B and T lymphocyte attenuator (BTLA) regulators and methods of use thereof

Abstract

BTLA binding agents, and immunoglobulin heavy and light chain polypeptides thereof, and methods of using the BTLA binding agents to treat disorders or diseases responsive to BTLA agonism, such as autoimmune or inflammatory diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 335,623, filed April 27, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Documents The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is hereby incorporated by reference in its entirety: a 339,641 byte XML file entitled "767170.XML", dated April 12, 2023. [Background technology]

[0003] T lymphocytes are activated from a naive state by a combination of T cell receptor (TCR) engagement and positive signals from costimulatory molecules. Conversely, co-inhibitory molecules play an important role in regulating T cell activity by sending negative signals to counteract positive costimulatory signals. Co-inhibitory molecules act as checkpoints to maintain immune tolerance to self and to control activated T cells after resolving immune insults such as infections and inflammation.

[0004] CD28 is the major costimulatory molecule in T cells, and upon engagement with its ligands B7.1 or B7.2 on the surface of antigen-presenting cells (APCs), it sends intracellular signals that promote T cell proliferation and differentiation upon TCR engagement. When the same ligand engages with the co-inhibitory molecule cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells, T cell proliferation and effector function are inhibited (Chambers et al., Ann. Rev. Immunol., 19:565-594, 2001; Egen et al., Nature Immunol., 3:611-618, 2002). Similarly, when cells expressing PD-L1 engage with the co-inhibitory molecule PD-1 on T cells, T cell proliferation and effector function are inhibited (Carter et al. Eur J Immunol., 32:634-43, 2002).

[0005] Dysfunction of negative checkpoint signals may contribute to chronic inflammatory states by interfering with inhibitory signals that normally control self-reactive B and T lymphocytes. In patients with autoimmune disorders, the immune system reacts to normal body tissues as if they were foreign, allowing activated T and B cells with broken tolerance to self-antigens to infiltrate the tissues. As a result, autoimmune T and B lymphocytes mediate inflammation and tissue damage. Lymphocytes expressing co-inhibitory molecules such as B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen-4 (CTLA-4), and PD-1 are normally suppressed by other immune or non-immune cells in the tissues that express the corresponding ligands.

[0006] In various animal models of cancer, knockout of co-inhibitory molecules or pharmacological blockade of co-inhibitory interactions has been shown to release inhibitory disruption and induce proliferation of tumor-specific T cell populations that are directed to attack and kill tumor cells. BTLA knockout mice have increased susceptibility to experimental autoimmune encephalomyelitis (Watanabe et al., Nat. Immunol., 4:670-679, 2003). Conversely, agonistic antibodies targeting mouse BTLA have been shown to suppress T cell activity and have demonstrated efficacy in a mouse model of graft-versus-host disease (GvHD) (Albring et al., J. Exp. Med., 207:2551-2559, 2010).

[0007] The ligand for BTLA is herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14). HVEM is also a positive costimulatory molecule that binds to two secreted growth factors, lymphotoxin alpha and LIGHT, and is also used by herpes simplex virus (HSV) to enter cells. When HVEM expressed on tumor cells or other immune cells engages with BTLA on T cells, a negative inhibitory signal is generated. Anti-BTLA antibodies that bind and agonize BTLA can induce a direct negative signal similar to that sent by the native ligand, HVEM, thereby suppressing autoreactive T and B cell responses in autoimmune and inflammatory diseases. Furthermore, agonistic antibodies that engage BTLA without interfering with the natural HVEM-BTLA interaction can enhance the natural co-inhibitory signal.

[0008] Thus, there is a need for agents that can bind to BTLA and modulate immune checkpoint signaling. Summary of the Invention

[0009] Provided herein are BTLA-binding agents comprising immunoglobulin heavy and light chain polypeptides for use in methods of modulating BTLA signaling, suppressing immune responses, or treating autoimmune or inflammatory diseases.

[0010] As will become apparent from the following detailed description, related compositions and methods are also provided. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the surface plasmon resonance binding kinetics results of the 6G3 antibody to the extracellular domains of human BTLA and cynomolgus BTLA. [Diagram 2] FIG. 2 is a graph depicting the binding kinetics results of an equilibrium exclusion assay of the 6G3 antibody to the extracellular domains of human BTLA and cynomolgus BTLA. [Diagram 3] FIG. 3 is a graph showing the results of 6G3 antibody binding to 293c18 cells stably transfected with human BTLA and cynomolgus BTLA. [Figure 4] FIG. 4 is a graph showing the results of 6G3 antibody binding to normal donor human peripheral blood CD4+ T cells, CD8+ T cells, and CD20+ B cells. [Figure 5A] FIG. 5A is a graph showing the results of 6G3 antibody binding to normal cynomolgus monkey peripheral blood CD3+ T cells. [Figure 5B] FIG. 5B is a graph showing the results of 6G3 antibody binding to normal cynomolgus monkey peripheral blood CD20+ B cells. [Figure 5C] FIG. 5C is a flow cytometry dot plot showing the results of binding of a reference anti-BTLA antibody to normal cynomolgus monkey peripheral blood CD3+ and CD3− cells. [Figure 5D] FIG. 5D is a flow cytometry dot plot showing the results of 6G3 antibody binding to normal cynomolgus monkey peripheral blood CD3+ and CD3- cells. [Figure 6]Figure 6 is a graph depicting the results of a competition assay, showing the ability of anti-BTLA antibodies to compete with HVEM-Fc and preformed HVEM-Fc / trimeric LIGHT complexes for binding to 293c18 cells stably transfected with human BTLA. [Figure 7A] Figure 7A shows a ribbon model representation (black) of the crystal structure of the human BTLA extracellular domain docked into a space-filling model (grey) of the crystal structure of the human HVEM extracellular binding domain. The model shows the results of a hydrogen-deuterium exchange experiment, which maps the peptide on human BTLA that is bound by the 6G3 antibody. [Figure 7B] Figure 7B is a ribbon model representation of the crystal structure of the human BTLA extracellular domain (black) docked into a space-filling model of the crystal structure of the human HVEM extracellular binding domain (grey). The molecule has been rotated 30° compared to the representation of the molecule shown in Figure 7A and shows the results of a hydrogen-deuterium exchange experiment that maps a peptide on human BTLA to which a reference anti-BTLA antagonist antibody binds. [Figure 8] FIG. 8 is a graph showing the inhibitory activity of 6G3 antibody in an HVEM-NF-κB HEK 293 luciferase reporter assay measuring LIGHT-induced HVEM signaling when BTLA and HVEM were expressed in the same cells. [Figure 9A] Figure 9A is a graph showing the results of a fluorescence resonance energy transfer assay measuring the association of BTLA and HVEM on the surface of transfected 293c18 cells, demonstrating the ability of anti-BTLA antibodies to compete with the binding of BTLA and HVEM on the same cell surface. [Figure 9B] Figure 9B is a graph showing the results of a fluorescence resonance energy transfer assay measuring the association of BTLA and HVEM on the surface of transfected 293c18 cells, demonstrating the ability of the fluorescent donor anti-BTLA antibody to generate an energy transfer signal together with the anti-HVEM acceptor antibody. [Figure 10]FIG. 10 is a graph showing the partial inhibitory activity of 6G3 antibody in an HVEM-NF-κB HEK 293 luciferase reporter assay measuring BTLA-induced HVEM signaling when BTLA and HVEM are expressed in different cells. [Figure 11] FIG. 11 is a graph showing the agonist activity of 6G3 antibody added as a soluble antibody in a SHP2-recruiting PathHunter Jurkat BTLA signaling assay. [Figure 12] FIG. 12 is a graph showing the inhibitory activity of anti-BTLA antibodies in a SHP2-recruited PathHunter Jurkat BTLA signaling assay, in which BTLA signaling was induced by HVEM in transfected U-2 OS cell lines. [Figure 13] FIG. 13 is a graph depicting agonist activity of 6G3 antibody in a SHP2-mobilized PathHunter Jurkat BTLA signaling assay supplemented with U-2 OS cells transfected with FcγRIa to provide FcγR engagement. [Figure 14A] FIG. 14A is a schematic diagram of a xenogeneic NSG / Hu-PBMC mouse model for graft-versus-host disease testing described herein according to an embodiment of the present invention. [Figure 14B] FIG. 14B is a schematic diagram showing the timeline, dosing schedule, and model groups of the NSG / Hu-PBMC graft-versus-host disease study described herein according to an embodiment of the present invention. [Figure 14C] FIG. 14C is a graph showing overall survival results in an NSG / Hu-PBMC graft-versus-host disease study for groups of animals administered either 1 mg / kg, 3 mg / kg, or 10 mg / kg of 6G3 antibody twice weekly. [Figure 15] Figure 15 is a plot showing individual and mean (SD) concentrations of human sBTLA by dose group in a humanized mouse model of GvHD following twice-weekly dosing with 6G3 antibody at 1 mg / kg, 3 mg / kg, and 10 mg / kg (IP). Plasma samples were collected by cardiac bleed at the midpoint of the study. [Figure 16] Figure 16 is a plot showing the mean (SD) serum concentration of cynomolgus sBTLA by dose group in cynomolgus monkeys following dosing with 6G3 IgG4. All animals received a single dose of 6G3 IgG4 either IV or SC and blood samples were collected from all animals in all groups pre-dose and at 3, 6, 12, 24, 48, 72, 96, 168, 240, 336, 504, 672, and 840 hours post-dose. [Figure 17] Figure 17 is a plot showing the mean (SD) serum concentrations of cynomolgus sBTLA by dose group in cynomolgus monkeys after dosing with 6G3 IgG4. All animals received a weekly dose of 6G3 IgG4 either IV or SC on days 1, 8, and 15, and blood samples were collected from all animals in all groups on days 1, 8, and 15: pre-dose, 3, 24, 48, 72, and 96 hours after dosing. [Figure 18] Figure 18 is a plot showing the mean (SD) serum concentration (μg / mL) of 6G3 IgG4 in cynomolgus monkeys by dose group after dosing with 6G3 IgG4. All animals received a weekly dose of 6G3 IgG4 either IV or SC on days 1, 8, and 15, and blood samples were collected from all animals in all groups on days 1, 8, and 15: pre-dose, 3, 24, 48, 72, and 96 hours after dosing. [Figure 19] FIG. 19 is four plots showing various data per treatment group in cynomolgus monkeys after treatment with 6G3 IgG4, isotype control, or CTLA-4-Ig control. The first plot shows BTLA expression (MFI) per treatment group. The second plot shows the percentage of BTLA+ T cells per treatment group. The third plot shows the number of human T cells per μL of blood per treatment group. The fourth plot shows the percentage of CD25 positivity per treatment group. [Figure 20]FIG. 20 is four plots showing receptor occupancy and BTLA surface expression on T and B cells by treatment group in cynomolgus monkeys after treatment with 6G3 IgG4. All animals received a weekly dose of 6G3 IgG4 or control either IV or SC on days 1, 8, and 15. The first plot shows the percent change from baseline in free receptors on T cells. The second plot shows the percent change from baseline in BTLA expression on T cells. The third plot shows the percent change from baseline in free receptors on B cells. The fourth plot shows the percent change from baseline in BTLA expression on B cells. [Figure 21A] FIG. 21A is two histograms of a healthy control and an atopic dermatitis donor presented as histograms of 6G3 IgG4 treated CD3+ T cells with overlaid isotype control. [Figure 21B] FIG. 21B is two plots showing the reduction of T cell proliferation by 6G3 IgG4 in healthy controls and atopic dermatitis donors, shown as the percentage reduction in proliferation (left) and mitotic index (right). [Figure 21C] FIG. 21C is two plots showing IFNγ levels in PBMC culture supernatants from healthy controls and atopic dermatitis donors 72 hours after anti-CD3 and anti-CD28 stimulation in the presence or absence of 100 nM 6G3 IgG4 or isotype control. [Figure 21D] FIG. 21D is a plot showing surface BTLA expression levels (plotted as mean fluorescent intensity (MFI)) on CD3+ T cells from healthy controls and atopic dermatitis donors. [Figure 22A] Figure 22A is a plot of receptor occupancy in CD3+ cells following a 400mg SC dose. The data show that a 400mg SC dose results in a half-life of approximately 9 days, with sufficient pharmacokinetic exposure (~7ug / mL) to fully occupy the receptors for over 30 days. [Figure 22B]Figure 22B is a plot of receptor occupancy on CD19+ cells following a 400mg SC dose. The data show that a 400mg SC dose results in a half-life of approximately 9 days, with sufficient pharmacokinetic exposure (~7ug / mL) to fully occupy the receptors for over 30 days. [Figure 23A] FIG. 23A is a plot of mouse survival versus study day following a single dose of 10 mg / kg of 6G3 antibody, following 3 mg / kg of 6G3 antibody administered every other week for 4 weeks, following a single dose of 10 mg / kg of IgG4 control antibody, following 3 mg / kg of IgG4 control antibody administered every other week for 4 weeks, or following untreated animals in a GvHD model. [Figure 23B] FIG. 23B is a plot of mouse body weight (% from baseline) versus study day following a single dose of 10 mg / kg of 6G3 antibody, a single dose of 10 mg / kg of IgG4 control antibody, a single dose of 3 mg / kg of IgG4 control antibody every other week for 4 weeks, or untreated animals in a GvHD model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Provided herein are BTLA-binding agents that include immunoglobulin heavy and light chain polypeptides. BTLA is a 30 kilodalton (kD) type 1 transmembrane protein with an immunoglobulin-like extracellular domain, an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine-based switch motif (ITSM). BTLA is expressed in B cells and T cells and acts as a negative regulator of both B cell and T cell activity through interaction with its receptor, herpes virus entry mediator (HVEM), expressed in tumor cells or APCs (Watanabe et al., Nat. Immunol., 4:670-679, 2003). In some embodiments, the BTLA-binding agent binds to BTLA without inhibiting the binding between BTLA and HVEM. In one aspect, the BTLA-binding agent enhances the binding between BTLA and HVEM.

[0013] The PD-1-binding agents comprise an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, each of which contains three complementarity determining regions (CDRs), usually referred to as CDR1, CDR2, or CDR3. The CDR regions can also be referred to in nomenclature using an "H" or "L" to refer to the heavy or light chain, respectively, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. The CDRs of a given Ig sequence can be determined by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo (these are commonly used names for numbering schemes that are well known in the art and are described in published literature, e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH (1991), which describes the "Kabat" numbering scheme; Chothia, et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901-917 (1987), which describes the "Chothia" numbering scheme; and Al-Lazikani et al., Standard Conformations for the Canonical Structures of Immunoglobulins, J. Mol. Biol., 273:927-948 (1997); Abhinandan et al., Analysis and Improvements to Kabat and Structurally Correct Numbering of Antibody Variable Domains, Mol. Immunol., 45:3832-3839 (2008), describing the "Martin" or "Enhanced Chothia" numbering scheme; Lefranc et al., describing the "IMGT" numbering scheme., The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domains, The Immunologist, 7:132-136 (1999); and Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and I superfamily V-like domains, Dev. Comp. Immunol., 27:55-77 (2003); and Honegger et al., Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol. 309:657-670 (2001), which describes the "AHo" numbering scheme. The BTLA-binding agents provided herein are man-made and do not occur in nature. They have been produced by laboratory techniques and are therefore properly considered to be recombinant or synthetic molecules that contain recombinant or synthetic amino acid sequences. Immunoglobulin heavy and light chain polypeptides may be "isolated" in the sense that they are removed from the environment in which they are produced (e.g., cell culture) and purified to any degree.

[0014] According to one embodiment of the disclosure, the BTLA-binding agent comprises an immunoglobulin heavy chain polypeptide of a BTLA-binding agent that comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218, or at least the CDRs thereof; or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218. The CDRs may be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, CDR1, CDR2, and CDR3 comprise residues 31-35 (CDRH1), 50-66 (CDRH2), and 99-106 (CDRH3) of SEQ ID NOs: 1-15, 207, 208, 217, or 218.

[0015] In some embodiments the immunoglobulin heavy chain has the following CDRs: (a) CDRH1 containing Asp Tyr Thr Ile His (SEQ ID NO: 27); (b) a CDRH2 (e.g., SEQ ID NO: 28, 30, 31, 212, or 222) that includes Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Asp Xaa1 Phe Lys Xaa2 (SEQ ID NO: 30), where Xaa1 is lysine (Lys) or glutamic acid (Glu) and Xaa2 is aspartic acid (Asp) or valine (Val); and (c) CDRH3 comprising Arg Xaa1 Xaa2 Tyr Xaa3 Met Xaa4 Tyr (SEQ ID NO: 32), where Xaa1 is asparagine (Asn) or serine (Ser), Xaa2 is tyrosine (Tyr) or histidine (His), Xaa3 is alanine (Ala) or valine (Val), and Xaa4 is glutamic acid (Glu) or aspartic acid (Asp). Examples of such CDRH3 sequences include, for example, SEQ ID NOs: 29, 33, 34, 213, or 223.

[0016] In some embodiments, the immunoglobulin heavy chain polypeptide has the amino acid sequence Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Xaa1 Thr Xaa2 Thr Asp Tyr Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Asp Xaa3 Phe Lys Xaa4 Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Xaa7 Xaa8 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa9 Cys Ala Arg Arg Xaa10 Xaa11 Tyr Xaa12 Met Xaa13 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala (SEQ ID NO: 26) or at least the CDR regions thereof, Xaa1 is phenylalanine (Phe) or tyrosine (Tyr); Xaa2 is phenylalanine (Phe) or leucine (Leu); Xaa3 is lysine (Lys) or glutamic acid (Glu); Xaa4 is aspartic acid (Asp) or valine (Val); Xaa5 is alanine (Ala) or arginine (Arg); Xaa6 is lysine (Lys) or threonine (Thr); Xaa7 is alanine (Ala) or serine (Ser); Xaa8 is serine (Ser) or threonine (Thr); Xaa9 is tyrosine (Tyr) or phenylalanine (Phe); Xaa10 is asparagine (Asn) or serine (Ser); Xaa11 is tyrosine (Tyr) or histidine (His); Xaa12 is alanine (Ala) or valine (Val); Xaa13 is glutamic acid (Glu) or aspartic acid (Asp). In some embodiments, the Ig heavy chain polypeptide comprises SEQ ID NO:26, but retains the same CDRs (CDR1, CDR2, and CDR3) of any of SEQ ID NOs:1-15, 207, 208, 217, or 218.

[0017] According to this aspect of the disclosure, the immunoglobulin light chain polypeptide of the BTLA binding agent can comprise the amino acid sequence of any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220, or at least the CDRs thereof; or an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220. Similar to Ig heavy chains, the CDRs may be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, CDR1, CDR2, and CDR3 comprise residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) of SEQ ID NOs: 16-25, 209, 210, 219, or 220.

[0018] In some embodiments, the Ig light chain has the following CDRs: (a) CDRL1 containing Lys Ala Ser Gln Asn Val Phe Thr Asn Val Ala (SEQ ID NO: 36); (b) a CDRL2 comprising Ser Ala Ser Tyr Arg Xaa Ser (SEQ ID NO: 39), where Xaa is tyrosine (Tyr) or serine (Ser) (e.g., SEQ ID NO: 37, 40, 215, or 225); and (c) a CDRL3 (e.g., SEQ ID NOs: 38, 41, 42, 216, or 226) that contains Gln Gln Tyr Xaa1 Xaa2 Tyr Pro Tyr Thr (SEQ ID NO: 41), where Xaa1 is serine (Ser) or asparagine (Asn) and Xaa2 is threonine (Thr) or serine (Ser). Includes.

[0019] In other embodiments, the immunoglobulin light chain polypeptide comprises the amino acid sequence Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asn Val Phe Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Xaa1 Pro Lys Xaa2 Leu Ile Tyr Ser Ala Ser Tyr Arg Xaa3 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Xaa4 Cys Gln Gln Tyr Xaa5 Xaa6 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg (SEQ ID NO: 35), or at least the CDR regions thereof, Xaa1 is serine (Ser) or proline (Pro); Xaa2 is proline (Leu) or leucine (Leu); Xaa3 is tyrosine (Tyr) or serine (Ser); Xaa4 is tyrosine (Tyr) or phenylalanine (Phe); Xaa5 is serine (Ser) or asparagine (Asn); Xaa6 is threonine (Thr) or serine (Ser). In some embodiments, the Ig light chain polypeptide comprises SEQ ID NO:35, but retains the same CDRs (CDR1, CDR2, and CDR3) of any one of SEQ ID NOs:16-25, 209, 210, 219, or 220.

[0020] In another embodiment of the disclosure, the BTLA binding agent comprises an immunoglobulin heavy chain polypeptide comprising any one of SEQ ID NOs: 43-156 or at least the CDRs thereof; or an amino acid sequence having at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOs: 43-156. The CDRs may be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, CDR1, CDR2, and CDR3 comprise residues 31-35 (CDRH1), 50-66 (CDRH2), and 99-113 (CDRH3) of SEQ ID NOs: 43-156, except that CDRH1 can be residues 50-67 and CDRH3 can be residues 100-114 in SEQ ID NO: 66, and CDRH3 can be residues 100-114 in SEQ ID NOs: 141, 150, 152, 153, 155, and 156.

[0021] In some embodiments, the immunoglobulin heavy chain polypeptide has the following CDRs: (a)X 1 SX 2 MN (SEQ ID NO: 195) (wherein 1 is N or T, and X 2 is W, F, H, G, P, R, K, D, S, L, V, N, or Y; (b)RIYPX 1 GX 2 X 3 DTNYX 4 GKFK (SEQ ID NO: 196), X 1 is absent or is A; X 2is D, Y, Q, G, L, F, H, S, P, R, or T; X 3 is G, Y, A, F, S, D, V, T, E, K, or R; X 4 is N, V, Q, R, A, F, Y, S, G, P, or T) CDRH2 containing; (c)X 1 SGTFX 2 X 3 GNYX 4 X 5 YFDV (SEQ ID NO: 197), X 1 is K or R; X 2 is N or D; X 3 is D, S, F, Y, F, V, S, G, T, R, I, L, or E; X 4 is R or H; X 5 is W, R, F, L, N, Y, P, I, V, A, S, G, R, or K) CDRH3 containing Includes.

[0022] In some embodiments, the Ig heavy chain comprises a CDRH1 comprising SEQ ID NO:201, a CDRH2 comprising SEQ ID NO:202, and a CDRH3 comprising SEQ ID NO:203.

[0023] In some embodiments, the BTLA-binding agent has the sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYX 1 FSX 2 SX 3 MNWVRQAPGQGLEWMGRIYPX 4 GX 5 X 6 DTNYX 7 GKFKGRVTITADKX 8 TX 9 TAYMELX 10 SLRSEX 11 TAVX 12 YX 13 CAX14 SGTFX 15 X 16 GNYX 17 X 18 YFDVWGKGTTVTVSSA (SEQ ID NO: 193), or at least the CDR regions thereof, X 1 is A or V; X 2 is N or T; X 3 is W, F, H, G, P, R, K, D, S, L, V, N, or Y; X 4 is absent or is A; X 5 is D, Y, Q, G, L, F, H, S, P, R, or T; X 6 is G, Y, A, F, S, D, V, T, E, K, or R; X 7 is N, V, Q, R, A, F, Y, S, G, P, or T; X 8 is S or F; X 9 is S, T, or N; X 10 is S or R; X 11 is D or V; X 12 is absent or Y; X 13 is Y or F; X 14 is K or R; X 15 is N or D; X 16 is D, S, F, Y, F, V, S, G, T, R, I, L, or E; X 17 is R or H; X 18is W, R, F, L, N, Y, P, I, V, A, S, G, R, or K. In some embodiments, an Ig heavy chain polypeptide comprises SEQ ID NO: 193, but retains the same CDRs (CDR1, CDR2, and CDR3) of any of SEQ ID NOs: 43-156.

[0024] According to this aspect of the disclosure, the binding agent further comprises an Ig light chain comprising any of SEQ ID NOs: 157-192 or at least the CDRs thereof; or an amino acid sequence having at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOs: 157-192. The CDRs may be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, CDR1, CDR2, and CDR3 comprise residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) of SEQ ID NOs: 157-192.

[0025] In some embodiments, the BTLA binding agent is (a)RX 1 SENIYX 2 X 3 LA (SEQ ID NO: 198), X 1 is A or V; X 2 is S or N; X 3 is H, N, or Y) CDRL1 containing; (b)X 1 AX 2 NLAX 3(SEQ ID NO: 199) (wherein X 1 is A or N; X 2 is T or K; X 3 is N, L, Q, G, F, V, K, S, R, T, H, or P) CDRL2 containing; (c)QX 1 FX 2 GPPLT (SEQ ID NO: 200), X 1 is L or H; X 2 is W, F, Y, P, N, V, K, M, L, G, or S) CDRL3 containing The immunoglobulin light chain polypeptide may comprise an immunoglobulin light chain polypeptide comprising:

[0026] In some embodiments, the Ig light comprises a CDRL1 comprising SEQ ID NO:204, a CDRL2 comprising SEQ ID NO:205, and a CDRL3 comprising SEQ ID NO:206.

[0027] In some embodiments the immunoglobulin light chain polypeptide has the sequence: X 1 IQX 2 TQSPSSLSASVGDRVTITCRX 3 SENIYX 4 X 5 LAWYQQKX 6 GKAPKLLIYX 7 AX 8 NLAX 9 GVPSRFSGSGSGTDX 10 TLTISSLQPEDFATYYCQX 11 FX 12 GPPLTFGGGTKVEIKR (SEQ ID NO: 194) or at least the CDRs thereof, X 1 is A or D; X 2 is L or M; X 3 is A or V; X 4is S or N; X 5 is H, N, or Y; X 6 is P or Q; X 7 is A or N; X 8 is T or K; X 9 is N, L, Q, G, F, V, K, S, R, T, H, or P; X 10 is F or Y; X 11 is L or H; X 12 are W, F, Y, P, N, V, K, M, L, G, and S). In some embodiments, the Ig light chain polypeptide comprises SEQ ID NO: 194, but retains the same CDRs (CDR1, CDR2, and CDR3) of any one of SEQ ID NOs: 157-192.

[0028] According to one embodiment, the BTLA-binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO: 144 or an amino acid sequence having at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO: 144, wherein the regions of the CDRs are as provided above (e.g., CDR1-SEQ ID NO:201, CDR2-SEQ ID NO:202, and CDR3-SEQ ID NO:203) or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced an immunoglobulin heavy chain variable region as determined according to any one of the methods set forth in claim 1, wherein the immunoglobulin heavy chain variable region is an immunoglobulin heavy chain variable region of SEQ ID NO: 174 or has at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 201%, at least 202%, at least 203%, at least 204%, at least 205%, at least 206%, at least 207%, at least 208%, at least 209%, at least 300%, at least 301%, at least 302%, at least 303%, at least 304%, at least 305%, at least 306%, at least 307%, at least 308%, at least 309%, at least 310%, at least 311%, at least 312%, at least 313%, at least 314%, at least 315%, at least 316%, at least 317%, at least 318%, at least 319%, at least 320%, at least 321%, at least 322%, at least 323%, at least 3 or an immunoglobulin light chain variable region comprising at least the CDRs of SEQ ID NO: 174, wherein the regions of the CDRs are as provided above (e.g., CDR1-SEQ ID NO:204, CDR2-SEQ ID NO:205, and CDR3-SEQ ID NO:206) or as determined according to any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo).In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174, or at least the CDRs thereof as determined by AHo.

[0029] Also provided are BTLA-binding agents that bind to the same epitope as a BTLA-binding agent that comprises an immunoglobulin heavy and light chain polypeptide described herein. In some embodiments, the BTLA-binding agent binds to the same epitope as a BTLA-binding agent that comprises a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 174. In some embodiments, the BTLA-binding agent binds to the same epitope as a BTLA-binding agent that comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region that comprises SEQ ID NO: 17; a BTLA-binding agent that comprises a heavy chain variable region of SEQ ID NO: 207 and a light chain variable region that comprises SEQ ID NO: 209; or a BTLA-binding agent that comprises a heavy chain variable region of SEQ ID NO: 217 and a light chain variable region that comprises SEQ ID NO: 219. A BTLA-binding agent is considered to bind to the same epitope if it competes for binding to BTLA with a BTLA-binding agent that comprises an immunoglobulin heavy and light chain polypeptide described herein. In some embodiments, provided herein are BTLA-binding agents that bind to amino acid residues 52-65 and / or 100-106 of human BTLA (e.g., SEQ ID NOs: 227 and / or 228) (reference sequence UniProt ID Q7Z6A9 or the corresponding sequence positions in naturally occurring variant human BTLA). In some embodiments, provided herein are BTLA-binding agents that bind to amino acid residues 46-65, 82-91, or 100-106 of human BTLA (e.g., SEQ ID NOs: 229, 230, and / or 231) (reference sequence UniProt ID Q7Z6A9 or the corresponding sequence positions in naturally occurring variant human BTLA).

[0030] When used in the context of a nucleic acid or amino acid sequence, sequence "identity" can be determined by comparing a subject nucleic acid or amino acid sequence to a reference nucleic acid or amino acid sequence. Percent identity is the percentage of nucleotides or amino acid residues that are the same (i.e., identical) between the subject and reference sequences when optimally aligned. Numerous mathematical algorithms for obtaining optimal alignment and calculating identity between two or more sequences are known and publicly available. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST2.1, BL2SEQ, and newer versions thereof, operated by the National Center for Biotechnology Information, Bethesda, MD), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Further, sequence alignment algorithms are described, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775(2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK(2009), Soding, Bioinformatics, 21(7):951-960(2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402(1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge This is disclosed in the UK (1997).

[0031] For sequences having less than 100% identity to the heavy and light chain sequences specifically set forth above, one or more amino acids of the heavy and / or light chain polypeptides of the aforementioned immunoglobulins may be replaced or substituted with different amino acids and / or one of more amino acids may be deleted from or inserted into the disclosed amino acid sequences, so long as the activity of the polypeptide (e.g., ability to bind BTLA when present as part of a BTLA-binding agent) is substantially retained. "Biological activity" of a BTLA-binding agent refers, for example, to a biological activity that does not inhibit binding of BTLA to its receptor and / or inhibits BTLA activity in vivo (e.g., IC 50 ), pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the BTLA protein, or with other proteins or tissues) for a particular BTLA epitope. In some embodiments, the biological activity of a BTLA-binding agent includes the ability of the agent to enhance binding of BTLA to its receptor(s) and / or otherwise increase BTLA activity in vivo. Other biological properties or characteristics of antigen-binding agents recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, and formulation. The foregoing properties or characteristics can be observed, measured, and / or assessed using standard techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or solution phase competition (KINEXA™), in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry assays.

[0032] Amino acid replacements or substitutions can be conservative, semi-conservative, or non-conservative. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that has common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such an analysis allows the definition of groups of amino acids that are preferentially exchanged with each other within a group, and thus are most similar to each other in their effect on the overall protein structure (Schulz and Schirmer, supra).

[0033] Amino acids are broadly classified as "aromatic" or "aliphatic". Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly classified as "aliphatic". Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine ​​(C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

[0034] The aliphatic amino acids can be subdivided into four subgroups: the "large aliphatic non-polar subgroup" consists of valine, leucine, and isoleucine; the "aliphatic slightly polar subgroup" consists of methionine, serine, threonine, and cysteine; the "aliphatic polar / charged subgroup" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine; and the "small residue subgroup" consists of glycine and alanine. The group of charged / polar amino acids can be subdivided into three subgroups: the "positively charged subgroup" consisting of lysine and arginine, the "negatively charged subgroup" consisting of glutamic acid and aspartic acid, and the "polar subgroup" consisting of asparagine and glutamine.

[0035] The aromatic amino acids can be subdivided into two subgroups: the "nitrogen ring subgroup", consisting of histidine and tryptophan, and the "phenyl subgroup", consisting of phenylalanine and tyrosine.

[0036] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, such as substitutions of arginine with lysine (or vice versa) so that a positive charge can be maintained, substitutions of aspartic acid with glutamic acid (or vice versa) so that a negative charge can be maintained, substitutions of threonine with serine so that a free -OH can be maintained, and substitutions of asparagine with glutamine so that a free -NH2 can be maintained. "Semi-conservative mutations" include amino acid substitutions of amino acids within the same group listed above but not within the same subgroup. For example, substitutions of asparagine with aspartic acid or lysine with asparagine involve amino acids within the same group but from different subgroups. "Non-conservative mutations" include amino acid substitutions between different groups, such as substitutions of tryptophan with lysine or serine with phenylalanine, etc.

[0037] The aforementioned mutations (e.g., substitutions) may be made in any region of the Ig chain. In some embodiments, an amino acid(s) in the CDRs (e.g., CDR1, CDR2, or CDR3) of the heavy and / or light chain polypeptides of the immunoglobulin are substituted; in other embodiments, an amino acid(s) in the framework regions are substituted but not in the CDRs; in still other embodiments, amino acids in both the framework regions and the CDRs are substituted. In some embodiments, the aforementioned mutations are made in regions other than the CDRs. In other words, the heavy and light chain variable regions can have a defined sequence identity to the sequences provided herein but can retain the CDRs of the sequences specifically provided.

[0038] Additionally, one or more amino acids may be inserted into the heavy and / or light chain polypeptides of the aforementioned immunoglobulins, so long as they do not abolish the function of the polypeptide in the context of the BTLA-binding agent (e.g., do not prevent a binding agent comprising the polypeptide from binding to BTLA without inhibiting binding of BTLA to its receptor). Any number of any suitable amino acids may be inserted into the amino acid sequence of the heavy or light chain polypeptides of the immunoglobulins. In some embodiments, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids) but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids) may be inserted into the amino acid sequence of the heavy and / or light chain polypeptides of the immunoglobulins. In other embodiments, between 1 and 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted into the amino acid sequence of the heavy and / or light chain polypeptides of the immunoglobulins. In this regard, the amino acid(s) may be inserted into any one of the aforementioned heavy and / or light chain polypeptides of the immunoglobulin at any suitable position. In some embodiments, the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the heavy and / or light chain polypeptide of the immunoglobulin; in other embodiments, the amino acid(s) are inserted into a framework region and not into a CDR; in still other embodiments, the amino acid(s) are inserted into both a framework region and a CDR.

[0039] The isolated immunoglobulin heavy and light chain polypeptides of the invention are not limited to polypeptides that contain the specific amino acid sequences described herein, but also include any heavy or light chain polypeptides that, when included in a BTLA-binding agent, compete with the immunoglobulin heavy or light chain polypeptides of the invention for binding to BTLA. In this regard, for example, the immunoglobulin heavy or light chain polypeptides can be any heavy or light chain polypeptides that, when included in a BTLA-binding agent, bind to the same epitope of BTLA that is recognized by the heavy and light chain polypeptides described herein. Antibody competition can be assayed using routine peptide competition assays utilizing ELISA, Western blot, or immunohistochemical methods (see, e.g., U.S. Pat. Nos. 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4:12 (2006)).

[0040] A BTLA-binding agent is a proteinaceous molecule (e.g., an antibody or antigen-binding fragment thereof) comprising a heavy chain variable region and a light chain variable region of an immunoglobulin described herein that specifically binds to a BTLA protein. In some embodiments, a BTLA-binding agent binds to BTLA without inhibiting, or in some embodiments, inhibiting, the binding of BTLA to its receptor. In some embodiments, a BTLA-binding agent enhances the binding of BTLA to HVEM so as to increase BTLA-mediated signaling. As used herein with respect to the binding of BTLA to its receptor or BTLA-mediated signaling, the term "inhibit" refers to the ability to substantially antagonize, prohibit, prevent, limit, slow down, impede, alter, eliminate, or stop such binding or signaling in the presence of the binding agent (in whole or in part) compared to the binding of BTLA to its receptor or BTLA-HVEM-mediated signaling in the absence of the binding agent. The terms "increase" or "enhance" when used in reference to binding of BTLA to its receptor or BTLA-mediated signaling means increasing or enhancing such binding or signaling in any manner or to any degree in the presence of the binding agent compared to such binding or signaling in the absence of the binding agent. In some embodiments, binding of BTLA to its receptor or BTLA-mediated signaling is increased sufficiently to reduce or alleviate any symptoms of a disease or condition associated with a lack of BTLA activity or that would benefit from enhanced BTLA activity, or to reverse the progression or severity of such a disease or condition. In some embodiments, the BTLA-binding agent does not inhibit BTLA-receptor binding by more than 25% (e.g., does not inhibit BTLA-receptor binding by more than 10% or more than 5%). In some embodiments, the BTLA-binding agent increases BTLA-receptor binding by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the above values, compared to the activity of BTLA in the absence of the BTLA-binding agent.

[0041] A BTLA-binding agent may be part of a multispecific (e.g., bispecific or "bireactive") construct (e.g., a multispecific antibody, such as a bispecific or bireactive antibody) that binds BTLA and another antigen. Such a construct may include immunoglobulin heavy and light chain polypeptides that bind BTLA as described herein, combined with immunoglobulin heavy and light chains from an immunoglobulin that binds an antigen other than BTLA. Such a bispecific BTLA-binding agent may, for example, bind BTLA and another negative regulator of the immune system, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4), T cell immunoglobulin and mucin domain-3 (TIM-3), programmed death 1 (PD-1), and / or lymphocyte activation gene 3 protein (LAG-3). Immunoglobulins that bind such other target antigens are known in the art.

[0042] Antibody conjugates are also provided herein. For example, a BTLA-binding agent can be a conjugate of (1) an anti-BTLA antibody, or a fragment thereof, and (2) a second protein or non-protein moiety. By way of further example, a BTLA-binding agent can include an anti-BTLA antibody, or a fragment thereof, conjugated to another peptide, a fluorescent molecule, or a chemotherapeutic agent.

[0043] In some embodiments, the BTLA-binding agent can be a "whole" immunoglobulin or an antigen-binding immunoglobulin "fragment." A "whole" immunoglobulin typically consists of four polypeptides: two heavy (H) chain polypeptides and two light (L) chain polypeptides. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H 1. C H 2, and C H 3) region, and each light chain comprises one N-terminal variable (V L ) region and one C-terminal constant (C L) region. The light chain of an antibody can be assigned to one of two different types, either kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. In a typical immunoglobulin, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. In this configuration, the light chain variable region generally aligns with the variable region of the heavy chain, and the light chain constant region generally aligns with the first constant region of the heavy chain. The remaining constant regions of the heavy chains generally align with each other.

[0044] The variable or hypervariable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H Area and V L The regions have the same general structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conserved amino acid sequence in the variable region located between the hypervariable region and the complementarity determining region (CDR). There are four framework regions in each variable domain, which are named FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides the structural framework of the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is generally considered to be involved in antigen binding.

[0045] The term "antibody fragment" and similar terms (e.g., "fragment of an antibody," "antibody fragment," "functional fragment of an antibody") are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). An antibody "fragment," as used herein, as routinely used in the art, includes not only fragments or pieces of whole antibodies in the literal sense, but also other known engineered antibody-like constructs that may contain linkers or other elements not present in naturally occurring antibodies in addition to antibody fragments. An antibody fragment may, for example, include one or more (or all) CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or some combination thereof. Examples of antibody fragments include: (i) V L , V H , C L (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V fragment of a single arm of an antibody. L and V H (iv) Fab' fragments resulting from the use of mild reducing conditions to disrupt the disulfide bridges of the F(ab')2 fragment; (v) disulfide stabilized Fv fragments (dsFv); and (vi) domain antibodies (dAbs), which are antibody single variable region domain (VH or VL) polypeptides that specifically bind to an antigen. A BTLA binder may also be a single chain antibody fragment. An example of a single chain antibody fragment is (i) an Fv fragment consisting of two domains (i.e., VH and VL) of an Fv fragment joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain. L and V H(ii) single-chain Fvs (scFvs), which are monovalent molecules consisting of a V domain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)), and (iii) single-chain Fvs (scFvs), which are monovalent molecules consisting of a V domain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998) ... H and V L V is a peptide linker that cannot pair with L V connected to H , whereby different V H -V L Antibody fragments include, but are not limited to, diabodies, which are dimers of polypeptide chains that drive pairing between complementary domains in the polypeptide chains to create a dimeric molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024 A1.

[0046] In some embodiments, the BTLA binder is fragment crystallizable (F c) region or a portion thereof. The Fc region can be any Ig class / subclass, including IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM, variants thereof. In some embodiments, the BTLA binding agent is a "whole" or "complete" Ig (i.e., antibody). In some embodiments, the BTLA binding agent comprises an IgG Fc region, such as an IgG1 or IgG4. For example, the BLTA binding agent can be an IgG1 or IgG4 antibody. In some embodiments, the BTLA binding agent comprises a variable heavy chain region and a variable light chain region comprising SEQ ID NOs: 144 and 174, respectively, or at least the CDRs thereof (as provided herein, or as determined according to Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo numbering), or sequences with 90% identity thereto, and the BTLA binding agent is an IgG1 antibody. In some embodiments, the BTLA-binding agent comprises a variable heavy chain region and a variable light chain region comprising SEQ ID NOs: 144 and 174, respectively, or at least the CDRs thereof (as provided herein, or as determined according to the Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo numbering), or sequences with 90% identity thereto, and the BTLA-binding agent is an IgG4 antibody.

[0047] A BTLA binding agent may be or be derived from a human, non-human, or chimeric antibody. By "chimeric" is meant an antibody or fragment thereof that contains both human and non-human regions. Preferably, the BTLA binding agent is a humanized antibody. A "humanized" antibody is a monoclonal antibody that comprises a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., mouse or rat). A humanized antibody may contain one, two, or three CDRs obtained or derived from a non-human antibody. In one embodiment of the invention, the CDRH3 of a BTLA binding agent of the invention is obtained or derived from a mouse monoclonal antibody, while the remaining variable and constant regions of the BTLA binding agent of the invention are obtained or derived from a human monoclonal antibody.

[0048] Human, non-human, chimeric, or humanized antibodies can be obtained by any means, including via in vitro sources (e.g., hybridomas or cell lines recombinantly producing antibodies) and in vivo sources (e.g., rodents). Methods of producing antibodies are known in the art and are described, for example, in Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). In certain embodiments, human or chimeric antibodies can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes have been effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005) and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). Humanized antibodies can be made using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), including, for example, grafting non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1):25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)).In one embodiment, humanized antibodies can be generated using the methods described, for example, in US Patent Application Publication No. 2011 / 0287485 A1.

[0049] A BTLA binding agent is not limited by any particular affinity for its epitope. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ). However, in some embodiments, BTLA may have an affinity for BTLA of about 1 picomolar (pM) to about 100 micromolar (μM) (e.g., about 1 picomolar (pM) to about 1 nanomolar (nM), about 1 nM to about 1 micromolar (μM), or about 1 μM to about 100 μM). In one embodiment, a BTLA binder has a K of 1 nanomolar or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). D In another embodiment, the BTLA binding agent can bind to the BTLA protein with a K of 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). DThe affinity of an immunoglobulin for an antigen or epitope of interest can be measured using any art-recognized assay. Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXA™), antigen panning, competitive binding assays, and / or ELISA (see, for example, Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001). In some embodiments, the BTLA-binding agent has an affinity for BTLA as described above (e.g., 1 nM or less or 200 pM or less) when measured using surface plasmon resonance (SPR). In some embodiments, the BTLA-binding agent has an affinity for BTLA as described above (e.g., 1 nM or less or 200 pM or less) when measured using surface plasmon resonance (SPR).

[0050] The BTLA-binding agents provided herein can be used for any purpose, such as modulating (e.g., promoting or enhancing) BTLA signaling in a mammal, modulating (e.g., inhibiting) an immune response in a mammal, and / or treating or preventing a disease or condition associated with defective BTLA signaling (e.g., associated with a BTLA-mediated immune response). Thus, in one aspect, the invention provides a method of promoting or enhancing BTLA signaling in a mammal, comprising administering to the mammal a BTLA-binding agent described herein, whereby the BTLA-binding agent promotes or enhances binding of BTLA to HVEM or otherwise promotes or enhances BTLA signaling.

[0051] The mammal may be a mammal suffering from a disease or condition associated with a deficiency in BTLA signaling or associated with a BTLA-mediated immune response. For example, the mammal may be suffering from a disease or condition that is ameliorated by immunosuppression. Such diseases are responsive to BTL agonism, such that the disease or condition is treated or prevented by administering a BTLA-binding agent to the mammal. A disease, condition, or disorder associated with BTLA signaling and responsive to BTLA agonism may be any disease or disorder in which increased BTLA activity has therapeutic benefit in a mammal, preferably a human, or in which underexpression or reduced activity of BTLA causes or contributes to the pathological effects of the disease or disorder. Without wishing to be bound by any theory or mechanism of action, it is believed that the BTLA-binding agent promotes immunosuppressive BTLA-HVEM signaling, thereby suppressing the immune response.

[0052] An "immune response" may involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells), production of inflammatory cytokines, or any of the indications or disorders described herein or otherwise known in the art. As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms that may result from said disease. To this end, the methods of the invention include administering a "therapeutically effective amount" of a BTLA-binding agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the state of the disease, the age, sex, and weight of the individual, and the ability of the BTLA-binding agent to elicit a desired response in the individual.

[0053] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention include administering a "prophylactically effective amount" of a BTLA-binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of the onset of a disease).

[0054] The BTLA-binding agents are useful for suppressing the immune response and treating any disease or condition associated with an aberrant or excessive immune response. The disease or disorder may be an inflammatory or autoimmune disorder (e.g., a T-cell or B-cell driven inflammatory disease or condition).Examples of inflammatory or autoimmune disorders include, for example, infections (viral, bacterial, fungal, and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Behcet's disease, Alzheimer's disease; inflammatory bowel disease including Crohn's disease and ulcerative colitis; Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, antineutrophil cytoplasmic antibody-associated (ANCA) vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, related disorders, and the like. immune-mediated inflammatory disorders of the central and peripheral nervous system, such as multiple sclerosis, lupus (including systemic lupus erythematosus and chronic discoid lupus erythematosus), and Guillain-Barré syndrome; atopic dermatitis, polymyositis, dermatomyositis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, pemphigoid, primary biliary cholangitis, hepatitis, sarcoidosis, scleroderma (localized, systemic, and progressive systemic scleroderma), polyhemorrhage, hepatitis, sarcoidosis, pulmonary embolism, and pulmonary edema. granulomatosis, other autoimmune disorders, cholangitis, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection; ischemic diseases such as myocardial infarction and heart disease including atherosclerosis; periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), allergic granulomatous vasculitis, hypersensitivity vasculitis, aortitis syndrome (Takayasu's arteritis), temporal arteritis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, Still's disease, Cogan's syndrome, RS3PE, polymyalgia rheumatica, fibromyalgia syndrome, antiphospholipid syndrome syndrome, eosinophilic fasciitis, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, hemolytic anemia, autoimmune neutropenia, Hashimoto's thyroiditis, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenal insufficiency), herpes gestationis, linear IgA bullous skin disease, epidermolysis bullosa acquisita, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent fetal loss, or infertility related to lack of fetal-maternal tolerance.

[0055] In some embodiments, the disease or disorder is arthritis (e.g., rheumatoid arthritis or TNF-refractory rheumatoid arthritis), giant cell arteritis, polymyalgia rheumatica, primary Sjogren's syndrome, alopecia areata, primary biliary cholangitis (PBC), vitiligo, ANCA vasculitis, type 1 diabetes, non-infectious uveitis psoriasis, graft-versus-host disease (GvHD), or inflammatory bowel disease.

[0056] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or relieves to any extent the adverse symptoms that may result from the disease. To this end, the method of the invention includes administering a "therapeutically effective amount" of a BTLA binding agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the state of the disease, the age, sex, and weight of the individual, and the ability of the BTLA binding agent to elicit a desired response in an individual. For example, a therapeutically effective amount of a BTLA binding agent of the invention is an amount that increases the bioactivity of BTLA in humans.

[0057] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention include administering a "prophylactically effective amount" of a BTLA-binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of the onset of a disease).

[0058] In some embodiments, the method of treating an inflammatory disease or condition comprises administering a BTLA binding agent at a dose of at least 7 mg per week, e.g., at least 10 mg per week (e.g., 20 mg to 800 mg per week, 70 mg to 800 mg per week, 70 mg to about 300 mg per week, or 70 mg to about 200 mg per week), which is administered intravenously. In some embodiments, the method of treating an inflammatory disease or condition comprises administering a BTLA binding agent at a dose of at least 20 mg per week, e.g., 20 mg to 800 mg per week or 70 mg to 800 mg per week (e.g., 70 mg to 300 mg per week or 70 mg to 200 mg per week), which is administered subcutaneously.

[0059] In some embodiments, the methods involve administering the BTLA-binding agent in a single intravenous dose of at least 150 mg or at least 200 mg, the dose being administered no more than once every 3 weeks or no more than once every 4 weeks (e.g., no more than once every 30 days, no more than once every 60 days, no more than once every 90 days, no more than once every 120 days, no more than once every 150 days, or no more than once every 180 days). In some embodiments, the method includes administering the BTLA-binding agent in a single subcutaneous dose of at least 300 mg (e.g., 300 mg to 800 mg or 300 mg to 500 mg), or at least 400 mg (e.g., 400 mg to 800 mg or 400 mg to 500 mg), administered no more than once every 3 weeks or no more than once every 4 weeks (e.g., no more than once every 30 days, no more than once every 60 days, no more than once every 90 days, no more than once every 120 days, no more than once every 150 days, or no more than once every 180 days). In some embodiments, the BTLA-binding agent is administered at a dosing regimen (dosages and dosing intervals such as those disclosed above) sufficient for the BTLA-binding agent to maintain BTLA receptor occupancy on CD19+ B cells and / or CD3+ T cells at 90% or greater. All dosing regimens may be continued as necessary to obtain the desired clinical result.

[0060] The BTLA binding agent may be part of a composition suitable for administration to a mammal. Preferably, the composition is a pharma- ceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, preferably a pharma- ceutically acceptable (e.g., physiologically acceptable) carrier, and an amino acid sequence, antigen binding agent, or vector of the invention. Any suitable carrier may be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier is determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition may also include any other excipients used in formulating therapeutic molecules (e.g., proteins or antibodies), particularly parenteral formulations, including, for example, buffers, osmolality adjusters, stabilizers, surfactants, and the like. The composition may optionally be sterile. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be produced according to conventional techniques as described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0061] The effectiveness of the treatment or prevention can be monitored by periodic evaluation of the treated patient. When administered repeatedly over several days or longer, depending on the condition, the treatment may be repeated until the symptoms of the disease are suppressed as desired, or may be continued for the life of the patient. However, other dosing regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus of the composition, multiple boluses of the composition, or a continuous infusion of the composition.

[0062] Administration can be performed using any standard administration technique, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Preferably, the composition is suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0063] Once administered to a mammal (e.g., a human), the biological activity of the BTLA-binding agent can be measured by any suitable method known in the art. The biological activity can be correlated with the stability of the BTLA-binding agent in the body. In one embodiment of the invention, the BTLA-binding agent (e.g., an antibody) has an in vivo half-life of about 30 minutes to 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the BTLA binding agent has an in vivo half-life of about 2 hours to about 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values). In another embodiment, the BTLA binding agent has an in vivo half-life of about 10 days to about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).

[0064] The BTLA-binding agent of the present invention may be administered alone or in combination with other drugs. For example, the BTLA-binding agent may be administered in combination with other agents for treating or preventing the diseases disclosed herein, such as other agents, anti-inflammatory agents or immunosuppressants. In this regard, for example, the BTLA-binding agent may be used in combination with at least one other agent, including, for example, any anti-inflammatory agent known in the art, glucocorticoids, small molecule immunosuppressants, vaccines, biological therapies (e.g., other monoclonal antibodies, viruses, gene therapies, and adoptive T cell transfer), and / or surgery. When treating an infectious disease by the method of the present invention, the BTLA-binding agent may be administered in combination with at least one antibacterial agent or at least one antiviral agent. In this regard, the antibacterial agent may be any suitable antibiotic known in the art. The antiviral agent may be any suitable type of vaccine (e.g., live attenuated vaccines, subunit vaccines, recombinant vector vaccines) that specifically targets a particular virus, and any small molecule antiviral therapy (e.g., viral replication inhibitors and nucleoside analogs).

[0065] In addition to therapeutic applications, the BTLA-binding agents described herein can also be used in diagnostic or research applications. In this regard, the BTLA-binding agents can be used in methods for diagnosing disorders or diseases in which inappropriate expression (e.g., underexpression) or reduced activity of BTLA causes or contributes to the pathological effects of the disease or disorder. Similarly, the BTLA-binding agents can be used in assays that monitor BTLA protein levels in subjects being tested for diseases or disorders that respond to BTLA stimulation. Research applications include, for example, methods that utilize the BTLA-binding agents and a label to detect BTLA protein in a sample, for example, in a human body fluid or in an extract of a cell or tissue. The BTLA-binding agents can be used with or without modifications, such as covalent or non-covalent labeling with a detectable moiety. For example, the detectable moiety can be a radioisotope (e.g., 3 H, 14 C. 32 P, 35S, or 125 I), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), or a prosthetic group. In the context of the present invention, any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed (see, e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982)).

[0066] The level of BTLA protein can be measured using the BTLA agents provided herein by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA) and FACS. The normal or standard expression value of BTLA can be established using any suitable technique, for example, by combining a sample containing or suspected of containing a BTLA polypeptide with a BTLA-specific antibody under conditions suitable for forming an antigen-antibody complex. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amount of BTLA polypeptide expressed in the sample is then compared with the standard value.

[0067] Without wishing to be bound by any particular theory or mechanism of action, it is believed that administration of the BTLA-binding agents described herein results in a portion of BTLA being shed from at least a portion of the cells in which BTLA is expressed, resulting in an increase in soluble BTLA (sBTLA) present in the blood, plasma, serum, or tissue (e.g., skin tissue) of a subject to which the BTLA-binding agent has been administered. Thus, provided herein are methods (e.g., in vitro methods) for detecting, measuring, or monitoring the pharmacological activity of a BTLA-binding agent in a subject, the methods including detecting soluble BTLA (sBTLA) in a sample of blood, plasma, serum, or tissue (e.g., skin tissue) from a subject to which the BTLA-binding agent has been administered. Also provided herein are methods of selecting a patient for treatment with a BTLA-binding agent by detecting or measuring (e.g., in vitro) sBTLA in a blood, plasma, serum, or tissue (e.g., skin tissue) sample from the patient, where the patient is selected for treatment if the level of sBTLA in the patient (sample from the patient) is decreased compared to the level of sBTLA in a normal, non-diseased subject (e.g., human subject) of the same type. sBTLA can be detected, and optionally quantified, using any of a number of techniques known in the art. In some embodiments, sBTLA is detected by contacting (e.g., in vitro) a blood, plasma, serum, or tissue sample from the subject with a BTLA-binding agent provided herein. In some embodiments, the sBTLA detected in the subject's blood, plasma, serum, or tissue sample is bound to a BTLA-binding agent (e.g., a previously administered BTLA-binding agent).

[0068] In some embodiments, the BTLA-binding agent administered to the subject that induces shedding of BTLA in the subject is a BTLA-binding agent that does not inhibit binding of BTLA to HVEM. The BTLA-binding agent can be any BTLA-binding agent, such as any of the BTLA-binding agents described herein. In some embodiments, the BTLA-binding agent comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto. For example, the BTLA-binding agent can comprise the CDRs represented by SEQ ID NOs: 27, 30, 32, 36, 39, and 41. In other embodiments, the BTLA binding agent comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 43-156, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 157-192, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto, and has any or all of the other features described herein. For example, the BTLA binding agent can comprise CDRs represented by SEQ ID NOs: 195-200; for example, the BTLA binding agent comprises CDRs comprising SEQ ID NOs: 201-206, or comprises the Ig heavy and light chains of SEQ ID NOs: 193 and 194 or SEQ ID NOs: 144 and 174, or at least the CDRs thereof, or an Ig heavy and light chains having 90% or more sequence identity to SEQ ID NOs: 193 and 194 or SEQ ID NOs: 144 and 174, respectively.

[0069] In some embodiments, sBTLA in the blood, plasma, serum, or tissues (e.g., skin tissue) of a subject to which BTLA has been administered is detected using a capture antibody that binds to sBTLA. The capture antibody can be any antibody that binds to BTLA. In some embodiments, the capture antibody is a BTLA antibody provided herein. The capture antibody can be the same or different from the BTLA binding agent administered to the subject. In some embodiments, the sBTLA capture antibody is different from the BTLA binding agent administered to the subject. In some embodiments, the capture antibody comprises a heavy chain variable region comprising any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218, or at least a CDR thereof, or an amino acid sequence having at least 90% sequence identity thereto; and a light chain variable region comprising any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220, or at least a CDR thereof, or an amino acid sequence having at least 90% sequence identity thereto; and has any or all of the other characteristics described herein. In further embodiments, the capture antibody may comprise heavy chain CDRs 1-3 represented by SEQ ID NOs: 27, 30, and 32; and light chain CDRs 1-3 represented by SEQ ID NOs: 36, 39, and 41; or any of the more specifically mentioned CDRs provided herein. For example, the capture antibody may comprise an Ig heavy chain of SEQ ID NO: 26 (e.g., any of SEQ ID NOs: 1-15, 207, 208, 217, or 218) and an Ig light chain of SEQ ID NO: 35 (e.g., any of SEQ ID NOs: 16-25, 209, 210, 219, or 220). Optionally, in combination with such embodiments, the BTLA-binding agent administered to a subject may comprise an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 43-156, or at least their CDRs, or an amino acid sequence having at least 90% sequence identity thereto; and an immunoglobulin light chain variable region of any of SEQ ID NOs: 157-192, or at least their CDRs, or an amino acid sequence having at least 90% sequence identity thereto; and having any or all of the other characteristics described herein.For example, a BTLA binding agent can comprise CDRs represented by SEQ ID NOs: 195-200; for example, a BTLA binding agent comprises CDRs comprising SEQ ID NOs: 201-206, or comprises the heavy and light chains of Ig of SEQ ID NOs: 193 and 194 or SEQ ID NOs: 144 and 174.

[0070] While not wishing to be bound by any particular theory or mechanism of action, as described herein, it is believed that binding of BTLA (or sBTLA) to an antibody that does not inhibit binding of BTLA to HVEM enhances binding of the capture antibody to sBTLA. Thus, also provided herein is an assay for detecting and / or quantifying sBTLA in blood, plasma, serum, or tissue (e.g., skin tissue), comprising contacting a sample of blood, plasma, serum, or tissue (e.g., skin tissue) with a capture antibody and a BTLA-binding agent that does not inhibit binding of BTLA to HVEM. In some embodiments, a BTLA-binding agent that does not inhibit binding of BTLA to HVEM is a BTLA-binding agent that comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 43-156, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 157-192, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and has any or all of the other characteristics described herein. For example, a BTLA-binding agent can comprise CDRs represented by SEQ ID NOs: 195-200; for example, a BTLA-binding agent comprises CDRs comprising SEQ ID NOs: 201-206, or comprises the heavy and light chains of Ig of SEQ ID NOs: 193 and 194 or SEQ ID NOs: 144 and 174. In some embodiments, the capture antibody comprises a heavy chain variable region comprising any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and a light chain variable region comprising any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and has any or all of the other characteristics described herein. For example, the capture antibody may comprise heavy chain CDRs 1-3 represented by SEQ ID NOs: 27, 30, and 32; and light chain CDRs 1-3 represented by SEQ ID NOs: 36, 39, and 41; or any of the more specifically mentioned CDRs provided herein.For example, the capture antibody may comprise an Ig heavy chain of SEQ ID NO: 26 (e.g., any of SEQ ID NOs: 1-15, 207, 208, 217, or 218) and an Ig light chain of SEQ ID NO: 35 (e.g., any of SEQ ID NOs: 16-25, 209, 210, 219, or 220).

[0071] In some embodiments of the aforementioned methods of detecting, measuring, or qualifying sBTLA in blood, plasma, serum, or tissue, the method may further include comparing the concentration of sBTLA in the blood, plasma, serum, or tissue sample to a reference sBTLA concentration. Any suitable reference concentration may be used. In some embodiments, the reference sBTLA concentration is the concentration of sBTLA in a blood, plasma, serum, or tissue sample from the same patient or subject prior to administration of the BTLA-binding agent. Alternatively or additionally, the reference sBTLA concentration may be provided by the concentration of sBTLA in the blood, plasma, serum, or tissue of another subject, e.g., a normal, non-diseased subject of the same type who has not been administered the BTLA-binding agent, or a reference sBTLA concentration established by statistical analysis of the concentration of sBTLA in the blood, plasma, serum, or tissue of a population of such subjects (e.g., the average concentration of sBTLA in blood, plasma, serum, or tissue samples from a population of normal, non-diseased subjects not treated with a BTLA-binding agent). In some embodiments, a reference sBTLA concentration is established by a method that includes contacting a blood, plasma, serum, or tissue sample from a subject, or a blood, plasma, serum, or tissue sample from a population of subjects, with a capture antibody described above; and optionally, simultaneously or sequentially in any order, contacting the blood, plasma, serum, or tissue sample with a BTLA binding agent that does not inhibit binding of BTLA to HVEM as described herein.

[0072] In other embodiments, the method includes comparing the concentration of sBTLA in a blood, plasma, serum, or tissue sample from a subject to which a BTLA-binding agent has been administered to the concentration of sBTLA in a blood, plasma, serum, or tissue sample from the same subject at a different time point, either before or after the BTLA-binding agent is administered to the subject. For example, the sBTLA concentration may be measured at two or more time points after administration of the BTLA-binding agent, and optionally compared to one or more additional intervening administrations of the BTLA-binding agent, to assess the effect of the BTLA-binding agent over time. In this manner, treatment with the BTLA-binding agent may be monitored.

[0073] Further provided herein are compositions comprising a BTLA-binding agent that does not inhibit binding of BTLA to HVEM and a second capture antibody that binds sBTLA, the compositions being useful in the aforementioned methods of detecting, measuring, or monitoring sBTLA in blood, plasma, serum, or tissue. In some embodiments, the BTLA-binding agent that does not inhibit binding of BTLA to HVEM comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 43-156, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 157-192, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and has any or all of the other characteristics described herein. For example, the BTLA-binding agent can comprise CDRs represented by SEQ ID NOs: 195-200; for example, the BTLA-binding agent comprises CDRs comprising SEQ ID NOs: 201-206, or comprises the heavy and light chains of Ig of SEQ ID NOs: 193 and 194 or SEQ ID NOs: 144 and 174. In some embodiments, the capture antibody comprises a heavy chain variable region comprising any one of SEQ ID NOs: 1-15, 207, 208, 217, or 218, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and a light chain variable region comprising any one of SEQ ID NOs: 16-25, 209, 210, 219, or 220, or at least the CDRs thereof, or an amino acid sequence having at least 90% sequence identity thereto; and has any or all of the other characteristics described herein. For example, the capture antibody may comprise heavy chain CDRs 1-3 represented by SEQ ID NOs: 27, 30, and 32; and light chain CDRs 1-3 represented by SEQ ID NOs: 36, 39, and 41; or any of the more specifically mentioned CDRs provided herein. For example, the capture antibody may comprise an Ig heavy chain of SEQ ID NO: 26 (e.g., any of SEQ ID NOs: 1-15, 207, 208, 217, or 218) and an Ig light chain of SEQ ID NO: 35 (e.g., any of SEQ ID NOs: 16-25, 209, 210, 219, or 220).

[0074] The BTLA binding agent, capture antibody, or composition can be provided as a kit, i.e., a packaged combination of predetermined amounts of reagents and instructions for carrying out the diagnostic assay. When the BTLA binding agent is labeled with an enzyme, the kit desirably includes a substrate and cofactor required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (e.g., blocking buffers or lysis buffers) may be included in the kit. The relative amounts of the various reagents may be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The reagents can be provided as dry powders (typically lyophilized) that include excipients that, when dissolved, provide a reagent solution having the appropriate concentration.

[0075] nucleic acid The present invention also provides one or more nucleic acids encoding the immunoglobulin heavy chain polypeptides, the immunoglobulin light chain polypeptides, and the BTLA-binding agents provided herein.

[0076] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or modified nucleotides. The terms "nucleic acid" and "polynucleotide" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA as well as double- and single-stranded RNA. The terms also include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically linked to form nucleic acid sequences or polynucleotides via phosphate linkages, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, etc.).

[0077] The nucleic acid encoding the immunoglobulin heavy chain polypeptide, the immunoglobulin light chain polypeptide, or the BTLA-binding agent may be part of a vector. The vector may be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or an adenovirus), or a phage. Suitable vectors and methods for preparing vectors are well known in the art (e.g., as reviewed in Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0078] A vector typically contains expression control sequences that direct the expression of a coding sequence in a host cell, such as a promoter, an enhancer, a polyadenylation signal, a transcription terminator, a signal peptide (e.g., osteonectin signal peptide), an internal ribosome entry site (IRES), etc. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0079] Numerous promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art. Representative sources of promoters include, for example, viral, mammalian, insect, plant, yeast, and bacterial, and suitable promoters from these sources can be readily available or synthetically produced, for example, based on publicly available sequences from depositories such as ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.), the LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27:4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144(2005)).

[0080] As used herein, the term "enhancer" refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. Many enhancers from a variety of different sources are known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as other commercial or private sources). Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain an enhancer sequence. Enhancers can be located upstream, within, or downstream of a coding sequence.

[0081] A vector may also contain a "selection marker gene." As used herein, the term "selection marker gene" refers to a nucleic acid sequence that allows for the specific selection of cells expressing the nucleic acid sequence in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art, see, e.g., WO 1992 / 008796 and WO 1994 / 028143; Wigler et al, Proc. Natl. Acad. Sci. USA, 77:3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1-14 (1981); Santerre et al., Gene, 30:147-156 (1984); Kent et al. al., Science, 237:901-903 (1987); Wigler et al., Cell, 11:223-232 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026-2034 (1962); Lowy et al., Cell, 22:817-823 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0082] In some embodiments, the vector is an "episomal expression vector" or "episome" that can replicate in a host cell and persist as an extrachromosomal segment of DNA in the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr virus nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA), and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use T-antigen and the SV40 origin of replication in place of EBNA1 and oriP.

[0083] Other suitable vectors include integrating expression vectors that can be randomly integrated into the DNA of a host cell or that can contain recombination sites that allow specific recombination between the expression vector and the host cell chromosome. Such integrating expression vectors can utilize the endogenous expression control sequences of the host cell chromosome to express the desired protein. Examples of site-specifically integrated vectors include, for example, the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, CA) or the components of the cre-lox system that can be found, for example, in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that can be randomly integrated into the host cell chromosome include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Life Technologies (Carlsbad, CA), UCOE from Millipore (Billerica, MA), and pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, WI).

[0084] Viral vectors may also be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentivirus-based pLP1 from Invitrogen (Carlsbad, Calif.), and the retrovirus vector pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.).

[0085] Nucleic acid sequences encoding the amino acid sequences of the invention can be provided to the cells on the same vector (i.e., in cis). A unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences. Alternatively, nucleic acid sequences encoding the amino acid sequences of the invention can be provided to a population of cells on separate vectors (i.e., in trans). Each nucleic acid sequence in each separate vector may contain the same expression control sequence or may contain different expression control sequences. The separate vectors can be provided to the cells simultaneously or sequentially.

[0086] The vector(s) containing the nucleic acid(s) encoding the amino acid sequence of the invention can be introduced into a host cell (including any suitable prokaryotic or eukaryotic cell) capable of expressing the polypeptide encoded thereby. Thus, the invention provides an isolated cell comprising the vector of the invention. Preferred host cells are those that can be grown easily and reliably, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected.

[0087] Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli, such as K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102.

[0088] In some embodiments, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerevisiae and Pichia pastoris.

[0089] Suitable insect cells are described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).

[0090] In some embodiments, the present invention utilizes mammalian cells. Many suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (e.g., CHO-K1 cells, ATCC No. CCL61), CHO DHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Regular diploid cells, cell lines obtained from in vitro culture of primary tissues, as well as primary explants are suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from ATCC. Methods for selecting suitable mammalian host cells, as well as methods for transforming, culturing, amplifying, screening, and purifying cells are known in the art.

[0091] In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell may be a human lymphocyte or lymphocyte-derived cell line, such as a cell line of pre-B lymphocyte origin. Examples of human lymphocyte cell lines include, but are not limited to, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85:1581-1585 (1988)), Raji cells (CCL-86), PER.C6 cells (Crucell Holland BV, Leiden, The Netherlands), and derivatives thereof.

[0092] Nucleic acid sequences encoding the amino acid sequences of the invention can be introduced into cells by any suitable method, such as "transfection," "transformation," or "transduction." "Transfection," "transformation," or "transduction," as used herein, refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many suitable techniques are known in the art, including calcium phosphate DNA co-precipitation (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle accelerated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). After growing the infectious particles in suitable packaging cells, many of which are commercially available, the phage or viral vector may be introduced into a host cell.

[0093] The nucleic acids and cells can be used for any purpose, including the manufacture of the BTLA-binding agents described herein. In this regard, the invention provides a method of preparing a BTLA-binding agent, comprising culturing a cell comprising a nucleic acid encoding a heavy and / or light immunoglobulin polypeptide of the BTLA-binding agent. Stated differently, the method comprises expressing a nucleic acid encoding an immunoglobulin heavy and / or light chain of the BTLA-binding agent in a cell (e.g., an in vitro cell, such as any of the cell lines discussed herein, including CHO cells and CHO-K1 cells). It will be understood that the immunoglobulin heavy and light chains may be expressed from a single nucleic acid in a given cell, or may be expressed from separate nucleic acids in the same cell. The method may further comprise harvesting and / or purifying the BTLA-binding agent from the cell or cell culture medium using known techniques.

[0094] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0095] Table 2 provides examples of BTLA binding agents (eg, antibodies or antibody fragments) that comprise an immunoglobulin heavy chain variable region of SEQ ID NOs:1-15 and a light chain variable region of SEQ ID NOs:16-25.

[0096] [Table 2]

[0097] For the antibodies or antibody fragments in Table 2, the Kabat numbered CDRs are as follows: CDRH1 is located at positions 31-35 of the respective VH sequence; CDRH2 is located at positions 50-66 of the respective VH sequence; CDRH3 is located at positions 99-106 of the respective VH sequence; CDRL1 is located at positions 24-34 of the respective VL sequence; CDRL2 is located at positions 50-56 of the respective VL sequence; CDRL3 is located at positions 89-97 of the respective VL sequence. BTLA binding agents having pairs of heavy and light chain variable regions or at least their CDRs as set forth in Table 2 provide specific embodiments of the disclosure. Additional pairs of heavy and light chain variable regions or at least their CDRs of Table 2 provide yet other BTLA binding agents and are contemplated to be within the scope of the disclosure.

[0098] Table 3 provides examples of BTLA binding agents (eg, antibodies or antibody fragments) comprising immunoglobulin heavy chain variable regions of SEQ ID NOs: 43-156 and light chain variable regions of SEQ ID NOs: 157-192.

[0099] [Table 3-1]

[0100] [Table 3-2]

[0101] [Table 3-3]

[0102] [Table 3-4]

[0103] [Table 3-5]

[0104] For the antibodies or antibody fragments in Table 3, the Kabat numbered CDR regions are as follows: CDRH1 is located at positions 31-35 of the respective VH sequence; CDRH2 is located at positions 50-66 of the respective VH sequence, except for the antibodies marked with an *, where CDRH2 is located at positions 50-67 of the respective VH sequence; CDRH3 is located at positions 99-113 of the respective VH sequence, except for the antibodies marked with a †, where CDRH3 is located at positions 100-114; CDRL1 is located at positions 24-34 of the respective VL sequence; CDRL2 is located at positions 50-56 of the respective VL sequence; CDRL3 is located at positions 89-97 of the respective VL sequence. BTLA binding agents having pairs of heavy and light chain variable regions or at least their CDRs as set forth in Table 3 provide specific embodiments of the disclosure. Additional pairs of heavy and light chain variable regions or at least their CDRs in Table 3 provide yet other BTLA binding agents and are contemplated to be within the scope of the disclosure.

[0105] In the examples below, the antibodies referred to are as follows:

[0106] JPEG2025515337000008.jpg60149

[0107] Example 1 Antibody 6G3 was derived from a mouse hybridoma generated by standard fusion techniques from spleen cells of mice immunized with BTLA. The antibody was humanized using standard techniques as described herein. The final optimized antibody was expressed in Chinese Hamster Ovary (CHO) cells using the vectors summarized in Table 1.

[0108] [Table 1]

[0109] Example 2 This example demonstrates that the 6G3 anti-BTLA antibody disclosed herein has a K DAbout 5 nM, and for cynomolgus BTLA, K D It is demonstrated to have a binding affinity of approximately 11 nM.

[0110] Surface plasmon resonance (SPR) analysis was performed using Biacore T200 (GE Healthcare Life Sciences). The kinetic constants were calculated using a 1:1 binding model in the Biacore T200 Evaluation Software, and the on-rate and off-rate (k a and k d ) and the dissociation constant (K D ) was calculated. Anti-human IgG (GE Healthcare Life Sciences) was immobilized on a CM5 chip using EDC-activated amine coupling chemistry (420 s contact at 10 μL / min flow rate). Antibodies (APE10585.04 6G3 IgG1 and APE10840.05 6G3 IgG4) at 0.5 μg / mL each were captured by contacting the flow cell for 60 s using a flow rate of 10 μL / min. Monomeric human BTLA-his or cynomolgus BTLA-his at concentrations of 60 nM, 20 nM, 6.7 nM, and 2.2 nM were flowed over the capture antibody (480 s association, 1800 s dissociation). Experiments were performed at 25°C, and the mobile phase and all dilutions were buffered with 10 mM HEPES, pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20 (HBS-EP+, pH 7.6; Teknova). At the end of each cycle, bound antigen was removed by regeneration with two successive exposures (60 s and 90 s) to 3 M MgCl2 (30 μL / min). The corresponding sensorgrams and binding constants are shown in Figure 1 (human BTLA) and Figure 1 (cynomolgus BTLA). Antibody capture levels (resonance units (RU)) are listed to the right of each panel.

[0111] Example 3 This example demonstrates that the 6G3 anti-BTLA antibody disclosed herein has a K D410 pM, and for cynomolgus BTLA, K D It is demonstrated to have a binding affinity of approximately 1.66 nM.

[0112] Solution-based affinity measurements of 6G3 IgG4 (APE10840.04 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)) binding to BTLA were performed on a KinExA 3000 (Sapidyne Instruments). Azlactone beads (ThermoFisher Scientific) were coated with human BTLA ECD-his (30 μg / mL) or cynomolgus BTLA ECD-his (20 μg / mL) in 50 mM Na2CO3 for 2 hours at room temperature and blocked with 10 mg / mL BSA. In both human and cynomolgus BTLA experiments, the concentration of 6G3 IgG4 APE10840.04 was kept constant at 100 pM for binding. Human or cynomolgus BTLA ECD-his was added at 2.5-fold dilutions from 1000 nM to 68 fM at 25°C. Sample sets were equilibrated at 4°C for 72 hours and then brought to room temperature for 6 hours before free antibody was captured with azlactone beads coupled to BTLA. The secondary antibody to quantify 6G3 bound to beads was Alexa-Fluor-647 AffiPure donkey anti-human IgG (250ng / mL, Jackson ImmunoResearch Laboratories). Maximum and background signals were determined for samples containing 6G3 only and buffer only, respectively. Data were analyzed using KinExA Pro Software 3.2.6. K for binding of 6G3 antibody to human BTLA and cynomolgus BTLA was D The 95% confidence intervals for the analysis of values ​​are shown in FIG.

[0113] Example 4 This example demonstrates that the 6G3 antibody disclosed herein exhibits saturable binding to human and cynomolgus BTLA expressed in stably transfected 293c18 cells.

[0114] 293c18 cells clone 1E4 stably expressing human BTLA constructs or clone 1G5 stably expressing cynomolgus BTLA were harvested with Accutase solution (Millipore Sigma / Sigma-Aldrich) and washed once with phosphate-buffered saline, 1% BSA. The lipophilic carbocyanine dye DiD (2 μM, 1,1'-dioctadecyl-3,3,3',3'-tetramentylindodicarbocyanine; ThermoFisher Scientific) was loaded into 293c18 cynomolgus BTLA cells by gentle rocking for 10 min at room temperature. DiD-stained cells were washed with PBS, 1% BSA, and equal volumes of human BTLA 293c18 cells and cynomolgus BTLA 293c18-DiD stained cells were mixed. Cells (2 × 10 ) were plated in FACS buffer (PBS, 1% BSA, 0.02% sodium azide) with gentle shaking for 10 min at 4°C. 5 Total cells / sample) were incubated with the indicated concentrations of purified 6G3 IgG4 (two production lots, APE10840.03 and APE10840.04 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)) or human IgG4 isotype control antibody specific for hen egg lysozyme. Cells were centrifuged, washed once with FACS buffer, resuspended in FACS buffer and incubated for an additional 20 min at 4°C with gentle shaking. Cells were centrifuged, washed once with FACS buffer without BSA and fixed with 100 μL / well of 2% paraformaldehyde in PBS for 10 min at room temperature. Cells were washed once with FACS buffer and incubated with secondary antibody (goat anti-human kappa-PE, 0.2 μg / mL in FACS buffer, SouthernBiotech) for 10 min at 4°C with gentle shaking. Cells were washed, resuspended in FACS buffer and fluorescence was analyzed on a BD FACSArray (BD Biosciences). DiD-stained cells expressing cynomolgus BTLA (Figure 3) were analyzed for median fluorescence intensity (MFI) separately from cells expressing human BTLA (Figure 3). Curves were fitted with a least-squares fit analysis of log(inhibitor) vs. response-variable slope (4 parameters) in GraphPad Prism (GraphPad Software, Inc).

[0115] The two lots of 6G3 antibody disclosed herein (APE10840.03 and APE10840.04) were cultured in human BTLA 293c18 cells (EC 50 Approximately 1.7 nM and 1.8 nM) and cynomolgus monkey BTLA 293c18 cells (EC 50 The antibody showed similar concentration-dependent saturation binding to the agonist (approximately 2.3 nM).

[0116] Example 5 This example demonstrates that the 6G3 antibody disclosed herein inhibits human peripheral blood CD4 + T cells, CD8 + T cells and CD20 + Demonstrated to exhibit saturable binding to B cells.

[0117] Human peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque (Sigma-Aldrich) density gradient centrifugation of normal donor blood obtained from the San Diego Blood Bank (San Diego, CA). PBMCs were washed and cultured at 2 × 10 in FACS buffer (PBS, 1% BSA, 0.02% sodium azide). 7 Human BD Fc Block (2.5μg / 1×10 cells / mL) 6 The cells were incubated with 1×10 IgG1 (BD Biosciences) and LIVE / DEAD® Fixable Yellow Dead Cell Stain (30 μL, ThermoFisher Scientific) on ice for 10 min. The cells were washed once with FACS buffer and diluted with 1×10 7The cells were resuspended in FACS buffer at 1 × 10 cells / mL and the following phenotyping antibodies were added: Alexa Fluor 488-anti-human CD3ε clone SK7, Brilliant Violet 421-anti-human CD4 clone OKT4, Brilliant Violet 785-anti-human CD8 clone SK1, and Brilliant Violet 570-anti-human CD20 clone 2H7 (50 μL each; all from BioLegend, Inc.). 6 Cells / sample) were plated in U-bottom 96-well plates and incubated with the indicated concentrations of DyLight650-labeled anti-BTLA antibodies or isotype control antibodies in FACS buffer for 20 min at 4°C with gentle shaking. 6G3 IgG4 (APE10916.02) was APE10840 labeled with DyLight650 (3.44 mol DyL650 per mol antibody). Human IgG4 isotype control antibody specific for hen egg lysozyme was labeled with DyLight650 (3.22 mol DyL650 per mol antibody). Reference anti-BTLA antibody MIH26 was purchased as an allophycocyanin (APC)-labeled antibody from BioLegend, Inc. Samples were washed once with FACS buffer, resuspended in 150 μL / well of FACS buffer, and further washed at 4°C for 10 min. Samples were centrifuged and fixed with 4% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature. Samples were washed twice with FACS buffer, resuspended in 150 μL / well of FACS buffer, and analyzed for fluorescence on a NovoCyte flow cytometer (ACEA Biosciences, Inc.). Data were analyzed using NovoExpress Software (ACEA Biosciences, Inc.). Gated CD4 + T cells (Figure 4), CD8 + T cells (Figure 4), or CD20 + Median fluorescence intensities of anti-BTLA or isotype staining in B cells (Figure 4) were graphed and analyzed using GraphPad Prism (GraphPad Software, Inc.). 50Curves were fitted with a log(agonist) vs. response (3-parameter) least-squares fit to calculate ΔΨ(A) = 0.01;

[0118] The 6G3 antibody disclosed herein, labeled with DyLight650, inhibits CD4+ + T cells (Figure 4), CD8 + The 6G3 antibody exhibits saturable binding to T cells (Figure 4) and B cells (Figure 4) in a concentration-dependent manner. 50 CD4 at approximately 2.4 nM + T cells, EC 50 CD8 at approximately 3.2 nM + T cells, EC 50 CD20 at approximately 0.5 nM + It binds to B cells. Staining with the positive control anti-BTLA antibody MIH26-APC is comparable to that with the 6G3 antibody. The DyLight650-labeled human IgG4 isotype control antibody shows no staining of these cell populations (Figure 4).

[0119] Example 6 This example demonstrates that the 6G3 antibody disclosed herein inhibits CD3 + T cells and CD20 + It is demonstrated that the antibody exhibits concentration-dependent binding to B cells.

[0120] 6G3 IgG4 (APE13308) produced from a pool of stably transfected CHO-K1 cells was labeled with Alexa Fluor 647 (AF647) (Alexa Fluor Antibody Labeling Kit; ThermoFisher Scientific / Molecular Probes) according to the manufacturer's instructions and designated APE13766.02 (6G3-AF647). Fresh peripheral blood from normal cynomolgus monkeys was obtained from Altasciences. Whole blood samples (800 μL) were incubated with FcR Blocking Reagent, human (Miltenyi Biotec, Inc.) for 10 min at room temperature and then stained with a mixture of fluorescently labeled antibodies to distinguish between cynomolgus monkey T and B cell populations [PerCP-Cy5.5 mouse anti-human CD3 (clone SP34-2; BD Biosciences), BD Horizon V450 mouse anti-human CD4 (clone L200; BD Biosciences), APC / Cy7 mouse anti-human CD8 (clone SK1; BD Biosciences), Brilliant Violet 785 anti-CD20 (clone 2H7; BioLegend, Inc.), BD Horizon V500 mouse anti-NHP CD45 (clone D058-1283; BD Biosciences)] in the dark for 20 min at room temperature. Blood was then dispensed into wells and incubated with the indicated concentrations of 6G3-AF647 (APE13766.02) or APC-mouse anti-human CD272 (BTLA) reference antibody (clone J168-540; BD Biosciences) for 30 min at room temperature in the dark. After incubation, red blood cells were lysed for 10 min by adding 2.0 mL of diluted BD Pharm Lyse (BD Biosciences), and samples were centrifuged at 200 × g for 5 min, washed with FACS buffer [Dulbecco's PBS, no calcium, no magnesium (Gibco / ThermoFisher Scientific), 25 mM HEPES, pH 7.2, 0.1% BSA, 0.1% sodium azide], and fixed with 4% paraformaldehyde (200 μL / sample) for 10 min at room temperature.Samples were washed twice with FACS buffer and analyzed for fluorescence on a NovoCyte Quanteon flow cytometer (ACEA Biosciences, Inc.). Data were analyzed using NovoExpress Software (ACEA Biosciences, Inc.). Mean fluorescence intensity (MFI) values ​​were graphed and fitted by nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc.). Total CD3. + The MFI values ​​of each anti-BTLA antibody against total CD20 cells are shown in Figure 5A. + The MFI values ​​of each anti-BTLA antibody against the cells are shown in Figure 5B. Figure 5C shows a dot plot analysis of anti-CD3 staining and 33 nM APC-labeled anti-BTLA reference antibody (clone J168-540) staining. Figure 5D shows a dot plot analysis of anti-CD3 staining and 100 nM 6G3-AF647 staining. - Binding of each anti-BTLA antibody to cells (green cells in the lower quadrant of each panel) was determined by CD20 + Reflects binding to B cells.

[0121] The 6G3 antibody disclosed herein labeled with AF647 inhibited peripheral blood CD3 + T cells and CD20 + Shows concentration-dependent binding to B cells. CD3 + Binding to peripheral blood T cells was not saturated at 100 nM antibody under the staining conditions used (Figure 5A) (estimated EC 50 Approximately 3.4 nM), 6G3-RF647 CD20 + Binding to peripheral blood B cells is saturated (estimated EC50 approx. 1.4 nM) (Figure 5B). The MFI of 6G3-AF647 staining of B cells is approx. 9-fold greater than the MFI of 6G3-AF647 staining of T cells. + and CD3 - (CD20 + ) The staining pattern of 6G3-AF647 at 100 nM in cynomolgus monkey cells closely resembles that of the commercially available reference APC-labeled anti-BTLA antibody J168 at 33 nM (Figures 5D and 5C, respectively).

[0122] Example 7 This example demonstrates that the anti-BTLA antibodies disclosed herein do not compete with HVEM or the HVEM / LIGHT complex for binding to cell surface BTLA.

[0123] 293c18 cell clone 1E4 stably expressing human BTLA was harvested with Accutase solution (Millipore Sigma / Sigma-Aldrich) and washed once with PBS, 1% BSA. 2 × 10 5 Cells were plated at 1000 x 1000 / well and placed on ice. Purified antibodies at 2x the indicated concentrations were serially diluted 3-fold in FACS buffer (PBS, 1% BSA, 0.02% sodium azide). Antibodies tested were a human IgG4 isotype control antibody specific for hen egg lysozyme, a reference BTLA IgG4 antagonist antibody, and 6G3 IgG4 (APE10840.06). Trimeric HVEM / LIGHT complexes were preformed by mixing equimolar (60 nM each) amounts of DyLight650-HVEM human IgG1 Fc and trimeric LIGHT-foldon-his and preincubating at room temperature for 15 min. Either DyLight650-HVEM-Fc (final concentration 100 nM; FIG. 6A) or preformed DyLight650-HVEM / LIGHT complexes (final concentration of HVEM / LIGHT, 30 nM each; FIG. 6B) were added to the diluted antibodies (final antibody concentrations as indicated) and incubated on ice for 15 min. Cells were centrifuged and gently resuspended in a mixture of antibody and HVEM (FIG. 6A) or antibody and HVEM / LIGHT complexes (FIG. 6B) and incubated on ice for 30 min. Cells were centrifuged, washed once with FACS buffer, and fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. Cells were washed once, resuspended in FACS buffer, and fluorescence was analyzed on a BD FACSArray (BD Biosciences). Median fluorescence intensity (MFI) was graphed with GraphPad Prism (GraphPad Software, Inc) and curves were fitted by least-squares fitting of log(agonist) versus response (3-parameter).

[0124] The 6G3 antibody (APE10840.06) disclosed herein does not compete with either HVEM-Fc binding (Figure 6A) or HVEM / LIGHT complex binding (Figure 6B) to cell surface BTLA. In the presence of 6G3 antibody, increased binding of HVEM-Fc to cell surface BTLA is observed (Figure 6A). The 6G3-dependent increase in HVEM binding is also observed with HVEM / LIGHT complex, but is more pronounced with HVEM-Fc alone (Figures 6A and 6B). The reference antagonist antibody shows concentration-dependent inhibition of HVEM and HVEM / LIGHT binding to BTLA (Figures 6A and 6B). An irrelevant isotype-matched IgG4 antibody does not affect HVEM or HVEM / LIGHT binding to BTLA.

[0125] Example 8 This example demonstrates that the epitopes on human BTLA to which the 6G3 and 10D8 antibodies disclosed herein bind are on the opposite side of BTLA to the HVEM binding site.

[0126] Recombinant human BTLA monomer was used to perform hydrogen-deuterium exchange mapping of peptides on BTLA bound by the 6G3 and 10D8 antibodies disclosed herein. The BTLA used in the experiment was a 6-his tag following amino acids 31-155 of hBTLA (UniProt ID# Q7Z6A9). Figures 7A, 7B, and 7C show a ribbon model of the crystal structure of the human BTLA extracellular domain in complex with a space-filling model of the crystal structure of the human HVEM extracellular binding domain (light grey) rendered in PyMOL from the Protein Data Bank structure (Compaan et al., J. Biol Chem 280:39553-39561 (2005)). FIG. 7A summarizes a hydrogen-deuterium exchange experiment with the 6G3 antibody (APE12839.05 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)), FIG. 7B summarizes a hydrogen-deuterium exchange experiment with the 10D8 antibody (APE11482.06), and FIG. 7C summarizes a hydrogen-deuterium exchange experiment with a reference BTLA antagonist antibody (APE10693.17). In each experiment, the BTLA / antibody mixture or BTLA alone was labeled with deuterium for 4, 10, or 60 minutes to determine the exchange rate. After incubation, the BTLA protein was rapidly enzymatically proteolyzed at acidic pH and deuterium incorporation into the resulting peptides was quantified by liquid chromatography-mass spectrometry.

[0127] Mapping showed the involvement of the following residues of BLTA: 6G3 combination Peptides 52-65 DPFELECPVKYCAN (SEQ ID NO: 227) Peptides 100 to 106 LHFEPVL (SEQ ID NO: 228) 10D8 bond Peptides 46-65 HSILAGDPFELECPVKYCAN (SEQ ID NO: 229) Peptides 82-91 LEDRQTSWKE (SEQ ID NO: 230) Peptides 100 to 106 LHFEPVL (SEQ ID NO: 231) Reference antagonist binding Peptides 39-41 YIK (SEQ ID NO: 232) Peptides 52 to 64 DPFELECPVKYCA (SEQ ID NO: 233) Peptides 100 to 106 LHFEPVL (SEQ ID NO: 228) Peptides 124-131 IESHSTTL (SEQ ID NO: 234)

[0128] Example 9 This example demonstrates that when BTLA and HVEM are expressed in the same cells, the 6G3 antibody disclosed herein inhibits LIGHT-induced HVEM signaling in an NF-κB luciferase reporter assay.

[0129] 293c18 cells were generated stably expressing full-length human BTLA, full-length human HVEM, and the pGL4.32-derived NF-κB luciferase reporter construct [luc2P / NF-κB-RE / Hygro] (Promega), single cell cloned, and designated huHVEM / huBTLA / NF-κB luciferase, clone 8. CHO-S cells were generated stably expressing full-length human LIGHT, and selected twice for highest LIGHT expression as a stable pool. huHVEM / huBTLA / NF-κB luciferase cells were harvested with Accutase solution (Sigma-Aldrich / Millipore Sigma), resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and plated (5 × 10 4 After 1 h incubation at 37° C. and 5% CO2, the indicated concentrations of 6G3 IgG4 (APE12839.07 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)), human IgG4 isotype control antibody specific for hen egg lysozyme, or human IgG4 reference anti-BTLA antagonist antibody were added to the cells. After 30 min incubation at room temperature, CHO-S LIGHT cells were harvested and added (0.55×10 4Cells / well; final condition, 9:1 HVEM-NF-κB responder:CHO-S LIGHT stimulator cells, which is an EC 50 After 4 h at 37 °C, 5% CO2, an equal volume of Steady-Glo Luciferase Assay System (Promega) was added to the wells and incubated for 10 min at room temperature. Samples were read for luminescence using an EnVision Multimode Plater Reader (PerkinElmer) with a measurement time of 0.1 s. Luminescence relative light units (RLU) were graphed and curves were fitted using a log(agonist) vs. response (3-parameter) least-squares fit in GraphPad Prism (GraphPad Software).

[0130] NF-κB luciferase reporter assays shown in Figure 8 demonstrate that the 6G3 antibody disclosed herein inhibits HVEM-dependent NF-κB signaling in response to CHO-S LIGHT in a concentration-dependent manner when BTLA and HVEM are expressed in the same cells, suggesting that under these conditions, the 6G3 antibody disclosed herein may promote the interaction between HVEM and BTLA in the same cells, thereby reducing LIGHT-driven HVEM-dependent NF-κB signaling.

[0131] NF-κB luciferase reporter assays shown in Figure 8 demonstrate that the reference antagonist antibody enhances LIGHT-mediated HVEM-dependent NF-κB signaling in a concentration-dependent manner in response to CHO-S LIGHT when BTLA and HVEM are expressed in the same cells. By disrupting the interaction of BTLA with HVEM in the same cells, the antagonist antibody can make more unbound HVEM molecules available for interaction with LIGHT, thereby increasing LIGHT-driven HVEM-dependent NF-κB signaling.

[0132] Example 10 This example demonstrates that the 6G3 antibody disclosed herein does not disrupt BTLA / HVEM complexes on the same cell surface in a fluorescence resonance energy transfer (FRET) assay.

[0133] In Figure 9A, 293c18 cell clones stably expressing full-length human BTLA and human HVEM were harvested with Accutase solution (Millipore Sigma / Sigma-Aldrich), washed with FACS buffer (PBS, 1% BSA, 0.02% sodium azide), and plated (2 × 10 5 Cells were centrifuged and 3-fold serially diluted antibodies at the indicated concentrations were added and cells were incubated for 1 hour at 4°C with gentle shaking. Antibodies tested were 6G3 IgG4 (APE12839.05 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)), a reference antagonist antibody IgG4, and a human IgG4P isotype control antibody specific for hen egg lysozyme. Cells were centrifuged, washed with FACS buffer and resuspended in FRET acceptor antibody APC-anti-HVEM (15 μg / mL; clone 122, BioLegend). The FRET donor antibody was biotin-humanized 10D8 (APE12774.02) and was pre-complexed at 10 μg / mL with 0.025 μg / mL streptavidin-Eu (LANCE Eu-W8044-streptavidin AD0060, PerkinElmer) by incubation at room temperature for 20 min. The donor complex, biotin-anti-BTLA / streptavidin-Eu, was added to the plate along with the acceptor, APC-anti-HVEM antibody, and incubated at 4°C for 24 h. The plate was washed with cold FACS buffer and the fluorescence of the samples was read on an EnVision Multimode Plater Reader (PerkinElmer, Santa Clara, CA). The ratio of fluorescence at 665 nm / 615 nm was graphed in GraphPad Prism (GraphPad Software). Each point is the mean ± SEM of three independent replicate experiments. Each sample condition in each experiment was in five replicate wells.

[0134] 9A demonstrates that BTLA / HVEM complexes are present on transfected 293c18 cells, and that the 6G3 antibody disclosed herein, like the isotype control antibody, shows no inhibition of the BTLA / HVEM FRET signal at concentrations up to 40 μg / mL, confirming that the 6G3 antibody disclosed herein does not inhibit the interaction of BTLA and HVEM on the same cell surface.

[0135] The FRET assay shown in Figure 9A shows that the reference antagonist antibody inhibited the FRET signal of the BTLA / HVEM complex in a concentration-dependent manner, with an IC 50 This demonstrates that the concentration of β-glucose (β-glucose) in the β-glucose (β-glucose)-containing IgG is approximately 0.65 μg / mL.

[0136] In Figure 9B, 293c18 cell clones stably expressing full-length human BTLA and human HVEM were harvested with Accutase solution (Millipore Sigma / Sigma-Aldrich), washed with FACS buffer, and plated (2 × 10 5The plates were then incubated at 37 °C for 20 min at 4 °C for 1 h (cells / well). Cells were centrifuged and 3-fold serially diluted FRET acceptor antibodies APC-anti-HVEM (clone 122, BioLegend) or APC-mouse IgG1 isotype control antibodies at the indicated concentrations were added to the plates. Biotinylated FRET donor antibodies were pre-complexed at 10 μg / mL with 0.025 μg / mL streptavidin-Eu (LANCE Eu-W8044-streptavidin AD0060, PerkinElmer) by incubation for 20 min at room temperature. The biotin-conjugated FRET donor antibodies tested were biotin-6G3 IgG4 (APE13124.01, which was biotin-conjugated APE12839.05 (H chain SEQ ID NO: 144, L chain SEQ ID NO: 174)), a biotin reference antagonist IgG4 antibody, and a biotin-human IgG4 isotype control antibody specific for hen egg lysozyme. The final concentrations of donor biotin-antibody / streptavidin-Eu complex in all wells were 0.3 μg / mL antibody and 0.75 ng / mL streptavidin-Eu. Plates were incubated at 4° C. for 24 hours, washed with cold FACS buffer, and the fluorescence of samples was read on an EnVision Multimode Plater Reader (PerkinElmer). The ratio of fluorescence at 665 nm / 615 nm was graphed in GraphPad Prism (GraphPad Software). Each point is the mean ± SEM of two replicate wells.

[0137] The FRET assay shown in Figure 9B demonstrates that in the presence of the biotin-6G3 antibody disclosed herein conjugated to streptavidin-Eu, increasing concentrations of APC-anti-HVEM generate a concentration-dependent increasing FRET signal. This example demonstrates that when the 6G3 antibody binds to BTLA on the cell surface, BTLA can still form a complex with HVEM on the same cell surface.

[0138] The FRET assay shown in Figure 9B demonstrates that in the presence of a biotin-reference antagonist antibody conjugated to streptavidin-EU, the FRET signal of the BTLA / HVEM complex is undetectable and similar to the biotin-isotype control antibody. This example demonstrates that the reference BTLA antagonist antibody disrupts the BTLA-HVEM complex on the same cell surface.

[0139] Example 11 This example demonstrates that when BTLA and HVEM are expressed in different cells, the 6G3 antibody disclosed herein partially inhibits BTLA-induced HVEM signaling in an NF-κB luciferase reporter assay.

[0140] 293c18 cells were generated stably expressing full-length human HVEM and NF-κB luciferase reporter constructs and designated HVEM / NF-κB luciferase, clone 11. 293c18 cells were generated stably expressing full-length human BTLA, single cell cloned, and designated huBTLA 293c18, clone 2. Human BTLA 293c18 cells were harvested with Accutase solution (Sigma-Aldrich / Millipore Sigma), resuspended in DMEM supplemented with 10% FBS, and plated (0.5 × 10 cells) in flat-bottom 96-well plates. 4 Cells were incubated at 100°C for 30 min with 5% CO2 and 10% TNF-κB (5 × 10 cells / well). The indicated concentrations of 6G3 IgG4 (APE13308.03) antibody produced from a pool of stably transfected CHO-K1 cells, a human IgG4 isotype control antibody specific for hen egg lysozyme, or an IgG4 reference anti-BTLA antagonist antibody were added to the cells. After a 30 min incubation at room temperature, HVEM / NF-κB cells were harvested and added (5 × 10 4After 5 h at 37°C, 5% CO2, an equal volume of Steady-Glo Luciferase Assay System (Promega) was added to the wells and incubated for 10 min at room temperature. Samples were read for luminescence in a GloMax Navigator Microplate Luminometer (Promega) with a measurement time of 0.3 s. Luminescence relative light units (RLU) were graphed and curves were fitted using a log(agonist) vs. response (3-parameter) least squares fit in GraphPad Prism (GraphPad Software).

[0141] The NF-κB luciferase reporter assay shown in Figure 10 demonstrates that the 6G3 antibody disclosed herein partially inhibits HVEM-dependent NF-κB signaling in a concentration-dependent manner in response to BTLA 293c18 cells when HVEM and BTLA are expressed on different cells. This suggests that under these conditions, the 6G3 antibody disclosed herein may partially inhibit HVEM signaling when BTLA is on different cells. The same results are obtained when human BTLA 293c18 cells are fixed with paraformaldehyde before incubation with the 6G3 antibody disclosed herein or when the cells are treated with an inhibitor of dynamin GTPase that blocks endocytosis.

[0142] NF-κB luciferase reporter assays shown in FIG. 10 demonstrate that when HVEM and BTLA are expressed in different cells, addition of a reference antagonist anti-BTLA antibody completely inhibits BTLA-mediated HVEM-dependent NF-κB signaling in a concentration-dependent manner.

[0143] Example 12 This example demonstrates direct BTLA agonist activity of the 6G3 antibody disclosed herein in a SHP2-recruiting PathHunter Jurkat BTLA signaling assay.

[0144] A clonal Jurkat cell line stably expressing β-galactosidase enzyme donor (ED)-tagged human BTLA and β-galactosidase enzyme acceptor (EA)-tagged human SHP2 was generated at Eurofins DiscoverX (Fremont, CA) and named Jurkat BTLA-ED SHP2-EA cells. For assay setup, Jurkat BTLA-ED SHP2-EA cells were harvested and plated (2 × 10) in 96-well plates. 4 Cells / well) were added to the cell assay plate. Antibody dilutions were prepared in a separate plate. Antibodies and proteins tested were 6G3 IgG4 (APE13308.03) antibody produced from a pool of stably transfected CHO-K1 cells, a human IgG4 isotype control antibody specific for hen egg lysozyme, a reference BTLA antagonist IgG4 antibody, and a soluble complex of human HVEM-IgG1 Fc / trimeric LIGHT (APE11989.16 and APE07872.05 at a molar ratio of 1 to 1.1). Antibodies and proteins were added to the cell assay plate and incubated for 2 hours at room temperature. PathHunter Bioassay Detection Reagent was added to all wells and incubated for 20 minutes at room temperature. Bioassay Detection Reagent 2 was then added to all wells and incubated for 1 hour at room temperature before measuring the luminescence signal with an integration time of 0.1 seconds in an EnVision Multimode Plater Reader (PerkinElmer). Data were graphed with GraphPad Prism (GraphPad Software). EC values ​​were calculated using an unconstrained sigmoidal dose-response curve fit with variable slope (4 parameters) and a least-squares fitting method. 50 Values ​​were calculated. Data represent the mean (± standard deviation) of triplicate samples for each point. Assays and data analysis were performed at Eurofins DiscoverX (Fremont, CA) under project ID: DRAX-ANAB-190724.

[0145] The SHP2-recruiting PathHunter Jurkat BTLA signaling assay shown in FIG. 11 demonstrates that the 6G3 antibody disclosed herein, as a soluble antibody, has direct BTLA agonist activity and inhibits concentration-dependent, low amplitude BTLA signaling (EC 50 = 125 ng / mL). By inducing recruitment of SHP2 to the BTLA cytoplasmic domain, the 6G3 antibody disclosed herein may function by initiating inhibitory signaling in activated T and B cells.

[0146] The SHP2-recruiting PathHunter Jurkat BTLA signaling assay shown in Figure 11 demonstrates that the reference BTLA antagonist antibody as a soluble antibody has direct BTLA agonist activity and suppresses concentration-dependent BTLA signaling (EC 50 We demonstrate that soluble HVEM / LIGHT complexes induce BTLA signaling at concentrations up to 1000 ng / mL (=28.8 ng / mL). In the SHP2-recruiting PathHunter Jurkat BTLA signaling assay shown in FIG. 11, soluble HVEM / LIGHT complexes induce a weak, concentration-dependent, non-saturating induction of BTLA signaling, likely due to the lower affinity of soluble HVEM for BTLA compared to the antibody.

[0147] Example 13 This example demonstrates that the 6G3 antibody disclosed herein does not inhibit HVEM-induced BTLA signaling in transfected U-2 OS cell lines in a SHP2-recruiting PathHunter Jurkat BTLA signaling assay.

[0148] A clonal Jurkat cell line stably expressing β-galactosidase enzyme donor (ED)-tagged human BTLA and β-galactosidase enzyme acceptor (EA)-tagged human SHP2 (Jurkat BTLA-ED SHP2-EA cells) and a U-2 OS osteosarcoma cell line stably expressing human HVEM (U-2 OS hHVEM cells) were generated at Eurofins DiscoverX (Fremont, CA). For the assay setup, Jurkat BTLA-ED SHP2-EA cells were harvested and plated (2 × 10) in 96-well plates. 4 Antibody dilutions were prepared in a separate plate. Antibodies tested were 6G3 IgG4 (APE13308.03) antibody produced from a pool of stably transfected CHO-K1 cells, a human IgG4 isotype control antibody specific for hen egg lysozyme, and a reference BTLA antagonist IgG4 antibody. The indicated concentrations of antibodies were added to the assay plate and incubated for 1 hour at 37°C in a humidified incubator with 5% CO2. U-2 OS hHVEM cells were harvested, resuspended, and added (5x10) to the assay plate along with Jurkat BTLA-ED SHP2-EA cells. 4 The wells were incubated at room temperature for 2 hours (cells / well). PathHunter Bioassay Detection Reagent was added to all wells and incubated at room temperature for 30 minutes. Bioassay Detection Reagent 2 was then added to all wells and incubated at room temperature for 1 hour before measuring the luminescence signal with an integration time of 0.1 seconds on an EnVision Multimode Plater Reader (PerkinElmer). Data were graphed in GraphPad Prism (GraphPad Software); IC was calculated using an unconstrained sigmoidal dose-response curve fit with variable slope (4 parameters) and least squares fitting. 50 Values ​​were calculated. Data represent the mean (± standard deviation) of triplicate samples for each point. Assays and data analysis were performed with Eurofins DiscoverX (Fremont, CA) under project ID: DRX-ANAB-191210.

[0149] The SHP2 recruitment PathHunter Jurkat BTLA signaling assay shown in FIG. 12 demonstrates that the 6G3 antibody disclosed herein does not affect SHP2 recruitment to BTLA induced by HVEM in different cells.

[0150] The SHP2-recruited PathHunter Jurkat BTLA signaling assay shown in FIG. 12 demonstrates that the reference BTLA antagonist antibody potently and concentration-dependently inhibits HVEM-induced BTLA signaling in different cells (IC 50 =8.9ng / mL).

[0151] Example 14 This example demonstrates increased direct BTLA agonist activity of the 6G3 antibody disclosed herein in a SHP2-mobilized PathHunter Jurkat BTLA signaling assay in which U-2 OS cells transfected with FcγRIa (CD64a) were added to provide FcγR engagement.

[0152] A clonal Jurkat cell line stably expressing β-galactosidase enzyme donor (ED)-tagged human BTLA and β-galactosidase enzyme acceptor (EA)-tagged human SHP2 (Jurkat BTLA-ED SHP2-EA cells) and a U-2 OS osteosarcoma cell line stably expressing human FcγRIa / CD64a (U-2 OS hFcγRIa cells) were generated at Eurofins DiscoverX (Fremont, CA). U-2 OS hFcγRIa cells were harvested and plated (1 × 10) in 96-well plates. 4Antibody dilutions were prepared in a separate plate and added to the assay plate and incubated for 1 hour in a humidified incubator at 37°C, 5% CO2. Antibodies tested were 6G3 IgG4 (APE13308.03) antibody produced from a pool of stably transfected CHO-K1 cells, a human IgG4 isotype control antibody specific for hen egg lysozyme, and a reference BTLA antagonist IgG4 antibody. Jurkat BTLA-ED SHP2-EA cells were harvested and added (2x106 cells / well) to the assay plate along with U-2 OS hFcγRIa cells. 4 The plates were incubated at room temperature for 2 hours. PathHunter Bioassay Detection Reagent was added to all wells and incubated at room temperature for 30 minutes. Bioassay Detection Reagent 2 was added to all wells and incubated at room temperature for 1 hour before measuring the luminescence signal with an integration time of 0.1 seconds on an EnVision Multimode Plater Reader (PerkinElmer). Data were graphed in GraphPad Prism (GraphPad Software); an unconstrained sigmoidal dose-response curve fit with variable slope (4 parameters) was used, and EC 50 Values ​​were calculated. Data represent the mean (± standard deviation) of triplicate samples for each point. Assays and data analysis were performed with Eurofins DiscoverX (Fremont, CA) under project ID: DRX-ANAB-191210.

[0153] The SHP2-recruiting PathHunter Jurkat BTLA signaling assay shown in FIG. 13 demonstrates that in the presence of cells that provide FcγRIa engagement, the 6G3 antibody disclosed herein as a soluble antibody has enhanced direct BTLA agonist activity and induces BTLA signaling in a concentration-dependent manner (EC 50 =9.3ng / mL).

[0154] The SHP2-recruiting PathHunter Jurkat BTLA signaling assay shown in FIG. 13 demonstrates that in the presence of cells that provide FcγRIa engagement, the reference BTLA antagonist antibody as a soluble antibody has direct BTLA agonist activity and induces BTLA signaling in a concentration-dependent manner (EC 50 =3.9ng / mL).

[0155] The SHP2 recruitment PathHunter Jurkat BTLA signaling assay shown in Figure 13 demonstrates that both the 6G3 antibody disclosed herein and the reference BTLA antagonist antibody exhibit increased potency of agonist activity in the presence of cell-associated FcγRIa (approximately 13-fold more potent for 6G3; approximately 7.4-fold more potent for the reference BTLA antagonist) compared to the Jurkat BTLA signaling assay without FcγRIa (compare Figures 11 and 13). Although the soluble 6G3 antibody disclosed herein can induce SHP2 recruitment to the BTLA cytoplasmic domain and initiate inhibitory signaling in activated T and B cells, the potential of 6G3 antibody bound to FcγRIa to directly induce inhibitory signaling may be enhanced.

[0156] Example 15 This example demonstrates that the 6G3 antibody disclosed herein, when administered at 1 mg / kg, 3 mg / kg, or 10 mg / kg twice weekly for four weeks, shows efficacy in vivo in a xenogeneic NSG / Hu-PBMC graft-versus-host disease (GvHD) model.

[0157] A xenogeneic NSG / Hu-PBMC GvHD model testing the efficacy of the 6G3 anti-BTLA antibody disclosed herein was performed at The Jackson Laboratory JAX® In Vivo Pharmacology Services (Sacramento, Calif.). As shown in FIG 14A, NOD-scid IL2rγ null (NSG) mice were irradiated with 1 Gy, and then each mouse was 6Human PBMCs were injected intravenously. Antibodies (human IgG4 isotype control antibody specific for hen egg lysozyme and 6G3 IgG4 APE13308.05 antibody produced from a pool of stably transfected CHO-K1 cells) were administered intraperitoneally twice weekly for 4 weeks starting the day after PBMC injection. The 6G3 antibody disclosed herein was administered at either 1 mg / kg, 3 mg / kg, or 10 mg / kg, and the human IgG4 isotype control antibody was administered at 10 mg / kg. There were 12 mice / group for each antibody treatment group. The belatacept biosimilar positive control was administered intraperitoneally at 75 μg / mouse three times weekly for 4 weeks. The belatacept biosimilar treatment group had 8 mice. The dosing regimen and treatment groups in this study are shown in FIG. 14B. Disease was monitored three times a week for weight loss, death, and GvHD scores (measured weight loss, activity, coat, pallor, and posture) over the 42-day study period. Animals with more than 10% weight loss were monitored daily for disease, and animals with more than 20% weight loss from starting weight were euthanized. Survival included animals confirmed dead and animals removed from the study due to endpoints defined in The Jackson Laboratory JAX® In Vivo Pharmacology Services study protocol. Survival data were graphed in GraphPad Prism (GraphPad Software) using Kaplan-Meier survival analysis, which allowed the calculation of median survival times for each group. Statistical significance of treatment groups in pairwise comparisons with isotype control groups was determined in GraphPad Prism using the Gehan-Breslow-Wilcoxon test to calculate p-values.

[0158] The survival results for the GvHD study shown in FIG. 14C demonstrate that the 6G3 anti-BTLA antibody disclosed herein exhibits a highly statistically significant effect in extending survival at all doses tested compared to the isotype control antibody. The efficacy of the 6G3 antibody was dose-responsive, with the 1 mg / kg dose treatment group exhibiting shorter survival times compared to the 3 mg / kg and 10 mg / kg treatment groups. Median survival times over the study period were 16 days for the isotype control group, 35 days for the 1 mg / kg 6G3 treatment group, and equivocal for the 3 mg / kg and 10 mg / kg 6G3 treatment groups. A reference antagonist anti-BTLA antibody was administered at 10 mg / kg as a secondary control. No survival benefit was observed in animals treated with the reference antagonist antibody compared to animals treated with the isotype control.

[0159] Example 16 This example describes the qualification of a 96-well electrochemiluminescence (ECL) sandwich assay to detect BTLA following 6G3 IgG4 anti-BTLA antibody administration in cynomolgus monkey serum.

[0160] During method development, assay parameters such as capture reagent concentration, MRD, assay buffer type, and detection reagent concentration were established. Method qualification of the assay was then performed by assessing intra- and interassay precision and accuracy, dilution linearity, specificity, and freeze-thaw stability. The final method will be used to analyze samples from non-GLP single- or multiple-dose PK, TK, and tolerability studies in cynomolgus monkeys.

[0161] A 96-well MSD Standard Bind assay plate (MSD part number L15XA-3) was coated with 50 μL of 1.0 μg / mL anti-BTLA clone 10D8 (APE10134) overnight at 4 °C. The next day, the coated plate was washed three times with 1x PBST and blocked with 250 μL of blocking buffer for 60-120 min. The calibration curve range of BTLA was 500-7.8 ng / mL, with a quantification range of 500-7.8 ng / mL. Standards were prepared using a two-fold dilution series with 100% cynomolgus serum. Quality controls (QCs) of five different concentrations spanning the quantification range with 100% cynomolgus serum were also prepared and frozen. All standards, samples, and controls were then diluted in assay buffer containing 100 μg / mL of 6G3 IgG4 at a minimum required dilution (MRD) of 1:10 and incubated on a shaker at room temperature for 1 h. After blocking, the plate was washed with 1x PBST and 50 μL of diluted standards, samples, and QCs were incubated on a shaker at 400–500 rpm for 2 h at room temperature. After sample incubation, the plate was washed three times with 1x PBST and 50 μL of 0.25 μg / mL biotinylated anti-BTLA polyclonal detection antibody (PA5-95592, ThermoFisher) was added to each well and incubated on a shaker at room temperature for 1 h. The plate was then washed three times with 1x PBST. Next, 50 μL of 0.2 μg / mL Streptavidin-SulfoTag secondary detection reagent per well was added to each well and incubated on a shaker at room temperature for 30 min. The plate was then washed a final three times with 1x PBST. 150 μL of 2x MSD Read Buffer T per well was added to each well and the plate was read on an MSD Quickplex Plate Reader. Replicates of standards, samples, and controls were tested in duplicate and serum concentrations were back-calculated based on the reference standard curve of the lead candidate using SoftMax Pro 7.01 and a four-parameter curve fit with 1 / y^2 weighting.

[0162] Before starting the method qualification, serum matrix interference for the 6G3 IgG4 antibody was evaluated. No matrix effects were observed and a minimum required dilution (MRD) of 1:10 was chosen.

[0163] Antibody 10D8 (APE10134) was used as a capture antibody. It was found that the binding of 10D8 to BTLA was increased in the presence of 6G3 IgG4 antibody, possibly through conformational changes of BTLA upon binding to 6G3 IgG4 in cynomolgus serum. To normalize the putative conformational changes of BTLA, 100 μg / mL of 6G3 was added to the dilution buffer, and all standards, samples, and controls were diluted according to the MRD of 1:10 and incubated on a shaker at room temperature for 1 h before adding the samples to the 10D8-coated MSD plate.

[0164] Standard curves of 6G3 IgG4 at 500 μg / mL, 50 μg / mL, 5 μg / mL, 0.5 μg / mL, and 0 μg / mL were diluted with assay buffer containing 100 μg / mL 6G3 IgG4 at MRD 1:10 to assess the (%RE) and precision (%CV) of BTLA. All five standard curve conditions incubated with 100 μg / mL 6G3 IgG4 had standard concentrations within the quantitative assay range and met the acceptance criteria of mean recovery (%RE) concentrations within 20% of the nominal concentration. The mean precision (%CV) results met the acceptance criteria and did not exceed 20% for any sample. The BTLA did not exceed a total percent error (%TE) of 30%.

[0165] Inter-assay accuracy (%RE) and precision (%CV) were evaluated in six assay runs performed on two different days and included five levels of QCs defining the calibration curve and quantification range for each plate. The five levels of QCs evaluated were ULOQ (500 ng / mL), HQC (400 ng / mL), MQC (62.4 ng / mL), LQC (15.6 ng / mL), and LLOQ (7.8 ng / mL). A total of three independent replicates were analyzed in duplicate at each QC level per run. The mean accuracy (%RE) results for all control and standard concentrations within the quantification assay range met the acceptance criterion of mean recovered concentrations within 20% of the nominal concentration. The mean precision (%CV) results met the acceptance criterion and did not exceed 20% for any sample. At any concentration within the quantitative range of the assay, BTLA never exceeded a percent total error (%TE) of 30% for either the standard curve or the quality controls.

[0166] The intra-assay accuracy (%RE) and precision (%CV) of the method were assessed in a single run of six independent replicates of five levels of QC. The mean control accuracy (%RE) results met the acceptance criterion that the mean recovered concentration was within 20% of the nominal concentration. The mean precision (%CV) results met the acceptance criterion and did not exceed 20%.

[0167] To demonstrate that high concentrations of test article can be diluted into the quantification range and that the assay does not have a front-zone effect, dilution linearity was evaluated by performing various dilutions on samples spiked with BTLA at concentrations above the ULOQ. Samples were diluted into the quantification range of the assay and back-calculated concentrations were evaluated. The average results show that it is possible to dilute samples into the quantification range with dilution factors of 1:1600 or less. There was no front-zone "hook effect" at the concentrations evaluated.

[0168] The results demonstrate that the assay has the sensitivity and reproducibility to assess BTLA concentrations in serum collected from a study of cynomolgus monkeys dosed with 6G3 IgG4.

[0169] Example 17 The following example describes the use of an electrochemiluminescence (ECL) sandwich assay to quantitatively determine soluble BTLA in cynomolgus monkey serum and NOD scid mouse plasma.

[0170] 96-well MSD Standard Bind assay plates (MSD part number L15XA-3) were coated overnight at 4°C with 50 μL of 1.0 μg / mL anti-BTLA capture reagent clone 10D8 (APE10134). The next day, the coated plates were washed three times with 1×PBST and blocked with 250 μL of blocking buffer for 60–120 min. The calibration curve range of sBTLA was 7.8–500 ng / mL and the quantification range was 7.8–500 ng / mL in cynomolgus monkey serum, while the calibration and quantification ranges were both 2.0–1000 ng / mL in CD1 mouse plasma. Standards were prepared using a two-fold dilution series in 100% species-specific matrix. Quality controls (QCs) spanning the quantification range in 100% species-specific matrix at five different concentrations were also prepared and frozen. All standards, samples, and controls were then diluted at a minimum required dilution (MRD) of 1:10 in assay buffer containing 100 μg / mL 6G3 and incubated for 1 hour at room temperature on a shaker.

[0171] After blocking, the plate was washed with 1x PBST and 50 μL of diluted standards, samples, and QCs were incubated on a shaker at 400-500 rpm for 2 hours at room temperature. After sample incubation, the plate was washed 3 times with 1x PBST and 50 μL of 0.25 μg / mL biotinylated anti-BTLA clone PA5-95592 (VC2963104B) detection antibody was added to each well and incubated on a shaker at room temperature for 1 hour. The plate was then washed 3 times with 1x PBST. Next, 50 μL of 0.2 μg / mL Streptavidin-SulfoTag secondary detection reagent per well was added to each well and incubated on a shaker at room temperature for 30 minutes. The plate was then washed a final 3 times with 1x PBST. 150 μL of 2x MSD Read Buffer T per well was added to each well and the plate was read on an MSD Quickplex Plate Reader. Replicates of standards, samples, and controls were run in duplicate and concentrations were back-calculated based on a BTLA reference standard curve using SoftMax Pro 7.01 and a 4-parameter curve fit with 1 / y^2 weighting.

[0172] NOD Scid mice (n=19) engrafted with human PBMC were dosed with 6G3 IgG4 antibody (APE13308) in three dose groups (1 mg / kg, 3 mg / kg, and 10 mg / kg IP). Animals dosed with isotype control IgG4 (10 mg / kg) or CTLA-4-Ig (75 μg) served as controls. Animals were dosed twice weekly and plasma samples were collected at the midpoint of the study via cardiac bleeding. Results are presented in FIG. 15. Additionally, circulating human T cells were analyzed by flow cytometry to characterize the expression of BTLA, enumerate human T cells, and characterize the activation marker CD25. Results are shown in FIG. 19. 6G3 IgG4 reduced BTLA expression on human T cells at all doses, inhibited T cell expansion in a dose-dependent manner, and reduced expression of the activation marker CD25.

[0173] Blood samples were analyzed from cynomolgus monkeys from two studies. The first study (n=180) consisted of three dosing groups (10 mg / kg IV; 10 mg / kg SC; and 1 mg / kg SC). All animals received a single dose of 6G3 IgG4 either IV or SC, and blood samples were collected from all animals in all groups pre-dose, 3, 6, 12, 24, 48, 72, 96, 168, 240, 336, 504, 672, and 840 hours post-dose. Pre-dose samples and animals that received vehicle control served as controls. Results are presented in FIG. 16.

[0174] In the second study, cynomolgus monkeys (n=380) were administered 6G3 in four dose groups (10 mg / kg, 50 mg / kg, and 100 mg / kg SC, and 100 mg / kg IV). All animals received weekly doses of 6G3 IgG4 either IV or SC on days 1, 8, and 15, and blood samples were collected from all animals in all groups on days 1, 8, and 15: pre-dose, 3 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours post-dose. Results are presented in FIG. 17. Serum levels of 6G3 IgG4 were also measured. Results are presented in FIG. 18.

[0175] No measurable levels of sBTLA were detected in samples from human PBMC-engrafted NOD scid mice treated with IgG4 isotype control or CTLA-4-Ig, or in pre-dose or vehicle control treated samples from cynomolgus monkeys from the cynomolgus monkey study. Shed sBTLA was detected in serum samples from all cynomolgus monkeys treated with 6G3 IgG4 and in all plasma samples from human PBMC-engrafted NOD scid mice, except for the lowest dose group (1 mg / kg) in the mouse study. This result suggests that BTLA is shed from the surface of B and T cells and that sBTLA serves as a pharmacodynamic marker of 6G3 IgG4 activity in vivo.

[0176] Example 18 This example demonstrates that 6G3 IgG4 achieves receptor occupancy and reduces BTLA expression on T and B cells in cynomolgus monkeys.

[0177] In a dose ranging study (DRFS), the effect of multiple doses of 6G3 IgG4 was analyzed using flow cytometry in peripheral blood of cynomolgus monkeys.Animals in four different treatment groups (10 mg / kg SC, 50 mg / kg SC, 100 mg / kg SC, 100 mg / kg IV) and one control group were administered 6G3 IgG4.

[0178] 6G3 IgG4 did not induce any significant changes in the absolute numbers or distribution of T, B, and NK cells compared to vehicle control test animals. Binding of the fluorescently labeled drug (6G3 IgG4-DyL488) was inhibited in all animals administered 6G3 IgG4 compared to the vehicle control treatment group, demonstrating that BLTA receptor occupancy by 6G3 IgG4 was greater than 80% on T cells and approximately 70% on B cells, as shown in Figure 20.

[0179] All animals treated with 6G3 IgG4 had reduced BTLA surface expression on T and B cells, as detected with a fluorochrome-labeled non-competitive anti-BTLA antibody (clone 10D8), compared to vehicle control treated animals. As shown in Figure 20, surface expression of BTLA on T and B cells was reduced by approximately 75% and 50%, respectively.

[0180] We found that the rate of BTLA expression in T and B cells decreased relatively slowly, whereas receptor occupancy in T and B cells occurred very rapidly, suggesting that BTLA is shed by a 6G3 IgG4-dependent mechanism. Two animals that developed anti-drug antibodies (ADA) showed a slight decrease in receptor occupancy and recovery of BTLA expression in T and B cells.

[0181] Example 19 This example demonstrates that the BTLA extracellular domain (ECD) is cleaved by serine protease 3 at a proposed recognition site near the transmembrane domain.

[0182] Proteinase 3 (PR3) is a serine protease in neutrophils that is released into the extracellular space upon neutrophil activation. PR3 has previously been shown to cleave the checkpoint receptor T-cell immunoglobulin and mucin domain 3 (TIM-3) and reduce the levels of TIM-3 on the cell surface.

[0183] Six different lanes were incubated with purified recombinant PR3 and / or BTLA-ECD for up to 60 minutes and consisted of: (1) PR3 control, (2) PR3 60m control, (3) BTLA control, (4) PR3+BTLA 0m, (5) PR3+BTLA 30m, and (5) PR3+BTLA 60m.

[0184] After co-incubation, soluble BLTA-ECD was cleaved into multiple smaller fragments, demonstrating the ability of PR3 to cleave BTLA. Sequence analysis suggested at least one PR2 recognition motif toward the C-terminus of BTLA-ECD.

[0185] Example 20 This example demonstrates that 6G3 IgG4 reduces T cell proliferation and BTLA surface expression in healthy control (HC) and atopic dermatitis (AD) donors.

[0186] PBMCs from HC or AD donors were labeled with 0.5 μM CFSE and then stimulated with soluble anti-CD3 (0.5 ng / mL, Biolegend, Cat. No. 300332) and soluble anti-CD28 (0.5 ng / mL, Biolegend, Cat. No. 302943) in the presence or absence of 100 nM 6G3 IgG4 or isotype control (IgG4-HyHel) for 72 h. Proliferating cells were determined by CFSE dilution.

[0187] FIG. 21A shows CD3 + CFSE histograms of HC and AD donors are provided, shown as histograms of T cells overlaid with isotype control. Figure 21B shows the T cell proliferation reduction rate (left) and mitotic index (right) during proliferation. Mitotic index is the sum of the number of divisions in each generation divided by the original number of cells, calculated by the automated cell cycle proliferation analysis in NovoExpress software. IFNγ levels in PBMC culture supernatants of HC and AD donors were measured by Mesoscale MDS assay after stimulation with anti-CD3 and anti-CD28 in the presence or absence of 100 nM 6G3 IgG4 or isotype control for 72 hours. Results are shown in Figure 21C. Figure 21D shows the CD3 and CD4 levels of HC and AD donors as determined by AF647-conjugated anti-BTLA (clone #10D8, AnaptysBio.) + BTLA surface expression on T cells (plotted as mean fluorescence intensity (MFI)) is shown.

[0188] The results show that 6G3 IgG4 reduces T cell proliferation and BTLA surface expression compared to controls.

[0189] Example 21 This example demonstrates that 6G3 IgG4 achieves high BTLA receptor occupancy at safe doses in human patients.

[0190] The study randomized / treated 96 subjects, including a single ascending dose (SAD) cohort of 72 and a multiple ascending dose (MAD) cohort of 24. The SAD cohort received a single dose of 7 mg, 20 mg, 70 mg, 200 mg, 400 mg, or 800 mg via either intravenous (IV) or subcutaneous (SC) administration. The MAD cohort received 70 mg, 200 mg, or 400 mg SC once weekly for 4 weeks. Each cohort included 8 subjects, 6 received the 6G3 IgG4 antibody and 2 received a placebo. 71 of 72 SAD subjects completed the study and 21 of 24 MAD subjects completed the study. Treatment was well tolerated and no safety signals were observed.

[0191] BTLA receptor occupancy was determined on CD3+ T cells and CD19+ B cells. Some results are presented in Figure 22A and B. Receptor occupancy was found to be maximal at 7 mg IV or 20 mg SC with increasing duration in a dose-dependent manner. Single doses of 200 mg IV and 400 mg SC resulted in >90% receptor occupancy sustained for >30 days. Maximal receptor occupancy can be maintained with multiple SC doses as demonstrated in the MAD cohort.

[0192] Example 22 This example demonstrates that the 6G3 antibody disclosed herein exhibits efficacy in vivo in a graft-versus-host disease (GvHD) model when administered as a single 10 mg / kg dose or at 3 mg / kg every other week for 4 weeks.

[0193] 1x10 for mouse 7 Human peripheral blood mononuclear cells (PBMCs) were transplanted. 10 mg / kg or 3 mg / kg of 6G3 antibody or IgG4 isotype control were then injected either as a single dose (SD) or every other week (BiW) for 4 weeks. A control group of untreated animals was also included to monitor engraftment levels and disease progression, excluding any treatment or placebo injections.

[0194] Animals treated with a single 10 mg / kg dose of 6G3 or 3 mg / kg BiW for 4 weeks showed a clear survival benefit up to day 46. Only one animal died from illness in each treatment group (SD or BiW), and all other animals survived to study day 46 (n=11). The median survival time for animals treated with a single 10 mg / kg dose of the IgG4 isotype control was 20.5 days, and 20 days for those treated with 3 mg / kg BiW. The median survival time for untreated control animals was 23 days. The results are shown in Figures 23A and 23B.

[0195] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0196] In the context of the description of the invention (particularly in the context of the claims which follow), use of the terms "a" and "an" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. Use of the term "at least one" after a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise specified herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention, and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0197] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of skill in the art, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A BTLA conjugate for suppressing the immune response and treating autoimmune or inflammatory diseases or conditions, (i) It is administered at a weekly dose of at least 7 mg IV; (ii) Administered at a weekly SC dose of at least 20 mg; (iii) administered at least once every 30 days in a single IV dose of at least 150 mg; or (iv) Administered as a single subcutaneous dose of at least 300 mg once every 30 days; (a) X １ SX ２ MN (Sequence No. 195) (wherein X １ is N or T, and X ２ CDRH1 (which is W, F, H, G, P, R, K, D, S, L, V, N, or Y); (b) RIYPX １ GX ２ X ３ DTNYX ４ GKFK (Sequence No. 196) (In the formula, X １ either does not exist or is A; X ２ is D, Y, Q, G, L, F, H, S, P, R, or T; X ３ is G, Y, A, F, S, D, V, T, E, K, or R; X ４ (The letters are N, V, Q, R, A, F, Y, S, G, P, or T.) CDRH2 including; and (c) X １ SGTFX ２ X ３ GNYX ４ X ５ YFDV (Sequence No. 197) (In the formula, X １ is either K or R; X ２ is N or D; X ３ is D, S, F, Y, F, V, S, G, T, R, I, L, or E; X ４ is R or H; X ５ (The letters are W, R, F, L, N, Y, P, I, V, A, S, G, R, or K.) CDRH3 including; Including; or any one immunoglobulin heavy chain variable region of SEQ ID NOs. 43 to 156 or at least their CDRs; or an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with respect to them; (a) RX １ SENIYX ２ X ３ LA (Sequence No. 198) (In the formula, X １ is A or V; X ２ is either S or N; X ３ (This is H, N, or Y) CDRL1 including; (b) X １ AX ２ NLAX ３ (Sequence No. 199) (In the formula, X １ is either A or N; X ２ is either T or K; X ３ (The letters are N, L, Q, G, F, V, K, S, R, T, H, or P.) CDRL2 including; and (c) QX １ FX ２ GPPLT (Sequence ID 200) (In the formula, X １ is either L or H; X ２ (The letters are W, F, Y, P, N, V, K, M, L, G, or S.) CDRL3 including; Including; or an immunoglobulin light chain variable region of any of SEQ ID NOs. 157 to 192 or at least their CDRs; or an immunoglobulin light chain variable region containing an amino acid sequence having at least 90% sequence identity with respect to them; BTLA binders, Alternatively, the BTLA binders listed in Table 3.

2. The immunoglobulin heavy chain polypeptide has the following sequence: QVQLVQSGAEVKKPGSSSVKVSCKASGYX １ FSX ２ SX ３ MNWVRQAPGQGLEWMGRIYPX ４ GX ５ X ６ DTNYX ７ GKFKGRVTITADKX ８ TX ９ TAYMELX １０ SLR SEX １１ TAVX １２ YX １３ CAX １４ SGTFX １５ X １６ GNYX １７ X １８ YFDVWGKGTTVTVSSA (Sequence No. 193) (In the formula, X １ is A or V; X ２ is N or T; X ３ is W, F, H, G, P, R, K, D, S, L, V, N, or Y; X ４ It either does not exist or is A; X ５ is D, Y, Q, G, L, F, H, S, P, R, or T; X ６ is G, Y, A, F, S, D, V, T, E, K, or R; X ７ is N, V, Q, R, A, F, Y, S, G, P, or T; X ８ is either S or F; X ９ is S, T, or N; X １０ is either S or R; X １１ is either D or V; X １２ is either nonexistent or Y; X １３ is either Y or F; X １４ is either K or R; X １５ is N or D; X １６ is D, S, F, Y, F, V, S, G, T, R, I, L, or E; X １７ is R or H; X １８ (The letters are W, R, F, L, N, Y, P, I, V, A, S, G, R, or K.) The BTLA binder according to claim 1, comprising:

3. The BTLA binder according to claim 1, wherein the immunoglobulin heavy chain polypeptide comprises one or at least one of SEQ ID NOs: 43 to 156; or an amino acid sequence having at least 90% sequence identity with respect to thereto.

4. The immunoglobulin light chain polypeptide has the following sequence: X １ IQX ２ TQSPSSLSSASVGDRVTITCRX ３ SENIYX ４ X ５ LAWYQQKX ６ GKAPKLLIYX ７ AX ８ NLAX ９ GVPSRFSGSGSGTDX １０ TLTISSLQPEDFATYYCQX １１ FX １２ GPPLTFGGGTKVEIKR (Sequence ID 194) (In the formula, X １ is either A or D; X ２ is L or M; X ３ is A or V; X ４ is S or N; X ５ is H, N, or Y; X ６ is either P or Q; X ７ is either A or N; X ８ is either T or K; X ９ is N, L, Q, G, F, V, K, S, R, T, H, or P; X １０ is either F or Y; X １１ is either L or H; X １２ (These are W, F, Y, P, N, V, K, M, L, G, S) The BTLA binder according to claim 1, comprising:

5. The BTLA binder according to claim 1, wherein the immunoglobulin light chain polypeptide comprises any or at least the CDRs of SEQ ID NOs: 157 to 192; or an amino acid sequence having at least 90% sequence identity with respect to thereto.

6. (a) CDRH1 containing Sequence ID No. 201; (b) CDRH2 containing Sequence ID No. 202; (c) CDRH3 containing Sequence ID No. 203: (d) CDRL1 containing sequence number 204; (e) CDRL2 containing Sequence ID No. 205; and (f) CDRL3 containing sequence number 206 The BTLA binder according to claim 1, comprising:

7. The BTLA binder according to claim 1, comprising the immunoglobulin heavy chain variable region of SEQ ID NO: 144 or at least its CDR; and the immunoglobulin light chain variable region of SEQ ID NO: 174 or at least its CDR; or comprising the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region of SEQ ID NO: 174 having 90% or more sequence identity with respect to SEQ ID NO:

144.

8. The BTLA conjugate according to any one of claims 1 to 7, wherein the disease is rheumatoid arthritis, graft-versus-host disease, psoriasis, or inflammatory bowel disease.

9. A method for selecting a subject for treatment with a BTLA binder, comprising: detecting soluble BTLA (sBTLA) in a blood, plasma, serum, or tissue sample from the subject; and selecting the subject for treatment if the sBTLA concentration in the sample from the subject is lower than the sBTLA level of a normal, non-disease subject of the same type.

10. The method according to claim 9, further comprising comparing the concentration of sBTLA in the blood, plasma, serum, or tissue sample with a reference sBTLA concentration.

11. The method according to claim 10, wherein the reference sBTLA concentration is the concentration of sBTLA in the blood, plasma, serum, or tissue of another normal, non-disease subject of the same species; or is a reference sBTLA concentration established by statistical analysis of sBTLA concentrations in the blood, plasma, serum, or tissue of a population of such subjects.

12. The method according to claim 9, further comprising the step of administering a BTLA binder to the subject.

13. The method according to claim 12, wherein the BTLA binder comprises the immunoglobulin heavy chain variable region of SEQ ID NO: 144 or at least its CDR; and the immunoglobulin heavy / light chain variable region of SEQ ID NO: 174 or at least its CDR; or comprises an immunoglobulin heavy chain variable region having 90% or more sequence identity with SEQ ID NO: 144 and an immunoglobulin light chain variable region having at least 90% or more sequence identity with SEQ ID NO:

174.

14. The method according to any one of claims 9 to 13, wherein the subject has an autoimmune or inflammatory disease.

15. The method according to claim 14, wherein the disease is rheumatoid arthritis, graft-versus-host disease, psoriasis, or inflammatory bowel disease.

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