WO2025109043A2 - Use of neutralizing anti-agr2 antibodies for preventing resistance to chemotherapy - Google Patents

Abstract

Most of therapeutic failures in chemotherapy during cancer invasion and metastasis are attributed to drug resistance. The inventors aimed to investigate the potential of targeting the secreted protein, eAGR2 as a novel adjuvant strategy to enhance the effectiveness of chemotherapy and overcoming chemoresistance in cancer therapy. Remarkably, extracellular AGR2 (eAGR2) demonstrated the ability to enhance resistance to both (doxorubicin and tamoxifen) treatments. Furthermore, even in the case of drug-resistant cancer cells that exhibited inherent resistance to chemotherapy, supplementation with an AGR2-blocking antibody restored sensitivity to tamoxifen in vitro. Thus, the inventor shed light on previously unknown chemotherapy resistance in resistant breast cancer cells, which can be effectively overcome by blocking eAGR2. Consequently, the inhibition of eAGR2 emerges as a promising adjunctive approach for concurrent administration alongside existing first- and second-line cancer therapies. This not only has the potential to bolster the efficacy of chemotherapy but also to prevent tumour chemoresistance.

Description

USE OF NEUTRALIZING ANTI-AGR2 ANTIBODIES FOR PREVENTING RESISTANCE TO CHEMOTHERAPY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology.

BACKGROUND OF THE INVENTION:

Currently, most of therapeutic failures in chemotherapy during cancer invasion and metastasis are attributed to drug resistance. Cancer drug resistance is a complex phenomenon, and numerous research studies suggest that the tumour microenvironment plays a role in initiating and maintaining resistance to treatments. In this context, we identify Anterior GRadient 2 (AGR2) protein, an endoplasmic reticulum (ER) resident protein that is also found outside of cells (extracellular AGR2 - eAGR2)^1>2, as well as in the blood and urine of cancer patients³. We have demonstrated that a fraction of AGR2 is present in the secretome (eAGR2), where it functions as a regulator of oncogenesis^1,4-6 and promotes pro-inflammatory phenotypes⁷ in epithelial tissues. Furthermore, studies have shown that increased AGR2 expression is associated with resistance to cancer treatments^8-11.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to the use of neutralizing anti-AGR2 antibodies for preventing resistance to chemotherapy.

DETAILED DESCRIPTION OF THE INVENTION:

The first object of the present invention relates to a method of treating chemotherapy-resistant cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a neutralizing anti-eAGR2 antibody.

A further object of the present invention relates to a method of preventing resistance to chemotherapy in a cancer patient comprising administering to the patient a therapeutically effective amount of a neutralizing anti-eAGR2 antibody.

As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a patient to be treated by the methods disclosed herein. In particular, the patient suffers from a cancer. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult. In some embodiments, the patient is elderly.

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes malignant diseases of any tissues/organs. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

As used herein; the term "chemotherapy-resistant cancer" refers to the clinical situation in a patient suffering from a cancer when the proliferation of tumor cells cannot be prevented or inhibited by means of a chemotherapeutic agent or a combination of chemotherapeutic agents usually used to treat cancer, at an acceptable dose to the patient. Thus, the expression "resistance to chemotherapy" is used in its broadest context to refer to the reduced effectiveness of chemotherapy to inhibit the growth of a tumor cell, kill a tumor cell or inhibit one or more cellular functions, and to the ability of a cell to survive exposure to an agent designed to inhibit the growth of the tumor cell, kill the tumor cell or inhibit one or more cellular functions. The leukemia can be intrinsically resistant prior to chemotherapy, or resistance may be acquired during treatment of leukemia that is initially sensitive to chemotherapy. The resistance displayed by a tumor cell may be complete in that the chemotherapy is rendered completely ineffective against the tumor cell, or may be partial in that the effectiveness of the chemotherapy is reduced. The phrase “preventing resistance to chemotherapy” or “overcoming resistance to chemotherapy” in context of the invention shall be effective if compared to a non-treated control, the tumor cells become more sensitive to chemotherapy. In particular, the patient becomes a responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the leukemia is eradicated, reduced or improved after immunotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after immunotherapy. A “non-responder” or “refractory patient” includes patients for whom the leukemia does not show reduction or improvement after chemotherapy. The term “non responder” also includes patients having a stabilized leukemia. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non-responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non-responder, the physician could take the decision to administer the patient with a therapeutically effective amount of a neutralizing anti-eAGR2 antibody.

As used herein, the term “chemotherapy” has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent. As used herein, the term "chemotherapeutic agent" refers to any chemical agent with therapeutic usefulness in the treatment of cancer. Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity upon which the tumor cell depends for continued survival. Categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these drugs are directly toxic to tumor cells and do not require immune stimulation. Suitable chemotherapeutic agents are described, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal medicine, 14th edition; Perry et al., Chemotherapeutic, Ch 17 in Abel off, Clinical Oncology 2nd ed., 2000 ChrchillLivingstone, Inc.; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Louis, mosby-Year Book, 1995; Fischer D. S., Knobf M. F., Durivage HJ. (eds): The Cancer Chemotherapeutic Handbook, 4th ed. St. Louis, Mosby- Year Handbook.

Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a 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 ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), 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, methotrexate, 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; an 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; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; 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., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In particular, chemotherapeutic agents include anthracy clines. Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin, N-trifluoro-acetyl doxorubicin- 14-val erate, aclacinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclarubicin).

In some embodiments, the method of the present invention is particularly suitable for preventing resistance to an anthracycline in a patient suffering from breast cancer. More particularly, the method of the present invention is particularly suitable for preventing resistance to doxorubicin in a patient suffering from breast cancer

In particular, chemotherapeutic agents also include selective estrogen receptor modulators (SERMs), also known as estrogen receptor agonist/antagonists (ERAAs). Examples of SERMs include Anordrin, Bazedoxifene, Broparestrol, Clomifene, Cyclofenil, Lasofoxifene, Ormeloxifene, Ospemifene, Raloxifene, Tamoxifen and Toremifene. In some embodiments, the method of the present invention is particularly suitable for preventing resistance to a selective estrogen receptor modulator in a patient suffering from breast cancer. More particularly, the method of the present invention is particularly suitable for preventing resistance to tamoxifen in a patient suffering from breast cancer.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

A further object of the present invention relates to a method of preventing relapse in a patient suffering from a cancer and who was treated by chemotherapy comprising administering to the patient a therapeutically effective amount of a neutralizing anti-eAGR2 antibody. As used herein, the term “relapse” refers to reappearance of the cancer after an initial period of responsiveness (e.g., complete response or partial response). The initial period of responsiveness may involve the level of tumor cells falling below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level of tumor cells rising above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. More generally, a response (e.g., complete response or partial response) can involve the absence of detectable MRD (minimal residual disease).

As used herein, the term “AGR2” has its general meaning in the art and refers to the gene encoding for the anterior gradient 2, protein disulphide isomerase family member (Gene ID: 10551). The genomic sequence is referenced in the NCBI database under the NC 000007.14 accession number. An exemplary amino acid sequence for the human AGR2 is represented by SEQ ID NO:1. Alternatives names for AGR2 include “Anterior Gradient 2 Protein”, “AG-2”, “AG2”, “HPC8”, “GOB-4”, “HAG-2”, “XAG-2”, “PADIA17”, “HEL-S-116”, “Protein Disulfide Isomerase Family A Member 17”, and “Secreted cement gland protein XAG-2 homolog” as non-limiting examples. Herein, the expressions “Anterior Gradient 2 Protein” and “AGR2” and “AG-2” are used indifferently.

MEKI PVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEE ALYKSKTSNKPLMI IHHLDECPHSQALKKVFAENKE IQKLAEQFVLLNLVYETTDKHLS P DGQYVPRIMFVDPSLTVRADITGRYSNRLYAYE PADTALLLDNMKKALKLLKTEL

As used herein, the term “eAGR2” refers to the secreted form of AGR2 such as described in Fessart, D., et al. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. Elife 5(2016). eAGR2 deems to have the same amino acid sequence as described for AGR2 (e g. SEQ ID NO: 1).

As used herein, the term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody is also “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four (a, 5, y) to five (p, s) domains, a variable domain (VH) and three to four constant domains (CHI, CH2, CH3 and CH4 collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (hereafter “Kabat et al ”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

As used herein, the term “affinity” means the strength of the binding of an antibody to a target molecule (e.g. an epitope). The affinity of a binding protein is given by the dissociation constant Kd. For an antibody said Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Preferred methods for determining the affinity of a binding protein can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of binding protein is the use of Biacore instruments.

As used herein, the term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of the binding of an antibody to a predetermined target molecule (e.g. an antigen or epitope) typically is a binding with an affinity corresponding to a KD of about 10'⁷ M or less, such as about 10'⁸ M or less, such as about 10'⁹ M or less, about 10’¹⁰ M or less, or about 10'¹¹ M or even less.

As used herein, the term “neutralizing antibody” or “blocking antibody” refers to an antibody that binds to eAGR2 and that reduces at least one biological activity of eAGR2. Assays for determining whether an antibody can neutralize the activity of eAGR2 may be performed as those disclosed in the EXAMPLE section of the present specification.

In some embodiments, the neutralizing antibody of the present invention does not specifically bind to AGR3.

The human AGR2 and AGR3 genes map to chromosome band 7p21.3. The AGR2 and AGR3 proteins are clustered together by phylogenetic analysis and share 65% sequence identity. AGR3 is the closest family member to AGR2.

As used herein, an antibody is said to be “specific for”, “immunospecific for”, or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., AGR2), preferably with an affinity constant (KA) of greater than or equal to about 10⁶ M’¹, preferably greater than or equal to about 10⁷ M’¹, 10⁸ M’¹, 5xl0⁸ M’¹, 10⁹ M’¹, 5xl0⁹ M'¹ or more. Affinity of an antibody or binding fragment thereof for its cognate antigen is also commonly expressed as the equilibrium dissociation constant (KD). An antibody is said to be “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., AGR2), preferably with a KD of less than or equal to 10'⁶ M, preferably less than or equal to 10'⁷ M, 5x1 O'⁸ M, 10'⁸ M, 5x1 O'⁹ M, 10'⁹ M or less. Affinities of antibodies or binding fragment thereof can be readily determined using conventional techniques, for example, those described by Scatchard, 1949. Ann NY Acad Sci. 51:660-672. Binding properties of an antibody or binding fragment thereof to antigens, cells or tissues may generally be determined and assessed using immunodetection methods including, for example, ELISA, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or 25 fluorescence-activated cell sorting (FACS) or by surface plasmon resonance (SPR, e.g., using BIAcore®).

In some embodiments, the antibody is a humanized antibody. As used herein, the term "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.

In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

In some embodiments, the antibody of the present invention an antibody fragment. As used herein, the term "antibody fragment" refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single chain antibody molecules, in particular scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as, for example, sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as, for example, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Fragments and derivatives of antibodies of this invention (which are encompassed by the term “antibody” as used in this application, unless otherwise stated or clearly contradicted by context), can be produced by techniques that are known in the art. “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single -chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods. For instance, Fab or F(ab')2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques. It will be appreciated that immunoreactive fragments can be modified using known methods, for example to slow clearance in vivo and obtain a more desirable pharmacokinetic profile the fragment may be modified with polyethylene glycol (PEG). Methods for coupling and site- specifically conjugating PEG to a Fab' fragment are described in, for example, Leong et al., Cytokines 16 (3): 106-119 (2001) and Delgado et al., Br. J. Cancer 5 73 (2): 175- 182 (1996), the disclosures of which are incorporated herein by reference.

In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also “nanobody®”.

In some embodiments, the antibody comprises human heavy chain constant regions sequences but will not induce antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody of the present invention does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD 16) polypeptide. In some embodiments, the antibody of the present invention lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody of the present invention consists of or comprises a Fab, Fab', Fab'-SH, F (ab¹) 2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the antibody of the present invention is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

In some embodiments, the antibody of the present invention is 18A4 or one of its derivative forms including the humanized form of said antibody as described in the following references, the contents of which are incorporated herein by reference:

Guo, Hao, et al. "A humanized monoclonal antibody targeting secreted anterior gradient 2 effectively inhibits the xenograft tumor growth. " Biochemical and biophysical research communications 475.1 (2016): 57-63.

Guo, H., et al. "Tumor-secreted anterior gradient-2 binds to VEGF and FGF2 and enhances their activities by promoting their homodimerization." Oncogene 36.36 (2017): 5098.

Qudsia, Sehar, et al. "A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies. " Biochemical and biophysical research communications 499.1 (2018): 71-77, and

US20140328829 Dawei Li, Zhenghua Wu, Hao GuoQi Zhu, Dhahiri S. Mashausi “Agr2 blocking antibody and use thereof”

In some embodiments, the antibody of the present invention is the murine anti-human monoclonal antibody 18A4 or humanized or chimeric form thereof. The 18A4 antibody is obtainable from the hybridoma cell line that was deposited in the China Center of Type Cell Collection (CCTCC) on Jan. 19, 2009 with a deposit number of CCTCC-C200902 at the address of the Wuhan University, Luojiashan, Wuchang, Wuhan, Hubei Province.

In some embodiments, the antibody of the present invention binds to an epitope that is located within the protein disulfide isomerase active domain of AGR2.

As used herein, the term “epitope” refers to a specific arrangement of amino acids located on a protein or proteins, serving as the binding site for an antibody. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, z.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

In some embodiments, the antibody of the invention binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 in the amino acid sequence as set forth in SEQ ID NO:2 (PLMIIHHLDECPHSQALKKVFA). In some embodiments, the antibody of the present invention binds to an epitope as set forth in SEQ ID NO:2.

In some embodiments, the antibody of the invention comprises a heavy chain variable region (VH) comprising at least one or at least two of the following CDRs:

H-CDR1 : DYNMD (SEQ ID NO:3)

H-CDR2: DINPNYDTTSYNQKFQG (SEQ ID NO:4)

H-CDR3: SMMGYGSPMDY (SEQ ID NO:5)

In some embodiments, the antibody of the invention comprises a light chain variable region (VL) comprising at least one or at least two of the following CDRs:

L-CDR1 : RASKSVSTSGYSYMH (SEQ ID NO:6)

L-CDR2: LASNLES (SEQ ID NO: 7)

L-CDR3: QHIRELPRT (SEQ ID NO: 8)

In some embodiment, the antibody of the invention comprises a heavy chain variable region (VH) comprising at least one of the following CDR i) the VH-CDR1 as set forth in SEQ ID NO:3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH-CDR3 as set forth in SEQ ID NO:5 (SMMGYGSPMDY) and/or a light chain variable region (VL) comprising at least one of the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKSVSTSGYSYMH), ii) the VL-CDR2 as set forth in SEQ ID NO:7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 (QHIRELPRT).

In some embodiment, the antibody of the invention comprises a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO:3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH- CDR3 as set forth in SEQ ID NO: 5 (SMMGYGSPMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKSVSTSGYSYMH), ii) the VL-CDR2 as set forth in SEQ ID NO:7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 (QHIRELPRT).

In some embodiments, the antibody of the present invention comprises the heavy chain as set forth in SEQ ID NO: 9:

SEQ ID NO : 9 :

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWI GDINPNYDTTSYNQKFKGKAT LTVDKSTSTAYMELSSLRSEDTAVYYCARSMMGYGS PMDYWGQGTLVTVSS

In some embodiments, the antibody of the present invention comprises a heavy chain as set forth in SEQ ID NO:9 mutated by four substitutions at positions 65, 67, 68 and 70, wherein said substitutions are characterized in that: lysine (K) at position 65 is changed to glutamine (Q), lysine (K) at position 67 is changed to arginine (R), alanine (A) at position 68 is changed to valine (V), and leucine (L) at position 70 is changed to methionine (M), and wherein the numbers of the positions correspond to the Kabat numbering system.

In some embodiments, the antibody of the present invention comprises the heavy chain as set forth in SEQ ID NO: 10:

SEQ ID NO : 10 :

E IVLTQS PATLSLS PGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGI PARFSGS

GSGTDFTLTI SRLE PEDFAVYYCQHIRELPRTFGGGTKLE IK In some embodiments, the antibody of the present invention is selected among the antibodies described in Arumugam, Thiruvengadam, et al. "New Blocking Antibodies against Novel AGR2- -C4. 4A Pathway Reduce Growth and Metastasis of Pancreatic Tumors and Increase Survival in Mice. "Molecular cancer therapeutics 14.4 (2015): 941-951, the content of which is incorporated herein by reference.

In some embodiments, the antibody of the invention binds to an epitope comprising 1, 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, or 28 in the amino acid sequence as set forth in SEQ ID NO: 11 (IHHLDECPHSQALKKVFAENKEIQKLAEQ). In some embodiments, the antibody of the present invention binds to an epitope as set forth in SEQ ID NO: 11.

In some embodiments, the antibody of the invention comprises a heavy chain variable region (VH) comprising at least one or at least two of the following CDRs:

H-CDR1 : NYGMN (SEQ ID NO: 12)

H-CDR2: WINTDTGKPTYTEEFKG (SEQ ID NO: 13)

H-CDR3: VTADSMDY (SEQ ID NO: 14)

In some embodiments, the antibody of the invention comprises a light chain variable region (VH) comprising at least one or at least two of the following CDRs:

L-CDR1 : RSSQSLVHSNGN (SEQ ID NO: 15)

L-CDR2: IYLH (SEQ ID NO: 16)

L-CDR3: SOSTHVPLT (SEQ ID NO: 17)

In some embodiment, the antibody of the invention comprises a heavy chain variable region (VH) comprising at least one of the following CDR i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and/or a light chain variable region (VH) comprising at least one of the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL-CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT). In some embodiment, the antibody of the invention comprises a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL- CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT).

In some embodiments, the antibody of the present invention cross-competes for binding to AGR2 with the antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO: 3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH-CDR3 as set forth in SEQ ID NO:5 (SMMGYGSPMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKSVSTSGYSYMH), ii) the VL-CDR2 as set forth in SEQ ID NO: 7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO:8 (QHIRELPRT).

In some embodiments, the antibody of the present invention cross-competes for binding to AGR2 with the antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL-CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT).

As used herein, the term “cross-competes” refers to monoclonal antibodies which share the ability to bind to a specific region of an antigen. In the present disclosure the monoclonal antibody that “cross-competes" has the ability to interfere with the binding of another monoclonal antibody for the antigen in a standard competitive binding assay. Such a monoclonal antibody may, according to non-limiting theory, bind to the same or a related or nearby (e.g., a structurally similar or spatially proximal) epitope as the antibody with which it competes. Cross-competition is present if antibody A reduces binding of antibody B at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said antibodies. As the skilled artisan appreciates competition may be assessed in different assay set-ups. One suitable assay involves the use of the Biacore technology (e.g., by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore, a high throughput process for "binning" antibodies based upon their cross-competition is described in International Patent Application No. WO2003/48731.

According to the present invention, the cross-competing antibody as above described retain the activity of antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO:3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH-CDR3 as set forth in SEQ ID NO:5 (SMMGYGSPMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKSVSTSGYSYMH), ii) the VL- CDR2 as set forth in SEQ ID NO: 7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO:8 (QHIRELPRT).

According to the present invention, the cross-competing antibody as above described retain the activity of antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL-CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT).

Any assay well known in the art would be suitable for identifying whether the cross-competing antibody retains the desired activity. The assays as those disclosed in the EXAMPLE section of the present specification would be suitable for determining whether the antibody retains said ability.

As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the antibody of the present invention for the treatment of the cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compound will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically, the antibody of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, the antibody of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Therapy-induced eAGR2 secretion during resistance.

(A) MCF-7 parental cells (MCF-7P) were treated with Doxorubicin until the emergence and growth of drug resistant clones (MCF-7R). Representative bright field pictures of the generated clones.

(B) Western blot analysis showing increased secretion levels of extracellular AGR2 protein (eAGR2) in MCF7-R cells compared to MCF-7P cells. One representative experiment (n = 3) is shown. (C) Evaluation of cell growth in response to treatment with IpM doxorubicin in the absence (Dox.) or presence of extracellular AGR2 (Dox. + eAGR2) compared to control cells. Data are presented as mean ± SEM of at least three independent experiments. The p-values (determined by Student's t-test) are relative to Control cells. ****p<0.0001.

Figure 2. Sensitizing cells to therapy with AGR2-blocking antibody.

(A) Cell growth of cells treated with IpM doxorubicin in the absence (Dox.) or presence of the AGR2 -blocking antibody (Dox. +Ac-AGR2) compared to control cells and antibody alone (Ac- AGR2). Data are presented as mean ± SEM of at least three independent experiments. The p- values (determined by Student's t-test) are relative to Control cells. ****p<0.0001, **p<0.01.

(B) Cell death of cells treated with IpM doxorubicin in the absence (Dox.) or presence of AGR2 -blocking antibody (Dox. +Ac-AGR2) compared to control cells and antibody alone (Ac- AGR2). Data are presented as mean ± SEM of at least three independent experiments. The p- values (determined by Student's t-test) are relative to Control cells. ****p<0.0001, **p<0.01.

Figure 3. AGR2-blocking antibody restores sensitivity to therapy-in resistant cells.

(A, C) Evaluation of cell growth in sensitive MCF-7 cells (A) or resistant cells (C) treated with IpM Tamoxifen (Tamox.) in absence or presence of eAGR2 (Tamox. + eAGR2) compared to control cells and eAGR2. Data are presented as mean ± SEM of at least three independent experiments. The p-values (determined by Student's t-test) are relative to Control cells. ****p<0 0001_anc[ ***p<0.001.

(B, D) Evaluation of cell growth in sensitive MCF-7 cells (B, left panel) or resistant cells (D, left panel) treated with IpM Tamoxifen (Tamox.) in absence or presence of AGR2 -blocking antibody (Tam. + Ac-AGR2) compared to control cells and antibody alone (Ac-AGR2). Evaluation of cell death in sensitive MCF-7 cells (B, right panel) or resistant MCF-7 cells (D, right panel) treated with IpM Tamoxifen (Tam.) in absence or presence of AGR2 -blocking antibody (Tam. +Ac-AGR2) compared to control cells and antibody alone. Data are presented as mean ± SEM of at least three independent experiments. The p-values (determined by Student's t-test) are relative to Control cells. **p<0.01.

Figure 4. AGR2-blocking antibodies restore sensitivity to doxorubicin treatment.

Evaluation of cell growth in MCF-7 cells treated with 100 nM doxorubicin following 72 h of treatment in the absence or presence of AGR2 -blocking antibody (Agtuzumab) or clone 1C3 (Abnova), compared to control cells and IgG antibody (IgG). Data are presented as mean ± SEM of at least three independent experiments. The p-values (determined by Student's t-test) are relative to doxorubicin-treated cells. *p<0.05.

Figure 5. AGR2-blocking antibody restores sensitivity to combine paclitaxel and cisplatin treatment.

Evaluation of cell growth in MCF-7 cells treated with the combined paclitaxel (5 nM) and cisplatin (2 pM) treatment following 72 h treatment in the absence or presence of AGR2- blocking antibody (clone 1C3 (Abnova)), compared to control cells and IgG antibody (IgG). Data are presented as mean ± SEM of at least three independent experiments. The p-values (determined by Student's t-test) are relative to treated cells (paclitaxel + cisplatin). ***p<0.001.

EXAMPLE:

Doxorubicin treatment induces the secretion of eAGR2 to support drug-resistant cells and enhances proliferation to promote the survival of drug-sensitive cells.

Soluble mediators from the tumour microenvironment can foster cancer growth and therapy resistance¹². We hypothesized that signals derived from sensitive cancer cells in response to chemotherapy, such as doxorubicin, drive the outgrowth of drug-resistant cell populations. To investigate this hypothesis, we developed an in vitro culture system using the human breast cancer cell line MCF-7 (MCF-7P) and monitored AGR2 secretion following doxorubicin treatment (Fig. 1A-B).

Remarkably, this treatment significantly promoted the growth of resistant cancer cells (Fig. 1A, MCF-7R), and led to an increased secretion of eAGR2 by these resistant cells (Fig. IB, MCF- 7R). Similarly, doxorubicin treatment in the presence of eAGR2 in the culture medium resulted in increased cell growth (Fig. 1C). Thus, the secretion of eAGR2 plays a pivotal role in driving the development of tumour-promoting doxorubicin-resistant cancer cells.

Blocking eAGR2 during doxorubicin treatment enhances cellular sensitivity to chemotherapy.

To assess the adjuvant effect of eAGR2 inhibition, we used a blocking antibody (Ac-AGR2, 1C3) to sequester eAGR2. Initially, we investigated the impact of doxorubicin on cell growth and cell death in MCF-7 breast-cancer cells (Fig. 2). After 48 hours, cells exhibited sensitivity to a IpM dose of doxorubicin (Fig. 2A-B), resulting in a 25% reduction in cell number. We then investigated the impact on cell viability of the AGR2 -blocking antibody in combination with chemotherapy. Our results demonstrated that the AGR2 -blocking antibody treatment, as adjuvant, significantly reduced cell viability beyond the effect of chemotherapy alone (Fig. 2A Doxo.+Ac-AGR2), compared to the antibody alone (Fig. 2A, Ac-AGR2). Similarly, the AGR2- blocking antibody enhanced cell death in doxorubicin-treated cells (Fig. 2B, Doxo.+Ac- AGR2). Therefore, inhibiting eAGR2 in MCF-7 cells enhanced their sensitivity to doxorubicin treatment.

Blocking eAGR2 during tamoxifen treatment restores sensitivity to therapy in resistant cells.

To determine whether the effect of eAGR2 is specific to doxorubicin treatment or applicable to others therapies, we decided to evaluate its impact in the context of tamoxifen treatment. Tamoxifen acts as an anti-estrogen in breast cancer cells, inhibiting proliferation. Given that a significant proportion of breast cancer patients are estrogen receptor (ER) positive and receive adjuvant endocrine therapy including tamoxifen, it is noteworthy that approximately one third of these patients develop chemoresistance⁸. Previous reports have established a correlation between AGR2 expression and drug resistance^8,11, prompting us to investigate the role of eAGR2 in both tamoxifen-sensitive and tamoxifen-resistant cells.

We conducted a comparative study between ER-positive MCF-7 sensitive cells and a tamoxifen-resistant cell line (TamR). The TamR cell line emerges as a result of prolonged exposure of MCF-7 to tamoxifen. In tamoxifen-sensitive cells, the presence of eAGR2 in the culture medium enhanced cell growth (Fig. 3A). Conversely, the AGR2-blocking antibody increased cell sensitivity to treatment by reducing cell growth (Fig. 3B, left panel) and increasing cell death (Fig. 3B, right panel).

Having evaluated the adjuvant anticancer properties of the AGR2-blocking antibody in tamoxifen-sensitive breast-cancer cells in vitro, we investigated whether the same effect could be replicated in tarn oxifen -resistant MCF-7 breast cancer cells (TamR). We evaluated the impact of combining tamoxifen treatment with either eAGR2 (Fig. 3C) or the blocking antibody (Fig. 3D). Firstly, we assessed the cytotoxic effects of a IpM tamoxifen dose over a 48-hour period. Figure 3C demonstrates that tamoxifen alone did not induce significant cytotoxic effects on TamR cells. However, despite the inherent tamoxifen resistance of TamR cells, the combination of tamoxifen treatment with the AGR2 -blocking antibody resulted in a significant reduction in cell growth at 48 hours (Fig. 3D, left panel) and a corresponding increase in cell death (Fig. 3D, right panel).

Blocking eAGR2 with different antibodies during doxorubicin treatment enhances cellular sensitivity to chemotherapy.

Next, we compared the effect of the antibody 1C3 (Abnova) to another source of antibody targeting eAGR2, such as Agtuzumab, in comparison to the IgG control antibody. After 72 hours, cells exhibited sensitivity to doxorubicin (Fig. 4), resulting in a 57-68% reduction in cell number. We then investigated the impact of the AGR2 -blocking antibody in combination with doxorubicin on cell viability. Our results demonstrated that AGR2 -blocking antibody treatment, whether 1C3 or Agtuzumab, significantly reduced cell viability beyond the effect of chemotherapy alone (Fig. 4), compared to the IgG control antibody (Fig. 4). Therefore, inhibiting eAGR2 with different AGR2 -targeted antiobodies enhanced sensitivity to doxorubicin treatment.

Blocking eAGR2 during combined palclitaxel and cisplatin treatment restores sensitivity to therapy in MCF-7cells.

To determine whether the effect of eAGR2 would be applicable to others therapies, we evaluated its impact in the context of combined treatment with paclitaxel and cisplatin. Combination chemotherapy, involving the administration of paclitaxel followed by a platinumbased regimen, is currently the first-line therapy for ovarian cancer and a common treatment for breast cancer. After 72 hours, cells exhibited sensitivity to the combined treatment (paclitaxel + cisplatin) (Fig. 5), resulting in a 53% reduction in cell number. We then investigated the impact of the AGR2-blocking antibody in combination with this combined treatment on cell viability. Our results demonstrated that AGR2 -blocking antibody treatment, as an adjuvant, significantly reduced cell viability (reaching a 75% reduction in cell number) beyond the effect of combined chemotherapy alone, compared to the control IgG antibody. Therefore, inhibiting eAGR2 in MCF-7 cells enhanced their sensitivity to various chemotherapies.

Conclusion

This study aimed to investigate the potential of targeting the secreted protein, eAGR2 as a novel adjuvant strategy to enhance the effectiveness of chemotherapy and overcoming chemoresistance in cancer therapy. Our initial focus was to evaluate the impact of eAGR2 on cancer cell growth. Remarkably, extracellular AGR2 (eAGR2) demonstrated the ability to enhance resistance to both (doxorubicin and tamoxifen) treatments. Furthermore, even in drugresistant cancer cells exhibiting inherent resistance to chemotherapy, supplementation with an AGR2 -blocking antibody successfully restored sensitivity to tamoxifen in vitro, as well as to combined therapy with paclitaxel and cisplatin.

In summary, our study sheds light on previously unknown chemotherapy resistance in tamoxifen-resistant breast cancer cells, which can be effectively overcome by blocking eAGR2. Consequently, the inhibition of eAGR2 emerges as a promising adjunctive approach for concurrent administration alongside existing first- and second-line cancer therapies. This not only has the potential to bolster the efficacy of chemotherapy but also to prevent tumour chemoresistance.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Fessart D, Domblides C, Avril T, et al. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. eLife 2016;5.

2. Fessart D, de Barbeyrac C, Boutin I, et al. Extracellular AGR2 triggers lung tumour cell proliferation through repression of p21(CIPl). Biochimica et biophysica acta Molecular cell research 2021; 1868: 118920.

3. Shi T, Gao Y, Quek SI, et al. A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum. Journal of proteome research 2014;13:875-82.

4. Chevet E, Fessart D, Delom F, et al. Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development. Oncogene 2013;32:2499-509.

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6. Higa A, Mulot A, Delom F, et al. Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. The Journal of biological chemistry 2011;286:44855-68. 7. Maurel M, Obacz J, Avril T, et al. Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation. EMBO molecular medicine 2019.

8. Hrstka R, Brychtova V, Fabian P, Vojtesek B, Svoboda M. AGR2 predicts tamoxifen resistance in postmenopausal breast cancer patients. Disease markers 2013;35:207-12. 9. Liu QG, Li YJ, Yao L. Knockdown of AGR2 induces cell apoptosis and reduces chemotherapy resistance of pancreatic cancer cells with the involvement of ERK/AKT axis. Pancreatology : official journal of the International Association of Pancreatology 2018;18:678- 88.

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Claims

CLAIMS:

1. A method of treating a chemotherapy-resistant cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a neutralizing anti-eAGR2 antibody.

2. A method of preventing resistance to chemotherapy in a cancer patient comprising administering to the patient a therapeutically effective amount of a neutralizing anti- eAGR2 antibody.

3. A method of preventing relapse in a patient suffering from a cancer and who was treated by chemotherapy comprising administering to the patient a therapeutically effective amount of a neutralizing anti-eAGR2 antibody.

4. The method according to any one of claims 1 to 3 wherein the chemotherapeutic agent is selected among anthracyclines such as daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin, N-trifluoro- acetyl doxorubicin- 14-val erate, aclacinomycin, morpholinodoxorubicin (morpholino- DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino- doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclarubicin).

5. The method of claim 4 for preventing resistance to an anthracy cline in a patient suffering from breast cancer.

6. The method of claim 5 for preventing resistance to doxorubicin in a patient suffering from breast cancer.

7. The method according to any one of claims 1 to 3 wherein the chemotherapeutic agent is selected among selective estrogen receptor modulators (SERMs) such as Anordrin, Bazedoxifene, Broparestrol, Clomifene, Cyclofenil, Lasofoxifene, Ormeloxifene, Ospemifene, Raloxifene, Tamoxifen and Toremifene.

8. The method of claim 7 for preventing resistance to a selective estrogen receptor modulator in a patient suffering from breast cancer.

9. The method of claim 8 for preventing resistance to tamoxifen in a patient suffering from breast cancer.

10. The method according to any one of claims 1 to 9 wherein the antibody binds to an epitope that is located within the protein disulfide isomerase active domain of AGR2.

11. The method according to any one of claims 1 to 9 wherein the antibody binds to an epitope as set forth in SEQ ID NO:2.

12. The method of claim 11 wherein the antibody comprises a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO:3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH-CDR3 as set forth in SEQ ID NO:5 (SMMGYGSPMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKS VSTSGYSYMH), ii) the VL-CDR2 as set forth in SEQ ID NO: 7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO:8 (QHIRELPRT).

13. The method of claim 11 wherein the antibody cross-competes for binding to AGR2 with the antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO:3 (DYNMD), ii) the VH-CDR2 as set forth in SEQ ID NO:4 (DINPNYDTTSYNQKFQG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 5 (SMMGYGSPMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO:6 (RASKSVSTSGYSYMH), ii) the VL-CDR2 as set forth in SEQ ID NO:7 (LASNLES) and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 (QHIRELPRT).

14. The method according to any one of claims 1 to 9 wherein the antibody binds to an epitope as set forth in SEQ ID NO: 11.

15. The method of claim 13 wherein the antibody comprises a heavy chain variable region (VH) comprising at least one of the following CDR i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and a light chain variable region (VH) comprising at least one of the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL-CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT).

16. The method of claim 13 wherein the antibody cross-competes for binding to AGR2 with the antibody comprising a heavy chain variable region (VH) comprising the following CDR: i) the VH-CDR1 as set forth in SEQ ID NO: 12 (NYGMN), ii) the VH-CDR2 as set forth in SEQ ID NO: 13 (WINTDTGKPTYTEEFKG) and iii) the VH-CDR3 as set forth in SEQ ID NO: 14 (VTADSMDY) and a light chain variable region (VH) comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 15 (RSSQSLVHSNGN), ii) the VL-CDR2 as set forth in SEQ ID NO: 16 (IYLH) and iii) the VL-CDR3 as set forth in SEQ ID NO: 17 (SOSTHVPLT).