Human nectin-4 binders

Abstract

The present disclosure provides isolated antibodies that specifically bind to human Nectin-4, antigen-binding fragments of such antibodies, and kits comprising the anti-Nectin-4 antibodies or binding fragments and a set of reagents for detecting a complex of the antibody or antigen-binding fragment thereof bound to human Nectin-4. The antibodies and antigen-binding fragments of this disclosure are useful for detection of human Nectin-4 expression in tissue samples and for treating cancer in a subject. Nucleic acid molecules encoding the antibodies and antigen-binding fragments of this disclosure, expression vectors, and host cells for expressing the antibodies and antigen-binding fragments are also provided.

Description

HUMAN NECTIN-4 BINDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S Provisional Application No. 63 / 599085 filed November 15, 2023.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 16, 2024, is named 25821-US-PSP_SL.xml and is 37,815 bytes in size.FIELD OF THE INVENTION

[0003] This disclosure relates generally to antibodies used in immunohistochemistry.BACKGROUND OF THE INVENTION

[0004] Nectin-4, also known as Nectin Cell Adhesion Molecule 4, poliovirus receptor-like protein 4 (PVRL4), PRR4. and LNIR, is a calcium ion-independent type I transmembrane protein that mediates cell adhesion junction formation. Nectin-4 includes an extracellular segment formed by three Ig-like domains, a transmembrane domain and an intracellular domain. The three Ig-like domains comprise one IgV domain and two IgC domains, and the C-terminal of the intracellular domain has a conservative region that binds the PDZ domain of the actin binding protein Afadin. thereby activating several cell-signaling cascades.

[0005] Nectin-4 can promote tumor cell survival and proliferation by inhibiting anoikis and activating PI3K-AKT signaling pathway. Studies have shown that Nectin-4 is expressed in gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer and esophageal cancer, and strongly expressed in non-small cell lung cancer, bladder cancer, breast cancer and pancreatic cancer. The high expression of Nectin-4 in tumors is also closely related to cancer progression and poor prognosis of patients having lung cancer, esophageal cancer, and pancreatic cancer (Bouleftour et al., Mol Cancer Ther. 2022 Apr 1 ;21(4):493-501).

[0006] Human Nectin-4 has been a challenging target for development of quality immunohistochemistry (IHC) reagents for use in formalin fixed paraffin embedded (FFPE) human tissue. Formalin fixation of tissue results in cross-linking of proteins which alternativeepitope structure, in many instances altenng the ability of antibodies to bind to them effectively. The formaldehyde in formalin fixatives react readily with various functional groups of biologic macromolecules with progressive cross-links forming over time; Thavarajah R, Mudimbaimannar VK, Elizabeth J, Rao UK, Ranganathan K. Chemical and Physical Basics of Formaldehyde Fixation. J Oral Maxillofac Pathol. 2012 Sep-Dec; 16(3): 400-405. As a result, many antibodies that bind native protein structure do not perform well as reagents in IHC assays, and antibodies that do perform well in IHC tend to bind epitopes that are minimally affected by formalin fixation. Such epitopes may be concealed or not strongly immunogenic, rendering generation of antibodies suitable for IHC applications difficult.

[0007] An anti-human Nectin-4 antibody for detecting Nectin-4 expression in biological samples is described in US11292837B2. However, a more sensitive antibody for detecting Nectin-4 in biological samples would be desirable.SUMMARY OF THE INVENTION

[0008] The present disclosure provides isolated antibodies that specifically bind to human Nectin-4, antigen-binding fragments of such antibodies, and kits comprising the anti-Nectin-4 antibodies or binding fragments and a set of reagents for detecting a complex of the antibody or antigen-binding fragment thereof bound to human Nectin-4.

[0009] In one aspect, the present disclosure provides a human Nectin-4 binder comprising: a) the three heavy chain complementarity determining regions (HC-CDRs) of a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 21; and b) the three light chain complementarity determining regions (LC-CDRs) of a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 22; wherein the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme.

[0010] In some embodiments, the human Nectin-4 binder comprises HC-CDRs and LC-CDRs according to the Kabat numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprises SEQ ID NO: 2, and HC-CDR3 comprises SEQ ID NO: 3; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 5. and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0011] In some embodiments, the human Nectin-4 binder comprises HC-CDRs and LC-CDRs according to the Chothia numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 8,and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 3; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0012] In some embodiments, the human Nectin-4 binder comprises HC-CDRs and LC-CDRs according to the AbM numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 10; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4. LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0013] 5 In some embodiments, the human Nectin-4 binder comprises HC-CDRs and LC- CDRs according to the IMGT numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 11, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 12, HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 13; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprises the amino acid sequence WTS, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0014] In some embodiments, the human Nectin-4 binder comprises HC-CDRs and LC-CDRs according to the Contact numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 15, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 17; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 20.

[0015] In any one of the foregoing embodiments, the human Nectin-4 binder comprises a VH comprising an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions to the amino acid sequence of SEQ ID NO: 21, and a VL comprising an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions to the amino acid sequence of SEQ ID NO: 22, wherein the antibody or antigen-binding fragment thereof does not vary at the CDR amino acid sequences of any one of claims 1-6.

[0016] In any one of the foregoing embodiments, the human Nectin-4 binder comprises a heavy chain variable region of SEQ ID NO: 21, and a light chain variable region of SEQ ID NO: 22. In any one of the foregoing embodiments, the human Nectin-4 binder comprises human Nectin-4 binder of claim 7, comprising a heavy chain region having the amino acid sequence of SEQ IDNO: 23, and a light chain having the amino acid sequence of SEQ ID NO: 24. In any one of the foregoing embodiments, the human Nectin-4 binder consists of a heavy chain region having the amino acid sequence of SEQ ID NO: 23, and a light chain having the amino acid sequence of SEQ ID NO: 24.

[0017] In any one of the foregoing embodiments, the human Nectin-4 binder comprises the VH comprises a framework selected from the group consisting of human VH1, VH2, VH3, VH4, VH5, and VH6, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and. the VL comprises a framework selected from the group consisting of human VKI, VK2, VK3, VK4, VK5, VK6, V I. VA2, VZ3. VZ4. V / .5. VA6, VZ7. VX8, VA9. and V 10, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In some embodiments, the VH comprises a framework selected from the group consisting of human VH1, VH2, VH3, VH4, VH5. and VH6; and the VL comprises a framework selected from the group consisting of human VKI. VK2, VK3, VK4, VK5, VK6. Vkl. V / .2. VL3. VA4. V / .5. VA6, VA7. V / .8. V / .9. and VMO.

[0018] In some embodiments of the human Nectin-4 binder, the first residue of the VH is pyroglutamate or pyroglutamic acid.

[0019] In some embodiments, the human Nectin-4 binder comprises a human IgGl, IgG2, IgG3, or IgG4 HC constant domain or variant thereof having 1. 2, 3, 4, 5. 6, 7, 8. 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgGl, IgG2, IgG3, or IgG4 isotype constant domain.

[0020] In some embodiments, the HC constant domain comprises a human IgGl constant domain comprising the amino acid sequence set forth in SEQ ID NO: 31.

[0021] In some embodiments, the HC constant domain lacks a C-terminal lysine or a C- terminal glycine-lysine dipeptide.

[0022] In some embodiments, the HC constant domain comprises one or more mutations that render the HC constant domain effector-silent.

[0023] In some embodiments, the effector-silent constant domain is an IgGl domain and comprises: (a) the amino acid sequence set forth in SEQ ID NO: 28; (b) the amino acid sequence set forth in SEQ ID NO: 29; (c) the amino acid sequence set forth in SEQ ID NO: 30; (d) the amino acid sequence set forth in SEQ ID NO: 31; (e) the amino acid sequence set forth in SEQ ID NO: 321 (f) the amino acid sequence set forth in SEQ ID NO: 33; (g) the amino acid sequence set forth in SEQ ID NO: 34; or (h) the amino acid sequence set forth in SEQ ID NO: 35.

[0024] In some embodiments, the human Nectin-4 binder comprises a Fab fragment, a F(ab)2 a Fab’ fragment, a F(ab’)2 fragment, an scFv, an Fv fragment, a single-domain antibody, or a multivalent antibody.

[0025] In another aspect, the disclosure provides a human Nectin-4 binder that cross-blocks or competes with the binding of an antibody or antigen-binding fragment thereof to human Nectin- 4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 22, and a light chain variable region of SEQ ID NO: 25.

[0026] In any one of the embodiments and aspects above, the human Nectin-4 binder binds to human Nectin-4 with a KD of 140pM ± 48.9pM.

[0027] In some embodiments, the human Nectin-4 binder is conjugated to a detectable moiety. The detectable moiety is detectable by microscopic imaging, magnetic resonance imaging (MRI), or X-ray imaging.

[0028] In some embodiments, the human Nectin-4 binder is conjugated to a therapeutic moiety.

[0029] The disclosure also provides an isolated nucleic acid encoding the VH domains, the VL domains, or both the VH and VL domains of a human Nectin-4 binder of any one of the foregoing embodiments. The disclosure provides an expression vector comprising such isolated nucleic acids. The disclosure provides a host cell comprising such expression vectors.

[0030] The disclosure also provides a method for producing a human Nectin-4 binder of any one of the embodiments herein, the method comprising: (a) culturing the host cell described above in culture medium under conditions wherein the nucleic acid sequence is expressed, thereby producing a polypeptide comprising the light and heavy chain variable regions; and (b) recovering the polypeptides from the host cell or culture medium.

[0031] The disclosure provides a method for assessing human Nectin-4 expression in a tissue sample from a human subject, comprising: (a) contacting the tissue sample with the human Nectin-4 binder of any one of the embodiments above; (b) detecting the binding of the human Nectin-4 binder to the tissue sample; (c) measuring the expression level of human Nectin-4 in the tissue sample; and (d) comparing the expression level of human Nectin-4 in the tissue sample with a reference expression level of human Nectin-4.

[0032] The disclosure also provides a method for assessing responsiveness of a cancer patient to an anti-cancer therapeutic agent, comprising: (a) contacting a tissue sample from the cancer patient with the human Nectin-4 binder of any one of claims 1-20; (b) detecting the binding of the human Nectin-4 binder to the tissue sample; (c) measuring the expression level of human Nectin-4 in the tissue sample; and (d) comparing the expression level of human Nectin-4 in the tissue sample with a reference expression level of human Nectin-4; wherein an increasedexpression level of human Nectin-4 compared to the reference expression level indicates responsiveness to the anti-cancer therapy.

[0033] The disclosure also provides a kit comprising the human Nectin-4 binder of any one of the above embodiments or the isolated nucleic acid of such embodiments.

[0034] The disclosure also provides a pharmaceutical composition comprising the human Nectin-4 binder of any one of the above embodiments and a pharmaceutically acceptable carrier or diluent.

[0035] The disclosure further provides a method for treating cancer in a subj ect in need thereof, comprising a therapeutically effective amount of a human Nectin-4 binder of any one of the foregoing embodiments or the pharmaceutical composition embodiment above.

[0036] The disclosure also provides a human Nectin-4 binder of any one of the above embodiments or the pharmaceutical composition embodiment for use in treating cancer.

[0037] The disclosure provides a human Nectin-4 binder of any one of the above embodiments or the pharmaceutical composition embodiment for the manufacture of a medicament for treating cancer.

[0038] The summary of the technology' described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 A shows a line graph of mAbl and binding to fixed human Nectin-4 in an ELISA. FIG. IB shows a line graph of mAbl binding to unfixed human Nectin-4 in an ELISA.

[0040] FIGs. 2A-2E show photomicrographs of formalin-fixed, paraffin embedded sections from cells transfected with various Nectin family members, then stained using mAbl. FIG. 2A is a section from cells transfected with null vector; FIG. 2B is a section from cells transfected with Nectin- 1: FIG. 2C is a section from cells transfected with Nectin-2; FIG. 2D is a section from cells transfected with Nectin-3; and FIG. 2E is a section from cells transfected with Nectin-4.

[0041] FIG. 3A is a schematic diagram of steps taken to prepare samples for comparing orthogonal methods of detecting Nectin-4 expression, such as in situ hybridization (ISH) and flow cytometry, to immunohistochemistry (IHC) performed using mAbl. FIG. 3B-3D are two- dimensional flow cytometry plots showing flow cytometry data for cells expressing Nectin-4 and untreated with mAbl (FIG. 3B), treated with isotype IgGl control antibody (FIG. 3C), and treated with mAbl. FIG. 3E shows a photomicrograph example of a mAbl -stained IHC section, and FIG. 3F shows an example ISH section. FIG. 3G show photomicrograph examples of Nectin-4 staining by IHC (left column) and Nectin-4 mRNA by 1SH (right column) for four Nectin-4- expressing cell lines. FIG. 3H shows a bar graph of the surface copy number of Nectin-4 detected by four cell lines using flow cytometry.

[0042] FIG. 4A shows example photomicrographs of normal Nectin-4-expressing tissue sections (bladder, top row; tonsil, middle row; and cervix, bottom row) treated by IHC using mAbl (left column) and a comparator mAb (comparator mAb 2) that also binds Nectin-4 (right column). FIG. 4B shows example photomicrographs of other normal Nectin-4-expressing tissue sections (esophagus, top row; skin, middle row; and breast, bottom row) treated by either IHC using mAbl (left column) or by situ hybridization (ISH).

[0043] FIG. 5 A shows a histogram for the number of samples for each of the IHC mAbl staining scores in TNBC, bladder cancer, and Cervical SCC. FIG. 5B shows examples of both mAbl (top row) and comparator mAb (bottom row) IHC staining intensity in bladder cancer tissue sections having high (left column), medium (middle column), and low (right column) expression of Nectin-4.DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure is directed to a monoclonal antibody and antigen-binding fragments thereof raised against human Nectin-4 protein which binds to human Nectin-4 protein in tissues, including cut tissue sections from formalin fixed paraffin embedded tissue blocks. The antibody may be used as a primary detection reagent for detecting human Nectin-4 protein in immunohistochemistry (IHC) and other IHC-like Nectin-4 assays, including but not limited to, imaging mass cytometry, cyclic multiplexed immunofluorescence, confocal microscopy, and barcode-mediated immunofluorescent tissue protein multiplexing systems.Abbreviations

[0045] Throughout the detailed description and examples of the invention the following abbreviations will be used:CDR Complementarity determining region in the immunoglobulin variable regionsCHO Chinese hamster ovaryEC50 Concentration resulting in 50% efficacy or bindingELISA Enzyme-linked immunosorbant assayFFPE Formalin-fixed, paraffin-embeddedFR Antibody framework region; the immunoglobulin variable regions excluding the CDR regionsIC50 Concentration resulting in 50% inhibitionIgG Immunoglobulin GIgV or V region The segment of IgG chains which is variable in sequence between different antibodies, extending to Kabat residue 109 in the light chain and Kabat residue 113 in the heavy chain.IHC immunohistochemistry mAb or Mab or MAb Monoclonal antibody VH or VH Immunoglobulin heavy chain variable regionVL or VL Immunoglobulin light chain variable regionDefinitions

[0046] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0048] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element" means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0049] As used herein, the term “about” refers to plus or minus up to 10% of the value it modifies (rounded up or down as applicable to the nearest whole number where appropriate for the situation, e.g., where you need a whole unit such as amino acids) and is meant to refer to a value within an acceptable error range for the particular value as determined by one skilled in the art depending in part on how the value is measured or determined. The term “about”, when modifying the quantity (e.g., mg) of a substance or composition, a parameter of a substance or composition or a parameter used in characterizing a step in a method, or the like, refers to variation in the numerical quantity that can occur. For example, such variation can occur through typical measuring, handling, and sampling procedures involved in the preparation, characterization and / or use of the substance or composition, through inadvertent error in theseprocedures, or through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures.

[0050] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg"’ is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0051] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s).” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of' and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0052] “Activation” as it applies to cells or to receptors refers to the activation or treatment of a cell or receptor with a ligand, unless indicated otherwise by the context or explicitly. “Ligand” encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins, and binding compounds derived from antibodies. “Ligand” also encompasses small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors.

[0053] “Activity” of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity , to the modulation of activities of other molecules, and the like. “Activity” of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. “Activity” can also mean specific activity, e.g., catalytic activity / mg protein, or immunological activity / mg protein, concentration in a biological compartment, or the like. “Activity” may refer to modulation of components of the innate or the adaptive immune systems.

[0054] As used herein, the term “affinity ” refers to the strength of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., anantigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity' of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including KINEXA® and BIACORE®. Specific illustrative and exemplary embodiments for measuring binding affinity are described in this disclosure.

[0055] As used herein, a “chromogen” or “chromogenic compound” is a substance that can be converted into a colored compound under specific conditions, e.g., when acted upon by an enzyme or under specific chemical / reaction conditions. Examples of enzyme-substrate combinations include: (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, where the hydrogen peroxidase oxidizes a dye precursor [e.g., orthophenylene diamine (OPD) or 3, 3 ',5, 5 '-tetramethyl benzidine hydrochloride (TMB)]; (ii) alkaline phosphatase (AP) with para- Nitrophenyl phosphate as chromogenic substrate; and (iii) 0-D-galactosidase 0-D-Gal) with a chromogenic substrate (e.g.. p-nitrophenyl-0-D-galactosidase) or fluorogenic substrate (e.g.. 4- methylumbelliferyl-0-D-galactosidase). Numerous other enzy me-substrate combinations are available to those skilled in the art. For a general review of chromogens, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

[0056] As used herein, the term “fluorophore” or “fluorescent label” refers to any label that absorbs light at a first wavelength (or range of wavelengths) and emits light at a second, different w avelength (or range of wavelengths). In general, the emitted light from a fluorescent labels has a longer wavelength, and therefore low er energy', than the absorbed w avelength. Examples of fluorescent labels include, but are not limited to, phycobiliproteins (e g., phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC)), rhodamine, fluorescein. Alexa Fluor™. Cascade Blue™, tetramethylrhodamine, Texas Red™, and the like.

[0057] As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissue section” refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate buffered saline) or Bouin solution, embedded in wax, cut into thin sections, and then mounted on a planar surface, e g., a microscope slide.

[0058] As used herein, the term “multiplexing” refers to using more than one label, stain, and / or chromogen for the simultaneous or sequential detection and measurement of a target in a sample, e.g., a tissue section.

[0059] As used herein, the term “Nectin-4” refers to a calcium ion-independent type I transmembrane protein that mediates cell adhesion junction formation. Nectin-4 includes anextracellular segment formed by three Ig-like domains, a transmembrane region and an intracellular tail region. Nectin-4 can promote tumor cell survival and proliferation by inhibiting anoikis and activating PI3K-AKT signaling pathway and is highly expressed in many tumor types. The amino acid sequences for the precursor and mature forms of human Nectin-4 are shown in SEQ ID NO: 25 (the mature form is amino acids 32-510 of SEQ ID NO: 25).

[0060] As used herein, the term ‘'human Nectin-4 binder” refers to an antibody or antigen binding fragment thereof that binds to soluble and / or membrane-bound human Nectin-4. A Nectin-4 binder includes but is not limited to a bivalent antibody tetramer (2H+2L), a monovalent antibody (H+L). a bi-specific antibody that targets human Nectin-4 and another target, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fv region, and an ScFv.

[0061] The terms “patient(s)” or “subject(s)” includes any organism, preferably an animal, more preferably a mammal (e.g., human, rat, mouse, dog, cat, rabbit). In a preferred embodiment, the terms “patient(s)” or “subject(s)” refers to a human.

[0062] As used herein, the term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s), approved by a regulatory’ agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes but is not limited to such sterile liquids as water and oils. The characteristics of the carrier will depend on the route of administration.

[0063] As used herein, the terms “primary antibody” and “secondary antibody” refer to different antibodies, where a primary antibody is a polyclonal or monoclonal antibody from one species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g.. a biomarker) in a sample (e g., a human tissue sample) under study, and a secondary antibody is an antibody (usually polyclonal) from a different species that specifically recognizes the primary antibody, e.g., in its Fc region.

[0064] Sometimes, the label may be indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody may be conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody. Thus, indirect conjugation of the label with the antibody can be achieved. In someembodiments of the invention one or more secondary antibody molecules may be conjugated with a label-conjugated polymer backbone. Thus, amplification of the signal may be achieved.

[0065] Indirectly and directly labeled secondary' antibodies are also commercially available. For example, one example of commercially available label-conjugated polymer backbone carrying secondary antibody molecules reagent is EnVision™ reagent (Agilent Technologies, Inc., Santa Clara CA). A secondary antibody carrying a label aimed for a particular type of detection may be obtained from numerous manufacturers.

[0066] The term ‘‘sample” refers to tissue, bodily fluid, or a cell (or a fraction of any of the foregoing) taken from a patient or a subject. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated. Other samples, including urine, tears, serum, plasma, cerebrospinal fluid, feces, sputum, cell extracts etc. can also be useful for detecting particular cancers.

[0067] The term “staining” includes binding a target (e.g., an antigen) in a cellular sample with a target-specific binding agent (e.g.. an antibody or a nucleic acid) and then detecting the presence of the target-specific binding agent on the cells of the cellular sample using a detectable label or chromogen. The detectable label can be directly conjugated to the target-specific binding agent (e.g., a primary antibody) or may be conjugated to a secondary reagent that binds specifically to an unlabeled target-specific reagent (e.g., a secondary antibody). In some cases, the target-specific reagent is itself detectable, and thus no additional attached label is needed.

[0068] As used herein, the term “target-specific binding agent” means any agent that specifically binds to a target or analyte of interest, e.g., a target of interest that is present in a tissue section as described herein (e.g., a polypeptide or polynucleotide). Examples of targetspecific binding agents include antibodies, receptors, and ligands, or target-binding fragments thereof, polynucleotide probes, and the like.

[0069] As used herein, the term “tissue section” refers to a piece of tissue that has been obtained from a subject and mounted on a planar surface, e.g., a microscope slide. The sample may be fixed and / or sectioned as desired. A “tumor tissue sample” or “tumor tissue biopsy sample” includes cells derived from a tumor in a subject, e g , a human subject having a malignancy. Such tissue samples are sometimes referred to simply as a “biopsy”Human Nectin-4 binders

[0070] The human Nectin-4 binders of the present invention are chimeric or fully human antibodies or antigen-binding fragments thereof that specifically bind human Nectin-4. Thehuman Nectin-4 binders compnse a Vjq domain and a VL domain, each domain comprising three CDRs and four Frameworks (FR) in the following arrangement.FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] These human Nectin-4 binders comprise six complementarity determining regions (CDRs) comprising a particular combination of three CDRs from a Vp and three CDRs from the VL that pairs with the V[-[. The CDR sequences may be defined according to any numbering scheme useful for defining CDR sequences including but not limited to the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme. Guidance for defining the CDR sequences may be found in the general rules disclosed in www.bioinf.org.uk: Prof. Andrew C.R. Martin's Group and reproduced in Table 1 below.

[0072] The antibodies and antigen-binding fragments of the present invention bind to the mature form of human Nectin-4 (lacking the presecretory leader sequence, also referred to as leader peptide) that is expressed on the surface of certain human cells. The terms “Nectin-4” and “mature Nectin-4” are used interchangeably herein and shall be understood to mean the same molecule unless otherwise indicated or readily apparent from the context. A mature human Nectin-4 molecule consists of amino acids 32-510 of the following sequence (SEQ ID NO: 25): MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDS GEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRN AVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGS PAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRI THILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRV DGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVG VIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPE ESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQ DEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV.

[0073] The extracellular domain of mature human Nectin-4 consists of the following sequence (SEQ ID NO: 26): GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGL HVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLV PPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEF HLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQ VTVDVLDPQEDSGKQVDLVSAS

[0074] In some embodiments, the human Nectin-4 binder is an antibody or antigen-binding fragment thereof that specifically binds to human Nectin-4. An antibody that “specifically binds to human Nectin-4,” or an antibody that “specifically binds to a polypeptide comprising the amino acid sequence of human Nectin-4,” is an antibody or antigen-binding fragment thereof that binds to target with a KD of about 1 nM or a higher affinity. For example, an antibody that specifically binds to a polypeptide comprising the target may bind to a FLAG®-tagged form of Nectin-4 but will not bind to other FLAG® -tagged proteins. In another embodiment, the antibody or antigen-binding fragment thereof binds to human Nectin-4 with a KD of about 100 pM or higher affinity. In one embodiment, the human Nectin-4 binder binds to human Nectin-4 with a KD of 140 pM ± 48.9 pM.

[0075] As used herein, the term “antibody,” “immunoglobulin,” or “Ig,” refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. Each HC is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain. Each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. In general, the basic antibody structural unit for antibodies is a Y-shaped tetramer comprising two HC / LC pairs (2H). Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa) (H+L). Each HC:LC pair comprises one VH: one VL pair. The one Vn:one VL pair may be referred to by the term “Fab”. Thus, each antibody tetramer comprises two Fabs, one per each arm of the Y-shaped antibody. An antibody provided herein may include post-translational modifications thereof, e g., C-terminal Lysine or Glycine-Lysine clipping in the heavy chain, conversion of glutamine or glutamic acid to pyroglutamate or pyroglutamic acid, which may occur when recombinantly expressed in host cells (e.g., CHO cells), or during purification / storage.

[0076] The human VH includes seven family members: VHI, VH2, VH3, VH4, VH5, VH6, and VH7; and the human VL includes 16 family members: VK1, VK2, VK3, VK4, VK5, VK6, V^l, Vy2. V^3, V .4, V;A, Vy6. V 7, V^S. Vy,9. and V^IO. Each of these family members can be further divided into particular subtypes. The VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining region (CDR) areas, interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDR regions and four FR regions, arranged from amino-terminus to carboxy -terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3. CDR3. FR4. Numbering of the amino acids in a VH may be determined using the Kabat numbering scheme. See Beranger, et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG, Created: 17 / 05 / 2001, Version: 08 / 06 / 2016, accessible at www.imgt.org / IMGTScientificChart / Numbering / Hu IGHGnber.html).

[0077] The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgGl (Eu), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgGl described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgGl, IgG2, IgG3, and IgG4 constant domains in Beranger et al., op. cit.

[0078] As used herein, the term ‘'antigen” as used herein refers to any foreign substance which induces an immune response in the body.

[0079] As used herein, unless otherw ise indicated, “antigen-binding fragment,” “antigenbinding domain,” or “antigen-binding region” refers to the portion of antibodies, i.e.. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab1, F(ab')2, and Fv fragments.

[0080] As used herein, the term “Fab fragment” refers to an antigen binder comprising one antibody light chain and the CHI and Vjq of one antibody heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.

[0081] As used herein, the term “F(ab')2 fragment” refers to an antigen binder comprising two antibody light chains and two heavy chains containing the Vjq and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the tw o heavy chains. An “F(ab')2 fragment” can be the product of pepsin cleavage of an antibody.

[0082] As used herein, the term "Fab1fragment" refers to an antigen binder comprising one antibody light chain and a portion or fragment of one antibody heavy chain that contains the Vjqand the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0083] As used herein, the term "F(ab')2 fragment" refers to an antigen binder comprising two antibody light chains and two heavy chains containing the V [ and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

[0084] As used herein, the term "Fv region" refers to an antigen binder comprising the variable regions from both the heavy and light chains of an antibody but lacks the constant regions.

[0085] As used herein, the term “ScFv” or “single-chain variable fragment"’ refers to a fusion protein comprising a V[_[ and VL fused or linked together by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the Vjq with the C-terminus of the VL. or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0086] As used herein, the term “diabody” or “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create tw o antigen-binding sites. Diabodies are described more fully in, e.g.. EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0087] These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for uti 1 ity in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0088] As used herein, the term “single-domain antibody” refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joinedwith a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0089] As used herein, the term “multivalent antibody” refers to an antigen binder comprising multiple antigen-binding domains, e g., a bivalent antibody having two binding domains, or a trivalent antibody having 3 binding domains. In some instances, the binding sites have the same antigen specificities. However, multivalent antibodies may be multi-specific and able to bind more than one antigen, e.g., a bispecific antibody.

[0090] In certain embodiments, monoclonal antibodies herein also include camelized single domain antibodies. See, e.g.. Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079). In one embodiment, the present invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

[0091] As used herein, the term “isolated” when referring to antigen binders, such as antibodies or antigen-binding fragments thereof, means antigen binders that are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

[0092] As used herein, the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, z.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495 or may be made by recombinant DNA methods (see. e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0093] As used herein, the term “chimeric antibody” refers to an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., rodent) antibody.

[0094] As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from both human and non-human (e.g., murine, rat) antibodies. In general, the humanized antibody will comprise substantially all of at least one, and typically two. variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0095] The term “fully human antibody” or “human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody that comprises mouse immunoglobulin sequences only. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat. in a rat cell, or in a hybridoma derived from a rat cell. Similarly, “rat antibody” refers to an antibody that comprises rat immunoglobulin sequences only.

[0096] The variable regions of the heavy and light chains contain a binding domain comprising the CDRs that interacts with an antigen. Several methods are available in the art for defining CDR sequences of antibody variable domains (see Dondelinger et al.. Frontiers in Immunol. 9: Article 2278 (2018)). The common numbering schemes include the following.• Kabat numbering scheme is based on sequence variability and is the most commonly used (See Kabat et al. Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence);• Chothia numbering scheme is based on the location of the structural loop region (See Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997));• AbM numbering scheme is a compromise between the two used by Oxford Molecular's AbM antibody modelling software (see Kara et al., IL AR Journal 37: 132-141 (1995);• Contact numbering scheme is based on an analysis of the available complex crystal structures (See www.bioinf.org.uk: Prof. Andrew C.R. Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008).• IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all the protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains as well as T cell receptor chains from different species and counts residues continuously from 1 to 128 based on the germline V sequence alignment (see Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509(1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003)).

[0097] The following general rules disclosed in www.bioinf.org.uk: Prof. Andrew C.R.Martin's Group and reproduced in Table 1 below may be used to define the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.

[0098] The entire nucleotide sequence of the heavy chain and light chain variable regions are commonly numbered according to Kabat while the three CDRs within the variable region may be defined according to any one of the above numbering schemes.

[0099] In general, the state of the art recognizes that in many cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on CDR3 of the heavy chain alone are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000).

[0100] As used herein, the term "Fc domain7; or "Fc " as used herein is the crystallizable fragment domain or region obtained from an antibody that comprises the CH2 and CH3 domains of an antibody. In an antibody, the two Fc domains are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The Fc domain may be obtained by digesting an antibody with the protease papain. Typically, amino acids in the Fc domain are numbered according to the Eu numbering convention (See Edelmann et al., Biochem. 63: 78-85 (1969)).

[0101] As used herein, “identity'’ or “sequence identity” refers to the degree to which the amino acid residues of two polypeptides or the bases of two nucleic acids are the same (i.e.. identical) at the equivalent positions when a query sequence is optimally aligned over the length of a reference sequence, introducing gaps as necessary if the reference and query sequence do not have the same length to achieve a maximum percent identity (called a “global alignment”). Forthis disclosure, a reference sequence is always an amino acid or nucleic acid sequence specifically disclosed herein, and a query sequence is any other sequence aligned to the reference sequence. Sequence identity is expressed as a percent sequence identity (% sequence identity or % identity). The global alignment and calculation of the percent identity is performed over a contiguous block of residue positions for the entire reference sequence. If the query sequence is longer than the reference sequence, then the percent identity is determined only using the residue positions of the reference sequence. Any residues of the query sequence with positions beyond the first and last positions of the reference sequence after an initial global alignment are removed and the reference and query sequences are re-aligned to determine percent identity. A query sequence has 100% identity to a reference sequence when the residues of both sequences are identical at the equivalent positions over the length of the reference sequence. Similarly, a query sequence has 50% identity to a reference sequence when 50% of the residues of the two amino acid sequences are identical at the equivalent positions over the length of the reference sequence.

[0102] A global alignment can be created using the global alignment tool "‘Needle” from the online European Molecular Biology Open Software Suite (EMBOSS) (www.ebi.ac.uk / Tools / psa / emboss_needle / ) or the global alignment tool “BLAST® » Global Alignment” from the National Center for Biotechnology Information (NCBI) (blast.ncbi.nlm.nih.gov / Blast. cgiPROGRAM=blastn&PAGE_TYPE=BlastSearch&PROG_DEF AULTS=on&BLAST_INIT=GlobalAln&BLAST_SPEC=GlobalAln&BLAST_PROGRAMS=bl astn). Both global alignment tools incorporate the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C D., “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453 (1970)). A global alignment of nucleotide sequences using BLAST Global Alignment in this disclosure uses the following default parameters: match score = 2; mismatch score = -3; Gap Cost Existence score = 5; Gap Cost Extension Score = 2. A global alignment of protein sequences in this disclosure using BLAST Global Alignment uses the following default parameters: Gap Cost Existence = 11; Gap Cost Extension = 1. A global alignment of nucleotide sequences using EMBOSS Needle in this disclosure uses the following default parameters: Output Format = pair; Matrix = DNAfull; Gap Open = 10; Gap Extend = 0.5; End Gap Penalty = false; End Gap Open = 10; End Gap Extend = 0.5. A global alignment of protein sequences using EMBOSS Needle in this disclosure uses the following default parameters: Output Format = pair; Matrix = BLOSUM62; Gap Open = 10; Gap Extend = 0.5; End Gap Penalty = false; End Gap Open = 10; End Gap Extend = 0.5.

[0103] “Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge,side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity’, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non- essential regions of a polypeptide do not substantially alter biological activity (see, e g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary' conservative substitutions are set forth in Table 2.

[0104] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. “Function-conservative variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and / or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table 2 above.Physical and Functional Properties of the Exemplary Anti-Nectin-4 Antibody

[0105] The present invention provides isolated anti-Nectin-4 antibodies, antigen-binding fragments thereof, and methods of using the antibodies or antigen-binding fragments thereof inthe detection of Nectin-4 expression on the surface of human cells. An example of an anti- Nectin-4 antibody of the invention includes but is not limited to mAbl (see Table 3 below).

[0106] These anti-Nectin-4 antibodies and antigen-binding fragments thereof comprise six complementarity determining regions (CDRs) comprising a particular combination of three CDRs from a VH and three CDRs from the Vi. that pairs with the VH. The CDR sequences may be defined according to any numbering scheme useful for defining CDR sequences including but not limited to the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme, as described above. The CDRs for each numbering scheme are shown in Table 4 below.

[0107] A particular CDR amino acid sequence determined using any one of the schemes for identifying CDR amino acid sequences (See Table 1 above) may have more or less amino acids than that of CDR amino acid sequences identified according to any other numbering scheme, but the CDR amino acid sequences will overlap to some extent. Thus, the CDR amino acid sequences defined according to Kabat are not to be construed as limiting and any anti-Nectin-4 antibody or antigen-binding fragment thereof in which the CDR amino acid sequences have been identified by another numbering scheme will fall within the scope of the anti-Nectin-4 antibody or antigen-binding fragment thereof of the present invention provided the amino acid sequences for such anti-Nectin-4 antibody or antigen-binding fragment thereof comprise the six CDR amino acid sequences as identified by Kabat. For all anti-Nectin-4 antibody or antigen-binding fragment thereof disclosed herein unless indicated otherwise, the amino acids comprising the variable domains are numbered according to the Kabat numbering scheme independently of how the amino acids comprising the CDR are defined. The heavy chain constant domains are numbered according to the Eu numbering scheme.

[0108] In some embodiments, provided herein is an anti-Nectin-4 antibody or antigen-binding fragment thereof which comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 22. The CDRs can be defined according to any numbering scheme know n in the art. In some embodiments, the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 4.

[0109] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 having the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 having the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 6, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0110] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof. In such embodiments, the antibody or antigen-binding fragment does not vary' at the CDR amino acid sequences.

[0111] In some embodiments, the variants of the VH and VL comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the VH or VL. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In such embodiments, the antibody or antigen-binding fragment does not vary- at the CDR amino acid sequences.

[0112] In some embodiments, the invention includes an antibody or antigen-binding fragment thereof that specifically binds human Nectin-4 and has VL domains and VH domains and shares 100% sequence identity to the light and heavy chain CDRs of Table 3, and at least 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98% or 99% sequence identity to the light and heavy chain variable regions of Table 4.

[0113] In some embodiments, an N-terminal E or Q of an anti-Nectin-4 antibodies or antigenbinding fragments thereof provided herein is substituted yvith pyroglutamate or pyroglutamic acid. In some embodiments, a C-terminal K of anti-Nectin-4 antibodies or antigen-binding fragments thereof provided herein is removed. In other embodiments, an N-terminal E or Q of anti-Nectin-4 antibody or antigen-binding fragment thereof provided herein is substituted with pyroglutamate or pyroglutamic acid and a C-terminal K of the anti-Nectin-4 antibody or antigenbinding fragment thereof (e.g.. heavy chain C terminal amino acid) is removed. The present disclosure includes any of the above-described post-translational modifications of any of the anti- Nectin-4 antibodies or antigen-binding fragments thereof provided herein. For example, in some embodiments, provided herein is an anti-Nectin-4 antibody or antigen-binding fragment thereof that comprises the same sequences as SEQ ID NO: 23 and SEQ ID NO: 24, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid and / or the C-terminal amino acid of the heavy chain is removed.

[0114] In some embodiments, provided herein is an antibody or antigen-binding fragment comprising an amino acid sequence of SEQ ID NO: 21, except that the first amino acid at the N- terminus is substituted with pyroglutamate or pyroglutamic acid.Epitope Binding

[0115] The present invention further provides antibodies or antigen-binding fragments thereof that block binding of antibody mAbl to human Nectin-4 by binding to the same epitope as mAbl. Such antibodies and binding fragments may be identified using any cross-blocking or competition analysis known in the art. such as an OCTET® competition analysis, followed by identifying the epitope on human Nectin-4 to which the cross-blocking antibody binds. A first antibody is considered to cross-block binding of a second antibody if prebinding the target with the first antibody to saturation increases the concentration of second antibody needed to achieve half-maximal binding of the target by 2-, 3-. 4-, 5-, 10-, 20-, 50-, 100- . 200-fold or more. The binding epitope for a cross-blocking antibody can be identified using techniques well-known in the art.

[0116] The term ‘‘epitope”, as used herein, is defined in the context of a molecular interaction between an "antigen-binding molecule", such as an antibody (Ab), and its corresponding “antigen” (Ag). Generally, “epitope” refers to the area or region on an Ag to which an Ab specifically binds, i.e., the area or region in physical contact with the Ab. Physical contact may be defined through distance criteria (e.g., a distance cut-off of 4 A) for atoms in the Ab and Ag molecules.

[0117] One such epitope mapping technique is hydrogen / deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS). This method relies on the accurate measurement and comparison of the degree of deuterium incorporation by an antigen when incubated in heavy water (D2O) on its own and in the presence of its antibody at various time intervals. Deuterium is exchanged with hydrogen on the amide backbone of the proteins in exposed areas whereas regions of the antigen bound to the antibody will be protected and will show less or no exchange after analysis by liquid chromatography -tandem mass spectrometry (LC-MS / MS) of proteolytic fragments.Nucleic Acids

[0118] The present invention also provides nucleic acids encoding the immunoglobulin chains of anti-Nectin-4 antibodies and antigen-binding fragments disclosed herein. For example, the present invention includes nucleic acids encoding the amino acids described in Tables 2 and 3, as well as nucleic acids which hybridize thereto.

[0119] In general, the nucleic acids hybridize under low, moderate or high strin ency conditions, and encode antibodies that maintain the ability to specifically bind to PD-L1. A first nucleic acid molecule is “hybridizable” to a second nucleic acid molecule when a single stranded form of the first nucleic acid molecule can anneal to the second nucleic acid molecule under theappropriate conditions of temperature and solution ionic strength (see Sambrook. et al., below). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Typical low stringency hybridization conditions include 55°C, 5X SSC, 0.1% SDS and no formamide; or 30% formamide, 5X SSC, 0.5% SDS at 42°C. Typical moderate stringency hybridization conditions are 40% formamide, with 5X or 6X SSC and 0.1% SDS at 42°C. High stringency hybridization conditions are 50% formamide, 5X or 6X SSC at 42°C or, optionally, at a higher temperature (e.g., 57°C, 59°C, 60°C, 62°C, 63°C, 65°C or 68°C). In general, SSC is 0. 15M NaCl and 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary’ sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity7or homology7between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook, et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, e.g., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, et al., below. 11.7-11.8).

[0120] In another embodiment, the invention provides an isolated nucleic acid or nucleic acids, for example DNA, encoding at least one of the polypeptide chains of the isolated anti-Nectin-4 antibodies or antigen-binding fragments described herein. In some embodiments the isolated nucleic acid encodes both a light chain and a heavy chain on a single nucleic acid molecule, and in other embodiments the light and heavy chains are encoded on separate nucleic acid molecules. In another embodiment the nucleic acids further encode a signal sequence.Methods for making human Nectin-4 binders

[0121] The present disclosure includes recombinant methods for making human Nectin-4 binders comprising introducing into a host cell (i) an expression vector that encodes the Vjq and VL of a human Nectin-4 binder or the HC and LC of a human Nectin-4 binder, or (ii) two expression vectors, one encoding the Vjq of a human Nectin-4 binder or the HC of a human Nectin-4 binder the other encoding the VL of a human Nectin-4 binder or the LC of a human Nectin-4 binder. The nucleic acid molecules or polynucleotides encoding the Vjq, V , HC, or LC are operably linked to a promoter and other transcription and translation regulatory sequences. The host cell is cultured under conditions and a time period suitable for expression of the nucleicacid molecules followed by isolating the mesothelin binder from the host cell and / or medium in which the host cell is grown. See e.g., W02004041862, WO2006122786, W02008020079, WO2008142164 or W02009068627. The expression vector may be a plasmid or viral vector. The disclosure also relates to hosts or host cells that contain such nucleic acid molecule encoding the Arginase 1 binders or components thereof, e.g., solely the Vjq or HC or solely the VL or HC.

[0122] Eukaryotic and prokary otic host cells, including mammalian cells as hosts for expression of the human Nectin-4 binder are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and several other cell lines. Thus, mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example. Pichia pastoris, Saccharomyces cerevisiae. and Trichoderma reesei. The present disclosure includes any host cell comprising a human Nectin-4 binder of the present disclosure or comprising one or more nucleic acid molecules encoding such a human Nectin-4 binder or comprising an expression vector that comprises one or more nucleic acid molecules encoding such human Nectin-4 binder.

[0123] Further, expression of a human Nectin-4 binder from production cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216846B1, 0256055B1, 0323997B1. and 0338841B1. Thus, in an embodiment of the disclosure, the mammalian host cells lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium wherein, however, the nucleic acid molecule encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell. Such host cells containing the human Nectin-4 binder or nucleic acid(s), or expression vector(s) as discussed herein as well as expression methods, as discussed herein, for making the human Nectin-4 binder using such a host cell are part of the present disclosure.

[0124] The present disclosure includes methods for purifying a human Nectin-4 binder comprising introducing a sample (e.g.. culture medium, cell lysate or cell lysate fraction, e.g., asoluble fraction of the lysate) comprising the human Nectin-4 binder to a purification medium (e.g., cation-exchange medium, anion-exchange medium and / or hydrophobic exchange medium) and either collecting purified human Nectin-4 binder from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound human Nectin-4 binder from the medium and collecting the eluate. In an embodiment of the disclosure, the medium is in a column to which the sample is applied. In an embodiment of the disclosure, the purification method is conducted following recombinant expression of the human Nectin-4 binder in a host cell, e.g., wherein the host cell is first lysed and. optionally, the lysate is purified of insoluble materials prior to purification on a medium; or wherein the human Nectin-4 binder is secreted into the culture medium by the host cell and the medium or a fraction thereof is applied to the purification medium.

[0125] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of a human Nectin-4 binder will depend on the particular cell line or transgenic animal used to produce the human Nectin-4 binder. Human Nectin-4 binders comprising only non-fucosylated N-glycans are part of the present disclosure and may be advantageous, because non-fucosylated antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These human Nectin-4 binders with non-fucosylated N-glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

[0126] The present disclosure includes human Nectin-4 binders comprising N-linked glycans that are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHO N- linked glycans) or to engineered yeast cells (engineered yeast N-linked glycans), such as, for example, Pichia pastoris. For example, in an embodiment of the disclosure, the mesothelin binder comprises one or more of the "engineered yeast N-linked glycans7’ or “CHO N-linked glycans” (e g., GO and / or GO-F and / or G1 and / or Gl-F and / or G2-F and / or Man5). In an embodiment of the disclosure, the human Nectin-4 binder comprises the engineered yeast N-linked glycans, i.e., GO and / or G1 and / or G2, optionally, further including Man5. In an embodiment of the disclosure, the human Nectin-4 binders comprise the CHO N-linked glycans. i.e., GO-F, Gl-F and G2-F, optionally, further including GO and / or G1 and / or G2 and / or Man5. In an embodiment of the disclosure, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90% or about 95%) of all N-linked glycans on the human Nectin-4 binders are engineered yeast N-linked glycans orCHO N-linked glycans. See Nett et al. Yeast. 28: 237-252 (2011); Hamilton et al. Science. 313: 1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in an embodiment of the disclosure, an engineered yeast cell is GFI5.0 or YGLY8316 or strains set forth in U.S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988: 31-43 (2013). See also international patent application publication no. WO2013066765.

[0127] The human Nectin-4 binders disclosed herein, such as anti-Nectin-4 antibodies or antigen-binding fragments thereof, may be produced recombinantly. In this embodiment, nucleic acid molecules encoding the antibody molecules may be inserted into an expression vector (plasmid or viral) and transfected or transformed into a host cell where the antibody molecules may be expressed and secreted from the host cell. There are several methods by which to produce recombinant antibodies which are know n in the art.

[0128] The present invention provides expression vectors comprising the isolated nucleic acids of the invention, wherein the nucleic acid is operably linked to control sequences that are recognized by a host cell when the host cell is transfected with the vector. Also provided are host cells comprising an expression vector of the present invention. The present invention also provides methods for producing the antibody or antigen-binding fragment thereof disclosed herein, comprising culturing a host cell comprising an expression vector encoding the antibody or antigen-binding fragment in culture medium, and isolating the antigen or antigen-binding fragment thereof from the host cell or culture medium.

[0129] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and / or the regulatory7sequences required for their expression. For example, "gene" refers to a nucleic acid fragment that expresses mRNA. functional RNA. or specific protein, including regulatory sequences. "Genes" also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. "Genes" can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences encoding a molecule of interest and synthetically derived nucleotide sequences encoding a molecule of interest, for example, complementary DNA (cDNA) obtained from a messenger RNA (mRNA) nucleotide sequence.

[0130] As used herein, the term ’germline" or "germline sequence" refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER® germline databases on the website for the National Institute ofArthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al., Nucleic Acids Res. 33: D256-D261 (2005).

[0131] As used herein, the term ‘‘library'’ as used herein is, typically, a collection of related but diverse polynucleotides that are. in general, in a common vector backbone. For example, a light chain or heavy chain immunoglobulin library may contain polynucleotides, in a common vector backbone, that encode light and / or heavy chain immunoglobulins, which are diverse but related in their nucleotide sequence; for example, which immunoglobulins are functionally diverse in their abilities to form complexes with other immunoglobulins, e.g., in an antibody display system of the present invention, and bind a particular antigen.

[0132] As used herein, the term “polynucleotides” discussed herein form part of the present invention. A "polynucleotide", “polynucleic acid”, "nucleic acid " or "nucleic acid molecule" include DNA and RNA, single- or double-stranded. Polynucleotides e.g., encoding an immunoglobulin chain or component of the antibody display system of the present invention, may, in an embodiment of the invention, be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non- coding regions, and the like.

[0133] Polynucleotides e.g., encoding an immunoglobulin chain or component of the antibody display system of the present invention, may be operably associated with a promoter. A “promoter” or “promoter sequence” is, in an embodiment of the invention, a DNA regulatory’ region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoterbound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., Nature 290: 304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22: 787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)); prokary otic expression vectors such as the (3-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)); see also "Useful proteins from recombinant bacteria" in Scientific American 242: 74-94 (1980); and promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0134] “Isolated nucleic acid molecule” or “isolated polynucleotide” means a DNA or RNA polynucleotide of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is (a) not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or (b) linked to a polynucleotide to which the isolated polynucleotide is not linked in nature. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and / or may include vector sequences or non-coding sequences.

[0135] As used herein, the term “control sequences” or “regulatory sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for expression in eukary otes, for example, include a promoter, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for expression of a messenger RNA encoding a protein and a ribosome binding site for facilitating translation of the messenger RNA.

[0136] As used herein, a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites donot exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0137] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0138] As used herein, the terms "vector", "cloning vector" and "expression vector" include a vehicle (e.g., a plasmid or virus) by which a DNA or RNA sequence can be introduced into a host cell to transform the host and, optionally, promote expression and / or replication of the introduced sequence. In one embodiment, polynucleotides encoding the human Nectin-4 binders disclosed here, such as an antibody or antigen-binding fragment thereof, may be in a vector.

[0139] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0140] As used herein, the expressions “cell,” “cell line.” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all cell progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant cell progenyhaving the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER® germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of theUnited States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

[0141] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “engineered cells,” “transformants.” and “transformed cells.” which include the primary engineered (e.g., transformed) cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein. As appropriate, the host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein, as described herein.

[0142] Hybridoma cells that produce parental (e.g., rodent) monoclonal anti-X antibodies may be produced by methods which are commonly known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler, etal., (1975) (Nature 256:495-497), as well as the trioma technique (Hering, et al., (1988) Biomed. Biochim. Acta. 47:211-216 and Hagiwara, et al., (1993) Hum. Antibod. Hybridomas 4: 15), the human B-cell hybridoma technique (Kozbor. et al., (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl. Acad. Sci. U.S.A 80:2026-2030), the EBV-hybridoma technique (Cole, et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), and electric field based electrofusion using a Cyto Pulse large chamber cull fusion electroporator (Cyto Pulse Sciences, Inc., Glen Bumie, MD). Preferably, mouse splenocytes are isolated and fused with PEG or by electrofusion to a mouse myeloma cell line based upon standard protocols.

[0143] The resulting hybridomas may then be screened for production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice may by fused to one-sixth the number of P3X63- Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately 2 x 105cells / mL in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% Origen (Roche Diagnostics Corp.), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM 2- mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells may be cultured in medium in which the HAT is replaced with HT. Individual wells may then be screened by ELISA for anti- X monoclonal IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomasmay be re-plated, screened again, and if still positive for human IgG. anti-X monoclonal antibodies, can be subcloned at least twice by limiting dilution.

[0144] The stable subclones may then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. For example, about 1 gram of the 22C3 antibody may be produced and purified from the mouse hybridoma cell line MEB037.22C3. 138 using the following procedure. Frozen MEB037.22C3.138 cells are thawed into are adapted into shake flask using hybridoma serum free media with 2 mM additional L-glutamine with or without 0.18% Pluronic F-68. The presence of Pluronic F-68 may improve the viability' of the shake flask culture. Once the cells are completely adapted into shake flask, a 20-liter production culture is performed in serum free media in a WAVE bioreactor (GE Healthcare Life Sciences) with the addition of 10% CHO CD efficient Feed B (Invitrogen, Catalogue#A10240-01). For cell expansion, a 1 -liter culture is initiated in a small WAVE bag, and then the IL WAVE culture is expanded into a 20L culture in the WAVE bioreactor. The 20-liter culture may be initiated at a cell density of 0.5 x 106viable cells / mL, fed with 10% CHO CD Efficient Feed B on Day 1. and pH adjusted daily with IN NaiCOv The cells are harvested after four days. Small samples may be collected daily for NOVA analysis.

[0145] Human Nectin-4 binders of the invention, such as antibodies and antigen-binding fragments thereof, may be purified from a hybridoma culture by the following process. The hybridoma culture is clarified by depth filtration using 1.2 micrometer glass fiber and 0.2 micrometer cellulose acetate filter. An equal volume of 2X ProSepA Buffer (100 mM Boric Acid, 5M NaCl, pH 8.5) is added to the clarified harvest and the diluted harvest is loaded onto a 170mL bed volume Protein-A column. The column is washed with 5 column volumes (CV) of IX ProSepA Buffer (50mM Boric Acid. 2.5M NaCl, pH 8.5). then washed with 2CV of IX PBS, and the anti -Nectin-4 antibody' or antigen-binding fragment eluted with 5CV of Elution Buffer (0. IM Glycine, pH 3.0). The elution fractions containing IgG are combined and the pH neutralized by adding 1 / 1 Oth volume of 1.0M Tris, pH buffer. The neutralized antibody composition is then sterile filtered using a 10 kDa disposable TFF cassette. The antibody may be formulated for storage by diafiltration against 10 liter of formulation buffer (20 mM sodium acetate, 9% sucrose, pH 5.0) and using 20 volume changes. Using this protocol, antibody 22C3 at a concentration of about 5.0 mg / ml can be prepared and having a purity of at least 98% by SDS- PAGE, SEC HPLC and C8 RP-HPLC measurements, with endotoxin levels of less than 0.1 EU / ml and less than 0.02 EU / mg.

[0146] The human Nectin-4 binders disclosed herein may' also be produced recombinantly (e.g., in an E. colH'Vl expression system as discussed above). In this embodiment, nucleic acidsencoding the antibody molecules of the invention (e.g., VH or VL) may be inserted into a pET- based plasmid and expressed in the E. coli / Tl system. There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

[0147] Human Nectin-4 binders can also be synthesized by any of the methods set forth in U.S. Patent No. 6.331,415.

[0148] Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells. NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Preferred cell lines of are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

[0149] Human Nectin-4 binders can be recovered from the culture medium using standard protein purification methods. Further, expression of human Nectin-4 binders of the invention (or other moieties therefrom) from production cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system isdiscussed in whole or part in connection with European Patent Nos. 0 216 846. 0 256 055. and 0 323 997 and European Patent Application No. 89303964.4.

[0150] A polyclonal antibody is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from collections of different B-lymphocytes, e.g.. the B-lymphocyte of an animal treated with an immunogen of interest, which produces a population of different antibodies that are all directed to the immunogen. Usually, polyclonal antibodies are obtained directly from an immunized animal, e.g., spleen, serum, or ascites fluid.

[0151] The present invention further includes antibody fragments of the anti-Nectin-4 antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain appended to a VH-CHI chain by a disulfide bridge. A F(ab)2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab)2 molecule includes a portion of the Fcregion between which disulfide bridges are located. An Fv fragment is the smallest fragment of a VL or VH region that retains antigen binding via contact with both the heavy and light chains.

[0152] Immunoglobulins may be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl. IgG2, IgG3 and IgG4; IgAl and IgA2. The invention comprises antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

[0153] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, e.g., a human constant region, such as yl, y2, y3, or y4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a human light chain constant region, such as lambda or kappa human light chain region (see SEQ ID NO: 36) or variant thereof. By way of example, and not limitation the human heavy chain constant region can be yl and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody is y4 with a Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38: 1-8, 2001). In some embodiments, different constant domains may be appended to humanized VL and VH regions derived from the CDRs provided herein.Human Nectin-4 binders comprising an effector-silent Fc domain

[0154] Effector-silent human Nectin-4 binders of the present invention that comprise full-sized antibodies may comprise an HC constant domain or Fc domain thereof that has been modified such that the antibody displays no measurable binding to one or more FcRs or displays reduced binding to one or more FcRs compared to that of an unmodified antibody of the same IgG isotype. The effector-silent antibodies may in further embodiments display no measurable binding to each of FcyRIIIa, FcyRIIa, and FcyRI or display reduced binding to each of FcyRIIIa, FcyRIIa, and FcyRI compared to that of an unmodified antibody of the same IgG isotype. In particular embodiments, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

[0155] In particular embodiments, the effector-silent antibody comprises an Fc domain of an IgGl isoty pe that has been modified to lack A'-glycosylation of the asparagine (Asn) residue at position 297 (Eu numbering system) of the HC constant domain. The consensus sequence for N- glycosylation is Asn-Xaa-Ser / Thr (wherein Xaa at position 298 is any amino acid except Pro); the A-glycosylation consensus sequence is Asn-Ser-Thr. The modification may be achieved by replacing the codon encoding the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, for example Ala, Asp, Gin, Gly, or Glu, e.g., N297A, N297Q, N297G, N297E. or N297D. Alternatively, the codon for Ser at position 298 may be replaced with the codon for Pro or the codon for Thr at position 299 may be replaced with any codon except the codon for Ser. In a further alternative each of the amino acids comprising the A-glycosylation consensus sequence is replaced with another amino acid. Such modified IgG molecules have no measurable effector function. In particular embodiments, these mutated HC molecules may further comprise 1. 2. 3, 4, 5. 6, 7, 8. 9. or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations. In further embodiments, such IgGs modified to lack N- glycosylation at position 297 may further include one or more additional mutations disclosed herein for eliminating measurable effector function.

[0156] An exemplary IgGl HC constant domain mutated at position 297, which abolishes the A-glycosylation of the HC constant domain, is set forth in SEQ ID NO: 34. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7. 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0157] In particular embodiments, the Fc domain of the IgGl HC constant domain comprising the effector-silent antibody is modified to include one or more amino acid substitutions selectedfrom E233P. L234A. L235A, L235E, N297A. N297D, D265S. and P331S (wherein the positions are identified according to Eu numbering) and wherein said HC constant domain is effector- silent. In particular embodiments, the modified IgGl further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0158] In particular embodiments, the HC constant domain comprises L234A, L235A, and D265S substitutions (wherein the positions are identified according to Eu numbering). In particular embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu numbering). These and other substitutions are disclosed in WO9428027; W02004099249; W020121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (2000)).

[0159] In particular embodiments of the above, the HC constant domain comprises an L234A / L235A / D265A; L234A / L235A / P329G; L235E; D265A; D265A / N297G; or V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions, wherein the positions are identified according to Eu numbering. In particular embodiments, the HC constant domain further comprises 1, 2, 3, 4, 5, 6. 7, 8, 9. or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0160] In particular embodiments, the effector-silent antibody comprises an IgGl isoty pe, in which the Fc domain of the HC constant domain has been modified to be effector-silent by substituting the amino acids from position 233 to position 236 of the IgGl with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgG4 HC, wherein the positions are identified according to Eu numbering (Armour et al., Eur. J. Immunol. 29(8):2613- 24 (1999); Shields et al.. J. Biol. Chem. 276(9):6591-604(2001)). In particular embodiments, the modified IgGl further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non- conservative mutations.

[0161] In particular embodiments, the effector-silent antibody comprises a VH fused or linked to a hybrid human immunoglobulin HC constant domain, which includes a hinge region, a CH2 domain and a CH3 domain in an / V-terminal to (" '-terminal direction, wherein the hinge region comprises an at least partial amino acid sequence of a human IgD hinge region or a human IgGlhinge region; and the CH2 domain is of a human IgG4 CH2 domain, a portion of which, at its Abterminal region, is replaced by 4-37 amino acid residues of an Ab-terminal region of a human IgG2 CH2 or human IgD CH2 domain. Such hybrid human HC constant domain is disclosed in U.S. Pat. No. 7,867,491, which is incorporated herein by reference in its entirety.

[0162] Exemplary IgGl HC constant domains include HC constant domains comprising an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0163] In particular embodiments of the human Nectin-4 binder, the human Nectin-4 binder is an antibody comprising an IgGl Fc domain as disclosed herein, which further comprises a C- terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0164] In any one of the embodiments disclosed herein, the light chain may comprise a human kappa light chain constant domain comprising SEQ ID NO: 36.Antibody Engineering

[0165] Further included are embodiments in which the anti-target antibodies are engineered to include modifications to framework residues within the variable domains of a parental (e.g., rodent) monoclonal antibody, e.g.. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e., framework residues) in a parental (e g., rodent) antibody with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g., human residues in the case of human therapeutics. Such an antibody is referred to as a “humanized’7antibody. In some cases, it is desirable to increase the affinity, or alter the specificity of an engineered (e.g., humanized) antibody. One approach is to “back-mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g., humanized) antibody, e.g.. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Patent No. 5,693,762, U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101.)

[0166] In certain embodiments, the anti-target antibodies and antigen-binding fragments thereof are engineered (e.g., humanized) to include modifications to in the framework and / or CDRs to improve their properties. Such engineered changes can be based on molecular modelling. A molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, MD; 5th ed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat. et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examined conformations of the loops in crystal structures of antibodies and proposed hypervariable loops. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are variations between regions classified as CDRs or hypervariable loops. Later studies (Raghunathan et al., (2012) J. Mol Recog. 25. 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen-binding regions in antibodies do not necessarily conform strictly to the CDR residues or hypervariable loops. The molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice the potential antigen-binding regions based on model differ from the conventional CDRs or '‘hypervariable loops. Commercial scientific software such as MOE (Chemical Computing Group) can be used for molecular modeling. Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs. For FR4 (framework 4) in VH, VJ regions for the human germlines are compared with the corresponding non-human region. In the case of FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non- human region. Once suitable human frameworks are identified, the CDRs are grafted into the selected human frameworks. In some cases, certain residues in the VL-VH interface can be retained as in the non-human (parental) sequence. Molecular models may also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation. Developability filters can be introduced early in the design stage to eliminate / minimize these potential problems.

[0167] In some embodiments of the antibody or antigen binding fragments disclosed herein, the antibody or antigen binding fragment comprises (a) a Vjq having a framework selected from thegroup consisting of human V[_[ 1 , Vj-[2, Vj-[3, Vj-[4, Vj-[5. and V p[6 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and. (b) a VL having a framework selected from the group consisting of human VK1, VK2, VK3, VK4, VK5, VK6, V / 1, V / 2, V / 3, V / 4, V / 5, V / 6, V / 7, V / 8, V / 9, and V / 10 family and variants thereof having 1, 2, 3. 4, 5, 6. 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments, the amino acid sequence differences are conserv ative changes / substitutions.

[0168] In some embodiments of the antibody or antigen binding fragments disclosed herein, the antibody or antigen binding fragment comprises (a) a Vjq having a framework selected from the group consisting of human Vjql, Vjq2, Vp . Vj_|4. Vjq5, and Vjq6 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions; and, (b) a VL having a framework selected from the group consisting of human VK1. VK2. VK3, VK4, VK5, VK6, V / 1, V / 2. V / 3. V / 4, V / 5, V / 6, V / 7, V / 8, V / 9, and V / 10 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In particular embodiments, the amino acid sequence differences are conserv ative changes / substitutions.

[0169] In some embodiments, the antibody or antigen binding fragment comprises (a) a Vjq having a human Vjql family framework or variant thereof having 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and, (b) a V having a human VK5 family framework or variant thereof having 1, 2, 3. 4, 5, 6. 7. 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes / substitutions.

[0170] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity7of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent No. 7,125,689.

[0171] In particular embodiments, it will be desirable to change certain amino acids containing exposed sidechains to another amino acid residue in order to provide for greater chemical stability of the final antibody, as follows. The deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). In certain embodiments, the antibodies of the present disclosure do not contain asparagine isomerism sites.

[0172] For example, an asparagine (Asn) residue may be changed to Gin or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR. A similar problem may occur at an Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60: 1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagine is changed to glutamine (Gin). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gin) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine residues (A pically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigenbinding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id. In one embodiment, the methionine is changed to alanine (Ala). Additionally, to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the antibody for the target, or other desired biological activity to unacceptable levels. Table 5 provides a list of exemplary stabilizing CDR variants.Human Nectin-4 binder conjugatesDetectable conjugates for diagnostics

[0173] The human Nectin-4 binders disclosed herein, such as anti-Nectin-4 antibodies and antigen-binding fragments thereof, may also be conjugated to a detectable moiety to allow detection of binding in a tissue sample. As used herein, the term “detectable moiety’" refers to a peptide, protein, or chemical moiety that is detectable by fluorescence, chemiluminescence,radioactive emissions, secondary antibody detection of a chemical or peptide tag (e.g.. streptavidin-biotin systems, hexa-histidine tags (SEQ ID NO: 37), hemagglutinin tags, or FLAG tags), or creation of a chromogen (e.g., oxidation of 3,3 '-Diaminobenzidine (DAB) by horseradish peroxidase leading to a brown precipitate). Detection modalities include but are not limited to microscopic imaging, magnetic resonance imaging (MRI), or X-ray imaging.

[0174] Chemical fluorescent or chemiluminescent labels may include fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine,152Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, an aequorin label, 2,3- dihydrophthalazinediones, biotin / avidin, spin labels and stable free radicals. In particular embodiments, the antibodies and antibody fragments disclosed herein may also be conjugated to radioisotopes such as "Tc.90Y,inIn,32P,14C.1251,3H,131I,nC,150,13N.18F,35S,51Cr,57To,226Ra,60Co,59Fe.57Se.152Eu,67CU,217Ci,21 1At.212Pb,47Sc.109Pd,234Th,40K,157Gd,55Mn.52Tr, and56Fe. Le Doussal et al. (1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889) disclose conjugation of various detectable labels to antibodies.

[0175] As used herein, the term "‘fluorescent protein” refers to a protein fluorophore that emits light at some wavelength after excitation by light at another wavelength. Exemplary fluorescent proteins that emit in the green spectrum range include but are not limited to: green fluorescent protein (GFP); enhanced GFP (eGFP); superfolder GFP; AcGFPl; and ZsGreenl. Exemplary fluorescent proteins that emit light in the blue spectrum range include but are not limited to: enhanced blue fluorescent protein (EBFP), EBFP2, Azurite, and mKalama. Exemplary fluorescent proteins that emit light in the cyan spectrum range include but are not limited to: cyan fluorescent protein (CFP); enhanced CFP (ECFP); Cerulean; mHoneydew; and CyPet.Exemplary fluorescent proteins that emit light in the yellow spectrum range include but are not limited to: yellow fluorescent protein (YFP); Citrine; Venus; mBanana; ZsYellow 1 ; and Ypet. Exemplary fluorescent proteins that emit in the orange spectrum range include but are not limited to: mOrange; tdTomato; LSSmOrange, PsmOrange and PsmOrange2. Exemplary fluorescent proteins that emit light in the red and far-red spectrum range include but are not limited to: DsRed: DsRed-monomer; DsRed-Express2; mRFPi; mCherry; mStrawberry; mRaspberry; niPluni; E2-Crimson; iRFP670; iRFP682; iRFP702; iRFP720. Exemplary listings of fluorescent proteins and their characteristics may be found in Day and Davidson, Chem Soc Rev 2009 October; 38(10): 2887-2921, incorporated herein by reference.

[0176] Fluorescent proteins may include chimeric combinations of fluorescent proteins that transfer and receive energy through fluorescent resonance energy transfer (FRET) when exposed to a particular wavelength of light. In some embodiments, an acceptor in a FRET pair may emit light at a certain wavelength after accepting energy from a donor molecule exposed to another wavelength of light. Exemplary chimeric FRET pairs include but are not limited to: ECFP-EYFP; mTurquoise2-SeYFP; EGFP-mCherry: and Clover-mRuby. In some embodiments, the acceptor molecule of chimeric fluorescent molecule may quench the light emission of a donor molecule exposed to its preferred wavelength of light. Quenching between different portions of chimeric fluorescent proteins may occur using a photoactivatable acceptor. For example, a chimeric fluorescent protein may include a photoactivatable GFP that can then quench photoemission by CFP. Examples of FRET proteins are discussed in Ehldebrandt et al., Sensors (Basel). 2016 Sep; 1 (9): 1488, incorporated herein by reference.

[0177] Any method known in the art for conjugating the human Nectin-4 binders to the various detectable moi eties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13: 1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. And Cytochem. 30:407. Methods for conjugating antibodies are conventional and well known in the art.Therapeutic conjugates

[0178] Human Nectin-4 binders disclosed herein may also be conjugated or recombinantly fused to one or more therapeutic moieties for cancer therapeutic applications, such as cellular toxins, radioisotopes, cytokines, or enzymes.

[0179] Therapeutic moieties include, but are not limited to, antimetabolites (e.g.. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine); alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP), and cisplatin); anthracy clines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (e g., d actinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); Auristatin molecules (e.g., 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 of which are incorporated herein by reference); hormones (e.g., glucocorticoids, progestins, androgens, andestrogens). DNA-repair enzyme inhibitors (e.g.. etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7):2167-76 (2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin. dihydroxy anthracin dione, mitoxantrone, mithramycin. actinomycin D, 1 -dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof and those compounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034.053, 5,985,877, 5.958,769, 5,925.376, 5,922,844, 5,911,995. 5,872,223, 5.863,904, 5,840.745, 5,728,868, 5,648,239, 5,587,459); famesyl transferase inhibitors (e.g., R115777, BMS-214662, and those disclosed by, for example, U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6.369,034, 6,362.188, 6,342,765, 6,342,487, 6,300,501, 6.268,363, 6,265.422, 6,248,756, 6.239,140, 6.232.338, 6,228.865, 6,228,856. 6,225,322. 6.218,406, 6.211.193, 6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051.574, and 6,040.305); topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211): DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA minor groove binders such as Hoechst dye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1; bisphosphonates (e.g.. alendronate, cimadronte, clodronate. tiludronate, etidronate, ibandronate. neridronate, olpandronate. risedronate. piridronate, pamidronate, zolendronate) HMG-CoA reductase inhibitors, (e.g., lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin); antisense oligonucleotides (e.g.. those disclosed in the U.S. Pat. Nos.6,277,832, 5.998,596, 5.885.834, 5,734.033, and 5,618.709); adenosine deaminase inhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine); ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) and pharmaceutically acceptable salts, solvates, clathrates, and prodrugs thereof.

[0180] Further, an antibody provided herein may be conjugated or recombinantly fused to a therapeutic moiety or drug moiety that modifies a given biological response. Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, y-interferon, a-interferon, nerve growth factor, platelet derived grow th factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-y, TNF-y, AIM I (see, International Publication No. WO 97 / 33899). AIM II (see, International Publication No. WO 97 / 34911), Fas Ligand (Takahashi et al., 1994, J. Immunol., 6: 1567-1574), and VEGF (see. International Publication No. WO 99 / 23105), an anti-angiogenic agent, e.g., angiostatin, endostatin or a component of the coagulation pathway (e.g., tissue factor); or, a biological response modifier such as, for example, a lymphokine (e.g., 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 coagulation 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 proteins-factors II (prothrombin), factor V, Xlla, VIII, Xllla, XI, Xia, IX, Ixa, X, phospholipid, and fibrin monomer).

[0181] Any method known in the art for conjugating the human Nectin-4 binders to the various therapeutic moi eties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13: 1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. And Cytochem. 30:407. Methods for conjugating antibodies are conventional and well known in the art.Pharmaceutical compositions and administration

[0182] “Administration” and “treatment,” as the terms apply to an animal, human, patient, subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. “Administration” and “treatment” also includes in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., human, rat, mouse, dog, cat, rabbit). In a preferred embodiment, the term “subjects” refers to a human.

[0183] As used herein, the term “treat’7or “treating” means to administer a therapeutic moiety, such as a composition containing any of the human Nectin-4 binders of the present invention, internally or externally to a subject or patient having one or more disease symptoms, for which the therapeutic moiety has therapeutic activity. In specific embodiments, the human Nectin-4 binders can be administered topically, subcutaneously, intramuscular, intradermally, intravenously, or systemically. Typically, the moiety is administered in an amount effective to: (i) alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree, or (ii) inhibiting or reducing the severity of a disease or disorder in an individual. The amount of a therapeutic moiety that is effective to alleviate any particular disease symptom, and / or to inhibit or reduce the severity of a disease or disorder, including in specific embodiments cancer or proliferative disease, in the individual may vary according to factors such as the injury or disease state, age, and / or weight of the individual, and the ability of the therapeutic moiety to elicit a desired response in the individual. Whether one or more disease symptoms have been alleviated or the severity of the disease or disorder inhibited or reduced can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of the symptom(s), disease or disorder. Thus, the terms denote that a beneficial result has been or will be conferred on a human or animal individual exhibiting one or more disease symptoms, for which the therapeutic moiety has therapeutic activity' and thus in need of the treatment (“in need thereof’). Treatment with human Nectin-4 binders could also be combined with other interventions (in specific embodiments, antibodies, nucleic acids, vaccines and small molecule compounds) to treat other symptoms, diseases or disorders.

[0184] As used herein, the term “therapeutically effective amount” refers to a quantity of a specific substance sufficient to achieve a desired effect (i.e., the alleviation of a particular disease symptom, and / or the inhibition or reduction of the severity of the disease or disorder) in an individual being treated. For instance, this may be the amount necessary to inhibit or reduce the severity of a disease or disorder in an individual.

[0185] The human Nectin-4 binder may be provided in suitable pharmaceutical compositions comprising the human Nectin-4 binder and a pharmaceutically acceptable carrier. The carrier may be a diluent, adjuvant, excipient, or vehicle with which the human Nectin-4 binder is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile andgenerally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules or of the disclosure in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21. sup. st Edition, Troy, D. B. ed., Lippincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

[0186] The mode of administration of the human Nectin-4 binder may be any suitable route such as parenteral administration, e.g.. intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.

[0187] The human Nectin-4 binder may be administered to an individual (e.g., patient) by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously, or intraperitoneally, i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0188] The dose given to an individual having cancer or malignancy is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount") and may be sometimes 0.005 mg / kg to about 100 mg / kg, e g., about 0.05 mg / kg to about 30 mg / kg or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg or about 24 mg / kg, or, e.g., about 1, 2, 3, 4, 5. 6, 7, 8, 9 or 10 mg / kg, but may even higher, for example about 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70. 80. 90 or 100 mg / kg.

[0189] A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg / n Usually between 1 and 8 doses, (e.g., 1, 2. 3, 4, 5. 6, 7 or 8) may be administered to treat cancer or malignancy, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

[0190] The administration of the human Nectin-4 binder may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, sixmonths or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.

[0191] The human Nectin-4 binder may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

[0192] The human Nectin-4 binder may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is know n to be present due to other biological factors.

[0193] The human Nectin-4 binder may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective w ith conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.Combination therapy treatments

[0194] As used herein, the term “combination therapy” refers to treatment of a human or animal individual comprising administering a first therapeutic agent and a second therapeutic agent consecutively or concurrently to the individual. In general, the first and second therapeutic agents are administered to the individual separately and not as a mixture; however, there may be embodiments where the first and second therapeutic agents are mixed prior to administration.

[0195] The combination therapy of the present disclosure comprises a human Nectin-4 binder and another therapeutic agent (small molecule or antibody) may be used for the treatment of any proliferative disease, in particular treatment of cancer. In particular embodiments, the combination therapy of the present disclosure may be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin ly mphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer. In another embodiment, the combination therapy of the present disclosure may be used to treat pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bow el or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.Combination therapy comprising a human Nectin-4 binder and a chemotherapy agent

[0196] The combination therapy of the present disclosure may be administered to an individual having a cancer in combination with chemotherapy. The individual may undergo the chemotherapy at the same time the individual is undergoing the combination therapy of the present disclosure. The individual may undergo the combination therapy of the present disclosure after the individual has completed chemotherapy. The individual may be administered the chemotherapy after completion of the combination therapy. The combination therapy of the present disclosure may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapse cancer and who is undergoing chemotherapy or who has completed chemotherapy.

[0197] The chemotherapy may include a chemotherapy agent selected from the group consisting of(i) alkylating agents, including but not limited to, bifunctional alkylators. cyclophosphamide, mechlorethamine, chlorambucil, and melphalan;(ii) monofunctional alkylators, including but not limited to, dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine);(iii) anthracy clines, including but not limited to. daunorubicin, doxorubicin, epirubicin. idarubicin, mitoxantrone, and valrubicin;(iv) cytoskeletal disruptors (taxanes), including but not limited to, paclitaxel, docetaxel, abraxane, and taxotere;(v) epothilones, including but not limited to. ixabepilone, and utidelone;(vi) histone deacetylase inhibitors, including but not limited to, vorinostat, and romidepsin;(vii) inhibitors of topoisomerase I, including but not limited to, irinotecan, and topotecan;(viii) inhibitors of topoisomerase II, including but not limited to, etoposide, teniposide, and tafluposide;(ix) kinase inhibitors, including but not limited to, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib;(x) nucleotide analogs and precursor analogs, including but not limited to, azacitidine, azathioprine, fluoropyrimidines (e g., such as capecitabine, carmofur. doxifluridine, fluorouracil, and tegafur) cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine (formerly thioguanine);(xi) peptide antibiotics, including but not limited to. bleomycin and actinomycin; a platinum-based agent, including but not limited to, carboplatin, cisplatin, and oxaliplatin;(xii) retinoids, including but not limited to, tretinoin, alitretinoin, and bexarotene; and xiii) vinca alkaloids and derivatives, including but not limited to, vinblastine, vincristine, vindesine, and vinorelbine.

[0198] Selecting a dose of the chemotherapy agent for chemotherapy depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. The dose of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each additional therapeutic agent will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g.. Wawrzynczak (1996) Antibody Therapy. Bios Scientific Pub. Ltd. Oxfordshire. UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) few Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of the appropriate dose regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the individual's clinical history (e g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0199] Thus, the present disclosure contemplates embodiments of the combination therapy of the present disclosure that further includes a chemotherapy step comprising platinum-containing chemotherapy, pemetrexed and platinum chemotherapy or carboplatin and either paclitaxel or nab-paclitaxel. In particular embodiments, the combination therapy with a chemotherapy step may be used for treating at least NSCLC and HNSCC.

[0200] The combination therapy further in combination with a chemotherapy step may be used for the treatment any proliferative disease, in particular, treatment of cancer. In particular embodiments, the combination therapy of the present disclosure may be used to treat melanoma,non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer.

[0201] In another embodiment, the combination therapy further in combination with a chemotherapy step may be used to treat pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

[0202] In particular embodiments, the combination therapy with a chemotherapy step may be used to treat one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced), and cutaneous squamous carcinoma.Combination Therapy Comprising a Human Nectin-4 Binder And a Therapeutic Antibody

[0203] The human Nectin-4 binder of the present disclosure may be administered in combination with one or more therapeutic agent, which is an antibody, for treatment of cancer or proliferative disease. The individual may undergo treatment with the therapeutic antibody at the same time the individual is undergoing the combination therapy of the present disclosure. The individual may undergo the combination therapy of the present disclosure after the individual has completed treatment with the therapeutic antibody. The individual may be administered the treatment with the therapeutic antibody after completion of the combination therapy. The combination therapy of the present disclosure may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapse cancer and who is undergoing chemotherapy or who has completed chemotherapy. In particular embodiments, the therapeutic agent targets the programmed death 1 receptor or ligand., PD-1 and PD-L1, respectively.

[0204] Exemplary anti -PD-1 antibodies that may be used in a combination therapy with the human Nectin-4 binder include any antibody that binds PD-1 and inhibits PD-1 from binding PD- Ll. In a further embodiment, the exemplary anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and cemiplimab-rwlc. Exemplary' antibodies includethe following anti-PD-1 antibodies and compositions comprising an anti-PDl antibody and a pharmaceutically acceptable salt.

[0205] Pembrolizumab, also known as KEYTRUDA, lambrolizumab, MK-3475 or SCH- 900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No. 8,354,509 and W02009 / 114335 and disclosed, e.g., in Hamid, et al.. New England J. Med. 369 (2): 134-144 (2013).

[0206] Nivolumab, also known as OPDIVO, MDX-1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in W02006 / 121168 and U.S. Pat. No. 8,008,449.

[0207] Cemiplimab-rwlc, also known as cemiplimab, LIBTAYO or REGN2810, is a recombinant human IgG4 monoclonal antibody that is described in WO2015112800 and U.S. Pat. No. 9,987,500.

[0208] In particular embodiments, the anti-PD-1 antibody comprises (i) a V[ [ comprising the three HC-CDRs of pembrolizumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of pembrolizumab fused or linked to a LC kappa or lambda constant domain.

[0209] In particular embodiments, the anti-PD-1 antibody comprises (i) a VH comprising the three HC-CDRs of nivolumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of nivolumab fused or linked to a LC kappa or lambda constant domain.

[0210] In particular embodiments, the anti-PD-1 antibody comprises (i) a Vj-[ comprising the three HC-CDRs of cemiplimab-rw lc fused or linked to an effector-silent HC constant domain and (ii) a V comprising the three LC-CDRs of cemiplimab-rwdc fused or linked to a LC kappa or lambda constant domain.

[0211] In particular embodiments, the anti-PD-1 antibody VH may be fused or linked to an IgGl, IgG2, IgG3, or IgG4 HC constant domain that is not currently linked to the particular Vpj or is linked to an IgGl, IgG2, IgG3, or IgG4 HC constant domain has been modified to include one or more mutations in the Fc domain that render the resulting anti-PD-1 antibody effecter- silent.Injection Device for Administering A Human Nectin-4 Binder

[0212] The present disclosure also provides an injection device compnsing a human Nectin-4 binder as set forth herein or a pharmaceutical composition thereof. An injection device is adevice that introduces a substance into the body of a patient via a parenteral route, e.g. intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the human Nectin-4 binder or a pharmaceutical composition thereof), a needle for piecing skin and / or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the disclosure, an injection device that comprises a human Nectin-4 binder or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the human Nectin-4 binder or a pharmaceutical composition thereof in a cannula or trocar / needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaC'b and optionally including glucose) introduced into the body of the subject through the cannula or trocar / needle.

[0213] The human Nectin-4 binder or a pharmaceutical composition thereof may, in an embodiment of the disclosure, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm): the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g. , a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the disclosure, an injection device is an autoinjector, a jet injector, or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the human Nectin-4 binder or a pharmaceutical composition thereof to a patient’s body. External infusion pumps are medical devices that deliver the human Nectin-4 binder or a pharmaceutical composition thereof into a patient's body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.Experimental and diagnostic uses of human Nectin-4 binders

[0214] The human Nectin-4 binders disclosed herein (e.g., antibodies and antibody fragments thereof) may be used as affinity purification agents. In this process, the antibodies or fragments are immobilized on a solid phase such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the target protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the target protein, which is bound to the immobilized antibody or fragment. Finally, the support is washed with a solvent which elutes the bound target from the column (e.g., protein A). Such immobilized antibodies form part of the present invention.

[0215] Further provided are antigens for generating secondary antibodies which are useful for example for performing Western blots and other immunoassays discussed herein. In particular, polypeptides are disclosed which comprise the variable regions and / or CDR sequences of a therapeutic antibody disclosed herein, and which may be used to generate an anti-idiotypic antibodies for use in specifically detecting the presence of the antibody, e.g., in a therapeutic context.

[0216] Human Nectin-4 binders disclosed herein (e.g., antibodies and antibody fragments) may also be useful in diagnostic assays for Nectin-4, e.g., detecting its expression in specific cells, tissues, or serum. Such diagnostic methods may be useful in various disease diagnoses.

[0217] For example, particular embodiments include ELISA assays (enzy me-linked immunosorbent assay) incorporating the use of an anti-Nectin-4 antibody or antigen-binding fragment thereof disclosed herein.

[0218] For example, such a method comprises the following steps:(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., a plastic plate) with antitarget antibody or antigen-binding fragment thereof;(b) apply a sample to be tested for the presence of the target to the substrate;(c) wash the plate, so that unbound material in the sample is removed;(d) apply detectably-labeled antibodies (e.g., enzyme-linked antibodies) which are also specific to the target;(e) wash the substrate, so that the unbound, labeled antibodies are removed;(I) if the labeled antibodies are enzyme linked, apply a chemical which is converted by the enzyme into a fluorescent signal; and(g) detect the presence of the labeled antibody.

[0219] In a further embodiment, the labeled antibody is labeled with peroxidase which react with ABTS (e g., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or 3, 3’, 5,5’- Tetramethylbenzidine to produce a color change which is detectable. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 3H) which can be detected by scintillation counter in the presence of a scintillant.

[0220] An anti-target antibody of the invention may be used in a Western blot or immune- protein blot procedure. Such a procedure forms part of the present invention and includes e.g.,(1) contacting a membrane or other solid substrate to be tested for the presence of bound target or a fragment thereof with an anti-target antibody or antigen-binding fragment thereof of the invention. Such a membrane may take the form of a nitrocellulose or vinylbased (e.g., polyvinylidene fluoride (PVDF)) membrane to which proteins to be tested for the presence of X in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel). Before contact of membrane with the anti-target antibody or fragment, the membrane is optionally blocked, e.g., with non-fat dry milk or the like to bind non-specific protein binding sites on the membrane.(2) washing the membrane one or more times to remove unbound anti-target antibody or fragment and other unbound substances; and(3) detecting the bound anti-target antibody or fragment.

[0221] Detection of the bound antibody or antigen-binding fragment thereof may occur bybinding the antibody or fragment with a secondary- antibody (an anti-immunoglobulin antibody) which is delectably labeled, and then detecting the presence of the secondary antibody.

[0222] The anti-target antibodies and antigen-binding fragments thereof disclosed herein may also be used for immunohistochemistry (IHC). Such a method forms part of the present invention and comprises, e g., (1) contacting a cell to be tested for the presence of target with an anti-target antibody or antigen-binding fragment thereof of the invention; and (2) detecting the antibody or fragment on or in the cell.

[0223] The antibodies and antigen-binding fragments of the present invention also bind to human Nectin-4 in preserved human tissue, e.g., FFPE tissue samples, which may be treated to unmask the Nectin-4 antigens using antigen retrieval techniques. The terms "antigen retrieval” or ■‘antigen retrieval techmque(s)” refer to a process of exposing a preserved tissue sample to heat and / or enzymes and partially reversing protein cross-linking caused by the tissue preservation process. Partial reversal of protein cross-linking using antigen retrieval techniques exposesantigens that were otherwise physically blocked from binding to antibody or antigen-binding fragments. Exemplary commercial antigen retrieval kits include Agilent Dako EnVision FLEX® High pH Target Retrieval Solution (Agilent Dako, Santa Clara, CA), Antigen Unmasking Solution (H-3300-250 or H-3300-251, Vector Laboratories, Newark CA), and eBioscience™ IHC Antigen Retrieval Solution (ThermoFisher Scientific, Waltham MA).

[0224] If the antibody or antigen-binding fragment thereof itself is detectably labeled, it can be detected directly. Alternatively, the antibody or antigen-binding fragment thereof may be bound by a detectably labeled secondary antibody which is detected.

[0225] Certain anti-target antibodies and antigen-binding fragments thereof disclosed herein may also be used for in vivo tumor imaging. Such a method may include injection of a radiolabeled anti-target antibody or antigen-binding fragment thereof into the body of a patient to be tested for the presence of a tumor associated with target expression followed by nuclear imaging of the body of the patient to detect the presence of the labeled antibody or fragment, e.g., at loci comprising a high concentration of the antibody or fragment which are bound to the tumor.

[0226] Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include e.g., iodine-123 (123I) and technetium-99m (99mTc), e.g., in conjunction with SPECT imaging ornC,13N.15O or18F, e.g., in conjunction with PET imaging or Indium-I l l (See e.g., Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).Detection Kits and Therapeutic Kits

[0227] As a matter of convenience, a human Nectin-4 binder disclosed herein can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. Where the antibody is labeled with an enzy me, the kit will include substrates and cofactors required by the enzyme (e.g.. a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

[0228] Also provided are diagnostic or detection reagents and kits comprising one or more such reagents for use in a variety7of detection assays, including for example, immunoassays such asELISA (sandwich-type or competitive format). The kit's components may be pre- attached to a solid support or may be applied to the surface of a solid support when the kit is used. In some embodiments, the signal generating means may come pre-associated with an antibody of the invention or may require combination with one or more components, e.g., buffers, antibodyenzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzy mes are well known in the art. Kits may comprise any of the capture agents and detection reagents described herein. Optionally the kit may also comprise instructions for carrying out the methods of the invention.

[0229] The detection kits disclosed herein may also be prepared that comprise at least one of the antibody or antigen-binding fragment disclosed herein and instructions for using the composition as a detection reagent. Containers for use in such kits may ty pically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection composition(s) may be placed, and preferably suitably aliquoted. The kits disclosed herein will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorigenic, or other ty pe of detectable label or detecting means is included within the kit. the labeling agent may be provided either in the same container as the detection composition itself or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent may be prepared in a single container means, and in most cases, the kit will also ty pically include a means for containing the vial(s) in close confinement for commercial sale and / or convenient packaging and delivery.

[0230] A device or apparatus for carry ing out the detection or monitoring methods described herein is also provided. Such an apparatus may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex.The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or by a combination of active and passive forces.

[0231] Further embodiments also provide a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be executed on the processor to perform any of the methods described herein. Examples of suitable computing systems, environments, and / or configurations include personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, or any other systems known in the art.

[0232] The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.General Methods

[0233] Standard methods in molecular biolog}’ are described Sambrook, Fritsch and Maniatis (1982 & 1989 2ndEdition, 2001 3rdEdition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA. Vol. 217, Academic Press. San Diego, CA). Standard methods also appear in Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4. John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0234] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 7, John Wiley and Sons, Inc., New York). Chemical analy sis,chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2. John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research. St. Louis, MO; pp. 45-89;Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0235] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g.. Shepherd and Dean (eds.) (2000) Monoclonal Antibodies. Oxford Univ. Press, New York, NY;Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160: 1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378: Baca et al. (1997) J. Biol. Chem. 272: 10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) 7 Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0236] An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309- 314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15: 146- 156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual , Academic Press, San Diego. CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0237] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry. 2nded:, Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available(Molecular Probes (2003) Catalogue, Molecular Probes, Inc.. Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0238] Standard methods of histology of the immune system are described (see, e.g., Muller- Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology. Lippincott. Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology:: Text and Atlas, McGraw-Hill, New York, NY).

[0239] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (see, e.g.. GenBank, Vector NTI® Suite (Informax, Inc. Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).EXAMPLES

[0240] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1: Antibody from Hybridoma Clone 1 binds to both fixed and unfixed Nectin-4 protein

[0241] Hybridoma clone 1 was identified from a hybridoma fusion cell generated in Lewis rats immunized with formalin-fixed human Nectin-4 protein. Preliminary hybridoma screening was performed by protein ELISA against fixed human Nectin-4 antigen. Nectin-4 binding for recombinantly-produced antibody from clone 1 (mAbl) was confirmed against fixed and unfixed human Nectin-4 proteins (FIGs. 1A and IB) using enzyme-linked immunosorbant assay (ELISA). FIG. 1 A shows the ELISA results for fixed Nectin-4 and FIG. IB shows the ELISA results for unfixed Nectin-4. ELISA plates were coated with 1 pg / mL fixed or unfixed human Nectin-4 protein and monoclonal antibodies were titrated in a 4-fold dilution series from starting concentration of 10 pg / mL. Positive controls in FIGs. 1 A- IB are a monoclonal antibody that specifically binds to human Nectin-4 antibody comparator mAb 1), and IgGl / Kappa (CX)).

[0242] Surface plasmon resonance (SPR) was used to measure the equilibrium dissociation constant (KD) of mAbl. SPR measurements were performed on a BIAcore T200 (Cytiva, USA)at 25°C. Buffers (Teknova cat#H8022. Lot# H802217H2101) were degassed and filter-sterilized through 0.2 gm filters prior to use. Anti-mouse Fc antibody (Cytiva Cat# 29234600) was immobilized onto a CM5 sensor chip using standard amine coupling chemistry.DAB019478 001 was diluted in assay buffer IX HBS-EP pH7.6 to Ipg / mL then captured on chip for 60s. For kinetic analysis, a concentration series of Nectin-4 (0-20nM) were injected over the antibody and reference surfaces at a flow rate of 30 pL / min. The association and dissociation were measured for 200s and 600s respectively. After each sample injection, the surface was regenerated by two 30s injection of lOmM Glycine pH1.7. Affinity and kinetic constants were determined by fitting the sensorgrams with the 1: 1 Langmuir model using Biacore T200 evaluation software (Cytiva). Table 6 below summarizes the association rate constant (ka), the dissociation rate constant (ka), and the KD (average and standard deviation). In this analysis, the KD was 1.4 x W10M (140 pM) ± 4.89 x 10’11M (48.9 pM).Example 2: mAbl binds to Nectin-4, but not its paralogs Nectin-1, Nectin-2, or Nectin-3

[0243] To confirm that mAbl was not cross-reactive with other Nectin family members, immunohistochemistry (IHC) with mAbl was performed on FFPE cell pellets generated from 3A9 cells transfected with empty vector, human Nectin-1 protein, human Nectin-2 protein, human Nectin-3 protein, and human Nectin-4 protein.Sectioning and epitope retrieval

[0244] FFPE blocks were sectioned at 5 pm and allowed to air dry overnight. The slides were baked at 60°C for 60 minutes, and deparaffinized using Leica Stainer XL (Wetzlar. Germany). Following deparaffinization, FFPE slides were subjected to heat induced epitope retrieval (HIER) using lx Target Retrieval Solution TRS pH 6.0, (Dako, S1699), with temperature of 120°C at 10- 15 psi for 4 minutes using a Biocare Antigen DeCloaker (Model DC2002, Pacheco, CA), then followed by a cool down step for 20-30 minutes. The slides were then immersed in deionized w ater, followed by immersion two times in EnVision FLEX Wash Buffer (Dako. FLEX Kit. K8002), being kept wet at all times. The slides were then loaded into the autostainer and stained by applying following steps: 1) EnVision Flex Peroxidase Block, 5 min incubation, lx rinse with TBST, 2) SV031.2. 1E8, 5.0 pg / mL, 60 min incubation, lx rinse with TBST, 3) EnVision Flex+MS Linker, 15 min incubation, lx rinse with TBST. 4) EnVision Flex / HRP, 20 min incubation, lx rinse with TBST, 5) EnVision Flex DAB, 10 min incubation, 3x rinse with deionized water, and 6) DAB enhancer, 7 min incubation, lx rinse with deionized water. Stained slides were immersed in deionized water and counterstained / dehydrated using Meyer’s Modified Hematoxylin (Poly Scientific, R&D Corp., Bay Shore, NY. USA) on Leica Stainer XL. Slides were cover-slipped with Micromount media (Leica Biosystems Inc., Buffalo Grove, IL, USA) and allowed to dry.

[0245] No mAbl staining was observed in cells transfected with null vector (FIG. 2A), Nectin- 1 (FIG. 2B), Nectin-2 (FIG. 2C), or Nectin-3 (FIG. 2D). However, mAbl staining was intense in cells transfected with Nectin-4 (FIG. 2E). Thus, mAbl is highly specific for Nectin-4 and does not have visually detectable cross-reactivity with Nectin paralog proteins.Example 3: Assessment of mAbl IHC relative to orthogonal methods

[0246] The specificity and performance of mAbl as an IHC reagent was assessed through comparison to findings applying orthogonal methods, such as in situ hybridization (ISH) and flow cytometry, to cell lines that express Nectin-4.Cell lines

[0247] Seventeen cell lines with varying levels of Nectin-4 expression were grown and partitioned, with a proportion of cells from each line analyzed by flow cytometry and the remainder being pelleted, formalin fixed, and embedded to generate an FFPE block for each line (see schematic diagram in FIG. 3A). Sections were cut from the FFPE blocks at 5pm thickness and used for IHC with mAbl and for Nectin-4 mRNA ISH (RNAScope™, Advanced Cell Diagnostics, a BioTechne brand. Newark. CA).Flow cytometry

[0248] Cell lines were acquired from internal cell bank, Merck Research Labs-South San Francisco, and cultured at 37°C and 5% CO2 until confluency was achieved. Attached cells were washed twice with PBS (Gibco; Cat# 10010-023) and detached from flask with non-enzymatic dissociation buffer (Gibco; Cat# 13150-016). Detached cells were transferred into a 15mL conical tube and centrifuged at 300xg for 5 minutes at 4°C. Supernatants were aspirated and cell pellets were washed with PBS. Viable cells were counted using Vi-Cell™ XR (Beckman Coulter) and diluted to a density of IxlO7viable cells per mL before transferring IxlO6viable cells into a V-bottom 96-well plate (Coming; Cat# 3894). Plated cells were centrifuged at 300xg for 5 minutes at 4°C, followed by supernatant aspiration, and cell pellets were resuspended in lOOpL of protein-free buffer, PBS. Cells were then treated with 5pL of ViaDye™ Red fixableviability dye (Cytek; Cat# SKU R7-60008). per manufacturer recommendation, resuspended and left at room temperature for 20 minutes protected from light. Cells were then washed twice with FACS buffer (2% Fetal Bovine Serum (Gibco; Cat# 16140-071), 1.5mM EDTA (Invitrogen; Cat# 15575-038) in PBS), incubated at room temperature with FcR blocking buffer (Human Trustain Fcx™; Biolegend; Cat# 422302). 5% mouse normal serum (Jackson Immuno Research; Cat# 015-000-120), in PBS) for 10 minutes, and then surface stained for 30 minutes over ice. Surface staining contained antibody raised against human Nectin-4 (Clone 337516) (R&D Systems; Cat# FAB2659G), or isotype control (Clone 133303) (R&D Systems; Cat# IC0041G), at a final concentration of 1.25pg / mL diluted in FACS buffer. Cells were then washed twice before fixation with freshly diluted 1.6% paraformaldehyde (Electron Microscopy Sciences; Cat# 15710) in PBS for 15 minutes over ice. After fixation, cells were washed twice in PBS before acquiring on a 5-Laser Cytek® Aurora. Unmixed FCS files were analyzed using FlowJo version 10.8.1 (Becton, Dickinson & Company).Copy number calculation

[0249] Fluorescent beads, Quantum™ AF488 MESF (Bangs Laboratories; Cat# 488B; Lot# 16165) were washed and fixed just like cells (See Flow Cytometry method). FCS files were unmixed together with cell files. FCS files were manually gated to remove debris and doublets, and dead cells for stained cells, before acquiring the median fluorescent Intensity (MFI). A linear regression was performed using GraphPad Prism version 9.0.0 and MESF values were normalized to the degree of labeling of the antibody for accurate copy numbers, per manufacturer recommendations.Results

[0250] FIGs. 3B-3E. show example results from one of the Nectin-4-expressing cell lines T- 47D. FIGs. 3B-3D show examples of two-dimensional histogram data from flow cytometry detection of mAbl (FIG. 3B, unstained control; FIG. 3C, isotype control; FIG. 3D, mAbl anti- Nectin-4). FIG. 3E shows a photomicrograph example of a mAbl -stained IHC section, and FIG. 3F shows an example ISH section. Cells in the IHC photomicrograph of FIG. 3E that show dark and black regions outlining the cells are positive for Nectin-4 protein. Cells in the ISH photomicrograph of FIG. 3F that show small dark dots within cells are positive for Nectin-4 mRNA. Nectin-4 prevalence by IHC using mAbl closely matched Nectin-4 prevalence by flow cytometry in cell lines exhibiting a wide range of Nectin-4 expression.

[0251] Correspondence between IHC, ISH, and surface receptor copy number by flow cytometry on evaluated cell lines was good. FIG. 3G-3H show results of Nectin-4 staining by IHC, Nectin-4 mRNA by ISH, and Nectin-4 surface copy number by flow cytometry for four ofthe 17 cell lines: 5367-HTB-9. BXPC-3. T-47D, and H322. Each of the cell lines has different Nectin-4 expression levels. FIG. 3G shows photomicrograph examples of IHC-treated (left column) and ISH-treated (right column) sections for the four cell lines. Cells in the IHC photomicrographs in the left column of FIG. 3G that show dark and black regions outlining the cells are positive for Nectin-4. Cells in the ISH photomicrographs in the right column of FIG. 3G that show small dark dots within cells are positive for Nectin-4.

[0252] FIG. 3H shows a bar graph of surface copy number of Nectin-4 detected by flow cytometry for mAbl. Cell lines 5367-HTB-9 and BXPC-3 had surface copy numbers of about 3000, T-47D had surface copy number of about 8000. and H322 had a surface copy number of about 20,000.

[0253] Table 7 below lists the correspondence between IHC, ISH, and flow cytometry in the full set of 17 cell lines. Semi-quantitative prevalence scores for IHC and ISH using a 0-5 scale (0 = negative, 1 = rare, 2 = low, 3 = moderate, 4 = high, 5 = very’ high, as reviewed by a pathologist) generally corresponded well, as did percent positivity for cells quantitated by flow cytometry (described as % frequency in live cells in Table 7). Occasional divergences were observed.Example 4: mAbl staining prevalence

[0254] Correspondence between the IHC staining of mAbl and the IHC staining of a comparator monoclonal antibody that specifically binds human Nectin-4 ( comparator mAb 2) was analyzed. Sectioning and epitope retrieval were conducted as described in Example 2.

[0255] As shown in FIG. 4A, mAbl stains known Nectin-4-positive normal human tissues (bladder, tonsil, and cervix) with a strong membranous pattern and sensitivity equivalent to or better than comparator mAb 2. Both IHC staining using mAbl and Nectin-4 ISH also stain other known Nectin-4-expressing normal tissues such as esophagus, skin, and breast tissue (see FIG. 4B, left column is mAbl IHC, right column is Nectin-4 ISH; arrows in ISH photomicrographs indicate cellular regions with ISH staining (dark dots).

[0256] Table 8 below lists the comparative scoring of 75 human tumors using mAbl and comparator mAb 2 (TNBC = triple negative breast cancer, Bladder = bladder cancer. Cervical SCC = cervical squamous cell carcinoma). Slides were stained using optimized assay conditions for each clone and scored independently by a board-certified pathologist using a 0-5 semi- quantitative prevalence scale (0 = no staining or less than 1 % of tumor cell staining, 1 = minimal, 2 = low, 3 = medium, 4 = high, 5 = very high). The slides stained with mAbl were scored first. During scoring of slides stained with comparator mAb 2, the pathologist was blinded to scores generated using mAbl. Scoring was generally highly concordant across the specimens. When there were discordances, scores with mAbl were consistently higher than scores for comparator mAb 2.Example 5: mA b l 1HC staining distribution

[0257] To assess the mAbl staining distribution, a set of 78 triple negative breast cancer (TNBC), bladder cancer, and cervical squamous cell carcinoma (Cervical SCC) specimens were selected to compare staining between mAbl and the comparator mAb and mRNA by ISH (RNAScope. ACD). The samples represented a broad range of Nectin-4 expression, many of which were used in Example 4 to assess correspondence of IHC staining with comparator mAb 2. Scoring was conducted using a 0-5 scale (0 = negative, 1 = rare, 2 = low, 3 = moderate, 4 = high, 5 = very' high, as reviewed by a pathologist). FIG. 5A shows a histogram the number of samples for each of the IHC mAbl staining scores in TNBC, bladder cancer, and Cervical SCC.

[0258] FIG. 5B shows examples of both mAbl and comparator mAb IHC staining patterns in bladder cancer tissue samples. mAbl shows IHC staining distribution in tumors comparable to that of the comparator clone show n in FIG. 4A, but with a trend tow ard greater sensitivity7in samples with lower levels of Nectin-4 expression (see FIG. 5B).

[0259] Given the stronger staining seen with mAbl IHC in medium and low expressing tumor samples, mAbl is more sensitive than the comparator antibody.

[0260] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., GenBank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every7individual publication, database entry (e.g., GenBank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0261] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0262] The foregoing writen specification is sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A human Nectin-4 binder comprising: a) the three heavy chain complementarity determining regions (HC-CDRs) of a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 21; and b) the three light chain complementarity determining regions (LC-CDRs) of a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 22; wherein the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme.

2. The human Nectin-4 binder of claim 1, comprising HC-CDRs and LC-CDRs according to the Kabat numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprises SEQ ID NO: 2, and HC-CDR3 comprises SEQ ID NO: 3; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

3. The human Nectin-4 binder of claim 1, comprising HC-CDRs and LC-CDRs according to the Chothia numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 3; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprises the amino acid sequence of SEQ ID NO:

5. and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

4. The human Nectin-4 binder of claim 1, comprising HC-CDRs and LC-CDRs according to the AbM numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 10; andb) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

5. The human Nectin-4 binder of claim 1, comprising HC-CDRs and LC-CDRs according to the IMGT numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 11, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 12, HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 13; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprises the amino acid sequence WTS, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

6. The human Nectin-4 binder of claim 1, comprising HC-CDRs and LC-CDRs according to the Contact numbering system, wherein: a) HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 15, HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 17; and b) LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 20.

7. The human Nectin-4 binder of any one of claims 1-6, comprising a VH comprising an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions to the amino acid sequence of SEQ ID NO: 21, and a VL comprising an amino acid sequence with 1, 2, 3, 4, 5, 6, 7,8. 9, or 10 amino acid substitutions to the amino acid sequence of SEQ ID NO: 22, wherein the antibody or antigen-binding fragment thereof does not vary at the CDR amino acid sequences of any one of claims 1-6.

8. The human Nectin-4 binder of claim 7, comprising a heavy chain variable region of SEQ ID NO:

21. and a light chain variable region of SEQ ID NO: 22.

9. The human Nectin-4 binder of claim 7, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 23, and a light chain having the amino acid sequence of SEQ ID NO: 24.

10. The human Nectin-4 binder of claim 7, consisting of a heavy chain region having the amino acid sequence of SEQ ID NO: 23, and a light chain having the amino acid sequence of SEQ ID NO: 24.

11. The human Nectin-4 binder of any one of claims 1 -6, wherein the Vjq comprises a framework selected from the group consisting of human VjqE Vjq2, Vjq3, Vjq4, Vjq5, and Vj-[6, and variants thereof having 1, 2. 3, 4, 5, 6. 7, 8, 9. or 10 amino acid substitutions, additions, deletions, or combinations thereof; and, the VL comprises a framework selected from the group consisting of human VK1, VK2, VK3, VK4, VK5. VK6, Vp , V 2, V 3, V; 4, ¥^5, V 6, V 7, VyS. Vy9. and VylO, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

12. The human Nectin-4 binder of claim 11, wherein the Vj j comprises a framework selected from the group consisting of human Vjql, Vjq2. Vjq3, Vjq4, Vj-[5, and Vjq6; and the VL comprises a framework selected from the group consisting of human VK1, VK2, VK3, VK4, VK5,13. The human Nectin-4 binder of any one of claims 1-12, wherein the first residue of the VH is pyroglutamate or pyroglutamic acid.

14. The human Nectin-4 binder of any one of claims 1-12, wherein the human Nectin-4 binder comprises a human IgGl. IgG2, IgG3, or IgG4 HC constant domain or variant thereof having 1. 2, 3, 4, 5, 6, 7, 8. 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgGl, IgG2, IgG3, or IgG4 isotype constant domain.

15. The human Nectin-4 binder of claim 14, wherein the HC constant domain comprises a human IgGl constant domain comprising the amino acid sequence set forth in SEQ ID NO: 31.

16. The human Nectin-4 binder of claim 14 or claim 15, wherein the HC constant domain lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

17. The human Nectin-4 binder of claim 15 or claim 16, wherein the HC constant domain comprises one or more mutations that render the HC constant domain effector-silent.

18. The human Nectin-4 binder of claim 17, wherein the effector-silent constant domain is an IgGl domain and comprises:(a) the amino acid sequence set forth in SEQ ID NO: 28;(b) the amino acid sequence set forth in SEQ ID NO: 29;(c) the amino acid sequence set forth in SEQ ID NO: 30;(d) the amino acid sequence set forth in SEQ ID NO: 31;(e) the amino acid sequence set forth in SEQ ID NO: 32;(f) the amino acid sequence set forth in SEQ ID NO: 33;(g) the amino acid sequence set forth in SEQ ID NO: 34; or(h) the amino acid sequence set forth in SEQ ID NO: 35.

19. The human Nectin-4 binder of any one of claims 1-14, wherein the human Nectin-4 binder comprises a Fab fragment, a F(ab)2 a Fab’ fragment, a F(ab’)2 fragment, an scFv, an Fv fragment, a single-domain antibody, or a multivalent antibody.

20. A human Nectin-4 binder that cross-blocks or competes with the binding of an antibody or antigen-binding fragment thereof to human Nectin-4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 22, and a light chain variable region of SEQ ID NO: 25.

21. The human Nectin-4 binder of any one of claims 1-20 conjugated to a detectable moiety.

22. The human Nectin-4 binder of claim 21. wherein the detectable moiety is detectable by microscopic imaging, magnetic resonance imaging (MRI), or X-ray imaging.

23. The human Nectin-4 binder of any one of claims 1-20 conjugated to a therapeutic moiety.

24. An isolated nucleic acid encoding the Vn domains, the VL domains, or both the VH andVL domains of a human Nectin-4 binder of anv one of claims 1-20.

25. An expression vector comprising the isolated nucleic acid of claim 24.

26. A host cell comprising the expression vector of claim 25.

27. A method for producing a human Nectin-4 binder of any one of claims 1-20 comprising:(a) culturing the host cell of claim 26 in culture medium under conditions wherein the nucleic acid sequence is expressed, thereby producing a polypeptide comprising the light and heavy chain variable regions; and(b) recovering the polypeptides from the host cell or culture medium.

28. A method for assessing human Nectin-4 expression in a tissue sample from a human subject, comprising:(a) contacting the tissue sample with the human Nectin-4 binder of any one of claims 1-20;(b) detecting the binding of the human Nectin-4 binder to the tissue sample;(c) measuring the expression level of human Nectin-4 in the tissue sample; and(d) comparing the expression level of human Nectin-4 in the tissue sample with a reference expression level of human Nectin-4.

29. A method for assessing responsiveness of a cancer patient to an anti-cancer therapeutic agent, comprising:(a) contacting a tissue sample from the cancer patient with the human Nectin-4 binder of any one of claims 1-20;(b) detecting the binding of the human Nectin-4 binder to the tissue sample;(c) measuring the expression level of human Nectin-4 in the tissue sample; and(d) comparing the expression level of human Nectin-4 in the tissue sample with a reference expression level of human Nectin-4; wherein an increased expression level of human Nectin-4 compared to the reference expression level indicates responsiveness to the anti-cancer therapy.

30. A kit comprising the human Nectin-4 binder of any one of claims 1-20 or the isolated nucleic acid of claim 24.

31. A pharmaceutical composition comprising the human Nectin-4 binder of any one of claims 1-20 and a pharmaceutically acceptable carrier.

32. A method for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of a human Nectin-4 binder of any one of claims 1-20 or the pharmaceutical composition of claim 29.

33. A human Nectin-4 binder of any one of claims 1-20 or the pharmaceutical composition of claim 29 for use in treating cancer.

34. Use of a human Nectin-4 binder of any one of claims 1-20 or the pharmaceutical composition of claim 29 for the manufacture of a medicament for treating cancer.

Images