BTLA antibodies

Abstract

The present invention relates generally to antibodies 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 the use of these agonistic antibodies in the treatment of inflammatory, autoimmune and proliferative diseases and disorders. Suitably, these antibodies also have Fc modifications that enhance signaling through FcyR2B.

Description

Technical Field

[0001] This invention relates generally to antibodies that bind to human B and T lymphocyte attenuators (BTLA) and their uses therein, these antibodies comprising antigen-binding fragments. More specifically, this invention relates to agonistic antibodies that bind to and modulate the activity of human BTLA, as well as agonistic antibodies having enhanced binding to and signaling via FcγR2B, and the use of these agonistic antibodies in the treatment of inflammatory, autoimmune, and proliferative diseases and disorders. Background Technology

[0002] The immune system must maintain a balance between the destruction of pathogens or dangerous mutant cells and the tolerance of healthy autologous tissues and harmless symbionts. To facilitate this balance, the activity of immune cells is influenced by integrating signals from a variety of stimulatory and inhibitory receptors that allow cells to adapt to their environment. These surface-expressed receptors present attractive targets for the therapeutic modulation of the immune response. Many human diseases are caused by abnormal or unwanted activation of the immune system, including autoimmune diseases, transplant rejection, and graft-versus-host disease. Agonists capable of inducing signaling through inhibitory receptors can suppress these unwanted immune responses.

[0003] The B and T lymphocyte attenuator (BTLA; also known as CD272) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and PD-1 (Watanabe et al., Nat Immunol., Vol. 4: pp. 670–679, 2003). This B and T lymphocyte attenuator is widely expressed on both bone marrow cells and lymphocytes throughout the immune system (Han et al., J Immunol., Vol. 172: pp. 5931–5939, 2004). Upon binding to its ligand, the herpesvirus entry mediator (HVEM), BTLA recruits the phosphatases SHP-1 and SHP-2 to its cytoplasmic domain (Sedy et al., Nat Immunol., Vol. 6: pp. 90–98, 2005), which in turn inhibits the signaling cascade of activating receptors. Mice lacking the complete BTLA gene exhibit hyperproliferative B and T cell responses in vitro, elevated DNP-KLH titers after immunization, and increased sensitivity to EAE (Watanabe et al., Nat. Immunol, Vol. 4: 670–679, 2003). If spontaneous production of autoantibodies is observed in aged BTLA knockout mice, autoimmune hepatitis-like disease and inflammatory cell infiltration in multiple organs occur (Oya et al., Arthritis Rheum, Vol. 58: 2498–2510, 2008). This evidence suggests that the BTLA inhibitory receptor plays a crucial role in maintaining immune homeostasis and suppressing autoimmunity. Furthermore, HVEM-BTLA signaling is involved in the regulation of mucosal inflammation and infectious immunity (Shui et al., J Leukoc Biol., Vol. 89: 517–523, 2011).

[0004] In cases of autoimmune disorders, therapeutic agents that can modulate BTLA function to suppress autoreactive lymphocytes will be in great need.

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

[0006] Furthermore, anti-mouse BTLA agonist antibodies have been shown to improve disease in mouse models of graft-versus-host disease (Sakoda et al., Blood., Vol. 117: pp. 2506–2514; Albring et al., J Exp Med., Vol. 207: pp. 2551–2559, 2010). Agonist antibodies targeting the human BTLA receptor have been shown to suppress T-cell responses in vitro (see Otsuki et al., Biochem Biophys Res Commun., Vol. 344: pp. 1121–1127, 2006; and WO2011 / 014438), but have not yet been used clinically.

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

[0008] On June 25, 2020, WO2020128446 (Oxford University Innovation Limited and MiroBio Ltd.) disclosed certain antibodies against BTLA.

[0009] In humans, there exists an inhibitory Fcγ receptor (FcγR2B), while other Fcγ receptors (FcγR1A, FcγR2A, FcγR3A, and FcγR3B) transmit immune activation signals. The important regulatory role of FcγR2B has been confirmed through studies in FcγR2B knockout mice with increased susceptibility to autoimmune diseases (Nakamura et al., Journal of Experimental Medicine, Vol. 191, No. 5: pp. 899–906, 2000). Furthermore, polymorphisms in the FcγR2B gene in humans are associated with the risk of autoimmune diseases, particularly systemic lupus erythematosus (SLE) (Floto et al., Nature Medicine, Vol. 11, No. 10, 2005). Therefore, FcγR2B is considered to play a crucial role in controlling the immune response and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0010] Antibodies with the Fc region have been reported to exhibit improved FcγR2B binding activity (Chu et al., Molecular Immunology, Vol. 45, No. 15: pp. 3926–3933, 2008). In this literature, FcγR2B binding activity was improved by adding alterations such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody. Among these, the antibody with the S267E / L328F mutation exhibited the strongest binding to FcγR2B and maintained the same binding levels to FcγR1A and FcγR2A (131H allotype) as the naturally occurring IgG1 binding level. However, another report indicates that this change enhances binding to FcγR2A 131R by hundreds of times, reaching the same level as FcγR2B binding, which means that the binding selectivity of FcγR2B is not improved compared to FcγR2A 131R (US Patent Publication No. 2009 / 0136485).

[0011] To enable BTLA agonist antibodies to effectively suppress the immune response without triggering inflammatory FcγR signaling, the inventors propose modulating the antibody to selectively bind Fc to FcγR2B. Molecules that bind more selectively to FcγR2B will promote bidirectional inhibitory signaling of BTLA on cells expressing BTLA and FcγR2B on cells expressing FcγR2B, thereby enhancing the immunosuppressive effect of the antibody. This is desirable in therapeutic antibodies intended for treating diseases of excessive immune activation. Summary of the Invention

[0012] This invention relates to BTLA agonist antibodies having one or more of the following desired properties, including antibody fragments thereof: high binding affinity in human subjects, high agonist potency, high agonist efficacy, favorable pharmacokinetics, and low antigenicity. In some embodiments, such molecules not only have increased binding to FcγR2B and thus drive FcγR2B signaling, but also have an in vivo half-life sufficient for suitable therapeutic use. Therefore, the antibodies of the present invention promote bidirectional inhibitory signaling of BTLA on cells expressing BTLA and FcγR2B on cells expressing FcγR2B. In some embodiments, such molecules have reduced binding to one or more activating Fcγ receptors (such as FcγR2A or FcγR1A) compared to the parental peptide. In some embodiments, such molecules have increased binding to FcγR2B / FcγR2A compared to the parental peptide. In some embodiments, such molecules have increased binding to FcγR2B / FcγR1A compared to the parental peptide. This invention also relates to the use of the antibodies of this 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 isolation antibody that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing a substitution that results in increased binding to FcγR2B compared to a parent molecule lacking substitution.

[0014] In some embodiments, the antibody has increased binding to FcγR2B compared to the parent molecule, such that the ratio of [KD value of parental peptide to FcγR2B] to [KD value of variant peptide to FcγR2B] is greater than 1, such as greater than 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 implementations, the antibody is more selective for binding FcγR2B than for binding FcγR2A.

[0016] In some embodiments, the antibody exhibits enhanced FcγR2B binding activity compared to the parent peptide and maintains or reduces binding activity to FcγR2A (R type) and / or FcγR2A (H type). In some embodiments, the ratio of [KD value of the variant peptide against FcγR2A (R type)] to [KD value of the variant peptide against FcγR2B] is 2 or greater, such as 3, 4, 5, 6, 7, 8, 9, 10 or greater. In some implementations, the ratio of [KD value of variant peptide against FcγR2A (H type)] to [KD value of variant peptide against FcγR2B] is 2 or greater, such as 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 greater.

[0017] In some embodiments, the antibody exhibits enhanced FcγR2B binding activity and maintains or reduces FcγR1A binding activity compared to the parent peptide. In some embodiments, the ratio of [KD value of the variant peptide against FcγR1A] to [KD value of the variant peptide against FcγR2B] is 0.05 or greater, such as at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or greater.

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

[0019] In some implementations, the antibody binds to residues selected from the following human BTLA:

[0020] (i) D52, P53, E55, E57, E83, Q86, E103, L106 and E92 (according to the position of SEQ ID NO:225).

[0021] (ii) Y39, K41, R42, Q43, E45 and S47 (according to the position of SEQ ID NO:225);

[0022] (iii) D35, T78, K81, S121 and L123 (according to the position of SEQ ID NO: 225);

[0023] (iv) N65 and A64 (according to the position of SEQ ID NO:225); or

[0024] (v)H68 (based on the position of SEQ ID NO: 225).

[0025] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain contains 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 numbers are based on the EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0026] Suitablely, the antibody is human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A.

[0027] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an aspartic acid residue at position 238 (EU index). Suitably, 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, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an aspartic acid residue at position 237 (EU index). Suitably, 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, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an aspartic acid residue at position 236 (EU index). Suitably, 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, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an alanine residue at position 235 (EU index). Suitably, 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, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an alanine residue at position 234 (EU index). Suitably, 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 invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing a glycine residue at position 271 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0033] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing a glutamic acid residue at position 267 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0034] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an alanine residue at position 265 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0035] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an alanine residue at position 297 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0036] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing an alanine residue at position 322 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0037] According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing arginine at position 330 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment. According to a variation of the first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing aspartic acid at position 237 (EU index), aspartic acid at position 238 (EU index), glycine at position 271 (EU index), and arginine at position 330 (EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0038] In a specific implementation, the antibody has a heavy chain and / or a light chain with at least one complementarity-determining region (CDR) present in antibodies 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 described herein. Suitably, the antibody also comprises an Fc region containing 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 numbers are based on the EU index).

[0039] In another embodiment, the antibody binding to human BTLA is selected from the group consisting of 6.2, 2.8.6, 3E8, or an antibody competing with any of 6.2, 2.8.6, or 3E8 for binding to human BTLA, wherein the antibody specifically binds to BTLA and induces signal transduction via the receptor. The antibody further comprises an Fc region containing 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 numbers are based on the EU index).

[0040] According to a variation of the first aspect of the invention, an isolating antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR sequences disclosed in SEQ ID NO: 1, 17, 3, 4, 12 and 6, respectively, and an Fc region comprising substitutions that result in increased binding to FcγR2B compared to an unsubstituted parent molecule. Suitably, the antibody comprises VH and VL sequences disclosed in SEQ ID NO: 18 and 14, respectively. Suitably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence disclosed in SEQ ID NO: 19, and / or the light chain comprises an amino acid sequence disclosed in SEQ ID NO: 16. In a specific embodiment, the antibody comprises an Fc region containing an aspartic acid residue at position 236 (EU index). Suitably, the antibody is an agonist antibody.

[0041] In a specific implementation, the antibody comprises an Fc region containing an aspartic acid residue at position 237 (EU index). Suitably, the antibody is an agonist antibody.

[0042] In a specific embodiment, the antibody comprises an Fc region containing an aspartic acid residue at position 238 (EU index). Suitably, the antibody is an agonist antibody.

[0043] In a specific implementation, the antibody comprises an Fc region containing an alanine at position 235 (EU index).

[0044] In a specific implementation, the antibody comprises an Fc region containing an alanine at position 234 (EU index).

[0045] In a specific implementation, the antibody comprises an Fc region containing an alanine at position 265 (EU index).

[0046] In a specific implementation, the antibody comprises an Fc region containing a glutamic acid at position 267 (EU index).

[0047] In a specific implementation, the antibody comprises an Fc region containing glycine at position 271 (EU index).

[0048] In a specific implementation, the antibody comprises an Fc region containing an alanine at position 297 (EU index).

[0049] In a specific implementation, the antibody comprises an Fc region containing an alanine at position 322 (EU index).

[0050] In a specific implementation, the antibody comprises an Fc region containing an arginine residue at position 330 (EU index).

[0051] In a specific implementation, the antibody comprises an Fc region containing aspartic acid at position 237 (EU index), aspartic acid at position 238 (EU index), glycine at position 271 (EU index), and arginine at position 330 (EU index).

[0052] In a specific embodiment of the first aspect of the invention, the antibody has increased binding to FcγR2B compared to a parent molecule lacking Fc region substitution, wherein the Fc region substitution is one or more of the following: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A.

[0053] In a specific embodiment of the first aspect of the invention, compared with a parent molecule lacking Fc region substitution, the antibody has increased binding to FcγR2B and reduced binding to one or more activating Fcγ receptors such as FcγR2A or FcγR1A.

[0054] In a specific embodiment of the first aspect of the invention, the antibody exhibits an increased binding rate to FcγR2B / FcγR2A compared to a parent molecule lacking Fc region substitution. Suitably, the FcγR2B / FcγR2A binding rate is increased by 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 times compared to a parent molecule lacking Fc region substitution.

[0055] In a specific embodiment of the first aspect of the invention, the antibody exhibits an increased binding rate to FcγR2B / FcγR1A compared to a parent molecule lacking an Fc region substitution relative to the wild-type sequence. Suitably, the FcγR2B / FcγR1A binding rate is increased by at least 1.1, 1.2, 1.5, 2, 5, 10, 50, 100, 150, 200, or 250 times compared to a parent molecule lacking an Fc region substitution.

[0056] Compared to a parental molecule lacking Fc region substitutions, we mean compared to an antibody molecule having the same amino acid sequence except for the amino acids representing Fc substitutions relative to the wild-type Fc as described in the claims. For example, this includes any of the following substitutions: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A. Therefore, the binding of antibody molecules with or without the said Fc substitution to FcγR2B can be measured, and optionally, the binding of antibody molecules with or without the said Fc substitution to activated Fcγ receptors (such as FcγR2A or FcγR1A) can be measured. Thus, for example, if the binding to FcγR2B increases by 1.5-fold compared to the unsubstituted parent molecule, the antibody exhibits an increased binding potency of up to 150% compared to the parent. If the binding to FcγR2A decreases to 2 / 3 compared to the unsubstituted parent molecule, the antibody exhibits a binding potency of 67% compared to the parent. For this exemplary antibody molecule, the change in FcγR2B / FcγR2A binding rate would be 150 / 67 = 2.24-fold. Any value greater than 1 indicates that the antibody molecule has enhanced selectivity for binding FcγR2B relative to binding FcγR2A compared to the parent molecule.

[0057] In a specific embodiment of the first aspect of the invention, the antibody has an increased [KD value for binding FcγR1A] / [KD value for binding FcγR2B] ratio compared to a parent molecule that lacks Fc region substitution relative to the wild-type sequence. Suitablely, the ratio of the [KD value for binding FcγR1A] to [KD value for binding FcγR2B] 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 10000 times that of the parent molecule lacking Fc region substitution.

[0058] In a specific embodiment of the first aspect of the invention, the antibody has an increased [KD value for binding FcγR2A 131R] / [KD value for binding FcγR2B] ratio compared to a parent molecule lacking Fc region substitution relative to the wild-type sequence. Suitably, the [KD value for binding FcγR2A 131R] / [KD value for binding FcγR2B] ratio of the variant molecule is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, or 100 times greater than the [KD value for binding FcγR1A] / [KD value for binding FcγR2B] ratio of the parent molecule lacking Fc region substitution.

[0059] According to a second aspect of the present invention, an isolated nucleic acid is provided, the isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain polypeptide and / or a light chain polypeptide of an isolated antibody of the first aspect of the present invention.

[0060] According to a third aspect of the present invention, a carrier is provided that comprises the nucleic acid of the second aspect of the present invention.

[0061] According to a fourth aspect of the invention, a host cell is provided, the host cell comprising a nucleic acid sequence according to a second aspect of the invention or a vector according to a third aspect of the invention.

[0062] According to a fifth aspect of the invention, a method for generating an antibody according to a first aspect of the invention is provided, the method comprising culturing a host cell according to a fourth aspect of the invention under conditions for generating the antibody, and optionally isolating and / or purifying the antibody.

[0063] According to a sixth aspect of the present invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody of the first aspect of the present invention or an antibody produced by the fifth aspect of the present invention.

[0064] According to a seventh aspect of the present invention, a method for preparing a pharmaceutical composition is provided, the method comprising formulating an antibody according to a first aspect of the present invention or an antibody produced according to a fifth aspect of the present invention into a composition comprising at least one additional component. In a specific embodiment, the at least one additional component is a pharmaceutically acceptable excipient.

[0065] According to an eighth aspect of the present invention, a kit is provided comprising an antibody according to a first aspect of the present invention or a pharmaceutical composition according to a sixth aspect of the present invention. Suitably, such a kit includes a package insert containing instructions for use.

[0066] According to a ninth aspect of the present invention, a method for treating BTLA-related diseases in a patient is provided, the method comprising administering to the patient a therapeutically effective amount of an antibody of the first aspect of the present invention or a pharmaceutical composition of the sixth aspect of the present invention.

[0067] Appropriately, BTLA-related diseases are autoimmune or inflammatory diseases. Detailed Implementation

[0068] The inventors have identified particularly potent agonist antibodies against BTLA, which are predicted to be more effective than existing antibodies in suppressing T-cell responses and are therefore particularly useful in the treatment of immune-mediated disorders. Compared to the parental peptide, such antibodies contain at least one substitution at a position in the Fc moiety of the molecule that selectively enhances binding to FcγR2B. Suitably, the antibody contains substitutions at one or more of the following positions (EU index positions): 234, 235, 236, 237, 238, 265, 267, 271, 297, 330, and 322. Suitablely, the antibody is a human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A, all using EU index numbers.

[0069] Modifications at one or more of the following locations: 236, 237, 238 and 267 (EU index) are particularly appropriate.

[0070] Combinations of Fc modifications are also suitable. In a specific implementation, a modification group called V9 is used, wherein the antibody heavy chain contains an Fc region comprising aspartic acid at position 237, aspartic acid at position 238, glycine at position 271, and arginine at position 330 (numbered according to the EU index).

[0071] By replacing the Fc portion of the molecule (i.e., the antibody or its antigen-binding fragment) with P238D (EU index), the molecule has enhanced binding to FcγR2B and signal transduction of the FcγR2B, but its level ensures that the antibody maintains a sufficient in vivo half-life.

[0072] As used in this specification and the appended claims, the singular forms “an,” “a,” and “the” include plural references unless otherwise expressly specified. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and so on.

[0073] It should be understood that wherever the language “includes” is used to describe aspects in this text, other similar aspects described as “consisting of” and / or “substantially consisting of” are provided.

[0074] It should be understood that one, some, or all of the characteristics of the various embodiments described herein are applicable to any aspect unless otherwise expressly stated. Furthermore, these various embodiments can be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the invention are further described below through a detailed description.

[0075] 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 relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide general dictionaries for those skilled in the art of the matter for many of the terms used in this disclosure.

[0076] As used herein, the term "approximately" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. The "approximately" values ​​or parameters mentioned herein include (and describe) embodiments involving that value or parameter itself.

[0077] Amino acids may be represented in this document by their commonly known three-letter symbols or by a single-letter symbol recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Similarly, nucleotides may be represented by their generally accepted single-letter codes.

[0078] The carboxyl-terminal portion of each chain defines the constant region primarily responsible for effector function. The amino-terminal portion of each chain defines the variable region responsible for antigen binding. Kabat et al. (NIH Publication No. 91 / 3242, pp. 679-687, 1991) collected numerous primary sequences of the variable regions of the heavy and light chains. Based on the degree of sequence conservation, they classified the individual primary sequences into CDRs and frames and compiled a list of them (see Kabat et al., "SEQUENCES OFIMMUNOLOGICAL INTEREST", 5th edition, NIH Publication No. 91-3242, 1991).

[0079] Unless otherwise stated, the identified CDRs of antibodies follow the Kabat definition as presented by Kabat et al. (“Sequences of Proteins of Immunological Interest”, 5th edition, National Institutes of Health Public Health Service, Bethesda, MD, 1991). The amino acid numbering within the variable domain is based on the sequence number provided in the sequence listing.

[0080] Unless otherwise stated, amino acids (such as C) in constant domains L C H 1. C H 2 and C H 3) The numbering follows the EU index numbering disclosed in Kabat et al. (NIH Publication No. 91 / 3242, pp. 679-687, 1991), referred to herein as the “EU index.” For example, the EU index is used to locate substitutions in the Fc region of the antibody / its antigen-binding fragment of the present invention. For example, glycine (G) is converted to aspartic acid (D) at position 236 (identified as G236D), or proline (P) is converted to aspartic acid (D) at position 238 (identified as P238D). Those skilled in the art will recognize that this numbering convention consists of non-contiguous numbering within specific regions of the immunoglobulin sequence, allowing for normalized reference to conserved positions within the immunoglobulin family. Therefore, the position of any given immunoglobulin defined by the EU index will not necessarily correspond to its contiguous sequence.

[0081] The terms “B and T lymphocyte attenuators” and “BTLA” are used interchangeably unless the context otherwise specifies, referring to a protein or gene (or other nucleic acid encoding all or part of BTLA). Human BTLA sequences include all human isotypes and variant forms. A representative example of full-length human BTLA is disclosed in Genbank with accession number AJ717664.1. Another representative polypeptide sequence of human BTLA is disclosed in SEQ ID NO: 225, which differs from AJ717664.1 only in two naturally occurring single nucleotide polymorphisms. Despite allelic variations, human BTLA polypeptide sequences will generally have at least 90% sequence identity with the human BTLA in SEQ ID NO: 225 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%).

[0082] A representative example of full-length cynomolgus (cyno) BTLA is disclosed in Genbank with accession number XP_005548224. A reference polypeptide sequence for cynomolgus BTLA is disclosed in SEQ ID NO: 226. The cynomolgus BTLA polypeptide sequence will generally have at least 90% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the cynomolgus BTLA disclosed in SEQ ID NO: 226.

[0083] The term sequence identity is well known in the art. For the purposes of this invention, when determining whether a target sequence satisfies a defined limit (e.g., 90% identity), if the target sequence is identified as satisfying the defined limit using the BLAST (Basic Local Alignment Search Tool) algorithm (see Altschul et al., J Mol. Biol., Vol. 215: pp. 403-410, 1990) or the Smith-Waterman algorithm (see Smith and Waterman, J Mol. Biol., Vol. 147: pp. 195-197, 1981), then the target sequence is considered to satisfy the defined limit.

[0084] Antibodies (including antigen-binding fragments of antibodies)

[0085] 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) through at least one antigen recognition site located in a variable domain of an immunoglobulin molecule. Specifically, as used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies (such as bispecific antibodies derived from at least two intact antibodies), chimeric antibodies, humanized antibodies, human antibodies, any other modified immunoglobulin molecule, and any fragment thereof containing an antigen recognition site, provided that such antibodies exhibit the desired biological activity. Antibodies can be derived from any species. Suitablely, an antibody is a human antibody.

[0086] 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 complete antibodies (such as IgG1, IgG4, etc.) and their antigen-binding fragments.

[0087] As used herein, BTLA agonist antibodies refer to antibodies (including antigen-binding fragments of the complete antibody) that bind to BTLA and enhance its co-inhibitory signaling against T and / or B cells.

[0088] An antigen-binding site is a portion of a molecule that binds to all or part of a target antigen. In an antibody molecule, this can be referred to as an antibody-antigen binding site, and it contains a portion of the antibody that specifically binds to all or part of the target antigen. When the antigen is large, the antibody may bind only to a specific portion of that antigen, which is called an epitope. Antibody-antigen binding sites can be provided by one or more antibody variable domains. Preferably, the antibody-antigen binding site comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain.

[0089] This invention also covers antibody fragments containing antigen-binding sites. Therefore, when referring to antibodies, the term "antigen-binding fragment thereof" means an antibody fragment having an antigen recognition site and thus the ability to bind to an antigen, such as Fab, Fab', F(ab')2, dimer, Fv fragments, and single-chain Fv (scFv) mutants. Antigen-binding immunoglobulin (antibody) fragments are well known in the art. Such fragments do not need to have a functional Fc receptor binding site. In a specific embodiment, the antigen-binding fragment comprises an Fc portion having substitutions 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 numbers are based on the EU index). In a specific embodiment, the antigen-binding fragment comprises an Fc portion having a G236D (EU index) substitution. In a specific embodiment, the antigen-binding fragment comprises an Fc portion having a P238D (EU index) substitution. In a specific implementation, the antigen-binding fragment comprises an Fc portion having aspartic acid at position 237 (EU index), aspartic acid at position 238 (EU index), glycine at position 271 (EU index), and arginine at position 330 (EU index).

[0090] As used herein, the terms "antibody fragment molecule of the present invention," "antibody fragment," and "antigen-binding fragment thereof" are used interchangeably. Antibodies or their antigen-binding fragments are collectively referred to as antigen-binding molecules.

[0091] As used in this article, the term "BTLA-binding molecule" refers to both the antibody and its binding fragment that can bind to BTLA.

[0092] There are five main classes (i.e., isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these immunoglobulins can be further subdivided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. Unless the context otherwise requires, the antibodies of this invention may be derived from one of these antibody classes or subclasses. The heavy chain constant domains corresponding to different classes of antibodies are generally represented by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chain of an antibody from any vertebrate species may be designated as one of two distinct types (designated as Kappa (κ) and Lambda (λ)) based on the amino acid sequence of its constant domain.

[0093] "Natural antibodies" are typically heterotetrameric Y-shaped glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies between heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the variable domains of the light and heavy chains. Each heavy chain contains a variable domain (VH) and a constant region, which, in the case of IgG, IgA, and IgD antibodies, contains a region called C. H 1. C H 2 and C H The three structural domains of 3 (IgM and IgE have a fourth structural domain C) H 4). Among the IgG, IgA, and IgD categories, C H 1 and C HThe 2 domains are separated by flexible hinges, which are proline- and cysteine-rich segments of variable length (approximately 10 to 60 amino acids in various IgG subclasses). The variable domains in both the light and heavy chains are connected to constant domains via “J” regions of approximately 12 or more amino acids, and the heavy chain also has a “D” region of approximately 10 additional amino acids. Each class of antibody also contains interchain 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 frame regions (FRs)). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0094] The antibodies of this invention can be derived from any animal species, including mice, rats, humans, or any other source (including chimeric antibodies or humanized antibodies). In some embodiments, the antibody is monoclonal, such as a monoclonal antibody. In some embodiments, the antibody is a human antibody or a humanized antibody or its antigen-binding fragment. Non-human antibodies or their antigen-binding fragments can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0095] As used herein, the term "monoclonal antibody" ("mAb") refers to an antibody derived from a substantially homologous population of antibodies, meaning that the individual antibodies constituting this population are identical except for possible mutations (e.g., naturally occurring mutations that may be present in small quantities). Therefore, the modifier "monoclonal" indicates the characteristic of the antibody or a fragment thereof, rather than a mixture of discrete antibodies or antigen-binding fragments. mAbs are typically highly specific, i.e., targeting a single antigenic site / epitope; however, monoclonal antibodies can also refer to a substantially homologous population of bispecific antibody molecules.

[0096] mAbs can be produced by hybridoma, recombination, transgenic, or other techniques known to those skilled in the art. For example, monoclonal antibodies or antigen-binding fragments thereof according to the invention can be prepared by the hybridoma method first described by Kohler and Milstein (Nature, Vol. 256, p. 495, 1975), or by recombinant DNA methods (such as those described in U.S. Patent Nos. 4,816,567 and 6,331,415). “Monoclonal antibodies” can also be isolated from phage antibody libraries using techniques described, for example, those described by Clackson et al. (Nature, 1991, Vol. 352, pp. 624-628) and Marks et al. (J. Mol. Biol. 1991, Vol. 222: pp. 581-597).

[0097] The term monoclonal can also be attributed to the antigen-binding fragment of the antibody of the present invention. This simply means that the molecule is produced or exists in a single clone.

[0098] A "human" antibody (HumAb) is an antibody with a variable region, wherein both the frame region and the CDR region are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0099] Human antibodies can be prepared by administering an immunogen / antigen to a transgenic animal that has been modified to produce fully human antibodies or complete antibodies with human variable regions responsive to antigen attack, but in which the endogenous loci have been disabled, such as in immunized xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 concerning XENOMOUSE (trademark) technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557-3562, 2006, concerning human antibodies produced via human B-cell hybridoma technology. Such animals typically contain all or part of the human immunoglobulin loci, which substitute for endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech., Vol. 23: pp. 1117-1125 (2005). See also, for example, a description of XENOMOUSE.TM U.S. Patent Nos. 6,075,181 and 6,150,584; describing HUMAB TM US Patent No. 5,770,429; describes KM MOUSE TM US Patent No. 7,041,870, and the description of VELOCIMOUSE TM The technology is described in U.S. Patent Application Publication No. US2007 / 0061900. The human variable region of the intact antibody produced from such animals can be further modified, for example, by combining it with different human constant regions.

[0100] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines used for the production of human monoclonal antibodies have been described. (See, for example, Kozbor, J. Immunol, Vol. 133: p. 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., Vol. 147: p. 86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557–3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (Description of Production of Monoclonal Human IgM Antibodies from Hybridoma Cell Lines) and Ni, Xiandai Mianyixue, Vol. 26: pp. 265-268 (2006) (Description of Human-Human Hybridoma). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, Vol. 20: pp. 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, Vol. 27: pp. 185-191 (2005).

[0101] The terms "human" antibody and "fully human" antibody are used synonymously. This definition of human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0102] As used herein, a “humanized antibody” refers to an antibody in which some, most, or all of the amino acids, outside the CDR of a non-human antibody, are replaced by corresponding amino acids derived from human immunoglobulins. In some embodiments, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the CDR from the receptor are replaced by residues of the CDR from a non-human species (donor antibody) such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. Humanized antibodies may contain residues that are not present in the receptor antibody or in the introduced CDR or frame sequence, but these residues are included to further improve and optimize antibody performance. In one embodiment of the humanized form of an Ab, some, most, or all of the amino acids outside the CDR have been replaced by amino acids derived from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted, as long as they do not eliminate the antibody’s ability to bind to a specific antigen. “Humanized” antibodies retain antigen specificity similar to that of the original antibody. Typically, humanized antibodies will contain substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). For further details, see, for example, Jones et al., Nature, Vol. 321: pp. 522–525 (1986); Riechmann et al., Nature, Vol. 332: pp. 323–329 (1988); and Presta, Curr. Op. Struct. Biol., Vol. 2: pp. 593–596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol., Vol. 1: pp. 105–115 (1998); Harris, Biochem. Soc. Transactions, Vol. 23: pp. 1035–1038 (1995); Hurle and Gross, Curr. Op. Biotech., Vol. 5: pp. 428–433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409. Suitablely, Fc will contain a P238D substitution mutation (using “EU Index” numbering) to enhance the specificity of binding to FcγR2B.

[0103] As used herein, Fc, Fc portion, or Fc region refers to the constant region of an antibody or antibody-like molecule other than the first constant region immunoglobulin domain. Therefore, Fc refers to the last two constant regions of IgA, IgD, and IgG immunoglobulin domains, and the last three constant regions of IgE and IgM immunoglobulin domains, as well as the N-terminus of the flexible hinge of these domains. For IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cγ2. For IgA and IgM, Fc may include the J chain.

[0104] As used herein, “engineered antibody” refers to an antibody, which may be a humanized antibody, wherein specific residues have been substituted with other residues to reduce adverse effects or properties. Such substitutions may be located within the CD domain. For example, as described herein (see Example 21), the CDRH2 of humanized antibody 3E8 is modified with an N57Q substitution to eliminate the possibility of deamidation, and / or modified with a K63S substitution to reduce predicted immunogenicity. In this case, the numbering is the ordinal number relative to the provided sequence identifier.

[0105] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse and the constant region is derived from a human, or vice versa. The term also encompasses antibodies containing a V region from one individual of a species (e.g., the first mouse) and a constant region from another individual of the same species (e.g., the second mouse). The term "antigen (Ag)" refers to a molecular entity used to immunize an immune-active vertebrate to generate antibodies (Abs) that recognize Ag or to screen expression libraries (e.g., phage, yeast, or ribosome display libraries). In this document, Ag is used more broadly and is generally intended to include target molecules specifically recognized by Abs, thus including portions or mimics of molecules used in the immunization process for generating Abs or in library screening for selecting Abs.

[0106] “Bispecific” or “bifunctional” antibodies are artificial hybrid antibodies with two distinct heavy / light chain pairs and two distinct binding sites. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, Vol. 305: pp. 537-539 (1983)). Methods for preparing bispecific antibodies are within the capabilities of those skilled in the art. For example, bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai et al. (1990), J. Biol. Exp. Immunol. Vol. 79: pp. 315-321; Kostelny et al. (1992), J Immunol. Vol. 148: pp. 1547-1553. Furthermore, bispecific antibodies can be formed as “biantibodies” (Holliger et al. (1993), PNAS USA, Vol. 90: 6444-6448) or as “bispecific single-chain molecules (Janusins)” (Traunecker et al. (1991), EMBO J., Vol. 10: 3655-3659 and Traunecker et al. (1992), Int. J. Cancer Suppl., Vol. 7: 51-52). Full-length bispecific antibodies can be generated, for example, by using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies, by introducing substitution at the heavy chain CH3 interface in each half-molecule to promote the formation of heterodimers of two antibody half-molecules with different specificities in a cell-free environment in vitro or by using co-expression. The Fab arm exchange reaction is a result of disulfide isomerization and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parental monospecific antibody are reduced. The resulting free cysteine ​​residue from one parental monospecific antibody forms an inter-heavy chain disulfide bond with a cysteine ​​residue from the second parental monospecific antibody molecule, while the CH3 domain of the parental antibody is released and reformed through dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding to a different epitope. A "knob-in-hole" strategy (see, for example, PCT International Publication No. WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids forming the interface of the CH3 domain in human IgG can be mutated at sites affecting CH3 domain interactions to promote heterodimer formation.Amino acids with small side chains (hole-like structures) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, and amino acids with large side chains (knob-like structures) are introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of both antibodies, a heterodimer is formed due to the preferential interaction between the heavy chains with "hole-like structures" and those with "knob-like structures." Exemplary CH3 substitution pairs forming the knob-like and hole-like structures are (represented as the modification position in the first CH3 domain of the first heavy chain / the modification 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.

[0107] Bispecific antibodies can also be generated in vitro in a cell-free environment according to the method described in International Patent Publication No. WO2011 / 131746: by introducing an asymmetric mutation in the CH3 region of two monospecific homodimer antibodies and forming a bispecific heterodimer antibody from the two parental monospecific homodimer antibodies under reducing conditions to allow disulfide bond isomerization. Another strategy for generating bispecific antibodies involves using electrostatic interactions to promote heavy chain heterodimerization by substituting positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in the following documents: US Patent Publication No. US2010 / 0015133; US Patent Publication No. US2009 / 0182127; US Patent Publication No. US2010 / 028637 or US Patent Publication No. US2011 / 0123532.

[0108] Suitably, one of the two antibody half-molecules in the bispecific molecule is the anti-BTLA antibody of the present invention. Suitably, the bispecific antibody comprises a binding arm and a second binding arm, the first binding arm comprising a BTLA antigen-binding region as disclosed herein, the second binding arm comprising a binding region against another antigen (e.g., against a different BTLA antigenic epitope or a completely different protein), and wherein 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 numbers are according to the EU index).

[0109] Generally, the term "epitope" refers to a region or area of ​​an antigen that specifically binds to an antibody, i.e., a region or area in physical contact with the antibody. Therefore, the term "epitope" refers to a molecular portion of one or more antigen-binding regions of an antibody that can be recognized and bound to that antibody. Typically, an epitope is defined in the context of molecular interactions between an antibody or its antigen-binding portion (Ab) and its corresponding antigen. Epitopes typically consist of surface groups of molecules (such as amino acids or sugar side chains) and have specific three-dimensional structural features and specific charge features. In some embodiments, an epitope may be a protein epitope. Protein epitopes can be linear epitopes or conformational epitopes. In a linear epitope, all interaction sites between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" includes a discontinuous polypeptide (or amino acid) within an antigen protein to which an antibody specifically binds. As used herein, the term "antigen epitope" is defined as a portion of an antigen that an antibody can specifically bind to, as determined by any method well known in the art (e.g., by conventional immunoassay).

[0110] Antibodies that "specifically bind" to epitopes are a well-known term 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 a greater affinity than it does with alternative cells, proteins, or substances.

[0111] Various assay methods can be used to select antibodies or peptides that specifically bind to the target molecule. Examples include solid-phase ELISA, immunoprecipitation, and Biacore assays. TM (GE Healthcare, Piscataway, NJ), KinExA, Fluorescence-Activated Cell Sorting (FACS), Octet TM (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that can be used to identify antibodies that specifically react with antigens, or receptors or their ligand-binding moieties that specifically bind to homologous ligands or binding partners. Typically, a specific or selective reaction 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. Additionally, when the equilibrium dissociation constant (K... D When the KD (or KD, which can be used interchangeably in this article) is <7nM, it is called "specifically binding" of the antigen by the antibody.

[0112] In some embodiments, this disclosure provides a chimeric antigen receptor comprising an antigen-binding fragment of the BTLA-binding antibody disclosed herein, a transmembrane domain, and an intracellular signaling domain. As used herein, the terms "chimeric antigen receptor" (CAR), "artificial T-cell receptor," "chimeric T-cell receptor," or "chimeric immune receptor" refer to an engineered receptor that can be specifically transplanted onto immune effector cells. CARs typically have an extracellular domain comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. The term "signaling domain" refers to the functional portion of a protein that functions by transmitting information within the cell to regulate cellular activity via defined signaling pathways that act as effectors by generating second messengers or by responding to such messengers.

[0113] Fcγ modification

[0114] In humans, isotypes of FcγR1A (CD64A), FcγR2A (CD32A), FcγR2B (CD32B), FcγR3A (CD16A), and FcγR3B (CD16B) have been reported as the FcγR protein family, and different allotypes of these receptors have also been reported (Jefferis and Lund, Immunology Letters, Vol. 82, Nos. 1-2: pp. 57-65, 2002). FcγR1A, FcγR2A, and FcγR3A are referred to as activating FcγRs due to their immunomodulatory functions, while FcγR2B is referred to as an immunosuppressive FcγR due to its immunosuppressive function (Smith and Clatworthy, Nat Rev Immunol, Vol. 10, No. 5, pp. 328-343, 2010). In the literature and in this article, FcγR1A may also be referred to as FcγR1.

[0115] When the activating FcγR is triggered by binding to the antibody Fc region, this leads to phosphorylation of the immunoreceptor tyrosine-based activating motif (ITAM) contained in the intracellular domain or the FcR common γ chain (interacting partner), which initiates an inflammatory immune response by launching an activation signaling cascade (Nimmerjahn and Ravetch, Nat Rev Immunol, Vol. 8, No. 1: pp. 34-47, 2008). When the inhibitory receptor FcγR2B is triggered by binding to the antibody Fc region, this leads to phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail, which subsequently recruits the SH2-containing inositol polyphosphate 5-phosphatase (SHIP1), which in turn inhibits the transduction of other activation signaling cascades and thus suppresses the inflammatory immune response (Ravetch and Lanier, Science, Vol. 290, No. 5489: pp. 84-89, 2000).

[0116] FcγR2B is the only FcγR expressed on B cells (Amigorena et al., European Journal of Immunology, Vol. 19, No. 8: pp. 1379-1385, 1989). The interaction between the antibody Fc region and FcγR2B has been reported to inhibit signaling via B cell receptors, suppressing B cell proliferation and antibody production (Nimmerjahn and Ravetch, Advances in Immunology, Vol. 96: pp. 179-204, 2007). In cell types expressing both activating and repressive FcγR (such as macrophages, dendritic cells, neutrophils, mast cells, and basophils), the threshold and outcome of FcγR-mediated signaling depend on the balance between the activation of activating and repressive FcγR (Nimmerjahn and Ravetch, Science, Vol. 310, No. 5753: pp. 1510-1512, 2005).

[0117] The important regulatory role of FcγR2B has been confirmed by studies in FcγR2B knockout mice with increased susceptibility to autoimmune diseases (Nakamura et al., Journal of Experimental Medicine, Vol. 191, No. 5: pp. 899–906, 2000). Furthermore, polymorphisms in the FcγR2B gene in humans are associated with the risk of autoimmune diseases, particularly systemic lupus erythematosus (SLE) (Floto et al., Nature Medicine, Vol. 11, No. 10, 2005). Therefore, FcγR2B is considered to play a crucial role in controlling the immune response and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0118] It is known that IgG1 and IgG4 (the most commonly used isotypes in commercially available antibody drugs) bind not only to FcγR2B but also strongly to activating FcγR (Bruhns et al., Blood, Vol. 113, No. 16: pp. 3716-3725, 2009). By utilizing Fc regions that exhibit enhanced or improved FcγR2B binding selectivity compared to activating FcyR, antibody drugs with stronger immunosuppressive properties than IgG1 or IgG4 can be developed.

[0119] Antibodies with the Fc region have been reported to exhibit improved FcγR2B binding activity (Chu et al., Molecular Immunology, Vol. 45, No. 15: pp. 3926–3933, 2008). In this literature, FcγR2B binding activity was improved by adding alterations such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody. Among these, the antibody with the S267E / L328F mutation exhibited the strongest binding to FcγR2B and maintained the same binding levels to FcγR1A and FcγR2A (131H allotype) as the naturally occurring IgG1 binding level. However, another report indicates that this change enhances binding to FcγR2A 131R by hundreds of times, reaching the same level as FcγR2B binding, which means that the binding selectivity of FcγR2B is not improved compared to FcγR2A 131R (US Patent Publication No. 2009 / 0136485). In addition to their pro-inflammatory effects, antibody binding to FcγR2A can lead to platelet activation, thereby causing thromboembolic events, as demonstrated by the therapeutic antibody bevacizumab (Meyer et al., Journal of Thrombosis and Haemostasis, Vol. 7, No. 1: 171-181, 2009; Scapmacalcci et al., Journal of the National Cancer Institute, Vol. 99, No. 16: 1232-1239, 2007), and as demonstrated by antibodies targeting the CD40 ligand (Boumpas et al., Arthritis and rheumatism, Vol. 48, No. 3: 719-727, 2003; Robles-Carrillo et al., Journal of Immunology (Baltimore, Md.: 1950), Vol. 185, No. 3: 1577-1583, 2010). Furthermore, antibodies with enhanced FcγR2A binding have been reported to enhance macrophage-mediated antibody-dependent phagocytosis (ADCP) (Richards et al., Molecular Cancer Therapeutics, Vol. 7, No. 8: pp. 2517-2527, 2008). When the antigen of an antibody is phagocytosed by macrophages, the antibody itself is also phagocytosed. In this case, peptide fragments derived from these antibodies are also presented as antigens, and their antigenicity may become more pronounced, thereby increasing the risk of generating antibodies against the antibody (antidrug antibodies). More specifically, enhanced FcγR2A binding will increase the risk of generating antibodies against the antibody, and this will significantly reduce their value as drugs.Therefore, antibodies that selectively bind to FcγR2B and reduce binding to FcγR2A may be more effective immunosuppressants and better-tolerated therapeutic agents with a lower risk of inducing thromboembolic events and lower immunogenicity.

[0120] Therefore, in order to enable BTLA agonist antibodies to effectively suppress the immune response without triggering inflammatory FcR signaling, the inventors proposed to regulate the antibody to selectively bind Fc to FcγR2B.

[0121] Molecules that bind more selectively to FcγR2B will promote bidirectional inhibitory signaling of BTLA on cells expressing BTLA and FcγR2B on cells expressing FcγR2B, which will enhance the immunosuppressive effect of the antibody. This is what is desired in therapeutic antibodies intended for the treatment of diseases with excessive immune activation.

[0122] However, the extremely high affinity for FcγR2B may adversely affect the antibody's half-life due to the receptor's metabolic turnover in hepatic sinusoidal epithelial cells (Ganesan et al., The Journal of Immunology, Vol. 189, No. 10: pp. 4981-4988, 2012), as exemplified by the FcγR2B-enhanced IgG1 antibody XmAb7195, which binds to FcγR2B with a KD of 7.74 nM (Chu et al., Journal of Allergy and Clinical). Immunology, Vol. 129, No. 4: pp. 1102-1115, 2012; https: / / linkinghub.elsevier.com / retrieve / pii / S0091674911018343 (May 13, 2020), and according to Xencor, compared to the average half-life of wild-type IgG1 of approximately 21 days (Morell, Terry, and Waldmann. Journal of Clinical...). Investigation, Vol. 49, No. 4: pp. 673-680, 1970; http: / / www.jci.org / articles / view / 106279 (May 16, 2020)), in a phase 1a trial, the mean in vivo half-life of this antibody was 3.9 days (American Thoracic Society (ATS), 2016 International Conference in San Francisco, California - A6476: Panel No. 407). Therefore, in the context of this invention, while selectivity for FcγR2B and adequate binding to a supporting agonist may be desirable for BTLA agonist antibodies, excessive affinity for FcγR2B may be undesirable in treatment, as the resulting shortened half-life would likely necessitate more frequent dosing.

[0123] Various mutations (including amino acid substitutions) can be incorporated into the heavy chain constant region of an antibody to modify signal transduction via one or more Fcγ receptors. WO2006 / 019447 (Xencor) discloses various Fc variant molecules (e.g., antibodies) with altered effector functions through amino acid substitutions in the Fc region.

[0124] The inventors have discovered that incorporating the P238D substitution mutation into the BTLA agonist of the present invention enhances the selectivity for binding to FcγR2B and signal transduction via FcγR2B without significantly reducing the in vivo half-life of the antibody.

[0125] Although the Fc portion can accommodate other modifications (such as amino acid substitutions), in this particular embodiment, the P238D modification is the only modification introduced into the BTLA-binding molecule of the present invention and relative to the wild-type Ig Fc sequence.

[0126] In one embodiment, the antibody comprises aspartic acid at position 238 of IgG1 (using EU index). Suitably, the antibody of the present invention comprises the hIgG1 constant region disclosed in SEQ ID NO: 227, or a constant region having up to 5 amino acid modifications, provided that P238D substitution is present.

[0127] In one embodiment, the antibody comprises aspartic acid at position 238 of IgG4 (using EU index). Suitably, the antibody of the present invention comprises the hIgG4 constant region disclosed in SEQ ID NO:235, or a constant region having up to 5 amino acid modifications, provided that P238D substitution is present.

[0128] The antibody of the present invention promotes bidirectional inhibitory signaling of BTLA on cells expressing BTLA and FcγR2B on cells expressing FcγR2B, and has an in vivo half-life sufficient for suitable therapeutic use. Suitably, the in vivo half-life in humans is at least 5 days, such as 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 a specific embodiment, the in vivo half-life in humans is at least 10 days, which would allow for suitable dosing regimens, such as three times a week. Suitably, the in vivo half-life is about 10 to 30 days, such as about 12 to 20 days or 14 to 25 days.

[0129] In a specific embodiment of the invention, the antibody of the present invention exhibits an in vivo half-life within ±3 days of the half-life of a comparable control antibody containing the wild-type Fc region. The comparable control antibody is a control antibody having the same heavy and light chains except for the addition of Fc modification for binding to FcγR2B, as described herein.

[0130] In a specific embodiment of the invention, the antibody of the present invention exhibits an in vivo half-life that retains at least 50%, such as at least 60%, at least 70%, at least 80%, or at least 90%, of the half-life of a comparable control antibody containing a wild-type Fc region. The comparable control antibody is a control antibody having the same heavy and light chains except for the addition of Fc modification for binding to FcγR2B, as described herein.

[0131] When the Fc modification that increases binding to FcγR2B is a P238D substitution, in a specific embodiment, the antibody of the present invention exhibits an in vivo half-life within ±3 days of the half-life of a comparable control antibody containing an Fc region containing a proline at position 238 (EU index).

[0132] In another specific embodiment, when the Fc modification that increases binding to FcγR2B is a P238D substitution, the antibody of the present invention exhibits an in vivo half-life that retains at least 50%, such as at least 60%, at least 70%, at least 80%, or at least 90%, of the half-life of the parent antibody, which contains an Fc region containing a proline at position 238 (EU index).

[0133] A longer half-life allows for a longer period of time with good receptor occupancy. This means longer intervals between doses, or, in alternatives to longer dose intervals, a longer half-life will allow for the administration of lower doses—which may be important if dose-limiting toxicities exist at higher peak doses.

[0134] Producing antibodies with long half-lives can also have certain benefits, such as reducing product costs, reducing the treatment burden on patients, and increasing patient compliance.

[0135] Suitably, the molecules of the present invention are capable of maintaining receptor occupancy >80% for at least 10 days, such as 14, 21, 28, 35, 42 or more days, after a single dose of 10 mg / kg.

[0136] Suitablely, the molecules of the present invention can be administered at dose intervals of 3 weeks, ideally 4 weeks or more (such as 6 weeks or 8 weeks).

[0137] According to a first aspect of the invention, an antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an Fc region containing substitutions that result in increased binding to FcγR2B compared to an unsubstituted parent molecule. Suitably, the antibody is a separating antibody.

[0138] In some embodiments, the antibody has increased binding to FcγR2B compared to the parent molecule, such that the ratio of [KD value of parental peptide to FcγR2B] to [KD value of variant peptide to FcγR2B] is greater than 1, such as greater than 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.

[0139] In some implementations, the antibody is more selective for binding FcγR2B than for binding FcγR2A.

[0140] In some embodiments, the antibody exhibits enhanced FcγR2B binding activity compared to the parent peptide and maintains or reduces binding activity to FcγR2A (R type) and / or FcγR2A (H type). In some embodiments, the ratio of [KD value of the variant peptide against FcγR2A (R type)] to [KD value of the variant peptide against FcγR2B] is 2 or greater, such as 3, 4, 5, 6, 7, 8, 9, 10 or greater. In some implementations, the ratio of [KD value of variant peptide against FcγR2A (H type)] to [KD value of variant peptide against FcγR2B] is 2 or greater, such as 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 greater.

[0141] In some embodiments, the antibody exhibits enhanced FcγR2B binding activity and maintains or reduces FcγR1A binding activity compared to the parent peptide. In some embodiments, the ratio of [KD value of the variant peptide against FcγR1A] to [KD value of the variant peptide against FcγR2B] is 0.05 or greater, such as at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or greater.

[0142] In some embodiments, the antibody has reduced Fcγ1 binding activity compared to the parent peptide. In some embodiments, the ratio of [KD value of variant peptide against FcγR1A] to [KD value of parent peptide against FcγR1A] is at least 10, 20, 50, 100, or 200.

[0143] In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residue H68 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO: 225).

[0144] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain contains 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 numbers are based on the EU index). Suitably, the antibody that specifically binds to human BTLA is an agonist antibody / antigen-binding fragment.

[0145] Suitablely, the antibody is human IgG1 or IgG4 with one or more amino acid substitutions selected from the group consisting of: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A. In a specific embodiment, the antibody binding to human BTLA has a heavy chain and / or light chain with at least one CDR, the at least one CDR being derived from antibodies selected from the following: 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 the binding of HVEM.

[0146] In a specific implementation, the isolating antibody binding to human BTLA is selected from the group consisting of 6.2, 2.8.6, 3E8, or an antibody competing with any of 6.2, 2.8.6, or 3E8 for binding to human BTLA, wherein the antibody specifically binds to BTLA and induces signal transduction via the receptor. The antibody also comprises an Fc region containing an aspartic acid residue at position 238 (EU index).

[0147] As disclosed in Table 1 and described herein, an antibody selected from the group consisting of the following: 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 means any antibody or antigen-binding fragment thereof that contains one or more of any antibodies disclosed in Table 1 or 2 (whether murine, humanized, or humanized / engineered) such as VH CDR 1, 2, and 3, or VL CDR 1, 2, and 3, or VH CDR 1, 2, and 3 and VL CDR 1, 2, and 3).

[0148] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA. The isolation antibody comprises at least one VH CDR having an amino acid sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:17, and having 0 to 3 amino acid modifications, such as 0, 1, 2, or 3 amino acid modifications. In some embodiments, the amino acid modifications include, but are not limited to, amino acid substitutions, additions, deletions, or chemical modifications, wherein the antibody-binding affinity or T-cell inhibitory effect of the modified amino acid sequence is not eliminated compared to the unmodified amino acid sequence.

[0149] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO:1, CDRH2 has the amino acid sequence shown in SEQ ID NO:2, 11 or 17, and CDRH3 has the amino acid sequence shown in SEQ ID NO:3.

[0150] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12, and having 0 to 3 amino acid modifications.

[0151] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 an amino acid sequence as shown in SEQ ID NO:4, CDRL2 has an amino acid sequence as shown in SEQ ID NO:5 or 12, and CDRL3 has an amino acid sequence as shown in SEQ ID NO:6.

[0152] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO:1, CDRH2 has the amino acid sequence shown in SEQ ID NO:2, 11, or 17, and CDRH3 has the amino acid sequence shown 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 shown in SEQ ID NO:4, CDRL2 has the amino acid sequence shown in SEQ ID NO:5 or 12, and CDRL3 has the amino acid sequence shown in SEQ ID NO:6.

[0153] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO: 1, CDRH2 has the amino acid sequence shown in SEQ ID NO: 17, and CDRH3 has the amino acid sequence shown 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 shown in SEQ ID NO: 4, CDRL2 has the amino acid sequence shown in SEQ ID NO: 12, and CDRL3 has the amino acid sequence shown in SEQ ID NO: 6; and wherein the heavy chain comprises aspartic acid at position 238 (EU index).

[0154] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, and having 0 to 3 amino acid modifications.

[0155] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO:20, CDRH2 has the amino acid sequence shown in SEQ ID NO:21 and CDRH3 has the amino acid sequence shown in SEQ ID NO:22.

[0156] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VL CDR having an amino acid sequence as shown in SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25, and having 0 to 3 amino acid modifications.

[0157] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO: 23, CDRL2 has the amino acid sequence shown in SEQ ID NO: 24 and CDRL3 has the amino acid sequence shown in SEQ ID NO: 25.

[0158] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO:20, CDRH2 has the amino acid sequence shown in SEQ ID NO:21, and CDRH3 has the amino acid sequence shown 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 shown in SEQ ID NO:23, CDRL2 has the amino acid sequence shown in SEQ ID NO:24, and CDRL3 has the amino acid sequence shown in SEQ ID NO:25.

[0159] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VH CDR having an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:48 or SEQ ID NO:32, and having 0 to 3 amino acid modifications.

[0160] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 an amino acid sequence as shown in SEQ ID NO: 30, CDRH2 has an amino acid sequence as shown in SEQ ID NO: 31, 40 or 48, and CDRH3 has an amino acid sequence as shown in SEQ ID NO: 32.

[0161] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VL CDR having an amino acid sequence as shown in SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35, and having 0 to 3 amino acid modifications.

[0162] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO: 33, CDRL2 has the amino acid sequence shown in SEQ ID NO: 34 and CDRL3 has the amino acid sequence shown in SEQ ID NO: 35.

[0163] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 shown in SEQ ID NO:30, CDRH2 has the amino acid sequence shown in SEQ ID NO:31, 40, or 48, and CDRH3 has the amino acid sequence shown 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 shown in SEQ ID NO:33, CDRL2 has the amino acid sequence shown in SEQ ID NO:34, and CDRL3 has the amino acid sequence shown in SEQ ID NO:35.

[0164] In each of these aspects, the antibody has an Fc region comprising at least one amino acid substitution, which, compared to an unsubstituted parent molecule, results in increased binding to FcγR2B. In some embodiments, the antibody exhibits better selectivity for binding to FcγR2B than for binding to FcγR2A compared to an unsubstituted parent molecule. In some embodiments, the antibody exhibits better selectivity for binding to FcγR2B than for binding to FcγR1A compared to an unsubstituted parent molecule.

[0165] In a specific implementation, the antibody contains an Fc region containing an aspartic acid residue at position 238 (EU index).

[0166] In a specific implementation, the antibody contains an Fc region containing aspartic acid at position 237 (EU index), aspartic acid at position 238 (EU index), glycine at position 271 (EU index), and arginine at position 330 (EU index).

[0167] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, wherein the antibody comprises a heavy chain and a light chain, wherein: the heavy chain comprises an Fc region and a heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs): CDRH1, CDRH2, and CDRH3, and the light chain comprises a light chain variable region, the light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3; wherein (1) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:17, and SEQ ID NO:3, respectively, having 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:12, and SEQ ID NO:6, respectively, having 0 to 3 amino acid modifications; or (2) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively, having 0 to 3 amino acid modifications; The amino acid sequence shown in NO:22 has 0 to 3 amino acid modifications, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25 respectively, with 0 to 3 amino acid modifications; or (3) CDRH1, CDRH2 and CDRH3 have the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32 respectively, with 0 to 3 amino acid modifications, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35 respectively, with 0 to 3 amino acid modifications, and the Fc region contains aspartic acid at position 238 (EU index).

[0168] A typical antibody consists of two heavy chains and two light chains, with the paired heavy chains containing Fc regions. Therefore, as used herein, "heavy chain containing Fc regions" refers to the region on the H-chain polypeptide that forms a functional Fc region together with the Fc region of another H-chain.

[0169] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VH CDR having an amino acid sequence as shown below: (1) SEQ ID NO: 45, 46 or 47, having 0 to 3 amino acid modifications; (2) SEQ ID NO: 53, 54 or 55, having 0 to 3 amino acid modifications; (3) SEQ ID NO: 61, 62 or 63, having 0 to 3 amino acid modifications; (4) SEQ ID NO: 61, 69 or 70, having 0 to 3 amino acid modifications; (5) SEQ ID NO: 76, 77 or 78, having 0 to 3 amino acid modifications; (6) SEQ ID NO: 45, 46 or 84, having 0 to 3 amino acid modifications; (7) SEQ ID NO: 88, 89 or 90, having 0 to 3 amino acid modifications; (8) SEQ ID NO: 95, 96 or 97, having 0 to 3 amino acid modifications; (9) SEQ ID NO: 45, 46 or 84, having 0 to 3 amino acid modifications; (10) SEQ ID NO: 103, 104 or 105, having 0 to 3 amino acid modifications; (11) SEQ ID NO: 76, 111 or 112, having 0 to 3 amino acid modifications; (12) SEQ ID NO: 126, 127 or 128, having 0 to 3 amino acid modifications; (13) SEQ ID NO: 133, 134 or 135, having 0 to 3 amino acid modifications; (14) SEQ ID NO: 103, 134 or 139, having 0 to 3 amino acid modifications; (15) SEQ ID NO: 143, 144 or 145, having 0 to 3 amino acid modifications; (16) SEQ ID NO: 151, 152 or 153, having 0 to 3 amino acid modifications; (17) SEQ ID (18) SEQ ID NO: 167, 168 or 169, having 0 to 3 amino acid modifications; (19) SEQ ID NO: 45, 46 or 177, having 0 to 3 amino acid modifications; (20) SEQ ID NO: 181, 182 or 183, having 0 to 3 amino acid modifications; (21) SEQ ID NO: 45, 191 or 192, having 0 to 3 amino acid modifications; (22) SEQ ID NO: 196, 197 or 198, having 0 to 3 amino acid modifications; (23) SEQ ID NO: 204, 205 or 206, having 0 to 3 amino acid modifications; (24) SEQ ID NO: 212, 213 or 214, having 0 to 3 amino acid modifications; (25) SEQ ID NO: 1, 2 or 3, having 0 to 3 amino acid modifications;(26) SEQ ID NO: 20, 163, or 22, having 0 to 3 amino acid modifications; (27) SEQ ID NO: 30, 48, or 32, having 0 to 3 amino acid modifications; (28) SEQ ID NO: 1, 11, or 3, having 0 to 3 amino acid modifications; (29) SEQ ID NO: 1, 17, or 3, having 0 to 3 amino acid modifications; (30) SEQ ID NO: 20, 21, or 22, having 0 to 3 amino acid modifications; (33) SEQ ID NO: 30, 31, or 32, having 0 to 3 amino acid modifications; or (34) SEQ ID NO: 30, 40, or 32, having 0 to 3 amino acid modifications. The antibody further comprises an Fc region containing an aspartic acid residue at position 238 (EU index).

[0170] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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, CDRH2, and CDRH3 have the following amino acid sequences: (1) SEQ ID NO: 45, 46, and 47, respectively; (2) SEQ ID NO: 53, 54, and 55, respectively; (3) SEQ ID NO: 61, 62, and 63, respectively; (4) SEQ ID NO: 61, 69, and 70, respectively; (5) SEQ ID NO: 76, 77, and 78, respectively; (6) SEQ ID NO: 45, 46, and 84, respectively; (7) SEQ ID NO: 88, 89, and 90, respectively; (8) SEQ ID NO: 95, 96, and 97, respectively; (9) SEQ ID NO: 103, 104, and 105, respectively; (10) SEQ ID NO: 45, 46, and 84, respectively; (11) SEQ ID NO: 45, 46, and 84, respectively; (12) SEQ ID NO: 45, 46, and 84, respectively; (13) SEQ ID NO: 45, 46, and 84, respectively; (14) SEQ ID NO: 45, 46, and 84, respectively; (15) SEQ ID NO: 45, 46, and 84, respectively; (16) SEQ ID NO: 45, 46, and 84, respectively; (17) SEQ ID NO: 45, 46, and 84, respectively; (18) SEQ ID NO: 45, 46, and 84 (11) SEQ ID NO:76, 111 and 112; (12) SEQ ID NO:118, 119 and 120; (13) SEQ ID NO:126, 127 and 128; (14) SEQ ID NO:133, 134 and 135; (15) SEQ ID NO:103, 134 and 139; (16) SEQ ID NO:143, 144 and 145; (17) SEQ ID NO:151, 152 and 153; (18) SEQ ID NO:159, 160 and 161; (19) SEQ ID NO:45, 46 and 177; (20) SEQ ID NO:181, 182 and 183; (21) SEQ ID NO:76, 111 and 112; (22) SEQ ID NO:118, 119 and 120; (23) SEQ ID NO:126, 127 and 128; (24) SEQ ID NO:133, 134 and 135; (25) SEQ ID NO:103, 134 and 139; (26) SEQ ID NO:143, 144 and 145; (27) SEQ ID NO:151, 152 and 153; (28) SEQ ID NO:159, 160 and 161; (29) SEQ ID NO:167, 168 and 169; (20) SEQ ID NO:181, 182 and 183; (21) SEQ ID NO:183, 184 and 185; (22) SEQ ID NO:183, 184 and 185; (23) SEQ ID NO: (22) SEQ ID NO: 45, 191 and 192; (23) SEQ ID NO: 196, 197 and 198; (24) SEQ ID NO: 204, 205 and 206; (25) SEQ ID NO: 212, 213 and 214; (26) SEQ ID NO: 1, 2 and 3; (27) SEQ ID NO: 20, 163 and 22; (28) SEQ ID NO: 30, 48 and 32; (29) SEQ ID NO: 1, 11 and 3; (30) SEQ ID NO: 20, 21 and 22; (33) SEQ ID NO: 30, 31 and 32; or (34) SEQ ID NO: 30, 40 and 32.The 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO:. The antibody also comprises an Fc region containing an aspartic acid residue at position 238 (EU index).

[0171] According to a variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising at least one VL CDR having an amino acid sequence as shown below: (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 92; (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: 121, 122 or 123; (13) SEQ ID NO: 79, 129 or 130; (14) SEQ ID NO: 106, 107 or 136; (15) SEQ ID NO: 146, 147 or 148; (16) SEQ ID NO: 154, 155 or 156; (17) SEQ ID NO: 4, 12 or 164; (18) SEQ ID NO: 170, 171 or 172; (19) SEQ ID NO: 154, 155 or 178; (20) SEQ ID NO: 184, 185 or 186; (21) SEQ ID NO: 154, 80 or 189; (22) SEQ ID NO: 154, 155 or 193; (23) SEQ ID NO: 199, 200 or 201; (24) SEQ ID NO: 207, 208 or 209; 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 may be present in any CDR / SEQ ID NO: and wherein the antibody further comprises an Fc region containing aspartic acid at position 238 (EU index).

[0172] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 have the following amino acid sequences: (1) SEQ ID NO: 33, 34 and 35, respectively; (2) SEQ ID NO: 56, 57 and 58, respectively; (3) SEQ ID NO: 64, 65 and 66, respectively; (4) SEQ ID NO: 71, 72 and 73, respectively; (5) SEQ ID NO: 79, 80 and 81, respectively; (6) SEQ ID NO: 33, 34 and 85, respectively; (7) SEQ ID NO: 91, 65 and 92, respectively; (8) SEQ ID NO: 98, 99 and 100, respectively; (9) SEQ ID NO: 106, 107 and 108, respectively; (10) SEQ ID NO: 33, 34 and 85, respectively; (10) SEQ ID NO: 33, 34 and 85, respectively; (11) SEQ ID NO: 33, 34 and 85, respectively; (12) SEQ ID NO: 33, 34 and 85, respectively; (13) SEQ ID NO: 33, 34 and 85, respectively; (14) SEQ ID NO: 33, 34 and 85, respectively; (15) SEQ ID NO: 33, 34 and 85, respectively; (16) SEQ ID NO: 33, 34 and 85, respectively; (17) SEQ ID NO: 33, 34 and 85, respectively; (18) SEQ ID NO: 33, 34 (11) SEQ ID NO: 113, 114 and 115; (12) SEQ ID NO: 121, 122 and 123 respectively; (13) SEQ ID NO: 79, 129 and 130 respectively; (14) SEQ ID NO: 106, 107 and 136 respectively; (15) SEQ ID NO: 146, 147 and 148 respectively; (16) SEQ ID NO: 154, 155 and 156 respectively; (17) SEQ ID NO: 170, 171 and 172 respectively; (18) SEQ ID NO: 154, 155 and 178 respectively; (19) SEQ ID NO: 184, 185 and 186 respectively; (20) SEQ ID NO: 79, 80 and 189 respectively; (21) SEQ ID NO: 154, 155 and 193 respectively; (22) SEQ ID NO: 154, 155 and 193 respectively; (23) SEQ ID NO: 199, 200 and 201; (24) SEQ ID NO: 207, 208 and 209; (25) SEQ ID NO: 215, 34 and 216; (26) SEQ ID NO: 4, 5 and 6; (27) SEQ ID NO: 23, 176 and 25; (28) SEQ ID NO: 33, 34 and 35; (29) SEQ ID NO: 4, 12 and 6; (30) SEQ ID NO: 23, 24 and 25; or (31) SEQ ID NO: 33, 34 and 35;The 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO:, and the antibody further comprises an Fc region containing an aspartic acid at position 238 (EU index).

[0173] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 amino acid sequences as shown in SEQ ID NO:45, 46, and 47, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:33, 34, and 35, respectively; (2) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:53, 54, and 55, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:56, 57, and 58, respectively; (3) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:33, 54, and 55, respectively. (3) The amino acid sequences shown in NO:61, 62 and 63, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:64, 65 and 66, respectively; (4) CDRH1, CDRH2 and CDRH3 have the amino acid sequences shown in SEQ ID NO:61, 69 and 70, respectively, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:71, 72 and 73, respectively; (5) CDRH1, CDRH2 and CDRH3 have the amino acid sequences shown in SEQ ID NO:76, 77 and 78, respectively, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:79, 80 and 81, respectively; (6) CDRH1, CDRH2 and CDRH3 have the amino acid sequences shown in SEQ ID NO:45, 46 and 84, respectively, and CDRL1, CDRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NO:64, 65 and 66, respectively. (7) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 88, 89, and 90, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 91, 65, and 92, respectively; (8) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 95, 96, and 97, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 98, 99, and 100, respectively;(9) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO: 103, 104, and 105, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO: 106, 107, and 108, respectively; (10) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO: 76, 111, and 112, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO: 113, 114, and 115, respectively; (11) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO: 118, 119, and 120, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO: 121, 122, and 123, respectively; (12) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO: 103, 104, and 105, respectively. (13) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 126, 127, and 128, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 79, 129, and 130, respectively; (14) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 103, 134, and 139, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 106, 107, and 136, respectively; (15) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 143, 144, and 145, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 79, 129, and 130, respectively. (16) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 151, 152, and 153, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 154, 155, and 156, respectively; (17) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 159, 160, and 161, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 4, 12, and 164, respectively;(18) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO:167, 168, and 169, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:170, 171, and 172, respectively; (19) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:170, 171, and 172, respectively; (20) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO:45, 46, and 177, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:154, 155, and 178, respectively; (21) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO:167, 168, and 169, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:170, 171, and 172, respectively; (22) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 181, 182, and 183, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 184, 185, and 186, respectively; (23) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 76, 77, and 78, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 79, 80, and 189, respectively; (24) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 45, 191, and 192, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 154, 155, and 193, respectively; (25) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 196, 197, and 198, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 184, 185, and 186, respectively. (25) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 204, 205, and 206, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 207, 208, and 209, respectively; (26) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 212, 213, and 214, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 215, 34, and 216, respectively;(27) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:4, 5, and 6, respectively; (28) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:20, 163, and 22, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:23, 176, and 25, respectively; (29) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:30, 48, and 32, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:33, 34, and 35, respectively; (30) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:1, 11, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:33, 34, and 35, respectively; (31) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:1, 11, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:4, 12, and 6, respectively; (32) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:1, 17, and 3, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:4, 12, and 6, respectively; (33) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:20, 21, and 22, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO:23, 24, and 25, respectively; (34) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO:4, 12, and 6, respectively. The amino acid sequences shown in NO:30, 31, and 32, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:33, 34, and 35, respectively; or (35) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO:30, 40, and 32, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO:33, 34, and 35, respectively; wherein 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO:, and wherein the antibody further comprises an Fc region containing an aspartic acid at position 238 (EU index).

[0174] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising: (1) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 45, 46 or 47, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35; (2) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 53, 54 or 55, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 56, 57 and 58; (3) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 61, 62 or 63, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 64, 65 or 66; (4) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 61, 69 or 70, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 71, 72 and 73; (5) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 45, 46 or 47, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35; (6) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 76, 77 or 78, and at least one VLCDR having an amino acid sequence as shown in SEQ ID NO: 79, 80 or 81; (7) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 45, 46 or 84, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 33, 34 or 85; (8) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 88, 89 or 90, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 91, 65 or 92; (9) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 95, 96 or 97, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 98, 99 or 100; (10) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 103, 104 or 105, and at least one VLCDR having an amino acid sequence as shown in SEQ ID NO: 79, 80 or 81. (10) A VL CDR having an amino acid sequence as shown in SEQ ID NO: 76, 111 or 112, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 113, 114 or 115; (11) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 118, 119 or 120, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 121, 122 or 123;(12) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 126, 127 or 128, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 79, 129 or 130; (13) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 133, 134 or 135, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 106, 107 or 136; (14) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 103, 134 or 139, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 106, 107 or 136; (15) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 143, 144 or 145, and at least one VLCDR having the amino acid sequence shown in SEQ ID NO: 146, 147 or 148; (16) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 146, 147 or 148. (17) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 151, 152 or 153, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 154, 155 or 156; (18) A VHCDR having an amino acid sequence as shown in SEQ ID NO: 167, 168 or 169, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 170, 171 or 172; (19) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 45, 46 or 47, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 170, 171 or 172; (20) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 45, 46 or 177. (21) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 181, 182 or 183, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 184, 185 or 186; (22) A VH CDR having an amino acid sequence as shown in SEQ ID NO: 76, 77 or 78, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 79, 80 or 189;(23) At least one VHCDR having the amino acid sequence shown in SEQ ID NO: 45, 191 or 192, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 154, 155 or 193; (24) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 196, 197 or 198, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 199, 200 or 201; (25) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 204, 205 or 206, and at least one VLCDR having the amino acid sequence shown in SEQ ID NO: 207, 208 or 209; (26) At least one VH CDR having the amino acid sequence shown in SEQ ID NO: 212, 213 or 214, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 215, 34 or 216; (27) At least one VHCDR having the amino acid sequence shown in SEQ ID NO: 45, 191 or 192, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 154, 155 or 193; (28) A VH CDR having the amino acid sequence shown in SEQ ID NO: 1, 2 or 3, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 4, 5 or 6; (29) A VH CDR having the amino acid sequence shown in SEQ ID NO: 30, 48 or 32, and at least one VL CDR having the amino acid sequence shown in SEQ ID NO: 33, 34 or 35; (30) A 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) A 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. CDR; (32) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 1, 17 or 3, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 4, 12 or 6; (33) at least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 20, 21 or 22, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 23, 24 or 25;(34) At least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 30, 31 or 32, and at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35; or (35) At least one VH CDR having an amino acid sequence as shown in SEQ ID NO: 30, 40 or 32, and at least one VL CDR having an amino acid sequence as shown 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 wherein the antibody further comprises an Fc region containing an aspartic acid at position 238 (EU index).

[0175] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 7, 13 or 18 or a sequence having at least 90% sequence identity with such amino acid sequences.

[0176] In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 7, 13 or 18, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0177] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:8 or 14 or a sequence having at least 90% sequence identity with such amino acid sequence.

[0178] In other embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8 or 14, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0179] In each of these embodiments, the antibody has an Fc region comprising at least one amino acid substitution, which results in increased binding to FcγR2B compared to an unsubstituted parental molecule. In some embodiments, the antibody exhibits better selectivity for binding to FcγR2B than for binding to FcγR2A compared to an unsubstituted parental molecule.

[0180] In a specific implementation, the antibody contains an Fc region containing an aspartic acid residue at position 238 (EU index).

[0181] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 with the amino acid sequence shown as 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 with the amino acid sequence shown as SEQ ID NO:8 or 14.

[0182] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:18 or SEQ ID NO:13, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14.

[0183] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:7, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:8.

[0184] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:13, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14.

[0185] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8.

[0186] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:18, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14.

[0187] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 9, 15 or 19 or a sequence having at least 90% sequence identity with such amino acid sequences.

[0188] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 9, 15 or 19 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0189] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 10, 16 or 29 or a sequence having at least 90% sequence identity with such amino acid sequences.

[0190] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 10, 16 or 29 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0191] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 9 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 10 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0192] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:9 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO:10 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0193] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:15 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:16 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0194] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 15 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 16 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0195] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 15 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 10 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0196] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 15 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 10 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0197] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 19 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 16 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0198] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO:16 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0199] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:26 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0200] In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0201] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:27 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0202] In other embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:27, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0203] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 27.

[0204] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 26, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 27.

[0205] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0206] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0207] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0208] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0209] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:28 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:29 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0210] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0211] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:36 or 41 or a sequence having at least 90% sequence identity with such amino acid sequence.

[0212] In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:36 or 41, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0213] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 37 or 43 or a sequence having at least 90% sequence identity with such amino acid sequence.

[0214] In other embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 37 or 43, having up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0215] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody 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 with the amino acid sequence shown as 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 with the amino acid sequence shown as SEQ ID NO:37 or 43.

[0216] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:36, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:37.

[0217] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 41, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 37.

[0218] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:36, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:43.

[0219] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:38 or 42 or a sequence having at least 90% sequence identity with such amino acid sequence.

[0220] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:38 or 42 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0221] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 39 or 44 or a sequence having at least 90% sequence identity with such amino acid sequence.

[0222] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO:39 or 44 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0223] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:38 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:39 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0224] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 38 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 39 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0225] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 42 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 39 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0226] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 42 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 39 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0227] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 38 or a sequence having at least 90% sequence identity with the amino acid sequence, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 44 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0228] According to another variation of the first aspect of the invention, an isolation antibody is provided that specifically binds to human BTLA, the isolation antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:38 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications), and wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO:44 or a sequence having up to 10 modifications (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications).

[0229] 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 with the sequence disclosed in SEQ ID NO: 18.

[0230] 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 with the sequence disclosed in SEQ ID NO: 26.

[0231] 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 with the sequence disclosed in SEQ ID NO:36.

[0232] 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 with the sequence disclosed in SEQ ID NO: 14.

[0233] 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 with the sequence disclosed in SEQ ID NO:27.

[0234] 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 with the sequence disclosed in SEQ ID NO:43.

[0235] According to another variation of the first aspect of the invention, a separating antibody having a primary VH domain and / or a primary VL domain, wherein the primary VH domain and / or primary VL domain has at least one CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any antibody clone as shown in Table 1 or Table 2. In some embodiments, a separating antibody selected from the antibody clones shown in Table 1 or Table 2 is provided herein.

[0236] Table 1: Exemplary BTLA agonist antibodies

[0237]

[0238]

[0239] Table 2: Humanized and engineered antibodies

[0240]

[0241] In each aspect of the first aspect of the invention, the antibody has an Fc region comprising at least one amino acid substitution, wherein the at least one amino acid substitution results in increased binding to FcγR2B compared to an unsubstituted parent molecule, and / or the at least one amino acid substitution results in increased selectivity for binding to FcγR2B relative to binding to FcγR2A compared to an unsubstituted parent molecule. In some embodiments, the antibody has enhanced selectivity for binding to FcγR2B relative to binding to FcγR1A compared to an unsubstituted parent molecule.

[0242] In a specific embodiment, each antibody according to the first aspect of the invention (which includes any variant of the first aspect) comprises an Fc region containing 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 numbers are based on the EU index).

[0243] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an aspartic acid at position 238 (EU index).

[0244] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an aspartic acid at position 237 (EU index).

[0245] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an aspartic acid at position 236 (EU index).

[0246] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an alanine at position 235 (EU index).

[0247] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an alanine at position 234 (EU index).

[0248] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an alanine at position 265 (EU index).

[0249] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having a glutamic acid at position 267 (EU index).

[0250] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having glycine at position 271 (EU index).

[0251] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an alanine at position 297 (EU index).

[0252] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an alanine at position 322 (EU index).

[0253] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc having an arginine at position 330 (EU index).

[0254] In a specific embodiment, each antibody according to the first aspect of the invention comprises an Fc containing aspartic acid at position 237 (EU index), aspartic acid at position 238 (EU index), glycine at position 271 (EU index), and arginine at position 330 (EU index).

[0255] In a specific embodiment, the antibody according to the first aspect of the invention comprises a substituted Fc isotype selected from the group consisting of: hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, and hIgG4L235A.

[0256] In specific implementations, the heavy or light chain also includes constant regions. If the molecule is a full-length IgG-type antibody molecule, the heavy chain may contain three constant structural domains. In some implementations, the isolated antibody that specifically binds to human BTLA exhibits up to approximately 10 × 10⁻⁶ domains. -9 M's combination of BTLA's K D In some implementations, the isolated antibody that specifically binds to human BTLA exhibits up to approximately 4 × 10⁻⁶. -9 M's combination of BTLA's K D In some implementations, the isolated antibody that specifically binds to human BTLA exhibits a maximum of approximately 1 × 10⁻⁶. -9 M's combination of BTLA's K D .

[0257] In some embodiments, the isolated antibody of the present invention (e.g., humanized) binds to human BTLA at 37°C, wherein KD Not exceeding approximately 10 nM (1 × 10 -8 M); preferably not exceeding about 1 nM; more preferably, the antibody has a concentration of not more than about 500 pM (5 × 10⁻⁶ mM) at 37°C. -10 K (M), 200pM, 100pM, 50pM, 20pM, 10pM, 5pM or even 2pM D The implementation plan for the value. As used in this context, the term "about" means + / - 10%.

[0258] In some embodiments, the isolating antibody of the present invention (e.g., humanized) is subjected to a temperature of at least 1.0 × 10⁻⁶ at 37°C. 5 Human BTLA binds at an association rate of (1 / Ms). In some embodiments, the isolating antibody of the present invention (e.g., humanized) binds at a rate of at least 2.0 × 10⁻⁶ 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 7.0×10 5 The association rate of (1 / Ms) with human BTLA.

[0259] In some embodiments, the isolating antibody of the present invention (e.g., humanized) is heated at 37°C at a concentration not exceeding or less than 1.0 × 10⁻⁶. -3 Human BTLA binds at a dissociation rate of (1 / s). In some embodiments, the separating antibody of the present invention (e.g., humanized) binds at a dissociation rate of no more than or less than 3.0 × 10⁻⁶ at 37°C. -4 Human BTLA binds at a dissociation rate of (1 / s). In some embodiments, the separating antibody of the present invention (e.g., humanized) binds at a dissociation rate of no more than or less than 2.0 × 10⁻⁶ at 37°C. -4 (1 / s) or 1.0 × 10 -4 The dissociation rate of human BTLA is (1 / s).

[0260] In a specific embodiment of the first aspect of the invention, this document provides isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuators (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, and wherein said 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 described in Example 2; does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined, for example, by surface plasmon resonance (SPR) using a method such as described in Example 4; and inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, said antibody has a KD of at least 5.0 × 10⁻⁶. 5 The antibody binds to human B and T lymphocyte attenuators (BTLA) at an association rate of (1 / Ms), 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 at an association rate of less than 3.0 × 10⁻⁶. -4 The antibody binds to human B and T lymphocyte attenuators (BTLA) at a dissociation rate of (1 / s), 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 dissociates at a rate of 3.0 × 10⁻⁶. -4 (1 / s) to 1.0×10 -3 The dissociation rate (1 / s) of binding to human B and T lymphocyte attenuators (BTLA) is determined, as measured 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 residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as that described in Example 5 (where the designation, e.g., D52, refers to the position in SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to residues selected from human BTLA: 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 residues selected from human BTLA: N65 and A64 (according to the position of SEQ ID NO: 225).

[0261] Methods for characterizing the properties of the antibodies of the present 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 the tested antibody / fragment inhibits the binding of BTLA to herpesvirus invasion mediators (HVEM) is described in Example 4; this also uses surface plasmon resonance (SPR). A suitable method for determining whether the tested antibody / fragment inhibits T cell proliferation in vitro is the mixed lymphocyte reaction assay, as described in Example 9. A suitable method for determining the binding site of the antibody / fragment to BTLA may utilize X-ray crystallography or flow cytometry of the mutant receptor, such as by the method described in Example 5.

[0262] In a specific embodiment of the first aspect of the invention, this document provides separation agonist antibodies, which are at least 5.0 × 10⁻⁶. 5 The antibody specifically binds to human B and T lymphocyte attenuators (BTLA) at an association rate of (1 / Ms), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined, for example, by surface plasmon resonance (SPR) using a method such as described in Example 4; and wherein the antibody inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds at an association rate of less than 3.0 × 10⁻⁶. -4The antibody binds to human B and T lymphocyte attenuators (BTLA) at a dissociation rate of (1 / s), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. In some embodiments, the antibody binds to human B and T lymphocyte attenuators (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as 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 described in Example 2. In some embodiments, the antibody binds to residues selected from human BTLA including D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA (at position SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from BTLA: N65 and A64 (at position SEQ ID NO: 225).

[0263] In a specific embodiment of the first aspect of the invention, this document provides a separation agonist antibody, which is 3.0 × 10 -4 (1 / s) to 1.0×10 -3The antibody specifically binds to the human B and T lymphocyte attenuator (BTLA) at a dissociation rate of (1 / s), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, wherein the antibody does not inhibit the binding of BTLA to the herpesvirus entry medium (HVEM), as determined, for example, by surface plasmon resonance (SPR) using a method such as described in Example 4; and wherein the antibody inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds to the human B and T lymphocyte attenuator (BTLA) at a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. In some embodiments, the antibody binds to cynomolgus monkey BTLA at a KD of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. In some embodiments, the antibody binds to cynomolgus monkey BTLA at a KD of at least 5.0 × 10⁻⁶. 5 The association rate (1 / Ms) of binding to human B and T lymphocyte attenuators (BTLA) was determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, antibody binding to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (at position according to SEQ ID NO: 225) was determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as that described in Example 5. In some embodiments, antibody binding to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at position according to SEQ ID NO: 225). In some embodiments, antibody binding to residues selected from human BTLA: D35, T78, K81, S121, and L123. In some embodiments, the antibody binds to residue H68 of human BTLA (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO: 225).

[0264] In a specific embodiment of the first aspect of the invention, this document provides separation agonist antibodies, which are less than 1.0 × 10⁻⁶. -3 A dissociation rate of (1 / s) and at least 5.0 × 10 5The antibody binds to human B and T lymphocyte attenuators (BTLA) at an association rate of (1 / Ms), as measured individually at 37°C by surface plasmon resonance (SPR) using a method such as described in Example 2, wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined, for example, by surface plasmon resonance (SPR) using a method such as described in Example 4; and wherein the antibody inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds to human B and T lymphocyte attenuators (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as 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 described in Example 2. In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position of SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody binds to residues selected from BTLA: Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody binds to residues selected from human BTLA: 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 residues selected from human BTLA: N65 and A64.

[0265] In a specific embodiment of the first aspect of the invention, this document provides a separating agonist antibody that specifically binds to human B and T lymphocyte attenuators (BTLA) at a concentration 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. This antibody inhibits the binding of BTLA to herpesvirus entry mediators (HVEMs), 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody has a concentration of less than 1.0 × 10⁻⁶ NM. 6The antibody binds to human B and T lymphocyte attenuators (BTLA) at an association rate of (1 / Ms), 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 at an association rate of less than 1.0 × 10⁻⁶. -3 The antibody binds to the human B and T lymphocyte attenuator (BTLA) at a dissociation rate of (1 / s), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. In some embodiments, the antibody binds to the cynomolgus monkey B and T lymphocyte attenuator (BTLA) at a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. In some embodiments, the antibody binds to residues selected from the human BTLA group consisting of D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position in SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as described in Example 5. In some embodiments, the antibody binds to residues selected from the human BTLA group consisting of Y39, K41, R42, Q43, E45, and S47 (according to the position in SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at the position of SEQ ID NO: 225). In some embodiments, the antibody binds to BTLA residue H68 (at the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at the position of SEQ ID NO: 225).

[0266] In a specific embodiment of the first aspect of the invention, this document provides a separating agonist antibody, which has a separation agonist antibody concentration of less than 1 × 10⁻⁶. -3The antibody specifically binds to the human B and T lymphocyte attenuator (BTLA) at an association-dissociation rate of (1 / s), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, wherein the antibody inhibits the binding of BTLA to the herpesvirus entry medium (HVEM), as determined by surface plasmon resonance (SPR) using a method such as described in Example 4; and inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds to the 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 described in Example 2. In some embodiments, the antibody binds to the 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 described in Example 2. In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (at position according to SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residue H68 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residues selected from N65 and A64 (at position according to SEQ ID NO: 225).

[0267] In a specific embodiment of the first aspect of the invention, this document provides isolated agonistic antibodies that specifically bind to human B and T lymphocyte attenuators (BTLA), wherein the antibody binds to cynomolgus monkey BTLA at a concentration of at least 5 nM KD, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, and wherein the antibody inhibits the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined by surface plasmon resonance (SPR) using a method such as described in Example 4; and inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position in SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residue H68 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO: 225).

[0268] In a specific embodiment of the first aspect of the invention, this document provides a dissociative agonist antibody that specifically binds to human B and T lymphocyte attenuators (BTLA), wherein the antibody binds to cynomolgus monkey BTLA at a concentration of at least 50 nM KD, as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, and wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined by surface plasmon resonance (SPR) using a method such as described in Example 4; and inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position in SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at the position of SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residue H68 (at the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at the position of SEQ ID NO: 225).

[0269] In a specific embodiment of the first aspect of the invention, this document provides a separating agonist antibody that specifically binds to human B and T lymphocyte attenuators (BTLA) at a KD concentration 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, and wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody has a concentration of at least 2.0 × 10⁻⁶ NM. 5 The antibody binds to human BTLA at an association rate of (1 / Ms), 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 at an association rate of less than 10.0 × 10⁻⁶. -1The dissociation rate (1 / s) of binding to human BTLA was determined, as measured at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2. In some embodiments, antibody binding to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position of SEQ ID NO: 225) was determined, as measured by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as that described in Example 5. In some embodiments, antibody binding to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (according to the position of SEQ ID NO: 225). In some embodiments, antibody binding to residues selected from human BTLA: D35, T78, K81, S121, and L123 (according to the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO: 225).

[0270] In a specific embodiment of the first aspect of the invention, this document provides a separation agonist antibody, which is 1.7 × 10 5 (1 / Ms) to 2.5×10 5 The antibody specifically binds to human B and T lymphocyte attenuators (BTLA) at an association rate of (1 / Ms), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2, and wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), as determined by surface plasmon resonance (SPR) using a method such as described in Example 4; and inhibits T cell proliferation in vitro, as determined, for example, by a mixed lymphocyte reaction assay using a method such as described in Example 9. In some embodiments, the antibody binds at a rate less than 3.0 × 10⁻⁶. -1 The antibody binds to human BTLA at a dissociation rate of (1 / s), 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 at a dissociation rate of 3.0 × 10⁻⁶. -1 (1 / s) to 5.0×10 -1The antibody binds to human BTLA at a dissociation rate of (1 / s), as determined by surface plasmon resonance (SPR) at 37°C using a method such as described in Example 2. 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 a method such as 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 a method such as described in Example 2. In some embodiments, the antibody binds to an epitope that blocks the binding of antibody 286. In some embodiments, the antibody binds to residues selected from human BTLA including D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to human BTLA residue H68 (at position according to SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO: 225).

[0271] In a specific embodiment of the first aspect of the invention, this document provides a separating agonist antibody that specifically binds to human B and T lymphocyte attenuators (BTLA) at a KD concentration 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 wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediators (HVEMs), 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody has a concentration of at least 1.0 × 10⁻⁶ NM. 5 The antibody binds to human BTLA at an association rate of (1 / Ms), 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 at an association rate of 1.0 × 10⁻⁶. 5 (1 / Ms) to 10×10 5The antibody binds to human BTLA at an association rate of (1 / Ms), 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 at an association rate of less than 6.0 × 10⁻⁶. -1 The antibody binds to human BTLA at a dissociation rate of (1 / s), 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 at a dissociation rate of 6.0 × 10⁻⁶. -1 (1 / s) to 10.0×10 -2 The dissociation rate (1 / s) binding to human BTLA was determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2. In some embodiments, the antibody binds to residues selected from human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position of SEQ ID NO: 225), as determined by X-ray crystallography of the mutant receptor or by flow cytometry using a method such as that described in Example 5. In some embodiments, the antibody binds to residues selected from human BTLA: Y39, K41, R42, Q43, E45, and S47 (according to the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residues selected from human BTLA: D35, T78, K81, S121, and L123 (according to the position of SEQ ID NO: 225). In some embodiments, the antibody binds to residue H68 of human BTLA (at position according to SEQ ID NO:225). In some embodiments, the antibody binds to residues selected from human BTLA: N65 and A64 (at position according to SEQ ID NO:225).

[0272] In some embodiments, the isolated antibody of the present invention that specifically binds to human BTLA enhances BTLA activity and / or signaling via receptor.

[0273] As described above, when used in relation to the first aspect of the invention, the term antibody encompasses both the complete antibody and its antigen-binding fragment.

[0274] Specific Fc receptor binding implementation scheme

[0275] In some embodiments of the invention, particularly when according to the first aspect of the invention, the heavy chain comprises an Fc region containing a substitution that confers increased binding of the antibody molecule to FcγR2B and thus enhances the signaling of FcγR2B. In some embodiments, such molecules have reduced binding to one or more activating Fcγ receptors (such as FcγR2A or FcγR1A) compared to the parental peptide. In some embodiments, such molecules have increased binding to FcγR2B / FcγR2A compared to the parental peptide. In some embodiments, such molecules have increased binding to FcγR2B / FcγR1A compared to the parental peptide.

[0276] As described above, in specific embodiments of the invention, particularly when 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 containing 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 numbers are according to EU index). Suitably, the Fc region and the antibody itself are thus capable of binding to the Fcγ receptor.

[0277] In a specific embodiment, the Fc region binds to FcγR2B with a higher affinity than a comparable control antibody containing an Fc region lacking one or more of the aforementioned Fc substitutions. In a specific embodiment, the antibody binds to FcγR2B with a dissociation constant (KD) of approximately 5 μM to 0.1 μM, as determined by surface plasmon resonance (SPR). Suitably, the antibody binds to FcγR2B via its Fc region.

[0278] In a specific implementation, the antibody binds to FcγR2B with up to 5 μM of KD, as determined by surface plasmon resonance (SPR).

[0279] In a specific implementation, the antibody binds to FcγR2A (131R allotype) with a lower or equal affinity to the parent molecule. The parent molecule is an equivalent antibody lacking the Fc substitution that confers increased binding to FcγR2B and thus enhances the signaling of FcγR2B.

[0280] In a specific implementation, when the antibody contains a P238D substitution, the antibody binds to FcγR2A (131R allotype) with a lower or equal affinity relative to a comparable control antibody containing an Fc region containing a proline at position 238 (EU index).

[0281] In a specific implementation, the antibody binds to FcγR2A (131R allotype) with at least 20 μM KD, as determined by surface plasmon resonance (SPR).

[0282] In a specific implementation, the antibody binds to FcγR2A (131R allotype) with a KD of approximately 25 μM to 35 μM, as determined by surface plasmon resonance (SPR).

[0283] In a specific implementation, the antibody binds to FcγR2A (131H allotype) with a lower or equal affinity to the parent molecule.

[0284] In a specific implementation, when the antibody contains a P238D substitution, the antibody binds to FcγR2A (131H allotype) with a lower or equal affinity relative to a comparable control antibody containing an Fc region containing a proline at position 238 (EU index).

[0285] In a specific implementation, the antibody binds to FcγR2A (131H allotype) with at least 50 μM KD, as determined by surface plasmon resonance (SPR).

[0286] In a specific embodiment, the antibody has a KD value of 3 or higher (such as at least 5) [antibody against FcγR2A (131R) / antibody against FcγR2B KD value]. Suitably, this is determined, as by surface plasmon resonance (SPR).

[0287] In a specific embodiment, the antibody has a KD value of 10 or higher (such as at least 15) for the ratio of [antibody against FcγR2A(131H)] to [antibody against FcγR2B]. Suitablely, this is determined, as by surface plasmon resonance (SPR).

[0288] In a specific embodiment, the antibody has a KD value of 3 or higher (such as at least 5) for [antibody against FcγR2A(131R) / KD value against FcγR2B] and / or a KD value of 10 or higher (such as at least 15) for [antibody against FcγR2A(131H)] / [antibody against FcγR2B]. Suitably, this is determined, as by surface plasmon resonance (SPR).

[0289] Appropriately, the antibodies of the present invention exhibit enhanced agonistic activity against human BTLA expressed on the surface of human immune cells relative to comparable control antibodies / parental antibodies, as measured by a BTLA agonist assay selected from: such as the T cell activation assay described in Example 24, such as the mixed lymphocyte reaction described in Example 25, or such as the B cell activation assay described in Example 26.

[0290] Therefore, if the antibody contains a P238D substitution, it exhibits enhanced agonistic activity against human BTLA expressed on the surface of human immune cells, as measured by a BTLA agonist assay selected from: such as the T cell activation assay described in Example 24, such as the mixed lymphocyte reaction described in Example 25, or such as the B cell activation assay described in Example 26.

[0291] In specific embodiments, the antibodies of the present invention are selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (such as bispecific antibodies).

[0292] In a specific embodiment, the antibody of the present invention is an antigen-binding fragment antibody, which is selected from the group consisting of: scFv, sc(Fv). 2 , dsFv, Fab, Fab′, (Fab′)2 and bivalent antibodies.

[0293] In a specific implementation, the heavy and light chain molecules that form the antigen-binding fragment are linked by a flexible linker. Many commonly used flexible linkers exist, and the selection of the linker can be made by someone skilled in the art.

[0294] The peptide linker connecting the VH and VL domains of scFv attaches the C-terminus of one variable domain to the N-terminus of another variable domain without significantly compromising the fidelity of VH-VL pairing and antigen-binding sites. The length of the peptide linker can vary from 10 to 25 amino acids and is typically, but not always, composed of hydrophilic amino acids such as glycine (G) and serine (S). The linker can be a type found in native multidomain proteins (see, for example, Argos PJ Mol Biol., Vol. 211: pp. 943–958, 1990; and Heringa G. Protein Eng., Vol. 15: pp. 871–879, 2002, or modifications thereof).

[0295] Commonly used flexible linkers have sequences consisting primarily of fragments of Gly and Ser residues (“GS” linkers). The most widely used example of a flexible linker is (Gly-Gly-Gly-Gly-Ser).n The sequence is (SEQ ID NO: 232). By adjusting the copy number "n", the length of this GS linker can be changed to achieve proper separation of functional domains or to maintain necessary interactions between domains. Typically, GS linkers with n = 3 peptides are used as scFv peptide linkers (Leith et al., Int. J. Oncol., Vol. 24: pp. 765-771, 2004; Holger et al., Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444-6448, 1993). This 15-amino acid linker sequence [named (GGGGS)3 linker] is used in recombinant phage antibody systems (RPAS kits) commercially available from Amersham. Several other connectors 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, Vol. 242: pp. 432-426, 1988).

[0296] Epitope combination

[0297] The inventors have mapped the epitopes on BTLA, in which the effective agonists and antibodies disclosed herein bind.

[0298] In a specific embodiment, the antibody of the present invention binds to residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, E92, Y39, K41, R42, Q43, E45, S47, D35, T78, K81, S121, L123, H68, N65, A64.

[0299] In a specific embodiment, the antibody of the present invention binds to residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0300] In a specific embodiment, the antibody of the present invention binds to at least two residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0301] In a specific embodiment, the antibody of the present invention binds to at least three residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

[0302] In a specific embodiment, the antibody of the present invention binds to at least five residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

[0303] In a specific embodiment, the antibody of the present invention binds to all residues selected from the following human BTLA: D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0304] In a specific embodiment, the antibody of the present invention binds to residues selected from the following human BTLA: Y39, K41, R42, Q43, E45, and S47.

[0305] In a specific embodiment, the antibody of the present invention binds to at least two residues selected from the following human BTLA: Y39, K41, R42, Q43, E45 and S47.

[0306] In a specific embodiment, the antibody of the present invention binds to all residues selected from the following human BTLA: Y39, K41, R42, Q43, E45, and S47.

[0307] In a specific embodiment, the antibody of the present invention binds to residues selected from the following human BTLA: D35, T78, K81, S121, and L123.

[0308] In a specific embodiment, the antibody of the present invention binds to at least two residues selected from the following human BTLA: D35, T78, K81, S121 and L123.

[0309] In a specific implementation, the antibody of the present invention binds to residue H68 of human BTLA.

[0310] In a specific embodiment, the antibody of the present invention binds to residues selected from the following human BTLA: N65 and A64.

[0311] In a specific implementation, the antibody of the present invention binds to both N65 and A64 residues of human BTLA.

[0312] Residue numbers such as K41 refer to the amino acid (K; lysine) at position 41; where the number refers to the position in the human BTLA polypeptide disclosed in SEQ ID NO:225.

[0313] In specific embodiments, the antibody of the present invention is an IgG1, IgG2, or IgG4 antibody. In specific embodiments, the antibody is a mammalian or human antibody.

[0314] In a specific implementation scheme, the antibody of the present invention is a humanized antibody.

[0315] In a specific implementation, the antibody of the present invention is a fully human antibody.

[0316] In a specific implementation, the antibody of the present invention acts as an agonist that induces signal transduction through the BTLA receptor.

[0317] The antibodies (including antigen-binding fragments) of the present invention are particularly effective agonists.

[0318] In a specific implementation, the antibody of the present invention has an EC50 of no more than 1 nM.

[0319] The agonist antibodies of the present invention (e.g., full-length / intact antibodies or their antigen-binding fragments) have particularly high efficacy.

[0320] In a specific embodiment, the antibody of the present invention inhibits the proliferation of T cells by at least 20%, suitably at least 30%, and more suitably at least 40%.

[0321] In a specific embodiment, the antibody of the present invention inhibits the production of IFN-γ on T cells by at least 50%, suitably at least 75%, and more suitably at least 95%, for example, by ELISA measurement of the supernatant in an in vitro mixed lymphocyte reaction.

[0322] In a specific embodiment, the antibody of the present invention inhibits the production of IL-2 by T cells by at least 50%, suitably at least 75%, and more suitably at least 95%, for example by ELISA measurement of the supernatant in an in vitro mixed lymphocyte reaction.

[0323] In a specific embodiment, the antibody of the present invention inhibits the production of IL-17 on T cells by at least 50%, suitably at least 75%, and more suitably at least 95%, for example by ELISA measurement of the supernatant in an in vitro mixed lymphocyte reaction.

[0324] In a specific implementation, using the method described in Example 12, the antibody of the present invention reduces mortality in a mouse GVHD model by at least 50%, suitably at least 75%, and more suitably at least 95%.

[0325] In a specific implementation, using the method described in Example 11, the antibody of the present invention reduces weight loss in a mouse T-cell colitis model by at least 50%, suitably at least 75%, and more suitably at least 95%.

[0326] In a specific implementation, using the method described in Example 11, the antibody of the present invention reduces colonic inflammation in a mouse T-cell colitis model by at least 50%, suitably at least 75%, and more suitably at least 95%.

[0327] In some respects, the present invention also relates to isolated polypeptides comprising the VL or VH domain of any of the antibodies described herein.

[0328] As described herein, in a specific implementation, the antibody that binds to BTLA comprises a heavy chain and a light chain, wherein the heavy chain contains an Fc region containing an aspartic acid residue at position 238 (EU index).

[0329] Nucleic acid molecules

[0330] The antibodies of the present invention will be encoded by nucleic acids. An antibody (including its antigen-binding fragment) may be encoded by a single nucleic acid molecule, or it may be encoded by two or more nucleic acid molecules. For example, since the antigen-binding site is typically formed by a combination of a heavy chain variable polypeptide region and a light chain variable polypeptide region, these two variable (heavy and light) polypeptide regions may be encoded by separate nucleic acid molecules. Alternatively, for example in the case of scFv, they may be encoded by the same nucleic acid molecule.

[0331] According to a second aspect of the invention, one or more nucleic acid molecules encoding an antibody according to a first aspect of the invention are provided.

[0332] Based on the primary amino acid sequence of the polypeptide encoding the antibody of the present invention, those skilled in the art can determine a suitable nucleotide sequence encoding the polypeptide, and, if necessary, a codon-optimized nucleotide sequence (see, for example, Mauro and Chappell., Trends Mol Med., Vol. 20, No. 11: pp. 604-613, 2014).

[0333] As used herein, when referring to a prior aspect of the invention, such as “according to a first (or second, etc.) aspect,” it should be understood that any variations of that aspect (e.g., variations of the first (or second, etc.) aspect are also covered. Furthermore, any embodiment applicable to a particular aspect of the invention is also applicable to any variation of that aspect, and therefore, any embodiment applicable to the first aspect of the invention is also applicable to variations of the first aspect of the invention.

[0334] According to a variation of the second aspect of the invention, an isolated nucleic acid is provided comprising a nucleotide sequence encoding a heavy-chain variable region polypeptide or a light-chain variable region polypeptide of the invention. The heavy-chain variable polypeptide or light-chain variable polypeptide of the invention refers to a single polypeptide chain comprising an amino acid constituting part of an antigen-binding site. Of course, the polypeptide may also comprise other domains, such as constant domains, hinge regions, and Fc regions, such as domains containing one or more Fc receptor binding sites.

[0335] According to another variation of the second aspect of the invention, an isolated nucleic acid is provided comprising one or more nucleotide sequences encoding a polypeptide capable of forming the antibody of the invention. In a specific embodiment, the polypeptide may also comprise other domains, such as constant domains, hinge regions, and Fc regions, such as domains containing one or more Fc receptor binding sites.

[0336] One nucleic acid molecule may encode only a polypeptide sequence containing the VL domain of an antibody or a fragment thereof. Another nucleic acid molecule may encode only a polypeptide sequence containing the VH domain of an antibody or a fragment thereof. However, a nucleic acid molecule may also encode both the VH and VL domains of a polypeptide sequence capable of forming the antibodies of the present invention (such as full-length / intact antibodies or antigen-binding fragments thereof).

[0337] The nucleic acid molecule encoding the antibody of the present invention (such as according to the first aspect of the invention) may be a vector (such as a plasmid vector, granular vector, or viral vector, or artificial chromosome) or may be a part thereof, which may contain other functional regions (elements), such as one or more promoters, one or more origins or replication sites, one or more selection markers, and one or more other elements commonly found in expression vectors. The cloning and expression of nucleic acids encoding proteins (including antibodies) are established within the scope of the skills of those skilled in the art.

[0338] According to a third aspect of the present invention, a vector is provided that comprises the nucleic acid of the second aspect of the present invention. In a specific embodiment, the vector is a plasmid vector, a granular vector, a viral vector, or an artificial chromosome.

[0339] The nucleic acid of the present invention includes a vector nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of forming the antibody of the present invention, which may be in a purified / isolated form.

[0340] The isolated / purified nucleic acids encoding the antibodies of the present invention will be free of or substantially free of substances naturally associated with them, such as other proteins or nucleic acids found in their natural environment, or other proteins or nucleic acids found in the environment in which they are prepared when such preparation is carried out by performing recombinant DNA technology in vitro or in vivo.

[0341] In specific embodiments, the purity of the nucleic acid of the present invention is greater than 80%, such as greater than 90%, greater than 95%, greater than 97%, and greater than 99%.

[0342] Therefore, according to another variation of the third aspect of the invention, a vector is provided comprising a nucleic acid or nucleotide sequence encoding the heavy-chain or light-chain variable polypeptide of the invention. In a specific embodiment, the vector comprises a nucleic acid encoding the heavy-chain and light-chain variable regions. In a specific embodiment, the polypeptide may also comprise other domains, such as constant domains, hinge regions, and Fc regions, such as domains containing one or more Fc receptor binding sites.

[0343] The nucleic acids and / or vectors of the present invention can be introduced into host cells. This introduction can be performed using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-glucan, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or, for insect cells, baculovirus). The introduction of nucleic acids into host cells, particularly eukaryotic cells, can be performed using virus-based or plasmid-based systems. Plasmid systems can be maintained in free form or incorporated into host cells or artificial chromosomes. Incorporation can be achieved by randomly or targetedly integrating one or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride conversion, electroporation, and transfection using bacteriophages.

[0344] In one embodiment, the nucleic acid of the present invention is integrated into the genome (e.g., chromosome) of a host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0345] host cells

[0346] Another aspect of the invention provides a host cell containing the nucleic acids disclosed herein. Such a host cell can be in vitro or in culture.

[0347] The host cell can be from any species, such as bacteria or yeast, but suitably, the host cell is a mammalian cell, such as human cells or rodent cells, such as HEK293T cells or CHO-K1 cells.

[0348] Therefore, according to a fourth aspect of the invention, a host cell comprising a nucleic acid sequence according to a second aspect of the invention or a vector according to a third aspect of the invention is provided.

[0349] Host cells can be treated to cause or allow the expression of the proteins of the present invention from nucleic acids, for example by culturing host cells under conditions that encode nucleic acids. Purification of the expressed product can be achieved by methods known to those skilled in the art.

[0350] Therefore, the nucleic acids of the present invention, including vector nucleic acids comprising a nucleotide sequence encoding a polypeptide capable of forming the antibodies of the present invention, can be present in isolated host cells. Host cells are generally part of a clonal population of host cells. As used herein, reference to host cells also encompasses a clonal population of said cells. A clonal population is a population that grows from a single parental host cell. Host cells can be derived from any suitable organism. Suitable host cells include bacterial, fungal, or mammalian cells.

[0351] Host cells can be used to assist in the amplification of vector nucleic acids (such as plasmids), or can be used as biofactories to express the polypeptides of the present invention that form the BTLA-binding antibody of the present invention. Suitable hosts for amplifying vector nucleic acids can be bacterial or fungal cells, such as *Escherichia coli* cells or *Saccharomyces cerevisiae* cells. Suitable hosts for expressing the proteins of the present invention (i.e., the polypeptides constituting the human BTLA-binding antibody of the present invention) will be mammalian cells, such as HEK293T or CHO-K1 cells. In a specific embodiment, the host cell is a mammalian cell, such as HEK293T or CHO-K1 cells.

[0352] A variety of host expression vector systems can be used to express BTLA-binding molecules as described herein (see, for example, U.S. Patent No. 5,807,715). For example, mammalian cells such as Chinese hamster ovary cells (CHO) combined with vectors such as major intermediate early gene promoter elements from human cytomegalovirus are efficient expression systems for CEA proteins (Foecking et al., Gene, Vol. 45: p. 101, 1986; and Cockett et al., Bio / Technology, Vol. 8: No. 2, 1990). Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the proper modification and processing of the proteins disclosed herein. For this purpose, eukaryotic host cells with cellular mechanisms for the appropriate processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. These mammalian host cells include, but are not limited to, CHO, HEK, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, T47D, NS0, CRL7O3O, and HsS78Bst cells.

[0353] Antibody production

[0354] According to a fifth aspect of the invention, a method for generating an antibody according to a first aspect of the invention is provided, the method comprising culturing a host cell according to a fourth aspect of the invention under conditions for generating the antibody, and optionally isolating and / or purifying the antibody.

[0355] According to a variation of the fifth aspect of the invention, a method for generating an antibody that binds to human BTLA is provided, the method comprising culturing a host cell containing a nucleic acid encoding a polypeptide that forms an antibody that binds to human BTLA under conditions for generating the antibody, optionally further comprising isolating / purifying the antibody.

[0356] By separation / purification, we mean that the antibodies or polypeptides that make up these molecules of the present invention will be free of or substantially free of substances naturally associated with them, such as other proteins or nucleic acids found in their natural environment, or other proteins or nucleic acids found in the environment in which they are prepared when such preparation is carried out by performing recombinant DNA technology in vitro or in vivo.

[0357] According to a variation of the fifth aspect of the present invention, a method for preparing an antibody that specifically binds to human BTLA is provided, the method comprising the following steps:

[0358] a) Provide a host cell containing one or more nucleic acid molecules that encode one or more polypeptides containing an amino acid sequence comprising a heavy chain variable domain and / or a light chain variable domain, wherein the one or more nucleic acid molecules can combine upon expression to produce a human BTLA-binding molecule;

[0359] b) Culturing host cells that express the encoded amino acid sequence; and c) Isolating antibody molecules.

[0360] The one or more nucleic acid molecules are those described above that encode one or more polypeptides of the present invention capable of forming an antibody that specifically binds to human BTLA.

[0361] In a specific embodiment, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO:1, CDRH2 has the amino acid sequence shown in SEQ ID NO:17, and CDRH3 has the amino acid sequence shown in SEQ ID NO:3; and

[0362] ii) A light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO:4, CDRL2 has the amino acid sequence shown in SEQ ID NO:12 and CDRL3 has the amino acid sequence shown in SEQ ID NO:6.

[0363] In a specific embodiment, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO:20, CDRH2 has the amino acid sequence shown in SEQ ID NO:21, and CDRH3 has the amino acid sequence shown in SEQ ID NO:22; and

[0364] ii) A light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO: 23, CDRL2 has the amino acid sequence shown in SEQ ID NO: 24 and CDRL3 has the amino acid sequence shown in SEQ ID NO: 25.

[0365] In a specific embodiment, the antibody comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO:30, CDRH2 has the amino acid sequence shown in SEQ ID NO:31, and CDRH3 has the amino acid sequence shown in SEQ ID NO:32; and

[0366] ii) A light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO:33, CDRL2 has the amino acid sequence shown in SEQ ID NO:34 and CDRL3 has the amino acid sequence shown in SEQ ID NO:35.

[0367] In a specific implementation plan, the antibody comprises:

[0368] 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 with the amino acid sequence; and

[0369] ii) Light chain variable region, which contains the amino acid sequence disclosed in SEQ ID NO:14 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0370] In a specific implementation plan, the antibody comprises:

[0371] 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 with the amino acid sequence; and

[0372] ii) Light chain variable region, which contains the amino acid sequence disclosed in SEQ ID NO:27 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0373] In a specific implementation plan, the antibody comprises:

[0374] 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 with the amino acid sequence; and

[0375] ii) Light chain variable region, which contains the amino acid sequence disclosed in SEQ ID NO:43 or a sequence having at least 90% sequence identity with the amino acid sequence.

[0376] The conditions for generating the antibodies of the present invention and purifying the molecules are well known in the art. One approach is to prepare a clonal population of cells capable of expressing the antibodies of the present invention or fragments thereof, and to culture them for a period of time in a suitable growth medium and at temperatures favorable to allow for the expansion / growth of the cell population and the expression of the protein of interest. If the protein of interest (e.g., the antibody of the present invention) is expressed within the host cells, the cells can be lysed (e.g., using a mild detergent or sonication) to release the cell contents (and thus the protein of interest) into the surrounding culture medium (which may be a culture medium or another medium in which the cells have been reconstituted), and then the purification process can be performed on that medium. If the target protein (e.g., the antibody of the present invention) is secreted into the growth medium, the purification process can be performed on that medium. Antibody purification typically involves isolating the antibody from the culture medium or culture supernatant of, for example, a hybridoma cell line using recognized methods, which typically include 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 unwanted host-derived proteins and other cellular contaminants (e.g., nucleic acids, carbohydrates, etc.). The purified protein may also undergo a virus inactivation step. Finally, the purified protein of interest can be, for example, lyophilized or formulated for storage, transport, and subsequent use. Preferably, the protein of interest (e.g., the intact antibody of the present invention or its antigen-binding fragment) will be substantially free of contaminating proteins initially present in the culture medium after expression or cell lysis.

[0377] In some embodiments, the purity of the antibody of the present invention will be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0378] The proteins of the present invention (e.g., the complete antibody or its antigen-binding fragment) can be formulated into suitable compositions.

[0379] Composition

[0380] While the BTLA-binding molecule (the antibody of the present invention) can be administered alone, in some embodiments, a pharmaceutical composition in which the BTLA-binding molecule is formulated with at least one pharmaceutically acceptable excipient is administered. The excipient can be a suitable pharmaceutical carrier solute. Such carriers are well known in the art and include phosphate-buffered saline solutions, water, liposomes, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using well-known conventional methods. These pharmaceutical compositions can be administered to the subject in appropriate doses. The dosing regimen will be determined by the attending physician and clinical factors.

[0381] According to a sixth aspect of the invention, a pharmaceutical composition is provided 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 specific embodiment, the composition comprises phosphate-buffered saline.

[0382] "Pharmaceutical composition" means a formulation which is present in a form that allows the bioactivity of the active ingredient to be effective and does not contain any additional components that would have unacceptable toxicity to a subject to be administered the formulation. A pharmaceutical composition will contain one or more pharmaceutically acceptable excipients. The term "excipient" as used herein refers to any additive, such as a filler, solubilizer, carrier, solvent, additive, etc.

[0383] Pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients, including, for example, water, ion exchangers, proteins, buffers, and salts. Preservatives and other additives may also be present. Excipients may be solvents or dispersion media. Suitable formulations for the treatments disclosed herein are described in: Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0384] "Pharmaceutically acceptable" excipients are those excipients for which the active ingredient is used can be rationally administered to test mammals to provide an effective dose. The pharmaceutical compositions of the present invention are prepared for storage as lyophilized formulations or aqueous solutions 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 non-toxic to receptors at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride); chlorhexidine; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. Low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Lyophilized HER2 antibody formulations are described in WO 97 / 04801.

[0385] Pharmaceutical compositions intended for internal administration must be sterile. This can be easily achieved by filtration through a sterile filter membrane.

[0386] The routes of administration for BTLA-binding molecules, such as antibodies or their antigen-binding fragments, can be, for example, oral, parenteral, inhalation, or topical. As used herein, the term parenteral includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0387] 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 such as ethyl oleate. Aqueous carriers include water, aqueous solutions, or suspensions, including saline and buffer media. Parenteral solvents include sodium chloride solution, Ringer's glucose, glucose and sodium chloride, lactated Ringer's, or fixed oil. Intravenous solvents include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's glucose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. Furthermore, in some embodiments of human origin, the composition may contain protein carriers, such as, for example, serum albumin or immunoglobulins. For intravenous injection or injection at the site of infection, the active ingredient will be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are able to prepare suitable solutions using, for example, isotonic solvents such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. As needed, preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included. As mentioned above, these all refer to excipients in this document.

[0388] The composition for injection can be administered using medical devices known in the art, such as a subcutaneous injection needle. Needle-free injection devices, such as those disclosed in U.S. Patent Nos. 6620135 and 5312335, can also be used.

[0389] Pharmaceutical compositions for oral administration can 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 typically contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. If desired, they may contain physiological saline solution, glucose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0390] Depending on the physicochemical properties of the molecules and the delivery route, the antibodies of the present invention can be formulated into liquid, semi-solid, or solid forms. Formulations may include excipients or combinations of excipients, such as sugars, amino acids, and surfactants. Liquid formulations may include a wide range of antibody concentrations and pH values. Solid formulations may be produced, for example, by lyophilization, spray drying, or drying using supercritical fluid technology.

[0391] Pharmaceutical compositions can be administered as a single dose, multiple doses, or infusion over a defined period of time. Dosing regimens can also be adjusted to provide the best expected response (e.g., therapeutic or preventative response). Specifically, parenteral formulations can be a single bolus dose, an infusion or loading bolus dose, followed by one or more maintenance doses. These compositions can be administered at specific fixed or variable intervals, such as once daily, or on an "on-demand" basis.

[0392] dose

[0393] The amount of a therapeutically effective BTLA-binding molecule or a pharmaceutical formulation containing that molecule can be determined using standard clinical techniques, such as through dose-range clinical trials. Alternatively, in vitro assays may be used to help identify the optimal dose range. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined based on the physician's judgment and each patient's individual circumstances. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems. Those skilled in the art can determine the dose of the composition to be administered without inappropriate experiments incorporating standard dose-response studies. Relevant factors to consider when making these determinations include one or more conditions to be treated, the choice of the composition to be administered, the individual patient's age, weight, and response, and the severity of the patient's symptoms. For example, the actual patient weight can be used to calculate the dose of the formulation to be administered in milliliters (mL). There may not be a downward adjustment for the "ideal" weight. In this case, the appropriate dose can be calculated using the following formula:

[0394] Dosage (mL) = [Patient weight (kg) × Dosage level (mg / kg) / Drug concentration (mg / mL)]

[0395] As discussed herein, the therapeutically effective dose of a pharmaceutical composition for treating BTLA-related diseases or disorders will vary depending on many different factors, including route of administration, target site, patient physiological status, patient weight or sex, patient age, whether the patient is human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. The therapeutically effective dose may have been determined from clinical trials and is the dose that the attending physician may determine using treatment guidelines. Typically, the patient is human, but non-human mammals may also be treated. The therapeutic dose may be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0396] In various implementation schemes, the BTLA-binding molecule is administered at concentrations 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.

[0397] The pharmaceutical compositions of the present invention can be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated. Such combinations may be administered with other immunosuppressants, such as those selected from: corticosteroids, cyclosporine, azathioprine, sulfasalazine, methotrexate, mycophenolate mofetil, tacrolimus, and fingolimod, or other biologics such as infliximab, adalimumab, uteroclosporine, tocilizumab, and rituximab.

[0398] According to a seventh aspect of the present invention, a method for preparing a pharmaceutical composition is provided, the method comprising formulating an antibody according to a first aspect of the present invention or an antibody produced according to a fifth aspect of the present invention into a composition comprising at least one additional component. In a specific embodiment, the at least one additional component is a pharmaceutically acceptable excipient.

[0399] Reagent test kit

[0400] In addition, products (e.g., BTLA-binding molecules or pharmaceutical compositions thereof) may be packaged and marketed as kits. Such products may have labels or instructions for use indicating the appropriate use of the product and for treating subjects with or susceptible to diseases or disorders.

[0401] Therefore, according to an eighth aspect of the present invention, a kit is provided comprising an antibody according to a first aspect of the present invention or a pharmaceutical composition according to a sixth aspect of the present invention. Suitably, such a kit includes a package insert containing instructions for use.

[0402] Therapeutic / Medical Use

[0403] The antibodies of the present invention or pharmaceutical compositions comprising said antibodies or their antigen-binding fragments may be used for treatment, typically as medicines.

[0404] In some embodiments, the antibodies of the present invention or pharmaceutical compositions comprising said antibodies may be used to treat or prevent any disease or condition in a subject in need.

[0405] BTLA involves downregulating the immune response, and many diseases or conditions can be treated by suppressing host T cells and / or B cells (see, for example, Crawford and Wherry, eds.: Therapeutic potential of targeting BTLA, J Leukocyte Biol., Vol. 86: pp. 5–8, 2009). Diseases or conditions that can benefit from treatment with anti-BTLA agonists are referred to herein as “BTLA-related diseases.” BTLA-related diseases include inflammatory or autoimmune diseases, as well as disorders of excessive immune cell proliferation.

[0406] Specific BTLA-related diseases that can be treated with the BTLA-binding molecules of this invention include: Addison's disease, allergic reactions, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behçet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuritis, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, and Guillain-Barré syndrome. Syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorder, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, aortitis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0407] In the specific implementation plan, the diseases to be treated are selected from the group consisting of the following: Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, graft-versus-host disease, transplant rejection, multiple sclerosis, vasculitis, Sjögren's syndrome, Behçet's disease, uveitis, type 1 diabetes mellitus, Hashimoto's thyroiditis, primary sclerosing cholangitis, and myasthenia gravis.

[0408] In the specific implementation plan, the disorder of excessive immune cell proliferation is selected from lymphoma, leukemia, systemic mast cell hyperplasia, myeloma, or lymphoproliferative disorder.

[0409] According to a ninth aspect of the present invention, an antibody according to a first aspect of the present invention or a pharmaceutical composition according to a sixth aspect of the present invention is provided for treatment.

[0410] In the specific implementation plan, the treatment is for the treatment or prevention of BTLA-related diseases.

[0411] In a specific implementation, BTLA-related diseases are diseases caused by decreased expression and / or activity of BTLA in the subject. Specifically, the BTLA agonist antibody of the present invention can be used to treat any disease or condition characterized by the presence or activity of T or B cells.

[0412] In one implementation, BTLA-related diseases are inflammatory diseases (such as rheumatoid arthritis), autoimmune diseases or disorders (such as graft-versus-host disease), or proliferative diseases or disorders (such as cancer).

[0413] In a specific implementation plan, the treatment is for the treatment or prevention of inflammatory or autoimmune diseases, as well as obstacles to the proliferation of excessive immune cells.

[0414] According to a variation of the ninth aspect of the present invention, a method of treating a patient in need is provided, the method comprising administering to the patient an antibody (or BTLA-binding molecule) according to the first aspect of the present invention or a pharmaceutical composition according to the sixth aspect of the present invention. In a specific embodiment, the patient requiring treatment or to be treated has (or is having) a BTLA-related disease. In a specific embodiment, the patient requiring treatment or to be treated has (or is having) an inflammatory disease, an autoimmune disease, or an impairment of excessive immune cell proliferation.

[0415] In a specific implementation, 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 in a pharmaceutically acceptable amount.

[0416] In a variation of this ninth aspect of the invention, use is provided of 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 a method of treating a patient in need. In a specific embodiment, the method is used to treat or prevent BTLA-related diseases. In a specific embodiment, the method is used to treat or prevent inflammatory or autoimmune diseases, as well as disorders of excessive immune cell proliferation.

[0417] In a further variation of this aspect, 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 preparation of a medicament for treating a patient in need thereof is provided.

[0418] In one implementation, the treatment is used to treat BTLA-related diseases. Suitablely, BTLA-related diseases are inflammatory diseases (such as asthma), autoimmune diseases or disorders (such as rheumatoid arthritis), or immune proliferative diseases or disorders (such as lymphoma).

[0419] In a specific embodiment, the antibody of the present invention or a pharmaceutical composition containing the antibody is used to inhibit T cells and / or B cells.

[0420] In specific embodiments, the antibodies of the present invention or pharmaceutical compositions comprising said antibodies are used to treat or prevent diseases or conditions in subjects who require them, the diseases or conditions being selected from the group consisting of: Addison's disease, allergic reactions, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behçet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuritis, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease. The main diseases include GVHD, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorder, multiple sclerosis (MS), myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, aortitis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0421] In a specific embodiment, the antibody of the present invention or a pharmaceutical composition comprising said antibody is used to treat or prevent a disease or condition in a subject in need, the disease or condition being selected from the group consisting of: Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, graft-versus-host disease, transplant rejection, multiple sclerosis, vasculitis, Sjögren's syndrome, Behçet's disease, uveitis, type 1 diabetes mellitus, Hashimoto's thyroiditis, primary sclerosing cholangitis, and myasthenia gravis. In one embodiment, the immunoproliferative disorder is cancer. Suitably, the cancer is leukemia or lymphoma.

[0422] In another embodiment, the antibody of the present invention or a pharmaceutical composition containing said antibody is used to prevent or treat transplant rejection.

[0423] In another embodiment, the present invention relates to the prevention or treatment of graft-versus-host disease.

[0424] In another embodiment, the antibody of the present invention or a pharmaceutical composition containing said antibody is used to treat rheumatoid arthritis.

[0425] In other embodiments, the antibodies of the present invention or pharmaceutical compositions comprising said antibodies are used to treat diabetes, such as type 1 diabetes.

[0426] In another embodiment, the antibody of the present invention or a pharmaceutical composition containing said antibody is used to treat psoriasis.

[0427] In another embodiment, the antibody of the present invention or a pharmaceutical composition containing said antibody is used to treat multiple sclerosis.

[0428] In another embodiment, the antibody of the present invention or a pharmaceutical composition containing said antibody is used to treat colitis.

[0429] The term "effective dose" or "therapeutic effective dose" refers to a dose or amount of a drug that is sufficient to improve a patient's symptoms or achieve desired biological outcomes, such as increased cancer, increased tumor cell death, reduced tumor size, increased progression-free survival, or increased overall survival. As disclosed elsewhere in this document, effective doses are typically evaluated through extensive human clinical studies.

[0430] Throughout this specification and claims, the words “comprising” and “containing,” and their variations, mean “including but not limited to,” and are not intended to (and do not) exclude other parts, additions, components, integrals, or steps. Throughout this specification and claims, the singular encompasses the plural unless the context otherwise requires. Specifically, where the indefinite article is used, the specification should be understood to consider both the plural and the singular unless the context otherwise requires.

[0431] The features, integrals, properties, compounds, chemical portions, or groups described in connection with specific aspects, embodiments, or examples of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination of steps of any method or process so disclosed.

[0432] The reader’s attention is directed to all papers and documents that were submitted concurrently with or prior to this specification and that are open to public examination of this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0433] The invention will now be further described with reference to the following non-limiting examples and accompanying drawings. Attached Figure Description

[0434] Figure 1

[0435] Binding of the antibody to soluble and cellularly expressed forms of human and cynomolgus monkey BTLA. (a) Surface plasmon resonance (SPR) binding curves of soluble monomeric human BTLA extracellular domains injected at increasing concentrations relative to immobilized anti-BTLA antibody; the figure shows the SPR signal after reference and blank subtraction. (b) Association and dissociation rates of binding to human or cynomolgus monkey BTLA calculated by curve fitting using BiaEvaluation software. (c) Binding of antibody 2.8.6 to Jurkat cell lines expressing human or cynomolgus monkey BTLA compared to the isotype control antibody (data points represent the mean + / - SD of three wells at each antibody concentration). (d) EC50 of antibody binding to transfected cell lines calculated by nonlinear curve fitting using GraphPad Prism software.

[0436] Figure 2

[0437] (a) Evaluation of anti-BTLA antibody blocking of ligand binding by SPR. The extracellular domain of human BTLA was immobilized on a sensor chip. Human HVEM was injected to confirm binding, followed by complete dissociation. Then, a saturated concentration of anti-BTLA antibody was injected, followed immediately by a second injection of HVEM. (b) Equilibrium binding of HVEM after antibody injection is expressed as the percentage of HVEM bound before antibody injection. Saturation of BTLA with clone 11.5.1, but not with clone 2.8.6, blocked subsequent ligand binding.

[0438] Figure 3

[0439] Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfected with BTLA constructs in a bicistronic vector also expressing GFP stained with Pacific Blue conjugated with anti-BTLA antibodies. Clone 11.5.1 binds to cells transfected with wild-type receptor (left), but not to cells transfected with BTLA containing the Y39R mutation (right). (b) Binding to each BTLA mutant construct is expressed as the percentage of binding of clones 2.8.6 and 11.5.1 to wild-type BTLA. (c) The Y39R and K41E mutations that selectively eliminate binding of clone 11.5.1 are mapped onto the crystal structure of human BTLA (black residues). Residues critical for binding to ligand HVEM are highlighted in gray.

[0440] Figure 4

[0441] (a) Crystal structure of the extracellular domain of human BTLA complexed with the Fab' fragment of clone 2.8.6. Residues on BTLA embedded at the interface are highlighted in black. (b) Relationship between the epitope (black residues) of antibody 2.8.6 and the HVEM binding site (gray residues) is shown.

[0442] Figure 5

[0443] (a) Strategy for generating chimeric BTLA genes in humanized BTLA mice. A human genomic DNA fragment from exon 2 to exon 3 is inserted into a mouse locus, replacing the mouse sequence from exon 2 to exon 4. The exon-intron junctions at the mouse exon 2 start and mouse exon 4 end are preserved intact to ensure proper splicing.

[0444] Figure 6

[0445] (a) Evaluation of the in vivo anti-BTLA antibody T-cell transfer assay protocol. A mixture of humanized and wild-type OVA-specific CD4 T cells was injected into recipient mice. The mice were immunized the next day with ovalbumin in alum to activate the transferred cells, and 24 hours later, anti-human BTLA antibody or an allotype control was administered. Eight days after the initial cell transfer, the ratio of humanized to wild-type cells in the spleen transfer population was assessed by flow cytometry. (b) Both clone 11.5.1 and the smaller version of 2.8.6 reduced the expansion of humanized cells compared to wild-type. The figure shows pooled data from two (for 11.5.1) or three (for 2.8.6) replicates. Each data point represents a single recipient mouse.

[0446] Figure 7

[0447] 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-stimulated cells in the presence of anti-BTLA antibody or an allotype control. After 96 hours, the proliferation of humanized CD4 cells was assessed and normalized to the proliferation in the absence of the antibody. Clone 2.8.6 inhibited the proliferation of humanized cells with an IC50 of 0.029 nM and a maximum inhibitory effect of 42%. Data points represent the mean + / - SD of three wells at each antibody concentration and represent 5 independent experiments.

[0448] Figure 8

[0449] (a) Role of clone 2.8.6 in a T-cell colitis model. RAG knockout receptor mice were injected with CD45RBhiCD25-CD4+ T cells from humanized BTLA mice and treated with 200 μg of 2.8.6 or an allotype control antibody on days 7, 21, and 35. Mice treated with the allotype control gradually lost weight starting at week 3, while mice treated with 2.8.6 were spared. (b) Eight weeks after cell transfer, the colon was treated to extract lamina propria lymphocytes, and the total number of inflammatory cells extracted from each colon was counted. Mice treated with the allotype control had significantly more infiltrative immune cells than mice treated with 2.8.6. (c) The colon weight-to-length ratio was calculated as a marker of inflammation and thickening. Treatment with 2.8.6 prevented the increase in weight-to-length ratio observed in mice treated with the allotype control.

[0450] Figure 9

[0451] (a) Role of BTLA antibody in a parent-to-F1 model of GVHD. C57BL / 6 spleen cells and bone marrow cells from humanized BTLA mice were injected into CB6F1 recipient mice and then treated with anti-BTLA antibody or an allotype control. Untreated mice developed clinical GVHD with progressive weight loss, dermatitis, and diarrhea and were sacrificed when they reached a pre-specified humanitarian endpoint. Mice treated with antibodies 2.8.6 and 11.5.1 were relatively redundant, with survival rates comparable to control mice reconstituted with homologous cells. (b) Mice were sacrificed 5 weeks after cell transfer, and the colon weight-to-length ratio was calculated as a marker of intestinal inflammation. Treatments 2.8.6 and 11.5.1 suppressed the colonic thickening observed in untreated mice.

[0452] Figure 10

[0453] (a) In vivo effect of D265A mutant clone 11.5.1 in 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 enhances proliferation due to receptor blockade. (b) The D265A mutant 11.5.1 antibody no longer inhibits T cell proliferation in mixed lymphocyte reactions.

[0454] Figure 11

[0455] Anti-BTLA antibody does not fix complement. Spleen cells from humanized BTLA mice were incubated with 10% rabbit complement at 37°C for 1 hour in the presence of 20 μg / mL BTLA antibody, an isotype control, or a positive control (depleted CD20 antibody). The anti-CD20 antibody depleted most B cells, confirming the activity of rabbit complement, but the BTLA antibody did not deplete either B cells or T cells, even though both populations were positive for BTLA staining.

[0456] Figure 12

[0457] Anti-BTLA antibodies do not induce antibody-dependent cell-mediated cytotoxicity. Spleen cells from humanized BTLA mice were incubated at 37°C for 24 hours in the presence of 20 μg / mL BTLA antibody, an allotype control, or a positive control (depleted CD20 antibody). Anti-CD20 antibody depleted most B cells by inducing ADCC from effector cells in the mixture, but BTLA antibody did not deplete either B cells or T cells, even if both populations were positive for BTLA staining.

[0458] Figure 13

[0459] Anti-BTLA antibodies did not deplete B cells or T cells in vivo. Humanized BTLA mice were injected with 200 μg of the 2.8.6 antibody. After 24 hours, spleen and bone marrow were collected, and cell populations were assessed by flow cytometry. 2.8.6 did not deplete B cells or T cells in the spleen or affect the frequency of different B cell precursor populations in the bone marrow (n = 3 mice / group).

[0460] Figure 14

[0461] Compared with BTLA expression in cells from mice injected with the same type of control antibody, B cells or CD4 cells from humanized mice incubated with antibody 2.8.6 or 11.5.1 for 6 days showed significantly higher BTLA expression. + BTLA expression level on T cells (n = 5 mice / group).

[0462] Figure 15

[0463] The agonist effect of BTLA antibodies in reporter gene assays is dependent on Fc receptor binding, and isoforms with greater FcγR2B binding are more potent agonists. Jurkat T cell lines expressing GFP under the control of NFkB-responsive transcriptional elements were transfected with human BTLA and stimulated by co-culturing with BW5147 cell lines expressing an anti-CD3scFv construct on their surface. NFkB signaling was detected by measuring the geometric mean of GFP by flow cytometry after 24 hours of culture. The inhibitory effect of adding different isoforms of BTLA agonist antibodies to the culture was evaluated under conditions where the BW5147 cell line was also transfected to express hFcγR2B (a) or under conditions where the Fc receptor was absent (b). Data points are the mean + / - SD of three wells at each antibody concentration and represent three independent experiments.

[0464] Figure 16

[0465] Humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype showed greater efficacy and potency in reporter gene assays compared to the Fc fusion protein of the BTLA ligand HVEM or the prior art BTLA agonist 22B3. Jurkat T cell lines expressing GFP under the control of NFkB-responsive transcriptional elements were transfected with human BTLA and stimulated by co-culturing with BW5147 cell lines expressing anti-CD3scFv constructs and hFcγR2B on their surfaces. NFkB signaling was detected by measuring the geometric mean of GFP by flow cytometry after 24 hours of culture. The inhibitory effect of the BTLA agonist antibodies added to the co-culture was evaluated. Data points are the mean + / - SD of three wells at each antibody concentration and represent three independent experiments.

[0466] Figure 17

[0467] Humanized anti-BTLA 2.8.6 inhibited CD4 T cell proliferation in a mixed leukocyte response. Purified primary human T cells from blood center donors were stained with cell proliferation tracer dyes in the presence of BTLA agonist antibody or hIgG1 P238D isotype controls and co-cultured for 5 days with allogeneic monocyte-derived dendritic cells from different donors at a 4:1 ratio. Cell populations were identified by flow cytometry, and proliferation was assessed by tracer dye dilution. CD4 proliferation in the presence of BTLA antibody was normalized to proliferation in the presence of an isotype control at an equal concentration. Data from six independent experiments were cross-referenced with different donor pairs. 2.8.6 as the P238D isotype significantly inhibited CD4 T cell proliferation, but not in other isotype forms. The prior art molecule 22B3 had no significant effect on CD4 proliferation.

[0468] Figure 18

[0469] Humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype inhibited primary B cell activation in response to the TLR9 agonist ODN2006. Primary human B cells were isolated from healthy donor PBMCs and stimulated with 0.01 μM ODN2006 in the presence or absence of different doses of P238D isotype control antibody or selected BTLA agonist antibody. After 5 days, IL-10 concentration in the supernatant was assessed by ELISA. Columns represent the mean + / - SD of three wells at each antibody concentration and represent three independent experiments.

[0470] Figure 19

[0471] In a xenograft-host disease model, humanized anti-BTLA agonist antibodies 2.8.6, 6.2_varC, and 3E8 expressed on the P238D isotype significantly reduced weight loss. Irradiated NSG mice were reconstituted with 10 million human PBMCs on day 0, followed by intravenous inoculation (IP) on day 1 with either 10 mg / kg BTLA antibody or a P238D isotype control. Mice were weighed periodically, and weights were plotted against starting weight (n = 9 mice / group, data points represent mean + / - SD).

[0472] Example

[0473] In subsequent 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 were shown to suppress T cell responses in vitro and in vivo upon binding to BTLA and to improve disease in mouse models of inflammatory bowel disease and graft-versus-host disease. While these agonist effects depend on Fc receptor binding, the antibodies do not cause the depletion of BTLA-expressing cells via cytotoxicity and do not induce receptor downregulation. The introduction of P238D modification into the heavy chain significantly enhances FcγR2B agonist signaling and increases the FcγR2B signaling ratio relative to FcγR2A. Such dual BTLA and FcγR2B agonist antibodies are expected to have therapeutic efficacy, particularly in the context of autoimmune and inflammatory diseases.

[0474] Example 1: Generation and sequencing of anti-BTLA antibodies

[0475] BioGenes GmbH, through its user BTLA (BTLA) K31-R151Mice were immunized with the extracellular domain of [a specific cell line] to produce antibodies recognizing the human immune cell receptor BTLA. Spleen cells from immunized mice were fused with Sp2 / O-Ag14 myeloma cells, and diluted clones were used. Hybridomas reacting with human BTLA were selected by ELISA of the supernatant. Antibodies were isotyped from the hybridoma supernatant using the Rapid Mouse Isotyping Kit (RayBiotech). Clones 2.8.6 and 11.5.1 were found to produce antibodies against IgG1k.

[0476] To sequence the variable domains of immunoglobulins, RNA was extracted from hybridomas using TRIzol reagent (ThermoFisher) according to the manufacturer's instructions. RNA was reverse transcribed to produce cDNA using primers specific to 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.

[0477] Then, as previously described (Tiller et al., J Immunol Methods, Vol. 350: pp. 183-193, 2009), PCR was performed using primers targeting conserved regions of immunoglobulin loci, and the PCR products were sequenced. In some cases, the large amount of nonfunctional κ light chain cDNA from fusion myeloma cell lines complicates the identification of functional light chains, and to address this issue, the previously described technique was employed, adding an excess of primers specific to the nonfunctional chain CDR3 to forcibly truncate the aberrant chain product (Yuan et al., J Immunol Methods, Vol. 294: pp. 39553-39561, 2005).

[0478] The NCBI IgBlast tool was used to evaluate the variable domain sequence to determine the location of the CDR.

[0479] Example 2: Binding with soluble human and cynomolgus monkey BTLA

[0480] The binding affinity and kinetics of the BTLA agonist antibodies (2.8.6 and 11.5.1) of this invention to human or cynomolgus monkey BTLA were determined using surface plasmon resonance with 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 onto the biosensor surface, and the negative control antibody (clone Mopc21; Biolegend) was captured into the reference channel. Various concentrations of monomeric soluble human BTLA extracellular domain (BTLA) were then subjected to a single-cycle kinetic analysis at 37°C. K31-R151 (from SEQ ID NO: 225) or soluble cynomolgus monkey BTLA extracellular domain (BTLA) K31-R151 (From SEQ ID NO: 226) Injected onto immobilized antibody in buffer 10 mM Hepes, 150 mM NaCl, 0.005% v / v surfactant P20, pH 7.4 (HBS-P) Figure 1 a). After reference and blank subtraction, the association rate and dissociation rate were fitted using BiaEvaluation software (GE Healthcare), and the dissociation constant was calculated. Figure 1 b). Clone 2.8.6 binds to human BTLA with 0.65 nM KD and to cynomolgus monkey BTLA with 7.89 nM KD. Clone 11.5.1 binds to human BTLA with 0.75 nM KD and to cynomolgus monkey BTLA with 0.99 nM KD. In a separate experiment targeting human BTLA only, clone 2.8.6 binds to human BTLA with 0.37 nM KD, and clone 11.5.1 binds to human BTLA with 0.53 nM KD.

[0481] Example 3: Binding to BTLA on cells

[0482] The ability of the BTLA agonist antibodies (2.8.6 and 11.5.1) of the present invention to bind to human or cynomolgus monkey BTLA expressed on the cell surface was evaluated by flow cytometry. Lentiviral transfection systems were used to express full-length human or cynomolgus monkey BTLA in Jurkat T cell lines. 1 × 10⁻⁶ 5Cells were seeded per well in 96-well U-shaped plates. Binding of the BTLA antibody to the mIgG1 isotype control (clone MOPC-21, Biolegend catalog number 400165) was assessed at twelve concentrations, starting at 90 μg / mL, using a 1:3 serial dilution in FACS buffer (PBS, 2% FCS, 0.05% sodium azide). Non-specific antibody binding was prevented by adding an Fc block (Biolegend catalog number 101319). Cells were incubated with the antibody on ice for 30 min, then washed twice with FACS buffer and stained with AF647-conjugated anti-mIgG1 secondary antibody (Biolegend catalog number 406618). Cells were incubated on ice for 30 min, washed, and resuspended in FACS buffer for flow cytometry analysis. The geometric mean fluorescence intensity of each secondary antibody concentration was plotted, and the EC50 of receptor binding was calculated using GraphPad Prism software via nonlinear curve fitting. Clone 11.5.1 bound to cells expressing human BTLA with an EC50 of 0.016 nM and cells expressing cynomolgus monkey BTLA with an EC50 of 0.0057 nM. Clone 2.8.6 bound to cells expressing human BTLA with an EC50 of 0.085 nM and cells expressing cynomolgus monkey BTLA with an EC50 of 0.16 nM. Figure 1 c to Figure 1 d).

[0483] Example 4: Competition with natural ligand HVEM for binding to BTLA

[0484] The ability of the BTLA agonist antibodies (2.8.6 and 11.5.1) of this invention to block the binding of natural ligands to BTLA was evaluated using surface plasmon resonance on a Biacore T200 (GE Healthcare) instrument. The extracellular domain of human BTLA (BTLA) was conjugated using amine coupling. 31K-151R The human HVEM extracellular domain fused with mouse IgG1Fc was then covalently coupled to the CM5 sensor chip. The human HVEM extracellular domain was then injected at 37°C onto immobilized hBTLA in HBS-P buffer and allowed to completely dissociate. A saturated amount of anti-BTLA antibody (2.8.6 or 11.5.1) was then injected, followed immediately by a second injection of human HVEM-mFc at the same concentration as the initial injection. Figure 2 A). The percentage of binding prior to antibody injection is expressed as the equilibrium HVEM binding (in resonance units) after antibody saturation with BTLA. Figure 2 B). If the binding of HVEM after antibody saturation is greater than 90% of the binding before antibody injection, then the antibody is considered non-blocking.

[0485] Example 5: Binding epitope of antibody 11.5.1 on human BTLA

[0486] The functional epitopes of antibody 11.5.1 on human BTLA were determined by flow cytometry to assess binding to a group of single-residue mutants of the receptor expressed on the cell surface. Constructs encoding the human extracellular region of BTLA and the transmembrane and intracellular regions of mouse CD28 were cloned into the bicistronic mammalian expression vector pGFP2-n2 (BioSignal Packard Ltd.), which also encodes GFP. Mutant constructs altered by one amino acid were prepared using a “violent” mutagenesis method (Davis et al., Proc Natl Acad Sci USA, Vol. 95: pp. 5490-5494, 1998). Plasmids (2 μg / well) were transfected into HEK293T cells in 6-well plates using Genejuice transfection reagent (Novagen; 6 μL / well). Each experiment included a dummy and a no-transfection control. Cells were harvested after 48 hours and stained with 10 μg / mL fluorescently conjugated anti-BTLA antibody and live / dead labels in PBS, 0.05% azide, and 2% FCS (FACS buffer) at 4°C for 1 h. Cells were washed, pelleted, and resuspended in 200 μL FACS buffer for analysis on a BD FACSCanto flow cytometer. An example of binding analysis of gated GFP-positive (transfected) live cells and anti-BTLA antibody was shown in clone 11.5.1. Figure 3 (a). For each mutant, the geometric mean of anti-BTLA antibodies binding to transfected cells is expressed as the percentage binding to the wild-type receptor ( Figure 3 b). A panel of anti-BTLA antibodies was evaluated, and any mutations that eliminated binding to all antibodies were excluded from the analysis, assuming such mutations caused drastic changes in protein folding or expression rather than indicating antibody epitopes. The mutations Y39R and K41E completely eliminated binding to antibody 11.5.1 without affecting binding to 2.8.6. These mutations were mapped to... Figure 3 The human BTLA crystal structure in c (Compaan et al., J Biol Chem., Vol. 280: pp. 39553-39561, 2005) (black residues) shows the binding epitope of 11.5.1. The residues required for HVEM binding (Gln37, Arg42, Pro59, His127; from patent publication number WO2017004213) are also mapped onto the gray structure, indicating that 11.5.1 binds to an epitope very close to the HVEM binding site.

[0487] Example 6: Crystal structure of the Fab' fragment of 2.8.6 complexed with human BTLA

[0488] The structural epitopes of antibody 2.8.6 on human BTLA were determined by resolving the crystal structure of antibody Fab complexed with the extracellular domains of human BTLA. 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 (with a 6x Histidine tag) of the mouse IgG1 heavy chain and the constant domain of the mouse Igκ chain, respectively. These vectors were transiently co-transfected into HEK293T cells to generate the Fab' fragment against BTLA 2.8.6, which was purified using Ni-NTA. The human BTLA Ig-V domain (BTLA...) was then... S33-D135 The BTLA inclusion bodies were cloned into the pGMT7 vector and expressed in BL21(DE3)pLysS *E. coli* cells (Novagen) to generate inclusion bodies. Inclusion bodies were isolated from the cell pellet by sonication and repeatedly washed with a washing solution containing 0.5% Triton X-100. The purified BTLA inclusion bodies were dissolved in a denaturing agent solution containing 6M guanidine hydrochloride. The dissolved protein solution was slowly diluted to a final protein concentration of 1-2 μM in refolding buffer [0.1M Tris-HCl (pH 8.0), 0.6M L-arginine, 2mM EDTA, 3.73mM cysteine, and 6.73mM cysteine] and 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 Superdex 75 column (GE Healthcare).

[0489] The purified BTLA and Fab' were mixed and purified into a complex by size exclusion chromatography. Crystals suitable for data collection were obtained by pendant drop vapor diffusion in 0.2 M calcium acetate, 0.1 M imidazole, pH 8.0, and 10% (w / v) PEG 8000 at 293°K. The final dataset was collected at Photon Factory, and the structures of BTLA (PDB ID: 2AW2 chain A) and anti-PD1-Fab (PDB ID: 5GGS chains C, D) were determined by molecular substitution as search probes.

[0490] The residues at the interface between BTLA and antibody 2.8.6 are A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, and D135.

[0491] Example 7: Development of humanized BTLA mice

[0492] To provide a platform for evaluating anti-human BTLA antibodies in mouse models, a knock-in strain of C57Bl / 6 mice expressing a chimeric form of BTLA containing both human extracellular and mouse transmembrane and signal transduction regions was developed. A human genomic DNA fragment from exon 2 to exon 3 was inserted into the mouse locus, replacing the mouse sequence from exon 2 to exon 4. The exon-intron junctions at the mouse exon 2 start and exon 4 end were preserved intact to ensure proper splicing. Figure 5 ).

[0493] Example 8: Inhibition of antigen-specific T cell proliferation in vivo

[0494] The ability of the BTLA agonist antibodies (2.8.6 and 11.5.1) of the present invention to inhibit antigen-specific T cell proliferation in vivo was evaluated using a sensitive T cell transfer assay. Figure 6 a). In this measurement, 5 × 10 5 T cells were transfected into non-transgenic C57BL / 6 receptors. These T cells contained purified OTII (TCR transgenic) CD4 cells specific to ovalbumin (OVA) from mice expressing homozygous human BTLA (hBTLA) and from OT-II mice expressing wild-type mouse BTLA receptor (The Jackson Laboratory). + A mixture of T cells. The transferred cells were distinguished from host cells using a CD45.2 (as opposed to CD45.1) allotype 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. On the second day after T cell transfer, recipient mice were immunized with 100 μg ovalbumin (Sigma-Aldrich) in 100 μL PBS mixed with 100 μL Imject Alum (ThermoFisher) to induce T cell expansion. On the second day, mice were administered 200 μg of antibody intraperitoneally. 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 expansion or contraction of humanized cells bound to anti-human BTLA antibodies relative to wild-type controls not bound to anti-human BTLA antibodies. Compared to wild-type controls, both antibodies 2.8.6 and 11.5.1 resulted in reduced proliferation of humanized BTLA cells, indicating that they induce signal transduction via the inhibitory BTLA receptor, leading to decreased T cell proliferation. Figure 6 b).

[0495] Example 9: Inhibition of T cell proliferation in mixed lymphocyte responses

[0496] The ability of the BTLA agonist antibodies (2.8.6 and 11.5.1) of the present invention to inhibit the proliferation of primary T cells from humanized mice in vitro was evaluated using a mixed lymphocyte reaction (MLR). Spleen cells from Balb / c mice were treated with mitomycin C at 37°C for 30 min, then washed and used as stimulatory cells. T cells were purified from humanized BTLA mouse spleens using negative selection via magnetically activated cell sorting (Mojosort Mouse CD3T Cell Isolation Kit, Biolegend Catalog No. 480023) and stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher) for use as responding cells. 4 × 10 5 1 stimulating cell / well and 2 × 10 5 One responder cell / well was mixed with various concentrations of anti-BTLA or allotype control antibody (clone MOPC-21, Biolegend catalog number 400165) in a 96-well U-shaped plate. The antibody was evaluated at 1:3 dilutions, starting at 1 μg / mL and continuing for 10 concentrations. A polyclonal anti-mHVEM antibody (R&D systems catalog number AF2516) was also added to all wells at 101 μg / mL to block any baseline signaling via the BTLA pathway and enhance the effect of the agonist antibody. After 96 hours, the dilution of CellTrace Violet in responder cells was assessed by flow cytometry as a marker of proliferation. Proliferation in the presence of anti-BTLA antibody or allotype control was compared with proliferation in the absence of the antibody. Gated CD4 + and CD8 + Populations were analyzed separately. Both antibodies 2.8.6 and 11.5.1 reduced the proliferation of T cells expressing human BTLA, indicating that they induced inhibitory signaling through the human BTLA receptor. Clone 2.8.6 inhibited CD4 T cells with an IC50 of 0.029 nM and had a maximum inhibitory effect of 42% proliferation. Figure 7 Clone 11.5.1 inhibited CD4T cells with an IC50 of 0.016 nM and had a maximum inhibitory effect of 33% on proliferation.

[0497] Example 10: Inhibition of NFkB signaling in Jurkat T cell lines transfected with human BTLA or cynomolgus monkey BTLA

[0498] The ability of the BTLA agonist antibodies (2.8.6 and 11.5.1) of the present invention to inhibit NF-κB signaling was evaluated using BTLA-transfected reporter T cell lines. Jurkat T cell lines stably transfected with an expression cassette including an NF-κB response transcriptional element (Source BioSciences catalog number TR850A-1) upstream of the minimal CMV promoter (mCMV)-GFP cassette were used as reporter cell lines for NF-κB signal transduction. A lentiviral transfection system was used to express full-length human or cynomolgus monkey BTLA in this reporter cell line. These cells were mixed with a stimulating cell line consisting of bw5147 cells expressing an anti-CD3scFv construct on their surface, as described in Leitner et al., J Immunol Methods, October 31, 2010, Vol. 362, Nos. 1-2: pp. 131-141. The stimulating cell line was also transfected with mouse FcγR2B to provide an Fc receptor for presenting the agonist antibody BTLA. In the presence of various concentrations of BTLA antibody or allotype control (clone MOPC-21, Biolegend catalog number 400165), 5×10 4 Reporter cells / well with 5 × 10 4 Stimulated cells / well were mixed in 96-well U-shaped plates. After incubation at 37°C for 24 hours, the cell pellet was stained with a viability dye (Zombie Aqua, Biolegend catalog 423101) and mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend catalog 109830) for flow cytometry to separate stimulated (mouse) cells from responder (human) cells. The geometric mean of GFP expression at each antibody concentration was assessed and normalized to GFP expression in the absence of the antibody. Clone 2.8.6 inhibited human BTLA-transfected cells with an IC50 of 0.06 nM and cynomolgus monkey 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 monkey BTLA-transfected cells with an IC50 of 0.14 nM.

[0499] Example 11: Treatment of a T-cell-driven colitis mouse model with antibody 2.8.6

[0500] The ability of BTLA agonist antibody 2.8.6 to improve a T-cell-driven colitis model was evaluated using humanized mice. This T-cell transfer model has previously been described as a mouse model of inflammatory bowel disease (Ostanin et al., Am J Physiol Gastrointest Liver Physiol., Vol. 296: G135-46, 2009). CD45RBs were sorted from the spleen and lymph nodes of humanized BTLA mice. hiCD25-CD4+ T cells were administered at a dose of 5 × 10⁻⁶. 5 Rag1KO receptor (Rag1) was injected intraperitoneally into mice using one cell / mouse cell. tm1Mom In Jackson Laboratory, the transfected T cells caused inflammatory colitis that developed after approximately 3 weeks, leading to diarrhea and weight loss. Rag1KO cagemates without transfected T cells served as non-disease controls. Recipient mice were intraperitoneally injected with 200 μg of either 2.8.6 or an isotype control antibody at days 7, 21, and 35 following T cell transfection. All mice were weighed periodically, and the colon was weighed and measured at 8 weeks. Inflammatory infiltration was assessed histologically and by flow cytometry using cell counting and extracted lamina propria leukocytes. Antibody 2.8.6 prevented weight loss. Figure 8 a) and significantly reduced inflammatory infiltration of the colon ( Figure 8 b). Inflammation of the colon in diseased mice led to an increased colon weight:length ratio, which was not observed in mice treated with 2.8.6. Figure 8 c).

[0501] Example 12: Treatment of a mouse model of graft-versus-host disease (GVHD)

[0502] The effect of anti-BTLA agonist antibodies was evaluated in a non-lethal parental GVHD F1 model. Bone marrow cells (BMCs) and spleen cells were harvested from humanized BTLA donor mice (C57BL / 6 background; H2B). 2 × 10⁶ cells were used to evaluate the effect of anti-BTLA agonist antibodies. 7 One BMC and 107 spleen cells were intravenously injected into CB6F1(H2B) cells that had already been lethally irradiated with 9 Gy of whole-body radiation. / d In the receptor. Irradiated CB6F1 mice reconstituted with syngeneic BMC and spleen cells served as non-disease controls. On the day of immune cell transfer, mice were intraperitoneally injected with 200 μg of anti-BTLA antibody or an allotype control. Mice were weighed regularly, and GVHD was monitored by calculating relative weight loss and clinical observation. Mice were sacrificed 5 weeks after immune cell transfer or when they reached a humanitarian endpoint, which included a weight loss of >20% relative to starting weight in the first 14 days, or a weight loss of >15% at any other time. At death, the colon was weighed and measured, and the colon weight:length ratio was calculated as a marker of colonic inflammation, a prominent clinical feature of GVHD. Both antibodies 2.8.6 and 11.5.1 significantly reduced weight loss, leading to increased survival. Figure 9 a) and prevented colitis ( Figure 9 b).

[0503] Example 13: The agonist activity of antibody 11.5.1 depends on Fc receptor binding.

[0504] Antibody 11.5.1 was recombinantly expressed as mIgG1k containing the D265A mutation, which had previously been described as significantly reducing Fc receptor binding (Clynes et al., Nat Med., Vol. 6: pp. 443-446, 2000). This mutated antibody was evaluated in the T-cell transfer assay described in Example 8. The parental 11.5.1 antibody inhibited the proliferation of humanized T cells because its net effect was BTLA receptor agonism. However, the FcR-null D265A mutation resulted in enhanced proliferation of humanized T cells, indicating that the FcR-null mutation removed the agonistic effect of the antibody, leaving only the receptor-blocking effect. Figure 10 a).

[0505] The D265A-mutated 11.5.1 antibody was also evaluated in the in vitro MLR assay described in Example 9. Similarly, the parental 11.5.1 antibody inhibited the proliferation of humanized T cells because its net effect was BTLA receptor agonism. The FcR-nullable D265A mutation removed the agonistic effect of the antibody, therefore this antibody did not show any effect in this assay. Figure 10 b). In this assay, the FcR ineffective 11.5.1 antibody did not enhance the proliferation of humanized cells because HVEM was blocked (by adding a polyclonal anti-HVEM antibody), and therefore there was no baseline signal through the pathway blocked by the BTLA blocking antibody.

[0506] Example 14: In vitro non-fixed complement of antibodies 2.8.6 and 11.5.1

[0507] Spleen cells from humanized mice were incubated with 10% young rabbit complement (BioRad) and 20 μg / mL of anti-BTLA antibody (or an allotype control or a positive control of depleted anti-CD20 antibody; clone SA271G2, from Biolegend) at 37°C for 15 minutes. Although the anti-CD20 antibody depleted most of the B220... + B cells, but anti-BTLA antibody does not deplete B220. + or CD4 + cell( Figure 11 Even if both groups are positive for BTLA staining.

[0508] Example 15: Antibodies 2.8.6 and 11.5.1 do not induce ADCC in vitro

[0509] Intact spleen cells (including myeloid effector cells) from humanized mice were incubated with 20 μg / mL of anti-BTLA antibody (or allotype control or depleted anti-CD20 antibody SA271G2) at 37°C for 24 hours. Although the anti-CD20 antibody depleted most of the B220... + Cells, but anti-BTLA antibody does not deplete B220 + or CD4 + cell( Figure 12 Even if both groups are positive for BTLA staining.

[0510] Example 16: Antibodies 2.8.6 and 11.5.1 do not deplete BTLA-expressing cells in vivo.

[0511] Humanized BTLA mice were intraperitoneally injected with 200 μg of anti-BTLA antibody or an allotype control. Spleens were harvested after 24 hours, and the frequencies of different cell populations were identified by flow cytometry. The anti-BTLA antibody had no effect on the frequency or absolute number of B cells or T cells in the spleen, or on the number of B cell precursors in the bone marrow. Figure 13 ).

[0512] Example 17: Stabilizing BTLA expression on immune cells in vivo using antibodies 2.8.6 and 11.5.1

[0513] Humanized mice were intraperitoneally injected 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 and processed into single-cell suspensions for evaluation by flow cytometry. Cells were stained with an antibody mixture to identify immune cell subsets and stained with a fluorescently conjugated anti-BTLA antibody that has a non-competitive epitope with the injected antibody. The geometric mean of BTLA staining after in vivo incubation with anti-BTLA antibody was normalized to the geometric mean of BTLA staining after incubation with an isotype control (using the same staining antibody). Compared with mice injected with the isotype control, mice injected with clones 2.8.6 or 11.5.1 showed significantly higher BTLA expression on B cells and CD4 T cells. Figure 14 This indicates 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, Vol. 215: pp. 570–578, 2010). For immunosuppressive purposes, agonist antibodies that stabilize receptor expression exhibit the benefit of prolonging high levels of inhibitory signaling through this pathway compared to downregulating antibodies.

[0514] Example 18: Tolerance and side effects in animal models

[0515] No tolerability issues or side effects were observed in any animal studies using antibody 2.8.6 or 11.5.1.

[0516] Example 19: Characterization of an exemplary BTLA antibody

[0517] Characterization of exemplary mIgG1BTLA antibodies provided herein, other than those described in 2.8.6 and 11.5.1, is described in this embodiment. The binding affinity of the various clones listed in Tables 1 and 2 to BTLA and their lymphocyte-inhibiting efficiency were evaluated (Table 3).

[0518] Table 1: Exemplary BTLA agonist antibodies

[0519]

[0520]

[0521] Table 2: Humanized and engineered antibodies

[0522]

[0523] For each antibody, the binding rate (“on rate”) and dissociation rate (“off rate”) of human BTLA, as well as the KD of binding human or cynomolgus monkey BTLA, were measured according to the method described in Example 2, and curves were fitted to the extracellular domain of BTLA injected at a single concentration. The inhibitory efficiency of individual antibodies against T cells was also evaluated at a single concentration of 10 μg / mL. MLR assays (two biological replicates as shown in Table 4) were performed on each individual antibody according to the method described in Example 9; anti-CD3 assays (two biological replicates, Table 4) were performed according to the method described below; and the inhibition of NFkB signaling in human BTLA-transfected Jurkat T cell lines by each antibody was determined according to the method described in Example 10 (Table 4). In various in vitro stimulation assays for each exemplary antibody, the mean inhibition of T cells relative to the isotype control was calculated as the average of the percentage inhibition of all assay results (Tables 3 and 4).

[0524] Table 3: Characterization of binding affinity and inhibitory activity of exemplary antibodies

[0525]

[0526]

[0527] Table 4: Results of Inhibitory Effect Assay of Exemplary Antibodies

[0528]

[0529] The following was an assessment of the ability of BTLA agonist antibodies to inhibit anti-CD3 and anti-CD28-induced T cell activation. Spleen cells from humanized BTLA mice were processed into single-cell suspensions and treated with ACK buffer to lyse erythrocytes. Cells were stained with CFSE (Biolegend catalog number 423801) to track cell proliferation. 2 × 10⁶ cells were then used. 5Cells were seeded per well in 96-well U-shaped plates at a concentration of 50 ng / mL each of soluble anti-CD3 antibody (clone 145.2C11; Biolegend catalog number 100339) and anti-CD28 (clone 37.51; Biolegend catalog number 102115), and 10 μg / mL of soluble anti-BTLA antibody or allotype control. After 72 hours, cells were analyzed by flow cytometry to assess proliferation (“anti-CD3 / CD28 (CD4 T cell proliferation)”) and T cell activation (“anti-CD3 / CD28 (CD69+CD4 T cells)”) by staining for surface-expressed activation markers. For each BTLA antibody, the percentage of inhibition compared to the allotype control antibody was calculated.

[0530] Furthermore, for each BTLA antibody, their ligand blocking ability was evaluated according to the method described in Example 4, for example, competing with HVEM for BTLA binding, and a result of "Yes" was given for more than 90% inhibition of HVEM-BTLA binding, and a result of "No" was given for less than 10% inhibition of HVEM-BTLA binding. The functional epitopes of each BTLA antibody were also determined according to the method described in Example 5. The "Epitopes" column in Table 3 summarizes the epitope sets 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 (referred to as “epitope 1” in the table), which contains at least one key residue selected from the following list: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 (according to the position in SEQ ID NO: 225). Antibodies binding to epitope 1 do not compete with ligand HVEM for binding to BTLA. Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, and 26F3 all bind to different second epitopes (“epitopes 2”), which contain at least one key residue selected from the following list: Y39, K41, R42, Q43, E45, and S47. Antibodies binding to epitopes 2 do indeed compete with the ligand HVEM for binding to BTLA. Antibody 26B1 binds to a third epitope (“epitope 3”), which contains at least one key residue selected from the following list: D35, T78, K81, S121, and L123. Antibodies binding to epitopes 3 do indeed compete with the ligand HVEM for binding to BTLA. Antibodies 24H7, 4B1, 8B4, and 4H4 all bind to different fourth epitopes (“epitopes 4”), which contain the key residue H68. Antibodies binding to epitopes 4 do not compete with the ligand HVEM for binding to BTLA. Antibody 21C7 binds to a distinct fifth epitope (“epitope 5”) containing at least one key residue selected from the following list: N65 and A64. Antibodies binding to epitope 5 do not compete with ligand HVEM for binding to BTLA.

[0531] Example 20: Humanization and CDR Engineering of BTLA Antibodies 6.2, 2.8.2 and 3E8

[0532] Antibody 2.8.6 was humanized by CDR grafting to the homologous human framework region (see SEQ ID NO: 26, 27). IGHV2-5*08 was used for the heavy chain, and IGKV3-11*01 for the light chain. Binding to BTLA was assessed by SPR after humanization. The humanized 2.8.6 binding to monomeric BTLA had a K+ of 0.73 nM. D .

[0533] The variable domains of 6.2 and 3E8 were humanized by germlining with homologous human germline framework regions (Seq ID Nos. 7, 8 and 36, 37). For 3E8, the selected receptor 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 receptor frameworks were VH3-3-21 and JH6 for the heavy chain and VK2-A19 and JK4 for the light chain.

[0534] Sometimes, certain residues in the CDR or variable framework region of an antibody may be substituted to remove unwanted features without significantly affecting target binding. The CDRH2 of humanized antibody 6.2 was modified with either N56Q alone (SEQ ID NO: 17) or N56Q and D54E substitution (SEQ ID NO: 11) to remove the deamidation and isomerization possibilities, respectively. The CDRL2 of humanized 6.2 was modified with D61E substitution to reduce predicted immunogenicity, as determined by Lonza's Epibase analysis (Seq ID NO: 12). In addition to the CDR, S77T substitution was introduced into the heavy variable framework region of humanized 6.2 to reduce predicted immunogenicity, and Q51K substitution was introduced into the light 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 describes the compositional CDR and variable domains of each of these variants. No engineered variants of antibody 6.2 containing only N56Q and no D54E substitution are disclosed in PCT / GB2019 / 053569 (e.g., engineered humanized 6.2 variant C).

[0535] Similarly, the CDRH2 domain of the humanized antibody 3E8 was modified with N57Q substitution to eliminate the possibility of deamidation, and with K63S substitution to reduce predicted immunogenicity (Seq ID No. 40). In addition to the CDR, G42D and A61S substitutions were introduced into the light chain variable framework of 3E8 to further reduce predicted immunogenicity. Furthermore, P15L and P81A substitutions were introduced into the light chain variable framework to restore these positions to the mouse sequence, rather than introducing proline residues that could potentially influence local conformation. The sequences of the engineered 3E8 light chain variable domains containing all four of these substitutions are given in Seq ID No. 43. Table 2 describes the compositional CDRs and variable domains of the engineered variants of humanized 3E8.

[0536] Example 21: Binding of humanized anti-BTLA antibody to soluble human and cynomolgus monkey BTLA

[0537] The binding affinity and kinetics of humanized BTLA agonist antibodies to human or cynomolgus monkey BTLA were determined using a Biacore 8K (GE Healthcare) biosensor via surface plasmon resonance. Series S CM5 sensor chips (GE Healthcare) were coated with polyclonal anti-human IgG using a human antibody capture kit (GE Healthcare catalog number 29234600). Anti-BTLA antibodies were then captured onto the biosensor surface, and a negative control antibody (human IgG1k isotype control; SinoBiological catalog number HG1K) was captured into the reference channel. Various concentrations of monomeric soluble human BTLA extracellular domains (BTLA) were then subjected to single-cycle kinetic analysis at 37°C. K31-R151 (generated through internal recombination) or soluble cynomolgus monkey BTLA extracellular domain (BTLA) K31-R151 (Internal recombination generated) was injected onto immobilized antibodies in buffer HBS-EP (GE Healthcare, catalog number BR100669), pH 7.4 (HBS-P). For human BTLA, sequential quadruple dilutions from 673 nM to 164 pM were used. For cynomolgus monkey BTLA, sequential quadruple dilutions from 1351 nM to 330 pM were used. After reference and blank subtraction, association and dissociation rates were fitted using BiaEvaluation software (GE Healthcare), and dissociation constants were calculated (Table 5). Humanized 2.8.6 bound human BTLA with a KD of 2.33 nM and cynomolgus monkey BTLA with a KD of 147 nM. Humanized 3E8 variant B (3E8_var_B) bound human BTLA with a KD of 141 nM and cynomolgus monkey BTLA with a KD of 1520 nM. Humanized 6.2 variant A, containing D54E and N56Q substitutions in its CDRH2 to eliminate the possibility of isomerization and deamidation, binds to human BTLA at a KD of 10.9 nM and to cynomolgus BTLA at a KD of 695 nM. This binding indicates a significant decrease in affinity for the parental clone 6.2 antibody, which binds to human BTLA at a KD of 1.7 nM and to cynomolgus BTLA at a KD of 9.71 nM (Table 5). Humanized 6.2 variant C (or 6.2_var_C) containing only N56Q substitution but not D54E substitution in CDRH2 binds to human BTLA at a KD of 1.25 nM and to cynomolgus BTLA at a KD of 15.4 nM, thus maintaining an affinity closer to the parental clone.

[0538]

[0539] Table 5: Binding kinetics and affinity of antibodies to soluble human or cynomolgus monkey BTLA, as shown by [method / method] at 37°C. Surface plasmon resonance measurements .

[0540] Example 22: Binding of humanized anti-BTLA antibody to BTLA on cells

[0541] The ability of the BTLA agonist antibody of the present invention to bind to human or cynomolgus monkey BTLA expressed on the cell surface was evaluated by flow cytometry. A lentiviral transfection system was used to express full-length human or cynomolgus monkey BTLA in Jurkat T cell lines. 1 × 10⁻⁶ 5 Cells were seeded per well in 96-well U-shaped plates. Binding of the BTLA antibody to the hIgG1k P238D isotype control (clone MOPC-21, generated via absolute antibody recombinant; heavy chain SEQ ID NO: 230, light chain SEQ ID NO: 231) was assessed at twelve concentrations, starting at 30 μg / mL, using a 1:3 serial dilution in FACS buffer (PBS, 2% FCS, 0.05% sodium azide). Non-specific antibody binding was prevented by adding an Fc block (BioLegend catalog number 101319). Cells were incubated with the antibody on ice for 60 min, then washed twice with FACS buffer and stained with AF647-conjugated anti-hIgG secondary antibody (clone HP6017; BioLegend catalog number 409320). Cells were incubated on ice for 30 min, then washed and resuspended in FACS buffer for flow cytometry analysis. Geometric mean fluorescence intensity was plotted for each concentration of secondary antibody, and the EC50 for receptor binding was calculated using GraphPad Prism software via nonlinear curve fitting. Humanized 2.8.6 cells expressing human BTLA had an EC50 of 0.066 nM. Figure 15 a) and the binding of cynomolgus monkey BTLA-expressing cells with an EC50 of 0.854 nM (a) Figure 15 b). Humanized 6.2_var_C bound to cells expressing human BTLA with an EC50 of 0.062 nM and cells expressing cynomolgus monkey BTLA with an EC50 of 0.148 nM. Humanized 3E8_var_B bound to cells expressing human BTLA with an EC50 of 0.177 nM and cells expressing cynomolgus monkey BTLA with an EC50 of 15.6 nM.

[0542] Example 23: Binding affinity of Fc variant antibodies to human Fc receptor

[0543] In Example 13, it was surprisingly demonstrated that the agonist function of the BTLA antibody may depend on Fc receptor binding of the antibody's Fc moiety. In humans, there exists an inhibitory Fcγ receptor (FcγR2B), while other Fcγ receptors (FcγR1A, FcγR2A, FcγR3A, and FcγR3B) transmit immune activation signals. To enable the BTLA agonist antibody to effectively suppress the immune response without inducing inflammatory FcR signaling, we propose that selective Fc binding to FcγR2B may be necessary. Furthermore, selective binding to FcγR2B would promote bidirectional inhibitory signaling via BTLA on BTLA-expressing cells and FcγR2B on FcγR2B-expressing cells, which would enhance the immunosuppressive effect of the antibody. This would be desirable in therapeutic antibodies intended for treating diseases of excessive immune activation. Conversely, the extremely high affinity for FcγR2B may adversely affect the antibody's half-life due to the receptor's metabolic turnover in hepatic sinusoidal epithelial cells (Ganesan et al., The Journal of Immunology, Vol. 189, No. 10: pp. 4981-4988, 2012), as exemplified by the FcγR2B-enhanced IgG1 antibody XmAb7195, which binds to FcγR2B with a KD of 7.74 nM (Chu et al., Journal of Allergy and Clinical). Immunology, Vol. 129, No. 4: pp. 1102-1115, 2012; https: / / linkinghub.elsevier.com / retrieve / pii / S0091674911018343 (May 13, 2020), and according to Xencor, compared to the average half-life of wild-type IgG1 of approximately 21 days (Morell, Terry, and Waldmann. Journal of Clinical...). Investigation, Vol. 49, No. 4: pp. 673-680, 1970; http: / / www.jci.org / articles / view / 106279 (May 16, 2020)), in a phase 1a trial, the mean in vivo half-life of this antibody was 3.9 days (American Thoracic Society (ATS), 2016 International Conference in San Francisco, California - A6476: Panel No. 407). Therefore, while selectivity for FcγR2B and adequate binding to supporting agonists may be desirable for BTLA agonist antibodies, excessive affinity for FcγR2B may be undesirable in therapy, as the resulting shortened half-life would likely necessitate more frequent dosing.

[0544] Recombination yielded a series of Fc-mutated antibody variants (containing the variable domain of humanized 2.8.6), and their binding to different human Fcγ receptors was assessed by surface plasmon resonance (at 37 °C, in buffer HBS-EP+, pH 7.4). The Fc variants were recombined on the hIgG1 or hIgG4 backbone and featured substitutions known to affect FcR binding or potentially affecting FcR binding based on their position at the Fc-FcR binding interface (hIgG1G236D, hIgG1G237D, hIgG1P238D, hIgG1D265A, hIgG1S267E, hIgG1P271G, hIgG1A330R, hIgG1K322A, hIgG1N297A, hIgG4P238D, hIgG4G237D, hIgG4P271G, hIgG4S330R, hIgG4F234A, hIgG4L235A). These mutations were evaluated as single substitutions or combinations. Variants containing sequence fragments converted from hIgG2, as described by Armour et al. (Molecular Immunology, Vol. 9, No. 40: 585–93, 2003) (designated δb, δc, δab, and δac), were also evaluated. The binding of mIgG1 and mIgG1D265A to human FcR was also assessed.

[0545] For low-affinity FcγRs (FcγR2A, FcγR2B, FcγR3A, and FcγR3B), the interactions were assessed by surface plasmon resonance using recombinant expressed FcRs (extracellular domain only) as analytes. In short, the interactions were assessed using the GE Healthcare Amine conjugation kit to express the recombinant human BTLA extracellular domain (BTLA). K31-R151 The 2.8.6 Fc variant to be evaluated (approximately 500 to 1000 response units) was covalently immobilized onto two flow cells in all channels of the CM5 series S sensor chip. The Fc variants were then captured in flow cell 2 of each channel (approximately 500 to 1000 response units). Steady-state affinity analysis was then performed by injecting different concentrations of FcR over multiple cycles and measuring equilibrium binding. A dual-reference method was used (subtracting the signal from reference Fc1 and also subtracting the signal from the blank zero-concentration injection). KD was calculated based on Langmuir curves (plotting equilibrium binding against analyte concentration to determine the concentration required for half-maximal binding).

[0546] For high-affinity FcR interactions (FcγR1A, and also FcRn evaluated at pH 6.0), binding was assessed in kinetic analysis using antibodies as analytes. Briefly, according to the provided protocol, biotinylated FcRs (SinoBiological, catalog number 10256-H08S-B for FcγR1A or catalog number CT009-H08H-B for FcRn) were captured in flow cell 2 on a streptavidin chip (Series S sensor chip SA-BR-1005-31). Reference flow cell 1 was empty in all channels. Purified antibodies were then injected at a single concentration, and association / dissociation rates were calculated by curve fitting on BiaEvaluation software. FcRn interactions at pH 6.0 do not induce inflammatory signal transduction but are necessary to maintain the antibody half-life in vivo; therefore, such interactions are desirable for therapeutic antibodies. IgG Fc has two FcRn binding sites, so this assessment using high-density immobilized FcRn provides an estimate of the affinity of the interaction rather than the true KD.

[0547] The KD values ​​for binding to each of the evaluated Fc variants and each of the human Fc receptors are provided in Table 6. The presence of the P238D mutation significantly enhanced selectivity for FcγR2B (by slightly increasing affinity for FcγR2B while sharply decreasing affinity for other FcγRs). Previously described mutant combinations including P238D (P238D G237D P271G A330R), termed V9 (Mimoto et al., Protein Engineering, Design and Selection, Vol. 26, No. 10: pp. 589-598, 2013), significantly increased binding affinity for FcγR2B but also preserved significant binding to the 131R polymorphic variant of FcγR2A. The same effect of increasing FcγR2B selectivity was observed when P238D was introduced alone or in combination into the hIgG4 backbone.

[0548]

[0549]

[0550] Table 6: Affinity (KD) of Fc variants to human FcR assessed by SPR at 37 °C. n / a = not assessed. NB = No binding detected .

[0551] Example 24: Inhibition of T cell activation by humanized BTLA agonist in NFkB reporter gene assay depends on Fc receptor binding .

[0552] BTLA is an inhibitory receptor expressed on T cells, therefore, agonist antibodies against BTLA are expected to inhibit T cell activation by inducing inhibitory signal transduction through the receptor. The ability of selected humanized BTLA agonist antibodies to inhibit T cell activation was evaluated using BTLA-transfected reporter T cell lines. Jurkat T cell lines stably transfected with an expression cassette including an NF-κB response transcriptional element (Source BioSciences catalog number TR850A-1) upstream of the minimal CMV promoter (mCMV)-GFP cassette were used as reporter cell lines for NF-κB signal transduction. A lentiviral transfection system was used to express full-length human BTLA in this reporter cell line. These cells were mixed with a stimulatory cell line consisting of bw5147 cells expressing an anti-CD3scFv construct on their surface, as described in Leitner et al. (J Immunol Methods, Vol. 362, Nos. 1-2: pp. 131-141, 2010). The stimulated cell lines were also transfected with human FcγR2B to provide Fc receptors for the presentation of the agonist BTLA antibody. In the presence of various concentrations of BTLA antibody or hIgG1k isotype control antibody (Sino Biologicals catalog number HG1K), 5 × 10⁻⁶ cells were used. 4 Reporter cells / well with 5 × 10 4 Stimulated cells / well were mixed in 96-well U-shaped plates. After incubation at 37°C for 24 hours, the cell pellet was stained with a viability dye (Zombie Aqua, Biolegend catalog 423101) and mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend catalog 109830) for flow cytometry to separate stimulated (mouse) cells from responder (human) cells. The geometric mean of GFP expression at each antibody concentration was assessed and normalized to GFP expression in the absence of the antibody.

[0553] Humanized 2.8.6 was tested on hIgG4 isotypes, as well as hIgG1 P238D and hIgG1V9 (P238D G237D P271G A330R) isotypes. 2.8.6 hIgG1 P238D resulted in more effective inhibition of NF-κB signaling than 2.8.6 hIgG4, and 2.8.6 hIgG1V9 still resulted in more effective inhibition (…). Figure 15 a). Therefore, under the condition that FcγR2B is the only present Fc receptor, increasing affinity for FcγR2B confers excellent agonistic activity to BTLA agonist antibodies. When the same antibody was tested in a modified version of an assay in which cells do not express FcγR2B, no inhibitory effect of the antibody was observed, confirming that the antibody agonistic activity of BTLA depends on antibody FcR binding (a). Figure 15b). In reporter gene assays, mouse IgG1 parental antibodies 2.8.6, 6.2, and 3E8 were also able to inhibit T cell activation when human FcγR2B was expressed on stimulated cells, which fits the cross-reactivity between mIgG1 and hFcγR2B observed in Example 23.

[0554] Humanized variants 2.8.6, 6.2_var_C, and 3E8_var_B were all generated on the hIgG1 P238D isotype and compared in the aforementioned T-cell reporter gene assays. They were also compared to prior art BTLA agonist 22B3 (expressed on the hIgG4 PAA isotype) as described in WO2018 / 213113 and a fusion protein of the native BTLA ligand HVEM fused to the mIgG1 Fc region (hHVEM-mFc, internally recombinant; the hHVEM-mFc fusion protein containing a signal peptide and a C-terminal His-tag has the sequence disclosed in SEQ ID NO:229). All three humanized P238D variant antibodies demonstrated significantly greater inhibition of NFkB signaling compared to 22B3 or hHVEM-mFc. Figure 16 a) 3E8_var_B inhibited NFkB signaling by 54%, with an IC50 of 65 pM. 6.2_var_C inhibited NFkB signaling by 47%, with an IC50 of 28 pM. 2.8.6 inhibited it by 42%, with an IC50 of 59 pM. 22B3 inhibited NFkB signaling by 18%, with an IC50 of 3.8 nM. hHVEM-mFc inhibited NFkB signaling by 27%, with an IC50 of 9.6 nM. Therefore, in the case where FcγR2B is the only Fc receptor present, humanized 2.8.6hIgG1P238D, 6.2_var_C hIgG1P238, and 3E8hIgG1P238D are all significantly more effective and potent BTLA agonists than the existing antibody 22B3hIgG4PAA, and deliver a stronger signal than the endogenous ligand HVEM, which is the Fc fusion protein.

[0555] Example 25: Inhibition of primary human T cell proliferation by humanized BTLA agonist in mixed lymphocyte response

[0556] The ability of selected BTLA agonist antibodies to inhibit human T cell proliferation was evaluated in the context of a mixed lymphocyte reaction (MLR). Briefly, primary human T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donors using the Human Pan T Cell Isolation Kit (Miltenyi Biotec catalog 130-096-535) and stained with the cell proliferation tracing dye Tag-itViolet (Biolegend catalog 425101). Allogeneic monocyte-derived dendritic cells (DCs) were generated by culturing CD14+ monocytes isolated from PBMCs using the CD14+ Isolation Kit (Miltenyi Biotec catalog 130-050-201). CD14+ monocytes were treated for 7 days with recombinant human IL-4 (Peprotech catalog 200-04) and GM-CSF (Biolegend catalog 572904). DC maturation was then induced by adding recombinant human TNF-α (Biolegend catalog number 717904) for an additional 2 days. Mature dendritic cells expressed both activated and inhibited FcγR (Guilliams et al., Nature Reviews Immunology, Vol. 14, No. 2: pp. 94-108, 2014, http: / / www.nature.com / articles / nri3582 (May 18, 2020)).

[0557] Then, 1×10⁶ cells were co-cultured in a flat-bottomed 96-well plate at a ratio of 4:1 (T:DC). 5Total T cells were used for MLR with allogeneic mature dendritic cells (DCs). T cells and DCs were incubated for 5 days in the absence of antibodies or in the presence of different doses of BTLA agonist antibodies (2.8.6hIgG1P238D, 2.8.6hIgG1V9, 2.8.6IgG4), hIgG1k isotype control antibody (SinoBiologicals catalog number HG1K), or the existing BTLA agonist 22B3hIgG4PAA. After 5 days, T cell proliferation was evaluated by flow cytometry. T cells were harvested and stained with anti-CD3 antibody (PerCP / Cy5.5 conjugated clone OKT3, Biolegend catalog number 317336), anti-CD4 antibody (BB515 conjugated clone RPA-T4, BD Horizon catalog number 564419), and anti-CD8 antibody (BV510 conjugated clone SK1, BD Horizon catalog number 563919) along with viability dye (Zombie NIR, Biolegend catalog number 423105) and collected on a BD FACSCelesta instrument. CD4 proliferation in the presence of antibodies (measured as the percentage of cells with low CTV) was normalized to the mean proliferation in the absence of antibodies. Figure 17 Data from six individual MLRs combined with different PBMC donors are presented. Antibody 2.8.6 on the hIgG1 P238D isotype significantly inhibited CD4 T cell proliferation, with a mean inhibition of 51% at 10 μg / mL. Antibody 2.8.6 on either the hIgG1 V9 or hIgG4 isotypes showed no inhibitory effect. Therefore, unexpectedly, in the presence of multiple Fc receptors, the hIgG1 P238D isotype, selectively binding to FcγR2B, conferred superior agonistic activity to the BTLA agonist compared to the other isotypes tested. The hIgG1 V9 isotype showed approximately 30-fold higher affinity for FcγR2B than the P238D isotype; its ineffectiveness in this case is likely due to activation signaling via FcγR2A (131R), which also retained significant binding. Alternatively, the ineffectiveness of the hIgG1V9 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 expressing both receptors), which may not induce signal transduction but will 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 if these cis interactions form, they are short-lived and do not completely block trans interactions.

[0558] 22B3hIgG4PAA showed no inhibitory effect in mixed lymphocyte responses and in fact tended to increase CD4T cell proliferation. This can be explained by the antibody blocking the natural inhibitory signaling through BTLA by interfering with its interaction with the ligand HVEM. Antibodies 6.2, 3E8, and 286 bind to epitopes on BTLA that do not overlap with the HVEM binding interface, therefore these antibodies do not block the BTLA-HVEM interaction (Example 19).

[0559] Example 26: Inhibition of primary human B cell activation by BTLA agonists

[0560] To evaluate the ability of BTLA agonist antibodies to inhibit the activation of primary human B cells. B cells expressed high levels of BTLA and FcγR2B.

[0561] Human primary B cells were isolated from peripheral blood mononuclear cells from healthy donors using a human B cell isolation kit (Miltenyi Biotec catalog number 130-050-301) and isolated using the cell proliferation tracing dye Tag-it Violet. TM (Biolegend catalog number 425101) staining.

[0562] Then, with or without different doses of isotype control antibody or selected BTLA agonist antibody, 1×10⁶ cells in a 96-well plate were stimulated with 0.01 μM of the TLR9 agonist ODN2006 (InvivoGen catalog number tlrl-2006-1). 5 B cells / well. BTLA agonists 2.8.6, 6.2_var_C, and 3E8_var_B (all hIgG1P238D isotypes) were tested and compared with the prior art BTLA agonist 22B3hIgG4PAA. Recombinant HVEM fusion protein (hHHEM-mFc, internally produced) was used as a positive control. After incubation at 37°C for 5 days, B cells were harvested and their proliferation was evaluated by flow cytometry using anti-CD20 antibody (PE-CF594 conjugated clone 2H7, BD Horizon catalog number 562295) and viability dye (Zombie NIR, Biolegend catalog number 423105). In addition, culture supernatant was collected to assess the production of IL-6 (rndsystems catalog number DY206) and IL-10 (rndsystems catalog number DY217B) by ELISA.

[0563] Following essentially the procedure described above, the BTLA agonist antibody was able to inhibit B cell proliferation as effectively as the hHVEM-mFc positive control. Furthermore, all three antibody variants showed more significant inhibition of B cell proliferation compared to 22B3. Additionally, the P238D BTLA agonist attenuated IL-10 production by activated B cells (…). Figure 18 ) and IL-6. Consistent with the proliferation data, the P238D BTLA antibody showed a greater ability to inhibit the production of IL-10 and IL-6 compared to the 22B3 antibody.

[0564] Example 27: Treatment of xenogeneic models of graft-versus-host disease (GVHD)

[0565] In vivo prevention of graft-versus-host disease (GvHD) driven by human PBMCs was established.

[0566] In short, female NSG mice (JAX Labs, breed number 05557), approximately 8-10 weeks old (n=10 mice per treatment group), received 2.4 Gy of whole-body radiation. Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopak (a HemaCare product ordered via Tissue Solutions) and analyzed at 50 × 10⁻⁶ cells / mL. 6 Resuspend the cells in PBS at 100 cells / mL. One day after irradiation, mice were intravenously (IV) injected with 200 μL of the cell suspension (10 × 10⁶ cells / mL). 6 (1 PBMC). The next day, mice were treated with 10 mg / kg of the test antibody via intraperitoneal injection. Mice were weighed periodically and euthanized when their body weight decreased by 15% or after 28 days. At the end of the study, the infiltration of human PBMCs into the lungs, liver, and spleen was quantified by flow cytometry using markers of hCD45, hCD4, hCD8, hCD20, hCD25, and FOXP3.

[0567] Following the procedure described above, compared with the hIgG1P238D isotype control, humanized 2.8.6 hIgG1P238D, 6.2_var_C hIgG1P238D, and 3E8_var_B hIgG1P238D all significantly reduced weight loss. Figure 19 This leads to a significant decrease in infiltrating human immune cells in the lungs, liver, and spleen. A trend toward increased frequency of regulatory T cells was also observed with all three BTLA agonists.

[0568] Example 28: Prediction of the in vivo half-life and human half-life of the p238D-mutated hIgG1 antibody in cynomolgus monkeys

[0569] The in vivo half-life of 6.2_var_C against the hIgG1P238D isotype in cynomolgus monkeys was evaluated. Two female cynomolgus monkeys were intravenously injected with 3 mg / kg of antibody, and two female cynomolgus monkeys were injected with 10 mg / kg of antibody. Blood samples were collected from the cynomolgus monkeys before antibody injection and at 1 hour, 6 hours, 24 hours, 48 ​​hours, 72 hours, 168 hours, 240 hours, 336 hours, 432 hours, and 504 hours after antibody injection. The serum concentration of 6.2_var_C at each time point was assessed by target capture ELISA. 96-well microplates (Thermoscientific catalog number 439454) were coated overnight at 4°C with 100 μL of human BTLA extracellular domain in 1 μg / mL PBS. The plate was then washed three times with wash buffer (PBS containing 0.05% Tween 20 (ThermoScientific catalog number 28320)) and the wells were blocked with 300 μL SuperBlock buffer (ThermoScientific catalog number 37515) at room temperature for 1 hour, followed by three more washes with wash buffer. Then, 100 μL of serum sample diluted in ELISA buffer (PBS, 1% bovine serum albumin, 0.05% Tween 20) was added, and the plate was incubated at room temperature for 1 hour. An 11-point standard curve at a known concentration of 6.2 var. C in ELISA buffer was performed in duplicate, with duplicate wells containing only ELISA buffer used as blanks. After incubation, the wells were washed three times with wash buffer, and then HRP-conjugated anti-human detection antibody (Abcam catalog number ab98624) diluted 1:20,000 in ELISA buffer was added, and the plate was incubated at room temperature for 1 hour. The wells were washed three times again with washing buffer, and then 100 μL of Ultra TMB-ELISA substrate solution (ThermoScientific catalo...

Claims

1. A separating antibody that specifically binds to B and T lymphocyte attenuators (BTLA), wherein the antibody comprises a heavy chain and a light chain, wherein (a) The heavy chain includes a heavy chain variable region, which comprises three complementarity-determining regions (CDRs): CDRH1, CDRH2, and CDRH3, wherein the amino acid sequences of CDRH1, CDRH2, and CDRH3 are as shown in SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 3, respectively. (b) The light chain includes a light chain variable region, which contains three CDRs: CDRL1, CDRL2 and CDRL3, wherein the amino acid sequences of CDRL1, CDRL2 and CDRL3 are as shown in SEQ ID NO: 4, 12 and 6, respectively.

2. The isolation antibody according to claim 1, wherein (i) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 18; and (ii) the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:

14.

3. The isolation antibody according to claim 1, wherein (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19; and (ii) the light chain comprises the amino acid sequence shown in SEQ ID NO:

16.

4. The isolation antibody according to any one of claims 1-3, wherein the heavy chain comprises an Fc region, the 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, numbered according to the EU index.

5. The separating antibody according to claim 4, wherein the heavy chain comprises an Fc region, the Fc region comprising an aspartic acid residue at position 238, numbered according to the EU index.

6. The isolation antibody according to claim 5, wherein... (i) The Fc region binds to FcγR2B with higher affinity than a comparable control antibody, the comparable control antibody comprising an Fc region having a proline residue at position 238, numbered according to the EU index; or (ii) The antibody binds to FcγR2B with an affinity of 5 µM to 0.1 µM, as determined by surface plasmon resonance (SPR); or (iii) The Fc region binds to the FcγR2A 131R allotype with a lower or equal affinity to a comparable control antibody, wherein the comparable control antibody contains an Fc region comprising a proline residue at position 238, numbered according to the EU index; or (iv) The antibody binds to the FcγR2A 131R isoform at a concentration of at least 20 µM KD, as determined by surface plasmon resonance (SPR); or (v) The antibody binds to the FcγR2A 131H allotype with a lower or equal affinity relative to a comparable control antibody, wherein the comparable control antibody comprises an Fc region containing a proline residue at position 238, numbered according to the EU index; or (vi) The antibody binds to the FcγR2A 131H isoform with at least 50 µM KD, as determined by surface plasmon resonance (SPR); or (vii) The antibody exhibits an in vivo half-life of at least 10 days.

7. The isolated antibody according to any one of claims 1-3 or 5-6, wherein: (i) is an IgG1, IgG2, or IgG4 antibody; and / or (ii) is selected from the group consisting of human antibodies, humanized antibodies, and chimeric antibodies; and / or (iii) is a monoclonal antibody.

8. A pharmaceutical composition comprising an isolating antibody according to any one of claims 1 to 7 and at least one pharmaceutically acceptable excipient.

9. A nucleic acid encoding an antibody isolated according to any one of claims 1 to 7.

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