Anti-BTN3A antibody for use in a method of treating gastrointestinal inflammatory disorders

Abstract

BTN3A inhibitory antibodies for use in the treatment of gastrointestinal inflammatory disorders such as inflammatory bowel disease are disclosed. More particularly, the present disclosure relates to specific anti-BTN3A antibodies that specifically bind to BTN3A and inhibit degranulation of Vγ9 / Vδ2 T cells, and their use in the manufacture of novel drugs for use in the treatment of gastrointestinal inflammatory disorders such as ulcerative colitis and Crohn's disease.

Description

Detailed Description of the Invention

[0001] Introduction Gastrointestinal disorders refer to diseases involving the gastrointestinal tract, namely the esophagus, stomach, small intestine, large intestine, and rectum, as well as the accessory organs of digestion, the liver, gallbladder, and pancreas. This term encompasses acute, chronic, recurrent, or functional disorders and covers a wide range of diseases of various origins, namely congenital, environmental, metabolic, functional, central nervous system processing, bacterial, autoimmune, etc. Several large-scale global studies have been conducted, showing that 24 - 40% of people worldwide have gastrointestinal disorders that affect the quality of life and the use and cost in health management (Sperber AD et al., Gastroenterology, January 2021; 160(1): 99 - 114 e3; Mathews C et al., Clin Gastroenterol Hepatol, July 1, 2021: S1542 - 3565(21)00711 - 4).

[0002] Among these gastrointestinal inflammatory disorders, inflammatory bowel disease (IBD) is one of the most influential and major public health problems. IBD is a wasting chronic inflammatory disorder of the gastrointestinal tract with a peak age of onset in adolescence and young adulthood. Inflammatory bowel disease includes two idiopathic GI disorders known as ulcerative colitis (UC) and Crohn's disease (CD). Despite intensive research efforts, the etiology(ies) of the disease are not fully understood. However, both genetic and environmental factors are thought to be involved in the IBD causal relationship, with a breakdown in the regulatory limitations of the mucosal immune response to intestinal bacteria, in other words, an immune (inflammatory) response that is easily induced and / or unduly prolonged, affecting the interaction between the intestinal mucosa and luminal bacteria. Both UC and CD are chronic diseases of the remitting and relapsing kind. Just as the initial cause of IBD is unknown, what brings about remission and relapse is also not clear (Tavakoli P et al., Public Health Rev, 2021, Public Health Rev. May 5, 2021; 42: 1603990).

[0003] The incidence of IBD is increasing globally, and in 2019, the highest prevalence values of IBD were reported in Europe, North America, and Australia (Tavakoli P et al., Public Health Rev, 2021, supra). The incidence of CD has increased by 70%, and the incidence of UC has increased by 60%. An estimated 6 to 8 million people worldwide were affected in 2017.

[0004] UC is characterized by chronic inflammation of the large intestine, accompanied by abnormal activation of the immune system. UC affects the innermost layer of the colon and rectum. CD can affect any level of the intestinal tract from the mouth to the anus, extending through all layers of the intestinal wall, but most affects the lower part of the small intestine (ileum) and the colon. The most common symptoms of IBD include diarrhea, rectal bleeding, intermittent nausea and vomiting, and abdominal pain or tenderness (Baumgart DC and Sandborn, WJ, The Lancet, May 12, 2007; 369(9573):1641 - 57; Strober, W, Fuss, I and Mannon, P, J Clin Invest, March 2007; 117(3):514 - 21). The symptoms are due to damage to the intestine resulting from an excessive inflammatory response. Complications of these immune-mediated diseases include anemia, malnutrition, intestinal obstruction, fistulas, infections, and an increased risk of colon cancer. Extraintestinal manifestations can also develop problems in joints (arthralgia, arthritis and ankylosing spondylitis), rashes and skin conditions (erythema nodosum, psoriasis), chronic liver disease (primary sclerosing cholangitis) and eye conditions (uveitis, etc.). Clinical management focuses on maintaining patients in remission and asymptomatic, with the first goal of reducing inflammation during relapses and the second goal of prolonging the time to endoscopic remission and mucosal healing, but currently, there is no cure for IBD patients. Current management options are based on various treatment categories: (i) anti-inflammatory drugs such as aminosalicylates or steroids, (ii) immunosuppressants such as azathioprine, cyclosporine, JAK inhibitors or (iii) biologics (anti-TNFa, anti-IL-12 / 23, anti-a4b7 monoclonal antibodies). Despite improvements in health management, IBD still strongly affects the lifespan of patients, and recent estimates indicate that IBD reduces life expectancy by 6.6 - 8.1 years in women and 5.0 - 6.1 years in men (Kuenzig Me et al., CMAJ, November 9, 2020; 192(45):E1394 - E1402). Thus, there are still significant, unmet medical needs (Danese S et al., Dig Dis, 2019, 37(4):266 - 83).

[0005] In humans and non-human primates (NHPs), the major peripheral γδ T cell subset expresses a T cell receptor (TCR) composed of Vγ9 and Vδ2 chains. This Vγ9Vδ2 T cell subset represents approximately 5% of CD3+ cells in peripheral blood and more than 80% of peripheral γδ T cells in healthy adults (Bonneville M et al., Nat Rev Immunol, 2010; Poggi A and Zocchi MR, Front Immunol, 2014). In vitro studies have shown that Vγ9Vδ2 T cells have the ability to detect stress signals from infected and malignant cells associated with the intracellular accumulation of organic pyrophosphate-containing molecules called phosphoantigens (pAgs). The identification of pAgs as potent and specific activators of Vγ9Vδ2 T cells has led to the understanding that BTN3A plays an essential role in the antigen activation of Vγ9Vδ2 T cells, which was an important advance in the understanding of Vγ9Vδ2 T cell biology. BTN3A is a member of the butyrophilin family of type I transmembrane proteins and also belongs to the Ig superfamily. Three isoforms of BTN3A have been described (BTN3A1, BTN3A2, and BTN3A3), and these isoforms are distinguished by differences in their intracellular domains (e.g., the presence or absence of the B30.2 / SPRY domain) and a few amino acids within their extracellular domains. Intracellular accumulation of pAgs induces a conformational change in BTN3A1 through the interaction of pAgs with the intracellular B30.2 domain. This interaction results in the specific recognition of BTN3A1 by Vγ9Vδ2 T cells, which are subsequently activated (Gu S et al., Semin Cell Dev Biol, December 2018; 84:65 - 74). BTN3A1 is the only molecule that can transmit the signal of intracellular pAg accumulation to the cell surface and induce Vγ9Vδ2 T cell recognition and activation (Gu S et al., Semin Cell Dev Biol, 2018, supra).

[0006] BTN3A expression is limited to humans and non-human primates (NHPs). Furthermore, BTN3A orthologs are not expressed in rodents, which also lack Vγ9Vδ2 T cells and are therefore not suitable for testing BTN3A / Vγ9Vδ2-based therapies.

[0007] Previous studies by the McCarthy laboratory confirmed that the human intestine contains two distinct subsets of Vδ2 T cells identified by differences in the expression of the "tissue residency" marker CD103. In healthy colon, C103+ Vδ2 T cells, which show only a weak cytokine response to microbial PAg, mostly aggregate in the mucosa, whereas the colon from patients with Crohn's disease (CD) is instead dominated by CD103-Vδ2 T cells that show enhanced inflammatory cytokine production upon PAg exposure in vitro. Furthermore, analysis of blood Vδ2 T cells from CD patients revealed that their frequency and gut-homing ability in circulation are associated with differences in the expression of TRM markers including CD69 and CD27, suggesting that cell mobilization / retention in the gut may play a major role in shaping mucosal Vδ2 T cell activity. Also, other groups have shown that circulating γδ T cells are more abundant in active IBD (CD and UC) and gut biopsies, particularly Vd2 T cells in biopsies from late-stage compared to early-stage IBD patients, and that these Vδ2 T cells produce more INFδ, TNFα, and IL-17 (Giacomelli R et al., Clin Exp Immunol, 1994; McCarthy NE et al., J Clin Invest, 2015; McCarthy Ne et al., J Immunol, 2013; Markovits N et al., Inflammopharmacology, 2017; Lo Presti E et al., J Crohns Colitis, 2019). Thus, targeting the Vδ2 population by blocking its function through anti-BTN3A monoclonal antibodies could be a potential therapeutic goal. There are currently no γδ-targeted therapies under development for IBD, and the medical unmet needs remain high.

[0008] International Publication No. WO 2012 / 080769 describes a specific murine monoclonal antibody called mAb103.2 that has the ability to inhibit the cytolytic function, production, and proliferation of Vγ9Vδ2 T cells. As a result, it was suggested that such murine antibody mAb103.2, as well as its corresponding chimeric and humanized forms or fragments thereof, may be potentially useful in the treatment of inflammatory disorders.

[0009] International Publication No. WO 2020 / 136218 further reports that the Fab fragment of mAb103.2 exhibits activation properties with the ability to activate the cytolytic function, production, and proliferation of Vγ9Vδ2 T cells.

[0010] The present invention now relies on the discovery that a particular humanized anti-BTN3A1 antibody can potently inhibit Vγ9Vδ2 T cell function in vitro in healthy donors, ex vivo in IBD patients, and in vivo in a cynomolgus animal model of gastroenteritis, and thus can be advantageously used for the treatment of gastrointestinal inflammatory diseases such as IBD.

Summary of the Invention

[0011] The present disclosure relates to an isolated anti-BTN3A antibody for use in the treatment of gastrointestinal inflammatory disorders such as inflammatory bowel disease in a human subject in need thereof, wherein the anti-BTN3A antibody specifically binds to BTN3A1 and the anti-BTN3A antibody inhibits degranulation of γδ T cells in vitro with an IC50 of 10 nM or less, preferably 1 nM or less, as determined, for example, by a CD107 degranulation assay by flow cytometry in co-culture with the Daudi Burkitt lymphoma cell line, and is selected from among anti-BTN3A antibodies that inhibit degranulation of γδ T cells in vitro.

[0012] The present disclosure also relates to a method for treating gastrointestinal inflammatory disorders, such as inflammatory bowel disease, in a human subject in need of treatment for a gastrointestinal inflammatory disorder, the method comprising administering to the subject a therapeutically effective amount of an isolated anti-BTN3A antibody, wherein the anti-BTN3A antibody specifically binds to BTN3A1 and the anti-BTN3A antibody inhibits degranulation of γδ T cells in vitro with an IC50 of 10 nM or less, preferably 1 nM or less, as determined, for example, by a CD107 degranulation assay by flow cytometry, in co-culture with the Daudi Burkitt lymphoma cell line, and is selected from anti-BTN3A antibodies that inhibit degranulation of γδ T cells in vitro.

[0013] The present disclosure further relates to the use of an isolated anti-BTN3A antibody in a method for preparing a medicament for treating gastrointestinal inflammatory disorders, such as inflammatory bowel disease, in a human subject in need of treatment for a gastrointestinal inflammatory disorder, wherein the anti-BTN3A antibody specifically binds to BTN3A1 and the anti-BTN3A antibody inhibits degranulation of γδ T cells in vitro with an IC50 of 10 nM or less, preferably 1 nM or less, as determined, for example, by a CD107 degranulation assay by flow cytometry, in co-culture with the Daudi Burkitt lymphoma cell line, and is selected from anti-BTN3A antibodies that inhibit degranulation of γδ T cells in vitro.

[0014] In certain embodiments of the anti-BTN3A antibodies and methods of use thereof described above, the antibody is (i) antibody mAb1 having a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 22, (ii) a variant of mAb1 having a heavy chain variable region (VH) of SEQ ID NO: 7 and a light chain variable region (VL) of SEQ ID NO: 8 but having a different constant region, (iii) a variant of mAb1 having HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6 but having a different framework region, or (iv) A variant of mAb1 that binds to the same epitope as mAb1, said epitope comprising or consisting essentially of SEQ ID NO:20, more preferably binding to at least residues 53, 62, 66 of BTN3A1, and said variant does not have an HCDR1 of SEQ ID NO:1, an HCDR2 of SEQ ID NO:2, an HCDR3 of SEQ ID NO:3, an LCDR1 of SEQ ID NO:4, an LCDR2 of SEQ ID NO:23 and an LCDR3 of SEQ ID NO:6. is selected from the group consisting of:

[0015] In a more specific embodiment, the selected isolated anti-BTN3A antibody as disclosed herein is a variant of mAb1 having an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6, wherein the VH amino acid sequence has at least 90% but less than 100% identity, preferably at least 95% identity, to SEQ ID NO: 7, and the VL amino acid sequence has at least 90% but less than 100% identity, preferably at least 95% identity, to SEQ ID NO: 8.

[0016] In certain embodiments, an isolated anti-BTN3A antibody for use in the present disclosure has a K of 10 nM or less as measured by surface plasmon resonance. D , preferably 1 nM or less K D , typically measured by surface plasmon resonance (SPR) assays, 1.10 -11 ~1.10 -9 K between M D and binds to human BTN3A1 isoform and / or binds to human peripheral blood mononuclear cells (PBMCs) with an EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, for example an EC50 of between 0.1 μg / mL and 0.005 μg / mL, such as about 0.02 μg / mL.

[0017] In certain embodiments, the isolated anti-BTN3A antibody for use in the present disclosure is a functional variant of mAb1 that retains at least a substantial proportion, preferably at least 90% of the affinity of mAb1 as measured by SPR assay, and has the following characteristics: (i) The property of inhibiting in vitro degranulation of γδ T cells with an EC50 of 10 nM or less, preferably 1 nM or less, as determined by, for example, the CD107 degranulation assay by flow cytometry in co-culture with the Daudi Burkitt lymphoma cell line; (ii) The property of substantially inhibiting phosphoantigen mediated activation, gut homing ability, proliferation and degranulation ability of patient-derived peripheral Vδ2+ T cells, as determined by, for example, an ex vivo assay of peripheral blood mononuclear cells (PBMCs) isolated from patients suffering from inflammatory bowel disease; and / or (iii) The property of substantially inhibiting phosphoantigen mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of patients with inflammatory bowel disease, as determined by, for example, CD25 or HLA-DR expression in an ex vivo assay in walked out cells having at least one or more of the above.

[0018] In certain embodiments, the isolated anti-BTN3A antibody for use in the present disclosure is a human or humanized antibody.

[0019] In certain embodiments, the isolated anti-BTN3A antibody for use in the present disclosure comprises a mutated or chemically modified IgG1 constant region, and the mutated or chemically modified IgG1 constant region does not confer or reduces binding to Fcγ receptors and / or ADCC mediating activity when compared to the corresponding antibody having wild-type IgG1, for example, a mutated or chemically modified IgG1 constant region having the following amino acid substitutions, L247F L248E and P350S.

[0020] In certain embodiments, the gastric inflammatory disorder is an inflammatory bowel disease, such as ulcerative colitis or Crohn's disease.

[0021] Typically, an isolated anti-BTN3A antibody for use in the present disclosure as described herein is administered intravenously to a subject at a dose of 1 to 100 mg.

[0022] In certain embodiments, an isolated anti-BTN3A antibody for use in the present disclosure is administered simultaneously or separately in combination with an anti-inflammatory treatment preferably selected from anti-cytokine antibodies (anti-IL-12, anti-IL-23, anti-TNFα), anti-α4β7 integrin antibodies, and JAK inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

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[0024] For a better understanding of the present disclosure, certain terms are first defined. Further definitions are provided throughout the detailed description. Definition

[0025] ​As used herein, the term "BTN3A" has its ordinary meaning in the art, and refers to a human BTN3A polypeptide, including any of BTN3A1 of SEQ ID NO: 17, BTN3A2 of SEQ ID NO: 18, or BTN3A3 of SEQ ID NO: 19, unless otherwise specified.

[0026] The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer broadly to polymers of amino acid residues of any length, regardless of modification (e.g., phosphorylation or glycosylation). The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. A polypeptide can be modified, for example, by the addition of carbohydrate residues, to become a glycoprotein. The terms "polypeptide", "peptide" and "protein" clearly include glycoproteins and non-glycoproteins. In certain embodiments, the terms "polypeptide" and "protein" refer to any polypeptide or protein that can be encoded by a gene and translated by recombinant means using a cell expression system such as a mammalian host cell, and include any polypeptide having post-translational or chemical modifications of the amino acid polymer.

[0027] The term "recombinant protein" as used herein includes proteins prepared, expressed, produced or isolated by recombinant means, for example, (a) antibodies isolated from hybridomas (further described below), (b) antibodies isolated from production cell lines transfected to express the corresponding heavy and light chains of said antibodies, such as transfectomas.

[0028] The term "antibody" as used herein refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an antigen.

[0029] In the natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity for antigens. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).

[0030] The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigen determinant. The antibody binding site is mainly composed of residues derived from the hypervariable regions or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) may participate in the antibody binding site or affect the overall domain structure and thus the binding site. The complementarity-determining region or CDR refers to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of the natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, the antigen binding site typically contains six CDRs, including a set of CDRs derived from each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequences sandwiched between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0031] The residues of the antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is shown in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter "Kabat et al.") by Kabat et al. in 1987. This numbering system is used herein. The Kabat residue designations do not necessarily directly correspond to the linear numbering of the amino acid residues of the sequences of SEQ ID NOs. The actual linear amino acid sequence may contain fewer or additional amino acids relative to the strict Kabat numbering to account for deletions, insertions of components in either the framework regions or complementarity determining regions (CDRs) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues in the antibody's sequence with a "standard" Kabat-numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0032] “K assoc ” or “K a ” when used herein is intended to refer to the association rate of a particular antibody-antigen interaction, while the term “K dis ” or “K d ” when used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

[0033] “K D ” when used herein refers to the ratio of K d to K a (i.e., K d / K a is intended to refer to the dissociation constant, obtained from (2) and expressed as a molar concentration (M). The K D value of an antibody can be determined using methods well established in the art. Methods for determining the K D of a protein or antibody include, for example, using a biosensor system such as the Biacore® system, by using surface plasmon resonance. A simple binding interaction analysis by surface plasmon resonance (SPR) requires immobilization of a ligand on the surface of a sensor chip and subsequent addition of the analyte of interest to the buffer flowing out from the ligand surface. The interaction between the ligand and the analyte is measured by an SPR instrument (typically the Biacore® system) as a change in refractive index over time. From this, the association (K a ) or dissociation (K d ) and equilibrium dissociation (K D ) constants can be obtained.

[0034] The terms "anti-BTN3A antibody" or "BTN3A antibody", as used herein, refer to an antibody having binding specificity for BTN3A.

[0035] As used herein, the term "binding specificity" means that an antibody has the ability to detectably bind to an antigen recombinant polypeptide such as a recombinant BTN3A1 polypeptide with a K D of 100 nM or less, 10 nM or less, 1 nM or less, as measured by surface plasmon resonance (SPR) measurement, as determined, for example, in the examples (Table 3). In some embodiments, the antibody has a K -3 between 10 -3 pM and 100 nM, particularly between 10 pM and 100 nM, particularly between 10 pM and 100 nM, or between 10 D pM and 10 nM, particularly between 1 pM and 10 nM, particularly between 10 pM and 10 nM, or between 1 pM and 5 nM, particularly between 10 pM and 5 nM or between 100 pM and 5 nM, as measured by SPR, and binds to the human BTN3A1 isoform.

[0036] An antibody that "cross-reacts with an antigen other than BTN3A" is intended to mean an antibody that binds to an antigen other than human BTN3A with a K of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody that "does not cross-react with a specific antigen" is an antibody that binds to that antigen with a K of 100 nM or more, or 1 μM or more, or 10 μM or more, and said affinity is measured, as disclosed in the examples, using, for example, similar surface plasmon resonance (SPR) measurements. In certain embodiments, such antibodies exhibit essentially undetectable binding to these proteins in a standard binding assay. D An antibody that "cross-reacts with an antigen other than BTN3A" is intended to mean an antibody that binds to an antigen other than human BTN3A with a K of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody that "does not cross-react with a specific antigen" is an antibody that binds to that antigen with a K of 100 nM or more, or 1 μM or more, or 10 μM or more, and said affinity is measured, as disclosed in the examples, using, for example, similar surface plasmon resonance (SPR) measurements. In certain embodiments, such antibodies exhibit essentially undetectable binding to these proteins in a standard binding assay. D or a K of 1 μM or more, D or a K of 10 μM or more D and said affinity is measured, as disclosed in the examples, using, for example, similar surface plasmon resonance (SPR) measurements. In certain embodiments, such antibodies exhibit essentially undetectable binding to these proteins in a standard binding assay.

[0037] Anti-BTN3A antibodies may have cross-reactivity to other antigens, such as related BTN3A molecules from other species, typically cynomolgus monkey BTN3A1. Furthermore, isolated anti-BTN3A antibodies may be substantially free of other cellular materials and / or chemicals.

[0038] The phrases "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

[0039] Specificity can be further demonstrated, for example, by the ratio of affinity / binding activity in the binding of a specific antigen (in this case, the specific antigen is the BTN3A polypeptide) to non-specific binding to other irrelevant molecules, such as about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more. The term "affinity", as used herein, means the strength of binding of an antibody to an epitope. Affinity is typically evaluated by the K D value of the antibody for BTN3A1.

[0040] As used herein, the term "humanized antibody" is used in a broad sense to include recombinant antibodies produced by non-natural cells, such as producer cell lines, having variable and constant regions that have been altered to resemble those of antibodies made by human cells. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies for use in the present disclosure may include, for example, in the CDRs, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or by somatic mutations in vivo).

[0041] In certain embodiments, the term "humanized antibody" also includes, as used herein, antibodies in which the CDR sequences from the germline of another mammalian species, such as a mouse, have been transplanted into a human framework sequence.

[0042] In other specific embodiments, the term "humanized antibody" also includes, as used herein, antibodies in which the H-CDR1 of SEQ ID NO: 1, the H-CDR2 of SEQ ID NO: 2, the H-CDR3 of SEQ ID NO: 3, the L-CDR1 of SEQ ID NO: 4, the L-CDR2 of SEQ ID NO: 5, and the L-CDR3 of SEQ ID NO: 6 have been transplanted into a human framework sequence.

[0043] As used herein, the term "inhibitory antibody" refers to an antibody that can directly or indirectly inhibit the immune functions of effector cells, such as inhibiting immune regulatory responses including proliferation and expansion, production of inflammatory molecules, cytolytic function against stressed cells, migration and trafficking properties, and / or antigen presentation. In a preferred embodiment, the inhibitory BTN3A antibody has an IC, preferably 10 nM or less, more preferably 1 nM or less, such as 0.1 nM or less, as determined by flow cytometry in a CD107 degranulation assay, at least in co-culture with cancer cells (such as the Daudi Burkitt lymphoma cell line, but other transformed or damaged cells may also be used), as described in the examples below.50 and has the ability to inhibit the degranulation of γδ T cells (typically Vγ9Vδ2 T cells) in vitro. IC 50 (50% inhibitory concentration) can be established in a dose-response curve and indicates the concentration of an inhibitory BTN3A antibody at which 50% of the maximum inhibitory effect (i.e., maximum inhibition of activated Vγ9Vδ2 T cells) is observed. As shown in the examples, the evaluation of Vγ9Vδ2 T cell cytotoxicity is performed by flow cytometry by evaluating the expression of CD107 molecules on the membrane surface of Vγ9Vδ2 T cells. The dose-response curve is typically established by quantifying CD107-positive Vγ9Vδ2 T cells after 4 hours of co-culture with Daudi cells in the presence of the antibody fragment at 37°C. In some embodiments, IC 50 is between 10 -4 nM and 10 nM, particularly between 10 -4 nM and 1 nM, particularly between 10 -4 nM and 0.1 nM, or between 10 -3 nM and 10 nM, between 10 -3 nM and 1 nM or between 10 -3 nM and 0.1 nM.

[0044] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc., including mammals and non-mammals.

[0045] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % identity = number of identical positions / total number of positions × 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm as described below.

[0046] The percent identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (NEEDLEMAN and Wunsch).

[0047] The percent identity between two nucleotide or amino acid sequences can also be determined using an algorithm such as, for example, EMBOSS Needle (pairwise alignment; www.ebi.au.uk available therein). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extension penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10, and an “end gap extension penalty” of 0.5. Generally, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For example, after alignment, if 6 out of 10 sequence positions are identical between two compared sequences, the identity is 60%. The % identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical regardless of any chemical and / or biological modifications.

[0048] Inhibitory BTN3A antibodies for use in the present disclosure The present disclosure relates to inhibitory BTN3A antibodies for use in the treatment of gastrointestinal inflammatory disorders, such as IBD, in a human subject in need thereof, wherein said inhibitory BTN3A antibody specifically binds to BTN3A and inhibits the cytolytic function of γδ T cells as determined in an in vitro degranulation assay; for example, a CD107 degranulation assay in the presence of Daudi cells as described in the Examples.

[0049] In certain embodiments, the inhibitory BTN3A antibody has a K D of 10 nM or less, preferably 1 nM or less, as measured by surface plasmon resonance, typically measured by a surface plasmon resonance (SPR) assay, of 1.10 D -11 ~1.10 -9 ​It binds to human BTN3A1 isoforms among M and / or binds to human peripheral blood mononuclear cells (PBMCs) with an EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, for example, an EC50 of about 0.02 μg / mL.

[0050] Preferably, the inhibitory BTN3A antibody for use in the present disclosure is a chimeric, humanized or human antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while having at least the same or similar affinity (or better affinity) as the parental non-human antibody.

[0051] Generally, a humanized antibody optionally has one amino acid substitution in one CDR (or a portion thereof) to reduce immunogenicity, such as in L-CDR2, for example, L-CDR2 of SEQ ID NO: 5 in which the isoleucine of the mouse L-CDR2 of SEQ ID NO: 24 is substituted with alanine, derived from a non-human antibody, such as mouse mAb103.2, and the FR (or a portion thereof) is derived from a mouse antibody sequence having mutations to reduce immunogenicity, and includes one or more variable domains.

[0052] The humanized antibody for use in the present disclosure also optionally includes at least a portion or all of the human constant region.

[0053] In certain embodiments, the inhibitory BTN3A antibody for use in the present disclosure includes an IgG Fc region, preferably a mutated or chemically modified IgG1 or IgG4 constant region, where the mutated or chemically modified IgG1 or IgG4 constant region does not confer or reduces binding to Fcγ receptors and / or ADCC-mediated activity as compared to a corresponding antibody having a wild-type IgG1 or IgG4 isotype constant region, such as a mutated IgG1 constant region having the following amino acid substitutions: L247F L248E P350S.

[0054] In certain embodiments, the inhibitory BTN3A antibody is further selected from BTN3A antibodies that inhibit phosphoantigen-mediated activation, gut homing ability, proliferation, and degranulation ability of peripheral Vδ2+ T cells, as measured in an ex vivo assay with PBMC isolated from, for example, patients suffering from IBD. Protocols for such ex vivo assays are described in more detail by way of example in the Examples. Briefly, inhibition of phosphoantigen-mediated ex vivo activation of peripheral Vδ2+ T cells can be determined by an ex vivo assay consisting of culturing PBMC isolated from the blood or gut biopsies of, for example, Crohn's disease or ulcerative colitis patients, and staining with 1 μM CTV dye for 4 days in the presence of (i) IL2 (20 IU / ml) / IL5 (20 ng / ml), (ii) 1 nM to 10 nM HDMAPP (phosphoantigen), and (iii) 1 μg / mL of said inhibitory BTN3A or control isotype. Activation can be determined by measuring several markers such as CD25, HLA-DR, b7 integrin, or CD49b. Proliferation can be determined by flow cytometry by measuring CTV dilution for Vδ2+ and Vδ2- T cells, and degranulation can be determined by determining CD107a expression by flow cytometry.

[0055] In certain embodiments, the inhibitory BTN3A antibody is further selected from BTN3A antibodies that substantially inhibit phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from gut biopsies of IBD patients, as determined by CD25 or HLA-DR expression in an ex vivo assay with walkout cells as described in the Examples below.

[0056] In preferred embodiments, the inhibitory BTN3A antibody also cross-reacts with cynomolgus BTN3A.

[0057] Reference antibody for use in the treatment of gastrointestinal inflammatory disorders In a preferred embodiment, the inhibitory BTN3A antibody for use in the treatment of gastrointestinal inflammatory disorders, such as IBD, in a human subject in need of treatment of a gastrointestinal inflammatory disorder is the mAb1 antibody having the full-length heavy chain of SEQ ID NO: 21 and the full-length light chain of SEQ ID NO: 22.

[0058] In other specific embodiments, the inhibitory BTN3A antibody for use in the treatment of gastrointestinal inflammatory disorders, such as IBD, in a human subject in need of treatment of a gastrointestinal inflammatory disorder is a variant of the mAb1 antibody having the heavy chain variable region (VH) of SEQ ID NO: 7 and the light chain variable region (VL) of SEQ ID NO: 8 but having a different constant region. In this embodiment, the variant of mAb1 has a variant constant region compared to mAb1, for example, a different isotype, or an isotype that is the same as mAb1 but has one or more amino acid mutations, compared to the corresponding Fc region of mAb1.

[0059] As used herein, a variant antibody is an antibody having a distinct primary amino acid sequence compared to a reference antibody, mAb1, for example, by one or more amino acid additions, deletions, and / or substitutions. Preferably, the variant antibody is an antibody having a distinct primary amino acid sequence of the heavy chain or light chain by one or more amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the heavy chain and / or light chain of mAb1.

[0060] In certain embodiments, the variant of mAb1 has a variant constant region compared to mAb1, for example, a different isotype than mAb1, or an isotype that is the same as mAb1 but has one or more amino acid mutations compared to mAb1, for example, having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the Fc region compared to mAb1.

[0061] In other specific embodiments, said inhibitory BTN3A antibody for use in the treatment of gastrointestinal inflammatory disorders such as IBD in a human subject is a variant of mAb1 having HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 but having a different framework region as compared to mAb1.

[0062] The CDR regions of the inhibitory BTN3A antibodies of the present disclosure are depicted using Kabat numbering (Kabat et al., 1992, hereinafter "Kabat et al."). For ease of understanding, H-CDR1, H-CDR2, and H-CDR3 refer to the three CDRs of the VH region, and L-CDR1, L-CDR2, and L-CDR3 refer to the three CDRs of the VL region.

[0063] In other specific embodiments, said inhibitory BTN3A antibody for use of the present disclosure is a variant of mAb1 antibody having HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, wherein the VH region has at least 90% homology, preferably at least 95% homology, to SEQ ID NO: 7, and the VL region has at least 90% homology, preferably at least 95% homology, to SEQ ID NO: 8. Preferably, said inhibitory BTN3A antibody is: (i) the heavy chain of an IgG antibody having VH of SEQ ID NO: 7, and (ii) the light chain of an IgG antibody having VL of SEQ ID NO: 8 and is a humanized BTN3A antibody.

[0064] In certain embodiments, new BTN3A antibodies can be created respectively using the inhibitory BTN3A antibodies of the present disclosure having VH and VL sequences by modifying the VH and / or VL sequences, or the constant regions associated therewith.

[0065] Thus, in another aspect of at least some embodiments of the present disclosure, structural features of inhibitory BTN3A antibodies are used to create structurally related BTN3A antibodies that retain the inhibitory properties of at least the reference inhibitory BTN3A antibody mAb1.

[0066] For example, six CDR regions of mAb1 or an antibody having H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, and H-CDR3 of SEQ ID NO: 3; L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6 can be recombinantly combined with known framework regions to create inhibitory BTN3A antibodies made by further recombinant techniques of at least some embodiments of the present disclosure as described above. The starting materials for the method of operation are one or more of the VH and / or VL sequences having SEQ ID NO: 7 and SEQ ID NO: 8.

[0067] The altered functional properties of the antibody can be evaluated using assays available in the art and / or described herein. In certain embodiments of methods of engineering antibodies according to at least some embodiments of the present disclosure, mutations can be introduced randomly or selectively along all or part of the inhibitory BTN3A antibody coding sequence, and the resulting modified inhibitory BTN3A antibodies can be screened for binding activity to peripheral Vδ2+ T cells isolated from patients suffering from IBD and / or other desirable functional properties such as in vitro inhibitory properties in a degranulation assay, or ex vivo activity, gut homing ability, inhibition of proliferation and degranulation ability (as described in the previous section).

[0068] Methods of mutagenesis are described in the art. For example, International Publication No. WO 02 / 092780, a PCT publication by Short, describes methods for creating and screening antibody variants using saturation mutagenesis, synthetic ligation assembly, or combinations thereof. Alternatively, International Publication No. WO 03 / 074679, a PCT publication by Lazar et al., describes methods for optimizing the physiochemical properties of antibodies using computer screening methods.

[0069] Isotype and Fc engineering In a preferred embodiment, inhibitory BTN3A antibodies for use in the present disclosure include the constant region of an immunoglobulin, preferably an IgG antibody.

[0070] As used herein, the terms "constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain and include the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined to include the amino acid residues from position C226 or P230 to the carboxyl-terminal of the IgG antibody, where the numbering follows the EU numbering system. The C-terminal lysine (K447 residue) of the Fc region may be removed, for example, during antibody production or purification, or its corresponding codon may be deleted in a recombinant construct. Thus, antibody compositions of the present disclosure may include an antibody population in which all K447 residues have been removed, an antibody population in which no K447 residues have been removed, and an antibody population having a mixture of antibodies with and without the K447 residue.

[0071] The constant region of an inhibitory BTN3A antibody for use in the present disclosure can be of any isotype. The choice of isotype is typically guided by the desired effector function, such as ADCC silencing. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human kappa or lambda light chain constant region can be used. If desired, the class of the antibodies of the present disclosure can be switched by known methods. Using typical class switch techniques, one IgG subclass can be converted to another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the present disclosure can be varied for various therapeutic uses by, for example, isotype switching to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In some embodiments, the antibodies of the present disclosure are full-length antibodies. In some embodiments, the full-length antibodies are IgG1 antibodies.

[0072] Inhibitory BTN3A antibodies for use in the present disclosure are typically engineered, typically by amino acid substitution, to contain modifications within the Fc region so as to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity activity, and more particularly, modifications of the Fc region of the IgG1 isotype.

[0073] In still other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be substituted with different amino acid residues such that the antibody has an altered affinity for an effector ligand while retaining the antigen-binding ability of the parental antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both to Winter et al.

[0074] In another embodiment, one or more amino acids selected from amino acid residues can be substituted with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0075] In another embodiment, one or more amino acids are altered such that the ability of the antibody to fix complement is changed. This approach is further described in International Publication No. WO 94 / 29351, a PCT publication by Bodmer et al.

[0076] In other embodiments, the Fc region is modified by modifying one or more amino acids such that the ability of the antibody to mediate antibody-dependent cell cytotoxicity (ADCC) is decreased and / or the affinity of the antibody for the Fcγ receptor is decreased. Such antibodies with reduced effector function, and in particular reduced ADCC, include silent antibodies.

[0077] In certain embodiments, the Fc domain of the IgG1 isotype is used. In some particular embodiments, a mutant variant of the IgG1 Fc fragment, for example, a silent IgG1 Fc that decreases or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cell cytotoxicity (ADCC) and / or bind to the Fcγ receptor is used.

[0078] In certain embodiments, the Fc domain of the IgG4 isotype is used. In some particular embodiments, a mutant variant of the IgG4 Fc fragment, for example, a silent IgG1 Fc that decreases or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cell cytotoxicity (ADCC) and / or bind to the Fcγ receptor is used.

[0079] The silent effector function can be obtained by mutation of the Fc constant region of an antibody and is described in the art (Baudino et al., 2008; Strohl, 2009). Examples of silent IgG1 antibodies include the triple mutant variant IgG1 L247F L248E P350S. Examples of silent IgG4 antibodies include the double mutant variant IgG4 S241P L248E.

[0080] In other specific embodiments, the antibody for use in the present disclosure comprises a constant region not selected from the double mutant variant IgG4 S241P L248E and / or the triple mutant IgG1 L247F L248E P350S.

[0081] In certain embodiments, the Fc domain is a silent Fc variant that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain comprises an amino acid substitution of aspartic acid at position 314. Examples of such amino acid substitutions are substitution of N314 with glycine or alanine.

[0082] In some embodiments, the full-length inhibitory BTN3A1 antibody of the present disclosure is an IgG1 antibody. In some embodiments, the full-length inhibitory BTN3A1 antibody of the present disclosure is an IgG1 antibody having the triple mutation L247F L248E P350S.

[0083] Furthermore, the inhibitory BTN3A antibodies for use in the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to again alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0084] The modifications of the antibodies contemplated by the present disclosure are PEGylation or HESylation or related techniques. PEGylation of an antibody can, for example, increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation can be effected by an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, European Patent Application Publication No. 0154316 by Nishimura et al. and European Patent Application Publication No. 0401384 by Ishikawa et al.

[0085] Another modification of the antibodies contemplated by the present disclosure is a conjugate or protein fusion of the antigen-binding region of the antibodies of the present disclosure to a serum protein, such as human serum albumin or a fragment thereof, at least for increasing the half-life of the resulting molecule.

[0086] Functional variant antibodies In yet another embodiment, the functional variant antibodies of the present disclosure have all six CDR region amino acid sequences that are homologous or more specifically identical to the full-length heavy and light chain amino acid sequences; or the variable region heavy and light chain amino acid sequences, or the corresponding amino acid sequences of the reference antibody mAb1 as described above, wherein such functional variant antibodies exhibit the desired functional properties of the reference antibody mAb1, preferably at substantially the same level.

[0087] In the context of the present disclosure, by a functional variant of the reference mAb1 antibody, particularly a functional variant of VL, VH, or CDR, it is still possible for the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity (measured typically by SPR assay as K D , and / or specificity / selectivity of the parental antibody (e.g., mAb1) as evaluated, and in some cases, such monoclonal antibodies of the present disclosure can bind with higher affinity, selectivity and / or specificity than the parental Ab (e.g., mAb1).

[0088] The desired functional characteristics of the reference mAb1 are the following characteristics: (i) K of 10 nM or less, preferably measured by surface plasmon resonance, as determined in a Biacore assay as described in the following examples, preferably K of 1 nM or less D , preferably K of 1 nM or less D , typically measured by SPR assay, K between 1.10 -11 ~1.10 -9 M, and the property of binding to human BTN3A1 isoform; D (ii) EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, such as about 0.02 μg / mL, such as EC50 between 0.1 μg / mL and 0.005 μg / mL, as determined in a PBMC binding assay as described in the following examples, preferably the property of binding to human PBMC; (iii) In co - culture with cancer cells such as Daudi Burkitt lymphoma cell line, IC of 10 nM or less, preferably 1 nM or less, such as 0.1 nM or less, as determined by a CD107 degranulation assay as described in the following examples, preferably the property of inhibiting degranulation of γδ T cells (typically Vγ9Vδ2 T cells) in vitro; (iii) In co - culture with cancer cells such as Daudi Burkitt lymphoma cell line, IC of 10 nM or less, preferably 1 nM or less, such as 0.1 nM or less, as determined by a CD107 degranulation assay as described in the following examples, preferably the property of inhibiting degranulation of γδ T cells (typically Vγ9Vδ2 T cells) in vitro; 50 , preferably 1 nM or less, such as 0.1 nM or less 50 IC, and the property of inhibiting degranulation of γδ T cells (typically Vγ9Vδ2 T cells) in vitro; (iv) For example, as described in the following examples, the properties of substantially inhibiting phosphoantigen-mediated activation, gut homing ability, proliferation, and degranulation ability of patient-derived peripheral Vδ2+ T cells, determined by ex vivo assays of PBMC isolated from IBD patients; and / or (v) For example, as described in the following examples, the properties of substantially inhibiting phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of IBD patients, determined by CD25 or HLA-DR expression in ex vivo assays with walkout cells may be selected from one or more (preferably all) of the above.

[0089] In certain embodiments, the isolated BTN3A antibody for use in the present disclosure is a functional variant of mAb1 that retains at least a substantial proportion, preferably at least 90% of the affinity of mAb1, as measured by SPR assay, and has the following properties: (i) The property of inhibiting in vitro degranulation of γδ T cells with an IC50 of 10 nM or less, preferably 1 nM or less, as determined by, for example, a CD107 degranulation assay by flow cytometry, in co-culture with the Daudi Burkitt lymphoma cell line; (ii) For example, the properties of substantially inhibiting phosphoantigen-mediated activation, gut homing ability, proliferation, and degranulation ability of patient-derived peripheral Vδ2+ T cells, determined by ex vivo assays of PBMC isolated from patients with IBD; and / or (iii) For example, as described in the following examples, the properties of substantially inhibiting phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of IBD patients, determined by CD25 or HLA-DR expression in ex vivo assays with walkout cells having at least one or more of the above.

[0090] For example, the present disclosure provides a variable heavy chain (VH) mAb1 antibody having CDR sequences, i.e., six CDR regions: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, all of which share at least 80, 90, or 100 percent sequence identity with the corresponding CDR sequences of mAb1. H ) and variable light chain (V L ) sequence, wherein said functional variant antibody specifically binds to BTN3A1, and said antibody has the following functional properties: (i) a K of 10 nM or less as measured by an SPR assay, e.g., as determined by a Biacore assay as described in the Examples below. D , preferably 1 nM or less K D , typically 1.10 -11 ~1.10 -9 K between M D and binds to the human BTN3A1 isoform; (ii) binds to human PBMCs with an EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, e.g., an EC50 of about 0.02 μg / mL, as determined, for example, in a PBMC binding assay such as that described in the Examples below; (iii) inhibiting γδ T cell degranulation in vitro in co-culture with a Daudi-Burkitt lymphoma cell line with an IC50 of 10 nM or less, preferably an IC50 of 1 nM or less, as determined, for example, by a flow cytometric CD107 degranulation assay; (iv) substantially inhibits phosphoantigen-mediated activation, gut-homing capacity, proliferation and degranulation capacity of peripheral Vδ2+ T cells from patients suffering from IBD, e.g., as determined in ex vivo assays of PBMCs isolated from patients suffering from IBD; (v) substantially inhibits phosphoantigen-mediated activation and / or proliferation of intestinal V52+ T cells from intestinal biopsies of IBD patients, as determined by CD25 or HLA-DR expression in ex vivo assays on walk-out cells, e.g., as described in the Examples below; and / or (vi) Cross-reacts with cynomolgus monkey BTN3A1 At least one of the following is shown.

[0091] The present disclosure further relates to a functional variant antibody of mAb1 comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95% or 100% identical to the heavy chain and light chain variable regions of the corresponding mAb1, wherein the functional variant antibody specifically binds to BTN3A1 and has the following functional characteristics: (i) A K of 10 nM or less, preferably 1 nM or less, typically between 1.10 D and 1.10 D M, as measured by SPR assay, such as determined by Biacore assay as described in the following examples, binding to human BTN3A1 isoforms; -11 ~1.10 -9 M binding to human BTN3A1 isoforms; D ; (ii) An EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, such as about 0.02 μg / mL, as determined in a PBMC binding assay as described in the following examples, binding to human PBMC; (iii) Inhibiting in vitro degranulation of γδ T cells with an IC50 of 10 nM or less, preferably 1 nM or less, as determined by, for example, a CD107 degranulation assay by flow cytometry in co-culture with the Daudi Burkitt lymphoma cell line; (iv) Substantially inhibiting phosphoantigen-mediated activation, gut homing ability, proliferation, and degranulation ability of patient-derived peripheral Vδ2+ T cells, as determined by, for example, an ex vivo assay of PBMC isolated from patients with IBD; (v) Substantially inhibiting phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of IBD patients, as determined by, for example, CD25 or HLA-DR expression in an ex vivo assay with walkout cells as described in the following examples, and / or (vi) Cross-reacting with cynomolgus BTN3A1 showing at least one (preferably all) of.

[0092] In certain embodiments, the sequences of the CDR functional variants can differ from the sequences of the CDRs of the parental / reference antibody sequence of mAb1, mostly through conservative substitutions; for example, at least 9, 8, 7, 6, 5, 4, 3, 2, or 1, etc., at least 10 of the substitutions of the variant are conservative amino acid residue substitutions. In the context of the present disclosure, conservative substitutions can be defined by substitutions within classes of amino acids represented as follows: Aliphatic residues I, L, V, and M Cycloalkenyl-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in turns A, C, D, E, G, H, K, N, Q, R, S, P, and T (formation T) Flexible residues Q, T, K, S, G, P, D, E, and R

[0093] Groupings of more conservative substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation with respect to hydropathic / hydrophilic characteristics and residue weight / size is also substantially retained in the variant CDRs as compared to the CDRs of mAb1.

[0094] The importance of the hydrophobic amino acid index in conferring biological functions of interactions with proteins is generally understood in the art. The relative hydrophobic nature of amino acids contributes to the resulting secondary structure of the protein, thereby defining the protein's interactions with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophobic index based on its hydrophobic and charge characteristics, which are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also be, or alternatively, determined by similarity scores measured by use of the BLAST program (e.g., BLAST 2.2.8 available through NCBI using standard settings BLOSUM62, open gap = 11 and extend gap = 1).

[0095] Suitable variants typically exhibit at least about 90%, such as 95%, identity to the parental polypeptide VH and VL sequences. According to the present disclosure, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% identity to the second amino acid sequence. According to the present disclosure, a first amino acid sequence having at least 50% identity to a second amino acid sequence means that the first sequence has 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% identity to the second amino acid sequence.

[0096] In some embodiments, the functional variant is a humanized antibody. In certain embodiments, the antibodies of the present disclosure are humanized antibodies that include the six CDRs of the mAb1 reference antibody and an alternative humanized framework region as compared to the humanized framework region of mAb1.

[0097] Functional variant antibodies having a mutant amino acid sequence can be obtained by mutagenesis of the encoding nucleic acid molecule (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), followed by testing for retention of function (i.e., the functions described above) of the encoded, altered antibody using the functional assays described herein.

[0098] An antibody that at least cross-competes with mAb1 and / or binds to the same epitope as mAb1 Additional antibodies having advantageous properties similar to the reference antibody mAb1 disclosed herein can be identified based on their ability to cross-compete (e.g., competitively inhibit binding) with mAb1 as described above, or their ability to bind to the same epitope as mAb1, in a standard BTN3A1 binding assay.

[0099] Test antibodies can first be screened for their binding affinity to the BTN3A1 antigen, for example, from a human recombinant antibody library using, for example, phage display technology, or from a transgenic mouse expressing human variable region antibodies immunized with the BTN3A1 antigen.

[0100] The ability of a test antibody to bind and cross-compete with, or inhibit the binding of, the antibodies of the present disclosure to human BTN3A1 indicates that the test antibody can compete with that antibody for binding to human BTN3A1; such antibodies may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitopes of human BTN3A1 as the competing antibody.

[0101] The epitope of mAb1 was determined and it was shown that mAb1 binds to at least residues 53, 62, and 66 of BTN3A1 (see, for example, FIG. 1), and thus it was shown that this epitope comprises or consists essentially of SEQ ID NO: 20 of BTN3A1.

[0102] In certain embodiments, the present disclosure provides inhibitory BTN3A antibodies that bind to the same epitope as at least the reference inhibitory BTN3A antibody mAb1 described herein.

[0103] To screen for the ability of anti-BTN3A1 antibodies to bind to the same epitope as one of the mAb1 reference antibodies, for example, BTN3A-KO HEK293 cells transfected with human BTN3A1 are stained with one of the reference antibodies mAb1 at a saturating concentration (10 μg / mL) for 30 minutes at 4° C. After two washes, different doses of the test anti-BTN3A1 mAb are tested for their ability to compete with any one of the mAb1 reference antibodies (30 minutes at 4° C.). mAbs that compete for the same binding site as the reference antibody cannot recognize BTN3A1 in the presence of such reference antibody. Data can be expressed as mean fluorescence intensity. Alternatively, the competition assay can be performed in a binning experiment using biolayer interferometry (BLI) by immobilizing recombinant human BTN3A1 on a biosensor and adding the reference antibody followed by the potentially competing antibody.

[0104] Selected antibodies will be further tested for the advantageous properties of mAb1, in particular with regard to inhibitory properties on phosphoantigen-mediated activation of Vγ2 T cells.

[0105] Thus, in one embodiment, the disclosure provides an isolated antibody that competes for binding of mAb1 to BTN3A1 or binds to the same epitope as mAb1 (typically including amino acids 52, 62 and 66 of BTN3A1), wherein the antibody has the following characteristics: (i) a K of 10 nM or less as measured by an SPR assay, e.g., as determined by a Biacore assay as described in the Examples below. D , preferably 1 nM or less, typically 1.10 -11 ~1.10 -9 K between M D , with binding properties to human BTN3A1 isoforms; (ii) the property of binding to human PBMCs with an EC50 of 0.1 μg / mL or less, preferably an EC50 of 0.05 μg / mL or less, for example about 0.02 μg / mL, as determined, for example, in a PBMC binding assay as described in the Examples below; (iii) The property of inhibiting in vitro degranulation of γδ T cells with an IC50 of 10 nM or less, preferably 1 nM or less, as determined by, for example, a CD107 degranulation assay by flow cytometry, in co-culture with a Daudi Burkitt lymphoma cell line; (iv) The property of substantially inhibiting phosphoantigen-mediated activation, gut homing ability, proliferation and degranulation ability of patient-derived peripheral Vδ2+ T cells, as determined by an ex vivo assay of PBMC isolated from, for example, patients suffering from IBD; and / or (v) The property of substantially inhibiting phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of IBD patients, as determined by CD25 or HLA-DR expression in an ex vivo assay with walkout cells, as described, for example, in the following examples showing at least one of.

[0106] Typically, the above-described functional properties of point (iii), (iv) and / or (v) of an antibody that competes with mAb1 for binding to BTN3A1 or binds to the same epitope as mAb1 are, as described above, substantially equal to or better than the corresponding functional properties of mAb1. As used herein, substantially equal means that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of the corresponding functional properties of the reference mAb1.

[0107] Typically, an antibody that competes with mAb1 for binding to BTN3A1 or binds to the same epitope as the disclosed mAb1 of the present disclosure still has at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity of the reference antibody and, in some cases, can bind with higher affinity, selectivity and / or specificity than the reference antibody mAb1.

[0108] In certain embodiments, the present disclosure provides an antibody, preferably a chimeric, humanized or human recombinant antibody, that binds to or cross-competes with the same epitope as the inhibitory BTN3A antibody mAb1.

[0109] Pharmaceutical composition In another aspect, the present disclosure provides a composition, such as a pharmaceutical composition, comprising an inhibitory BTN3A antibody disclosed above formulated with a pharmaceutically acceptable carrier.

[0110] In another aspect, the present disclosure provides a composition, such as a pharmaceutical composition, comprising one or a combination of the antibodies disclosed herein, such as mAb1 or a variant thereof, or an antigen-binding fragment, formulated with a pharmaceutically acceptable carrier. Such a composition may comprise one or a combination of the above-described (e.g., two or more different) antibodies.

[0111] As used herein, "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous route or intravenous injection.

[0112] Sterile phosphate buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. Compositions containing such carriers are formulated by well-known conventional methods (e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000) (see). The formulation may further comprise one or more excipients, preservatives, solubilizing agents, buffering agents, albumin to prevent protein loss on the surface of the virus, etc.

[0113] The form, route of administration, dosage, and regimen of the pharmaceutical composition will, of course, depend on the condition being treated, the severity of the disease, the age, weight, and sex of the patient, etc.

[0114] The pharmaceutical compositions of the present disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0115] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injectable formulations. These can be, in particular, isotonic, sterile saline (sodium monophosphate or disodium phosphate, sodium chloride, potassium, calcium, or magnesium, etc. or a mixture of such salts), or, optionally, a dry, especially lyophilized composition that allows the constitution of an injection solution upon addition of sterile water or physiological saline.

[0116] The dosage used for administration can be adapted as a function of various parameters and, in particular, can be adapted as a function of the mode of administration used, the associated pathology, or, alternatively, the desired duration of treatment.

[0117] To prepare the pharmaceutical composition, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0118] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders or lyophilized products for the immediate preparation of sterile injection solutions or dispersions. In all cases, this form must be sterile and fluid to the extent that easy syringeability exists. This form must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0119] Solutions of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.

[0120] The dispersion can also be prepared in glycerin, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0121] The antibodies of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. (formed with the free amino groups of the protein). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium, or ferric salts, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0122] The carrier can also be a solvent or dispersion medium, including, for example, water, ethanol, polyols (such as glycerin, propylene glycol, and liquid propylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, the use of coating agents such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants can maintain appropriate fluidity. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

[0123] Aseptic injectable solutions are prepared by incorporating the active compound in the required amount into a suitable solvent along with the various other ingredients enumerated above, followed by filtration sterilization if necessary. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from its previously sterile filtered solution.

[0124] The preparation of more or highly concentrated solutions for direct injection is also contemplated where the use of DMSO as a solvent is expected to result in extremely rapid penetration and delivery of high concentrations of the active agent to small inflamed tissue regions.

[0125] Upon formulation, the solution is administered in a therapeutically effective amount in a dosage form compatible with the dosage form. The formulations are readily administered in a variety of dosage forms such as the types of injectable solutions described above, but drug release capsules etc. may also be employed.

[0126] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media which may be employed are known to those skilled in the art in light of the present disclosure. For example, a single dosage may be either dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of a liquid for subcutaneous injection or injected at the proposed site of infusion (see, for example, "Remington’s Pharmaceutical Sciencse", 15th Edition, pages 1035 - 1038 and 1570 - 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. It is the responsibility of the administering person to determine the appropriate dosage for each individual subject in every instance.

[0127] The antibodies of the present disclosure can be formulated in a therapeutic mixture such that each dose contains from about 1 to 100 mg. Multiple doses can also be administered.

[0128] Suitable formulations of solutions for intravenous or subcutaneous injection of antibodies are described in the art and are reviewed, for example, by Cui et al. (Drug Dev Ind Phram 2017, 43(4):519-530).

[0129] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of the antibody into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0130] Use and methods of the BTN3A inhibitory antibodies of the present disclosure The inhibitory BTN3A antibodies of the present disclosure have diagnostic and therapeutic utility in vitro and in vivo.

[0131] The inhibitory BTN3A antibodies disclosed above are in fact useful in a method for preparing a medicament for use in the treatment of gastrointestinal inflammation, such as IBD, in a human subject in need of treatment for gastrointestinal inflammation.

[0132] In particular, the inhibitory BTN3A antibody can inhibit in vivo the cytolytic function, cytokine production and / or proliferation of phosphoantigen-mediated Vδ2 T cells, as suggested in vitro or ex vivo using isolated immune cells from IBD patients.

[0133] Accordingly, the inhibitory BTN3A antibodies of the present disclosure can be used in a method for inhibiting the activation of Vδ2 T cells in a subject in need of inhibition of the activation of Vδ2 T cells, particularly with respect to migration, antigen presentation, cytokine secretion or cytolytic function, said method comprising administering to a subject in need of inhibition of the activation of Vδ2 T cells an inhibitory effective amount of said inhibitory BTN3A antibody of the present disclosure, such as mAb1 disclosed herein.

[0134] Accordingly, another object of the present disclosure is a method of inhibiting an immune response in a subject in need of inhibition of an immune response, particularly a method of inhibiting the cytolytic properties of activated Vδ2T cells in a subject in need of inhibition of an immune response, comprising the step of administering to the subject a therapeutically effective amount of the inhibitory BTN3A antibody of the present disclosure, such as mAb1 disclosed herein.

[0135] In certain embodiments, the subject in need of inhibition of an immune response is suffering from a gastrointestinal disorder, such as IBD, and exhibits a higher blood cell count of activated δ2T cells prior to treatment with the inhibitory BTN3A antibody as compared to a control, and is selected from among subjects. Such a control can be, for example, the average blood cell count of activated δ2T cells of a healthy subject.

[0136] In other certain embodiments, the subject in need of inhibition of an immune response is suffering from a gastrointestinal disorder, such as IBD, based on the BTN3A expression level in the intestine, and typically exhibits a high level of BTN3A expression in the intestine as compared to a control, and is selected from among subjects. Such a control can be, for example, the average BNT3A expression in a healthy subject.

[0137] In certain embodiments, the inhibitory BTN3A antibody of the present disclosure is particularly useful for treating, preventing or diagnosing gastrointestinal inflammatory disorders such as IBD, particularly gastrointestinal inflammatory disorders involving activated δ2T cells, such as ulcerative colitis or Crohn's disease.

[0138] Other gastrointestinal inflammatory disorders involving activated δ2T cells that can be advantageously treated with the inhibitory BTN3A antibody include, but are not limited to, peptic ulcerative disease, gastritis, gastroenteritis, celiac disease, appendicitis, pancreatitis, Whipple's disease, hepatitis, enteritis, enterocolitis, duodenitis, jejunitis and ileitis, cholangitis.

[0139] The present disclosure also relates to a method of manufacturing a medicament for use in the treatment of gastrointestinal inflammatory disorders such as IBD, such as ulcerative colitis or Crohn's disease, the medicament comprising the inhibitory BTN3A antibody of the present disclosure, such as mAb1 disclosed herein, as described in the previous paragraph.

[0140] The antibodies of the present disclosure can be administered, for example, for the treatment or prevention of the above-mentioned diseases, as a single active ingredient, or in combination with other drugs, such as immunosuppressive agents or immunomodulatory agents or other anti-inflammatory agents, for example as an adjuvant, or in combination (simultaneously or sequentially).

[0141] Typically, the anti-inflammatory agent may include, but is not limited to, anti-inflammatory cytokines, which may optionally be selected from interleukin (IL)-12, IL-22, IL-23, tumor necrosis factor (TNF) α. In other embodiments, the anti-inflammatory agent may include, but is not limited to, steroids such as glucocorticoids, prednisone, hydrocortisone, 5-ASA, cyclosporin A (CsA), or immunomodulatory substances including anti-a4 integrin, anti-a4b7 integrin, JAK inhibitors, azathioprine, mercaptopurine or methotrexate.

[0142] As used herein, the terms "treatment" or "treating" refer to both prophylactic or preventive treatment and curative or disease-modifying treatment, including suppression of clinical recurrence, of a subject at risk of having a disease or suspected of having a disease, and of a subject diagnosed as being ill or suffering from a disease or medical condition. Treatment can be administered to a subject having a medical disorder or a subject who may ultimately acquire a disorder to prevent, treat, delay the onset of, reduce the severity of, or improve one or more symptoms of the disorder or recurring disorder, or to extend the lifespan of the subject beyond that expected in the absence of such treatment.

[0143] As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages, over a period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the antibodies of the present disclosure can vary depending on factors such as the disease state, the age, sex and weight of the individual, and the ability of the antibodies of the present disclosure to induce a desired response in the individual. A therapeutically effective amount is also an amount where the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody portion. The effective dosage and administration regimen of the antibodies of the present disclosure depend on the disease or condition being treated and can be determined by one of ordinary skill in the art. A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start with a dosage of the antibodies of the present disclosure employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate dosage of the compositions of the present disclosure is the amount of the compound at the lowest dosage effective to produce a therapeutic effect according to a particular dosing regimen. Such effective dosages generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by the ability to stabilize the progression of the disease. Typically, the ability of a compound to treat an inflammatory disorder is evaluated, for example, in an animal model system that predicts efficacy in the treatment of inflammatory disorders. Alternatively, this property of the composition can be evaluated by in vitro assays known to those of ordinary skill in the art by examining the ability of the compound to inhibit the induction of an immune response. A therapeutically effective amount of a therapeutic compound can reduce an immune response or inflammatory response or otherwise improve the symptoms of a subject. One of ordinary skill in the art can determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected. Exemplary, non-limiting ranges of the therapeutically effective amount of the antibodies of the present disclosure are from about 0.1 to 50 mg / kg, such as from about 0.1 to 10 mg / kg, from about 0.1 to 20 mg / kg, such as about 0.3, about 0.5, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg, etc., from about 0.1 to 100 mg / kg.Exemplary, non-limiting ranges for a therapeutically effective amount of the antibodies of the present disclosure are from about 0.05 to 10 mg / kg or 0.1 to 3 mg / kg, such as about 0.5 to 2 mg / kg, etc., from about 0.01 to 30 mg / kg, etc., 0.01 to 100 mg / kg.

[0144] In accordance with the foregoing, the present disclosure, in a further aspect: Comprises co-administration of a therapeutically effective amount of an inhibitory BTN3A antibody of the present disclosure (e.g., mAb1), and at least one second prodrug, for example simultaneously or sequentially, wherein said second prodrug is, for example, an antiviral, anti-inflammatory or antimicrobial agent as described above, the method as set forth above is provided.

[0145] In one embodiment, the antibodies of the present disclosure can also be used to detect the level of BTN3A-expressing cells. This can be achieved, for example, by incubating a sample (such as an in vitro sample) and a control sample with an anti-BTN3A antibody under conditions that allow for the formation of a complex between the antibody and BTN3A (e.g., expressed on the surface of cells in a blood sample). Any complex formed between the antibody and BTN3A is detected and compared in the sample and the control. For example, standard detection methods well known in the art, such as ELISA and flow cytometry assays, can be performed using the compositions of the present disclosure.

[0146] Accordingly, in one aspect, the present disclosure further provides a method for detecting the presence of BTN3A, or BTN3A-expressing cells (e.g., human BTN3A antigen), or measuring the amount of BTN3A in a sample, comprising incubating a sample and a control sample with an antibody of the present disclosure that specifically binds to BTN3A under conditions that allow for the formation of a complex between the antibody and BTN3A. Then, the formation of the complex is detected, wherein a difference in complex formation between the sample compared to the control sample indicates the presence of BTN3A in the sample.

[0147] Also, a kit comprising the composition disclosed herein (e.g., mAb1) and instructions for use is within the scope of the present disclosure. The kit may further comprise at least one additional reagent, or one or more additional antibodies or proteins. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any writing or recording material on, supplied with, or otherwise attached to the kit. The kit may further comprise a tool for diagnosing whether a patient belongs to the group responsive to treatment with an inhibitory BTN3A antibody as defined above.

[0148] The present disclosure is further illustrated by the following drawings and examples. However, these examples and drawings should in no way be construed as limiting the scope of the invention.

Examples

[0149] Functional assay for selecting candidate BTN3A antibodies having γδ inhibitory properties for their use in the treatment of gastrointestinal inflammatory disorders SPR Biacore assay Multi-cycle kinetic analysis can be performed on candidate BTN3A using a Biacore T200 instrument (serial number 1909913) operating Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).

[0150] Dilute the purified candidates to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, capture each antibody onto Protein A at a density of approximately 146.5 RU (RL) (theoretical value to obtain an RMax of approximately 50 RU). After capture, stabilize the surface prior to injection of the BTN3A1 antigen (Sino Biological catalog number 15973-H08H). BTN3A1 is titrated in 0.1% BSA / HBS-P+ (running buffer) in a two-fold serial dilution range of 25 to 0.78 nM. The association phase is monitored for 400 seconds and the dissociation phase is monitored for 35 minutes (2100 seconds). Use a flow rate of 50 μl / min to minimize any potential mass transfer effects and obtain kinetic data. Regeneration of the Protein A surface is performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Perform two blanks (without BTN3A1) and replicates of a single concentration of analyte for each test antibody to examine the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 is subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface. Additionally, a blank run is subtracted for each Fc to correct for any antigen-independent signal variations such as drift. Sensorgrams are fitted using a one-to-one binding mathematical model that uses the global RMax parameter and does not use the bulk signal (RI constant = 0 RU).

[0151] PBMC Binding Assay Human PBMCs were isolated from healthy community donor buffy coats (derived from blood collected within 24 hours) using Lymphoprep (Axis-shield, Dandy, UK) density centrifugation method. Cynomolgus (Cyno) whole blood was obtained from Envigo (Huntingdon, UK). On the day of the experiment, frozen PBMCs were thawed and counted. Cells were stained for live and dead cells by incubating for 30 minutes in the dark using the LIVE / DEAD (registered trademark) Far Red Dead Cell staining kit (ThermoFisher, Paisley, UK) according to the manufacturer's instructions. During this staining, one in three titration curves of the test antibodies were prepared in flow buffer (0.5% BSA, 2 mM EDTA, 1×DPBS, pH 7.4) in a 96-well dilution plate. After viability staining, the cells were harvested, washed twice, and resuspended in flow buffer at 1×10 6 cells / mL. 1×10 6100 μL of cells at [number of cells] / mL were transferred to each well of a fresh U-bottom 96-well plate. The plate was centrifuged, the supernatant discarded, and the cells resuspended in 50 μL of a serial dilution of pre-prepared test antibody titrations. After incubation for 30 minutes at 4 °C in the dark, the plate was centrifuged, washed twice, and resuspended in 50 μL of PE-labeled goat anti-human antibody (Sigma, pool, UK) diluted 1 / 100 in flow buffer. After incubation for 15 minutes at 4 °C in the dark, the plate was centrifuged, washed, and the cells resuspended in 200 μL of flow buffer. The cells were then analyzed on an Attune NxT focusing cytometer (ThermoFisher Scientific, Loughborough, UK), collecting 10,000 events per sample using two laser channels: RL1 for live / dead cells and BL2 for PE. Data were analyzed using the instrument's statistical tool for gating on the viable population of lymphocytes or FlowJo software (version 10, FlowJo, LLC, Ashland, USA). The median X of the BL2 channel (PE signal) was then calculated and plotted against concentration.

[0152] CD107 degranulation assay The assay consists of measuring the inhibitory effect of BTN3A candidate antibodies on the γδT cell degranulation ability against the Daudi Burkitt lymphoma cell line (Harly C et al., Blood, 2012, Vol.120 Issue 11, pp.2269 - 79). γδT cells are expanded by culturing from PBMC of healthy donors with zoledronic acid (1 μM) and IL2 (200 IU / ml) for 11 - 13 days. IL2 is added on day 5, day 8, and then every 2 days thereafter. The percentage of γδT cells is determined at the beginning of the culture and evaluated during the culture by flow cytometry until it reaches at least 80%. Subsequently, cryopreserved γδT cells are used in a degranulation assay against the Daudi cell line (1:1 E:T ratio), whereby the cells are co - cultured at 37°C for 4 hours in the presence of increasing concentrations of the candidate antibody or control antibody. Activation by PMA (20 ng / ml) + ionomycin (1 μg / ml) serves as a positive control for γδT cell degranulation, and medium alone serves as a negative control. After 4 hours of incubation, the cells are analyzed by flow cytometry to evaluate the percentage of γδT cells positive for CD107a (LAMP - 1, lysosomal membrane protein - 1) + CD107b (LAMP - 2). CD107 accumulates on the cell surface after activation - induced granule exocytosis, so measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytotoxic T cells.

[0153] Ex vivo assay of PBMC - derived Vδ2 + T cell activation PBMC derived from blood of patients with Crohn's disease or ulcerative colitis are isolated, stained with 1 μM CTV dye, and cultured for 4 days in the presence of IL - 2 (20 IU / ml) / IL - 15 (20 ng / ml), 1 nM HDMAPP or HMBPP (phosphoantigen), and 1 μg / ml control isotype or candidate BTN3A antibody.

[0154] The expression of the following T cell activation markers: CD25, HLA - DR, β7 integrin, CD49b can be evaluated by flow cytometry for Vδ2 + and Vδ2 - cells.

[0155] Proliferation can be evaluated by measuring CTV dilution, and degranulation ability can be evaluated by measuring CD107a expression of Vδ2+ and Vδ2-, and also by flow cytometry.

[0156] Ex vivo assay of Vδ2+ T cell activation from intestinal biopsy Intestinal biopsies from Crohn's disease of patients with ulcerative colitis are added to the culture for "walk-out" overnight culture. The biopsies are removed the next day, and then the walk-out cells are stained with 1 μM CTV dye. Then, the CTV-stained cells are incubated for 7 days in the presence of IL-2 (20 IU / ml) / IL-15 (20 ng / ml), 10 nM HDMAPP or HMBPP (phosphoantigen), and 1 μg / ml control isotype or candidate BTN3A antibody. The expression of the following T cell activation markers: CD25, HLA-DR can also be evaluated for Vδ2+ by flow cytometry. Proliferation can be evaluated by measuring CTV dilution, and degranulation ability can be measured by CD107a expression of Vδ2+ by flow cytometry.

[0157] Example 1: Generation of inhibitory BTN3A1 suitable for use as a drug in human subjects Design of the variable region sequences of Composite Human Antibody™ The crystal structure of the mouse 103.2 antibody V region (WO 2012 / 80351) was generated using Swiss PDB and analyzed to identify important "constraining" amino acids in the V region that are likely essential for the binding properties of the antibody.

[0158] Most residues contained within the CDRs along with some framework residues (using both Kabat and Chothia definitions) were considered important. The VH and V Κ sequences contain typical framework residues, and the CDR1, 2, and 3 motifs are equivalent to those of many mouse antibodies.

[0159] Based on structural analysis, a large preliminary set of array segments that can be used to generate 103.2 humanized variants was selected, and iTope™ technology for in silico analysis of peptides that bind to human MHC class II alleles was used (Perry et al., 2008, Drugs R D 9(6):385-396), and TCED™ of known antibody sequence-related T cell epitopes was used (Bryson et al., 2010, Biodrugs 24(1):1-8) for analysis. Array segments that were identified as significant non-human germline binders to human MHC class II or that yielded significant hits to TCED™ were discarded. This resulted in a reduction in the set of segments, and these combinations were re-analyzed as described above to confirm that the linkages between the segments did not contain potential T cell epitopes. The selected array segments were assembled into a complete V region sequence predicted to lack significant T cell epitopes.

[0160] Then, five heavy chains (VH1-VH5) and four light chains (V Κ 1-V Κ 4) sequences were selected for gene synthesis and mammalian expression.

[0161] Construction of humanized variant plasmids Twenty humanized variants, each combining one of the five VH regions with one of the four Vk regions, were synthesized with adjacent restriction enzyme sites for cloning into expression vector systems for human IgG4 (S241P, L248E) heavy chains and kappa light chains. All constructs were confirmed by sequencing.

[0162] To evaluate the binding of all Composite Human Antibody (trademark) variants and to select antibodies with appropriate affinity for BTN3A compared to the original mouse antibody, a Biacore T200 (serial number 1909913) using Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden) was used to perform single-cycle kinetic analysis on supernatants from transfected cell cultures.

[0163] The results are shown in Table 1 below:

[0164]

Table 1

[0165] The selected humanized variants, along with their chimeric forms and the most conservatively humanized variant (VH1 / V Κ 1), were subjected to purification for further assay. Antibodies were purified from cell culture supernatants using a Protein A Sepharose column followed by size exclusion chromatography (SEC) (GE Healthcare, Little Chalfont, UK) with 10 mM sodium acetate, 100 mM NaCl, pH 5.5 as the mobile phase and final formulation buffer. Samples were quantified by OD 280nm using the extinction coefficient (Ec(0.1%)) based on the predicted amino acid sequence.

[0166] Antibodies were analyzed using SDS-PAGE by loading 2 μg of each antibody onto the gel, and bands corresponding to the profile of a typical antibody were observed. Thermal stability analysis To evaluate the thermal stability of six selected composite human antibody (trademark) variants, a fluorescence-based thermal shift assay was used to determine the melting temperature (the temperature at which 50% of the protein domain is in the unfolded state).

[0167] All six purified humanized antibodies, along with chimeric (VH0 / V Κ 0) antibodies and humanized variants (VH1 / V Κ 1), were diluted to a final concentration of 0.1 mg / ml in a formulation buffer (10 mM sodium acetate, 100 mM NaCl, pH 5.5) containing SYPRO® Orange (ThermoFisher, Loughborough, UK) diluted 1:1000 and subjected to a temperature gradient from 25 °C to 99 °C for 56 minutes using a StepOnePlus real-time PCR system (ThermoFisher, Loughborough, UK). 10 mM sodium acetate, 100 mM NaCl, pH 5.5 was used as a negative control. The melting curves were analyzed using Protein Thermal Stability Software (version 1.2).

[0168] All antibody variants showed two distinct unfolding events with higher T m values as the degree of human-likeness increased, as shown in Table 2.

[0169]

Table 2

[0170] Multi-cycle kinetic analysis Using a Biacore T200 instrument (serial number 1909913) operating Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden), six selected humanized 103.2 variants (VH4 / V Κ 2, VH4 / V Κ 3, VH4 / V Κ 4, VH5 / V Κ 2, VH5 / V Κ 3, VH5 / V ΚRegarding (4), a multi-cycle kinetic analysis was performed with the chimeric antibody and the humanized variant VH1 / V Κ 1 together.

[0171] The purified antibody was diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured on Protein A at a density of approximately 146.5 RU (RL) (the theoretical value for obtaining an RMax of approximately 50 RU). After capture, the surface was stabilized prior to injection of the BTN3A1 antigen (Sino Biological catalog number 15973-H08H). BTN3A1 was titrated in 0.1% BSA / HBS-P+ (running buffer) in a two-fold serial dilution range of 25 to 0.78 nM. The binding phase was monitored for 400 seconds and the dissociation phase was monitored for 35 minutes (2100 seconds). A flow rate of 50 μl / min was used to minimize any potential mass transfer effects and obtain kinetic data. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Two blanks (without BTN3A1) and repeats of a single concentration of analyte were performed for each test antibody to examine the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface. Additionally, a blank run was subtracted for each Fc to correct for any antigen-independent signal variations such as drift. Sensorgrams were fitted using a one-to-one binding mathematical model that uses the global RMax parameter and does not use the bulk signal (RI constant = 0 RU).

[0172] The K of the 103.2 composite human antibody (TM) variant D was divided by the K of the chimeric on the same chip D to calculate the relative K compared to 103.2 chimeric (VH0 / V Κ 0). All selected composite human antibody (TM) variants showed an affinity within 2-fold of the chimeric antibody (Table 3). D

[0173]

Table 3

[0174] In vitro functional efficacy: γδT cell degranulation assay The assay consists of measuring the inhibitory effects of 103.2 humanized variants and their chimeric forms on the γδT cell degranulation ability against the Daudi Burkitt lymphoma cell line (Harly C et al., 2012, supra). γδT cells were expanded by culturing from PBMC of healthy donors with zoledronic acid (1 μM) and IL2 (200 IU / ml) for 11 - 13 days. IL2 was added on day 5, day 8, and then every 2 days thereafter. The percentage of γδT cells was determined at the beginning of the culture and evaluated during the culture by flow cytometry until at least 80% was reached. The cryopreserved γδT cells were then used in a degranulation assay against the Daudi cell line (1:1 E:T ratio), whereby the cells were co - cultured at 37°C for 4 hours in the presence of increasing concentrations of 103.2 humanized variants as well as their chimeric and murine forms. Activation by PMA (20 ng / ml) + ionomycin (1 μg / ml) served as a positive control for γδT cell degranulation, and medium alone served as a negative control. After 4 hours of incubation, the cells were analyzed by flow cytometry to evaluate the percentage of γδT cells positive for CD107a (LAMP - 1, lysosomal membrane protein - 1) + CD107b (LAMP - 2). CD107 accumulates on the cell surface after activation - induced granule exocytosis, so measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytotoxic T cells.

[0175] All tested humanized variants maintained their inhibitory effects in the degranulation assay compared to chimeric and murine antibodies (see Table 4).

[0176] Surprisingly, VH4V Κ 4, which contained amino acid mutations in CDR2 compared to the original murine CDR2 of mouse mAb 103.2, was still highly capable in this assay.

[0177] [Table 4]

[0178] Selection of humanized candidates In summary, surprisingly, VH4V Κ 4 humanized variants showed the highest Biacore affinity and the highest binding affinity for human PBMC and were found to be most capable with respect to inhibitory properties in the degranulation assay at a concentration of 10 μg / ml compared to other humanized candidates.

[0179] Also, the thermal stability of VH4V Κ 4 was improved compared to chimeric antibodies with murine VH and VL regions.

[0180] Therefore, humanized candidate VH4V Κ 4 was selected for further in vivo and in vitro evaluation for its suitability for the treatment of IBD.

[0181] Cross-reactivity with human and cynomolgus PBMC Human PBMC were isolated from healthy community donor buffy coats (derived from blood collected within 24 hours) obtained with consent from commercial suppliers. Cynomolgus whole blood was obtained from Envigo (Huntingdon, UK). PBMC were isolated using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. The PBMC were then frozen and stored in the gas phase of nitrogen until needed.

[0182] The frozen PBMCs were thawed and added to pre-warmed AIM-V (ThermoFisher Scientific, Loughborough, UK) medium and counted. Cells were harvested and washed by centrifugation at 500×g for 10 minutes and then resuspended in 1×DPBS, pH 7.4. This step was repeated once and the cells were resuspended in 1×DPBS, pH 7.4 at 1×10 6 cells / mL. The cells were stained for viable and dead cells by incubating for half an hour in the dark using the LIVE / DEAD® Far Red Dead Cell Staining Kit (ThermoFisher, Paisley, UK) according to the manufacturer's instructions. A fixed amount of cells heated at 70°C for 10 minutes was included as a dead cell control. During this staining, one titration curve was prepared in flow buffer (0.5% BSA, 2 mM EDTA, 1×DPBS, pH 7.4) in triplicate for the test antibody (starting at 0.063 μg / mL for the selected antibody) in a 96-well dilution plate. After viability staining, the cells were harvested, washed twice as described above and then resuspended in flow buffer at 1×10 6 cells / mL. 1×10 6Cells were transferred at 100 μL of cells at [number of cells] / mL into each well of a fresh U-bottom 96-well plate (columns 1-12). The plate was centrifuged, the supernatant discarded, and the cells resuspended in 50 μL of a serial dilution of the pre-prepared test antibody titration. After incubation for 30 minutes at 4°C in the dark, the plate was centrifuged, washed twice with 150 μL / well of flow buffer, and the cells resuspended in 50 μL of PE-labeled goat anti-human antibody (Sigma, pool, UK) diluted 1 / 100 in flow buffer. After incubation for 15 minutes at 4°C in the dark, the plate was centrifuged, washed once with 150 μL / well of flow buffer, and the cells resuspended in 200 μL of flow buffer. Cells were then analyzed on an Attune NxT focusing cytometer (ThermoFisher Scientific, Loughborough, UK), collecting 10,000 events per sample using two laser channels: RL1 for live / dead cells and BL2 for PE. Data were analyzed using the instrument's statistical tool for gating on the viable population of lymphocytes or FlowJo software (version 10, FlowJo, LLC, Ashland, USA). The median X of the BL2 channel (PE signal) was then calculated and plotted against concentration.

[0183] Next, a full titration was performed with a 1 in 3 of 11-point full titration curve of the test antibody (starting at 5 μg / mL).

[0184] Table 5 below shows that binding was observed to both human and cynomolgus macaque PBMCs for both test antibodies. The binding pattern was consistent between the two species and between different donors.

[0185]

Table 5

[0186] Epitope mapping To determine the epitope of a high-precision antibody / antigen complex, the protein complex was incubated with a deuterated crosslinker and subjected to cleavage by multiple enzymes. After enrichment of the crosslinked peptides, the sample was analyzed by high-resolution mass spectrometry (nLC-Q Exactive MS), and the generated data were analyzed using XQuest and Stavrox software.

[0187] Mixtures of BTN3A1 / mAb1 were prepared at the following concentrations:

[0188] [Table 6]

[0189] For reductive alkylation, 20 μL of the prepared antibody / antigen mixture was mixed with 2 μL of DSS d0 / d12 (2 mg / mL; DMF) prior to incubation at room temperature for 180 minutes. After incubation, the reaction was stopped by adding 1 μL of ammonium bicarbonate (final concentration 20 mM) prior to incubation at room temperature for 1 hour.

[0190] Then, H 2 The solution was dried using a SpeedVac prior to an 8 M urea suspension in H2O (20 μL). After mixing, 2 μl of DTT (500 mM) was added to the solution. The mixture was then incubated at 37 °C for 1 hour. After incubation, 2 μl of iodoacetamide (1 M) was added in the dark room prior to an incubation time of 1 hour at room temperature. After incubation, 80 μl of proteolysis buffer was added. The trypsin buffer contains 50 mM Ambic pH8.5, 5% acetonitrile. The chymotrypsin buffer contains 100 mM Tris HCl, CaCl 2 10 mM pH7.8. The ASP-N buffer contains 50 mM phosphate buffer pH7.8. The elastase buffer contains 50 mM Tris HCl pH8.0, and the thermolysin buffer contains 50 mM Tris HCl, 0.5 mM CaCL2 pH9.0.

[0191] For trypsin proteolysis, 100 μl of the reduced / alkylated antibody / antigen mixture was mixed with 0.9 μl of trypsin (Roche Diagnostic) at a 1 / 100 (w / w) ratio. The proteolysis mixture was incubated overnight at 37 °C.

[0192] For chymotrypsin proteolysis, 100 μl of the reduced / alkylated antibody / antigen mixture was mixed with 0.45 μl of chymotrypsin (Roche Diagnostic) at a 1 / 200 (w / w) ratio. The proteolysis mixture was incubated overnight at 25 °C.

[0193] For Asp-N proteolysis, 100 μl of the reduced / alkylated antibody / antigen mixture was mixed with 0.45 μl of AspN (Roche Diagnostic) at a 1 / 200 (w / w) ratio. The proteolysis mixture was incubated overnight at 37 °C.

[0194] For elastase proteolysis, 100 μl of the reduced / alkylated antibody / antigen mixture was mixed with 0.9 μl of elastase (Roche Diagnostic) at a 1 / 100 (w / w) ratio. The proteolysis mixture was incubated overnight at 37 °C.

[0195] For thermolysin proteolysis, 100 μl of the reduced / alkylated antibody / antigen mixture was mixed with 1.8 μl of thermolysin (Roche Diagnostic) at a 1 / 50 (w / w) ratio. The proteolysis mixture was incubated overnight at 70 °C. After digestion, 1% formic acid final concentration was added to the solution.

[0196] After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the antibody / antigen protein complex with deuterated d0d12, five cross-linked peptides between BTN3A1 (protein 2) and mAb1 (protein 1) were detected by nLC-Orbitrap MS / MS analysis. The sequences and positions of the cross-links are shown in Table 6 below.

[0197]

Table 7

[0198] Using chemical crosslinking, high-quality MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry, the inventors were able to characterize the epitope of mAb1 on BTN3A1.

[0199] As shown in Figure 1, the epitope contains the following amino acids on the antigen BTN3A1: 53, 62, 66, such as those contained in SEQ ID NO: 20. Off-target Using cell microarray technology (Retrogenix), the specific off-target binding of Fc-silent humanized 103.2 IgG1 (referred to as mAb1, targeting the human BTN3A protein) was screened.

[0200] From the investigation of the binding levels of the test antibody to untransfected HEK293 cells and cells overexpressing BTN3A1, either before or after cell fixation, it was shown that 2 μg / ml for fixed cells is an appropriate screening condition. Under this condition, the test antibody was screened for binding to human HEK293 cells individually expressing 5528 human proteins composed of cell surface membrane proteins and cell surface-tethered secreted proteins. From this, 11 major hits were revealed. Each major hit was re-expressed together with two control receptors (CD20 and EGFR) and retested with 2 μg / ml of the test antibody, 2 μg / ml of the isotype control antibody, and other positive and negative control treatments. After excluding one hit with very low intensity and non-reproducible, and five non-specific hits, five specific interactions with the test antibody remained. These were two isoforms of BTN3A1, two isoforms of BTN3A2, and one isoform of BTN3A3, which are its major targets.

[0201] No off-target interactions of mAb1 were identified, and high specificity of mAb1 for its main target was shown (data not shown).

[0202] Example 2: In Vitro Pharmacology Evaluation of the helper function of peripheral Vγ9Vδ2 T cells upon microbial activation and the ability of mAb1 to regulate the Vγ9Vδ2 T cell "helper" function Vγ9Vδ2 T cells are known to express several markers upon phosphoantigen stimulation, particularly in IBD (Mann ER et al., Clin Exp Immunol, November 2012;170(2):122-30; Tyler CJ et al., J Immunol, May 1, 2017;198(9):3417-3425; McCarthy NE et al., J Clin Invest, August 3, 2015;125(8):3215-25). To determine whether humanized 103.2Fc silent IgG1 (hereinafter referred to as mAb1) can affect the phenotype of Vγ9δ2 T cells and other immune populations, PBMCs from six healthy donors were stimulated with phosphoantigen (Hmbpp) for 2 days at 0.5 μM + / - 50 IU / ml IL-2 in the presence of increasing concentrations of humanized 103.2 or the corresponding control isotype. The expression of costimulatory markers (CD40, CD80, and CD86), mature APC markers (CD83), antigen-presenting molecules (HLA-DR), adhesion molecules (CD11a, CD11b, CD11c, and CD54), gut and lymph node homing molecules (α4 and β7 integrins, CCR9, and CCR7), and B cell costimulatory molecules (OX40, ICOS, and CD70) was evaluated by flow cytometry of Vγ9Vδ2 T cells and other immune cells (B cells, monocytes, and Vδ2-T cells), and the IC50 was calculated.

[0203] mAb1 inhibits all the markers evaluated in a dose-dependent manner (similar inhibition observed in the frequency of positive cells and median fluorescence intensity), suggesting that mAb1 has the ability to inhibit the APC function, gut homing ability and adhesion ability of peripheral Vγ9Vδ2T cells from healthy donors stimulated with phosphoantigen.

[0204] The results are equivalent in the presence or absence of IL-2 in the medium. Table 7 shows different IC50s calculated through the sigmoid 4PL equation for the MFI of each marker.

[0205]

Table 8

[0206] The effect of mAb1 is specific to Vδ2+ T cells and was not observed in other immune cell populations: Vδ2- T cells, B cells, monocytes. This indicates good prospects for the safe use of mAb1 as an anti-inflammatory treatment, particularly in gastrointestinal inflammatory disorders where activated δ2T cells are particularly involved.

[0207] Kinetics of the in vitro effect of mAb1 on phosphoantigen-mediated Vγ9Vδ2T cell activation PBMC from three healthy donors were stimulated with 0.5 μM phosphoantigen (Hmbpp) + 50 IU / ml IL-2 in the presence of 10 μg / ml mAb1 or the corresponding control isotype at the indicated time points. The same markers were evaluated by flow cytometry.

[0208] The results show that mAb1 can also inhibit the expression of all the markers evaluated from 24 hours after the start of in vitro treatment until the end of the 6-day culture, indicating the long-term in vitro effect of the antibody.

[0209] Evaluation of the potential effect of mAb1 on various peripheral immune compartments with or without stimulation PBMCs from 3 healthy donors or erythrocyte-depleted whole blood from 8 healthy donors were cultured with 10 μg / ml of Hu103.2 or the corresponding control isotype with or without various stimuli for 2 days and 5 days. The stimuli used were phosphoantigen (HmBPP) known to activate Vγ9Vδ2 T cells; IL-15 known to activate Vγ9Vδ2 T cells, NK cells and CD8+ αβ T cells; phytohemagglutinin known to activate lymphocytes, anti-IgA / G / M known to activate B cells, TLR agonists (lipopolysaccharide known to activate monocytes, macrophages, dendritic cells and B cells, R848 known to activate myeloid cells, plasmacytoid dendritic cells and B cells, CpGB known to activate B cells and monocytes). After 2 days of culture for marker expression and 5 days for proliferation capacity, the expression of HLA-DR (antigen-presenting molecule), costimulatory molecules (CD40 and CD86), activation markers (CD69 and CD25), CCR9 (intestinal homing receptor), CD11c (adhesion molecule) and the proliferation capacity of various peripheral immune cell compartments (Vγ9Vδ2 T cells, B cells, monocytes, NK cells, CD8+ and CD8-αβ T cells) were evaluated by flow cytometry. Proliferation was evaluated using CTV-stained PBMCs.

[0210] The results show that mAb1 inhibits the expression of all markers evaluated and the proliferation capacity of Vγ9Vδ2 T cells only in the response to phosphoantigen. Interestingly, the observed inhibition was specific to Vγ9Vδ2 T cells as mAb1 did not affect the proliferation capacity of any of the other immune cell compartments evaluated in the response to either activation markers or any of the tested stimuli.

[0211] These results show that mAb1 can specifically inhibit phosphoantigen-driven activation of Vγ9Vδ2 T cells. These results were confirmed using erythrocyte-depleted whole blood from 8 healthy donors.

[0212] Example 3: In vitro and in vivo efficacy mAb1 inhibits the phosphoantigen-induced Vγ9Vδ2 T cell activation phenotype, proliferation, and degranulation from IBD patients PBMCs were isolated from the blood of patients with Crohn's disease or ulcerative colitis, stained with 1 μM CTV dye, and cultured for 4 days in the presence of IL-2 / IL-5, 1 nM HDMAPP (phosphoantigen), and 1 μg / ml control isotype or humanized 103.2 mAb. The expression of several markers (CD25, HLA-DR, β7 integrin, CD49b), the proliferation and degranulation ability of Vδ2+ and Vδ2- (measured by CTV dilution and CD107a expression, respectively) were evaluated by flow cytometry. The frequency of Vδ2+ T cells was equivalent between non-IBD individuals and IBD individuals (data not shown). Peripheral Vδ2+ T cells from non-IBD patients and IBD patients similarly responded well to phosphoantigen and were not affected when an isotype control was added to the culture.

[0213] In contrast, the addition of mAb1 similarly inhibits phosphoantigen-mediated activation of Vδ2+ T cells from non-IBD patients and IBD patients (Figures 2A and B). This indicates that mAb1 can inhibit the ex vivo activation, gut homing ability, proliferation, and degranulation ability of peripheral Vδ2+ T cells from IBD patients. Therefore, mAb1 has the ability to reduce the migration of peripheral Vδ2+ T cells to the gut, thus reducing the inflammatory potential of peripheral Vδ2+ T cells.

[0214] These results were confirmed in intestinal biopsies from non-IBD patients and IBD patients. Intestinal biopsies were placed in culture for an "overnight walkout" culture. The next day, the biopsies were removed and the walkout cells were stained with 1 μM CTV dye. The CTV-stained cells were then incubated for 7 days in the presence of IL-2 / IL-5, 10 nM HDMAPP (phosphoantigen), and 1 μg / ml control isotype or mAb1.

[0215] mAb1 was also able to inhibit phosphoantigen-mediated activation of Vδ2+ T cells derived from intestinal biopsies of IBD patients, which led to a reduction in intestinal inflammation and suppression of the disease (Figure 3). mAb1 inhibits in vivo the clinical signs associated with DSS-induced colitis in cynomolgus monkeys The DSS-induced colitis model in non-human primates is known to resemble the characteristics of gastrointestinal disorders such as human ulcerative colitis, and thus this model has become a tool for studying the important immunological mechanisms and efficacy of new treatments (Takahashi N et al., 2020, Heliyon, 6(1):e03178; McQueen P et al., 2019, Mucosal Immunol, 12(6):1327).

[0216] BTN3A orthologs and Vγ9Vδ2 T cells are present in non-human primates. Therefore, the therapeutic efficacy of mAb1 was tested in an acute DSS-induced colitis model in cynomolgus monkeys.

[0217] In this study, a total of 14 naive male cynomolgus monkeys (3 - 5 kg) were enrolled in two groups. DSS was dissolved in saline to a concentration of 40 mg / mL and protected from light. From day 0, each monkey was orally administered 0.65 g DSS (dissolved in water) twice a day for 7 days. mAb1 or its corresponding isotype control was administered intravenously at a dose of 0.6 mg / kg on days 3, 10, and 17. Throughout the study, weight loss and disease activity index (DAI) were monitored daily. The clinical severity of colitis was evaluated by diarrhea (0, normal stool consistency; 1, soft stool; 2, pasty stool; and 3, watery stool), intestinal bleeding (0, no bleeding; 1, fecal occult blood positive; 2, visible bloody stool; 3, massive rectal bleeding), and weight loss (0, ≤1%; 1, 1 - 5%; 2, 5 - 10%; 3, 10 - 15%; and 4, >15%). To calculate the DAI, the above scores were summed and then divided by 3. Serum samples were collected at the indicated time points and stored until cytokine analysis. Cytokine analysis was performed using the MSD technology and the V-Plex NHP cytokine 24Plex kit. The study continued until day 21 (Figure 4).

[0218] As shown in FIG. 5, the clinical signs associated with DSS-induced colitis (intestinal bleeding, diarrhea, weight loss, disease activity index) decreased after injection of mAb1 compared to monkeys injected with the corresponding isotype control, indicating that mAb1 has potential therapeutic benefits in the treatment of IBD.

[0219] Furthermore, as shown in FIG. 6, the circulating levels of the inflammatory cytokines IL-6 and IL-8 were lower in animals treated with mAb1 compared to those treated with the isotype control. At the same time, the levels of the circulating immune defenses IL-17 and VEGF were increased in animals treated with mAb1 compared to those treated with the isotype control, confirming that mAb1 can bring about a less inflammatory environment in IBD.

[0220] Sequence Listing Tables 8 and 9: Brief description of useful amino acid and nucleotide sequences for carrying out the present invention [Table 9] [Table 10] TIFF2025516472000011.tif158149 TIFF2025516472000012.tif138149

Claims

1. An isolated anti-BTN3A antibody for use in the treatment of gastrointestinal inflammatory disorders in a human subject in need of treatment for a gastrointestinal inflammatory disorder, such as inflammatory bowel disease, wherein said anti-BTN3A antibody specifically binds to BTN3A1, and said anti-BTN3A antibody inhibits degranulation of γδ T cells in vitro with an IC50 of 10 nM or less, preferably 1 nM or less, as determined, for example, by a CD107 degranulation assay by flow cytometry, and is selected from among anti-BTN3A antibodies that inhibit degranulation of γδ T cells in vitro.

2. An isolated anti-BTN3A antibody for use in the treatment of inflammatory bowel disease in a human subject in need of treatment for inflammatory bowel disease, wherein said antibody is (i) antibody mAb1 having a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 22, (ii) a variant of mAb1 having a heavy chain variable region (VH) of SEQ ID NO: 7 and a light chain variable region (VL) of SEQ ID NO: 8 but having a different constant region, (iii) a variant of mAb1 having an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5 and an LCDR3 of SEQ ID NO: 6 but having a different framework region, or (iv) a variant of mAb1 that binds to the same epitope as mAb1, wherein said epitope comprises SEQ ID NO: 20 or consists essentially of SEQ ID NO: 20, and said variant does not have an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 23 and an LCDR3 of SEQ ID NO: 6 and is selected from the group consisting of isolated anti-BTN3A antibodies.

3. A variant of mAb1 having an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5 and an LCDR3 of SEQ ID NO: 6, wherein the VH amino acid sequence has at least 90% but less than 100% identity, preferably at least 95% identity, to SEQ ID NO: 7, and the VL amino acid sequence has at least 90% but less than 100% identity, preferably at least 95% identity, to SEQ ID NO: 8, for use according to Claim 2.

4. K of 10 nM or less as measured by surface plasmon resonance Ｄ , preferably 1 nM or less K Ｄ , typically measured by surface plasmon resonance (SPR) assay, 1.10 －１１ ~1.10 －９ K between M Ｄ 4. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 3, which binds to the human BTN3A1 isoform with an EC50 of 0.1 μg / mL or less, preferably 0.05 μg / mL or less, for example an EC50 of between 0.1 μg / mL and 0.005 μg / mL, such as about 0.02 μg / mL.

5. A functional variant of mAb1 that retains at least a substantial proportion, preferably at least 90% of the affinity of mAb1 as measured by SPR assay, having the following characteristics: (i) The property of inhibiting in vitro degranulation of γδ T cells with an EC50 of 10 nM or less, preferably 1 nM or less, as determined by, for example, a CD107 degranulation assay by flow cytometry in co-culture with the Daudi Burkitt lymphoma cell line; (ii) The property of substantially inhibiting phosphoantigen-mediated activation, gut homing ability, proliferation and degranulation ability of patient-derived peripheral Vδ2+ T cells, as determined by, for example, an ex vivo assay of peripheral blood mononuclear cells (PBMCs) isolated from patients suffering from inflammatory bowel disease; and / or (iii) The property of substantially inhibiting phosphoantigen-mediated activation and / or proliferation of gut Vδ2+ T cells derived from intestinal biopsies of patients with inflammatory bowel disease, as determined by, for example, CD25 or HLA-DR expression in an ex vivo assay with walkout cells An isolated anti-BTN3A antibody for use according to any one of claims 1 to 4, having at least one or more of the above characteristics.

6. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 5, which is a human or humanized antibody.

7. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 6, wherein the anti-BTN3A antibody has a mutated or chemically modified IgG1 constant region that does not confer or reduces binding to Fcγ receptors and / or ADCC-mediated activity when compared to the corresponding antibody having an IgG Fc region, preferably wild-type IgG1, for example a mutated or chemically modified IgG1 constant region having the following amino acid substitutions: L247F, L248E and P350S.

8. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 7, wherein the gastrointestinal inflammatory disorder is inflammatory bowel disease.

9. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 8, wherein the gastrointestinal inflammatory disorder is ulcerative colitis or Crohn's disease.

10. An isolated anti-BTN3A antibody for use according to any one of claims 1 to 8, wherein a dose of 1 to 100 mg is administered intravenously to a subject.

11. The isolated anti-BTN3A antibody according to any one of claims 1 to 10, wherein the anti-BTN3A antibody is preferably administered simultaneously or separately in combination with an anti-inflammatory treatment selected from anti-cytokine antibodies (anti-IL-12, anti-IL-23, anti-TNFα), anti-α4β7 integrin antibodies, and JAK inhibitors.

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