BTLA agonist antibodies and uses thereof

Abstract

The present invention relates to anti-human BTLA agonist antibodies and their use to prevent or treat inflammatory or autoimmune diseases or disorders, such as systemic lupus erythematosus or graft-versus-host disease.

Description

[Technical Field]

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

[0002] BTLA, also known as cluster of differentiation 272 or CD272, is a member of the Ig superfamily and part of a family of checkpoint receptors that negatively regulate immune cell activation. The natural ligand for BTLA is herpesvirus entry mediator (HVEM, or CD270), a member of the TNF receptor superfamily. Engagement of BTLA by HVEM negatively affects the proliferation and activation of B and T cells. Dysregulation of the BTLA / HVEM pathway is associated with inflammatory and autoimmune diseases and disorders. Therefore, agonists directed against BTLA may be useful for preventing and / or treating autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

[0003] BTLA agonist antibodies have been disclosed, for example, in PCT Publication Nos. WO 2018 / 213113, WO 2021 / 250419, and WO 2022 / 087441. However, no BTLA agonist antibodies have been approved for therapy, and therefore, 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

[0004] Thus, the present disclosure provides novel BTLA agonist antibodies. The antibodies of the present disclosure 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 association and dissociation rates, 2) they are non-HVEM-blocking BTLA agonists that result 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 monotherapy for the treatment and / or prevention of disorders such as autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders, and 6) they inhibit the proliferation and proliferation of human peripheral blood mononuclear cells tested, including T cells, B cells, monocytes, myeloid DCs, PDCs, and NK cells. 6) they exhibit very limited internalization in all subpopulations of human leukocytes (human leukocytes, PBMCs); 7) they have lower immunogenicity; 8) they are therapeutically effective at lower doses or less frequent dosing; and / or 9) they demonstrate in vivo stability, physical stability, and chemical stability, including but not limited to, thermal stability, solubility, low self-association, and other pharmacokinetic properties acceptable for development, manufacture, formulation, storage, administration, and use in the treatment of autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

[0005] In some embodiments, the 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 heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), and the LCVR comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3), 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; 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 comprises RASQGISSWLA (SEQ ID NO: 1); e. LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and f. LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

[0006] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises LIFWNGDKR (SEQ ID NO: 12), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0007] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises QEFWTGDKR (SEQ ID NO: 13), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0008] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 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).

[0009] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 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).

[0010] In some embodiments, the 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.

[0011] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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.

[0012] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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 the antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region and a light chain constant region.

[0013] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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 the antibody, or antigen-binding fragment thereof, comprises a light chain constant region, and a human IgG2, human IgG4, or modified human IgG4 subtype heavy chain constant region.

[0014] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is a human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

[0015] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is an engineered human IgG4 subtype comprising a S228P substitution (EU numbering) in the hinge region of human IgG4, also referred to as IgG4P (Labrijn et al., Nat. Biotechnol. 2009, 27(8):767).

[0016] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is an engineered human IgG4 subtype comprising the amino acid sequence of SEQ ID NO: 23.

[0017] In some embodiments, the 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.

[0018] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody comprises two HCs and two LCs, each heavy chain comprising the amino acid sequence of SEQ ID NO:24 and each light chain comprising the amino acid sequence of SEQ ID NO:5.

[0019] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an HC comprising the amino acid sequence of SEQ ID NO:26, and an LC comprising the amino acid sequence of SEQ ID NO:5.

[0020] In some embodiments, the disclosure provides antibodies that bind to human BTLA, wherein the antibodies comprise two HCs and two LCs, each heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and each light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0021] In another aspect, provided herein are nucleic acids encoding the heavy or light chains, or HCVRs or LCVRs, of the novel human BTLA agonist antibodies described herein, and vectors or cells comprising such nucleic acids.

[0022] In another aspect, provided herein are pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, described herein, or nucleic acids encoding same. Pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding same, described herein may be used to treat a variety of conditions, including acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjogren'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-Barré syndrome (Guillain-Barré syndrome), and other conditions. The present invention may be used to treat autoimmune disorders, allergic diseases, or other inflammatory disorders, including, but not limited to, glaucoma, glaucoma 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

[0023] 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 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, the preferred methods and materials are described herein.

[0024] Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0025] definition As used herein, "about" means within a statistically significant range of a value or values, e.g., a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0026] As used herein, and when used with respect to one or more receptors, "activity," "activate," "activating," and the like refer to the ability of a compound, such as a BTLA agonist antibody described herein, to bind to, induce, or increase the response, activity, or function of a target protein, such as the BTLA receptor, as measured using assays known in the art, such as the in vitro assays described below.

[0027] As used herein, "amino acid" refers to a molecule that is characterized, from a chemical standpoint, 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 20 amino acids that are referred to as standard amino acids and can be used as building blocks for peptides / proteins produced by any organism. The amino acid sequences of the present disclosure include the standard one-letter or three-letter codes for the 20 naturally occurring amino acids.

[0028] The term "antibody," as used herein, refers to an engineered, non-naturally occurring polypeptide complex comprising an intact 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. Antibody embodiments include monoclonal, polyclonal, human, humanized, chimeric, bispecific or multispecific, or conjugated antibodies. An exemplary antibody of the present disclosure is an immunoglobulin G (IgG)-type antibody comprising four polypeptide chains, i.e., 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 (consisting 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. HCVRs and LCVRs can be further subdivided into regions of high variability called complementarity-determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each HCVR and LCVR consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain binding domains that interact with antigens.The assignment of amino acid residues to CDRs can be performed using the sequences of 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 CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international standard available at www.imgt.org). This can be done according to existing schemes, including those described in the ImMunoGeneTics database; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212. A combination of the IMGT and North CDR definitions was used for the exemplary anti-human BTLA antibody described herein.

[0029] In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of a hinge, CH1 domain, CH2 domain, and CH3 domain. The antibody constant region 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.

[0030] Antibodies can be derived from any of the commonly known isotypes, including, but not limited to, IgA, IgG, and IgM. The IgG isotype is 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.

[0031] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" refers to a portion of an antibody containing a binding domain that interacts with an antigen, and includes, but is not limited to, a "Fab fragment" containing the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. A "F(ab')2 fragment" generally comprises a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them. Other chemical bonds of antibody fragments are also known in the art, such as single-chain variable fragments (scFvs) containing the HCVR and LCVR of an antibody, with these two domains connected by a flexible linker peptide.

[0032] BTLA is a co-inhibitory receptor that plays a role in downregulating immune responses and preventing autoimmunity. Thus, as used herein, a "BTLA agonist antibody" refers to an antibody or antigen-binding fragment thereof that binds to human BTLA, enhances co-inhibitory signals to T cells and / or B cells, and, when administered in vivo, results in a significant reduction in at least one autoimmune activity, such as a reduction in anti-double-stranded DNA (ds-DNA) titers, a reduction in disease scores, or a reduction in inflammatory cytokines.

[0033] The agonist antibodies of the invention can be used to prevent or treat autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

[0034] As used herein, the terms "autoimmune disease" or "autoimmune disorder" are used interchangeably herein and refer to an undesirable condition resulting from an inappropriate or unwanted immune response against self-cells and / or tissues or transplanted cells and / or tissues. The terms "autoimmune disease" or "autoimmune disorder" are meant to include such conditions, whether mediated by a humoral immune response and / or a cellular immune response. Exemplary autoimmune diseases or disorders include acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE), scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, and Addison's disease. , dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré 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.

[0035] As used herein, "hBTLA" or "human BTLA" refers to wild-type human BTLA, preferably wild-type BTLA having the amino acid sequence set forth in SEQ ID NO: 29. The amino acid sequence of Balbc mouse BTLA is set forth in SEQ ID NO: 30, the amino acid sequence of C57BL / 6 mouse BTLA is set forth in SEQ ID NO: 31, and the amino acid sequence of cynomolgus monkey BTLA is set forth in SEQ ID NO: 32.

[0036] The terms "bind" and "binds," as used herein, unless otherwise specified, are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in proximity of the two proteins or molecules as determined by common methods known in the art.

[0037] The terms "cyno," "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 BTLA, preferably wild-type cynomolgus BTLA having the amino acid sequence set forth in SEQ ID NO:32.

[0038] As used herein, "effective amount" refers to an amount or dose of one or more of the BTLA agonist antibodies disclosed herein, or pharmaceutically acceptable salts thereof, that, upon single or multiple administration to an individual in need thereof, provides the desired effect in such individual during diagnosis or treatment (i.e., can make a clinically measurable difference in the individual's condition, such as, for example, a decrease in proinflammatory cytokines or an alteration in lymphocyte activation). An effective amount can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, numerous factors are taken into account, including, but not limited to, the species of mammal, its size, age, and general health, the particular disease or disorder involved, the extent, involvement, or severity of the disease or disorder, the individual's response, the particular antibody administered, the mode of administration, the bioavailability characteristics of the administered preparation, the selected dosing regimen, 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.

[0039] A BTLA polypeptide "extracellular domain" or "ECD" refers to a form of a BTLA polypeptide that is essentially free of transmembrane and cytoplasmic domains. Preferably, the BTLA ECD has less than 1% of transmembrane and cytoplasmic domains, 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 can be prepared using methods known in the art. Alternatively, human BTLA polypeptides or human BTLA ECD polypeptides are purchased commercially from various suppliers, such as Sino Biological (Houston, Texas; see, e.g., catalog numbers 11895-H02H or 29982-H38H).

[0040] "Fc region" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an antibody heavy chain, which can be obtained by papain digestion of an intact antibody. The Fc region may comprise a native Fc sequence or a 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 comprise a portion or the entire hinge region of an antibody heavy chain. The Fc 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 (C1q) of the classical complement system.

[0041] When expressed in a certain biological system, antibodies with a human Fc sequence are glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of antibodies at highly conserved N-glycosylation sites. N-glycans are typically linked to asparagine. Antibodies can also be glycosylated at other positions.

[0042] The term "epitope" as used herein 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 hybrid epitope. The term "epitope" can be used in reference to a structural epitope, which, according to some embodiments, can be used to describe the region of an antigen that is covered by an antibody (e.g., the footprint of the antibody when bound to the antigen). In some embodiments, a structural epitope can describe amino acid residues of an antigen that are within a specific proximity (e.g., within a specific number of angstroms) of amino acid residues of an antibody. The term "epitope" can also be used in reference to a functional epitope, which, according to some embodiments, can be used to describe amino acid residues of an antigen that interact with amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody. Epitopes can be determined according to different experimental techniques, also referred to as "epitope mapping techniques." It is understood that the determination of an epitope may vary based on the different epitope mapping techniques used, and may also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by the 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, 3 rd ed. 2018; Holst et al., Molecular Pharmacology 1998, 53(1):166-175), for example, 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.

[0043] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. Polynucleotides of the present disclosure can also include substrates incorporated therein, for example, by DNA or RNA polymerase or a synthetic reaction.

[0044] As used herein, the term "subject" refers to a mammal, 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.

[0045] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease symptom disclosed herein, but does not necessarily indicate the complete disappearance of all disorder or disease 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.

[0046] 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 provided herein that bind to human BTLA and are agonists may induce or increase one or more activities or functions associated with human BTLA, such as one or more of the activities or functions described in the Examples.

[0047] 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 heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), and the LCVR comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3); 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; 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 comprises RASQGISSWLA (SEQ ID NO: 1); e. LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and f. LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

[0048] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises LIFWNGDKR (SEQ ID NO: 12), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0049] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprises QEFWTGDKR (SEQ ID NO: 13), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

[0050] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 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).

[0051] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an 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 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).

[0052] In some embodiments, the 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.

[0053] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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.

[0054] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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 the antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region and a light chain constant region.

[0055] In some embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds to human BTLA, wherein the antibody, or antigen-binding fragment thereof, comprises an HCVR and an 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 the antibody, or antigen-binding fragment thereof, comprises a light chain constant region, and a human IgG2, human IgG4, or modified human IgG4 subtype heavy chain constant region.

[0056] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is a human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

[0057] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is an engineered human IgG4 subtype comprising a S228P substitution (EU numbering) in the hinge region of human IgG4, also referred to as IgG4P (Labrijn et al., Nat. Biotechnol. 2009, 27(8):767).

[0058] In some embodiments, the 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 the antibody or antigen-binding fragment thereof comprises a light chain constant region and a heavy chain constant region that is an engineered human IgG4 subtype comprising the amino acid sequence of SEQ ID NO: 23.

[0059] In some embodiments, the 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.

[0060] In some embodiments, the disclosure provides antibodies that bind to human BTLA, wherein the antibodies comprise two HCs and two LCs, each heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and each light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0061] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an HC comprising the amino acid sequence of SEQ ID NO:26, and an LC comprising the amino acid sequence of SEQ ID NO:5.

[0062] In some embodiments, the disclosure provides antibodies that bind to human BTLA, wherein the antibodies comprise two HCs and two LCs, each heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and each light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0063] In some embodiments, the novel BTLA agonist antibodies, or antigen-binding fragments thereof, provided herein comprise an HCVR comprising a sequence having at least 95% sequence identity to an HCVR in Table 1. In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein comprise an LCVR comprising a sequence having at least 95% sequence identity to an LCVR in Table 1. In some embodiments, the novel BTLA agonist antibodies, or antigen-binding fragments thereof, provided herein comprise an HCVR and / or LCVR in Table 1, or a sequence having at least 95% sequence identity to the HCVR and / or LCVR in Table 1.

[0064] In another aspect, provided herein are nucleic acids encoding the heavy or light chains, or HCVRs or LCVRs, of the novel human BTLA agonist antibodies described herein, and vectors or cells comprising such nucleic acids.

[0065] In another aspect, provided herein are pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, described herein, or nucleic acids encoding same. Pharmaceutical compositions comprising the novel human BTLA agonist antibodies, or antigen-binding fragments thereof, or nucleic acids encoding same, 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 (such as 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.

[0066] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding the amino acid sequence of SEQ ID NO:5, SEQ ID NO:24, or SEQ ID NO:26.

[0067] In some embodiments, the disclosure provides a vector comprising 1) a first nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and 2) a second nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:5. In some embodiments, a composition comprises a first vector comprising a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and a second vector comprising a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:5.

[0068] The nucleic acids of the present disclosure can be expressed in host cells, for example, after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked nucleic acid are well known in the art. The expression vector may include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. The expression vector containing the nucleic acid of interest (e.g., a nucleic acid encoding the heavy or light chain of an antibody) can be transferred into the host cell by known methods, such as stable or transient transfection, transformation, transduction, or infection. Additionally, the expression vector may include one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired nucleic acid sequence.

[0069] In another aspect, provided herein are cells, e.g., host cells, comprising a nucleic acid, vector, or nucleic acid composition described herein. Host cells can be cells 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, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing heavy and light chain polypeptides of an antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing a heavy chain polypeptide and a second vector expressing a light chain polypeptide of an antibody described herein. Such host cells, e.g., mammalian host cells, can express an antibody or antigen-binding fragment thereof that binds to human BTLA 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 to express the human BTLA agonist antibodies disclosed herein.

[0070] 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 SEQ ID NO:26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:5, and the cell is a mammalian cell. In some such embodiments, the mammalian cell is a CHO cell or a derivative thereof.

[0071] 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 SEQ ID NO: 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, and the cell is a mammalian cell. In some such embodiments, the mammalian cell is a CHO cell.

[0072] The present disclosure further provides a process for producing an antibody or antigen-binding fragment thereof that binds to human BTLA described herein by culturing the above-described host cells, e.g., mammalian host cells, under conditions such that the antibody or antigen-binding fragment thereof is expressed, and recovering the expressed antibody from the culture medium. The culture medium into which the antibody or antigen-binding fragment thereof is secreted can be purified by conventional techniques. Various methods of protein purification can be used, 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).

[0073] The present disclosure further provides an antibody or antigen-binding fragment thereof produced by any of the processes described herein.

[0074] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein. Such a pharmaceutical composition may also comprise one or more pharmaceutically acceptable excipients, diluents, or carriers. Pharmaceutical compositions can be prepared by methods known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0075] The human BTLA agonist antibodies or antigen-binding fragments thereof described herein, or pharmaceutical compositions comprising such antibodies or antigen-binding fragments thereof, 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 (such as 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.

[0076] In some embodiments, provided herein are methods for treating an inflammatory 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 antigen-binding fragment thereof disclosed herein. The BTLA agonist antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions comprising same, can be administered parenterally (e.g., subcutaneously and intravenously). In some embodiments, the BTLA-associated disease or disorder is an autoimmune disorder, allergic disease, or other inflammatory disorder, including, but not limited to, acute or chronic GvHD, chronic allergic disease (such as, for example, 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.

[0077] 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]

[0078] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0079] Expression and purification of engineered BTLA agonist antibodies The BTLA agonist antibodies or antigen-binding fragments thereof of the present invention can be expressed and purified essentially as follows: Suitable host cells, such as HEK293 or CHO, can be either transiently or stably transfected with an expression system for antibody secretion using an optimal predetermined HC:LC vector ratio (e.g., 1:1, 1:2, or 1:3), or with a single vector system encoding both the HC and LC. The clarified medium into which the antibody or antigen-binding fragment thereof is secreted can be purified using any of a number of commonly used techniques. For example, the medium can be applied to a MabSelect column (Cytiva) or a KappaSelect column (Cytiva) for Fab fragments equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column can be washed to remove nonspecifically bound components. Bound antibodies or antigen-binding fragments can 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 can be detected by SDS-PAGE and analytical size-exclusion chromatography, and then pooled. Depending on the intended use, further purification is optional. Antibodies or antibody fragments can be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques, including size-exclusion chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, multimodal chromatography, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is about 95% to about 99%. The product can be stored refrigerated, immediately frozen at -70°C, or lyophilized. SEQ ID NOs for the amino acid sequences of certain exemplary BTLA agonist antibodies of the present invention are shown in Table 1 below.

[0080] [Table 1] *All five exemplary antibodies share the same light chain.

[0081] Binding affinity and kinetics The binding affinity and kinetics of the disclosed BTLA agonist antibodies (M10825 and M10824) to BTLA are measured by surface plasmon resonance using a Biacore 8k (Cytiva). Binding affinity is measured by capturing the BTLA agonist antibody via an anti-human Fc antibody (Cytiva) immobilized via amine coupling onto a CM4 sensor chip (Cytiva) and running it over either recombinant human or cynomolgus BTLA at concentrations made up of four-fold serial dilutions starting at 100 nM down to 0.1 nM. Experiments are 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, a BTLA agonist antibody prepared at 5 μg / mL is flowed through the active flow cell at 10 μL / min to achieve approximately 120 RU capture, followed by a 2-minute injection of either human or cynomolgus BTLA at 100 μL / min, followed by a 20-minute dissociation period. The chip surface is regenerated with a 3M MgCl2 solution at 10 μL / min for 30 seconds. The data are fitted to a 1:1 Langmuir binding model, k on and k off Derive K D Following procedures essentially as described above, the following parameters (shown in Table 2) are observed: The data shown below are the average of three experiments.

[0082] [Table 2]

[0083] BTLA agonist antibody-induced signaling To determine the ability of the BTLA agonist antibodies of the invention (M10825 and M10824) to induce BTLA activation, the PathHunter® Jurkat BTLA signaling cell line, which overexpresses BTLA, and the PathHunter® Bioassay Detection Kit (Eurofins DiscoverX) can be used. PathHunter® Jurkat BTLA cells co-express a ProLink™-tagged BTLA receptor and an enzyme acceptor-tagged SH2 domain. Receptor activation and phosphorylation result in recruitment of SH2 to the receptor, forcing the complementation of two β-galactosidase enzyme fragments (Enzyme Acceptor and ProLink™). The resulting functional enzyme hydrolyzes the substrate, generating a chemiluminescent signal.

[0084] Immobilization plates are prepared by incubating with anti-human IgG, Fcγ (Jackson Immunoresearch) at 200 nM, followed by a blocking step. Isotype control or a titration of BTLA antibodies (0-300 nM) are captured and incubated with shaking at room temperature for 2 hours, followed by a PBS wash step. PathHunter® Jurkat BTLA signaling cell line is plated at 10 x 10 3 Cells / well may be added to wells and incubated for 1 hour in a humidified tissue culture incubator at 37°C and 5% CO2. Phosphorylated BTLA is detected with the PathHunter® Bioassay Detection Kit using Enzyme Fragment Complementation (EFC) technology, and luminescence is read on an EnVision (Perkin Elmer). Luminescence values ​​are used to calculate IC using graphing software (GraphPad Prism). 50 A value can be determined.

[0085] Antibody M10825 was tested essentially following the procedure described above, with a 2.4-fold increase in signal compared to the isotype control at the highest dose tested, 300 nM, and an IC 50Similarly, the BTLA agonist antibody M10824, at the highest dose tested, 300 nM, produced a 2.3-fold increase in signal compared to the isotype control, with an IC 50 The value was 2.4 nM. These data demonstrate that the BTLA agonist antibodies (M10825 and M10824) induce BTLA phosphorylation in PathHunter® Jurkat BTLA cells, an immortalized human T lymphocyte cell line that overexpresses BTLA.

[0086] HVEM Non-Blocking To determine the ability of the BTLA agonist antibodies M10825 and M10824 of the present invention to block HVEM ligand activation of BTLA, the PathHunter® Jurkat BTLA signaling cell line, which overexpresses BTLA, the PathHunter® U2OS HVEM ligand cell line, and the PathHunter® Bioassay Detection Kit (Eurofins DiscoverX) can be used. PathHunter® Jurkat BTLA cells co-express a ProLink™-tagged BTLA receptor and an enzyme acceptor-tagged SH2 domain. Receptor activation and phosphorylation result in recruitment of SH2 to the receptor, forcing the complementation of two β-galactosidase enzyme fragments (Enzyme Acceptor and ProLink™). The resulting functional enzyme hydrolyzes the substrate to generate a chemiluminescent signal.

[0087] Assay plates are 20 x 10 3 Assay plates are prepared by seeding PathHunter® Jurkat BTLA cells / well for 15 minutes at room temperature, followed by the addition of a titration of isotype control or BTLA antibody (0-10 μg / mL) and incubating the assay plate for 1 hour in a 37°C, 5% CO2 incubator. PathHunter® U2OS HVEM Ligand cells are then plated at 50 x 10 3Cells / well are added and the plate is incubated at room temperature for 2 hours. Phosphorylated BTLA is detected with the PathHunter® Bioassay Detection Kit using Enzyme Fragment Complementation (EFC) technology, and luminescence is read on an EnVision (Perkin Elmer). Luminescence values ​​for the 3.3 μg / mL titration point are expressed as a percentage of the signal obtained with a known HVEM non-blocker at that dose and compared to known HVEM blockers.

[0088] The signal of antibody M10825 was 104% (100% signal) of the known HVEM blocker at 3.3 μg / mL, compared to 92% for the isotype control. Similarly, the BTLA agonist antibody M10824 at 3.3 μg / mL yielded 104% (100% signal) of the known HVEM blocker, compared to 93% of the isotype control. The known HVEM blocker had 61% of the signal of the known HVEM non-blocker at the 3.3 μg / mL dose. These data demonstrate that the BTLA agonist antibodies (M10825 and M10824) do not block HVEM-induced BTLA phosphorylation in PathHunter® Jurkat BTLA cells, an immortalized human T lymphocyte cell line that overexpresses BTLA.

[0089] Inhibition of human primary B cell proliferation The in vitro potency of BTLA agonist antibodies of the present invention is assessed by their ability to inhibit human primary B cell proliferation. Human primary B cells are isolated from healthy human peripheral blood mononuclear cells using a human B cell isolation kit (EasySep) and resuspended in appropriate human primary cell medium. Anti-IgM is coated onto plates along with an isotype control or a titration of BTLA antibodies (0-40 nM) and incubated at 37°C for 1 hour, followed by a PBS wash step. Isolated human B cells are added to each well and incubated at 37°C in 5% CO2 for 72 hours, followed by a final 18-hour incubation. 3After incubation, plates are removed and stored at -80°C until ready for harvest. Cells are lysed by thawing and harvested with a FilterMate Universal Harvester (Perkin Elmer). 2 2450 Microplate Counter (Perkin Elmer) 3 Proliferation is assessed by measuring [H]-thymidine incorporation.

[0090] Counts were used to assess the relative proliferative response in this assay, and percent inhibition was calculated using the equation [% Inhibition = (AVG Maximum Signal - Signal Sample) / AVG Maximum Signal x 100], which was calculated as an IC using graphing software (GraphPad Prism). 50 can be used to determine the value.

[0091] The BTLA agonist antibody M10825, at the highest dose tested, was able to inhibit in vitro primary B cell proliferation by 96% compared to 61% inhibition for the isotype control (average of three human blood donors) using essentially the procedure described above, with a mean IC 50 Similarly, the BTLA agonist antibody M10824, at the highest dose tested, was able to inhibit in vitro primary B cell proliferation by 98% compared to 40% inhibition for the isotype control (average of three human blood donors), resulting in a calculated IC 50 The BTLA agonist antibody M10825 and M10824 were found to inhibit B cell proliferation in vitro.

[0092] Efficacy of BTLA agonist antibodies in a humanized mouse model of GvHD Because the BTLA agonist antibodies of the invention do not agonize mouse BTLA, the in vivo efficacy of the BTLA agonist antibodies of the invention can be tested in NOD SCID gamma 2 chain- / - (NSG) humanized mice to assess their ability to inhibit human T and B cell function in an in vivo setting. More specifically, in vivo efficacy is assessed in a humanized NSG mouse model of GvHD, a mouse model transplanted with human peripheral blood mononuclear cells (PMBCs), whereby human immune cells recognize the mouse as foreign, become activated, and drive GvHD. A hallmark phenotype of T cell and B cell activation is circulating human pro-inflammatory cytokines and immunoglobulins (Igs) in mouse plasma. Therefore, this model can be used to assess the ability of BTLA agonist antibodies (which do not agonize mouse BTLA) to inhibit the production of these circulating factors.

[0093] Briefly, studies using the NSG mouse model of GvHD can be performed as follows. Female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, stock #05557) were housed four per cage under a 12-hour light / dark cycle at 72°F and 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 SepMate 50 Ficol preparation tubes according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC). Freshly isolated PBMCs were diluted to 1.2 x 10 in PBS. 8 The PBMCs may be suspended in 100 μL of cells / mL and mice may be implanted intravenously with 100 μL of the PBMC suspension on day 0 (1.2 × 10 7On day 1, mice may be divided into weight groups and administered 10 mg / kg of isotype control or test BTLA antibody subcutaneously (200 μL / mouse) at doses ranging from 0.001 to 10.0 mg / kg. Administration may continue weekly for 15 days. Physical examinations and weight measurements may be performed periodically. On day 15, mice may be sacrificed and blood may be collected by cardiac puncture under isoflurane anesthesia.

[0094] Plasma analysis: Blood from cardiac puncture may be collected into EDTA-coated tubes, clarified by centrifugation, and the resulting plasma may be stored at -80°C for future processing. Plasma human cytokines and Igs may be measured using the Mesoscale Discovery (MSD) Human Th1 / Th2 10-Vplex and Human Isotyping Panel (Rockville, Maryland), respectively.

[0095] In two separate experiments performed essentially as described above, antibody M10825 significantly reduced levels of the T cell-associated human pro-inflammatory 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 isotype control-treated animals. Similarly, animals treated with antibody M10825 demonstrated a reduction in TNF-α 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 the isotype control, and the reductions resulting from the 0.1, 0.5, 1.0, 5.0, and 10.0 mg / kg antibody doses were statistically significant compared to the isotype control (data not shown).

[0096] In a second study performed essentially as above but using PBMCs from a different donor plasma, animals treated with antibody M10825 demonstrated a reduction in INF-γ, IL-10, and TNF-α 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 a statistically significant reduction in INF-γ compared to the isotype control (data not shown).

[0097] Furthermore, 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), resulting in a statistically significant decrease in IgM at doses of 1.0 and 10.0 mg / kg antibody compared to the isotype control (data not shown).

[0098] Taken together, these data demonstrate that the BTLA agonist antibody M10825 is effective in preventing GvHD in vivo.

[0099] 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 when p < 0.05 compared with isotype control using one-way ANOVA with Dunnett's post hoc test.

[0100] Absent or low internalization in a subpopulation of human PBMCs Biotherapeutics that are highly immunogenic in the clinic exhibit higher levels of internalization than biotherapeutics that are less immunogenic (Melendez, et al., BIOANALYSIS, Vol. 14, No. 10 (2022)). Therefore, an in vitro assay measuring antibody internalization in human peripheral blood mononuclear cells (PBMCs) can be used to assess the immunogenicity risk of antibodies. To analyze the internalization of the BTLA agonist antibodies of the present disclosure, human PBMCs are isolated from the buffy coats of three donors according to the instructions provided by StemCell Technologies for the SepMate-50. PBMCs are counted using a Vi-Cell cell counter and cultured at 2 x 10 in pre-warmed complete medium (X-Vivo15, Lonza #04-418Q) supplemented with 10% FBS. 6 Adjust the cell density to 2 x 10 cells / ml. Cells are plated in 96-well plates at 100 μl / well. 5 Aliquot cells / well. Place plates in a humidified incubator at 37° C. and 5% CO2.

[0101] 4x BTLA antibody (i.e., 60 nM) and 4x TAMRA-QSY7-Fab antibody (i.e., 180 nM) are prepared and mixed in equal volumes. The anti-BTLA / TAMRA-QSY7-Fab complex is formed by incubating at 4°C for 30 minutes. The anti-BTLA / TAMRA-QSY7-Fab complex is then aliquoted at 100 μl / well onto the PBMCs prepared above. The cells are incubated for 3 hours at 37°C and 5% CO2 in a humidified incubator. The cells are centrifuged at 500 g for 5 minutes and then washed three times in BD Biosciences FACS staining buffer. Cells are then stained with a 15-color panel of surface markers, including Aqua Live / Dead cell dye and fluorophore-conjugated antibodies against CD45, CD3, CD4, CD8, CD19, CD14, CD16, HLA-DR, CD123, IgD, CD27, CD11c, and CD56. This panel can identify T cells and their subtypes, B cells and their subtypes, classical and non-classical monocytes, pDCs, myeloid DCs, and NK cells. FMO control staining is included for gating. Secondary antibody-only controls and internal positive controls are set up. An LSR Fortessa X-20 is used for data acquisition. Instrument calibration is performed according to the manufacturer's instructions. A compensation panel is set up using antibody-conjugated compensation beads. FlowJo 10 is used for data analysis, measuring internalization by MFI values ​​for each subpopulation. Dead cells are excluded from the analysis. Gates are set based on the FMO staining.

[0102] The internalization of the BTLA agonist antibodies M10825 and M10824, as well as an isotype control, was analyzed essentially following the procedures described above. Both antibodies, M10825 and M10824, showed low to no internalization in all cell types, including T cells, B cells, monocytes, myeloid DCs, pDCs, and NK cells, based on mean fluorescent intensity (MFI) values. The internal positive control internalizing antibody showed very strong internalization across different cell types. No internalization was observed for the isotype control. The internalizing BTLA antibody clones identified during the initial internalization screen showed consistent internalization profiles. These data demonstrate that the BTLA agonist antibodies M10825 and M10824 exhibit limited internalization in all subpopulations of human PBMCs tested, posing a low risk of immunogenicity.

[0103] Low immunogenicity risk of BTLA agonist antibodies M10825 and M10824 BTLA agonist antibodies can be characterized for their relative risk of clinical immunogenicity using in silico and in vitro methods, including, but not limited to, T cell proliferation assays, existing reactivity assays, and MHC-associated peptide proteomics (MAPPs) assays.

[0104] MAPPs assay MAPPs profile human leukocyte antigen class II (HLA-II)-presented peptides on human dendritic cells previously treated with a test molecule. Briefly, primary human dendritic cells from 10 normal human donors were prepared from buffy coats by isolation of CD-14+ cells 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 number R0278) containing 5% serum replacement at 37°C and 5% CO2 for 4 days. Three micromolar test antibodies were then added to approximately 5 x 10 6Fresh medium containing 5 μg / ml lipopolysaccharide (LPS) can be added to the cells and replaced after approximately 5 hours of culture to transform the cells into mature dendritic cells. The next day, mature cells can be lysed in 1 mL of RIPA buffer with protease inhibitors and DNAse. A biotinylated anti-pan-HLA class II antibody (clone Tu39) can be used to isolate HLA-II molecules from the thawed lysate using an automated liquid handling system. Bound receptor-peptide complexes can be eluted with 5% acetic acid, 0.1% trifluoroacetic acid (TFA). The eluted HLA-II peptides can be passed through a pre-washed 10k MWCO filter to remove high-molecular-weight proteins. The isolated HLA-II peptides can be analyzed by nanoLC / MS using a Thermo easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. The separation was performed using a 75 μm × 15 cm PepMap RSLC c18 column with a flow rate of 300 nL / min and a 65-minute gradient of 0.1% formic acid in water as solvent A and 80% acetonitrile containing 0.1% formic acid as solvent B. Mass analysis was performed in full scan mode at 240,000 resolution, followed by a 3-second data-dependent MS / MS cycle consisting of an ion trap rapid scan with higher-energy collisional dissociation (HCD) and electron-transfer / higher-energy collision dissociation (EThcD) fragmentation.

[0105] Peptide identifications can be generated by an internal proteomics pipeline using multiple search algorithms without enzyme search parameters against bovine / human databases containing test antibody sequences (see, e.g., Higgs, RE, et al., 428, 209-230 (2008)). A KNIME workflow can be used to process the sample identification file. Peptides identified from the test material can be aligned to the parent sequence. A summary of all donors can be generated, annotating the percentage of donors presenting non-germline residues, the number of different regions presenting peptides with non-germline residues, and the depth of peptide presentation in each region with non-germline residues.

[0106] In a MAPPs analysis performed essentially as described above, only 10% of donors displayed peptides containing non-germline residues for the heavy chain CDR3 of antibody M10825. None of the other displayed peptides derived from M10825 contain non-germline residues and therefore do not pose an immunogenicity risk.

[0107] Taken together, the compilation of data from such various in silico and in vitro methods, including but not limited to the MAPPs assay essentially described above (data not shown), strongly supports that the BTLA agonist antibodies M10825 and M10824 each pose a low to moderate risk of inducing clinical immunogenicity.

[0108] Amino acid and nucleotide sequences LCDR1: <SEQ ID NO: 1, AA, synthetic construct>

[0109] RASQGISSWLA LCDR2: <SEQ ID NO: 2, AA, synthetic construct> YAASGLQS

[0110] LCDR3: <SEQ ID NO: 3, AA, synthetic construct> QQANSPFFT

[0111] LCVR:<SEQ ID NO:4, AA, synthetic construct> DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK

[0112] Full length LC: <SEQ ID NO: 5, AA, synthetic construct> DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0113] DNA encoding full-length LC (DNA of SEQ ID NO: 6, synthetic construct) GACATCCAGATGACTCAGTCGCCGAGCTCGGTGTCGGCATCCGTGGGCGACAGAGTGACCATTACTTGTCGGGCCAGTCAGGGAATTTCCAGCTGGCTCGCCTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTCATCTATGCTGCGTCAGGGCTTCAGTCCGGAGTGCCCTCAAGGTTCAGCGGTTCCGGATCCGGCACCGACTTCACCCTGACAATTTCTAGCCTGCAACCGGAGGATTTCGCGACTTACTACTGCCAGCAGGCCAACAGCTTCCCTTTTACCTTCGGACCTGGTACCAAGGTCGACATCAAGCGGACTGTCGCTGCACCCTCCGTGTTTATCTTCCCGCCCTCCGACGAACAGCTCAAGTCCGGGACCGCCTCAGTCGTGTGCTTGCTGAACAACTTCTACCCTCGGGAGGCCAAAGTGCAATGGAAGGTCGACAACGCCCTGCAAAGCGGCAATTCCCAAGAGTCCGTGACTGAGCAGGACTCCAAGGATTCCACCTACTCGCTGAGCTCCACCCTGACCCTGTCGAAGGCCGATTACGAAAAGCACAAAGTGTACGCCTGCGAAGTGACCCATCAGGGACTTTCCTCGCCCGTGACCAAGTCCTTCAACCGCGGCGAATGCTAATGA

[0114] HCDR1 <SEQ ID NO: 7, AA, synthetic construct> TFSGFSLSTXXVGVG X at position 10 is S, G, or P, and X at position 11 is G or A.

[0115] <SEQ ID NO: 8, AA, synthetic construct> TFSGFSLSTSGVGVG

[0116] <SEQ ID NO: 9, AA, synthetic construct> TFSGFSLSTGGVGVG

[0117] <SEQ ID NO: 10, AA, synthetic construct> TFSGFSLSTPAVGVG

[0118] HCDR2: <SEQ ID NO: 11, AA, synthetic construct> XXFWXGDKR 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.

[0119] <SEQ ID NO: 12, AA, synthetic construct> LIFWNGDKR

[0120] <SEQ ID NO: 13, AA, synthetic construct> QEFWTGDKR

[0121] <SEQ ID NO: 14, AA, synthetic construct> QIFWTGDKR

[0122] <SEQ ID NO: 15, AA, synthetic construct> LEFWTGDKR

[0123] HCDR3: <SEQ ID NO: 16, AA, synthetic construct> THKLGMNYFDY

[0124] HCVR: <SEQ ID NO: 17, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTXXVGVGWIRQPPGKALEWLAXXFWXGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCTHKLGMNYFDYWGQGTLVTVSS 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, and X at position 56 is N or T.

[0125] M7944: <SEQ ID NO: 18, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIFWNGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYWGQGTLVTVSS

[0126] M10825 and M10824: <SEQ ID NO:19, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWTGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYWGQGTLVTVSS

[0127] M10782: <SEQ ID NO:20, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTGGVGVGWIRQPPGKALEWLAQIFWTGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYWGQGTLVTVSS

[0128] M10469: <SEQ ID NO:21, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTPAVGVGWIRQPPGKALEWLALEFWTGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYWGQGTLVTVSS

[0129] Constant region (IgG2): <SEQ ID NO:22, AA, Homo sapiens> ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0130] Constant region (IgG4P): <SEQ ID NO: 23, AA, synthetic construct> ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0131] Full-length heavy chain M10825: <SEQ ID NO: 24, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWTGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCTHKLGMNYFDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0132] DNA encoding the heavy chain of M10825: <SEQ ID NO: 25, DNA, synthetic construct>

[0133] M10824: <SEQ ID NO: 26, AA, synthetic construct> QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWTGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCTHKLGMNYFDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0134] DNA encoding the heavy chain of M10824: <SEQ ID NO: 27, DNA, synthetic construct>

[0135] Human BTLA ECD (SEQ ID NO: 28) KESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMAS

[0136] Human BTLA (SEQ ID NO: 29) MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPS KDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS

[0137] Mouse Balbc BTLA (SEQ ID NO: 30) MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECEVQLNIKRNSKHSAWTGELFKIECPVKYCVHRPNVTWCKHNGTIWVPLEVGPQLYTSWEENRSVPVFVLHFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVRERTQNSSEHP LITVSDIPDATNASGPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHCVIGRNPRQENNMQEAPTEYASICVRS

[0138] Mouse C57BL6 BTLA (SEQ ID NO: 31) MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECPVQLTITRNSKQSARTGELFKIQCPVKYCVHRPNVTWCKHNGTICVPLEVSPQLYTSWEENQSVPVFVLHFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVTERTQNSSEHP LITVSDIPDATNASGPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHCVIGRNPRQENNMQEAPTEYASICVRS

[0139] Cynomolgus BTLA (SEQ ID NO: 32) MKTLPAMLGSGRLFWVVFLIPYLDIWNIHGKESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASERPS KDEMASRPWLLYSLLPLGGLPLLITTCFCLFCFLRRHQGKQNELSDTTGREITLVDVPFKSEQTEASTRQNSQVLLSETGIYDNEPDFCFRMQEGSEVYSNPCLEENKPGIIYASLNHSIIGLNSRQARNVKEAPTEYASICVRS

[0140] Cynomolgus BTLA ECD mutant (SEQ ID NO: 33) KESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEGHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASERPSKDEMASRLWLLYS

[0141] Cynomolgus BTLA ECD (SEQ ID NO: 34) KESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASERPSKDEMASRLWLLYS

Claims

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

2. The LCVR comprises an LCDR1 comprising RASQGISSWLA (SEQ ID NO: 1), an LCDR2 comprising YAASGLQS (SEQ ID NO: 2), and an LCDR3 comprising QQANSSFPFT (SEQ ID NO: 3), and the HCVR comprises a. an HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), an HCDR2 comprising LIFWNGDKR (SEQ ID NO: 12), and an HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16); or b. an HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), an HCDR2 comprising QEFWTGDKR (SEQ ID NO: 13), and an HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16); or c. an HCDR1 comprising TFSGFSLSTGGGVGVG (SEQ ID NO: 9), an HCDR2 comprising QIFWTGDKR (SEQ ID NO: 14), and an HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16); or d. The antibody of claim 1, comprising an HCDR1 comprising TFSGFSLSTPAVGVG (SEQ ID NO: 10), an HCDR2 comprising LEFWTGDKR (SEQ ID NO: 15), and an 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, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and the LCVR comprises the amino acid sequence of SEQ ID NO:

4.

5. The antibody of any one of claims 1 to 4, comprising a heavy chain constant region and a light chain constant region.

6. The antibody of claim 5 , wherein the heavy chain constant region is a human IgG2 subtype, a human IgG4 subtype, or a modified human IgG4 subtype.

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

22.

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

9. 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, each heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and each light chain comprising the amino acid sequence of SEQ ID NO:

5.

12. The antibody of claim 9, wherein the antibody comprises 1) an HC comprising the amino acid sequence of SEQ ID NO: 26, and 2) an 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, each heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and each light chain comprising 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, SEQ ID NO:24, or SEQ ID NO:

26.

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

5.

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

20. 20. A process for producing an antibody, comprising culturing a 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. 19. An antibody produced by culturing a cell according to any one of claims 16 to 18 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

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

24. The inflammatory disease or the autoimmune disease may be 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, Hashimoto's disease, Addison's disease, psoriasis, 24. The method of claim 23, wherein the condition is dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré 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. An antibody according to any one of claims 1 to 13 and claim 21 for use in therapy.

26. An antibody according to any one of claims 1 to 9, 11 to 13 and 21 for use in the treatment of an inflammatory or autoimmune disease.

27. 27. The antibody for use according to claim 26, wherein the inflammatory disease or the 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. 28. The use of claim 27, wherein the inflammatory disease or the 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.

Images