Novel Anti-nectin-4 antibodies, conjugated biological molecules, pharmaceutical compositions and uses thereof

Abstract

The present invention provides an antibody or an antigen-binding portion thereof that binds to Nectin-4. Also provided is an antibody-drug conjugate (ADC) including a drug moiety conjugated to the anti-Nectin-4 antibody or antigen-binding portion thereof. Also disclosed are methods of using the antibody or ADC for inhibiting cancer cell proliferation.

Description

NOVEL ANTI-NECTIN-4 ANTIBODIES, CONJUGATED BIOLOGICAL MOLECULES, PHARMACEUTICAL COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Patent Applications No. 63 / 688,885 (filed on August 30, 2024) and 63 / 725,554 (filed on November 27, 2024). The entirety of the aforementioned application is incorporated herein by reference.FIELD

[0002] The present disclosure relates to antibodies, antibody-drug conjugates (ADCs) and methods of use thereof to treat cancer. Particularly, the present disclosure relates to anti-Nectin-4 antibodies and antigen-binding portions thereof, ADCs derived therefrom, pharmaceutical compositions including the antibodies or ADCs, and methods of using the same to inhibit cancer cell proliferation.BACKGROUND

[0003] Nectins are a novel class of cell adhesion proteins, which regulate cell-cell adhesions either cooperatively with or independently of cadherins. Nectins comprise a family of four members, i.e., Nectin-1, -2, -3, and -4. All the nectins have one extracellular region with three Ig- like loops, one transmembrane segment, and one cytoplasmic tail. Among those members, Nectin-4 is a type I transmembrane polypeptide and a member of the immunoglobulin superfamily; it is encoded by the poliovirus receptor related-4 (PVRL4) gene and is 510 amino acids in full length. Nectin-4 has an extracellular region with three immunoglobulin-like domains and serves a role in the formation and maintenance of adhesion connection together with cadherin. Nectin-4 is specifically expressed in embryo and placenta as well as tumor cells and has been found in some studies to be closely related to the generation and development of a variety of tumor cells (Li et al., Front Oncol, 14: 1354543 (2024). Therefore, Nectin-4 has become an important target for the diagnosis and treatment of many tumors or cancers.

[0004] Some earliest antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with antigens. Antibodies derived from non-human species are humanized to enhance the effector function and / or reduce the adverse reactions. Antibody-drug conjugate (ADC) technology can precisely deliver anti-tumor drugs to target tumor cells by utilizing the ability of antibodies to specifically recognize specific antigens on the surface of tumor cells and accumulate and intracellularly release the anti-tumor drugs, thereby precisely killing tumors. An antibody-drug conjugate is generally composed of three parts: an antibody, a small molecule drug, and a linker coupling the two together. Antibody-drug conjugates have been considered as one of the most promising anti -neoplastic drugs due to their appropriatemolecular weights, high stability, strong targeting properties, and minor toxic and side effects.

[0005] Exatecan [IUPAC name: (lS,9S)-l-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl- l,2,3,9,12,15-hexahydro-10H,13H-benzo[de] pyrano[3',4':6,7] indolizino[l,2-b] quinoline-10,13- dione; CAS No.: 171335-80-1; Molecular Formula: C24H22FN3O4) is a topoisomerase I inhibitor which is a structural analog of camptothecin with antineoplastic activity. In the past, exatecan has been clinically evaluated as a stand-alone chemotherapy drug (exatecan mesylate, DX-8951f), as preclinical studies indicated it was more potent than SN-38-based irinotecan (CPT-11) against various tumor xenograft models, including CPT-11 -resistant tumors. The development of exatecan mesylate as a free drug has been discontinued, but exatecan is still a candidate for cytotoxic drug moiety of antibody drug conjugates with an excellent anti-tumor effect.SUMMARY OF THE INVENTION

[0006] The present disclosure provides novel antibodies that bind to Nectin-4, antigen-binding portions thereof, and antibody conjugates, such as antibody-drug conjugates (ADCs), derived from the antibodies.

[0007] In one aspect, the disclosure provides an isolated anti-Necctin-4 antibody or an antigenbinding portion thereof, including a heavy chain variable region that includes three heavy chain complementarity determining regions (HCDRs) and a light chain variable region that includes three light chain complementarity determining regions (LCDRs), wherein the HCDRs and the LCDRs have amino acid sequences of (a) SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (b) SEQ ID NOs: 29, 30, 31, 32, 33, and 34 , respectively; (c) SEQ ID NOs: 35, 36, 37, 38, 39, and 40, respectively; (d) SEQ ID NOs: 41, 42, 43, 44, 45, and 46, respectively; (e) SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively; (f) SEQ ID NOs: 53, 54, 55, 56, 57, and 58, respectively; (g) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively; (h) SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (i) SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; or (j) SEQ ID NOs: 77, 78, 79, 80, 81, and 82, respectively.

[0008] In certain embodiments, the anti-Nectin-4 antibody or the antigen-binding portion thereof includes the heavy chain variable region and the light chain variable region including amino acid sequences having at least 90% sequence identity to (a) SEQ ID NOs: 1 and 2, respectively; (b) SEQ ID NOs: 3 and 4, respectively; (c) SEQ ID NOs: 5 and 6, respectively; (d) SEQ ID NOs: 7 and 8, respectively; (e) SEQ ID NOs: 9 and 10, respectively; (f) SEQ ID NOs: 11 and 12, respectively; (g) SEQ ID NOs: 13 and 14, respectively; (h) SEQ ID NOs: 15 and 16, respectively; (i) SEQ ID NOs: 17 and 18, respectively; or (j) SEQ ID NOs: 19 and 20, respectively.

[0009] In another aspect, the disclosure provides an antibody-drug conjugate (ADC) specific to Nectin-4, including a drug moiety conjugated to the antibody or the antigen-binding portion thereof as described herein via a linker moiety. The drug moiety may be a cytotoxic agent such as achemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0010] In certain embodiments, the drug moiety is the cytotoxic agent selected from pyrrolobenzodiazepine compounds or derivatives thereof (e.g., PBD), auristatin compounds or derivatives thereof (e.g., MMAE, MMAF), maytansinoid compounds or derivatives thereof (e.g., maytansine, DM1, DM4, DM21), duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof (e.g., calicheamicin), anthracycline compounds or derivatives thereof (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof (e.g., cryptophycin 52), drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof (e.g., SN-38, belotecan, exatecan, deruxtecan).

[0011] In certain embodiments, the drug moiety is a topoisomerase inhibitor, including a topoisomerase I inhibitor and a topoisomerase II inhibitor.

[0012] In certain embodiments, the drug moiety is a topoisomerase I inhibitor, including but not limited to Camptothecin (CPT) or Non-camptothecins. Examples of topoisomerase I inhibitors include Irinotecan, Topotecan, Camptothecin, Rubitecan, MLN576, Exatecan, Belotecan, Seconeolitsine, SN-38, Genz-644282, Betulinic acid, P-Lapachone, Karenitecin, Gimatecan, Namitecan, Edotecarin, SW044248, LMP744, T-2513, Podocarpusflavone A, Indimitecan, Lurtotecan, TP3011 and 10-hydroxy camptothecin.

[0013] In another aspect, the present disclosure provides a pharmaceutical composition including the antibody, the antigen-binding portion thereof, or the ADC as described herein, and a pharmaceutically acceptable carrier.

[0014] In another aspect, the present disclosure provides a method for inhibiting proliferation of cancer cells or treating cancer, including administering to a subject in need thereof an effective amount of the antibody, the antigen-binding portion thereof, or the ADC (for example, OBI-904) as described herein.

[0015] In certain embodiments, the method for treating cancer includes administering to a subject in need thereof an effective amount of the ADC (for example, OBI-904) as described herein.

[0016] In certain embodiments, the cancer is a Nectin-4 expressing cancer, which includes but are not limited to breast cancer, bladder cancer, urothelial cancer, lung cancer, ovarian cancer, pancreas cancer, esophageal cancer, gallbladder cancer, hepatocellular cancer, gastric cancer, renal cell cancer, colorectal cancer, colon cancer, cervical cancer and prostate cancer (Li et al., FrontOncol, 14: 1354543 (2024).

[0017] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the detailed description of preferred embodiments and the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs. 1 A and IB illustrated the amino acid sequences of the heavy chain variable regions (FIG. 1A) and light chain variable regions (FIG. IB) of ten anti-Nectin-4 antibodies designated as 08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, and 13C24.

[0019] FIGs. 2A and 2B illustrated the pharmacokinetic profiles of the anti-Nectin-4 antibodies in mouse. FIG. 2A illustrated serum concentration vs. time curves of anti-Nectin-4 antibodies in naive mice. FIG. 2B illustrated pharmacokinetic (PK) parameters of six anti-Nectin-4 antibodies.

[0020] FIG. 3 illustrated the ADCP (antibody-dependent cellular phagocytosis) of the anti- Nectin-4 antibodies.

[0021] FIG. 4 showed the reducing PAGE of 10K06 and 10K06-DAR8-ADC (OBI-904).

[0022] FIG. 5 showed the hydrophobic interaction chromatography (HIC) results of 10K06- DAR8-ADC (OBI-904).

[0023] FIGs. 6A and 6B showed the in-vitro cytotoxicity IC50 values of anti-Nectin-4 ADCs in human colorectal cancer cell line (DLD-1; FIG. 6A) and human hypopharyngeal squamous cancer cell line (FaDu; FIG. 6B).

[0024] FIGs. 7A to 7C showed the in-vivo efficacy assay of anti-Nectin-4 ADCs in Nectin-4 overexpression PC-3 human prostate cancer cell-derived xenograft in BLAB / c nude mice (FIG. 7 A), Nectin-4 middle expressed FaDu human head and neck cancer cell-derived xenograft in BLAB / c nude mice (FIG. 7B) and Nectin-4 low expressed TFK-1 human cholangiocarcinoma cell- derived xenograft in BLAB / c nude mice (FIG. 7C).DETAILED DESCRIPTION OF THE INVENTIONAbbreviations

[0025] ACN: acetonitrile; ADC: Antibody-drug conjugate; ADCC: antibody-dependent cellular cytotoxicity; BCN: Bicyclononyne; bsADC: bispecific ADC; CDR: complementarity-determining region; CE-SDS: capillary electrophoresis-sodium dodecyl sulfate; DAR: drug-to-antibody ratio; DBCO: Dibenzocyclooctyne; DL: linker-payload; DMSO: dimethyl sulfoxide; FA: formic acid; GlcNAc: N-acetylglycosamine; HIC: hydrophobic interaction chromatography; HRMS: high resolution mass spectrometry; mAb: monoclonal antibody; NaOAc: sodium acetate; NaOH: sodium hydroxide; NSCT: sialylated complex type N-glycan; PAB: Para-aminobenzyl alcohol; PBS:phosphate buffered saline; TFA: trifluoroacetic acid; T785: l-(4-aminobutyl)-2-butylimidazo[4,5- c]quinolin-4-amine; PSar20: polysarcosine 20; PDAC: pancreatic ductal adenocarcinoma; SMCC: succinimidyl-trans-4-(N-maleimidylmethyl)cyclohexane-l -carboxylate; MMAE: monomethyl auristatin E; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.Definitions

[0026] As used herein, the articles “a” and “an” refer to one or more than one (z.e., at least one) grammatical object of the article. For example, “an element” means one element or more than one element.

[0027] As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and have a linear or branched structure. A glycan may include natural sugar residues (e.g., glucose, N- acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2’ -fluororibose, 2’ -deoxyribose, phosphomannose, 6’ sulfo N-acetylglucosamine, etc.).

[0028] As used herein, the terms “fucose”, “core fucose”, and “core fucose residue” are used interchangeably and refer to a fucose in a-l,6-position linked to the N-acetylglucosamine.

[0029] As used herein, the terms “N-glycan” and “N-linked glycan” are used interchangeably and refer to an N-linked oligosaccharide attached via an N-acetylglucosamine (GlcNAc) to the amide nitrogen of an asparagine residue in a protein or an antibody.

[0030] As used herein, the terms “glycosylation pattern” and “glycosylation profile” are used interchangeably and refer to the characteristic “fingerprint” of the N-glycan species on a glycoprotein or antibody. The glycosylation profile can be obtained by collecting a N-glycan species released from a glycoprotein through enzymatic digestion or chemical hydrolysis, and then analyzing the carbohydrate structure, for example, LC-HPLC, or MALDI-TOF MS, and the like.

[0031] As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

[0032] As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0033] As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

[0034] As used herein, the term “specific binding” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10'6mol / L, about 10'7mol / L, or about 10'8mol / L, or less.

[0035] The term “antibody” is intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies thatmimic the structure and / or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof, each containing at least one CDR derived from an antibody disclosed herein. Antibodies include antibody fragments, antibody variants, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies and the like. Antibodies can be generated in mice, rabbits or humans.

[0036] The antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), Facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al., Nature, 341 :544-546 (1989)), an isolated CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, bispecific and multispecific antibodies formed from antibody fragments.

[0037] Multispecific or bi-specific antibodies or fragments thereof may be specific for different epitopes of one target antigen (e.g., Nectiin-4) or may contain antigen-binding domains specific for more than one target antigens. The antibodies disclosed herein can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity. In one embodiment, the bi-specific antibody comprises a first binding domain that binds to Nectin-4 antigen and a second binding domain that specifically binds to a R0R1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfrl, TNF alpha, tissue factor, folate receptor alpha, c-MET, HER3, EGFR, HER2, or TROP2.

[0038] An antibody light or heavy chain variable region includes a framework region (FW) interrupted by three hypervariable regions, referred to as complementarity determining regions or CDRs. According to one aspect of the invention, the antibody or the antigen-binding portion thereof may include the following structure:Leader Sequence-FWl-CDRl-FW2-CDR2-FW3-CDR3- wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the antibodies disclosed herein are shown in Tables 1 and 2.

[0039] The heavy chain and light chain variable regions of the antibodies disclosed herein or the antigen-binding portions thereof can be from a non-human or human source. The framework of thepresent antibodies or antigen-binding portions thereof can be human, humanized, non-human (e.g., a murine framework modified to decrease antigenicity in humans), or a synthetic framework (e.g., a consensus sequence).

[0040] Antibodies disclosed herein also include chimerized or humanized monoclonal antibodies, generated from non-human (e.g., murine) antibodies of a hybridoma. Also encompassed by the present disclosure are antibodies or antigen-binding portions thereof including one or more variable regions, and certain sequences of the variable region, such as the framework sequence, may be replaced by sequences from at least one different species including, but not limited to, human, rabbits, sheep, dogs, cats, cows, horses, goats, pigs, monkeys, apes, gorillas, chimpanzees, ducks, geese, chickens, amphibians, reptiles and other animals.

[0041] The term “humanized antibody” refers to an antibody including at least one human framework and at least one, preferably all CDRs from a non-human antibody, and in which any constant region present is substantially identical to a human antibody constant region, i.e., about 85-90%, at least about 90%, at least about 95% identical. Hence, all parts of a humanized antibody, except possibly the CDR, are substantially identical to corresponding parts of one or more human antibody sequences. Humanized antibodies can be generated by replacing sequences of the variable region that are not directly involved in antigen binding (e.g., framework) with equivalent sequences from human variable regions.

[0042] All antibody isotypes (including all classes and subclasses) are encompassed by the present disclosure, including IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD or IgE. The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.

[0043] The terms "wild type antibody" and “unmodified antibody” are used interchangeably and as used herein refer to an antibody including an amino acid sequence which lacks one or more of amino acid substitutions disclosed herein.

[0044] Antibodies, antigen-binding portions, fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., a tumor associated antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0045] In one aspect, the present disclosure provides an antibody or an antigen-binding portion thereof that binds to Nectin-4. The antibody or antigen-binding portion thereof includes a heavy chain variable region that includes three heavy chain complementarity determining regions (HCDRs) and a light chain variable region that includes three light chain complementarity determining regions (LCDRs), wherein the HCDRs and the LCDRs have amino acid sequences of (a) SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (b) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; (c) SEQ ID NOs: 35, 36, 37, 38, 39, and 40, respectively; (d) SEQ ID NOs: 41, 42, 43, 44, 45, and 46, respectively; (e) SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively; (f) SEQ ID NOs: 53, 54, 55, 56, 57, and 58, respectively; (g) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively; (h) SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (i) SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; or (j) SEQ ID NOs: 77, 78, 79, 80, 81, and 82, respectively.

[0046] In certain embodiments, the anti-Nectin-4 antibody or the antigen-binding portion thereof includes the heavy chain variable region and the light chain variable region including amino acid sequences having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to (a) SEQ ID NOs: 1 and 2, respectively; (b) SEQ ID NOs: 3 and 4, respectively; (c) SEQ ID NOs: 5 and 6, respectively; (d) SEQ ID NOs: 7 and 8, respectively; (e) SEQ ID NOs: 9 and 10, respectively; (f) SEQ ID NOs: 11 and 12, respectively; (g) SEQ ID NOs: 13 and 14, respectively; (h) SEQ ID NOs: 15 and 16, respectively; (i) SEQ ID NOs: 17 and 18, respectively; or (j) SEQ ID NOs: 19 and 20, respectively.

[0047] All numbers of sequence identity are approximations and may be modified by “about”, which is defined as ± 1%.

[0048] Tables 1 and 2 show the amino acid sequences of the heavy chain variable regions, the light chain variable regions, and the CDRs of the anti-Nectin-4 antibodies.

[0049] Table 1. Sequences of the heavy chain and light chain variable regions of anti-Nectin-4 antibodiesTable 2. CDR and constant region sequences of anti-Nectin-4 antibodies*Human lambda light chain constant region was used in 14B21, 05004, 08C24, 12E03 and 13C24 antibodies; human kappa light chain constant region was used in 14108, 08B04, 02P14, 11023, and 10K06 antibodies as well as and Enfortumab. Human IgGi heavy chain constant region was used in all the anti- Nectin-4 antibodies.

[0050] The term “single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0051] In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is an anti-Nectin-4 antibody selected from 08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, 13C24 or Enfortumab.

[0052] The term “tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0053] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, multiple myeloma and leukemia. More particular examples of such cancers include lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0054] As used herein, “treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0055] An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

[0056] A “combination therapy” refers to a combination of an amount of an ADC and an amount of other biological or chemical drugs that when administered together (either as coadministration and / or co-formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

[0057] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracycline, pyrrolobenzodiazepine, a- amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone), camptothecin (including the synthetic analogue topotecan), bryostatin, callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues), cryptophy cins (particularly cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1), eleutherobin, pancrati statin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall), dynemicin, including dynemicin A; aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara- C”), cyclophosphamide, thiotepa, taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil, GEMZAR® gemcitabine, 6- thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin; vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine (XELODA®, Roche), and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0058] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceuticallyacceptable organic or inorganic salts of a cytotoxic agent or an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.Antibody-drug conjugates

[0059] In certain embodiments, the antibody-drug conjugates (ADC) is represented by Formula (I):Ab-(DL)n(I); wherein Ab is an antibody or an antigen-binding fragment thereof capable of binding to one or more of tumor-associated antigens or cell-surface receptors;DL is a linker-payload as described herein where the payload is a drug moiety, and DL forms a covalent linkage with the antibody or the antigen-binding fragment thereof; and n is a drug-to-antibody ratio (DAR) ranging from 2 to 32.

[0060] In some embodiments, the present disclosure provides the linker-payload DL having the structure of Formula (II):EC - L - P(n). wherein E is a hydrophilic moiety including: polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly (glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), saccharides, or any combination thereof;C is a bioorthogonal group for conjugation to an antibody or an antigen-binding fragment thereof; L is a linker unit including a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; andP is a payload, wherein the payload is a drug moiety.

[0061] In certain embodiments, the hydrophilic moiety E has a molecular weight ranging from 60 Da to 13 kDa, from 100 Da to 10 kDa, from 200 Da to 9 kDa, from 250 Da to 8 kDa, from 300 Da to 7 kDa, from 350 Da to 6 kDa, from 400 Da to 5 kDa, from 450 Da to 4 kDa, from 500 Da to 3 kDa, from 600 Da to 3 kDa, from 700 Da to 3 kDa, from 800 Da to 3 kDa, from 900 Da to 3 kDa, or from 1 kDa to 3 kDa, or from 500 Da to 2.5 kDa. In certain embodiments, the hydrophilic moiety E includes ethylene glycol or polyethylene glycol (PEG) with or without other chemical moieties at one or two ends of the PEG, and the PEG may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ethylene glycol units, or the ethylene glycol units may range between any two of the numbers listed above.

[0062] The term “bioorthogonal group C” refers to a non-native chemical group that can be used to conjugate the linker-payload with an antibody or an antigen-binding fragment thereof under the conditions of living systems without affecting the activity of the antibody or antigen-binding fragment thereof. In certain embodiments, the bioorthogonal group C is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a maleimide group, a a,P-unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4-dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group. In certain embodiments, the bioorthogonal group C is an alkyne group, which can react with an azide introduced at a specific site on an antibody through metal-free azide-alkyne cycloaddition, resulting in antibody conjugation via a triazole linkage. In other embodiments, the bioorthogonal group C is a cyclooctyne, DBCO, or BCN group, which can react with an azide on an antibody (e.g., an azide on an N-glycan at an asparagine residue in an antibody heavy chain) without a catalyst via strain- promoted azide-alkyne cycloaddition to achieve antibody conjugation via a triazoline linkage. In other embodiments, the bioorthogonal group C is a tetrazine group, which can react with a transcyclooctene (TCO) on an antibody to form a dihydropyridazine linkage, resulting in antibody conjugation.

[0063] The term “linker unit L” refers to an element connecting the bioorthogonal group C with the payload P and including a cleavable moiety. In certain embodiments, the linker unit L is a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety or a hydrolysable moiety, each of which being directly linked with the bioorthogonal group C and / or directly linked with the payload P. In certain embodiments, the linker unit L includes any one ormore of the listed cleavable moieties and an intervening spacer (usually with a molecular weight of between 15 Da and 1800 Da) for connecting with the bioorthogonal group or the payload. The spacer may be a di-functional or tri -functional chemical moiety that is capable of covalently linking together the cleavable moiety of the linker unit L and any one or two of the bioorthogonal group C, the payload P, and the hydrophilic moiety E. Examples of the spacer include but not limited to amino acids and aminobenzyl alcohols.

[0064] The protease-cleavable peptide moiety may be a peptide composed of two or more natural or non-natural amino acids that can be cleaved by a peptidase. Preferably, the protease-cleavable peptide moiety may be a dipeptide selected from valine-alanine (VA), valine-cysteine (VC), phenylalanine-glycine (FG), phenylalanine-lysine (FK), alanine-alanine (AA), glycine- valine (GV), or glycine-cysteine (GC); alternatively, a peptide may be composed of said dipeptide and one to eight additional amino acids, for example, a tetrapeptide where two additional glycine or alanine residues are attached to the N-terminal of said dipeptide.

[0065] The glycosidase-cleavable sugar moiety may a sugar residue linked via an oxygen glycosidic bond to a self-immolative group, for example, a glucuronic acid linked via an oxygen glycosidic bond to a p-aminobenzyl alcohol linker, which can be cleaved by P-glucuronidase.

[0066] The pH sensitive moiety may be an acid-liable moiety that can be hydrolyzed at acidic pH and optionally an intracellularly-cleavable moiety cleavable by the low pH environment of endosomal and lysosomal vesicles. The acidic pH condition is beneficial to release payload from the ADC. Examples of the pH sensitive moiety may be one or more amino acids spacers between the drug and the linker.

[0067] The hydrolysable moiety may be a chemical moiety that can be cleaved by hydrolase. Preferably, the hydrolase is an esterase.

[0068] The term “payload” as used herein refers to a molecule to be carried and delivered by an antibody or an antigen-binding fragment thereof. The term “drug moiety” refers to a drug molecule (e.g., a cytotoxic agent), an inhibitor of an enzyme, a ligand of a receptor, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or a prodrug thereof. In certain embodiments, the payload is selected from a chemotherapeutic agent, a toxin, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent.

[0069] In certain embodiments, the toxin is selected from pyrrolobenzodiazepine compounds or derivatives thereof (e.g., PBD), auristatin compounds or derivatives thereof (e.g., MMAE, MMAF), maytansinoid compounds or derivatives thereof (e.g., maytansine, DM1, DM4, DM21), duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds orderivatives thereof (e.g., calicheamicin), anthracycline compounds or derivatives thereof (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophy cin compounds or derivatives thereof (e.g., cryptophy cin 52), drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof (e.g., SN-38, belotecan, exatecan, deruxtecan).

[0070] In certain embodiments, the linker unit (L) of Formula (II) has the structure of LB-QCL-BP, and the linker-payload DL is further represented by Formula (III):wherein:(a) C is a bioorthogonal group for conjugation to an antibody or an antigen-binding fragment thereof;(b) E is a hydrophilic moiety as defined in Formula (II);(c) Bpis a branch unit that covalently connects QCL, E and P, wherein the branch unit includes aR3J®® functional group defined as1, whereinA is an aromatic group;R3 is linked to E and is selected from -C(O)-, -C(O)O-, -C(O)NH-, alkyl-O-, alkyl-NH-, alkyl- C(O)-, alkyl-C(O)-O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-SO2-, alkyl-S-, alkyl-O-P(O)-O2-, alkyl-O-C(O)-NH-, or triazole;(d) QCLis a cleavable unit including a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety;(e) LBis a bridge unit that covalently connects C and QCL; and(f) P is a payload, wherein the payload is a drug moiety.

[0071] In certain embodiments, the hydrophilic moiety E of Formula (III) includes PEG and may have the formula of:wherein the wavy line indicates the site of covalent attachment to Bp,Ri is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, orR2 is selected from H, SO3H, PO3H2, a sugar derivative, C1-C10 (hetero) alkyl group, C3-C10 (hetero) cycloalkyl group, C2-C10 alkyl-NEh, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(Ci-C3 alkyl), C2-C10 alkyl-N (C1-C3 alkyl)2, or sarcosines, and the subscript n is an integer ranging from 2 to 72.

[0072] In another aspect, the present disclosure provides a method for preparing a glycoengineered antibody, including conjugating an antibody with a glycan moiety in the presence of a glycosynthase to obtain the glycoengineered antibody, wherein the antibody has a fucosylated or non-fucosylated N-acetylglucosamine linked to an asparagine residue (e.g., N297 of human IgG heavy chain constant region) of the antibody. The conjugation between the antibody and the glycan moiety disclosed herein is accomplished through deglycosylation and transglycosylation reactions catalyzed by one or more glycosynthases and variants thereof. In certain embodiments, the glycosynthase variants are EndoSd-D232M and EndoSz-D234M. Exemplary EndoSd-D232M and EndoSz-D234M are as described in PCT patent publication W02020006176A1, the content of which is incorporated herein by reference in its entirety.

[0073] In another embodiments, the linker-payload DL is further represented by a structure of the following formula (IV):wherein z is an integer ranging from 6 to 48.

[0074] In one embodiment, the linker-payload DL is further represented by a structure of Formula (V) (referred to as DBCO-GGVA-Hydra-PAB-PEG24-Exatecan or DL-5):

[0075] In certain embodiments, the linker-payload DL is further represented by a structure of the following formula (VI):wherein z is an integer ranging from 6 to 48.

[0076] In one embodiment, the linker-payload DL is further represented by a structure of Formula (VII) (referred to as BCN-GGVA-Hydra-PAB-PEG24-Exatecan or DL-1):

[0077] In another aspect, the present disclosure provides a glycosite-specific ADC as formula (VIII):wherein Ab is a glycoengineered antibody that binds to Nectin-4;G represents a glycan moiety connected to an Neu5Ac and the glycoengineered antibody, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose, N-acetyl-glucosamine, glucose, mannose, fucose, and derivatives thereof;Y is a connector moiety independently comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combinations thereof; Z is independently selected from a triazole or imine linkage;L is a linker connecting D and Z;D is a drug moiety; m is a number of Neu5Ac units branching from G, and is an integer from 1 to 4; and p is a number of Y-Z-L-D units attached to each of the Neu5Ac units, and is an integer from 1 to 4.

[0078] In certain embodiments, the glycosite-specific ADC is represented as formula (IX):Q = Af-acetylglacosamsne (GlcIMAcJ= ALacetylneurarnin add (NeuSAc) wherein Ab is a glycoengineered antibody capable of binding to Nectin-4; L is a linker connecting exatecan and the glycoengineered antibody; and a, b are independently 0 or 1. The gly cositespecific ADC represented by formula (IX) includes a glycoengineered antibody carrying two glycan moieties, whereby the two glycan moieties are conjugated with four units of linker-exatecan.

[0079] In another aspect, the present disclosure provides a method for preparing an antibody conjugate, including reacting a glycoengineered antibody disclosed herein with a linker-payload to obtain the antibody conjugate, wherein the linker-payload includes a biorthogonal group, such as a bicyclononyne (BCN) group or a dibenzocyclooctyne (DBCO) group. The glycoengineered antibody was modified to carry out conjugation with the linker-payload by click chemistry. Thismethod offers a unique glycosite-specific antibody conjugate (e.g., a glycosite-specific ADC) platform that is distinct from conventional ones by high product homogeneity. Therefore, sitespecific ADCs with homogeneous DAR (drug-to-antibody ratio) can be prepared readily. These glycosite-specific ADCs have favorable manufacturing, quality control, and in vivo pharmacokinetic profiles.

[0080] The present disclosure further provides a method for preparing a glycan-engineered antibody conjugate, including reacting a glycan-engineered antibody with the linker-payload disclosed herein to obtain the glycan-engineered antibody conjugate, wherein the glycan- engineered antibody includes a fucosylated or non-fucosylated N-acetylglucosamine (GlcNAc) at an asparagine residue coupled to a glycan.

[0081] In some embodiments, the glycan-engineered antibody is obtained by contacting an antibody with a glycosynthase and a glycan oxazoline to couple the glycan with the fucosylated or non-fucosylated GlcNAc. In some embodiments, the glycosynthase includes EndoSd-D232M and EndoSz-D234M.Therapeutic Applications

[0082] The present disclosure also provides methods for inhibiting cell growth or proliferation in vitro, ex vivo or in vivo. The cell may be a cancer cell or a cell prone to tumorigenesis. The methods include contacting the cells with an effective amount of the antibody, the antigen-binding portion thereof, or the ADC as described herein. Pathological cells or tissue such as hyperproliferative cells or tissue may be treated by contacting the cells or tissue with an effective amount of the antibody, the antigen-binding portion thereof, or the ADC of the present disclosure. The cells, such as cancer cells, can be primary cancer cells or can be cultured cells available from tissue banks such as the American Type Culture Collection (ATCC). In one embodiment, the cancer is a Nectin-4 expressing cancer, including one or more of breast cancer, bladder cancer, urothelial cancer, lung cancer, ovarian cancer, pancreas cancer, esophageal cancer, gallbladder cancer, hepatocellular cancer, gastric cancer, renal cell cancer, colorectal cancer, colon cancer, cervical cancer, and prostate cancer (Li et al., Front Oncol, 14: 1354543 (2024).

[0083] In vitro therapeutic efficacy of the present antibody, the antigen-binding portion thereof, or the ADC can be determined using methods well known in the art. The cytotoxicity of the antibody, the antigen-binding portion thereof, or the ADC may be studied by colony formation assay. Functional assays for binding Nectin-4 antigen may be performed via ELISA or SPR. Cell cycle block by the antibody or the antigen-binding thereof may be studied by standard propidium iodide (PI) staining and flow cytometry. Invasion inhibition may be studied by Boyden chambers. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells withinvasive capacity can cross the Matrigel barrier. Other assays include, but are not limited to cell viability assays, apoptosis assays, and morphological assays. Assays can also be done in vivo using a murine model.

[0084] The present disclosure also provides pharmaceutical compositions including the antibody, the antigen-binding portion thereof, or the antibody conjugate (such as an ADC) described herein, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. In certain embodiment, the pharmaceutical composition is effective to inhibit cancer cell proliferation in a subject.

[0085] Routes of administration of the present pharmaceutical compositions include, but are not limited to, intravenous, intramuscular, intranasal, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subdermal, parenteral, rectal, spinal, or epidermal administration.

[0086] The pharmaceutical composition may be administered alone or mixed with another therapeutic agent, for example, a second monoclonal or polyclonal antibody or the antigen-binding portion thereof, a cancer vaccine, or an anti-cancer agent such as DNA damaging or tubulin binding agents, or agents which inhibit angiogenesis, signal transduction pathways or mitotic checkpoints. The combination product may be a mixture of the two ingredients, or they may be covalently attached. In one example, the antibody or antigen-binding portion thereof specifically binds to Nectin-4 is combined with an antibody (monoclonal or polyclonal) or antigen-binding portion thereof specifically binds VEGF. In another example, the second agent is a chemotherapy agent (e.g., cyclophosphamide, 5 -fluorouracil or Actinomycin-D). The additional therapeutic agent may be administered simultaneously with, optionally as a component of the same pharmaceutical preparation, or before or after administration of the claimed antibody of the invention. Actual methods of preparing such dosage forms are known, or will be modified, to those skilled in the art.

[0087] The pharmaceutical compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular composition used, and the route of administration, whether the pharmaceutical composition is used for prophylactic or curative purposes, etc. For example, in one embodiment, the pharmaceutical composition according to the invention is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0088] For ease of administration and uniformity of dosage, oral or parenteral pharmaceutical compositions in dosage unit form may be used. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0089] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In one embodiment, the dosage of such compounds lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. In another embodiment, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.

[0090] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody, an antigen-binding portion, or an ADC of the invention is from about 0.001 to about 60 mg / kg body weight, about 0.01 to about 30 mg / kg body weight, about 0.01 to about 25 mg / kg body weight, about 0.5 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, about 10 to about 20 mg / kg body weight, about 0.75 to about 10 mg / kg body weight, about 1 to about 10 mg / kg body weight, about 2 to about 9 mg / kg body weight, about 1 to about 2 mg / kg body weight, about 3 to about 8 mg / kg body weight, about 4 to about 7 mg / kg body weight, about 5 to about 6 mg / kg body weight, about 8 to about 13 mg / kg body weight, about 8.3 to about 12.5 mg / kg body weight, about 4 to about 6 mg / kg body weight, about 4.2 to about 6.3 mg / kg body weight, about 1.6 to about 2.5 mg / kg body weight, about 2 to about 3 mg / kg body weight, or about 10 mg / kg body weight.

[0091] The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.EXAMPLESExample 1. Characterization of anti-Nectin-4 antibodies

[0092] The immunogen construct, mouse immunization, hybridoma screening and humanization of anti-Nectin-4 antibodies were performed by ALIVAMAB DISCOVERY SERVICES, LLC. FIG. 1 illustrated the sequences of ten anti-Nectin-4 monoclonal antibodies (08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, and 13C24) and the commercially available Enfortumab. All mAbs were screened using hybridomas derived from a humanized mouse that was immunized with human Nectin-4 DNA and human Nectin-4 protein. Fully humanized Abs were recombinant with hlgGi constant region.Example 2. Binding affinity of anti-Nectin-4 antibodies assessed by Octet BLI (BiolayerInterferometry) analysis

[0093] 2.1 Experimental Material(a) Biosensor: Anti-Human IgG Fc Capture (AHC) Analyte: Human Nectin-4 protein (Aero Biosystems)(b) Ligand: Anti-Nectin-4 antibody(c) Assay buffer: PBS (pH7.4) with 0.02% Tween 20

[0094] 2.2 Experimental Method

[0095] BLI analysis2.2.1. Anti-Nectin-4 antibodies were first captured on Anti-Human IgG Fc Capture (AHC) biosensors with 5 / / g / mL in assay buffer and loading about 1.0 nm signal onto the biosensors.2.2.2. Human Nectin-4 protein (Aero Biosystems) was prepared in two fold serial dilution of analyte with assay buffer. The final concentrations of Nectin-4 were 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 nM.2.2.3. Running condition:Association: 300 secondsDissociation time: 600 seconds2.2.4. Dissociation constant (KD) values were calculated using a 1 : 1 global fit model using ForteBio’s Data Analysis software.2.2.5. Experiment parameters of each analyte are listed in below.(a) Sensor: AHC(b) Analyte: Human Nectin-4(c) Analyte concentration: 100 nM follow with two-fold serial dilution(d) Assay Buffer: PBS (pH 7.4) with 0.02% Tween 20(e) Association: 300 seconds(f) Dissociation: 600 seconds

[0096] Table 3 listed the binding affinity of ten anti-Nectin-4 antibodies and commercialized Enfortumab. The dissociation constant (KD) value was ranging from 2.2E-9 to 1.1E-7 M. However, the 10K06 antibody exhibited the lowest KD value (2.2E-9 M).

[0097] Table 3. Binding affinity of anti-Nectin-4 antibodies assessed by Bilayer Interferometry (BLI)Example 3. Pharmacokinetic (PK) study of anti-Nectin-4 antibodies in naive mouse

[0098] 3.1 Animal and Study Design

[0099] The six anti-Nectin-4 antibodies, 05004, 12E03, 08B04, 10K06, 14108 and Enfortumab, were prepared in formulation buffer (25 mM Na-citrate, 100 mM NaCl, pH 6.5). The antibody preparation and administration were performed by TRINEO BIOTECHNOLOGY Co. Ltd, Taiwan. Female nude (nu / nu) mice, aged 6-8 weeks, were obtained from BioLASCO Taiwan Co. Ltd. The mice were first allocated into six groups based on the administered antibody. Each group were further subdivided into two batches and mice were given 10 mg / kg of indicated anti-Nectin-4 antibodies via the tail vein on Day 1. Blood samples were collected at Ih, 4h and 8h of Day 1, Day 2, Day 4, Day 8, Day 15, Day 22 and Day 29 post IV injection. Sera were harvested after clotting- off and stored at -70 °C until analyzed. Serum samples were measured for antibody concentrations by Nectin-4 coating ELISA and data was subjected for non-compartmental analysis (NCA) to derive PK parameters. The PK study design was listed in Table 4.

[0100] Table 4. PK study design of anti-Nectin-4 antibodies in naive mouse■ : Non-terminal blood collections• : Sacrifice and drain all the blood

[0101] 3.2 Non-compartmental Analysis

[0102] PK parameters were estimated by Phoenix pharmacokinetic software (Winnonlin 8.4 Certara, USA) using, non-compartmental analysis approach with sparse sampling strategy and IV bolus administration. All PK parameters were generated from serum concentrations of anti-Nectin- 4 antibodies and estimated using nominal sampling times relative to the start of dose administration. The area under the serum concentration curve vs. time (AUC) was calculated using the linear trapezoidal method with linear interpolation. The AUC was not calculated for PK profiles with less than three quantifiable concentrations of anti-Nectin-4 antibodies at separate time points. Whenpractical, the terminal elimination phase of each concentration versus time curve was identified using at least the final three observed concentration values. The slope of the terminal elimination phase was determined using log linear regression with uniform weighting. Parameters relying on the determination of the terminal elimination phase were not reported if the coefficient of determination was less than 0.700, or if the extrapolated AUC to infinity was more than 20% of total area, or the time span of elimination phase over half-life was less than 2.0. Table 5 described the PK parameters applied for the study.

[0103] Table 5. PK parameters described for anti-Nectin-4 antibodiesFIG. 2A illustrated serum concentration vs. time curves of anti-Nectin-4 antibodies in naive mice. FIG. 2B illustrated PK parameters of six anti-Nectin-4 antibodies. The result indicated that 08B04, 14108 and 10K06 exhibited comparable PK profiles to the benchmark antibody, Enfortumab, with the half lives of 10.96 days (TI / 2=263 hours), 10.00 days (TI / 2=240 hours) and 9.71 days (TI / 2=233 hours), respectively, in naive mice.Example 4. ADCP (antibody-dependent cellular phagocytosis) of anti-Nectin-4 antibodies

[0104] 4.1 Reagent(a) CD14 Microbeads human (MACS, Cat. No. 130-050-201)(b) MACS Rinsing buffer (MACS, Cat. No. 130-091-022)(c) PBMC (Lonza, Cat. No. CC-2702): Thawed PBMC in 3 mL Rinsing buffer and centrifuged with 300g for five minutes. Removed supernatant solution and repeated three times.(d) LS Column (MACS, Cat. No. 130-042-401)(e) RRMI-1640 (Gibco, Cat. No. A10491-01)(f) FBS (Gibco, Cat. No. 16000044)(g) Recombinant Human M-CSF (Peprotech, Cat. No. 300-25)(h) Bio-Gio (Promega, Cat. No. G7941)(i) CellTracker™ Green CMFDADye (Invitrogen, Cat. No. C7025)(j) CellTracker™ Deep Red (Invitrogen, Cat. No. C34565)(k) Cellstripper (Corning, Cat. No. 25-056-CI)

[0105] 4.2 Buffer preparation(a) Binding buffer: 0.5% FBS in MACS Rinsing buffer(b) MCSF stock (100 ng / pL): 100 pg MCSF powder+1 mL serum free RRMI-1640(c) Macrophage culture medium: 10% FBS in RRMI-1640 + 50 ng / mL MCSF(d) Assay medium: 2.5% FBS in RPMI-1640(e) Prepare CellTracker™ Green and CellTracker™ Deep Red:Dissolved CellTracker™ Green in DMSO to a final concentration of 1 mM (1000X) in 100 pL DMSO. Dissolved CellTracker™ Deep Red in 20 pL DMSO per vial to make a 1 mM (1000X) solution.

[0106] 4.3 Purification of PBMCPBMC was purified by using Ficoll® Paque Plus (Merck, Cat. No. GE17-1440-02) solution from fresh whole blood (Taipei Blood Center).

[0107] 4.4 Positive selection of CD14 Monocyte4.4.1. Added CD14 beads into darkened tube with binding buffer for binding twenty minutes (1E7 cells 15 pL CD14 beads+35 pL Binding buffer=50 pL; 2E7 cells 30 pL CD14 beads+70 pL Binding buffer=100 pL; 3E7 cells 45 pL CD14 beads+105 pL Binding buffer=150 pL).4.4.2. Added binding buffer and centrifuged with 300g for five minutes. Removed supernatant solution and repeated three times.4.4.3. Used 3 mL binding buffer to re-suspend cell pellet and added into column (Macrophage culture density: 2E6 / 2 mL per well in 6 well plate; Monocyte :PBMC=1 : 10 to 1 :20).

[0108] 4.5 Primary ADCP by M0 Macrophage cells4.5.1 Trypsinized the target cell and Macrophage (Human CD14+monocytes are differentiated after cultured for seven days with MCSF’s final concentration: 50 ng / mL) cells with 2 mL of trypsin- EDTA for five minutes at 37 °C.4.5.2 Labeled the fluorescence dye on the target cell and macrophages:(a) Cell accounting(b) Staining the 2xl06of target cells with 2 pL of Cell tracker green CMFDA dye in total 2 mL of RPMI 1640 at 37 °C for thirty minutes. (CMFDA final concentration: 1 pM).(c) Staining the 2xl06of macrophages with 2 pL of cell tracker deep red dye in total 2 mL of RPMI 1640 at 37 °C for thirty minutes (Deep red final concentration: 1 pM).4.5.3 Washed with 12 mL of lx PBS and centrifuging at 300g for five minutes.4.5.4 Aspirated the supernatant, mix the antibody (Antibody concentration is 0.3 nM) with targetcell and macrophage in total 500 pL of RPMI medium with 2.5 % FBS. (Target cell: Macrophage = 1 : 1).4.5.5 Analyzed by flow cytometer after incubating at 37 °C and 5% CO2 for one hour.

[0109] FIG. 3 illustrated the phagocytosis of the anti-Nectin-4 antibodies. Antibody pre-treated target cells and healthy donor macrophage were co-cultured in serum free medium for three hours. It indicated that all ten anti-Nectin-4 antibodies exhibited comparable ADCP capacity to Enfortumab under suspension co-culture of E / T cells in serum free medium condition.Example 5. Formulation study of OBI-904 (10K06-DAR8-ADC)

[0110] 5-1. pH value and buffer screening (buffer type)

[0111] 5-1-1. Material and method

[0112] OBI-904 samples were buffer-exchanged by centrifuge filtration into the formulations for stability screening. OBI-904 samples were placed in an Amicon Ultra-3 OK Da centrifugal filter (Millipore Sigma, Burlington, MA) and exchanged the buffer into the desired formulation at least three times. Then, OBI-904 concentration was adjusted to target concentration with same formulation buffer. We tried to evaluate the stability indicators by stress conditions and get the suitable pH and buffer for OBI-904. Eight buffers were tested in this study. Two stress conditions were conducted in this study: mechanical stress condition with agitation and thermal stress at 50±3°C. OBI-904 stored at 5±3°C was used as the control samples in this study. The details are listed below.(a) Buffer conditionNo. 1. 20 mM Histidine pH 5.0+10 mg / mL OBI-904No. 2. 20 mM Histidine pH 6.0+10 mg / mL OBI-902No. 3. 20 mM sodium acetate pH 5.0+10 mg / mL OBL904No. 4. 20 mM sodium acetate pH 5.5+10 mg OBL904No. 5. 20 mM sodium citrate pH 5.0+10 mg / mL OBL904No. 6. 20 mM sodium citrate pH 6.0+10 mg / mL OBL904No. 7 20 mM sodium phosphate pH 6.0+10 mg / mL OBL904No. 8 20 mM sodium phosphate pH 6.5+10 mg / mL OBL904(b) Stress condition1. Control: 5+3 °C2. Thermal stress condition: 50+3 °C for five days3. Agitation: uu mode, 50 rpm, four hours(c) Test item1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg^mLcm'1)2. Drug load distribution and DAR by HIC-UV3. Aggregation by SEC-UV4. Linker payload related substance by fluorescence detection

[0113] 5-1-2. Study result

[0114] Results for this study are shown in Table 6. There was no significant difference observed among the eight buffer conditions for OBI-904 at 5±3°C. Key quality attributes of OBI-904 such as protein concentration, drug-to-antibody ratio (DAR) and free drug level (DLD) remained comparable across all buffer conditions, even after four hours of agitation.

[0115] However, thermal stress at 50 ±3°C for five days was able to differentiate the impact of the variant formulation. After thermal treatment, most samples exhibited varying degrees of white coloration, turbidity, and even precipitation. Once turbidity or precipitation occurred, further analytical tests were not performed on those samples. Only OBI-904 formulated in histidine, pH 6.0 buffer solution and sodium acetate, pH 5.5 buffer solution remained clear and colorless.

[0116] Table 6. Summary table of pH and buffer screening test (buffer type)*CC: clear, colorless and no particles; WT: White and turbid; P: Precipitation; NA: Not available.

[0117] 5-2. pH value and buffer screening (buffer concentration)

[0118] 5-2-1. Material and method

[0119] A follow-up study was conducted to further investigate the impact of buffer concentration and pH range using histidine and sodium acetate buffer systems. Eight different formulations were designed. The details are listed below.(a) Buffer conditionNo. 1. 20 mM sodium acetate pH 5.2+10 mg / mL OBI-904No. 2. 20 mM sodium acetate pH 5.6+10 mg / mL OBI-902No. 3. 50 mM sodium acetate pH 5.6+10 mg / mL OBL904No. 4. 20 mM Histidine pH 5.5+10 mg OBL904No. 5. 10 mM Histidine pH 6.0+10 mg / mL OBL904No. 6. 20 mM Histidine pH 6.0+10 mg / mL OBL904No. 7 50 mM Histidine pH 6.0+10 mg / mL OBL904No. 8 20 mM Histidine pH 6.5+10 mg / mL OBL904(b) Stress condition1. Control: 5±3 °C2. Thermal stress condition: 50±3 °C for five days3. Agitation: uu mode, 50 rpm, four hours(c) Test item1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg^mLcm'1)2. Drug load distribution and DAR by HIC-UV3. Aggregation by SEC-UV4. Linker payload related substance by fluorescence detection

[0120] 5-2-2. Study result

[0121] As shown in Table 7, no obvious change of OBI-904 was observed in these eight variant buffers after mechanical stress condition with agitation (uu mode, 50rpm) for four hours. All the quality attributes of these test articles were still qualified, met the spec and had no degradation observed under the conditions.

[0122] After thermal treatment, an increase of aggregation and a decrease of %DAR8 were observed in thermal treatment group. Eight formulations included sodium acetate (20 to 50 mM, pH 5.2 to5.6) and histidine (10 to 50 mM, pH 5.5 to 6.5). Among the tested conditions, 10 mM Histidine at pH 6.0 showed the most favorable profile, with %DAR8 at 90.07, monomer content at 95.43%, and HMWS at 4.19%, indicating superior stability under this condition. In general, histidine buffers at pH 6.0 and 6.5 demonstrated higher %DAR8 and monomer levels, as well as lower HMWS, compared to sodium acetate buffers.

[0123] Table 7. Summary table of pH and buffer screening test (buffer concentration)

[0124] 5-3. Excipient screening

[0125] 5-3-1. Material and method

[0126] In this study, different excipients with the function of protein stabilizer and cryoprotectant were studied to enhance stability OBI-904 in liquid solution. Sucrose, trehalose, glycine and polysorbate 80 were evaluated. Two stress conditions were conducted in this study: mechanical stress condition with agitation for six hours (uu mode, 50rpm, four hours and UU mode, 99rpm, two hours) and thermal stress at 50±3°C for five days to evaluate the formulation for OBI- 904. OBI-904 stored at 5±3°C was used as the control samples in this study. The details are listedbelow.(a) Buffer and excipient condition1. 10 mM Histidine pH 6.2+10 mg / mL OBI-9042. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+150 mM sucrose3. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+250 mM sucrose4. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+250 mM trehalose5. 10 mM Histidine pH 6.2+10 mg / mL OBL904+0.05% (w / v) Polysorbate 806. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+100 mM glycine7. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+250 mM sucrose+0.05% (w / v) Polysorbate 808. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+150 mM sucrose +100 mM glycine9. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+150 mM sucrose+100 mM glycine+0.05% (w / v) Polysorbate 8010. 10 mM Histidine pH 6.2+10 mg / mL OBI-904+100 mM glycine +0.05% (w / v) Polysorbate 80(b) Stress condition1. Control: 5+3 °C2. Thermal stress condition: 50+3 °C for five days3. Agitation: uu mode, 50 rpm, four hours+ uu mode, 99 rpm, two hours(c) Test item1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg-lmLcm-1)2. Drug load distribution and DAR by HIC-UV3. Aggregation by SEC-UV4. Linker payload related substance by fluorescence detection

[0127] 5-3-2. Study result

[0128] As shown in Table 8, no significant changes were observed in OBL904 after mechanical stress with agitation. All tested articles met specifications, remained qualified, and showed no degradation under these conditions. Based on these results, the recommended formulations are sample 5, 7, 9 and 10, which contain 0.05 % polysorbate.

[0129] Following thermal stress treatment, formulations containing sucrose or trehalose helped prevent aggregation, with sample 2, 3, 4, 7, and 8 maintaining %HMWS not more than 3.0 % based on SEC-UV results. Other formulations exceeded this threshold, with sample 10 reaching 4.08 %.

[0130] Table 8. Summary table of excipient screeningExample 6. Preparation of NSCT derivates6-1. Preparation of NSCT-3 (NSCT-Tetra-N3)

[0131] NSCT (CAS No. 58902-60-2, 384 mg, 0.19 mmol; purchased from Glytech, Inc.; HPLC purity > 90%), amino linker-3 (560 mg, 1.14 mmol) and benzotriazol- 1-yl-oxy -tri pyrrolidino- phosphonium hexafluorophosphate (CAS No. 128625-52-5, 593 mg, 1.14 mmol) were mixed with DMSO (4 mL). Triethanolamine (CAS No. 102-71-6, 506 pL, 3.8 mmol) was added, and the mixture was heated at 40 °C overnight. Added water (10 mL) and washed with DCM (60 mL) twice. The aqueous layer was collected and concentrated under reduced pressure. The residual mixture was purified by C-18 preparative HPLC (eluent: ACN / Water). Fractions with the desired product were combined and freeze-dried to afford NSCT-3 (339 mg) as a white solid. 1H NMR (D2O): 5 5.22 (d, J= 3.3 Hz, 1H, Hl of GlcNAc), 5.14 (s, 1H, Hl of Man), 4.95 (s, 1H, Hl of Man), 4.73 (br, 1H, Hl of Man), 4.61 (d, J= 7.3 Hz, 2H, Hl of two GlcNAc), 4.45 (d, J= 7.9 Hz, 2H, Hl of twoGal), 4.27 (s, 1H, H2 of P-form Man), 4.20 (s, 1H, H2 of a-form Man), 4.13 (s, 1H, H2 of a-form Man), 3.99-3.48 (m), 2.80-2.76 (m, 4H, CH2 next to carbonyl on amino linker), 2.70 (dd, Jl= 12.9 Hz, J2= 4.5 Hz, 2H, H3eq of two Neu5 Ac), 2.10-2.00 (m, 15H, five NHAc), 1.85 (dd, J1=J2= 12.3 Hz, 2H, H3ax of two Neu5Ac). MASS [M+3Na]3+1011.08.NSCT-36-2. Preparation of NSCT-4 (oxazoline-NSCT-tetra-Ns)

[0132] NSCT-3 (339 mg, 0.114 mmol) and triethylamine (715 pL, 5.12 mmol) were dissolved in water (3.7 mL) and cooled to 0 °C. 2-chloro-l,3-dimethyl-lH-benzimidazol-3-ium chloride aqueous solution (1 M, 1.71 mL) was added slowly and the resulting mixture was stirred at -2 to 0 °C for four hours. NaOH solution was added (0.01 M, 1 mL) and the resulting mixture was concentrated under reduced pressure. After most of the triethylamine was evaporated, the residual mixture was purified by Sephadex® G-25 column. 0.01M NaOH as eluent is used to stabilize the product. Fractions with the desired product were combined and freeze-dried to afford NSCT-4 (324 mg) as a white solid. 1H NMR (D2O): 5 6.10 (d, J= 7.4 Hz, 1H, Hl of oxazoline), 5.14 (s, 1H, Hl of Man), 4.97 (s, 1H, Hl of Man), 4.76 (s, 1H, Hl of Man), 4.63-4.60 (m, 2H, Hl of two GlcNAc), 4.45 (d, J= 7.5 Hz, 2H, Hl of two Gal), 4.40 (s, 1H, H3 of oxa-GlcNAc), 4.20-4.16 (br, 4H, H2 ofthree Man and H2 of oxa-GlcNAc), 3.99-3.48 (m), 2.80-2.76 (m, 4H, CH2 next to carbonyl on amino linker), 2.70 (dd, Jl= 12.2 Hz, J2= 3.6 Hz, 2H, H3eq of two Neu5Ac), 2.08-2.02 (m, 15H, five NHAc), 1.85 (dd, J1=J2= 12.4 Hz, 2H, H3ax of two Neu5Ac).NSCT6-3. Preparation of amino linker-3

[0133] Fmoc-P-Ala-OH (CAS No. 35737-10-1, 382 mg, 1.23 mmol) and HATU (CAS No. 148893-10-1, 544 mg, 1.43 mmol), and NH-Bis(PEG3-azide) amino linker-2 (CAS No. 1258939- 39-7, 500 mg, 1.19 mmol) were dissolved in DMF (5 mL) and cooled to 0 °C. N-methyl morpholine (393 pL, 3.57 mmol) was added slowly. The resulting solution was stirred at room temperature for twelve hours.

[0134] The reaction was monitored by reversed-phase C-18 TLC, indicating a complete conversion (eluent: ACN / water= 6 / 4, Rf value= 0.25). Add Diethylamine (CAS NO. 109-89-7) 1.5 mL to the intermediate solution and stir at room temperature for two hours. The reaction wasmonitored by SiCh TLC, indicating a complete conversion (eluent: DCM / Me0H / NH40H= 90 / 10 / 1, Rf value= 0.3). After being concentrated under reduced pressure to evaporate most of the DMF, the residual mixture was purified by silica gel chromatography (eluent: DCM / Me0H / NH40H= 920 / 80 / 8). Fractions with the desired product were combined, concentrated, and then freeze-dried to afford amino linker-3 (580 mg, 1.18 mmol) as a light-yellow oil. 1. 1H NMR 600 Hz, (CDC13): 5 3.66-3.38 (m, 28H, PEGs), 3.37 (t, J= 5.0 Hz, 4H, CH2 next to N), 2.96 (t, J= 5.6 Hz, 2H, CH2 next to NH2), 2.54 (t, J= 6.0 Hz, 2H, CH2 next to carbonyl), 1.94 (Br, NH2). Mass: [M+l]+= 491.30.amirs© ln er- : amino mfer-3Example 7. deglycosylation, transglycosylation and purification of monoclonal antibodies7-1. Deglycosylation of mAbs by EndoSz-D234M

[0135] The monoclonal antibody 10K06 was used to illustrate the process to obtain glycoengineered antibody. 10K06 was deglycosylated with EndoSz-D234M at a weight ratio of 1 :250 (enzyme: mAbs) and 10U Endo H / mg mAb in 100 mM Sodium phosphate, pH 7.0 at 37 °C for 35-42 hours to remove glycans and generate 10k06-GlcNAc(Fuc). The complete cleavage of Fc N-glycans were analyzed by SDS-PAGE and CE-SDS.

[0136] The EndoSz-D234M was applied to other mAbs for further deglycosylation and transglycosylation investigation. For the deglycosylation studies, the 10K06 mAb was incubated with EndoSz-D234M at a weight ratio of 1 :30 (EndoSz-D234M: mAb) and the mAb-GlcNAc(Fuc) reached to >90% by EndoSz-D234M cleavage. For the transglycosylation investigation with NSCT-4, 38 equivalents of NSCT-4 were added to mAb-GlcNAc(Fuc) for incubation with EndoSz- D234M at 37 °C for two hours. Table 9 showed that the yield of 10K06-(NSCT-tetra-N3)2 was 95.5%.

[0137] Table 9. Deglycosylation and transglycosylation of 10K06 mAbmAb 10K067-2. Transglycosylation of mAb-GlcNAc(Fuc)

[0138] 10K06-GlcNAc(Fuc) was incubated with 8-10 equivalents of NSCT-4 at 15 °C for eight hours to generate 10K06-(NSCT-tetra-N3)2. The transglycosylation efficiency was monitored by SDS-PAGE and CE-SDS.7-3. Purification of mAb-(NSCT-tetra-N3)2

[0139] The transglycosylation mixture was applied to the pre-equilibrated PrismA column (Cytiva). The non-bound contaminations were washed by 3CV of 3 M NaCl PBS, pH 7.2 buffer, 5CV of 100 mM pH 6.0 Citrate Buffer, and 5CV of 100 mM pH 5.5 Citrate Buffer. 10K06-(NSCT- trtra-N3)2 was eluted with 5CV of 50 mM pH 3.0 Citrate Buffer with 150 mM NaCl and the eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 to natural pH. The neutralized sample was diluted to reduce conductivity and concentration, then activated carbon was added at a 3: 1 weight ratio relative to the antibody (AC= 3: 1 w / w). Stirred the mixture and filtered after two hours to collect the clarified sample. The purified 10K06-(NSCT-tetra-N3)2 was concentrated with 7 volumes of storage buffer (20 mM Sodium Acetate, pH 5.0) using a 30 kDa cutoff cassette (PES, Sartorius). The purified 10K06-(NSCT-tetra-N3)2 was also stored at -80 °C.Example 8. Synthesis of drug-linker compound8-1. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-1)

[0140] Step l :A solution of H-PAB-5 (57.0 mg, 0.0289 mmol) in CH2Q2 (1.2 mL) was cooled to 0 °C. TFA (285.0 pL) was added dropwise. The reaction mixture was stirred at 0-4 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solution was concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (5.1 mg, 0.0289 mmol) and HATU (13.2 mg, 0.0347 mmol) were dissolved in anhydrous DMF (0.58 mL). EtsN (12.1 pL, 0.0868 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, theresulting mixture was purified with preparative HPLC to obtain 40.4 mg of H-PAB-7 with 68.9% yield.

[0141] Table 10. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)

[0142] Step 2:A solution of H-PAB-7 (38.5 mg, 0.0190 mmol) in CH2Q2 (0.77 mL) was cooled to 0 °C. TFA (192.5 pL) was added dropwise. The reaction mixture was stirred at 0-4 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solution was concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (3.4 mg, 0.0190 mmol) and HATU (8.7 mg, 0.0228 mmol) were dissolved in anhydrous DMF (0.38 mL). EtsN (7.9 pL, 0.0570 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, theresulting mixture was purified with preparative HPLC to obtain 28.7 mg of H-PAB-8 with 72.6% yield.

[0143] Table 11. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)

[0144] Step 3:A solution of H-PAB-8 (25.0 mg, 0.0120 mmol) in CH2Q2 (0.50 mL) was cooled to 0 °C. TFA (125.0 pL) was added dropwise. The reaction mixture was stirred at 0 °C for four hours. After the reaction was completed, the reaction mixture was diluted with MeOH (5 mL) and then concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. Theresulting material was dried in high vacuum to obtain crude deprotected intermediate. A solution of crude intermediate (0.0120 mmol) in anhydrous DMF (0.24 mL), BCN-OSu (3.5 mg, 0.0120 mmol) and EtsN (5.1 pL, 0.0360 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 15.7 mg of DL-1 with 60.9% yield.

[0145] Table 12. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)8-2. Preparation of BCN-GGVA-Hydra-PAB-PEG24-MMAE (also termed DL-2) and BCN- GGVA-Hydra-PAB-PEG24-T785 (also termed DL-3)

[0146] Step l:A solution of N-DT-0024 (50.0 mg, 0.065 mmol) and payload (MMAE or T785, 1.1 eq) in anhydrous DMF (0.65 mL), DIPEA (33.4 pL, 0.192 mmol) was added slowly. The resulting mixture was stirred at room temperature until N-DT-0024 was consumed. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (6.5 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude HL-1 (X=MMAE with 75.6% yield) or HL-2 (X=T785 with 79.1% yield). This crude product was used in next step without further purification.

[0147] Step 2:A stirring suspension of HL-1 or HL-2 (83.4 mg) in DCM (1.46 mL) was cooled to 0 °C. TFA (0.63 mL) was added at 0 °C slowly. The reaction mixture was stirred at 0-4 °C for 20-24 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (20.9 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate. This crude product was used in next step without further purification.

[0148] The intermediate and BCN-OSu (1 eq) were dissolved in anhydrous DMF (0.05 M). EtsN (3 eq) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (10 times amount of intermediate) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude BCN intermediate. This crude product was used in next step without further purification.

[0149] The BCN intermediate (1 eq), NH2-PEG24-Ome (2 eq) and HATU (1.2 eq) were dissolved in anhydrous DMF (0.05 M). NMM (5 eq) was added. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain DL-2 or DL-3.

[0150] Table 13. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)DL-2 was obtained with 26.0% yield. MS (ESI) m / z found [(M+H+NH4 / 2]+, 1229.810C120H210N12O402, required 1229.738.DL-3 was obtained with 27.7% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1018.0743C99Hi6sNiiO332+, required 1018.0785.8-3. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Eribulin (also termed DL-4)

[0151] Step l :

[0152] Fmoc-VA-PAB(COOtBu)-OH (202.8 mg, 0.329 mmol) in anhydrous CH2CI2 (3.8 mL) was cooled to 0 °C. After five minutes, TFA (1.27 mL) was added slowly. The reaction mixture was stirred at 0-4 °C overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring TBME (50.0 mL) to get precipitate. The solids were collected byfiltration and followed with high vacuum drying to obtain crude intermediate (75.71 mg ). The previous intermediate (75.71 mg, 0.135 mmol), NH2-PEG24-OMe (161.9 mg, 0.149 mmol) and HATU (61.7 mg, 0.162 mmol) were dissolved in anhydrous DMF (1.35 mL). NMM (44.8 pL, 0.406 mmol) was added and the reaction mixture was stirred at room temperature for eight hours. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, the residue was purified by flash silica gel column (CTECh / MeOH = 12 / 1 to 9 / 1) to obtain 212.7 mg of HL-3 with 39.6% yield.

[0153] Step 2:

[0154] HL-3 (22.57 mg, 0.014 mmol) and Bis(4-nitrophenyl) carbonate (8.42 mg, 0.028 mmol) in anhydrous CH2Q2 (0.28 mL) was cooled to 0-4 °C. 2,6-lutidine (2.4 pL, 0.021 mmol) and DIPEA (2.4 pL, 0.014 mmol) were added sequentially at 0-4 °C. The resulting mixture was stirred at 0-4 °C for 20-24 hours. Et2NH (1.4 pL, 0.014 mmol) was added slowly at 0-4 °C to quench excess Bis(4-nitrophenyl) carbonate. Anhydrous DMF (0.55 mL), Eribulin (15.2 mg, 0.021 mmol), DIPEA (7.2 pL, 0.042 mmol) were added into previous reaction solution at 0-4 °C. The reaction mixture was stirred at 0-4 °C overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 7.54 mg of HL-4 with 22.8% yield.

[0155] Table 14. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)

[0156] Step 3:

[0157] HL-4 (7.54 mg, 0.003 mmol) was dissolved in CH2Cl2 / MeOH (226.0 pL, 1 / 1) and then cooled to 0 °C. Et2NH (45.2 pL) was added and the reaction mixture was stirred at 0-4 °C overnight. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, and azeotroped with Toluene (1 mL x 3) to remove excess Et2NH. The crude compound was high vacuum drying to obtain crude intermediate (8.2 mg). The intermediate (8.2 mg), Fmoc-GG-OH (1.61 mg, 0.004 mmol) and HATU (2.16 mg, 0.005 mmol) were dissolved in anhydrous DMF (0.2 mL). NMM (1.25 pL, 0.011 mmol) was added at room temperature. The resulting mixture was stirred at room temperature overnight. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, the residue was purified by flash silica gel column(CthCb / MeOH = 100 / 0 to 87 / 13) to obtain Fmoc-GG-intermediate. Fmoc-GG-intermediate was dissolved in CFFCh / MeOH (0.6 mL, 1 / 1) and then cooled to 0 °C. Et2NH (0.13 mL) was added and the reaction mixture was stirred at 0-4 °C for 20-24 hours. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35 °C, and azeotroped with Toluene (2 mL x 3) to remove excess Et2NH. The crude compound was high vacuum drying to obtain crude intermediate. The intermediate (19.3 mg), BCN-OSu (2.51 mg, 0.008 mmol) were dissolved in anhydrous DMF 0.17 mL. EtsN (3.61 pL, 0.026 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 2.85 mg of DL-3 with 37.4% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1227.1713 Ci2iHi99N7O442+, required 1227.1697.

[0158] Table 15. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)Step 2H L-38-4. Preparation of DBCO-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-5)

[0159] Step l :DBCO-acid (265.7 mg, 0.87 mmol), tert-butyl 2-(2-aminoacetamido)acetate (163.8 mg, 0.87 mmol) and HATU (363.9 mg, 0.96 mmol) were dissolved in CH2Q2 (8.7 mL) under room temperature. NMM (287.0 pL, 2.61 mmol) was added. After the addition, the resulting mixture was stirred at room temperature for 16-18 hours. The reaction solution was concentrated in vacuo at 30-35 °C, and the residue was purified by flash silica gel column (CH2Q2 / EA = 1 / 2 to 1 / 4) to obtain 342.2 mg of DBCO-4 with 82.7% yield.Step 1

[0160] Step 2:A solution of DBCO-4 (340.0 mg, 0.71 mmol) in CH2Q2 (6.8 mL) was cooled to 0 °C. TFA (1.7 mL) was added dropwise. The reaction mixture was warmed to room temperature (rt) slowly and then stirred for two hours. After the reaction was completed, the reaction mixture was concentrated in the vacuo at 30-35 °C and then azeotroped with Toluene (5 mL x 3) to remove excess TFA. The resulting mixture was purified with preparative HPLC to obtain 168.4 mg of DBCO-5 with 56.1% yield.

[0161] Table 16. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)

[0162] Step 3:DBCO-5 (3.98 mg, 0.0095 mmol), H-PAB-6 (17.8 mg, 0.0095 mmol) and HATU (3.97 mg, 0.0104 mmol) were dissolved in anhydrous DMF (0.32 mL). EtsN (4.0 pL, 0.0285 mmol) was added. The reaction mixture was stirred at room temperature for four hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.7 mg of DL-5 with 49.6% yield.

[0163] Table 17. HPLC condition (Column: YMC-Actus Trait C18 250 x 20 mm, 5 pm, 12 nm)Example 9. ADC preparation and analysis9-1. 10K06-DAR8-ADC preparation

[0164] BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed as DL-1) was dissolved in DMSO to form a solution. The solution was added slowly to 10K06-(NSCT-tetra-N3)2 in 20 mM NaOAc, pH 5.0 buffer and stirred at 25 °C for 16-24 hours. After conjugation, the crude ADC was purified using a 30 kDa cut-off cassette, with a 40-volume buffer exchange (10 mM Histidine, pH 6.2). The concentration of ADC was adjusted to 10 mg / mL and sterilized by passing through Millex-GP Syringe Filter (PES, 0.22 um). The drug-to-antibody ratio (DAR) value of final ADC is determined by hydrophilic interaction chromatography (HIC).9-2. 10K06-DAR8-ADC analysis

[0165] The CE-SDS analysis was conducted under reducing condition. Beckman Coulter PA800Plus system equipped with a UV photodiode array detector (220 nm wavelength employed) was used in this test. A bare fused-silica capillary (50 m ID * 30 cm total length) with the 20 cm effective capillary separation length was rinsed with 0.1 M NaOH, 0.1 M HC1 and SDS gel buffer prior to injection. Electrokinetic injection mode was applied at -5 kV for 20 seconds in reverse polarity and followed by applying a -15 kV voltage for capillary separation. The total separation time was 35 minutes. 60 pg of Test article was sampled and diluted in 120pL sample buffer which was 1% SDS in diluted PBS, pH 7.0. Then diluted sample was mixed with 5 pL of 2ME, and 2 pLof 10 kDa internal standard, followed by incubation at 65 °C for 10 minutes. Finally, the sample was cooled down at room temperature for CE-SDS analysis.

[0166] EndoSz-D234M EndoSz-D234M showed high deglycosylation and transglycosylation activity Anti-Nectin-4 (10K06) antibody pretreated with EndoSz-D234M were used to produce 10K06-DAR8-ADC (also termed OBI-904), as the model of glycosite-specific ADC. FIG. 4 indicated the non-reducing and reducing PAGEs results of 10K06 antibody and OBI-904 ADC. Furthermore, FIG. 5 indicated hydrophobic interaction chromatography (HIC) result of OBI-904. The DAR distributions of OBI-904 were DAR0 (0.28%), DAR4 (6.92%) and DAR8 (92.8%).Example 10. In-vitro cytotoxicity comparison of anti-Nectin-4 ADCs

[0167] Tumor cells (2 x 103cells / well) were seeded in 96 well plate and treated with 10K06- DAR8-ADC (also termed OBI-904) for six days. CellTiter-Glo® Reagent (Cat. G7572, Promega) was prepared by adding CellTiter-Glo® Buffer into lyophilized CellTiter-Glo® Substrate. Reconstituted CellTiter-Glo® Reagent was added into the culture medium with cells at 1 : 1 ratio after treatment for six days. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording the luminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). Percentage of the cytotoxicity was calculated through dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50 was determined by plotting x (concentration in nM) - y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.

[0168] The in vitro efficacy of OBI-904 and commercial anti-Nectin-4 ADCs in human colorectal cancer cell line DLD-1 cells (Cat. No. 60132, Bioresource Collection and Research Center) and human hypopharyngeal squamous cell line FaDu (Cat. No. 60214, Bioresource Collection and Research Center) were evaluated by cytotoxicity assay. The IC50 of ETx-22 was 32 nM and OBI-904 was 143 nM in DLD-1 cancer cell (FIG. 6A). It indicated the IC50 of these two ADCs were better than Padcev®. Furthermore, the IC50 of ETx-22 was 14 nM, OBL904 was 67 nM, and Padcev® was 153 nM in FaDu cancer cell (FIG. 6B). It also demonstrated OBL904 is competitive with other commercial anti-Nectin-4 ADCs.Example 11. In vivo tumor inhibition efficacy comparison of anti-Nectin-4 ADCs

[0169] 11.1. Nectin-4 overexpression PC-3 human prostate cancer cell-derived xenograft in BLAB / c nude mice

[0170] 11.1.1 Test Substances and Dosing Pattern(a) OBL904: 3 mg / kg(b) EV (Enfortumab Vedotin): 3 mg / kg

[0171] Table 18. Study Design and sampling

[0172] 11.1.2 Cell line: Nectin-4 overexpression PC-3 cells (High Nectin-4 expressed) (Cat. No. 60122, Bioresource Collection and Research Center)

[0173] 11.1.3 Animal(a) Species: Mus musculus(b) Strain: CAnN.Cg-Foxw7"7CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained four mice. A total of twelve mice were involved in the study.

[0174] 11.1.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-900 IV)(e) Matrigel (BD / Cat. No. : 356234)

[0175] 11.15.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: IxlO7Nectin-4 overexpression PC3 cells were mixed with the equal volume of matrigel (volume ratio 1 : 1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 200 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume reach 150-200 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenouslyadministered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 49). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0176] 11.1.6 Result

[0177] FIG. 7 A indicated the in-vivo efficacy result in Nectin-4 overexpression PC3 CDX model. The mean+SEM of tumor volume to vehicle group was 1036.52+145.39 mm3on Day 14. The mean+SEM of tumor volume to treated groups were 168.48+83.98 mm3to OBI-904 on Day 49 and 585.06+467.77 mm3to EV (Enfortumab Vedotin) on Day 43. It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0178] 11.2. Nectin-4 middle expressed FaDu human head and neck cancer cell-derived xenograft in BLAB / c nude mice

[0179] 11.2.1 Test Substances and Dosing Pattern(a) OBI-904: 6 mg / kg(b) EV (Enfortumab Vedotin): 6 mg / kg

[0180] Table 19. Study Design and sampling

[0181] 11.2.2 Cell line: FaDu cells (Middle Nectin-4 expressed) (Cat. No. 60214, BioresourceCollection and Research Center)

[0182] 11.2.3 Animal(a) Species: Mus musculus(b) Strain: CAnN.Cg-Foxw7"7CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained five mice. A total of fifteen mice were involved in the study.

[0183] 11.2.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-900 IV)(e) Matrigel (BD / Cat. No. : 356234)

[0184] 11.2.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 5xl06FaDu cells were mixed with the equal volume of matrigel (volume ratio 1 : 1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume around 150 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 6 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 14). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0185] 11.2.6 Result

[0186] FIG. 7B indicated the in-vivo efficacy result in FaDu head and neck CDX model. The mean+ SEM of tumor volume to vehicle group was 1050.83+305.98 mm3on Day 14. The mean+SEM of tumor volume to treated groups were 241.70+193.64 mm3to OBI-904 and 931.14+431.90 mm3to EV (Enfortumab Vedotin). It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0187] 11.3. Nectin-4 low expressed TFK-1 human cholangiocarcinoma cell-derived xenograft in BLAB / c nude mice

[0188] 11.3.1 Test Substances and Dosing Pattern(a) OBI-904: 8 mg / kg(b) EV (Enfortumab Vedotin): 8 mg / kg

[0189] Table 20. Study Design and sampling

[0190] 11.3.2 Cell line: TFK-1 cells (Low Nectin-4 expressed) (Cat. No. ACC344, DSMZ)

[0191] 11.3.3 Animal(a) Species: Mus musculus(b) Strain: CAnN.Cg-Foxw7"7CrlBltw (BALB / c nude)(c) Source: BioLasco Taiwan(d) Sex: Female(e) Age at initiation of study: seven weeks(f) Body weight range at start of study: 15-25 g(g) Animal Grouping: The mice were divided into three groups and each group contained six mice. A total of eighteen mice were involved in the study.

[0192] 11.3.4 Equipment and Material(a) Biosafety cabinet (NUAIRE / NU-620-400)(b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-900 IV)(e) Matrigel (BD / Cat. No. : 356234)

[0193] 11.3.5 Method(a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: IxlO7TFK-1 cells were mixed with the equal volume of matrigel (volume ratio 1 : 1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 200 pL / mouse.(b) Route and administration of test article:The first dosing day was denoted as Day 1 when average tumor volume reach 150-200 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 8 mg / kg and the injection volume was 5 mL / kg.(c) Tumor growth inhibition rate calculationTumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 - (Ti - Tl) / (Ci - Cl)] x 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 62). Whereas TI and Cl indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0194] 11.3.6 Result

[0195] FIG. 7C indicated the in-vivo efficacy result in TFK-1 human cholangiocarcinoma CDX model. The mean+SEM of tumor volume to vehicle group was 916.80+320.79 mm3, 204.62+319.50 mm3to OBI-904 and 669.04+237.85 mm3to EV (Enfortumab Vedotin) on Day 62. It demonstrated that the tumor inhibitions efficacy of OBI-904 was higher than EV.

[0196] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0197] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

What is claimed is:

1. An antibody or an antigen-binding portion thereof that binds to Nectin-4, comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs); wherein the HCDRs and the LCDRs have amino acid sequences of:(a) SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively;(b) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively;(c) SEQ ID NOs: 35, 36, 37, 38, 39, and 40, respectively;(d) SEQ ID NOs: 41, 42, 43, 44, 45, and 46, respectively;(e) SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively;(f) SEQ ID NOs: 53, 54, 55, 56, 57, and 58, respectively;(g) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively;(h) SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively;(i) SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; or(j) SEQ ID NOs: 77, 78, 79, 80, 81, and 82, respectively.2 The antibody or the antigen-binding portion thereof according to Claim 1, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 90% sequence identity to:(a) SEQ ID NOs: 1 and 2, respectively;(b) SEQ ID NOs: 3 and 4, respectively;(c) SEQ ID NOs: 5 and 6, respectively;(d) SEQ ID NOs: 7 and 8, respectively;(e) SEQ ID NOs: 9 and 10, respectively;(f) SEQ ID NOs: 11 and 12, respectively;(g) SEQ ID NOs: 13 and 14, respectively;(h) SEQ ID NOs: 15 and 16, respectively;(i) SEQ ID NOs: 17 and 18, respectively; or(j) SEQ ID NOs: 19 and 20, respectively.3 The antibody or the antigen-binding portion thereof according to Claim 1, wherein the antibody is: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab’)2; or (e) a disulfide link Fv.4 The antibody or the antigen-binding portion thereof according to Claim 1 or 2, wherein the antibody is a humanized antibody.

5. The antibody or the antigen-binding portion thereof according to Claim 4, wherein the humanized antibody is an IgG or IgM.

6. An antibody-drug conjugate (ADC) having a structure of Formula (I):Ab-(DL)n (I); wherein:(a) Ab is an antibody or an antigen-binding fragment thereof capable of binding to Nectin-4;(b) DL is a linker-payload of Formula (III) that forms a covalent linkage with the antibody or the antigen-binding fragment thereof:wherein:C is a bioorthogonal group for conjugation to the antibody or the antigen-binding fragment thereof;QCLis a cleavable unit, wherein the cleavable unit comprises a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety;R3Bpis a branch unit comprising a functional group defined as, whereinA is an aromatic group;R3is linked to E and is selected from -C(O)-, -C(O)O-, -C(O)NH-, alkyl-O-, alkyl-NH-, alkyl- C(O)-, alkyl-C(O)-O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-SO2-, alkyl-S-, alkyl-O-P(O)- O2-, alkyl-O-C(O)-NH-, or triazole;E is a hydrophilic moiety comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly (glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), saccharides, or any combination thereof;P is a payload, wherein the payload is a drug moiety; andLBis a bridge unit; and(c) n is a drug-to-antibody ratio (DAR) ranging from 2 to 32.7 The ADC according to claim 6, wherein the hydrophilic moiety E has a formula of:wherein the wavy line indicates the site of covalent attachment to the branch unit Bp,Ri is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, orR2 is H, SO3H, PO3H2, a sugar derivative, C1-C10 (hetero) alkyl group, C3-C10 (hetero) cycloalkyl group, C2-C10 alkyl-NH2, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(Ci-C3 alkyl), C2-C10 alkyl-N (C1-C3 alkyl)2, or sarcosines; and n is an integer ranging from 2 to 72. The ADC according to claim 6, wherein the bioorthogonal group C is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a mal eimide group, a a,P-unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4- dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group. The ADC according to claim 6, wherein the payload P is selected from a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope. The ADC according to claim 9, wherein the cytotoxic agent is selected from pyrrolobenzodiazepine compounds or derivatives thereof, auristatin compounds or derivatives thereof, maytansinoid compounds or derivatives thereof, duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof, anthracycline compounds or derivatives thereof, pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof, drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof. The ADC according to claim 9, wherein the cytotoxic agent is exatecan, MMAE, T785, or Eribulin. The ADC according to claim 6, wherein the linker-payload DL of formula (III) is further represented by a structure of Formula (IV) or Formula (VI):

13. The ADC according to claim 6, wherein the linker-payload DL of formula (III) is further represented by a structure of Formula (V) or Formula (VII):

14. An antibody-drug conjugate (ADC) having a structure of Formula (VIII):(VIII); wherein Ab is a glycoengineered antibody that binds to Nectin-4;G represents a glycan moiety connected to an Neu5Ac and the glycoengineered antibody, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose, N-acetyl-glucosamine, glucose, mannose, fucose, and derivatives thereof; Y is a connector moiety independently comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly (glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combinations thereof;Z is independently selected from a triazole or imine linkage;L is a linker connecting D and Z;D is a drug moiety; m is a number of Neu5Ac units branching from G, and is an integer from 1 to 4; and p is a number of Y-Z-L-D units attached to each of the Neu5Ac units, and is an integer from 1 to 4.

15. The ADC of claim 14, wherein the glycoengineered antibody Ab is selected from 08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, 13C24 or Enfortumab.

16. The ADC of claim 14, wherein the drug moiety D is selected from MMAE, T785, camptothecin, SN-38, belotecan, exatecan, or deruxtecan.

17. An antibody-drug conjugate (ADC) having a structure of Formula (IX):= Fucose ( ) = Mannose = Galactose ] - / V-acetylglucosamine (GkHAcJ< / >~ J -acetylneuraminic add (I euSAc} wherein Ab is a glycoengineered antibody capable of binding to Nectin-4; L is a linker connecting exatecan and the glycoengineered antibody; and a, b are independently 0 or 1.

18. A pharmaceutical composition, comprising: the antibody or the antigen-biding portion thereof according to any one of Claims 1-5 or the ADC according to any one of Claims 6-17; and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to Claim 18, further comprising a therapeutic agent.

20. A method for inhibiting proliferation of cancer cells or treating cancer of a patient, comprising administering to the patient in need thereof an effective amount of the antibody or antigenbinding portion thereof according to any one of Claims 1-5 or the ADC according to any one of Claims 6-17.

21. The method according to Claim 20, wherein the cancer is a Nectin-4 expressing cancer.

22. The method according to Claim 21, wherein the Nectin-4 expressing cancer is selected from the group consisting of breast cancer, bladder cancer, urothelial cancer, lung cancer, ovarian cancer, pancreas cancer, esophageal cancer, gallbladder cancer, hepatocellular cancer, gastric cancer, renal cell cancer, colorectal cancer, colon cancer, cervical cancer, and prostate cancer.

23. A liquid formulation comprising the ADC according to any one of claims 6-17.

24. The liquid formulation of claim 23, further comprising a buffer solution, wherein the buffer solution comprises a buffer and / or an excipient.

25. The liquid formulation of claim 24, wherein the buffer is selected from histidine, sodium citrate buffer, sodium acetate buffer or sodium phosphate buffer.

26. The liquid formulation of claim 24, wherein the excipient is selected from sucrose, trehalose, glycine or polysorbate 80.

27. The liquid formulation of claim 24, wherein the buffer has a pH value range from pH 4.5 to 7.0.

28. The liquid formulation of claim 24, wherein the buffer has a concentration range from 1 mM to 100 mM.

29. The liquid formulation of claim 26, wherein the sucrose has a concentration range from 10 mM to 500 mM.

30. The liquid formulation of claim 26, wherein the polysorbate 80 has a concentration range from 0.01% (w / v) to 0.1% (w / v).

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