BTLA antibody

Abstract

To provide BTLA antibodies.SOLUTION: The present invention relates generally to antibodies that bind to human B and T lymphocyte attenuator (BTLA) and uses thereof. More specifically, the invention relates to agonist antibodies that bind to and modulate the activity of human BTLA, and their use in the treatment of inflammatory, autoimmune, and proliferative diseases and disorders. Suitably, the antibodies also have Fc modifications that enhance signaling through Fc γ R2B. In certain embodiments, such molecules have reduced binding to one or more activating Fc gamma receptors, such as Fc γ R2A or Fc γ R1A, as compared to the parent polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to antibodies, including antigen-binding fragments, that bind to human B and T lymphocyte attenuator (BTLA), and uses thereof. More specifically, the present invention relates to agonistic antibodies that bind to and modulate the activity of human BTLA and have enhanced binding to and signaling through FcγR2B, and their use in the treatment of inflammatory, autoimmune, and proliferative diseases and disorders. [Background technology]

[0002] The immune system must balance the destruction of pathogens or dangerous mutated cells with the tolerance of healthy self-tissues and harmless commensals. To facilitate this balance, immune cell activity is influenced by the integration of signals from multiple stimulatory and inhibitory receptors that align cells with their environment. These surface-expressed receptors present attractive targets for therapeutic modulation of the immune response. Many human diseases result from aberrant or unwanted activation of the immune system, including autoimmune diseases, graft rejection, and graft-versus-host disease. Agonistic agents capable of inducing signaling through inhibitory receptors can attenuate these unwanted immune responses.

[0003] B and T lymphocyte attenuator (BTLA; also called CD272) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and PD-1 (Watanabe et al., Nat Immunol. 4:670-679, 2003). It is widely expressed on both myeloid and lymphoid cells throughout the immune system (Han et al., J Immunol. 172:5931-9, 2004). Following binding 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. Mice lacking an intact BTLA gene exhibit 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 infiltration in multiple organs (Oya et al., Arthritis Rheum, 58:2498-2510, 2008). This evidence indicates that the BTLA inhibitory receptor plays an important role in maintaining immune homeostasis and suppressing autoimmunity. Furthermore, HVEM-BTLA signaling is involved in regulating mucosal inflammation and infection immunity (Shui et al., J. Leukoc. Biol., 89:517-523, 2011).

[0004] Therapeutic agents that can modulate BTLA function to suppress autoreactive lymphocytes associated with autoimmune disorders are highly desirable.

[0005] It has previously been shown that monoclonal antibodies that bind to murine BTLA can act as agonists, inducing receptor-mediated signaling and suppressing immune cell responses. In the presence of agonistic anti-BTLA antibodies (mAbs), anti-CD3 and anti-CD28 activated T cells exhibit reduced IL-2 production and proliferation (Kreig et al., J. Immunol., 175, 6420-6472, 2005).

[0006] Furthermore, anti-mouse BTLA agonist antibodies have been shown to ameliorate disease in mouse models of graft-versus-host disease (Sakoda et al., Blood. 117:2506-2514; Albring et al., J Exp Med. 207:2551-9, 2010). Agonist antibodies targeting the human BTLA receptor have been shown to suppress T cell responses ex vivo (Otsuki et al., Biochem Biophys Res Commun 344:1121-7, 2006, and WO 2011 / 014438), but have not yet been translated into the clinic.

[0007] WO 2018 / 213113 (Eli Lilly & Co.) discloses certain antibodies against BTLA.

[0008] International Publication No. 2020 / 128446 published on June 25, 2020 (Oxford University Innovation Limited and MiroBio Limited discloses certain antibodies against BTLA.

[0009] In humans, there is one inhibitory Fc gamma receptor (FcγR2B), while the other Fc gamma receptors all deliver immune activation signals (FcγR1A, FcγR2A, FcγR3A, and FcγR3B). The important regulatory role of FcγR2B has been demonstrated through studies of FcγR2B knockout mice, which have increased susceptibility to autoimmune diseases (Nakamura et al. Journal of Experimental Medicine 191(5):899-906, 2000). Furthermore, polymorphisms in the FcγR2B gene in humans are associated with the risk of autoimmune diseases, particularly systemic lupus erythematosus (Floto et al. Nature Medicine 11(10), 2005). Therefore, FcγR2B is thought to play an important role in regulating immune responses and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0010] Antibodies with Fc having improved FcγR2B binding activity have been reported (Chu et al. (Molecular Immunology 45(15):3926-33, 2008). In this paper, FcγR2B binding activity was improved by introducing alterations such as S267E / L328F, G236D / S267E, and S239D / S267E into the antibody Fc region. Among these, antibodies with the S267E / L328F mutation introduced exhibited the strongest binding to FcγR2B, maintaining the same level of binding to FcγR1A and FcγR2A (131H allotype) as naturally occurring IgG1. However, another report showed that this alteration enhanced binding to FcγR2A 131R by several hundred times, to the same level as FcγR2B binding, suggesting that FcγR2B binding selectivity was enhanced by the FcγR2A allotype. This means that there is no improvement over 131R (U.S. Patent Publication No. 2009 / 0136485).

[0011] For a BTLA agonist antibody to be effective in suppressing immune responses without inducing inflammatory FcγR signaling, the inventors propose tailoring the antibody for selective Fc binding to FcγR2B. Molecules with more selective binding to FcγR2B would promote bidirectional inhibitory signaling through BTLA on BTLA-expressing cells and through FcγR2B on FcγR2B-expressing cells, enhancing the immunosuppressive effects of the antibody. This is desirable in therapeutic antibodies intended to treat diseases of immune hyperactivation. Summary of the Invention [Means for solving the problem]

[0012] The present invention relates to BTLA agonist antibodies (including antibody fragments thereof) that possess one or more desirable properties, including high binding affinity in human subjects, high agonist potency, high agonist efficacy, favorable pharmacokinetics, and low antigenicity. In certain embodiments, such molecules also have increased binding to FcγR2B, thus driving FcγR2B signaling, and have sufficient in vivo half-life for suitable therapeutic applications. Thus, antibodies of the present invention promote bidirectional inhibitory signaling via BTLA on BTLA-expressing cells and via FcγR2B on FcγR2B-expressing cells. In certain embodiments, such molecules have reduced binding to one or more activating Fc gamma receptors, such as FcγR2A or FcγR1A, compared to the parent polypeptide. In certain embodiments, such molecules have an increased binding ratio to FcγR2B / FcγR2A compared to the parent polypeptide. In certain embodiments, such molecules have an increased binding ratio to FcγR2B / FcγR1A compared to the parent polypeptide. The present invention also relates to the use of the antibodies of the present invention in the treatment of diseases such as autoimmune and / or inflammatory diseases.

[0013] According to a first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region that comprises a substitution that results in increased binding to FcγR2B compared to the parent molecule lacking the substitution.

[0014] In some embodiments, the binding to FcγR2B is increased compared to the parent polypeptide such that the value of [KD value of parent polypeptide for FcγR2B] / [KD value of variant polypeptide for FcγR2B] is greater than 1 (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100).

[0015] In some embodiments, the antibody has a preference for binding to FcγR2B over FcγR2A.

[0016] In some embodiments, the antibody has enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR2A (R type) and / or FcγR2A (H type) compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR2A (R type)] / [KD value of the variant polypeptide for FcγR2B] is 2 or greater (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the value of [KD value of variant polypeptide for FcγR2A (H type)] / [KD value of variant polypeptide for FcγR2B] is 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or more).

[0017] In some embodiments, the antibody has enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR1A compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR1A] / [KD value of the variant polypeptide for FcγR2B] is 0.05 or greater (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, or more).

[0018] In some embodiments, the variant polypeptide has reduced Fcγ1 binding activity compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR1A] / [KD value of the parent polypeptide for FcγR1A] is at least 10, 20, 50, 100, or 200.

[0019] In some embodiments, the antibody binds to a residue of human BTLA selected from the following: (i) D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225); (ii) Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225); (iii) D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225); (iv) N65 and A64 (positions according to SEQ ID NO: 225), or (v) H68 (position according to SEQ ID NO: 225).

[0020] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, or alanine (A) at position 297 (all numbering according to the EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0021] Preferably, the antibody is a human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A.

[0022] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an aspartic acid at position 238 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0023] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an aspartic acid at position 237 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0024] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an aspartic acid at position 236 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0025] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an alanine at position 235 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0026] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an alanine at position 234 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0027] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising a glycine at position 271 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0028] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising a glutamic acid at position 267 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0029] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an alanine at position 265 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0030] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an alanine at position 297 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0031] According to a variation of the first aspect of the invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an alanine at position 322 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0032] According to a variation of the first aspect of the present invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an arginine at position 330 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonistic antibody / antigen-binding fragment. According to a variation of the first aspect of the present invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, the heavy chain comprising an Fc region comprising an aspartic acid at position 237 (EU index), an aspartic acid at position 238 (EU index), a glycine at position 271 (EU index), and an arginine at position 330 (EU index). Preferably, the antibody that specifically binds to human BTLA is an agonistic antibody / antigen-binding fragment.

[0033] In certain embodiments, the antibody has a heavy chain and / or a complementarity-determining region (CDR) present in an antibody selected from the group consisting of 6.2, 2.8.6, 3E8, 11.5.1, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, and 831, as identified in Table 1 or Table 2, and as described herein. Preferably, the antibody also comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index).

[0034] In a further embodiment, the antibody that binds to human BTLA is selected from the group consisting of 6.2, 2.8.6, 3E8, or an antibody that competes with any one of 6.2, 2.8.6, or 3E8 for binding to human BTLA, wherein the antibody specifically binds to BTLA and induces receptor-mediated signaling. The antibody also comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index).

[0035] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, the antibody comprising heavy and light chains comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR sequences disclosed in SEQ ID NOs: 1, 17, 3, 4, 12, and 6, respectively, and an Fc region comprising substitutions that result in increased binding to FcγR2B compared to the parent molecule lacking the substitutions. Preferably, the antibody comprises the VH and VL sequences disclosed in SEQ ID NOs: 18 and 14, respectively. Preferably, the antibody comprises a heavy and light chain, wherein the heavy chain comprises the amino acid sequence disclosed in SEQ ID NO: 19 and / or the light chain comprises the amino acid sequence disclosed in SEQ ID NO: 16. In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 236 (EU index). Preferably, the antibody is an agonist antibody.

[0036] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 237 (EU index). Preferably, the antibody is an agonist antibody.

[0037] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 238 (EU index). Preferably, the antibody is an agonist antibody.

[0038] In a specific embodiment, the antibody comprises an Fc region comprising an alanine at position 235 (EU index).

[0039] In a specific embodiment, the antibody comprises an Fc region comprising an alanine at position 234 (EU index).

[0040] In a specific embodiment, the antibody comprises an Fc region comprising an alanine at position 265 (EU index).

[0041] In a specific embodiment, the antibody comprises an Fc region comprising a glutamic acid at position 267 (EU index).

[0042] In a specific embodiment, the antibody comprises an Fc region comprising a glycine at position 271 (EU index).

[0043] In a specific embodiment, the antibody comprises an Fc region comprising an alanine at position 297 (EU index).

[0044] In a specific embodiment, the antibody comprises an Fc region comprising an alanine at position 322 (EU index).

[0045] In a specific embodiment, the antibody comprises an Fc region comprising an arginine at position 330 (EU index).

[0046] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 237 (EU index), an aspartic acid at position 238 (EU index), a glycine at position 271 (EU index), and an arginine at position 330 (EU index).

[0047] In certain embodiments of the first aspect of the invention, the antibody has increased binding to FcγR2B compared to a parent molecule lacking one or more of the Fc region substitutions, e.g., hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A.

[0048] In certain embodiments of the first aspect of the invention, the antibodies have increased binding to FcγR2B and decreased binding to one or more activating Fcγ receptors, such as FcγR2A or FcγR1A, compared to the parent molecule lacking the Fc region substitution.

[0049] In certain embodiments of the first aspect of the invention, the antibody has an increased ratio of binding to FcγR2B / FcγR2A compared to the parent molecule lacking the Fc region substitution. Suitably, the increased ratio to FcγR2B / FcγR2A is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10 fold compared to the parent molecule lacking the Fc region substitution.

[0050] In certain embodiments of the first aspect of the invention, the antibody has an increased ratio of binding to FcγR2B / FcγR1A relative to the wild-type sequence, compared to the parent molecule lacking the Fc region substitution. Suitably, the increased ratio to FcγR2B / FcγR1A is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, 100, 150, 200, 250 fold, compared to the parent molecule lacking the Fc region substitution.

[0051] Compared to a parent molecule lacking an Fc region substitution means compared to an antibody molecule having the same amino acid sequence except for the amino acids recited in the claims that represent Fc substitutions relative to wild-type Fc, for example, including any of the following substitutions: hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A. Thus, binding of antibody molecules with or without the listed Fc substitutions to FcγR2B can be measured, and optionally, binding of antibody molecules with or without the listed Fc substitutions to an activating Fcγ receptor such as FcγR2A or FcγR1A can be measured. Thus, for example, a 1.5-fold increase in binding to FcγR2B compared to a parent molecule without the substitutions indicates a 150% increase in binding efficiency compared to the parent. A 1.5-fold decrease in binding to FcγR2A compared to a parent molecule without the substitutions indicates a 67% binding efficiency compared to the parent. For this exemplary antibody molecule, the change in the FcγR2B / FcγR2A binding ratio is 150 / 67 = 2.24-fold. Any value greater than 1 indicates that the compound has enhanced selectivity for binding to FcγR2B over FcγR2A compared to the parent compound.

[0052] In certain embodiments of the first aspect of the invention, the antibody has an increased ratio of [KD value for FcγR1A binding] / [KD value for FcγR2B binding] compared to the parent molecule lacking the Fc region substitution relative to the wild-type sequence. Suitably, the ratio of [KD value for FcγR1A binding] / [KD value for FcγR2B binding] of the variant molecule is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 times greater than the ratio of [KD value for FcγR1A binding] / [KD value for FcγR2B binding] of the parent molecule lacking the Fc region substitution.

[0053] In certain embodiments of the first aspect of the invention, the variant molecule has an increased ratio of [KD value for FcγR2A 131R binding] / [KD value for FcγR2B binding] compared to the parent molecule lacking the Fc region substitution relative to the wild-type sequence. Suitably, the ratio of [KD value for FcγR2A 131R binding] / [KD value for FcγR2B binding] of the variant molecule is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, or 100-fold greater than the ratio of [KD value for FcγR1A binding] / [KD value for FcγR2B binding] of the parent molecule lacking the Fc region substitution.

[0054] According to a second aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding the heavy and / or light chain polypeptides of the isolated antibody of the first aspect of the invention.

[0055] According to a third aspect of the invention, there is provided a vector, comprising a nucleic acid according to the second aspect of the invention.

[0056] According to a fourth aspect of the invention there is provided a host cell, comprising a nucleic acid sequence according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0057] According to a fifth aspect of the invention there is provided a method of producing an antibody according to the first aspect of the invention, comprising culturing a host cell of the fourth aspect of the invention under conditions for the production of said antibody, and optionally isolating and / or purifying said antibody.

[0058] According to a sixth aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody of the first aspect of the invention or an antibody produced according to the fifth aspect of the invention.

[0059] According to a seventh aspect of the invention there is provided a method for preparing a pharmaceutical composition comprising formulating an antibody according to the first aspect of the invention or an antibody produced according to the fifth aspect of the invention into a composition comprising at least one additional component, in certain embodiments the at least one additional component is a pharmaceutically acceptable excipient.

[0060] According to an eighth aspect of the present invention there is provided a kit comprising an antibody according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention. Suitably such a kit comprises a package insert containing instructions for use.

[0061] According to a ninth aspect of the invention there is provided a method of treating a BTLA-associated disease in a patient, comprising administering to the patient a therapeutically effective amount of an antibody of the first aspect of the invention or a pharmaceutical composition of the sixth aspect of the invention.

[0062] Preferably, the BTLA-associated disease is an autoimmune disease or an inflammatory disease. MODE FOR CARRYING OUT THE INVENTION

[0063] The present inventors have identified particularly potent agonistic antibodies against BTLA that are more effective than current antibodies in suppressing T cell responses and are therefore predicted to be particularly useful in the treatment of immune-mediated disorders. Such antibodies contain at least one substitution in the Fc portion of the molecule that selectively enhances binding to FcγR2B compared to the parent polypeptide. Preferably, the antibodies contain a substitution at one or more of the following positions (EU index positions): 234, 235, 236, 237, 238, 265, 267, 271, 297, 330, and 322. Suitably, the antibody is a human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A (all using EU index numbering).

[0064] Alterations at one or more of the following positions are particularly preferred: 236, 237, 238, and 267 (EU index).

[0065] Combinations of Fc modifications are also suitable. In a specific embodiment, a set of modifications designated V9 is used, in which the antibody heavy chain comprises an Fc region comprising an aspartic acid at position 237, an aspartic acid at position 238, a glycine at position 271, and an arginine at position 330 (EU index numbering).

[0066] By introducing the P238D (EU index) substitution into the Fc portion of the molecule (i.e., antibody or antigen-binding fragment thereof), the molecule enhanced binding to and FcγR2B signaling at a level other than that which ensured sufficient in vivo half-life.

[0067] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally refers to two or more Combinations of the above molecules, etc.

[0068] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided. will be done.

[0069] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect unless the content clearly dictates otherwise. Moreover, various embodiments may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0071] The term "about" as used herein refers to a normal error range for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments relating to that value or parameter itself.

[0072] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes.

[0073] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The amino-terminal portion of each chain defines a variable region responsible for antigen binding. Kabat et al. (NIH Publication No. 91 / 3242, pp. 679-687; 1991) collected a large number of primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, they classified each primary sequence into CDRs and frameworks and compiled a list of these (see Kabat et al., SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, 1991).

[0074] Identified CDRs of antibodies, unless otherwise indicated, follow the definition of Kabat as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Amino acid numbering in variable domains is ordinal based on the sequences provided in the sequence listing.

[0075] C L, C H 1. C H 2, and C H The numbering of amino acids in constant domains such as 3 follows the EU index numbering system (referred to herein as the "EU index") disclosed in Kabat et al. (NIH Pub. No. 91 / 3242, pp. 679-687; 1991) unless otherwise indicated. For example, the EU index is used to locate substitutions in the Fc region of an antibody / antigen-binding fragment thereof (e.g., a glycine (G) to aspartic acid (D) substitution at position 236 (identified as G236D) or a proline (P) to aspartic acid (D) substitution at position 238 (identified as P238D)). Those skilled in the art of antibodies will understand that this numbering convention consists of non-contiguous numbering in certain regions of an immunoglobulin sequence, allowing for normalized reference to conserved positions in the immunoglobulin family. Thus, the positions of any given immunoglobulin defined by the EU index do not necessarily correspond to its contiguous sequence.

[0076] The terms "B and T lymphocyte attenuator" and "BTLA" are used interchangeably and, unless the context dictates otherwise, are used with reference to either the protein or the gene (or other nucleic acid encoding all or a portion of BTLA). Human BTLA sequences encompass all human isotypes and variant forms. A representative example of full-length human BTLA is disclosed in Genbank under accession number AJ717664.1. Another representative polypeptide sequence of human BTLA is disclosed in SEQ ID NO:225, which differs from the sequence of AJ717664.1 by only two naturally occurring variant single nucleotide polymorphisms. Despite allelic diversity, human BTLA polypeptide sequences typically have at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the human BTLA of SEQ ID NO:225.

[0077] A representative example of full-length cynomolgus (cyno) BTLA is disclosed in Genbank under accession number XP_005548224. A reference polypeptide sequence for cynomolgus BTLA is disclosed in SEQ ID NO: 226. Cynomolgus BTLA polypeptide sequences typically have at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the cynomolgus BTLA disclosed in SEQ ID NO: 226.

[0078] The term sequence identity is well known in the art. For the purposes of the present invention, when determining whether a target sequence meets a specified limit (e.g., 90% identity), it is considered to meet the specified limit if it is identified as such using the BLAST (Basic local alignment search tool) algorithm (see Altschul et al. J Mol Biol 215:403-410, 1990) or the Smith-Waterman algorithm (see Smith and Waterman. J Mol. Biol. 147:195-197, 1981).

[0079] Antibodies (including antigen-binding fragments of antibodies) An antibody is an immunoglobulin molecule capable of specifically binding to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. In particular, as used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies generated from at least two intact antibodies), chimeric antibodies, humanized antibodies, human antibodies, any other modified immunoglobulin molecule, and any fragment thereof that contains an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies can be derived from any species. Preferably, the antibody is a human antibody.

[0080] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen and contains an FcR binding site, which may or may not be functional. The term encompasses whole antibodies (e.g., IgG1, IgG4, etc.) and antigen-binding fragments thereof.

[0081] As used herein, a BTLA agonist antibody refers to an antibody (including an antigen-binding fragment of an intact antibody) that binds to BTLA and enhances its co-inhibitory signal on T cells and / or B cells.

[0082] An antigen-binding site refers to the part of a molecule that binds to all or part of a target antigen. In an antibody molecule, it may be called the antigen-binding site of the antibody, and includes the part of the antibody that specifically binds to all or part of the target antigen. If the antigen is large, the antibody can only bind to a specific part of the antigen, which part is called an epitope. The antigen-binding site of an antibody can be provided by one or more antibody variable domains. Preferably, the antigen-binding site of an antibody includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0083] The present invention also encompasses antibody fragments that contain an antigen-binding site. Thus, the term "antigen-binding fragment thereof," when referring to an antibody, refers to antibody fragments such as Fab, Fab', F(ab')2, diabody, Fv fragments, and single-chain Fv (scFv) variants that have an antigen recognition site (and thus the ability to bind to an antigen). Antigen-binding immunoglobulin (antibody) fragments are well known in the art. Such fragments do not necessarily have a functional Fc receptor binding site. In certain embodiments, the antigen-binding fragment comprises an Fc portion having a substitution selected from one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index). In certain embodiments, the antigen-binding fragment comprises an Fc portion having a G236D (EU index) substitution. In certain embodiments, the antigen-binding fragment comprises an Fc portion having a P238D (EU index) substitution. In a specific embodiment, the antigen-binding fragment comprises an Fc portion having an aspartic acid at position 237 (EU index), an aspartic acid at position 238 (EU index), a glycine at position 271 (EU index), and an arginine at position 330 (EU index).

[0084] As used herein, the terms "antibody fragment molecule of the invention," "antibody fragment," and "antigen-binding fragment thereof" are used interchangeably herein. Collectively, antibodies or antigen-binding fragments thereof may be referred to as antigen-binding molecules.

[0085] As used herein, the term "BTLA-binding molecule" refers to both antibodies and binding fragments thereof that are capable of binding to BTLA.

[0086] There are five major classes of immunoglobulins (i.e., isotypes): IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Unless context dictates otherwise, antibodies of the present invention may be derived from one of these classes or subclasses of antibodies. The heavy-chain constant domains corresponding to the different classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0087] "Native antibodies" are typically heterotetrameric, Y-shaped glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD antibodies is C H 1. C H 2, and C H 3 (IgM and IgE contain a fourth domain, C H4). In the IgG, IgA, and IgD classes, C H 1 and C H The two domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length (approximately 10 to 60 amino acids in various IgG subclasses). The variable domains in both the light and heavy chains are linked to the constant domain by a "J" region of approximately 12 or more amino acids, with the heavy chain also having a "D" region of approximately 10 additional amino acids. Each class of antibody further contains inter- and intrachain disulfide bonds formed by paired cysteine ​​residues. The heavy-chain variable region (VH) and light-chain variable region (VL) can each be further subdivided into hypervariable regions called CDRs, interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0088] The antibodies of the present invention may be derived from any animal species, including mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is monoclonal, e.g., a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody, or an antigen-binding fragment thereof. A non-human antibody or antigen-binding fragment thereof may be humanized by recombinant methods to reduce its immunogenicity in humans.

[0089] As used herein, the term "monoclonal antibody" ("mAb") refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible minor mutations (e.g., naturally occurring mutations). Thus, the modifier "monoclonal" indicates the character of the antibody or fragment thereof as not being a mixture of distinct antibodies or antigen-binding fragments. mAbs are typically highly specific, directed against a single antigenic site / epitope, although monoclonal antibody can also refer to a substantially homogeneous population of bispecific antibody molecules.

[0090] mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art. For example, the monoclonal antibodies or antigen-binding fragments thereof of the invention may be made by the hybridoma method first described by Kohler and Milstein (Nature 256:495, 1975), or may be made by recombinant DNA methods described, for example, in U.S. Pat. Nos. 4,816,567 and 6,331,415. "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 1991;352:624-628 and Marks et al., J. Mol. Biol. 1991;222:581-597.

[0091] The term monoclonal can also be applied to antigen-binding fragments of antibodies of the present invention, which simply means that the molecule is produced or exists in a single clonal form.

[0092] A "human" antibody (HumAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0093] Human antibodies can be prepared by administering an immunogen / antigen to a transgenic animal (e.g., an immunized xenogeneic mouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology) that has been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge, but whose endogenous gene loci have been disabled. See, e.g., Li et al. See, e.g., K. et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) (regarding human antibodies produced via human B cell hybridoma technology). Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584, describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, describing HUMAB® technology; and K.M. See also U.S. Patent No. 7,041,870, which describes MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0094] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26:265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20:927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27:185-91 (2005).

[0095] The terms "human" antibody and "fully human" antibody are used interchangeably. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0096] As used herein, a "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDRs of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In some embodiments, a humanized antibody comprises a human immunoglobulin (recipient antibody) in which residues from the recipient's CDRs have been replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capacity. A humanized antibody can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In one embodiment of a humanized form of an Ab, some, most, or all amino acids outside the CDRs have been replaced with amino acids from a human immunoglobulin, while some, most, or all amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abolish the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions are framework regions from a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409. Preferably, the Fc will contain a P238D substitution mutation (using "EU index" numbering) to enhance specificity for binding to FcγR2B.

[0097] As used herein, Fc, Fc portion, or Fc region refers to the constant region of an antibody or antibody-like molecule, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In the case of IgG, Fc includes immunoglobulin domains C gamma 2 and C gamma 3 (C gamma 2 and C gamma 3), as well as the hinge between C gamma 1 (C gamma 1) and C gamma 2. In the case of IgA and IgM, Fc may include the J chain.

[0098] As used herein, an "engineered antibody" refers to an antibody, which may be a humanized antibody, in which certain residues have been substituted for other residues to reduce adverse effects or properties. Such substitutions may be within the CD domain. For example, as described herein (see Example 21), the CDRH2 of humanized antibody 3E8 was modified with an N57Q substitution to eliminate the possibility of deamidation and / or a K63S substitution to reduce predicted immunogenicity. Numbering in this case is ordinal, referring to the sequence identifiers provided.

[0099] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, e.g., the variable region is derived from a mouse antibody and the constant region is derived from a human antibody, or vice versa. The term also encompasses antibodies comprising V regions from one individual from one species (e.g., a first mouse) and a constant region from another individual from the same species (e.g., a second mouse). The term "antigen (Ag)" refers to a molecular entity used to immunize an immunocompetent vertebrate to produce antibodies (Abs) that recognize the Ag or to screen an expression library (e.g., phage, yeast, or ribosome display libraries, among others). As used herein, Ag is referred to more broadly and is generally intended to include the target molecule specifically recognized by an Ab, and thus includes portions or mimetics of molecules used in the immunization process to generate Abs or in library screening to select Abs.

[0100] A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Methods for producing bispecific antibodies are within the knowledge of those skilled in the art. For example, bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79:315-321; Kostelny, et al., (1992) J. Immunol. 148:1547-1553. Additionally, bispecific antibodies may be used as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al. (1992) Int. J. Cancer Suppl. 7:51-52). Full-length bispecific antibodies can be generated in vitro in a cell-free environment or using co-expression, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, by introducing substitutions at the heavy chain CH3 interface of each half molecule to favor heterodimer formation of two antibody half molecules with distinct specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domains of the parent antibody are released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a distinct epitope. A "knob-in-hole" strategy (see, e.g., PCT Publication WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain of human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to one antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After coexpression of the two antibodies, heterodimers form as a result of preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob."Exemplary CH3 substitution pairs that form knobs and holes are (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0101] Bispecific antibodies can also be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization according to the method described in International Patent Publication No. 2011 / 131746. Another strategy for generating bispecific antibodies can be used, which involves promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface (as described in U.S. Patent Publication No. US2010 / 0015133, U.S. Patent Publication No. US2009 / 0182127, U.S. Patent Publication No. US2010 / 028637, or U.S. Patent Publication No. US2011 / 0123532).

[0102] Preferably, one of the two antibody half molecules of the bispecific molecule is an anti-BTLA antibody of the invention. Preferably, the bispecific antibody comprises one binding arm comprising a BTLA antigen-binding region disclosed herein and a second binding arm comprising a binding region for another antigen (e.g., for a different BTLA antigen epitope or an entirely different protein), and the molecule comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index).

[0103] Generally, the term "epitope" refers to the area or region of an antigen to which an antibody specifically binds (i.e., the area or region that makes physical contact with the antibody). Thus, the term "epitope" refers to the portion of a molecule that can be recognized and bound by an antibody at one or more of the antibody's antigen-binding regions. Typically, an epitope is defined in terms of the molecular interactions between an antibody or its antigen-binding portion (Ab) and its corresponding antigen. Epitopes often consist of surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, an epitope may be a protein epitope. Protein epitopes may be linear or conformational. A linear epitope refers to an epitope in which all of the points of interaction between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or sugars within an antigenic protein to which an epitope-specific antibody binds. As used herein, the term "antigenic epitope" is defined as a portion of an antigen to which an antibody can specifically bind, as determined by any method well known in the art (e.g., conventional immunoassay).

[0104] The term "specifically binds" to an epitope is well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell, protein, or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than with alternative cells, proteins, or substances.

[0105] A variety of assay formats can be used to select antibodies or peptides that specifically bind to a molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA), and Western blot analysis are among the many assays that can be used to identify antibodies or peptides that specifically bind to an antigen or receptor, or a ligand-binding portion thereof that specifically binds to a cognate ligand or binding partner. Typically, a specific or selective response will be at least twice the background signal or noise, more typically greater than 10 times the background, even more typically greater than 50 times the background, more typically greater than 100 times the background, even more typically greater than 500 times the background, even more typically greater than 1000 times the background, and even more typically greater than 10,000 times the background. Antibodies also have an equilibrium dissociation constant (K D or KD, used interchangeably herein) is less than 7 nM.

[0106] In some embodiments, the present disclosure provides a chimeric antigen receptor, comprising an antigen-binding fragment of a BTLA-binding antibody disclosed herein, a transmembrane domain, and an intracellular signaling domain. As used herein, a "chimeric antigen receptor" (CA) is a chimeric antigen receptor (CAR). The terms "artificial T cell receptor," "chimeric T cell receptor," or "chimeric immune receptor" refer to engineered receptors that transfer any specificity onto immune effector cells. CARs typically have an extracellular domain (ectodomain) containing an antigen-binding domain, a transmembrane domain, and an intracellular domain (endodomain). The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information intracellularly to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector in response to such messengers.

[0107] Fc gamma engineered In humans, the FcγR protein family includes FcγR1A (CD64A), FcγR2A (CD32A), FcγR2B (CD32B), FcγR3A (CD16A), and FcγR3B (CD16B) isoforms, and distinct allotypes of these receptors have also been reported (Jefferis and Lund, Immunology Letters 82(1-2):57-65, 2002). FcγR1A, FcγR2A, and FcγR3A are called activating FcγRs because of their immunologically active functions, whereas FcγR2B is called inhibitory FcγR because of its immunosuppressive functions (Smith and Clatworthy, Nat Rev Immunol, 10(5), 328-343, 2010). In the literature and herein, FcγR1A may also be referred to as FcγR1.

[0108] When activating FcγRs are triggered by antibody binding to the Fc region, they are activated by phosphorylation of immunoreceptor tyrosine-based activating motifs (ITAMs) contained in the intracellular domain or the FcR common γ chain (the interaction partner). This leads to the induction of inflammatory immune responses by initiating the activation of signaling cascades (Nimmerjahn and Ravetch, Nat Rev Immunol 8(1):34-47, 2008). When the inhibitory receptor FcγR2B is induced by binding to the Fc region of an antibody, it is activated by phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail. This leads to the activation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP1), which then suppresses the transmission of other activating signaling cascades and thus the inflammatory immune response (Ravetch and Lanier, Science 290(5489):84-89, 2000).

[0109] FcγR2B is the only FcγR expressed on B cells (Amigorena et al. European Journal of Immunology 19(8):1379-85, 1989). It has been reported that the interaction of the Fc region of an antibody with FcγR2B inhibits signal transduction via the B cell receptor, suppressing B cell proliferation and antibody production (Nimmerjahn and Ravetch, Advances in immunology 96:179-204, 2007). In cell types that express both activating and inhibitory FcγRs (e.g., macrophages, DCs, neutrophils, mast cells, and basophils), the signal transduction threshold and outcome of FcγR binding are determined by the balance of activation of activating and inhibitory FcγRs (Nimmerjahn et al. European Journal of Immunology 19(8):1379-85, 1989). and Ravetch, Science 310(5753):1510-12, 2005).

[0110] The important regulatory role of FcγR2B has been demonstrated through studies of FcγR2B knockout mice, which show increased susceptibility to autoimmune diseases (Nakamura et al. Journal of Experimental Medicine 191(5):899-906, 2000). Furthermore, polymorphisms in the FcγR2B gene in humans are associated with the risk of autoimmune diseases, particularly systemic lupus erythematosus (Floto et al. Nature Medicine 11(10), 2005). Therefore, FcγR2B is thought to play an important role in regulating immune responses and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0111] IgG1 and IgG4 are the antibody isotypes most commonly used in commercially available antibody pharmaceuticals, and are known to strongly bind not only to FcγR2B but also to activating FcγRs (Bruhns et al. Blood 113(16):3716-25, 2009). By utilizing an Fc region with enhanced FcγR2B binding or improved FcγR2B binding selectivity compared to activating FcγRs, it may be possible to develop antibody pharmaceuticals with greater immunosuppressive properties than those of IgG1 or IgG4.

[0112] Antibodies with Fc having improved FcγR2B binding activity have been reported (Chu et al. (Molecular Immunology 45(15):3926-33, 2008). In this paper, FcγR2B binding activity was improved by introducing alterations such as S267E / L328F, G236D / S267E, and S239D / S267E into the antibody Fc region. Among these, antibodies with the S267E / L328F mutation introduced exhibited the strongest binding to FcγR2B, maintaining the same level of binding to FcγR1A and FcγR2A (131H allotype) as naturally occurring IgG1. However, another report showed that this alteration enhanced binding to FcγR2A 131R by several hundred times, to the same level as FcγR2B binding, suggesting that FcγR2B binding selectivity was enhanced by the FcγR2A allotype. This means that there is no improvement over 131R (U.S. Patent Publication No. 2009 / 0136485). In addition to its pro-inflammatory effects, antibody binding to FcγR2A can lead to platelet activation resulting in thromboembolic events, as seen with the therapeutic antibody bevacizumab (Meyer et al. Journal of Thrombosis and Hemostasis 7(1):171-81, 2009; Scappaticci et al. Journal of the National Cancer Institute 99(16):1232-39, 2007) and antibodies targeting CD40 ligand (Boumpas et al. Arthritis and rheumatism 48(3):719-27, 2003; Robles-Carrillo et al. Journal of Immunology (Baltimore, Md.: 1950) 185(3):1577-83, 2010). Furthermore, antibodies with enhanced FcγR2A binding are associated with macrophage-mediated antibody-dependent cellular phagocytosis (ADC). It has been reported that FcγR2B enhances immunosuppressive therapy (ADCP) (Richards et al., Molecular Cancer Therapeutics 7(8):2517-27, 2008). When an antibody's antigen is phagocytosed by a macrophage, the antibody itself is also phagocytosed. In this case, peptide fragments derived from the antibody are also presented as antigens, increasing their antigenicity and potentially increasing the risk of producing antibodies against the antibody (anti-drug antibodies). More specifically, enhanced FcγR2A binding increases the risk of producing antibodies against the antibody, significantly reducing their value as a pharmaceutical. Therefore, antibodies with selective binding to FcγR2B and reduced binding to FcγR2A may be more effective immunosuppressants and may also be better tolerated therapeutic agents with a lower risk of inducing thromboembolic events and lower immunogenicity.

[0113] Therefore, for BTLA agonistic antibodies to be effective in suppressing immune responses without inducing inflammatory FcR signaling, we propose to adapt the antibodies for selective Fc binding to FcγR2B.

[0114] Molecules with more selective binding to FcγR2B would promote bidirectional inhibitory signaling through BTLA on BTLA-expressing cells and through FcγR2B on FcγR2B-expressing cells, enhancing the immunosuppressive effects of the antibody, which is desirable in therapeutic antibodies intended to treat diseases of immune hyperactivation.

[0115] However, very high affinity for FcγR2B can adversely affect antibody half-life due to receptor turnover in liver sinusoidal epithelial cells (Ganesan et al. The Journal of Immunology 189(10):4981-88, 2012). This is demonstrated by the FcγR2B-enhancing IgG1 antibody XmAb7195, which binds to FcγR2B with a KD of 7.74 nM (Chu et al. Journal of Allergy and Clinical Immunology 129(4):1102-15, 2012, https: / / linkinghub.elsevier.com / retrieve / pii / S0091674911018343 (May 13, 2020)). According to Xencor, the average half-life of wild-type IgG1 is approximately 21 days (Morell, Terry, and Waldmann. Journal of Clinical Investigation 49(4):673-80, 1970; http: / / www.jci.org / articles / view / 106279(May 16, 2020)), and a Phase 1a trial reported a mean in vivo half-life of 3.9 days (American Thoracic Society (ATS) 2016 International Conference in San Francisco, CA-A6476: Poster Board Number 407). Thus, in the context of the present invention, sufficient binding to support selectivity and agonism for FcγR2B may be desirable for a BTLA agonist antibody, but excessively high affinity for FcγR2B may be undesirable in therapy, as the resulting shortened half-life is likely to require more frequent dosing.

[0116] Various mutations, including amino acid substitutions, can be incorporated into the heavy chain constant region of an antibody to alter signaling through one or more Fcγ receptors. WO 2006 / 019447 (Xencor) discloses various Fc variant molecules (e.g., antibodies) with altered effector function via amino acid substitutions in the Fc region.

[0117] The inventors have found that incorporation of the P238D substitution mutation into the BTLA agonist antibodies of the invention enhances selectivity for binding to and signaling through FcγR2B without significantly reducing the in vivo half-life of the antibody.

[0118] Although the Fc portion can accommodate other modifications (such as amino acid substitutions), in certain embodiments, the P238D modification is the only modification introduced into the BTLA binding molecules of the present invention compared to the wild-type Ig Fc sequence.

[0119] In one embodiment, the antibody comprises an aspartic acid at a position corresponding to position 238 of IgG1 (using the EU index). Suitably, the antibody of the invention comprises an hIgG1 constant region disclosed in SEQ ID NO: 227 or an hIgG1 constant region with up to five amino acid alterations so long as the P238D substitution is present.

[0120] In one embodiment, the antibody comprises an aspartic acid at a position corresponding to IgG4 position 238 (using the EU index). Suitably, the antibody of the invention comprises an hIgG4 constant region as disclosed in SEQ ID NO: 235, or an hIgG4 constant region with up to five amino acid alterations so long as the P238D substitution is present.

[0121] The antibodies of the present invention promote bidirectional inhibitory signaling via BTLA on BTLA-expressing cells and FcγR2B on FcγR2B-expressing cells and have an in vivo half-life sufficient for appropriate therapeutic use. Preferably, the in vivo half-life is at least 5 days in the human body (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, or more). In specific embodiments, the in vivo half-life in humans is at least 10 days, allowing for a suitable dosing regimen (e.g., every 3 weeks). Preferably, the in vivo half-life is about 10-30 days (e.g., about 12-20 days or 14-25 days).

[0122] In certain embodiments of the invention, the antibodies of the invention exhibit an in vivo half-life within ±3 days of the half-life of a control antibody comprising a wild-type Fc region, the control antibody having the same heavy and light chains except for Fc modifications that increase binding to FcγR2B as described herein.

[0123] In certain embodiments of the invention, antibodies of the invention exhibit an in vivo half-life that retains at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%) of the half-life of a reference antibody comprising a wild-type Fc region, the reference antibody being an antibody having the same heavy and light chains except for Fc modifications that increase binding to FcγR2B as described herein.

[0124] When the Fc modification that increases binding to FcγR2B is a P238D substitution, in certain embodiments, the antibodies of the invention exhibit an in vivo half-life within ±3 days of the half-life of a control antibody comprising an Fc region containing a proline at position 238 (EU index).

[0125] In another specific embodiment, when the Fc modification that increases binding to FcγR2B is a P238D substitution, the antibody of the invention exhibits an in vivo half-life that retains at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%) of the half-life of a parent antibody comprising an Fc region comprising a proline at position 238 (EU index).

[0126] The longer the half-life, the longer the period over which good receptor occupancy is achieved. This therefore means that a longer half-life allows for lower doses to be administered, either with longer intervals between doses or instead of longer intervals between doses, which can be important if there is dose-limiting toxicity at higher peak doses.

[0127] Producing antibodies with long half-lives may also have benefits such as reduced cost of goods, reduced burden on the patient, and increased patient compliance.

[0128] Suitably, the molecules of the invention are capable of greater than 80% receptor occupancy for at least 10 days (eg, 14, 21, 28, 35, 42 days, or more) following a single dose of 10 mg / kg.

[0129] Suitably, the molecules of the invention may be administered at 3 week, ideally 4 week or longer, dosing intervals (eg, every 6 or 8 weeks).

[0130] According to a first aspect of the present invention, there is provided an antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising a substitution that results in increased binding to FcγR2B compared to the parent molecule lacking the substitution. Preferably, the antibody is an isolated antibody.

[0131] In some embodiments, the binding to FcγR2B is increased compared to the parent polypeptide such that the value of [KD value of parent polypeptide for FcγR2B] / [KD value of variant polypeptide for FcγR2B] is greater than 1 (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100).

[0132] In some embodiments, the antibody has a preference for binding to FcγR2B over FcγR2A.

[0133] In some embodiments, the antibody has enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR2A (R type) and / or FcγR2A (H type) compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR2A (R type)] / [KD value of the variant polypeptide for FcγR2B] is 2 or greater (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the value of [KD value of variant polypeptide for FcγR2A (H type)] / [KD value of variant polypeptide for FcγR2B] is 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or more).

[0134] In some embodiments, the antibody has enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR1A compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR1A] / [KD value of the variant polypeptide for FcγR2B] is 0.05 or greater (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, or more).

[0135] In some embodiments, the variant polypeptide has reduced Fcγ1 binding activity compared to the parent polypeptide. In some embodiments, the value of [KD value of the variant polypeptide for FcγR1A] / [KD value of the parent polypeptide for FcγR1A] is at least 10, 20, 50, 100, or 200.

[0136] In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0137] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index). Preferably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0138] Preferably, the antibody is a human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A. In certain embodiments, the antibody that binds to human BTLA has a heavy chain and / or light chain with at least one CDR from an antibody selected from the group consisting of 6.2, 2.8.6, 3E8, 11.5.1, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, and 831, as disclosed in Table 1 or Table 2 and described herein. In one embodiment, the antibody competes with its natural ligand, HVEM, for binding to BTLA. In another embodiment, the antibody does not interfere with HVEM binding.

[0139] In a specific embodiment, the isolated antibody that binds to human BTLA is selected from the group consisting of 6.2, 2.8.6, 3E8, or an antibody that competes with any one of 6.2, 2.8.6, or 3E8 for binding to human BTLA, wherein the antibody specifically binds to BTLA and induces signaling through the receptor. The antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0140] An antibody selected from the group consisting of 6.2, 2.8.6, 3E8, 11.5.1, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, and 831 disclosed in Table 1 and described herein refers to an antibody selected from the group consisting of VH CDR1, 2, and 3, or VL CDR1, 2, and 3, or VH CDR1, 2, and 3 and VL CDR1, 2, and 3 from any of the antibodies disclosed in Table 1 or Table 2 (whether murine, humanized, or humanized / engineered). It refers to any antibody or antigen-binding fragment thereof comprising one or more of CDR1, 2, and 3, etc.

[0141] According to a variation of the first aspect of the invention, an isolated antibody that specifically binds to human BTLA is provided, comprising at least one VH CDR having an amino acid sequence set forth in any of SEQ ID NOs: 1, 2, 3, 11, or 17, with zero to three amino acid modifications (e.g., zero, one, two, or three amino acid modifications). In certain embodiments, the amino acid modifications include, but are not limited to, amino acid substitutions, additions, deletions, or chemical modifications that do not eliminate the antibody binding affinity or T cell inhibitory effect of the modified amino acid sequence compared to the unmodified amino acid sequence.

[0142] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO:1, CDRH2 has the amino acid sequence set forth in SEQ ID NO:2, 11, or 17, and CDRH3 has the amino acid sequence set forth in SEQ ID NO:3.

[0143] According to a variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 4, 5, 6, or 12, with 0 to 3 amino acid alterations.

[0144] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5 or 12, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6.

[0145] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, 11, or 17, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3; and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 5 or 12, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 6.

[0146] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 17, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3; the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 6; and the heavy chain comprises an aspartic acid at position 238 (EU index).

[0147] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 20, 21, or 22, with 0 to 3 amino acid alterations.

[0148] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO:20, CDRH2 has the amino acid sequence set forth in SEQ ID NO:21, and CDRH3 has the amino acid sequence set forth in SEQ ID NO:22.

[0149] According to a variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 23, 24, or 25, with 0 to 3 amino acid alterations.

[0150] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO:23, CDRL2 has the amino acid sequence set forth in SEQ ID NO:24, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:25.

[0151] According to another variant of the twentieth aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO:20, CDRH2 has the amino acid sequence set forth in SEQ ID NO:21, and CDRH3 has the amino acid sequence set forth in SEQ ID NO:22; and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO:23, CDRL2 has the amino acid sequence set forth in SEQ ID NO:24, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:25.

[0152] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 30, 31, 40, 48, or 32, with 0 to 3 amino acid modifications.

[0153] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 30, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 31, 40, or 48, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 32.

[0154] According to a variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 33, 34, or 35, with 0 to 3 amino acid alterations.

[0155] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 33, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 34, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 35.

[0156] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO:30, CDRH2 has the amino acid sequence set forth in SEQ ID NO:31, 40, or 48, and CDRH3 has the amino acid sequence set forth in SEQ ID NO:32; and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO:33, CDRL2 has the amino acid sequence set forth in SEQ ID NO:34, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:35.

[0157] In each of these aspects, the antibody has an Fc region comprising at least one amino acid substitution that results in increased binding to FcγR2B relative to the parent molecule lacking the substitution. In some embodiments, the antibody has preferential binding to FcγR2B over FcγR2A relative to the parent molecule lacking the substitution. In some embodiments, the antibody has preferential binding to FcγR2B over FcγR1A relative to the parent molecule lacking the substitution.

[0158] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0159] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 237 (EU index), an aspartic acid at position 238 (EU index), a glycine at position 271 (EU index), and an arginine at position 330 (EU index).

[0160] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region and a heavy chain variable region comprising three complementarity determining regions (CDRs): CDRH1, CDRH2, and CDRH3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (1) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 1, 17, and 3, respectively, and have 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 4, 12, and 6, respectively, and have 0 to 3 amino acid modifications, and (2) the CDRs (3) H1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively, and have 0 to 3 amino acid alterations; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and have 0 to 3 amino acid alterations; or (4) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and have 0 to 3 amino acid alterations; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and have 0 to 3 amino acid alterations; and the Fc region portion comprises an aspartic acid at position 238 (EU index).

[0161] A typical antibody comprises two heavy chains and two light chains, and the paired heavy chains comprise an Fc region; therefore, as used herein, a "heavy chain comprising an Fc region" refers to a region on an H chain polypeptide that, together with the Fc region of another H chain, forms a functional Fc region.

[0162] According to another variation of the first aspect of the present invention, an isolated antibody that specifically binds to human BTLA is provided, comprising at least one VH CDR having the amino acid sequence set forth below: (1) SEQ ID NO: 45, 46, or 47 with 0 to 3 amino acid alterations; (2) SEQ ID NO: 53, 54, or 55 with 0 to 3 amino acid alterations; (3) SEQ ID NO: 61, 62, or 63 with 0 to 3 amino acid alterations; (4) SEQ ID NO: 61, 69, or 70 with 0 to 3 amino acid alterations; (5) SEQ ID NO: 76, 77, or 78 with 0 to 3 amino acid alterations; (6) SEQ ID NO: 45, 46, or 84 with 0 to 3 amino acid alterations; (7) SEQ ID NO: 53, 54, or 55 with 0 to 3 amino acid alterations; (8) SEQ ID NO: 95, 96, or 97 having 0 to 3 amino acid alterations; (9) SEQ ID NO: 103, 104, or 105 having 0 to 3 amino acid alterations; (10) SEQ ID NO: 76, 111, or 112 having 0 to 3 amino acid alterations; (11) SEQ ID NO: 118, 119, or 120 having 0 to 3 amino acid alterations; (12) SEQ ID NO: 126, 127, or 128 having 0 to 3 amino acid alterations; (13) SEQ ID NO: 130, 131, or 132 having 0 to 3 amino acid alterations; (14) SEQ ID NO: 103, 134, or 139 having 0 to 3 amino acid alterations; (15) SEQ ID NO: 143, 144, or 145 having 0 to 3 amino acid alterations; (16) SEQ ID NO: 151, 152, or 153 having 0 to 3 amino acid alterations; (17) SEQ ID NO: 159, 160, or 161 having 0 to 3 amino acid alterations; (18) SEQ ID NO: 167, 168, or 169 having 0 to 3 amino acid alterations. 9, (19) SEQ ID NO: 45, 46, or 177 having 0 to 3 amino acid alterations, (20) SEQ ID NO: 181, 182, or 183 having 0 to 3 amino acid alterations, (21) SEQ ID NO: 45, 191, or 192 having 0 to 3 amino acid alterations, (22) SEQ ID NO: 196, 197, or 198 having 0 to 3 amino acid alterations, (23) SEQ ID NO: 204, 205, or 206 having 0 to 3 amino acid alterations, (24) SEQ ID NO: 212, 213 having 0 to 3 amino acid alterations,or 214, (25) SEQ ID NO: 1, 2, or 3 having 0 to 3 amino acid alterations, (26) SEQ ID NO: 20, 163, or 22 having 0 to 3 amino acid alterations, (27) SEQ ID NO: 30, 48, or 32 having 0 to 3 amino acid alterations, (28) SEQ ID NO: 1, 11, or 3 having 0 to 3 amino acid alterations, (29) SEQ ID NO: 1, 17, or 3 having 0 to 3 amino acid alterations, (30) SEQ ID NO: 20, 21, or 22 having 0 to 3 amino acid alterations, (33) SEQ ID NO: 30, 31, or 32 having 0 to 3 amino acid alterations, or (34) SEQ ID NO: 30, 40, or 32 having 0 to 3 amino acid alterations. The antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0163] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, and CDRH1, CDRH2, and CDRH3 are: (1) SEQ ID NOs: 45, 46, and 47, respectively; (2) SEQ ID NOs: 53, 54, and 55, respectively; (3) SEQ ID NOs: 61, 62, and 63, respectively; (4) SEQ ID NOs: 61, 69, and 70, respectively; (5) SEQ ID NOs: 76, 77, and 78, respectively; and (6) SEQ ID NOs: 80, 81, and 82, respectively. , SEQ ID NOs: 45, 46, and 84, (7) SEQ ID NOs: 88, 89, and 90, respectively, (8) SEQ ID NOs: 95, 96, and 97, respectively, (9) SEQ ID NOs: 103, 104, and 105, respectively, (10) SEQ ID NOs: 76, 111, and 112, respectively, (11) SEQ ID NOs: 118, 119, and 120, respectively, (12) SEQ ID NOs: 126, 127, and 128, respectively, (13) SEQ ID NOs: 133, 134, and 135, respectively, (14) SEQ ID NOs: 103, 134, and 139, respectively, (15) SEQ ID NOs: 143, 144, and 145, respectively. 4, and 145, (16) SEQ ID NOs: 151, 152, and 153, respectively, (17) SEQ ID NOs: 159, 160, and 161, respectively, (18) SEQ ID NOs: 167, 168, and 169, respectively, (19) SEQ ID NOs: 45, 46, and 177, respectively, (20) SEQ ID NOs: 181, 182, and 183, respectively, (21) SEQ ID NOs: 45, 191, and 192, respectively, (22) SEQ ID NOs: 196, 197, and 198, respectively, (23) SEQ ID NOs: 204, 205, and 206, respectively, (24) SEQ ID NOs: 212 and 213, respectively. , and 214, (25) SEQ ID NOs: 1, 2, and 3, respectively; (26) SEQ ID NOs: 20, 163, and 22, respectively; (27) SEQ ID NOs: 30, 48, and 32, respectively; (28) SEQ ID NOs: 1, 11, and 3, respectively; (29) SEQ ID NOs: 1, 17, and 3, respectively; (30) SEQ ID NOs: 20, 21, and 22, respectively; (33) SEQ ID NOs: 30, 31, and 32, respectively; or (34) SEQ ID NOs: 30, 40, and 32, respectively; and 0 to 3 amino acid alterations may be present in any CDR / SEQ ID NO.The antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0164] According to a variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, the antibody being selected from the group consisting of: (1) SEQ ID NO: 33, 34, or 35; (2) SEQ ID NO: 56, 57, or 58; (3) SEQ ID NO: 64, 65, or 66; (4) SEQ ID NO: 71, 72, or 73; (5) SEQ ID NO: 79, 80, or 81; (6) SEQ ID NO: 33, 34, or 85; (7) SEQ ID NO: 91, 65; or (8) SEQ ID NO: 98, 99, or 100, (9) SEQ ID NO: 106, 107, or 108, (10) SEQ ID NO: 113, 114, or 115, (11) SEQ ID NO: 121, 122, or 123, (12) SEQ ID NO: 79, 129, or 130, (13) SEQ ID NO: 106, 107, or 136, (14) SEQ ID NO: 146, 147, or 148, (15) SEQ ID NO: 154, 155, or 156, (16) SEQ ID NO: 4, 12, or 164, (17) SEQ ID NO: 170, 171, or 172, (18) SEQ ID NO: 154, 155, or 178, (19) SEQ ID NO: 184, 185, or 186, (20) SEQ ID NO: 79, 80, or 189, (21) SEQ ID NO: 154, 155, or 193, (22) SEQ ID NO: 199, 200, or 201, (23) SEQ ID NO: 207, (20) at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 208, or 209, (24) SEQ ID NO: 215, 34, or 216, (25) SEQ ID NO: 4, 5, or 6, (26) SEQ ID NO: 23, 176, or 25, (27) SEQ ID NO: 33, 34, or 35, (28) SEQ ID NO: 4, 12, or 6, (29) SEQ ID NO: 23, 24, or 25, or (30) SEQ ID NO: 33, 34, or 35, wherein 0 to 3 amino acid modifications can be present in any CDR / SEQ ID NO:, and the antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0165] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 are selected from the group consisting of: (1) SEQ ID NOs: 33, 34, and 35, respectively; (2) SEQ ID NOs: 56, 57, and 58, respectively; (3) SEQ ID NOs: 64, 65, and 66, respectively; (4) SEQ ID NOs: 71, 72, and 73, respectively; (5) SEQ ID NOs: 79, 80, and 81, respectively; and (6) SEQ ID NOs: 82, 83, and 84, respectively. (7) SEQ ID NOs: 33, 34, and 85, respectively; (8) SEQ ID NOs: 91, 65, and 92, respectively; (9) SEQ ID NOs: 106, 107, and 108, respectively; (10) SEQ ID NOs: 113, 114, and 115, respectively; (11) SEQ ID NOs: 121, 122, and 123, respectively; (12) SEQ ID NOs: 79, 129, and 130, respectively; (13) SEQ ID NOs: 106, 107, and 136, respectively; (14) SEQ ID NOs: 146, 147, and 148, respectively; (15) SEQ ID NOs: 154 and 155, respectively; and 156, (16) SEQ ID NOs: 4, 12, and 164, respectively; (17) SEQ ID NOs: 170, 171, and 172, respectively; (18) SEQ ID NOs: 154, 155, and 178, respectively; (19) SEQ ID NOs: 184, 185, and 186, respectively; (20) SEQ ID NOs: 79, 80, and 189, respectively; (21) SEQ ID NOs: 154, 155, and 193, respectively; (22) SEQ ID NOs: 199, 200, and 201, respectively; (23) SEQ ID NOs: 207, 208, and 209, respectively; (24) SEQ ID NOs: 215, 34, and 216, respectively; (2 (27) SEQ ID NOs: 33, 34, and 35, respectively; (28) SEQ ID NOs: 4, 5, and 6, respectively; (29) SEQ ID NOs: 4, 12, and 6, respectively; (30) SEQ ID NOs: 23, 24, and 25, respectively; or (31) SEQ ID NOs: 33, 34, and 35, respectively; wherein 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO; and the antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0166] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3; and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (1) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; and (2) CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively. (2) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 56, 57, and 58, respectively; (3) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively; (4) CDRH1, CDRH2 (4) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 61, 69, and 70, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively; (5) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively; and (6) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 84, respectively. (7) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 85, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 91, 65, and 92, respectively; (8) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 95, 96, and 97, respectively, and CDRL1, CDRL2,and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 98, 99, and 100, respectively; (9) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 103, 104, and 105, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 106, 107, and 108, respectively; (10) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 76, 111, and 112, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: (11) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 118, 119, and 120, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 121, 122, and 123, respectively; (12) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 126, 127, and 128, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 79, 129, and 130, respectively. (13) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 133, 134, and 135, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 106, 107, and 136, respectively; (14) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 103, 134, and 139, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 106, 107, and 136, respectively; (15) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 143, 144, and 145, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 146, 147, and 148, respectively; (16) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 151, 152, and 153, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 154, 155, and 156, respectively; (17) CDRH1, CDRH2,(17) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively; CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 4, 12, and 164, respectively; (18) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively; CDRL1, CDRL2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively; (19) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: (20) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively; (21) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 181, 182, and 183, respectively. (21) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 184, 185, and 186, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 189, respectively, and (22) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 189, respectively, and (23) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 45, 191, and 192, respectively, and CDRL1, CDRL2, and and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 154, 155, and 193, respectively; (24) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 196, 197, and 198, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 199, 200, and 201, respectively; (25) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 204, 205, and 206, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs:(26) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 212, 213, and 214, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 215, 34, and 216, respectively; (27) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. (28) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 20, 163, and 22, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 23, 176, and 25, respectively; (29) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs: 30, 48, and 32, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, respectively; (30) CDRH1, CDRH2, CDRH3 (31) CDRH1, CDRH2, CDRH3 have the amino acid sequences shown in SEQ ID NOs: 1, 11, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs: 4, 12, and 6, respectively; (32) CDRH1, CDRH2, CDRH3 have the amino acid sequences shown in SEQ ID NOs: 1, 17, and 3, respectively. wherein CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 4, 12, and 6, respectively; (33) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively; and (34) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively; and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs:(35) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 30, 40, and 32, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO:, and the antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0167] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising: (1) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO:45, 46, or 47, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO:33, 34, or 35; (2) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO:53, 54, or 55, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO:56, 57, and 58; (3) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO:61, 62, or 63, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO:64, 65, or 66; (4) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO:61, 69, or 70, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO:71, 72, and 73; (5) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO:76, 77, or 78. (6) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 45, 46, or 84, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 33, 34, or 85; (7) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 88, 89, or 90, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 91, 65, or 92; (8) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 95, 96, or 97, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 98, 99, or 100; (9) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 103, 104, or 105, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 106, 107, or 108. CDR, (10) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 76, 111, or 112, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 113, 114, or 115; (11) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 118, 119, or 120, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 121, 122, or 123; (12) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 126, 127, or 128, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 79, 129, or 130; (13) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 133, 134, or 135, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 106, 107, or 136. CDR, (14) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 103, 134, or 139, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 106, 107, or 136.CDR, (15) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 143, 144, or 145, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 146, 147, or 148; (16) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 151, 152, or 153, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 154, 155, or 156; (17) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 159, 160, or 161, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 4, 12, or 164; (18) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 167, 168, or 169, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 170, 171, or 172. CDR, (19) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 45, 46, or 47, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 170, 171, or 172; (20) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 45, 46, or 177, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 154, 155, or 178; (21) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 181, 182, or 183, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 184, 185, or 186; (22) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 76, 77, or 78, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 79, 80, or 189. CDR, (23) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 45, 191, or 192, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 154, 155, or 193, (24) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 196, 197, or 198(24) at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 199, 200, or 201; (25) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 204, 205, or 206; (26) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 212, 213, or 214, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 215, 34, or 216; (27) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6; (28) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 20, 163, or 22, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 23, 176, or 25; (29) at least one VH CDR having the amino acid sequence set forth in SEQ ID NO: 30, 48, or 32, and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 33, 34, or 35. CDR, (30) at least one VH CDR having the amino acid sequence shown in SEQ ID NO: 1, 11, or 3, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 4, 12, or 6; (31) at least one VH CDR having the amino acid sequence shown in SEQ ID NO: 1, 11, or 3, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 4, 5, or 6; (32) at least one VH CDR having the amino acid sequence shown in SEQ ID NO: 1, 17, or 3, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 4, 12, or 6; (33) at least one VH CDR having the amino acid sequence shown in SEQ ID NO: 20, 21, or 22, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 23, 24, or 25; (34) at least one VH CDR having the amino acid sequence shown in SEQ ID NO: 30, 31, or 32, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 33, 34, or 35. (35) at least one VH having the amino acid sequence set forth in SEQ ID NO: 30, 40, or 32;and at least one VL CDR having the amino acid sequence set forth in SEQ ID NO: 33, 34, or 35, wherein 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO:, and the antibody also comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0168] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7, 13, or 18, or a sequence having at least 90% sequence identity thereto.

[0169] In other embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, 13, or 18, with up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0170] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8 or 14, or a sequence having at least 90% sequence identity thereto.

[0171] In other embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or 14 with up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0172] In each of these embodiments, the antibody has an Fc region comprising at least one amino acid substitution that results in increased binding to FcγR2B relative to the parent molecule lacking the substitution, hi some embodiments, the antibody has selectivity for binding to FcγR2B over FcγR2A relative to the parent molecule lacking the substitution.

[0173] In a specific embodiment, the antibody comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0174] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, 13, or 18, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8 or 14.

[0175] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 13, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0176] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0177] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14.

[0178] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0179] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14.

[0180] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, 15, or 19, or a sequence having at least 90% sequence identity thereto.

[0181] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, 15, or 19, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0182] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, 16, or 29, or a sequence having at least 90% sequence identity thereto.

[0183] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, 16, or 29, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0184] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:10, or a sequence having at least 90% sequence identity thereto.

[0185] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0186] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90% sequence identity thereto.

[0187] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0188] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence having at least 90% sequence identity thereto.

[0189] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0190] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90% sequence identity thereto.

[0191] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0192] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:26, or a sequence having at least 90% sequence identity thereto.

[0193] In other embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 26 with up to 10 alterations therein (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0194] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27, or a sequence having at least 90% sequence identity thereto.

[0195] In other embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27 with up to 10 alterations therein (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0196] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:26, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:27.

[0197] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:26, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27.

[0198] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28, or a sequence having at least 90% sequence identity thereto.

[0199] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0200] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:29, or a sequence having at least 90% sequence identity thereto.

[0201] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:29 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0202] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:29, or a sequence having at least 90% sequence identity thereto.

[0203] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO:29 or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0204] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 36 or 41, or a sequence having at least 90% sequence identity thereto.

[0205] In other embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 36 or 41 with up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0206] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37 or 43, or a sequence having at least 90% sequence identity thereto.

[0207] In other embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37 or 43 with up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0208] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36 or 41, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37 or 43.

[0209] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:36, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:37.

[0210] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:41, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:37.

[0211] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:36, and the light chain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:43.

[0212] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38 or 42, or a sequence having at least 90% sequence identity thereto.

[0213] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38 or 42, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0214] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39 or 44, or a sequence having at least 90% sequence identity thereto.

[0215] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39 or 44, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0216] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto.

[0217] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0218] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:42, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:39, or a sequence having at least 90% sequence identity thereto.

[0219] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:42, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO:39, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0220] According to another variation of the first aspect of the present invention, there is provided an isolated antibody that specifically binds to human BTLA, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 44, or a sequence having at least 90% sequence identity thereto.

[0221] According to another variation of the first aspect of the invention, there is provided an isolated antibody that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations), and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 44, or a sequence having up to 10 alterations therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations).

[0222] In other embodiments, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:18.

[0223] In other embodiments, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:26.

[0224] In other embodiments, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:36.

[0225] In other embodiments, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:14.

[0226] In other embodiments, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:27.

[0227] In other embodiments, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the sequence disclosed in SEQ ID NO:43.

[0228] According to another variation of the first aspect of the present invention, an isolated antibody is provided, having a primary VH domain and / or a primary VL domain having at least one CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any antibody clone set forth in Table 1 or Table 2. In certain embodiments, provided herein is an isolated antibody selected from the antibody clones set forth in Table 1 or Table 2.

[0229] [Table 1]

[0230] [Table 2]

[0231] In each of the first aspects of the invention, the antibody has an Fc region comprising at least one amino acid substitution that results in increased binding to FcγR2B relative to the parent molecule lacking the substitution, and / or increased selectivity for binding to FcγR2B over FcγR2A relative to the parent molecule lacking the substitution, hi some embodiments, the antibody has increased selectivity for binding to FcγR2B over FcγR1A relative to the parent molecule lacking the substitution.

[0232] In certain embodiments, the antibodies according to the first aspect of the invention (including any variation of the first aspect) each comprise an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index).

[0233] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an aspartic acid at position 238 (EU index).

[0234] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an aspartic acid at position 237 (EU index).

[0235] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an aspartic acid at position 236 (EU index).

[0236] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an alanine at position 235 (EU index).

[0237] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an alanine at position 234 (EU index).

[0238] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an alanine at position 265 (EU index).

[0239] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with a glutamic acid at position 267 (EU index).

[0240] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with a glycine at position 271 (EU index).

[0241] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an alanine at position 297 (EU index).

[0242] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an alanine at position 322 (EU index).

[0243] In certain embodiments, the antibodies according to the first aspect of the invention each comprise an Fc with an arginine at position 330 (EU index).

[0244] In a specific embodiment, each of the antibodies according to the first aspect of the invention comprises an Fc comprising an aspartic acid at position 237 (EU index), an aspartic acid at position 238 (EU index), a glycine at position 271 (EU index), and an arginine at position 330 (EU index).

[0245] In a particular embodiment, the antibody according to the first aspect of the invention comprises an Fc isotype with a substitution selected from the group consisting of: hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, and hIgG4 L235A.

[0246] In certain embodiments, the heavy or light chain also comprises a constant region. If the molecule is a full-length IgG antibody molecule, the heavy chain may comprise three constant domains. In certain embodiments, an isolated antibody that specifically binds to human BTLA has a constant domain size of up to about 10 x 10 for binding to human BTLA. -9 K of M D In certain embodiments, an isolated antibody that specifically binds human BTLA exhibits an antibody activity of up to about 4 x 10 for binding to human BTLA. -9 K of M D In certain embodiments, an isolated antibody that specifically binds human BTLA exhibits an antibody activity of up to about 1 x 10 for binding to human BTLA. -9 K of M D Shows.

[0247] In certain embodiments, an isolated antibody (e.g., a humanized antibody) of the invention binds to human BTLA at 37° C. at a concentration of about 10 nM (1×10 -8 M) K below D and preferably with a Kd of about 1 nM or less, and in a more preferred embodiment, the antibody binds with a Kd of up to about 500 pM (5 x 10) at 37°C. -10 M), 200 pM, 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or even 2 pM or less K D The term "about" as used in this context means + / - 10%.

[0248] In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) has a cytoplasmic affinity of at least 1.0 x 10 at 37°C. 5 (1 / Ms). In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an association rate of at least 2.0 x 10 at 37°C. 5 (1 / Ms), 3.0 × 10 5 (1 / Ms), 4.0 × 10 5 (1 / Ms), 5.0 × 10 5 (1 / Ms), 6.0 × 10 5 (1 / Ms), or 7.0 x 10 5 It binds to human BTLA with an on-rate of (1 / Ms).

[0249] In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) has a cytotoxicity of 1.0 x 10 at 37°C. -3 In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an off rate of 3.0 x 10 at 37°C or less (1 / s). -4 In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an off rate of 2.0 x 10 at 37°C or less (1 / s). -4 (1 / s) or 1.0×10 -4 (1 / s) or less.

[0250] In certain embodiments of the first aspect of the invention, isolated agonist antibodies are provided herein, having a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37° C. using methods such as those described in Example 2. Specifically binds human B and T lymphocyte attenuator (BTLA), and the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. Does not inhibit binding of BTLA to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR), e.g., using a method such as that described in Example 4, and inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay, e.g., using a method such as that described in Example 9. In some embodiments, the antibody has a KD of at least 5.0 x 10, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 (1 / Ms)). In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with an on-rate of 3.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with an off rate of less than 3.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 (1 / s)~1.0×10 -3(1 / s) as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (where numbering, e.g., D52, refers to a position in SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA. In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0251] Methods for characterizing the properties of antibodies of the invention are well known in the art. A suitable method for determining binding specificity using surface plasmon resonance (SPR) at 37°C is described in Example 2. A suitable method for determining whether a tested antibody / fragment thereof inhibits BTLA binding to herpesvirus entry mediator (HVEM) is described in Example 4, which also uses surface plasmon resonance (SPR). A suitable method for determining whether a tested antibody / fragment thereof inhibits T cell proliferation in vitro is a mixed lymphocyte reaction assay, such as that described in Example 9. A suitable method for determining the binding site of an antibody / fragment thereof to BTLA can utilize X-ray crystallography or flow cytometry of mutant receptors, such as by the method described in Example 5.

[0252] In certain embodiments of the first aspect of the invention, the solubility of at least 5.0 x 10 ions is determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5Provided herein are isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with an on-rate of 1 / Ms, wherein the antibody does not inhibit BTLA binding to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and the antibody inhibits T-cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody has an on-rate of 3.0 x 10, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 (1 / s) or less. In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C using methods such as those described in Example 2. In some embodiments, the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C using methods such as those described in Example 2. In some embodiments, the antibody binds to residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 as determined by x-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of BTLA selected from N65 and A64 (position according to SEQ ID NO: 225).

[0253] In certain embodiments of the first aspect of the invention, the concentration of 3.0×10 -4 (1 / s)~1.0×10 -3 Provided herein are isolated agonist antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with an off rate of (1 / s), wherein the antibody does not inhibit binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and the antibody inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody has a denaturing activity of at least 5.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5(1 / Ms). In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0254] In certain embodiments of the first aspect of the invention, 1.0 x 10 -3 (1 / s) and an off-rate of at least 5.0×10 5Provided herein are isolated agonist antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with an on-rate of (1 / Ms), wherein the antibody does not inhibit binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and the antibody inhibits T-cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody binds to a residue in human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue in BTLA selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to a residue in human BTLA selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA. In some embodiments, the antibody binds to a residue in human BTLA selected from N65 and A64.

[0255] In certain embodiments of the first aspect of the invention, provided herein are isolated agonist antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with a KD of less than 2 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, the antibody inhibits BTLA binding to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and the antibody inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody has a KD of less than 1.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 6 In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with an on-rate of less than 1.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -3(1 / s) or less. In some embodiments, the antibody binds to cynomolgus monkey B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using methods such as those described in Example 2. In some embodiments, the antibody binds to residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to residues of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of BTLA (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0256] In certain embodiments of the first aspect of the invention, the concentration of 1×10 -3Provided herein are isolated agonist antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with an on / off rate of less than (1 / s), where the antibody inhibits binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody binds to cynomolgus monkey B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody binds to human B and T lymphocyte attenuator (BTLA) with a KD of less than 2 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0257] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonist antibody that specifically binds to human B and T lymphocyte attenuator (BTLA), wherein the antibody binds to cynomolgus monkey BTLA with a KD of at least 5 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, inhibits binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody binds to residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225) as determined by x-ray crystallography or by flow cytometry of mutant receptors using a method such as that described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0258] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonist antibody that specifically binds to human B and T lymphocyte attenuator (BTLA), wherein the antibody binds to cynomolgus monkey BTLA with a KD of at least 50 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, does not inhibit binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody binds to residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225) as determined by x-ray crystallography or by flow cytometry of mutant receptors using a method such as that described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0259] In certain embodiments of the first aspect of the invention, provided herein are isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with a KD of 1400 nM to 3500 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, wherein the antibody does not inhibit BTLA binding to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody has a KD of at least 2.0 x 10, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 (1 / Ms). In some embodiments, the antibody binds to human BTLA with an on-rate of 10.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s) or less. In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0260] In certain embodiments of the first aspect of the invention, the concentration of 1.7×10 5 (1 / Ms)~2.5×10 5 Provided herein are isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with an on-rate of 1 / Ms, wherein the antibody does not inhibit binding of BTLA to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T-cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody has an on-rate of 3.0 x 10, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s) or less. In some embodiments, the antibody binds to human BTLA with an off rate of less than 3.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s)~5.0×10 -1(1 / s). In some embodiments, the antibody binds to human BTLA with a KD of at least 150 nM, as determined by surface plasmon resonance (SPR) at 37°C using methods such as those described in Example 2. In some embodiments, the antibody binds to human BTLA with a KD of 150 nM to 1500 nM, as determined by surface plasmon resonance (SPR) at 37°C using methods such as those described in Example 2. In some embodiments, the antibody binds to an epitope that blocks binding of the 286 antibody. In some embodiments, the antibody binds to residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by x-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0261] In certain embodiments of the first aspect of the invention, provided herein are isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuator (BTLA) with a KD of 40 nM to 1200 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, and the antibody does not inhibit BTLA binding to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, and inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay using, for example, a method such as that described in Example 9. In some embodiments, the antibody has a KD of at least 1.0 x 10, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 (1 / Ms). In some embodiments, the antibody binds to human BTLA with an on-rate of 1.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. 5 (1 / Ms)~10×10 5 (1 / Ms). In some embodiments, the antibody binds to human BTLA with an on-rate of 6.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. -1 In some embodiments, the antibody binds to human BTLA with an off rate of less than 6.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s)~10.0×10 -2(1 / s). In some embodiments, the antibody binds to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), as determined by X-ray crystallography or by flow cytometry of mutant receptors using methods such as those described in Example 5. In some embodiments, the antibody binds to a residue of human BTLA selected from Y39, K41, R42, Q43, E45, and S47 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from D35, T78, K81, S121, and L123 (positions according to SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (position according to SEQ ID NO: 225). In some embodiments, the antibody binds to a residue of human BTLA selected from N65 and A64 (positions according to SEQ ID NO: 225).

[0262] In certain embodiments, an isolated antibody of the invention that specifically binds to human BTLA increases BTLA activity and / or receptor-mediated signaling.

[0263] As stated above, the term antibody when used in connection with the first aspect of the invention includes whole antibodies as well as antigen-binding fragments thereof.

[0264] Specific Fc Receptor Binding Embodiments In certain embodiments of the invention, particularly according to the first aspect of the invention, the heavy chain comprises an Fc region comprising a substitution that confers increased binding to FcγR2B and thus enhanced FcγR2B signaling. In certain embodiments, such molecules have decreased binding to one or more activating Fc gamma receptors, such as FcγR2A or FcγR1A, compared to the parent polypeptide. In certain embodiments, such molecules have an increased binding ratio to FcγR2B / FcγR2A compared to the parent polypeptide. In certain embodiments, such molecules have an increased binding ratio to FcγR2B / FcγR1A compared to the parent polypeptide.

[0265] As mentioned above, in certain embodiments of the invention, particularly according to the first aspect of the invention, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, and alanine (A) at position 297 (all numbering according to the EU index). Suitably, the Fc region, and therefore the antibody itself, is capable of binding to an Fcγ receptor.

[0266] In certain embodiments, the Fc region binds to FcγR2B with higher affinity compared to a control antibody comprising an Fc region lacking one or more of the above Fc substitutions. In certain embodiments, the antibody binds to FcγR2B with a dissociation constant (KD) of about 5 μM to 0.1 μM as determined by surface plasmon resonance (SPR). Preferably, the antibody binds to FcγR2B via the Fc region.

[0267] In a specific embodiment, the antibody binds to FcγR2B with a KD of up to 5 μM as determined by surface plasmon resonance (SPR).

[0268] In a specific embodiment, the antibody binds to FcγR2A (131R allotype) with lower or equal affinity compared to the parent molecule, which is a comparable antibody lacking Fc substitutions that allow the antibody molecule to increase binding to FcγR2B and thus enhance FcγR2B signaling.

[0269] In a specific embodiment, when an antibody comprises a P238D substitution, it binds to FcγR2A (131R allotype) with less or equal affinity than a control antibody comprising an Fc region containing a proline at position 238 (EU index).

[0270] In a specific embodiment, the antibody binds to FcγR2A (131R allotype) with a KD of at least 20 μM as determined by surface plasmon resonance (SPR).

[0271] In a specific embodiment, the antibody binds to FcγR2A (131R allotype) with a KD of approximately 25 μM to 35 μM as determined by surface plasmon resonance (SPR).

[0272] In a specific embodiment, the antibody binds to FcγR2A (131H allotype) with less or equal affinity compared to the parent molecule.

[0273] In a specific embodiment, when an antibody comprises a P238D substitution, it binds to FcγR2A (131H allotype) with less or equal affinity than a control antibody comprising an Fc region containing a proline at position 238 (EU index).

[0274] In a specific embodiment, the antibody binds to FcγR2A (131H allotype) with a KD of at least 50 μM as determined by surface plasmon resonance (SPR).

[0275] In a specific embodiment, the antibody has a [KD value of the antibody for FcγR2A(131R) / KD value of the antibody for FcγR2B] ratio of 3 or greater (e.g., at least 5), preferably as determined by surface plasmon resonance (SPR).

[0276] In certain embodiments, the antibody has a KD value of the antibody for FcγR2A(131H) / KD value of the antibody for FcγR2B of 10 or greater (e.g., at least 15), preferably as determined by surface plasmon resonance (SPR).

[0277] In certain embodiments, the antibody has a [KD value of the antibody for FcγR2A(131R)] / [KD value of the antibody for FcγR2B] of 3 or more (e.g., at least 5) and / or a [KD value of the antibody for FcγR2A(131H)] / [KD value of the antibody for FcγR2B] of 10 or more (e.g., at least 15), preferably as determined by surface plasmon resonance (SPR).

[0278] Preferably, the antibodies of the invention exhibit increased agonism of human BTLA expressed on the surface of human immune cells compared to a control / parent antibody, as measured by a BTLA agonist assay selected from a T cell activation assay such as that described in Example 24, a mixed lymphocyte reaction such as that described in Example 25, or a B cell activation assay such as that described in Example 26.

[0279] Thus, when an antibody contains the P238D substitution, the antibody exhibits increased agonism of human BTLA expressed on the surface of human immune cells, as compared to a control antibody comprising an Fc region containing a proline at position 238, as measured by a BTLA agonist assay selected from a T cell activation assay such as that described in Example 24, a mixed lymphocyte reaction such as that described in Example 25, or a B cell activation assay such as that described in Example 26.

[0280] In certain embodiments, the antibody of the present invention is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody (such as a bispecific antibody).

[0281] In certain embodiments, the antibodies of the invention are scFv, sc(Fv) 2 , dsFv, Fab, Fab', (Fab')2, and diabody.

[0282] In certain embodiments, the heavy and light chain molecules that form the antigen-binding fragment are linked by a flexible linker. There are many commonly used flexible linkers, and the linker can be selected by one of skill in the art.

[0283] The peptide linker connecting the scFv VH and VL domains connects the carboxyl terminus of one variable region domain to the amino terminus of another variable domain without significantly impairing the fidelity of the VH-VL pairing and antigen-binding site. Peptide linkers can vary in length from 10 to 25 amino acids and are typically, but not always, composed of hydrophilic amino acids such as glycine (G) and serine (S). Linkers can be those found in natural multidomain proteins (see, e.g., Argos PJ Mol Biol. 211:943-958, 1990 and Heringa G. Protein Eng. 15:871-879, 2002), or adapted therefrom.

[0284] Commonly used flexible linkers have sequences consisting primarily of a stretch of Gly and Ser residues ("GS" linkers). An example of the most widely used flexible linker is (Gly-Gly-Gly-Gly-Ser). n (SEQ ID NO: 232). By adjusting the copy number "n", the length of this GS linker can be varied to achieve appropriate separation of functional domains or to maintain necessary inter-domain interactions. Generally, GS linkers with n = 3 peptides are used as scFv peptide linkers (Leith et al., Int. J. Oncol. 24:765-771, 2004; Holiger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). This 15 amino acid linker sequence [referred to as the (GGGGS)3 linker] is used in the commercially available Recombinant Phage Antibody System (RPAS kit) from Amersham. Several other linkers have also been used to generate scFv molecules (e.g., KESGSVSSEQLAQFRSLD (SEQ ID NO: 233) and EGKSSGSGSESKST (SEQ ID NO: 234); Bird et al., Science 242:432-426, 1988).

[0285] Epitope Binding The inventors have mapped the epitopes on BTLA to which the potent agonists and antibodies disclosed herein bind.

[0286] In specific embodiments, antibodies of the invention bind to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, E92, Y39, K41, R42, Q43, E45, S47, D35, T78, K81, S121, L123, H68, N65, A64.

[0287] In specific embodiments, antibodies of the invention bind to a residue of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, E92.

[0288] In certain embodiments, antibodies of the invention bind to at least two residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, E92.

[0289] In certain embodiments, antibodies of the invention bind to at least three residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0290] In certain embodiments, antibodies of the invention bind to at least five residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0291] In certain embodiments, antibodies of the invention bind to all of the residues of human BTLA selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0292] In certain embodiments, antibodies of the invention bind to a residue in human BTLA selected from Y39, K41, R42, Q43, E45, and S47.

[0293] In certain embodiments, antibodies of the invention bind to at least two residues in human BTLA selected from Y39, K41, R42, Q43, E45, and S47.

[0294] In certain embodiments, antibodies of the invention bind to all of the residues in human BTLA selected from Y39, K41, R42, Q43, E45, and S47.

[0295] In certain embodiments, antibodies of the invention bind to a residue in human BTLA selected from D35, T78, K81, S121, and L123.

[0296] In certain embodiments, antibodies of the invention bind to at least two residues in human BTLA selected from D35, T78, K81, S121, and L123.

[0297] In certain embodiments, antibodies of the invention bind to residue H68 of human BTLA.

[0298] In certain embodiments, antibodies of the invention bind to a residue in human BTLA selected from N65 and A64.

[0299] In certain embodiments, the antibodies of the invention bind to both residues N65 and A64 of human BTLA.

[0300] Residue numbering, e.g., K41, refers to the amino acid (K41; lysine), and the numbering refers to the position in the human BTLA polypeptide disclosed in SEQ ID NO:225.

[0301] In certain embodiments, the antibody is an IgG1, IgG2, or IgG4 antibody. In certain embodiments, the antibody is a murine antibody or a human antibody.

[0302] In certain embodiments, the antibodies of the invention are humanized antibodies.

[0303] In certain embodiments, the antibodies of the invention are fully human antibodies.

[0304] In certain embodiments, the antibodies of the invention act as agonists to induce signaling through the BTLA receptor.

[0305] The antibodies (including antigen-binding fragments) of the present invention are particularly potent agonists.

[0306] In certain embodiments, the antibodies of the invention have an EC50 of 1 nM or less.

[0307] The agonist antibodies of the present invention (eg, full-length / whole antibodies or antigen-binding fragments thereof) have particularly high efficacy.

[0308] In certain embodiments, the antibodies of the invention inhibit T cell proliferation by at least 20%, preferably at least 30%, and more preferably at least 40%.

[0309] In certain embodiments, the antibodies of the invention inhibit T cell IFN-γ production by at least 50%, preferably at least 75%, and more preferably at least 95%, as measured, for example, by ELISA of supernatants in an in vitro mixed lymphocyte reaction.

[0310] In certain embodiments, antibodies of the invention inhibit IL-2 production by T cells by at least 50%, preferably at least 75%, and more preferably at least 95%, as measured, for example, by ELISA of supernatants in an in vitro mixed lymphocyte reaction.

[0311] In certain embodiments, the antibodies of the invention inhibit IL-17 production by T cells by at least 50%, preferably at least 75%, and more preferably at least 95%, as measured, for example, by ELISA of supernatants in an in vitro mixed lymphocyte reaction.

[0312] In a specific embodiment, the antibodies of the invention reduce mortality in a mouse GVHD model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 12.

[0313] In certain embodiments, antibodies of the invention reduce weight loss in a murine T-cell colitis model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 11.

[0314] In certain embodiments, antibodies of the invention reduce colonic inflammation in a murine T-cell colitis model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 11.

[0315] In certain aspects, the present invention also relates to an isolated polypeptide comprising the VL domain or VH domain of any of the antibodies described herein.

[0316] As described herein, in certain embodiments, an antibody that binds to BTLA comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising an aspartic acid at position 238 (EU index).

[0317] nucleic acid molecule The antibodies of the present invention will be encoded by nucleic acids. Antibodies (including antigen-binding fragments thereof) may be encoded by a single nucleic acid molecule, or by two or more nucleic acid molecules. For example, because an antigen-binding site is typically formed by combining a heavy chain variable polypeptide region and a light chain variable polypeptide region, the two variable (heavy and light chain) polypeptide regions may be encoded by separate nucleic acid molecules. Alternatively, in the case of, for example, an ScFv, they may be encoded by the same nucleic acid molecule.

[0318] According to a second aspect of the invention there is provided one or more nucleic acid molecules encoding an antibody according to the first aspect of the invention.

[0319] From the primary amino acid sequence of a polypeptide encoding an antibody of the present invention, one skilled in the art can determine a suitable nucleotide sequence encoding the polypeptide, and, if necessary, one that is codon-optimized (see, e.g., Mauro and Chappell. Trends Mol Med. 20(11):604-613, 2014).

[0320] As used herein, when there is a reference to a previous aspect of the invention (e.g., "in accordance with the first (or second, etc.) aspect of the invention"), it is understood to encompass any recited variations of that aspect (e.g., variations of the first (or second, etc.) aspect). Furthermore, any embodiment that applies to a particular aspect of the invention also applies to any variations of that aspect, and thus an embodiment that applies to the first aspect of the invention also applies to variations of the first aspect of the invention.

[0321] According to a variant of the second aspect of the present invention, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region polypeptide or a light chain variable region polypeptide of the present invention. A heavy chain variable polypeptide or a light chain variable polypeptide of the present invention refers to an individual polypeptide chain comprising amino acids that form part of an antigen-binding site. Of course, the polypeptide may also comprise constant domains, hinge region, and other domains of an Fc region (such as one comprising one or more Fc receptor binding sites).

[0322] According to another variation of the second aspect of the present invention, there is provided an isolated nucleic acid comprising one or more nucleotide sequences encoding a polypeptide capable of forming an antibody of the present invention. In certain embodiments, the polypeptide may comprise a constant domain, a hinge region, and other domains of an Fc region (such as one comprising one or more Fc receptor binding sites).

[0323] One of the nucleic acid molecules may encode only the polypeptide sequence comprising the VL domain of an antibody or fragment thereof. One of the nucleic acid molecules may encode only the polypeptide sequence comprising the VH domain of an antibody or fragment thereof. However, the nucleic acid molecule may also encode both the VH and VL domains comprising polypeptide sequences capable of forming an antibody of the invention (such as a full-length / whole antibody or an antigen-binding fragment thereof).

[0324] A nucleic acid molecule encoding an antibody of the invention, e.g., an antibody according to the first aspect of the invention, may be, or may be part of, a vector (e.g., a plasmid, cosmid, or viral vector, or an artificial chromosome), which may contain other functional regions (elements), such as one or more promoters, one or more origins or replication, one or more selectable markers, and one or more other elements typically found in expression vectors. The cloning and expression of nucleic acids encoding proteins, including antibodies, is well established and well within the skill of one of ordinary skill in the art.

[0325] According to a third aspect of the present invention, there is provided a vector, comprising the nucleic acid of the second aspect of the invention. In certain embodiments, the vector is a plasmid vector, a cosmid vector, a viral vector, or an artificial chromosome.

[0326] The nucleic acids of the present invention, including vector nucleic acids comprising a nucleotide sequence encoding a polypeptide capable of forming an antibody of the present invention, may be in purified / isolated form.

[0327] Isolated / purified nucleic acids encoding an antibody of the invention are free or substantially free from materials with which they are naturally associated (e.g., other proteins or nucleic acids with which they are found in their natural environment or in the environment (e.g., cell culture) in which they are prepared), whether such preparation is carried out by recombinant DNA techniques in vitro or in vivo.

[0328] In certain embodiments, the nucleic acids of the invention are more than 80% pure, such as more than 90%, more than 95%, more than 97%, and more than 99% pure.

[0329] Thus, according to another variant of the third aspect of the invention, there is provided a vector comprising a nucleic acid or nucleotide sequence encoding a heavy chain variable polypeptide or a light chain variable polypeptide of the invention. In certain embodiments, the vector comprises nucleic acid encoding both a heavy chain variable region and a light chain variable region. In certain embodiments, the polypeptide may comprise a constant domain, a hinge region, and other domains of an Fc region (such as one comprising one or more Fc receptor binding sites).

[0330] The nucleic acids and / or vectors of the present invention can be introduced into host cells. Introduction can be by any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction (using retroviruses or other viruses, e.g., vaccinia or baculovirus for insect cells). Introduction of nucleic acids into host cells (particularly eukaryotic cells) can use virus- or plasmid-based systems. Plasmid systems can be maintained episomally or integrated into the host cell or artificial chromosomes. Integration can be by either random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection (using bacteriophage).

[0331] In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0332] host cell A further aspect of the present invention provides a host cell, comprising a nucleic acid as disclosed herein. Such a host cell may be in vitro or in culture.

[0333] The host cell may be from any species, such as bacteria or yeast, but preferably the host cell is a mammalian cell, such as a human cell or a rodent cell (eg, HEK293T cell or CHO-K1 cell).

[0334] Thus, according to a fourth aspect of the invention, there is provided a host cell, comprising a nucleic acid sequence according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0335] Host cells can be treated to cause or allow expression of a protein of the invention from a nucleic acid, for example, by culturing the host cells under conditions for expression of the encoding nucleic acid. Purification of the expressed product can be achieved by methods known to those skilled in the art.

[0336] Thus, the nucleic acids of the invention (including vector nucleic acids comprising a nucleotide sequence encoding a polypeptide that can form an antibody of the invention) can be present in an isolated host cell. The host cell is typically part of a clonal population of host cells. As used herein, reference to a host cell also encompasses a clonal population of such cells. A clonal population is one that has been propagated from a single parent host cell. The host cell can be from any suitable organism. Suitable host cells include bacterial, fungal, or mammalian cells.

[0337] Host cells can serve to support the amplification of vector nucleic acids (e.g., using plasmids) or can serve as biological factories for expressing the polypeptides of the invention that form the BTLA antibodies of the invention. Suitable hosts for amplifying vector nucleic acids can be bacterial or fungal cells (e.g., Escherichia coli cells or Saccharomyces cerevisiae cells). Suitable hosts for expressing proteins of the invention (i.e., polypeptides that constitute human BTLA-binding antibodies of the invention) will be mammalian cells (e.g., HEK293T or CHO-K1 cells). In certain embodiments, the host cells are mammalian cells, such as HEK293T or CHO-K1 cells.

[0338] A variety of host-expression vector systems can be utilized to express the BTLA-binding molecules described herein (see, e.g., U.S. Pat. No. 5,807,715). Mammalian cells (e.g., Chinese hamster ovary cells, CHO) is an effective expression system for CEA protein, in combination with a vector such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking et al., Gene, 45:101 (1986), and Cockett et al., Bio / Technology, 8:2 (1990)). Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the proteins of the present disclosure. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, HEK, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O, and HsS78Bst cells.

[0339] antibody production According to a fifth aspect of the invention there is provided a method of producing an antibody according to the first aspect of the invention, comprising culturing a host cell of the fourth aspect of the invention under conditions for the production of said antibody, and optionally isolating and / or purifying said antibody.

[0340] According to a variation of the fifth aspect of the invention, there is provided a method of producing an antibody that binds human BTLA, comprising culturing a host cell comprising nucleic acid encoding a polypeptide that forms an antibody that binds human BTLA under conditions for the production of the antibody, and optionally further comprising the step of isolating / purifying the antibody.

[0341] By isolated / purified is meant that the antibodies of the invention or the polypeptides constituting these molecules are free or substantially free from materials with which they are naturally associated (e.g., other proteins or nucleic acids with which they are found in their natural environment or in the environment (e.g., cell culture) from which they are prepared), where such preparation is carried out by in vitro or in vivo recombinant DNA techniques.

[0342] According to a fifth variant of the invention, there is provided a method for preparing an antibody that specifically binds to human BTLA, comprising: a) providing a host cell comprising one or more nucleic acid molecules encoding one or more polypeptides comprising amino acid sequences of heavy chain variable domains and / or light chain variable domains that, when expressed, can combine to produce a human BTLA-binding molecule; b) culturing the host cell that expresses the encoded amino acid sequence; and c) isolating the antibody molecule.

[0343] The one or more nucleic acid molecules are those described above that encode one or more polypeptides capable of forming an antibody of the invention that specifically binds to human BTLA.

[0344] In certain embodiments, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 17, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3; ii) a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 6.

[0345] In certain embodiments, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 20, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 21, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 22; ii) a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 25.

[0346] In certain embodiments, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 30, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 31, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 32; ii) a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 33, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 34, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 35.

[0347] In certain embodiments, the antibody i) a heavy chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 18 or a sequence having at least 90% sequence identity thereto; ii) a light chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 14 or a sequence having at least 90% sequence identity thereto.

[0348] In certain embodiments, the antibody i) a heavy chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 26 or a sequence having at least 90% sequence identity thereto; ii) a light chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 27 or a sequence having at least 90% sequence identity thereto.

[0349] In certain embodiments, the antibody i) a heavy chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 36 or a sequence having at least 90% sequence identity thereto; ii) a light chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 43 or a sequence having at least 90% sequence identity thereto.

[0350] Conditions for producing antibodies of the present invention and purifying such molecules are well known in the art. One way to address this is to prepare a clonal population of cells capable of expressing the antibody of the present invention or a fragment thereof and culture them in a suitable growth medium for a period and temperature conducive to expansion / proliferation of the cell population and expression of the protein of interest. If the protein of interest (e.g., an antibody of the present invention) is expressed in host cells, the cells can be lysed (e.g., using a mild detergent or sonication) to release the contents of the cells (and thus the protein of interest) into the surrounding medium (which may be the culture medium in which the cells were reconstituted or another medium), and this medium is then subjected to the purification process. If the protein of interest (e.g., an antibody of the present invention) is secreted into the growth medium, the medium is subjected to the purification process. Antibody purification typically involves isolating the antibody from, for example, the culture medium of a hybridoma cell line or from the culture supernatant using well-established methods that typically involve chromatography (e.g., using affinity chromatography, anion and / or cation exchange chromatography, size exclusion chromatography, or other separation techniques) to separate the protein of interest from undesired host-derived proteins and other cellular contaminants (e.g., nucleic acids, carbohydrates, etc.). The purified protein may also be subjected to a viral inactivation step. Finally, the purified protein of interest may be, for example, lyophilized or otherwise formulated for storage, transport, and (ready for subsequent use). Preferably, the protein of interest (e.g., a whole antibody or antigen-binding fragment thereof of the present invention) will be substantially free of contaminating proteins originally present in the culture medium after expression or cell lysis.

[0351] In certain embodiments, antibodies of the invention will be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0352] The proteins of the invention (eg, whole antibodies or antigen-binding fragments thereof of the invention) can be formulated into suitable compositions.

[0353] composition Although the BTLA-binding molecule (antibody of the invention) may be administered alone, in certain embodiments, the administration is of a pharmaceutical composition in which the BTLA-binding molecule is formulated with at least one pharmaceutically acceptable excipient. The excipient can be a suitable pharmaceutical carrier solute. Such carriers are well known in the art and include phosphate-buffered saline solution, water, liposomes, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at a suitable dose. The dosing regimen is determined by the attending physician and clinical factors.

[0354] According to a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of an antibody of the first aspect of the invention or an antibody produced by the fifth aspect of the invention. In a particular embodiment, the composition comprises phosphate buffered saline.

[0355] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Pharmaceutical compositions will contain one or more pharmaceutically acceptable excipients. The term excipient in this context refers to any additive, such as a filler, solubilizer, carrier, vehicle, or additive.

[0356] Pharmaceutical compositions can include one or more pharmaceutically acceptable excipients, including, for example, water, ion exchangers, proteins, buffer substances, and salts. Preservatives and other additives can also be present. The excipient can be a solvent or dispersion medium. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0357] A "pharmaceutically acceptable" excipient is an excipient that can be reasonably administered to a target mammal to provide an effective dose of the active ingredient employed. The pharmaceutical compositions of the present invention are prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the composition with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., TWEEN™, PLURONICS™, or polyethylene glycol (PEG)). Lyophilized HER2 antibody formulations are described in WO 97 / 04801.

[0358] Pharmaceutical compositions to be used for in vivo administration must be sterile, which may readily be accomplished by filtration through sterile filtration membranes.

[0359] The route of administration of a BTLA-binding moiety molecule (e.g., an antibody or antigen-binding fragment thereof) can be, for example, oral, parenteral, inhalation, or topical. As used herein, the term parenteral includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0360] Pharmaceutical compositions for parenteral administration include sterile aqueous or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, aqueous solutions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose). Preservatives and other additives (e.g., antimicrobial agents, antioxidants, chelating agents, and inert gases) may also be present. Furthermore, in certain embodiments of human origin, compositions may include proteinaceous carriers, such as serum albumin or immunoglobulin. For intravenous injection or injection at the affected site, the active ingredient will be in the form of a pyrogen-free, parenterally acceptable aqueous solution having suitable pH, isotonicity, and stability. Those of skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included, as desired, all of which, as noted above, are referred to herein as excipients.

[0361] Injectable compositions can be administered using medical devices known in the art, such as hypodermic needles. Needle-free injection devices such as those disclosed in U.S. Patent Nos. 6,620,135 and 5,312,335 can also be used.

[0362] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, liquid, or semi-solid form. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Optionally, saline, dextrose or other sugar solution, or glycols (e.g., ethylene glycol, propylene glycol, or polyethylene glycol) may be included.

[0363] The antibodies of the present invention can be formulated in liquid, semi-solid, or solid form depending on the physicochemical properties of the molecule and the delivery route. The formulation may also contain excipients or combinations of excipients (e.g., sugars, amino acids, and surfactants). Liquid formulations can cover a wide range of antibody concentrations and pH. Solid formulations can be produced, for example, by freeze-drying, spray-drying, or drying by supercritical fluid techniques.

[0364] Pharmaceutical compositions can be administered as a single dose, multiple doses, or over an established period of time in an infusion. Dosage regimens can also be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). In particular, parenteral formulations can be a single bolus dose, an infusion, or a loading bolus dose, followed by one or more maintenance doses. These compositions can be administered at specific fixed or variable intervals, for example, once a day or "as needed."

[0365] Dosage The amount of a BTLA-binding molecule or pharmaceutical formulation containing such a molecule that will be therapeutically effective can be determined by standard clinical techniques, such as a dose-ranging clinical trial. Additionally, in vitro assays can optionally be used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of the composition to be administered can be determined by one of ordinary skill in the art without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to consider when making these determinations include the condition(s) being treated, the choice of composition to be administered, the age and weight of the individual patient, and the severity of the patient's symptoms. For example, the actual patient weight can be used to calculate the dosage of the formulation to be administered in milliliters (mL). Adjusting downward to the "ideal" weight may not be necessary. In such circumstances, the appropriate dosage can be calculated using the following formula: Dose (ml) = [patient weight (kg) × dose level (mg / kg) / drug concentration (mg / mL)]

[0366] The therapeutically effective dose of a pharmaceutical composition for treating a BTLA-associated disease or disorder will vary depending on many different factors, including the means of administration, the target site, the patient's physiological condition, the weight or condition of the patient, the patient's gender, the patient's age, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic, as discussed herein. Therapeutically effective amounts will likely have been determined from clinical trials and can be determined by the attending physician using treatment guidelines. Typically, the patient is a human, although non-human mammals can also be treated. Therapeutic dosages can be titrated to optimize safety and efficacy using routine methods known to those of skill in the art.

[0367] In various embodiments, the BTLA binding molecule is administered at a concentration of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg.

[0368] The pharmaceutical compositions of the present invention can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. Such combinations can be with other immunosuppressants (e.g., selected from the following): corticosteroids, cyclosporine, azathioprine, sulfasalazine, methotrexate, mycophenolate, tacrolimus, and fingolimod, or other biologics (e.g., infliximab, adalimumab, ustekinumab, tocilizumab, and rituximab).

[0369] According to a seventh aspect of the invention there is provided a method for preparing a pharmaceutical composition comprising formulating an antibody according to the first aspect of the invention or an antibody produced according to the fifth aspect of the invention into a composition comprising at least one additional component, in certain embodiments the at least one additional component is a pharmaceutically acceptable excipient.

[0370] kit Additionally, articles of manufacture (e.g., BTLA-binding molecules or pharmaceutical compositions thereof) can be packaged and sold in the form of a kit. Such articles of manufacture can have a label or insert indicating instructions for the product and the proper use of the product for the treatment of a subject suffering from or susceptible to a disease or disorder.

[0371] Thus, according to an eighth aspect of the invention there is provided a kit comprising an antibody according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention. Suitably, such a kit comprises a package insert containing instructions for use.

[0372] Therapeutic / Medical Uses The antibodies of the invention or pharmaceutical compositions comprising said antibodies or antigen-binding fragments thereof can be used in therapy, typically as medicines.

[0373] In certain embodiments, the antibodies of the invention or pharmaceutical compositions comprising said antibodies may be used to treat or prevent any disease or condition in a subject in need thereof.

[0374] BTLA is involved in downregulating the immune response, and there are many diseases or conditions that can be treated by suppressing host T cells and / or B cells (see, e.g., Crawford & Wherry. Editorial: Therapeutic potential of targeting BTLA. J Leukocyte Biol. 86:5-8, 2009). Diseases or conditions that can benefit from treatment with anti-BTLA agonists are referred to herein as "BTLA-associated diseases." BTLA-associated diseases include inflammatory or autoimmune diseases and disorders of excessive immune cell proliferation.

[0375] Specific BTLA-associated diseases that can be treated with the BTLA-binding molecules of the invention include Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, and venous ulcers. cerebrovascular disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrotic diseases (e.g., scleroderma, pulmonary fibrosis, and liver cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoma These include proliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, graft rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0376] In certain embodiments, the disease being treated is selected from the group consisting of Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, graft-versus-host disease, graft rejection, multiple sclerosis, vasculitis, Sjogren's syndrome, Behcet's disease, uveitis, type 1 diabetes, Hashimoto's thyroiditis, primary sclerosing cholangitis, and myasthenia gravis.

[0377] In certain embodiments, the disorder of excessive immune cell proliferation is selected from lymphoma, leukemia, systemic mastocytosis, myeloma, or a lymphoproliferative disorder.

[0378] According to a ninth aspect of the present invention there is provided an antibody according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention for use in therapy.

[0379] In certain embodiments, the therapy is the treatment or prevention of a BTLA-associated disease.

[0380] In certain embodiments, the BTLA-associated disease is one caused by decreased expression and / or activity of BTLA in a subject. In particular, any disease or disorder characterized by the presence or activity of T cells or B cells can be treated with the BTLA agonist antibodies of the invention.

[0381] In one embodiment, the BTLA-associated disease is an inflammatory disease (e.g., rheumatoid arthritis), an autoimmune disease or disorder (e.g., graft versus host), or a proliferative disease or disorder (e.g., cancer).

[0382] In particular embodiments, the treatment is treatment or prevention of inflammatory or autoimmune diseases and disorders of excessive immune cell proliferation.

[0383] According to a variation of the ninth aspect of the invention, there is provided a method of treating a patient in need of treatment, comprising administering to the patient an antibody (or BTLA-binding molecule) according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention. In certain embodiments, the patient in need of treatment or the patient to be treated has (or is suffering from) a BTLA-associated disease. In certain embodiments, the patient in need of treatment or the patient to be treated has (or is suffering from) an inflammatory disease, an autoimmune disease, or a disorder of excessive immune cell proliferation.

[0384] In a particular embodiment, the antibody according to the first aspect of the invention or the pharmaceutical composition according to the sixth aspect of the invention is administered to a patient in need thereof in a pharmaceutically acceptable amount.

[0385] In a variation of this ninth aspect of the invention, there is provided an antibody (or BTLA-binding molecule) according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention for use in a method of treating a patient in need of treatment. In certain embodiments, the method is for treating or preventing a BTLA-associated disease. In certain embodiments, the method is for treating or preventing an inflammatory or autoimmune disease, and a disorder of excessive immune cell proliferation.

[0386] In a further variation of this aspect, there is provided the use of an antibody according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention in the manufacture of a medicament for the treatment of a patient in need thereof.

[0387] In one embodiment, the therapy is for treating a BTLA-associated disease. Preferably, the BTLA-associated disease is an inflammatory disease (e.g., asthma), an autoimmune disease or disorder (e.g., rheumatoid arthritis), or an immunoproliferative disease or disorder (e.g., lymphoma).

[0388] In a specific embodiment, the antibodies of the invention or pharmaceutical compositions comprising said antibodies are used to suppress T cells and / or B cells.

[0389] In certain embodiments, the antibodies of the invention or pharmaceutical compositions comprising the antibodies are used to treat or prevent a disease or condition selected from the group consisting of Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, and graft versus host disease in a subject in need thereof. disease, GVHD), Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and liver cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis (MS), myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis , primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, graft rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0390] In certain embodiments, an antibody of the invention or a pharmaceutical composition comprising the antibody is used to treat or prevent a disease or condition selected from the group consisting of Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, graft-versus-host disease, graft rejection, multiple sclerosis, vasculitis, Sjogren's syndrome, Behcet's disease, uveitis, type 1 diabetes, Hashimoto's thyroiditis, primary sclerosing cholangitis, and myasthenia gravis in a subject in need of such treatment or prevention. In one embodiment, the immunoproliferative disorder is cancer. Preferably, the cancer is leukemia or lymphoma.

[0391] In another embodiment, the antibody of the invention or a pharmaceutical composition comprising said antibody is for use in the prevention or treatment of transplant rejection.

[0392] In another embodiment, the present invention relates to the prevention or treatment of graft-versus-host disease.

[0393] In another embodiment, the antibody of the invention or a pharmaceutical composition comprising the antibody is for use in treating rheumatoid arthritis.

[0394] In another embodiment, the antibody of the invention or pharmaceutical composition comprising the antibody is for use in treating diabetes, such as type 1 diabetes.

[0395] In another embodiment, the antibody of the invention or a pharmaceutical composition comprising said antibody is for use in the treatment of psoriasis.

[0396] In another embodiment, the antibody of the invention or a pharmaceutical composition comprising said antibody is for use in the treatment of multiple sclerosis.

[0397] In another embodiment, the antibody of the invention or a pharmaceutical composition comprising said antibody is for use in treating colitis.

[0398] The term "effective amount" or "therapeutically effective amount" refers to the dosage or amount of drug that is sufficient to ameliorate symptoms or achieve a desired biological outcome in a patient (e.g., in the case of cancer, increased tumor cell death, decreased tumor size, increased progression-free survival or overall survival, etc.). As disclosed elsewhere herein, effective amounts are typically assessed through extensive human clinical trials.

[0399] Throughout this description and the claims, the terms "comprise" and "contain," and variations thereof, mean "including but not limited to," and include other moieties, additives, ingredients, integers, etc. It is not intended (and does not exclude) any step or step. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0400] It should be understood that any feature, integer, property, compound, chemical moiety, or chemical group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless inconsistent therewith. All features disclosed herein (including any accompanying claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiment. The invention extends to any novel feature or combination of features disclosed herein (including any accompanying claims, abstract, and drawings), or any novel step or combination of steps of any method or process so disclosed.

[0401] The reader's attention is directed to all articles and documents related to this application that have been filed contemporaneously or earlier herewith and that are open to public inspection herewith, the contents of all such articles and documents being incorporated herein by reference.

[0402] The invention will now be further described with reference to the following non-limiting examples and accompanying drawings. [Brief explanation of the drawings]

[0403] [Figure 1] Binding of antibodies to human and cynomolgus BTLA in soluble and cell-expressed forms. (a) Surface plasmon resonance (SPR) binding curves for soluble monomeric human BTLA extracellular domain injected at increasing concentrations against immobilized anti-BTLA antibody; the graph shows the SPR signal after reference and blank subtraction. (b) Association and dissociation rates for binding to human or cynomolgus BTLA calculated by curve fitting using BiaEvaluation software. (c) Binding of antibody 2.8.6 to Jurkat cell lines expressing human BTLA or cynomolgus BTLA compared to an isotype control antibody (data points represent the mean + / - SD of triplicate wells at each antibody concentration). (d) EC50 for antibody binding to transfected cell lines calculated by nonlinear curve fitting using GraphPad Prism software. [Figure 2] (a) Blockade of ligand binding by anti-BTLA antibody was assessed by SPR. Human BTLA extracellular domain was immobilized on a sensor chip. Human HVEM was injected to confirm binding and then allowed to dissociate completely. A saturating concentration of anti-BTLA antibody was then injected, immediately followed by a second injection of HVEM. (b) Equilibrium binding of HVEM after antibody injection was expressed as a percentage of HVEM binding before antibody injection. Saturation of BTLA with clone 11.5.1, but not clone 2.8.6, blocked subsequent binding of the ligand. [Figure 3]Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfected with a BTLA construct in a bicistronic vector that also expresses GFP were stained with Pacific Blue-conjugated anti-BTLA antibody. Clone 11.5.1 binds to cells transfected with the wild-type receptor (left) but not to cells transfected with BTLA containing the Y39R mutation (right). (b) Binding to each BTLA mutant construct was expressed as a percentage of binding to wild-type BTLA for clones 2.8.6 and 11.5.1. (c) Mutations Y39R and K41E, which selectively eliminate binding of clone 11.5.1, were mapped onto the crystal structure of human BTLA (black residues). Residues important for binding of the ligand HVEM are highlighted in gray. [Figure 4] (a) Crystal structure of the human BTLA extracellular domain in complex with the Fab' fragment of clone 2.8.6. Residues on BTLA that are buried in the interface are highlighted in black. (b) The epitope of antibody 2.8.6 (black residues) relative to the HVEM binding site (gray residues) is shown. [Figure 5] (a) Strategy for generating a chimeric BTLA gene in humanized BTLA mice. A section of human genomic DNA from the beginning of exon 2 to the end of exon 3 was inserted into the mouse locus, replacing the mouse sequence from the beginning of exon 2 to the end of exon 4. The exon-intron junction sequences at the beginning of mouse exon 2 and the end of mouse exon 4 were left intact to ensure proper splicing. [Figure 6]Protocol for T cell transfer assay to evaluate anti-BTLA antibodies in vivo. A mixture of humanized and wild-type OVA-specific CD4 T cells was injected into recipient mice. The following day, mice were immunized with ovalbumin in Alum to activate the transferred cells, and 24 hours later, anti-human BTLA antibody or isotype control was administered. Eight days after the initial cell transfer, the ratio of humanized to wild-type cells in the transferred population within the spleen was assessed by flow cytometry. (b) Clone 11.5.1, and to a lesser extent, clone 2.8.6, both reduced the proliferation of humanized cells compared to wild-type. Graphs show pooled data from two (11.5.1) or three (2.8.6) replicate experiments. Each data point represents an individual recipient mouse. [Figure 7] Effect of anti-BTLA clone 2.8.6 on CD4 T cell proliferation in an in vitro mixed lymphocyte reaction. T cells from humanized C57BL / 6 mice were stained with CellTraceViolet and added to mitomycin C-treated Balb / c stimulator cells in the presence of anti-BTLA antibody or isotype control. After 96 hours, proliferation of humanized CD4 cells was assessed and normalized to proliferation in the absence of antibody. Clone 2.8.6 inhibited humanized cell proliferation with an IC50 of 0.029 nM, with a maximal effect of 42% inhibition of proliferation. Data points represent the mean + / - SD of triplicate wells at each antibody concentration and are representative of five independent experiments. [Figure 8](a) Effect of clone 2.8.6 in a T-cell colitis model. RAG knockout recipient mice were injected with CD45RBhiCD25-CD4+ T cells from humanized BTLA mice and treated with 200 μg of 2.8.6 or isotype control antibody on days 7, 21, and 35. Isotype control-treated mice gradually lost weight from week 3 onward, while 2.8.6-treated mice were spared. (b) Eight weeks after cell transfer, colons were processed to extract lamina propria lymphocytes, and the total number of inflammatory cells extracted per colon was calculated. Isotype control-treated mice had significantly more infiltrating immune cells than 2.8.6-treated mice. (c) Colon weight-to-length ratio was calculated as a marker of inflammation and thickening. 2.8.6 treatment prevented the increase in weight-to-length ratio seen in isotype control-treated mice. [Figure 9] (a) Effect of BTLA antibodies in a parent-to-F1 transfer model of GVHD. C57BL / 6 splenocytes and bone marrow cells derived from humanized BTLA mice were injected into CB6F1 recipient mice, which were then treated with anti-BTLA antibodies or isotype control. Untreated mice developed clinical GVHD with progressive weight loss, dermatitis, and diarrhea and were culled when they reached a pre-specified humane endpoint. 2.8.6 and 11.5.1 antibody-treated mice were relatively spared, with survival rates comparable to control mice reconstituted with syngeneic cells. (b) Five weeks after cell transfer, mice were culled, and colon weight-to-length ratios were calculated as a marker of intestinal inflammation. 2.8.6 and 11.5.1 treatment prevented the colon thickening seen in untreated mice. [Figure 10] (a) Effect of D265A mutant clone 11.5.1 in an in vivo T cell transfer assay. This mutant antibody, which does not bind to the Fc receptor, no longer inhibits the proliferation of humanized BTLA cells, but instead leads to enhanced proliferation due to receptor blockade. (b) The D265A mutant 11.5.1 antibody no longer inhibits T cell proliferation in a mixed lymphocyte reaction. [Figure 11]Anti-BTLA antibodies do not fix complement. Splenocytes from humanized BTLA mice were incubated with 10% rabbit complement in the presence of 20 μg / ml of BTLA antibody, isotype control, or positive control (depleting CD20 antibody) for 1 hour at 37°C. The anti-CD20 antibody depleted the majority of B cells, confirming rabbit complement activity, whereas the BTLA antibody did not deplete either B or T cells, even though both of these populations stained positive for BTLA. [Figure 12] Anti-BTLA antibodies do not induce antibody-dependent cell-mediated cytotoxicity. Splenocytes from humanized BTLA mice were incubated for 24 hours at 37°C in the presence of 20 μg / ml of BTLA antibody, an isotype control, or a positive control (depleting CD20 antibody). The anti-CD20 antibody depleted the majority of B cells by inducing ADCC by effector cells in the mixture, whereas the BTLA antibody did not deplete either B or T cells, even though both of these populations stained positive for BTLA. [Figure 13] Anti-BTLA antibodies do not deplete B or T cells in vivo. Humanized BTLA mice were injected with 200 μg of 2.8.6 antibody. At 24 hours, spleens and bone marrow were harvested and cell populations were assessed by flow cytometry. 2.8.6 did not deplete B or T cells in the spleen or affect the frequencies of different B cell precursor populations in the bone marrow (n=3 mice per group). [Figure 14] BTLA expression levels in B cells or CD4+ T cells from humanized mice after 6 days of in vivo incubation with antibody 2.8.6 or 11.5.1 compared to BTLA expression in cells from mice injected with an isotype control antibody (n=5 mice per group). [Figure 15]The agonistic effect of BTLA antibodies in reporter assays depends on Fc receptor binding and isotype; the greater the FcγR2B binding, the more potent the agonist. A Jurkat T cell line expressing GFP under the control of an NFκB-responsive transcription element was transfected with human BTLA and stimulated by coculture with the BW5147 cell line expressing an anti-CD3 ScFv construct on its surface. NFκB signaling was detected by measuring the geometric mean GFP concentration by flow cytometry after 24 hours of culture. The inhibitory effect of adding BTLA agonist antibodies of different isotypes to the cultures was assessed (a) in the presence of either the BW5147 cell line, which was also transfected to express hFcγR2B, or (b) in the absence of Fc receptors. Data points are the mean + / - SD of triplicate wells at each antibody concentration and represent three independent experiments. [Figure 16] The humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype exhibited greater potency and efficacy in reporter assays compared with an Fc fusion protein of the BTLA ligand HVEM or the prior art BTLA agonist 22B3. A Jurkat T cell line expressing GFP under the control of an NFκB-responsive transcription element was transfected with human BTLA and stimulated by co-culture with the BW5147 cell line expressing an anti-CD3 ScFv construct and hFcγR2B on its surface. NFκB signaling was detected by measuring the geometric mean GFP concentration by flow cytometry after 24 hours of culture. The inhibitory effects of BTLA agonist antibodies added to the co-culture were assessed. Data points are the mean + / - SD of triplicate wells at each antibody concentration and represent three independent experiments. [Figure 17]Humanized anti-BTLA 2.8.6 suppresses CD4 T cell proliferation in a mixed leukocyte reaction. Purified primary human T cells from blood bank donors were stained with a cell proliferation tracking dye and cocultured with allogeneic monocyte-derived dendritic cells from a different donor at a 4:1 ratio for 5 days in the presence of a BTLA agonist antibody or hIgG1 P238D isotype control. Cell populations were identified by flow cytometry, and proliferation was assessed by dilution of the tracking dye. CD4 proliferation in the presence of BTLA antibody was normalized to proliferation in the presence of an equivalent concentration of isotype control. Data were collated from six independent experiments using different donor pairs. 2.8.6 significantly suppressed CD4 T cell proliferation in the P238D isotype but not in other isotype formats. The prior art molecule 22B3 had no significant effect on CD4 proliferation. [Figure 18] Humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype inhibit primary B cell activation in response to the TLR9 agonist ODN2006. Primary human B cells were isolated from PBMCs of healthy donors and stimulated with 0.01 μM ODN2006 in the presence or absence of different doses of P238D isotype control antibody or selected BTLA agonist antibodies. Five days later, IL-10 concentrations in the supernatants were assessed by ELISA. Bars represent the mean + / - SD of triplicate wells at each antibody concentration, representing three independent experiments. [Figure 19] Humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype significantly reduce weight loss in a xenograft-versus-host disease model. Irradiated NSG mice were reconstituted IV with 10 million human PBMCs on day 0 and then treated IP with 10 mg / kg of BTLA antibody or P238D isotype control on day 1. Mice were weighed periodically, and weights were plotted against starting body weights (n=9 mice per group, data points represent mean + / - SD). [Example]

[0404] In the following examples, antibodies such as 11.5.1 and 2.8.6 are shown to bind human BTLA with high affinity. Using transgenic mice expressing the human receptor, these antibodies are shown to suppress T cell responses in vitro and in vivo after binding to BTLA and to ameliorate disease in mouse models of inflammatory bowel disease and graft-versus-host disease. While these agonistic effects depend on Fc receptor binding, the antibodies do not cause depletion of BTLA-expressing cells via cytotoxicity or induce receptor downregulation. Introducing the P238D modification into the heavy chain significantly enhances FcγR2B agonist signaling, increasing the ratio of FcγR2B signaling to FcγR2A. Such dual BTLA and FcγR2B agonist antibodies are expected to be therapeutically useful, particularly in the context of autoimmune and inflammatory diseases.

[0405] Example 1. Generation and sequencing of anti-BTLA antibodies The antibody that recognizes the human immune cell receptor BTLA is the extracellular domain of human BTLA (BTLA K31-R151 BTLA was generated by BioGenes GmbH via immunization of mice with 2.8.6. Splenocytes from immunized mice were fused with Sp2 / 0-Ag14 myeloma cells, and the resulting hybridomas were selected for reactivity with human BTLA by ELISA of the supernatants in conjunction with dilution cloning. Antibodies from hybridoma supernatants were isotyped using a rapid mouse isotyping kit (RayBiotech). Antibodies produced by clones 2.8.6 and 11.5.1 were both found to be IgG1k.

[0406] To sequence the immunoglobulin variable domains, RNA was extracted from the hybridomas using TRIzol Reagent (ThermoFisher) according to the manufacturer's instructions. The RNA was reverse transcribed to cDNA using primers specific for the first constant domain of the heavy chain or the constant domain of the light chain and SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

[0407] PCR was then performed using primers targeting conserved regions of the immunoglobulin locus, as previously described (Tiller et al., J Immunol Methods. 350:183-193, 2009), and the PCR products were sequenced. In some cases, identification of functional light chains was complicated by the abundance of nonfunctional kappa light chain cDNAs derived from fused myeloma cell lines. To resolve this, we used a previously described technique in which primers specific for the nonfunctional chain CDR3 were added in excess to force cleavage of the aberrant chain products (Yuan et al., J Immunol Methods. 294:39553-61, 2005).

[0408] The variable domain sequences were evaluated using the NCBI IgBlast tool to determine the locations of the CDRs.

[0409] Example 2. Binding to soluble human and cynomolgus BTLA The binding affinity and kinetics of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to human or cynomolgus monkey BTLA were determined by surface plasmon resonance using a Biacore T200 (GE Healthcare). A series S CM5 sensor chip (GE Healthcare) was coated with polyclonal anti-mouse IgG using a mouse antibody capture kit (GE Healthcare). The anti-BTLA antibody was then captured on the biosensor surface, and a negative control antibody (clone Mopc21; Biolegend) was captured in the reference channel. Varying concentrations of monomeric soluble human BTLA extracellular domain (BTLA) were then captured on the biosensor surface. K31-R151 ) (from SEQ ID NO: 225) or soluble cynomolgus monkey BTLA extracellular domain (BTLA K31-R151) (from SEQ ID NO: 226) was injected over immobilized antibody in buffer (10 mM Hepes, 150 mM NaCl, 0.005% (v / v) surfactant P20, pH 7.4 (HBS-P) at 37°C in a single cycle kinetic analysis (Figure 1a). After reference and blank subtraction, the analysis was performed using BiaEvaluation software (GE The association and dissociation rates were fitted using a FTIR (Finite Fluorescence Integrity Modeling) and dissociation constants were calculated (Figure 1b). Clone 2.8.6 bound to human BTLA with a KD of 0.65 nM and to cynomolgus monkey BTLA with a KD of 7.89 nM. Clone 11.5.1 bound to human BTLA with a KD of 0.75 nM and to cynomolgus monkey BTLA with a KD of 0.99 nM. In a separate experiment with human BTLA alone, clone 2.8.6 bound to human BTLA with a KD of 0.37 nM, and clone 11.5.1 bound to human BTLA with a KD of 0.53 nM.

[0410] Example 3. Binding to BTLA on cells The ability of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to bind to cell surface-expressed human or cynomolgus monkey BTLA was assessed by flow cytometry. Full-length human or cynomolgus monkey BTLA was expressed in Jurkat T cell lines using a lentiviral transfection system. 1 x 10 cells per well were used. 5Cells were seeded into 96-well U-bottom plates. BTLA antibody binding versus mIgG1 isotype control (clone MOPC-21, Biolegend #400165) was assessed at 12 concentrations by serial 1 / 3 dilutions (starting at 90 μg / ml) in FACS buffer (PBS, 2% FCS, 0.05% sodium azide). Nonspecific antibody binding was prevented by adding Fc block (Biolegend #101319). The antibody was incubated with the cells for 30 minutes on ice, and then the cells were washed twice with FACS buffer before staining with AF647-conjugated anti-mIgG1 secondary antibody (Biolegend #406618). After the secondary antibody incubation for 30 minutes on ice, the cells were washed, resuspended in FACS buffer, and analyzed on a flow cytometer. The geometric mean fluorescence intensity of the secondary antibodies was plotted for each concentration, and the EC50 for receptor binding was calculated by nonlinear curve fitting using GraphPad Prism software. Clone 11.5.1 bound to human BTLA-expressing cells with an EC50 of 0.016 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.0057 nM. Clone 2.8.6 bound to human BTLA-expressing cells with an EC50 of 0.085 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.16 nM (Figure 1c-d).

[0411] Example 4. Competition with the natural ligand HVEM for binding to BTLA The ability of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to block the binding of the natural ligand to BTLA was assessed by surface plasmon resonance using a Biacore T200 (GE Healthcare). 31K-151R) was covalently coupled to a CM5 sensor chip using amine coupling. Human HVEM extracellular domain fused to mouse IgG1 Fc was then injected over immobilized hBTLA in HBS-P buffer at 37°C and allowed to dissociate completely. A saturating amount of anti-BTLA antibody (2.8.6 or 11.5.1) was then injected, immediately followed by a second injection of human HVEM-mFc at the same concentration as the first injection (Figure 2A). Equilibrium HVEM binding (in resonance units) after saturation of BTLA with antibody was expressed as a percentage of binding before antibody injection (Figure 2B). An antibody was considered non-blocking if HVEM binding after saturation with antibody was greater than 90% of binding before antibody injection.

[0412] Example 5. Binding Epitope of Antibody 11.5.1 on Human BTLA The functional epitope of antibody 11.5.1 on human BTLA was determined by flow cytometric assessment of binding to a panel of single-residue mutants of the cell surface-expressed receptor. A construct encoding the human extracellular domain of BTLA along with the transmembrane and intracellular domains of mouse CD28 was cloned into pGFP2-n2 (BioSignal Packard Ltd), a bicistronic mammalian expression vector that also encodes GFP. A "dramatic" mutagenesis approach (Davis et al., Proc Natl Acad Sci USA. 95, 5490-4 (1998)) was used to prepare mutant constructs differing by a single amino acid. Plasmids (2 μg / well) were transfected into HEK-293T cells in six-well plates using Genejuice transfection reagent (Novagen; 6 μl / well). Mock and no-transfection controls were included in each experiment. Cells were harvested at 48 hours and stained with 10 μg / ml of fluorochrome-conjugated anti-BTLA antibody in PBS, 0.05% azide, and 2% FCS (FACS buffer) along with a Live / Dead marker for 1 hour at 4°C. Cells were washed, pelleted, resuspended in 200 μl of FACS buffer, and then analyzed on a BD FACSCanto flow cytometer. GFP-positive (transfected) viable cells were gated and analyzed for anti-BTLA antibody binding (Figure 3a shows an example of binding analysis for clone 11.5.1). For each mutant, the geometric mean of anti-BTLA antibody binding to transfected cells was expressed as a percentage of binding to the wild-type receptor (Figure 3b). A panel of anti-BTLA antibodies was evaluated, and any mutations that eliminated binding of all antibodies were excluded from the analysis, under the assumption that such mutations result in dramatic changes in protein folding or expression rather than revealing an antibody epitope. Mutations Y39R and K41E completely abolish binding of antibody 11.5.1, while binding of 2.8.6 remains unaffected.These mutations are mapped onto the human BTLA crystal structure (Compaan et al., J. Biol. Chem. 280:39553-61, 2005) (residues in black) in Figure 3c, showing the binding epitope of 11.5.1. Residues required for HVEM binding (Gln37, Arg42, Pro59, His127; from patent publication WO 2017 / 004213) are mapped onto the structure in gray, showing that 11.5.1 binds to an epitope very close to the HVEM binding site.

[0413] Example 6. Crystal structure of the Fab' fragment of 2.8.6 in complex with human BTLA The structural epitope of antibody 2.8.6 on human BTLA was determined by solving the crystal structure of the antibody Fab complexed with the human BTLA extracellular domain. The heavy and light chain variable domains of antibody 2.8.6 were cloned into the pOPINVH and pOPINVL expression vectors (Addgene), which encode the first constant domain of the mouse IgG1 heavy chain (with a 6x histidine tag) and the constant domain of the mouse Ig kappa chain, respectively. These vectors were transiently co-transfected into HEK293T cells to produce the anti-BTLA 2.8.6 Fab' fragment, which was purified by Ni-NTA purification. The human BTLA Ig-V set domain (BTLA) S33-D135) was cloned into the pGMT7 vector and expressed in BL21(DE3)pLysS E. coli cells (Novagen) to produce inclusion bodies. Inclusion bodies were isolated from the cell pellet by sonication and washed repeatedly with a washing solution containing 0.5% Triton X-100. Purified BTLA inclusion bodies were solubilized in a denaturant solution containing 6 M guanidine hydrochloride. The solubilized protein solution was slowly diluted to a final protein concentration of 1-2 μM in refolding buffer [0.1 M Tris-HCl (pH 8.0), 0.6 M L-arginine, 2 mM ethylenediaminetetraacetic acid, 3.73 mM cystamine, and 6.73 mM cysteamine] and then stirred at 4 °C for 48 h. The refolded BTLA mixture was then concentrated using a VIVA FLOW50 system (Sartorius). BTLA was purified by gel filtration on a Superdex75 column (GE Healthcare).

[0414] Purified BTLA and Fab' were mixed and purified as a complex by size-exclusion chromatography. Crystals suitable for data collection were obtained by hanging-drop vapor diffusion at 293°K in 0.2 M calcium acetate, 0.1 M imidazole (pH 8.0), and 10% (w / v) PEG 8000. The final data set was collected using Photon Factory, and the structure was determined by molecular replacement using the structure of BTLA (PDB ID: 2AW2 A chain) and anti-PD1-Fab (PDB ID: 5GGS chains C and D) as search probes.

[0415] Residues on BTLA at the interface with antibody 2.8.6 are A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, D135.

[0416] Example 7. Development of humanized BTLA mice To provide a platform for evaluating anti-human BTLA antibodies in a mouse model, we developed a knock-in strain of C57B1 / 6 mice expressing a chimeric form of BTLA with a human extracellular domain and mouse transmembrane and signaling domains. A section of human genomic DNA from the beginning of exon 2 to the end of exon 3 was inserted into the mouse locus, replacing the mouse sequence from the beginning of exon 2 to the end of exon 4. The exon-intron junction sequences at the beginning of mouse exon 2 and the end of mouse exon 4 were left intact to ensure proper splicing (Figure 5).

[0417] Example 8. Suppression of antigen-specific T cell proliferation in vivo The ability of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to suppress antigen-specific T cell proliferation in vivo was assessed using a sensitive T cell transfer assay (Figure 6a). In this assay, purified OTII (TCR transgenic) CD4 T cells specific for ovalbumin (OVA) from mice expressing homozygous human BTLA (hBTLA) and from OT-II mice expressing the wild-type murine BTLA receptor were transfected with BTLA. + A mixture of T cells containing 5 x 10 5T cells were transferred into non-transgenic C57BL / 6 recipients. Transferred cells were distinguished from host cells using the CD45.2 (vs. CD45.1) allotypic marker. Wild-type donor cells also expressed green fluorescent protein under the control of the human ubiquitin C promoter, allowing them to be distinguished from humanized donor cells by flow cytometry. The day after T cell transfer, recipient mice were immunized with 100 μg of ovalbumin (Sigma-Aldrich) in 100 μl of PBS mixed with 100 μl of Imject Alum (ThermoFisher) to induce T cell proliferation. On day 2, mice were intraperitoneally administered 200 μg of antibody. Eight days after the initial T cell transfer, the ratio of humanized BTLA-expressing T cells to wild-type OVA-specific T cells in the spleen was determined by flow cytometry. In this way, it was possible to track the proliferation or shrinkage of humanized cells that bound anti-human BTLA antibodies compared to non-binding wild-type controls. Antibodies 2.8.6 and 11.5.1 both resulted in reduced proliferation of humanized BTLA cells compared to wild-type controls, indicating that they induce signaling through the inhibitory BTLA receptor, which results in reduced T cell proliferation (Figure 6b).

[0418] Example 9. Inhibition of T cell proliferation in mixed lymphocyte reaction The ability of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to suppress proliferation of primary T cells from humanized mice in vitro was assessed using a mixed lymphocyte reaction (MLR). Splenocytes from Balb / c mice were The cells were treated with mitomycin C for 30 minutes at 37°C, then washed and used as stimulator cells. T cells were purified from the spleens of humanized BTLA mice by negative selection using magnetic-activated cell sorting (Mojosort Mouse CD3 T Cell Isolation Kit, Biolegend #480023), stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher), and used as responder cells. 4 × 10 cells per well were used. 5 stimulator cells and 2 x 105 Responder cells were mixed in 96-well U-bottom plates containing various concentrations of anti-BTLA antibody or isotype control antibody (clone MOPC-21, Biolegend #400165). Serial dilutions of the antibody were evaluated, starting at 1 μg / ml, for a total of 10 concentrations. Polyclonal anti-mHVEM antibody (R&D Systems #AF2516) was also added to all wells at 101 μg / ml to block any baseline signaling through the BTLA pathway and highlight the effects of agonist antibodies. After 96 hours, responder cells were assessed by flow cytometry for dilution of CellTrace Violet, a marker of proliferation. Proliferation in the presence of anti-BTLA antibody or isotype control was compared to proliferation in the absence of antibody. CD4 + Population and CD8 + Populations were gated out and analyzed separately. Antibodies 2.8.6 and 11.5.1 both reduced proliferation of human BTLA-expressing T cells, indicating that they induce inhibitory signaling through the human BTLA receptor. Clone 2.8.6 suppressed CD4 T cells with an IC50 of 0.029 nM, with a maximal effect of 42% suppression of proliferation (Figure 7). Clone 11.5.1 suppressed CD4 T cells with an IC50 of 0.016 nM, with a maximal effect of 33% suppression of proliferation.

[0419] Example 10. Inhibition of NFκB signaling in human BTLA or cynomolgus monkey BTLA-transfected Jurkat T cell lines The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit NFκB signaling was evaluated using a BTLA-transfected reporter T cell line. A Jurkat T cell line stably transfected with an expression cassette containing an NF-κB-responsive transcription element upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences No. TR850A-1) was used as a reporter cell line for NFκB signaling. Full-length human or cynomolgus monkey BTLA was expressed in this reporter cell line using a lentiviral transfection system. These cells were mixed with a stimulator cell line consisting of bw5147 cells expressing an anti-CD3 ScFv construct on their surface, as described in Leitner et al. J Immunol Methods. 2010 Oct 31;362(1-2):131-41. The stimulator cell line was also transfected with mouse FcγR2B to provide Fc receptors for presentation of the agonist BTLA antibody. 5 × 10 cells per well were cultured. 4 5 × 10 reporter cells were plated in 96-well U-bottom plates in the presence of various concentrations of BTLA antibody or isotype control (clone MOPC-21, Biolegend #400165). 4 The cells were incubated at 37°C for 24 hours, then pelleted and stained with a viability dye for flow cytometry. Stimulator (mouse) cells were separated from responder (human) cells by staining with a mouse CD45 antibody (Zombie Aqua, Biolegend #423101) and a mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830). The geometric mean of GFP expression was assessed for each antibody concentration and normalized to GFP expression in the absence of antibody. Clone 2.8.6 inhibited human BTLA-transfected cells with an IC50 of 0.06 nM and cynomolgus BTLA-transfected cells with an IC50 of 0.22 nM. Clone 11.5.1 inhibited human BTLA-transfected cells with an IC50 of 0.033 nM and cynomolgus BTLA-transfected cells with an IC50 of 0.14 nM.

[0420] Example 11. Treatment of a T cell-driven mouse model of colitis with antibody 2.8.6 The ability of the BTLA agonist antibody 2.8.6 to ameliorate a T cell-driven model of colitis was evaluated using humanized mice. This T cell transfer model has been previously described as a murine model of inflammatory bowel disease (Ostanin et al., Am J Physiol Gastrointest Liver Physiol. 296:G135-46, 2009). CD45RB T cells sorted from the spleen and lymph nodes of humanized BTLA mice were analyzed. hi CD25-CD4+ T cells were cultured in Rag1 KO recipients (Rag1 tm1Mom ; Jackson Laboratory) at 5 × 10 per mouse. 5 The mice were injected intraperitoneally at a dose of 2.8.6 cells. The transferred T cells induce inflammatory bowel disease, which develops approximately 3 weeks later, resulting in diarrhea and weight loss. Rag1 KO cagemates that did not receive the transferred T cells served as unaffected controls. Recipient mice were injected intraperitoneally with 200 μg of 2.8.6 or isotype control antibody at 7, 21, and 35 days after T cell transfer. All mice were weighed periodically, and at 8 weeks, colons were weighed and measured to assess inflammatory infiltrates histologically, as well as by cell counting and flu counting of extracted lamina propria leukocytes. Flow cytometry was performed. Antibody 2.8.6 prevented weight loss (Figure 8a) and significantly reduced inflammatory infiltrates in the colon (Figure 8b). Colon inflammation in diseased mice resulted in an increased colon weight:length ratio, but not in 2.8.6-treated mice (Figure 8c).

[0421] Example 12. Treatment of a mouse model of graft-versus-host disease (GVHD) The effects of anti-BTLA agonist antibodies were evaluated in a non-lethal parent-into-F1 model of GVHD. Bone marrow cells (BMC) and splenocytes were harvested from humanized BTLA donor mice (C57BL / 6 background; H2B). 2 × 10 7 10 BMCs and 10 splenocytes were lethally irradiated with 9 Gy total body irradiation. / d ) were injected intravenously into recipients. Irradiated CB6F1 mice reconstituted with syngeneic BMCs and splenocytes served as unaffected controls. On the day of immune cell transfer, mice were intraperitoneally injected with 200 μg of anti-BTLA antibody or isotype control. GVHD was monitored by weighing mice periodically and calculating relative weight loss and by clinical observation. Mice were culled 5 weeks after immune cell transfer or when they reached a humane endpoint (including a weight loss of more than 20% relative to baseline weight within the first 14 days or a weight loss of more than 15% at any other time point). Colons were weighed at the time of death, and the colon weight:length ratio was calculated as a marker of colonic inflammation, which is a hallmark clinical feature of GVHD. Both antibodies 2.8.6 and 11.5.1 significantly reduced weight loss, resulting in increased survival (Figure 9a), and prevented colonic inflammation (Figure 9b).

[0422] Example 13. Agonist activity of antibody 11.5.1 is dependent on Fc receptor binding Antibody 11.5.1 was recombinantly expressed as mIgG1k containing the D265A mutation, previously described to significantly reduce Fc receptor binding (Clynes et al., Nat Med. 6:443-446, 2000). This mutant antibody was evaluated in the T cell transfer assay described in Example 8. The parental 11.5.1 antibody inhibited proliferation of humanized T cells, as its net effect is agonism of the BTLA receptor. However, the FcR null D265A mutation resulted in enhanced proliferation of humanized T cells, suggesting that the FcR null mutation removes the agonistic effect of the antibody, leaving only the effect of receptor blockade (Figure 10a).

[0423] The D265A mutant 11.5.1 antibody was also evaluated in the in vitro MLR assay described in Example 9. Here, too, the parental 11.5.1 antibody inhibited proliferation of humanized T cells because its net effect is agonism of the BTLA receptor. Because the FcR-null D265A mutation eliminates the agonist effect of the antibody, this antibody had no effect in this assay (Figure 10b). The FcR-null 11.5.1 antibody did not enhance proliferation of humanized cells in this assay because HVEM was blocked (by the addition of a polyclonal anti-HVEM antibody), and therefore there was no baseline signaling through the pathway blocked by the BTLA-blocking antibody.

[0424] Example 14. Antibodies 2.8.6 and 11.5.1 do not fix complement in vitro. Splenocytes from humanized mice were incubated with 10% baby rabbit complement (BioRad) and 20 μg / ml anti-BTLA antibody (or an isotype control or a positive control depleting anti-CD20 antibody; clone SA271G2 from Biolegend) for 15 minutes at 37°C. + Although anti-BTLA antibodies depleted the majority of B cells, they also depleted B220 + or CD4 + Neither of these cells was depleted, even though both stained positive for BTLA (Fig. 11).

[0425] Example 15. Antibodies 2.8.6 and 11.5.1 do not induce ADCC in vitro Whole splenocytes (including myeloid effector cells) from humanized mice were incubated with 20 μg / ml of anti-BTLA antibody (or isotype control or depleting anti-CD20 antibody SA271G2) for 24 hours at 37°C. + Although anti-BTLA antibodies depleted most of the cells, B220 + or CD4 + Neither of these cells was depleted, even though both stained positive for BTLA (FIG. 12).

[0426] Example 16. Antibodies 2.8.6 and 11.5.1 do not deplete BTLA-expressing cells in vitro. Humanized BTLA mice were injected intraperitoneally with 200 μg of anti-BTLA antibody or isotype control. At 24 hours, spleens were harvested and the frequencies of different cell populations were determined by flow cytometry. Anti-BTLA antibody had no effect on the frequency or absolute numbers of splenic B or T cells or on the number of B cell precursors in the bone marrow ( FIG. 13 ).

[0427] Example 17. Antibodies 2.8.6 and 11.5.1 stabilize BTLA expression in immune cells in vivo Humanized mice were injected intraperitoneally with 10 mg / kg of antibody 2.8.6 or 11.5.1. Six days after injection, mice were humanely sacrificed, and spleens were harvested, processed into single-cell suspensions, and evaluated by flow cytometry. Cells were stained with a cocktail of antibodies to identify immune cell subsets and with a fluorescently conjugated anti-BTLA antibody bearing an epitope that does not compete with the injected antibody. The geometric mean of BTLA staining after in vivo incubation with the anti-BTLA antibody was normalized to the geometric mean of BTLA staining after incubation with the isotype control (using the same staining antibody). BTLA expression was significantly higher in B cells and CD4 T cells from mice injected with either clone 2.8.6 or 11.5.1 compared to mice injected with the isotype control (Figure 14). This suggests that clones 2.8.6 and 11.5.1 stabilize BTLA expression on the cell surface in vivo rather than inducing receptor downregulation, as observed with other BTLA antibodies in the prior art (M.-L. del Rio et al. / Immunobiology 215(2010)570-578). For immunosuppressive purposes, agonistic antibodies that stabilize receptor expression offer the advantage over downregulating antibodies of allowing prolonged, high-level inhibitory signaling through the pathway.

[0428] Example 18. Tolerability and side effects in animal models There were no tolerability issues or side effects in any animal studies with antibody 2.8.6 or 11.5.1.

[0429] Example 19. Characterization of exemplary BTLA antibodies This Example describes the characterization of exemplary mIgG1 BTLA antibodies provided herein in addition to 2.8.6 and 11.5.1. Various clones listed in Tables 1 and 2 were evaluated for their binding affinity to BTLA and efficacy in suppressing lymphocytes (Table 3).

[0430] [Table 1-1]

[0431] [Table 2-1]

[0432] For each antibody, the association rate ("on rate") and dissociation rate ("off rate") for binding to human BTLA, as well as the KD for binding to human or cynomolgus monkey BTLA, were measured according to the method described in Example 2, and curves were fitted for injection of the BTLA extracellular domain at a single concentration. The inhibitory efficiency of each antibody on T cells was also evaluated at a single concentration of 10 μg / ml. MLR assays were performed for each individual antibody according to the method described in Example 9 (Table 4, two biological repeats). Anti-CD3 assays were performed according to the method described below (Table 4, two biological repeats). Inhibition of NFκB signaling in a human BTLA-transfected Jurkat T cell line by each antibody was determined according to the method described in Example 10 (Table 4). The average inhibition of T cells compared to the isotype control in various in vitro stimulation assays for each exemplary antibody was calculated as the average of the percent inhibition across all assay results (Tables 3 and 4).

[0433] [Table 3]

[0434] [Table 4]

[0435] The ability of BTLA agonist antibodies to inhibit anti-CD3 and anti-CD28-induced T cell activation was assessed as follows. Splenocytes from humanized BTLA mice were processed into a single cell suspension and treated with ACK buffer to lyse red blood cells. Cells were stained with CFSE (Biolegend catalog number 423801) to allow tracking of cell proliferation. 2 x 10 cells per well were cultured. 5Cells were seeded into 96-well U-bottom plates containing soluble anti-CD3 antibody (clone 145.2C11; Biolegend #100339) and anti-CD28 antibody (clone 37.51; Biolegend #102115) at a concentration of 50 ng / ml each, and soluble anti-BTLA antibody or isotype control at a concentration of 10 μg / ml. After 72 hours, cells were analyzed by flow cytometry to assess proliferation ("anti-CD3 / CD28 (CD4 T cell proliferation)") and by staining for surface-expressed activation markers to assess T cell activation ("anti-CD3 / CD28 (CD69+ CD4 T cells)"). The percent inhibition was calculated for each BTLA antibody compared to the isotype control antibody.

[0436] Additionally, for each BTLA antibody, their ligand-blocking ability, e.g., competition with HVEM for binding to BTLA, was assessed according to the method described in Example 4, and the results are presented as "yes" for greater than 90% inhibition of HVEM-BTLA binding and "no" for less than 10% inhibition of HVEM-BTLA binding. The functional epitope of each BTLA antibody was also determined according to the method described in Example 5. The "Epitope" column in Table 3 summarizes the epitopes bound by each individual BTLA antibody. Antibodies 2.8.6, 6.2, 831, 16H2, 7A1, 16F10, 6G8, 3E8, 4E8, 15C6, 12F11, 10B1, 15B6, 4D3, 16E1, 4D5, and 3A9 all bind to a first epitope (designated "Epitope 1" in the table) that includes at least one key residue selected from the list: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225). Antibodies that bind to Epitope 1 do not compete with the ligand HVEM for binding to BTLA. Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, and 26F3 all bind to a different, second epitope ("epitope 2") that includes at least one key residue selected from the list: Y39, K41, R42, Q43, E45, and S47. Antibodies that bind to epitope 2 compete with the ligand HVEM for binding to BTLA. Antibody 26B1 binds to a third epitope ("epitope 3") that includes at least one key residue selected from the list: D35, T78, K81, S121, and L123. Antibodies that bind to epitope 3 compete with the ligand HVEM for binding to BTLA. Antibodies 24H7, 4B1, 8B4, and 4H4 all bind to a different, fourth epitope ("epitope 4") that includes the key residue H68. Antibodies that bind to epitope 4 do not compete with the ligand HVEM for binding to BTLA. Antibody 21C7 binds to a different fifth epitope ("epitope 5") that includes at least one key residue selected from the list: N65 and A64. Antibodies that bind to epitope 5 do not compete with the ligand HVEM for binding to BTLA.

[0437] Example 20. Humanization and CDR Engineering of BTLA Antibodies 6.2, 2.8.2, and 3E8 Antibody 2.8.6 was humanized by CDR grafting into homologous human germline framework regions (see SEQ ID NOs: 26, 27). IGHV2-5 * 08 was used for the heavy chain, and IGKV3-11 * 01 was used for the light chain. After humanization, binding to BTLA was assessed by SPR. Humanized 2.8.6 had a K of 0.73 nM. D bound to monomeric BTLA.

[0438] The variable domains of 6.2 and 3E8 were humanized by germlining to homologous human germline framework regions (SEQ ID NOs: 7, 8, and 36, 37). For 3E8, the selected acceptor frameworks were VH1-1-08 and JH6 for the heavy chain and VK3-L6 and JK2 for the light chain. For 6.2, the selected acceptor frameworks were VH3-3-21 and JH6 for the heavy chain and VK2-A19 and JK4 for the light chain.

[0439] It may be possible to substitute certain residues in the CDRs or variable domain framework regions of an antibody to eliminate undesirable characteristics without significantly affecting target binding. CDRH2 of humanized antibody 6.2 was modified with N56Q alone (SEQ ID NO: 17) or with N56Q and D54E substitutions (SEQ ID NO: 11) to eliminate deamidation and isomerization potential, respectively. CDRL2 of humanized 6.2 was modified with a D61E substitution to reduce predicted immunogenicity as determined by Lonza's Epibase analysis (SEQ ID NO: 12). Outside the CDRs, a S77T substitution was introduced into the heavy variable framework region of humanized 6.2 to reduce predicted immunogenicity, and a Q51K substitution was introduced into the light chain variable framework region to reduce immunogenicity. Three engineered variants of humanized 6.2 containing different combinations of these substitutions were generated (engineered humanized 6.2 "Variant A," "Variant B," and "Variant C"). Table 2 lists the constituent CDRs and variable domains for each of these variants. Engineered variants of antibody 6.2 containing a CDRH2 that has only the N56Q and no D54E substitution (e.g., engineered humanized 6.2 variant C) are not disclosed in PCT / GB2019 / 053569.

[0440] Similarly, CDRH2 of humanized antibody 3E8 was modified with an N57Q substitution to eliminate deamidation potential and a K63S substitution to reduce predicted immunogenicity (SEQ ID NO: 40). Outside the CDRs, G42D and A61S substitutions were introduced into the light chain variable framework of 3E8 to reduce predicted immunogenicity. Additionally, P15L and P81A substitutions were introduced into the light chain variable framework to revert these positions to the mouse sequence instead of introducing a proline, which may affect local conformation. The sequence of the engineered 3E8 light chain variable domain containing all four of these substitutions is shown in SEQ ID NO: 43. Table 2 lists the constituent CDRs and variable domains for the engineered variants of humanized 3E8.

[0441] Example 21. Binding of humanized anti-BTLA antibodies to soluble human and cynomolgus monkey BTLA The binding affinity and kinetics of humanized BTLA agonist antibodies to human or cynomolgus monkey BTLA were determined by surface plasmon resonance using a Biacore 8K (GE Healthcare). A series S CM5 sensor chip (GE Healthcare) was coated with polyclonal anti-human IgG using a human antibody capture kit (GE Healthcare catalog number 29234600). The anti-BTLA antibody was then captured on the biosensor surface, and a negative control antibody (human IgG1k isotype control; Sino Biological catalog number HG1K) was captured in the reference channel. Various concentrations of monomeric soluble human BTLA extracellular domain (BTLA) were then applied to the biosensor surface. K31-R151 , in-house recombinant production) or soluble cynomolgus monkey BTLA extracellular domain (BTLA K31-R151, recombinantly produced in-house) was injected over immobilized antibody in buffer HBS-EP (GE Healthcare, catalog no. BR100669), pH 7.4 (HBS-P) at 37°C in a single-cycle kinetic analysis. For human BTLA, concentrations ranging from 673 nM to 164 pM were used in a four-fold serial dilution. For cynomolgus BTLA, concentrations ranging from 1351 nM to 330 pM were used in a four-fold serial dilution. After reference and blank subtraction, the association and dissociation rates were fitted and dissociation constants were calculated using BiaEvaluation software (GE Healthcare) (Table 5). Humanized 2.8.6 binds to human BTLA with a KD of 2.33 nM and to cynomolgus BTLA with a KD of 147 nM. Humanized 3E8 variant B (3E8_var_B) binds to human BTLA with a KD of 141 nM and to cynomolgus BTLA with a KD of 1520 nM. Humanized 6.2 variant A, which contains both D54E and N56Q substitutions in CDRH2 to eliminate deamidation and isomerization potential, respectively, binds to human BTLA with a KD of 10.9 nM and to cynomolgus BTLA with a KD of 695 nM. This binding represents a significant reduction in affinity from the parent clone 6.2 antibody, which binds to human BTLA with a KD of 1.7 nM and to cynomolgus BTLA with a KD of 9.71 nM (Table 5). The humanized variant of 6.2, designated humanized 6.2 variant C (or 6.2_var_C), contains only the N56Q substitution in CDRH2, but not the D54E substitution, and binds to human BTLA with a KD of 1.25 nM and to cynomolgus BTLA with a KD of 15.4 nM, thus retaining affinity closer to that of the parent clone.

[0442] [Table 5]

[0443] Example 22. Binding of humanized anti-BTLA antibodies to BTLA in cells The ability of the BTLA agonist antibodies of the present invention to bind to cynomolgus monkey BTLA was assessed by flow cytometry. A lentiviral transfection system was used to express full-length human or cynomolgus monkey BTLA in Jurkat T cell lines. 1 x 10 cells per well were used. 5 Cells were seeded into 96-well U-bottom plates. BTLA antibody binding versus hIgG1k P238D isotype control (clone MOPC-21, recombinantly produced by Absolute Antibody; heavy chain SEQ ID NO: 230, light chain SEQ ID NO: 231) was assessed at 12 concentrations by serial 1 / 3 dilutions (starting at 30 μg / ml) in FACS buffer (PBS, 2% FCS, 0.05% sodium azide). Nonspecific antibody binding was prevented by adding Fc block (Biolegend #101319). The antibody was incubated with the cells for 60 minutes on ice, and then the cells were washed twice with FACS buffer before staining with AF647-conjugated anti-hIgG secondary antibody (clone HP6017; BioLegend catalog #409320). After a 30-minute incubation with the secondary antibody on ice, the cells were washed, resuspended in FACS buffer, and analyzed on a flow cytometer. The geometric mean fluorescence intensity of the secondary antibody was plotted for each concentration, and the EC50 for receptor binding was calculated by nonlinear curve fitting using GraphPad Prism software. Humanized 2.8.6 binds to human BTLA-expressing cells with an EC50 of 0.066 nM (Figure 15a) and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.854 nM (Figure 15b). Humanized 6.2_var_C binds to human BTLA-expressing cells with an EC50 of 0.062 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.148 nM. Humanized 3E8_var_B binds to human BTLA-expressing cells with an EC50 of 0.177 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 15.6 nM.

[0444] Example 23. Binding affinity of Fc variant antibodies to human Fc receptors Example 13 surprisingly demonstrated that the agonistic function of BTLA antibodies may depend on Fc receptor binding by the Fc portion of the antibody. In humans, one inhibitory Fc gamma receptor (FcγR2B) exists, while the other Fc gamma receptors all deliver immune activation signals (FcγR1A, FcγR2A, FcγR3A, and FcγR3B). We propose that selective Fc binding to FcγR2B may be necessary for BTLA agonist antibodies to be effective in suppressing immune responses without inducing inflammatory FcR signaling. More selective binding to FcγR2B would promote bidirectional inhibitory signaling through BTLA on BTLA-expressing cells and through FcγR2B on FcγR2B-expressing cells, enhancing the immunosuppressive effects of the antibody. This is desirable in therapeutic antibodies intended to treat diseases of immune hyperactivation. Conversely, very high affinity for FcγR2B can negatively impact antibody half-life due to receptor turnover in liver sinusoidal epithelial cells (Ganesan et al. The Journal of Immunology 189(10):4981-88, 2012). This is demonstrated by the FcγR2B-enhancing IgG1 antibody XmAb7195, which binds to FcγR2B with a KD of 7.74 nM (Chu et al. Journal of Allergy and Clinical Immunology 129(4):1102-15, 2012, https: / / linkinghub.elsevier.com / retrieve / pii / S0091674911018343 (May 13, 2020)), and according to Xencor, the average half-life of wild-type IgG1 is approximately 21 days (Morell, Terry, and Waldmann. Journal of Clinical Investigation 49(4):673-80, 1970; http: / / www.jci.org / articles / view / 106279 (May 16, 2020)), and a phase 1a trial reported a mean in vivo half-life of 3.9 days (American Thoracic Society (ATS) 2016 International Conference in San Francisco, CA-A6476: Poster Board Number 407). Thus, while sufficient binding to support selectivity and agonism for FcγR2B may be desirable for a BTLA agonist antibody, excessively high affinity for FcγR2B may be undesirable in therapy, as the resulting shortened half-life would likely require more frequent dosing.

[0445] A series of Fc-mutated antibody variants (containing the variable domains of humanized 2.8.6) were recombinantly produced and their binding to different human Fc gamma receptors was assessed by surface plasmon resonance (in HBS-EP+ buffer at pH 7.4 at 37°C). Fc variants were recombinantly produced into either the hIgG1 or hIgG4 backbone based on their location in the Fc-FcR binding interface, with substitutions known to affect FcR binding or likely to do so (hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, hIgG4 L235A). These mutations were evaluated as single substitutions or combinations of substitutions. Variants containing sections of sequence switched from hIgG2 (termed delta b, delta c, delta ab, and delta ac) were also evaluated, as described by Armour et al. (Molecular Immunology 40(9):585-93, 2003). Binding of mIgG1 and mIgG1 D265A to human FcR was also evaluated.

[0446] For low affinity FcγRs (FcγR2A, FcγR2B, FcγR3A, and FcγR3B), the interaction was assessed by surface plasmon resonance using recombinantly expressed FcR (extracellular domain only) as the analyte. Briefly, recombinant human BTLA extracellular domain (BTLA) was coupled to the FcγR1 protein using a GE Healthcare amine coupling kit. K31-R151) was covalently immobilized on both flow cells of all channels of a CM5 Series S sensor chip. The 2.8.6 Fc variant to be evaluated was then captured on flow cell 2 of each channel (approximately 500-1000 response units). Steady-state affinity analysis was then performed by injecting various concentrations of FcR for multiple cycles and measuring equilibrium binding. Double referencing was used (subtracting the signal from the reference FcR and also subtracting the signal from a blank injection of concentration 0). KD was calculated from the Langmuir curve (equilibrium binding was plotted against analyte concentration to determine the concentration required for half-maximal binding).

[0447] For high-affinity FcR interactions (FcγR1A and FcRn assessed at pH 6.0), binding was assessed by kinetic analysis using antibodies as analytes. Briefly, biotinylated FcR (Sino Biological, catalog number 10256-H08S-B for FcγR1A or catalog number CT009-H08H-B for FcRn) was captured on a streptavidin chip (Series S Sensor Chip SA-BR-1005-31) in flow cell 2 according to the provided protocol. Reference flow cell 1 was left empty in all channels. Purified antibodies were then injected at a single concentration, and on / off rates were calculated by curve fitting in BiaEvaluation software. FcRn interaction at pH 6.0 does not trigger inflammatory signaling but is required for sustained antibody half-life in vivo, and therefore, this interaction is desirable for therapeutic antibodies. Because IgG Fc has two binding sites for FcRn, this assessment, performed with high density immobilized FcRn, provides an avidity estimate for the interaction rather than the true KD.

[0448] The KD values ​​for each of the Fc variants binding to each of the human Fc receptors for which they were evaluated are shown in Table 6. The presence of the P238D mutation significantly enhanced selectivity for FcγR2B (by slightly increasing affinity for FcγR2B while dramatically decreasing affinity for other FcγRs). A previously described mutation combination containing P238D (P238D G237D P271G A330R), designated V9 (Mimoto et al., Protein Engineering, Design). and Selection 26(10):589-98, 2013) significantly increased binding affinity to FcγR2B, while retaining significant binding to the 131R polymorphic variant of FcγR2A. The same effect of increasing FcγR2B selectivity was seen when the P238D single substitution or combination substitutions were introduced into the hIgG4 backbone.

[0449] [Table 6]

[0450] Example 24. Inhibition of T cell activation by humanized BTLA agonists in an NFκB reporter assay is dependent on Fc receptor binding. BTLA is an inhibitory receptor expressed on T cells. Therefore, agonistic antibodies against BTLA are expected to suppress T cell activation by inducing inhibitory signaling through the receptor. The ability of selected humanized BTLA agonistic antibodies to suppress T cell activation was assessed using a BTLA-transfected reporter T cell line. A Jurkat T cell line stably transfected with an expression cassette containing an NF-κB-responsive transcription element upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences No. TR850A-1) was used as a reporter cell line for NFκB signaling. Full-length human BTLA was expressed in this reporter cell line using a lentiviral transfection system. These cells were mixed with a stimulator cell line composed of bw5147 cells expressing an anti-CD3 ScFv construct on their surface, as described by Leitner et al. (J Immunol Methods. 362(1-2):131-41, 2010). The stimulator cell line was also transfected with human FcγR2B to provide Fc receptors to present the agonist BTLA antibody. 4 5 × 10 reporter cells were cultured in 96-well U-bottom plates in the presence of various concentrations of BTLA antibody or hIgG1k isotype control antibody (Sino Biologicals cat#HG1K). 4 The cells were mixed with stimulator cells. Cells were incubated at 37°C for 24 hours, then pelleted and stained with a viability dye (Zombie Aqua, Biolegend #423101) and mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830) for flow cytometry to separate stimulator (mouse) from responder (human) cells. The geometric mean of GFP expression was assessed for each antibody concentration and normalized to GFP expression in the absence of antibody.

[0451] Humanized 2.8.6 was tested with the hIgG4 isotype, as well as the hIgG1 P238D and hIgG1 V9 (P238D G237D P271G A330R) isotypes. 2.8.6 hIgG1 P238D provided more effective inhibition of NFκB signaling than 2.8.6 hIgG4, and 2.8.6 hIgG1 V9 provided even more effective inhibition (Figure 15a). Thus, increased affinity for FcγR2B confers superior agonistic activity to BTLA agonist antibodies in conditions where FcγR2B is the only Fc receptor present. When the same antibodies were tested in a modified version of the assay in which stimulator cells did not express FcγR2B, no inhibitory effect of either antibody was observed, confirming that antibody agonism of BTLA is dependent on FcR binding by the antibody (Figure 15b). The 2.8.6, 6.2, and 3E8 mouse IgG1 parent antibodies were also able to suppress T cell activation in reporter assays when human FcγR2B was expressed on stimulator cells, consistent with the cross-reactivity between mIgG1 and hFcγR2B observed in Example 23.

[0452] Humanized 2.8.6, 6.2_var_C, and 3E8_var_B were all hIgG1 These humanized P238D variant antibodies were produced on the P238D isotype and compared in the T cell reporter assay described above. They were also compared to the prior art BTLA agonist 22B3 (expressed on the hIgG4PAA isotype) described in WO 2018 / 213113, and a fusion protein of the native BTLA ligand HVEM fused to the mIgG1 Fc region (hHVEM-mFc, uniquely recombinantly produced; the hHVEM-mFc fusion protein, including the signal peptide and C-terminal His tag, has the sequence disclosed in SEQ ID NO: 229). All three of the humanized P238D variant antibodies demonstrated significantly greater inhibition of NFκB signaling compared to 22B3 or hHVEM-mFc (Figure 16a). 3E8_var_B inhibited NFκB signaling by up to 54% with an IC50 of 65 pM. 6.2_var_C inhibited NFκB signaling by up to 47% with an IC50 of 28 pM. 2.8.6 inhibited NFκB signaling by up to 42% with an IC50 of 59 pM. 22B3 inhibited NFκB signaling by up to 18% with an IC50 of 3.8 nM. hHVEM-mFc inhibited NFκB signaling by up to 27% with an IC50 of 9.6 nM. Therefore, under conditions where FcγR2B was the only Fc receptor present, humanized 2.8.6 hIgG1 P238D, 6.2_var_C hIgG1 P238 and 3E8 hIgG1 P238D are all significantly more effective and potent agonists of BTLA than the prior art antibody 22B3 hIgG4PAA, and deliver a stronger signal than the endogenous ligand HVEM as an Fc fusion protein.

[0453] Example 25. Suppression of primary human T cell proliferation in a mixed lymphocyte reaction by humanized BTLA agonists The ability of selected BTLA agonist antibodies to suppress human T cell proliferation was assessed in the context of a mixed lymphocyte reaction (MLR). Briefly, human primary T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) using a Human Pan T Cell Isolation Kit (Miltenyi Biotec Catalog No. 130-096-535) and stained with the cell proliferation tracking dye Tag-it Violet (Biolegend Catalog No. 425101). Allogeneic monocyte-derived dendritic cells (DDCs) were then cultured. DCs were generated by culturing CD14+ monocytes isolated from PBMCs using a CD14+ isolation kit (Miltenyi Biotec catalog number 130-050-201). The CD14+ monocytes were treated with human recombinant IL-4 (Peprotech catalog number 200-04) and GM-CSF (Biolegend catalog number 572904) for 7 days. DC maturation was then induced for 2 days by further addition of human recombinant TNF-α (Biolegend catalog number 717904). Mature dendritic cells express both activating and inhibitory FcγRs (Guilliams et al.Nature Reviews Immunology 14(2):94-108,2014.http: / / www.nature.com / articles / nri3582(May 18,2020)).

[0454] Then, in a flat-bottom 96-well plate, 1 x 10 5MLR was performed by co-culturing total T cells with allogeneic mature DCs at a 4:1 (T:DC) ratio. T cells and DCs were incubated for 5 days without antibody or in the presence of different doses of BTLA agonist antibodies (2.8.6 hIgG1 P238D, 2.8.6 hIgG1 V9, 2.8.6 IgG4), hIgG1k isotype control antibody (Sino Biologicals catalog no. HG1K), or the prior art BTLA agonist 22B3 hIgG4PAA. After 5 days, T cell proliferation was assessed by flow cytometry. T cells were harvested and stained with anti-CD3 (PerCP / Cy5.5 conjugated clone OKT3, Biolegend catalog no. 317336), anti-CD4 (BB515 conjugated clone RPA-T4, BD Horizon catalog no. 564419), and anti-CD8 (BV510 conjugated clone SK1, BD Horizon catalog no. 563919) antibodies along with a viability dye (Zombie NIR, Biolegend catalog no. 423105) and acquired on a BD FACS Celesta instrument. CD4 proliferation in the presence of antibody (measured as the percentage of CTV-low cells) was normalized to the average proliferation in the absence of antibody. Figure 17 shows combined data from six separate MLRs using different PBMC donors. Antibody 2.8.6 on the hIgG1 P238D isotype significantly suppressed CD4 T cell proliferation, with a mean inhibitory effect of 51% at 10 μg / ml. Antibody 2.8.6, directed against either the hIgG1 V9 or hIgG4 isotype, had no inhibitory effect. Thus, unexpectedly, in the presence of multiple Fc receptors, the hIgG1 P238D isotype, which selectively binds to FcγR2B, confers superior agonistic activity to BTLA agonists compared with the other isotypes tested. The lack of efficacy in this setting of the hIgG1 V9 isotype, which binds to FcγR2B with approximately 30-fold greater affinity than the P238D isotype, may be due to activation signaling through FcγR2A(131R), to which it still retains significant binding.Alternatively, the ineffectiveness of the hIgG1 V9 isotype may be due to the stable formation of cis interactions between the antibody, BTLA, and FcγR2B on the same cell surface (e.g., on dendritic cells that express both receptors), which may not induce signaling but block the formation of productive trans interactions between the antibody, BTLA, and FcγR2B on different cells. The lower affinity of the P238D isotype for FcγR2B may mean that these cis interactions, if formed, are shorter-lived and do not completely block trans interactions.

[0455] 22B3 hIgG4PAA also has no suppressive effect in the mixed lymphocyte reaction and, in fact, tends to increase CD4 T cell proliferation, which may be explained by the antibody blocking natural inhibitory signaling through BTLA by interfering with its interaction with the ligand HVEM. Antibodies 6.2, 3E8, and 286 do not block BTLA-HVEM interaction because they bind to epitopes on BTLA that do not overlap with the HVEM-binding interface (Example 19).

[0456] Example 26. Inhibition of primary human B cell activation by BTLA agonists The ability of BTLA agonist antibodies to inhibit activation of primary human B cells was evaluated. B cells express high levels of both BTLA and FcγR2B.

[0457] Human primary B cells were isolated from healthy donor peripheral blood mononuclear cells using a human B cell isolation kit (Miltenyi Biotec cat# 130-050-301) and stained with the cell proliferation tracking dye Tag-it Violet™ (Biolegend cat#425101).

[0458] Then, 1 x 10 cells were cultured per well of a 96-well flat-bottom plate. 5B cells were stimulated with 0.01 μM of the TLR9 agonist ODN2006 (InvivoGen catalog no. tlrl-2006-1) in the presence or absence of different doses of an isotype control antibody or selected BTLA agonist antibodies. BTLA agonists 2.8.6, 6.2_var_C, and 3E8_var_B (all hIgG1 P238D isotype) were tested and compared with the prior art BTLA agonist 22B3 hIgG4PAA. Recombinant HVEM fusion protein (hHVEM-mFc, proprietary production) was used as a positive control. After 5 days of incubation at 37°C, B cells were harvested and stained with an anti-CD20 antibody (PE-CF594 conjugated clone 2H7, BD Horizon no. 562295) and a viability dye (Zombie NIR, Biolegend no. 423105), and their proliferation was assessed by flow cytometry. Additionally, culture supernatants were collected and the production of IL-6 (rndsystems catalog number DY206) and IL-10 (rndsystems catalog number DY217B) was assessed by ELISA.

[0459] Essentially following the procedure described above, the BTLA agonist antibodies were able to suppress B cell proliferation as efficiently as the hHVEM-mFc positive control. In addition, all three antibody variants demonstrated significantly greater suppression of B cell proliferation compared to 22B3. Furthermore, the P238D BTLA agonist impaired the production of IL-10 (FIG. 18) and IL-6 by activated B cells. Consistent with the proliferation data, the ability of the P238D BTLA antibody to suppress IL-10 and IL-6 production was greater compared to the 22B3 antibody.

[0460] Example 27. Treatment of xenogeneic models of graft-versus-host disease (GVHD) Prevention of human PBMC-induced graft-versus-host disease (GvHD) was determined in vivo.

[0461] Briefly, approximately 8-10 week old female NSG mice (JAX Labs, stock no. 05557) (n = 10 mice per treatment group) were irradiated with 2.4 Gy total body irradiation. Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks (HemaCare products ordered from Tissue Solutions) at 50 × 10 per ml of PBS. 6 One day after irradiation, mice were resuspended in 200 μl of cell suspension (10 × 10 6 PBMCs) are injected via tail vein (IV). The next day, mice are treated with 10 mg / kg of test antibody via intraperitoneal injection. Mice are weighed periodically and euthanized when they lose 15% weight or after 28 days. At the end of the study, human PBMC infiltration into the lungs, liver, and spleen is quantified by flow cytometry using the markers hCD45, hCD4, hCD8, hCD20, hCD25, and FOXP3.

[0462] Following the procedure described above, humanized 2.8.6 hIgG1 P238D, 6.2_var_C hIgG1 P238D, and 3E8_var_B hIgG1 P238D all significantly reduced weight loss compared to the hIgG1 P238D isotype control ( FIG. 19 ), resulting in a significant reduction in infiltrating human immune cells in the lung, liver, and spleen. A trend toward increased frequencies of regulatory T cells was also observed with all three BTLA agonists.

[0463] Example 28. In vivo half-life of P238D mutant hIgG1 antibody in cynomolgus monkeys and prediction of human half-life The in vivo half-life of 6.2_var_C against the hIgG1 P238D isotype was evaluated in cynomolgus monkeys. Two female macaques were intravenously injected with 3 mg / kg of antibody, and two female macaques were injected with 10 mg / kg of antibody. The macaques were bled before antibody infusion and at 1, 6, 24, 48, 72, 168, 240, 336, 432, and 504 hours after antibody infusion. The concentration of 6.2_var_C in serum at each of these time points was assessed by target capture ELISA. A 96-well microplate (Thermoscientific catalog number 439454) was coated with 100 μl of human BTLA extracellular domain at 1 μg / ml in PBS overnight at 4°C. The plate was then washed three times with wash buffer (PBS containing 0.05% Tween 20 (ThermoScientific catalog no. 28320)), and the wells were blocked with 300 μl of SuperBlock buffer (Thermoscientific catalog no. 37515) for 1 hour at room temperature, followed by washing three times again with wash buffer. 100 μl of serum samples diluted in ELISA buffer (PBS, 1% bovine serum albumin, 0.05% Tween 20) were then added and incubated for 1 hour at room temperature. An 11-point standard curve of known concentrations of 6.2_var_C in ELISA buffer was run in duplicate, with duplicate wells containing only ELISA buffer used as a blank. After incubation, the wells were washed three times with wash buffer, and then an HRP-conjugated anti-human detection antibody (Abcam catalog no. ab98624) diluted 20,000-fold in ELISA buffer was added and incubated for 1 hour at room temperature. The wells were again washed three times with wash buffer, and then 100 μL of Ultra TMB-ELISA substrate solution (ThermoScientific catalog number 34028) was added per well. The plate was covered with foil to ensure it was not under direct light and incubated for 90 seconds, and then 50 μL of stop solution (ThermoScientific catalog number SS04) was added per well. The absorbance at 450 nm was then read on a Thermo MultiSkan FC.Concentrations were interpolated from the standard curve using GraphPad Prism software.

[0464] Using serum antibody concentrations at each time point, pharmacokinetics in each monkey was fitted with a two-compartment model (Dirks et al. Clin. Pharmacokinet 49(10):633-659, 2010). The mean terminal half-life in macaques was calculated to be 5.4 days (130 hours). The model parameters (volumes of distribution V1 and V2, clearance C1, and intercompartmental exchange coefficient Q) were then scaled to humans using allometric scaling. Using allometric scaling, parameter 1 (body weight BW1) for one species was estimated from parameter 2 (body weight BW2) for another species using the following equation:

[0465]

number

[0466] For humans, a body weight of 70 kg was assumed. For cynomolgus monkeys, a reference body weight of 3 kg was used. Theoretical scaling exponents for large molecules were used: β = 1 for V1 and V2, β = 0.75 for Cl (as described by Kleiber et al. Hilgardia 6(11):315-333. 1932), and β = 2 / 3 for Q. For scaling of the intercompartmental exchange coefficient Q, it was assumed that the exchange rate of the compound depends on the surface area of ​​the vascular endothelium. This assumption is based on the implementation of intercompartmental exchange, which is written as follows: Q (c p-c t )=P·S·(c p -c t ) In the formula, c p -c t is the concentration difference across the vascular boundary, P is the vascular permeability coefficient (units: m / s), and S is the surface area of ​​the vasculature involved in the exchange (units: m 2 ) where vascular permeability P is assumed to be a molecular property and is species independent. The only difference between species is the surface area of ​​the blood vessels, which is scaled by body mass by a factor of 2 / 3. Using these arguments, the assumed scaling value for Q is 2 / 3. The predicted terminal half-life in humans was then calculated from the scaled parameters using a two-compartment model. The mean predicted half-life in humans was calculated to be 12.5 days (300 hours).

[0467] Certain embodiments of the present invention 1. An isolated antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising a substitution that results in increased binding to FcγR2B compared to the parent molecule lacking the substitution. 2. The antibody of embodiment 1, which has selectivity for binding to FcγR2B over FcγR2A compared to the parent molecule lacking the substitution. 3.Antibodies (i) enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR2A (R type) and / or FcγR2A (H type) compared to the parent polypeptide; and / or (ii) a value of [KD value of polypeptide variant for FcγR2A (R type)] / [KD value of polypeptide variant for FcγR2B] of 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more); and / or (iii) a value of [KD value of the polypeptide variant for FcγR2A (H type)] / [KD value of the polypeptide variant for FcγR2B] of 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more); and / or (iv) enhanced FcγR2B binding activity and maintained or reduced binding activity to FcγR1A compared to the parent polypeptide; and / or (v) An antibody described in embodiment 1 or 2, having a value of [KD value of polypeptide variant for FcγR1A] / [KD value of polypeptide variant for FcγR2B] of 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more). 4.Antibodies (i) D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (positions according to SEQ ID NO: 225), or (ii) Y39, K41, R42, Q43, E45, and S47, or (iii) D35, T78, K81, S121, and L123, or (iv) H68, or (v) binds to a residue in human BTLA selected from N65 and A64; The antibody of any one of embodiments 1 to 3, wherein each position is relative to the amino acid sequence disclosed in SEQ ID NO: 225. 5. An antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region comprising one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, or alanine (A) at position 297 (numbering according to the EU index). 6. The antibody of embodiment 5, wherein the heavy chain then comprises an Fc region comprising an aspartic acid at position 238 (EU index). 7. The antibody of any one of embodiments 1 to 6, which is an agonistic antibody. 8. The antibody of embodiment 6, wherein the antibody binds to FcγR2B with higher affinity than a control antibody comprising an Fc region containing a proline at position 238 (EU index). 9. The antibody of any one of embodiments 1 to 8, wherein the antibody binds to FcγR2B with an affinity of between about 5 μM and 0.1 μM as determined by surface plasmon resonance (SPR). 10. The method of any one of embodiments 1 to 9, wherein the antibody binds to FcγR2B with an affinity of up to 5 μM as determined by surface plasmon resonance (SPR). 11. The antibody of any one of embodiments 6 to 10, wherein the antibody binds to FcγR2A (131R allotype) with an affinity that is lower than or equal to that of a control antibody comprising an Fc region containing a proline at position 238 (EU index). 12. The antibody of any one of embodiments 1 to 11, wherein the antibody binds to FcγR2A (131R allotype) with a KD of at least 20 μM as determined by surface plasmon resonance (SPR). 13. The antibody of any one of embodiments 6 to 12, wherein the antibody binds to FcγR2A (131H allotype) with an affinity that is lower than or equal to that of a control antibody comprising an Fc region containing a proline at position 238 (EU index). 14. The antibody of any one of embodiments 1 to 13, wherein the antibody binds to FcγR2A (131H allotype) with a KD of at least 50 μM as determined by surface plasmon resonance (SPR). 15. The antibody of any one of embodiments 1-14, wherein the antibody exhibits increased agonism of human BTLA expressed on the surface of human immune cells, as measured by a BTLA agonism assay selected from a T cell activation assay such as that described in Example 24, a mixed lymphocyte reaction such as that described in Example 25, or a B cell activation assay such as that described in Example 26. 16. An isolated antibody that specifically binds to human BTLA, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an Fc region and a heavy chain variable region comprising three complementarity determining regions (CDRs): CDRH1, CDRH2, and CDRH3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRH2, and CDRL3, (1) CDRH1, CDRH2, and CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 1, 17, and 3, respectively, and have 0 to 3 amino acid alterations, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 4, 12, and 6, respectively, and have 0 to 3 amino acid alterations, and (2) CDRH1,...

Claims

1. An isolated antibody that specifically binds to B and T lymphocyte attenuator (BTLA), comprising a heavy chain and a light chain, (a) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 18; (b) the light chain comprises a light chain variable region comprising an amino acid sequence having at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 14; Isolated antibodies. (I) (a) the heavy chain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19; and (b) the light chain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16; or (II) The isolated antibody of claim 1, wherein (a) the heavy chain comprises an amino acid sequence having up to 10 modifications relative to the amino acid sequence of SEQ ID NO: 19, and (b) the light chain comprises an amino acid sequence having up to 10 modifications relative to the amino acid sequence of SEQ ID NO:

16.

3. The isolated antibody described in claim 1, wherein the heavy chain further comprises an Fc region.

4. The isolated antibody of claim 3, wherein the Fc region comprises one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, or alanine (A) at position 297 (numbering according to the EU index).

5. An isolated antibody as described in claim 4, wherein the heavy chain comprises an Fc region including an aspartic acid at position 238 (EU index). (i) the Fc region binds to FcγR2B with higher affinity than a control antibody comprising an Fc region having a proline at position 238 (EU index); or (ii) the antibody binds to FcγR2B with an affinity of between 5 μM and 0.1 μM, as determined by surface plasmon resonance (SPR); or (iii) the Fc region binds to FcγR2A (131R allotype) with less or equal affinity than a control antibody comprising an Fc region containing a proline at position 238 (EU index); or (iv) the antibody binds to FcγR2A (131R allotype) with a K of at least 20 μM as determined by surface plasmon resonance (SPR); or (v) the antibody binds to FcγR2A (131H allotype) with less or equal affinity to a control antibody comprising an Fc region containing a proline at position 238 (EU index); or (vi) the antibody binds to FcγR2A (131H allotype) with a K of at least 50 μM as determined by surface plasmon resonance (SPR); or (vii) The isolated antibody of claim 5, wherein the antibody exhibits an in vivo half-life of at least 10 days.

7. The isolated antibody of any one of claims 1 to 6, wherein (i) the antibody is an IgG1, IgG2, or IgG4 antibody; and / or (ii) the antibody is selected from the group consisting of a humanized antibody, a chimeric antibody, and a multispecific antibody; and / or (iii) the antibody is monoclonal. (i) the antibody exhibits increased agonism of human BTLA expressed on the surface of human immune cells; and / or (ii) the antibody agonizes human BTLA expressed on the surface of immune cells; and / or (iii) binding of the antibody to human BTLA expressed on the surface of an immune cell reduces proliferation of the cell compared to a comparable immune cell not bound by the antibody. An isolated antibody according to any one of claims 1 to 7. (i) the antibody specifically binds to human B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM; and / or (ii) the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM, and / or (iii) the antibody does not inhibit BTLA binding to herpesvirus entry mediator (HVEM), and / or (iv) the antibody inhibits T-cell proliferation in vitro as determined by a mixed lymphocyte reaction assay; and / or (v) The isolated antibody of any one of claims 1 to 8, wherein the antibody binds to human B and T lymphocyte attenuator (BTLA) with an on rate of at least 5.0 x 10 5 (1 / Ms) at 37°C and / or an off rate of less than 3.0 x 10 -3 (1 / s) at 37°C, as determined by surface plasmon resonance (SPR) at 37°C.

10. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 9 and at least one pharmaceutically acceptable excipient.

11. A nucleic acid comprising one or more nucleotide sequences encoding a polypeptide capable of forming an antibody described in any one of claims 1 to 9.

12. Use of an antibody described in any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of inflammatory or autoimmune diseases, and disorders in which immune cells proliferate.

13. The inflammatory disease or autoimmune disease and the disorder in which immune cells proliferate are selected from the group consisting of Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and chemokines.

13. The use of claim 12, wherein the disease is selected from the group consisting of hidradenitis suppurativa, inflammatory fibrosis, juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, graft rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, systemic mastocytosis, inflammatory bowel disease, and inflammatory bowel disease.

14. A composition for treating a BTLA-associated disease in a patient, comprising an antibody described in any one of claims 1 to 9, wherein the BTLA-associated disease is an inflammatory disease or an autoimmune disease, or a disorder in which immune cells proliferate.

15. The inflammatory disease or autoimmune disease, or disorder in which immune cells proliferate, is selected from the group consisting of Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and suppurative hepatitis.

15. The composition of claim 14, wherein the disease is selected from the group consisting of hidradenitis urinary tract infection, inflammatory fibrosis, juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, graft rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, systemic mastocytosis, inflammatory bowel disease, and inflammatory bowel disease.

16. A composition comprising an antibody described in any one of claims 1 to 9 for the treatment or prevention of inflammatory or autoimmune diseases, and disorders in which immune cells proliferate.

17. The inflammatory disease or autoimmune disease, and disorder in which immune cells proliferate, is selected from the group consisting of Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and suppurative hepatitis.

17. The composition of claim 16, wherein the disease is selected from the group consisting of hidradenitis urinary tract infection, inflammatory fibrosis, juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, graft rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, systemic mastocytosis, inflammatory bowel disease, and inflammatory bowel disease.

18. The use described in claim 13, wherein the inflammatory or autoimmune disease and disorder in which immune cells proliferate is selected from the group consisting of systemic lupus erythematosus, juvenile arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

19. The composition described in claim 15 or 17, wherein the inflammatory or autoimmune disease and disorder in which immune cells proliferate is selected from the group consisting of systemic lupus erythematosus, juvenile arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

20. The use of claim 13, wherein the inflammatory or autoimmune disease is rheumatoid arthritis.

21. The composition described in claim 15 or 17, wherein the inflammatory disease or autoimmune disease is rheumatoid arthritis.

Images