Anti-nectin-4 antibodies and methods of use thereof

Abstract

Provided herein are antibodies which specifically binds Nectin-4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-L1 is SEQ ID NO: 1, CDR-L2 is amino acid sequence GA, and CDR-L3 is SEQ ID NO: 3, CDR-H1, CDR-H2, CDR-H3 are SEQ ID NOs 4-6, respectively; CDR-L1 is SEQ ID NO: 68, CDR-L2 is amino acid sequence DA, and CDR-L3 is SEQ ID NO: 70, CDR-H1, CDR-H2, CDR-H3 are SEQ ID NOs 71-73, respectively; or CDR-L1 is SEQ ID NO: 74, CDR-L2 is amino acid sequence AA, and CDR-L3 is SEQ ID NO: 76, CDR-H1, CDR- H2, CDR-H3 are SEQ ID NOs 77-79, respectively, and methods of use thereof.

Description

TITLE: ANTI-NECTIN-4 ANTIBODIES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 536,823, filed September 6, 2023, the contents of which is incorporated herein by reference in its entirety. INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “30344- P72848PC00_Sequence_Listing” (113,700 bytes) created on September 5, 2024, is herein incorporated by reference. FIELD

[0003] The present disclosure relates to the development of antibodies to Nectin-4 and the use of these antibodies for radio-imaging and radiotherapy for cancers that are Nectin-4 expressing, such as triple negative breast cancer. BACKGROUND

[0004] Breast cancer (BC) is the most common cancer in women, with 1 in 8 women likely to be diagnosed with the disease in their lifetime. Triple negative breast cancer (TNBC) represents 10% – 20% of invasive BC and has been associated with African-American race, deprivation status, younger age at diagnosis, more advanced disease stage at diagnosis, higher grade, high mitotic indices, family history of breast cancer and BRCA1 mutations, and is aggressive with poor prognosis [1]. Taxanes and anthracyclines alone, or in combination constitute the first- line of treatment for metastatic BC with response rates of 38% and 46% for single agent and combination-based therapies, respectively [2]. However, de novo resistance to taxanes is common in over 50% of patients and most patients acquire resistance [2]. Sacituzumab govitecan (SG) is the only antibody-based therapeutics recently approved for the treatment of TNBC. Sacituzumab govitecan (SG) is an anti-TROP2 antibody drug conjugate (ADC) in which the anti-TROP2 antibody is conjugated to cytotoxic drug SN38, and is the only antibody-based therapeutic approved for TNBC, and shows significantly improved benefits compared with chemotherapy[3]. However, objective response rate (ORR) was just 33% for SG (complete response 4%, partial response 27%) compared with 4% ORR for chemotherapy[3]. Despite this promising outcome, novel approaches are required. The poliovirus receptor-related protein 4 (PVRL4 / nectin-4) is uniquely overexpressed in most cancers of epithelial origin (includinggastric, breast, lung, pancreatic ovarian, bladder and esophageal) but not in non- malignant or healthy tissues [4, 5]. Since its identification less than two decades ago, nectin-4 has become a highly sought-after target in precision oncology. Nectin-4 is a member of the nectin family of immunoglobulin-like adhesion molecules that are proposed to mediate Ca2+-independent cell–cell adhesion via both homophilic and heterophilic trans-interactions at adherens junctions where they recruit cadherins and modulate cytoskeleton rearrangements [6]. Notably, large clinical studies have consistently confirmed 60 – 70% of all TNBC overexpress nectin-4 [5, 7, 8]. Immunohistochemical analysis of a panel of 294 healthy tissue specimens from 36 human (healthy) organs showed very weak homogenous staining mainly in human skin keratinocytes, skin appendages (sweat glands and hair follicles), transitional epithelium of bladder, salivary gland (ducts), esophagus, breast, and stomach, with no staining from other tissues [8].

[0005] Enfortumab vedotin (PADCEV^) is an anti-nectin-4 antibody drug conjugate (ADC) in which the antibody is conjugated to monomethyl auristatin (MMAE) via a cleavable linker is approved for the treatment of nectin-4 positive advanced urothelial cancers

[0015] . In mice bearing nectin-4 positive TNBC xenografts treated with this ADC, tumors re-grew after periods of no- treatment.

[0006] Given that current standard of care is ineffective, novel targeted therapies are much needed for TNBC treatment. Targeted therapies for TNBC and other Nectin-4 expressing cancers are desirable. SUMMARY

[0007] Provided herein are fully human monoclonal anti-Nectin-4 antibodies that can be used for PET imaging or targeted radioimmunotherapy of Nectin-4 expressing cancers.

[0008] Nectin-4 (PVRL4) is a tumor biomarker that is overexpressed in ~62% of triple-negative breast cancer (TNBC) and basal subtypes. TNBC is the most aggressive subtype that accounts for ~20% of breast cancers and is associated with poor prognosis. The overexpression of nectin- 4 in TNBC and its lack of expression in normal adult tissues make it a target for both therapy and non-invasive PET / CT imaging of TNBC using a targeted antibody. PVRL4 / Nectin-4 is uniquely overexpressed in most cancers of epithelial origin (including gastric, breast, lung, pancreatic, ovarian, bladder and esophageal) but not in non-malignant tissues.

[0009] The inventors have developed a fully human anti-nectin-4 (anti-N4) antibody using phage display and used it to develop anti-N4 theranostics. Anti-N4 diagnostic agents were developed by conjugating the antibody with deferoxamine (using p-SCN-Bn-DFO) and radiolabeling withzirconium-89 (89Zr) to obtain a PET imaging agent. Anti-N4 radiotherapeutics were developed by conjugating the antibody with 18-membered ring macrocyclic chelator macropa (using p-SCN- macropa) followed by radiolabeling with actinium-225 (225Ac) for alpha particle therapy. The theranostic agents were evaluated in TNBC models with different levels of nectin-4 expression.

[0010] The89Zr-anti-N4 radioimmunoconjugate (RIC) was an effective PET / CT imaging probe with specific uptake in tumor. The225Ac- anti-N4 RIC was effective as a therapeutic agent resulting in complete remission of tumors in mouse xenograft models.

[0011] Described herein includes the development and evaluation of a fully human anti-nectin-4 antibody for the non- invasive PET imaging and the targeted RIT of TNBC. Also described herein is the development of a225Ac-labeled anti-nectin-4 α-particle therapeutic agent. The anti-N4 antibody is fully human, and therefore is less immunogenic compared with murine based antibodies which is beneficial for translation into effective pharmaceuticals. The antibodies described herein show for example targeting specificity and high tumor uptake of89Zr-anti-N4 antibody.

[0012] Also herein, the immune inducing effects of high linear energy transfer (LET) alpha (α) particle-labeled [225Ac]Ac-Nectin-4 have been compared with beta-emitter (β-) / Auger electron- labeled antibody [161Tb]Tb-N4MU01 against Nectin-4-positive TNBC and NSCLC. The effectiveness of these radioimmunotherapeutics (RIT) in vitro, and in vivo in patient-derived xenograft (PDX) mouse models has been evaluated.

[0013] An aspect of the disclosure includes an antibody which specifically binds Nectin-4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs, as defined using IMGT numbering, are: a) CDR-L1 QSV X1 X2 X3Y (SEQ ID NO: 1); CDR-L2 GA; CDR-L3 QQR X4 X5 X6PPT (SEQ ID NO: 3); CDR-H1 GF X7F X8SYG (SEQ ID NO: 4); CDR-H2IWYDG X9NK(SEQ ID NO: 5); andCDR-H3AR D X10W X11X12G X13Y(SEQ ID NO: 6)wherein, X1is an amino acid selected from S, K, F, and R, X2is an amino acid selected from N, H, A, T, S, and M, X3is an amino acid selected from S, G, and R, X4is an amino acid selected from S, Y, W, and T, X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6is an amino acid selected from W, F, and Y, X7is an amino acid selected from T, A, and N, X8is an amino acid selected from R, M, K, and G, X9 is an amino acid selected from S and T, X10 is an amino acid selected from D and G, X11 is an amino acid selected from T, D, N, A, S, and R, X12 is an amino acid selected from N, K, Y, H, R, and D, and / or X13 is an amino acid selected from W, F, and Y; b) CDR-L1 QSVSRY (SEQ ID NO: 68); CDR-L2 DA; CDR-L3 QQRYNWPPD (SEQ ID NO: 70); CDR-H1 GFTFSSYG (SEQ ID NO: 71); CDR-H2 ISYDGSNK (SEQ ID NO: 72); and CDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73); or c) CDR-L1 QDISNY (SEQ ID NO: 74); CDR-L2 AA; CDR-L3 QQSYTTRTT (SEQ ID NO: 76); CDR-H1 GFRFSGYP (SEQ ID NO: 77);CDR-H2IWYDGRNR(SEQ ID NO: 78); and CDR-H3AKEGKWGEWYFDL(SEQ ID NO: 79).

[0014] Another aspect of the disclosure includes a nucleic acid molecule encoding any antibody described herein.

[0015] Another aspect of the disclosure includes a vector comprising any nucleic acid molecule described herein.

[0016] Another aspect of the disclosure includes a cell comprising any nucleic acid molecule described herein, any vector described herein, or expressing any antibody described herein.

[0017] Another aspect of the disclosure includes an immunoconjugate comprising any antibody described herein and a therapeutic agent and / or detectable label.

[0018] Another aspect of the disclosure includes a composition comprising any antibody described herein, any nucleic acid molecule described herein, any vector described herein, any cell described herein, or any immunoconjugate described herein, and a diluent or pharmaceutically acceptable carrier.

[0019] Another aspect of the disclosure includes any antibody described herein, any immunoconjugate described herein, or any composition described herein, for use in treating a cancer.

[0020] Another aspect of the disclosure includes an antibody described herein, an immunoconjugate described herein, or a composition described herein, for use in the manufacture of a medicament for treating a cancer. The antibodies can be used for treating cancers that are Nectin-4 expressing, for example cancers that are positive for Nectin-4 by imaging, in situ hybridization or ELISA.

[0021] Another aspect of the disclosure includes use of any antibody described herein, any immunoconjugate described herein, or any composition described herein, for treating cancer. The antibodies can be used for treating cancers that are Nectin-4 expressing.

[0022] Another aspect of the disclosure includes a method for detecting Nectin-4 expression in a biological sample, the method comprising a) obtaining a biological sample suspected of containing Nectin-4, b) contacting the sample with any antibody or immunoconjugate described herein under conditions permissive for forming an antibody:Netctin-4 complex, and c) detecting the presence of any antibody: Nectin-4 complex, wherein the detection of antibody: Nectin-4complex is indicative that the sample expresses or contains Nectin-4. The biological sample may comprise or be a cancer biopsy or comprise cancer cells.

[0023] Another aspect of the disclosure includes a method of detecting whether a subject has cancer, the method comprising obtaining a biological sample suspected of containing a cancer cell, the biological sample having been obtained from the subject, contacting the sample with any antibody or immunoconjugate described herein under conditions permissive for forming an antibody:Nectin-4 complex, and detecting the presence of any antibody:Nectin-4 complex, wherein the presence of antibody: Nectin-4 complex indicates that the subject has or has an increased likelihood of having cancer

[0024] Another aspect of the disclosure includes a method of determining if a subject has a Nectin-4-expressing cancer, the method comprising administering an immunoconjugate comprising a detectable label described herein, or any composition comprising the immunoconjugate described herein to the subject, and detecting the presence of the label by imaging, optionally the subject has or is suspected of having a cancer, optionally cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer.

[0025] Another aspect of the disclosure includes a method for imaging a Nectin-4-expressing tumor in a subject, the method comprising administering any immunoconjugate comprising a detectable label or composition thereof described herein to the subject, and detecting the presence of the label.

[0026] Another aspect of the disclosure includes a method of treating a cancer, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, and lung cancer, in a subject in need thereof, the method comprising administering an effective amount of an antibody, immunoconjugate comprising an antibody described herein and a therapeutic agent, or composition described herein to the subject.

[0027] Another aspect includes a method of treating cancer in a subject in need thereof, the method comprising administering an immunoconjugate comprising comprising an antibody described herein and a therapeutic agent, or a composition comprising the immunoconjugate to the subject, wherein the subject has been previously determined to have a Nectin-4 expressingcancer. The subject can for example have been determined to have a Nectin-4 expressing cancer by imaging, or by testing a biopsy of the cancer.

[0028] A further aspect includes a method of treating cancer in a subject in need thereof, the method comprising I) a) administering an immunoconjugate comprising a detectable label, or a composition comprising the immunoconjugate; or b) testing a sample of the cancer for Nectin-4, optionally using an antibody described herein; and II) administering an immunoconjugate comprising an antibody described herein and a therapeutic agent or a composition comprising the immunoconjugate to the subject, when the subject is determined in I) to have a Nectin-4 expressing cancer.

[0029] Another aspect of the disclosure includes use of an antibody to receive information about whether or not the patient is suitable for receiving treatment with an antibody, immunoconjugate comprising an antibody described herein and a therapeutic agent, or a composition described herein.

[0030] Another aspect of the disclosure includes use of an antibody described herein, immunoconjugate comprising the antibody and therapeutic agent described herein, or a composition comprising the antibody or immunoconjugate and a pharmaceutically acceptable carrier for treating cancer in a subject in need thereof, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, and lung cancer, in a subject in need thereof. BRIEF DESCRIPTION OF DRAWINGS

[0031] An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0032] Figs.1A-D: In vitro flow cytometry binding assay results of anti-nectin4 (anti-N4) antibody binding and internalization using live-cell imaging. Different concentration of anti-N4 were incubated with A) MDA-MB-468 B) MCF-7 cells and C) MDA-MB-231 cells. Dose dependent binding was observed in nectin-4 expressing cell lines, MDA-MB-468 and MCF7. No binding was observed in MDA-MB-231 control cell line. D) Internalization of anti-N4 antibody: MDA-MB-468, MCF-7 and MDA-MB-231 cells were treated with IncuCyte FabFluor- labeled N4 antibody (4μg / mL); HD phase and red fluorescence images (10×) were captured every 2 h for 48 h. All data are shown as a mean of three wells ± SEM.

[0033] Figs.2A-E: Saturation binding assay by flow cytometry. Nectin-4 positive MCF-7 cells were titrated with decreasing concentrations of the naked anti-N4 antibody (A and B), and DFO-anti-N4 antibody (C and D) toallow for the estimation of KDand EC50values. Mean fluorescence intensity (MFI) were plotted against concentration. A non-linear curve fitting was used to estimatethe KDvalues. E) Saturation radioligand bindingassay with different concentrations of89Zr -anti-N4 radioimmunoconjugate (RIC) was performed to evaluate the binding in nectin-4 positive MCF- 7 cells.

[0034] Fig.3: Pharmacokinetics of89Zr-anti-N4 antibody in normal CD-1 nude mice.89Zr-anti-N4 antibody showed a bi-phasic half-life with a fast distribution and slow elimination.

[0035] Figs. 4A-D: PET / CT imaging: (A) Maximum intensity projection (MIP) microPET / CT images of a representative CD-1 nude mouse bearing nectin-4-positive MDA-MB-468 xenograft at different time points post89Zr-anti-N4 antibody injection. (B) MIP images of an athymic nude mouse bearing medium expression BT-474 and MCF-7 xenografts located in the same mouse on the right and left flanks, respectively. (C) MIP microPET / CT images at different time points of a representative CD-1 nude mouse bearing nectin-4 positive MDA-MB-468 xenograft on the right flank, blocked with 900 µg unlabeled anti-N4 antibody prior to administration of89Zr-anti-N4 injection. Significantly higher tumor uptake was seen in nectin-4 positive MDA-MB-468, BT-474 and MCF-7 xenografts compared with negative control blocked xenograft. (D) MicroPET images of an immunocompetent mouse bearing nectin-4 positive 4T1-nectin-4 xenograft at 24 and 120 h post injection

[0036] Figs.5A-B: Biodistribution of89Zr-labeled anti-nectin-4 antibody (89Zr-anti-N4 antibody) in nectin-4 expressing MDA-MB-468 (high receptor density), and BT-474 and MCF-7 (medium receptor density) xenografts at 24 and 120 h post injection. (A) female CD-1 nude mice bearing MDA-MB-468 xenografts were injected intravenously with 5 - 6 MBq (10-12 μg) of89Zr-anti-N4 antibody followed by biodistribution studies at 24 and 120 h. In addition, female CD-1 nude mice bearing N4 positive MDA-MB-468 xenografts were injected intravenously with 900 μg of cold anti- N4 antibody 4 h prior to the injection of 5 - 6 MBq (10 - 12 μg) of89Zr-anti-N4 antibody followed by biodistribution studies at 120 h. Tumor uptake in MDA-MB-468 xenograft was significantly higher than in the blocked (negative control) MDA-MB-468 xenograft at 120 h (***p < 0.0001) post injection. (B) female athymic nude mice bearing BT-474 and MCF-7 xenografts were injectedintravenously with 5 – 6 MBq (10 – 12 μg) of89Zr-anti-N4 antibody followed by biodistribution studies at 120 h.

[0037] Figs.6A-E: Efficacy of225Ac-labeled anti-nectin-4 (N4) radioimmunoconjugate. (A) Tumor growth curve of 350 nCi225Ac- anti-N4 antibody, 500 nCi225Ac- anti-N4 antibody, 350 nCi225Ac- rituximab, PBS control and unlabeled anti-N4 antibody treated mice bearing MDA-MB-468 xenograft represented as normalized tumor volume versus days post treatment (B) Kaplan-Meier survival curve of mice bearing MDA-MB-468 xenograft. (C) Body weights of mice (± SEM) in the different study groups. Study endpoint was when xenograft reached ≥400 mm3. (D) Tumor volumes of mice bearing nectin-4 positive 4T1-nectin-4 xenograft after treatment using two doses of 350 nCi225Ac- anti-N4 antibody or vehicle (saline). Mice reached end point after tumors exceeded 1500mm3. (E) Corresponding Kaplan Meier survival curves of mice with 4T1-nectin-4 xenograft.

[0038] Figs.7A-B: Phage ELISA showing specific binding of selected clones to Nectin-4. A) Light chain variants B) Heavy chain variants.

[0039] Figs.8A-C: The alignment of selected antibodies with respective germline V genes and J genes

[0040] Fig.9: Phage ELISA of N4-Fab1 showing specific binding to Nectin-4-Fc fusion protein.

[0041] Figs.10A-C: Bioanalyzer ladder (A) and chromatograms (B) of ladder, unconjugated anti- N4 antibody, DFO-anti-N4 antibody and Macropa-anti-N4 antibody. (C) Representative size 89 exclusion (SEC) HPLC chromatograms showing stability of DFO-anti-N4 antibody and Zr-DFO- anti-N4 antibody. UV channel (280) and radiometric channels are shown.

[0042] Figs.11A-D: Saturation binding assay by flow cytometry. Nectin4 expressing MDA-MB- 468 cells were titrated with decreasing concentrations of (A &B) unconjugated anti-N4 and (C & D) macropa conjugated anti-N4 antibody to allow for the estimation of Kd and EC50 values. Mean fluorescence intensity (MFI) were plotted against concentration. A non-linear curve fitting was used to estimate the Kd values.

[0043] Fig.12: Stability studies of 89Zr-DFO-anti-N4 antibody at room in human plasma and PBS at 37°C.

[0044] Figs.13A-13B are graphs depicting efficacy of [225Ac]Ac-Macropa-N4MU01monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model. A) Average tumor growth curves. B) Kaplan-Meier survival curves.

[0045] Fig.14A-B are graphs depicting efficacy of [225Ac]AC-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H358 in an athymic BALB / c mouse xenograft model. A) Average tumor growth curves. B) Kaplan Meier survival curve of treatment groups shows Log-rank (mantel- Cox) test for survival comparison.

[0046] Figs.15A-B are graphs depicting efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H2170 in an athymic BALB / c mouse xenograft model. A) Average tumor growth curves. B) Average body weights.

[0047] Figs.16A-C are graphs depicting efficacy of [161Tb]Tb-DOTA-N4MU01 monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model. A) Average tumor growth curves with a t-test performed on d13 following start of treatment. B) Kaplan Meier survival curve of treatment groups shows Log-rank (mantel-Cox) test for survival comparison. C) Average body weight of treatment groups over time.

[0048] Figs.17A-17B are graphs that show the efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy in 4T1.nectin-4 syngeneic BALB / c mouse model (n = 5-6 mice / group) in an exemplary embodiment of the disclosure. Each treatment group received two doses of the treatment on d0 and on d10. FIG.17A shows average tumor growth curves that show complete remission for all mice in [225Ac]Ac-Macropa-N4MU01 treatment group. FIG.17B shows a Kaplan- Meier survival curve. Study endpoint was considered as tumor volume ≥1500 mm3 or survival for 30 days. DETAILED DESCRIPTION Definitions

[0049] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0050] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term "a cell" includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized inconnection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York.).

[0051] Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0052] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds.

[0053] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0054] As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0055] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, (such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0056] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0057] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0058] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of' or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0059] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0060] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0061] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of antibodies that to contribute to epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, Martin (AbM), AHo and IMGT. The CDRs listed in the present disclosure are identified using the Kabat definition or the IMGT definition, as indicated. A person skilled in the art having regard to the sequences comprised herein would also be able to identify CDR sequences based on IMGT, Kabat, and Chothia etc (e.g., see Giudicelli et al., 2022, Swindells et al., 2017). Such antibodies are similarly encompassed.

[0062] The term “framework region” or “FR” as used herein refers to regions interspersed before and between CDRs. For example, the light chain or the heavy chain can comprise FR1 N-terminal to CDR1, FR2 between CDR1 and CDR2, FR3 between CDR2 and CDR3, and FR4 C-terminal to CDR3.

[0063] The term "antibody" as used herein is intended to encompass for example monoclonal antibodies, polyclonal antibodies, fully human antibodies, humanized and other chimeric antibodies, and binding fragments thereof, including for example a single chain Fab fragment, Fab’2 fragment, or single chain Fv fragment. The antibody may be from recombinant sources and / or produced in transgenic animals. Also included are human antibodies that can be produced in transgenic animals or using biochemical techniques, or can be isolated from a library such as a phage display library. Antibody backbones may comprise any suitable variable heavy chain or variable light chain sequences. Antibodies, including humanized and / or other chimeric antibodies may include sequences from one or more than one isotype, class, or species. Antibodies may be any class of immunoglobulins including: IgG, IgM, IgD, IgA, or IgE; and any isotype thereof, including IgG1, IgG2 (e.g. IgG2a, IgG2b), IgG3 and IgG4. Further, these antibodies can be produced as antigen binding fragments such as Fab, Fab' F(ab')2, Fd, Fv and single domain antibody fragments, or as single chain antibodies in which the heavy and light chains are linked by a spacer.

[0064] The term "binding fragment" as used herein refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain and which binds the antigen or competes with intact antibody. Exemplary binding fragments include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, and diabodies, and includes multimers thereof. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. For example, F(ab')2 fragments can be generated by treatingthe antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be constructed by recombinant expression techniques.

[0065] The term “cell” as used herein refers to a single cell or a plurality of cells.

[0066] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.

[0067] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to any chain of two or more natural or unnatural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). Included are proteins that are a single polypeptide chain and multisubunit proteins (e.g., composed of 2 or more polypeptides).

[0068] The term "sequence identity" as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = [number of identical overlapping positions] / [total number of positions] X 100%). The determination of percent identity between two sequences can also be accomplished using amathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0069] For antibodies, percentage sequence identities can be determined when antibody sequences are maximally aligned by Kabat, IMGT, or other numbering conventions. The terms “Kabat numbering”, ”IMGT numbering”, etc., which are recognized in the art, refer to systems of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen binding portion thereof. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. Accordingly, Kabat, IMGT, and other alignment systems can also be used to identify or annotate CDRs in an antibody sequence.

[0070] The term "nucleic acid” or “nucleic acid molecule", as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. Unless otherwise indicated, standard IUPAC-IUB nomenclature is used herein. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid molecule" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term "isolated nucleic acid molecules" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0071] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, Adeno Associated viral vectors and the like.

[0072] The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.

[0073] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, inhibiting spread or progression of disease, delay or slowing of disease progression or onset, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0074] As used herein, the term “cancer” refers to “Nectin-4-expressing cancers” also referred to as “Nectin-4 positive cancers”. Cancer refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. Cancer can include solid tumors (e.g., cancer cells can form a solid tumor, in which thecancer cells are massed together), or cancer cells which exist as dispersed cells, such as in blood cancers. “Nectin-4-expressing cancers” also referred to as “Nectin-4 positive cancers”, exhibit increased expression of nectin-4 in cancer cells or tissue as compared to corresponding and / or surrounding normal cells or tissue, such as cancers of epithelial origin (e.g., breast cancer, triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, lung cancer, bladder cancer, for example urothelial cancer, gastric cancer among others). For example, a Nectin-4- expressing cancer comprises cancer cells that have a level of nectin-4 expression that is detectable by immunohistochemistry, e.g., as compared to normal cells. For example, a Nectin- 4-expressing cancer may comprise cancer cells that have a level of nectin-4 expression that is 2X-3X higher as compared to corresponding normal cells. Normal cells typically show undetectable or background levels of nectin-4 detectable by immunohistochemistry.

[0075] The term “cancer cell” refers to a Nectin-4-expressing cancer cell, e.g., a cell characterized by uncontrolled, abnormal growth and the ability to invade another tissue or a cell derived from such a cell, which exhibits increased expression of nectin-4 as compared to corresponding and / or surrounding normal cells or tissue. Cancer cells include, for example, a primary cancer cell obtained from a patient with cancer or cell line derived from such a cell. For example, a Nectin-4- expressing cancer cell has a level of nectin-4 expression that is detectable by immunohistochemistry, e.g., as compared to a normal cell which may not have a level of nectin- 4 detectable by immunohistochemistry.

[0076] The term "administered" as used herein means administration of a therapeutically effective dose of an antibody, immunoconjugate, or composition of the disclosure to a cell or subject.

[0077] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example in the context of treating cancer, an effective amount is an amount that for example induces remission, reduces tumor burden, and / or prevents tumor spread or growth of cancer cells compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given antibody or immunoconjugate, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0078] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Optionally, the term “subject” includes mammals that have been diagnosed with cancer, such as breast cancer, ovarian cancer, triple negative breast cancer, lung cancer, gastric cancer, bladder cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, esophageal cancer or pancreatic cancer, or are in remission. In one embodiment, the term “subject” refers to a human having, or suspected of having, cancer such as breast cancer, ovarian cancer, triple negative breast cancer, lung cancer, gastric cancer, bladder cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, esophageal cancer or pancreatic cancer.

[0079] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0080] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about".

[0081] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0082] An aspect of the disclosure includes an antibody which specifically binds Nectin-4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs, as defined using IMGT numbering, are: a) CDR-L1 QSV X1X2X3Y (SEQ ID NO: 1);CDR-L2 GA CDR-L3 QQR X4 X5 X6PPT (SEQ ID NO: 3); CDR-H1 GF X7F X8SYG (SEQ ID NO: 4); CDR-H2 IWYDG X9NK (SEQ ID NO: 5); and CDR-H3AR D X10W X11X12G X13Y(SEQ ID NO: 6)wherein, X1is an amino acid selected from S, K, F, and R, X2is an amino acid selected from N, H, A, T, S, and M, X3is an amino acid selected from S, G, and R, X4is an amino acid selected from S, Y, W, and T, X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6is an amino acid selected from W, F, and Y, X7is an amino acid selected from T, A, and N, X8is an amino acid selected from R, M, K, and G, X9is an amino acid selected from S and T, X10is an amino acid selected from D and G; X11is an amino acid selected from T, D, N, A, S, and R; X12is an amino acid selected from N, K, Y, H, R, and D; and / or X13. is an amino acid selected from W, F, and Y; b) CDR-L1 QSVSRY (SEQ ID NO: 68); CDR-L2 DA; CDR-L3 QQRYNWPPD (SEQ ID NO: 70); CDR-H1 GFTFSSYG (SEQ ID NO: 71); CDR-H2 ISYDGSNK (SEQ ID NO: 72); and CDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73); or c)CDR-L1 QDISNY (SEQ ID NO: 74); CDR-L2 AA; CDR-L3 QQSYTTRTT (SEQ ID NO: 76); CDR-H1GFRFSGYP(SEQ ID NO: 77); CDR-H2 IWYDGRNR (SEQ ID NO: 78); and CDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).

[0083] In some embodiments, the CDRs, as defined using IMGT, are CDR-L1 QSV X1X2X3Y (SEQ ID NO: 1); CDR-L2 GA CDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3); CDR-H1 GF X7F X8SYG (SEQ ID NO: 4); CDR-H2 IWYDG X9NK (SEQ ID NO: 5); and CDR-H3 AR D X10W X11X12G X13Y (SEQ ID NO: 6) wherein, X1 is an amino acid selected from S, K, F, and R, X2 is an amino acid selected from N, H, A, T, S, and M, X3 is an amino acid selected from S, G, and R, X4 is an amino acid selected from S, Y, W, and T, X5 is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6 is an amino acid selected from W, F, and Y, X7 is an amino acid selected from T, A, and N, X8 is an amino acid selected from R, M, K, and G, X9 is an amino acid selected from S and T, X10 is an amino acid selected from D and G; X11 is an amino acid selected from T, D, N, A, S, and R; X12 is an amino acid selected from N, K, Y, H, R, and D; and / or X13. is an amino acid selected from W, F, and Y.

[0084] In some embodiments, X1is S. In some embodiments, X1is K. In some embodiments, X1is F. In some embodiments, X1is R. In some embodiments, X2is N. In some embodiments, X2is H. In some embodiments, X2is A. In some embodiments, X2is T. In some embodiments, X2is S. In some embodiments, X2is M.

[0085] In some embodiments, X3is S. In some embodiments, X3is G. In some embodiments, X3is R. In some embodiments, X4is S. In some embodiments, X4is Y. In some embodiments, X4is W. In some embodiments, X4is T.

[0086] In some embodiments, X5is N. In some embodiments, X5is G. In some embodiments, X5is D. In some embodiments, X5is S. In some embodiments, X5is Y. In some embodiments, X5is T. In some embodiments, X5 is H. In some embodiments, X5 is F. In some embodiments, X5 is E.

[0087] In some embodiments, X6 is W. In some embodiments, X6 is F. In some embodiments, X6 is Y. In some embodiments, X7 is T. In some embodiments, X7 is A. In some embodiments, X7 is N. In some embodiments, X8 is R. In some embodiments, X8 is M. In some embodiments, X8 is K. In some embodiments, X8 is K. In some embodiments, X8 is G. In some embodiments, X9 is S. In some embodiments, X9 is T.

[0088] In some embodiments, X10 is D. In some embodiments, X10 is G. In some embodiments, X11 is T. In some embodiments, X11 is D. In some embodiments, X11 is N. In some embodiments, X11 is A. In some embodiments, X11 is S. In some embodiments, X11 is R. In some embodiments, X12 is N. In some embodiments, X12 is K. In some embodiments, X12 is Y. In some embodiments, X12 is H. In some embodiments, X12 is R. In some embodiments, X12 is D. In some embodiments, X13 is W. In some embodiments, X13 is F. In some embodiments, X13 is Y.

[0089] In some embodiments, X1 is an amino acid selected from S, K, F, and R. In some embodiments, X2 is an amino acid selected from N, H, A, T, S, and M. In some embodiments, X3 is an amino acid selected from S, G, and R. In some embodiments, X4 is an amino acid selected from S, Y, W, and T. In some embodiments, X5 is an amino acid selected from N, G, D, S, Y, T, H, F, and E. In some embodiments, X6 is an amino acid selected from W, F, and Y. In some embodiments, X7 is an amino acid selected from T, A, and N. In some embodiments, X8 is an amino acid selected from R, M, K, and G. In some embodiments, X9 is an amino acid selected from S and T. In some embodiments, X10 is selected from D and G. In some embodiments, X11 is an amino acid selected from T, D, N, A, S, and R. In some embodiments, X12 is an amino acid selected from N, K, Y, H, R, and D. In some embodiments, X13is an amino acid selected from W, F, and Y. In some embodiments, each are individually selected.

[0090] In some embodiments, the CDRs are those identified in Table 1. In some embodiments, position L53 as defined by Kabat is R, V, K, G, S, T or M (CDR-L2). Position L53 as defined by Kabat is position L67 as defined by IMGT. In some embodiments, position H58 as defined by Kabat is Y, L or F (CDR-H2).

[0091] In some embodiments, the CDRs, as defined using IMGT, are: CDR-L1 QSVSNSY (SEQ ID NO: 80); CDR-L2 GA ; CDR-L3 QQRSNWPPT (SEQ ID NO: 81); CDR-H1 GFTFRSYG (SEQ ID NO: 82); CDR-H2 IWYDGSNK (SEQ ID NO: 83); and CDR-H3 ARDDWTNGWY (SEQ ID NO: 84).

[0092] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 7-35 or 62; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 7-35 or 62, wherein the CDR-L1 is or comprises SEQ ID NO: 1, CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 1, CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3.

[0093] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 36-61 or 63; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 36-61 or 63, wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6.

[0094] In some embodiments, the CDRs, as defined using IMGT, are: CDR-L1 QSVSRY (SEQ ID NO: 68); CDR-L2 DA CDR-L3 QQRYNWPPD (SEQ ID NO: 70); CDR-H1 GFTFSSYG (SEQ ID NO: 71);CDR-H2ISYDGSNK(SEQ ID NO: 72); and CDR-H3AKSTLHSSGWYMDY(SEQ ID NO: 73).

[0095] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 64; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 64, wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO: 70; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO: 70.

[0096] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 65; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 65, wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73.

[0097] In some embodiments, the CDRs, as defined using IMGT, are: CDR-L1 QDISNY (SEQ ID NO: 74); CDR-L2 AA CDR-L3 QQSYTTRTT (SEQ ID NO: 76); CDR-H1 GFRFSGYP (SEQ ID NO: 77); CDR-H2 IWYDGRNR (SEQ ID NO: 78); and CDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).

[0098] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 66; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 66, wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO: 76; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO: 76.

[0099] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 67; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID Ns: 67, wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79.

[0100] In some embodiments, the antibody is a biphasic antibody (e.g., having a biphasic pharmacokinetic profile). In some embodiments, the biphasic antibody comprises an initial phase (e.g., a distribution phase) and a second phase (e.g., an elimination phase). In some embodiments, the initial phase (e.g., distribution phase) of the biphasic antibody comprises between about 3 and 5 hours. In some embodiments, the initial phase (e.g., distribution phase) of the biphasic antibody comprises about 3, 4, or 5 hours. In some embodiments, the initial phase (e.g., distribution phase) of the biphasic antibody comprises about 3 hours. In some embodiments, the second phase (e.g., elimination phase) of the biphasic antibody comprises between about 50 to 70 hours. In some embodiments, the second phase (e.g., elimination phase) of the biphasic antibody comprises about 50, 60, or 70 hours. In some embodiments, the second phase (e.g., elimination phase) of the biphasic antibody comprises about 60 hours. In some embodiments, the second phase (e.g., elimination phase) of the biphasic antibody comprises about 60—65 or about 63 hours.

[0101] In some embodiments, the antibody is a humanized or human antibody.

[0102] In some embodiments, the antibody is a single chain antibody.

[0103] In some embodiments, the antibody is an antibody fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, and diabodies,. In some embodiments, the antibody is a multimer.

[0104] Antibodies may be any class of immunoglobulins including: IgG, IgM, IgD, IgA, or IgE; and any isotype thereof, including IgG1, IgG2 (e.g. IgG2a, IgG2b), IgG3 and IgG4. In some embodiments, the antibody is an IgG, optionally IgG1.

[0105] In an embodiment, the antibody or immunoconjugate has a binding affinity of at least 3nM. Assays for determining binding affinity are well known in the art and binding affinity can be for example determined using the methods described in the Examples section.

[0106] Another aspect of the disclosure includes a nucleic acid molecule encoding any antibody described herein.

[0107] Another aspect of the disclosure includes a vector comprising any nucleic acid molecule described herein.

[0108] Another aspect of the disclosure includes a cell comprising any nucleic acid molecule described herein, any vector described herein, or expressing any antibody described herein.

[0109] Another aspect of the disclosure includes an immunoconjugate comprising any antibody described herein and a therapeutic agent and / or detectable label.

[0110] In some embodiments, the detectable label and / or therapeutic agent is a radionuclide, optionally an alpha- beta- or gamma-emitting radionuclide. In some embodiments, the detectable label comprises a sub-therapeutic dose of a radionuclide, optionally an alpha- beta- or gamma-emitting radionuclide. In some embodiments, the antibody is labeled with the radionuclide using a bifunctional chelator, optionally desferrioxamine (DFO), NOTA, DOTA, NODAGA, TETA, 3p-C-NETA, HYNIC, DTPA, CB-TE2A, H2dedpa, H4octapa, NODAGA, HBED- CC, 3p-C-DEPA, HOPO. In some embodiments, the chelator is 18-membered macrocyclic bifunctional chelator 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16- diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid (macropa-SCN). In some embodiments, the chelator-antibody ratio (CAR) is 4:1 to 1:1, preferably 3:1 to1:1. . In some embodiments, the CAR is 1:1. In some embodiments, the CAR is 3:1. In some embodiments, the CAR is 2:1. In some embodiments, the CAR is 4:1. In some embodiments, the chelator is DFO. In some embodiments, the chelator is DFO and the CAR is 3:1. In some embodiments, the chelator is macropa-SCN . In some embodiments, the chelator is macropa-SCN and the CAR is 1:1.In some embodiments, the radionuclide is selected from Fluorine-18 (half-life 109.8 min), iodine-131 (8half-life 8 days), idodine-123 (half-life 13.2 hours), copper-67 (half-life 61.8 days), indium-111 (half-life 2.8 days), thorium-227 (half-life 18.7 days), astatine-211 (half-life 7.2 hours), bismutt-212 (half-life 45 minutes), lead-212 (half-life 10.6 hours), radium-223 (half-life 11.4 days), rhenium-188 (half-life 16.9 hours), technecium-99m (half-life 6.6 hours), yttrium-90 (half-life 64.6 hours), leutetium-177 (half-life 6.8 days), copper-61 (half-life 3.3 hours), copper-64 (half-life 12.7h), In-111 (half-life 2.8 days), terbium-152 (half-life 17.5 hours), terbium-156 (half-life 5.3 days), terbium-161 (half-life 6.8 days), terbium-149 (half-life 4.1 hours), iodine-124, gallium-68 (half-life 68 min), gallium-67 (half-life 78.3 hours), actinium-225 (half-life 10 days) and zirconium- 89 (half-life 78.4 h).

[0111] In some embodiments, the radionuclide is225Actinium. In some embodiments, the radionuclide is89Zirconium. In some embodiments, the radionuclide is161Terbium.

[0112] In some embodiments, the chelator is DFO or macropa-SCN . In some embodiments, the chelator is DFO. In some embodiments, the chelator is macropa-SCN.

[0113] Another aspect of the disclosure includes a composition comprising any antibody described herein, any nucleic acid molecule described herein, any vector described herein, any cell described herein, or any immunoconjugate described herein, and a diluent or pharmaceutically acceptable carrier. Mixtures of antibodies, mixtures of nucleic acids and / or vectors, mixtures of cells or mixtures of immunoconjugates as well as mixtures of antibodies and immunoconjugates are also contemplated.

[0114] In another embodiment any antibody described herein is further conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin. In some embodiments, any antibody described herein is further conjugated to a cytotoxin to form an antibody drug conjugate (ADC) to treat cancer cells in vitro and vivo. In some embodiments, the cytotoxin is selected from the group comprising or consisting of maytansine and derivatives thereof (maytansinoids), auristatins, calicheamicins, duocarmycins, doxorubicin, anthracyclines, amanitins and camptothecins. In some embodiments, the antibody drug conjugate comprises a plurality of cytotoxins conjugated to the antibody, preferably 2 – 8 cytotoxins.

[0115] In another embodiment any antibody described herein is further conjugated to a therapeutic agent and / or an detectable label, such as gamma emitters or therapeutic isotopes such as an alpha emitter, an auger electron emitter, and / or a beta-emitting isotope to treat cancer cells in vitro and vivo. Therapeutic agents can be of different classes. For example, therapeutic agents include chemotherapeutic drug (maytansinoids), auristatins, calicheamicins, duocarmycins, doxorubicin, anthracyclines, amanitins, and camptothecins and / or any of the isotopes mentioned above. For example, examples of alpha emitters include 213-Bismuth (half- life 46 minutes), 223-Radium (half-life 11.3 days), 224-Radium (half-life 3.7 days), 225-Radium (half-life 14.8 days), 225-Actinium (half-life 9.9 days), 212-Lead (half-life 10.6 hours), 212-Bismuth (half-life 60 minutes), 211-Astatin (half-life 7.2 hours), and 255-Fermium (half-life 20 hours), 149- Terbium (half-life 4.12 hours), 227-Thorium (half-life 18.7 days), 212-Lead (half-life 10.6 hours). Examples of beta emitters include 188-Rhenium (half-life 16.7 hours), 90-Yttrium (half-life 2.7 days), 32-Phosphorous (half-life 14.3 days), 47-Scandium (half-life 3.4 days), 67-Copper (half-life 62 hours), 64-Copper (half-life 13 hours), 77-Arsenic (half-life 38.8 hours), 89-Strontium (half-life 51 days), 105-Rhodium (half-life 35 hours), 109-Palladium (half-life 13 hours), 111-Silver (half-life 7.5 days), 131-Iodine (half-life 8 days), 177-Lutetium (half-life 6.7 days), 153-Samarium (half-life 46.7 hours), 159-Gadolinium (half-life 18.6 hours), 186-Rhenium (half-life 3.7 days), 166-Holmium(half-life 26.8 hours), 166-Dysprosium (half-life 81.6 hours), 140-Lantanum (half-life 40.3 hours), 194-Irridium (half-life 19 hours), 198-Gold (half-life 2.7 days), and 199-Gold (half-life 3.1 days), 161-Terbium (half-life 6.89 days). Examples of Auger electron emitters include 161-Terbium (half- life 6.89 days), 111-Indium (half-life 2.8 days), 67-Gallium (half-life 78.3 hours), 99m-Technetium (half-life 6.2 hours), 125-Iodine (half-life 60 days) and 123-Iodine (half-life 13.2 hours). For example, detectable labels can comprise sub-therapeutic doses gamma emitters or isotopes such as an alpha emitter, an auger electron emitter, and / or a beta-emitting isotope that can also be used as therapeutic agents at higher doses.

[0116] Alpha particle therapeutics are up to 1000-fold more potent than beta-emittingtherapeutics, are effective in both normoxic andhypoxic environments. 225Ac has the followingcharateristics: t1 / 210.0 days; energy range of 4 αs is 6–8 MeV (cumulatively28 MeV / decay) decayswith the emission of 4 αs with a range of 50–80 μm and linear energy transfer (LET) of 0.16 MeV / μm .

[0117] Other detectable labels, such as fluorescent dyes, enzymes, or biotin may be used depending on the application, and are contemplated herein.

[0118] Immunoconjugates may be generated using any suitable technique. Common conjugation techniques include N-hydroxysuccinimide ester (NHS ester) or maleimide crosslinking, but other techniques are known in the art. In the case of radionuclide-conjugated antibodies, bifunctional chelating agents may be used. Suitable bifunctional chelating agents are known in the art, including desferrioxamine (DFO). Other suitable chelating agents include DTPA (diethylenetriamine pentaacetic acid), TCMC (1,4,7,10-tetraaza-1,4,7,10-tetra(2- carbamoylmethyl)cyclododecane), TETA (1,4,8,11-Tetraazacyclotetradecane-1,4,8,11- tetraacetic acid), DOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid), macropa (N,N′-bis[(6- carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6).

[0119] As will be understood by the skilled person, the molar ratio or chelator-antibody ratio (CAR) refers to the ratio of chelator to antibody in the conjugate. The CAR for example may depend on various factors such as the antibody and the linker used, as well as the initial molar ratio of each used in the conjugation reaction.

[0120] Another aspect of the disclosure includes any antibody described herein, any immunoconjugate described herein, or any composition described herein, for use in treating a cancer. The immunoconjugates are useful for treating cancers that are Nectin-4 expressing.

[0121] Another aspect of the disclosure includes any antibody described herein, any immunoconjugate described herein, or any composition described herein, for use in the manufacture of a medicament for treating a cancer. The immunoconjugates are useful for treating cancers that are Nectin-4 expressing.

[0122] Another aspect of the disclosure includes use of any antibody described herein, any immunoconjugate described herein, or any composition described herein, for treating cancer. The antibodies are useful for treating cancers that are Nectin-4 expressing.

[0123] In some embodiments, the cancer is triple negative breast cancer.

[0124] In some embodiments, the subject is a human.

[0125] Another aspect of the disclosure includes a method for detecting Nectin-4 expression in a biological sample, the method comprising a) obtaining a biological sample suspected of containing Nectin-4, b) contacting the sample with any antibody or immunoconjugate described herein under conditions permissive for forming an antibody:Netctin-4 complex, and c) detecting the presence of any antibody: Nectin-4 complex, wherein the detection of antibody: Nectin-4 complex is indicative that the sample expresses Nectin-4.

[0126] Another aspect of the disclosure includes a method of detecting whether a subject has cancer, the method comprising obtaining a biological sample suspected of containing a cancer cell, the biological sample having been obtained from the subject, contacting the sample with an antibody or immunoconjugate described herein under conditions permissive for forming an antibody:Nectin-4 complex, and detecting the presence of any antibody:Nectin-4 complex, wherein the presence of antibody: Nectin-4 complex indicates that the subject has or has an increased likelihood of having cancer. The method of detecting described herein may be performed prior to, after, or concurrently with other methods detection of cancer e.g., the biological sample may also be subject to a pathology assessment. The biological sample can be further assessed. For example, the biological sample may be assessed morphologically, or stained for one or more markers indicative of cell type such as breast cells, ovarian cells esophageal cells, intestinal cells, liver cells, gallbladder cells, head and neck cells, bladder cells, gastric cells, pancreatic cells or lung cancer. Alternatively, or in addition, the biological sample may be assessed for the presence of one or more tumor associated antigens. Depending on the biological sample taken, the presence of antibody:Nectin-4 complex may be indicative of a cancer selected from, breast cancer, such as triple negative breast cancer, ovarian cancer, esophageal cancer,bladder cancer, gastric cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer.

[0127] In some embodiments, the biological sample is obtained from a subject having or suspected of having a cancer, optionally wherein the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer, optionally wherein the biological sample is a tumor sample.

[0128] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma.

[0129] In some embodiments, the method further comprises identifying the subject that has or has an increased likelihood of having cancer as being likely to benefit from treatment with an antibody described herein, an immunoconjugate described herein, or a composition described herein.

[0130] In some embodiments, the method further comprises selecting the subject that has or has an increased likelihood of having cancer for treatment with an antibody described herein, an immunoconjugate described herein, or a composition described herein.

[0131] Another aspect of the disclosure includes a method of determining if a subject has a Nectin-4-expressing cancer, the method comprising administering an immunoconjugate comprising a detectable label described herein, or any composition comprising the immunoconjugate described herein to the subject, and detecting the presence of the label by imaging, optionally the subject has or is suspected of having a cancer, optionally cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer.

[0132] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma.

[0133] Another aspect of the disclosure includes a method for imaging a Nectin-4- expressing tumor in a subject, the method comprising administering an immunoconjugate comprising a detectable label or composition thereof described herein to the subject, and detecting the presence of the label.

[0134] Another aspect of the disclosure includes a method of treating a cancer, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, and lung cancer, in a subject in need thereof, the method comprising administering an effective amount of an antibody, immunoconjugate comprising an antibody described herein and a therapeutic agent, or composition described herein to the subject.

[0135] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma.

[0136] In some embodiments, the method of treatment further comprises a plurality of doses of an effective amount of the antibody, immunoconjugate, or composition described being administered to the subject. In some embodiments, the method of treatment further comprises at least 4 doses of an effective amount of the antibody, immunoconjugate, or composition described being administered to the subject. In some embodiments, 1, 2, 3, 4, 5, or 6 doses of an effective amount of the antibody, immunoconjugate, or composition described is administered to the subject. In some embodiments 3, 4, 5, or 6 doses of an effective amount of the antibody, immunoconjugate, or composition described is administered to the subject. In some embodiments 4, 5, or 6 doses of an effective amount of the antibody, immunoconjugate, or composition described is administered to the subject.

[0137] After imaging is performed to confirm the presence of Nectin-4 in a cancer cell, a subject can be treated using an antibody, immunoconjugate comprising an antibody described herein and a therapeutic agent, or composition described herein. Accordingly, in some embodiments, a method of imaging is followed by a method of treating a cancer. In some embodiments, the method further comprises, determining if a subject has a Nectin-4-expressing cancer according to a method described herein prior to treatment. In some embodiments, the method further comprises imaging a Nectin-4-expressing tumor in a subject according to a method described herein prior or after treatment. In some embodiments, the method furthercomprises detecting whether a subject has cancer according to a method described herein prior or after treatment.

[0138] Another aspect includes a method of treating cancer in a subject in need thereof, the method comprising administering an immunoconjugate comprising comprising an antibody described herein and a therapeutic agent, or a composition comprising the immunoconjugate to the subject, wherein the subject has been previously determined to have a Nectin-4 expressing cancer. The subject can for example have been determined to have a Nectin-4 expressing cancer by imaging, or by testing a biopsy of the cancer.

[0139] A further aspect includes a method of treating cancer in a subject in need thereof, the method comprising I) a) administering an immunoconjugate comprising a detectable label, or a composition comprising the immunoconjugate; or b) testing a sample of the cancer for Nectin-4, optionally using an antibody described herein; and II) administering an immunoconjugate comprising an antibody described herein and a therapeutic agent or a composition comprising the immunoconjugate to the subject, when the subject is determined in I) to have a Nectin-4 expressing cancer.

[0140] Another aspect of the disclosure includes use of an antibody to receive information about whether or not the patient is suitable for receiving treatment with an antibody, immunoconjugate comprising an antibody described herein and a therapeutic agent, or a composition described herein.

[0141] Another aspect of the disclosure includes use of an antibody described herein, immunoconjugate comprising the antibody and therapeutic agent described herein, or a composition comprising the antibody or immunoconjugate and a pharmaceutically acceptable carrier for treating cancer in a subject in need thereof, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, and lung cancer, in a subject in need thereof.

[0142] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In someembodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma.

[0143] In some embodiments, treating comprises administering the antibody, immunoconjugate, or composition described herein as part of treatment regimen. In some embodiments, the treatment regimen comprises administering about 8 MBq / cycle, about 9 MBq / cycle, about 10 MBq / cycle, about 11 MBq / cycle or about 12 MBq / cycle or for example any dose between 8-12 MBq / cycle. In an embodiment, the number of cycles is 4 cycles, 5 cycles or 6 cycles or for example 4-6 cycles.

[0144] In some embodiments, the method further comprises a) detecting Nectin-4 expression in a biological sample according to the method described herein, wherein the biological sample is obtained from the subject, and / or b) imaging an nectin-4 -expressing tumor in the subject according to the method described herein, wherein the detecting and / or imaging is done before, during, or following administering the antibody, immunoconjugate, or composition.

[0145] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma.

[0146] In some embodiments, the subject is a human.

[0147] In some embodiments, the antibody is fully human.

[0148] Uses of the methods described herein are also contemplated.

[0149] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0150] The following non-limiting examples are illustrative of the present disclosure: EXAMPLES Example 1

[0151] Fully human monoclonal antibodies against nectin-4 (anti-N4) were developed using phage display. The anti-N4 antibody N4-Fab1 (SEQ ID NOs: 62 and 63) was conjugated with p-SCN-Bn-DFO or macropa and radiolabeled with89Zr or225Ac, respectively, for imaging or radiotherapy using TNBC mouse xenograft models. Characterization of binding affinity of chelator-conjugated and radiolabeled antibodies was performed using flow cytometry and radioligand binding assay in Nectin4 expressing cell lines. Pharmacokinetic profile, biodistribution and microPET / CT imaging of89Zr-anti-Nectin4 radioimmunoconjugate (RIC) was studied in mice bearing nectin-4 positive xenografts. Cytotoxicity of the225Ac-anti-N4 antibody was evaluated in MDA-MB-468 cells. Biodistribution of225Ac-anti-N4 RIC was studied in healthy mice, and the therapeutic efficacy was evaluated in mice bearing MDA- MB-468 xenograft. Mice were treated with two doses of 350 nCi or 500 nCi administered at 10 days apart. Tumor growth was monitored using a digital caliper.

[0152] Macropa and DFO conjugated anti-Nectin4 antibodies retained high affinity binding (3nM) to cell lines and appeared to have better binding affinity compared to the radiolabeled antibody (10 nM) which was not expected. The pharmacokinetic profile of89Zr-anti-N4 RICshowed fast distribution half-life t1 / 2α of 3.4 h and a moderate elimination t1 / 2ß of 63 h. MicroPET / CT imaging and biodistribution of89Zr-anti-N4 in mice bearing MDA-MB-468 xenograft showed high tumor uptake of (14.14 ± 5.1 %IA / g) and (13.2 ± 1.12 %IA / g) injected dose per gram (%IA / g) at 24 h and 120 h post injection (p.i.), respectively. Tumor uptake of89Zr-anti-N4 RIC in BT-474 and MCF-7 xenografts was (13.2 ± 8.2 % IA / g; 16.4 ± 7.0 % IA / g), respectively at 120 h p.i.225Ac–anti-N4 RIC was effectively internalized in MDA-MB-468,and was cytotoxic to the cells with an IC50of(1.2 kBq / mL). The therapeutic efficacy of 225Ac–anti-N4 RIC in TNBC tumor bearing mice for 90days was evaluated. Treatment with225Ac–anti-N4 (350 nCi or 500 nCi) led to complete tumor remission in all mice in these groups.

[0153] In addition to the MDA-MB-468 cell line that has endogenous expression of nectin- 4, murine cancer cell line 4T1 was transduced to express human nectin-4 (4T1-nectin-4, syngeneic model). Radiotherapy was studied in mice bearing 4T1-nectin-4 xenograft after injection of 350 nCi (two doses at 10 days apart). All mice treated with vehicle alone reached end point (1500 mm3) after 22 days while 7 / 8 mice treated with 225Ac-anti-N-4 had complete tumor remission, and the remaining 1 / 8 had a tumor volume of 36 mm3. Materials and methods Cell lines and xenografts

[0154] Human breast cancer cell lines MDA-MB-468, MCF-7 and BT-474 that express nectin-4 and MDA-MB-231 known for nectin-4 negative expression were purchased from ATCC (Rockville, MD). MDA-MB-468 and MDA- MB-231 cells were propagated in Leibovitz's L-15 medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% FBS. MCF-7 cells were cultured in DMEM medium (HyClone Laboratories, Logan, UT, USA), supplemented with 10 % fetal bovine serum (FBS) (Biochrom). BT-474 cells were cultured in ATCC Hybri-Care medium supplemented with 1.5 g / L sodium bicarbonate and 10% FBS. MCF-7 and BT-474 cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2, while MDA-MB-468 and MDA-MB-231 cells did not require CO2. Female CD-1 nude and athymic nude mice of 4 weeks of age wereobtained from Charles RiverCanada (St-Constant, QC, Canada). Animals used in this study were maintained following the guidelines of the University of Saskatchewan Animal Care Committee (protocol # 20170084 and 20220021). At five weeks of age, CD-1 nude mice were subcutaneously injected at the right flank with 10 × 106MDA-MB-468 cells in 100 μL suspension of a 1:1 mixture of medium and matrigel matrix basement membrane (Discovery Laboware, Inc. Bedford, MA). Athymic nude mice were subcutaneous implanted with 17 beta-Estradiol 1.7 mg / 90 days release pellets, as per manufacturer recommendations (Innovative Research of America, Sarasota, FL), using a 10-gauge precision trochar. Seven days later, the mice were injected with 1.4 × 107MCF- 7 and BT-474 cells in the left and right flanks, respectively. Tumor growth was measured using a digital calliper. Mammalian expression and purification of recombinant nectin-4 domain I

[0155] The DNA sequence encoding IgV-type domain of nectin-4 (amino acids 32-144, Uniprot reference Q96NY8) was amplified from cDNA (GenBank Accession #BC010423) using the primers; AS53-5’-CTTGTCACGAATTCGATAGGTGAGCTGGAGACCTCAG-3’ (SEQ ID NO: 102), AS60-5’-CAGATCTAACCATGGC AGGCACCAGCACTCGGAG-3’ (SEQ ID NO: 103).

[0156] The sequence was cloned into pFUSE-hIgG1-Fc2 (InvivoGen, San Diego, CA, USA) using restriction enzymes NcoI and EcoRI. The recombinant protein for nectin-4 IgV-Fc fusion was expressed and purified using transient transfection of sequence verified plasmids in Expi293F suspension cells (Thermo Fisher Scientific Waltham, MA, USA). Expi293F cells were cultured using Expi293F complete media (Thermo Fisher Scientific Waltham, MA, USA).75 x 106Expi293F cells were transfected with 30 ug of nectin-4 IgV-Fc plasmid DNA at 37 °C. Cells were harvested and pelleted after five days, and recombinant protein was purified from the supernatant using affinity chromatography (MabSelectSure resin, GE Healthcare Chicago, IL, USA) followingmanufacturer recommended protocol. The integrity and the purity of the protein was confirmed using SDS-PAGE. Screening naïve antibody libraries using phage display

[0157] A naïve antibody library previously developed was used

[0021] . This library was panned against the recombinant nectin-4 IgV-Fc fusion protein using previously described protocols

[0022] . Three rounds of selection were performed, and the selection pool was tested for specific binding to nectin-4 IgV-Fc fusion protein. Clonal ELISA was performed using 24 clones isolated from round 3 pool to test for specific binding to the target protein following standard ELISA protocols

[0023] . Clones specific to target recombinant nectin-4 IgV-Fc fusion protein were identified by DNA sequencing.

[0158] Expression of anti-N4 IgG1 antibody N4-Fab1 To express the full-length IgG1 version of nectin-4 antibody, the VL and VH coding sequences were cloned into pFUSE2ss-CLIg- hK and pFUSE2ss-CHIg-hG1 vectors (InvivoGen, San Diego, CA, USA), respectively. The verified clones were transiently transfected into Expi293F cells for secretory expression of full length anti-N4 IgG1 following previously described protocols

[0024] . The purification of the anti-N4 antibody N4-Fab1 (Seq IDs 62 and 63) was performed using MabSelectSure (GE Healthcare, Chicago, IL, USA) affinity resin using manufacturer’s recommended protocols. Bioanalyzer (Agilent 2100 Bioanalyzer using Agilent High Sensitivity Protein 230 Kit, Santa Clara, CA, USA) was used to confirm the purity of the protein following manufacturer's protocol.Characterization of anti-N4 binding using flow cytometry

[0159] In a 96 well non-binding plate, different concentrations of the anti-N4 antibody N4- Fab1 in 1 x PBS of (500 nM, 250 nM, 125 nM, 62 nM, 30 nM, 10 nM and 3 nM) were added to nectin-4 expressing breast cancer cells MDA-MB-468, MCF-7 and nectin-4 negative MDA-MB- 231 cells at 3 x 105cells / well. After 30 min of binding at 4 °C, cells were washed two times and re-suspended in ice-cold 1x PBS. FITC labeled goat F(ab')2 fragment anti-human IgG (H + L) antibody (Beckman Coulter, Brea, CA, USA) was added to cells in (1 in 50) dilution and allowed to bind for 30 min at 4 °C. Cells were washed with ice-cold 1 x PBS three times before reading the plate using CytoFLEX (Beckman Coulter) on the FL1 channel. Flow cytometry data were acquired using CytoFLEX and analyzed using FlowJo (version 10.7.2; FlowJo LLC, Ashland, OR, USA). GraphPad Prism v9 (GraphPad software, San Diego, CA, USA) was used to determine the binding constant (Kd) and half maximal effective concentration (EC50)

[0025] .Internalization of anti-N4

[0160] In a flat-bottom 96-well plate, nectin-4 expressing MDA-MB-468, MCF-7 and nectin-4 negative MDA-MB-231 cells were seeded and incubated at 37 °C overnight. Twenty-four hours later, anti-N4 antibody N4-Fab1 (Seq IDs 62 and 63) was added with a final concentration of 4 ug / mL to a 3x molar-excess of FabFluor pH red antibody internalization reagent (Essen Bioscience Ann Arbor, MI, USA) and incubated for 15 minutes at 37 °C. The labeled antibody was then added to the cells, and images were taken at 10x magnification every 2 hours with phase contrast and red fluorescence filters using the Incucyte live-cell imaging system (Essen BioScience Ann Arbor, MI, USA). The mean red object area (μm2 / well) was calculated using the Incucyte software and was used to quantify antibody internalization.Conjugation with bifunctional chelators, and radiolabeling with 89Zr and 225Ac

[0161] The anti-N4 antibody N4-Fab1 was conjugated with p-SCN-Bn-deferoxamine (DFO) for labeling with89Zr as described previously

[0026] . Quality control of the immunoconjugate was done using SEC HPLC, flow cytometry, and automated electrophoresis (2100 Bioanalyzer, Agilent, Santa Clara, CA, USA). Radiolabeling and purification of DFO-N4 with89Zr was done as reported previously

[0026] . 18-membered macrocyclic bifunctional chelator 6-((16-((6- carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4- isothiocyanatopicolinic acid (macropa-SCN) (p-SCN- macropa) was synthesized as reported by Thiele et al

[0027] . To conjugate the antibody with the chelator, a stock of 20 mg / mL of macropa- SCN was prepared for conjugation reactions. Briefly, 1 mg each of the above antibodies were buffer exchanged with 0.1 M sodium bicarbonate buffer containing 0.15 M sodium chloride using a spin cap column. After buffer exchange, the desired volume of macropa-SCN (15-fold excess) was added to the antibody solution and incubated at 4˚C for 18 h. Following incubation, excess unreacted macropa was removed using Amicon Ultra-410K centrifugal filters (EMD Millipore, Burlington, MA, USA). Radiolabeling with225Ac was performed as previously reported

[0026] .

[0162] The stability of89Zr-labeled and225Ac-labeled anti-N4 antibody conjugates at 37 °C was investigated using iTLC by analyzing aliquots of the radiolabeled over time.89Zr-labeled or225Ac-labeled conjugates was mixed with human plasma or 1x PBS solution at a final concentration of 15 MBq / mL and incubated at 37 °C. Aliquots of the solution were taken every 24 h for five days and analyzed for radiochemical purity using iTLC.Radioligand binding assay of 89Zr-anti-N4

[0163] The binding of89Zr-labeled anti-N4 antibody to nectin-4 positive MCF-7 cells was determined using a saturation radioligand binding assay. Briefly, 106cells were incubated withincreasing concentrations of89Zr-nectin-4 antibody (0.17 to 375 nmol / L in 100 μL of PBS) for 4 h at 4°C. Non-specific binding was determined in a similar assay using a 10-fold molar excess (3750 nmol / L) of unlabeled anti-nectin-4 antibody. A non-linear regressionanalysis with one-site - totaland non-specific binding equation was used to determine the Kdand BmaxusingGraphPad Prismversion 9 (La Jolla, CA).MicroPET / CT imaging and biodistribution of 89Zr-anti-N4

[0164] Female CD-1 nude mice (n = 4 / group) bearing nectin-4 positive MDA-MB-468 xenograft and female Balb-C athymic nude mice (n = 4) bearing nectin-4 positive BT-474 and MCF-7 xenografts were injected intravenously using 12 ± 1 MBq (21 – 26 µg)89Zr-anti-N4 antibody. Additionally, mice bearing MDA-MB-468 xenograft (n = 4) were injected intravenously with 900 ug of unlabeled anti-N4 antibody four hours prior to the injection of89Zr- anti-N4 antibody to pre-block nectin-4 receptors. Additionally, to understand uptake of89Zr- anti-N4 in immunocompetent mice murine 4T1 breast cancer cell line were transduced to stably express nectin-4 (4T1-nectin-4). 4T1-nectin-4 was injected subcutaneously in healthy Balb-C mice to grow xenografts for microPET and radioimmunotherapy studies. MicroPET / CT imaging was performed at 24, 48, 72, 96, and 120 h post-injection using the Vector4CT scanner (MILabs B.V., Utrecht). PET scans were acquired in a list- mode data format using a high-energy ultra-high resolution (HE-UHR-1.0 mm) mouse / rat pinhole collimator. Images were reconstructed using PMOD 3.8 software (PMOD, Switzerland). Immediately after imaging, mice were sacrificed at 120 h post injection for biodistribution studies. To complete the biodistribution studies, additional group (n=4) of tumor-bearing mice were sacrificed 24 h post injection. All major organs and blood were harvested and collected in tared tubes and weighted. The radioactivity in all organs and blood were measured using an automated gamma counter (Wallac Wizard 1480, PerkinElmer, Waltham, MA), and expressed as percent injected activity per gram (%IA / g).Pharmacokinetic profile of 89Zr-anti-N4

[0165] Female CD-1 nude mice (n = 4) were injected intravenously with 5 – 6 MBq of89Zr- anti-N4 antibody (10 – 12 µg of antibody). Blood was collected from a saphenous vein in heparinized capillary tubes at different time points (0.5 – 120 h). The height of the capillary tube occupied by blood was determined using a digital caliper. Blood volume in the capillary tube (mL) was measured using V = ^^ ^^ r2h. The radioactivity in the samples was measured using a gamma counter and expressed as % injected activity / mL (%IA / mL). Pharmacokinetic parameters, including the distribution and elimination half-lives (t1 / 2α and t1 / 2β), volume of distribution at steady-state (Vss), clearance (CL) and volume of the central compartment (V1), were calculated by fittingthe blood radioactivity versus time curve to a two-compartment model with i.v. bolus input.In vitro cytotoxicity

[0166] The cytotoxicity (IC50 values) of225Ac-anti-N4 and control immunoconjugates(225Ac-rituximab, non-labeledanti-N4) in nectin-4 positive MDA-MB-468, MCF-7, and nectin-4 negative MDA-MB-231 was determined using IncuCyte Cytotox Red reagent in an IncuCyte S3 live-cell imager (Essen BioScience, AnnArbor, MI) as previously reported

[0028] . Live-cell images were captured every 2 h using a 10Å~ objective lens using phase contrast and fluorescence channel. Images were processed and analyzed using IncuCyte S3 software. The redfluorescentvalues were generated, and IC50 values for individual compounds were calculated usingGraphPadprism v9.Biodistribution and dosimetry of 225Ac-anti-N4

[0167] To estimate radiation dose to healthy tissues, normal BALB / c nude mice (n ≥ 4 / group) were administered 350 nCi of225Ac–anti-N4 antibody via a tail vein and sacrificed at 1, 24, 48, 120, and 264 post injection followed by biodistribution studies. Carcasses were collected and analyzed using a gamma counter and activity was expressed as injected activity per gram organ weight (% IA / g) and % IA. For each organ or tissue, the effective data (non- decay-corrected % IA / g) were plotted against sampling time. This data was used to obtain an estimate of the microcurie-hours per microcurie administered, represented by the area under the time-activity function integrated to infinity (complete decay) of the225Ac. Absorbed doses in units of cGy (centi- gray) per millicurie (cGy / mCi) of225Ac administered were calculated. Mouse organ doses were extrapolated hypothetically to the human and recalculated by recalculating the residence times for the human model from the mouse model using OLINDA1.1 (Hermes Medical Solutions,Montreal QC) [29, 30].225Ac-anti-nectin-4 radioimmunotherapy

[0168] In vivo efficacy studies were done using athymic BALB / c mice (Charles River Laboratories, Hartford, CT) aged 4 – 6 weeks bearing MDA-MB-468 xenograft. Mice were obtained and housed in accordance with University Animal Care Committee (UACC) guidelines (protocol # 20220021). All the experiments and euthanasia were performed in accordance with UACC guidelines. Mice bearing MDA-MB-468 xenograft were divided into 5 groups (n ≥ 4 / group) namely;225Ac-anti-N4 antibody (two doses of 350 nCi / dose),225Ac-anti-N4 antibody (two doses of 500 nCi / dose), PBS treatment, unlabeled anti-N4 antibody, and225Ac-rituximab (an anti-CD20non- specific control IgG, two doses of 350 nCi / dose). Additionally, mice (n=8 / group) bearing 4T1- nectin-4 xenografts were treated using225Ac-anti-N4 antibody (two doses of 350 nCi / dose) or saline. Treated mice received two doses of treatment via a tail vein on days 0 and 10. Tumor growth was monitored by measuring the greatest length and width using a digital caliper (tumor volume = length × width2 / 2). The MDA-MB-468 xenograft groups were terminated when xenograft reached a volume ≥ 400 mm3(compared with the 1500 mm3endpoint we routinely employ for therapy studies, the ≥ 400 mm3was chosen due to the slow-growing nature of this xenograft), and this was used to determine survival in the different groups using Kaplan Meier curves. For 4T1-nectin- 4 xenograft, the study was terminated when tumors reached >= 1500 mm3. The body weight of each mouse was recorded during the quarantine and experimental period.Statistical analysis

[0169] All data were expressed as the mean ± standard deviation of at least three independent experiments. A two- tailed Student's t-test or analysis of variance (ANOVA) with Bonferoni post hoc test were used to assess the statistical significance between the groups. All graphs were prepared and analyzed using GraphPad Prism (version 9; GraphPad, La Jolla, CA).ResultsGeneration and in vitro characterization of anti-nectin-4 antibody

[0170] Panning of naïve antibody Fab-phage library on recombinant nectin-4 IgV-Fc protein resulted in three clones that bind specifically to the recombinant protein. N4-Fab1 was selected as the lead clone after ranking by binding affinity in a competitive ELISA. Phage ELISA showed specific binding of N4-Fab1 phage to nectin-4 IgV-Fc and not to negative control Fc fusion proteins (Fig. 9). Full-length human IgG1 version of N4- Fab1 was expressed in human origin Expi293F cells by transient transfection and purified using affinity chromatography. The purity and integrity of the purified full length anti-N4 antibody was confirmed using the bioanalyzer and HPLC (Figs.10A-C). In vitro characterization using flow cytometry was performed to determine the binding of anti-N4 antibody to nectin-4 positive MDA-MB-468, MCF-7, and nectin- 4 negative MDA-MB-231 cell lines. Dose dependent specific binding was observed with both MDA-MB-468 and MCF-7 and not with MDA-MB-231 cells (Figs.1A-C).

[0171] Table 1: CDRs and Heavy Chain and Light Chains Variable Domains of Fabs 1-3 Variable Domain Kabat CDRs IMGT CDRsN4-Fab1 EIVLTQSPGTLSLSPGERATLSC RASQSVSNSYLA QSVSNSY (SEQ ID RASQSVSNSYLAWYQQKPGQA (SEQ ID NO: 85) NO: 80) Light PGLLIYGASRRATGIPDRFSGS GASRRAT(SEQ ID GA Chain GSGTDFTLTISSLEPEDFAVYYC NO: 86) QQRSNWPPTFGQGTKVEIK QQRSNWPPT(SE QQRSNWPPT (SEQ ID (SEQ ID NO: 62) Q ID NO: 87) NO: 81) N4-Fab1 QVQLVESGGGVVQPGGSLRLS SYGMH (SEQ ID GFTFRSYG(SEQ ID CAASGFTFRSYGMHWVRQAP NO: 88) NO: 82) Heavy GKGLEWVALIWYDGSNKYYAD LIWYDGSNKYYA IWYDGSNK (SEQ ID Chain SVKGRFTISRDNSKNTLYLQMD DSVKG (SEQ ID NO: 83) SLRAEDTAVYYCARDDWTNGW NO: 89) YFGQGTLVTVSS (SEQ ID NO: DDWTNGWY ARDDWTNGWY (SEQ 63) (SEQ ID NO: 90) ID NO: 84) N4-Fab2 EIVLTQSPATLSVSPGERATLSCR RASQSVSRYLA QSVSRY (SEQ ID NO: ASQSVSRYLAWYQQKPGQAPRL (SEQ ID NO: 91) 68) Light LISDASNRATGIPARFSGSGSGT DASNRAT (SEQ DA Chain DFTLTISRLEPEDFAVYYCQQRY ID NO: 92) NWPPDFGQGTRLEIK (SEQ ID QQRYNWPPD QQRYNWPPD (SEQ ID NO: 64) (SEQ ID NO: 93) NO: 70) N4-Fab2 EVQLVESGGGVVQPGRSLRLS SYGMH (SEQ ID GFTFSSYG (SEQ ID CAASGFTFSSYGMHWVRQAP NO: 88) NO: 71) Heavy GKGLEWVAVISYDGSNKYYADS VISYDGSNKYYAD ISYDGSNK (SEQ ID Chain VKGRFTISRDNSKNTLYLQMNS SVKG (SEQ ID NO: 72) LRAEDTAVYYCAKSTLHSSGWY NO: 94) MDYWGQGTLVTVSS(SEQ ID STLHSSGWYMDY AKSTLHSSGWYMDY NO: 65) (SEQ ID NO: 95) (SEQ ID NO: 73) N4-Fab3 DIQMTQSPSSLSASVGDRVTI QASQDISNYLN QDISNY (SEQ ID NO: TCQASQDISNYLNWYQQKP (SEQ ID NO: 96) 74) Light GKAPKLMIYAATSLQSGVPSR AATSLQS (SEQ ID AA Chain FSGSGSGTDFTLTISNLQPED NO: 97) FATYYCQQSYTTRTTFGQGT QQSYTTRTT QQSYTTRTT (SEQ ID KLEIK(SEQ ID NO: 66) (SEQ ID NO: 98) NO: 76) N4-Fab3 QVQLVESGGGVVQPGRSLRL GYPMH (SEQ ID GFRFSGYP (SEQ ID SCEASGFRFSGYPMHWVRQ NO: 99) NO: 77) Heavy APGKGLEWVAVIWYDGRNR VIWYDGRNRHYA IWYDGRNR (SEQ ID Chain HYADSVKGRFTISRDNSKNTL DSVKG (SEQ ID NO: 78) YLQMNSLRAEDTAIYYCAKE NO: 100) GKWGEWYFDLWGRGTLVTV EGKWGEWYFDL AKEGKWGEWYFDL SS (SEQ ID NO: 67) (SEQ ID NO: 101) (SEQ ID NO: 79) Anti-nectin-4 antibody internalization

[0172] A time-dependent increase in red fluorescence was observed in nectin-4 expressing MDA-MB-468 (high expression) and MCF-7(moderate expression) cell lines, respectively, but not in the negative control MDA-MB- 231 cells or media control at 4 – 48 h post incubation (Fig. 1D). The anti-N4 antibody showed 72- and 1100- fold higher internalization in nectin-4 positive MDA-MB-468 cells compared with the negative control MDA- MB-231 cells at 24 and 48 h, respectively. In MCF-7 cells, the anti-N4 antibody showed 71- and 365-fold higher internalization compared to the negative control MDA-MB-231 cells. Conjugation and quality control of anti-nectin-4 radioimmunoconjugates

[0173] HPLC analysis showed that the conjugation of p-SCN-DFO to anti-N4 antibody resulted in ~100% pure DFO- anti-N4 antibody with no aggregates (Figs.10A-C). Similarly, the conjugation of macropa to anti-N4 antibody resulted in 100% pure immunoconjugate with no aggregates. The size and purity of DFO-anti-N4 and macropa-anti-N4 antibodies were evaluated using bioanalyzer (Figs.10A-C). Automated electrophoresis using Bioanalyzer showed that DFO- anti-N4 and macropa-anti-N4 immunoconjugates was 90% and 92.4% pure with molecular weight of 153.2 kDa and 150.3 kDa, respectively (vs 150.2 kDa for unconjugated anti-N4 antibody). This indicates that there were 4.0 DFO and one macropa chelator molecule(s) / antibody molecule.

[0174] In vitro saturation binding assay of unconjugated anti-N4 and DFO-anti-N4antibody was done usingnectin-4 positive MCF-7 cells using flow cytometry. The estimated Kdvalues for unconjugated anti-N4 antibody and DFO-anti-N4 were 2.9 and 3 nM, respectively. Theestimated EC50 values for unconjugated anti-N4 antibodyand DFO-anti-N4 were 7.3 and 20.0 nM,respectively (Figs.2A-D). Additionally, a saturation binding assay of unconjugated anti-N4 was done using nectin-4 positive TNBC MDA-MB-468 cells using flow cytometry (Fig. 11A). This indicated that the conjugation did not affect the binding affinity of the antibody.Similarly, the Kd andEC50 of macropa-anti-N4 antibody were 0.3 and 1.6 nM, respectively (Fig.11B).

[0175] The anti-N4 antibodies conjugated with DFO or macropa were labeled with89Zr or225Ac respectively. The radiochemical yield (RCY) of89Zr-anti-N4 antibody was ~100% at a specific activity of 0.5 MBq / μg. Similarly, the RCY of225Ac-anti-N4 was > 95% at a specific activity of 10 kBq / μg. The stability of89Zr-anti-N4 antibody was determined at different time points at 37 °C in PBS and human plasma using iTLC. More than 95% of89Zr- anti-N4 antibody remained intact for 96 h in human serum and for 72 h in PBS, respectively (Fig. 12). The saturation radioligand binding assay using89Zr-anti-N4 antibody on MCF7 cells showed dose-dependentincrease in specific binding (Fig.2E). The estimated Kdof89Zr-anti-N4 antibody was 10 nM whichis 4-fold lower affinity (p < 0.05) than naked anti-nectin-4 antibody and 2.9-fold lower affinity (p < 0.05) than the DFO-anti-N4 antibody. Pharmacokinetics, biodistribution and microPET / CT of89Zr-anti-N4

[0176] The pharmacokinetic profile of89Zr-anti-N4 injected in CD-1 nude mice is shown in Fig.3 and the analyzed parameters are shown in Table 2.

[0177] Table 2: Pharmacokinetics of89Zr- anti-N4 antibody in healthy nude mice .

[0178] MicroPET / CT imaging experiments were conducted with89Zr-anti-N4 in mice carrying xenografts of MDA-MB-468, MCF7, BT-474 and 4T1-nectin-4 cells. High tumor uptake was delineated in all nectin-4 positive xenografts as shown by microPET / CT imaging at 24 – 120 h p.i.(Figs.4A and B). There was visibly very low tumor uptake of89Zr-anti-N4 antibody when pre- blocked with unlabeled antibody (Fig.4C). Uptake of89Zr-anti-N4 in nectin-4 positive 4T1-nectin- 4 xenograft (immunocompetent mice) was similar to those observed in MDA-MB-468, MCF7, BT- 474 as delineated by microPET imaging (Fig.4D) and ex vivo biodistribution studies.

[0179] Biodistribution of89Zr-anti-N4 antibody was performed at 24 and 120 h p.i. (Fig.5) in female athymic CD-1 nude mice bearing nectin-4 positive MDA-MB-468 (high expression) xenograft, and at 120 h in BALB / c athymic nude mice bearing nectin-4 positive BT-474 and MCF- 7 (medium expression) xenografts, and in a syngeneic mouse model bearing 4T1-nectin-4 xenograft. To investigate specificity, mice bearing MDA-MB-468 xenografts were pre-blocked using 900 µg of unlabeled anti-N4 antibody 4 h prior to injection of89Zr-anti-N4 antibody. In athymic CD-1 nude mice, tumor uptake of MDA-MB-468 was highest at 24 h p.i. (14.1 ± 5.1 % IA / g) and this decreased slightly (insignificantly, p > 0.05) to 13.2 ± 1.1 %IA / g at 120 h p.i.. When pre-blocked using unlabeled antibody, tumor uptake of MDA-MB-468 decreased significantly (p < 0.0005) to 2.8 ± 1.3 %IA / g at 120 h p.i. There was a slightly high uptake of89Zr-anti-N4 in the lungs and heart (6.0 ± 1.5 % IA / g and 4.7 ± 2.3 % IA / g, respectively) at 24 h p.i., likely reflecting blood pool activity. However, this decreased to 3.4 ± 1.5 %IA / g and 2.7 ± 0.5 %IA / g) at 120 h p.i., respectively. Tumor to blood ratio for MDA-MB-468 was 1.0 and 3.1 at 24 and 120 h p.i., respectively. Tumor to muscle ratio for MDA-MB-468 was 13.2 compared with 1.7 for pre-blocked at 120 h p.i. Tumor uptake in BT-474, MCF-7 and 4T1-nectin-4 xenografts was 13.2 ± 8.2 % IA / g, 16.4 ± 7.0 % IA / g and 11%± 2.46 % IA / g, respectively, at 120 h p.i. Tumor to blood ratio was 6.9, 8.6 and 5.5 for BT- 474, MCF-7 and 4T1-nectin-4, respectively at 120 h p.i. In vitro cytotoxicity of225Ac-anti-N4

[0180] Live cell imaging was used to study the in vitro cytotoxicity of unlabeled anti-N4 antibody and225Ac- anti-N4 antibody on MDA-MB-468, MCF7 and MDA-MB-231 cells (Table 3). Phase contrast images showed potent cytotoxicity with225Ac- anti-N4 antibody when comparedwith unlabeled anti-N4 antibody. Increasedcytotoxicity was observed in MDA-MB-468 at 24 husing225Ac- anti-N4 antibody with IC50of 1.2 kBq / ml whereas unlabeled N4 antibody had no effecton MDA-MB-468 cells. The IC50 values for MCF7 and MDA-MB-231 cells were consistent withmoderate and no expression of nectin-4 respectively.

[0181] Table 3: IC50 values of immunoconjugates in different in nectin-4 expressing TNBC cell lines. Biodistribution of225Ac-anti-N4

[0182] Biodistribution of225Ac- anti-N4 antibody was studied in healthy BALB / c mice. The uptake of225Ac- anti-N4 antibody was high in the kidney, liver, spleen, lungs, and blood at early time points but this uptake decreased overtime. Ten days post injection, the highest uptake was observed in the spleen (4 ± 1.3 %IA / g), lungs (6 ± 1.6%IA / g) and the blood (5.8 ± 0.9 %IA / g) (Table 4).

[0183] Table 4: Biodistribution of 225Ac-anti-N4 antibody in healthy BALB / c mice at different time points post injection expressed as % injected activity per gram (%IA / g) ± SD.Efficacy of225Ac-anti-N4

[0184] The efficacy of225Ac-anti-N4 RIT, and control225Ac-rituximab in TNBC cell line MDA-MB-468 mouse xenograft model was studied (Fig. 6A). Tumor growth inhibition was evaluated using a digital caliper. All mice treated with two doses of 350 nCi225Ac-anti-N4 or 500 nCi225Ac-anti-N4 antibody had complete tumor remission by day 22 or 24, respectively. There was no tumor re-growth in the two groups at the end of the study period of 90 days. Two out of four mice treated with two doses of 350 nCi225Ac-rituximab control IgG reached study endpoint ( ^ 400 mm3) by day 66 and 78, respectively, while the other two mice reached 200 mm3by day 90. All the mice in PBS group reached the study endpoint of ^ 400 mm3by day 73. Two out of four mice treated with unlabeled N4 antibody reached the study endpoint by day 72 and the rest of the mice reached the study endpoint by day 90. The Kaplan Meier survival curve showed that mice treated with225Ac-anti-N4 antibody survived for the whole period of the therapy study of 90 days. However, the median survival of the PBS and unlabeled N4 antibody treated mice was 72.5 and 77.5 days, respectively. The median survival for mice treated with225Ac- rituximab was 84 days (Fig.6B).

[0185] Two doses of225Ac-anti-N4 administered 10 days apart led to complete tumor remission in 7 / 8 immunocompetent mice bearing 4T1-nectin-4 tumor by day 26, with the remaining 1 / 8 xenograft having a tumor volume of 36 mm3at day 26 (Fig.6D). In contrast, all 6saline-treated mice bearing 4T1-nectin-4 tumor reached end point of >= 1500 mm3by day 23 following start of treatment. No regrowth of 4T1-nectin-4 has been observed. Median survival was 21 days for saline-treated 4T1-nectin-4 tumor bearing mice, and had not reached for the225Ac- anti-N4 treated group (Fig.6E).

[0186] For clinical application, fully human antibodies are advantageous for therapy for which the concern of immunogenicity is eliminated

[0032] . This study describes the development of a fully human anti-nectin-4 antibody and its evaluation as a PET imaging probe for the early detection of TNBCs and its development as a radioimmunotherapy (RIT) agent. Using phage display, an anti-N4 antibody was developed, which was selected for further evaluation as a PET imaging probe based on its low Kdof 2.9 nM in nectin-4 positive TNBC cell line (MDA-MB-468). 89Zr isconsidered an ideal PET isotope because of its long half-life of 78.4 h and high resolution due to the emission of positrons

[0035] . To evaluate anti-N4 IgG as immunoPET imaging probe, the antibody was conjugated with p-SCN- Bn-DFO, and the immunoconjugate had similar low nanomolar affinity (3 nM) compared with the unconjugated antibody. As expected, radiolabeling with89Zr resulted in a stable probe. The imaging89Zr-anti-N4 probe was evaluated (microPET and biodistribution studies) using nectin-4 positive TNBC cell lines with high andmediumreceptor densities. Shao et al

[0034] studied the tumor uptake of99mTc-HYNIC-mAbNectin-4in nectin-4 positiveMDA-MB-468 xenograft. Maximum tumor uptake in MDA-MB-468 xenograft was 15.32 ± 1.04% ID / g and when pre-blocked was 4.33 ± 0.48% ID / g which is similarto the present results. Additionally, the tumor-to-blood andtumor-to-muscle ratios for the presentdata are comparable with those for99mTc-HYNIC-mAbNectin-4tracer. Campbell etal evaluated89Zr-AGS-22M6 in tumor bearing mice and cynomolgus monkeys

[0033] . Tumor uptake of89Zr-AGS- 22M6 in nectin-4 transduced MDA-MB-231 cells (MDA-MB-231-Nectine-4) transduced to express the receptor ranged from an average of 38.8 ± 2.8 % ID / g on day 1 to an average of 39.9 ± 5.9 %ID / g on day 6 with an average high of 45.3 ± 2.4 %ID / g on day 3 compared with non-transduced receptor negative MDA-MB-231-Neo) that ranged from ranged from an average of 16.3 ± 1.8 %ID / g on day 1 to an average of 17.2 ± 1.3 %ID / g on day 6 with an average high of 18.2 ± 2.8 % ID / g on day 2. Similar tumor uptake ratios were found for89Zr-AGS-22M6 in nectin-4 positive patient-derived xenografts (PDX) compared with nectin-4 negative PDXs, In the current study, tumor uptake of89Zr-anti-N4 in MDA-MB-468 decreased from 13.2 %IA / g to 2.8 %IA / g when pre- blocked with cold antibody which is indicative for better specificity compared with89Zr-AGS-22M6in spite of its apparentlower Kd of 0.01 nM [8]. In addition,89Zr-anti-N4 displayed fast distributionhalf-life t1 / 2αof 3.37 h and a relatively moderate clearance t1 / 2βof 63 h compared with other veryslow-clearing IgGs such as trastuzumab which is advantageous for imaging.

[0187] Eighteen-membered ring macrocylic chelator macropa forms a highly stable and inert complex with225Ac [26, 27]. To investigate the antitumor effects of the radioimmunoconjugate, the in vitro cytotoxicity was studied using IncuCyte S3 live-cell imaging with Cytotox Red reagent, that allows for real-time quantification of dead cells.225Ac-anti-N4 displayed enhanced cytotoxicity to nectin-4 expressing cells while the unlabeled N4 antibodywasnot cytotoxic to nectin-4 expressing TNBC MDA-MB-468. The IC50of the presentradioimmunoconjugate(1.2 kBq / mL) was similar to otherhighly cytotoxic225Ac-labeledradioimmunoconjugates [36, 37].

[0188] The therapeutic efficacy of the unlabeled N4 antibody and225Ac-anti-N4 antibody at controlling the growth of the TNBC MDA-MB-468 xenograft or 4T1-nectin-4 syngeneic mouse model were then investigated. For MDA-MB-468 xenograft mice were administered two doses of 350 nCi (low dose) or 500 nCi (high dose) on days 0, and 10, while for 4T1-nectin-4 syngeneic model mice were only administered two doses of 350 nCi . Interestingly, all mice bearing MDA- MB-468 xenograft treated with both the low and the high dose had complete tumor remission following the administration of only two doses of225Ac-anti-N4. Tumors in mice treated with PBS, unlabeled N4 antibody and225Ac-rituximab controls continued to grow until they reached the study end point of ^400 mm3, which was chosen as an end point for the study as MDA-MB-468 xenograft tumor growth is relatively slow

[0034] , compared to other cell lines.

[0189] All mice treated with225Ac-anti-N4 RIT that had complete tumor remission on day 20 with no regrowth until the end of the study (90 days). There was also no apparent toxicity evident from weight gain throughout in the RIT treated group. M-Rabet et al [7] evaluated an anti- nectin-4 ADC in which the antibody is conjugated to monomethyl auristatin E (MMAE) via a valine- citruline cleavable linker in cell line-derived TNBC xenograft SUM190 and several PDX models, where, in contrast to the present results, regrowth of tumors was observed following period of complete regression. Continued regrowth was observed after re-treatment ended. Example 2: Affinity Maturation of Anti-N4 (N4-Fab1) Materials and Methods: Affinity maturation library construction

[0190] The phagemid encoding the parent clone N4-Fab1, was used as a template for site-directed mutagenesis and stop codons were introduced in CDRL3 and CDRH3 using the following oligonucleotides (Integrated DNA Technologies):

[0191] 5’- GTTTATTACTGTCAGCAGTAATAAACTTTTGGCCAGGGG-3’ (CDRL3) (SEQ ID NO: 104)

[0192] 5’- GTATTACTGTGCAAGATAATAAGGCCAGGGAACCCTG-3’ (CDRH3) (SEQ ID NO: 105)

[0193] This step eliminates the high prevalence of parent clone in the library, as site- directed mutagenesis usually results in >10% of the library being the unmutated template. Using the CDRH3-stop codon plasmid as a template, two libraries were constructed using the following mutagenic random oligonucleotides (Integrated DNA technologies): CDRL1:5’-GCCAGTCAGAGTGTT (N1:70101010)(N3:10107010)(N2:10701010) (N1)(N1)(N2) (N1)(N3)(N4:10101070) (N4)(N1)(N2) TTAGCCTGGTACCAG-3' (SEQ ID NO: 106) CDRL2:5’- GGCCTCCTCATCTAT (N3:10107010)(N3)(N3) GCCTCC (N1:70101010)(N3)(N3) CGC GCCACTGGCATCCCAG -3’ (SEQ ID NO: 107) CDRL3: 5’- GTTTATTACTGTCAGCAG (N2:10701010)(N3:10107010)(N4:10101070) (N1:70101010)(N3)(N2) (N1)(N1)(N2) (N4)(N3)(N3) (N2)(N2)(N4) (N2)(N2)(N4) ACTTTTGGCCAGGGG-3' (SEQ ID NO: 2) CDRH1: 5’- GCAGCGTCTGGATTC (N1:70101010)(N2:10701010)(N2) TTC (N1)(N3:10107010)(N3) (N4:10101070)(N2)(N2) TATGGCATGCACTGG-3’ (SEQ ID NO: 109) CDRH2: 5’- GTGGGTGGCACTTATA (N4:10101070)(N3:10107010)(N3) (N4)(N1:70101010)(N4) (N3)(N1)(N4) GGA (N1)(N3)(N4) (N1)(N1)(N4) AAA (N4)(N1)(N2:10701010) TATGCAGACTCCGTG-3’ (SEQ ID NO: 69 CDRH3: 5’- GTATTACTGTGCAAGA (N3:10107010)(N1:70101010)(N4:10101070) (N3)(N1)(N4) (N4)(N3)(N3) (N1)(N2:10701010)(N2) (N1)(N1)(N4) (N3)(N3)(N2) (N4)(N3)(N3) TAC (N4)(N4)(N4) GGCCAGGGAACCCTG- 3’ (SEQ ID NO: 75).

[0194] For library 1, CDRL1, CDRL2 and CDRL3 were mutated. For Library 2, CDRH1, H2 and H3 were mutated. Both libraries were constructed using established protocols as described previously (Fellouse et.al.) and resulted in ~ 3 x 109transformants for each library. Panning of libraries and characterization of individual clones

[0195] Three rounds of library panning were performed using standard protocols. The recombinant protein Nectin-ECD-Fc protein was immobilized at 2µg / ml concentration of NUNC maxisorp plates and each library was panned separately. After three rounds, 48 clones from each library were picked and characterized by clonal phage ELISA followed by sequencing. Results and Discussion Library Design

[0196] Upon sequence alignment with germline genes in the IMGT repertoire, N4-Fab1 is closely aligned with genes IGKV3-20*2 and IGHV3-33 for VL and VH respectively. The crystal structure PDB 5I1C (representing an antibody derived from IGHV3-33) and 5GGU (representing an antibody derived from IGKV3-20) were used as a guide to ascertain solvent exposed residues in CDRH1, H2, L1 and L2. Based on these structures, positions 29-32 (IMGT numbering) in CDRL1, positions 50,53 in CDRL2, positions 91-96 in CDRL3, positions 28, 30, 31 in CDRH1, positions 52, 52A, 53, 55, 56, 58 in CDRH2, loop positions (95-101) and flanking residue 103 (mutation from IgHJ4 gene) in CDRH3 were selected for mutagenesis. A soft-randomization scheme was used where nucleotide sequences for the corresponding amino acids were biased with hand-mixed nucleotides where: A was replaced with a mix of (70%A, 10%C, 10%G, 10%T)-Named (N1), C was replaced with a mix of (10%A, 70%C, 10%G, 10%T)-Named (N2), G was replaced with a mix of (10%A, 10%C, 70%G, 10%T)-Named (N3), T was replaced with a mix of (10%A.10%C, 10%G, 70%T)-Named (N4).

[0197] This allowed for a bias of ~40% for native amino acid found in the parent clone while still allowing for other 19 amino acids to occur in this position in the library of mutants. Library panning and characterization of individual clones

[0198] Both libraries showed enhanced binding to Nectin4-ECD-Fc when compared to control proteins in each round of selection and 48 clones from Round 3 selection were picked and analyzed for specific binding to Nectin-4 in a phage ELISA assay (Figs.8A-B). Positive clones were sequenced and shown below. Table 5: Sequences of specific unique clones from light chain library. Differences from parent clone N4-Fab1-LC are bolded and underlined. 1. EIVLTQSPGTLSLSPGERATLSCRASQSVSHSYLAWYQQKPGQA SEQ ID NO: 7 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSGFPPTFGQGTKVEIK2. EIVLTQSPGTLSLSPGERATLSCRASQSVKNSYLAWYQQKPGQA SEQ ID NO: 8 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSDYPPTFGQGTKVEIK 3. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQA SEQ ID NO: 9 PGLLIYGASVRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSGFPPTFGQGTKVEIK 4. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQA SEQ ID NO: 10 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSGFPPTFGQGTKVEIK 5. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQA SEQ ID NO: 11 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRYDYPPTFGQGTKVEIK 6. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQA SEQ ID NO: 12 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRWSFPPTFGQGTKVEIK 7. EIVLTQSPGTLSLSPGERATLSCRASQSVSAGYLAWYQQKPGQA SEQ ID NO: 13 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRWGYPPTFGQGTKVEIK 8. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQA SEQ ID NO: 14 PGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSDFPPTFGQGTKVEIK 9. EIVLTQSPGTLSLSPGERATLSCRASQSVSTSYLAWYQQKPGQA SEQ ID NO: 15 PGLLIYGASKRATGIPDRFSGSGSGTDFTLTISSL EPEDFAVYYCQQRTYFPPTFGQGTKVEIK 10. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 16 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSGYPPTFGQGTKVEIK 11. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 17 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRTNFPPTFGQGTKVEIK 12. EIVLTQSPGTLSLSPGERATLSCRASQSVSASYLAWYQQKPGQ SEQ ID NO: 18 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSSFPPTFGQGTKVEIK 13. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 19 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSYFPPTFGQGTKVEIK 14. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 20 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRWTFPPTFGQGTKVEIK 15. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQ SEQ ID NO: 21 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNFPPTFGQGTKVEIK 16. EIVLTQSPGTLSLSPGERATLSCRASQSVFHRYLPWYQQKPGQ SEQ ID NO: 22 APGLLIYGASKRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRWGYPPTFGQGTKVEIK 17. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 23 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNFPPTFGQGTKVEIK 18. EIVLTQSPGTLSLSPGERATLSCRASQSVRMGYLAWYQQKPG SEQ ID NO: 24 QAPGLLIYGASRRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQRWGFPPTFGQGTKVEIK 19. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 25 APGLLIYGASGRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSDFPPTFGQGTKVEIK 20. EIVLTQSPGTLSLSPGERATLSCRASQSVSTSYLAWYQQKPGQ SEQ ID NO: 26 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSHFPPTFGQGTKVEIK 21. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 27 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRWDFPPTFGQGTKVEIK 22. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 28 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRWGFPPTFGQGTKVEIK 23. EIVLTQSPGTLSLSPGERATLSCRASQSVSAGYLAWYQQKPGQ SEQ ID NO: 29 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSYFPPTFGQGTKVEIK 24. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 30 APGLLIYGASSRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSDFPPTFGQGTKVEIK 25. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 31 APGLLIYGASTRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSYYPPTFGQGTKVEIK 26. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 32 APGLLIYGASRRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRTFFPPTFGQGTKVEIK 27. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 33 APGLLIYGASGRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSEFPPTFGQGTKVEIK 28. EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQ SEQ ID NO: 34 APGLLIYGASMRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSGFPPTFGQGTKVEIK 29. SEQ ID NO: 35 EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPGL LIYGASKRATGIPDRFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSSFPPTFGQGTKVEIK

[0199] Table 6. Summary of mutations in light-chain positions (IMGT numbering) compatible with binding to Nectin-4 (first row are parent clone amino acids).

[0200] Positions 38, 56, 107, 115 and 116 are conserved. Position 114 highly favors F, Y over W found in N4-Fab1-LC. Position L67 is a mutation in the framework close to CDRL2 as defined by IMGT (and in CDR-L2 as defined by Kabat) and was randomized.

[0201] Table 7: Sequences of specific unique clones from heavy chain library. Differences from parent clone N4-Fab1-HC are underlined and bolded. 1. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 36 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWDNGWYFGQGTLVTVSS 2. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 37 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWNKGWYFGQGTLVTVSS 3. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 38 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWTKGWYFGQGTLVTVSS 4. QVQLVESGGGVVQPGGSLRLSCAASGFAFRSYGMHWVRQA SEQ ID NO: 39 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWANGWYFGQGTLVTVSS 5. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 40 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWNNGWYFGQGTLVTVSS 6. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 41 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWSNGWYFGQGTLVTVSS 7. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 42 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTNGWYFGQGTLVTVSS 8. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 43PGKGLEWVALIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWNNGWYFGQGTLVTVSS 9. QVQLVESGGGVVQPGGSLRLSCAASGFTFMSYGMHWVRQA SEQ ID NO: 44 PGKGLEWVALIWYDGSNKLYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWTNGWYFGQGTLVTVSS 10. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 45 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWANGWYFGQGTLVTVSS 11. QVQLVESGGGVVQPGGSLRLSCAASGFNFRSYGMHWVRQ SEQ ID NO: 46 APGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMD SLRAEDTAVYYCARDDWNKGWYFGQGTLVTVSS 12. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 47 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWSNGWYFGQGTLVTVSS 13. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 48 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTNGFYFGQGTLVTVSS 14. QVQLVESGGGVVQPGGSLRLSCAASGFTFKSYGMHWVRQA SEQ ID NO: 49 PGKGLEWVALIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWTNGWYFGQGTLVTVSS 15. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 50 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWAYGWYFGQGTLVTVSS 16. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 51 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTYGYYFGQGTLVTVSS 17. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 52 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTYGFYFGQGTLVTVSS 18. QVQLVESGGGVVQPGGSLRLSCAASGFTFGSYGMHWVRQ SEQ ID NO: 53 APGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMD SLRAEDTAVYYCARDDWANGWYFGQGTLVTVSS 19. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 54 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWSYGWYFGQGTLVTVSS 20. QVQLVESGGGVVQPGGSLRLSCAASGFTFGSYGMHWVRQ SEQ ID NO: 55 APGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMD SLRAEDTAVYYCARDDWTHGWYFGQGTLVTVSS 21. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 56 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWSRGWYFGQGTLVTVSS 22. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 57 PGKGLEWVALIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTNGFYFGQGTLVTVSS 23. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 58 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWRNGWYFGQGTLVTVSS 24. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 59 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDDWDHGWYFGQGTLVTVSS25. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 60 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTHGYYFGQGTLVTVSS 26. QVQLVESGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQA SEQ ID NO: 61 PGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMDS LRAEDTAVYYCARDGWTDGWYFGQGTLVTVSS

[0202] Table 8. Summary of mutations in heavy-chain positions (IMGT numbering) compatible with binding to Nectin-4 (first row are parent clone amino acids).

[0203] Positions 36, 57, 58, 59, 64, 107, 109, 115 and 118 are conserved. Positions 29, 35, 63, 66, 116 seem to favor amino acids found in the parent clone N4-Fab1-HC. Positions 113 and 114 seem to be highly variable.

[0204] Position H66 (Kabat position 58) is part of CDRH2 in Kabat scheme. Position H118 is a mutation in the framework close to CDRH3 in IMGT and was randomized.Table 9A: Light Chain IMGT Numbering and CDR Annotation for N4-Fab1 (SEQ ID NO: 62).Table 9B Heavy Chain IMGT Numbering and CDR Annotation for N4-Fab1 (SEQ ID NO: 63).

[0205] The alignment of selected antibodies with respective germline V genes and J genes. Mutations are highlighted and CDR regions are underlined (Figs.8A-C). Example 3: Effectiveness of radiolabeled conjugates of the fully human anti-Nectin-4 antibody described herein (denoted N4MU01), [161Tb]Tb-DOTA-N4MU01 and [225Ac]Ac- Macropa-N4MU01monotherapy, against non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC)

[0206] Figs. 13A-B demonstrates the efficacy of [225Ac]Ac-Macropa- N4MU01monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model. Mice were either treated with a repeated dose of each treatment, injected intravenously on d0 and on d10 (n = 4-7 mice / group). Fig. 13A shows average tumor growth curves. Fig.13B shows Kaplan-Meier survival curves. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 90 days.

[0207] Figs 14A-B demonstrate efficacy of [225Ac]AC-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H358 in an athymic BALB / c mouse xenograft model. Mice were either treated with a repeated dose of [225Ac]Ac-Macropa-N4MU01 (n = 6 mice) injected intravenously on d0 and on d10 or with saline (n = 4 mice). Fig.14A shows average tumor growth curves. Fig. 14B shows Kaplan Meier survival curve of treatment groups shows Log-rank (mantel-Cox) test for survival comparison. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume ≥ 650 mm3or survival for 50 days. [225Ac]Ac-N4MU01 = [225Ac]Ac-Macropa- N4MU01.

[0208] Figs 15A-B demonstrate the efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H2170 in an athymic BALB / c mouse xenograft model. Mice were either treated with a repeated dose of [225Ac]Ac-Macropa-N4MU01 (n = 8 mice) injected intravenously on d0 and on d10 or with saline (n = 3 mice). Fig.15A shows average tumor growth curves. Fig.15B shows average body weights. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 37 days. [225Ac]Ac-N4MU01 = [225Ac]Ac-Macropa-N4MU01

[0209] Figs16A-C demonstrate the efficacy of [161Tb]Tb-DOTA-N4MU01 monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model. Mice were either treated with a repeated dose of [161Tb]Tb-DOTA-N4MU01 injected intravenously on d0and on d7 or with saline (n = 7 mice / group). Fig.16A shows average tumor growth curves with a t-test performed on d13 following start of treatment. Fig.16B shows Kaplan Meier survival curve of treatment groups shows Log-rank (mantel-Cox) test for survival comparison. Fig.16C shows average body weight of treatment groups over time. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 97 days. [161Tb]Tb-N4MU01 = [161Tb]Tb-DOTA-N4MU01.

[0210] The efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy was investigated in a 4T1.nectin-4syngeneic BALB / c mouse model (n = 5-6 mice / group). Each treatment group received two doses of the treatment on d0 and on d10. Average tumor growth curves showed complete remission for all mice in [225Ac]Ac-Macropa-N4MU01 treatment group (FIG.17A). Kaplan-Meier survival curve (FIG.17B). The study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 30 days. Example 4: Pharmacokinetics of fully human anti-Nectin-4 antibody (N4MU01)

[0211] Pharmacokinetic (PK) profile of [89Zr]Zr-DFO-Nectin-4, comprising the fully human anti-nectin-4 antibody described herein, injected in CD-1 nude mice was biphasic with fast distribution half-life t1 / 2α of 3.4 h and a moderate elimination t1 / 2ß of 63 h. Over 85% of the antibody (% injected activity %IA) was eliminated in the first 10 hours, followed by a slow elimination half-life. The area under the curve AUC (%IA.h / mL), clearance Cl (mL / h) and volume of distribution at steady state Vss (mL / h*102) was 729.4, 0.14 and 9.57, respectively.

[0212] In contrast the PK profile of enfortumab antibody AGS-22ME in humans showed a very slow monophasic half-life. The t1 / 2 of AGS-22ME in humans was 3.6 days

[0040] . 9MW2821 is another anti-Nectin-4 antibody that is currently in clinical development. The preclinical characteristics including pharmacokinetics of enfortumab vedotin and 9MW2821 has been evaluated by Zhou et al.,

[0041] . In summary, Zhou et al., shows that the pharmacokinetics of enfortumab vedotin and 9MW2821 are similar and that their monophasic PK is significantly slower than that exhibited by the fully human anti-nectin-4 antibody described herein, N4MU01. As a result of the slower PK and differences in the binding characteristics of enfortumab vedotin and 9MW2821, non-target tissues uptake are significantly higher particularly in highly vascularized non-target tissues such as the lungs, heart, spleen and liver compared with those of N4MU01. The faster PK of N4MU01 allows for significantly reduced radiation doses to healthy tissues at an optimized dosing to nectin-4 expressing tumors.

[0213] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0214] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers for proteins and / or nucleic acid provided in the Tables or elsewhere, are incorporated by reference in its entirely.

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Claims

CLAIMS 1. An antibody which specifically binds Nectin-4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs, as defined using IMGT, are: a) CDR-L1 QSV X1X2X3Y (SEQ ID NO: 1); CDR-L2 GA; CDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3); CDR-H1 GF X7F X8SYG (SEQ ID NO: 4); CDR-H2 IWYDG X9NK (SEQ ID NO: 5); and CDR-H3 AR D X10W X11X12G X13Y (SEQ ID NO: 6) wherein, X1 is an amino acid selected from S, K, F, and R, X2 is an amino acid selected from N, H, A, T, S, and M, X3 is an amino acid selected from S, G, and R, X4 is an amino acid selected from S, Y, W, and T, X5 is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6 is an amino acid selected from W, F, and Y, X7 is an amino acid selected from T, A, and N, X8 is an amino acid selected from R, M, K, and G, X9 is an amino acid selected from S and T, X10 is an amino acid selected from D and G, X11 is an amino acid selected from T, D, N, A, S, and R, X12 is an amino acid selected from N, K, Y, H, R, and D; and / or X13 is an amino acid selected from W, F, and Y; b)CDR-L1 QSVSRY (SEQ ID NO: 68); CDR-L2 DA; CDR-L3 QQRYNWPPD (SEQ ID NO: 70); CDR-H1GFTFSSYG(SEQ ID NO: 71); CDR-H2 ISYDGSNK (SEQ ID NO: 72); and CDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73); or c) CDR-L1 QDISNY (SEQ ID NO: 74); CDR-L2 AA; CDR-L3 QQSYTTRTT (SEQ ID NO: 76); CDR-H1 GFRFSGYP (SEQ ID NO: 77); CDR-H2 IWYDGRNR (SEQ ID NO: 78); and CDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).

2. The antibody of claim 1, wherein the the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR- H3, wherein the amino acid sequences of said CDRs, as defined using IMGT, are: CDR-L1 QSV X1 X2 X3Y (SEQ ID NO: 1); CDR-L2 GA; CDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3); CDR-H1 GF X7F X8SYG (SEQ ID NO: 4); CDR-H2 IWYDG X9NK (SEQ ID NO: 5); and CDR-H3 AR D X10W X11X12G X13Y (SEQ ID NO: 6) wherein, X1 is an amino acid selected from S, K, F, and R, X2 is an amino acid selected from N, H, A, T, S, and M, X3 is an amino acid selected from S, G, and R, X4 is an amino acid selected from S, Y, W, and T,X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6is an amino acid selected from W, F, and Y, X7is an amino acid selected from T, A, and N, X8is an amino acid selected from R, M, K, and G, X9is an amino acid selected from S and T, X10is an amino acid selected from D and G, X11is an amino acid selected from T, D, N, A, S, and R, X12is an amino acid selected from N, K, Y, H, R, and D; and / or X13is an amino acid selected from W, F, and Y.

3. The antibody of claim 1 or 2, wherein the the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR- H3, wherein the amino acid sequences of said CDRs, as defined using IMGT, are: CDR-L1 QSVSNSY (SEQ ID NO: 80); CDR-L2 GA; CDR-L3 QQRSNWPPT (SEQ ID NO: 81); CDR-H1 GFTFRSYG (SEQ ID NO: 82); CDR-H2 IWYDGSNK (SEQ ID NO: 83); and CDR-H3 ARDDWTNGWY (SEQ ID NO: 84).

4. The antibody of any one of claims 1 to 3, wherein the light chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 7-35 or 62; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 7-35 or 62, wherein CDR-L1 is SEQ ID NO: 1, CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is SEQ ID NO: 1, CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3.

5. The antibody of any one of claims 1 to 4, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 36-61 or 63; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 36-61 or 63, wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6.

6. The antibody of claim 1, wherein the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR- H3, wherein the amino acid sequences of said CDRs, as defined using IMGT, are: CDR-L1 QSVSRY (SEQ ID NO: 68); CDR-L2 DA; CDR-L3 QQRYNWPPD (SEQ ID NO: 70); CDR-H1 GFTFSSYG (SEQ ID NO: 71); CDR-H2 ISYDGSNK (SEQ ID NO: 72); and CDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73).

7. The antibody of claim 1 or 6, wherein the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 64; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 64, wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO: 70; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO:

70.

8. The antibody of any one of claims 1 or 6-7, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 65; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 65, wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73.

9. The antibody of claim 1, wherein the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR- H3, wherein the amino acid sequences of said CDRs, as defined using IMGT, are: CDR-L1 QDISNY (SEQ ID NO: 74); CDR-L2 AA; CDR-L3 QQSYTTRTT (SEQ ID NO: 76); CDR-H1 GFRFSGYP (SEQ ID NO: 77); CDR-H2 IWYDGRNR (SEQ ID NO: 78); and CDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).

10. The antibody of claim 1 or 9, wherein the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 66; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 66, wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO: 76; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO:

76.

11. The antibody of any one of claims 1 or 9-10, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 67; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID Ns: 67, wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79.

12. The antibody of any one of claims 1 to 11, wherein the antibody is a humanized or human antibody.

13. The antibody of any one of claims 1 to 12, wherein the antibody is a single chain antibody.

14. The antibody of any one of claims 1 to 13, wherein the antibody is an antibody fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, and diabodies.

15. The antibody of any one of claims 1 to 14, wherein the antibody is an IgG, optionally IgG1.

16. A nucleic acid molecule encoding the antibody of any one of claims 1 to 15.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A cell comprising the nucleic acid molecule of claim 16, the vector of claim 17, or expressing the antibody of any one of claims 1 to 15.

19. An immunoconjugate comprising the antibody of any one of claims 1 to 15 and a therapeutic agent and / or detectable label.

20. The immunoconjugate of claim 19, wherein the detectable label and / or therapeutic agent is a radionuclide, optionally an alpha- beta- or gamma-emitting radionuclide.

21. The immunoconjugate of claim 19 or 20, wherein the antibody is labeled with the radionuclide using a bifunctional chelator.

22. The immunoconjugate of claim 21, wherein the bifunctional chelator is desferrioxamine (DFO) or 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7- yl)methyl)-4-isothiocyanatopicolinic acid (macropa-SCN).

23. The immunoconjugate of any one of claims 19 to 22, wherein the radionuclide is225Actinium.

24. The immunoconjugate of any one of claims 19 to 22, wherein the radionuclide is89Zirconium.

25. The immunoconjugate of any one of claims 19 to 22, wherein the radionuclide is161Terbium.

26. A composition comprising the antibody of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17, the cell of claim 18, or the immunoconjugate of any one of claims 19 to 25, and a diluent or pharmaceutically acceptable carrier.

27. The antibody of any one of claims 1 to 15, the immunoconjugate of any one of claims 19 to 25, or the composition of claim 26, for use in treating cancer in a subject.

28. The antibody, immunoconjugate or composition for use of claim 27, wherein the cancer is breast cancer.

29. The antibody, immunoconjugate or composition for use of claim 27 or 28, wherein the cancer is triple negative breast cancer.

30. The antibody, immunoconjugate or composition for use of claim 27, wherein the cancer is lung cancer.

31. The antibody, immunoconjugate or composition for use of claim 27 or 30, wherein the cancer is non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer.

32. The antibody, immunoconjugate, or composition for use of any one of claims 27 to 31, wherein the subject is a human.

33. A method for detecting Nectin-4 expression in a biological sample, the method comprising a) obtaining a biological sample suspected of containing Nectin-4, b) contacting the sample with the antibody of any one of claims 1 to 15 or the immunoconjugate of any one of claims 19 to 25 under conditions permissive for forming an antibody:Netctin-4 complex, and c) detecting the presence of any complex, wherein the presence of detectable complex is indicative that the sample expresses Nectin-4.

34. A method of detecting whether a subject has cancer, the method comprising obtaining a biological sample suspected of containing a cancer cell, the biological sample having been obtained from the subject, contacting the sample with the antibody of any one of claims 1 to 15 or the immunoconjugate of any one of claims 19 to 25 under conditions permissive for forming an antibody:Nectin-4 complex, and detecting the presence of an antibody complex, wherein the presence of an antibody complex indicates that the subject has cancer, optionally selected from, breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, bladder cancer, gastric cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer.

35. The method of claim 33, wherein the biological sample is obtained from a subject having or suspected of having a cancer, optionally wherein the cancer is selected from breast cancer,preferably triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer, optionally wherein the biological sample is a tumor sample.

36. A method of determining if a subject has an Nectin-4-expressing tumor, the method comprising administering the immunoconjugate of any one of claims 19 to 25, or the composition of claim 26 to the subject, and detecting the presence of the label by imaging, optionally the subject has or is suspected of having cancer, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, and lung cancer.

37. The method of any one of claims 34 to 36, wherein the cancer is breast cancer.

38. The method of any one of claims 34 to 36, wherein the cancer is triple negative breast cancer.

39. The method of any one of claims 34 to 36, wherein the cancer is lung cancer.

40. The method of any one of claims 34 to 36, wherein the cancer non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer.

41. A method for imaging a tumor in a subject, the method comprising administering the immunoconjugate of any one of claims 19 to 25, or the composition of claim 26 to the subject, and detecting the presence of the label.

42. The method of claim 37, wherein the method further comprises a) detecting Nectin-4 expression in a biological sample according to the method of claim 34 or 35, wherein the biological sample is obtained from the subject, and / or b) imaging an Nectin-4 -expressing tumor in the subject according to the method of claim 37, wherein the detecting and / or imaging is done before, during, or following administering the antibody, immunoconjugate, or composition.

43. Use of the immunoconjugate of any one of claims 19 to 25, or a composition comprising the immunoconjugate and a pharmaceutically acceptable carrier for treating cancer in a subject in need thereof, optionally the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer,gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, and lung cancer, in a subject in need thereof.

44. The use of claim 43, wherein the cancer is breast cancer, optionally triple negative breast cancer.

45. The use of claim 43, wherein the cancer is lung cancer, optionally non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer.

46. The method or use of any one of claims 33 to 45, wherein the subject is a human. The method or use of any one of claims 33 to 46, wherein the antibody is fully human.

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