Humanized anti-AGR2 antibody

Abstract

The present invention relates to the field of immunotherapy. In particular, the present invention relates to an antibody or a binding fragment thereof that specifically binds to anti-gradient protein 2 (AGR2). The present invention further relates to the use of the antibody or a binding fragment thereof in therapy.

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. In particular, the present invention relates to an antibody or a binding fragment thereof that specifically binds to Anterior Gradient 2 Protein (AGR2). The present invention further relates to the use of the antibody or a binding fragment thereof in therapy. [Background technology]

[0002] Antigradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein belonging to the protein disulfide isomerase superfamily. AGR2 exhibits two localizations: intracellular and extracellular. In healthy cells expressing AGR2, the predominant form is the intracellular form present in the endoplasmic reticulum. In contrast, cancer cells express AGR2 on the cell surface and secrete it into the extracellular environment. Thus, AGR2 is found not only in the ER but also in other locations, such as the nucleus, cytoplasm, plasma membrane, and extracellular space.

[0003] It has been shown that AGR2 not only exists as a monomer but can also form homodimers, indeed, AGR2 forms dimers via residues E60 and C81.

[0004] The AGR2 protein possesses the relatively unique chaperone properties in that it can bind to a specific peptide motif (TTIYY-SEQ ID NO: 48) in a sequence-specific manner. Furthermore, AGR2 possesses an ER retention signal sequence (KTEL-SEQ ID NO: 49). In normal tissues, AGR2 may contribute to regulating the total protein load within cells. AGR2 is also involved in pathways of ER stress, protein folding, transcriptional regulation, and exosome formation. Basal levels of AGR2 expression were observed across different tissue types, particularly for tissues of epithelial origin. In adult tissues, the highest levels of AGR2 expression were observed in the gastrointestinal tract (from the stomach to the rectum) and urogenital tract (the urinary bladder and female and male reproductive tract), as well as in the respiratory epithelium of the nasopharynx and bronchi (i.e., all mucosal epithelia). In mammals, AGR2 is generally present in mucus-secreting epithelial cells, highly expressed in Paneth and goblet enterocytes, with the highest levels in the ileum and colon.

[0005] AGR2 is a marker of tumor aggressiveness expressed by many solid tumor types. In the prostate, AGR2 is overexpressed in cancer cells compared with normal luminal cells, and the majority of primary prostate tumors are AGR2-positive. This pattern is also found in pancreatic, oral, and breast cancers. AGR2 is also highly expressed in non-small cell lung cancer, and high expression is associated with decreased survival.

[0006] Studies in mammals have revealed a role for AGR2 as a prometastatic protein essential for cancer progression and drug resistance. Recent data highlight the extracellular role of AGR2 in promoting cancer growth. The cancer-related functions of AGR2 appear to derive from its ability to promote cell adhesion, stimulate cell migration via its extracellular activity, and catalyze plasma membrane receptor trafficking via its intracellular function. Recently, a specific protein-protein interaction between AGR2 and the oncogenic membrane receptor EpCAM has been demonstrated.

[0007] The role of extracellular AGR2 (eAGR2) in tumorigenesis clearly demonstrates that the eAGR2 protein acts as an extracellular regulator of tumor morphogenesis, tumorigenicity, and inflammation-related phenotypes through gain of extracellular function. Dysregulation of intracellular (iAGR2) and extracellular (eAGR2) AGR2 localization can result in distinct pro-oncogenic gain-of-function interactions.

[0008] Due to its near ubiquitous expression in solid tumors, expression in premalignant lesions and its involvement in metastatic disease, the AGR2 protein is a relevant target for cancer therapy.

[0009] Furthermore, AGR2 is also involved in other diseases, such as asthma and inflammatory bowel disease. Indeed, studies in transgenic mice have shown that AGR2-null animals have defective mucin production, altered asthma incidence, and are primed to develop inflammatory bowel disease. The development of these pathologies in AGR2-null animals is due to the essential role of intracellular AGR2, which acts as a chaperone in mucin folding and transport.

[0010] Several anti-AGR2 antibodies are commercially available. The epitopes recognized by these commercially available antibodies have not been described. These antibodies are mostly sold for Western blot or immunohistochemistry applications without any guarantee of functionality in ELISA or in vitro functional assays. Furthermore, they are not described as cross-reactive with other species, preventing their use in animal models. Furthermore, these antibodies are rarely mentioned in any publications.

[0011] The anti-AGR2 clone ≪1C3≫, commercially available from Abnova (H00010551-M03), was obtained by immunization of mice with a full-length recombinant AGR2 protein containing a GST tag, but its epitope is unknown. This monoclonal antibody is a mouse IgG2b kappa antibody.

[0012] Liu et al. generated two anti-human AGR2 antibodies, P1G4 and P3A5, intended for use as therapeutic treatments for cancer. The authors demonstrated in vivo enhancement of gemcitabine inhibition of tumor growth by the P1G4 monoclonal antibody, but not by P3A5.

[0013] A humanized anti-AGR2 antibody (18A4Hu) and its murine version (18A4) were reported to have inhibitory effects on the AGR2+ ovarian cancer xenograft SK-OV-3.

[0014] It should be noted that all of these monoclonal antibodies were developed solely for oncological indications, and the data obtained with these antibodies, when available, only demonstrate inhibitory effects on tumors.

[0015] Additionally, AGR2 has been shown to be involved in other diseases, such as inflammatory diseases.

[0016] AGR2 expression is increased in biopsies taken from patients with active ulcerative colitis disease compared with patients in remission and non-IBD controls. AGR2 has also been found to be associated with disruption of homeostasis in pediatric ulcerative colitis disease. Furthermore, in surgical specimens of ulcerative colitis, AGR2 is highly expressed in colonic epithelium associated with histological evidence of fibrosis, whereas the immunohistochemical signal for AGR2 is significantly lower in isolated normal surgical margins in non-IBD colons.

[0017] Furthermore, immunohistological staining of colon sections from patients with inflammatory bowel disease shows that AGR2 expression is highly upregulated compared to non-inflammatory controls and is localized to all epithelial surfaces (Al-Shaibi et al., Cell Mol Gastroenterol Hepatol, 2021;12(5):1809-1830).

[0018] In inflammatory bowel diseases, particularly Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, which correlates with disease severity. AGR2 dimers act as sensors of ER homeostasis and are disrupted by ER stress, promoting the secretion of AGR2 monomers. The latter may represent a systemic warning signal for proinflammatory responses (Maurel et al., EMBO Mol Med, 2019, 11(6):e10120).

[0019] AGR2 release in the extracellular environment enhances monocyte recruitment and a proinflammatory phenotype. Regulation of AGR2 dimerization is associated with proinflammatory responses and macrophage enrichment in the colonic mucosa, which can be observed in Crohn's disease (Maurel et al., EMBO Mol Med, 2019, 11(6):e10120).

[0020] Secretion of AGR2 by epithelial cells may be involved in the development of fibrosis in Crohn's disease. In the ileum, there was a significant increase in AGR2 in tissues with fibrotic compartments compared to purely inflammatory samples, highlighting its involvement in the fibrostenotic process. AGR2 overexpression at the mRNA level correlated with the fibrosis grade in Crohn's disease patients (Vieujean et al., J Crohns Colitis, 2021, 15(10):1737-1750).

[0021] In its monomeric form, eAGR2 has been shown to selectively promote monocyte attraction, thereby linking eAGR2 to a proinflammatory phenotype and elucidating the extracellular gain of function of AGR2 as a proinflammatory chemokine (Maurel et al., EMBO Mol Med, 2019, 11(6):e10120). AGR2-blocking antibodies have been shown to prevent monocyte migration and may therefore inhibit the very early stages of inflammation by blocking local monocyte recruitment.

[0022] Furthermore, the differentiation of fibroblasts into myofibroblasts obtained in the presence of supernatant from intestinal epithelial cells preconditioned by ER stress and when cultured with recombinant AGR2 can be attenuated after blocking AGR2 with anti-AGR2 antibodies. Thus, AGR2 appears to have a profibrotic role and act as a paracrine inducer of intestinal fibroblast-to-myofibroblast differentiation (Vieujean et al., J Crohns Colitis, 2021, 15(10):1737-1750).

[0023] By blocking the differentiation of fibroblasts into myofibroblasts, anti-AGR2 antibodies may block the establishment of fibrosis in patients and prevent strictures.

[0024] The pathogenesis of chronic inflammatory bowel diseases such as Crohn's disease or ulcerative colitis is poorly understood, which leads to therapeutic strategies that focus on treating the inflammatory symptoms without being able to act on the initial cause of the disease.

[0025] Less severe cases are treated with aminosalicylates or corticosteroids, which have local effects on inflammation. Patients with more severe forms of these diseases or who develop dependence on corticosteroids are treated with so-called biologic therapies. Management of inflammatory bowel disease often includes immunosuppressive treatments such as corticosteroids, immunomodulators, small molecules, and biologics (e.g., anti-TNF antibodies) that inhibit pro-inflammatory cytokine pathways.

[0026] Anti-TNF (tumor necrosis factor) antibodies are first-line treatments, but are effective in only 30-40% of patients who achieve remission. Furthermore, 15% of patients who respond to anti-TNF develop insensitivity per year, forcing gastroenterologists to use second-line anti-interleukin (usketinumab, STELARA®, Janssen) and anti-integrin (vedolizumab, ENTYVIO®, Takeda) antibodies.

[0027] Although the efficacy of these biologic therapies is good, it rapidly diminishes over time. Notably, these treatments, which target the patient's immune system, have numerous side effects, including skin disorders, opportunistic infections, and cancer risk. New therapeutic approaches, particularly small molecules targeting the ubiquitous JAK or S1P pathways, are currently being developed, but their efficacy or safety in patients has not been demonstrated.

[0028] Given the high associated risks of cancer and serious infections, the use of more traditional immunosuppressants is becoming increasingly rare. Surgery remains the most frequent outcome to avoid severe complications (especially in colorectal cancer), causing serious changes in the patient's life.

[0029] When patients experience relapse of inflammation, current treatments can induce remission. However, these treatments must be administered chronically for life to avoid recurrence. However, very serious and rapid treatment failure has been observed in patients, and serious side effects urge utmost caution during long-term administration.

[0030] Therefore, the discovery of a treatment that can maintain patients in long-term remission without presenting the risk of side effects would be a breakthrough in the field of inflammatory diseases of the mucosa.

[0031] AGR2 is a promising therapeutic target in the setting of inflammatory diseases, however, anti-AGR2 antibodies have not been developed or shown to be effective for the prevention or treatment of inflammatory diseases.

[0032] Thus, there remains a need for novel anti-AGR2 antibodies suitable for use as therapeutics, particularly in the context of inflammatory diseases.

[0033] As a result, the present inventors aimed to develop novel tools that target the AGR2 protein, particularly extracellular AGR2. More specifically, the present inventors disclose novel antibodies and binding fragments thereof that specifically bind to AGR2. The antibodies and binding fragments thereof of the present invention that specifically bind to AGR2 may be useful therapeutic tools for treating, for example, mucosal inflammatory diseases or cancer.

[0034] By targeting a pathologically abnormal secreted protein (eAGR2), we aim to abrogate a unique mechanism of action: early crosstalk between the epithelium and the immune system in mucosal inflammatory diseases, while having a secretory profile that is much less aggressive than competing immunotherapies.

[0035] Compared to standard products currently used to treat mucosal inflammatory diseases, anti-eAGR2 is the only product that combines anti-inflammatory and anti-fibrotic effects, has no risk of side effects, and has a targeted effect on affected tissues.

[0036] We have identified several humanized monoclonal antibodies that can specifically bind to human AGR2 with high affinity. These humanized antibodies may be used to prevent or treat inflammatory diseases and cancer. [Prior art documents] [Non-patent literature]

[0037] [Non-Patent Document 1] Al-Shaibi et al.,Cell Mol Gastroenterol Hepatol,2021;12(5):1809-1830 [Non-patent document 2] Maurel et al.,EMBO Mol Med,2019,11(6):e10120 [Non-patent document 3] Vieujean et al.,J Crohns Colitis,2021,15(10):1737-1750 Summary of the Invention

[0038] The present invention provides an isolated antibody or binding fragment thereof that specifically binds to anterior gradient protein 2 (AGR2), comprising: The following three complementarity determining regions (CDRs): - CDR1: RSWMN (SEQ ID NO: 8); - CDR2: WIYPGDGDTNYNGKXKD (SEQ ID NO: 9) (wherein X is F or V); - CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (VH) comprising: The following three CDRs: - CDR1: KASQDINSYLS (SEQ ID NO: 13); CDR2: RANRLVD (SEQ ID NO: 14); and - CDR3: LQYDEFPFT (SEQ ID NO: 15) A light chain variable region (VL) containing The present invention relates to an isolated antibody or binding fragment thereof comprising:

[0039] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 16; and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 17; and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 16; and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 20; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 17; and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 20; or a heavy chain variable region (VH) comprising a framework region that shares at least 80% sequence identity with the framework region of SEQ ID NO: 18; and a light chain variable region (VL) comprising a framework region that shares at least 80% sequence identity with the framework region of SEQ ID NO: 21. Includes.

[0040] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is a heavy chain variable region (VH) having a sequence consisting of the sequence SEQ ID NO: 16; and a light chain variable region (VL) having a sequence consisting of the sequence SEQ ID NO: 19; or a heavy chain variable region (VH) having a sequence consisting of the sequence SEQ ID NO: 17; and a light chain variable region (VL) having a sequence consisting of the sequence SEQ ID NO: 19; or a heavy chain variable region (VH) having a sequence consisting of the sequence SEQ ID NO: 16; and a light chain variable region (VL) having a sequence consisting of the sequence SEQ ID NO: 20; or a heavy chain variable region (VH) having a sequence consisting of the sequence SEQ ID NO: 17; and a light chain variable region (VL) having a sequence consisting of the sequence SEQ ID NO: 20; or a heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 18; and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO: 21 Includes.

[0041] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is an immunoconjugate.

[0042] The present invention also relates to nucleic acids encoding antibodies or binding fragments thereof that specifically bind to AGR2.

[0043] The present invention also relates to an expression vector comprising the nucleic acid.

[0044] The present invention also relates to cells containing the nucleic acid or expression vector.

[0045] The present invention also relates to pharmaceutical compositions comprising an isolated antibody or binding fragment thereof that specifically binds to AGR2, a nucleic acid, an expression vector, or a cell, and at least one pharmaceutically acceptable excipient.

[0046] The present invention also relates to an isolated antibody or binding fragment thereof that specifically binds to AGR2, a nucleic acid, an expression vector, a cell, or a pharmaceutical composition for use as a medicament.

[0047] The present invention also relates to an isolated antibody or binding fragment thereof that specifically binds to AGR2, a nucleic acid, an expression vector, a cell, or a pharmaceutical composition for use in treating a mucosal inflammatory disease or cancer in a subject in need thereof.

[0048] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 neutralizes the pro-inflammatory activity of eAGR2 and / or the pro-fibrotic activity of eAGR2.

[0049] In some embodiments, the mucosal inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, chronic pancreatitis, microscopic colitis, inflammatory bowel disease (IBD), endometriosis, appendicitis, inflammatory bowel syndrome, idiopathic pulmonary fibrosis, systemic sclerosis, systemic sclerosis associated with interstitial lung disease, asthma, and chronic obstructive pulmonary disease.

[0050] In some embodiments, the cancer is selected from the group consisting of colon cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, oral cancer, breast cancer, lung cancer, ovarian cancer, thyroid cancer, bile duct cancer, head and neck squamous cell carcinoma, brain glioblastoma, adrenocortical carcinoma, bladder cancer, kidney cancer, penile cancer, renal cancer, testicular cancer, urethral cancer, colorectal cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, gestational trophoblastic disease (GTD), and primary peritoneal cancer.

[0051] The present invention also relates to an in vitro method for detecting or quantifying AGR2 expression in a biological sample, comprising contacting the biological sample with an isolated antibody or binding fragment thereof that specifically binds to AGR2, as described herein.

[0052] In some embodiments, the methods are for diagnosing or monitoring an AGR2-associated disease in a subject, or for selecting a subject suffering from an AGR2-associated disease for disease-targeted treatment. definition

[0053] In the present invention, the following terms have the following meanings:

[0054] "Affinity" is used to define the strength of an antibody-antigen complex. Affinity measures the strength of the interaction between an epitope and the antigen-binding site on an antibody. This is expressed as the affinity constant, K A or the dissociation constant K D It can be represented by:

[0055] As used herein, "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin (IgG) molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. The term "antibody" also includes multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, so long as they exhibit the desired biological activity. An antibody can be a multimer of immunoglobulin molecules, such as a tetramer of immunoglobulin molecules. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. L chains from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains (CL). Depending on the amino acid sequence of the constant domains (CH) of the heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the [α] and [γ] chains, and four CH domains for the [μ] and [ε] isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain.Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units with an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, whereas secretory IgA antibodies can polymerize to form multivalent aggregates containing two to five basic four-chain units with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons.

[0056] As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody, that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of the VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments, such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting their ability to readily crystallize. Fab fragments consist of the entire L chain along with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0057] As used herein, the term "binding fragment" refers to a portion or region of an antibody according to the present invention that contains fewer amino acid residues than the whole antibody. A "binding fragment" binds to an antigen and / or competes with the whole antibody from which it was derived for antigen binding. Antibody-binding fragments include, but are not limited to, single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies.

[0058] "Antigen" or "Ag" refers to a molecule that elicits an immune response, which may include either antibody production and / or activation of specific immunocompetent cells.

[0059] "Cancer" generally refers to a disease caused by the uncontrolled division of abnormal cells. The term "cancer" specifically refers to any disease associated with tumor formation. The term "cancer" encompasses solid tumors and hematological cancers, and includes both primary and metastatic cancers.

[0060] "CDR" or "complementarity determining region" refers to the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. The precise amino acid sequence boundaries of a given CDR are described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest." The CDRs can be determined using any of several well-known schemes, including those described in the 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. More recently, a universal numbering system has been developed and widely adopted, known as the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. 27:209-212 1999). Herein, CDRs are referred to both in terms of amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides within structures called loops, CDR and framework residues can be readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and substitute CDR residues from an immunoglobulin of one species into an acceptor framework, typically from a human antibody. In some embodiments, by CDR regions or CDRs is intended to refer to the hypervariable regions of the immunoglobulin heavy and light chains as defined by Kabat et al. (1991) (the "Kabat" numbering scheme).

[0061] "Epitope" refers to a specific arrangement of amino acids located on one or more proteins to which an antibody binds. Epitopes often consist of chemically active surface groupings 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 contiguous) or conformational, i.e., they can include two or more sequences of amino acids from different regions of an antigen, which need not necessarily be contiguous.

[0062] The "framework region" or "FR region" comprises amino acid residues of an antibody or binding fragment thereof that are part of the variable region but not part of the CDR. In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically arranged to form an antigen-binding site when the antibody assumes its three-dimensional shape in an aqueous environment. The remaining parts of the heavy and light chain variable domains show less inter-molecular variability in amino acid sequence and are called framework regions.

[0063] "Fc domain," "Fc portion," and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain, or the corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or naturally occurring allotypes thereof.

[0064] A "heavy chain region" comprises an amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A protein comprising a heavy chain region is H 1 domain, a hinge (e.g., upper hinge region, middle hinge region, and / or lower hinge region) domain, C H 2 domains, C H In one embodiment, the antibody according to the present invention comprises at least one of the Fc region of an immunoglobulin heavy chain (e.g., hinge region, C H 2 domain and C H In some embodiments, antibodies according to the invention may comprise a heavy chain region comprising all of the constant domains derived from a human immunoglobulin 1 (IgG1) heavy chain. In some embodiments, the constant domains of the heavy chain region may be modified so that they differ in amino acid sequence from a naturally occurring (wild-type) immunoglobulin molecule. That is, antibodies according to the invention may comprise one or more heavy chain constant domains (C H 1. Hinge, C H 2 or C H 3) and / or the light chain constant domain (C L) Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0065] Within an antibody, the term "hinge region" refers to the C H 1 domain to C H The hinge region comprises the region of the heavy chain molecule that connects the two N-terminal antigen-binding domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding domains to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0066] "Identity" or "identical," as used herein in the context of the relationship between the sequences of two or more amino acid sequences or two or more nucleic acid sequences, refers to the degree of sequence relatedness between the amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid or nucleic acid sequences can be readily calculated by known methods. Preferred methods for determining identity are designed to produce the largest match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, which includes GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984, Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0067] As used herein, the term "immune cells" generally refers to white blood cells derived from hematopoietic stem cells (HSCs) produced in the bone marrow. Examples of immune cells include, but are not limited to, lymphocytes (T cells, B cells, and natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0068] As used herein, the terms "isolated" or "non-naturally occurring" with respect to a biological component (such as a nucleic acid molecule, protein, or cell) refer to a biological component that has been altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is. An isolated nucleic acid or peptide can exist in a substantially purified form or can exist in a non-native environment, such as a host cell. Typically, preparations of isolated nucleic acids or peptides contain nucleic acids or peptides that are at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure. "Non-naturally occurring" or "isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells as well as chemically synthesized nucleic acids. An "isolated polypeptide" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0069] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies or binding fragments thereof according to the present invention can be prepared by the hybridoma method first described by Kohler et al., 1975. Nature. 256(5517):495-7, or can be produced using recombinant DNA methodologies in bacterial, eukaryotic, or plant cells (U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97.

[0070] As used herein, the terms "nucleic acid" or "polynucleotide" refer to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0071] As used herein, the terms "prevent," "preventing," and "prevention" refer to prophylactic and preventative measures aimed at reducing the likelihood that a subject will develop a pathological condition or disorder over a given period of time. Such reduction may be reflected, for example, in a delay in the onset of at least one symptom of the pathological condition or disorder in the subject.

[0072] "Single-chain variable fragment," also abbreviated "sFv" or "scFv," refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, for example, via a synthetic linker, e.g., a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions in either order; for example, with respect to the N- and C-termini of the polypeptide, the scFv may comprise VL-linker-VH or VH-linker-VL. Bivalent, trivalent, and higher-level scFvs, referred to as tandem di-scFvs, tandem tri-scFvs, etc., can be engineered by linking two, three, or more scFvs together. Alternatively, a V H Domains and V L Using a linker peptide that is too short (typically about 5-10 amino acids) between the domains allows two scFvs to dimerize into diabodies. Alternatively, an even shorter linker peptide (typically about 1-2 amino acids) leads to the formation of scFv trimers, also called triabodies or tribodies.

[0073] "Subject" is intended to include any organism in which an immune response can be elicited. Preferably, the term "subject" refers to a warm-blooded animal, more preferably a mammal. The term "mammal," as used herein, refers to any mammal, including humans, livestock, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Preferably, the mammal is a primate, more preferably a human. In some embodiments, the subject may be a "patient," who is awaiting medical treatment, is undergoing medical treatment, has been / is / will be the subject of medical treatment, or is being monitored for the development of a target disease or condition, such as a mucosal inflammatory disease. In some embodiments, the subject is an adult (e.g., a subject over 18 years of age). In some embodiments, the subject is a child (e.g., a subject under 18 years of age). In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is suffering from, and preferably has been diagnosed with, a mucosal inflammatory disease. In some embodiments, the subject is at risk for developing a mucosal inflammatory disease. Examples of risk factors include, but are not limited to, a genetic predisposition or family history of mucosal inflammatory disease.

[0074] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0075] A "therapeutically effective amount" refers to a level or amount of an antibody or binding fragment thereof described herein that is intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or halt the progression, progression, or worsening of one or more symptoms of a disease, disorder, or condition; (3) result in amelioration of the symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition, without causing significant negative or adverse side effects to the target. A therapeutically effective amount can be administered prior to the onset of a disease, disorder, or condition for a prophylactic or preventative effect. Alternatively, or in addition, a therapeutically effective amount can be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0076] "Treating" or "treatment" or "alleviating" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those already with the disorder, as well as those prone to having the disorder or those in whom the disorder is to be prevented. In some embodiments, a subject is successfully "treated" for a disease or disorder if, after receiving a therapeutic amount of an antibody or binding fragment thereof in accordance with the present invention, the subject exhibits at least one of the following: relief to some extent of one or more symptoms associated with the disease or disorder to be treated; reduced morbidity and mortality; and improved quality of life issues. The above parameters for assessing successful treatment and improvement of a disease are readily measurable by routine procedures familiar to physicians.

[0077] As used herein, the terms "variable," "variable region," or "variable domain" refer to a variable domain V H and V L This refers to the fact that certain regions of the V domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the V domains of antibodies. This is due to the fact that the V domains, which form part of the antigen-binding site, are not uniformly distributed throughout the V LDomains and V H Each of the domains is concentrated into three segments called "complementarity determining regions" or "CDRs." DETAILED DESCRIPTION OF THE INVENTION

[0078] Human AGR2 typically refers to the protein referenced as AAY84776.1 in the NCBI database on January 19, 2007. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human AGR2 gene sequence corresponds to NCBI Gene ID: 10551, updated on November 28, 2021. The human AGR2 gene consists of eight exons on chromosome 7p21.1. The AGR2 transcript encompasses 1697 nucleotides and encodes a 175-amino acid protein. The reference human AGR2 protein sequence corresponds to SEQ ID NO: 1.

[0079] Mouse AGR2 typically refers to the protein referenced as NP_035913.1 in the NCBI database on December 4, 2021. In the NCBI database, the reference mouse AGR2 gene sequence corresponds to NCBI Gene ID: 23795, updated on November 30, 2021. The mouse AGR2 gene consists of 8 exons on chromosome 12; 12A. The AGR2 transcript encompasses 760 nucleotides and encodes a 175-amino acid protein. The reference mouse AGR2 protein sequence corresponds to SEQ ID NO: 2.

[0080] Rhesus AGR2 typically refers to the protein referenced as NP_001181233.1 in the NCBI database as of July 11, 2020. In the NCBI database, the reference rhesus AGR2 gene sequence corresponds to NCBI Gene ID: 709127, updated on June 24, 2020. The rhesus AGR2 gene consists of eight exons on chromosome 3. The AGR2 transcript encompasses 1808 nucleotides and encodes a 175-amino acid protein. The reference rhesus AGR2 protein sequence corresponds to SEQ ID NO: 3.

[0081] Chimpanzee AGR2 typically refers to the protein referenced as XP_003318381.1 in the NCBI database as of March 20, 2018. In the NCBI database, the reference chimpanzee AGR2 gene sequence corresponds to NCBI Gene ID: 463277, updated on March 19, 2021. The chimpanzee AGR2 gene consists of eight exons on chromosome 7. The AGR2 transcript encompasses 2197 nucleotides and encodes a 175-amino acid protein. The reference chimpanzee AGR2 protein sequence corresponds to SEQ ID NO: 4.

[0082] Rat AGR2 typically refers to the protein referenced as NP_001100195.1 in the NCBI database as of February 1, 2021. In the NCBI database, the reference rat AGR2 gene sequence corresponds to NCBI Gene ID: 298961, updated on December 15, 2021. The rat AGR2 gene consists of nine exons on chromosome 6q16. The AGR2 transcript encompasses 961 nucleotides and encodes a 175-amino acid protein. The reference rat AGR2 protein sequence corresponds to SEQ ID NO: 5.

[0083] Canine AGR2 typically refers to the protein referenced as XP_038542570.1 in the NCBI database on January 7, 2021. In the NCBI database, the reference canine AGR2 gene sequence corresponds to NCBI Gene ID: 482333, updated on December 15, 2021. The canine AGR2 gene consists of 9 exons on chromosome 14. The AGR2 transcript encompasses 2657 nucleotides and encodes a 175-amino acid protein. The reference canine AGR2 protein sequence corresponds to SEQ ID NO: 6.

[0084] Alternative names for AGR2 include, by way of non-limiting example, "anterior gradient protein 2," "AG-2," "AG2," "HPC8," "GOB-4," "HAG-2," "XAG-2," "PADIA17," "HEL-S-116," "protein disulfide isomerase family A member 17," and "secretory cement gland protein XAG-2 homolog." The terms "anterior gradient protein 2," "AGR2," and "AG-2" are used interchangeably herein.

[0085] In the context of the present invention, AGR2 refers to human AGR2 as well as any homologous protein in any animal species. For example, AGR2 refers to any homologous sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in any other species.

[0086] The AGR2 protein contains an N-terminal signal peptide sequence, a catalytically active thioredoxin domain, and a C-terminal ER retention sequence. AGR2 plays a role in cell migration, cell transformation, and metastasis, and acts as a p53 inhibitor. As an ER-localized molecular chaperone, AGR2 plays a role in the folding, transport, and assembly of cysteine-rich transmembrane receptors and cysteine-rich intestinal glycoprotein mucins. Extracellular (or secreted) AGR2 also exhibits pro-inflammatory and pro-fibrotic activities.

[0087] The AGR2 protein can be found in different locations: it can be present intracellularly or extracellularly, either bound to the cell surface or in a circulating (secreted) form.

[0088] The present invention first relates to an isolated antibody or binding fragment thereof that specifically binds to anterior gradient protein 2 (AGR2).

[0089] As used herein, an "isolated antibody" is intended to refer to an antibody that has been modified or removed from its natural state. In particular, an isolated antibody may be substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to AGR2 may be substantially free of antibodies that specifically bind to antigens other than AGR2). However, an isolated antibody that specifically binds to AGR2 may have cross-reactivity to other antigens, such as AGR2 molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals, particularly, but not limited to, enzymes, hormones, and other proteinaceous or non-proteinaceous components, that would interfere with the therapeutic use of the antibody.

[0090] Preferably, the isolated antibody or binding fragment thereof is purified, for example, the isolated antibody or binding fragment thereof is more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95% or more purified by weight of the protein.

[0091] The isolated antibody or binding fragment thereof of the present invention specifically binds to AGR2.

[0092] The isolated antibodies or binding fragments thereof of the present invention specifically bind to all forms of AGR2, particularly intracellular AGR2, extracellular AGR2 (i.e., expressed on the cell surface), and circulating AGR2 (i.e., secreted in the extracellular environment).

[0093] In particular, the isolated antibody or binding fragment thereof of the present invention does not specifically bind to AGR3.

[0094] The human AGR2 and AGR3 genes are mapped to chromosome band 7p21.3. 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 or a binding fragment thereof is a molecule that binds to an antigen (e.g., AGR2) at a detectable level, preferably about 10 6 M -1 or more, preferably about 10 7 M -1 , 10 8 M -1 , 5x10 8 M -1 , 10 9 M -1 , 5x10 9 M -1 The affinity constant (K A ) is said to be "specific," "immunospecific," or "specifically binds" to an antigen. The affinity of an antibody or binding fragment thereof for its cognate antigen is also generally measured by the equilibrium dissociation constant (K D The antibody or binding fragment thereof can detectably bind to the antigen (e.g., AGR2), preferably at a concentration of 10 -6 M or less, preferably 10 -7 M or less, 5x10 -8 M, 10 -8 Medium, 5x10 -9 M, 10 -9 K below M D An antibody or binding fragment thereof is said to be "specific for" or "specifically binds" to an antigen when it reacts with the antigen. The affinity of an antibody or binding fragment thereof can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NY Acad Sci. 51:660-672. The binding properties of an antibody or binding fragment thereof for an antigen, cell, or tissue can generally be determined and evaluated using immunodetection methods including, for example, ELISA, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS), or by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0095] In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention is capable of recognizing and binding to soluble (i.e., not membrane-bound) AGR2 protein. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention is capable of recognizing and binding to membrane-bound AGR2 protein.

[0096] In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to monomeric AGR2. In some embodiments, an isolated antibody or binding fragment thereof recognizes and binds to human AGR2 of SEQ ID NO: 1, which contains a mutation consisting of the glutamic acid residue at position 60 being substituted with an alanine residue. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to dimeric AGR2. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to both the monomeric and dimeric forms of the human AGR2 protein with the same affinity.

[0097] The isolated antibody or binding fragment thereof of the present invention recognizes and binds to human AGR2 protein. Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 also binds to one or more homologous AGR2 proteins from other species. For example, the isolated antibody or binding fragment thereof that specifically binds to AGR2 may also bind to one or more AGR2 proteins selected from mouse AGR2 protein, rhesus monkey AGR2 protein, chimpanzee AGR2 protein, rat AGR2 protein, or canine AGR2 protein.

[0098] In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to mouse AGR2 protein. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to rhesus monkey AGR2 protein. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to chimpanzee AGR2 protein. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to rat AGR2 protein. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to canine AGR2 protein.

[0099] The isolated antibody or binding fragment thereof of the invention recognizes and binds to the human AGR2 protein of sequence SEQ ID NO: 1. Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 also binds to one or more AGR2 proteins of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6.

[0100] In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the mouse AGR2 protein of SEQ ID NO: 2. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the rhesus monkey AGR2 protein of SEQ ID NO: 3. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the chimpanzee AGR2 protein of SEQ ID NO: 4. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the rat AGR2 protein of SEQ ID NO: 5. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the canine AGR2 protein of SEQ ID NO: 6. In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention recognizes and binds to the monomeric AGR2 protein of SEQ ID NO: 7.

[0101] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention can recognize and bind to AGR2 variants, preferably variants of human AGR2 protein, variants of mouse AGR2 protein, variants of rhesus monkey AGR2 protein, variants of chimpanzee AGR2 protein, variants of rat AGR2 protein, and / or variants of canine AGR2 protein.

[0102] A "variant" or "derivative" protein is defined as having a sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even at least 95%, 96%, 97%, 98% or 99% identical to a reference sequence.

[0103] These variant sequences may differ from the reference sequence by one or more amino acid substitutions, deletions and / or insertions. Substitutions may in particular correspond to conservative substitutions or substitutions of natural amino acids with non-natural or pseudo-amino acids.

[0104] "An amino acid sequence having (for example) at least 80% identity to a reference sequence" as used herein means a sequence that is identical to the reference sequence, but which may contain up to 20 mutations (substitutions, deletions, and / or insertions) per 100 amino acid portion of the reference sequence. Thus, for a 100 amino acid reference sequence, an 80 amino acid fragment and a 100 amino acid sequence that contains 20 substitutions compared to the reference sequence are two examples of sequences that have 80% sequence identity to the reference sequence.

[0105] The percent identity is generally determined using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). The amino acid sequences to be compared are aligned to obtain the maximum percent identity. For this purpose, it may be necessary to artificially add gaps to the sequence. Alignment can be performed manually or automatically. Automatic alignment algorithms for nucleotide sequences are well known to those skilled in the art, for example, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389 and implemented by software such as Blast software. One algorithm that can be separated is, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48:443-53). Once optimal alignment is achieved, the percent identity is established by recording all positions where the amino acids of the two compared sequences are identical, relative to the total number of positions.

[0106] In certain embodiments, the sequence of the AGR2 protein differs from the reference sequence only by the presence of conservative substitutions. Conservative substitutions are substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, cysteine, and tyrosine), substitutions of amino acids with basic side chains (such as lysine, arginine, and histidine), substitutions of amino acids with acidic side chains (such as aspartic acid and glutamic acid), and substitutions of amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan).

[0107] Preferably, a variant of AGR2 refers to an AGR2 protein having a sequence in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 35 amino acids have been deleted, added, or substituted compared to the original protein sequence. In some embodiments, a variant of AGR2 refers to an AGR2 protein having a sequence in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 35 amino acids have been deleted, added, or substituted compared to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0108] According to the present invention, polypeptides can be chemically or enzymatically modified to improve their stability or bioavailability. Such chemical or enzymatic modifications are well known to those skilled in the art. The following modifications: - modifications of the C-terminus or N-terminus of the polypeptide, such as deamination or acylation (preferably acetylation) of the N-terminus or amidation or esterification, for example, of the C-terminus; - modification of the amide bond between two amino acids, for example acylation (preferably acetylation) or alkylation at the nitrogen or alpha carbon; - a change in chirality, for example, replacement of a natural amino acid (L-enantiomer) with the corresponding D-enantiomer; this modification may optionally involve inversion of the side chain (from C-terminus to N-terminus); -Azapeptide alteration in which one or more alpha carbons are replaced by nitrogen atoms; and / or -Beta peptides are formed by adding one or more carbon atoms to the N-alpha or C-alpha side of the main chain. These may include, but are not limited to:

[0109] In this regard, one or more of the lysine amino acids (K) of the polypeptide may be, in particular: - amidation: this modification is easy to achieve and involves replacing the positive charge of lysine with a hydrophobic group (e.g., acetyl or phenylacetyl); -Amination: Primary amine R = (CH2)4-NH3 + by formation of a secondary amide from, for example, by formation of an N-methyl, N-allyl or N-benzyl group; and - Formation of N-oxide, N-nitroso, N-dialkylphosphoryl, N-sulfenyl or N-glycosidic groups It is possible to modify it by

[0110] Similarly or alternatively, one or more threonine (T) and / or serine (S) amino acids of a polypeptide can be modified by adding an ester or ether group, particularly to the OH group of the threonine and / or serine side chain. Esterification is a simple procedure and can be carried out using carboxylic acids, anhydrides, crosslinking, etc. to form acetates or benzoates. Etherification, which gives more stable compounds, can be carried out using alcohols, halides, etc. to form, for example, methyl ethers or O-glycosides.

[0111] Similarly or alternatively, it is also possible to modify one or more glutamine (Q) amino acids, for example by amidation, by forming secondary or tertiary amines, in particular with groups of the methyl, ethyl type, whether functionalized or not.

[0112] Similarly or alternatively, one or more glutamic (E) and / or aspartic (D) amino acids may be added, for example: by esterification, forming methyl esters, ethyl esters, benzyl esters, thiols (activated esters), whether substituted or not; and by amidation, in particular forming N,N-dimethyl groups, nitroanilides, pyrrolidinyls, It is possible to modify it.

[0113] In some embodiments, the isolated antibody or binding fragment thereof of the present invention comprises one or more of: - methionine (M) and / or tryptophan (W) by oxidation, - asparagine (N) by deamidation, and / or -Aspartic acid (D) by isomerization This includes chemical modifications of the

[0114] On the other hand, proline amino acids that are involved in the secondary structure of the polypeptide are preferably not modified.

[0115] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention can recognize and bind to an AGR2 fragment, preferably a fragment of the human AGR2 protein, a fragment of the mouse AGR2 protein, a fragment of the rhesus monkey AGR2 protein, a fragment of the chimpanzee AGR2 protein, a fragment of the rat AGR2 protein, and / or a fragment of the canine AGR2 protein.

[0116] As used herein, the term "fragment" of an antigen refers to any subset of the antigen as a shorter peptide. In some embodiments, a fragment of an antigen is a peptide at least 6 amino acids in length. In some embodiments, a fragment of an antigen is a peptide 6 to 50 amino acids in length, 6 to 30 amino acids in length, or 6 to 20 amino acids in length.

[0117] The isolated antibodies or binding fragments thereof that specifically bind to AGR2 of the present invention can be polyclonal or monoclonal.

[0118] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is monoclonal.

[0119] The isolated antibody that specifically binds to AGR2 of the present invention can be a whole antibody or a binding fragment of the antibody. Preferably, the isolated antibody that specifically binds to AGR2 is a whole antibody.

[0120] The antigen-binding fragment of the present invention that specifically binds to AGR2 can be a molecule selected from the group comprising or consisting of a single-chain antibody, a dimeric single-chain antibody, a single-domain antibody, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody, and a tetrabody.

[0121] The antigen-binding fragments of the present invention that specifically bind to AGR2 can be single-chain antibodies. In some embodiments, the single-chain antibodies are selected from the group consisting of single-chain variable fragments (scFvs), tandem di-scFvs, tandem tri-scFvs, scFv-Fc, (scFv-CH3)2 (also called minibodies), (scFv-CH2-CH3)2 (also called maxibodies), diabodies, and triabodies.

[0122] The term "binding fragment" as used herein refers to a portion or region of an antibody according to the present invention that contains fewer amino acid residues than the whole antibody. A "binding fragment" binds to an antigen and / or competes with the whole antibody from which it is derived for antigen binding (e.g., specific binding to AGR2). Antibody-binding fragments include, but are not limited to, single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies.

[0123] As used herein, "single-chain antibody" refers to any antibody or fragment thereof that is a protein having a primary structure comprising or consisting of a single uninterrupted sequence of contiguous amino acid residues, including, but not limited to, (1) a single-chain Fv molecule (scFv); (2) a single-chain protein containing only one light-chain variable domain, or a fragment thereof containing the three CDRs of the light-chain variable domain and no associated heavy-chain portion; and (3) a single-chain protein containing only one heavy-chain variable region, or a fragment thereof containing the three CDRs of the heavy-chain variable region and no associated light-chain portion.

[0124] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a VF linked to a single amino acid chain. H and V L scFv refers to an antibody fragment containing an antibody domain. Preferably, the scFv amino acid sequence is V H Domains and V L It further comprises a peptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0125] As used herein, "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one HCVR and one LCVR in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy chain and light chain) that contribute to antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0126] As used herein, "diabody" refers to small antibody fragments prepared by constructing scFv fragments with a short linker (approximately 5-10 residues) between the HCVR and LCVR such that inter-chain, rather than intra-chain, pairing of the variable domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the HCVRs and LCVRs of the two antibodies are present on different polypeptide chains.

[0127] Antibody-binding fragments can be obtained using standard methods. For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies in accordance with conventional techniques.

[0128] In some embodiments, an antibody or binding fragment thereof according to the invention is a molecule selected from the group comprising or consisting of a unibody, a domain antibody, and a nanobody.

[0129] "Unibody" refers to an antibody fragment that lacks the hinge region of an IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than the bivalent binding region of an IgG4 antibody.

[0130] "Domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of either the heavy or light chain of an antibody.

[0131] "Single domain antibody" refers to an antibody-derived protein that contains the unique structural and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (V H H) - one such example is a Nanobody® - or a single variable domain (V H H) and two constant domains (C H 2 and C H 3)—e.g., camelid antibodies—or a single variable domain (V H H) and five constant domains (C H 1. CH 2. C H 3. C H 4 and C H 5) - for example, shark antibodies.

[0132] In one embodiment, the antibody or binding fragment thereof according to the invention is a mimetic selected from the group comprising or consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.

[0133] "Affibody" refers to an affinity protein based on a 58 amino acid residue protein domain derived from one of the IgG binding domains of Staphylococcus protein A.

[0134] "DARPin" (designed ankyrin repeat protein) refers to antibody-mimetic DRP (designed repeat protein) technology developed to harness the binding capacity of non-antibody proteins.

[0135] "Anticalins" refer to another antibody mimetic technology whose binding specificity is derived from lipocalins. Anticalins can also be formatted as dual-targeting proteins called "duocalins."

[0136] "Avimer" refers to another antibody mimetic technology.

[0137] "Versabodies" refer to another antibody-mimetic technology. They are small proteins of 3-5 kDa with >15% cysteines, forming a high-disulfide-density scaffold that replaces the hydrophobic core found in typical proteins. Replacing the many hydrophobic amino acids that comprise the hydrophobic core with a small number of disulfides results in proteins that are smaller, more hydrophilic (less aggregation and nonspecific binding), more resistant to proteases and heat, and have a lower density of T-cell epitopes, since the residues most responsible for MHC presentation are hydrophobic. All four of these properties are known to affect immunogenicity, and together they are expected to cause a significant reduction in immunogenicity.

[0138] In some embodiments, antibodies or binding fragments thereof according to the invention also include multispecific antibodies or binding fragments thereof, i.e., multispecific antibodies or binding fragments thereof that are immunospecific for two or more, e.g., at least two, different antigens, one of which is AGR2 according to the invention.

[0139] In some embodiments, antibodies or binding fragments thereof according to the present invention also include polymers of antibodies or binding fragments thereof, i.e., two or more, e.g., at least two, antibodies or binding fragments thereof, whether identical or different, that are covalently linked together, directly or indirectly.

[0140] The isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention is humanized.

[0141] Complementarity determining regions (CDRs) are determined herein using the Kabat numbering system.

[0142] The isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention is -The following three CDRs: VH CDR1: RSWMN (SEQ ID NO: 8); VH CDR2: WIYPGDGDTNYNGKXKD (SEQ ID NO: 9), (wherein X is F or V), VH CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (abbreviated herein as VH) comprising: -The following three CDRs: VL CDR1: KASQDINSYLS (SEQ ID NO: 13); VL CDR2: RANRLVD (SEQ ID NO: 14); VL CDR3: LQYDEFPFT (SEQ ID NO: 15) a light chain variable region (abbreviated herein as VL) comprising Includes.

[0143] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is -The following three CDRs: VH CDR1: RSWMN (SEQ ID NO: 8); VH CDR2: WIYPGDGDTNYNGKFKD (SEQ ID NO: 10) or WIYPGDGDTNYNGKVKD (SEQ ID NO: 11); and VH CDR3: GGYDGSPWLSY (SEQ ID NO: 12) or a set of three CDRs having an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NOs: 8, 10 or 11 and 12. a heavy chain variable region (abbreviated herein as VH) comprising: -The following three CDRs: VL CDR1: KASQDINSYLS (SEQ ID NO: 13); VL CDR2: RANRLVD (SEQ ID NO: 14); VL CDR3 LQYDEFPFT (SEQ ID NO: 15); or a set of three CDRs having amino acid sequences at least 85%, 90% or 95% identical to SEQ ID NOs: 13, 14 and 15. a light chain variable region (abbreviated herein as VL) comprising Includes.

[0144] "A set of three CDRs having at least 85%, 90%, or 95% amino acid identity to a SEQ ID NO" means - VH CDR1 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 8; - VH CDR2 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 10 or 11; and - VH CDR3 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 12 The term "CDRs" refers to a set of three CDRs.

[0145] "A set of three CDRs having at least 85%, 90%, or 95% amino acid identity to a SEQ ID NO" means - VL CDR1 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 13; - VL CDR2 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 14; and - VL CDR3 as an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 15 The term "CDRs" refers to a set of three CDRs.

[0146] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is -The following three CDRs: VH CDR1: RSWMN (SEQ ID NO: 8); VH CDR2:WIYPGDGDTNYNGKFKD (SEQ ID NO: 10) or WIYPGDGDTNYNGKVKD (SEQ ID NO: 11); VH CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (abbreviated herein as VH) comprising: -The following three CDRs: VL CDR1: KASQDINSYLS (SEQ ID NO: 13); VL CDR2: RANRLVD (SEQ ID NO: 14); VL CDR3: LQYDEFPFT (SEQ ID NO: 15) a light chain variable region (abbreviated herein as VL) comprising Includes.

[0147] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is -The following three CDRs: VH CDR1: RSWMN (SEQ ID NO: 8); VH CDR2:WIYPGDGDTNYNGKFKD (SEQ ID NO: 10); VH CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (abbreviated herein as VH) comprising: -The following three CDRs: VL CDR1: KASQDINSYLS (SEQ ID NO: 13); VL CDR2: RANRLVD (SEQ ID NO: 14); VL CDR3: LQYDEFPFT (SEQ ID NO: 15) a light chain variable region (abbreviated herein as VL) comprising Includes.

[0148] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is -The following three CDRs: VH CDR1: RSWMN (SEQ ID NO: 8); VH CDR2:WIYPGDGDTNYNGKVKD (SEQ ID NO: 11); VH CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (abbreviated herein as VH) comprising: -The following three CDRs: VL CDR1: KASQDINSYLS (SEQ ID NO: 13); VL CDR2: RANRLVD (SEQ ID NO: 14); VL CDR3: LQYDEFPFT (SEQ ID NO: 15) a light chain variable region (abbreviated herein as VL) comprising Includes.

[0149] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is - a VH comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 16; and a VL comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 19; or - a VH comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 17; and a VL comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 19; or - a VH comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 16; and a VL comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 20; or - a VH comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 17; and a VL comprising or consisting of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 20; or - a VH comprising, or consisting of, a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 18; and a VL comprising, or consisting of, a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 21. Includes.

[0150] In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is a heavy chain variable region (VH) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 16; and a light chain variable region (VL) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 17; and a light chain variable region (VL) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 16; and a light chain variable region (VL) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 20; or a heavy chain variable region (VH) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 17; and a light chain variable region (VL) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 20; or - a heavy chain variable region (VH) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 18; and a light chain variable region (VL) comprising framework regions that share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the framework regions of SEQ ID NO: 21. Includes.

[0151] Preferably, the isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention comprises: - a VH comprising or consisting of the sequence SEQ ID NO: 16 and a VL comprising or consisting of the sequence SEQ ID NO: 19; or - a VH comprising or consisting of the sequence SEQ ID NO: 17 and a VL comprising or consisting of the sequence SEQ ID NO: 19; or - a VH comprising or consisting of the sequence SEQ ID NO: 16 and a VL comprising or consisting of the sequence SEQ ID NO: 20; or a VH comprising or consisting of the sequence SEQ ID NO: 17 and a VL comprising or consisting of the sequence SEQ ID NO: 20; or a VH comprising or consisting of the sequence SEQ ID NO: 18 and a VL comprising or consisting of the sequence SEQ ID NO: 21 Includes.

[0152] The fragment crystallizable (Fc) region of the isolated antibody that specifically binds to AGR2 can comprise at least one mutation that reduces an antibody effector function.

[0153] In some embodiments, the isolated antibody that specifically binds to AGR2 is an Fc silencing antibody.

[0154] As used herein, "Fc silencing antibody" refers to an antibody having modifications in the immunoglobulin Fc region to eliminate binding of the immunoglobulin Fc to Fc gamma receptors (FcγRs).

[0155] As used herein, "EU numbering" refers to the numbering of the heavy and light chain constant domains of human gamma G1 immunoglobulin (IgG1) as defined by Edelman et al. (Proc Natl Acad Sci USA, 63(1):78-85, 1969). In this numbering, the first amino acid residue in the heavy chain constant domain is an alanine residue and is numbered at position 118, while the first amino acid residue in the light kappa chain constant domain is an arginine residue and is numbered at position 108.

[0156] In some embodiments, an isolated antibody of the present invention that specifically binds to AGR2 comprises an IgG1 heavy chain constant region, wherein the heavy chain constant region sequence comprises mutations (L234A and L235A) consisting of the leucine residue at position 234 (corresponding to position 117 of SEQ ID NO: 22) substituted with an alanine residue and the leucine residue at position 235 (corresponding to position 118 of SEQ ID NO: 22) substituted with an alanine residue.

[0157] In some embodiments, an isolated antibody of the present invention that specifically binds to AGR2 comprises an IgG1 heavy chain constant region, wherein the heavy chain constant region sequence comprises mutations (L234F, L235E, and P331S) consisting of the leucine residue at position 234 (EU numbering) (corresponding to position 117 of SEQ ID NO: 22) being substituted with a phenylalanine residue, the leucine residue at position 235 (EU numbering) (corresponding to position 118 of SEQ ID NO: 22) being substituted with a glutamic acid residue, and the proline residue at position 331 (EU numbering) (corresponding to position 214 of SEQ ID NO: 22) being substituted with a serine residue.

[0158] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises an IgG4 heavy chain constant region.

[0159] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of SEQ ID NO: 22, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 22; and a light chain constant region having a sequence comprising or consisting of SEQ ID NO: 26, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 26; Includes.

[0160] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 22; and a light chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 26 Includes.

[0161] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of SEQ ID NO: 23, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 23; and a light chain constant region having a sequence comprising or consisting of SEQ ID NO: 26, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 26; Includes.

[0162] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 23; and a light chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 26 Includes.

[0163] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of SEQ ID NO: 24, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 24; and a light chain constant region having a sequence comprising or consisting of SEQ ID NO: 26, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 26; Includes.

[0164] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 24; and a light chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 26 Includes.

[0165] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of SEQ ID NO: 25, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 25; and a light chain constant region having a sequence comprising or consisting of SEQ ID NO: 26, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 26; Includes.

[0166] In some embodiments, the isolated antibody that specifically binds to AGR2 of the present invention comprises: - a heavy chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 25; and a light chain constant region having a sequence comprising or consisting of the sequence of SEQ ID NO: 26 Includes.

[0167] In some embodiments, the isolated antibody that specifically binds to AGR2 is a heavy chain having a sequence consisting of any one of SEQ ID NOs: 27-32, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to one of SEQ ID NOs: 27-32; and a light chain having a sequence consisting of SEQ ID NOs: 33 to 35 or a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NOs: 33 to 35 Includes.

[0168] In some embodiments, the isolated antibody that specifically binds to AGR2 is - a heavy chain having a sequence consisting of the sequence SEQ ID NO: 27 or SEQ ID NO: 30; and a light chain having a sequence consisting of the sequence SEQ ID NO: 33 Includes.

[0169] In some embodiments, the isolated antibody that specifically binds to AGR2 is - a heavy chain having a sequence consisting of SEQ ID NO: 28 or SEQ ID NO: 31; and a light chain having a sequence consisting of the sequence SEQ ID NO: 33 Includes.

[0170] In some embodiments, the isolated antibody that specifically binds to AGR2 is - a heavy chain having a sequence consisting of the sequence SEQ ID NO: 27 or SEQ ID NO: 30; and a light chain having a sequence consisting of the sequence SEQ ID NO: 34 Includes.

[0171] In some embodiments, the isolated antibody that specifically binds to AGR2 is - a heavy chain having a sequence consisting of SEQ ID NO: 28 or SEQ ID NO: 31; and a light chain having a sequence consisting of the sequence SEQ ID NO: 34 Includes.

[0172] In some embodiments, the isolated antibody that specifically binds to AGR2 is - a heavy chain having a sequence consisting of SEQ ID NO: 29 or SEQ ID NO: 32; and a light chain having a sequence consisting of the sequence SEQ ID NO: 35 Includes.

[0173] The isolated antibody or binding fragment thereof of the present invention has anti-inflammatory function, particularly, the isolated antibody or binding fragment thereof can inhibit eAGR2-induced monocyte recruitment.

[0174] The isolated antibody or binding fragment thereof of the present invention has anti-fibrotic function, particularly, the isolated antibody or binding fragment thereof can inhibit the differentiation of fibroblasts into myofibroblasts.

[0175] The isolated antibody or binding fragment thereof of the present invention has reduced effector function, particularly, the isolated antibody or binding fragment thereof exhibits reduced binding to Fc receptors and / or reduced binding to complement components.

[0176] As used herein, the term "reduced effector function" refers, for example, to reduced antibody-dependent cellular cytotoxicity (ADCC) and reduced complement-dependent cytotoxicity (CDC).

[0177] Thus, the isolated antibodies or binding fragments thereof of the present invention have the following (non-exhaustive) advantages: -specifically binds to AGR2 with high affinity and does not cross-react with AGR3; -specifically binds to AGR2 of various species (e.g. human, mouse, dog, macaque, rat...), - specifically binds to both soluble and membrane-bound AGR2 protein as well as intracellular AGR2 protein; - specifically binds to both the monomeric and dimeric forms of AGR2, - recognizing an epitope comprising at least one amino acid residue located in the catalytic site of AGR2, - exhibiting anti-inflammatory and / or anti-fibrotic activity, exhibiting antiproliferative and / or anti-metastatic activity, - Preserves mucus-secreting cells and / or participates in epithelial regeneration It has.

[0178] The isolated antibodies or binding fragments thereof of the present invention may also exhibit, compared to other anti-AGR2 antibodies, particularly compared to Agtuzumab, for example: - Easier production and better yields (typically 0.11-0.25 compared to 0.05 for Agtuzumab); - the epitope bound by the isolated antibody or binding fragment thereof of the present invention is located in the catalytic domain of the human AGR2 protein, whereas the epitope bound by Agtuzumab is not located in the functional domain, suggesting that the isolated antibody or binding fragment thereof of the present invention may be able to inhibit AGR2 catalytic activity, but Agtuzumab cannot; Better binding capacity (typically with an EC50 of 520-2373 pM compared to 3073 pM for Agtuzumab); Better affinity (typically a K<150 pM compared to the nanomolar range of Agtuzumab) D has a value); - Lower Kdapp (typically 0.62 nM compared to 1.3 nM for Agtuzumab); Better thermal stability (higher melting temperature) (typically 80.36-83.28°C compared to 76.28°C for Agtuzumab); and -Better inhibition of AGR2-induced monocyte migration The present invention exhibits improved features, including:

[0179] Another object of the invention is an isolated nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the invention.

[0180] As used herein, "isolated nucleic acid" refers to a nucleic acid that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the natural sequence. This term encompasses nucleic acid sequences that have been removed from their natural environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or biologically synthesized analogs in heterologous systems. Substantially pure nucleic acid includes nucleic acids in isolated form. Of course, this refers to the nucleic acid that was originally isolated, and does not exclude genes or sequences that were later added to the isolated nucleic acid by the hand of man.

[0181] Preferably, the isolated nucleic acid is purified.

[0182] For example, the isolated nucleic acid may be (1) greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more, most preferably greater than 96%, 97%, 98%, or 99% by weight of nucleic acids as determined by absorbance or fluorescence (e.g., by measuring the ratio of absorbance at 260 nm and 280 nm (A260 / 280)); or (2) Homogeneity as demonstrated by agarose gel electrophoresis and the use of intercalating agents such as ethidium bromide, SYBR Green, and GelGreen. It is refined into

[0183] In some embodiments, the nucleic acid or set of nucleic acids according to the invention comprises: a sequence encoding the VH of an antibody or binding fragment thereof according to the invention; and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention It comprises or consists of:

[0184] In some embodiments, the nucleic acid or set of nucleic acids according to the invention comprises: a sequence encoding the heavy chain of an antibody according to the invention; and - a sequence encoding the light chain of an antibody according to the invention It comprises or consists of:

[0185] In some embodiments, the nucleic acid or set of nucleic acids according to the invention comprises: a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 16 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 16, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 19 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 19; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 17 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 19 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 19; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 16 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 16, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 20 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 17 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 20 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 18 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 18, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 21 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 21. It comprises or consists of:

[0186] In some embodiments, the nucleic acid or set of nucleic acids according to the invention comprises: a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 36 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 36; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 37 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 37; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 38 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 38; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 39 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 39; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 40 or SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 40 or SEQ ID NO: 42; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 41 or SEQ ID NO: 43, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 41 or SEQ ID NO: 43; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 44 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 44; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 45 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 45; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 46 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 46; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 47 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 47. It comprises or consists of:

[0187] Another object of the invention is an expression vector comprising a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the invention.

[0188] In some embodiments, the expression vector or set of expression vectors according to the invention comprises: a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, preferably operably linked to a regulatory element; a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, preferably operably linked to a regulatory element; Includes.

[0189] In some embodiments, the expression vector according to the invention comprises: a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 16 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 16, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 19 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 19; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 17 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 19 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 19; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 16 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 16, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 20 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 17 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 17, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 20 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 20; or a sequence encoding the VH of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 18 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 18, and a sequence encoding the VL of an antibody or binding fragment thereof according to the invention, which is SEQ ID NO: 21 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 21. Includes.

[0190] In some embodiments, the expression vector or set of expression vectors according to the invention comprises: a sequence encoding the heavy chain of an isolated antibody that specifically binds to AGR2 according to the invention, preferably operably linked to a regulatory element; and a sequence encoding the light chain of an isolated antibody that specifically binds to AGR2 according to the invention, preferably operably linked to a regulatory element; Includes.

[0191] In some embodiments, the expression vector according to the invention comprises: a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 36 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 36, preferably operably linked to a regulatory element; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 37 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 37, preferably operably linked to a regulatory element; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 38 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 38, preferably operably linked to a regulatory element; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 39 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 39, preferably operably linked to a regulatory element; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 40 or SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 40 or SEQ ID NO: 42, and preferably operably linked to a regulatory element; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 41 or SEQ ID NO: 43, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 41 or SEQ ID NO: 43, and preferably operably linked to a regulatory element; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 44 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 44, preferably operably linked to a regulatory element; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 45 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 45, preferably operably linked to a regulatory element; or a sequence encoding a heavy chain of an antibody according to the invention, which is SEQ ID NO: 46 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 46, and preferably operably linked to a regulatory element; and a sequence encoding a light chain of an antibody according to the invention, which is SEQ ID NO: 47 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 47, and preferably operably linked to a regulatory element. Includes.

[0192] In some embodiments, an expression vector according to the invention is monocistronic.

[0193] "Monocistronic" means that a single nucleic acid encoding a single protein is expressed on a single expression vector.

[0194] In some embodiments, an expression vector according to the invention is polycistronic.

[0195] "Polycistronic" means that at least two or more nucleic acids, each encoding a single protein, are expressed in a single expression vector.

[0196] Another object of the invention is a cell comprising a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the invention, or an expression vector comprising a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the invention.

[0197] In some embodiments, the cells are Escherichia coli (E. coli) cells. In other embodiments, the cells are Chinese hamster ovary (CHO) cells.

[0198] Another object of the present invention is a method for producing and purifying an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the present invention.

[0199] In some embodiments, the method comprises: - culturing a host cell transformed with a nucleic acid or an expression vector according to the invention under conditions suitable for the expression of the isolated antibody or binding fragment thereof that specifically binds to AGR2; - recovering the expressed antibody or binding fragment thereof that specifically binds to AGR2. Includes.

[0200] This recombinant process can be used for the large-scale production of antibodies or binding fragments thereof, including monoclonal antibodies intended for therapeutic and / or diagnostic use in vitro, ex vivo and / or in vivo.

[0201] These processes are well known in the art (Subramanian (Ed.), 2004. Antibodies (1st ed., Vol. 1: Production and Purification). New York, NY: Springer US).

[0202] In some embodiments, the expressed antibody or binding fragment thereof is further purified.

[0203] Methods for purifying antibodies or binding fragments thereof according to the invention are well known in the art (Subramanian (Ed.), 2004. Antibodies (1st ed., Vol. 1: Production and Purification). New York, NY: Springer US) and include, but are not limited to, chromatography, preferably affinity chromatography, more preferably affinity chromatography on protein L agarose.

[0204] In some embodiments, the antibody or binding fragment thereof according to the invention is an immunoconjugate.

[0205] In some embodiments, the immunoconjugates according to the invention are antibodies or binding fragments thereof linked to a therapeutic moiety, i.e., a drug. In some embodiments, the therapeutic moiety is selected from a cytokine, an immunomodulator, an immunosuppressant, a cytotoxin, a chemotherapeutic agent, a cytolytic peptide, and a radioisotope. Such conjugates are referred to herein as "antibody drug conjugates" or "ADCs."

[0206] In some embodiments, an immunoconjugate according to the present invention is a labeled antibody or binding fragment thereof.

[0207] By "labeled" is meant that the isolated antibody or binding fragment thereof has at least one element, isotope, or compound bound or attached to it that allows, for example, for detection of the isolated antibody or binding fragment thereof.

[0208] Examples of labels include, but are not limited to, luminescent dyes (also called fluorophores or photodetectable labels), isotopic labels (also called radioactive labels, radioisotope labels or heavy isotopes), contrast agents, magnetic labels, electrical labels, thermal labels and colored labels.

[0209] Another object of the present invention is to provide - at least one isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention; - at least one immunoconjugate as described herein; - at least one nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein; - at least one expression vector containing such a nucleic acid; or - at least one host cell containing such an expression vector A composition comprising, consisting essentially of, or consisting of:

[0210] In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient.

[0211] Therefore, another object of the present invention is to - at least one isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention; - at least one immunoconjugate as described herein; - at least one nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein; - at least one expression vector containing such a nucleic acid; or - at least one host cell containing such an expression vector and at least one pharmaceutically acceptable excipient A pharmaceutical composition comprising, consisting essentially of, or consisting of:

[0212] As used herein, "consisting essentially of" with respect to the compositions of the invention means that the at least one isolated anti-AGR2 antibody or binding fragment thereof is the only agent having biological or therapeutic activity within the composition.

[0213] The term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The excipient does not produce adverse, allergic, or other untoward reactions when administered to animals, preferably mammals, and more preferably humans. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards required by regulatory authorities, such as the FDA Office or EMA.

[0214] Examples of pharmaceutically acceptable excipients that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0215] In some embodiments, the pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable vehicle for the formulation to be injected into a subject. These may be, in particular, isotonic sterile saline (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), or a dried composition, in particular a lyophilized composition, which, when added with sterile water or saline, allows the constitution of an injectable solution.

[0216] Another object of the present invention is to provide - at least one isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention; - at least one immunoconjugate as described herein; - at least one nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein; - at least one expression vector containing such a nucleic acid; or - at least one host cell containing such an expression vector A pharmaceutical comprising, consisting essentially of, or consisting of:

[0217] Another object of the present invention is to provide - at least one isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the present invention; - at least one immunoconjugate as described herein; - at least one nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein; - at least one expression vector containing such a nucleic acid; or - at least one host cell containing such an expression vector and instructions for use The kit includes:

[0218] By "kit" is intended any article of manufacture (e.g., a package or container) that includes at least an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the invention.

[0219] AGR2 has been shown to be overexpressed in mucosal inflammatory diseases. Furthermore, secretion of AGR2 monomers has been observed in inflammatory bowel diseases, particularly Crohn's disease. Furthermore, extracellular AGR2 exhibits pro-inflammatory and pro-fibrotic properties.

[0220] Therefore, AGR2 is a promising therapeutic target in the context of inflammatory diseases. Indeed, AGR2 blockers, such as anti-AGR2 antibodies, can act at two levels in the inflammatory processes involved in mucosal inflammatory diseases: 1) blocking local monocyte recruitment and 2) blocking the differentiation of fibroblasts into myofibroblasts, thus preventing the establishment of fibrosis.

[0221] The present invention further relates to an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein for use as a medicament.

[0222] Another object of the present invention is a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid molecule, or a host cell comprising such an expression vector, for use as a medicament.

[0223] The present invention further relates to an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein for use in treating and / or preventing a mucosal inflammatory disease in a subject in need thereof.

[0224] Another object of the present invention is a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein, for use in treating and / or preventing a mucosal inflammatory disease in a subject in need thereof.

[0225] As used herein, the term "inflammatory disease" refers to a vast number of disorders and conditions characterized by inflammation. Symptoms of inflammatory disease may include chronic pain, swelling, redness, joint and muscle stiffness, and loss of function and movement in affected areas. Inflammatory disorders are characterized by the following pathophysiological features: -inflammatory responses to unidentified agents involving various tissues and organs, -responses that depend on the genetic variability of the response properties of immune cells such as antigen-presenting cells, B lymphocytes and T lymphocytes, - production of autoantibodies (natural and pathogenic autoantibodies) against exogenous or endogenous antigens, - production of antigen-specific inflammatory cells, e.g. lymphocytes and T cells, -production and deposition of abnormal proteins and other inflammatory products in various tissues, which induce further inflammatory and immune responses, -These responses include inflammation of blood vessels in surrounding tissues (i.e., vasculitis); and / or -Production of pro- and anti-inflammatory mediators refers to the many heterogeneous states that can have

[0226] The persistence of chronic inflammation is thought to reflect an imbalance between pro-inflammatory cytokines, such as TNF-α, IL-1, IL-6, and GM-CSF, and anti-inflammatory cytokines, including IL-10 and TGF-β. Consequently, a lack of anti-inflammatory cytokines is consistent with impaired immune regulation. Chronic inflammatory changes may result from the inability of regulatory T cells to downregulate the inflammatory process. Non-limiting examples of inflammatory diseases include allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, and transplant rejection.

[0227] As used herein, the term "mucosa" refers to a moist tissue lining a body cavity that secretes mucous membranes and is covered with epithelium. Examples of mucosal tissues include, but are not limited to, oral mucosa (e.g., buccal and sublingual), nasal mucosa, ocular mucosa, genital mucosa, rectal mucosa, pulmonary mucosa, bronchial mucosa, gastric mucosa, intestinal mucosa, olfactory mucosa, uterine mucosa, and esophageal mucosa.

[0228] As used herein, the term "mucosal inflammatory disease" refers to specific inflammatory diseases involving mucosal inflammation. The etiology is unknown and is likely multifactorial, involving genetic susceptibility, environmental factors, microbiota, and the immune system. Mucosal inflammation is characterized by squamous epithelial atrophy, vascular damage, ulceration, and inflammatory infiltrates, leading to fibrosis. It typically affects the mucosal layers of the mouth, gastrointestinal tract, or respiratory system. Non-limiting examples of mucosal inflammatory diseases include Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, chronic pancreatitis, microscopic colitis, inflammatory bowel disease (IBD), irritable bowel syndrome, endometriosis, appendicitis, asthma, idiopathic pulmonary fibrosis, systemic sclerosis associated with interstitial lung disease, and chronic obstructive pulmonary disease. For example, inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease, which are characterized by idiopathic inflammation of the gastrointestinal tract.

[0229] As used herein, "Crohn's disease" refers to a condition involving chronic inflammation of the gastrointestinal tract. Crohn's disease-related inflammation usually affects the intestines, but can occur anywhere from the mouth to the anus. Crohn's disease differs from ulcerative colitis in that the inflammation penetrates all layers of the intestinal wall, involving the mesentery and lymph nodes. The disease is often discontinuous, i.e., severely diseased areas of the intestine are separated from areas that are apparently disease-free. In Crohn's disease, the intestinal wall may also thicken, causing obstruction, and the development of fistulas and fissures is not uncommon. Crohn's disease can be one or more of several types of Crohn's disease, including, but not limited to, ileocolitis (affecting the ileum and large intestine), ileitis (affecting the ileum), gastroduodenal Crohn's disease (inflammation of the stomach and duodenum), jejunoileitis (patchy inflammatory patches in the jejunum), and Crohn's (granulomatous) colitis (affecting only the large intestine).

[0230] As used herein, "ulcerative colitis" refers to a condition involving inflammation of the large intestine and rectum. In patients with ulcerative colitis, there is an inflammatory reaction primarily involving the colonic mucosa. The inflammation is typically uniform and continuous, with no intervening areas of normal mucosa. Surface mucosal cells as well as crypt epithelium and submucosa are involved in the inflammatory reaction, accompanied by neutrophil infiltration. Ultimately, this reaction typically progresses to epithelial damage and epithelial cell loss, resulting in multiple ulcers, fibrosis, metaplasia, and longitudinal regression of the colon.

[0231] AGR2 is a marker of tumor aggressiveness expressed by many solid tumor types. Due to its nearly ubiquitous expression in solid tumors, its expression in premalignant lesions, and its involvement in metastatic disease, the AGR2 protein is a relevant target for cancer therapy. AGR2, particularly extracellular AGR2, is also involved in chemotherapy drug resistance, mesenchymal transition (EMT), angiogenesis, metastasis, and cancer cell proliferation, migration, and invasion.

[0232] Accordingly, the present invention further relates to an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, for use in treating and / or preventing cancer in a subject in need thereof.

[0233] Another object of the present invention is an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein, for use in treating and / or preventing cancer in a subject in need thereof.

[0234] As used herein, the term "cancer" has its general meaning in the art and specifically refers to diseases caused by the uncontrolled division of abnormal cells. The term "cancer" encompasses solid tumors and blood cancers, and includes both primary and metastatic cancers.

[0235] Examples of cancers include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, pancreas, skin, stomach, testes, tongue, or uterus.

[0236] In some embodiments, the cancer is a tumor, such as a solid tumor.

[0237] In some embodiments, the cancer is an epithelial cancer.

[0238] In some embodiments, the cancer is a genitourinary cancer. Examples of genitourinary cancer include, but are not limited to, adrenocortical carcinoma, bladder cancer, kidney cancer, penile cancer, prostate cancer, renal cancer, testicular cancer, urethral cancer, colorectal cancer, cervical cancer, ovarian cancer, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, gestational trophoblastic disease (GTD), and primary peritoneal cancer.

[0239] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a hematological malignancy. Examples of blood cancer include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma) and blood cancer, such as acute or chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, and myelodysplastic syndrome.

[0240] Examples of cancer include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoma / rhabdoid tumor, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, spinal cord cancer, Chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, osteofibrohistiocytoma and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, embryonal Cell tumors, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant histiocytoma of bone, medulloblastoma, epithelioma, melanoma, Merkel cell carcinoma , mesothelioma, metastatic squamous cell neck cancer with occult primary midline duct carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer,Oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Cesarean section These include, but are not limited to, leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor, bile duct cancer, squamous cell carcinoma of the head and neck, glioblastoma of the brain, adrenocortical carcinoma, bladder cancer, kidney cancer, renal cancer, colorectal cancer, cervical cancer, gestational trophoblastic disease (GTD), and primary peritoneal cancer.

[0241] Preferably, the cancer to be prevented or treated using an isolated antibody or binding fragment thereof that specifically binds to AGR2 is selected from the group consisting of colon cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, oral cancer, breast cancer, lung cancer, ovarian cancer, thyroid cancer, bile duct cancer, head and neck squamous cell carcinoma, brain glioblastoma, adrenocortical carcinoma, bladder cancer, kidney cancer, penile cancer, renal cancer, testicular cancer, urethral cancer, colorectal cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, gestational trophoblastic disease (GTD), and primary peritoneal cancer.

[0242] The present invention further relates to a method for treating and / or preventing a mucosal inflammatory disease or cancer in a subject in need thereof, comprising administering to a subject an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or a nucleic acid encoding the isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein.

[0243] The present invention further relates to the use of an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein for the manufacture of a medicament for treating and / or preventing a mucosal inflammatory disease or cancer in a subject in need thereof.

[0244] The present invention also relates to the use of a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein, for the manufacture of a medicament for treating and / or preventing a mucosal inflammatory disease or cancer in a subject in need thereof.

[0245] The present invention further relates to the use of an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein for treating and / or preventing a mucosal inflammatory disease or cancer in a subject in need thereof.

[0246] The present invention also relates to the use of a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein, for treating and / or preventing a mucosal inflammatory disease or cancer in a subject in need thereof.

[0247] In some embodiments, an isolated antibody or binding fragment thereof that specifically binds to AGR2 according to the present invention, a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, an expression vector comprising such a nucleic acid, a host cell comprising such an expression vector, a composition as described herein, or a pharmaceutical composition as described herein may be used to treat and / or prevent mucosal inflammatory disease or cancer in a subject in need thereof.

[0248] The present invention further relates to a method for reducing mucosal inflammation in a subject in need thereof, comprising administering to the subject an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein.

[0249] The present invention also relates to a method for reducing mucosal inflammation in a subject in need thereof, comprising administering to the subject a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition as described herein, or a pharmaceutical composition as described herein.

[0250] Examples of mucosal inflammatory diseases include, but are not limited to, Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, chronic pancreatitis, microscopic colitis, inflammatory bowel disease (IBD), endometriosis, appendicitis, inflammatory bowel syndrome, idiopathic pulmonary fibrosis, systemic sclerosis, particularly systemic sclerosis associated with interstitial lung disease, asthma, and chronic obstructive pulmonary disease.

[0251] The present invention further relates to a method for reducing the pro-inflammatory and / or pro-fibrotic activity of extracellular AGR2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated antibody or binding fragment thereof that specifically binds to AGR2 of the present invention.

[0252] The present invention also relates to a method for reducing the pro-inflammatory and / or pro-fibrotic activity of extracellular AGR2 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding an isolated antibody or binding fragment thereof that specifically binds to AGR2 as described herein, or an expression vector comprising such a nucleic acid, or a host cell comprising such an expression vector, or a composition described herein, or a pharmaceutical composition described herein.

[0253] The composition, pharmaceutical composition or medicament is formulated for use in administration to a subject.

[0254] In some embodiments, a composition, pharmaceutical composition or medicament according to the invention is administered parenterally, orally, by inhalation, by spray, rectally, nasally, or via an implanted reservoir.

[0255] In some embodiments, the composition, pharmaceutical composition or medicament is administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0256] Examples of forms suitable for injection include, but are not limited to, liquid solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for use in preparing solutions or suspensions by adding a liquid prior to use, such as powders, liposomal forms, etc.

[0257] In some embodiments, the isolated anti-AGR2 antibody or binding fragment thereof, composition, pharmaceutical composition or medicament according to the invention should be administered to a subject in need thereof in a therapeutically effective amount.

[0258] However, it will be understood that the total daily usage amount of the isolated anti-AGR2 antibody or binding fragment thereof, composition, pharmaceutical composition, or medicament according to the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on various factors, including the disease being treated and the severity of the disease; the activity of the isolated antibody or binding fragment thereof that specifically binds to AGR2 used; the age, weight, general health, sex, and diet of the subject; the administration time, route of administration, and excretion rate of the particular therapeutic agent used; the duration of treatment; drugs used in combination with or simultaneously with the particular therapeutic agent used; and similar factors well known in the medical field. For example, it is well within the skill of the art to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or in a single dose.

[0259] In some embodiments, the daily dosage of an isolated antibody or binding fragment thereof that specifically binds to AGR2 can vary over a wide range, from 0.01 to 1,000 mg per adult per day. The composition can 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 active ingredient, allowing for symptomatic adjustment of dosage to the subject being treated. Pharmaceutical compositions or medicaments typically contain from about 0.01 mg to about 500 mg of active ingredient. Therapeutically effective amounts of the drug are usually supplied at dosage levels of 0.0002 mg / kg to about 20 mg / kg of body weight per day. For example, the antibody or binding fragment thereof present in the composition, pharmaceutical composition, or medicament of the present invention can be provided at a concentration ranging from 1 mg / mL to about 100 mg / mL, e.g., 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, or 100 mg / mL. In some embodiments, the isolated antibody or binding fragment thereof that specifically binds to AGR2 is provided at a concentration of about 10 mg / mL in either a 100 mg (10 mL) or 500 mg (50 mL) single-use vial. It will be understood that these dosages are exemplary and that optimal dosages can be adapted taking into account the affinity and tolerability of a particular therapeutic agent, which must be determined in clinical trials.

[0260] In some embodiments, the isolated antibody or binding fragment thereof, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention is to be administered before, simultaneously with, or after a therapeutic agent.

[0261] Some examples of therapeutic agents suitable for co-administration with an isolated antibody or binding fragment thereof, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention include, but are not limited to, immunosuppressants, cytokines, and immunomodulatory agents.

[0262] It will be understood by those skilled in the art that co-administration of an isolated antibody or binding fragment thereof, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention with a particular therapeutic agent, which may be selected from, but is not limited to, those listed herein, will depend on the disease or condition to be prevented and / or treated.

[0263] Examples of immunosuppressants include, but are not limited to, corticosteroids, mTOR inhibitors, such as sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, and zotarolimus; IL-1 receptor antagonists, such as anakinra; antimetabolites, such as azathioprine, leflunomide, methotrexate, mycophenolate, and teriflunomide; IMiDs, such as apremilast, lenalidomide, pomalidomide, and and thalidomide; antibodies, such as vedolizumab, eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, elsilimomab, lebrikizumab, ustekinumab, secukinumab, muromonab-CD3, otelixizumab, teplizumab, bisilizumab, clenoliximab, keliximab, zanolimumab, efalizumab, erlidomide; Ipilimumab, obinutuzumab, rituximab, ocrelizumab, pascolizumab, gomiliximab, lumiliximab, teneliximab, toralizumab, aselizumab, galiximab, gavilimomab, ruplizumab, belimumab, blisibimod, ipilimumab, tremelimumab, bertilimumab, lerdelimumab, metelimumab, natalizumab, tocilizumab, odulimomab, basiliximab, daclizumab, inolimomab, zolimomab These include alitox, atollimumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, siplizumab, talizumab, terimomab, alitox, bapaliximab, bepalimomab, abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, and rilonacept, as well as small molecules targeting the ubiquitous JAK or S1P pathways.

[0264] The CCCP / M20CCL1 (including CLCP / M20CCL1) was also activated. L3 / MIP1α、CCL4 / MIP1β、CCL5 / RANTES、CCL6、CCL7、CCL8、CC L9. CL23、CCL24、CCL25、CCL26、CCL27、CCL28、CXCL1 / KC、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、XX8C CL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16、CX CL17、CX3CL1、XCL1〈よびXCL2)、is in the TNFA database. TNFSF4、TNFSF5 / CD40LG、TNFSF6、TNFSF7、TNFSF8、TNFSF9、 TNFSF10、TNFSF11、TNFSF13、TNFSF13B (EDA) has been described in the text.キン(IL-1α、IL-1β、IL-1Ra、IL-2、IL-3、IL-4、IL-5、IL- 6、IL-7、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、I L-16. 32、IL-33、IL-34、IL-35、IL-36α、IL-36β、IL-36γ、IL-36Ra IFNκ (IFNβ (GM-CSF) has been reported.

[0265] Examples of immunomodulatory agents include, but are not limited to, filgrastim, pegfilgrastim, lenograstim, molgramostim, sargramostim, ancestim, albinterferon, interferon alpha, peginterferon alpha, interferon beta, peginterferon beta, interferon gamma, aldesleukin, oprelvekin, growth hormone, immunocyanin, pegademase, prolactin, tasonermin, histamine dihydrochloride, poly ICLC, vitamin D, lentinan, plerixafor, roquinimex, mifamurtide, glatiramer acetate, thymopentin, thymosin alpha 1, thymulin, polyinosinic:polycytidylic acid, pidotimod, Bacillus Calmette-Guerin vaccine, melanovaccine, and sipuleucel-T vaccine.

[0266] Other therapeutic agents suitable for co-administration with an isolated antibody or binding fragment thereof, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention include anti-cancer agents. For example, a therapeutic agent suitable for co-administration can be a cytotoxin, a chemotherapeutic agent, a cytolytic peptide or a radioisotope.

[0267] Examples of cytotoxins include, but are not limited to, radionuclides (e.g., 35 S, 14 C. 32 P, 125 I, 131 I, 90 Y, 89 Zr, 201 Tl, 186 Re, 188 Re, 57 Cu, 213 Bi and 211At), complex radionuclides, and chemotherapeutic agents. Further examples of cytotoxins include antimetabolites (e.g., 5-fluorouricil (5-FU), methotrexate (MTX), fludarabine, etc.), antimicrotubule agents (e.g., vincristine, vinblastine, colchicine, taxanes (e.g., paclitaxel and docetaxel, etc.), alkylating agents (e.g., cyclophosphamide, melphalan, bischloroethylnitrosourea (BCNU), etc.), platinum agents (e.g., cisplatin (also called cDDP), carboplatin, oxaliplatin, JM- 216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide, tenoposide, and camptothecin), or other cytotoxic agents such as ricin, diphtheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed virus protein, ethidium bromide, glucocorticoids, anthrax toxin, etc.

[0268] Examples of chemotherapeutic agents include, but are not limited to, platinum coordination compounds (such as cisplatin, carboplatin, or oxaliplatin); taxane compounds (such as paclitaxel or docetaxel); topoisomerase I inhibitors (such as irinotecan or topotecan); topoisomerase II inhibitors (such as etoposide or teniposide); vinca alkaloids (such as vinblastine, vincristine, or vinorelbine); antitumor nucleoside derivatives (such as 5-fluorouracil, gemcitabine, or capecitabine); alkylating agents (such as nitrogen mustard or nitrosoureas, cyclophosphamide, chlorambucil, carmustine, or lomustine); antitumor anthracycline derivatives (such as daunorubicin, doxorubicin, anti-HER2 antibodies (e.g., trastuzumab); estrogen receptor antagonists or selective estrogen receptor modulators (e.g., tamoxifen, toremifene, droloxifene, faslodex, or raloxifene); aromatase inhibitors (e.g., exemestane, anastrozole, letrazole, or vorozole); differentiation inducers (e.g., retinoids, vitamin D, and retinoic acid metabolism blockers [RAMBAs] such as accutane); DNA methyltransferase inhibitors (e.g., azacitidine); kinase inhibitors (e.g., flaboperidol, imatinib mesylate, or gefitinib); farnesyltransferase inhibitors; and HDAC inhibitors.

[0269] Examples of cytolytic peptides include, but are not limited to, toxins such as diphtheria toxin or Pseudomonas exotoxin.

[0270] Examples of radioisotopes include, but are not limited to, technetium (e.g., Tc-99 and Tc-97), potassium (e.g., K-40), rubidium (e.g., Rb-82), iodine (e.g., I-123, I-124, I-125, I-129, I-131), cesium (e.g., Cs-135, Cs-137), cobalt (e.g., Co-60), palladium (e.g., Pd-103, Pd-107), cadmium (e.g., Cd-113), strontium (e.g., Sr-89, Sr-90), and the like. ), europium (e.g., Eu-55), tin (e.g., Sn-121, Sn-126), phosphorus (e.g., P-32, P-33), thallium (e.g., Tl-201), indium (e.g., In-111), gallium (e.g., Ga-67, Ga-68), yttrium (e.g., Y-90), iridium (e.g., Ir-192), bismuth (e.g., Bi-213), radium (e.g., Ra-223, Ra-225), and ruthenium (e.g., Ru-106) radionuclides.

[0271] The present invention further relates to the use of an isolated antibody or binding fragment thereof according to the invention for detecting or quantifying AGR2 expression in a biological sample, and to a method for detecting or quantifying AGR2 expression in a biological sample, comprising contacting the biological sample with the isolated antibody or binding fragment thereof of the invention.

[0272] As used herein, a "biological sample" may be, for example, a cell, a tissue, or an organ. Examples of biological samples include, but are not limited to, body fluids (preferably blood, more preferably serum), plasma, urine, feces, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophages, tissue lysates, biopsies, and extracts prepared from diseased tissues.

[0273] In some embodiments, the uses and methods for detecting or quantitating AGR2 can be in vitro or in vivo.

[0274] As used herein, the term "expression" may alternatively refer to the translation of AGR2 (i.e., expression of the protein), the presence of AGR2 protein within a cell, on the cell surface, or the presence of secreted or circulating AGR2 protein.

[0275] Suitable assays for detecting or quantifying protein level in sample are well known in the art.Examples of such assays include but are not limited to mass spectrometry, immunohistochemistry, multiplex method (Luminex), Western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), flow cytometry (FACS), immunofluorescence, immunoprecipitation etc.

[0276] In some embodiments, determining the expression level of AGR2 specifically corresponds to the detection and quantification of AGR2 protein in cells. Methods for analyzing the presence of a protein in cells are well known to those skilled in the art and include, but are not limited to, FACS analysis, immunohistochemistry, mass spectrometry, Western blot associated with cell fractionation, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), or image analysis, such as high-content analysis.

[0277] In some embodiments, the isolated antibody or binding fragment thereof according to the invention is labeled for detection or diagnostic purposes, or for patient monitoring purposes, as described above.

[0278] In some embodiments, the isolated antibody or its binding fragment according to the present invention is used in combination with one or several other antibodies. For example, another anti-AGR2 antibody can be used as a capture antibody, while the isolated antibody or its binding fragment according to the present invention can be used as a detection antibody, or vice versa. Non-limiting examples of anti-AGR2 antibodies include, for example, the antibody provided by Abcam under the reference Ab244826.

[0279] In some embodiments, the sample is taken or collected from the subject prior to any analysis. Thus, in this embodiment, the uses and methods for detecting or quantifying AGR2 are in vitro uses and methods.

[0280] The present invention further relates to the use of an isolated antibody or binding fragment thereof of the present invention for diagnosing or monitoring an AGR2-related disease in a subject, as well as to a method of diagnosing or monitoring an AGR2-related disease in a subject, comprising contacting a sample from the subject with an isolated antibody or binding fragment thereof of the present invention.

[0281] The present invention further relates to the use of the isolated antibody or binding fragment thereof of the present invention to select a subject suffering from an AGR2-associated disease for a treatment that targets the AGR2-associated disease, and to a method of selecting a subject suffering from an AGR2-associated disease for a treatment that targets the AGR2-associated disease, comprising contacting a sample from the subject with the isolated antibody or binding fragment thereof of the present invention.

[0282] The present invention also relates to the use of the isolated antibody or binding fragment thereof of the present invention for monitoring a subject's response to a therapy that targets an AGR2-associated disease, and to a method of monitoring a subject's response to a therapy that targets an AGR2-associated disease, comprising contacting a sample from the subject with the isolated antibody or binding fragment thereof of the present invention.

[0283] In some embodiments, the isolated antibodies or binding fragments thereof of the present invention may be used in a companion diagnostic test, for example, in connection with a treatment targeting an AGR2-associated disease.

[0284] As used herein, the term "AGR2-related disease" refers to any disease in which AGR2 is involved. For example, an "AGR2-related disease" may be a disease in which AGR2 is dysregulated, e.g., overexpressed (upregulated) or downregulated, preferably overexpressed. An "AGR2-related disease" may also be a disease in which abnormal extracellular (membrane-bound or secreted) AGR2 can be detected. An "AGR2-related disease" may also be a disease in which AGR2 monomers or dimers can be detected.

[0285] In particular, the "AGR2-associated disease" may be, for example, a mucosal inflammatory disease, including but not limited to, Crohn's disease, inflammatory bowel disease, and ulcerative colitis disease.

[0286] The uses and methods for diagnosing or monitoring AGR2-associated diseases can be in vitro or in vivo, preferably in vitro. In some embodiments, the diagnostic methods of the present invention are in vitro diagnostic methods, i.e., the methods of the present invention are performed on a biological sample obtained from a patient before performing the methods of the present invention. As a result, in some embodiments, the methods of the present invention do not involve obtaining a sample from a patient, i.e., the methods of the present invention are non-invasive.

[0287] In some embodiments, the AGR2-associated disease is 高 According to this embodiment, if AGR2 is detected in a sample from a subject at a level, amount, or concentration higher than in a reference subject (e.g., a substantially healthy subject, or a subject known to be unaffected or not suffering from an AGR2-related disease), the subject may be diagnosed as being affected by or suffering from an AGR2-related disease.

[0288] The phrase "monitoring a subject's response to a therapy targeting an AGR2-associated disease" can mean, for example, adapting the therapy. Preferably, "monitoring a subject's response to a therapy targeting an AGR2-associated disease" means changing the drug used to treat the subject, or increasing or decreasing the dose, frequency of administration, or changing the route of administration of the therapy.

[0289] When the method is used to monitor the progression of an AGR2-associated disease or to monitor a subject's response to treatment, it is repeated at least two different time points (e.g., before and after initiation of treatment).

[0290] In some embodiments, the uses and methods for diagnosing or monitoring an AGR2-associated disease further comprise treating the subject if the subject is diagnosed as being affected or suffering from an AGR2-associated disease.

[0291] In some embodiments, the method of selecting a subject suffering from an AGR2-associated disease for a therapy that targets the AGR2-associated disease further comprises, if the subject is selected to receive the therapy that targets the AGR2-associated disease, subjecting the subject to the therapy that targets the AGR2-associated disease. [Brief explanation of the drawings]

[0292] [Figure 1] FIG. 1 is a schematic representation of the three-dimensional structure of the human AGR2 protein, showing the locations of the epitopes bound by Agtuzumab and the TH-106 humanized monoclonal antibody with dotted circles.

[0293] [Figure 2]Figure 2 is a schematic diagram of the structure of the human AGR2 protein, which contains, from N- to C-terminus: a cleavable signal peptide, an adhesive domain, a dimerization motif, a pseudo-thioredoxin motif, a peptide-binding loop, and an endoplasmic reticulum retention signal.

[0294] [Figure 3A-F] Figures 3A-F are a set of graphs showing the cross-reactivity and specificity of Agtuzumab IgG1-LALA and humanized anti-AGR2 monoclonal antibodies of the present invention (i.e., TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-109 IgG1-LALA, and TH-115 IgG1-LALA). The binding ability of the antibodies to human AGR2 WT, mouse AGR2, canine AGR2, macaque AGR2, human AGR2 E60A (i.e., monomeric AGR2), human AGR3, and BSA was assessed by ELISA. Antibodies were tested at an initial concentration of 10 μg / mL and serially diluted in quarter steps to levels as low as 0.15 ng / mL. Figure 3A shows Agtuzumab IgG1-LALA. Figure 3B shows the TH-101 IgG1-LALA humanized monoclonal antibody. Figure 3C shows the TH-104 IgG1-LALA humanized monoclonal antibody, Figure 3D shows the TH-106 IgG1-LALA humanized monoclonal antibody, Figure 3E shows the TH-109 IgG1-LALA humanized monoclonal antibody, and Figure 3F shows the TH-115 IgG1-LALA humanized monoclonal antibody.

[0295] [Figure 4]4 is a graph showing the binding of Agtuzumab IgG1-LALA and humanized anti-AGR2 monoclonal antibodies of the invention (i.e., TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-109 IgG1-LALA, TH-115 IgG1-LALA) to human AGR2 WT, as assessed by ELISA. Antibodies were tested at an initial concentration of 10 μg / mL and serially diluted in quarter steps to levels as low as 0.15 ng / mL.

[0296] [Figure 5A-B] Figures 5A-B are a set of graphs showing the cross-reactivity and specificity of the TH-106 IgG1 and TH-106 IgG1-LALA humanized monoclonal antibodies. The binding ability of the antibodies to human AGR2 WT, mouse AGR2, canine AGR2, macaque AGR2, human AGR2 E60A (i.e., monomeric AGR2), human AGR3, and BSA was assessed by ELISA. Antibodies were tested at an initial concentration of 10 μg / mL and serially diluted in 1 / 4 increments to levels as low as 0.15 ng / mL. Figure 5A shows the TH-106 IgG1 humanized monoclonal antibody. Figure 5B shows the TH-106 IgG1-LALA humanized monoclonal antibody.

[0297] [Figure 6A-F]Figures 6A-F are a set of histograms showing parameters measured in a DSS-induced mouse model of acute colitis treated with PBS as a control or anti-AGR2 antibody administered intraperitoneally or intravenously at doses of 5 μg / mouse, 10 μg / mouse, 20 μg / mouse, or 40 μg / mouse, as indicated. Data are expressed as mean ± sem. p values ​​were calculated compared to DSS + PBS. *p<0.05; **p<0.01; ***p<0.001. Figure 6A shows the Disease Activity Index (DAI) score at the time of euthanasia (day 12). Figure 6B shows stool consistency at the time of sacrifice. Figure 6C shows the presence of occult blood in the stool. Figure 6D shows the colon weight / size ratio. Figure 6E shows the overall histological score of inflammation. Figure 6F shows quantification of myeloperoxidase in the whole colon.

[0298] [Figure 7A-D] Figures 7A-D are a set of graphs showing that blocking eAGR2 using antibodies of the invention down-regulates circulating cytokines and chemokines involved in inflammation (particularly TNFα and IL-6) in a DSS-induced mouse model of acute colitis. Figure 7A shows systemic levels of TNFα (p<0.0001). Figure 7B shows systemic levels of IL-6 (p<0.0001). Figure 7C shows mRNA levels of TNFα (p<0.05). Figure 7D shows mRNA levels of IL-6 (p<0.05).

[0299] [Figure 8A] Figure 8A is a graph showing a summary of immunohistochemical results (median ± Q) for macrophages (F4 / 80) in PBS-treated control mice (CTL), PBS-treated DSS mice (DSS), and DSS mice treated with anti-AGR2 antibody at a dose of 5 μg or 20 μg, obtained using digital morphometric analysis. [Figure 8B]Figure 8B is a graph showing a summary of immunohistochemical results (median ± Q) for T cells (CD3) in PBS-treated control mice (CTL), PBS-treated DSS mice (DSS), and DSS mice treated with anti-AGR2 antibody at doses of 5 μg or 20 μg, obtained using digital morphometric analysis. [Figure 8C] Figure 8C is a graph showing a summary of immunohistochemical results (median ± Q) for neutrophils (LY6G) in control mice treated with PBS (CTL), DSS mice treated with PBS (DSS), and DSS mice treated with anti-AGR2 antibody at a dose of 5 μg or 20 μg, obtained using digital morphometric analysis. Statistical significance was calculated using the Kruskal-Wallis test followed by the Mann-Whitney U test. *p<0.001.

[0300] [Figure 9A-M] Figures 9A-M are a set of histograms showing parameters measured in a DSS-induced mouse model of chronic colitis treated with PBS as a control or with anti-AGR2 antibody administered intraperitoneally prophylactically or therapeutically at a dose of 10 μg / mouse. Data are expressed as mean + / - sem. *p<0.05; **p<0.01; ***p<0.001. Figure 9A shows the Disease Activity Index (DAI) score at the time of euthanasia (day 42). Figure 9B shows the stool consistency at the time of euthanasia (day 42). Figure 9C shows the presence of occult blood in the stool. Figure 9D shows the macroscopic score of fibrosis. Figure 9E shows the colon weight / size ratio. Figure 9F shows the overall histological score of inflammation. Figure 9G shows collagen deposition in mice administered a therapeutic anti-AGR2 antibody. Figure 9H shows mice administered a prophylactic anti-AGR2 antibody. Figure 9I shows α-SMA mRNA levels. Figure 9J shows fibronectin mRNA levels. Figure 9K shows Col1A mRNA levels. Figure 9L shows TGF-β mRNA levels. Figure 9M shows quantification of α-SMA staining by immunofluorescence.

[0301] [Mode for Carrying Out the Invention] [Sequence table] [Table 1] TIFF2025539142000002.tif225159TIFF2025539142000003.tif223159TIFF2025539142000004.tif220159 TIFF2025539142000005.tif220159TIFF2025539142000006.tif228159TIFF2025539142000007.tif217159 TIFF2025539142000008.tif219159TIFF2025539142000009.tif227159TIFF2025539142000010.tif217159 TIFF2025539142000011.tif226159TIFF2025539142000012.tif216159TIFF2025539142000013.tif194159 [Example]

[0302] The present invention is further illustrated by the following examples. Table 1: Example antibody sequences [Table 2]

[0303] Example 1: Generation of anti-AGR2 humanized monoclonal antibodies material and method Plasmid preparation The target DNA sequence was designed, optimized, and synthesized. The complete sequence was subcloned into a proprietary vector from GenScript. Transfection-grade plasmids were maxi-prepared for expression in TurboCHO-HT cells.

[0304] Cell culture and transient transfection Cells were maintained at 37°C and 5% CO2 on an orbital shaker. Cells were seeded at the appropriate density one day before transfection. On the day of transfection, DNA and reagents were mixed in the optimal ratio and then added to the cells ready for transfection. Approximately 24 hours after transfection, feed was added to each sample.

[0305] Purification and analysis The cell culture broth was centrifuged and subsequently filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with an appropriate buffer, the eluted fractions were pooled and the buffer was exchanged with the final formulation buffer. The purified protein was analyzed by SDS-PAGE and SEC-HPLC analysis to determine molecular weight and purity. The concentration was determined by the A280 method (i.e., the absorbance of the protein solution at 280 nm).

[0306] result The TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, and TH-109 IgG1-LALA antibodies were easily produced. Furthermore, the TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, and TH-109 IgG1-LALA antibodies all had better yields than Agtuzumab IgG1-LALA (as shown in Table 2 below). However, the TH-115 IgG1-LALA antibody had a lower yield compared to the other humanized antibodies (as shown in Table 2). Table 2: Production characteristics of Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, TH-109 IgG1-LALA and TH-115 IgG1-LALA antibodies. [Table 3]

[0307] Example 2: Epitope Mapping material and method DMS DNA library preparation A DMS library of human AGR2 protein was generated by PCR, with each mutation position having a degenerate "NNS" or "NNK" codon encoding 20 amino acids / 32 codons.

[0308] Construction of antigen expression plasmids The gene corresponding to human AGR2 WT (SEQ ID NO: 1) was synthesized and cloned into a plasmid that allows galactose-inducible expression on the surface of yeast. In this construct, the expressed antigen (i.e., human AGR2 WT) has a C-terminal HA tag. The expression plasmid was then transformed into the yeast strain S. cerevisiae EBY100.

[0309] Induction of antigen expression in yeast surface display Induction of the transformed yeast in SG-CAA induction medium (6.7 g / L casamino acid-free yeast nitrogen base medium, 20 g / L, 5 g / L casamino acids, 100 mM sodium phosphate, pH 6.0) allows expression of the antigen (i.e., human AGR2 WT) on the yeast surface.

[0310] Flow cytometry sorting 10 7 The induced cells were washed with 1 mL of PBSF (PBS containing 0.1% BSA). The cells were then resuspended in an appropriate volume of solution containing 3 nM Agtuzumab or 500 pM TH-106 humanized monoclonal antibody. After 2 hours of incubation at 20°C with agitation, the cells were washed with 1 mL of ice-cold PBSF (to avoid dissociation). The cells were then incubated with anti-human PE fluorescent reporter on ice for 15 minutes. The cells were then sorted on a BD FACSAria™ III cytometer using BD FACSDiva™ software.

[0311] Next-generation sequencing and data analysis Plasmids from each selected yeast population were extracted and prepared for sequencing. Two-step PCR was performed: the first step amplifies the region of interest, and the second step adds the Illumina adapters required for sequencing. Sequencing was performed on an Illumina iSeq100 instrument (2 × 150 bp, 300 cycles) with at least 150,000 reads per population. Data were then processed through an analysis pipeline using dedicated proprietary scripts. Low-quality sequences (Q<30) were removed, and single mutants were then detected and counted.

[0312] result Deep mutation scanning (DMS) is a mutagenesis method that aims to perform all possible single substitutions for all selected residues in a given protein sequence. A DMS library is obtained in the form of DNA encoding the protein under study (i.e., human AGR2 protein in this case). In this library, each DNA strand contains a mutated codon relative to the parent sequence.

[0313] This DMS DNA library is incorporated into an expression plasmid specifically designed to express recombinant proteins on the yeast surface. The yeast is then transformed and induced to allow the expression of a single mutant protein on its surface. This new library (called a display library) is screened by flow cytometry using a fluorescent reporter to reveal the expression of the protein (anti-tag fluorescent antibody) and the binding of the protein to its partner (fluorescent partner).

[0314] For epitope mapping, the ideal case is to have two antibodies with compatible epitopes that can bind together on the same antigen. In this way, each of the two antibodies acts as a conformational control of the mutated antigen for the other antibody. In fact, a single substitution made to an antigen can have four types of effects: 1. Loss of affinity for the first antibody while retaining binding to the second antibody: This is a mutation made within the epitope of the first antibody. 2. Loss of affinity for the second antibody while retaining binding to the first antibody: This is a mutation in the epitope of the second antibody. 3. Loss of affinity for both antibodies: this is a so-called "deconstruction" mutation that affects the conformation of the antigen and therefore prevents binding of both antibodies. 4. No effect: The mutation is not present in the epitope of one of the two antibodies and does not cause a significant change in the conformation of the antigen.

[0315] After flow cytometry analysis, a yeast population that retains binding to the second antibody but loses affinity for the antibody of interest is selected. The plasmids contained in this yeast population are extracted and sequenced by high-throughput sequencing. Analysis of the sequencing data allows identification of mutations that affect the binding of the antibody to its target. Thus, this analysis allows identification of the important position on the antigen for binding of the antibody of interest, i.e., its epitope.

[0316] The epitopes bound by Agtuzumab and the TH-106 humanized monoclonal antibody were identified using DMS. As shown in Figure 1, the epitopes bound by Agtuzumab and the TH-106 humanized monoclonal antibody are located in two different domains of the human AGR2 protein. The Agtuzumab epitope contains the histidine residue at position 117 (H117), the aspartic acid residue at position 121 (D121), and the tyrosine residue at position 124 as the most critical amino acid residues. On the other hand, the epitope bound by the TH-106 humanized monoclonal antibody contains the proline residue at position 82 (P82) and the glutamic acid residue at position 153 (E153) as the most critical amino acid residues. As shown in Figure 2, which shows the structure of the AGR2 protein, the proline residue at position 82 is located in the pseudothioredoxin domain. Thus, the epitope bound by the TH-106 humanized monoclonal antibody is located in the catalytic domain of the human AGR2 protein, whereas amino acid residues 117, 121, and 124 bound by Agtuzumab are not located in the functional domain (as shown in Figure 2). This suggests that the TH-106 humanized monoclonal antibody may be able to inhibit AGR2 catalytic activity, whereas Agtuzumab may not.

[0317] Example 3: Cross-reactivity and specificity of anti-AGR2 humanized monoclonal antibodies material and method ELISA ELISA-optimized plates were coated with human AGR2 WT (SEQ ID NO: 1), mouse AGR2 (SEQ ID NO: 2), canine AGR2 (SEQ ID NO: 6), macaque AGR2 (SEQ ID NO: 3), human AGR2 E60A (SEQ ID NO: 7), human AGR3, or BSA at a concentration of 1 μg / mL in 50 μL per well and incubated overnight at 4° C. The plates were then washed with 300 μL / well of PBS containing 0.05% Tween 20. The plates were blocked with 150 μL / well of PBS containing 2.5% milk for 1 hour at room temperature. The plates were then washed with 300 μL / well of PBS containing 0.05% Tween 20. Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, TH-109 IgG1-LALA, and TH-115 IgG1-LALA antibodies were diluted to an initial concentration of 10 μg / mL and serially diluted 1 / 4 to 0.15 ng / mL. The antibodies were added to the plate and incubated for 2 hours at room temperature. The plate was washed three times with 300 μL / well of PBS containing 0.05% Tween 20. A 1 / 2000 dilution of anti-human IgG1 (Sigma A0170-1mL, lot: 0000088179) secondary antibody conjugated to HRP was added to the plate and incubated for 1 hour at room temperature. Detection was performed using TMB (KPL 52-00-01; lot 10602343). The reaction was stopped with H2SO4 solution and the optical density (OD) was read at 450 nm.

[0318] result The binding ability to human AGR2 WT, mouse AGR2, canine AGR2, macaque AGR2, human AGR2 E60A, human AGR3, or BSA was assessed for each of the Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, TH-109 IgG1-LALA, and TH-115 IgG1-LALA humanized monoclonal antibodies using ELISA.

[0319] As shown in Figures 3A-F, all antibodies were able to recognize and bind to both human AGR2 WT and human AGR2 E60A. Furthermore, all antibodies were able to cross-react with mouse AGR2, canine AGR2, and macaque AGR2, but none of them were able to bind to human AGR3 or BSA, which was used as a control. However, as shown by the EC50 values ​​presented in Table 3 below, the TH-101, TH-104, TH-106, and TH-115 humanized monoclonal antibodies were better binders than Agtuzumab, especially for binding to human AGR2 WT (Figure 4) and human AGR2 E60A. Conversely, the TH-109 humanized monoclonal antibody showed lower binding capacity than the other humanized monoclonal antibodies.

[0320] As shown in Table 4 below, Agtuzumab binds to human AGR2 with affinity in the nanomolar range. All other antibodies (i.e., TH-101, TH-104, TH-106, TH-109, and TH-115) have K values ​​less than 150 pM. D values ​​indicate very strong binding to AGR2.

[0321] Furthermore, the binding ability was compared between the TH-106 IgG1-LALA antibody and the TH-106 IgG1 antibody. As shown in Figures 5A-B and Table 3, the scaffold change had minimal effect on the binding properties of the TH-106 humanized monoclonal antibody. Table 3: EC50 of Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, TH-109 IgG1-LALA and TH-115 IgG1-LALA humanized monoclonal antibodies against human AGR2 WT, mouse AGR2, canine AGR2, macaque AGR2, human AGR2 E60A and human AGR3. [Table 4] [Table 5]

[0322] Example 4: Thermal stability of anti-AGR2 humanized monoclonal antibodies material and method The thermal stability of Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-106 IgG1, TH-109 IgG1-LALA, and TH-115 IgG1-LALA humanized monoclonal antibodies was assessed by differential scanning calorimetry (DSC).

[0323] Using MicroCal PEAQ DSC analysis software, the data were fitted to a non-two-state unfolding model, including buffer subtraction. The thermal transition midpoint (Tm), representing the point at which equal amounts of folded and unfolded protein undergo a transition, was then determined. The calorimetric enthalpy (ΔHcal) was calculated from the integrated area under the transition peak and represents the total heat energy uptake by the sample undergoing the transition. This heat absorption depends on the concentration of sample in the instrument's sample cell undergoing the transition and is a model-free absolute measure of the enthalpy of the process involved.

[0324] result As shown in Table 5, all humanized monoclonal antibodies, except for the TH-115 IgG1-LALA antibody, exhibited better thermal stability than Agtuzumab. Table 5: Melting temperatures (Tm) of the Fab fragments of Agtuzumab IgG1-LALA, TH-101 IgG1-LALA, TH-104 IgG1-LALA, TH-106 IgG1-LALA, TH-109 IgG1-LALA and TH-115 IgG1-LALA humanized monoclonal antibodies. [Table 6]

[0325] Example 5: Effect of anti-AGR2 humanized monoclonal antibodies on monocyte migration material and method Monocyte chemoattraction assay Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. PBMCs were washed with RPMI 1% FCS (Life Technologies) and placed on top of a Boyden chamber system (5 × 10 in RPMI 1% FCS). 5 (Cells / chamber; Millipore, France). The upper part of the Boyden chamber was placed in RPMI containing recombinant human AGR2 or tumor cell culture supernatant containing eAGR2, in the presence or absence of either Agtuzumab or TH-106 humanized monoclonal antibody. The Boyden chamber was then incubated at 37°C for 24 hours. The migrated PBMCs (lower part of the Boyden chamber) were collected and washed with PBS. The cells were stained for monocyte, T cell, B cell, and NK cell markers (anti-CD14, anti-CD3, anti-CD19, and anti-CD56, respectively) and analyzed by flow cytometry. The data were then analyzed using a FACSDiva (BD Biosciences). The relative number of migrated cells was estimated by flow cytometry by counting the absolute number of cells.

[0326] result Monocyte migration induced by either recombinant human AGR2 or eAGR2-containing tumor cell (Mz-Cha-1) supernatant was blocked by both Agtuzumab and the TH-106 humanized monoclonal antibody. Residual migration in the presence of the TH-106 humanized monoclonal antibody was comparable to that of the negative control (medium alone). However, this blockade was more efficient with the TH-106 humanized monoclonal antibody than with Agtuzumab. These data demonstrate that the TH-106 anti-AGR2 humanized monoclonal antibody can inhibit monocyte migration induced by Mz-Cha-1 conditioned medium.

[0327] Example 6: Effect of anti-AGR2 humanized monoclonal antibody on myofibroblast differentiation material and method Differentiation of CCD-18Co fibroblasts into myofibroblasts CCD-18Co cells were seeded in a 6-well plate (1 × 10 6 Cells (cells / well) were grown for 48 hours before treatment. Endoplasmic reticulum (ER) stress was induced in CCD-18Co cells using 10 μg / mL tunicamycin (Tm, Sigma, solubilized in DMSO). DMSO was used as a control condition (at the same final concentration) to monitor the response of CCD-18Co cells to Tm. A 48-hour treatment with 10 ng / mL TGF-β1 (R&D Systems) added to the CCD-18Co cell culture medium (after 24 hours of serum starvation and the addition of 1% FBS during stimulation) provided a positive control for CCD-18Co cell differentiation. Supernatants from HT-29 cells, harvested 8, 24, and 32 hours after medium change, with or without preconditioning with Tm (and thus with or without transient ER stress), were applied to CCD-18Co cells for a further 48 hours of incubation as the induction condition. HT-29 supernatants were used without a freeze cycle. The ability of recombinant human AGR2 (rAGR2) to induce differentiation of fibroblasts into myofibroblasts was investigated by applying 40 ng / mL rhAGR2 to the culture medium of CCD-18Co cells (after 24 h of serum starvation and the addition of 1% FBS during stimulation), with the same conditions without rhAGR2 added as a control. The effects of AGR2 blockade were assessed using Agtuzumab or the TH-106 anti-AGR2 humanized monoclonal antibody added to HT-29 supernatants preconditioned with rhAGR2 or Tm. HT-29 and CCD-18Co cells were harvested and processed for either total protein extracts (stored at -20°C) or RNA extraction (stored at -80°C). HT-29 supernatants collected for analysis were stored at -20°C. Samples were used for immunofluorescence analysis, Western blot, and RNA extraction and RT-qPCR.

[0328] result Fibroblasts can differentiate into myofibroblasts upon exposure to AGR2, the fibrogenic factor. Myofibroblast differentiation is induced by eAGR2. Both Agtuzumab and the TH-106 humanized monoclonal antibody can block this differentiation. However, the blockade of myofibroblast differentiation is more efficient with the TH-106 humanized monoclonal antibody than with Agtuzumab.

[0329] Example 7: In vivo efficacy of anti-AGR2 monoclonal antibodies in an acute DSS model material and method Animal models and colitis induction Animal experiments were performed in accredited facilities at the Institut Pasteur in Lille (license number B59-35009) in accordance with government guidelines (European Directive 2010 / 63 / UE, updated February 13, 2013, code rural R214-87 to R214-137) and the guidelines of the Nord-Pas de Calais Ethical Committee for Animal Use. Animals were housed five per cage and provided with standard mouse chow and tap water ad libitum.

[0330] For this study, C57Bl / 6 mice were used. To induce colitis, C57Bl / 6 mice were administered 2.5% dextran sulfate sodium (DSS) (45 kD; MP Biomedicals, reference number 160110) in drinking water for 5 days (days 0 to 5), followed by 3 or 7 days of regular water. Inflammation peaked on day 8 after the start of DSS administration. Mice were euthanized on day 12.

[0331] 110 C57B1 / 6 mice were assigned to the following groups, as described in Table 6 below: Table 6: Groups of mice for in vivo study of DSS-induced acute colitis [Table 7] Antibody treatment Table 7: Sequences of anti-AGR2 antibodies used in Example 7 [Table 8]

[0332] Anti-AGR2 monoclonal antibodies were administered in the drinking water at doses ranging from 5 μg to 40 μg per mouse by intraperitoneal (IP) or intravenous (IV) route, starting from the day of the first DSS administration, every 2 days (equivalent to a total of 6 administrations (D0, D2, D4, D6, D8, and D10)) until the day of euthanasia, which occurred 7 days after the last DSS administration.

[0333] Laboratory tests Mortality and clinical signs During the experiment, each animal was examined for mortality once a day. The body weight of each animal was recorded daily from day 0 (the first day of colitis induction with DSS) to day 12 (the time of euthanasia). Body weight was uniform among the different groups before colitis induction.

[0334] Disease Activity Index (DAI) To evaluate the effect of anti-AGR2 antibodies on colonic inflammation, various parameters were monitored daily throughout the experimental period. The disease activity index (DAI) is a simple scoring system used to determine the severity of colitis in mice. The disease activity index (DAI) was determined at the time of euthanasia (D12) based on changes in body weight, stool consistency, and the presence of blood in the stool, assessed using an occult blood test. Briefly, the DAI was assessed by an investigator blinded to the protocol according to a standard scoring system. Body weight (BW), stool consistency (scored 0–3, where 0 = normal, 1 = loose stool, 2 = diarrhea, and 3 = watery diarrhea), and the presence of visible blood (rectum of mice) were recorded daily. At the time of euthanasia, the presence of occult blood (OB) was recorded using the fecal occult blood method. BW loss was scored as follows: 0, no weight loss; 1, <10% weight loss from baseline; 2, >10% weight loss from baseline. For stool consistency, a score of 0 was assigned for well-formed pellets, 1 for pasty and semi-formed stool that did not adhere to the anus, and 2 for liquid stool that adhered to the anus. For OB, a score of 0 was assigned for no blood and 1 for positive OB or gross bleeding. These scores were added together. BW loss was calculated as the percent difference between the original BW (day 0) and the BW on any particular day.

[0335] Colon weight / size ratio After euthanasia, the colon was carefully excised and its weight and size were measured. Indeed, a decrease in colon size was induced by severe inflammation, and an increase in colon weight was also observed due to edema / inflammatory infiltrates caused by severe inflammation.

[0336] Blood and tissue sampling Serum from each animal was collected by cardiac puncture at the time of euthanasia and placed in a 1.1 ml Z-Gel microtube (Sarstedt). The tubes were inverted five times, and the samples were then allowed to clot at ambient temperature for 1 hour. The tubes were centrifuged at 2300 x g for 10 minutes at 20°C.

[0337] Serum (supernatant) was collected into fresh pre-chilled tubes and stored at -80°C until analysis. Samples from the distal colon were divided into four portions and snap-frozen (one portion for protein analysis, one portion for histological evaluation, one portion for MPO measurement, and one portion for mRNA extraction in 500 μl of RNAlater). The spleen, a portion of the liver (right lobe), MLN (if available), and a portion of the distal ileum were snap-frozen, and a portion of the distal ileum was excised for histological evaluation.

[0338] Histological evaluation of colonic lesions Paraffin-embedded colonic samples were analyzed to assess the level of inflammation. For histological evaluation, sections of colonic tissue (4 μm) and distal ileum were stained with May-Grünwald-Giemsa and evaluated. Multiparametric scoring (0–18) was performed blindly by two examiners as described by Dieleman et al. (1998). Histological examination graded the severity and extent of inflammation, the intensity of the cellular infiltrate in the mucosa, its extension in the submucosa, and the presence of epithelial lesions.

[0339] Quantification of MPO Myeloperoxidase (MPO), an enzyme contained in primary granules of polymorphonuclear neutrophils, is used as a marker of neutrophil infiltration. MPO protein levels are a quantitative and objective marker used to assess neutrophil recruitment in tissues and, indirectly, neutrophil-mediated colonic inflammation. MPO levels were quantified in colonic samples by ELISA (reference HK210-01, Clinisciences) according to the manufacturer's recommendations. MPO activity was measured to monitor the degree of neutrophil infiltration in colonic lesions during chemically induced colitis. Colonic specimens were homogenized with an Ultra Turrax T8 (Ika-Werke, Staufen, Germany) in phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium and subjected to two sonication and freeze-thaw cycles. The suspension was centrifuged at 14,000 × g for 15 minutes at 4°C, and the supernatant was reacted with 1 mg / mL o-dianisidine hydrochloride and 0.0005% hydrogen peroxide. The optical density of each sample was read at 450 nm using a Versamax microplate reader (MDS Analytical Technologies, Saint-Gregoire, France). One unit of MPO activity was defined as the amount that decomposes 1 μmol of peroxidase per minute at 25°C. Results were expressed as absorbance per total amount of protein, as determined by the Bradford method (Pineton de Chambrun G et al. Mucosal Immunol. 2014).

[0340] Cytokine and chemokine analysis Analytes in mouse serum were quantified by ELISA using the Cytokine & Chemokine Convenience 26-Plex Mouse ProcartaPlex™ Panel 1 (ThermoFischer), TGF beta 1 Mouse ProcartaPlex™ Simplex Kit (ThermoFischer), and ProcartaPlex Mouse Basic Kit (part 2 of the TGF-b1 kit) (ThermoFischer). The following analytes were quantified: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A (CTLA-8), IL-18, IL-22, IL-23, IL-27, TNF-α, eotaxin (CCL11), GRO-α (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1α (CCL3), MIP-1β (CCL4), MIP-2α (CXCL2), RANTES (CCL5), and TGF-β1. The Bio-Plex® 200 System, a flow-based, dual-laser system for simultaneously identifying and quantifying up to 100 different analytes in a single biomolecule assay (xMAP technology), was used. Analyte concentrations are proportional to the mean fluorescence intensity (MFI) collected from 50 beads per region, with one specific microparticle region assigned per analyte, and back-calculated from interpolation with a calibration curve.

[0341] Analyte mRNA levels were assessed by qRT-PCR. Mouse colon tissue samples were obtained from mice, stabilized in RNALater (ThermoFisher Scientific, AM7020), and stored at -80°C. Tissues were thawed, cleaned from excess RNALater in PBS, and subjected to bead tissue homogenization using a MiniLys tissue homogenizer (Bertin Technologies, ref. P000673-MLYS0-A) containing 1.4 mm ceramic beads in a 2 mL tube (Bertin Technologies, CK14-2 mL, ref. P000933-LYSK0-A) filled with 350 μL of Tripure Isolation Reagent (Sigma-Aldrich, catalog no. 11667157001) at 5000 rpm for three cycles of 15 seconds. 650 μL of Tripure® was added to the homogenized tissue, and extraction was performed using the Maxwell® RSC miRNA Plasma and Serum Kit (Promega, Catalog No. AS1680) in combination with the Maxwell RSC48 Instrument (Promega, Catalog No. AS8500). Sample pretreatment was performed according to an in-house developed protocol. Briefly, 200 μL of bromochloropropane (BCP) (Sigma-Aldrich, Catalog No. B9673) was added to 1 mL of Tripure® homogenized sample and mixed by vortexing for 10–15 seconds until the solution was bleached. This was followed by a 5-minute incubation at room temperature and 15-minute centrifugation at 12,000 rcf in a benchtop centrifuge at 4°C. The aqueous phase containing the RNA (approximately 500 μL) was then transferred to a new 1.5 mL Eppendorf tube, and 230 μL of Lysis Buffer C from the Maxwell® RSC miRNA Plasma and Serum Kit was added. Mixing was performed by vortexing for 5 seconds. Cartridge preparation from the Maxwell RSC miRNA Kit and preparation and running of the Maxwell RSC 48 instrument were performed according to the steps outlined by Promega. Elution was performed in 75 μL of nuclease-free water.Reverse transcription of 2.5 μg of RNA per sample was performed using SuperScript™ IV VILO™ Master Mix (ThermoFisher Scientific, catalog number 11756050) according to the user guide provided by the manufacturer. Briefly, 4 μL of the master mix was combined with 2.5 μg of total RNA and up to 20 μL of nuclease-free water on ice and mixed by vortexing. The reverse transcription reaction was then performed in a thermocycler under the following program: 25°C / 10 min; 50°C / 10 min; 85°C / 5 min. RT-PCR was performed using a custom-designed Taqman Array Fast 96-well plate with 45 ng of cDNA per well according to the user guide provided by ThermoFisher Scientific. Briefly, the cDNA and nuclease-free water were combined with Taqman Fast Advance Master Mix (catalog number 4444963) in a microcentrifuge tube and then vortex mixed. 10 μL of the cDNA-Master Mix was added to the wells of a Taqman Array Fast plate, which was then sealed and centrifuged. RT-PCR was performed in a Quantstudio 5 Real-Time PCR System, 96-well 0.2 mL (Cat. No. A28139) under the following cycling conditions: 1 cycle (50°C / 2 min; 95°C / 20 sec), 40 cycles (95°C / 1 sec; 60°C / 20 sec).

[0342] immune infiltrate Immunohistochemistry (IHC) and digital morphometry analysis were used to quantify T cells, macrophages, and neutrophils in mouse colons. Paraffin sections approximately 4 microns thick were cut and placed on glass slides. Using IHC, they were stained with CD3 for T cells, F4 / 80 for macrophages, and LY6G for neutrophils. Slides were then subjected to histological evaluation and digital morphometry. Prepared stained slides were scanned with a KF-BIO-40 scanner. The number of stained cells per colonic section was counted using Image Pro Ver. 10. Only the most intensely brown-stained cells were considered positive and were counted using the Smart Segmentation method based on color, background, and morphology. The table below shows the number of positive cells per section for each stain. The method was the same for all three stains. Statistical analysis was performed using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test. A significance level of p<0.05 was considered significant.

[0343] statistical analysis All comparisons were analyzed using the permutation test for two independent samples, which is the most powerful statistical test suitable for small sample sizes. Statistics were calculated using StatXact software (Cytel Inc, Cambridge, MA, USA). Differences were considered statistically significant if p-value was <0.05.

[0344] result To evaluate the anti-inflammatory and / or wound healing properties of anti-AGR2 monoclonal antibodies, four doses of anti-AGR2 monoclonal antibodies were administered intraperitoneally or two doses were administered intravenously in a model of acute colitis induced by dextran sulfate sodium (DSS) in C57Bl / 6 mice.

[0345] mortality rate Mortality in each group of mice was monitored daily. Very low mortality rates were recorded in this study. One mouse died in the group of DSS mice receiving vehicle, one died in the group of mice receiving anti-AGR2 antibody via IP at doses of 5 μg and 20 μg, and two died in the group of DSS mice receiving anti-AGR2 antibody via IP at 40 μg and IV at 20 μg. Three mice died in the group of DSS mice receiving anti-AGR2 antibody via IP at a dose of 10 μg / mouse. The deaths may be due to severe colitis induced by DSS, which results in significant weight loss of up to 20% of initial body weight. However, these results indicate that intraperitoneal or intravenous administration of AGR2 every two days is safe even under inflammatory conditions.

[0346] Disease Activity Index (DAI) A significant improvement in DAI scores was observed in all groups of DSS mice administered anti-AGR2 antibody at different test doses by both intraperitoneal (IP) and intravenous (IV) administration (Figure 6A). Anti-AGR2 antibody, administered by both the IP and IV routes, induced a strong improvement in clinical parameters, ranging from 42% to 58%.

[0347] The improvement in DAI score can be explained by analyzing different parameters independently (i.e., change in body weight, stool consistency, and presence of blood in the stool).

[0348] Stool consistency A significant increase in stool consistency score was recorded in vehicle-treated colitic mice, indicating that the mice suffered from severe diarrhea-inducing colitis, exemplified by a score of 1.43 ± 0.17, compared to healthy control mice with no signs of colitis and normal stool consistency (score 0, p = 0.001634) (Figure 6B).

[0349] Significant improvements in stool consistency scores were recorded in all groups of DSS mice administered anti-AGR2 antibody by IV or IP, except for the group of DSS mice administered anti-AGR2 at a dose of 10 μg by IP, in which a significant reduction of 42% in stool consistency score was still observed ( Figure 6B ).

[0350] For all other groups receiving anti-AGR2 antibody via IP, the percentage of improvement ranged from 55% to 68%, demonstrating significant improvements in stool consistency. Higher percentages of improvement were recorded in the groups receiving anti-AGR2 antibody via IV, with a 72% reduction in stool consistency score at the 5 μg / mouse IV dose and a 78% reduction at the 20 μg / mouse IV dose (Figure 6B).

[0351] Presence of blood in the stool In DSS mice administered vehicle, a significant increase in the score for the presence of occult blood in the stool was recorded, indicating that the mice suffered from strong colitis compared to healthy control mice without signs of colitis (scores of 0.79 ± 0.11 vs. 0.00 ± 0.00, respectively, p = 0.01144) (Figure 6C).

[0352] A significant reduction in the presence of occult blood in the feces was recorded in the groups of DSS mice that received anti-AGR2 antibody by IP at a dose of 10 μg / mouse (0.33 ± 0.14 vs. 0.79 ± 0.11, p = 0.04474) and 40 μg / mouse (0.23 ± 0.12 vs. 0.79 ± 0.11, p = 0.007) compared to the group of DSS mice that received vehicle (Figure 6C). This corresponds to a mean reduction in the presence of occult blood of 58% at the 10 μg / mouse dose and up to 70% at the 40 μg / mouse IP dose.

[0353] A trend toward a decrease was observed in DSS mice receiving IP anti-AGR2 antibody at doses of 5 and 20 μg / mouse. With IV administration of anti-AGR2 antibody, a trend toward a decrease in the presence of occult blood was observed in DSS mice at doses of 5 μg or 20 μg (41% and 52% decrease, respectively) (Figure 6C).

[0354] These results demonstrated that anti-AGR2 antibody improved clinical parameters of colitis by significantly reducing the presence of occult blood in a dose-dependent manner, with greater efficacy when administered intraperitoneally. Anti-AGR2 antibody also improved stool consistency, but with greater efficacy in DSS-treated mice administered anti-AGR2 antibody via the intravenous route.

[0355] Colon weight / size ratio At the time of euthanasia (day 12), the colons were carefully excised and their sizes were recorded. Then, the luminal contents were removed from the colons before weighing them. Indeed, a decrease in colon size was induced by severe inflammation, and an increase in colon weight was also observed due to edema / inflammatory infiltrates caused by severe inflammation.

[0356] In healthy mice, the average colon size was 8.80 ± 0.17 cm, and the average weight was 163.00 ± 4.06 mg. The colon weight / size ratio was significantly increased in the vehicle-treated DSS mice compared with the healthy control group without colitis: weight / size ratios of 37.30 ± 1.68 vs. 18.56 ± 0.70, respectively, p = 0.00033 (Figure 6D). This result confirmed that colon inflammation remained elevated 7 days after the last DSS administration in the C57BL / 6 genetic background.

[0357] A significant decrease in colon weight / size ratio was observed in the treated groups of mice administered anti-AGR2 antibody by IP at doses of 10, 20, and 40 μg / mouse (FIG. 6D). Compared to DSS mice administered vehicle, IV administration of anti-AGR2 antibody at doses of 5 and 20 μg / mouse induced a significant decrease in colon weight / length ratio (FIG. 6D).

[0358] These results demonstrated a strong anti-inflammatory effect of anti-AGR2 antibodies administered by both IP and IV routes.

[0359] Assessment of colonic inflammation at the histological level Assessment of inflammation and colonic lesions at the histological level was performed according to a validated score for DSS-induced colitis. Results are expressed as mean ± SEM scores. As described above, May-Grünwald-Giemsa-stained sections of colonic tissue were evaluated for inflammation using a multiparametric scoring system (0–18). The system assessed the severity of inflammation (0–3) and extent of inflammation (0–3), the level of regeneration (0–4), crypt damage (0–4), and the percentage of involvement (expansion) (1–4).

[0360] Seven days after the last DSS administration (day 12), persistent and significant colonic inflammation was still recorded at the histological level in the vehicle-treated DSS mice group compared to the healthy control group without colitis (7.79 ± 1.53 vs. 1.00 ± 0.00 p = 0.02843) (Figure 6E).

[0361] In DSS-treated mice receiving anti-AGR2 antibody via IP at a dose of 10 μg / mouse, a significant decrease in histological inflammation was observed compared to DSS-treated mice receiving vehicle alone, with scores of 2.42 ± 0.58 vs. 7.79 ± 1.53, p = 0.0064 (Figure 6E), corresponding to a 69% improvement in inflammatory lesions.

[0362] A similar improvement in inflammation at the histological level was observed in DSS mice administered anti-AGR2 at a dose of 40 μg / mouse by IP (2.77 ± 0.91 vs. 7.79 ± 1.53, p = 0.01272), which corresponded to a 65% reduction in inflammatory lesions (Figure 6E).

[0363] On the other hand, a tendency to reduce inflammation at the histological level was recorded in the groups of DSS mice administered anti-AGR2 antibody by IP at doses of 5 μg / mouse (5.50 ± 1.27 vs. 7.79 ± 1.53, not significant) and 20 μg / mouse (5.21 ± 1.29 vs. 7.79 ± 1.53, not significant) (Figure 6E).

[0364] For anti-AGR2 antibodies administered by the IV route, the lowest tested dose of 5 μg induced a significant decrease in inflammation at the histological level compared to DSS mice receiving vehicle alone (mean score 3.53 ± 1.33 vs. 7.79 ± 1.53, respectively, p = 0.047), corresponding to a 55% reduction in inflammatory lesions (Figure 6E). The highest tested dose of anti-AGR2 antibodies (20 μg / mouse by the IV route) only induced a tendency to decrease the score of inflammatory lesions at the histological level (4.23 ± 1.51 vs. 7.79 ± 1.53, not significant) (Figure 6E).

[0365] In conclusion, anti-AGR2 antibodies can exert strong anti-inflammatory properties at the histological level, and IP administration of the antibodies at a dose of 10 μg / mouse exerted the strongest anti-inflammatory properties.

[0366] Myeloperoxidase levels Myeloperoxidase (MPO) levels are a quantitative marker (an enzyme in polymorphonuclear neutrophil granules) used to assess neutrophil recruitment in tissues and, indirectly, neutrophil-mediated colonic inflammation. Total protein was extracted from defined regions of the entire colon, and cytokine expression was determined using an ELISA method.

[0367] At the stage of inflammatory wound healing / onset of chronic colitis, which occurs 7 days after the last DSS administration, a significant increase in MPO was measured in the colons of vehicle-treated DSS mice compared to vehicle-treated healthy control mice (179.34 ± 43.77 vs. 16.36 ± 2.64, respectively, p = 0.02696) (Figure 6F). The results, expressed as ng MPO / mg total protein extracted from a limited area of ​​the colon, indicate that DSS-induced inflammation was still persistent on day 12 (sacrifice). Significant numbers of inflammatory cell infiltrates were still present in the colons of DSS-treated mice.

[0368] A significant decrease in MPO levels was recorded in DSS-treated mice receiving anti-AGR2 either via IP or IV routes, reaching 66% at a dose of 20 μg / mouse (IP) compared with DSS-treated mice receiving vehicle alone (mean doses 60.31 ± 21.52 vs. 179.34 ± 43.77, respectively, p = 0.01164) (Figure 6F).

[0369] Similar efficacy was noted in the group of DSS-treated mice receiving anti-AGR2 IP at a dose of 5 μg (reduction in MPO mass of 56.42 ± 9.05 vs. 179.34 ± 43.77, respectively, p = 0.006134), corresponding to a 69% reduction in inflammatory cell infiltrates (Figure 6F). With IV administration, a significant 60% reduction in MPO mass was noted in DSS-treated mice compared to DSS mice receiving vehicle alone (71.68 ± 26.97 vs. 179.34 ± 43.77 in ng MPO / mg total protein, p = 0.02628) (Figure 6F).

[0370] This improvement in inflammatory cell infiltrates reached a maximum of 80% in the group of DSS-treated mice that received anti-AGR2 at a dose of 5 μg by IV (34.98 ± 7.44 vs. 179.34 ± 43.77 in ng MPO / mg total protein, p = 0.0009017) (Figure 6F).

[0371] Cytokine and chemokine levels As shown in Figures 7A-D, blocking extracellular AGR2 (eAGR2) using anti-AGR2 antibodies of the present invention down-regulates circulating cytokines and chemokines involved in inflammation, particularly TNFα (Figures 7A and 7C) and IL-6 (Figures 7B and 7D) in a mouse model of DSS-induced acute colitis. This reduction in cytokine and chemokine levels is observed for TNFα and IL-6 at both the systemic level (Figures 7A and 7B) and mRNA level (Figures 7C and 7D).

[0372] immune infiltrate Colon cross sections from 46 mice were subjected to IHC staining for the presence and quantity of T cells (Figure 8B), macrophages (Figure 8A), and neutrophils (Figure 8C) using digital morphometric analysis. As shown in Figure 8B, the naive mouse group (CTL) had significantly lower T cell numbers compared to the other groups, especially the DSS + PBS group (DSS), which showed significantly higher T cell numbers. The anti-inflammatory effect of anti-AGR2 antibody is evidenced by the strong reduction in immune infiltrating cells, namely macrophages (p<0.01) (Figure 8A) and T cells (p<0.001) (Figure 8B), at two anti-AGR2 doses (5 μg and 20 μg, as indicated) administered via the IV route.

[0373] conclusion Taken together, these results demonstrated significant anti-inflammatory properties of anti-AGR2 antibodies administered by intraperitoneal or intravenous routes on clinical parameters, inflammation at the histological and cellular levels.

[0374] Example 8: In vivo efficacy of anti-AGR2 monoclonal antibodies in a chronic DSS model material and method Animal models and colitis induction Animal experiments were performed in accredited facilities at the Institut Pasteur from Lille in accordance with government guidelines. All studies were approved by the local institutional review board (Nord-Pas-de-Calais CEEA No. 75, Lille, France; protocol references 352012 and 19-2009R) and French government agreement No. APAFIS#7542-20 17030609233680). Animals were housed five per cage and provided with standard mouse chow and tap water ad libitum.

[0375] Groups of 20 randomly assigned, 8-week-old male C57Bl / 6 mice were used. Mice were exposed to 2.5% DSS (40 kDa; MP Biomedicals) for 7 days, followed by 7 days of exposure to regular drinking water. The DSS exposure and recovery cycle was repeated three times (i.e., mice received DSS from days 0 to 7, 14 to 21, and 28 to 35). Mice were sacrificed on day 42, 7 days after the third DSS cycle.

[0376] Sixty-five C57B1 / 6 mice were assigned to the following groups, as described in Table 8 below:

[0377] Table 8: Mouse groups for in vivo study of DSS-induced chronic colitis [Table 9] Antibody treatment Table 9: Sequences of anti-AGR2 antibodies used in Example 8 [Table 10]

[0378] Anti-AGR2 monoclonal antibodies were administered intraperitoneally (IP) at a dose of 10 μg per mouse every 2 days either (1) for prophylactic treatment starting on day 0 and until euthanasia on day 42, or (2) for therapeutic treatment starting on day 18 and until euthanasia on day 42.

[0379] Laboratory tests Mortality and clinical signs The effects of anti-AGR2 antibodies administered by the intraperitoneal route were recorded for daily mortality and bi-day body weight change over the course of the study (days 0 to 42) and compared to the effects recorded in DSS mice receiving vehicle alone.

[0380] Disease Activity Index (DAI) DAI was assessed as described in Example 7 above.

[0381] Clinical Score: Fibrosis Score At the time of euthanasia, the presence of fibrotic signs (dilatation, thickness, stenosis, and adhesions) was determined. The macroscopic score of fibrosis included four different parameters of the colon: the presence of adhesions, colon thickness, and the presence of colonic dilatation and stenosis. For each of these parameters, a score of 0–3 was assigned (0 → absent, 1 → mild, 2 → moderate, 3 → severe).

[0382] Histological evaluation of colonic lesions Assessment of inflammation levels was performed as described above in Example 7.

[0383] Assessment of gene expression by qRT-PCR Frozen colon samples were homogenized, and mRNA expression of key profibrotic mediators, including TGF-β, α-SMA, Col1A1, and fibronectin, was assessed by quantitative RT-PCR. Briefly, total RNA was extracted using the Nucleospin RNA kit (Macherey-Nagel, Hoerdt, France). After RNAse inactivation, traces of genomic DNA were removed from the total RNA by DNAse treatment and eluted in RNAse-free, DEPC-free water. RNA purity was assessed by UV spectroscopy in a Nanodrop system at 220–350 nm. Quantitative RT-PCR was performed using 1 μg of total RNA using the LightCycler FastStart DNA Master SYBR Green I kit from Roche Diagnostics (Indianapolis, IN) according to the manufacturer's protocol. Primer set sequences and relative NCBI references are listed in Table 10 below. For each reaction, the critical threshold cycle (Ct) value indicates the cycle number at which DNA amplification was determined. Relative gene expression values ​​were calculated as E=2-ΔCt, where ΔCt is the difference in the intersection point between GAPDH and each gene. Table 10: Sequences of primers used for qRT-PCR [Table 11]

[0384] Assessment of colonic fibrosis Collagen deposition was assessed by picrosirius red staining of serial 4-μm sections of each colon sample according to the manufacturer's protocol. Briefly, colon tissues were collected, fixed in fresh 4% paraformaldehyde (PFA) / PBS solution, dehydrated, and paraffin-embedded according to standard methods. After deparaffinization and rehydration, 4-μm sections of dried colon were stained with 0.1% Direct Red Stain (Sigma-Aldrich) / 0.5% Picric Acid (Sigma-Aldrich) for 60 minutes. Slides were analyzed using web-based Image J software. Quantitative analysis of connective tissue deposition was performed using a threshold detection method for grayscale images.

[0385] α-Smooth muscle actin (α-SMA) IHC Alpha-smooth muscle actin (α-SMA) is an actin isoform that predominates in vascular smooth muscle cells and plays a key role in fibrogenesis. To detect myofibroblast activation, colon sections were blocked with 5% bovine serum albumin for 30 minutes, then deparaffinized and rehydrated through graded alcohols to water. Slides were incubated with the primary monoclonal antibody α-SMA (Abcam-ab5694). α-SMA-positive cells were detected by incubating each section with a green-fluorescent Alexa-Fluor-conjugated secondary antibody (AlexaFluor 488, Thermo Fisher Scientific). An irrelevant isotype-matched antibody was used as a control. Nuclei were visualized as blue fluorescence obtained by adding 4',6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) to the mounting medium. α-SMA-positive cells were counted in three randomly selected high-power fields in each section at 20x magnification. Quantitative analysis of the percentage of α-SMA positive cells was performed by using threshold detection method of grayscale images using web-based Image J software.

[0386] Blood and tissue sampling Serum and samples were collected and processed as described in Example 7 above.

[0387] statistical analysis All comparisons were analyzed using a permutation test for two independent samples. Statistics were calculated using StatXact software (Cytel Inc, Cambridge, MA, USA). Differences were considered statistically significant when p-value was <0.05.

[0388] result To evaluate the antifibrotic properties of anti-AGR2 monoclonal antibodies, anti-AGR2 monoclonal antibodies were administered intraperitoneally at a dose of 10 μg / mouse as a prophylactic or therapeutic treatment in a model of chronic colitis induced by dextran sulfate sodium (DSS) in C57Bl / 6 mice.

[0389] mortality rate Mortality in each group of mice was monitored daily. Due to severe weight loss and severe colitis induced by DSS administration, mice died in different groups at the end of the first cycle of DSS. Nine mice died in the group of DSS mice administered vehicle, six died in the group of mice administered anti-AGR2 antibody by IP at a dose of 10 μg in the prophylactic treatment mode, and five died in the group of DSS mice administered anti-AGR2 antibody by IP at a dose of 10 μg in the therapeutic treatment mode. The deaths may be due to the severe colitis induced by DSS, which induces significant weight loss of up to 20% of the initial body weight. No further deaths were recorded after two additional cycles of colitis induction until euthanasia after the end of the first cycle. In this model, animals that survive the first DSS cycle are known to be more "resistant" to subsequent DSS cycles, resulting in greater weight recovery after each new DSS cycle.

[0390] In addition, these results demonstrated that intraperitoneal administration of anti-AGR2 antibodies every 2 days in a prophylactic or therapeutic treatment mode is safe even under inflammatory conditions.

[0391] Disease Activity Index (DAI) DAI scores were recorded on the day of euthanasia on day 42, 7 days after the final DSS administration of the third cycle, which corresponds to the wound healing phase. The DAI scores were significantly increased in the vehicle-treated colitis mice compared with the healthy control group (1.82 ± 0.26 vs. 0.00 ± 0.00, respectively, p = 0.00023) (Figure 9A). This indicated that colitis remained at a persistent level in C57BL6 mice, even though inflammation was less severe after three cycles of DSS administration. The decrease in inflammation was due to the development of fibrosis, a direct result of the repeated inflammation induced by three cycles of DSS.

[0392] A trend toward improvement in DAI scores was observed in all groups of DSS mice intraperitoneally administered with anti-AGR2 antibody in either the prophylactic or therapeutic mode (Figure 9A). Compared to DSS mice administered vehicle alone, a 16% reduction in clinical scores was observed for the prophylactic treatment and a 25% reduction in clinical scores for the therapeutic treatment (Figure 9A). The therapeutic treatment mode of anti-AGR2 antibody at a dose of 10 μg / mouse resulted in the greatest improvement in DAI scores, with a 25% reduction in DAI scores (1.40 ± 0.30 vs. 1.87 ± 0.26, not significant) 7 days after the third cycle of DSS (i.e., day 42 before euthanasia).

[0393] The improvement in DAI score can be explained by analyzing different parameters independently (i.e., change in body weight, stool consistency, and presence of blood in the stool).

[0394] Stool consistency A significant increase in stool consistency scores was recorded in vehicle-treated colitic mice, indicating that the mice suffered from severe diarrhea-inducing colitis, as indicated by a score of 1.00 ± 0.13 vs. 0.00 ± 0.00, p = 0.0014, compared to healthy control mice with no signs of colitis and normal stool consistency (score 0) (Figure 9B).

[0395] A similar trend toward decreased stool consistency scores was recorded in the DSS mice receiving anti-AGR2 antibody in either a preventive or therapeutic mode (Figure 9B). In fact, a decrease of approximately 14% was recorded in the group of DSS mice receiving anti-AGR2 antibody in a preventive mode, and a decrease of 20% was recorded in the group of DSS mice receiving anti-AGR2 in a therapeutic mode (Figure 9B).

[0396] Presence of blood in the stool During the wound healing phase of colitic mice administered vehicle after three cycles of DSS-induced colitis, a moderate but significant increase in the score for the presence of occult blood in the stool was recorded, indicating that the mice had colitis compared to healthy control mice without signs of colitis, with scores of 0.55 ± 0.16 for DSS + vehicle and 0.00 ± 0.00 for control + vehicle, p = 0.057 (Figure 9C).

[0397] When anti-AGR2 antibody was administered in a therapeutic treatment mode, a trend toward a reduction in the presence of occult blood was observed with higher efficacy (Figure 9C). These results indicated that anti-AGR2 antibody moderately improved the clinical parameters of colitis. The lack of a significant effect of anti-AGR2 antibody may be due to the fact that in the model of chronic colitis induced by three cycles of DSS, inflammation levels were lower after the third cycle of colitis induction by DSS, as fibrosis began to establish in the colon of DSS mice.

[0398] Fibrosis Clinical Score Intestinal fibrosis is characterized by shortening and thickening of the colon and adhesions. Measurement of the colon weight / size ratio is also an indicator of the level of inflammation and fibrosis.

[0399] At the time of euthanasia, before removal of the colon, a macroscopic score of fibrosis was assessed. This score included four different parameters of the colon: the presence of adhesions, colon thickness, colonic dilatation, and the presence of strictures. For each of these parameters, a score of 0–3 was assigned (0 = none, 1 = mild, 2 = moderate, 3 = severe).

[0400] Chronic inflammation induced by three cycles of DSS induced significant intestinal fibrosis compared with the group of healthy mice administered vehicle only, with a mean score of 6.18 ± 0.40 vs. 0.00 ± 0.00, p = 0.0002289, respectively (Figure 9D).

[0401] In the group of DSS mice administered anti-AGR2 antibody in a preventive treatment mode, a significant reduction in macroscopic signs of fibrosis was recorded compared to the colitis mice administered the vehicle, with a mean fibrosis score of 3.64 ± 0.40 vs. 6.18 ± 0.040, p = 0.0003318, corresponding to a 41% reduction in the fibrosis score (Figure 9D).

[0402] Similar results were observed in the group of DSS mice treated with anti-AGR2 antibodies in a therapeutic treatment mode: Indeed, a significant reduction in macroscopic signs of fibrosis was observed compared to DSS mice receiving vehicle, with a mean score of 3.13 ± 0.40 vs. 6.18 ± 0.40, respectively, p < 0.0001, corresponding to a 51% reduction in fibrosis score (Figure 9D).

[0403] Colon weight / size ratio Since intestinal fibrosis is characterized by shortening and thickening of the colon and adhesions, measurement of the colon weight / size ratio is another indicator of the level of inflammation and fibrosis.

[0404] At the time of euthanasia, the colons were carefully dissected and their sizes recorded. The luminal contents were then removed from the colons before weighing them. Indeed, a decrease in colon size was induced by severe inflammation, and an increase in colon weight was also observed due to edema / inflammatory infiltrates caused by severe inflammation. In healthy mice, the average colon size was 9.16 ± 0.27 cm and the average weight was 200.60 ± 8.82 mg (Figure 9E).

[0405] The colon weight / size ratio was significantly increased in the vehicle-treated DSS mice compared with the healthy control group without colitis (weight / size ratios of 54.25 ± 2.76 vs. 21.87 ± 0.49, respectively, p = 0.0002289) (Figure 9E). This result confirmed that colon inflammation remained elevated 7 days after the last DSS administration in the C57BL / 6 genetic background and that fibrosis was present after repeated cycles of DSS-induced inflammation.

[0406] In mice in both treatment groups that received anti-AGR2 antibody at a dose of 10 μg / mouse in either the prophylactic or therapeutic treatment modes, a significant decrease in colon weight / size ratio was observed compared to colitic mice that received vehicle alone (Figure 9E).

[0407] This result confirmed that anti-AGR2 antibodies have not only anti-inflammatory but also anti-fibrotic properties.

[0408] Assessment of colonic inflammation at the histological level Assessment of inflammation and colonic lesions at the histological level was performed according to a validated score for DSS-induced colitis. Results are expressed as mean ± SEM scores. As described above, May-Grünwald-Giemsa-stained sections of colonic tissue were evaluated for inflammation using a multiparametric scoring system (0–18). The system assessed the severity (0–1) and extent (0–3) of inflammation, the level of regeneration (0–4), crypt damage (0–4), and the rate of involvement (expansion) (1–4).

[0409] Seven days after the last DSS administration of the third cycle of DSS, persistent and significant colonic inflammation was still recorded at the histological level in the group of DSS mice administered vehicle compared to the healthy control group without colitis (Figure 9F) (11.27 ± 1.28 vs. 1.00 ± 0.00 p = 0.0002289).

[0410] In colitic mice receiving anti-AGR2 antibody at a dose of 10 μg / mouse in the therapeutic treatment mode, a significant decrease in inflammation levels at the histological level was observed compared to colitic mice receiving vehicle alone (scores 7.87 ± 1.16 vs. 11.27 ± 1.28, respectively, p = 0.0035), corresponding to a 30% improvement in inflammatory lesions (Figure 9F).

[0411] No improvement in inflammation at the histological level was observed in DSS mice administered anti-AGR2 at a dose of 10 μg / mouse in a prophylactic treatment mode (10.64 ± 1.16 vs. 11.27 ± 1.28) (Figure 9F).

[0412] In conclusion, anti-AGR2 antibodies administered in a therapeutic mode by IP at a dose of 10 μg / mouse showed strong anti-inflammatory properties at the histological level.

[0413] Histological evaluation of collagen deposition Collagen fiber deposition was assessed in mouse colons by histological staining with picrosirius red. Quantification of collagen deposition was performed using web-based Image J software. Three different fields were analyzed for each section to measure collagen deposition. Data are presented as mean ± sem.

[0414] Collagen deposition levels were significantly increased in the group of colitic mice administered vehicle after three cycles of colitis induction with DSS compared to the respective healthy control groups (226.38 ± 10.67 vs. 62.80 ± 4.18, p = 0.0016) (Figures 9G and 9H).

[0415] A significant reduction in the level of collagen deposition was observed in colitic mice treated with anti-AGR2 prophylactically compared to treatment with anti-AGR2, with greater efficacy (100.21 ± 5.99 vs. 226.38 ± 10.67, p < 0.0001), corresponding to a 56% improvement in fibrosis (Figures 9G and 9H).

[0416] Indeed, a significant decrease in collagen deposition levels in the colon was also recorded in colitic mice therapeutically treated with anti-AGR2 (mean levels 142.87 ± 8.40 vs. 226.38 ± 10.67, p = 0.0003), corresponding to a 37% reduction in collagen deposition ( Figure 9G ).

[0417] Evaluation of fibrosis marker gene expression Gene expression of fibrotic markers (TGF-β, α-SMA, fibronectin, and type I collagen expression) was assessed using qRT-PCR in mouse colon sections. Data are presented as mean ± SEM.

[0418] Regarding α-SMA gene expression, there was a tendency for the levels of this gene to be elevated in DSS mice treated with vehicle compared to control healthy mice treated with vehicle, but the difference was not significant (mean levels 2473.72 ± 590.81 vs. 1713.67 ± 185.85, respectively, not significant). No effect of anti-AGR2 administration in preventive or therapeutic treatment modes was recorded on this marker of fibrosis at the mRNA level (Figure 9I).

[0419] For the other three fibrosis markers tested, i.e., Col1A, TGF-β, and fibronectin, significant increases in gene expression levels at the mRNA level were observed in the colons of vehicle-treated colitis mice compared with healthy control mice after three cycles of DSS (Figures 9J, 9K, and 9L).

[0420] Indeed, significantly increased expression levels were recorded in colitic mice compared to healthy mice, with mean levels of COL1A 3509.06 ± 1895.68 vs. 333.42 ± 45.08, p = 0.0002289 (Figure 9K), TGF-β 107.25 ± 30.76 vs. 22.71 ± 2.73, p = 0.0002289 (Figure 9L), and fibronectin 986.39 ± 238.86 vs. 333.54 ± 50.60, p = 0.0006868 (Figure 9J), respectively.

[0421] In DSS mice that received prophylactic anti-AGR2 antibody, no significant effect on gene expression of the four tested fibrotic markers was measured compared to DSS mice that received vehicle alone (Figures 9I-9L).

[0422] On the other hand, in the colons of DSS mice treated with anti-AGR2 antibody, significant decreases in the gene expression levels of COL1A (Figure 9K) (1041.50 ± 176.83 vs. 3509.06 ± 1895.68, p = 0.02367), TGF-β (Figure 9K) (59.61 ± 8.60 vs. 107.25 ± 30.76, p = 0.0397), and fibronectin (Figure 9J) (519.05 ± 57.47 vs. 986.39 ± 238.86, p = 0.009682) were recorded.

[0423] These results confirmed that prolonged inflammation induces fibrosis and increases the expression of fibrotic markers. Anti-AGR2 antibody administered at a dose of 10 μg by IP in a therapeutic treatment mode was able to exert antifibrotic properties in the colon of DSS mice.

[0424] Evaluation of α-SMA protein expression by IHC staining Fibrosis is characterized by the proliferation of myofibroblasts, and α-SMA is a specific marker of the activation of these cells. The expression of α-SMA in the different groups of treated mice was assessed at the protein level by immunohistochemistry.

[0425] In a model of chronic colitis induced by three cycles of DSS, a significant increase in α-SMA at the protein level was measured in the colons of vehicle-treated DSS mice compared with vehicle-treated control healthy mice, with mean levels of 1.54 ± 0.62 vs. 0.10 ± 0.04, respectively, p = 0.0002289 (Figure 9M).

[0426] In the group of DSS mice receiving anti-AGR2 antibody in a prophylactic treatment mode, a significant decrease in α-SMA protein levels was recorded compared to the colitis mice receiving vehicle, with mean amounts of 0.25 ± 0.09 vs. 1.54 ± 0.62, respectively, p = 0.0005981, corresponding to an 81% decrease in the presence of fibrotic markers (Figure 9M).

[0427] In the colons of colitic mice receiving anti-AGR2 antibodies in a therapeutic treatment mode, a higher efficacy for reducing α-SMA at the protein level was observed compared to colitic mice receiving vehicle, reaching an improvement of up to 99%, with mean α-SMA amounts of 0.09 ± 0.02 vs. 1.54 ± 0.62, respectively, p < 0.0001 (Figure 9M).

[0428] These results confirmed that anti-AGR2 antibody exerted strong antifibrotic properties in a model of chronic colitis induced by three cycles of DSS in C57BL6 mice, with a therapeutic treatment mode exerting a higher antifibrotic effect at a dose of 10 μg.

[0429] conclusion Efficacy was demonstrated at the clinical, inflammatory, fibrotic, histological and molecular levels on day 42, which corresponds to the fibrotic phase of the model.

[0430] Taken together, these results demonstrated important and significant anti-inflammatory, wound healing and anti-fibrotic properties of anti-AGR2 antibodies in a therapeutic treatment mode.

Claims

1. An isolated antibody or binding fragment thereof that specifically binds to anterior gradient protein 2 (AGR2), The following three complementarity determining regions (CDRs): CDR1: RSWMN (SEQ ID NO: 8), CDR2: WIYPGDGDTNYNGKXKD (SEQ ID NO: 9) (wherein X is F or V), CDR3: GGYDGSPWLSY (SEQ ID NO: 12) a heavy chain variable region (VH) comprising: The following three CDRs: CDR1: KASQDINSYLS (SEQ ID NO: 13), CDR2: RANRLVD (SEQ ID NO: 14), and CDR3: LQYDEFPFT (SEQ ID NO: 15) a light chain variable region (VL) comprising 1. An isolated antibody or binding fragment thereof comprising:

2. the antibody or binding fragment thereof a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 16, and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 17, and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 19; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 16, and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 20; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 17, and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 20; or a heavy chain variable region (VH) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 18, and a light chain variable region (VL) comprising framework regions that share at least 80% sequence identity with the framework regions of SEQ ID NO: 21; 2. The isolated antibody or binding fragment thereof of claim 1, comprising:

3. the antibody or binding fragment thereof a heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 16, and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO: 19, or a heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 17, and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO: 19, or a heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 16, and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO: 20, or a heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 17, and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO: 20, or A heavy chain variable region (VH) having a sequence consisting of the sequence of SEQ ID NO: 18, and a light chain variable region (VL) having a sequence consisting of the sequence of SEQ ID NO:

21.

3. The isolated antibody or binding fragment thereof of claim 1 or 2, comprising:

4. The isolated antibody or binding fragment thereof of any one of claims 1 to 3, wherein the antibody or binding fragment thereof is an immunoconjugate.

5. A nucleic acid encoding the antibody or binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid of claim 5.

7. A cell comprising the nucleic acid of claim 5 or the expression vector of claim 6.

8. A pharmaceutical composition comprising an isolated antibody or binding fragment thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5, an expression vector according to claim 6, or a cell according to claim 7, and at least one pharmaceutically acceptable excipient.

9. 9. The isolated antibody or binding fragment thereof of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, the cell of claim 7, or the pharmaceutical composition of claim 8 for use as a medicament.

10. 9. The isolated antibody or binding fragment thereof of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, the cell of claim 7, or the pharmaceutical composition of claim 8 for use in treating a mucosal inflammatory disease or cancer in a subject in need thereof.

11. 11. The isolated antibody or binding fragment thereof of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, the cell of claim 7, or the pharmaceutical composition of claim 8, for use according to claim 10, wherein the isolated antibody or binding fragment thereof neutralizes the pro-inflammatory activity of eAGR2 and / or the pro-fibrotic activity of eAGR2.

12. 12. The isolated antibody or binding fragment thereof of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, the cell of claim 7, or the pharmaceutical composition of claim 8, for use according to claim 10 or 11, wherein the mucosal inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, chronic pancreatitis, microscopic colitis, inflammatory bowel disease (IBD), endometriosis, appendicitis, inflammatory bowel syndrome, idiopathic pulmonary fibrosis, systemic sclerosis, systemic sclerosis associated with interstitial lung disease, asthma, and chronic obstructive pulmonary disease.

13. 12. The isolated antibody or binding fragment thereof of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, the cell of claim 7, or the pharmaceutical composition of claim 8 for use according to claim 10 or 11, wherein the cancer is selected from the group consisting of colon cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, oral cancer, breast cancer, lung cancer, ovarian cancer, thyroid cancer, bile duct cancer, head and neck squamous cell carcinoma, brain glioblastoma, adrenocortical carcinoma, bladder cancer, kidney cancer, penile cancer, renal cancer, testicular cancer, urethral cancer, colorectal cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, gestational trophoblastic disease (GTD), and primary peritoneal cancer.

14. An in vitro method for detecting or quantifying AGR2 expression in a biological sample, comprising contacting the biological sample with an isolated antibody or binding fragment thereof according to any one of claims 1 to 4.

15. The in vitro method of claim 14, wherein the method is for diagnosing or monitoring an AGR2-related disease in a subject, or for selecting a subject suffering from an AGR2-related disease for a treatment that targets the disease.

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