BTLA agonist antibodies and uses thereof

Abstract

The present invention relates to anti-human BTLA agonist antibodies and their use for the prevention or treatment of inflammatory or autoimmune diseases or disorders such as systemic lupus erythematosus or graft versus host disease.

Description

Field of the Invention

[0001] The present disclosure belongs to the field of medicine. More specifically, the present disclosure relates to agonistic antibodies against human B and T lymphocyte attenuator (BTLA), compositions comprising such BTLA agonistic antibodies, and methods of using such BTLA agonistic antibodies for treating autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders. Background of the Invention

[0003] BTLA, also known as cluster of differentiation 272 or CD272, is a member of the Ig superfamily and is part of a family of checkpoint receptors that negatively regulate immune cell activation. The natural ligand for BTLA is a member of the TNF receptor superfamily, herpes virus entry mediator (HVEM or CD270). BTLA exerts a negative effect on the proliferation and activation of B cells and T cells through the engagement of HVEM. Dysregulation of the BTLA / HVEM pathway is associated with inflammatory and autoimmune diseases and disorders. Therefore, agonists for BTLA may be useful in preventing and / or treating autoimmune disorders, allergic diseases, asthma or other inflammatory disorders.

[0004] BTLA agonist antibodies have been disclosed, for example, in PCT patent application publications WO 2018 / 213113, WO 2021 / 250419, and WO 2022 / 087441. However, no BTLA agonist antibodies have been approved for therapeutic use, and thus there remains a need to develop alternative BTLA agonist antibodies that can be used to treat autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders. SUMMARY OF THE INVENTION

[0006] Accordingly, the present disclosure provides novel BTLA agonist antibodies. The antibodies of the present invention are particularly advantageous over prior art BTLA antibodies for a variety of reasons, including, but not limited to, the following: 1) they bind to human BTLA and cynomolgus monkey BTLA with comparable affinity and desirable on- and off-rates, 2) they are non-HVEM blocking BTLA agonists, resulting in enhanced agonism in the presence of HVEM, 3) they inhibit primary human T cell proliferation, 4) they do not cause significant cytokine release, 5) they exhibit enhanced efficacy as a monotherapy for the treatment and / or prevention of conditions such as autoimmune conditions, allergic diseases, asthma or other inflammatory conditions, 6) they are resistant to HVEM blocking BTLA agonists, 7) they are resistant to HVEM blocking BTLA agonists, 8) they are resistant to HVEM blocking BTLA agonists, 9) they are resistant to HVEM blocking BTLA agonists, 10) they are resistant to HVEM blocking BTLA agonists, 11) they are resistant to HVEM blocking BTLA agonists, 12) they are resistant to HVEM blocking BTLA agonists, 13) they are resistant to HVEM blocking BTLA agonists, 14) they are resistant to HVEM blocking BTLA agonists, 15) they are resistant to HVEM blocking BTLA agonists, 16) they are resistant to HVEM blocking BTLA agonists, 17) they are resistant to HVEM blocking BTLA agonists, 18) they are resistant to HVEM blocking BTLA agonists, 19) they are resistant to HVEM blocking BTLA agonists, 20) they are resistant to HVEM blocking BTLA agonists, 21) they are resistant to HVEM blocking BTLA agonists, 22) they are resistant to HVEM blocking BTLA ) they have very limited internalization in all subsets of human peripheral blood mononuclear cells (PBMCs) tested, including T cells, B cells, monocytes, myeloid DCs, pDCs, and NK cells, 7) they have low immunogenicity, 8) they are therapeutically effective at lower doses or less frequent dosing, and / or 9) they demonstrate in vivo stability, physical and chemical stability including, but not limited to, thermal stability, solubility, low self-association, and other pharmacokinetic properties that are acceptable for development, manufacture, formulation, storage, administration, and use in autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

[0007] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), and the LCVR comprises a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein:

[0008] a. HCDR1 comprises TFSGFSLSTXXVGVG (SEQ ID NO: 7), wherein X at position 10 is S, G, or P; and X at position 11 is G or A;

[0009] b. HCDR2 comprises XXFWXGDKR (SEQ ID NO: 11), wherein X at position 1 is L or Q; X at position 2 is I or E; and X at position 5 is N or T;

[0010] c. HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16);

[0011] d. LCDR1 contains RASQGISSWLA (SEQ ID NO: 1);

[0012] e. LCDR2 comprises YAASGLQS (SEQ ID NO: 2); and

[0013] f. LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

[0014] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises LIFWNGDKR (SEQ ID NO: 12), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0015] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises QEFWTGDKR (SEQ ID NO: 13), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0016] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprises QIFWTGDKR (SEQ ID NO: 14), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0017] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprises LEFWTGDKR (SEQ ID NO: 15), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0018] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4.

[0019] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, or 21, and the LCVR comprises the amino acid sequence of SEQ ID NO: 4.

[0020] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

[0021] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region of human IgG2, human IgG4, or modified human IgG4 subtype.

[0022] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region, the heavy chain constant region is of the human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

[0023] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region, the heavy chain constant region being a modified human IgG4 subtype comprising an S228P substitution in the hinge region of human IgG4 (EU numbering), also known as IgG4P (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767).

[0024] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is a modified human IgG4 subtype comprising the amino acid sequence of SEQ ID NO: 23.

[0025] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 24, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 5.

[0026] In some embodiments, the present disclosure provides antibodies that bind to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and each light chain comprises the amino acid sequence of SEQ ID NO: 5.

[0027] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HC comprising the amino acid sequence of SEQ ID NO:26, and a LC comprising the amino acid sequence of SEQ ID NO:5.

[0028] In some embodiments, the present disclosure provides antibodies that bind to human BTLA, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and each light chain comprises the amino acid sequence of SEQ ID NO: 5.

[0029] In another aspect, provided herein are nucleic acids encoding the heavy or light chains, or HCVR or LCVR of the novel human BTLA agonist antibodies described herein, and vectors or cells comprising such nucleic acids.

[0030] In another aspect, provided herein are pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding the same as described herein. Pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding the same as described herein can be used to treat autoimmune disorders, allergic diseases, or other inflammatory disorders, including but not limited to acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (Guillain-Barre syndrome), and inflammatory bowel disease. syndrome (GBS), multiple sclerosis (MS), myasthenia gravis, progressive systemic sclerosis (pSS), atopic dermatitis (AtD), enzyme replacement therapy (ERT), factor VIII deficiency, myositis, lupus nephritis (LN), organ and tissue transplantation, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, and vasculitis. Detailed Description of the Invention

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the human BTLA agonist antibodies, pharmaceutical compositions, and methods provided herein.

[0033] Furthermore, referring to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. The indefinite article "a" or "an" therefore usually means "at least one".

[0034] definition

[0035] As used herein, "about" means within a statistically significant range of one or more values ​​(e.g., as specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc.). Such values ​​or ranges may typically be on the order of 20%, more typically 10%, and even more typically 5% of a given value or range. The permissible variations encompassed by "about" will depend on the specific system under study and can be readily understood by those skilled in the art.

[0036] As used herein, and with reference to one or more receptors, "activity," "activate," "activating," and the like refer to the ability of a compound, such as a BTLA agonist antibody as described herein, to bind to and induce or increase a response, activity, or function of a target protein, such as receptor BTLA, as measured using assays known in the art, such as the in vitro assays described below.

[0037] As used herein, "amino acid" means a molecule that, from a chemical point of view, is characterized by the presence of one or more amine groups and one or more carboxylic acid groups, and may contain other functional groups. As is known in the art, there is a set of twenty amino acids that are designated as standard amino acids and can be used as building blocks for peptides / proteins produced by any organism. The amino acid sequences in the present disclosure contain the standard one-letter or three-letter codes for the twenty naturally occurring amino acids.

[0038] As used herein, the term "antibody" refers to a modified, non-naturally occurring polypeptide complex, including a complete antibody and any antigen-binding fragment thereof (i.e., the "antigen-binding portion" of an antibody). As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Exemplary antibodies of the present disclosure are immunoglobulin G (IgG) type antibodies comprising four polypeptide chains (two heavy chains and two light chains interconnected by disulfide bonds). Each heavy chain is composed of an N-terminal heavy chain variable region (HCVR) and a heavy chain constant region (composed of three domains, CH1, CH2, and CH3). Each light chain is composed of an N-terminal light chain variable region (LCVR) and a light chain constant region. The HCVR and LCVR can be further subdivided into highly variable regions designated as complementarity determining regions (CDRs), which are separated by more conserved regions designated as framework regions (FRs). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.Assignment of amino acid residues to CDRs may be accomplished according to well-known schemes, including those described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDRLoop Conformations," Journal of Molecular Biology, 274, 927-948 (1997)), and North et al., "A New Clustering of Antibody CDRLoop Conformations," Journal of Molecular Biology. Biology, 406, 228-256 (2011)), or IMGT (International ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212). The combination of IMGT and North CDR definitions is used for the exemplified anti-human BTLA antibodies as described herein.

[0039] In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of a hinge, a CH1 domain, a CH2 domain, and a CH3 domain. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0040] Antibodies may be of any of the commonly known isotypes, including but not limited to IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the antibodies described herein are of the human IgG2 or IgG4 subtype, or modified human IgG2 or IgG4 subtype.

[0041] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" refers to the portion of an antibody that contains a binding domain that interacts with an antigen, including, but not limited to, a "Fab fragment" containing the variable and constant domains of a light chain and the variable and first constant domain (CH1) of a heavy chain; and a "F(ab')2 fragment" comprising a pair of Fab fragments covalently linked near their carboxyl termini, typically by hinge cysteines therebetween. Other chemical couplings of antibody fragments are also known in the art, such as single-chain variable region fragments (scFv) comprising the HCVR and LCVR of an antibody, wherein the two domains are connected by a flexible linker peptide.

[0042] BTLA is a co-inhibitory receptor that plays a role in downregulating immune responses and preventing autoimmunity. Therefore, as used herein, "BTLA agonist antibody" refers to an antibody or antigen-binding fragment thereof that binds to human BTLA and enhances its co-inhibitory signaling for T cells and / or B cells, and when administered in vivo, results in at least one significantly attenuated autoimmune activity, such as a decrease in anti-double-stranded DNA (ds-DNA) titer, a decrease in disease score, or a decrease in inflammatory cytokines.

[0043] The agonist antibodies of the present invention can be used to prevent or treat autoimmune disorders, allergic diseases, asthma or other inflammatory disorders.

[0044] As used herein, the terms "autoimmune disease" or "autoimmune disorder" are used interchangeably herein and refer to an undesirable condition arising from an inappropriate or unwanted immune response against one's own cells and / or tissues or transplanted cells and / or tissues. The terms "autoimmune disease" or "autoimmune disorder" are intended to include such conditions, whether mediated by a humoral or cellular immune response. Exemplary autoimmune diseases or disorders include, but are not limited to, acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), multiple sclerosis (MS), myasthenia gravis, progressive systemic sclerosis (pSS), atopic dermatitis (AtD), enzyme replacement therapy (ERT), factor VIII deficiency, myositis, lupus nephritis (LN), organ and tissue transplantation, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, and vasculitis.

[0045] As used herein, "hBTLA" or "human BTLA" refers to wild-type human BTLA, preferably wild-type human BTLA having the amino acid sequence set forth in SEQ ID NO: 29. The amino acid sequence of Balbc mouse BTLA is given by SEQ ID NO: 30, the amino acid sequence of mouse C57BL6 is given by SEQ ID NO: 31, and the amino acid sequence of cynomolgus monkey BTLA is given by SEQ ID NO: 32.

[0046] As used herein, unless otherwise indicated, the terms "bind" and "binds" are intended to mean the ability of a protein or molecule to form a chemical bond or mutual attraction with another protein or molecule that results in proximity of the two proteins or molecules as determined by common methods known in the art.

[0047] The terms "cynomolgus," "cynomolgus," or "cynomolgus monkey" are used interchangeably herein. When used in reference to a BTLA polypeptide, the term is intended to refer to wild-type cynomolgus monkey BTLA, and preferably wild-type cynomolgus monkey BTLA having the amino acid sequence set forth in SEQ ID NO: 32.

[0048] As used herein, an "effective amount" means an amount or dosage of one or more BTLA agonist antibodies disclosed herein, or a pharmaceutically acceptable salt thereof, which, following single or multiple dose administration to an individual in need thereof, provides the desired effect in such individual under diagnosis or treatment (i.e., is likely to produce a clinically measurable difference in the individual's condition, such as a reduction in proinflammatory cytokines or a change in lymphocyte activation). An effective amount can be readily determined by one skilled in the art by using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, many factors are taken into consideration, including, but not limited to, the species of mammal, its size, age, and general health, the specific disease or condition involved, the extent, involvement, or severity of the disease or condition, the response of the individual, the specific antibody being administered, the mode of administration, the bioavailability characteristics of the administered formulation, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.

[0049] A BTLA polypeptide "extracellular domain" or "ECD" refers to a form of the BTLA polypeptide that is substantially free of the transmembrane domain and the cytoplasmic domain. Preferably, the BTLA ECD has less than 1% transmembrane domain and cytoplasmic domain, and more preferably, the BTLA ECD has less than 0.5% of such domains. Even more preferably, the human BTLA ECD polypeptide is as set forth in SEQ ID NO:28, and the cynomolgus monkey BTLA ECD polypeptide is as set forth in SEQ ID NO:33 or SEQ ID NO:34. BTLA polypeptide ECDs may be prepared using methods known in the art. Alternatively, human BTLA polypeptides or human BTLA ECD polypeptides are commercially available from various suppliers, such as Sino Biological (Houston, Texas; see, e.g., Catalog Nos. 11895-H02H or 29982-H38H).

[0050] "Fc region" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an antibody heavy chain, wherein the C-terminal polypeptide sequence is the sequence that can be obtained by papain digestion of an intact antibody. The Fc region may comprise a native or variant Fc sequence. The Fc sequence of an antibody generally comprises two constant domains: a CH2 domain and a CH3 domain. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of an antibody heavy chain. The Fc region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0051] When expressed in certain biological systems, antibodies with human Fc sequences are glycosylated in the Fc region. Typically, glycosylation occurs at highly conserved N-glycosylation sites in the Fc region of the antibody. N-glycans are typically attached to asparagine. Antibodies may also be glycosylated at other positions.

[0052] As used herein, the term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a mixed epitope. The term "epitope" may be used to refer to a structural epitope. According to some embodiments, a structural epitope may be used to describe the region of an antigen covered by an antibody (e.g., the footprint of an antibody when it binds to an antigen). In some embodiments, a structural epitope may be described as an amino acid residue of an antigen within a specified proximity of the amino acid residues of the antibody (e.g., within a specified number of angstroms). The term "epitope" may also be used to refer to a functional epitope. According to some embodiments, a functional epitope may be used to describe the amino acid residues of an antigen that interact with the amino acid residues of the antibody in a manner that contributes to the binding energy between the antigen and the antibody. An epitope can be determined according to different experimental techniques, also referred to as "epitope mapping technology." It should be understood that the determination of an epitope may vary based on the different epitope mapping technologies used, and may also vary due to the different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed., 2018; Holst et al., Molecular Pharmacology 1998, 53(1): 166-175), and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0053] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, including molecules containing single-stranded and / or double-stranded nucleotides, such as DNA, cDNA, and RNA molecules, which incorporate natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or synthesis reactions.

[0054] As used herein, the term "subject" refers to mammals, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is a human.

[0055] As used herein, the term "treatment" or "treating" refers to all processes in which there may be a slowing, control, delay or cessation of the progression of a disease or condition disclosed herein, or an amelioration of disease or condition symptoms, but does not necessarily indicate a complete elimination of all disease or condition symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a disease or condition in a patient, particularly a human.

[0056] In one aspect, provided herein are novel antibodies or antigen-binding fragments thereof that bind to human BTLA. In some embodiments, the novel antibodies or antigen-binding fragments thereof that bind to human BTLA are agonists of BTLA. In some embodiments, the novel antibodies or antigen-binding fragments thereof that bind to human BTLA and are agonists provided herein can induce or increase one or more activities or functions associated with human BTLA, such as one or more activities or functions described in the Examples.

[0057] In some embodiments, the novel antibodies or antigen-binding fragments thereof that bind to human BTLA comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), and the LCVR comprises a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein:

[0058] a. HCDR1 comprises TFSGFSLSTXXVGVG (SEQ ID NO: 7), wherein X at position 10 is S, G, or P; and X at position 11 is G or A;

[0059] b. HCDR2 comprises XXFWXGDKR (SEQ ID NO: 11), wherein X at position 1 is L or Q; X at position 2 is I or E; and X at position 5 is N or T;

[0060] c. HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16);

[0061] d. LCDR1 contains RASQGISSWLA (SEQ ID NO: 1);

[0062] e. LCDR2 comprises YAASGLQS (SEQ ID NO: 2); and

[0063] f. LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

[0064] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises LIFWNGDKR (SEQ ID NO: 12), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0065] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises QEFWTGDKR (SEQ ID NO: 13), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0066] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprises QIFWTGDKR (SEQ ID NO: 14), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0067] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a LCVR and a HCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprises LEFWTGDKR (SEQ ID NO: 15), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0068] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4.

[0069] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, or 21, and the LCVR comprises the amino acid sequence of SEQ ID NO: 4.

[0070] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

[0071] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region of human IgG2, human IgG4, or modified human IgG4 subtype.

[0072] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region, the heavy chain constant region is of the human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

[0073] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region, the heavy chain constant region being a modified human IgG4 subtype comprising an S228P substitution in the hinge region of human IgG4 (EU numbering), also known as IgG4P (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767).

[0074] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO: 4, and wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is a modified human IgG4 subtype comprising the amino acid sequence of SEQ ID NO: 23.

[0075] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 24, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 5.

[0076] In some embodiments, the present disclosure provides antibodies that bind to human BTLA, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and each light chain comprises the amino acid sequence of SEQ ID NO: 5.

[0077] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises a HC comprising the amino acid sequence of SEQ ID NO:26, and a LC comprising the amino acid sequence of SEQ ID NO:5.

[0078] In some embodiments, the present disclosure provides antibodies that bind to human BTLA, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and each light chain comprises the amino acid sequence of SEQ ID NO: 5.

[0079] In some embodiments, the novel BTLA agonist antibodies or antigen-binding fragments thereof provided herein include a HCVR comprising a sequence having at least 95% sequence identity to a HCVR in Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein include a LCVR comprising a sequence having at least 95% sequence identity to a LCVR in Table 1. In some embodiments, the novel BTLA agonist antibodies or antigen-binding fragments thereof provided herein include a HCVR and / or LCVR in Table 1, or a sequence having at least 95% sequence identity to a HCVR and / or LCVR in Table 1.

[0080] In another aspect, provided herein are nucleic acids encoding the heavy or light chains, or HCVR or LCVR of the novel human BTLA agonist antibodies described herein, and vectors or cells comprising such nucleic acids.

[0081] In another aspect, provided herein are pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding the same as described herein. Pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding the same as described herein can be used to treat autoimmune disorders, allergic diseases, or other inflammatory disorders, including but not limited to acute or chronic GVHD, chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, pSS, AtD, ERT, factor VIII deficiency, myositis, LN, organ and tissue transplantation, T1DM, autoimmune vasculitis, pernicious anemia, and vasculitis.

[0082] In some embodiments, the present disclosure provides a nucleic acid comprising a sequence encoding the amino acid sequence of SEQ ID NO: 5, 24, or 26.

[0083] In some embodiments, the present disclosure provides a vector comprising 1) a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. In some embodiments, the composition includes a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5.

[0084] For example, after the nucleic acid has been operably linked to an expression control sequence, the nucleic acid of the present disclosure may be expressed in a host cell. Expression control sequences capable of expressing the nucleic acids to which they are operably linked are well known in the art. An expression vector may include a sequence encoding one or more signal peptides that promote the secretion of the polypeptide from the host cell. An expression vector containing a nucleic acid of interest (e.g., a nucleic acid encoding the heavy or light chain of an antibody) may be transferred into a host cell by well-known methods (e.g., stable or transient transfection, transformation, transduction, or infection). In addition, an expression vector may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to help detect host cells transformed with the desired nucleic acid sequence.

[0085] In another aspect, provided herein are cells, e.g., host cells, comprising a nucleic acid, vector, or nucleic acid composition described herein. The host cell may be stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the heavy and light chain polypeptides of an antibody disclosed herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the heavy chain polypeptide of an antibody described herein and a second vector expressing the light chain polypeptide. Such host cells, e.g., mammalian host cells, can express antibodies or antigen-binding fragments thereof that bind to human BTLA as described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells. Preferably, CHO host cells or derivatives thereof, such as CHO-K1, CHO-S, GS-CHO, CHO-DG44, CHOK1SV, or GS-CHOK1SV, are used, for example, to express the human BTLA agonist antibodies disclosed herein.

[0086] In some embodiments, the host cell comprises a vector comprising: 1) a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, wherein the cell is a mammalian cell. In some such embodiments, the mammalian cell is a CHO cell or a derivative thereof.

[0087] In some embodiments, the host cell comprises 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, wherein the cell is a mammalian cell. In some such embodiments, the mammalian cell is a CHO cell.

[0088] The present disclosure further provides methods for producing an antibody or antigen-binding fragment thereof that binds to human BTLA as described herein by culturing a host cell, e.g., a mammalian host cell, under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody from the culture medium. The culture medium into which the antibody or antigen-binding fragment thereof has been secreted may be purified by conventional techniques. Various methods for protein purification may be employed, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0089] The present disclosure further provides antibodies or antigen-binding fragments thereof produced by any of the methods described herein.

[0090] In another aspect, provided herein are pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents, or carriers. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0091] The human BTLA agonist antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions comprising such antibodies or antigen-binding fragments described herein can be used to treat autoimmune disorders, allergic diseases, or other inflammatory disorders, including, but not limited to, acute or chronic GVHD, chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, pSS, AtD, ERT, factor VIII deficiency, myositis, LN, organ and tissue transplantation, T1DM, autoimmune vasculitis, pernicious anemia, and vasculitis.

[0092] In some embodiments, provided herein are methods for treating an inflammatory disease or autoimmune disease in a subject (e.g., a human patient) in need thereof, comprising administering to the subject a therapeutically effective amount of a BTLA agonist antibody, or an antigen-binding fragment thereof disclosed herein. The BTLA agonist antibody, or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, described herein may be administered parenterally (e.g., subcutaneously and intravenously). In some embodiments, the BTLA-associated disease or disorder is an autoimmune disorder, an allergic disease, or other inflammatory disorder, including but not limited to acute or chronic GvHD, chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, pSS, AtD, ERT, factor VIII deficiency, myositis, LN, organ and tissue transplantation, T1DM, autoimmune vasculitis, pernicious anemia, and vasculitis.

[0093] Also provided herein are human BTLA agonist antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions comprising at least one such human BTLA agonist antibody or antigen-binding fragment thereof, for use in therapy. Example

[0094] The following examples are offered to illustrate but not to limit the invention.

[0095] Expression and purification of engineered BTLA agonist antibodies

[0096] The BTLA agonist antibodies of the present invention, or antigen-binding fragments thereof, can be expressed and purified essentially as follows. Appropriate host cells, such as HEK293 or CHO, can be transiently or stably transfected with an expression system for secreting the antibody or a single vector system encoding both the HC and LC using an optimal predetermined HC:LC vector ratio (e.g., 1:1, 1:2, or 1:3). The clarified culture medium into which the antibody or antigen-binding fragment thereof has been secreted may be purified using any of a variety of commonly used techniques. For example, the culture medium may be applied to a MabSelect column (Cytiva) or a KappaSelect column (Cytiva) for Fab fragments, which has been equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column may be washed to remove non-specifically bound components. The bound antibody or antigen-binding fragment may be eluted, for example, by a pH gradient (e.g., 20 mM tris buffer, pH 7.0 to 10 mM acetate / sodium citrate buffer, pH 3.0, or phosphate buffered saline, pH 7.4 to 100 mM glycine buffer, pH 3.0). Antibody fractions may be detected, for example, by SDS-PAGE and analytical size exclusion chromatography, and may then be pooled. Further purification is optional and depends on the intended use. The antibody or antibody fragment may be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is about 95% to about 99%. The product may be kept refrigerated, immediately frozen at -70°C, or may be lyophilized. The SEQ ID NOs for the amino acid sequences of certain exemplary BTLA agonist antibodies of the present invention are shown in Table 1 below.

[0097] Table 1. Amino acid sequences of exemplary BTLA agonist antibodies

[0098]

[0099] *All five exemplified antibodies share the same light chain.

[0100] Binding affinity and kinetics

[0101] The binding affinity and kinetics of the BTLA agonist antibodies (M10825 and M10824) disclosed herein to BTLA were measured by surface plasmon resonance using Biacore 8k (Cytiva). Binding affinity was measured by capturing the BTLA agonist antibodies with an anti-human Fc antibody (Cytiva) immobilized on a CM4 sensor chip (Cytiva) via amine coupling and flowing over recombinant human or cynomolgus monkey BTLA at a concentration prepared by 4-fold serial dilutions starting from 100 nM down to 0.1 nM. The experiments were performed at 37° C. in HBS-EP buffer (Teknova, H8022; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.6) supplemented with 0.01% BSA (Gibco). For each cycle, BTLA agonist antibody prepared at 5 μg / mL was flowed through the active flow cell at 10 μL / min to achieve approximately 120 RU of capture, followed by a 2-minute injection of human or cynomolgus monkey BTLA at 100 μL / min, followed by a 20-minute dissociation period. The chip surface was regenerated using 3M MgCl2 solution at 10 μL / min for 30 seconds. The data were fitted to a 1:1 Langmuir binding model to derive k on 、k off And calculate K D Following a procedure essentially as described above, the following parameters were observed (shown in Table 2). The data shown below are the average of three experiments.

[0102] Table 2: In vitro binding affinities of M10824 and M10825 for human and cynomolgus monkey BTLA

[0103] Antibody Analytes <![CDATA[K a(on) (1 / Ms)]]> <![CDATA[K d(off) (1 / s)]]> <![CDATA[K D (M)]]> M10824 BTLA 2.76E+06 5.33E-04 1.93E-10 Cynomolgus monkey BTLA 1.65E+06 3.91E-04 2.38E-10 M10825 BTLA 2.96E+06 4.14E-04 1.40E-10 Cynomolgus monkey BTLA 1.62E+06 3.81E-04 2.35E-10

[0104] BTLA agonist antibody-induced signaling

[0105] To determine the ability of the BTLA agonist antibodies (M10825 and M10824) of the present invention to induce BTLA activation, BTLA-overexpressing cells can be used. Jurkat BTLA signaling cell line and Bioassay Detection Kit (Eurofins DiscoverX). Jurkat BTLA cells co-expressed with ProLink TM The BTLA receptor is tagged with the SH2 domain of the enzyme receptor tag. Receptor activation and phosphorylation lead to the recruitment of SH2 to the receptor and force the two β-galactosidase fragments (enzyme receptor and ProLink) to TMThe resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0106] Immobilized plates were prepared by incubating anti-human IgG, Fcγ (Jackson Immunoresearch) at 200 nM followed by a blocking step. Titration of isotype control or BTLA antibody (0-300 nM) was captured and incubated for 2 hours at room temperature with shaking followed by a PBS wash step. Jurkat BTLA signaling cell line may be expressed at 10 x 10 3 Cells / well were added to the wells and incubated for 1 hour in a humidified tissue culture incubator at 37°C and 5% CO2. The phosphorylated BTLA was detected using the Bioassay Detection Kit and luminescence was read on an EnVision (Perkin Elmer). Luminescence values ​​were used to determine the IC using graphing software (GraphPad Prism). 50 value.

[0107] Following essentially the procedure described above, antibody M10825 resulted in a 2.4-fold increase in signal compared to the isotype control at the highest dose tested, 300 nM, and an IC of 2.3 nM. 50 Similarly, the BTLA agonist antibody M10824 resulted in a 2.3-fold increase in signal compared to the isotype control at the highest dose tested of 300 nM and an IC of 2.4 nM. 50 These data confirm that BTLA agonist antibodies (M10825 and M10824) BTLA phosphorylation was induced in JurkatBTLA cells, an immortalized human T lymphocyte cell line overexpressing BTLA.

[0108] HVEM non-blocking

[0109] To determine the ability of the BTLA agonist antibodies of the present invention (M10825 and M10824) to block HVEM ligand activation of BTLA, BTLA-overexpressing cells were used. Jurkat BTLA signaling cell line, U2OS HVEM Ligand cell line and Bioassay Detection Kit (Eurofins DiscoverX). Jurkat BTLA cells co-expressed with ProLink TMThe BTLA receptor is tagged with the SH2 domain of the enzyme receptor tag. Receptor activation and phosphorylation lead to the recruitment of SH2 to the receptor and force the two β-galactosidase fragments (enzyme receptor and ProLink) to TM The resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0110] Assay plates were prepared by seeding 20 x 10 3 indivual Jurkat BTLA cells / well were added for 15 minutes, after which a titration of isotype control or BTLA antibody (0-10 μg / mL) was added and the assay plates were incubated for 1 hour at 37° C. and 5% CO 2 incubator. U2OS HVEM Ligand cells were then cultured at 50 x 10 3 Cells / well were added and the plates were incubated for 2 hours at room temperature. The Bioassay Detection Kit detected phosphorylated BTLA and luminescence was read on an EnVision (PerkinElmer).Luminescence values ​​for the 3.3 μg / mL titration point are shown as a percentage of the signal obtained by a known HVEM non-blocker at that dose and compared to a known HVEM blocker.

[0111] Following essentially the procedure described above, antibody M10825 resulted in 104% of the signal of a known HVEM blocker (100% signal) at 3.3 μg / mL compared to an isotype control at 92%. Similarly, BTLA agonist antibody M10824 resulted in 104% of the signal of a known HVEM blocker (100% signal) at 3.3 μg / mL compared to an isotype control at 93%. The known HVEM blocker had 61% of the signal of a known HVEM non-blocker at a dose of 3.3 μg / mL. These data confirm that the BTLA agonist antibodies (M10825 and M10824) do not block HVEM-induced BTLA phosphorylation in Jurkat BTLA cells, an immortalized human T lymphocyte cell line overexpressing BTLA.

[0112] Inhibition of proliferation of primary human B cells

[0113] The in vitro efficacy of the BTLA agonist antibodies of the present invention was assessed by their ability to inhibit the proliferation of human primary B cells. Human primary B cells were isolated from healthy human peripheral blood mononuclear cells using a human B cell isolation kit (EasySep) and resuspended in an appropriate human primary cell culture medium. Anti-IgM was coated onto the plate along with a titration of isotype control or BTLA antibody (0-40 nM) and incubated at 37°C for 1 hour, followed by a PBS wash step. Isolated human B cells were added to each well and incubated at 37°C with 5% CO2 for 72 hours, followed by a PBS wash step for the final 18 hours. 3 [H]-thymidine pulse. After incubation, the plate was removed and stored at -80°C until ready to harvest. Cells were lysed by thawing and harvested using a FilterMate Universal Harvester (Perkin Elmer). 2 2450 Microplate Counter (Perkin Elmer) was used to measure 3 H]-thymidine incorporation was used to assess proliferation.

[0114] Counts were used to evaluate the relative proliferative response in the assay, and percent inhibition was calculated using the equation [% inhibition = (AVG maximum signal - sample signal) / AVG maximum signal x 100], which can be used to determine the IC using graphing software (GraphPad Prism). 50 value.

[0115] Following essentially the above-described procedure, the BTLA agonist antibody M10825 was able to inhibit primary B cell proliferation in vitro by 96% at the highest dose tested, with a calculated mean IC of 10.5 nM, compared to 61% inhibition of the isotype control (mean from 3 human blood donors). 50 Similarly, the BTLA agonist antibody M10824 was able to inhibit primary B cell proliferation in vitro by 98% at the highest dose tested, with a calculated IC of 196.40 nM, compared to 40% inhibition of the isotype control (average from 3 human blood donors). 50 These data demonstrate that the BTLA agonist antibodies M10825 and M10824 are able to inhibit B cell proliferation in vitro.

[0116] Efficacy of BTLA agonist antibodies in a humanized mouse model of GvHD

[0117] Since the BTLA agonist antibodies of the present invention do not excite murine BTLA, the in vivo efficacy of the BTLA agonist antibodies of the present invention may be tested in NOD SCID γ2 chain- / - (NSG) humanized mice to evaluate their ability to inhibit human T cell and B cell function in an in vivo environment. More specifically, the in vivo effect is evaluated in a humanized NSG mouse model of GvHD. The model is a mouse model that is engrafted with human peripheral blood mononuclear cells (PMBCs), whereby human immune cells recognize the mouse as foreign, become activated, and drive GvHD. The hallmark phenotypes of T cell and B cell activation are circulating human proinflammatory cytokines and immunoglobulins (Ig) in mouse plasma. Therefore, this model can be used to evaluate the ability of BTLA agonist antibodies (which do not excite murine BTLA) to inhibit the production of these circulating factors.

[0118] Briefly, studies using the NSG mouse model of GvHD might be performed as follows:

[0119] Female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, Stock#05557) were housed 4 / cage at 72°F under a 12-hour light: dark cycle and were allowed free access to food and water. Human peripheral blood mononuclear cells (PBMCs) were isolated from LRS tubes obtained from two donors (San Diego Blood Bank, San Diego, CA) using a SepMate 50Ficol preparation tube according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC). Freshly isolated PBMCs were isolated at 1.2 x 10 8 cells / mL suspended in PBS, and mice may be implanted on day 0 with 100 μL PBMC suspension (1.210 7 / mouse). On day 1, mice may be divided into weight groups and dosed subcutaneously with 10 mg / kg isotype control or a test BTLA antibody in a dose range of 0.001 to 10.0 mg / kg (200 μL / mouse). Dosing may be performed once a week for 15 days. Health checks and body weight measurements may be performed as usual. On day 15, mice may be sacrificed and blood may be collected by cardiac puncture under isoflurane anesthesia.

[0120] Plasma analysis: Blood from cardiac puncture can be collected into EDTA-coated tubes, clarified by centrifugation, and the resulting plasma can be stored at -80°C for future processing. Plasma human cytokines and Ig can be measured using the Mesoscale Discovery (MSD) Human Th1 / Th2 10-Vplex and Human Isotyping Panels, respectively (Rockville, Maryland).

[0121] In two separate experiments performed essentially as described above, antibody M10825 significantly reduced the levels of T cell-associated human proinflammatory cytokines interferon gamma (INF-γ), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) in a dose-dependent manner. More specifically, animals treated with antibody M10825 demonstrated statistically significant reductions in INF-γ and IL-10 at all doses tested (i.e., 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 mg / kg antibody) compared to animals treated with the isotype control. Similarly, animals treated with antibody M10825 demonstrated reductions in TNF-α compared to isotype controls at all doses tested (i.e., 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 mg / kg antibody), with the reductions resulting from doses of 0.1, 0.5, 1.0, 5.0, and 10.0 mg / kg antibody being statistically significant compared to isotype controls (data not shown).

[0122] In a second study, conducted essentially as described above but using PBMCs from plasma of different donors, animals treated with antibody M10825 demonstrated reductions in INF-γ, IL-10, and TNF-α compared to isotype controls at all doses tested (i.e., 0.001, 0.01, 0.1, 1.0, and 10.0 mg / kg antibody), with doses (i.e., 0.1, 1.0, and 10.0 mg / kg antibody) resulting in statistically significant reductions in INF-γ compared to isotype controls (data not shown).

[0123] Additionally, in a second study, plasma IgA and IgM levels were shown to be attenuated by the BTLA agonist antibody M10825 at all doses tested (i.e., 0.001, 0.01, 0.1, 1.0, and 10.0 mg / kg antibody) compared to isotype controls, with doses of 1.0 and 10.0 mg / kg antibody resulting in statistically significant reductions in IgM compared to isotype controls (data not shown).

[0124] Collectively, these data demonstrate that the BTLA agonist antibody M10825 is effective in preventing GvHD in vivo.

[0125] Plasma cytokines and plasma human IgA and IgM were measured by MSD assay and expressed as mean ± standard error of the mean. Differences were considered significant if p < 0.05 compared to isotype control using one-way ANOVA with Dunnett's post hoc test.

[0126] No or low internalization in subpopulations of human PBMCs

[0127] Antibody-based biotherapeutics with higher immunogenicity in the clinic have greater internalization levels than antibodies with lower immunogenicity (Melendez, et al., BIOANALYSIS, Vol. 14, No. 10 (2022)). Therefore, in vitro assays measuring antibody internalization in human peripheral blood mononuclear cells (PBMCs) may be used to evaluate the immunogenicity risk of antibodies. To analyze the internalization of the BTLA agonist antibodies of the present disclosure, human PBMCs were isolated from buffy coats of three donors following the guidance provided by StemCell Technologies for SepMate-50. PBMCs were counted with a Vi-Cell cell counter and adjusted to 2 x 10 in pre-warmed complete medium (X-Vivo 15, Lonza #04-418Q) supplemented with 10% FBS. 6 Cells were aliquoted into 96-well plates at 100 μl / well for a total of 2 x 10 5 The plate was placed in a humidified incubator at 37°C and 5% CO2.

[0128] Prepare 4XBTLA antibody (ie, 60nM) and 4X TAMRA-QSY7-Fab antibody (ie, 180nM) and mix with equal volume. Anti-BTLA / TAMRA-QSY7-Fab complex is formed by incubation at 4°C for 30 minutes. Anti-BTLA / TAMRA-QSY7-Fab complex is then aliquoted into PBMCs prepared above at 100 μl / well. The cells are incubated for 3 hours at 37°C and 5% CO2 in a humidified incubator. The cells are centrifuged at 500g for 5 minutes and then washed three times in BD Biosciences FACS staining buffer. The cells are then stained with a surface marker panel comprising the following 15 colors: Aqua live / dead cell dye, fluorophore-conjugated antibodies for CD45, CD3, CD4, CD8, CD19, CD14, CD16, HLA-DR, CD123, IgD, CD27, CD11c and CD56. This panel allows identification of T cells and their subtypes, B cells and their subtypes, classical and nonclassical monocytes, pDCs, myeloid DCs, and NK cells. An FMO control stain was included for gating. A secondary antibody-only control and an internal positive control were set up. An LSRFortessa X-20 was used for data acquisition. Instrument calibration was performed according to the manufacturer's instructions. A complementary panel was set up using complementary beads bound to the antibodies. FlowJo 10 was used for data analysis, and internalization was measured using MFI values ​​for each subpopulation. Dead cells were excluded from the analysis. Gating was set based on FMO staining.

[0129] Following essentially the same procedure as described above, BTLA agonist antibodies M10825 and M10824 and isotype controls were analyzed for internalization. Based on mean fluorescence intensity (MFI) values, both antibodies M10825 and M10824 showed low or no internalization in all cell types, including T cells, B cells, monocytes, myeloid DCs, pDCs, and NK cells. An internal positive control internalizing antibody showed very strong internalization across different cell types. No internalization was observed for the isotype control. Internalizing BTLA antibody clones identified in the earlier internalization screening process showed consistent internalization profiles. These data confirm that BTLA agonist antibodies M10825 and M10824 have limited internalization in all subpopulations of human PBMCs tested, indicating a low risk of immunogenicity.

[0130] Low immunogenicity risk of the BTLA agonist antibodies M10825 and M10824

[0131] BTLA agonist antibodies may be characterized for relative risk of clinical immunogenicity using in silico and in vitro methods, including but not limited to T cell proliferation assays, pre-existing reactivity assays, and MHC-associated peptide proteomics (MAPPs) assays.

[0132] MAPPs assay

[0133] MAPPs profile peptides presented by human leukocyte antigen class II (HLA-II) on human dendritic cells previously treated with the test molecule. Briefly, primary human dendritic cells from 10 normal human donors may be prepared by isolating CD-14 positive cells from buffy coats and differentiated into immature dendritic cells by incubation with 20 ng / ml IL-4 and 40 ng / ml GM-CSF in complete RPMI medium (Sigma-Aldrich, catalog # R0278) containing 5% Serum Replacement at 37°C and 5% CO2 for 4 days, essentially as described in, for example, Knierman, MD, et al., 2020. Then, 3 micromolar test antibodies may be added to approximately 5 x 10 cells on day 4. 6 cells, and fresh medium containing 5 μg / ml lipopolysaccharide (LPS) may be replaced after approximately 5 hours of incubation to allow the cells to convert into mature dendritic cells. The mature cells may be lysed the next day with 1 mL of RIPA buffer with protease inhibitors and DNase. An automated liquid handling system may be used to isolate HLA-II molecules from the thawed lysate using a biotinylated anti-pan-HLA class II antibody (clone Tu39). Bound receptor-peptide complexes may be eluted with 5% acetic acid, 0.1% trifluoroacetic acid (TFA). The eluted HLA-II peptides may be passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated HLA-II peptides may be analyzed by nano-LC / MS using a Thermo easy 1200n LC-HPLC system with a Thermo LUMOS mass spectrometer. Separation may be performed using a 75 μm x 15 cm PepMap RSLC c18 column for a 65 minute gradient with a flow rate of 300 nL / min and 0.1% formic acid in water as solvent A and 80% acetonitrile containing 0.1% formic acid as solvent B. Mass spectrometry may be run in full scan mode with a resolution of 240,000, followed by a 3 second data-dependent MS / MS cycle consisting of an ion trap rapid scan with high energy collisional dissociation (HCD) and electron transfer / high energy collisional dissociation fragmentation (EThcD) fragmentation.

[0134] A multiple search algorithm that may use enzyme search parameters excluding bovine / human databases containing the test antibody sequence may be used to generate peptide identifications through an internal proteomics process (see, e.g., Higgs, R.E., et al., Methods Mol. Biol., 428, 209 - 230 (2008)). A KNIME workflow may be used to process the identification files for the samples. The peptides identified from the test article may be aligned against the parental sequences. A summary may be created for all donors, annotating the percentage of donors showing non - germline residues, the number of different regions of peptides showing non - germline residues, and the peptide display depth at each region with non - germline residues.

[0135] In the MAPPs analysis performed essentially as described above, only 10% of the donors showed peptides containing non - germline residues in the heavy - chain CDR3 of antibody M10825. None of the other displayed peptides from M10825 contained non - germline residues and thus did not present an immunogenic risk.

[0136] Overall, the compilation of data from such various computer chips and in vitro methods (data not shown), including but not limited to the MAPPs assays performed essentially as described above, strongly supports that each of the BTLA agonist antibodies M10825 and M10824 has a low to moderate risk of inducing clinical immunogenicity.

[0137] Amino acid sequence and nucleotide sequence LCDR1: <SEQ ID NO:1; AA; synthetic construct>

[0138]

[0139] LCDR2: <SEQ ID NO:2; AA; synthetic construct>

[0140]

[0141] LCDR3: <SEQ ID NO:3; AA; synthetic construct>

[0142]

[0143] LCVR: <SEQ ID NO:4; AA; synthetic construct>

[0144]

[0145] Full - length LC: <SEQ ID NO:5; AA; synthetic construct>

[0146] <000037​DNA encoding full-length LC: <SEQ ID NO:6; DNA; synthetic construct>

[0148]

[0149] HCDR1s

[0150] <SEQ ID NO:7; AA; synthetic construct>

[0151]

[0152] where X at position 10 is S, G or P; X at position 11 is G or A. <SEQ ID NO:8; AA; synthetic construct>

[0153]

[0154] <SEQ ID NO:9; AA; synthetic construct>

[0155]

[0156] <SEQ ID NO:10; AA; synthetic construct>

[0157]

[0158] HCDR2s :

[0159] <SEQ ID NO:11; AA; synthetic construct>

[0160]

[0161] where X at position 1 is L or Q; X at position 2 is I or E; X at position 5 is N or T.

[0162] <SEQ ID NO:12; AA; synthetic construct>

[0163]

[0164] <SEQ ID NO:13; AA; synthetic construct>

[0165]

[0166] <SEQ ID NO:14; AA; synthetic construct>

[0167]

[0168] <SEQ ID NO:15; AA; synthetic construct>

[0169]

[0170] HCDR3 :

[0171] <SEQ ID NO:16; AA; Synthetic construct>

[0172]

[0173] HCVRs :

[0174] <SEQ ID NO:17; AA; Synthetic construct>

[0175]

[0176] where X at position 32 is S, G or P; X at position 33 is G or A; X at position 52 is L or Q; X at position 53 is I or E; X at position 56 is N or T.

[0177] M7944: <SEQ ID NO:18; AA; Synthetic construct>

[0178]

[0179] M10825 and M10824: <SEQ ID NO:19; AA; Synthetic construct>

[0180]

[0181] M10782: <SEQ ID NO:20; AA; Synthetic construct>

[0182]

[0183] M10469: <SEQ ID NO:21; AA; Synthetic construct>

[0184]

[0185] Constant region (IgG2): <SEQ ID NO:22; AA; Homo sapiens>

[0186]

[0187] Constant region (IgG4P): <SEQ ID NO:23; AA; Synthetic construct>

[0188]

[0189] Full-length heavy chain

[0190] M10825: <SEQ ID NO:24; AA; Synthetic construct>

[0191]

[0192] DNA encoding the heavy chain of M10825: <SEQ ID NO:25; DNA; Synthetic construct>

[0193]

[0194] M10824: <SEQ ID NO:26; AA; Synthetic construct>

[0195]

[0196] DNA encoding the heavy chain of M10824: <SEQ ID NO:27; DNA; Synthetic construct>

[0197]

[0198] Human BTLA ECD (SEQ ID NO:28)

[0199]

[0200] Human BTLA (SEQ ID NO:29)

[0201]

[0202] Mouse Balbc BTLA (SEQ ID NO:30)

[0203]

[0204] Mouse C57BL6 BTLA (SEQ ID NO:31)

[0205]

[0206] Cynomolgus monkey BTLA (SEQ ID NO:32)

[0207]

[0208] Cynomolgus monkey BTLA ECD variant (SEQ ID NO:33)

[0209]

[0210] Cynomolgus monkey BTLA ECD (SEQ ID NO:34)

[0211]

Claims

1. An antibody that binds to human B and T lymphocyte attenuator (BTLA), comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), and the LCVR comprises a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein: a. HCDR1 comprises TFSGFSLSTXXVGVG (SEQ ID NO: 7), wherein X at position 10 is S, G or P; X at position 11 is G or A; b. HCDR2 comprises XXFWXGDKR (SEQ ID NO: 11), wherein X at position 1 is L or Q; X at position 2 is I or E; and X at position 5 is N or T; c. HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16); d. LCDR1 contains RASQGISSWLA (SEQ ID NO: 1); e. LCDR2 comprises YAASGLQS (SEQ ID NO: 2); and f. LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

2. The antibody of claim 1 , wherein the LCVR comprises a LCDR1 comprising RASQGISSWLA (SEQ ID NO: 1), a LCDR2 comprising YAASGLQS (SEQ ID NO: 2), and a LCDR3 comprising QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises a. HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprising LIFWNGDKR (SEQ ID NO: 12), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or b. HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprising QEFWTGDKR (SEQ ID NO: 13), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or c. HCDR1 comprising TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprising QIFWTGDKR (SEQ ID NO: 14), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or d. HCDR1 comprising TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprising LEFWTGDKR (SEQ ID NO: 15), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16).

3. The antibody of claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 17 and the LCVR comprises the amino acid sequence of SEQ ID NO:

4.

4. The antibody of claim 3, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, or 21, and the LCVR comprises the amino acid sequence of SEQ ID NO:

4.

5. The antibody of any one of claims 1-4, wherein the antibody comprises a heavy chain constant region and a light chain constant region. The antibody according to claim 5 , wherein the heavy chain constant region is of human IgG2 subtype, human IgG4 subtype or modified human IgG4 subtype.

7. The antibody of claim 6, wherein the heavy chain constant region is of the human IgG2 subtype comprising the amino acid sequence of SEQ ID NO:

22.

8. The antibody of claim 6, wherein the heavy chain constant region is a modified human IgG4 subtype comprising an S228P (EU numbering) substitution in the hinge region of the human IgG4 subtype.

9. The antibody of claim 8, wherein the heavy chain constant region is a modified human IgG4 subtype comprising the amino acid sequence of SEQ ID NO:

23. 10 . The antibody of claim 7 , wherein the antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 24, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:

5.

11. The antibody of claim 10, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and each light chain comprises the amino acid sequence of SEQ ID NO:

5.

12. The antibody of claim 9, wherein the antibody comprises 1) a HC comprising the amino acid sequence of SEQ ID NO: 26, and 2) a LC comprising the amino acid sequence of SEQ ID NO:

5.

13. The antibody of claim 12, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and each light chain comprises the amino acid sequence of SEQ ID NO:

5.

14. A nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, 24 or 26.

15. A vector comprising 1) a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

5.

16. A composition comprising: 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

5.

17. A cell comprising: 1) a vector comprising a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

5.

18. A cell comprising: 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

5.

19. The cell according to claim 17 or 18, wherein the cell is a mammalian cell.

20. A method of producing an antibody, comprising culturing the cell according to any one of claims 17 to 19 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

21. An antibody produced by culturing the cell according to any one of claims 16 to 18 under conditions allowing expression of the antibody, and recovering the expressed antibody from the culture medium.

22. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 13 and claim 21, and one or more pharmaceutically acceptable carriers, diluents or excipients.

23. A method of treating an inflammatory disease or an autoimmune disease, comprising administering to a patient in need thereof an effective amount of the antibody according to any one of claims 1 to 13 and claim 21.

24. The method of claim 23, wherein the inflammatory disease or autoimmune disease is acute or chronic graft-versus-host disease (GVHD), chronic allergy, asthma, hay fever, allergic rhinitis, psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, bridging Benedict's disease, Addison's disease, psoriasis, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), multiple sclerosis (MS), myasthenia gravis, progressive systemic sclerosis (pSS), atopic dermatitis (AtD), enzyme replacement therapy (ERT), factor VIII deficiency, myositis, lupus nephritis (LN), organ and tissue transplantation, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, or vasculitis.

25. The antibody of any one of claims 1 to 13 and claim 21 for use in therapy.

26. The antibody of any one of claims 1 to 9, claims 11 to 13 and claim 21 for use in treating an inflammatory disease or an autoimmune disease.

27. The antibody for use according to claim 26, wherein the inflammatory disease or autoimmune disease is acute or chronic GVHD, chronic allergic asthma, hay fever, allergic rhinitis, psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, pSS, AtD, ERT, factor VIII deficiency, myositis, LN, organ and tissue transplantation, T1DM, autoimmune vasculitis, pernicious anemia or vasculitis.

28. The use according to claim 27, wherein the inflammatory disease or autoimmune disease is acute or chronic GVHD, chronic allergy, asthma, hay fever, allergic rhinitis, psoriatic arthritis, psoriasis, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, psoriasis, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, vasculitis, T1DM, autoimmune vasculitis, pernicious anemia or vasculitis.

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