EA043600B1 - BTLA FUSION PROTEINS AGONISTS AND THEIR APPLICATIONS - Google Patents

Abstract

The invention is based on the breakthrough discovery that BTLA agonist fusion proteins modulate the immune response. Specifically, the invention relates to fusion proteins that bind BTLA, enhancing BTLA signaling. The invention further relates to methods for treating cancer and immune and inflammatory diseases and disorders using a BTLA agonist fusion protein, as described herein.

Description

Cross reference to related applications

This application claims priority under 35 USC 119(e) to the US patent application.

No. 62/187105, filed June 30, 2015. The disclosure of the prior application is deemed to be a part of, and is incorporated by reference in its entirety, the disclosure of this application.

Governmental support

This invention was made in part with government support through grant no. R01CA164679 from the National Institutes of Health. The US Government has certain rights to this invention.

Including a sequence list

The material in the accompanying sequence listing is incorporated by reference into this application. The attached text file with a list of sequences, called BURN1680_1WO_ Sequence_Listing, was created on June 29, 2016, and has a size of 19 kb. The file can be evaluated using Microsoft Word on a computer that uses Windows OS.

Field of invention

The invention generally relates to fusion proteins and more particularly to the development and use of BTLA agonist fusion proteins and their use for modulating the immune response and treating diseases and disorders.

BACKGROUND OF THE INVENTION

The tumor necrosis factor (TNF) receptor herpes virus entry mediator (HVEM; TNFRSF14) is a major focus for manipulation by viral pathogens, and for mutation in cancers and autoimmune diseases. In humans, HVEM interacts with the TNF, LIGHT and lymphotoxin α superfamily of cytokines and the immunoglobulin (Ig) family containing B and T lymphocyte attenuator (BTLA) and CD160 receptors. Membrane-bound LIGHT, BTLA, and CD160 all activate the NF-κB downstream signaling pathway of HVEM upon receptor interaction, while HVEM activates BTLA and CD160 receptors, leading to bidirectional signaling between neighboring cells. Cells coexpressing HVEM and BTLA or CD 160 may also form membrane complexes of these proteins that prevent these receptors from being accessible to extracellular ligands due to steric hindrance.

BTLA-mediated inhibition has been shown to regulate a number of different cellular signaling pathways, including antigen receptor signaling in T and B cells. In T cells, BTLA was first shown to be involved in inhibitory signal transduction pathways, including activation of SH2 domain-containing protein tyrosine phosphatases (SHP)-1 and -2. Recently, BTLA was shown to regulate Toll-like receptor signaling in dendritic cells, and IL-7 signaling in γδ T cells. However, in human natural killer (NK) cells, HVEM can also stimulate cytolytic and proinflammatory signaling pathways through CD160 as a host response to human cytomegalovirus (HCMV). Additionally, recent work describing CD160 deficiency in mice confirmed its proinflammatory function in NK cells.

There is a large unmet need for new therapies designed to inhibit T cell activity in patients suffering from immune-mediated pathologies such as graft-versus-host disease and autoimmune diseases. For many types of these diseases, there are a limited range of approved treatments, and some patients do not respond to available treatments. An attractive target for new therapies is the agonistic activation of inhibitory receptors expressed by pathogenic lymphocytes that cause autoimmune disease. Attempts to develop antibody therapies targeting human inhibitory receptors have largely failed, despite promising results in animal models.

Essence of the invention

The present invention is based on the breakthrough discovery that BTLA agonist fusion proteins modulate the immune response. Specifically, the present invention relates to fusion proteins that bind BTLA, enhancing BTLA signaling. The present invention further relates to methods of treating cancer and immune and inflammatory diseases and disorders using a BTLA agonist fusion protein as described herein.

In one embodiment, the present invention provides a fusion protein including a non-naturally occurring HVEM protein and an Fc protein, wherein the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68t, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In one aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In another aspect, the fusion protein includes at least two, three, four, or more mutations in the HVEM protein, for example, S58R, S58K,

- 1 043600

S58Q, G68T, L70D, L70E, L70N, L70W, L90A and their combination. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, for example, S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; and S58R, G68T, L70W and L90A. In one aspect, Fc protein refers to IgA, IgG, IgD, IgE and IgM. In another aspect, Fc protein refers to IgG1, IgG2, IgG3 and IgG4. In a specific aspect, the IgG Fc protein is human.

In a further embodiment, the present invention provides a pharmaceutical composition comprising a fusion protein, for example, a non-naturally occurring HVEM protein and an Fc protein, and a pharmaceutically acceptable carrier. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68t, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, for example, S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; and S58R, G68T, L70W and L90A. In one aspect, Fc protein refers to IgA, IgG, IgD, IgE and IgM. In another aspect, Fc protein refers to IgG1, IgG2, IgG3 and IgG4. In a specific aspect, the IgG Fc protein is a human protein.

In one embodiment, the present invention provides a method for treating BTLA-related disorders, including administering a fusion protein, for example, a non-natural HVEM protein and an Fc protein, to an individual in need thereof, thereby providing treatment for BTLA-related disorders. In one aspect, the disorder associated with BTLA is cancer and an autoimmune disease and disorder. In an additional aspect, the autoimmune disease and disorder is Addison's disease, amyotrophic lateral sclerosis, Crohn's disease, Cushing's syndrome, type 1 diabetes mellitus, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis , rheumatoid arthritis, sarcoidosis, scleroderma, systemic lupus erythematosus, transplant rejection and vasculitis. In another aspect, the cancer is cancer of the prostate, colon, abdominal organs, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvic organs, skin, soft tissues, spleen, chest and genitourinary tract. In an additional aspect, BTLA signaling is enhanced. In another aspect, the fusion protein is a BTLA agonist.

In one aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In a further aspect, the fusion protein includes amino acid residues 39-161 of SEQ ID NO:2 and an Fc protein. In a further aspect, Fc protein refers to IgA, IgG, IgD, IgE and IgM. In certain aspects, Fc protein refers to IgG1, IgG2, IgG3 and IgG4. In a specific aspect, the IgG Fc protein is a human protein. In one aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In one aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70w, L90A, and a combination thereof. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, for example, S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; and S58R, G68T, L70W and L90A.

In one aspect, the method also includes administering an immune response modulator and a chemotherapeutic agent. In another aspect, immune response modulators include eicosanoids, cytokines, prostaglandins, interleukins, chemokines, checkpoint regulators, members of the TNF superfamily, members of the TNF receptor superfamily, and/or interferons. In an additional aspect, the immune response modulator is CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL15, IL17, IL17, IFN-α, IFN-β, IFN-ε, IFN-γ, G-CSF, TNF-α, CTLA4, CD20, PD1, PD1L1, PD1L2, ICOS, CD200, CD52, LTa, ΚΓαβ, LIGHT, CD27L, 41BBL, FasL, Ox40L, April, TL1A, CD30L, TRAIL, RANKL, BAFF, TWEAK, CD40L, EDA1, EDA2, APP, NGF, TNFR1, TNFR2, LTeR, HVEM, CD27, 4-1BB, Fas, 0x40, AITR, DR3, CD30, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, BAFFR, TACI, BCMA, Fn14, CD40, EDAR XEDAR, DR6, DcR3, NGFR-p75 and/or Taj . In certain aspects, the immune response modulator is tocilizumab (Actemra), CDP870

- 2 043600 (Cimzia), etanercept (Enbrel), adalimumab (Humira), Kineret, abatacept (Orencia), infliximab (Remicade), rituximab (Rituxan), golimumab (Simponi), Avonex, Rebif, Recigen, Plegridy, betaseron, Copaxone , Novatron, natalizumab (Tysabri), fingolimod (Gilenya), teriflunomide (Aubagio), BG12, tecfidera and/or alemtuzumab (Campas, Lemtrada).

In an additional aspect, the chemotherapeutic agent is actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine , hydroxyurea, idarubicin , imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumumab, erbitux (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), Rituximab, Synagis (palivizumab), Mylotarg (gemtuzumab oxogamicin), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (dacliximab) ), NeutroSpec (technetium (99mTc) fanolezomab), tocilizumab, ProstaScint (indium-III labeled capromab pendetide), Bexar (tositumomab), Zevalin (ibritumomab tiuxetan (IDEC-Y2B8) conjugated with yttrium 90), Xolair (omalizumab), Mabthera (rituximab), Reopro (abciximab), Mabcampat (alemtuzumab), Simulect (basiliximab), Leukoscan (sulezomab), CEA-scan (arcitumomab), Verluma (nofetumomab), Panorex (edrecolomab), alemtuzumab, CDP 870 and/or natalizumab. In one aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In another aspect, total cellular phosphorylation and phosphorylation of SHP2 are increased.

In a further embodiment, the present invention relates to a method of modulating an immune response in an individual, including administering a fusion protein, for example, a non-natural HVEM protein and an Fc protein to the individual, thereby modulating the immune response. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In another aspect, the fusion protein includes at least two, three, four, or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, for example, S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; and S58R, G68T, L70W and L90A. In one aspect, Fc protein refers to IgA, IgG, IgD, IgE and IgM. In another aspect, Fc protein refers to IgG1, IgG2, IgG3 and IgG4. In a specific aspect, the IgG Fc protein is a human protein. In one aspect, BTLA signaling is enhanced. In another aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In an additional aspect, global cellular phosphorylation and SHP2 phosphorylation are induced. In a further aspect, the individual has a disease and disorder associated with BTLA. In certain aspects, a disease associated with BTLA is cancer and an autoimmune disease and disorder.

In one embodiment, the present invention provides a method for modulating BTLA signaling in a cell, including contacting a cell expressing BTLA with a fusion protein, for example, a non-natural HVEM protein and an Fc protein, thereby modulating BTLA signaling. In one of the BTLA variants, signaling is increased. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In another aspect, the fusion protein includes at least two, three, four, or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, and a combination thereof. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, for example, S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R, L70D and L90A; S58R and G68T; S58R and L70W; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; and S58R, G68T, L70W and L90A. In one aspect, Fc protein refers to IgA, IgG, IgD, IgE and IgM. In another aspect, Fc protein refers to IgG1, IgG2, IgG3 and IgG4. In a specific aspect, the IgG Fc protein is a human protein. In one aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In another aspect, global cellular phosphorylation and SHP2 phosphorylation are induced.

Brief description of the figures

In fig. 1A-C show the identification of the BTLA binding surface of UL144. In fig. Figure 1A shows images of human CMV UL144 indicating the mutated surface residues. In fig. 1B shows the binding curves for the indicated UL144 mutants. In fig. 1C presented Kd

- 3 043600 BTLA-Fc binding to UL144 proteins.

In fig. 2A-H show the identification of HVEM ligand binding mutants in human lymphoma. In fig. Figure 2A shows human HVEM images indicating mutated surface residues. In fig. 2B shows HVEM binding in cells transduced with LIGHT. In fig. 2C shows HVEM binding in cells transduced with BTLA-Fc. In fig. 2D shows HVEM binding in cells transduced with CD160-FC. In fig. Figure 2E shows the Kd of LIGHT binding to HVEM mutant proteins. In fig. 2F shows the Kd of binding of BTLA-Fc to HVEM mutant proteins. In fig. 2G shows the Kd of CD160-FC binding to HVEM mutant proteins. In fig. 2H shows a series of individual DLBCL biopsies, where each column represents one DLBCL sample.

In fig. 3A-E show that HVEM and UL144 bind the same BTLA surface. In fig. Figure 3A shows two BTLA images. In fig. 3B shows typical curves after injection of human HVEM-Fc. In fig. 3C shows typical curves after injection of human CMV UL144-FC. In fig. 3D shows BJAB cells transduced with BTLA, incubated with anti-BTLA, and then stained with HVEM-Fc. In fig. 3E shows BJAB cells transduced with BTLA, incubated with anti-BTLA, and then stained with human CMV UL144-FC.

In fig. 4A-C show that CD160 limits HVEM activation of BTLA. In fig. 4A-C show JTAg cells transduced with the indicated HVEM ligands and cultured with microspheres coupled with anti-CD3 with and without Fc proteins. In fig. 4A shows cells stained for phosphoEKK1/2 (T202/Y204). In fig. 4B shows cells stained for phospho-ZAP70/Syk (Y319/Y352). In fig. Figure 4C shows cells stained for phosphotyrosine.

In fig. 5A-B show that selective BTLA agonists inhibit IL-2 signaling. In fig. Figure 5A shows Western blots of whole-cell extracts of phospho-JAKR phospho-STAT5 and actin. In fig. 5B shows graphs showing quantitative analysis of band intensity normalized to actin.

In fig. 6A-C show that de novo mutant HVEM inhibits ZAP70/Syk activation. In fig. 6A-C show JTAg cells transduced with HVEM ligands cultured with microspheres coupled with anti-CD3 with and without Fc proteins. In fig. 6A shows staining for PhosPho-EKK1/2 (T202/Y204). In fig. 6B shows staining for phospho-ZAP70/Syk (Y319/Y352). In fig. 6C shows staining for phospho-SHP2 (Y542).

In fig. 7A-E show mutations that affect UL144 binding to BTLA. In fig. 7A shows the extracellular domains of human HVEM and human CMV UL144. In fig. 7B shows histograms of 293T cells transduced with mutated and wild-type human CMV UL144 stained with anti-UL144 (2F11). In fig. 7C shows 293T cells transduced with mutated and wild-type human CMV UL144 or HVEM stained with LIGHT. In fig. 7D shows 293T cells transduced with mutated and wild-type human CMV UL144 or HVEM stained with BTLA-Fc. In fig. 7E shows 293T cells transduced with mutated and wild-type human CMV UL144 or HVEM stained with CD160-FC.

In fig. 8A-B show the identification of HVEM ligand binding mutants in human lymphoma. In fig. Figure 8A shows a summary of TNFRSF14 mutations (spots) observed in human FL and DLBCL biopsies. In fig. Figure 8B shows 293T cells transduced with mutated and wild-type human HVEM stained with anti-HVEM.

In fig. 9A-F show that HVEM and UL144 bind the same BTLA surface. In fig. Figure 9A shows MFI-specific staining for 6F4, J168, or MIH26 using anti-BTLA. In fig. Figure 9B shows MFI-specific staining using HVEM-Fc or UL144-FC. In fig. 9C shows 293T cells transfected with BTLA alone or together with HVEM or human CMV UL144 stained with HVEM-Fc. In fig. 9D shows 293T cells transfected with BTLA alone or together with HVEM or human CMV UL144 stained with CMV UL144-FC. In fig. 9E shows 293T cells transfected with HVEM stained with BTLA-Fc. In fig. 9F shows 293T cells transfected with human HVEM or CMV UL144 stained with BTLA-Fc.

In fig. 10A-B show that CD160 restricts HVEM activation of BTLA. In fig. 10A shows JTAg cells transduced with the indicated HVEM ligands or control vector stained with anti-human BTLA (top), anti-human CD160 (middle), or HVEM-Fc followed by species-specific secondary antibodies (bottom). In fig. 10B shows BJAB cells cultured with microspheres coupled with anti-IgM with or without titrated Fc proteins prior to intracellular staining.

In fig. 11 A-C show that selective BTLA agonists inhibit IL-2 signaling. In fig. 11A are graphs showing the percentage of CD69 expression in CD19+ B cells, CD4+ and CD8+ T cells, CD3+CD56+ cells, CD56 dim and CD56 bright NK cells in PBMCs pretreated with the indicated Fc proteins. In fig. 11B shows Western blots of NK92 cells stimulated with titrated IL-2. In fig. 11C shows Western blots of NK92 cells stimulated with titrated IFN-β.

In fig. 12 A-B show that de novo mutant HVEM inhibits ZAP70/Syk activation. In fig.

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12A-B show JTAg cells transduced with the indicated HVEM ligands cultured with microspheres coupled with anti-CD3 with or without Fc proteins. In fig. 12A shows phospho-NF-KB staining. In fig. Figure 12B shows phosphotyrosine staining.

In fig. 13 A-G show that various pathogen-associated and de novo bioengineered HVEM mutein BTLA agonists inhibit T cell signaling. JTAg cells transduced with the indicated HVEM ligands were cultured with microspheres coupled with anti-CD3 with or without Fc proteins. In fig. 13A-B shows staining for phospho-ERK1/2 (T202/Y204). In fig. 13C shows staining for phospho-NF-KB p65 (S529). In fig. 13D shows staining for phospho-BTK/ITK (Y551/Y511). In fig. 13E shows staining for PhosPho-PLCyI (Y783). In fig. 13F shows staining for phospho-ZAP70/Syk (Y319/Y352). In fig. 13G shows phosphotyrosine staining.

In fig. 14A-B show that BTLA agonists inhibit B cell signaling. BJAB cells were cultured with microspheres coupled with anti-IgM with or without titrated Fc proteins. In fig. 14A shows culture for 10 minutes. In fig. 14B shows culture for 60 minutes.

In fig. 15A-D show that BTLA agonists inhibit B cell activation via interferon. Human B cells were stimulated with interferon-β in the presence of microspheres coupled with control immunoglobulin or BTLA agonist fusion proteins. In fig. 15A shows HVEMFc coupled to microspheres. In fig. 15B shows UL144 Fc coupled to microspheres. In fig. 15C shows HVEM ^R109w Fc coupled to microspheres. In fig. 15D shows an HVEM ^RTWA Fc coupled to microspheres.

In fig. 16A-B show that selective BTLA agonists limit IL-2 signaling in NK cells. In fig. 16A shows Western blots of whole cell extracts of phospho-JAKQ phospho-STAT5 and actin. In fig. 16B shows graphs of quantification of band intensity normalized to actin.

In fig. 17A-C show identification of ligand selectivity in mouse HVEM. HVEM-Fc muteins, including variants that either blocked binding to LIGHT (BTLA/CD160-sp) or both BTLA and CD160 (LIGHT-sp), were titrated onto 293T cells. In fig. 17A shows cells transfected with CD160. In fig. 17B shows cells transfected with mouse BTLA. In fig. 17C shows cells transfected with mouse LIGHT.

In fig. 18A-C show that selective HVEM-Fc inhibits skin inflammation in vivo. Mouse HVEM-Fc muteins were injected intraperitoneally into imiquimod-treated animals as a model of skin inflammation. In fig. 18A shows histological analysis. In fig. 18B-C shows thickening of the epidermis.

In fig. 19A-B show the nucleic acid sequence and amino acid sequence for human HVEM. In fig. 19A shows the nucleic acid sequence for human HVEM. In fig. 19B shows the amino acid sequence for human HVEM.

Detailed description of the invention

The present invention is based on the breakthrough discovery that BTLA agonist fusion proteins modulate the immune response. Specifically, the present invention relates to fusion proteins that bind BTLA, enhancing BTLA signaling. The present invention further relates to methods of treating cancer and immune and inflammatory diseases and disorders using a BTLA agonist fusion protein as described herein.

Before reading the description of the present compositions and methods, it should be understood that this invention is not limited to the specific compositions, methods and experimental conditions described, thus the compositions, methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods and materials are described below. The following definitions are intended to assist in understanding the description of the invention, but should not be construed as a substitute for the understanding of the terms of those skilled in the art.

As used in this specification and the appended claims, the singular forms include plural references unless the context clearly requires otherwise. Thus, for example, references to a method include one or more methods and/or steps of the type described herein, which will become apparent to those skilled in the art upon reading this invention, and so on.

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Antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a series of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the light chain constant domain is mapped to the first heavy chain constant domain, and the light chain variable domain is mapped to the heavy chain variable domain. Specific amino acid residues are believed to form the boundary between the light and heavy chain variable domains. Each variable region includes three segments called complementarity determining regions (CDRs) or hypervariable regions, and more highly conserved portions of the variable domains called framework regions (FR). The heavy and light chain variable domains each comprise four FR regions, generally adopting a β-sheet configuration, connected through three CDRs that form a loop junction, and in some cases form part of the β-sheet structure. CDRs on each chain are linked in close proximity by FRs and, with CDRs from the other chain, facilitating the formation of the antigen-binding site of antibodies. Constant domains are not directly involved in antibody-antigen binding but exhibit a variety of effector functions, such as involvement of the antibody in antibody-dependent cellular cytotoxicity.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can belong to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that belong to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and spatial configurations of various classes of immunoglobulins are well known.

The Fc region of an antibody is the tail region of an antibody that interacts with cell surface receptors and certain complement proteins. This feature allows antibodies to activate the immune system. In the IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two antibody heavy chains; The Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc regions of IgGs carry a highly conserved N-site of glycosylation.

Glycosylation of the Fc moiety is required for Fc receptor-mediated activity. The Nglycans attached to this site are predominantly diantennary structures with a complex-type fucosylated core. In addition, small amounts of these N-glycans also carry bisected GlcNAc and α-2,6-linked sialic acid residues.

The term antibody, as used herein, refers to intact monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided they exhibit the desired biological activity .

Antibody fragments include a portion of an intact antibody, preferably an antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fc, Fab, Fab', F(ab') ₂ and Fv fragments; diabodies, tribodies, etc.; linear antibodies; single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments.

The term monoclonal antibody, as used herein, refers to an antibody derived from a collection of essentially homogeneous antibodies, i.e. individual antibodies, including a collection of identical antibodies, with the exception of possible natural mutations that may be present in small quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are interesting in that they are synthesized through a hybridoma that is not contaminated by other immunoglobulins. For example, monoclonal antibodies for use in accordance with the present invention can be made through hybridomas, through recombinant DNA technologies, or isolated from phage antibody libraries.

The terms fusion molecule and fusion protein are used interchangeably and are intended to refer to a biologically active polypeptide, e.g., HVEM or antibody, or fragment thereof (e.g., Fc region), with or without an additional effector molecule, typically a protein or peptide sequence, covalently linked (i.e. ie fused) by recombinant, chemical or other suitable method. If desired, the fusion molecule may be fused in one or

- 6 043600 several sites via a peptide linker sequence. Alternatively, a peptide linker can be used to assist in the construction of the fusion molecule. Particularly preferred fusion molecules are fusion proteins. Typically, the fusion molecule may also include conjugated molecules.

Fc fusion proteins (also known as Fc chimeric fusion protein, Fc-Ig, Ig-based chimeric fusion protein, and Fc-tagged protein) consist of an IgG Fc domain genetically linked to a peptide or protein of interest. Fc fusion proteins have become valuable reagents for in vivo and in vitro studies.

The Fc fusion binding partner may be a single peptide, a ligand that is activated upon binding to a cell surface receptor, signaling molecules, an extracellular domain of the receptor that is activated upon dimerization, or a decoy protein that is used to identify binding partners in a protein microarray. .

One of the most valuable characteristics of the Fc domain in vivo is that it can significantly prolong the plasma half-life of a protein of interest, which in the case of biotherapeutics leads to improved therapeutic efficacy; a parameter that has made Fc fusion proteins attractive biotherapeutics.

The Fc fusion protein may be part of a pharmaceutical composition comprising the Fc fusion protein and pharmaceutically acceptable excipients or a carrier. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; complex compounds with metals (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

As used herein, the term modulation of the immune response refers to either enhancing or inhibiting the immune response. In some aspects, the fusion proteins of the present invention inhibit or attenuate the immune response.

As used herein, the term modulation of BTLA signaling refers to either increasing or decreasing BTLA signaling. In some aspects, the fusion proteins of the present invention increase BTLA signaling.

As used herein, the terms treatment or treatment or alleviation of symptoms refer to therapeutic treatment, prophylactic and/or preventive measures, where the object is intended to prevent or arrest (alleviate) a particular pathological condition or disorder. These, if treatment is needed, include those already with the disorder, as well as those who are predisposed to have the disorder, or those in whom the disorder is to be prevented.

The term therapeutic agent, as used herein, includes a chemical compound or composition capable of inducing a desired therapeutic effect when administered to a patient or individual. An example of a therapeutic agent of the present invention is a BTLA agonist fusion protein.

As used herein, the terms effective amount or therapeutically effective amount of a drug used to treat a disease is an amount that can reduce the severity of the disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more severe symptoms or more serious illness, which may occur with some frequency after the condition requiring treatment. The effective amount can be determined empirically and by common practice, depending on the intended use.

The therapeutic agent can be administered by any suitable means, including topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, intravenous and/or intralesional administration for the purpose of treating an individual. However, in illustrative embodiments, the therapeutic agent is formulated for topical use, such as in the form of a liquid, cream, gel, ointment, spray foam, or the like.

As used herein, the terms BTLA disorder or BTLA disease refer to any condition that is effectively treated

- 7 043600 when using a BTLA agonist fusion protein. Examples of diseases and disorders that are effectively treated with a BTLA agonist fusion protein include cancer, immune, autoimmune and inflammatory diseases and disorders.

An immune disease or disorder is a malfunction of the immune system. These disorders can be characterized in several different ways: by the immune system components involved; by whether the immune system is overactive or hypoactive, and by whether the condition is congenital or acquired. Autoimmune diseases result from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity). Major insights into the mechanisms underlying the pathophysiology of autoimmune diseases have been achieved through the use of genome-wide scans, which have revealed a striking degree of genetic inheritance among autoimmune diseases.

Autoimmune disorders include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis (Lou Gehrig's disease), ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia , autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticarial rash, autoimmune uveitis, disease Balo/concentric sclerosis Balo, Behçet's disease, Berger's disease, Bickerstaff's encephalitis, Blau's syndrome, bullous pemphigoid, cancer, Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic obstructive pulmonary disease, chronic relapsing multiple osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Dego's disease, Dercum's disease, herpetiformis dermatitis, dermatomyositis, diabetes mellitus type 1, diffuse cutaneous systemic sclerosis, discoid lupus erythematosus, Dressler's syndrome, drug-induced lupus, eczema, endometriosis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, hemolytic jaundice of newborns, primary cryoglobus lineemia mixed type, Evans syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schönlein purpura, gestational herpes also known as gestational pemphigoid, hidradenitis suppurativa, Hughes-Stovin syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, interstitial cystitis , juvenile idiopathic arthritis also known as juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, IgA-dependent linear dermatosis, lupus hepatitis also known as autoimmune hepatitis, lupus erythematosus, Majid's syndrome, microscopic colitis, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, limited scleroderma, Mucha-Haberman disease also known as acute lichenoid and varioliform parapsoriasis, multiple sclerosis, myasthenia gravis, myositis, Meniere's disease, narcolepsy, neuromyelitis optica, neuromyotonia , cicatricial ocular pemphigoid, opsomyoclonal syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, parsplanitis, Personage-Turner syndrome, usual novena pemphigus , perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, periarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, true red cell aplasia, pyoderma gangrenosum ia, Rasmussen's encephalitis, Raynaud's phenomenon, syndrome Reuter's, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic attack, rheumatoid arthritis, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, serum sickness, Sjögren's syndrome, spondyloarthropathy, stiff muscle syndrome, Still's disease, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu syndrome, temporal arteritis, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated

- 8 043600 spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, Wegener's granulomatosis.

The term immunomodulator as used herein refers to any therapeutic agent that modulates the immune system. Examples of immunomodulators include eicosanoids, cytokines, prostaglandins, interleukins, chemokines, checkpoint regulators, members of the TNF superfamily, members of the TNF receptor superfamily, and interferons. Specific examples of immunomodulators include PGI2, PGe2, PGF2, CCL14, CCL19, cCl20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL15, IL17, IL17, INF-α, INF-β, INF-ε, INF-γ, G-CSF, TNF-α, CTLA, CD20, PD1, PD1L1, PD1L2, ICOS, CD200, CD52, LTa, Παβ, LIGHT, CD27L, 41BBL, FasL, Ox40L, April, TL1A, CD30L, TRAIL, RANKL, BAFF, TWEAK, CD40L, EDA1, EDA2, APP, NGF, TNFR1, TNFR2, LTPR, HVEM, CD27, 4-1BB, Fas, Ox40, AITR, DR3, CD30, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, BAFFR, TACI, BCMA, Fn14, CD40, EDAR XEDAR, DR6, DcR3, NGFR-p75 and Taj. Other examples of immunomodulators include tocilizumab (Actemra), CDP870 (Cimzia), etanercept (Enbrel), adalimumab (Humira), Kineret, abatacept (Orencia), infliximab (Remicade), rituximab (Rituxan), golimumab (Simponi), Avonex, Rebif, Recigen, Plegridy, Betaseron, Copaxone, Novatron, Natalizumab (Tysabri), Fingolimod (Gilenia), Teriflunomide (Abagio), BG12, Tecfidera and Alemtuzumab (Campas, Lemtrada).

Cancer is a group of diseases characterized by abnormal cellular growth and the potential to invade or metastasize to other parts of the body. Examples of cancers described by the National Cancer Institute include: adult acute lymphoblastic leukemia; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; bile duct cancer, extrahepatic; bladder cancer; bladder cancer in children; bone cancer, osteosarcoma/malignant fibrous histiocytoma; brainstem glioma in children; brain tumor in adults; brain tumor, brain stem glioma in children; brain tumor, cerebellar astrocytoma in children; brain tumor, cerebral astrocytoma/malignant glioma in children; brain tumor, ependymoma in children; brain tumor, medulloblastoma in children; brain tumor, supratentorial primitive neuroectodermal tumors in children; brain tumor, glioma of the optic pathway and hypothalamus in children; brain tumor in children (others); mammary cancer; breast cancer and pregnancy; breast cancer in children; breast cancer in men; bronchial adenomas/carcinoids in children: carcinoid tumor in children; carcinoid tumor of the gastrointestinal tract; adrenocortical carcinoma; islet cell carcinoma; carcinoma with unknown primary focus; primary lymphoma of the central nervous system; cerebellar astrocytoma in children; cerebral astrocytoma/malignant glioma in children; cervical cancer; cancer in children; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; clear cell sarcoma of the tendon sheath; colon cancer; colorectal cancer in children; T-cell lymphoma of the skin; endometrial cancer; ependymoma in children; epithelial ovarian cancer; esophageal carcinoma; esophageal cancer in children; Ewing tumor family; extracranial germ cell tumor in children; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; stomach cancer; stomach cancer in children; carcinoid tumor of the gastrointestinal tract; germ cell tumor, extracranial in children; germ cell tumor, extragonadal; germ cell tumor of the ovary; gestational trophoblastic tumor; brainstem glioma in children; glioma of the optic pathway and hypothalamus in children; hairy cell leukemia; head and neck cancer; hepatocellular cancer (liver cancer) in adults (primary); hepatocellular cancer (liver cancer) in children (primary); Hodgkin's lymphoma in adults; Hodgkin's lymphoma in children; Hodgkin's lymphoma during pregnancy; hypopharyngeal cancer; glioma of the hypothalamus and optic pathway in children; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; kidney cancer; laryngeal cancer; laryngeal cancer in children; leukemia, acute lymphoblastic in adults; leukemia, acute lymphoblastic in children; leukemia, acute myeloid in adults; leukemia, acute myeloid in children; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; cancer of the lips and oral cavity; liver cancer in adults (primary); liver cancer in children (primary); lung cancer, non-small cell; lung cancer, small cell; acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; lymphocytic leukemia, chronic; lymphoma, AIDS-related; lymphoma of the central nervous system (primary); lymphoma, cutaneous T-cell; Hodgkin's lymphoma in adults; Hodgkin's lymphoma in children; Hodgkin's lymphoma during pregnancy; lymphoma, non-Hodgkin's in adults; non-Hodgkin's lymphoma in children; lymphoma, non-Hodgkin's during pregnancy; lymphoma of the central nervous system, primary; macroglobulinemia, Waldenström; breast cancer in men; malignant mesothelioma in adults; malignant mesothelioma in de

- 9 043600 tey; malignant thymoma; medulloblastoma in children; melanoma; melanoma, intraocular; Merkel cell carcinoma; mesothelioma, malignant; metastatic squamous cell carcinoma of the neck with a primary lesion of unknown origin; multiple endocrine neoplasia syndrome in children; multiple myeloma/plasma cell neoplasia; fungoid mycosis; myelodysplastic syndromes; myelogenous leukemia, chronic; myeloid leukemia in children, acute; myeloma, multiple; myeloproliferative disorders, chronic; cancer of the nasal cavity and paranasal sinuses; nasopharyngeal cancer; nasopharyngeal cancer in children; neuroblastoma; non-Hodgkin's lymphoma in adults; non-Hodgkin's lymphoma in children; non-Hodgkin's lymphoma during pregnancy; non-small cell lung cancer; oral cancer in children; cancer of the mouth and lips; oropharyngeal cancer; osteosarcoma/malignant fibrous histiocytoma of bone; ovarian cancer in children; epithelial ovarian cancer; germ cell tumor of the ovaries; ovarian tumor with low malignant potential; pancreas cancer; childhood pancreatic cancer, pancreatic cancer, islet cells; cancer of the nasal cavity and paranasal sinuses; parathyroid cancer; penial cancer; pheochromocytoma; pineal and supratentoreal primitive neuroectodermal tumors in children; pituitary tumor; plasma cell neoplasia/multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin's lymphoma; pregnancy and non-Hodgkin's lymphoma; primary lymphoma of the central nervous system; primary liver cancer in adults; primary liver cancer in children; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); renal cell cancer in children; transitional cell carcinoma of the renal pelvis and ureter; retinoblastoma; rhabdomyosarcoma in children; salivary gland cancer; salivary gland cancer in children; Ewing's sarcoma family of tumors; Kaposi's sarcoma; sarcoma (osteosarcoma)/malignant fibrous histiocytoma of bone; sarcoma, rhabdomyosarcoma in children; soft tissue sarcoma in adults; soft tissue sarcoma in children; Sézary syndrome; skin cancer; skin cancer in children; skin cancer (melanoma); skin carcinoma, Merkel cells; small cell lung cancer; small intestine cancer; soft tissue sarcoma in adults; soft tissue sarcoma in children; squamous cell carcinoma of the neck with a primary focus of unknown origin, metastatic; stomach cancer; stomach cancer in children; supratentorial primitive non-iroectodermal tumors in children; T-cell lymphoma, cutaneous; testicular cancer; thymoma in children; thymoma, malignant; thyroid cancer; thyroid cancer in children; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, gestational; cancer in children with an unknown primary site; unusual cancers in children; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; glioma of the optic pathway and hypothalamus in children; vulvar cancer; Waldenström's macroglobulinemia; and Wilms tumor.

The term chemotherapeutic agent, as used herein, refers to any therapeutic agent used to treat cancer. Examples of the chemotherapeutic agent include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil , gemcitabine, hydroxyurea , idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumumab, erbitux (cetuximab), matuzumab , IMC-IIF 8 , TheraCIM hR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), Rituximab, Synagis (palivizumab), Mylotarg (gemtuzumab ozogamicin), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (daclizumab), NeutroSpec (technetium (99mTc) fanolezomab), tocilizumab, ProstaScint (indium-III labeled capromab pendetide), Bexar (tositumomab), Zevalin (ibritumomab tiuxetan (IDEC-Y2B8) conjugated with yttrium 90), Xolair (omalizumab) , Mabthera (rituximab), Reopro (abciximab), Mabcampas (alemtuzumab), Simulect (basiliximab), Leukoscan (sulezomab), CEA-scan (arcitumomab), Verluma (nofetumomab), Panorex (edrecolomab), alemtuzumab, CDP 870 and natalizumab.

The fusion protein of the invention can be used in combination, for example, with an immunomodulator or chemotherapeutic agent. Treatment using the fusion protein of the invention includes administration before, after, or at substantially the same time as other drugs, such as, for example, immunomodulators or chemotherapeutic agents.

The immune system is a system of biological structures and processes within the body that protects against disease. This system is a diffuse, complex network of interacting cells, cell products and cell-forming tissues that protect the body from pathogens and other foreign substances, destroy infected and malignant cells and remove cellular debris: the system includes the thymus, spleen, lymph nodes and lymphatic tissue, stem cells , leukocytes, antibodies and lymphokines. B cells or B lymphocytes are a type of lymphocyte involved in the humoral immunity of the adaptive immune system and are important for immune surveillance. T cells or T lymphocytes are a type of lymphocyte that play a central role in cellular immunity. There are two main subtypes of T cells: killer T cells and helper cells. In addition, there are T-suppressor cells that play a role in modulating

- 10 043600 general immune response. Killer T cells recognize only antigens bound to MHC class I molecules, while T helper cells recognize only antigens bound to MHC class II molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cells. The third minor subtype is γδ T cells, which recognize intact antigens that are not bound to MHC receptors. In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface that recognizes whole pathogens without any need for antigen processing. Each B cell lineage expresses a different antibody, so the full complement of B cell antigen receptors represents all the antibodies the body can produce.

B and T cell attenuator (BTLA or CD272) is an integral part of the immune system. BTLA expression is induced during T cell activation, and BTLA expression is maintained on Th1 cells but not Th2 cells. Like programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte associated protein 4 (CTLA4), BTLA activates inhibitory signaling pathways that regulate T cell activation. However, unlike PD-1 and CTLA-4, BTLA exhibits T cell inhibition through interaction with the tumor necrosis factor family of receptors (TNF-R) rather than the B7 cell surface receptor family. BTLA is a ligand for tumor necrosis factor superfamily (receptor) member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses.

Tumor necrosis factor receptor superfamily (TNFRSF) member herpes virus entry mediator (HVEM) (TNFRSF14) binds the canonical TNF-related ligands, lymphotoxin-α (LT-α) and LIGHT; however, a distinctive feature of HVEM is its interaction with members of the immunoglobulin superfamily, B and T lymphocyte attenuator (BTLA) and CD160. The ability of HVEM to interact with multiple ligands in different configurations creates a functionally distinct set of intrinsic and bidirectional signal transduction pathways. The ability to bind these different ligands is shared by two distinct topographic regions in the extracellular domain of HVEM. These different sites give HVEM the ability to activate both proinflammatory and inhibitory pathways. Given the interrelationship of HVEM with multiple signal transduction pathways, it is not surprising that it plays important roles in the immune system, such as T cell costimulation, regulation of dendritic cell (DC) homeostasis, autoimmune-mediated inflammatory responses, as well as host response against pathogens. HVEM may also play significant roles outside the immune system, in regulating sensory neuron development and adipocyte metabolism. The human HVEM protein has 283 amino acids (SEQ ID NO:2). The extracellular domain includes 164 amino acid residues, for example amino acids 39-202. The fusion protein of the present invention includes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110 , 120, 130, 140, 150, 160, 170, 180, 190 or more residues of the HVEM protein (SEQ ID NO:2). The fusion protein of the present invention may include, for example, amino acid residues 1-283, 1-202, 1-184, 1-161, 39-202 or 39-161 of the HVEM protein (SEQ ID NO:2).

As used herein, the term unnatural HVEM protein refers to an HVEM protein (SEQ ID NO:2, FIG. 19B) containing at least one or more mutations. The fusion protein of the present invention includes at least one mutation, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. The fusion protein of the present invention includes at least one additional mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. The fusion protein of the present invention may include at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. The fusion protein of the present invention includes at least one mutation in the HVEM protein, such as S58R; S58K; S58Q; G68T; L70D; L70E; L70N; L70W; L90A; S58R and L90A; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A.

BTLA uses a special surface to interface with HVEM. The BTLA/HVEM pathway plays an important role in maintaining immunological tolerance and preventing autoimmune diseases. BTLA-deficient mice develop rheumatoid arthritis, lymphocytic infiltration, autoimmune hepatitis (AIH)-like diseases, and EAE. HVEM-deficient mice have been shown to be susceptible to MOG peptide-induced EAE and increased T cell proliferation and cytokine production. Antagonistic HVEM-Ig exacerbates the autoimmune response in collagen-induced arthritis in mice with a DBA1 genetic background. Thus, enhanced expression of BTLA in activated T cells would be a promising strategy for the treatment of autoimmune diseases.

In relation to antitumor immunity, CD8+ T cells specific for tumor antigens begin to constitutively express BTLA. CpG vaccination has been reported to partially suppress BTLA expression in tumor antigen-specific CD8+ T cells and block BTLA/HVEM-mediated inhibitory signaling. Although the BTLA/HVEM path blocking looks

- 11 043600 appropriate as a means to enhance the functions of effector T cells, special attention must be paid to the complexity of HVEM-interacting molecules. CD160, an IgSF inhibitory receptor, also binds HVEM. In addition, LIGHT, a member of the TNF family, transmits a co-stimulatory signal when interacting with HVEM. These multiple pathways make it difficult for us to establish new therapeutic targets for malignancies.

Manipulating the BTLA/HVEM pathway may be a promising strategy for treating patients with infections. BTLA is induced during P. berghei ANKA infection in mice, and an anti-BTLA antagonistic mAb significantly reduces the incidence of cerebral malaria caused by protozoa. Thus, pathogens that distort the BTLA/HVEM pathway may represent ideal targets for anti-BTLA mAb immunotherapy.

Successful activation of inhibitory receptor signaling depends on the ability of receptor agonists to interact with inhibitory receptor configurations in the activated state, similar to the activated receptor-ligand configuration. A receptor agonist in the form of an antibody will interact with specific epitopes of inhibitory receptors such as B-T-lymphocyte attenuator (BTLA), which can stimulate this activated configuration, resulting in enhanced BTLA signaling. The epitope of these antibodies will not overlap with the BTLA receptor binding site for herpes virus entry mediator (HVEM). The function of these antibodies will be to enhance signaling by activating phosphorylation of the BTLA cytoplasmic domain and recruiting and phosphorylating associated proteins, including SHP1 and 2, other signaling proteins recruited by the BTLA cytoplasmic domain as an activation marker. Activation of the inhibitory downstream signaling pathway by BTLA is expected to negatively regulate the normal downstream signaling pathways of the T cell receptor in T cells, and the B cell receptor in B cells. Additionally, BTLA inhibitory signaling negatively regulates IL-7 cytokine signaling and type I interferon in intrinsic cells such as γδ T cells and natural killer (NK) cells.

Much of the β-herpesvirus cytomegalovirus (CMV) genome is designed to evade the host immune response, and many of these genes are used as the virus progresses toward establishing a state of latent infection. CMV expresses an HVEM mimic (ORF UL144) that binds BTLA and inhibits T cell proliferation to a greater extent than HVEM. Recently, it was shown that UL144 does not bind the HVEM receptor CD160, preventing NK cell activation. Activation of cytolytic cells is also critical in immune responses to cancer, and tumors with mutations in signaling pathways associated with immunological recognition are associated with more aggressive tumor growth and mortality. In human follicular lymphoma (FL), the most common secondary mutation occurs in TNFRSF14, while in diffuse large B-cell lymphoma (DLBCL), TNFRSF14 is also frequently mutated, resulting in gene deletion or loss of expression. However, for a subpopulation of lymphomas, several mutations have been predicted that affect HVEM ligand binding. It remains unclear how these mutations or altered ligand interactions may influence lymphoma fitness within the tumor microenvironment.

Similar to the viral UL144 protein, several HVEM mutant lymphomas exhibit ligand selection for BTLA without CD160 binding. CD160 expression inhibits wild-type HVEM from engaging with BTLA and inhibiting T cell receptor signaling. In contrast, BTLA activation triggered by viral and mutant HVEM is sterically unhindered by CD160 expression. Ultimately, we found that BTLA activation inhibited phosphorylation of cytokine-induced signal transporter and activator of transcription (STAT) in CD160-expressing NK cells, and that the viral UL144 protein inhibited cytokine signaling more effectively than wild-type HVEM. Thus, ligand competition prevents HVEM from uniformly engaging inhibitory signals, while ligand selectivity allows viral and mutant HVEM to trigger a unique inhibitory function, pointing the way toward the development of a BTLA-selective agonist. Together, these data indicate potential selective pressure for the evolution of UL144 in CMV and for the acquisition of somatic TNFRSF14 mutations in lymphoma, promoting inhibitory and rate-limiting inflammatory signaling in infection and cancer.

In one embodiment, the present invention provides a fusion protein including a non-naturally occurring HVEM protein and an Fc protein, wherein the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In one aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, e.g.

- 12 043600

S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A or a combination thereof. In certain aspects, the fusion protein includes at least one mutation in an HVEM protein, such as S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A. In one aspect, the Fc protein is IgA, IgG, IgD, IgE, or IgM. In another aspect, the Fc protein is IgG1, IgG2, IgG3 or IgG4. In a specific aspect, the IgG Fc protein is human.

In a further embodiment, the present invention provides a pharmaceutical composition comprising a fusion protein, such as a non-naturally occurring HVEM protein and an Fc protein, and a pharmaceutically acceptable carrier. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, l90A, or a combination thereof. In certain aspects, the fusion protein includes at least one mutation in an HVEM protein, such as S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A. In one aspect, the Fc protein is IgA, IgG, IgD, IgE, or IgM. In another aspect, the Fc protein is IgG1, IgG2, IgG3 or IgG4. In a specific aspect, the IgG Fc protein is human.

In one embodiment, the present invention provides a method of treating a BTLA-related disorder, including administering a fusion protein, such as a non-naturally occurring HVEM protein and an Fc protein, to an individual in need thereof, thereby providing treatment for a BTLA-related disorder. In one aspect, the BTLA-related disorder is cancer or an autoimmune disease or disorder. In an additional aspect, the autoimmune disease or disorder is Addison's disease, amyotrophic lateral sclerosis, Crohn's disease, Cushing's syndrome, diabetes mellitus type 1, graft-versus-host disease, Graves' disease, Guillain-Barre syndrome, lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis , rheumatoid arthritis, sarcoidosis, scleroderma, systemic lupus erythematosus, transplant rejection or vasculitis. In another aspect, the cancer is cancer of the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneal cavity, endocrine glands (adrenal glands), parathyroid glands, pituitary gland, testes, ovary, thymus, thyroid gland) , eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvic organs, skin, soft tissues, spleen, chest organs, or genitourinary tract. In an additional aspect, BTLA signaling is enhanced. In another aspect, the fusion protein is a BTLA agonist.

In one aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In a further aspect, the fusion protein includes amino acid residues 39-161 of SEQ ID NO:2 and an Fc protein. In a further aspect, the Fc protein is IgA, IgG, IgD, IgE or IgM. In certain aspects, the Fc protein is IgG1, IgG2, IgG3, or IgG4. In a specific aspect, the IgG Fc protein is human. In one aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In one aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In certain aspects, the fusion protein includes at least one mutation in an HVEM protein, such as S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A.

In one aspect, the method also includes administering an immune response modulator or chemotherapeutic agent. In another aspect, the immune response modulator includes eicosanoids, cytokines, prostaglandins, interleukins, chemokines, checkpoint regulators, members of the TNF superfamily, members of the TNF receptor superfamily, and/or interferons. In an additional aspect, the immune response modulator is CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL15, IL17, IL17, IFN-a, IFN-β, IFN-ε, IFN-γ, G-CSF, TNF-α, CTLA4, CD20, PD1, PD1L1, PD1L2, ICOS, CD200, CD52, LTa, ΓΓαβ, LIGHT, CD27L, 41BBL, FasL, Ox40L, April, TL1A, CD30L, TRAIL, RANKL, BAFF, TWEAK, CD40L, EDA1, EDA2, APP, NGF, TNFR1, TNFR2, LTJR, HVEM, CD27, 4-1BB, Fas, Ox40, AITR, DR3, CD30, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, BAFFR, TACI, BCMA, Fn14, CD40, EDAR XEDAR, DR6, DcR3, NGFR-p75

- 13 043600 and/or Taj. In certain aspects, the immune response modulator is tocilizumab (Actemra),

CDP870 (Cimzia), etanercept (Enbrel), adalimumab (Humira), Kineret, abatacept (Orencia), infliximab (Remicade), rituximab (Rituxan), golimumab (Simponi), Avonex, Rebif, Recigen, Plegridy, betaseron, Copaxone, Novatron , natalizumab (Tysabri), fingolimod (Gilenya), teriflunomide (Abagio),

BG12, tecfidera and/or alemtuzumab (Campas, Lemtrada).

In an additional aspect, the chemotherapeutic agent is actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine , hydroxyurea, idarubicin , imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumumab, erbitux (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), Rituximab, Synagis (palivizumab), Molotarg (gemtuzumab ozogamicin), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (daclizumab) ), NeutroSpec (technetium (99mTc) fanolezomab), tocilizumab, ProstaScint (indium-III labeled capromab pendetide), Bexar (tositumomab), Zevalin (ibritumomab tiuxetan (IDEC-Y2B8) conjugated with yttrium 90), Xolair (omalizumab), Mabthera ( rituximab), Reopro (abciximab), Mabcampas (alemtuzumab), Simulect (basiliximab), Leukoscan (sulezomab), CEA-scan (arcitumomab), Verluma (nofetumomab), Panorex (edrecolomab), alemtuzumab, CDP 870 and/or natalizumab. In one aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In another aspect, global cellular phosphorylation and SHP2 phosphorylation are induced.

In a further embodiment, the present invention provides a method of modulating an immune response in an individual, including administering a fusion protein, such as a non-natural HVEM protein and an Fc protein, to the individual, thereby modulating the immune response. In one aspect, the fusion protein is a BTLA agonist. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In certain aspects, the fusion protein includes at least one mutation in an HVEM protein, such as S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A. In one aspect, the Fc protein is IgA, IgG, IgD, IgE, or IgM. In another aspect, the Fc protein is IgG1, IgG2, IgG3 or IgG4. In a specific aspect, the IgG Fc protein is human. In one aspect, BTLA signaling is enhanced. In another aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In an additional aspect, global cellular phosphorylation and SHP2 phosphorylation are induced. In a further aspect, the individual has a BTLA-related disease or disorder. In certain aspects, the BTLA-related disease is cancer or an autoimmune disease or disorder.

In one embodiment, the present invention provides a method for modulating BTLA signaling in a cell, including contacting a BTLA expressing cell with a fusion protein, such as a non-natural HVEM protein and an Fc protein, thereby modulating BTLA signaling. In one option, BTLA signaling is enhanced. In another aspect, the fusion protein includes an extracellular domain of an HVEM protein and an Fc protein. In another aspect, the fusion protein includes at least one mutation in the HVEM protein. In a further aspect, the mutation is S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In an additional aspect, the fusion protein further includes at least one mutation in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In another aspect, the fusion protein includes at least two, three, four or more mutations in the HVEM protein, for example, S58R, S58K, S58Q, G68T, L70D, L70E, L70N, L70W, L90A, or a combination thereof. In certain aspects, the fusion protein includes at least one mutation in the HVEM protein, such as S58R; S58K; S58Q; L70D; L70E; L70N; L90A; S58R and L90A; S58R and G68T; S58R and L70W; S58R, L70D and L90A; S58R, G68T and L90A; S58R, L70W and L90A; S58R, G68T, L70D and L90A; or S58R, G68T, L70W and L90A. In one aspect, the Fc protein is IgA, IgG, IgD, IgE, or IgM. In another aspect, the Fc protein is IgG1, IgG2, IgG3 or IgG4. In a specific aspect, the IgG Fc protein is human. In one aspect, phosphorylation of ERK1/2 and/or ZAP70/Syk is reduced. In another aspect, global cellular phosphorylation and SHP2 phosphorylation are induced.

The invention, taking all these aspects into account, is further illustrated in the following examples. The examples, however, do not limit the scope of the invention, which is defined by the attached claims.

Examples

Example 1.

UL144 residues homologous to HVEM are required for binding to BTLA

Human CMV-encoded UL144 selectively binds BTLA but not CD160 and inhibits T cell proliferation activated by T cell receptor signaling to a greater extent than HVEM proteins. To determine whether BTLA selection by UL144 results from a unique interaction between the surfaces of these two proteins, the BTLA structure of the UL144 contact surface was modeled on an HVEM-BTLA cocrystal with a predefined structure ( Figure 1A ). Next, surface residues of UL144 that differed from HVEM were targeted by alanine- and homology-scanning mutagenesis to screen for potential BTLA binding surfaces (Fig. 1). It was observed that mutation at several UL144 residues disrupted or reduced BTLA binding, which defines a surface centered on one side of the CRD1 region similar to the BTLA binding surface of HVEM. The most critical mutations that are reliably expressed on the surface occur in Gly41 and Tyr42, homologous to Gly60 and Tyr61 in HVEM (Fig. 7). None of the homology scanning mutations in UL144 showed additional ligand binding to CD160 or to the TNFSF ligand, LIGHT, although the G46K and N61A mutants increased binding to BTLA (Fig. 1B, C and Fig. 7). Thus, no single mutation restores CD160 binding to the UL144 protein, indicating that CMV likely shaped UL144 to select for BTLA binding through multiple genetic modifications. Specific residues affect BTLA binding: Glu ²⁷ , Gln ³³ , Pro ³⁶ , Gly ⁴¹ , Tyr ⁴² , Thr ⁵² , Leu ⁶⁸ appear to be essential; Arg ⁴³ , Thr ⁴⁵ have reduced binding; Asn ³² , Gln ⁴⁷ , Gly ⁴⁹ , Gln ⁵⁰ apparently have no effect; Gly ⁴⁶ , Asn ⁶¹ increase binding.

Cells and surface protein expression: EL4 and 293T cells were cultured in DMEM with 10% heat-inactivated FBS, antibiotics, L-glutamine and 50 μM 2-ME. NK92 cells were cultured in RPMI with 8% heat-inactivated FBS, 8% horse serum, antibiotics, L-glutamine and 50 μM 2-ME supplemented with 100 U/ml IL-2. BJAB and Jurkat TAg (JTAg) cells were cultured in RPMI with 10% heat-inactivated FBS, antibiotics, L-glutamine, and 50 μM 2ME. EL4 cells and BJAB cells transduced with retroviruses containing wild-type and mutant human BTLA (Watanabe et al. (2003); Nat Immunol 4:670) were sorted for GFP expression to increase the frequency of BTLA-expressing cells. Single-infection pseudotyped retrovirus was generated by cotransfection of retroviral plasmid, pCG VSVg envelope protein and Hit60 gag-pol as described previously (Sedy et al. (2005), Nat Immunol 6:90). UL144 and BTLA mutants were generated by round-the-world PCR mutagenesis. For Fc binding studies, 293T cells transduced by calcium phosphate transfection using a pND vector containing mutant or wild type UL144 were used. All oligonucleotides used for PCR amplification and site-specific mutagenesis are listed in Table. 1. For transient expression of HVEM ligands in JTAgs, cells were electroporated using 10 μg of the indicated DNA constructs with a control vector at 230V for 65 ms using a T820 square wave electroporator.

Antibodies and Fc fusion proteins: Human anti-HVEM was purchased from BD Biosciences. Anti-human BTLA antibodies, clones MIH26 and J168, were obtained from eBioscience and BD Biosciences. Anti-human BTLA clone 6F4 and anti-UL144 clone 2F11 were prepared as previously described (Cheung et al. (2005), Proc Natl Acad Sci 102:11318; Cheung et al. (2009a), J Immunol 183:7286) . Donkey anti-Fcy and human anti-Fcμ, F(ab') ₂ were obtained from Jackson Immunoresearch.

Antibodies to identify the human PBMC population include CD19 FITC, CD56 PE-Cy7, CD8 PE, CD3 PE 610, CD4 eFluor450 and CD69 PerCP-Cy5.5. Phospho-specific antibodies include phospho-tyrosine PE, phospho-Akt (S473) PE and phospho-SHP-2 (Y542) Alexa647, phospho-ERK1/2 (pT202/pY204) PerCPe710 and phospho-NF-KB p65 (S529) PE. Fc fusion proteins were designed and produced as follows: an ectodomain (residues 1-184) comprising the native human HVEM signal sequence was fused at the 3' end to a human IgG Fc sequence. For human BTLA and CMV UL144, residues 26-150 and 19-132, respectively, were fused at the 5' end to the human Ig signal sequence and at the 3' end to the human IgG Fc regions. Fc fusion proteins were prepared in transfected 293T cells grown in serum-free CellGro Free medium and purified by affinity chromatography on protein A columns. Human IgG1 control protein was obtained from Sigma-Aldrich. BTLA selective HVEM mutant proteins were constructed de novo through three rounds of mutagenesis as follows: first, alanine mutagenesis of surface-located residues to identify ligand-binding hotspots; second, saturation mutagenesis at hot spots to optimize effects on ligand binding; third, combinatorial mutagenesis to achieve BTLA selectivity and affinity enhancement.

Binding assay: Flow cytometry binding assay was performed as previously described ( Sedy et al., 2013 ). Briefly, cells were incubated with Fc ligands for 30 minutes.

- 15 043600 on ice in buffer (PBS with 2% FBS), washed twice, incubated with anti-Fc secondary antibodies for 15 minutes on ice in buffer, washed twice and analyzed. Specific mean fluorescence intensity (MFI) was calculated by subtracting the experimental cell

MFI from control cell MFI.

Kinetic affinity analysis by surface plasmon resonance: human BTLA-Fc ligand was immobilized on a CM5 sensor chip up to 150 response units using amine coupling. Sensograms were collected at a flow rate of 30 μl/min, 25°C. Specific binding was determined by subtracting the control channel from the ligand channel. The indicated concentrations of the analyte were injected from test tubes cooled to 7°C. Data acquisition involves 3 minutes of 90 μl analyte followed by 15 minutes of disassociation. Regeneration after each cycle was carried out using 30 seconds of injection of 15 μl of 10 mM glycine pH 2.5. The first 10 seconds after analyte injection and disassociation were used for affinity measurements using both Langmuir and Bivalent models in the kinetic analysis module of BIAevaluation software (version 4.1).

Phosphoflow Cell Activated Microsphere Assay: Aldehyde/sulfate latex microspheres (5 μm) were covalently coupled to 100 μg/ml anti-Fcμ, F(ab') ₂ alone or with the indicated concentrations of human IgG1, HVEM-Fc, or UL144- FC as described previously (Sperling et al. (1998) J immunol 161:6459). Briefly, microspheres were washed in PBS, incubated with the indicated proteins at 37°C for 90 min, blocked in buffer (PBS with 1% BSA and 0.1% glycine), washed twice, resuspended in medium, and counted. Microspheres were used to stimulate JTAg and BJAB cells in a 3:1 ratio. Microspheres were first added to 96-well flat-bottom plates, followed by washing the cells in PBS and resuspending to a concentration of 1.5 x 106 cells/ml. The plates were quickly unscrewed and incubated for the indicated time at 37°C, followed by fixation with 2% paraformaldehyde in PBS and additional incubation at 37°C for 10 min. Cells were washed in buffer (PBS with 2% FBS) and permeabilized in Perm III Buffer (BD Bioscience) for 30 min on ice, washed twice, incubated with phospho-specific antibodies for 30 min on ice, washed twice and analyzed.

Western Blot: IL-2-dependent NK92 model cell line was activated using the indicated concentrations of IL-2 or interferon-β. In experiments testing IL-2 responses, NK92 cells were cultured overnight without IL-2 to restore basal signaling. For experiments using fusion proteins and antibodies, NK92 cells were coated with control human IgG1, HVEM-Fc or UL144-Fc or control mouse IgG2a or anti-BTLA for at least 15 minutes on ice before activation. NK92 cells were aliquoted to 2x106 cells per 100 μl condition and activated at 37°C for the indicated times, blocked using ice-cold PBS and lysed in RIPA buffer at 4°C for 20 minutes followed by centrifugation at 14,000 rpm, 4 °C. Extracts were heated in SDS loading buffer containing 1% βmercaptoethanol for 5 minutes and separated by SDS-PAGE on 10% Bis-Tris gels. Proteins were transferred using the reservoir method to a PVDF membrane and blocked using 1% ovalbumin in TBS-T buffer, and labeled with antibodies against phospho-JAK, phospho-STAT5, phospho-STATR phospho-Akt (S473) and total actin, followed by staining rabbit anti-HRP or mouse anti-HRP and visualized by enhanced chemiluminescence.

Cytokine activation of human PBMCs: Fresh human blood was collected and obtained from healthy donors as described previously (Sedy et al., 2013). Briefly, PBMCs were isolated from the buffy coat on a Ficoll gradient, incubated at a concentration of 106 cells/ml with the indicated Fc proteins on ice for 15 min, followed by cross-linking with 5 μg/ml anti-Fcy F(ab') ₂ people for 6 hours before flow cytometry analysis.

Example 2.

Somatic TNFRSF14 mutations upon target ligand binding in lymphoma.

It has been observed that somatic TNFRSF14 mutations acquired by FL and DLBCL have the potential to disrupt conserved HVEM interactions with its ligands, and subsequent function (Fig. 8). Lymphoma-associated mutations that were not predicted to disrupt HVEM structure were selected and the binding of the mutant receptor to BTLA, CD160, and LIGHT was assessed. HVEM mutants that were reliably expressed on the surface could be classified into three groups according to their effects on ligand binding (Fig. 8). Mutations containing P59S, A102P, or R109W disrupted interactions with CD160 individually. The Y61C and G72D mutations abolished interactions with both BTLA and CD160 but had little effect on LIGHT binding. Ultimately, the G60D and T82P mutants, or the insertion of Ile between Ser91 and Lys92, disrupted all ligand interactions (Fig. 2). HVEM Tyr61 was identified as critical for binding of both BTLA and CD160. Together, these somatic mutations define a ligand binding hierarchy with preferential loss of interactions with CD160 and BTLA, and either unaffected or also altered interactions with LIGHT. Genetic alterations in TNFSF14 were further confirmed in an additional DLBCL cohort, and additional tumors with deletions in

- 16 043600

BTLA or TNFSF14 (Fig. 2H). The presence of multiple genetic damage to the HVEM network in FL and

Human DLBCL indicates that these pathways may contribute significantly to cellular selection within the tumor microenvironment. Specific residues are important for ligand binding: Pro ⁵⁹ , Ala ¹⁰² , Arg ¹⁰ no binding to CD160; Tyr ⁶¹ and Gly ⁷² do not bind to BTLA or CD160; Gly ⁶⁰ , Thr ⁸² , and ins91I (indicated at Ser ⁹¹ and Lys ⁹² ) - no binding to LIGHT, BTLA, or CD160 (Fig. 2C-H).

Example 3.

HVEM and UL144 bind overlapping BTLA surfaces.

To determine whether the altered BTLA activity of viral UL144 or mutant HVEM was caused by contact of different surfaces, BTLA residues at contact surfaces were compared for these agonists. It was found that the binding of an anti-BTLA mAb (clone MIH26), which was previously shown to have agonist activity, was impaired by mutation in either Glu57 or Pro59, while the binding of a competing anti-BTLA mAb (clone J168) was disrupted by mutation in Arg42 (Fig. 3A and Fig. 9A). Namely, the MIH26-binding residue Glu57 is homologous to Gln63 in mouse BTLA, which is contained within the epitope of the agonistic anti-BTLA mAb (clone 6A6). In comparison, similar requirements were observed for both HVEMFc and UL144-Fc binding to Gln37, Arg42, Pro59, and His127, given previous studies of human and mouse BTLA. Similar affinities for BTLA to HVEM or UL144 were confirmed using surface plasmon resonance (Figure 3B,C). The avidity (KD, 1:1 binding model) of UL144-FC for BTLA (295 nM) was slightly less than HVEM-Fc (177 nM) (Table 2). While subtle avidity and binding differences were observed between HVEM and UL144, in general human CMV UL144 closely mimics HVEM binding to BTLA. In fig. 9A-B show EL4 cells transduced with wild-type or mutant human BTLA stained with polyclonal or monoclonal anti-human BTLA antibodies or with human HVEM-Fc or CMV UL144-FC, followed by species-specific secondary antibodies. From the top to the bottom of the graphs, MFI-specific 6F4, J168, or MIH26 anti-BTLA staining (A), or HVEM-Fc or UL144-FC (B) staining on cells gated for GFP expression are shown.

Coexpression of HVEM and BTLA in lymphocytes leads to the formation of a characteristic complex in cis configuration located on the cell surface, which competitively blocks activation by external ligands in trans configuration. It was determined whether UL144 coexpressed with BTLA formed complexes in cis to prevent ligand access in trans, or whether UL144 ligating BTLA in trans likely circumvented steric hindrance of BTLA access via HVEM. Both HVEM and UL144 expressed with BTLA blocked binding to both HVEM-Fc and UL144-FC, indicating that HVEM and UL144 form similar complexes with BTLA in cis configuration and that UL144 cannot bypass the BTLA-formed cis complex on cell surfaces (Fig. 9C, D). However, coexpression of the BTLA R42D mutant prevented the binding of cis-expressed HVEM, but not UL144, to BTLA-Fc in the trans configuration, indicating that a single mutation may not be sufficient to disrupt the BTLA-UL144 cis complex (Fig. 9E,F).

The antibody epitopes recognized by mAbs J168 and MIH2 6 overlap the BTLA surface capped by HVEM molecules in a tetrameric asymmetric structure and are predicted to block HVEM binding to BTLA (Fig. 3A). While titration of both of these anti-BTLA clones prevented HVEM-Fc and UL144-Fc from binding to BTLA, a third anti-BTLA mAb (clone 6F4) enhanced HVEM-Fc binding to BTLA but had no effect on UL144-FC binding (Fig. .3D, E). Its epitope has not been identified using these BTLA mutants, and this clone does not show any reactivity against HVEM. Together, these data indicate that while the same surface of BTLA appears to be used for binding of HVEM and UL144, there may be additional structural elements that contribute to ligand binding. Additionally, epitope mapping of BTLA antibodies indicates that agonistic activity is associated with Glu57 or Pro59 on human BTLA (for clone MIH26) and Gln63 on mouse BTLA (for clone 6A6). In fig. 3, left image, specific residues indicate the requirements for antibody binding: Glu45, Glu57, Pro59 are required for MIH26 binding, Arg42 is required for J168 binding. In fig. 3, right image, specific residues indicate HVEM/UL144 binding epitope: Gln37, Arg42, Pro59, His127 appear to be required for HVEM/UL144 binding, Glu45, Glu57, Phe119, Ser121 appear to be dispensable for HVEM/UL144 binding.

Example 4.

CD160 limits BTLA-mediated inhibition by competing for HVEM.

Expression of CD160 and LIGHT in different lymphocyte subsets may influence the ability of viral and cancer mutant HVEM to participate in BTLA inhibitory signaling. To assess whether targeting the expression of HVEM ligands affected BTLA agonism, Jurkat T cells expressing ectopic isoforms of BTLA or CD160 were activated (Fig. 4 and Fig. 10A). Control cells activated with immobilized anti-CD3 antibodies induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, zeta chain associated protein kinase 70 kD (ZAP70)/Syk, and total cellular tyrosine, while ERK1/2 Phosphorylation was reduced in cells activated with co-immobilized aHTu-CD3 and HVEM or UL144-FC (~50-70% reduction). Ectopic BTLA expression enhanced the ability of HVEM and UL144 to inhibit ERK1/2 phosphorylation to background levels, correlating with significantly reduced ZAP70/Syk phosphorylation (∼15–25%) (Figure 4A,B). Tyrosine phosphorylation was increased following stimulation with HVEM or UL144, reflecting activation of BTLA and associated signaling proteins (Fig. 4C). Importantly, in cells expressing ectopic CD160 (glycophosphoinositide or transmembrane isoforms), HVEM was unable to inhibit ERK1/2 phosphorylation as long as BTLA was additionally presented. In contrast, UL144 inhibited ERK1/2 phosphorylation despite expression of the CD160 isoform. The agonist activity of HVEM and UL144 was confirmed in the human non-Hodgkin's lymphoma, BJAB, which expresses high levels of human BTLA in the absence of other HVEM ligands and activates Syk-dependent ERK and Akt phosphorylation in response to IgM stimulation (Fig. 10B). Specifically, in cells activated with co-immobilized anti-IgM and titrated HVEM or UL144-FC, an approximately 50% reduction in phosphorylation of ERK, Akt, and cellular phosphotyrosines was observed compared to control cells. Together, these results illustrate that the ability of HVEM to activate BTLA signaling depends on the relative ratio of BTLA to CD160, and that selectivity of the viral mimic receptor results in sterically unhindered BTLA agonism.

Example 5.

BTLA agonist activation inhibits proinflammatory cytokine stimulation.

It was previously shown that in PBMCs cultured with cytokines or CMV-infected fibroblasts in vitro, soluble HVEM-Fc, but not UL144-Fc, uniquely co-stimulated the activation of CD160-expressing NK cells. In parallel studies, it was observed that inhibition of IL-2-induced CD69 expression by HVEM and UL144-FC in different PBMC subsets correlated with BTLA expression (Fig. 11A). The ability of cellular and viral HVEM to inhibit IL-2 signaling in the presence of CD160 was further tested (Fig. 5). The human NK cell line NK92 responds to IL-2 in a titratable manner through phosphorylation of JAK1 kinase, leading to activation of STAT5, Akt and ERK pathways. In IL-2-treated cells, decreased phosphorylation of JAK1, STAT5, and Akt proteins was observed following activation of BTLA with UL144-FC or anti-BTLA mAb (clone MIH26), but not HVEM-Fc, indicating that the targeting of BTLA to UL144 was steric unhindered by the presence of excess CD160 (Fig. 5A, 5B and Fig. 11B). Additionally, it was tested whether BTLA regulates type I interferon signaling, since this pathway is also regulated by SHP-1 inhibition. Anti-BTLA mAb (clone MIH26) reduced the magnitude of STAT1 phosphorylation at early and late exposure times, indicating that in SHP-1 sensitive cytokine signaling pathways, BTLA exhibits broad inhibitory function (Fig. 11C). Additionally, CD160 restricted HVEM, but not its viral mimic, from binding and activating BTLA. In fig. 11 shows PBMCs pretreated with the indicated Fc proteins and stimulated with the indicated concentrations of IL-2 for 6 hours before staining for CD69 expression within cell subsets. The graphs show the percentage of CD69 expression in CD19+ B cells, CD4+ and CD8+ T cells, CD3+CD56+ cells, CD56 dim and CD56 bright NK cells. V.-S. NK92 cells were stimulated with titrated IL-2 (B) and IFNβ (C) at the indicated times after pretreatment with anti-BTLA (MIH26) or control Ig. Western blots show whole cell extracts of phospho-JAKR STAT5 and Akt (S473) to monitor IL-2 signaling, or PhosPho-STATI to monitor IFN-β signaling, and actin to monitor total protein levels.

Example 6.

Bioengineered HVEM selectively activates BTLA signaling.

The selectivity of UL144 ligand binding and mutations in lymphoma suggested that de novo construction of HVEM would result in the formation of a BTLA-specific agonist. Mutant HVEM-Fc proteins were constructed by alanine-scanning, saturation, and combinatorial mutagenesis. We found that HVEM muteins containing mutations in S58R and L90A (HVEM-RA) conferred BTLA selectivity, while additional mutations in G68T and L70W enhanced BTLA affinity 10-fold (HVEM-RTWA). Notably, both RA and RTWA mutants inhibited anti-CD3-induced PhosPho-EKK1/2 to a greater extent than parental HVEMFc proteins in control cells, and all HVEM-Fc proteins reduced ERK1/2 phosphorylation to background levels in ectopically expressing cells. BTLA (Fig. 6A). Only the high-affinity HVEMRTWA mutant significantly reduced ZAP-70/Syk phosphorylation to background levels in all cells (Fig. 6B). In cells expressing ectopic BTLA, the inhibitory activity of HVEM-RA and HVEM-RTWA mutants correlated with the exclusive induction of phospho-SHP2 signaling as well as total cellular phosphotyrosine (Fig. 6C and Fig. 12B). Thus, bioengineered HVEMFc recapitulate the selective and sterically unhindered agonism of viral and mutant HVEM.

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Example 7.

Various pathogen-associated and de novo bioengineered HVEM mutein BTLA agonists inhibit T cell signaling.

HVEM mutein BTLA agonists have been shown to inhibit T cell signaling. JTAg cells were transduced with control BTLA, CD160-GPI, CD160-TM, BTLA CD160-GPI and BTLACD160-TMHVEM ligands by electroporation and left alone for 4-8 hours before activation, or stably transduced with retroviruses expressing the indicated HVEM ligands. To activate JTAg cells, aldehyde sulfate microspheres were combined with anti-CD3 at a concentration of 100 μg/ml with or without the indicated HVEM or UL144 Fc proteins at a concentration of 1 μM in PBS for 90' at 37°C. Microspheres were washed twice and incubated with JTAg cells at a ratio of 4:1 microspheres to cells for 5 minutes. Cells were immediately fixed with 2% paraformaldehyde, permeabilized with 90% methanol, and stained with the indicated phosphospecific antibodies. Cells were then examined for intracellular staining of phospho-EKK1/2 (T202/Y204) (Fig. 13A, B), phospho-NF-KB p65 (S529) (Fig. 13C), phospho-BTK/ITK (Y551/ Y511) (Fig. 13D), phospho-ZAP70/Syk (Y319/Y352) (Fig. 13F), and phosphotyrosine (Fig. 13G). In fig. 13A shows MFI plots of stained cells. In fig. 13A.-G. the percentage of positive events among stained cells is shown (mean ± SEM of three experiments). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Example 8.

BTLA agonists inhibit B cell signaling.

BTLA agonists have been shown to inhibit B cell signaling. To activate BJAB cells, aldehyde sulfate microspheres were coupled with anti-IgM at a concentration of 100 μg/ml with or without the indicated HVEM or UL144 Fc proteins at 1 μM in PBS for 90' at 37°C. Microspheres were washed twice and incubated with BJAB cells at a ratio of 4:1 microspheres to cells for 10 or 60 minutes. Cells were immediately fixed with 2% paraformaldehyde, permeabilized with 90% methanol, and stained with the indicated phospho-specific antibodies. BJAB cells were then examined for intracellular phospho-ERK1/2 (pT202/pY204), phospho-Akt (S473) or phosphotyrosine staining (Fig. 14A-B). In fig. 14A-B show the percentage of cells positive for Phospho-EIK1/2 (pT202/pY204), phospho-Akt (S473) or phosphotyrosine.

BTLA agonists inhibit interferon activation of B cells. To activate human B cells, aldehyde sulfate microspheres were coupled with the indicated HVEM or UL144 Fc proteins at 1 pM in PBS for 90' at 37°C. The microspheres were washed twice and incubated with human B cells purified from normal human donor blood PBMCs at a 4:1 microspheres to cells ratio and stimulated with 10 U/ml interferon-β for 6 hours before lysis and RNA isolation. In fig. 15 shows the fold reduction in the levels of each of the indicated interferon-stimulated genes when cells were treated with each of the BTLA agonists (HVEM, UL144 Fc, HVEM ^R109w Fc and HVEM ^RTWA Fc) compared to control. (mean ± SEM, pooled data from two experiments). *, p<0.05; **, p<0.01.

BTLA agonists have been shown to limit IL-2 signaling in NK cells. NK92 cells were cultured without serum for at least four hours and then stimulated with 20 U/ml human IL-2 at 0, 1, 5, 15, 30 and 60 minutes after pretreatment with the indicated Fc and antibodies at a concentration of 2 μg/ml on ice for 15 minutes (Fig. 16). After activation, cells were lysed in RIPA buffer, and protein phosphorylation was analyzed by Western blotting. In fig. 16A shows Western blots of whole-cell extracts of phosphoJAK1, phospho-STAT5, and actin to monitor total protein levels. In fig. 16B shows graphs indicating the quantification of band intensity normalized to actin.

Example 9.

Identification of ligand selectivity in mouse HVEM.

Ligand selectivity was determined in mouse HVEM. A panel of mouse HVEM-Fc muteins was generated by transient transfection into 293T cells, including wild-type HVEM and two variants containing single amino acid substitutions predicted to block binding to LIGHT (BTLA/CD160-sp) or both BTLA and CD160 (LIGHT-sp) based on homology with human HVEM (Fig. 17). HVEM-Fc proteins were titrated onto 293T cells transiently transfected with either mouse CD160 (FIG. 17A), or mouse BTLA (FIG. 17B), or mouse LIGHT, and binding was detected using human anti-Fc. Specific binding was measured using flow cytometry analysis (Figure 17C). The graphs show the MFI of HVEM-Fc protein staining on cells expressing the ligands.

Example 10.

Selective HVEM-Fc inhibits skin inflammation in vivo.

HVEM-Fc has been shown to inhibit skin inflammation in vivo. Mouse HVEM-Fc muteins were injected intraperitoneally into animal models of skin inflammation treated with imiquimod.

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Skin tissue was collected after three treatments with imiquimod (Aldara formulation) at 50 mg on the shaved back of each animal for each day of treatment, and sectioned for histological analysis. Epidermal thickness was quantified in H&E-stained skin sections at 10 sites along the length of each tissue. Representative images showed thickening of the epidermis in different groups of animals treated with HVEM muteins (Fig. 18A). In fig. 18B shows a quantification of epidermal thickness. *, p<0.05; * *, p<0.01; ***, p<0.001; ****, p<0.0001.

Example 11.

Summary.

The data presented here indicate that expression of BTLA selective agonists by viruses and malignant cells is a common strategy to target the HVEM signaling network. Evolution and mutations of HVEM viruses that prevent CD160 binding appear to provide a selective advantage for the pathogen and the malignant cell. The activity of the viral HVEM mimic in CMV has been shown to be sterically unhindered in CD160-expressing cells compared to HVEM, directly inhibiting the activation of ZAP70/Syk and downstream ERK1/2 pathways following antigen receptor stimulation in lymphocytes. Additionally, in CD160-expressing NK cells, activation of BTLA by the viral HVEM mimic directly limits inflammatory cytokine signaling. Together, these data illustrate how the potential to limit inflammatory signaling through inhibitory receptors can provide selective pressure against various intracellular pathogens such as viruses and tumors. This knowledge base of viral and tumor mutations has pushed HVEM bioengineering to achieve selectivity and high affinity for BTLA, which may be practical in altering inflammatory and proliferative processes.

The UL144 protein was originally modeled using the deciphered HVEM structure in part due to its homology (Fig. 1A). However, there are subtle differences in binding between UL144 and HVEM with certain BTLA mutants, with BTLA bound to the 6F4 antibody, and with BTLA in cis configuration, indicating non-identical interactions (Figs. 3, 4). UL144 could not be folded into the CD160-binding protein by an individual point mutation, likely reflecting multiple changes in specificity that arose during host virus evolution. As a result, the UL144 protein is highly antigenic despite the persistence of BTLA binding between strains. In contrast, each tumor-associated HVEM contained only one mutation, which alone could not abrogate all CD160 binding. There are other examples of TNF receptors that interact with Ig domains, such as NGFRp75 with Longol. Notably, HVEM itself was originally identified through its interaction with the Ig domain of herpes simplex virus glycoprotein D. CMV expresses another protein, ORF UL141, which binds TNF-related apoptosis-inducing receptor ligands (TRAIL-R). Recently, a cooperative complex of these two proteins was deciphered, demonstrating that UL141 folds as an Ig domain, however, contact between these proteins occurs at the surface where the TRAIL ligand comes into contact with TRAIL-R, as opposed to HVEM interaction with BTLA or CD160. HVEM may be unique in accepting multiple protein species of ligands, and complexes between the N-terminal cysteine-rich domain 1 (CRD1) of TNFR proteins and Ig domains do not appear to be the preferred type of protein-protein interaction among eukaryotes. It is interesting to note the increased use of these unconventional interactions by different viruses, leading to the temptation to speculate that modulation of immune receptors may enhance pathogen survival.

We found that BTLA inhibited IL-2 and type I interferon signaling in human NK cells and confirmed the role of BTLA as an immune checkpoint inhibitor that regulates T- and B-cell receptor signaling. The mechanism of BTLA downstream signaling inhibition is thought to involve activation of SHP-1 or 2, and likely involves additional pathways. The inhibitory effect of bioengineered HVEM-RTWA on ZAP70/Syk phosphorylation is consistent with previous studies, demonstrating that CD3Z and Syk are direct targets of BTLA activity in T and B cells. JAK/STAT signaling is regulated in part through activation of SHP-1 and dephosphorylation of target proteins, including JAK kinases and STAT proteins. BTLA agonists may directly target cytokine signaling through involvement of SHP-1 or through coupling to unknown inhibitory pathways.

In contrast to selective viral and mutant BTLA agonists, HVEM interacts with BTLA, LIGHT, and CD160 in activated T cells, while in NK cells, abundant CD160 expression blocks HVEM, potentially activating proinflammatory signaling. In the context of lymphoma, mutated TNFRSF14 often pairs with a nonfunctional allele, leading to worse prognosis. The experiments described above predict that the HVEM mutation, in addition to CD160 binding, prevents cytolytic cell activation, while the persistence of BTLA binding activates inhibitory signaling in neighboring cells. The identification of DLBCL deletions in LIGHT and BTLA, both of which can activate HVEM signaling in trans configuration, further supports the hypothesis that the major selective factor is tumor activation

- 20 043600 infiltrating lymphocytes. Additionally, tumors themselves may respond to LIGHT and BTLA to activate the downstream HVEM survival signaling pathway. Notably, follicular T helper cells prominently express BTLA and LIGHT and may contribute to the maintenance of HVEM functionality in lymphoma. Continued research is warranted to determine how HVEM contributes to lymphoma adaptability to the tumor microenvironment through ligand selection. Expression of BTLA, LIGHT, and CD160 varies significantly between different cell types, activation, and differentiation states. Thus, the dynamic regulation of HVEM ligands provides a mechanism for controlling activating and inhibitory signals depending on the cellular context. Determining the factors that regulate receptor and ligand expression will allow us to better understand the role of these proteins in immune responses and how these pathways can be manipulated for therapeutic intervention. The development of targeted agonists for BTLA or other inhibitory receptors may serve to expand the repertoire of drugs to treat inflammatory disease.

Although the invention has been described with reference to the above-described examples, it should be understood that modifications and variations are intended within the spirit and scope of the invention. Thus, the invention is limited only by the claims.

Table 1

Primers used for cloning and site-directed mutagenesis

Gene Primer Subsequence CMV UL144 human E27A mutant Fiala UL144 E46 For AAACCCGAAGCAGTGСAATTAGGAAATCAGTG E27A mutant Fiala UL144 E46 Rev TAATTGCACTGCTTCGGGTTTGCATATTTCAG V28Y mutant UL144_V28YTop AC C C GAAGAATAT CAAT TAGGAAAT CAGT GT T GT C V28Y mutant UL144_V28YBot TTCCTAATTGATATTCTTCGGGTTTGCATATTTC Q29P mutant UL144_Q29PTop GAAGAAGT GC CAT TAGGAAAT CAGT GTTGTCCC Q29P mutant UL144_Q29PBot ATTTCTAATGGCACTTCTTCGGGTTTGCATATT TC N32S mutant UL144_N32STop GCAAT TAGGAAGT CAGT GTTGTCCCC CAT GTAAA C N32S mutant UL144_N32SBot ACAACACTGACTTCCTAATTGCACTTCTTCGG

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Q33A mutant Fiala UL144 Q52 For TTAGGAAATGCGTGTTGTTCCCCCATGTAAACAAG Q33A mutant Fiala UL144 Q52 Rev GGGACAACACGCATTTCCTAATTGCACTTCTTC P36A mutant Fiala UL144 P55 For CAGT GTTGTGCCC CAT GTAAACAAGGATAT C GT G P36A mutant Fiala UL144 P55 Rev TTTACATGGGGCACAACACTGATTTTCCTAATTG P37K mutant UL144_P37KTop GTGTTGTCC CAAAT GTAAACAAGGATAT C GT GT T A.C. P37K mutant UL144_P37KBot TTGTTTACATTTGGGACAACACTGATTTCCTAAT TG K39S mutant K39STop T C C C C CAT GT T CACAAGGATAT C GT GT TACAGG K39S mutant K39SBot ATATCCTTGTGAACATGGGGGACAACACTGATTT C G41A mutant Fiala UL144 G60 For T GTAAACAAGGATAT C GT GT TACAGGACAAT GTA C G41A mutant Fiala UL144 G60 Rev AACACGATATGCTTGTTTACATGGGGGACAACAC TG Y42A mutant UL144F-Y42A-F CCCCCATGTAAACAAGGAGCTCGTGTTACAGGAC AATG Y42A mutant UL144F-Y42A-R CATTGTCCTGTAACACGAGCTCCTTGTTTACATG GGGG R43A mutant Fiala UL144 R62A For CAAGGATAT GCT GTTACAGGACAAT GTACGCAAT ATAC R43A mutant Fiala UL144 R62A Rev TCCTGTAACAGCATATCCTTGTTTACATGGGGG T45A mutant Fiala UL144 T64 For TAT CGTGTTGCAG GACAAT GTAC G CAATATAC G T45A mutant Fiala UL144 T64 Rev ACAT T GT C C T G CAACAC GATAT C C T T GT T TACAT GG G46A mutant Fiala UL144 G65 For CGTGTTACAGCACAATGTACGCAATATACGAGTA C G46A mutant Fiala UL144 G65 Rev CGTACATTGTGCTGTAACACGATATCCTTGTTTA C G46K mutant FUL144-G46K 5' AAACAAGGATAT C GT GT TACAAAACAAT GTAC GC AATATACGAGT G46K mutant FUL144-G46K 3' ACT C GTATAT T GC GTACAT T GT T T T GTAACAC GA TATCCTGTTTT Q47A mutant UL144_Q47ATop GTTACAGGAGCATGTACGCAATATACGAGTACAA C Q47A mutant UL144_Q47ABot TGCGTACATGCTCCTGTAACACGATATCCTTG T49G mutant UL144_T49GTop GGACAAT GT GGGCAATATACGAGTACAACAT GTA C T49G mutant UL144_T49GBot CGTATATTGCC CACAT TGTCCTGTAACAC GATAT C Q50A mutant Fiala UL144 Q69 For CAAT GTAC GGCATATAC GAGTACAACAT GTACAG

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Q50A mutant Fiala UL144 Q69 Rev ACT C GTATAT GC C GTACAT T GT C CT GTAACAC GA TATC T52A mutant Fiala UL144 T71 For ACGCAATATGCGAGTACAACATGTACACTTTGCC C T52A mutant Fiala UL144 T71 Rev TGTTGTACTCGCATATTGCGTACATTGTTCTGTA A.C. N61A mutant UL144_N61ATop CTTTGCCCTGCCGGTACGTATGTATCAGGGC N61A mutant UL144_N61ABot CGTACCGGCAGGGCAAAGTGTACATGTTGTAC L68A mutant Fiala UL144 L86 For GTATCAGGGGCTTACAATTGTACCAATTGCACTG L68A mutant Fiala UL144 L86 Rev ACAAT T GTAAGC C C CT GATACATAC GTAC C GT TA G E76M mutant UL144_E76MTop CAATT GCACTAT GT GTAAT GACACT GAGGTTAC E76M mutant UL144_E76MBot CAATT GCACTAT GT GTAAT GACACT GAGGTTAC P106A mutant Fiala UL144 P124 For TTTTCCGTTGCAGGCGTCCAACATCACAAGCAAC G P106A mutant Fiala UL144 P124 Rev TTGGACGCCTGCAACGGAAAATGACGTATAATTC Human HVEM P59S mutant hHVEM_P59STop CAAGTGCAGTTCAGGTTATCGTGTGAAGGAG P59S mutant hHVEM_P59SBot CGATAACCTGAACTGCACTTGGGCAGCAC G60D mutant HuHVEMG60Dfor tgcagtccagattatcgtgtgaaggaggcctg G60D mutant HuHVEMG60Drev ACACGATAATCTGGACTGCACTTGGGGC Y61C mutant HuHVEMY61Cfor GTCCAGGTTGTCGTGTGAAGGAGGCCTGC Y61C mutant HuHVEMY61Crev CTTCACACGACAACCTGGACTGCACTTGGG G72D mutant hHVEM_G72DTop GCTGACGGACACAGTGTGTGAACCCTGC G72D mutant hHVEM_G72DBot ACACACTGTGTCCGTCAGCTCCCCGCAG T82P mutant hHVEM_T82PTop TCCAGGCCCCTACATTGCCCACCTCAATG T82P mutant hHVEM_T82PBot AATGTAGGGGCCTGGAGGGCAGGGTTC Ins91I mutant hHVEM_ins91ITop CTCAATGGCCTAATAAGCAAGTGTCTGCAGTGC Ins91I mutant hHVEM_ins91IBot CACTTGCTTATTAGGCCATTGAGGTGGGCAATG A102P mutant hHVEM_Al02PTop GTGACCCACCCATGGGCCTGCGCGCG A102P mutant hHVEM_Al02PBot GGCCCATGGGTGGGTCACACATTTGGCACTG R109W mutant HuHVEMR109Wfor CGCGAGCTGGAACTGCTCCAGGACAGAG R109W mutant HuHVEMRl09Wrev GAGCAGTTCCAGCTCGCGCGCAGGCCC BTLA person Q37A mutant HuBTLAQ37Atop catgtgatgtAGCGCTTTATATAAAGAGACAATC TGAACACTC Q37A mutant HuBTLAQ37Abot CTTTATATAAAAGCGCTACATCACATGATTCTTTC CCATG

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L38H mutant HuBTLAL38Htop AT GT GAT GTACAGCATTATATAAAAGAGACAAT CT GAACACTCC L38H mutant HuBTLAL38Hbot TT GT CT CTTTATATAAT GCT GTACAT CACAT GAT TCTTTCC R42D mutant HuBTLAR42Dtop CTTTATATAAAAGGACCAATCTGAACACTCCATCT TAGC R42D mutant HuBTLAR42Dbot GTGTTCAGATTGGTCCTTTATATAAAAGCTGTACA TCACATGATTC E45A mutant HuBTLAE45Atop AGAGACAAT CT GCACACT CCAT CTTAGCAGGAGA TCC E45A mutant HuBTLAE45Abot AAGAT GGAGT GT GGAGATT GT CT CTTTATATAAAA GCTGTAC E57A mutant HuBTLAE57Atop CTTTGAACTAGCATGCCCTGTGAAATACTGTGCT A.A.C. E57A mutant HuBTLAE57Abot TCACAGGGCATGCTAGTTCAAAGGGATCTCCTGC TAAG P59A mutant HuBTLAP59Atop GAACTAGAATGCGCTGTGAAATACTGTGCTAACA GGC P59A mutant HuBTLAP59Abot GTATTTCACAGCGCATTCTAGTTCAAAGGGATCT C K90A mutant HuBTLAK90Atop ACAAGTTGGGCGGAAGAAGAACATTTCATTTT TCATTC K90A mutant HuBTLAK90Abot CTTCTCTTCCGCCCAACTTGTTTGTCTATCTTCA AGTTTTAC V117A mutant HuBTLAV117Atop TGTTCTGCAAATTTTCAGTCTAATCTCATTGAAA GC V117A mutant HuBTLAV117Abot GATTAGACTGAAAATTTGCAGAACAGCGGTATGA CCC N118F mutant HuBTLAN118Ftop GCTGTTCTGCATTTTTTCAGTCTAATCTCATTGA AAGC N118F mutant HuBTLAN118Fbot TAGACT GAAAAAAT GCAGAACAGCGGTAT GAC F119A mutant HuBTLAF119Atop GTTCTGCAAATGCTCAGTCTAATCTCATTGAAAG CCAC F119A mutant HuBTLAF119Abot GAGATTAGACTGAGCATTTGCAGAACAGCGGTAT G S121H mutant HuBTLAS12IHtop CAAAT T T T CAG CATAAT C T CAT T GAAAG C CAC T C A.A.C. S121H mutant HuBTLAS121Hbot CAAT GAGAT TAT G C T GAAAAT T T G CAGAACAG C G H127D mutant HuBTLAH127Dtop CATTGAAAGCGACTCAACAACTCTTTATGTGACA GATG H127D mutant HuBTLAH127Dbot GTTGTTGAGTCGCTTTCAATGAGATTAGACTGAA AATTTG S128H mutant HuBTLAS128Htop T GAAAG CCAC CATACAAC TCTTTATGT GACAGAT GTAAAAAG S128H mutant HuBTLAS128Hbot AAGAGTTGTATGGTGGCTTTCAATGAGATTAGAC TG

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Claims (16)

table 2 Monovalent and bivalent kinetic rate constants for Fc fusion protein binding Analyst HVEM-Fc HuCMV UL144-FC Monovalent analysis k _a (x 10 ⁴ M^s- ¹ ) 3.74 1.61 k _d (x 10~ ³ s~ ⁴ ) 6.6 4.76 K _D (nM) 177 295 Bivalent analysis k _al (x 10 ⁴ M^s- ¹ ) 1.53 0.781 k _dl (x IQ- ³ s- ¹ ) 9.02 5.1 k _a2 (x 10 ^-3 M ^-1 s ^-1 ) 57 .3 0.0139 k _d2 (s^ ¹ ) 2.05 0.00289 CLAIM 1. A fusion protein comprising a herpes virus entry mediator (HVEM) protein and an Fc protein, wherein the HVEM protein consists of a human HVEM ectodomain that is fused to the 3' end of the Fc protein, and wherein the HVEM protein has the mutation S58R and L90A relative to SEQ ID NO: 2, where the fusion protein is a B and T lymphocyte attenuator agonist (BTLA). 2. The fusion protein according to claim 1, wherein the HVEM protein further contains the mutation G68T and L70W relative to SEQ ID NO: 2. 3. The fusion protein according to claim 1 or 2, wherein the HVEM protein is fused to the Fc protein through a peptide linker sequence. 4. The fusion protein according to claim 1, 2 or 3, wherein the Fc protein is selected from the group consisting of IgA, IgG, IgD, IgE and IgM. 5. The fusion protein of claim 4, wherein the Fc protein is an IgG protein selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 6. The fusion protein according to claim 5, wherein the IgG Fc protein is human. 7. The fusion protein of claim 6, wherein the Fc protein is a human IgG1 protein comprising two identical protein fragments derived from a second constant domain and a third constant domain of two heavy chains of a human IgG1 antibody. 8. The fusion protein according to claim 1, wherein the human HVEM ectodomain consists of amino acids 39-184 in accordance with SEQ ID NO: 2. 9. A herpes virus entry mediator (HVEM) protein consisting of the ectodomain of human HVEM, wherein the HVEM protein has the mutation S58R and L90A relative to SEQ ID NO: 2, and wherein the HVEM protein is a BTLA agonist. 10. A herpes virus entry mediator (HVEM) protein consisting of the ectodomain of human HVEM, wherein the HVEM protein has the mutation S58R, G68T, L70W and L90A relative to SEQ ID NO: 2, and wherein the HVEM protein is a BTLA agonist. 11. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 12. Use of a fusion protein according to any one of claims 1 to 8 or an HVEM protein according to claim 9 or 10 in the manufacture of a medicament for use in a method of treating a BTLA-related disorder in a patient. 13. Use according to claim 12, wherein the BTLA-related disorder is cancer or an autoimmune disease or disorder. 14. Use according to claim 12 or 13, further comprising administering an immune response modulator or a chemotherapeutic agent. 15. The use of claim 14, wherein the immune response modulator is selected from the group consisting of: eicosanoids, cytokines, prostaglandins, interleukins, chemokines, checkpoint regulators, TNF superfamily members, TNF receptor superfamily members, and interferons. 16. Use according to claim 14, where the immune response modulator is selected from the group consisting of: CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9 , IL10, IL11, IL12, IL13, IL15, IL17, IL17, IFN-α, IFN-β, IFN-ε, IFN-γ, G-CSF, TNF-α, CTLA4, CD20, PD1, PD1L1, PD1L2, ICOS , CD200, CD52, LTa, LTae, LIGHT, CD27L, 41BBL, FasL, Ox40L, April, TL1A, CD30L, TRAIL, RANKL, BAFF, TWEAK, CD40L, EDA1, EDA2, APP, NGF, TNFR1, TNFR2, LTeR, HVEM , CD27, 4-1BB, Fas, Ox40, AITR, DR3, CD30, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, BAFFR, TACI, -