EP4626929A2 - Btla agonist antibodies and uses thereof - Google Patents

Abstract

The present invention relates to anti-human BTLA agonist antibodies and uses thereof for preventing or treating inflammatory or autoimmune diseases or disorders such as systemic lupus erythematosus or graft-versus-host disease.

Description

BTLA AGONIST ANTIBODIES AND USES THEREOF

FIELD OF THE INVENTION

The present disclosure is in the field of medicine. More particularly, the present disclosure relates to agonistic antibodies directed to human B and T lymphocyte attenuator (BTLA), compositions comprising such BTLA agonistic antibodies, and methods of using such BTLA agonistic antibodies for the treatment of autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.

BACKGROUND

BTLA, also known as cluster of differentiation 272 or CD272, is an Ig superfamily member and part of a family of checkpoint receptors that negatively regulate immune cell activation. The natural ligand for BTLA is the TNF receptor superfamily member, herpes virus entry mediator (HVEM, or CD270). Engagement of BTLA by HVEM exerts negative effects on 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 to BTLA may be useful in preventing and/or treating autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.

BTLA agonist antibodies have been disclosed in PCT Patent Application Publications WO 2018/213113, WO 2021/250419, and WO2022/087441, for example. However, no BTLA agonist antibodies have been approved for therapy so there remains a need to develop alternative BTLA agonist antibodies, which can be used for treating autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.

SUMMARY OF INVENTION

Accordingly, the present disclosure provides novel BTLA agonist antibodies. The antibodies of the present disclosure are particularly advantageous over prior art BTLA antibodies for various reasons, including, but not limited to, the following: 1) they bind human BTLA and cynomolgus monkey BTLA with comparable affinity and desirable association and dissociation rates, 2) they are non HVEM blocking BTLA agonists leading to 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 display enhanced potency as a monotherapy for the treatment and/or prevention of disorders such as autoimmune disorders, allergic disease, asthma, or other inflammatory disorders, 6) they have very limited internalization in all subpopulations of human peripheral blood mononuclear cells (PBMCs) tested including T cells, B cells, monocy tes, myeloid DCs, pDCs, and NK cells, 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 and chemical stability⁷ including, but not limited to, thermal stability, solubility, low self-association, and other pharmacokinetic characteristics which are acceptable for development, manufacturing, formulation, storage, administration and use in the treatment of autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.

In some embodiments, the present disclosure provides an antibody, or antigen binding fragment thereof, that binds 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 complementarity⁷ determining region 3 (LCDR3), wherein: a. the 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. the 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. 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. the LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

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

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

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

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

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 NOs: 18, 19, 20, or 21, and the LCVR comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 human IgG2, a human IgG4, or a modified human IgG4 subtype heavy chain constant region.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 a S228P substitution in the hinge region of human IgG4 (EU Numbering), also referred to as IgG4P (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767).

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody that binds human BTLA wherein the antibody comprises two HCs and tw o 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.

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

In another aspect, provided herein are pharmaceutical compositions comprising a novel human BTLA agonist antibody, or antigen binding fragment thereof, described herein or a nucleic acid encoding the same. The pharmaceutical compositions comprising a novel human BTLA agonist antibody, or antigen binding fragment thereof, described herein or nucleic acid(s) encoding the same can be used for treating an autoimmune disorder, allergic disease, or other inflammatory disorder, including, but not limited to, 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-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 transplant, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, and vasculitis.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains. Although any methods and materials similar to 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.

Moreover, reference to an element by the indefinite article “a” or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”

Definitions

As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such a value or range 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 upon the particular system under study, and can be readily appreciated by one of skill in the art.

As used herein, and in reference io one or more receptors, “activity," “activate,” “activating” and the like means a capacity of a compound, such as aBTLA agonist antibody as described herein, to bind to and induce or increase a response, activity’, or function of a target protein such as the receptor BTLA, as measured using assays known in the art, such as the in vitro assays described below.

As used herein, “amino acid” means a molecule that, from a chemical standpoint, is characterized by a 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 that can be used as building blocks for peptides/proteins produced by any living being. The amino acid sequences in the disclosure contain the standard single letter or three letter codes for the twenty naturally occurring amino acids.

The term “antibody” as used herein refers to an engineered, non-naturally occurring polypeptide complex, including an intact antibody and any antigen binding fragments thereof (i.e.. “antigen binding portions” of an antibody). The term “antibody,” as used herein, refers to an immunoglobulin molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, bispecific or multispecific antibody, or conjugated antibody. An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) type antibody comprising four polypeptide chains, two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is comprised of an N-terminal heavy chain variable region (HCVR) and a heavy' chain constant region (consisting of three domains, CHI, CH2 and CH3). Each light chain is comprised of an N-terminal light chain variable region (LCVR) and a light chain constant region. HCVR and LCVR can be further subdivided into regions of high variability named as complementarity determining regions (CDRs) that are spaced by more conserved regions named as framework regions (FRs). Each HCVR and LCVR consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2. CDR2. FR3, CDR3, FR4. The variable regions of the heavy chain and light chain contain binding domains that interacts with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well- known schemes, including those described in 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); ALLazikani 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 ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). A combination of IMGT and North CDR definitions were used for the exemplified anti-human BTLA antibodies as described herein.

In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of a hinge, a CHI domain, a CH2 domain and a CH3 domain. The constant regions of the antibodies 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.

An antibody may be from any of the commonly known isotypes, including but not limited to IgA, IgG, and IgM. The IgG isotype is divided in subclasses in certain species: IgGl. IgG2, IgG3 and IgG4 in humans, and IgGl, 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.

The term “antigen binding fragment” or “antigen-binding portion”, as used herein, is the portion of an antibody that contains a binding domain that interacts with an antigen, including, but not limited to: “Fab fragments” containing a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavychain); “F(ab’)2 fragments” comprising a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments also are know n in the art, and single-chain variable fragments (scFvs) comprising a HCVR and a LCVR of an antibody, wherein these two domains are connected by a flexible linker peptide.

BTLA is a co-inhibitory receptor, playing a role in down-regulating immune responses and preventing autoimmunity. Therefore, as used herein, “BTLA agonist antibody” refers to an antibody or an antigen binding fragment thereof that binds to human BTLA and enhances its co-inhibitory signal to T and/or B cells, and, when administered in vivo, results in at least one significantly lessened autoimmune activity such as reduction in anti-double stranded DNA (ds-DNA) titers, reduction in disease scores or reduction in inflammatory cytokines.

The agonist antibodies of the present invention can be used for preventing or treating autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.

As used herein, the term “autoimmune disease” or “autoimmune disorder” are used interchangeably herein and refer to undesirable conditions that arise from an inappropriate or unwanted immune reaction against self-cells and/or tissues or transplanted cells and/or tissues. The term “autoimmune disease” or “autoimmune disorder” is meant to include such conditions, whether they be mediated by humoral and/or cellular immune responses. Exemplary^ autoimmune diseases or disorders include, but are not limited to, 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-Bane 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 transplant, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, and vasculitis. As used herein, “hBTLA” or “human BTLA” refers to a wild-ty pe human BTLA, preferably, a wild-type human BTLA that has 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 murine C57BL6 is given by SEQ ID NO: 31 , and the amino acid sequence of cynomolgus monkey BTLA is given by SEQ ID NO: 32.

The terms “bind’' and “binds” as used herein, are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.

The terms “cyno”, “cynomolgus” or “cynomolgus monkey” are used interchangeably herein. When used in reference to a BTLA polypeptide it is intended that the terms refer to wild-type cynomolgus monkey BTLA, and. preferably, a wild-type cynomolgus monkey BTLA that has the amino acid sequence set forth in SEQ ID NO: 32.

As used herein, “effective amount” means an amount or dose of one or more of the BTLA agonist antibodies disclosed herein, or a pharmaceutically acceptable salt thereof that, upon single or multiple dose administration to an individual in need thereof, provides a desired effect in such an individual under diagnosis or treatment (z.e., may produce a clinically measurable difference in a condition of the individual such as a reduction in pro-inflammatory cytokines, or changes in lymphocyte activation. An effective amount can be readily determined by one of skill in the art by using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the species of mammal, its size, age and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual, the particular antibody administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, 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.

A BTLA polypeptide “extracellular domain” or “ECD” refers to a form of the BTLA polypeptide that is essentially free of the transmembrane and cytoplasmic domains. Preferably, a BTLA ECD has less than 1% of the transmembrane and cytoplasmic domain, more preferably, a BTLA ECD has less than 0.5% of such domains. Even more preferably, the human BTLA ECD polypeptide is as shown in SEQ ID NO: 28 and cynomolgus monkey BTLA ECD polypeptide is as shown in SEQ ID NO: 33 or SEQ ID NO:34. BTLA polypeptide ECD may prepared using methods known in the art.

Alternatively, human BTLA polypeptide or human BTLA ECD polypeptide be purchased commercially from various vendors such as Sino Biological (Houston, Texas; see, for example, catalog nos. 11895-H02H or 29982-H38H).

“Fc region’’ refers to a dimer complex comprising the C-terminal polypeptide sequences of an antibody heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences. 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 the antibody heavy' chain. The Fc region of an antibody can 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.

When expressed in certain biological systems, antibodies having human Fc sequences are glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of the antibody at a highly conserved N-glycosylation site. N-glycans ty pi cal ly attach to asparagine. Antibodies may be glycosylated at other positions as well.

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” may be used in reference to a structural epitope. A structural epitope, according to some embodiments, may be used to describe the region of an antigen which is covered by an antibody (e.g., an antibody’s footprint when bound to the antigen). In some embodiments, a structural epitope may describe the amino acid residues of the antigen that are w ithin a specified proximity (e.g., within a specified number of Angstroms) of an amino acid residue of the antibody. The term “epitope” may also be used in reference to a functional epitope. A functional epitope, according to some embodiments, may be used to describe amino acid residues of the antigen that interact with amino acid residues of the antibody in a manner contributing to the binding energy⁷ between the antigen and the antibody. An epitope can be determined according to different experimental techniques, also called “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' with the different experimental conditions used, e.g., due to the conformational changes or cleavages 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, 3^rd ed. 2018; Holst et al.. Molecular Pharmacology 1998, 53(1): 166-175), including but not limited to, X-ray⁷ crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine-scanning mutagenesis, steric hindrance mutagenesis, hydrogendeuterium exchange (HDX). and cross-blocking assays.

The terms “nucleic acid” as used herein, refer to polymers of nucleotides, including single-stranded and/ or double-stranded nucleotide-containing molecules, such as DNA, cDNA and RNA molecules, incorporating native, modified, and/ or analogs of, nucleotides. Polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or a synthetic reaction.

The term “subject” as used herein, refers to a mammal, including, but are not limited to, a human, chimpanzee, ape, monkey, cattle, horse, sheep, goat, swine, rabbit, dog, cat, rat, mouse, guinea pig, and the like. Preferably, the subject is a human.

The term “treatment” or “treating” as used herein, refers to all processes wherein there may be a slowing, controlling, delaying or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease sy mptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.

In one aspect, provided herein are novel antibodies or antigen binding fragments thereof that binds human BTLA. In some embodiments, the novel antibodies or antigen binding fragments thereof that bind human BTLA are agonists of BTLA. In some embodiments, the novel antibodies or antigen binding fragments thereof provided herein that bind human BTLA and are agonists can induce or increase one or more activities or functions associated with human BTLA, e.g., one or more activities or functions described in the Examples.

In some embodiments, the novel antibodies, or antigen binding fragments thereof, that bind 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: a. the 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. the 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. 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 the LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

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

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

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 the LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), the LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and the LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises TFSGFSLSTGGVGVG (SEQ ID NO: 9). the HCDR2 comprises Q1FWTGDKR (SEQ ID NO: 14). and the HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

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

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 NOs: 18, 19, 20, or 21, and the LCVR comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 human IgG2. a human IgG4. or a modified human IgG4 subtype heavy chain constant region.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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 a S228P substitution in the hinge region of human IgG4 (EU Numbering), also referred to as IgG4P (see Labrijn. et al., Nat. Biotechnol. 2009, 27(8):767).

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody that binds 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.

In some embodiments, the present disclosure provides an antibody, or an antigen binding fragment thereof, that binds 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.

In some embodiments, the present disclosure provides an antibody that binds 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.

In some embodiments, a novel BTLA agonist antibody, or antigen binding fragment thereof, provided herein comprises a HCVR comprising a sequence having at least 95% sequence identity to a HCVR in Table 1. In some embodiments, the antibody, or antigen binding fragment thereof, provided herein comprises a LCVR comprising a sequence having at least 95% sequence identity to a LCVR in Table 1 . In some embodiments, the novel BTLA agonist antibody, or antigen binding fragment thereof, provided herein comprises a HCVR and/or a LCVR in Table 1 or a sequence having at least 95% sequence identity to a HCVR and/or a LCVR in Table 1.

In another aspect, provided herein are nucleic acids encoding a heavy chain or light chain, or a HCVR or LCVR, of the novel human BTLA agonist antibodies described herein, and vectors or cells comprising such nucleic acids. In another aspect, provided herein are pharmaceutical compositions comprising a novel human BTLA agonist antibody, or antigen binding fragment thereof, described herein or a nucleic acid encoding the same. The pharmaceutical compositions comprising a novel human BTLA agonist antibody, or antigen binding fragment thereof, described herein or nucleic acid encoding the same can be used for treating an autoimmune disorder, allergic disease, or other inflammatory disorder, 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 transplant, T1DM. autoimmune vasculitis, pernicious anemia, and vasculitis.

In some embodiments, the present disclosure provides nucleic acids comprising a sequence encoding the amino acid sequence of SEQ ID NOs: 5. 24, or 26.

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

Nucleic acids of the present disclosure may be expressed in a host cell, for example, after the nucleic acids have been operably linked to an expression control sequence. Expression control sequences capable of expression of nucleic acids to which they are operably linked are well known in the art. An expression vector may include a sequence that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectors containing a nucleic acid of interest (e.g., a nucleic acid encoding a heavy chain or light chain of an antibody) may be transferred into a host cell by w ell-known methods, e.g., stable or transient transfection, transformation, transduction or infection. Additionally, expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aide in detection of host cells transformed with the desired nucleic acid sequences.

In another aspect, provided herein are cells, e.g.. host cells, comprising the nucleic acids, vectors, or nucleic acid compositions described herein. A host cell may be a cell 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, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the heavy chain and light chain polypeptides of an antibody of the present disclosure. In some embodiments, a host cell may be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the heavy chain polypeptide and a second vector expressing the light chain polypeptide of an antibody described herein. Such host cells, e.g., mammalian host cells, can express the antibodies, or antigen binding fragments thereof, that bind human BTLA as described herein. Mammalian host cells know n to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NSO cells. Preferably, CHO host cells or derivatives thereof such as CHO-K.1, CHO-S, GS-CHO, CHO-DG44, CHOK1SV or GS-CHOK1SV, for example, are used for expressing human BTLA agonist antibodies disclosed herein.

In some embodiments, a host cell comprises a vector comprising 1) a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 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 derivatives thereof.

In some embodiments, a host cell comprises 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 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.

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

The present disclosure further provides antibodies, or antigen binding fragments thereof, produced by any of the processes described herein.

In another aspect, provided herein are pharmaceutical compositions comprising an antibody, or antigen binding fragments thereof, described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier. 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).

The human BTLA agonist antibodies, or antigen binding fragments thereof, or pharmaceutical compositions comprising such an antibody, or antigen binding fragment thereof, described herein, can be used for treating an autoimmune disorder, allergic disease, or other inflammatory disorder, 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 transplant, T1DM, autoimmune vasculitis, pernicious anemia, and vasculitis

In some embodiments, provided herein are methods of 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. A BTLA agonist antibody, or antigen binding fragment thereof, described herein or a pharmaceutical composition comprising the same may be administered by parenteral routes (e.g., subcutaneous, and intravenous). 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 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 transplant. T1DM. autoimmune vasculitis, pernicious anemia, and vasculitis.

Also provided herein are human BTLA agonist antibodies, or antigen binding fragments thereof, or pharmaceutical compositions comprising at least one of such human BTLA agonist antibodies, or antigen binding fragments thereof, for use in therapy.

EXAMPLES

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

Expression and purification of eneineered BTLA asonist antibodies

BTLA agonist antibodies, or antigen binding fragments thereof, of the present invention can be expressed and purified essentially as follows. An appropriate host cell, such as HEK 293 or CHO, can be either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio (such as 1 : 1, 1 :2, or 1 :3) or a single vector system encoding both the HC and the LC. Clarified media, into which the antibody, or antigen binding fragment thereof, has been secreted, may be purified using any of many commonly used techniques. For example, the medium may be applied to a MabSelect column (Cytiva), or KappaSelect column (Cytiva) for Fab fragment, that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column may be washed to remove nonspecific binding components. The bound antibody, or antigen binding fragment, may be eluted, for example, by pH gradient (such as 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, such as by SDS-PAGE and analytical size exclusion chromatography, and then may be pooled. Further purification is optional, depending on intended use. The antibody, or antibody fragment, may be concentrated and/or sterile filtered using common techniques. Soluble aggregate 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 between about 95% to about 99%. The product may be held refrigerated, immediately frozen at -70°C, or may be lyophilized. SEQ ID NOs for amino acid sequences of certain exemplified BTLA agonist antibodies of the present invention are shown below in Table 1.

Table 1. Amino acid sequences of exemplified BTLA agonist antibodies

*A11 five exemplified antibodies share the same light chain.

Binding affinity and kinetics

The binding affinity' and kinetics of the BTLA agonist antibodies of the present disclosure (M10825 and M10824) to BTLA is measured by surface plasmon resonance using Biacore 8k (Cytiva). The binding affinity is measured by capturing BTLA agonist antibodies through an anti -human Fc antibody (Cytiva) immobilized via amine coupling onto a CM4 sensor chip (Cytiva) and flowing over either recombinant human or cynomolgus BTLA at concentrations made from a 4-fold serial dilution starting at 100 nM down to 0. 1 nM. The experiments are carried out 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 pg/mL is flowed the active flow cell at 10 pL/min to achieve a capture around 120RU. followed by injection of either human or cynomolgus BTLA at 100 pL/min for 2 minutes, followed by dissociation for 20 minutes. Chip surface is regenerated using 3M MgCh solution for 30 seconds at 10 pL/min. The data are fit to a 1: 1 Langmiur binding model to derive k_on, foil. and to calculate KD . Following procedures essentially as described above, the following parameters (shown in Table 2) are observed. Data shown below are the average of three experiments.

Table 2: In vitro binding affinity of M10824 and M10825 to human and cynomolgus BTLA

BTLA agonist antibody-induced signaling

To determine the ability of BTLA agonist antibodies of the present invention (M10825 and M10824) to induce activation of BTLA, the PathHunter® Jurkat BTLA signaling cell line which overexpresses BTLA and PathHunter® Bioassay Detection Kit (Eurofms DiscoverX) can be used. The PathHunter® Jurkat BTLA cells co-express ProLink™ tagged BTLA receptor and an Enzyme Acceptor tagged SH2 domain. Receptor activation and phosphorylation results in SH2 recruitment to the receptor and forces complementation of the two P-galactosidase enzy me fragments (Enzy me Acceptor and ProLink™). The resulting functional enzyme hydroly zes substrate to generate a chemiluminescent signal.

Immobilized plates are prepared by incubating anti-human IgG, Fey (Jackson Immunoresearch) at 200 nM followed by a blocking step. Titrations of isotype control or BTLA antibody (0-300 nM) are captured and incubated for 2 hours at room temperature with shaking followed by a PBS wash step. The PathHunter® Jurkat BTLA signaling cell line may be added to the wells at 10 x 10³ cells/well an incubated in a 37°C and 5% CO2 humidified tissue culture incubator for 1 hour. Phosphorylated-BTLA is detected with a PathHunter® Bioassay Detection Kit using Enzy me Fragment Complementation (EFC) technology and luminescence is read on an EnVision (Perkin Elmer). Luminescence values can be used to determine ICso values using graphing software (GraphPad Prism).

Following procedures essentially⁷ as described above, antibody Ml 0825 resulted in a 2.4-fold increase in signal compared to isotype control at the highest dose tested of 300 nM and an ICso value of 2.3 nM. Similarly, the BTLA agonist antibody Ml 0824 resulted in a 2.3-fold increase in signal compared to isotype control at the highest dose tested of 300 nM and an ICso value of 2.4 nM. These data demonstrate that the BTLA agonist antibodies (Ml 0825 and Ml 0824) induce BTLA phosphorylation in PathHunter® Jurkat BTLA cells, an immortalized human T lymphocyte cell line overexpressing BTLA.

HVEM non-blocking

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

Assay plates are prepared by seeding 20 x 10³ PathHunter® Jurkat BTLA cells/well at room temperature for 15 minutes followed by addition of titrations of isotype control or BTLA antibody (0-10 pg/mL) and incubating the assay plate at 37°C and 5% CO2 incubator for 1 hour. PathHunter® U2OS HVEM Ligand cells are then added at 50 x 10³ cells/well and the plates are incubated at room temperature for 2 hours. Phosphorylated-BTLA is detected with a 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 pg/mL titration point are shown as percent of signal obtained by a known HVEM non-blocker at that dose and compared to a known HVEM blocker.

Following procedures essentially as described above, antibody Ml 0825 resulted in a signal at 3.3 pg/mL of 104% of known HVEM blocker (100% signal) compared to isotype control at 92%. Similarly, the BTLA agonist antibody Ml 0824 resulted in a signal at 3.3 pg/mL of 104% of known HVEM blocker (100% signal) compared to isotype control at 93%. The known HVEM blocker had a signal of 61% of the known HVEM non-blocker at the 3.3 pg/mL dose. These data demonstrate that the BTLA agonist antibodies (M10825 and M10824) do not block the HVEM induced BTLA phosphorylation in PathHunter® Jurkat BTLA cells, an immortalized human T lymphocyte cell line overexpressing BTLA.

Inhibition of human primary B cell proliferation

The in vitro potency of BTLA agonist antibodies of the present invention is evaluated by the abi 1 i ty to inhibit human primary B cell proliferation. Human primary B cells are isolated from healthy human peripheral blood mononuclear cells using human B cell isolation kit (EasySep) and are resuspended in appropriate human primary cell media. Anti-IgM is coated to plates along with titrations of isotype control or BTLA antibody (0-40 nM) and incubated for one hour at 37°C followed by PBS wash step. Isolated human B cells are added to each well and incubated for 72 hours at 37 °C with 5% CO2 followed by [³H] -thymidine pulse for the last 18 hours. Post incubation plates are removed and then stored at -80°C until ready to harvest. Cells are lysed by thawing and harvested with FilterMate Universal Harvester (Perkin Elmer). Proliferation is assessed by measuring [³H] -thymidine incorporation with a MicroBeta² 2450 Microplate Counter (Perkin Elmer).

Counts are used to assess relative proliferative response in this assay, and percent inhibition is calculated using the equation [% Inhibition= (AVGmaxsignaL signalsample)/AVGmaxsignal x 100], which can be used to determine IC50 values using graphing softw are (GraphPad Prism).

Following procedures essentially as described above, the BTLA agonist antibody M10825 was able to inhibit primary B cell proliferation in vitro at the highest dose tested by 96% compared to isotype control inhibition of 61% (average from 3 human blood donors) with a calculated average IC50 of 10.5 nM. Similarly, the BTLA agonist antibody M10824 was able to inhibit primary B cell proliferation in vitro at the highest dose tested by 98% compared to isotype control inhibition of 40% (average from 3 human blood donors) with a calculated IC50 of 196.40 nM. These data demonstrate that the BTLA agonist antibodies M10825 and M10824 are able to inhibit B cell proliferation in vitro. antibodies in a humanized mouse model of GvHD

Because BTLA agonist antibodies of the present invention do not agonize murine BTLA. the in vivo potency of BTLA agonist antibodies of the present invention may be tested in NOD SCID Gamma2 chain-/- (NSG) humanized mice to assess its abi 1 i ty to inhibit human T and B cell function in an in vivo setting. More specifically, in vivo effects were assessed in a humanized NSG mouse model of GvHD, a human peripheral blood mononuclear cell (PMBC) engrafted mouse model whereby the human immune cells recognize the mouse as foreign, become activated, and drive GvHD. Hallmark phenotypes indicative of T-cell and B-cell activation are circulating human pro- inflammatory cytokines and immunoglobulins (Ig’s) in the mouse plasma. Thus, the model can be used to evaluate the ability of BTLA agonist antibodies (which do not agonize murine BTLA), to inhibit the production of these circulating factors.

Briefly described, studies using the NSG mouse model of GvHD may be conducted as follows:

Female NSG mice (NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ, J AX Labs, Stock # 05557) may be housed 4/cage at 72° F under a 12 hr. lightdark cycle and allowed food and water ad libitum. Human peripheral blood mononuclear cells (PBMC’s) may be 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 PBMC’s may be suspended in PBS at 1.2 x 10⁸ cells/mL and mice may be engrafted with 100 pL PBMC’s suspension intravenously on day 0 (1.2 10⁷/mouse). On day 1, mice may be divided into weight groups dosed with 10 mg/kg isotype control or test BTLA antibody at doses ranging from 0.001 to 10.0 mg/kg subcutaneously (200 pL/mouse). Dosing may be continued weekly for 15 days. Health checks and body weight measurements may be performed routinely. On day 15, mice may be sacrificed, and blood may be collected by cardiac puncture under isoflurane anesthesia.

Plasma analyses: Blood from the cardiac puncture may be collected into EDTA coated tubes, clarified by centrifugation, and the resultant plasma may be stored at -80° C for future processing. Plasma human cytokines and Ig’s may be measured using the Mesoscale Discovery (MSD) Human Thl/Th2 10-Vplex and Human Isotyping Panel, respectively (Rockville Maryland). In two separate experiments conducted essentially as described above, antibody M10825 markedly reduced levels of the T cell associated human pro- inflammatory cytokines interferon gamma (INF-y), interleukin- 10 (IL- 10) and tumor necrosis factor alpha (TNF-a) in a dose dependent manner. More specifically, animals treated with antibody Ml 0825 demonstrated statistically significant reduction in INF-y 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 with isotype control treated animals. Similarly, animals treated with antibody M10825 demonstrated reductions in TNF-a 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 isot pe control 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 control (data not shown).

In a second study conducted essentially as described above but with PBMCs from a different donor plasma, animals treated with antibody M10825 demonstrated reductions in INF-y, IL-10, and TNF-a at all doses tested (i.e, 0.001, 0.01, 0.1, 1.0, and 10.0 mg/kg antibody) compared to isotype control with the doses of (i.e, 0.1, L0, and 10.0 mg/kg antibody) resulting in statistically significant reductions of INF-y compared to isotype control (data not shown).

Additionally, in the second study plasma IgA and IgM levels were shown to be attenuated by 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 control with the doses of 1.0 and 10.0 mg/kg antibody resulting in statistically significant reductions of IgM as compared to isotype control (data not shown).

Taken together, these data demonstrate that BTLA agonist antibody Ml 0825 is efficacious in preventing GvHD in vivo.

Plasma cytokines and plasma human IgA and IgM w ere measured by an MSD assay and expressed as mean ± standard error of the mean. Differences were considered significant if p < 0.05 compared to isotype control using a one-way ANOVA with Dunnett’s post hoc test. No or low Internalization in Subpopulations of Human PBMCs

Antibody-based biotherapeutics with higher immunogenicity in the clinic have a greater level of internalization than antibodies with lower immunogenicity (Melendez, et al., BIOANALYSIS, Vol. 14, No. 1 (2022)). Thus, in vitro assays measuring antibody internalization in human peripheral blood mononuclear cells (PBMCs) may be used to assess immunogenicity risk of antibodies. To analyze internalization of BTLA agonist antibodies of the present disclosure, human PBMC, PBMCs are isolated from three donors of buffy coats following directions provided by StemCell Technologies for SepMate-50. PBMCs are counted with Vi-Cell cell counter and adjusted to 2 x 10⁶ cells/ml in pre-warmed complete media (X-Vivol5, Lonza #04-418Q) supplemented with 10% FBS. Cells are aliquoted 100 pl/well in 96-well plates for 2 x 10⁵ cells/well. Plates are placed in a humidified incubator at 37°C and 5% CO2.

4X BTLA antibody (i.e., 60 nM) and 4X TAMRA-QSY7-Fab antibody (i.e., 180 nM) are prepared and mixed at equal volume. 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 100 pl/well to the PBMCS prepared above. The cells are incubated at 37°C and 5% CO2 for 3 hours in a humidified incubator. Cells are centrifuged at 500g for 5 minutes followed by washing in BD Biosciences FACS staining buffer three times. The cells are then stained with a 15-color panel of surface markers including Aqua live/dead cell dye, fluorophore conjugated antibodies against CD45, CD3, CD4, CD8. CD19, CD14, CD16, HLA-DR, CD123. IgD, CD27, CDl lc, and CD56. This panel can identify T cell and its subtype. B cell and its subtypes, classical/non-classical monocytes, pDCs, myeloid DCs, and NK cells. FMO control staining is included for gating. Secondary antibody only control and an internal positive control are set up. LSRFortessaX-20 is used for data acquisition. Instrument calibration is performed according to the manufacturer’s instruction. Compensation panel is set up using compensation beads bound with antibodies. FlowJo 10 is used for data analysis and internalization is measured by MFI value for each subpopulation. Dead cells are excluded from analysis. Gates are set up based on FMO staining.

Following procedures essentially as described above, BTLA agonist antibodies Ml 0825 and Ml 0824 and isotype control were analyzed for internalization. Both antibody M10825 and M10824 showed low or no internalization in all cell types including T cells, B cells, monocytes, myeloid DCs. pDCs, and NK cells based on the mean fluorescent intensity (MFI) value. The internal positive control internalizing antibody showed very strong internalization across different cell types. No internalization was observed for isotype controls. An internalizing BTLA antibody clone identified during earlier internalization screening showed consistent internalization profile. These data demonstrate that the BTLA agonist antibodies M10825 and M10824 have limited internalization in all subpopulations of human PBMC tested, indicating low risk of immunogenicity .

Low Immunogenicity Risk of BTLA agonist antibodies M10825 and M10824

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.

MAPPs Assay

MAPPs profiles the human leukocyte antigen class II (HLA-II) presented peptides on human dendritic cells previously treated with test molecules. Briefly described, primary human dendritic cells from 10 normal human donors may be prepared from buffy coats by isolation of CD- 14 positive cells and differentiated into immature dendritic cells by incubation with 20 ng/ml IL-4 and 40 ng/ml GM-CSF in complete RPMI media (Sigma-Aldrich, catalog # R0278) containing 5% Serum Replacement for 4 days at 37°C and 5% CO2 essentially as described in Knierman, M.D., et al., 2020, for example. Then three micromolar of test antibody may be added to approximately 5 x 10⁶ cells on day 4 and fresh media containing 5 pg/ml of lipopolysaccharide (LPS) to transform the cells into mature dendritic cells may be exchanged after about a 5-hour incubation. The matured cells may be lysed in 1 mL of RIP A buffer with protease inhibitors and DNAse the following day. An automated liquid handling system may be used to isolate the HLA- II molecules from thawed lysate using biotinylated anti-pan HLA class II antibody (clone Tu39). The bound receptor-peptide complex may be eluted with 5% acetic acid, 0.1% trifluoroacetic acid (TFA). The eluted HLA-II peptides were passed over a prewashed 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 1200 nLC-HPLC system with a Thermo LUMOS mass spectrometer. The separation may use a 75 pm x 15 cm PepMap RSLC cl 8 column for 65-minute gradient with a 300 nL/min flow rate and 0.1 % formic acid in water as A solvent and 80% acetonitrile with 0.1% formic acid as B solvent. Mass spectrometry may be run in full scan mode with 240,000 resolution followed by a 3 second data dependent MS/MS cycle comprised of ion trap rapid scans with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation fragmentation (EThcD) fragmentation.

Peptide identifications may be generated by an internal proteomics pipeline (see, for example, Higgs, R.E., et al., Methods Mol. Biol., 428. 209-230 (2008)) using multiple search algorithms with no enzyme search parameter against a bovine/human database containing the test antibody sequences. A KNIME workflow may be used to process the identification files for the samples. Peptides identified from the test articles may be aligned against the parent sequence. A summary may be created for all donors that annotates the percent of donors that display non-germline residues, the number of different regions that display peptides with non-germline residues and the depth of peptide display at each region with non-germline residues.

In 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 from M10825 contained non- germline residues and therefore do not present an immunogenicity risk.

Taken together, a compilation of data from such various in silico and in vitro methods (data not shown), including, but not limited to, the MAPPs assay essentially as described above, strongly supports BTLA agonist antibodies Ml 0825 and Ml 0824 each having a low-to-moderate risk of inducing clinical immunogenicity. Amino Acid and Nucleotide Sequences

LCDR1: <SEQ ID NO: 1; AA; synthetic construct>

RASQGISSWLA

LCDR2: <SEQ ID NO: 2; AA; synthetic construct

YAASGLQS

LCDR3: <SEQ ID NO: 3; AA; synthetic construct

QQANSFPFT

LCVR: <SEQ ID NO: 4; AA; synthetic construct

DIQMTQSPSSVSASVGDRVT1TCRASQG1SSWLAWYQQKPGKAPKLL1YAASGLQ

SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK

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

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASGLQ

SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIKRTVAA

PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

DNA encoding Full-length LC: <SEQ ID NO: 6; DNA; synthetic construct

GACATCCAGATGACTCAGTCGCCGAGCTCGGTGTCGGCATCCGTGGGCGACA

GAGTGACCATTACTTGTCGGGCCAGTCAGGGAATTTCCAGCTGGCTCGCCTGG

TACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTCATCTATGCTGCGTCAG

GGCTTCAGTCCGGAGTGCCCTCAAGGTTCAGCGGTTCCGGATCCGGCACCGA

CTTCACCCTGACAATTTCTAGCCTGCAACCGGAGGATTTCGCGACTTACTACT

GCCAGCAGGCCAACAGCTTCCCTTTTACCTTCGGACCTGGTACCAAGGTCGAC

ATCAAGCGGACTGTCGCTGCACCCTCCGTGTTTATCTTCCCGCCCTCCGACGA

ACAGCTCAAGTCCGGGACCGCCTCAGTCGTGTGCTTGCTGAACAACTTCTACC

CTCGGGAGGCCAAAGTGCAATGGAAGGTCGACAACGCCCTGCAAAGCGGCA

ATTCCCAAGAGTCCGTGACTGAGCAGGACTCCAAGGATTCCACCTACTCGCTG

AGCTCCACCCTGACCCTGTCGAAGGCCGATTACGAAAAGCACAAAGTGTACG

CCTGCGAAGTGACCCATCAGGGACTTTCCTCGCCCGTGACCAAGTCCTTCAAC

CGCGGCGAATGCTAATGA HCDRls

<SEQ ID NO: 7; AA; synthetic construct

TFSGFSLSTXXVGVG wherein X at position 10 is S, G or P; X at position 11 is G or A.

<SEQ ID NO: 8; AA; synthetic construct

TFSGFSLSTSGVGVG

<SEQ ID NO: 9; AA; synthetic construct

TFSGFSLSTGGVGVG

<SEQ ID NO: 10; AA; synthetic construct

TFSGFSLSTPAVGVG

HCDR2s:

<SEQ ID NO: 11; AA; synthetic construct

XXFWXGDKR wherein X at position 1 is L or Q; X at position 2 is I or E; X at position 5 is N or T.

<SEQ ID NO: 12; AA; synthetic construct

LIFWNGDKR

<SEQ ID NO: 13; AA; synthetic construct

QEFWTGDKR

<SEQ ID NO: 14; AA; synthetic construct

QIFWTGDKR

<SEQ ID NO: 15; AA; synthetic construct

LEFWTGDKR

HCDR3:

<SEQ ID NO: 16; AA; synthetic construct

THKLGMNYFDY HCVRs:

<SEQ ID NO: 17; AA; synthetic construct

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTXXVGVGWIRQPPGKALEWLAXXFW

XGDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDY WGQGTLVTVSS wherein 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.

M7944: <SEQ ID NO: 18; AA; synthetic construct>

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIFWN

GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW GQGTLVTVSS

M10825 and M10824: <SEQ ID NO: 19; AA; synthetic construct>

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWT

GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW GQGTLVTVSS

M10782: <SEQ ID NO: 20; AA; synthetic construct

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTGGVGVGWIRQPPGKALEWLAQIFWT

GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW GQGTLVTVSS

M10469: <SEQ ID NO: 21; AA; synthetic construct

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTPAVGVGWIRQPPGKALEWLALEFWT

GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW GQGTLVTVSS

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP

APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVH

NAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT

KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Constant region (IgG4P): <SEQ ID NO: 23; AA; synthetic construct

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH

NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA

KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

Full-length Heavy Chains

M10825: <SEQ ID NO: 24; AA; synthetic construct

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWT

GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW

GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPG

DNA encoding the heavy chain of M10825: <SEQ ID NO: 25; DNA; synthetic construct

CAAATCACACTGAAGGAGTCCGGCCCAACGCTCGTGAAGCCGACCCAGACCC

TGACCCTCACATGTACCTTCTCCGGGTTCAGTCTGAGCACTTCCGGTGTCGGA

GTGGGCTGGATCAGACAGCCCCCTGGAAAGGCCCTTGAATGGTTGGCACAGG

AATTCTGGACGGGCGATAAGCGGTACAGCCCGAGCCTTAAGTCACGGCTGAC

TATCACCAAGGACACTTCCAAGAACCAGGTCGTGCTCACCATGACTAACATG

GACCCAGTGGACACCGCAACCTACTACTGCACCCATAAGCTGGGGATGAACT

ACTTTGACTACTGGGGCCAGGGCACTCTGGTCACTGTGTCGTCTGCGTCAACC

AAGGGTCCGTCCGTGTTTCCCCTGGCCCCGTGCTCGCGGAGCACCTCCGAGTC

CACTGCCGCCTTGGGCTGCCTGGTCAAAGACTACTTCCCTGAACCCGTGACTG

TCAGCTGGAACTCCGGAGCTCTGACCTCGGGAGTGCACACCTTCCCGGCCGTG

CTGCAATCGAGCGGCCTCTACTCCCTGTCCTCCGTCGTGACCGTGCCATCATC

AAACTTCGGAACCCAAACTTATACGTGCAACGTCGACCACAAGCCCTCCAAT

ACCAAAGTCGACAAGACCGTGGAGAGGAAATGCTGCGTGGAGTGTCCGCCTT

GCCCCGCGCCGCCGGTGGCCGGACCTAGCGTGTTCCTGTTCCCGCCGAAGCCA

AAGGACACTCTCATGATCTCCCGCACCCCTGAAGTCACTTGCGTCGTGGTGGA

CGTTTCCCACGAGGATCCCGAAGTGCAGTTCAATTGGTACGTGGACGGGGTG

GAAGTACATAACGCCAAGACCAAGCCCAGGGAAGAACAGTTTAACTCCACCT

TCCGGGTGGTGTCGGTGCTCACTGTGGTGCATCAGGATTGGCTCAATGGAAA

GGAGTACAAGTGCAAAGTGTCGAACAAGGGTCTGCCCGCTCCTATTGAAAAG

ACCATTTCCAAAACCAAGGGACAGCCCAGAGAGCCTCAGGTCTACACCCTGC

CTCCGAGCCGCGAGGAAATGACCAAGAACCAAGTGTCTCTGACTTGCCTCGT

GAAGGGATTCTACCCCTCCGATATCGCGGTGGAGTGGGAGAGCAACGGGCAG

CCAGAGAACAACTATAAGACCACCCCGCCTATGCTGGACTCCGATGGCTCCTT

CTTCTTGTACTCGAAGCTGACCGTGGACAAGTCCCGCTGGCAACAGGGAAAC GTGTTCAGCTGTAGCGTGATGCACGAAGCCCTGCACAACCACTACACCCAGA

AGTCCCTGTCGCTTTCCCCCGGG

M10824: <SEQ ID NO: 26; AA; synthetic construct

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAQEFWT GDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTHKLGMNYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLG

DNA encoding the heavy chain of M10824: <SEQ ID NO: 27; DNA; synthetic construct

CAAATCACACTCAAGGAGTCGGGTCCCACTCTCGTGAAGCCGACCCAGACCC TCACCCTGACTTGCACCTTTTCGGGATTCTCTCTGTCAACCTCCGGAGTGGGA GTCGGCTGGATCAGACAGCCTCCCGGAAAGGCCCTGGAGTGGCTGGCGCAGG AGTTCTGGACCGGCGACAAGCGGTACAGCCCCTCGCTGAAGTCACGCCTGAC TATCACCAAGGACACCTCCAAAAACCAAGTGGTGCTGACTATGACCAACATG GACCCAGTCGATACCGCCACTTACTACTGCACCCACAAGCTGGGCATGAACT ACTTCGACTACTGGGGCCAGGGTACTCTTGTCACCGTGTCCTCCGCCTCAACC AAGGGACCCAGCGTGTTTCCTCTTGCCCCTTGCTCCCGGTCAACATCTGAATC GACCGCAGCTCTGGGATGCCTCGTGAAGGACTATTTCCCCGAACCCGTGACG

GTATCCTGGAACAGCGGGGCTCTGACCTCGGGCGTGCACACCTTCCCCGCGGT GTTGCAGAGTTCCGGCCTTTACTCGCTGTCGTCCGTCGTGACTGTGCCGAGCT CCAGCTTGGGAACCAAGACGTACACTTGCAACGTGGACCACAAGCCGTCGAA CACCAAGGTCGATAAGAGAGTGGAGTCCAAATATGGACCTCCGTGTCCCCCT TGCCCTGCACCTGAATTCCTCGGCGGGCCTTCAGTGTTCCTCTTCCCGCCAAA GCCGAAGGACACCCTGATGATCAGCCGGACCCCGGAAGTGACCTGTGTGGTG GTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAATTGGTACGTCGATG GGGTCGAAGTGCATAACGCCAAGACTAAGCCCCGCGAAGAACAGTTCAACTC CACTTACCGCGTGGTGTCAGTCCTGACTGTGTTGCACCAGGACTGGCTGAACG GAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGACTGCCTAGCTCCATCGA AAAGACCATTAGCAAGGCCAAGGGACAACCGAGGGAACCCCAGGTCTACAC TCTGCCACCAAGCCAGGAAGAGATGACCAAGAACCAAGTGTCCCTGACCTGT

CTGGTCAAGGGGTTCTACCCGTCCGATATTGCCGTGGAATGGGAGTCCAACG GCCAGCCGGAAAACAATTACAAGACCACTCCGCCCGTGCTGGATAGCGACGG TTCCTTCTTCCTCTACTCCCGGCTCACCGTGGACAAGTCGAGGTGGCAGGAGG GGAACGTGTTCTCGTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACG C AGAAGTC CCTGAGC CTTTC GCTCGGC

Human BTLA ECD (SEQ ID NO: 28) KESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDR

QTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASER

PSKDEMAS

Human BTLA (SEQ ID NO: 29)

MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFEL ECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNG SYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLL ITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSET GIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEA

PTEYASICVRS

Mouse Balbc BTLA (SEQ ID NO: 30)

MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECEVQLNIKRNSKHSAW TGELFKIECPVKYCVHRPNVTWCKHNGTIWVPLEVGPQLYTSWEENRSVPVFVL HFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVRERTQNSSEHPLITVSDIPDATNAS GPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDT NLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLN

HCVIGRNPRQENNMQEAPTEYASICVRS

Mouse C57BL6 BTLA (SEQ ID NO: 31)

MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECPVQLTITRNSKQSART GELFKIQCPVKYCVHRPNVTWCKHNGTICVPLEVSPQLYTSWEENQSVPVFVLHF KPIHLSDNGSYSCSTNFNSQVINSHSVTIHVTERTQNSSEHPLITVSDIPDATNASGP STMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNL VDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHC VIGRNPRQENNMQEAPTEYASICVRS

Cynomolgus Monkey BTLA (SEQ ID NO: 32)

MKTLPAMLGSGRLFWVVFLIPYLDIWNIHGKESCDVQLYIKRQSYHSIFAGDPFK LECPVKYCAHRPQVTWCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPVLPSD NGSYRCSANFLSAIIESHSTTLYVTDVKSASERPSKDEMASRPWLLYSLLPLGGLP LLITTCFCLFCFLRRHQGKQNELSDTTGREITLVDVPFKSEQTEASTRQNSQVLLSE

TGIYDNEPDFCFRMQEGSEVYSNPCLEENKPGIIYASLNHSIIGLNSRQARNVKEA PTEYASICVRS Cynomolgus Monkey BTLA ECD variant (SEQ ID NO: 33)

KESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEG HTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASER PSKDEMASRLWLLYS Cynomolgus Monkey BTLA ECD (SEQ ID NO: 34)

KESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEG RHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSA1IESHSTTLYVTDVKSASE RPSKDEMASRLWLLYS

Claims

WE CLAIM:

1. An antibody that binds 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. the HCDR1 comprises TFSGFSLSTXXVGVG (SEQ ID NO: 7), wherein X at position 1 is S, G or P; X at position 11 is G or A, b. the HCDR2 comprises XXFWXGDKR (SEQ ID NO: 11), wherein X at position 1 is L or Q; X at position 2 is I or E; 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. the LCDR3 comprises QQANSFPFT (SEQ ID NO: 3).

2. The antibody of Claim 1, wherein the LCVR comprises a LCDR1 compnses RASQGISSWLA (SEQ ID NO: 1), a LCDR2 comprises YAASGLQS (SEQ ID NO: 2), and a LCDR3 comprises QQANSFPFT (SEQ ID NO: 3); and wherein the HCVR comprises a. a HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), a HCDR2 comprising LIFWNGDKR (SEQ ID NO: 12). a HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 1 ), or b. a HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), a HCDR2 comprising QEFWTGDKR (SEQ ID NO: 13), a HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or c. a HCDR1 comprising TFSGFSLSTGGVGVG (SEQ ID NO: 9), a HCDR2 comprising QIFWTGDKR (SEQ ID NO: 14), a HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or d. a HCDR1 comprising TFSGFSLSTPAVGVG (SEQ ID NO: 10), a HCDR2 comprising LEFWTGDKR (SEQ ID NO: 15), a 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 NOs: 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 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. 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 a 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. 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. The antibody of Claims 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. A nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 5, 24, or 26. A vector comprising 1) a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. A composition comprising 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. A cell comprising 1) a vector comprising a first nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 24 or 26, and 2) a second nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. A cell comprising 1) a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. The cell of Claim 17 or 18. wherein the cell is a mammalian cell. A process of producing an antibody comprising culturing the cell of any one of Claims 17-19 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. An antibody produced by culturing the cell of any one of claims 16-18 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. A pharmaceutical composition comprising the antibody of any one of Claims 1 to 13 and Claim 21. and one or more pharmaceutically acceptable carriers, diluents, or excipients. A method of treating an inflammatory or autoimmune disease comprising administering to a patient in need thereof, an effective amount of the antibody of any one of Claims 1 to 13 and Claim 21. The method of Claim 23. wherein the inflammatory or autoimmune disease is acute or chronic graft-versus-host disease (GVHD), chronic allergies, asthma, hay fever, 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, 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 transplant, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, or vasculitis. The antibody of any one of Claims 1 to 13 and Claim 21, for use in therapy. The antibody of any one of Claims 1 to 9, Claims 11 to 13 and Claim 21, for use in treating an inflammatory or autoimmune disease. The antibody for use of Claim 26, wherein the inflammatory or autoimmune disease is acute or chronic GVHD, chronic allergies 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 transplant, T1DM, autoimmune vasculitis, pernicious anemia, or vasculitis The use of Claim 27, wherein the inflammatory or autoimmune disease is acute or chronic GVHD, chronic allergies, 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.