Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• 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.
• BTLA human B and T lymphocyte attenuator
• BTLA also known as cluster of differentiation 272 or CD272
• 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.
• 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/
• 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).
• 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).
• 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).
• 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.
• 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).
• EU Numbering also referred to as IgG4P
• 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.
• 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.
• 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).
• GVHD graft-versus-host disease
• chronic allergic diseases such as asthma, hay fever, or allergic rhinitis
• psoriatic arthritis psoriasis
• pemphigus vulgaris idiopathic pulmonary' fibrosis
• idiopathic pulmonary' fibrosis hidradenitis suppurativa
• RA rheumatoid arthritis
• SjS Sjogren’s syndrome
• SLE systemic lupus erythematosus
• scleroderma Crohn’s disease
• celiac disease ulcerative colitis
• 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).
• CIDP demyelinating polyneuropathy
• GSS Guillain-Barre syndrome
• MS multiple sclerosis
• pSS progressive systemic sclerosis
• AtD atopic dermatitis
• ERT enzyme replacement therapy
• Factor VIII deficiency myositis, lupus nephritis (LN), organ and tissue transplant, type 1 diabetes mellitus (T1DM), autoimmune vasculitis, pernicious anemia, and vasculitis.
• activity 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.
• 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.
• amino acids 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.
• antibody 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).
• antibody 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.
• IgG immunoglobulin G
• 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).
• CDRs complementarity determining regions
• FRs framework regions
• 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.
• 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..
• 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.
• the antibodies described herein are of the human IgG2 or IgG4 subtype or modified human IgG2 or IgG4 subtype.
• 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.
• 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.
• 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.
• ds-DNA anti-double stranded DNA
• the agonist antibodies of the present invention can be used for preventing or treating autoimmune disorders, allergic disease, asthma, or other inflammatory disorders.
• 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.
• 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 (
• 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.
• cyno cynomolgus or cynomolgus monkey
• cynomolgus or “cynomolgus monkey” are used interchangeably herein.
• 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.
• an 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• antibodies having human Fc sequences 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.
• epitope refers to the amino acid residues of an antigen, that are bound by an antibody.
• An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope.
• epitope may be used in reference to a structural epitope.
• a structural epitope 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).
• 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.
• epitope may also be used in reference to a functional epitope.
• a functional epitope 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 energy 7 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-ray 7 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.
• NMR nuclear magnetic resonance
• HDX hydrogendeuterium exchange
• nucleic acid refers 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.
• subject 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.
• the subject is a human.
• treatment 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.
• novel antibodies or antigen binding fragments thereof that binds human BTLA are novel antibodies or antigen binding fragments thereof that binds human BTLA.
• the novel antibodies or antigen binding fragments thereof that bind human BTLA are agonists of BTLA.
• 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.
• 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.
• HCVR heavy chain complementarity determining region 1
• HCDR2 heavy chain complementarity determining region 2
• HCDR3 heavy chain complementarity’ determining region 3
• 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).
• 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).
• the HCVR comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises RASQGISSWLA (SEQ ID NO: 1), the LCD
• 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).
• 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).
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• EU Numbering also referred to as IgG4P
• 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.
• 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.
• HC heavy chain
• LC light chain
• 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.
• 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.
• 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.
• pharmaceutical compositions comprising a novel human BTLA agonist antibody, or antigen binding fragment thereof, described herein or a nucleic acid encoding the same.
• 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.
• acute or chronic GVHD chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis)
• psoriatic arthritis such as asthma, hay fever, or allergic rhinitis
• CIDP CIDP
• GBS GBS
• MS myasthenia gravis
• pSS pSS.
• AtD ERT.
• ERT Factor VIII deficiency
• myositis LN
• organ and tissue transplant T1DM.
• autoimmune vasculitis pernicious anemia, and vasculitis.
• the present disclosure provides nucleic acids comprising a sequence encoding the amino acid sequence of SEQ ID NOs: 5. 24, or 26.
• 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.
• 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.
• the present disclosure further provides antibodies, or antigen binding fragments thereof, produced by any of the processes described herein.
• 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
• kits for treating an inflammatory or autoimmune disease in a subject 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).
• 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.
• acute or chronic GvHD chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis)
• chronic allergic diseases such as asthma, hay fever, or allergic rhinitis
• psoriatic arthritis such as asthma, hay fever, or allergic rhinitis
• psoriasis psori
• CIDP CIDP
• GBS GBS
• MS myasthenia gravis
• pSS AtD
• ERT Factor VIII deficiency
• myositis LN
• organ and tissue transplant T1DM. autoimmune vasculitis, pernicious anemia, and vasculitis.
• 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.
• 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.
• 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).
• 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).
• mice 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).
• 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).
• MSD Mesoscale Discovery
• IL- 10 interleukin- 10
• TNF-a tumor necrosis factor alpha
• mice 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).
• 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).
• 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
• PBMCs peripheral blood mononuclear cells
• PBMCs are counted with Vi-Cell cell counter and adjusted to 2 x 10 6 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 10 5 cells/well. Plates are placed in a humidified incubator at 37°C and 5% CO2.
• 4X BTLA antibody i.e., 60 nM
• 4X TAMRA-QSY7-Fab antibody i.e., 180 nM
• 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.
• 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 MHC-associated peptide proteomics
• test antibody may be added to approximately 5 x 10 6 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.
• HCD collisional dissociation
• EhcD electron-transfer/higher-energy collision dissociation 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.
• LCDR1 ⁇ SEQ ID NO: 1; AA; synthetic construct>
• LCDR2 ⁇ SEQ ID NO: 2; AA; synthetic construct
• TFSGFSLSTXXVGVG wherein X at position 10 is S, G or P; X at position 11 is G or A.
• XFWXGDKR 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.
• 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.
• M10825 and M10824 ⁇ SEQ ID NO: 19; AA; synthetic construct>
• M10469 ⁇ SEQ ID NO: 21; AA; synthetic construct
• Constant region (IgG2): ⁇ SEQ ID NO: 22; AA; Homo sapiens>
• DNA encoding the heavy chain of M10824 ⁇ SEQ ID NO: 27; DNA; synthetic construct

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