Antibodies to BTLA and uses thereof

Abstract

Provided are monoclonal antibodies and antigen-binding fragments thereof specific for BTLA, and methods of using same to treat autoimmune diseases, including combination therapies.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of the filing date of International Application No. PCT / CN2022 / 075109, filed January 29, 2022, the entire contents of which, including any drawings and sequence listing, are incorporated herein by reference.

[0002] Sequence Listing This application has been submitted electronically in XML format and contains a Sequence Listing XML file, which is incorporated herein by reference in its entirety. The XML copy, created on Jan. 23, 2023, is named 131206-01219_SL_XML.xml and is 170,638 bytes in size. [Background technology]

[0003] B and T lymphocyte attenuator (BTLA; also known as CD272) is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and PD-1 (Watanabe et al., Nat Immunol. 4:670-679, 2003). BTLA is widely expressed throughout the immune system on both myeloid and lymphoid cells (Han et al., J Immunol. 172:5931-9, 2004). Following engagement by its ligand herpesvirus entry mediator (HVEM), BTLA recruits the phosphatases SHP-1 and SHP-2 to its cytoplasmic domain (Sedy et al., Nat Immunol. 6:90-8, 2005), which then inhibit the signaling cascade of activated receptors.

[0004] BTLA knockout mice lacking an intact BTLA gene show hyperproliferative B and T cell responses in vitro, higher titers to DNP-KLH after immunization, and increased susceptibility to EAE (Watanabe et al., Nat. Immunol. 4:670-679, 2003). When observed to old age, BTLA knockout mice spontaneously develop autoantibodies, autoimmune hepatitis-like disease, and inflammatory cell infiltrates in multiple organs (Oya et al., Arthritis Rheum. 58:2498-2510, 2008). Thus, BTLA inhibitory receptors appear to play an important role in maintaining immune homeostasis and inhibiting autoimmunity. Furthermore, HVEM-BTLA signaling is involved in regulating mucosal inflammation and infection immunity (Shui et al., J Leukoc Biol. 89:517-523, 2011).

[0005] Monoclonal antibodies that bind to mouse BTLA can act as agonists, inducing signal transduction through the receptor and inhibiting immune cell responses. In the presence of agonistic anti-BTLA antibodies (mAh), anti-CD3 and anti-CD28 activated T cells show reduced IL-2 production and proliferation (Kreig et al., J.Immunol.175,6420-6472,2005). Anti-mouse BTLA agonistic antibodies have been shown to ameliorate disease in mouse models of graft-versus-host disease (Sakoda et al., Blood117:2506-2514; Albring et al., J Exp Med.207:2551-9,2010). Although BTLA agonist antibodies targeting the human BTLA receptor have been shown to inhibit T cell-mediated responses ex vivo (Otsuki et al., Biochem Biophys Res Commun 344:1121-7, 2006; and WO 2011 / 014438), such antibodies have not been approved for treating any disease.

[0006] Thus, there is a need to develop therapeutic reagents that can modulate BTLA function to inhibit autoreactive lymphocytes associated with autoimmune disease. Summary of the Invention

[0007] One aspect of the invention is an isolated monoclonal antibody, or antigen-binding fragment thereof, that is specific for and activates human BTLA (B- and T-lymphocyte attenuator) and, optionally, is cross-reactive with cynomolgus monkey BTLA, the monoclonal antibody comprising (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3, and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6, or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 61, the HCVR CDR2 sequence of SEQ ID NO: 62, and the HCVR CDR3 sequence of SEQ ID NO: 63, and (2b) a light chain variable region (LCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 64, The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising the CDR1 sequence, the LCVR CDR2 sequence of SEQ ID NO: 65, and the LCVR CDR3 sequence of SEQ ID NO: 66, and optionally, the monoclonal antibody is non-naturally occurring.

[0008] In certain embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, (1A) the HCVR sequence is SEQ ID NO: (7+2n), or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region; and (1B) the LCVR sequence is SEQ ID NO: (8+2n), or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region, where n is 1 , 0, and any one of 2 to 24; or (2A) the HCVR sequence is SEQ ID NO: (67+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region; and (2B) the LCVR sequence is SEQ ID NO: (68+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region, where n is any one of 0 to 24.

[0009] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0010] In certain embodiments, the isolated monoclonal antibody is an IgG1 antibody.

[0011] In certain embodiments, the IgG1 antibody comprises a heavy chain constant region sequence of SEQ ID NO:57 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising amino acid changes therein (e.g., insertions, deletions, and / or substitutions), and a light chain constant region sequence of SEQ ID NO:58 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising amino acid changes therein (e.g., insertions, deletions, and / or substitutions).

[0012] In certain embodiments, the monoclonal antibody is capable of (1) activating antibody-dependent cell mediated cytotoxicity (ADCC) and / or antibody-dependent cell mediated phagocytosis (ABPC). and / or (2) an IgG1 antibody that comprises mutations in the heavy chain constant region that modulate ADCC (e.g., F243L, G236A, S239D / I332E, S239D / A330L / I332E, S298A / E333A / K334A, F243L / R292P / Y300L / V305I / P396L, or afucosylated (non-fucosylated) antibodies (e.g., afucosylated N297 in the Fc region)) and / or that enhance serum half-life (e.g., T250Q / M428L, M252Y / S254T / T256E).

[0013] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a murine antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0014] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a humanized antibody.

[0015] In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab'), F d , single chain Fv or scFv, disulfide bond F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0016] In certain embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, cross-reacts with cynomolgus / rhesus BTLA but does not substantially cross-react with mouse BTLA.

[0017] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM or less. D Binds human BTLA at 100 ng / mL.

[0018] In certain embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, is an agonist of human BTLA and activates downstream signaling from BTLA upon binding to BTLA. For example, in certain embodiments, downstream signaling from BTLA inhibits B cell proliferation and / or inhibits T cell (e.g., CD4, CD8, Th1, THF, αβ, or γδ T cells) and / or plasma cell activation.

[0019] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof does not block / interfere / quench BTLA binding to HVEM.

[0020] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof does not result in significant weight loss when administered (e.g., ip to mice) at a dose of 10 mg / kg BIW×6 (e.g., for 21 days).

[0021] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits CD45 when administered in vivo (e.g., ip to mice at a dose of 10 mg / kg BIW×6 for 14 days). + Significantly reduces T cells.

[0022] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof significantly reduces disease severity (e.g., mean total disease score of less than 3 at 3 weeks) and / or overall survival (e.g., at least 80% overall survival at 4 weeks) in GvHD or an animal model thereof when the monoclonal antibody or antigen-binding fragment thereof is administered in vivo (e.g., ip to mice for 14 days at a dose of 10 mg / kg BIW×6) compared to an isotype-matched control Ab.

[0023] Another aspect of the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes with an isolated monoclonal antibody, or antigen-binding fragment thereof, of the invention for binding to human BTLA.

[0024] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof, upon binding to BTLA, inhibits B cell proliferation and / or inhibits T cell (e.g., CD4, CD8, Th1, THF, αβ, or γδ T-cell) activation.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises (1) an HCVR sequence of SEQ ID NO: 9, a heavy chain constant region sequence of SEQ ID NO: 57, an LCVR sequence of SEQ ID NO: 10, and a light chain constant region sequence of SEQ ID NO: 58, or (2) a heavy chain amino acid sequence of SEQ ID NO: 59, and a light chain amino acid sequence of SEQ ID NO: 60.

[0026] Another aspect of the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising / consisting essentially of / consisting of: (1) an HCVR sequence of SEQ ID NO: 9, a heavy chain constant region sequence of SEQ ID NO: 57, an LCVR sequence of SEQ ID NO: 10, and a light chain constant region sequence of SEQ ID NO: 58; or (2) a heavy chain amino acid sequence of SEQ ID NO: 59, and a light chain amino acid sequence of SEQ ID NO: 60.

[0027] Another aspect of the invention provides polynucleotides encoding the heavy or light chains of the invention, or antigen-binding portions thereof.

[0028] In certain embodiments, the polynucleotides of the invention are codon optimized for expression in human cells.

[0029] Another aspect of the present invention provides a vector comprising a polynucleotide of the present invention.

[0030] In certain embodiments, the vector is an expression vector (eg, a mammalian, yeast, insect, or bacterial expression vector).

[0031] Another aspect of the present invention provides a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, or the vector of the present invention.

[0032] In certain embodiments, the pharmaceutical compositions are formulated for intravenous (iv) injection or administration or subcutaneous (sc) administration.

[0033] Another aspect of the present invention provides a method of downregulating a B cell- or T cell-mediated immune response or treating an autoimmune disease in a patient in need thereof, the method comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention.

[0034] In certain embodiments, the method is for treating an autoimmune disease.

[0035] In certain embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE); ulcerative colitis (UC), including pediatric ulcerative colitis; rheumatoid arthritis (RA); psoriasis (Ps), including chronic plaque psoriasis; psoriatic arthritis (PsA); Crohn's Disease (CD), including pediatric Crohn's Disease; inflammatory bowel disease (IBD); ankylosing spondylitis; juvenile idiopathic arthritis (JIA), including polyarticular juvenile idiopathic arthritis; hidradenitis abscess; non-infectious intermediate, posterior, and panuveitis; autoimmune hepatitis-like disease; experimental autoimmune encephalomyelitis (EAE). encephalomyelitis, EAE); MHC-mismatched cardiac allotransplant; lung inflammation in acute airway allergy; graft versus host disease (GvHD); or allogeneic hematopoietic stem cell transplantation (hSCT).

[0036] In certain embodiments, the method further comprises administering to the patient an additional agent effective to treat the autoimmune disease.

[0037] Another aspect of the present invention provides the use of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for downregulating a B cell- or T cell-mediated immune response or for treating an autoimmune disease in a patient in need thereof.

[0038] Another aspect of the present invention provides a composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention for use in downregulating a B cell-mediated or T cell-mediated immune response or for treating an autoimmune disease.

[0039] It is to be understood that any one embodiment of the invention described herein (including those described only in the examples or claims) may be combined with any other one or more embodiments of the invention, unless such combination is expressly prohibited or inappropriate. [Brief description of the drawings]

[0040] [Figure 1] Binding of anti-BTLA antibodies to HEK293 cells overexpressing human BTLA (HEK293.hBTLA) or Expi293 cells overexpressing cynomolgus BTLA (HEK293.cynoBTLA) is shown. Anti-BTLA antibody binding to target cells was quantified by flow cytometry with an AF647-conjugated anti-human IgG Fc secondary antibody (readout: mean fluorescence intensity (MFI)). [Figure 2A]Shown is the inhibition of primary B cell proliferation after 4 days of anti-IgM stimulation by control and candidate anti-BTLA antibodies. HFB6-4 and HFB6-5 were selected based on their ability to inhibit anti-IgM-mediated B cell proliferation to a similar or greater extent than benchmark 22B3. B cell proliferation was quantified by CFSE dilution relative to unstimulated B cells (CFSElo%) and expressed as % division. [Figure 2B] The inability of HFB6-4 and HFB6-5 to displace HVEM (the natural ligand of HVEM) when bound to BTLA is shown. The top panel is an illustrative scheme showing a CHO cell line engineered to express TCR activator (TCRa) and HVEM (the ligand of BTLA) co-cultured with a Jurkat reporter cell line expressing BTLA and a luciferase reporter driven by an NFAT-binding promoter. The positive control anti-BTLA blocking antibody JS004, as well as many other tested anti-BTLA antibodies (see bottom panel), were able to dose-dependently reverse HVEM-mediated T cell inhibition. However, the ability of HFB6-4 and HFB-5 to reverse HVEM-mediated T cell inhibition was virtually abolished (see bottom panel). [Diagram 3] Shows inhibition of TCR signaling by HFB6-4 and HFB6-5 compared to benchmark antibody 22B3. The left panel depicts the assay: CHO cells expressing FcγRIIb on the cell surface were first incubated with anti-BTLA antibody, isotype control MGO, or benchmark antibody 22B3 in the presence or absence of anti-CD3 antibody. The antibody-coated CHO cells were then incubated with Jurkat reporter cells expressing surface TCR and BTLA. TCR signaling was measured in the reporter cells via activity of a luciferase reporter gene under the transcriptional control of the NFAT promoter downstream of TCR signaling. The right panel shows the luminescent readout from testing different antibodies in the assay. [Figure 4]Inhibition of primary B cell proliferation after anti-IgM stimulation (10 μg / ml) by isotype control (MGO53), benchmark antibodies 22B3, HFB6-4, and HFB6-5 antibodies. The effect of the antibodies was tested on primary B cells from three different donors (left panel) and at various concentrations (0.41-100 nM) (right panel). [Diagram 5] Binding of humanized variants of the HFB6-4 and HFB6-5 antibodies to HEK293 cells overexpressing human BTLA (HEK293.hBTLA) or Expi293 cells overexpressing cynomolgus monkey BTLA (HEK293.cynoBTLA) is shown. Anti-BTLA antibody binding to target cells was quantified by flow cytometry with an AF647-conjugated anti-human IgG Fc secondary antibody (readout: mean fluorescence intensity, MFI). [Figure 6A] Figure 1 shows the inhibition of anti-IgM-induced primary B cell proliferation by an isotype control antibody (MGO53), the benchmark antibody 22B3, and humanized variants of HFB6-4 and HFB6-5 at concentrations of 1 and 10 μg / ml. B cell proliferation was quantified by CFSE dilution relative to unstimulated B cells (CFSElo%) and expressed as % division. [Figure 6B] Figure 2 shows inhibition of anti-CD3-induced Jurkat cell activation by an isotype control antibody (MGO53), the benchmark antibody 22B3, and humanized variants of HFB6-4 and HFB6-5 as measured by the luminescence signal produced by activity of a luminescent reporter gene in Jurkat reporter cells (relative light units (RLU)). [Figure 7] Pharmacokinetic profiles of humanized variants of HFB6-4 and HFB6-5 in C57BL6 mice after intravenous injection at a dose of 10 mg / kg. Blood was collected 1, 24, and 72 hours after injection. [Figure 8A]The therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD) is shown. Figure 8A shows a schematic of the experiment. [Figure 8B] The therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD) is shown. Figure 8B shows the detailed experimental design and experimental readouts. [Figure 8C] The therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD). Preliminary results are shown from 14 to 21 days post-inoculation, including total disease scores (Figure 8C). [Figure 8D] Figure 8 shows the therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD). Preliminary results are shown up to 14-21 days post-inoculation, including percent of RCBW (Figure 8D). [Figure 8E] Figure 8 shows the therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD). Preliminary results are shown up to 14-21 days post-inoculation, including the percentage of hCD45+ cells under the lymphocyte population (Figure 8E). [Figure 8F]The therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD) is shown. Preliminary results are shown up to 14-21 days post-inoculation, including percent survival (Figure 8F). [Figure 8G] The therapeutic efficacy of the antibodies of interest in inhibiting immune responses following inoculation with human peripheral blood mononuclear cells (hPBMCs) in an NSG mouse model of acute graft-versus-host disease (GvHD) is shown. Preliminary results are shown up to 14-21 days post-inoculation, including percentage of survival (Figure 8G). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] 1. Overview The invention described herein provides isolated monoclonal antibodies or antigen-binding fragments thereof that are specific for and activate human BTLA (B- and T-lymphocyte attenuator), i.e., BTLA agonist antibodies of the invention that optionally cross-react with non-human primate BTLA, such as monkey (e.g., rhesus and / or cynomolgus) BTLA, but may not cross-react with rodent (e.g., mouse or rat BTLA).

[0042] In accordance with the invention described herein, certain BTLA agonist antibodies of the invention were first obtained by immunizing mice with a recombinant extracellular domain (ECD) of human BTLA to generate a panel of diverse antibodies, which were then characterized for their binding, cross-reactivity, selectivity, and / or other functional activities. Certain such antibodies were then selected for their ability to inhibit IgM-induced proliferation of primary B and / or T cells. Functionally selected antibodies may also exhibit cross-reactivity to non-human primate (such as monkey) orthologs of human BTLA, which may be a beneficial feature for toxicity testing of human therapeutic agents in non-human host (e.g., non-human primate or NHP) animals.

[0043] Two exemplary murine antibodies, designated herein as HFB6-4 and HFB6-5, with subnanomolar or single-digit nanomolar binding affinities for human BTLA were initially identified for further characterization and humanization.

[0044] Several humanized variants of these murine antibodies, including HFB6-4hz1-hG1, were generated as IgG1 isotypes and retained the binding and cross-reactivity profiles of their respective murine-human chimeric parent antibodies, as well as the inhibitory effects on immune cells, including B cells, T cells, and / or plasma cells.

[0045] Preliminary in vitro and in vivo efficacy evaluations of these antibodies in mouse models of immune disorder have been performed, as well as initial toxicity and developability analyses. Preliminary data indicate that the subject monoclonal antibodies are more effective as BTLA agonists than certain benchmark antibodies, and more effectively inhibit B and T cells.

[0046] The functional profile of these antibodies, together with their favorable developability and pharmacokinetic profiles, support their development as novel therapeutic agents for treating immune disorders such as various autoimmune diseases (e.g., graft-versus-host disease or GvHD).

[0047] Accordingly, one aspect of the invention is an isolated monoclonal antibody, or antigen-binding fragment thereof, that is specific for and activates human BTLA (B- and T-lymphocyte attenuator) and, optionally, is cross-reactive with cynomolgus monkey BTLA, the monoclonal antibody comprising (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3, and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6, or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 61, the HCVR CDR2 sequence of SEQ ID NO: 62, and the HCVR CDR3 sequence of SEQ ID NO: 63, and (2b) a light chain variable region (LCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 64, the LCVR CDR2 sequence of SEQ ID NO: 65, and (2c) a light chain variable region (LCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 65, and (2d) a light chain variable region (LCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 67, and (2e) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 68, the LCVR CDR2 sequence of SEQ ID NO: 69, and (2f) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 70, the LCVR CDR2 sequence of SEQ ID NO: The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising the CDR2 sequence of SEQ ID NO: 66, and the LCVR CDR3 sequence of SEQ ID NO: 67, and optionally, the monoclonal antibody is non-naturally occurring.

[0048] In certain embodiments, the CDR region sequences are based on the IMGT numbering scheme. CDR sequences corresponding to other numbering schemes, such as the Kabat, Chothia, or Martin (enhanced Chothia) schemes, can be readily derived by antibody sequence alignment.

[0049] In a related aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising / consisting essentially of / consisting of: (1) an HCVR sequence of SEQ ID NO:9, a heavy chain constant region sequence of SEQ ID NO:57, an LCVR sequence of SEQ ID NO:10, and a light chain constant region sequence of SEQ ID NO:58; or (2) a heavy chain amino acid sequence of SEQ ID NO:59, and a light chain amino acid sequence of SEQ ID NO:60.

[0050] In a related aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes for binding to human BTLA (such as SEQ ID NO: 121) with an isolated monoclonal antibody, or antigen-binding fragment thereof, having defined CDR and / or VH / VL region sequences as defined herein.

[0051] Another aspect of the invention provides a polynucleotide encoding a heavy or light chain or an antigen-binding portion thereof of any of the monoclonal antibodies or antigen-binding fragments thereof as described herein. In a related aspect, the invention also provides a polynucleotide that hybridizes to the polynucleotide or to the complement of the polynucleotide under stringent conditions.

[0052] Another aspect of the present invention provides a vector comprising a polynucleotide of the present invention as described herein.

[0053] Another aspect of the present invention provides a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, or the vector of the present invention.

[0054] Another aspect of the present invention provides a method for downregulating a B cell- or T cell-mediated immune response or treating an autoimmune disease in a patient in need thereof, the method comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention.

[0055] Another aspect of the present invention provides the use of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for downregulating a B cell- or T cell-mediated immune response or for treating an autoimmune disease in a patient in need thereof.

[0056] Another aspect of the present invention provides a composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention for use in downregulating a B cell-mediated or T cell-mediated immune response or for treating an autoimmune disease.

[0057] Detailed aspects of the invention are further described separately in various sections below, however, it should be understood that any one embodiment of the invention (including embodiments described only in the examples or drawings and embodiments described only in one section below) can be combined with any other embodiment of the invention.

[0058] 2.Definition The term "antibody" in its broadest sense encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term "antibody" can also broadly refer to a molecule comprising a complementarity determining region (CDR)1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain, the molecule being capable of binding to an antigen. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, such as mouse, human, and cynomolgus monkey.

[0059] However, in a narrower sense, "antibody" refers to a variety of monoclonal antibodies, including chimeric, humanized, and human monoclonal antibodies, particularly the humanized monoclonal antibodies of the present invention.

[0060] In some embodiments, the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In some embodiments, the antibody comprises at least one heavy chain (heavy chain, HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (light chain, LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains, each heavy chain comprising at least a portion of a heavy chain variable region and a heavy chain constant region, and two light chains, each light chain comprising at least a portion of a light chain variable region and a light chain constant region.

[0061] As used herein, a single chain Fv (scFv), or any other antibody that comprises, for example, a single polypeptide chain that comprises all six CDRs (three heavy chain CDRs and three light chain CDRs), is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs, and the light chain is the region of the antibody that comprises the three light chain CDRs.

[0062] The term "heavy chain variable region (HCVR)" as used herein refers to a region comprising at least heavy chain CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3), and CDR3 (CDR-H3). In some embodiments, the heavy chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (HFR1) that is N-terminal to CDR-H1, and / or at least a portion (e.g., the entirety) of FR4 (HFR4) that is C-terminal to CDR-H3.

[0063] The term "heavy chain constant region" as used herein refers to a region that includes at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, an antibody that includes an alpha constant region is an IgA antibody, an antibody that includes an epsilon constant region is an IgE antibody, and an antibody that includes a mu constant region is an IgM antibody.

[0064] Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies. IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies, and IgM antibodies include, but are not limited to, IgM1 (containing a μ1 constant region) and IgM2 (containing a μ2 constant region).

[0065] In certain embodiments, the antibodies of the invention are IgG1 antibodies.

[0066] The term "heavy chain" as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine.

[0067] The term "light chain variable region (LCVR)" as used herein refers to a region comprising light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3). In some embodiments, the light chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (LFR1) and / or at least a portion (e.g., the entirety) of FR4 (LFR4).

[0068] The term "light chain constant region" as used herein refers to a light chain constant domain, C L Non-limiting exemplary light chain constant regions include lambda and kappa.

[0069] The term "light chain" as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0070] The terms "antibody fragment," "antigen-binding portion," or "antigen-binding fragment" (of an antibody) include, but are not limited to, fragments capable of binding to an antigen, such as Fv, single chain Fv (scFv), Fab, Fab', and (Fab')2. In certain embodiments, an antibody fragment is a Fab, Fab', F(ab')2, F d , single chain Fv or scFv, disulfide bond F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0071] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 daltons and having antigen-binding activity. It contains about the N-terminal half of the heavy chain and the entire light chain linked by disulfide bridges. Fab can be obtained by treatment of immunoglobulins with the protease, papain, among others.

[0072] The term "F(ab')2" designates a fragment of about 100,000 daltons and antigen-binding activity. This fragment is slightly larger than two Fab fragments linked via disulfide bridges in the hinge region. These fragments are obtained by treating immunoglobulins with the protease, pepsin. Fab fragments can be obtained from F(ab')2 fragments by cleavage of the disulfide bridges in the hinge region.

[0073] A single Fv chain "scFv" corresponds to a VH:VL polypeptide synthesized using genes encoding the VL and VH domains and a sequence encoding a peptide intended to link these domains. An scFv according to the invention contains the CDRs held in the proper conformation, e.g., using recombinant genetic techniques.

[0074] A "scFv" dimer corresponds to two scFv molecules linked together by a peptide bond. The Fv chain is often the result of expression of a fusion gene comprising VH and VL encoding genes linked by a peptide-encoding linker sequence. Human scFv fragments may contain the CDR regions maintained in the proper conformation, preferably by the use of recombinant gene technology.

[0075] A "dsFv" fragment is a VH-VL heterodimer stabilized by disulfide bridges and can be bivalent (dsFV2). Bivalent Sc(Fv)2 or multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be produced by linking scFv fragments by peptide bond sequences.

[0076] The Fc fragment is the bearer of the biological properties of the antibody, in particular its ability to be recognized by immune effectors or its ability to activate complement. It consists of the constant fragment of the heavy chain beyond the hinge region.

[0077] The term "diabody" refers to small antibody fragments with two antigen-fixing sites. These fragments contain a variable heavy domain VH linked to a variable light domain VL in the same VH-VL polypeptide chain. Using a linking sequence that is too short to allow the two domains of the same chain to fit, a match with the two complementary domains of another chain must occur, thus creating two antigen-fixing sites.

[0078] An "antibody that binds to the same epitope" or an "antibody that competes for binding with (another) antibody" refers to an antibody that can be determined by an antibody competition assay. It refers to an antibody that blocks the binding of a reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. The term "competes", when used in the context of antibodies competing for the same epitope, means that the competition between the antibodies is determined by an assay in which the antibody being tested prevents or inhibits specific binding of the reference antibody to a common antigen.

[0079] There are many types of competitive binding assays, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assays; solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); 125 Solid-phase direct labeling RIA using labels (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol.) can be used.

[0080] Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either of these unlabeled test antigen binding proteins and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually, the test antibody is present in excess. Antibodies identified by competitive assays (competitor antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody for steric hindrance to occur. In some embodiments, when a competitor antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0081] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody or an immunologically functional fragment thereof, and that can be used in a mammal to generate antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that can interact with an antibody.

[0082] The term "epitope" is the portion of an antigen molecule that is bound by a selective binding agent such as an antibody or fragment thereof. The term includes any determinant capable of specific binding to an antibody. Epitopes may be contiguous or non-contiguous (e.g., in a polypeptide, amino acid residues that are not adjacent to each other in the polypeptide sequence but are bound by an antigen binding protein in the context of the molecule). In some embodiments, an epitope may be a mimetic in that it comprises a three-dimensional structure similar to the epitope used to generate the antibody, but contains none or only some of the amino acid residues found in the epitope used to generate the antibody. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0083] In some embodiments, an "epitope" is defined by the method used to determine it. For example, in some embodiments, an antibody binds to the same epitope as a reference antibody if they bind to the same region of an antigen, as determined by hydrogen-deuterium exchange (HDX).

[0084] In certain embodiments, an antibody binds to the same epitope as a reference antibody if they bind to the same region of the antigen as determined by x-ray crystallography.

[0085] "Chimeric antibody," as used herein, refers to an antibody that comprises at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, cynomolgus monkey, chicken, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, all of the variable regions of the chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.

[0086] "Humanized antibody," as used herein, refers to an antibody in which at least one amino acid in the framework region of a non-human variable region (e.g., mouse, rat, cynomolgus monkey, chicken, etc.) has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody fragment is a Fab, scFv, (Fab')2, etc.

[0087] "CDR-grafted antibody", as used herein, refers to a humanized antibody in which one or more complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework region (FR) of a second (human) species.

[0088] "Human antibody," as used herein, refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, such as the XENOMOUSE®, and antibodies whose antibody repertoires are selected using in vitro methods, such as phage display, based on human immunoglobulin sequences.

[0089] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells and their derivatives, such as 293-6E and DG44 cells, respectively.

[0090] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components with which it is typically found in nature or from at least some of the components from which it is typically produced. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the producing cell is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide that is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or, for example, in the case of an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated" as long as the polynucleotide is not naturally found in the vector.

[0091] The terms "subject" and "patient" are used interchangeably herein to refer to a mammal, such as a human. In some embodiments, methods of treating other non-human mammals, including but not limited to rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided. In some instances, "subject" or "patient" refers to a (human) subject or patient in need of treatment for a disease or disorder.

[0092] The terms "sample" or "patient sample," as used herein, refer to material obtained or derived from a subject of interest that contains cellular and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological properties. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is suspected of or known to contain the cellular and / or molecular entities to be characterized.

[0093] "Tissue or cell sample" refers to a collection of similar cells obtained from a subject's or patient's tissue. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen, and / or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; bodily fluids, such as sputum, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time point in the subject's pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0094] A "reference sample", "reference cell", or "reference tissue", as used herein, refers to a sample, cell, or tissue obtained from a source known or believed to be free of the disease or condition that the method or composition of the present invention is being used to identify. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of the same subject or patient whose disease or condition has been identified using the composition or method of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient whose disease or condition has been identified using the composition or method of the present invention. In some embodiments, the reference sample, reference cell, or reference tissue is previously obtained from the patient before the onset of the disease or condition or at an early stage of the disease or condition.

[0095] A "disorder" or "disease" is any condition that would benefit from treatment with one or more BTLA antagonists of the present invention. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders that may be treated herein include autoimmune diseases.

[0096] "Hyperactive immune disorder" refers to any disease in which the immune system unnecessarily attacks the subject's cells, organs, or transplants, or in which cells of the immune system proliferate uncontrollably. Common hyperactive immune disorders include autoimmune diseases and allograft rejection, such as graft-versus-host disease.

[0097] "Treatment" refers to, for example, therapeutic treatments in which the objective is to slow (reduce) the targeted pathological condition or disorder, as well as therapeutic treatments in which the objective is, for example, to inhibit the recurrence of the condition or disorder. "Treatment" encompasses any administration or application of a therapeutic agent to a disease (also referred to herein as a "disorder" or "condition") in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, halting its onset, partially or completely alleviating the disease, partially or completely alleviating one or more symptoms of the disease, or restoring or repairing a lost, missing, or defective function, or stimulating an inefficient process. The term "treatment" also includes reducing the severity of any phenotypic trait and / or reducing the incidence, extent, or likelihood of the trait. Those in need of treatment include those who already have the disorder, as well as those at risk of recurrence of the disorder, or those in whom recurrence of the disorder is to be prevented or delayed.

[0098] The term "effective amount" or "therapeutically effective amount" refers to an amount of drug effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the subject antibody are outweighed by the therapeutically beneficial effects.

[0099] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0100] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent together comprising a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. If the therapeutic agent is administered subcutaneously, the carrier is ideally not irritating to the skin and does not cause injection site reactions.

[0101] An "article of manufacture" is any article of manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a drug product for the treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0102] 3. Methods for Treating Hyperactive Immune Disorders The invention described herein provides anti-BTLA antibodies for use in methods of treating humans and other non-human mammals.

[0103] In pathological situations, the immune system may be overactive and attack normal and healthy tissues, resulting in autoimmune disease. In other pathological situations, cells of the immune system may be hyperproliferative, such as lymphomas (e.g., B-cell lymphomas, etc.). In other situations, the immune system attacks the foreign entity as intended, but acceptance of the allogeneic entity is the desired outcome, such as in allogeneic transplantation.

[0104] Thus, in some non-limiting embodiments, the anti-BTLA agonist antibodies or antigen-binding fragments thereof of the invention can be used in subjects who would benefit from a treatment that dampens the immune response, such as subjects with an autoimmune disease or subjects who have received an allograft. The anti-BTLA antibodies of the invention provide suppression of the immune response, for example, by inhibiting the proliferation and / or activity of T cells and / or B cells and plasma cells.

[0105] That is, the invention described herein provides a method of downregulating a B cell- or T cell-mediated immune response or treating an autoimmune disease in a patient in need thereof, the method comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, a polynucleotide of the invention, a vector of the invention, or a pharmaceutical composition of the invention.

[0106] A related aspect provides the use of the isolated monoclonal antibody or antigen-binding fragment thereof, the polynucleotide of the invention, the vector of the invention, or the pharmaceutical composition of the invention in the manufacture of a medicament for downregulating a B cell- or T cell-mediated immune response or for treating an autoimmune disease in a patient in need thereof.

[0107] Another related aspect provides a composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the invention, the polynucleotide of the invention, the vector of the invention, or the pharmaceutical composition of the invention for use in downregulating a B cell-mediated or T cell-mediated immune response or for treating an autoimmune disease.

[0108] In some embodiments, methods are provided for treating or preventing a hyperactive immune disorder, comprising administering an effective amount of any of the subject anti-BTLA antibodies, or antigen-binding fragments thereof, to a subject in need of such treatment.

[0109] In some embodiments, methods are provided for treating a hyperactive immune disorder, comprising administering an effective amount of any of the subject anti-BTLA agonist antibodies, or antigen-binding fragments thereof, to a subject in need thereof, e.g., a subject diagnosed with, at risk of having, or exhibiting symptoms of having a hyperactive immune disorder, e.g., an autoimmune disease.

[0110] Non-limiting exemplary hyperactive immune disorders, e.g., autoimmune diseases, that can be treated with a subject anti-BTLA antibody or antigen-binding fragment thereof are provided herein and include systemic lupus erythematosus (SLE); ulcerative colitis (UC), including pediatric ulcerative colitis; rheumatoid arthritis (RA); psoriasis (Ps), including chronic plaque psoriasis; psoriatic arthritis (PsA); Crohn's disease (CD), including pediatric Crohn's disease; inflammatory bowel disease (IBD); ankylosing spondylitis; juvenile idiopathic arthritis (JIA), including polyarticular juvenile idiopathic arthritis; hidradenitis suppurativa; non-infectious intermediate uveitis, posterior uveitis, and panuveitis; autoimmune hepatitis-like disease; experimental autoimmune encephalomyelitis (EAE); MHC-mismatched cardiac allotransplantation; pulmonary inflammation in acute airway allergy; graft-versus-host disease (GvHD); or allogeneic hematopoietic stem cell transplantation (aHSCT).

[0111] In certain embodiments, the hyperactive immune disorder is systemic lupus erythematosus (SLE or lupus). Lupus is characterized by the pathological formation of pathogenic autoantibodies against nuclear, cytoplasmic, and / or cell surface molecules resulting from B-cell and T-cell immune dysregulation. Local formation and / or deposition of circulating antigen-antibody immune complexes induces an inflammatory response that is responsible for a wide range of systemic and organ-specific clinical findings characterized by remissions and exacerbations, leading to multi-organ system damage and potentially end-organ failure. Lupus is a multifaceted autoimmune disease characterized by disabling symptoms and progressive organ damage.

[0112] In certain embodiments, the hyperactive immune disorder is inflammatory bowel disease (IBD). IBD encompasses two conditions characterized by chronic inflammation that leads to damage to the gastrointestinal (GI) tract: Crohn's disease (CD) and ulcerative colitis (UC). Some common symptoms of IBD are persistent diarrhea, abdominal pain, rectal bleeding / bloody stool, weight loss, and fatigue.

[0113] In certain embodiments, the hyperactive immune disorder is a condition in which the subject's immune system attacks the allograft obtained from the donor, such as graft-versus-host disease (GvHD). GvHD can occur at any time after transplantation and can be acute or chronic. In bone marrow transplantation, GvHD more commonly occurs after the bone marrow begins to make cells. Symptoms vary based on how long the patient has had the condition, but can include mouth ulcers, abdominal pain, and rashes.

[0114] In some embodiments, methods are provided for preventing a hyperactive immune disorder, comprising prophylactically administering an effective amount of any of the subject anti-BTLA antibodies, or antigen-binding fragments thereof, to a subject at risk of developing symptoms of a hyperactive immune related disorder. For example, a subject anti-BTLA antibody, or antigen-binding fragment thereof, can be administered to a subject prior to receiving an allogeneic transplant.

[0115] In some embodiments, the anti-BTLA antibodies, or antigen-binding fragments thereof, of the invention can be used alone, or alternatively, in combination with any other suitable compound known to be capable of treating the disease or indication.

[0116] Thus, according to a particular embodiment of the invention, an antibody directed against BLTA and inhibiting T and / or B cells and plasma cells, as defined above, is used in combination with a second therapeutic agent for treating a disease associated with an overactive immune system, e.g. an autoimmune disease such as lupus.

[0117] In such combination therapy, the antibodies of the invention may be used before, after, or simultaneously with the second therapeutic agent, see further section below regarding combination therapy.

[0118] 4. Route of Administration and Carriers In various embodiments, the subject agonist anti-BTLA monoclonal antibodies may be administered subcutaneously or intravenously. For brevity, "the subject anti-BTLA monoclonal antibodies" refers to the mouse-human chimeric anti-BTLA antibodies of the invention, as well as humanized variants thereof.

[0119] In some embodiments, the subject anti-BTLA monoclonal antibodies may be administered in vivo by a variety of routes, including, but not limited to, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intravenous, and subcutaneous administration, inhalation, intradermal, topical, transdermal, and intrathecal, or other, such as by implantation.

[0120] In some embodiments, the subject anti-BTLA monoclonal antibodies may be administered via intravenous (iv) or subcutaneous (sc) administration.

[0121] The subject antibody compositions may be formulated into solid, semi-solid, liquid, or gaseous form preparations including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0122] In various embodiments, compositions comprising the subject anti-BTLA monoclonal antibodies are provided in formulations with a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). A variety of pharma-ceutically acceptable carriers, including vehicles, adjuvants, and diluents, are available. In addition, a variety of pharma-ceutically acceptable auxiliary substances (e.g., pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like) are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0123] In various embodiments, compositions comprising the subject anti-BTLA monoclonal antibodies can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if desired, with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0124] In various embodiments, the compositions can be formulated for inhalation using pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0125] The composition may also be formulated in various embodiments into sustained release microcapsules using biodegradable or non-biodegradable polymers and the like. Non-limiting exemplary biodegradable formulations include poly lactic acid-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. Certain methods of making such formulations are described, for example, in EP 1125584 A1.

[0126] Also provided are pharmaceutical dosage packs comprising one or more containers, each containing one or more doses of a subject anti-BTLA monoclonal antibody. In some embodiments, unit doses are provided, the unit doses containing a predetermined amount of a composition comprising a subject anti-BTLA monoclonal antibody, with or without one or more additional agents. In some embodiments, such unit doses are supplied in single-use pre-filled syringes for injection. In various embodiments, the composition contained in the unit dose may include saline, sucrose, and the like; buffers such as phosphates, and the like, and / or may be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid (e.g., sterile water). In some embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the compositions of the invention include heparin and / or proteoglycan.

[0127] The pharmaceutical compositions are administered in an amount effective for treating or preventing a particular indication. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated.

[0128] 5. Combination Therapy The subject anti-BTLA monoclonal antibodies (including functional fragments thereof) of the present invention can be administered to a subject in need thereof in combination with other biologically active substances or other treatment procedures for the treatment of disease. For example, the subject anti-BTLA monoclonal antibodies can be administered alone or together with other treatment modalities. They can be provided prior to, substantially simultaneously with, or subsequent to other treatment modalities, such as immunosuppressants.

[0129] For the treatment of hyperactive immune disorders, the subject anti-BTLA monoclonal antibodies may be administered in conjunction with one or more immunosuppressants, such as corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, IMDH inhibitors, and other immunosuppressants.

[0130] In certain embodiments, a subject anti-BTLA monoclonal antibody is administered to a subject, e.g., a subject with an autoimmune disease, either simultaneously or sequentially with another treatment. For example, a subject anti-BTLA monoclonal antibody can be administered with one or more immunosuppressants.

[0131] In certain embodiments, a method of treating a subject with a hyperactive immune disorder comprises administering to the subject an anti-BTLA monoclonal antibody of the invention and one or more immunosuppressants, such as a corticosteroid (e.g., a steroid).

[0132] Immunosuppressants that may be administered in combination with a subject anti-BTLA monoclonal antibody include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, and high-dose aspirin; antimalarials such as hydroxychloroquine and chloroquine phosphate; corticosteroids such as prednisone, budesonide, and prednisolone; Janus kinase inhibitors such as tofacitinib; calcineurin inhibitors such as cyclosporine and tacrolimus; mTOR inhibitors such as sirolimus and everolimus; and IMDH inhibitors such as azathioprine, leflunomide, and mycophenolate.

[0133] 6. Exemplary Anti-BTLA Monoclonal Antibodies The invention described herein provides agonistic monoclonal antibodies, or antigen-binding fragments thereof, specific for BTLA.

[0134] Thus, one aspect of the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes with any of the isolated monoclonal antibodies, or antigen-binding fragments thereof, described herein for binding to the epitope bound by HFB6-4 or HFB6-5.

[0135] Another related aspect of the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, specific for human BTLA, the monoclonal antibody comprising: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO:1, the HCVR CDR2 sequence of SEQ ID NO:2, and the HCVR CDR3 sequence of SEQ ID NO:3; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO:4, the LCVR CDR2 sequence of SEQ ID NO:5, and the LCVR CDR3 sequence of SEQ ID NO:6; or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO:61, the HCVR CDR2 sequence of SEQ ID NO:62, and the HCVR CDR3 sequence of SEQ ID NO:63; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO:64, the LCVR CDR2 sequence of SEQ ID NO:65, and the LCVR CDR3 sequence of SEQ ID NO:66.

[0136] In certain embodiments, the CDR region sequences described herein are based on the IMGT numbering scheme. CDR sequences corresponding to other numbering schemes, such as the Kabat, Chothia, or Martin (enhanced Chothia) schemes, can be readily derived by antibody sequence alignment.

[0137] For any of the aspects of the invention described above, in some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof: (1A) the HCVR sequence is SEQ ID NO:7, and / or (1B) the LCVR sequence is SEQ ID NO:8, or (2A) the HCVR sequence is SEQ ID NO:67, and / or (2B) the LCVR sequence is SEQ ID NO:68.

[0138] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:7 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:8 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region.

[0139] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 9 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 10 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0140] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 11 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 12 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0141] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 13 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 14 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0142] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 15 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 16 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0143] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 17 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 18 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0144] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 19 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 20 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0145] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:21 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:22 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0146] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:23 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:24 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0147] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:25 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:26 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0148] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:27 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:28 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0149] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:29 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:30 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0150] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 31 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 32 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0151] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 33 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 34 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0152] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 35 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 36 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0153] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 37 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 38 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0154] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 39 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 40 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0155] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 41 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 42 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0156] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 43 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 44 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0157] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 45 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 46 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0158] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 47 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 48 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0159] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:49 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:50 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0160] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:51 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:52 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0161] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO:53 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO:54 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0162] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 55 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 56 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0163] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 67 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 68 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region.

[0164] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 69 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 70 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0165] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 71 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 72 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0166] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 73 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 74 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0167] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 75 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 76 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0168] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 77 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 78 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0169] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 79 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 80 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0170] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 81 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 82 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0171] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 83 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 84 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0172] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 85 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 86 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0173] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 87 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 88 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0174] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 89 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 90 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0175] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 91 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 92 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0176] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 93 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 94 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0177] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 95 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 96 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0178] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 97 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 98 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0179] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 99 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 100 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0180] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 101 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 102 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0181] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 103 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 104 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0182] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 105 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 106 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0183] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 107 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 108 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0184] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 109 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 110 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0185] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 111 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 112 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0186] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 113 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 114 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0187] In some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, the HCVR sequence is SEQ ID NO: 115 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region, and / or the LCVR sequence is SEQ ID NO: 116 or a variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the light chain framework region.

[0188] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof has (1a) the heavy chain sequence of SEQ ID NO: 117 and / or the light chain sequence of SEQ ID NO: 118, or (2a) the heavy chain sequence of SEQ ID NO: 119 and / or (2b) the light chain sequence of SEQ ID NO: 120.

[0189] Some of the sequences of the antibodies of the invention are provided below. HFB6-4-hG1 (human-mouse chimeric monoclonal antibody) CDR-H1: CTTSGFNIKDTYIHW (SEQ ID NO: 1) CDR-H2: GRNDPANGNTKYD (SEQ ID NO: 2) CDR-H3: CASYEGYYERFGYW (SEQ ID NO: 3) CDR-L1: CKSSQSLLDRDGKTYLNW (SEQ ID NO: 4) CDR-L2: LVSKLDS (SEQ ID NO: 5) CDR-L3: CWQDTHFPYTF (SEQ ID NO: 6) HCVR: EVQLQQSGAELVNPGASVKLSCTTSGFNIKDTYIHWVKQRPEQGLEWIGRNDPANGNTKYDPKFQGKATITADTSSSTAYLQLSSLTSEDTAVYYCASYEGYYERFGYWGQGTLVTVSA (SEQ ID NO: 7) LCVR: DVVMTQAPLTLSVTIGQPASISCKSSQSLLDRDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQDTHFPYTFGGGTKLEIK (SEQ ID NO: 8) HC:EVQLQQSGAELVNPGASVKLSCTTSGFNIKDTYIHWVKQRPEQGLEWIGRNDPANGNTKYDPKFQGKATITADTSSSTAYLQLSSLTSEDTAVYYCASYEGYYERFGYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117) LC:DVVMTQAPLTLSVTIGQPASISCKSSQSLLDRDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQDTHFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 118) HFB6-5-hG1 (human-mouse chimeric monoclonal antibody) CDR-H1: CTASGVNIKDTYMYW (SEQ ID NO: 61) CDR-H2: GRIDPANGYTRYD (SEQ ID NO: 62) CDR-H3: CAVYDGYYESFDVW (SEQ ID NO: 63) CDR-L1: CKSSQSLLDSDGKTYLNW (SEQ ID NO: 64) CDR-L2: LVSKLDS (SEQ ID NO: 65) CDR-L3: CWQGTLFPRTF (SEQ ID NO: 66) HCVR: EVQLQQSGAELVKPGASVKLSCTASGVNIKDTYMYWVKQRPEQGLEWIGRIDPANGYTRYDPKFQGKATITADTSSNTAHLQLSSLTSEDAAVYYCAVYDGYYESFDVWGAGTTVTVSS (SEQ ID NO: 67) LCVR: DVVMTQAPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKINRVEAEDLGVYYCWQGTLFPRTFGGGTKLEIK (SEQ ID NO: 68) HC:EVQLQQSGAELVKPGASVKLSCTASGVNIKDTYMYWVKQRPEQGLEWIGRIDPANGYTRYDPKFQGKATITADTSSNTAHLQLSSLTSEDAAVYYCAVYDGYYESFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 119) LC:DVVMTQAPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKINRVEAEDLGVYYCWQGTLFPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 120)

[0190] [Table 1]

[0191] [Table 2]

[0192] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention is a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0193] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0194] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an IgG1 antibody.

[0195] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an IgG1 antibody comprising a heavy chain constant region sequence of SEQ ID NO:57 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and containing amino acid changes therein (e.g., insertions, deletions, and / or substitutions), and a light chain constant region sequence of SEQ ID NO:58 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and containing amino acid changes therein (e.g., insertions, deletions, and / or substitutions).

[0196] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of: (1) an antibody that modulates antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) (e.g., F243L, G236A, S239D / I332E, S239D / A330L ... 298A / E333A / K334A, F243L / R292P / Y300L / V305I / P396L, or an afucosylated (non-fucosylated) antibody (e.g., afucosylated N297 in the Fc region), and / or (2) an IgG1 antibody that contains mutations in the heavy chain constant region that enhance serum half-life (e.g., T250Q / M428L, M252Y / S254T / T256E).

[0197] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a murine antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0198] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a humanized antibody.

[0199] In some embodiments, the antigen-binding fragment is a Fab, Fab', F(ab'), F d , single chain Fv or scFv, disulfide bond Fv , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0200] In certain embodiments, the monoclonal antibodies, or antigen-binding fragments thereof, of the invention are specific for human BTLA and do not substantially cross-react with, for example, mouse BTLA.

[0201] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention cross-react with a primary non-human BTLA, such as monkey BTLA (e.g., cynomolgus or rhesus BTLA, e.g., SEQ ID NO: 122). In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention do not cross-react with rodent BTLA, such as mouse BTLA (e.g., SEQ ID NO: 123). Exemplary amino acid sequences of human, monkey, and mouse BTLA known in the art are listed below.

[0202] [Table 3]

[0203] In some embodiments, the monoclonal antibodies, or antigen-binding fragments thereof, of the invention have an affinity for human BTLA of ≦1 μM, ≦100 nM, ≦50 nM, ≦25 nM, ≦20 nM, ≦15 nM, ≦10 nM, ≦5 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D ).

[0204] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention bind to the epitope bound by HFB6-4. In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention bind to the epitope bound by HFB6-5.

[0205] In some embodiments, the monoclonal antibodies, or antigen-binding fragments thereof, of the invention are agonists of human BTLA and, upon binding to BTLA, activate downstream signaling from BTLA.

[0206] In some embodiments, such downstream signaling from BTLA inhibits B cell proliferation and / or inhibits T cell (e.g., CD4, CD8, Th1, THF, αβ, or γδ T cells) and / or plasma cell activation.

[0207] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention do not block / interfere / quench BTLA binding to HVEM.

[0208] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, upon binding to BTLA, inhibit B cell proliferation and / or inhibit T cell (e.g., CD4, CD8, Th1, THF, αβ, or γδ T-cell) activation.

[0209] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises (1) an HCVR sequence of SEQ ID NO: 9, a heavy chain constant region sequence of SEQ ID NO: 57, an LCVR sequence of SEQ ID NO: 10, and a light chain constant region sequence of SEQ ID NO: 58, or (2) a heavy chain amino acid sequence of SEQ ID NO: 59, and a light chain amino acid sequence of SEQ ID NO: 60.

[0210] In a related aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising / consisting essentially of / consisting of: (1) an HCVR sequence of SEQ ID NO:9, a heavy chain constant region sequence of SEQ ID NO:57, an LCVR sequence of SEQ ID NO:10, and a light chain constant region sequence of SEQ ID NO:58; or (2) a heavy chain amino acid sequence of SEQ ID NO:59, and a light chain amino acid sequence of SEQ ID NO:60.

[0211] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention, including humanized monoclonal antibodies or antigen-binding fragments thereof, have a good developability profile, including being stable under high temperatures (e.g., stable upon storage at 25° C. or 40° C.), at low pH conditions (e.g., pH 3.5, near room temperature), and / or after several freeze / thaw cycles.

[0212] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, including humanized monoclonal antibodies or antigen-binding fragments thereof, contain one or more point mutations in the amino acid sequence that are designed to improve the developability of the antibody. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS116(10):4025-4030, 2019) describe the Therapeutic Antibody Profiler (TAP), a computational tool for building downloadable homology models of variable domain sequences, testing them against five developability guidelines, and reporting potential sequence roles and classical forms. The authors further provide freely available TAP at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php.

[0213] In addition to achieving the desired affinity to the antigen, there are many barriers to therapeutic mAb development. These include inherent immunogenicity, chemical and conformational instability, self-association, high viscosity, multispecificity, and poor expression. For example, high levels of hydrophobicity, especially in the hypervariable complementarity determining regions (CDRs), have been repeatedly implicated in aggregation, viscosity, and multispecificity. Net charge asymmetry of the heavy and light chain variable domains also correlates with self-association and viscosity at high concentrations. Patches of positive and negative charges in the CDRs are associated with high rates of clearance and low expression levels. Product heterogeneity (e.g., due to oxidation, isomerization, or glycosylation) often results from specific sequence motifs that are susceptible to post-translational or co-translational modifications. Computational tools are available to facilitate the identification of the role of sequences. Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Biol 15(8):e1007207. https: / / doi.org / 10.1371 / journal.pcbi.1007207) also described a method for optimizing antibody affinity and stability by automated design of the variable light-heavy chain interfaces. Additional methods are available for identifying potential developability problems of a candidate antibody, and in a preferred embodiment of the invention, one or more point mutations can be introduced into the candidate antibody via conventional methods to address such problems and result in an optimized therapeutic antibody of the invention.

[0214] 7. Humanized antibodies In some embodiments, the antibodies of the invention are humanized antibodies. Humanized antibodies are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies (e.g., human anti-mouse antibody (HAMA) responses), which can result in a reduced immune response to antibody therapeutics and the efficacy of the therapeutics.

[0215] Antibodies can be humanized by any standard method.Non-limiting exemplary methods of humanization include, for example, those described in U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370; Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-27 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and U.S. Patent Application Publication No. 2009 / 0136500. All are incorporated by reference.

[0216] A humanized antibody is an antibody in which at least one amino acid in a framework region of a non-human variable region is replaced with an amino acid from the corresponding position in a human framework region, hi some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 15, or at least 20 amino acids in the framework regions of a non-human variable region are replaced with an amino acid from one or more corresponding positions in one or more human framework regions.

[0217] In some embodiments, some of the corresponding human amino acids used for substitutions are from framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a first human antibody or encoded by a first human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a second human antibody or encoded by a second human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a third human antibody or encoded by a third human immunoglobulin gene, etc. Furthermore, in some embodiments, all of the corresponding human amino acids used for substitutions in a single framework region (e.g., FR2) need not be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitutions are from the same human antibody or encoded by the same human immunoglobulin gene.

[0218] In some embodiments, an antibody is humanized by replacing one or more entire framework regions with the corresponding human framework regions. In some embodiments, the human framework region with the highest level of homology to the non-human framework region being replaced is selected. In some embodiments, such a humanized antibody is a CDR-grafted antibody.

[0219] In some embodiments, following CDR grafting, one or more framework amino acids are returned to the corresponding amino acid in the mouse framework region. Such "backmutations" are made in some embodiments to retain one or more mouse framework amino acids that may contribute to the structure of one or more of the CDRs and / or may be involved in antigen contact and / or may be involved in the overall structural integrity of the antibody. In some embodiments, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or no backmutations are made to the framework regions of the antibody after CDR grafting.

[0220] In some embodiments, a humanized antibody also comprises a human heavy chain constant region and / or a human light chain constant region.

[0221] In some embodiments, the humanized anti-BTLA antibody or antigen-binding fragment thereof of the present invention is one of the antibodies listed in Tables 1-2 and the accompanying Sequence Listing, which are incorporated herein by reference.

[0222] 8. Human antibodies In some embodiments, the antibodies of the present invention are human antibodies. Human antibodies can be produced by any suitable method. Non-limiting exemplary methods include producing human antibodies in transgenic mice that contain human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-55 (1993); Jakobovits et al, Nature 362:255-8 (1993); Onberg et al, Nature 368:856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

[0223] Non-limiting exemplary methods also include generating human antibodies using phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227:381-8 (1992); Marks et al., J. Mol. Biol. 222:581-97 (1991); and PCT Publication WO 99 / 10494.

[0224] Antibody constant region In some embodiments, the humanized, chimeric, or human antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region, e.g., human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or Fc fusion partner comprises, for example, a C237S mutation in the IgG1 constant region. In some embodiments, the antibodies described herein comprise a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region comprises a P331S mutation as described in U.S. Pat. No. 6,900,292. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein comprise a S241P mutation in the human IgG4 constant region. See, e.g., Angal et al. Mol. Immunol. 30(1):105-108 (1993). In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

[0225] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a human IgG1 constant region comprising a heavy chain constant region sequence of SEQ ID NO:57 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising amino acid changes therein (e.g., insertions, deletions, and / or substitutions), and a light chain constant region sequence of SEQ ID NO:58 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and comprising amino acid changes therein (e.g., insertions, deletions, and / or substitutions).

[0226] The choice of heavy chain constant region can determine whether an antibody has effector function in vivo. Such effector function can, in some embodiments, include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), resulting in the killing of a cell to which the antibody binds. Typically, antibodies that contain human IgG1 or IgG3 heavy chains have effector function.

[0227] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention is an IgG1 antibody that comprises mutations in the heavy chain constant region that modulate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) (e.g., F243L, G236A, S239D / I332E, S239D / A330L / I332E, F243L / R292P / Y300L / V305I / P396L, S298A / E333A / K334A, or an afucosylated (non-fucosylated) antibody (e.g., afucosylated N297 in the Fc region) with enhanced ADCC via increased binding to FcγRIIIa.

[0228] In some embodiments, the isolated antibodies of the invention are IgG1 antibodies that contain mutations in the heavy chain constant region (eg, T250Q / M428L, and M252Y / S254T / T256E).

[0229] In some embodiments, effector function is undesirable. For example, in some embodiments, effector function may be undesirable in the treatment of inflammatory conditions and / or autoimmune diseases. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, the IgG4 constant region comprises a S241P mutation.

[0230] Any of the antibodies described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and / or epitope to which the antibody binds, as well as ligands that bind the antibody constant region. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind the constant region and purify the antibody.

[0231] In some embodiments, hydrophobic interactive chromatography (HIC), such as butyl or phenyl columns, is also used to purify some polypeptides. Many methods of purifying polypeptides are known in the art.

[0232] Alternatively, in some embodiments, the antibodies described herein are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0233] 9. Nucleic acid molecules encoding antibodies of the invention The present invention also provides nucleic acid molecules comprising polynucleotides encoding one or more chains of an antibody described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0234] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides, either from one nucleic acid molecule or from two separate nucleic acid molecules, in some embodiments, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together, such as when the antibody is an scFv.

[0235] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody described herein comprises a nucleotide sequence encoding a leader sequence that, when translated, is located at the N-terminus of the heavy or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0236] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell, such as a mammalian cell.

[0237] In some embodiments, polynucleotides encoding the heavy or light chains of the antibodies described herein are codon optimized for expression in human cells.

[0238] 10. Vector A vector is provided that comprises a polynucleotide encoding the heavy and / or light chain of the antibody described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is an scFv.

[0239] In some embodiments, the vector comprising a polynucleotide encoding the heavy and / or light chain of an antibody described herein is an expression vector (e.g., a mammalian, yeast, insect, or bacterial expression vector).

[0240] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into the host cell in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of the first vector and the second vector of 5:1 to 1:5 is transfected into the host cell. Some embodiments use a mass ratio of 1:1 to 1:5 for the vector encoding the heavy chain and the vector encoding the light chain. Some embodiments use a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain.

[0241] In some embodiments, a vector is selected that is optimized for expression of a polypeptide in CHO or CHO-derived cells, or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol.Prog.20:880-889 (2004). In some embodiments, a vector is selected for in vivo expression of the subject antibodies in animals, including humans. In some such aspects, expression of one or more polypeptides is under the control of one or more promoters that function in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT Publication WO 2006 / 076288.

[0242] 11.Host cells In various embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in prokaryotic cells, such as bacterial cells, or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Cru cells); and NSO cells. In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045(A1). In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications to the heavy and / or light chains of the anti-BTLA antibody. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0243] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into a desired host cell according to any suitable method.

[0244] In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method. EXAMPLES

[0245] Example 1 Identification of Human BTLA Agonist Antibodies that Cross-React with Cynomolgus Monkey BTLA This example presents the results of the characterization of two murine lead antibody candidates (HFB6-4 and HFB6-5) based on their ability to inhibit anti-IgM-mediated B cell proliferation to a similar or greater extent than a benchmark BTLA agonist antibody (referred to herein as 22B3) ( FIG. 2A ) and their inability to reverse HVEM-mediated T cell inhibition ( FIG. 2B ). These murine antibodies are therefore sometimes referred to as "agonist non-blockers" due to their ability to bind to BTLA and initiate immunosuppressive functions downstream of BTLA, as well as their inability to displace the natural ligand of BTLA (i.e., HVEM) when bound to BTLA.

[0246] In Figure 1, HEK293 cells overexpressing human BTLA and Expi293 cells overexpressing cynomolgus BTLA were incubated with different concentrations of anti-BTLA parent antibodies. Anti-BTLA antibody binding to target cells was quantified by flow cytometry with an AF647-conjugated anti-human IgG Fc secondary antibody. HFB6-4 and HFB6-5 parent antibodies were found to bind human BTLA and cynomolgus BTLA with single-digit nM affinity.

[0247] In Figure 2A, primary B cells were isolated from healthy donor PBMCs using the EasySep™ Human B Cell Isolation Kit and labeled with CFSE. B cells were stimulated with 10 μg / mL anti-IgM in the presence of 100 nM anti-BTLA antibody or control antibody. After 4 days, B cell proliferation was quantified by CFSE dilution relative to unstimulated B cells (CFSElo%).

[0248] In the HVEM-BTLA blocking reporter assay, a CHO cell line engineered to express a TCR activator (TCRα) and the BTLA ligand HVEM was co-cultured with a Jurkat reporter cell line expressing BTLA and a luciferase reporter driven by an NFAT-binding promoter. Application of a benchmark anti-BTLA blocking antibody (herein referred to as JS004) was able to reverse HVEM-mediated T cell inhibition, whereas HFB6-4 and HFB6-5 were not (Figure 2B).

[0249] Based on binning experiments, it was further confirmed that the HFB6-4 and 22B3 epitopes overlap at the BTLA / HVEM binding interface. It is therefore particularly surprising that different antibodies bind to seemingly the same or substantially overlapping epitopes, yet appear to have substantially different biological activities.

[0250] Specifically, in ELISA binning experiments, ELISA plates were coated with 5 μg / mL of each capture antibody (HFB6-4 or 22B3) or ligand (recombinant Fc-tagged HVEM). After blocking, recombinant his-tagged BTLA (BTLA-his) was administered together with an excess (30 μg / mL) of competing or isotype control antibody (MGO53) in solution. Captured BTLA-his was detected with an HRP-conjugated anti-his secondary antibody. The percentage of BTLA-his capture inhibition was quantified relative to the use of MGO53 as the competing antibody (see table below).

[0251] [Table 4]

[0252] The data showed that HFB6-4 prevented the binding of recombinant BTLA (BTLA-his) to immobilized 22B3 and vice versa, and conversely, to recombinant HVEM (HVEM-Fc). These data indicate that the HFB6-4 and 22B3 epitopes overlap at the BTLA / HVEM binding interface.

[0253] Example 2 hBTLA agonist antibodies HFB6-4 and HFB6-5 inhibit T cell activation This experiment demonstrates that the isolated HFB6-4 and HFB-6-5 antibodies are functional hBTLA agonists, as evidenced by their ability to inhibit T cell activation.

[0254] In this assay, CHO cells expressing FcγRIIb on the cell surface were first incubated with a test antibody, such as HFB6-4 or HFB6-5 (or a positive or negative control antibody), in the presence or absence of an anti-CD3 antibody, allowing the test antibody (and anti-CD3 antibody, if added) to bind to surface FcγRIIb via the Fc region.

[0255] The test antibody coated CHO cells were then incubated with Jurkat reporter cells expressing surface TCR and BTLA. If anti-CD3 antibody was also present on the CHO cells, upon TCR activation by anti-CD3 antibody, BTLA agonist antibodies such as HFB6-4 and HFB6-5 engaged BTLA and stimulated downstream BTLA signaling, thus inhibiting TCR signaling as measured in the reporter cells by a luciferase reporter gene under the transcriptional control of the TCR downstream NFAT promoter.

[0256] No luciferase activity was detected in negative controls, in which no anti-CD3 antibody was added to stimulate TCR activation. Luciferase activity was not attenuated in the presence of the control antibody MGO, which did not bind to BTLA.

[0257] In contrast, the benchmark BTLA agonist antibody 22B3 reduced / inhibited TCR-mediated luciferase activity in a dose-dependent manner. Both HFB6-4 and HFB6-5, as well as another BTLA agonist antibody H6S8-9A10A, inhibited TCR-mediated luciferase activity to a greater extent than the benchmark antibody 22B3. See FIG. 3.

[0258] The data showed that HFB6-4, HFB6-5, and H6S8-9A10A were at least as good (if not better) at inhibiting TCR signaling as the benchmark agonist antibody 22B3.

[0259] Example 3 hBTLA agonist antibodies HFB6-4 / HFB6-5 inhibit B cell proliferation This example demonstrates that the BTLA agonistic antibodies HFB6-4 and HFB6-5 inhibit primary B cell proliferation to a degree comparable to the benchmark agonistic antibody 22B3 in all donors tested.

[0260] Primary B cells were isolated from healthy donor PBMCs using the EasySep™ Human B Cell Isolation Kit and labeled with CFSE. B cells were stimulated with 10 μg / mL of anti-IgM in the presence of anti-BTLA or control antibodies. After 4 days, B cell proliferation was quantified by CFSE dilution relative to unstimulated B cells (CFSElo%).

[0261] HFB6-4 and HFB6-5 inhibited proliferation of primary B cells from three different donors at levels similar to (donor 015 and donor 017) or better (donor 019) than the benchmark 22B3 antibody (Figure 4, left panel). Increasing concentrations of HFB6-4 and HFB6-5 increased inhibition of primary B cell proliferation (Figure 4, right panel).

[0262] Example 4 Binding and cross-reactivity of humanized HFB6-4 / HFB6-5 mutants This experiment demonstrates that all humanized variants of HFB6-4 and HFB6-5 bind hBTLA, like the parental HFB6-4 and HFB6-5 antibodies, and cross-react with cynomolgus BTLA with high affinity.

[0263] Briefly, 24 individual humanized variants of HFB6-4 and HFB6-5 (e.g., HFB6-4-hz1 to -hz24, and HFB6-5-hz1 to -hz24) were generated based on standard humanization protocols, and the resulting humanized variants were tested to identify high-affinity binders to hBTLA and cynoBTLA.

[0264] HEK293 cells overexpressing human BTLA and Expi293 cells overexpressing cynomolgus monkey BTLA were incubated with different concentrations of humanized anti-BTLA antibodies. Anti-BTLA antibody binding to target cells was quantified by flow cytometry with an AF647-conjugated anti-human IgG Fc secondary antibody. See Figure 5.

[0265] The results show that the HFB6-4 and HFB6-5 humanized variants bind to human and cynomolgus BTLA with single-digit nM affinity.

[0266] Example 5 HFB6-4 and HFB6-5 humanized variants inhibit B cell proliferation and T cell reporter activity The potency of selected HFB6-4 and HFB6-5 humanized variants in inhibiting B cell proliferation and T cell reporter activity was compared to that of the parental antibody and benchmark controls.

[0267] For the B cell proliferation inhibition assay, primary B cells were isolated from healthy donor PBMCs using the EasySep™ Human B Cell Isolation Kit and labeled with CFSE. B cells were stimulated with 10 μg / mL anti-IgM in the presence of 1 μg / mL and 10 μg / mL anti-BTLA antibody or control antibody. After 4 days, B cell proliferation was quantified by CFSE dilution relative to unstimulated B cells (CFSElo%).

[0268] In a T cell suppression reporter assay, anti-CD3 and anti-BTLA antibodies were cross-linked using a CHO cell line engineered to express FcγRIIb. Upon Fc receptor cross-linking, the anti-BTLA agonist antibody was able to inhibit the luciferase reporter signal driven by TCR-NFAT pathway activation in a Jurkat reporter cell line expressing BTLA and an NFAT luciferase reporter.

[0269] The results show that the selected HFB6-4 and HFB6-5 humanized variants maintain the parental HFB6-4 and HFB6-5 activity in inhibiting B cell proliferation (FIG. 6A) and T cell reporter activity (FIG. 6B).

[0270] Example 6 Favorable PK Profiles of Selected HFB6-4 and HFB6-5 Humanized Variants in Wild-Type C57BL / 6 Mice To investigate the pharmacokinetic profile of HFB6-4 and HFB6-5 humanized variants, a single dose (10 mg / kg) of the selected variants was intravenously injected into wild-type C57BL / 6 mice. Blood was collected at 1, 24, and 72 hours, and the plasma concentrations of the selected antibodies were measured by ELISA.

[0271] The data show that selected humanized variants, such as HFB6-4 humanized variants HFB6-4hz1-hG1, HFB6-4hz6-hG1, and HFB6-5 humanized variants HFB6-5hz12-hG1, HFB6-5hz19-hG1, and HFB6-5hz24-hG1, exhibited prolonged exposure in mice when administered as a single dose of 10 mg / kg.

[0272] Example 7 Favorable developability of selected humanized variants The developability of selected humanized variants of HFB6-4 and HFB6-5 was investigated, and the variants exhibit favorable developability characteristics in multiple assays, including melting temperature assays, stability at 4°C and 37°C, and stability at low pH (e.g., pH 3.8).

[0273] Example 8 Therapeutic efficacy in the hPBMC-NSG acute GvHD model This example demonstrates that the subject BTLA agonist antibodies are effective in inhibiting immune responses in an NSG mouse model of acute GvHD (graft-versus-host disease).

[0274] In this experiment, the in vivo efficacy of the subject BTLA agonist antibodies is evaluated in a xenogeneic humanized mouse model of acute graft-versus-host disease (GvHD), a T cell-mediated disease induced by engagement of human PBMCs in sublethally irradiated NSG mice. A schematic of the experimental protocol is illustrated in Figure 8A.

[0275] Figure 8B shows a more detailed experimental design, including the doses and measured readouts for each treatment group. Each of the four treatment groups has eight mice. They include a PBS negative control group (G1), an IgG1 isotype-matched control group (MGO53-hG1, G2), a BTLA agonist antibody (HFB6-4-hz1-hG1) group (G3), and a 22B3 benchmark control group (G4).

[0276] Preliminary data showed that at day 14 after hPBMC inoculation, G3 mice treated with the subject BTLA agonist antibody HFB6-4-hz1-hG1 exhibited a statistically significant reduction in the percentage of hCD45+ immune cells compared to the isotype-matched control group G2. Meanwhile, the benchmark group G4 did not appear to have a statistically significant difference, if any, at the same time point. See Figure 8E.

[0277] Furthermore, the Kaplan-Meier survival curves in FIG. 8F show that more than 85% of G3 mice treated with the subject BTLA agonist antibody HFB6-4-hz1-hG1 survived at the end of the experiment on day 28. This is statistically significantly higher than the less than 20% survival rate of the PBS control group (G1) and the isotype-matched MGO53-hG1 control group (G2).

[0278] On the other hand, survival rates declined steadily after 15 days in the G4 control treatment group treated with the 22B3 benchmark antibody, with less than 40% of treated mice surviving. The difference in survival rates between the G3 and G4 groups is also statistically significant.

[0279] In addition, the HFB6-4hz1-hG1 treated group (G3) showed significantly reduced total disease scores (see FIG. 8C) and relative control body weight (RCBD) loss (FIG. 8D) compared to the PBS control group G1, the isotype-matched control group G2, and the 22B3 benchmark group G4.

[0280] The dose-response efficacy of HFB6-4hz1-hG1 was also evaluated in the same hPBMC-NSG acute GvHD model with low doses of HFB6-4hz1-hG1 compared to high doses of the control antibody 22B3 (Figure 8G). Specifically, treatment with 1, 3, and 10 mg / kg HFB6-4hz1-hG1 all resulted in greater survival benefit than 10 mg / kg 22B3-hG4 and 10 mg / kg IgG4 version of HFB6-4hz1 (HFB6-4hz1-hG4P). Even in the IgG4 format, HFB6-4hz1-hG4P is superior to 22B3-hG4P at 10 mg / kg. Furthermore, HFB6-4hz1-hG1 (10 mg / kg) treatment demonstrated a long-term survival benefit over abatacept (6.13 mg / kg, molar equivalent to 10 mg / kg antibody), the approved prophylaxis for acute GvHD in humans.

[0281] These data demonstrated that the subject BTLA agonist antibody HFB6-4-hz1-hG1 was superior to the benchmark 22B3 agonist in this mouse model of acute GvHD under experimental conditions, at least in terms of better therapeutic efficacy over the entire treatment course, longer and better overall survival rates, as well as reduced toxicity.

[0282] Overall, the above data demonstrate that subject BTLA agonist antibodies such as HFB6-4 and HFB6-5 and their derived humanized variants have single-digit binding affinity to human and cynomolgus BTLA, that multiple humanized variants of these antibodies (such as HFB6-4hz1-hG1) retained parental antibody function in B cell proliferation inhibition and T cell reporter assays, and that the antibodies exhibited favorable PK profiles in wild-type mice, supporting dosing at frequencies of q3d or less for efficacy evaluation. Furthermore, in an in vivo hPBMC-NSG mouse model of acute GvHD, the subject humanized variant HFB6-4hz1-hG1 proved to be more effective and less toxic than the benchmark agonist antibody 22B3, even at doses one-tenth (1 / 10) or one-third (1 / 3) of the dose of the control antibody.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for and activates human B- and T-lymphocyte attenuator (BTLA), and optionally is cross-reactive with cynomolgus monkey BTLA, the monoclonal antibody comprising: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; Or, (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 61, the HCVR CDR2 sequence of SEQ ID NO: 62, and the HCVR CDR3 sequence of SEQ ID NO: 63; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 64, the LCVR CDR2 sequence of SEQ ID NO: 65, and the LCVR CDR3 sequence of SEQ ID NO: 66; Optionally, the monoclonal antibody is an isolated monoclonal antibody or antigen-binding fragment thereof that is not naturally occurring.

2. (1A) the HCVR sequence is SEQ ID NO: (7+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in a heavy chain framework region; (1B) the LCVR sequence is SEQ ID NO: (8+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and comprising / consisting essentially of / consisting of at least one amino acid alteration (e.g., insertion, deletion, and / or substitution) in the light chain framework region; n is any one of 1, 0, and 2 to 24, or (2A) the HCVR sequence is SEQ ID NO: (67+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and comprising / consisting essentially of / consisting of at least one amino acid change (e.g., insertion, deletion, and / or substitution) in the heavy chain framework region; (2B) the LCVR sequence is SEQ ID NO: (68+2n), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and comprising / consisting essentially of / consisting of at least one amino acid alteration (e.g., insertion, deletion, and / or substitution) in the light chain framework region; 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein n is any one of 0 to 24.

3. the monoclonal antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody; The monoclonal antibody may be an IgG1 antibody. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

4. 4. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the IgG1 antibody comprises a heavy chain constant region sequence of SEQ ID NO: 57 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and containing amino acid changes (e.g., insertions, deletions, and / or substitutions) therein, and a light chain constant region sequence of SEQ ID NO: 58 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and containing amino acid changes (e.g., insertions, deletions, and / or substitutions) therein.

5. The monoclonal antibody (1) modulate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) (e.g., F243L, G236A, S239D / I332E, S239D / A330L / I332E, S298A / E333A / K334A, F243L / R292P / Y300L / V305I / P396L, or afucosylated (non-fucosylated) antibodies (e.g., afucosylated N297 in the Fc region) with enhanced ADCC via increased binding to FcγRIIIa); and / or (2) The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, which is an IgG1 antibody containing mutations in the heavy chain constant region that enhance serum half-life (e.g., T250Q / M428L, M252Y / S254T / T256E).

6. a murine antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody, The antigen-binding fragment thereof may be a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc; The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

7. the monoclonal antibody or antigen-binding fragment thereof cross-reacts with cynomolgus / rhesus BTLA but does not substantially cross-react with mouse BTLA; and / or the monoclonal antibody or antigen-binding fragment thereof binds human BTLA with a K of less than about 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM or less; The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

8. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising: (i) is an agonist of human BTLA, and upon binding to BTLA, activates downstream signaling from BTLA; wherein further said downstream signaling from BTLA may inhibit B cell proliferation and / or inhibit T cell (e.g., CD4, CD8, Th1, TFH, αβ, or γδ T cells) and / or plasma cell activation; (ii) does not block / interfere / eliminate BTLA binding to HVEM; (iii) does not result in significant weight loss when administered (e.g., i.p. to mice) at a dose of 10 mg / kg BIW x 6 (e.g., for 21 days); (iv) when administered in vivo (e.g., i.p. to mice at a dose of 10 mg / kg BIW x 6 for 14 days), significantly reduces CD45+ T cells; and / or (v) significantly reduces disease severity (e.g., a mean total disease score of less than 3 at 3 weeks) and / or overall survival (e.g., at least 80% overall survival at 4 weeks) in GvHD or an animal model thereof compared to an isotype-matched control Ab; The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

9. competes for binding to human BTLA with the isolated monoclonal antibody or antigen-binding fragment thereof of claim 1; Furthermore, binding to BTLA may inhibit B cell proliferation and / or inhibit T cell (e.g., CD4, CD8, Th1, TFH, αβ, or γδ T-cell) activation; An isolated monoclonal antibody or antigen-binding fragment thereof.

10. (1) the HCVR sequence of SEQ ID NO: 9, the heavy chain constant region sequence of SEQ ID NO: 57, the LCVR sequence of SEQ ID NO: 10, and the light chain constant region sequence of SEQ ID NO: 58; or (2) An isolated monoclonal antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO:

60.

11. 10. A polynucleotide encoding the heavy or light chain or antigen-binding portion thereof of claim 1, The polynucleotide may be codon-optimized for expression in a human cell. Polynucleotide.

12. A vector comprising the polynucleotide of claim 9, The vector may be an expression vector (e.g., a mammalian, yeast, insect, or bacterial expression vector), vector.

13. 13. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, the polynucleotide of claim 11, or the vector of claim 12, The pharmaceutical composition may be formulated for intravenous (i.v.) infusion or administration, or subcutaneous (s.c.) administration. Pharmaceutical compositions.

14. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, a polynucleotide according to claim 11, a vector according to claim 12, or a pharmaceutical composition according to claim 13 for use in down-regulating a B cell-mediated or T cell-mediated immune response or for use in treating an autoimmune disease in a patient in need thereof, comprising: The autoimmune disease may be systemic lupus erythematosus (SLE); ulcerative colitis (UC), including pediatric ulcerative colitis; rheumatoid arthritis (RA); psoriasis (Ps), including chronic plaque psoriasis; psoriatic arthritis (PsA); Crohn's disease (CD), including pediatric Crohn's disease; inflammatory bowel disease (IBD); ankylosing spondylitis; juvenile idiopathic arthritis (JIA), including polyarticular juvenile idiopathic arthritis; hidradenitis suppurativa; non-infectious intermediate uveitis, posterior uveitis, and panuveitis; autoimmune hepatitis-like disease; experimental autoimmune encephalomyelitis (EAE); MHC-mismatched cardiac allotransplantation; lung inflammation in acute airway allergy; graft-versus-host disease (GvHD); or allogeneic hematopoietic stem cell transplantation (aHSCT).

14. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, a polynucleotide according to claim 11, a vector according to claim 12, or a pharmaceutical composition according to claim 13.

15. The autoimmune disease of claim 1, wherein the autoimmune disease is lupus.

15. An isolated monoclonal antibody or antigen-binding fragment thereof for use according to claim 14.

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