KR20250115437A - BTLA agonist antibodies and their uses - Google Patents

Abstract

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

Description

BTLA agonist antibodies and their uses

The present disclosure relates to the medical field. More particularly, the present disclosure relates to agonistic antibodies directed against human B and T lymphocyte attenuating factor (BTLA), compositions comprising such agonistic BTLA antibodies, and methods of using such agonistic BTLA antibodies for the treatment of autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

BTLA, also known as cluster of differentiation 272 or CD272, is a member of the Ig superfamily and a checkpoint receptor family that negatively regulates immune cell activation. The natural ligand for BTLA is herpes virus entry mediator (HVEM, or CD270), a member of the TNF receptor superfamily. Binding of BTLA to 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.

For example, BTLA agonist antibodies are disclosed in PCT patent application publications WO2018/213113, WO2021/250419, and WO2022/087441. However, BTLA agonist antibodies are not approved for therapeutic use, and thus there is still a need to develop alternative BTLA agonist antibodies that can be used to treat autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders.

Accordingly, 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: 1) binding to human BTLA and cynomolgus monkey BTLA with similar affinity and favorable association and dissociation rates, and/or 2) being non-HVEM blocking BTLA agonists, resulting in enhanced agonism in the presence of HVEM, and/or 3) inhibiting primary human T cell proliferation, and/or 4) not causing significant cytokine release, and/or 5) exhibiting enhanced efficacy as a monotherapy for the treatment and/or prevention of disorders such as autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders, and/or 6) showing very limited internalization in all subpopulations of human peripheral blood mononuclear cells (PBMCs) tested, including T cells, B cells, monocytes, myeloid DCs, pDCs, and NK cells, and/or 7) being less immunogenic, and/or 8) requiring lower doses or less 9) exhibits in vivo stability, physical and chemical stability, including but not limited to, acceptable thermal stability, solubility, low self-association and other pharmacokinetic properties for development, manufacture, formulation, storage, administration and use in the treatment of autoimmune disorders, allergic diseases, asthma, or other inflammatory disorders, and/or is therapeutically effective in frequent dosing.

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

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

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

c. HCDR3 contains THKLGMNYFDY (SEQ ID NO: 16);

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

e. LCDR2 contains YAASGLQS (SEQ ID NO: 2);

f. LCDR3 contains QQANSFPFT (SEQ ID NO: 3).

An antibody or antigen-binding fragment thereof is provided.

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprises QIFWTGDKR (SEQ ID NO: 14), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprises LEFWTGDKR (SEQ ID NO: 15), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an HCVR, 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 of the human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

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

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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.

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

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

In another aspect, provided herein is a pharmaceutical composition comprising a novel human BTLA agonist antibody or antigen-binding fragment thereof, or a nucleic acid encoding the same, as described herein. The pharmaceutical composition comprising the novel human BTLA agonist antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding the same, as described herein, is useful for the treatment of acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), 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 (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, including but not limited to autoimmune disorders, allergic diseases, or other inflammatory disorders.

Unless defined otherwise, 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.

Furthermore, referring to an element in the singular does not exclude the possibility of more than one element, unless the context clearly requires that there be only one element. Therefore, the singular generally means "at least one."

definition

As used herein, "about" means within a statistically significant range of 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 acceptable variation encompassed by "about" will depend on the particular system under study and will be readily understood by those skilled in the art.

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

As used herein, "amino acid" refers to a molecule characterized by the presence of at least one amine group and at least one carboxylic acid group, and may contain other functional groups. As is known in the art, there is a set of 20 amino acids designated as standard amino acids and which can be used as building blocks for peptides/proteins produced by any organism. The amino acid sequences of the present disclosure contain the standard single-letter or three-letter codes for the 20 naturally occurring amino acids.

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., an "antigen-binding portion" of an antibody). The term "antibody," as used herein, refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Exemplary antibodies of the present disclosure are immunoglobulin G (IgG) type antibodies comprising four polypeptide chains: two heavy chains and two light chains joined 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, CH1, CH2, and CH3). Each light chain is comprised of an N-terminal light chain variable region (LCVR) and a light chain constant region. HCVRs and LCVRs can be further subdivided into highly variable regions, termed complementarity determining regions (CDRs), separated by more conserved regions, termed framework regions (FRs). Each HCVR and LCVR consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen. Assignment of amino acid residues to CDRs is done by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia et al. (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 (at www.imgt.org). This can be done according to well-known systems, including those described in the available International ImmunoGeneTics database; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). A combination of IMGT and North CDR definitions was used for the exemplified anti-human BTLA antibodies described herein.

In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of a hinge, a CH1 domain, a CH2 domain, and a CH3 domain. The constant region of an antibody can mediate 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.

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

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

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

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

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 to self-cells and/or tissues or transplanted cells and/or tissues. The terms "autoimmune disease" or "autoimmune disorder" are intended to encompass such conditions, regardless of whether they are mediated by humoral and/or cellular immune responses. Exemplary autoimmune diseases or disorders include acute or chronic graft-versus-host disease (GVHD), chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-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 Including, but not limited to, vasculitis, pernicious anemia, and vasculitis.

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

As used herein, unless otherwise indicated, the terms "bind" and "binds" are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which brings the two proteins or molecules into proximity as determined by conventional methods known in the art.

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

As used herein, "effective amount" means an amount or dosage of one or more of the BTLA agonist antibodies disclosed herein or pharmaceutically acceptable salts thereof, which, when administered in single or multiple doses to a subject in need thereof, produces a desired effect in the subject being diagnosed or treated (i.e., capable of producing a clinically measurable difference in the condition of the subject, such as a decrease in proinflammatory cytokines or a change in lymphocyte activation). An effective amount can be readily determined by one of ordinary skill in the art using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for a subject, a number of factors are taken into consideration, including but not limited to the species of mammal, its size, age, and general health, the particular disease or disorder involved, the extent or involvement or severity of the disease or disorder, the response of the subject, the specific antibody administered, the mode of administration, the bioavailability characteristics of the formulation administered, the selected dosage regimen, the use of concomitant medications, and other relevant circumstances. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody.

BTLA polypeptide "extracellular domain" or "ECD" refers to a form of the BTLA polypeptide that is essentially free of transmembrane and cytoplasmic domains. Preferably, the BTLA ECD has less than 1% 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 polypeptide or human BTLA ECD polypeptide can be purchased commercially from a variety of sources, such as Sino Biological (Houston, TX; see, e.g., catalog number 11895-H02H or 29982-H38H).

The term "Fc region" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an antibody heavy chain, wherein the C-terminal polypeptide sequence is obtainable by papain digestion of an intact antibody. The Fc region may comprise a native or variant Fc sequence. The Fc sequence of an antibody typically 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 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.

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

As used herein, the term "epitope" refers to an amino acid residue of an antigen that is 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 connection with a conformational epitope. In some embodiments, a conformational epitope can be used to describe a region of an antigen covered by an antibody (e.g., the footprint of an antibody when bound to an antigen). In some embodiments, a conformational epitope can describe an amino acid residue of an antigen that is within a certain proximity (e.g., within a certain number of angstroms) of an amino acid residue of an antibody. The term "epitope" can also be used in connection with a functional epitope. In some embodiments, a functional epitope can be used to describe an amino acid residue of an antigen that interacts with an amino acid residue of an antibody in a manner that contributes to the binding energy between the antigen and the antibody. Epitopes can be determined using various 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 employed, and may also vary depending on the different experimental conditions employed, for example, due to conformational changes or cleavage of the antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology , Humana Press, 3 ^rd ed. 2018; Holst et al., Molecular Pharmacology 1998, 53(1): 166-175) and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine-scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

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

The term "subject" as used herein 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, and the like. Preferably, the subject is a human.

The terms "treatment" or "treating," as used herein, refer to any process that may result in slowing, controlling, delaying, or stopping the disorder or disease described herein, or in ameliorating the symptoms of the disorder or disease, but does not necessarily indicate complete elimination of all of the disorder or disease symptoms. Treatment includes administering a protein or nucleic acid or a vector or composition to a patient, particularly a human, for the treatment of a disease or condition.

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 can induce or increase one or more activities or functions associated with human BTLA, e.g., one or more of the activities or functions described in the Examples.

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

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

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

c. HCDR3 contains THKLGMNYFDY (SEQ ID NO: 16);

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

e. LCDR2 contains YAASGLQS (SEQ ID NO: 2);

f. LCDR3 contains QQANSFPFT (SEQ ID NO: 3).

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprises QIFWTGDKR (SEQ ID NO: 14), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprises LEFWTGDKR (SEQ ID NO: 15), and HCDR3 comprises THKLGMNYFDY (SEQ ID NO: 16).

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises an LCVR and an HCVR, 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 of the human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22.

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

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

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

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BTLA, wherein the antibody or antigen-binding fragment thereof comprises 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.

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

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

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

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

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

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

The nucleic acids of the present disclosure can be expressed in a host cell, for example, after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing the nucleic acid to which they are operably linked 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(s) 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 well-known methods, such as stable or transient transfection, transformation, transduction, or infection. Additionally, the expression vector may contain one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired nucleic acid sequence.

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

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

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

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

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

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 include one or more pharmaceutically acceptable excipients, diluents, or carriers. The pharmaceutical composition may be prepared by methods well known in the art (see, 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 antibodies or antigen-binding fragments thereof, described herein can be used to treat autoimmune disorders, allergic diseases, or other inflammatory disorders, including but not limited to acute or chronic GVHD, chronic allergic diseases (e.g., asthma, hay fever, or allergic rhinitis), psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, pSS, AtD, ERT, factor VIII deficiency, myositis, LN, organ and tissue transplantation, T1DM, autoimmune vasculitis, pernicious anemia, and vasculitis.

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

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

The following examples are provided to illustrate the claimed invention and are not intended to limit it.

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 HEK 293 or CHO, can be transiently or stably transfected with an expression system for antibody secretion using an optimal pre-determined HC:LC vector ratio (e.g., 1:1, 1:2, or 1:3) or a single vector system encoding both HC and LC. The clarified medium containing the antibody or antigen-binding fragment thereof 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 for Fab fragments, a KappaSelect column (Cytiva), equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column can be washed to remove non-specific binding components. The bound antibody or antigen-binding fragment can be eluted, for example, using a pH gradient (e.g., 20 mM Tris buffer, pH 7.0 → 10 mM acetate/sodium citrate buffer, pH 3.0, or phosphate buffered saline, pH 7.4 → 100 mM glycine buffer, pH 3.0). Antibody fractions can be detected, for example, by SDS-PAGE and analytical size exclusion chromatography, and then pooled. Further purification is optional, depending on the intended use. The antibody or antibody fragment can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by conventional techniques, including size exclusion, hydrophobic interaction, ion exchange, multimode, or hydroxyapatite chromatography. The purity of the antibody after such chromatographic steps is about 95% to about 99%. The product may be stored refrigerated, immediately frozen at -70°C, or lyophilized. The sequence identification numbers for the amino acid sequences of specific exemplified BTLA agonist antibodies of the present invention are provided in Table 1 below.

Table 1. Amino acid sequences of exemplified BTLA agonist antibodies.

Binding affinity and kinetics

The binding affinity and kinetics of the BTLA agonist antibodies (M10825 and M10824) of the present disclosure to BTLA are measured by surface plasmon resonance using a Biacore 8k (Citiva). The BTLA agonist antibodies are captured via an anti-human Fc antibody (Citiva) immobilized on a CM4 sensor chip (Citiva) via amine coupling, and the binding affinity is measured by flowing recombinant human or cynomolgus BTLA at concentrations prepared from 4-fold serial dilutions starting at 100 nM and going down to 0.1 nM. Experiments are performed 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) at 37°C. For each cycle, BTLA agonist antibodies prepared at 5 μg/mL are flowed through the active flow cell at 10 μL/min to achieve approximately 120 RU of capture, after which human or cynomolgus BTLA is injected at 100 μL/min for 2 min and then dissociated for 20 min. The chip surface is regenerated using 3 M MgCl ₂ solution at 10 μL/min for 30 s. By fitting the data to a 1:1 Langmuir coupling model, k _on and k _off are derived, and K _D is calculated. Following essentially the same procedure as described above, the following parameters (shown in Table 2) are observed. The data presented below are the average of three experiments.

Table 2. In vitro binding affinities of M10824 and M10825 to human and cynomolgus BTLA.

BTLA agonist antibody-induced signal transduction

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

Fixed plates are prepared by a blocking step followed by incubation with 200 nM anti-human IgG, Fcγ (Jackson Immunoresearch). Titration of isotype control or BTLA antibody (0-300 nM) is captured and incubated with shaking at room temperature for 2 hours, followed by a PBS wash step. PathHunter® Jurkat BTLA signaling cell line is added to the wells at 10 x 10 ³ cells/well and incubated for 1 hour in a humidified tissue culture incubator at 37°C and 5% CO ₂ . Phospho-BTLA is detected with the PathHunter® Bioassay Detection Kit using enzyme fragment complementation (EFC) technology and luminescence is read on an EnVision (Perkin Elmer). Using the luminescence values, IC ₅₀ values can be determined using graphing software (GraphPad Prism).

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

HVEM non-blocking

To determine the ability of the BTLA agonist antibodies of the present invention (M10825 and M10824) to block HVEM ligand activation of BTLA, the Pathhunter® Jurkat BTLA signaling cell line overexpressing 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 the BTLA receptor tagged with a Prolink™ tag and an SH2 domain tagged with an enzyme acceptor. Receptor activation and phosphorylation result in recruitment of SH2 to the receptor, forcing the complementation of two β-galactosidase enzyme fragments (the enzyme acceptor and the Prolink™). The resulting functional enzyme hydrolyzes the substrate, generating a chemiluminescent signal.

Assay plates are prepared by seeding 20 x 10 ³ PathHunter® Jurkat BTLA cells/well for 15 minutes at room temperature, followed by addition of isotype control or titer of BTLA antibody (0-10 μg/mL) and incubation of the assay plates in a 37°C, 5% CO ₂ incubator for 1 hour. PathHunter® U2OS HVEM ligand cells are then added at 50 x 10 ³ cells/well and the plates are incubated for 2 hours at room temperature. Phospho-BTLA is detected with the PathHunter® Bioassay Detection Kit using enzyme fragment complementation (EFC) technology and luminescence is read on an Envision (PerkinElmer). Luminescence values for the 3.3 μg/mL titration point are presented as a percentage of the signal obtained by a known HVEM non-blocker at this dose and compared to a known HVEM blocker.

Essentially following the procedure described above, antibody M10825 resulted in 104% of the signal of the known HVEM blocker (100% signal) at 3.3 μg/mL, compared to the isotype control at 92%. Similarly, the BTLA agonist antibody M10824 resulted in 104% of the signal of the known HVEM blocker (100% signal) at 3.3 μg/mL, compared to the isotype control at 93%. 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.

Inhibition of human primary B cell proliferation

The in vitro efficacy of the 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 a suitable human primary cell medium. Anti-IgM is coated on plates along with an isotype control or a titer 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 a 5% CO ₂ atmosphere for 72 hours, followed by a [ ³ H]-thymidine pulse for the final 18 hours. After incubation, the plates are removed and stored at -80°C until ready for harvest. Cells are lysed by thawing and harvested using a FilterMate Universal Harvester (PerkinElmer). Proliferation is assessed by measuring [ ^3H ]-thymidine incorporation using a MicroBeta ² 2450 microplate counter (PerkinElmer).

The relative proliferative response in these assays can be assessed using counts, and the percent inhibition can be determined using the formula [% inhibition = (AVGmax signal - signal samples) / AVGmax signal x 100], which can then be used to determine IC ₅₀ values using graphing software (GraphPad Prism).

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

Efficacy of BTLA agonist antibodies in a humanized mouse model of GvHD

Because the BTLA agonist antibody of the present invention does not exert agonism on murine BTLA, the in vivo efficacy of the BTLA agonist antibody of the present invention can be tested in NOD SCID gamma2 chain-/- (NSG) humanized mice to evaluate its ability to suppress human T and B cell function in an in vivo setting. More specifically, the in vivo efficacy was evaluated in a humanized NSG mouse model of GvHD, a mouse model in which human peripheral blood mononuclear cells (PMBCs) are transplanted, whereby human immune cells recognize the mouse as foreign, become activated, and drive GvHD. Characteristic phenotypes indicative of T and B cell activation are the circulating human proinflammatory cytokines and immunoglobulins (Ig) in mouse plasma. Therefore, using this model, the ability of the BTLA agonist antibody (which does not exert agonism on murine BTLA) to suppress the production of these circulating factors can be evaluated.

Briefly, studies using the NSG mouse model of GvHD can be conducted as follows.

Female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, JAX Labs, Stock # 05557) are housed four per cage at 72°F under a 12-h light:dark cycle, with food and water available ad libitum. Human peripheral blood mononuclear cells (PBMCs) are isolated from LRS tubes (San Diego Blood Bank, San Diego, CA) obtained from two donors using SepMate 50 Ficoll preparation tubes according to the manufacturer's instructions (StemCell Technologies, Vancouver, British Columbia). Freshly isolated PBMCs can be suspended in PBS at 1.2 x 10 ⁸ cells/mL, and 100 μL of the PBMC suspension can be transplanted intravenously into mice on day 0 (1.2 10 ⁷ /mouse). On day 1, mice can be divided into weight groups administered subcutaneously with 10 mg/kg isotype control or test BTLA antibody (200 μL/mouse) at doses ranging from 0.001 to 10.0 mg/kg. Weekly dosing can continue for 15 days. Health checkups and body weight measurements can be performed regularly. On day 15, mice can be sacrificed, and blood can be collected by cardiac puncture under isoflurane anesthesia.

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

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

In a second study performed essentially as described above, but with PBMCs from different donor plasma, animals treated with antibody M10825 exhibited a decrease 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) compared to the isotype control, with doses (i.e., 0.1, 1.0, and 10.0 mg/kg antibody) resulting in a statistically significant decrease in INF-γ compared to the isotype control (data not shown).

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

Overall, these data demonstrate that the BTLA agonist antibody M10825 is effective in preventing GvHD in vivo.

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

No or low internalization in a subset of human PBMCs

In the clinic, antibody-based biotherapeutics with higher immunogenicity have higher levels of internalization than antibodies with lower immunogenicity (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 in human PBMCs, PBMCs are isolated from the buffy coat of three donors according to the instructions provided by StemCell Technologies for Sepmate-50. PBMCs are counted with a Vi-Cell cell counter and adjusted to 2 x ¹⁰⁶ cells/ml in pre-warmed complete medium (X-Vivo15, Lonza #04-418Q) supplemented with 10% FBS. Aliquot the cells into 96-well plates at 2 x 10 ⁵ cells/well in 100 μl/well. Place the plates in a humidified incubator at 37°C and 5% CO ₂ .

Prepare 4X BTLA antibody (i.e., 60 nM) and 4X TAMRA-QSY7-Fab antibody (i.e., 180 nM) and mix in equal volumes. Incubate at 4°C for 30 minutes to form anti-BTLA/TAMRA-QSY7-Fab complexes. Then, aliquot 100 μl/well of the anti-BTLA/TAMRA-QSY7-Fab complex into the PBMCs prepared above. Incubate the cells in a humidified incubator at 37°C and 5% CO ₂ for 3 hours. Centrifuge the cells at 500 g for 5 minutes and then wash them three times in FACS staining buffer from BD Biosciences. Cells are then stained with a 15-color surface marker panel including Aqua Live/Dead dye and fluorophore-conjugated antibodies to 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/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 established. LSRFortessa X-20 is used for data acquisition. Instrument calibration is performed according to the manufacturer's instructions. A compensation panel is established using antibody-coupled compensation beads. FlowJo 10 is used for data analysis, and internalization is measured by MFI values for each subpopulation. Apoptotic cells are excluded from the analysis. Gates are set based on FMO staining.

Essentially following the procedure described above, the BTLA agonist antibodies M10825 and M10824 and the isotype control were assayed for internalization. Both antibodies M10825 and M10824 demonstrated low or no internalization in all cell types, including T cells, B cells, monocytes, myeloid DCs, pDCs, and NK cells, based on mean fluorescence intensity (MFI) values. The internalizing antibody, an internal positive control, demonstrated very strong internalization across different cell types. No internalization was observed for the isotype control. The internalizing BTLA antibody clones identified during the preliminary internalization screening exhibited consistent internalization profiles. These data demonstrate that the BTLA agonist antibodies M10825 and M10824 exhibit limited internalization in all human PBMC subsets tested, indicating a low immunogenicity risk.

Low immunogenicity risk of BTLA agonist antibodies M10825 and M10824

BTLA agonist antibodies can be characterized for their relative clinical immunogenicity risk using in silico and in vitro methods, including but not limited to T cell proliferation assays, conventional reactivity assays, and MHC-associated peptide proteomics (MAPP) assays.

MAPP test method

MAPP profiles human leukocyte antigen class II (HLA-II)-presenting peptides on human dendritic cells previously treated with test molecules. Briefly, primary human dendritic cells from 10 normal human donors can be prepared from the leukocyte buffy coat by isolation of CD14-positive cells, essentially as described in the literature [Knierman, MD, et al., 2020], and differentiated into immature dendritic cells by incubation with 20 ng/ml IL-4 and 40 ng/ml GM- _CSF in complete RPMI medium (Sigma-Aldrich, catalog # R0278) containing 5% serum replacement for 4 days at 37°C and 5% CO 2 . Afterwards, 3 micromolar test antibodies can be added to about 5 x ¹⁰⁶ cells on day 4, and after about 5 hours of incubation, the cells can be replaced with fresh medium containing 5 μg/mL lipopolysaccharide (LPS) to convert them into mature dendritic cells. The following day, the matured cells can be lysed in 1 mL of RIPA buffer with protease inhibitors and DNAse. Using an automated liquid handling system, HLA-II molecules can be isolated from the thawed lysate using a biotinylated anti-pan-HLA class II antibody (clone Tu39). The 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-cleaned 10k MWCO filter to remove high molecular weight proteins. Isolated HLA-II peptides can be analyzed by nano LC/MS using a Thermo easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. Separation can be achieved using a 75 μm x 15 cm PepMap RSLC c18 column for a flow rate of 300 nL/min and a 65-minute gradient from 0.1% formic acid in water as solvent A to 80% acetonitrile + 0.1% formic acid as solvent B. Mass spectrometry can be run in full scan mode at a resolution of 240,000, followed by a 3-second data-dependent MS/MS cycle consisting of an ion trap rapid scan with high-energy collisional dissociation (HCD) and electron-transfer/higher-energy collisional dissociation fragmentation (EThcD) fragmentation.

Peptide identifications can be generated by an in-house proteomics pipeline (see, e.g., Higgs, RE, et al., Methods Mol. Biol., 428 , 209-230 (2008)) using a multi-search algorithm without enzyme search parameters against bovine/human databases containing test antibody sequences. Identification files can be processed for the samples using the KNIME workflow. Identified peptides from the test article can be aligned to the parent sequences. An annotated summary can be generated for all donors, including the percentage of donors displaying non-germline residues, the number of different regions displaying peptides with non-germline residues, and the depth of peptide display in each region with non-germline residues.

In a MAPP analysis performed essentially as described above, only 10% of donors displayed peptides containing non-germline residues in the heavy chain CDR3 of antibody M10825. None of the other displayed peptides from M10825 contained non-germline residues and therefore do not pose an immunogenicity risk.

In summary, the compilation of data from these various in silico and in vitro methods, including but not limited to the MAPP assay described above (data not shown), strongly supports that the BTLA agonist antibodies M10825 and M10824 each have a low to moderate risk of inducing clinical immunogenicity.

Attach an electronic file of the sequence list

Claims (28)

An antibody that binds to human B and T lymphocyte attenuator (BTLA), comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementary determining region 1 (HCDR1), heavy chain complementary determining region 2 (HCDR2), and heavy chain complementary determining region 3 (HCDR3), and the LCVR comprises light chain complementary determining region 1 (LCDR1), light chain complementary determining region 2 (LCDR2), and light chain complementary determining region 3 (LCDR3), wherein:
a. HCDR1 contains TFSGFSLSTXXVGVG (SEQ ID NO: 7),
Here, X at position 10 is S, G or P; X at position 11 is G or A,
b. HCDR2 contains XXFWXGDKR (SEQ ID NO: 11),
Here, 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. HCDR3 contains THKLGMNYFDY (SEQ ID NO: 16),
d. LCDR1 contains RASQGISSWLA (SEQ ID NO: 1),
e. LCDR2 contains YAASGLQS (SEQ ID NO: 2),
f. LCDR3 contains QQANSFPFT (SEQ ID NO: 3).
Antibodies. In claim 1, an antibody comprising LCDR1 comprising RASQGISSWLA (SEQ ID NO: 1), LCDR2 comprising YAASGLQS (SEQ ID NO: 2), and LCDR3 comprising QQANSFPFT (SEQ ID NO: 3), and wherein HCVR comprises:
a. HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprising LIFWNGDKR (SEQ ID NO: 12), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or
b. HCDR1 comprising TFSGFSLSTSGVGVG (SEQ ID NO: 8), HCDR2 comprising QEFWTGDKR (SEQ ID NO: 13), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or
c. HCDR1 comprising TFSGFSLSTGGVGVG (SEQ ID NO: 9), HCDR2 comprising QIFWTGDKR (SEQ ID NO: 14), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16), or
d. HCDR1 comprising TFSGFSLSTPAVGVG (SEQ ID NO: 10), HCDR2 comprising LEFWTGDKR (SEQ ID NO: 15), HCDR3 comprising THKLGMNYFDY (SEQ ID NO: 16). An antibody according to claim 1, wherein HCVR comprises an amino acid sequence of SEQ ID NO: 17 and LCVR comprises an amino acid sequence of SEQ ID NO: 4. An antibody according to claim 3, wherein HCVR comprises an amino acid sequence of SEQ ID NO: 18, 19, 20, or 21, and LCVR comprises an amino acid sequence of SEQ ID NO: 4. An antibody according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain constant region and a light chain constant region. An antibody in claim 5, wherein the heavy chain constant region is of a human IgG2 subtype, a human IgG4 subtype, or a modified human IgG4 subtype. An antibody in claim 6, wherein the heavy chain constant region is a human IgG2 subtype comprising the amino acid sequence of SEQ ID NO: 22. An antibody in 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. An antibody according to claim 8, wherein the heavy chain constant region is a modified human IgG4 subtype comprising the amino acid sequence of SEQ ID NO: 23. An antibody according to claim 7, wherein the antibody comprises a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 24 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 5. An antibody according to 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. An antibody according to claim 9, wherein the antibody comprises 1) an HC comprising an amino acid sequence of SEQ ID NO: 26, and 2) an LC comprising an amino acid sequence of SEQ ID NO: 5. An antibody according to claim 12, wherein the antibody comprises two HCs and two LCs, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and each light chain comprises the amino acid sequence of SEQ ID NO: 5. A nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, 24, or 26. A vector comprising 1) a first nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding an 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 NO: 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 a vector comprising 1) a first nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 5. 1) A cell comprising a first vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 or 26, and 2) a second vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5. A cell according to claim 17 or 18, wherein the cell is a mammalian cell. A method for producing an antibody, comprising culturing a cell of any one of claims 17 to 19 under conditions that allow an antibody to be expressed, and recovering the expressed antibody from the culture medium. An antibody produced by culturing the cells of any one of claims 16 to 18 under conditions that allow the antibody to be expressed, and recovering the expressed antibody from the culture medium. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 13 and 21, and one or more pharmaceutically acceptable carriers, diluents or excipients. A method for treating an inflammatory or autoimmune disease, comprising administering to a patient in need of treatment for an inflammatory or autoimmune disease an effective amount of an antibody according to any one of claims 1 to 13 and 21. In claim 23, the inflammatory or autoimmune disease is acute or chronic graft-versus-host disease (GVHD), chronic allergy, asthma, hay fever, allergic rhinitis, psoriatic arthritis, psoriasis, pemphigus vulgaris, idiopathic pulmonary fibrosis, hidradenitis suppurativa, rheumatoid arthritis (RA), 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-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), How to treat autoimmune vasculitis, pernicious anemia, or vasculitis. An antibody for use in therapy according to any one of claims 1 to 13 and 21. An antibody for use in the treatment of an inflammatory or autoimmune disease according to any one of claims 1 to 9, 11 to 13, and 21. An antibody for use in claim 26, wherein the inflammatory or autoimmune disease is acute or chronic GVHD, chronic allergy, 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. The use of claim 27, wherein the inflammatory or autoimmune disease is acute or chronic GVHD, chronic allergy, asthma, hay fever, allergic rhinitis, psoriatic arthritis, psoriasis, RA, SjS, SLE, scleroderma, Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's disease, Addison's disease, psoriasis, dermatomyositis, CIDP, GBS, MS, myasthenia gravis, vasculitis, T1DM, autoimmune vasculitis, pernicious anemia, or vasculitis.