0 2+ 3+ BL802 0VK8 E3 TCC G2 0 25 M, C 50 C 20 C 5 195 95 N.S. 0 1+ 0 1+ 2+ BL802 0VK8 E4 TCC G2 0 100 M, C 0 BUT. 0 BUT. 0 300 100 N.S. 0 3+ 0 3+ 3+ BL802 0VK8 E5 TCC G2 0 70 M, C 10 M, C 5 M, C 15 235 85 N.S. 0 N.S. 0 2+ 2+ BL802 0VK8 E6 TCC G2 0 80 M, C 20 M, C 0 BUT. 0 280 100 N.S. 0 N.S. 0 2+F 0 BL802 0VK8 E7 TCC G3 0 65 M, C 20 M, C 5 M, C 10 240 90 N.S. 0 0 0 0 0 BL802 0VK8 E8 TCC G2 0 thirty M, C 50 M, C 15 C 5 205 95 N.S. 0 N.S. 0 0 0 BL802 0VK8 E9 TCC G2 0 75 M, C 20 M, C 5 C 0 270 100 N.S. 0 N.S. 0 0 0 BL802 0VK8 E10 TCC G2 0 2 M, C 18 M, C thirty M, C 50 72 50 N.S. 0 N.S. 0 0 0 BL802 0VK8 F1 TCC G2 0 100 C 0 BUT. 0 BUT. 0 300 100 N.S. 0 N.S. 0 2+ 0 BL802 0VK8 F2 TCC G2-G3 0 80 M, C 20 M, C 0 BUT. 0 280 100 N.S. 0 N.S. 0 3+ 3+ BL802 0VK8 F3 TCC G3 0 10 C 85 C 3 C 2 203 98 N.S. 0 1+ 0 0 2+ BL802 0VK8 F4 TCC G3 0 50 C thirty C 18 C 2 228 98 N.S. 0 N.S. 0 1+ 2+ BL802 0VK8 F5 TCC G3 0 40 M, C 40 M, C 20 C 0 220 100 N.S. 0 N.S. 0 1+ 2+ BL802 0VK8 F6 TCC* 0 20 C 10 C 10 C 60 90 40 N.S. 0 N.S. 0 0 0 BL802 0VK8 F7 TCC G3 0 0 BUT. 0 BUT. 0 BUT. 100 0 0 N.S. 0 N.S. 0 0 0 BL802 0VK8 F8 AdenoCa G2 0 0 BUT. 3 M, C 0 BUT. 97 6 3 N.S. 0 N.S. 0 0 0 BL802 0VK8 F9 AdenoCa (SM & BU) 0 0 BUT. 5 M, C 5 M, C 90 15 10 N.S. 0 0 0 0 0 BL802 0VK8 F10 TCC G2 0 95 C 5 C 0 BUT. 0 295 100 3+ 0 N.S. 0 1+ 2+ BL802 0VK8 G1 NAT 0 0 BUT. 10 C thirty C 60 50 40 BUT. 0 0 0 0 0 BL802 0VK8 G2 NAT 0 40 M, C thirty M, C 10 C 20 190 80 BUT. 0 0 0 0 0 BL802 0VK8 G3 NAT 0 0 BUT. 0 BUT. 0 BUT. 100 0 0 BUT. 0 N.S. 0 0 0 BL802 0VK8 G4 NAT (SM&BV) 0 15 C thirty C 45 C 10 150 90 BUT. 0 0 0 0 0 BL802 0VK8 G5 NAT (chronic)
inflamed
slimy.) 0 80 M, C 20 M, C 0 BUT. 0 280 100 BUT. 0 0 0 0 0 BL802 0VK8 G6 NAT (chronic)
inflamed
slimy.) 0 25 M, C 15 M, C 40 C 20 145 80 BUT. 0 0 0 0 0 BL802 0VK8 G7 NAT 0 thirty M, C 60 M, C 10 M, C 0 220 100 BUT. 0 0 0 0 0 BL802 0VK8 G8 NAT 0 20 M, C 20 M, C thirty M, C thirty 130 70 BUT. 0 0 0 0 0 BL802 0VK8 G9 NAT 0 60 M, C 20 M, C 15 C 5 235 95 BUT. 0 0 0 0 0 BL802 0VK8 G10 NAT (fibrosis)
textile
and blood
vessels) 0 BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. urothelium absent. BL802 0VK8 H1 NAT (Sparse) norm 0 0 BUT. 15 M, C 15 C 70 45 thirty BUT. 0 0 0 0 0 Calcium-ficats BL802 0VK8 H2 NAT (SM & BV) norm 0 BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. Fabric destroyed BL802 0VK8 H3 NAT (Sparse) norm 0 0 BUT. 70 M, C thirty C 0 170 100 BUT. 0 0 0 0 0 BL802 0VK8 H4 NAT (SM & BV) norm 0 BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. BUT. Textile
destroyed BL802 0VK8 H5 NAT (SM & BV) norm 0 0 BUT. 80 M, C 15 M, C 5 175 95 BUT. 0 0 0 0 0 BL802 0VK8 H6 NAT (Sparse) norm 0 0 BUT. 0 BUT. 10 C 90 10 10 BUT. 0 0 0 0 0 Scanty urothelium BL802 0VK8 H7 NAT norm 0 0 BUT. 25 M, C 15 C 60 65 40 BUT. 0 0 0 0 0 Missing basal layer BL802 0VK8 H8 NAT norm 0 70 M, C 20 M, C 0 BUT. 10 250 90 BUT. 0 0 0 0 0 BL802 0VK8 H9 NAT norm 0 0 BUT. 0 BUT. 40 C 60 40 40 BUT. 0 0 0 0 0 BL802 0VK8 H10 NAT norm 0 0 BUT. 0 BUT. thirty M, C 70 thirty thirty BUT. 0 0 0 0 0 Vessel urothelium General comments/notes:
G=level BUT=not defined Occ= sometimes N= normal F= focal positive NS=not visible Sc=ship C=cytoplasmic staining TCC=transitional cell carcinoma Ca=carcinoma BLA=bladder M=membrane staining SM=smooth muscle * = chronic inflammation of fibrous tissue and blood vessels BV=blood vessel NAT=normal next to tumor

[00626] The specificity and sensitivity of M22-321b41.1 were further determined in all 33 normal human samples contained in triplicate in FDA999d. As shown in Table 21, IHC staining with anti-nectin-4 antibody M22-321b41.1 was consistent with nectin-4 expression in normal cells reported in the literature (Rabet et al., 2016.), except that healthy breasts showed partial and weak or moderate immunoreactivity. Erythrocytes stained for nectin-4 with moderate intensity. The squamous epithelium was strongly stained, but the basal layer was not stained.

Table 21. Sensitivity of anti-nectin-4 antibody M22-321b41.1 in IHC staining of various tissues.

General information about the sample TEST ARTICLE STAINING OF DISTINCTIVE CELLS TEST ARTICLE STAINING OF OTHER CELL TYPES number
access number
test Place
TMA Cell. line/
Textile Place
i-type H-Score % nectin
4 Endothelium Ch.
musk Fibroblast Stroma Cl.
playback Comm. 3+
% 3+
sub 2+
% 2+
sub 1+
% 1+
sub 0
% TISSUE FDA999d 0VK5 A1 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A2 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A3 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A4 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A5 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A6 Brain (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A7 Cerebellum(N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A8 Cerebellum (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 A9 Cerebellum (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT N.S. 0 N.S. FDA999d 0VK5 B1 Adrenal (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B2 Adrenal (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B3 Adrenal (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. Presence of endogenous pigment FDA999d 0VK5 B4 Egg (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B5 Egg (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B6 Egg (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B7 Pankr. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B8 Pankr. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 B9 Pankr. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. Having painted. artifact cells 2+ FDA999d 0VK5 C1 Parashield (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C2 Parashield (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C3 Parashield (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C4 Pituitary gland (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C5 Pituitary gland (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C6 Pituitary gland (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. RBC 2+ FDA999d 0VK5 C7 Testicle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C8 Testicle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 C9 Testicle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. OCC interotitis. cells 2+ FDA999d 0VK5 D1 Shchitovit (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 D2 Shchitovit (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. FDA999d 0VK5 D3 Shchitovit (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 N.S. Stralny2+F colloidal2+ FDA999d 0VK5 D4 Milk yellow
(HOT) 0 0 BUT 20 M, C thirty C 50 70 50 0 N.S. 0 0-1 + 0 FDA999d 0VK5 D5 Milk yellow (HOT) 0 0 BUT 15 C 25 C 60 55 40 0 N.S. 0 0 0 FDA999d 0VK5 D6 Milk yellow (HOT) 0 10 M 25 M, C 25 M, C 40 105 60 0 N.S. 0 0 0 FDA999d 0VK5 D7 Spleen (N) 0 0 M 0 M, C 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 D8 Spleen (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 1-2+ 0 FDA999d 0VK5 D9 Spleen (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 1-2+ 0 FDA999d 0VK5 E1 Mind. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 BUT 0 0 0 RBC 3+ FDA999d 0VK5 E2 Mind. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E3 Mind. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0-1+F 0 FDA999d 0VK5 E4 Thymus (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E5 Thymus (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E6 Thymus (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E7 Bone brain. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E8 Bone brain. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 E9 Bone brain. (N) 0 25 C 5 C 0 BUT 70 85 thirty 0 N.S. 0 0 0 FDA999d 0VK5 F1 Lung(N) 0 0 BUT 10 M, C 20 M, C 70 40 thirty 0 0 0 0 0 Bronchial. epithelium (points) FDA999d 0VK5 F2 Lung(N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 F3 Lung(N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 F4 Heart muscle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. N.S. 0 N.S. FDA999d 0VK5 F5 Heart muscle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. N.S. 0 N.S. FDA999d 0VK5 F6 Heart muscle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. N.S. 0 N.S. FDA999d 0VK5 F7 Esophagus(N) 0 70 M, C 10 M, C 0 BUT 20 230 80 0 2+ 0 0 2+ Basal layer Neg: RBC's=3+ FDA999d 0VK5 F8 Esophagus(N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 F9 Esophagus(N) 0 thirty M, C thirty M, C 10 M, C thirty 160 70 0 3+ 0 0 3+ Basal layer Neg FDA999d 0VK5 G1 Stomach (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G2 Stomach (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G3 Stomach (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G4 Tonk.
intestinal (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G5 Tonk. intestinal (N) 0 BUT BUT BUT BUT BUT BUT BUT BUT BUT BUT BUT BUT BUT BUT No epithelium seen FDA999d 0VK5 G6 Tonk. intestinal (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G7 Thick. intestinal (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 2+ FDA999d 0VK5 G8 Thick. intestinal (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 G9 Thick. intestinal (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 H1 Liver (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 H2 Liver (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 H3 Liver (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 FDA999d 0VK5 H4 Saliva. iron (N) 0 0 BUT 15 C 10 C 75 40 25 0 N.S. 0 0 0 Mostly duct cells are stained FDA999d 0VK5 H5 Saliva. iron (N) 0 0 BUT 0 BUT 10 C 90 10 10 0 N.S. 0 0 2+ FDA999d 0VK5 H6 Saliva. iron (N) 0 10 C 10 C 0 BUT 80 50 20 0 N.S. 0 0 0 Painted
serous cells + ductal cells FDA999d 0VK5 H7 Kidneys (N) 0 0 BUT 5 C 0 BUT 95 10 5 0 N.S. N.S. 0 N.S. Proxim painted. Tubules FDA999d 0VK5 H8 Kidneys (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. N.S. 0 0 FDA999d 0VK5 H9 Kidneys (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. N.S. 0 0 FDA999d 0VK5 I1 Prostate (N) 0 0 BUT 10 C thirty C 60 50 40 0 3+ 0 0 0 FDA999d 0VK5 I2 Prostate (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 2+ 0 0 0 FDA999d 0VK5 I3 Prostate (N) 0 0 BUT 10 C 20 C 70 40 thirty 0 3+ 0 0 0 FDA999d 0VK5 I4 Endometrium (N) 0 20 M, C 20 M 0 BUT 60 100 40 0 0 0 0 N.S. FDA999d 0VK5 I5 Endometrium (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 I6 Endometrium (N) 0 0 BUT 20 M 20 M 60 60 40 0 0 0 0 N.S. Painted
mostly apical FDA999d 0VK5 I7 Neck
uterus
(HOT) 0 0 BUT 20 M 10 M 70 50 thirty 0 0 0 0 0 FDA999d 0VK5 I8 Neck
uterus
(HOT) 0 70 M, C 20 M, C 0 BUT 10 250 90 0 0 0 0 0 cytoplasmic basal 3+ FDA999d 0VK5 I9 Neck
uterus (HOT) 0 45 M, C 5 M, C 5 C 45 150 55 0 0 0 0 0 Not painted
ecto-cervical. basal FDA999d 0VK5 J1 Skeletal muscle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 J2 Skeletal muscle (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 J3 Muscle skeleton. (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 J4 Leather
(N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 Pigment.
Leather FDA999d 0VK5 J5 Leather (N) 0 80 M, C 10 M, C 0 BUT 10 260 90 0 0 0 0 0 Negative
basal layer FDA999d 0VK5 J6 Leather (N) 0 70 M, C 20 M, C 0 BUT 10 250 90 0 0 0 0 3+ FDA999d 0VK5 J7 Peripheral nerve (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 J8 Peripheral nerve (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. FDA999d 0VK5 J9 Perf. nerve (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 N.S. Occ IC=2+ FDA999d 0VK5 K1 Mesothelium (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K2 Meso-tholium (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K3 Mesothelium (N) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K4 Eye (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K5 Eye (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K6 Eye (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 N.S. 0 0 0 FDA999d 0VK5 K7 Larynx(HOT) 0 0 BUT 5 M, C 5 M, C 90 15 10 0 0 0 0 0 FDA999d 0VK5 K8 Larynx (HOT) 0 25 M, C 15 M, C 0 BUT 60 105 40 0 0 0 0-1 + 0 Negative
basal layer. FDA999d 0VK5 K9 Larynx (HOT) 0 0 BUT 0 BUT 0 BUT 100 0 0 0 0 0 0 0 Epithelium is rarely present. fragments General comments/notes: BUT=not defined Occ=sometimes N=normal F=focal put NS=Not Seen Sc=occasionally C=cytoplasmic staining HOT=Malignant tissue next to normal tissue IC=inflammatory cells M=membrane staining

[00627] Therefore, the IHC assay for nectin-4 expression based on M22-321b41.1 is sensitive and specific for nectin-4 expression. IHC analysis with M22-321b41.1 showed excellent accuracy and reproducibility.

EXAMPLE 7: FUNCTIONAL ANALYSIS: IMMUNOBLOTING

[00628] The functional properties and specificity of mAb M22-321b41.1 were further assessed using immunoblotting, such as Western blotting. Briefly, protein lysates prepared from various nectin-4 mRNA-positive and nectin-4 mRNA-negative samples were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probe M22-321b41.1 was added. Anti-human GAPDH antibody was used as a control to confirm that appropriate and equal amounts of total cell lysate were separated in each lane of the SDS-PAGE gel. As shown in FIG. 12A, M22-321b1.1 recognized and blotted a specific band of ~58-60 kDa, consistent with the predicted molecular mass of human nectin-4, in nectin-4 mRNA lysates of NCI H322M, AG-B1, and Rat1(E) positive cells , engineered to overexpress nectin-4. Smaller nectin-4 degradation products were detected in the AG-B1 xenograft samples as well as in the NCIH322M cell line, consistent with reports of nectin-4. (Buchanan et al., 2017.) No bands were detected in cells lacking nectin-4 MDA-MB-231 and Rat1(E)-Neo mRNAs. M22-321b41.1 also did not detect any of the nectin-1, nectin-2, and nectin-3 overexpressed by engineered Rat1(E) cells in Western blot analysis, although all members of the nectin family (nectin 1-4) were overexpressed in engineered Rat1(E) cells. Rat1(E) cells, as shown by FACS in Fig. 12B. Therefore, M22-321b41.1 specifically detected nectin-4 in immunoblotting analysis.

EXAMPLE 8: EPITOPES MAPPING

[00629] To further map the epitopes on nectin-4 recognized by the M22-321b41.1 antibody, various fragments of nectin-4 were cloned, transfected into cells, and expressed in cells. Cells expressing various nectin-4 fragments were cultured and lysed in RIPA buffer. Total protein concentration in RIPA lysates was quantified using a BSA standard, and a total of 20 μg of reduced (50 mM TCEP) lysates was subjected to SDS-PAGE on a 4-12% BT 15-lane gel in 1x MES buffer transferred from the I -blot a 2 μg/ml M22-321b41.1 antibody probe in 1:1 LI-COR blocking buffer was added to a nitrocellulose membrane blocked with LI-COR blocking buffer. A 1:10,000 dilution of α-GAPDH antibody (Chicken pAb, Millipore) was used to detect GAPDH as a control. Antibodies associated with protein bands were then detected at a 1:5000 dilution of goat anti-mouse antibody conjugated to IRDye 680 (Odyssey) or 1:5000 dilution of donkey anti-α-chicken conjugated to IRDye 800 (Odyssey) and visualized in Odyssey system. Antibody M22-321b41.1 recognized fragment V of nectin-4 (amino acid residues 1-150) (Figure 13). Therefore, antibody M22-321b41.1 recognized an epitope located at amino acid residues 1-150 (SEQ ID NO: 45) of human nectin-4. In addition, since antibody M22-321b41.1 was produced against a fragment of nectin-4 from amino acid residues 31-346 (SEQ ID NO: 2) of human nectin-4, as shown in Examples 1, 2 and 3, then antibody M22-321b41 .1 can recognize an epitope located at amino acid residues 31-150 (SEQ ID NO: 46) of human nectin-4 (overlapping region of fragment 1-150 and fragment 31-346).

EXAMPLE 9: Diagnostic and Prognostic Use of Nectin-4 IHC Assay

[00630] Nectin-4 expression was first examined in normal tissue samples using IHC staining with the M22-321b41.1 antibody. Variable, but generally weak to moderate, expression of nectin-4 was found in the skin (epidermis, sweat glands and hair follicles), transitional epithelium of the bladder, salivary glands (ducts), esophagus, mammary gland and stomach. To correlate nectin-4 expression with different cancers, nectin-4 expression was then examined in different tumor samples using IHC staining with the M22-321b41.1 antibody. Nectin-4 was highly expressed in bladder cancer cells and more moderately expressed in breast, pancreatic, lung, and ovarian tissue microarrays (TMAs). A study of the correlation between nectin-4 expression and bladder cancer using the same IHC assay found that 83% (434 of 524) of bladder cancers were TMA positive and 60% were strongly or moderately stained by IHC with M22-321b41. 1.

[00631] The effectiveness of nectin-4 expression in patient tissue, as determined by IHC staining with M22-321b41.1, to predict the patient's response to anticancer therapy (e.g., anticancer therapy using an anti-nectin-4 antibody conjugated to a cytotoxic or cytostatic agent) , then assessed, for example, by analyzing the response to anticancer therapy in patients who satisfy the following criteria: (1) have histologically confirmed metastatic solid tumor cancers (excluding sarcoma) that are refractory or recur; (2) have tumors positive for nectin-4 expression and a nectin-4 IHC assay H-score equal to or greater than 150; (3) who have failed any previous chemotherapy regimen for metastatic disease (patients with urethral and bladder cancer may respond to a previous chemotherapy regimen if they are considered eligible for cisplatin-based chemotherapy) (Galsky et al., Journal of Clinical Oncology, vol. 29, no. 17, 2432-2438 (2011)); (4) have documented disease progression if the most recent systemic treatment was an investigational immunotherapy drug; (5) have an Eastern Cooperative Oncology Group (ECOG) status of 0 or 1; (7) have measurable disease according to RECIST (version 1.1) (Eisenhauer, et. Al. European Journal of Cancer; 45(2): 228-247 (2009)). A total of 184 clinical trial samples were examined for nectin-4 expression using an IHC assay with antibody M22-321b41.1; of the 184 samples examined, 180 (98%) clinical trial samples had an H-score greater than 0; and 171 (93%) had moderate or high nectin-4 expression (H-score was ≥150). A total of 44 clinical subjects met the inclusion criteria. The location of tumor and/or metastases in these 44 patients is listed in Table 22.

Table 22. Location of primary tumors and metastases

Site of primary tumor N (%) Bladder 28 (63.6) Pelvis 10 (22.7) Ureter 3 (6.8) Bladder, other histology 2 (4.5) Other 1 (2.3) Place of metastasis N (%) Visceral 25 (56.8) Liver 10 (22.7) Lung 20 (45.5)

[00632] These 44 patients with moderate to high nectin-4 expression (H-score was ≥150) were administered an antitumor therapeutic agent containing a fully human anti-nectin-4 antibody (IgG1κ) conjugated to the microtubule disruptor monomethyl auristatin-E. All patients received a single 30-minute infusion of anti-nectin-4 antibody ADC once weekly for 3 weeks of each 4-week cycle (eg, days 1, 8, and 15) until disease progression, treatment intolerance, investigator decision, or revocation of informed consent. The doses administered were 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, and 1.25 mg/kg. Patient response was assessed by the investigator using RECIST version 1.1 once every 8 weeks (±7 days) (RECIST as described in Eisenhauer, et al. European Journal of Cancer; 45(2): 228–247 (2009)). A total of 36 patients showed results that could be assessed.

[00633] An anti-nectin-4 antibody ADC produced antitumor activity in treated patients, all of whom had tumors expressing nectin-4. Ten of 36 patients had partial responses, a 27.8% response rate (with partial or complete responses considered a response). The anti-nectin-4 antibody ADC also induced antitumor activity in 4 of 10 patients with liver metastases (40% response rate) and in 3 of 12 patients previously treated with checkpoint inhibitors (25% response rate).

[00634] Consequently, the anti-nectin-4 antibody ADC induced antitumor activity in patients with tumors expressing nectin-4.

EXAMPLE 10: Diagnostic and Prognostic Use of Nectin-4 IHC Assays - Distinct Cohort A

[00635] The effectiveness of nectin-4 expression in patient tissue, as determined by IHC staining with M22-321b41.1, to predict the patient's response to anticancer therapy (e.g., anticancer therapy using an anti-nectin-4 antibody conjugated to a cytotoxic or cytostatic agent) , then assessed, for example, by analyzing the response to anticancer therapy in patients in another cohort who met the following criteria: (1) had histologically confirmed metastatic solid tumor cancers, including urothelial cancer; (2) have tumors positive for nectin-4 expression and a nectin-4 IHC assay H-score equal to or greater than 150; (3) those who have failed any previous chemotherapy regimen for metastatic disease and/or patients with urothelial cancer who are not suitable for cisplatin-based chemotherapy (Galsky et al., Journal of Clinical Oncology, vol. 29, no. 17, 2432 -2438 (2011)); (4) have documented disease progression following immune checkpoint inhibitor (CPI) therapy immediately prior to the study; (5) have an Eastern Cooperative Oncology Group (ECOG) status of 0 or 1; (7) have measurable disease according to RECIST (version 1.1) (Eisenhauer, et. Al. European Journal of Cancer; 45(2): 228-247 (2009)). A total of 295 clinical trial samples were examined for nectin-4 expression using an IHC assay with antibody M22-321b41.1; of the 295 samples examined, 278 (94%) clinical trial samples had an H-score equal to or greater than 1; and 245 (83%) had moderate or high nectin-4 expression (H-score was ≥150). A total of 58 clinical subjects met inclusion criteria. The location of tumor and/or metastases in these 58 patients is listed in Table 23.

Table 23. Location of primary tumors and metastases

Site of primary tumor N (%) Bladder 39 (67.2) Pelvis 14 (24.1) Ureter 4 (6.9) Other, right kidney 1 (1.7) Site of metastasis N (%) Visceral 34 (58.6) Liver 16 (27.6) Lung 26 (44.8)

[00636] These 58 patients with moderate to high nectin-4 expression (H-score was ≥150) were administered an antitumor therapeutic agent containing a fully human anti-nectin-4 antibody (IgG1κ) conjugated to the microtubule disruptor monomethyl auristatin-E. All patients received a single 30-minute infusion of anti-nectin-4 antibody ADC once weekly for 3 weeks of each 4-week cycle (eg, days 1, 8, and 15) until disease progression, treatment intolerance, investigator decision, or withdrawal of informed consent. The doses administered were 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, and 1.25 mg/kg. Patient response was assessed by the investigator using RECIST version 1.1 once every 8 weeks (±7 days) (RECIST as described in Eisenhauer, et al. European Journal of Cancer; 45(2): 228–247 (2009)). A total of 49 patients showed results that could be assessed.

[00637] An anti-nectin-4 antibody ADC produced antitumor activity in treated patients, all of whom had tumors expressing nectin-4. One patient had a complete response and 17 patients had partial responses, resulting in an overall response rate of 36.7% (with both partial and complete responses considered responses). In the group receiving 1.25 mg/kg anti-nectin-4 ADC, 10 of 17 patients responded, for a response rate of 58.8%. The anti-nectin-4 antibody ADC also induced antitumor activity in 5 of 12 patients with liver metastases (response rate 41.7%), 6 of 16 patients previously treated with checkpoint inhibitors (response rate 37.5%), and 8 of 20 patients previously treated with taxanes (40% response rate).

[00638] Consequently, the anti-nectin-4 antibody ADC induced antitumor activity in patients with tumors expressing nectin-4.

EXAMPLE 11: Therapeutic use of anti-nectin-4 antibody ADC.

[00639] The effectiveness of the anti-nectin-4 antibody ADC of the present invention in treating patients with various types of cancer was assessed, for example, by comparing the results of treating patients with an anti-nectin-4 antibody ADC, including the human or humanized antibody M22-321b41.1, and a control with patients treated with isotype antibody placebo. Briefly, patients were randomized and assigned to a control or treatment group. Patients in the treatment group received anti-nectin-4 ADC antibodies at a dose range that was determined according to the mg (antibody) per kg (body weight) dose range optimized in animal models and/or in Examples 9 and 10. The injection was given once or several times over a period of time. Patient outcomes were periodically analyzed and compared between the treatment group and the placebo group.

[00640] Outcome was improved in patients receiving anti-nectin-4 ADC compared to patients receiving placebo. Compared with placebo treatment, ADC treatment with anti-nectin-4 antibody resulted in higher survival at 6 months, 1 year, 2 years, 3 years, or 5 years, increased survival time, decreased levels of cancer markers, decreased mean or average tumor size, and /or slower cancer progression.

EXAMPLE 12: Additional diagnostic and prognostic applications of nectin-4 IHC assays.

[00641] The effectiveness of nectin-4 expression in patient tissue, as determined by IHC staining with M22-321b41.1, to predict the patient's response to anticancer therapy (e.g., anticancer therapy using an anti-nectin-4 antibody conjugated to a cytotoxic or cytostatic agent) , was assessed, for example, by correlating the response of patients to cancer treatment with the expression of nectin-4 in the patient's tumor tissues, determined by IHC staining with a specific anti-nectin-4 antibody. Briefly, all patients were treated with the same cancer treatment regimen, and nectin-4 expression levels in tissues of cancer patients were determined in IHC assays using the M22-321b41.1 antibody as described herein. Patients are divided into two, three, four or more groups according to the expression of nectin-4 in their tumor tissues. For example, when divided into four groups, patients with high expression of nectin-4 in tumor tissues are placed in the first group, the second group with moderate expression, the third group with low expression, and the fourth group with no expression. Response to a cancer treatment regimen that is reflected in patient outcome such as patient weight, patient tumor size, patient survival rate of 6 months, 1 year, 2 years, 3 years, or 5 years, or patient survival rate in each group are summarized and correlated with nectin-4 expression in patients in each group. Correlation is performed using the mean or median value for each group or performed for each patient in each group.

[00642] Expression of nectin-4 in a patient's tumor tissue indicates how the patient responds to an anti-tumor treatment regimen, for example, anti-tumor treatment with an anti-nectin-4 antibody conjugated to cytotoxic or cytostatic agents (referred to as an anti-nectin-4 antibody ADC) . Patients with higher expression of nectin-4 in tumor tissues respond better to the antitumor treatment regimen, have higher survival of 6 months, 1 year, 2 years, 3 years or 5 years and/or live longer than patients with reduced nectin expression -4 in tumor tissues. The higher the expression of nectin-4 in a patient's tumor tissues, the more effective antitumor therapy, such as anti-nectin-4 antibody ADC therapy, is for the patient.

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LIST OF SEQUENCES

<110> AGENCY, INC.

<120> NECTIN-4 BINDING PROTEINS AND METHODS OF THEIR APPLICATION

<130> 14369-208-228

<140> Will be assigned

<141> here

<150> 62/515,454

<151> 2017-06-05

<160> 46

<170> Patent In version 3.5

<210> 1

<211> 510

<212> Protein

<213> Homo sapiens

<220>

<223> GenBank accession number NP_112178

<400> 1

Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly

20 25 30

Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala

35 40 45

Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln

50 55 60

Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala

65 70 75 80

Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly

85 90 95

Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val

100 105 110

Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg

115 120 125

Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg

130 135 140

Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu

145 150 155 160

Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser

165 170 175

Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser

180 185 190

Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe

195 200 205

His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val

210 215 220

Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu

225 230 235 240

His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln

245 250 255

Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser

260 265 270

Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro

275 280 285

Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro

290 295 300

Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu

305 310 315 320

Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln

325 330 335

Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val

340 345 350

Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val

355 360 365

Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln

370 375 380

Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg

385 390 395 400

Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val

405 410 415

Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser

420 425 430

Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu

435 440 445

Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly

450 455 460

Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln

465 470 475 480

Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro

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Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val

500 505 510

<210> 2

<211> 316

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 2

Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln

1 5 10 15

Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val

20 25 30

Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu

35 40 45

Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr

50 55 60

Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly

65 70 75 80

Ser Val Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu

85 90 95

Cys Arg Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg

100 105 110

Leu Arg Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala

115 120 125

Leu Glu Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu

130 135 140

Gly Ser Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr

145 150 155 160

Thr Ser Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser

165 170 175

Glu Phe His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr

180 185 190

Cys Val Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His

195 200 205

Ile Leu His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu

210 215 220

Asp Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys

225 230 235 240

Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp

245 250 255

Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe

260 265 270

Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser

275 280 285

Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp

290 295 300

Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val

305 310 315

<210> 3

<211> 133

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 3

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr His Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly

130

<210> 4

<211> 136

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Thr Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala

115 120 125

Pro Val Cys Gly Asp Thr Thr Gly

130 135

<210> 5

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 5

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr His Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Thr Phe Asn Arg Asn Glu Cys

210 215

<210> 6

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 6

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Thr Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala

115 120 125

Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu

130 135 140

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly

145 150 155 160

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp

165 170 175

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro

180 185 190

Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys

195 200 205

Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro

210 215 220

Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro

245 250 255

Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val

260 265 270

Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala

290 295 300

Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys

305 310 315 320

Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser

325 330 335

Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro

340 345 350

Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val

355 360 365

Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly

370 375 380

Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp

405 410 415

Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His

420 425 430

Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 7

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 7

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 8

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 8

Ile Thr Tyr Leu Tyr Trp Tyr

15

<210> 9

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 9

Leu Leu Ile Tyr His Met Ser Asn Leu Ala Ser

1 5 10

<210> 10

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 10

His Met Ser Asn Leu Ala Ser

15

<210> 11

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 11

Leu Leu Ile Tyr His Met Ser Asn Leu Ala

1 5 10

<210> 12

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 12

Ala Gln Asn Leu Glu Leu Pro Phe Thr

15

<210> 13

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 13

Ala Gln Asn Leu Glu Leu Pro Phe

15

<210> 14

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 14

Gly Tyr Thr Phe Thr Thr Tyr Trp Met Gln

1 5 10

<210> 15

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 15

Thr Tyr Trp Met Gln

15

<210> 16

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 16

Gly Tyr Thr Phe Thr Thr Tyr

15

<210> 17

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 17

Thr Thr Tyr Trp Met Gln

15

<210> 18

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 18

Trp Ile Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln

1 5 10 15

Lys Phe Lys Gly

20

<210> 19

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 19

Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys

1 5 10 15

Gly

<210> 20

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 20

Tyr Pro Gly Asp Gly Asp

15

<210> 21

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 21

Trp Ile Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg

1 5 10

<210> 22

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 22

Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr

15

<210> 23

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 23

Glu Tyr Tyr Gly Leu Asp Tyr

15

<210> 24

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 24

Ala Arg Glu Tyr Tyr Gly Leu Asp

15

<210> 25

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 25

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys

20

<210> 26

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 26

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

1 5 10 15

<210> 27

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 27

Gly Val Pro Asp Arg Phe Thr Ser Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 28

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 28

Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg

1 5 10

<210> 29

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 29

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

<210> 30

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 30

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10

<210> 31

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 31

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Ile Gln

1 5 10 15

Leu Ser Thr Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 32

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 32

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 33

<211> 86

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 33

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

1 5 10 15

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

20 25 30

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

35 40 45

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

50 55 60

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Thr

65 70 75 80

Phe Asn Arg Asn Glu Cys

85

<210> 34

<211> 310

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 34

Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

1 5 10 15

Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

20 25 30

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val

35 40 45

Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val

50 55 60

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg

65 70 75 80

Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn

85 90 95

Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp

100 105 110

Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp

115 120 125

Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn

130 135 140

Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn

145 150 155 160

Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp

165 170 175

Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro

180 185 190

Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala

195 200 205

Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys

210 215 220

Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile

225 230 235 240

Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn

245 250 255

Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys

260 265 270

Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys

275 280 285

Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe

290 295 300

Ser Arg Thr Pro Gly Lys

305 310

<210> 35

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 35

Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala

1 5 10 15

Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

20 25 30

Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

50 55 60

Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

85 90 95

Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp

100 105 110

Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val

115 120 125

Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr

130 135 140

Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu

145 150 155 160

Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr

165 170 175

Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr

180 185 190

Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr

195 200 205

Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys

210 215 220

Ser Phe Ser Arg Thr Pro Gly Lys

225 230

<210> 36

<211> 1533

<212> DNA

<213> Homo sapiens

<220>

<223> GenBank accession number NM_030916

<400> 36

atgcccctgt ccctgggagc cgagatgtgg gggcctgagg cctggctgct gctgctgcta

60

ctgctggcat catttacagg ccggtgcccc gcgggtgagc tggagacctc agacgtggta

120

actgtggtgc tgggccagga cgcaaaactg ccctgcttct accgagggga ctccggcgag

180

caagtggggc aagtggcatg ggctcgggtg gacgcggggcg aaggcgccca ggaactagcg

240

ctactgcact ccaaatacgg gcttcatgtg agcccggctt acgagggccg cgtggagcag

300

ccgccgcccc cacgcaaccc cctggacggc tcagtgctcc tgcgcaacgc agtgcaggcg

360

gatgagggcg agtacgagtg ccgggtcagc accttccccg ccggcagctt ccaggcgcgg

420

ctgcggctcc gagtgctggt gcctcccctg ccctcactga atcctggtcc agcactagaa

480

gagggccagg gcctgaccct ggcagcctcc tgcacagctg agggcagccc agcccccagc

540

gtgacctggg acacggaggt caaaggcaca acgtccagcc gttccttcaa gcactcccgc

600

tctgctgccg tcacctcaga gttccacttg gtgcctagcc gcagcatgaa tgggcagcca

660

ctgacttgtg tggtgtccca tcctggcctg ctccaggacc aaaggatcac ccacatcctc

720

cacgtgtcct tccttgctga ggcctctgtg aggggccttg aagaccaaaa tctgtggcac

780

attggcagag aaggagctat gctcaagtgc ctgagtgaag ggcagccccc tccctcatac

840

aactggacac ggctggatgg gcctctgccc agtggggtac gagtggatgg ggacactttg

900

ggctttcccc cactgaccac tgagcacagc ggcatctacg tctgccatgt cagcaatgag

960

ttctcctcaa gggattctca ggtcactgtg gatgttcttg acccccagga agactctggg

1020

aagcaggtgg acctagtgtc agcctcggtg gtggtggtgg gtgtgatcgc cgcactcttg

1080

ttctgccttc tggtggtggt ggtggtgctc atgtcccgat accatcggcg caaggcccag

1140

cagatgaccc agaaatatga ggaggagctg accctgacca gggagaactc catccggagg

1200

ctgcattccc atcacacgga ccccaggagc cagccggagg agagtgtagg gctgagagcc

1260

gagggccacc ctgatagtct caaggacaac agtagctgct ctgtgatgag tgaagagccc

1320

gagggccgca gttactccac gctgaccacg gtgagggaga tagaaacaca gactgaactg

1380

ctgtctccag gctctgggcg ggccgaggag gaggaagatc aggatgaagg catcaaacag

1440

gccatgaacc attttgttca ggagaatggg accctacggg ccaagcccac gggcaatggc

1500

atctacatca atgggcgggg acacctggtc tga

1533

<210> 37

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 37

gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc

60

atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg

120

tatctgcaga agccaggcca gtctcctcag ctcctgattt atcatatgtc caaccttgcc

180

tcaggagtcc cagacaggtt cactagcagt gggtcaggaa ctgatttcac actgagaatc

240

agcagagtgg aggctgagga tgtgggtgtt tattactgcg ctcaaaatct agaacttccg

300

ttcacgttcg gaggggggac caagctggaa acaaaacggg ctgatgctgc accaactgta

360

tccatcttcc caccatccag tgagcagtta acatctgga

399

<210> 38

<211> 408

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 38

caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaaattg

60

tcctgcaagg cttctggcta tacctttact acctactgga tgcagtgggt aaaacagagg

120

cctggacagg gtctggaatg gattgggtct atttatcctg gagatggtga tactaggtac

180

actcagaagt tcaagggcaa ggccacattg actgcagata aatcctccag cacagcctac

240

attcaactca gcaccttggc atctgaggac tctgcggtct attactgtgc aagagaatac

300

tacggtcttg actactgggg ccaaggcacc actctcacag tctcctcagc caaaacaaca

360

gccccatcgg tctatccact ggcccctgtg tgtggagata caactggc

408

<210> 39

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 39

gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc

60

atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg

120

tatctgcaga agccaggcca gtctcctcag ctcctgattt atcatatgtc caaccttgcc

180

tcaggagtcc cagacaggtt cactagcagt gggtcaggaa ctgatttcac actgagaatc

240

agcagagtgg aggctgagga tgtgggtgtt tattactgcg ctcaaaatct agaacttccg

300

ttcacgttcg gaggggggac caagctggaa acaaaacggg ctgatgctgc accaactgta

360

tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc

420

ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga

480

caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg

540

agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag

600

gccactcaca agacatcaac ttcacccatt gtcaagacct tcaacaggaa tgagtgt

657

<210> 40

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 40

caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaaattg

60

tcctgcaagg cttctggcta tacctttact acctactgga tgcagtgggt aaaacagagg

120

cctggacagg gtctggaatg gattgggtct atttatcctg gagatggtga tactaggtac

180

actcagaagt tcaagggcaa ggccacattg actgcagata aatcctccag cacagcctac

240

attcaactca gcaccttggc atctgaggac tctgcggtct attactgtgc aagagaatac

300

tacggtcttg actactgggg ccaaggcacc actctcacag tctcctcagc caaaacaaca

360

gccccatcgg tctatccact ggcccctgtg tgtggagata caactggctc ctcggtgact

420

ctaggatgcc tggtcaaggg ttatttccct gagccagtga ccttgacctg gaactctgga

480

tccctgtcca gtggtgtgca caccttccca gctgtcctgc agtctgacct ctacaccctc

540

agcagctcag tgactgtaac ctcgagcacc tggcccagcc agtccatcac ctgcaatgtg

600

gcccacccgg caagcagcac caaggtggac aagaaaattg agcccagagg gcccacaatc

660

aagccctgtc ctccatgcaa atgcccagca cctaacctct tgggtggacc atccgtcttc

720

atcttccctc caaagatcaa ggatgtactc atgatctccc tgagccccat agtcacatgt

780

gtggtggtgg atgtgagcga ggatgaccca gatgtccaga tcagctggtt tgtgaacaac

840

gtggaagtac acacagctca gacacaaacc catagagagg attacaacag tactctccgg

900

gtggtcagtg ccctccccat ccagcaccag gactggatga gtggcaagga gttcaaatgc

960

aaggtcaaca acaaagacct cccagcgccc atcgagagaa ccatctcaaa acccaaaggg

1020

tcagtaagag ctccacaggt atatgtcttg cctccaccag aagaagagat gactaagaaa

1080

caggtcactc tgacctgcat ggtcacagac ttcatgcctg aagacattta cgtggagtgg

1140

accaacaacg ggaaaacaga gctaaactac aagaacactg aaccagtcct ggactctgat

1200

ggttcttact tcatgtacag caagctgaga gtggaaaaga agaactgggt ggaaagaaat

1260

agctactcct gttcagtggt ccacgagggt ctgcacaatc accacacgac taagagcttc

1320

tcccggactccgggtaaa

1338

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 41

ggctggagtt caatgaggtt tattt

25

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 42

tccagcagat ttcagactaa gaaga

25

<210> 43

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 43

agaacatcat ccctgcctct actg

24

<210> 44

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 44

aaatgagctt gacaaagtgg tcgt

24

<210> 45

<211> 150

<212> Protein

<213> Homo sapiens

<400> 45

Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly

20 25 30

Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala

35 40 45

Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln

50 55 60

Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala

65 70 75 80

Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly

85 90 95

Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val

100 105 110

Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg

115 120 125

Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg

130 135 140

Val Leu Val Pro Pro Leu

145 150

<210> 46

<211> 120

<212> Protein

<213> Homo sapiens

<400> 46

Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln

1 5 10 15

Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val

20 25 30

Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu

35 40 45

Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr

50 55 60

Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly

65 70 75 80

Ser Val Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu

85 90 95

Cys Arg Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg

100 105 110

Leu Arg Val Leu Val Pro Pro Leu

115 120

<---

Claims (77)

1. An antibody or antigen-binding fragment that binds to human nectin-4 containing (a) a light chain variable region (VL) containing: (1) CDR1 VL with the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (2) CDR2 VL with the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; And (3) CDR3 VL with the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; And (b) a heavy chain variable region (VH) containing: (1) CDR1 VH with the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; (2) CDR2 VH with the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; And (3) CDR3 VH with the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. 2. Antibody or antigen-binding fragment thereof according to claim 1, where: (a) VL contains VL framework 1 (FR1) containing the amino acid sequence SEQ ID NO: 25, FR2 VL containing the amino acid sequence SEQ ID NO: 26, FR3 VL containing the amino acid sequence of SEQ ID NO: 27, and FR4 VL containing the amino acid sequence SEQ ID NO: 28; and/or (b) VH contains VH framework 1 (FR1) containing the amino acid sequence SEQ ID NO: 29, FR2 VH containing the amino acid sequence SEQ ID NO: 30, FR3 VH containing the amino acid sequence of SEQ ID NO: 31, and FR4 VH containing the amino acid sequence SEQ ID NO: 32. 3. Antibody or antigen-binding fragment thereof according to claim 1 or 2, where CDR1 VL, CDR2 VL and CDR3 VL contain the amino acid sequences SEQ ID NO: 7, 10 and 12, respectively and CDR1 VH, CDR2 VH and CDR3 VH contain the amino acid sequences of SEQ ID NO: 15, 19 and 23, respectively. 4. Antibody or antigen-binding fragment thereof according to any one of claims 1-3, where VL contains the amino acid sequence SEQ ID NO: 3 and/or VH contains the amino acid sequence SEQ ID NO: 4. 5. An antibody or an antigen-binding fragment thereof according to any one of claims 1-4, where the antibody or an antigen-binding fragment thereof contains: a) Fc fragment of mouse IgG2, Fc fragment of human IgG1 or its mutated variant; b) a heavy chain Fc fragment containing the amino acid sequence of SEQ ID NO: 35, and/or a light chain constant region containing the amino acid sequence of SEQ ID NO: 33; and/or c) a light chain containing the amino acid sequence of SEQ ID NO: 5, and/or heavy chain containing the amino acid sequence SEQ ID NO: 6. 6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein human nectin-4 is expressed by the cell and the antibody or antigen-binding fragment thereof binds to the cell. 7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the cell is a malignant tissue cell. 8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody or antigen-binding fragment thereof binds to said cancer cell at a higher level compared to its binding to a control cell. 9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the control cell is a non-malignant cell of the same tissue. 10. The antibody or antigen-binding fragment according to claim 7, where the specified tissue includes tissue of the bladder, ureter, breast, lung, colon, rectum, ovary, fallopian tube, esophagus, cervix, endometrium of the uterus, skin, larynx, bone brain, salivary gland, kidney, prostate gland, brain, spinal cord, placenta, adrenal gland, pancreas, parathyroid gland, pituitary gland, testicle, thyroid gland, spleen, tonsil, thymus, heart, stomach, small intestine, liver, skeletal muscles, peripheral nerve, mesothelium or eye. 11. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, where i) the antibody is a) monoclonal antibody and/or b) a humanized antibody, a human antibody or a chimeric antibody; and/or ii) the antigen binding fragment is a Fab, Fab', F(ab') ₂ , variable fragment (Fv), single chain variable fragment (scFv) or disulfide linked variable fragment (dsFv). 12. A polynucleotide for encoding a polypeptide, containing a nucleotide sequence encoding the VH of an antibody or an antigen-binding fragment according to any one of claims 1 to 11. 13. A polynucleotide for encoding a polypeptide, containing a nucleotide sequence encoding the VL of an antibody or an antigen-binding fragment according to any one of claims 1 to 11. 14. A polynucleotide for encoding a polypeptide, comprising a first nucleotide sequence encoding a VH and a second nucleotide sequence encoding a VL of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. 15. A polynucleotide for encoding a polypeptide, containing a nucleotide sequence encoding the heavy chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11. 16. A polynucleotide for encoding a polypeptide, containing a nucleotide sequence encoding a light chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11. 17. A polynucleotide for encoding a polypeptide, comprising a first nucleotide sequence encoding a heavy chain and a second nucleotide sequence encoding a light chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11. 18. A vector for introducing a nucleic acid into a host cell, containing a polynucleotide according to any one of claims 12-17. 19. A cell for expressing a polynucleotide, comprising a polynucleotide according to any one of claims 12 to 17 or a vector according to claim 18. 20. A method for producing an antibody or an antigen-binding fragment according to any one of claims 1 to 11, including cultivating a cell containing a polynucleotide containing a) a nucleotide sequence encoding a VH antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, and b) a nucleotide sequence encoding a VL antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, to express an antibody or an antigen-binding fragment thereof. 21. A kit for predicting the susceptibility of a patient with a malignant neoplasm to antitumor therapy, containing an antibody or its antigen-binding fragment according to claims 1-11 and a label or insert. 22. The kit of claim 16, wherein a cancer patient is predicted to be susceptible to anticancer therapy if the patient's cancer cell is positive for nectin-4 expression and/or if the level of nectin-4 expression in the tissue sample is higher than the control level of nectin expression -4. 23. A method for determining the expression of nectin-4 in a tissue sample of an individual suspected of having a malignant neoplasm, comprising: (a) bringing the tissue sample into contact with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; (b) detecting binding of an antibody or an antigen-binding fragment thereof to the tissue sample; And (c) determination of nectin-4 expression in a tissue sample. 24. A method for determining the expression of nectin-4 in a tissue sample of an individual suspected of having a malignant neoplasm, comprising: (a) performing an immunohistochemical (IHC) analysis of a tissue sample with an antibody or an antigen-binding fragment according to any one of claims 1 to 11; And (b) determination of nectin-4 expression in a tissue sample. 25. A method for assessing the susceptibility of an individual with a malignant disease to an antitumor therapeutic agent, including: (a) bringing a tissue sample of an individual with a malignant disease into contact with an antibody or an antigen-binding fragment according to any one of claims 1 to 11; (b) detecting binding of an antibody or an antigen-binding fragment thereof to a tissue sample; (c) determining the expression level of nectin-4 in the tissue sample; And (d) comparing the level of expression of nectin-4 in the tissue sample determined in step (c) with the control level of expression of nectin-4, wherein the increased level of expression of netin-4 determined in step (c) compared with the control indicates the individual's susceptibility to antitumor therapy. 26. A method for assessing the susceptibility of an individual with a malignant disease to an antitumor therapeutic agent, including: (a) performing an immunohistochemical analysis (IHC) of a tissue sample from an individual with a malignant tumor with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11; (b) determining the expression level of nectin-4 in the tissue sample; And (c) comparing the level of expression of nectin-1 in the tissue sample measured in step (b) with the control level of expression of nectin-4, wherein an increased level of expression of netin-4 in the tissue sample compared to the control indicates susceptibility to the indicated antitumor therapy. 27. The method of claim 25 or 26, wherein the antitumor therapeutic agent comprises an anti-nectin-4 antibody or an antibody-drug conjugate comprising an anti-nectin-4 antibody. 28. The method according to any one of claims 23-27, where the malignancy is selected from the group consisting of endometrial cancer, urothelial cancer, bladder cancer, ureteral cancer, urethral cancer, lung cancer, ovarian cancer, breast cancer, esophageal cancer , pancreatic cancer, head and neck cancer, prostate cancer, penile cancer, anal cancer, vulvar cancer, cancer of the primary urachal duct (urachus), and cancers of epithelial origin in which nectin-4 is expressed. 29. The method according to any one of claims 25 to 28, wherein the control level of nectin-4 expression is the level of expression of nectin-4 in non-cancerous cells of an individual or in non-cancerous cells of another individual. 30. The method according to any one of claims 23-29, where the individual is a person.

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