Multi-domain immunomodulatory proteins and methods of use thereof

Abstract

Abstract Provided herein are multi-domain immunomodulatory proteins, nucleic acids encoding such immunomodulatory proteins, cells engineered to express the immunomodulatory proteins and infectious agents containing nucleic acid encoding the immunomodulatory proteins. The immunomodulatory proteins bind both an inhibitory receptor and a receptor involved in activation signaling cascades in an immune cell, such as a T cell. The immunomodulatory proteins, engineered cells and infectious agents provide therapeutic utility for a variety of immunological diseases or conditions. Compositions and methods for making and using such proteins are provided. Abstract Provided herein are multi-domain immunomodulatory proteins, nucleic acids encoding such immunomodulatory proteins, cells engineered to express the immunomodulatory proteins and infectious agents containing nucleic acid encoding the immunomodulatory proteins. The immunomodulatory proteins bind both an inhibitory receptor and a receptor involved in activation signaling cascades in an immune cell, such as a T cell. The immunomodulatory proteins, engineered cells and infectious agents provide therapeutic utility for a variety of immunological diseases or conditions. Compositions and methods for making and using such proteins are provided. 20 24 20 45 59 01 J ul 2 02 4 2 0 2 4 2 0 4 5 5 9 0 1 J u l 2 0 2 4

Description

[0001] This application is a divisional of Australian Application No. 2019205273, filed 3 January 2019, is related to PCT / US2019 / 012222, and claims priority from U.S. provisional patent application No. 62 / 613,378, filed 3 January 2018, and U.S. provisional application No. 62 / 733,622, filed 19 September 2018, the disclosure of each of which is incorporated by reference in its entirety herein for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] Preceding applications contained a Sequence Listing which was originally submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The present application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. FIELD

[0003] The present disclosure provides multi-domain immunomodulatory proteins, nucleic acids encoding such immunomodulatory proteins, cells engineered to express the immunomodulatory proteins and infectious agents containing nucleic acid encoding the immunomodulatory proteins. The immunomodulatory proteins bind both an inhibitory receptor and a receptor involved in activation signaling cascades in an immune cell, such as a T cell. The immunomodulatory proteins, engineered cells and infectious agents provide therapeutic utility for a variety of immunological diseases or conditions. Compositions and methods for making and using such proteins are provided. BACKGROUND

[0004] Modulation of the immune response by intervening in the processes that occur in the immunological synapse (IS) formed by and between antigen-presenting cells (APCs) or target cells and lymphocytes is of increasing medical interest. Currently, biologics used to enhance or suppress immune responses have generally been limited to immunoglobulins (e.g., anti-PD-1 2024204559   07 Aug 2026 antibodies) or soluble receptors against a single cell surface molecule (e.g., Fc-CTLA4). Improved therapeutic agents that can modulate the IS are needed. Provided are embodiments that meet such needs. [0004a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. [0004b] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. SUMMARY [0004c] In one aspect, the present disclosure provides an immunomodulatory protein comprising: (1) at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor, wherein the inhibitory receptor is PD-1; and (2) at least one activating receptor binding molecule (ARBM) that binds to a ligand of an activating receptor, wherein the IRBM comprises a variant PD-L1 polypeptide comprising an N45D amino acid modification and one or more amino acid modifications selected from the group comprising I20L, K28E, D43G, L56Q, V58A, N78I, Q89R, and G101G-ins, with reference to numbering set forth in SEQ ID NO: 309, and wherein the variant PD-L1 polypeptide comprises a sequence of amino acids that is at least 90% identical to SEQ ID NO: 309, and wherein the ARBM comprises a variant CTLA-4 polypeptide comprising amino acid modification L63P and one or more amino acid modifications selected from the group consisting of G29W, T53S, M56K, N58S, Q82R, M87V, L98Q, and Y105L, with reference to numbering set forth in SEQ ID NO:36 or 2655, and wherein the variant CTLA-4 polypeptide comprises a sequence of amino acids that is at least 90% identical to SEQ ID NO: 36 or 2655. [0004d] In another aspect, the present disclosure provides a nucleic acid molecule(s), encoding the immunomodulatory protein of the invention, optionally wherein the nucleic acid is a synthetic nucleic acid, optionally wherein the nucleic acid molecule is a cDNA. 2024204559   07 Aug 2026 [0004e] In another aspect, the present disclosure provides a vector, comprising the nucleic acid molecule of the invention, optionally wherein the vector is an expression vector, optionally wherein the vector is a mammalian expression vector or a viral vector. [0004f] In another aspect, the present disclosure provides a cell, comprising the nucleic acid of the invention or the vector of the invention, optionally wherein the cell is a mammalian cell, optionally wherein the cell is a human cell. [0004g] In another aspect, the present disclosure provides a method of producing an immunomodulatory protein, comprising introducing a nucleic acid molecule of the invention or a vector of the invention into a host cell under conditions to express the protein in the cell, optionally wherein the method further comprises isolating or purifying the immunomodulatory protein from the cell. [0004h] In another aspect, the present disclosure provides a pharmaceutical composition, comprising an immunomodulatory protein of the invention, optionally wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, optionally wherein the pharmaceutical composition is sterile. [0004i] In another aspect, the present disclosure provides an article of manufacture comprising a pharmaceutical composition of the invention in a vial or container, optionally wherein the vial or container is sealed. [0004j] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition of the invention or an article of manufacture of the invention, and instructions for use. [0004k] In another aspect, the present disclosure provides a method of decreasing an immune response in a subject, comprising administering an immunomodulatory protein of the invention or a pharmaceutical composition of the invention to the subject, optionally wherein modulating the immune response treats a disease or condition in the subject. [0004l] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject, comprising administering an immunomodulatory protein of the invention or a pharmaceutical composition of the invention to the subject, wherein the disease or condition is Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, a thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, an autoimmune 2024204559   07 Aug 2026 hematological disease, an autoimmune demyelinating disease, an autoimmune disease involving a systemic autoimmune disorder, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, lupus erythematosus, celiac disease, Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Graves' disease, DeQuervains thyroiditis, Vasculitis, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune thrombocytopenia, sympathetic opthalmia, chronic aggressive hepatitis, membranous glomerulopathy, primary idiopathic myxedema, chronic hepatitis, hypogonadism, pernicious anemia, autoimmune enteropathy syndrome, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sensoneural hearing loss, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, and idiopathic facial paralysis, giant cell arteritis (GCA), cancer, Addison’s Disease, Allergy, Alopecia Areata, Alzheimer’s, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), Arthritis, Atherosclerosis, Atherosclerotic plaque, autoimmune disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet’s Disease, Berger’s Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue / Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan’s Syndrome, Exopthalmos, Fibromyalgia, Goodpasture’s Syndrome, graft-related disease or disorder, GVHD, Hashimoto’s Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki’s Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, Lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere’s Disease, Miller Fish Syndrome / acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, 2024204559   07 Aug 2026 Pemphigus Vulgaris, Pernicious Anaemia, Polychondritis, Polyglandular Syndromes (Whitaker’s syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis / Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud’s Phenomenon, Reiter’s Syndrome / Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt’s syndrome, Scleroderma, Sjorgen’s Syndrome, Solid-organ transplant rejection, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis / Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Uveitis, Vitiligo, Wegener’s Granulomatosis, an immune response associated with rejection of a donor tissue, cell, graft, or organ transplant by a recipient subject, graft versus host disease (GVDH), or interstitial bowel disease. [0004m] In another aspect, the present disclosure provides use of an immunomodulatory protein of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for: decreasing an immune response in a subject; or treating a disease or condition in a subject, wherein the disease or condition is Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, a thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, an autoimmune hematological disease, an autoimmune demyelinating disease, an autoimmune disease involving a systemic autoimmune disorder, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, lupus erythematosus, celiac disease, Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Graves' disease, DeQuervains thyroiditis, Vasculitis, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune thrombocytopenia, sympathetic opthalmia, chronic aggressive hepatitis, membranous glomerulopathy, primary idiopathic myxedema, chronic hepatitis, hypogonadism, pernicious anemia, autoimmune enteropathy syndrome, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sensoneural hearing loss, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, and idiopathic facial paralysis, giant cell arteritis (GCA), cancer, Addison’s Disease, Allergy, Alopecia Areata, Alzheimer’s, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), Arthritis, Atherosclerosis, Atherosclerotic plaque, autoimmune disease, 2c 2024204559   07 Aug 2026 Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet’s Disease, Berger’s Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue / Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan’s Syndrome, Exopthalmos, Fibromyalgia, Goodpasture’s Syndrome, graft-related disease or disorder, GVHD, Hashimoto’s Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki’s Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, Lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere’s Disease, Miller Fish Syndrome / acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polychondritis, Polyglandular Syndromes (Whitaker’s syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis / Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud’s Phenomenon, Reiter’s Syndrome / Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt’s syndrome, Scleroderma, Sjorgen’s Syndrome, Solid-organ transplant rejection, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis / Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Uveitis, Vitiligo, Wegener’s Granulomatosis, an immune response associated with rejection of a donor tissue, cell, graft, or organ transplant by a recipient subject, graft versus host disease (GVDH), or interstitial bowel disease.

[0005] Provided are immunomodulatory proteins containing at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor; and at least one activating receptor binding molecule (ARBM) that binds to (i) an activating receptor (ii) a ligand of the 2d 2024204559   07 Aug 2026 activating receptor, wherein the inhibitory receptor and the activating receptor are expressed on the same immune cell; the IRBM and ARBM are different polypeptides in which (a) one of the at least one of the IRBM or ARBM is a not an antibody or an antigen-binding antibody fragment; and / or (b) the at least one of the IRBM or the at least one of the ARBM contains a non-antibody immunoglobulin superfamily (IgSF) domain; and the IRBM exhibits higher binding activity, optionally higher binding affinity, for the inhibitory receptor than the ARBM exhibits for the activating receptor or a ligand of the activating receptor.

[0006] Provided are immunomodulatory proteins containing at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor; and at least one activating receptor binding molecule (ARBM) that binds to (i) an activating receptor or (ii) a ligand of an activating receptor, wherein the inhibitory receptor and the activating receptor are expressed on the same immune cell; the IRBM and ARBM are different polypeptides in which (a) one of the at least one of the IRBM or ARBM is a not an antibody or an antigen-binding antibody fragment; and / or (b) the at least one of the IRBM or the at least one of the ARBM contains a non-antibody immunoglobulin superfamily (IgSF) domain; and the immunomodulatory protein is a monomeric polypeptide and / or contains a single polypeptide chain.

[0007] In some of any of the provided embodiments, the ARBM binds to a ligand of an activating receptor. In some examples, the ligand of an activating receptor is CD80, CD86, ICOSL or an MHC molecule. In some embodiments, the ARBM binds to an activating receptor.

[0008] Provided are immunomodulatory proteins containing at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor; and at least one activating receptor binding molecule (ARBM) that binds to an activating receptor, wherein the inhibitory receptor and the activating receptor are expressed on the same immune cell; and the IRBM and ARBM are different polypeptides in which (a) one of the at least one of the IRBM or ARBM is a 2024204559   01 Jul 2024 not an antibody or an antigen-binding antibody fragment; and / or (b) the at least one of the IRBM or the at least one of the ARBM contains a non-antibody immunoglobulin superfamily (IgSF) domain. In some of any such embodiments, the immune cell is a T cell.

[0009] In some of any of the provided embodiments, the activating receptor contains an immunoreceptor tyrosine-based activation motif (ITAM) or interacts with an adaptor protein comprising a binding domain specific to a phosphotyrosine residue. In some embodiments, the activating receptor is selected from a TCR, CD3, CD4, CD8, CD28, ICOS or CD2. In some embodiments, the activating receptor is CD28. In some embodiments, the activating receptor is CD2.

[0010] Provided are immunomodulatory proteins containing at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor, wherein the inhibitory receptor is PD-1 and at least one activating receptor binding molecule (ARBM) that binds to an activating receptor or to a ligand of the activating receptor, wherein the IRBM and ARBM are different polypeptides.Provided are immunomodulatory proteins containing at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor, wherein the inhibitory receptor is TIGIT and at least one activating receptor binding molecule (ARBM) that binds to an activating receptor or to a ligand of the activating receptor, wherein the IRBM and ARBM are different polypeptides. In some of any such embodiments, the activating receptor contains an immunoreceptor tyrosine-based activation motif (ITAM) or interacts with an adaptor protein comprising a binding domain specific to a phosphotyrosine residue. In some embodiments, the activating receptor is selected from a TCR, CD3, CD4, CD8, CD28, ICOS or CD2. In some examples, the activating receptor is CD28. In some embodiments, the activating receptor is CD2.

[0011] Provided are immunomodulatory proteins comprising at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor; and at least one activating receptor binding molecule (ARBM) that binds to an activating receptor or to a ligand of the activating receptor, wherein the activating receptor is CD28, wherein the IRBM and ARBM are different polypeptides.

[0012] Provided are immunomodulatory proteins comprising at least one inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor; and at least one activating receptor binding molecule (ARBM) that binds to an activating receptor or to a ligand of the 2024204559   01 Jul 2024 activating receptor, wherein the activating receptor is CD2, wherein the IRBM and ARBM are different polypeptides.

[0013] In some of any of the provided embodiments, the inhibitory receptor and the activating receptor are expressed on the same immune cell. In some embodiments, the immune cell is a T cell.

[0014] In some of any of the provided embodiments, one of the at least one of the IRBM or ARBM is a not an antibody or an antigen-binding antibody fragment; and / or the at least one of the IRBM or the at least one of the ARBM contains a non-antibody immunoglobulin superfamily (IgSF) domain.

[0015] In some of any of the provided embodiments, the IRBM exhibits higher binding activity, optionally higher binding affinity, for the inhibitory receptor than the ARBM exhibits for the activating receptor or a ligand of the activating receptor. In some embodiments, the IRBM exhibits 1.2-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0 fold or higher binding activity, optionally higher binding affinity, for the inhibitory receptor than the ARBM exhibits for the activating receptor or a ligand of the activating receptor.

[0016] In some of any such embodiments, the at least one of the IRBM and the at least one of the ARBM is not an antibody or an antigen-binding antibody fragment. In some embodiments, the at least one of the IRBM or the at least one of the ARBM contains a nonantibody immunoglobulin superfamily (IgSF) domain. In some embodiments, the at least one of the IRBM and the at least one of the ARBM each individually contains at least one non-antibody IgSF domain.

[0017] In some of any of the provided embodiments, the ARBM binds to a ligand of the activating receptor and the ligand of the activating receptor is CD80 or CD86. In some embodiments, the ARBM is an antibody that specifically binds to the ligand of an activating receptor or is an antigen-binding antibody fragment. In some embodiments, the ARBM contains at least one IgSF domain of a binding partner of the ligand of the activating receptor. In some such embodiments, the binding partner of a ligand of an activating receptor is a wild-type CTLA-4 or is a variant thereof that binds to the ligand of the activating receptor, optionally wherein the ligand is CD80 or CD86. In some cases, the wild-type protein is a human protein. In some embodiments, the ARBM is not the full-length sequence of the binding partner; and / or the ARBM only contains the extracellular domain or an IgSF domain or specific binding 2024204559   01 Jul 2024 fragment thereof of the binding partner. In some embodiments, the IgSF domain is an IgV domain. In some of any such embodiments, the binding partner of the ligand of an activating receptor is a CTLA-4 polypeptide or a variant thereof and the ARBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 36, 2655 or 2947, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36, 2655 2947; or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the binding partner is a variant of a CTLA-4 polypeptide and the variant exhibits increased binding activity, optionally binding affinity, to the ligand of the activating receptor compared to the binding of unmodified or wild-type binding partner to the same ligand. In some cases, the binding activity, optionally binding affinity, to the ligand of the activating receptor is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold.

[0018] In some of any of the provided embodiments, the ARBM is an antibody that specifically binds to the activating receptor or is an antigen-binding antibody fragment.

[0019] In some of any of the provided embodiments, the ARBM contains at least one IgSF domain of a binding partner of the activating receptor. In some of any such embodiments, the ARBM is not the full-length sequence of the binding partner; and / or the ARBM only contains the extracellular domain or at least one IgSF domain or specific binding fragment thereof of the binding partner. In some embodiments, the IgSF domain is an IgV domain or an IgC domain. In some aspects, the at least one IgSF domain contains an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both. In some embodiments, the at least one IgSF domain consists of an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both.

[0020] In some embodiments, the activating receptor is CD28. In some embodiments, the binding partner of the activating receptor is selected from a wild-type ICOSL, CD80 or CD86 or is a variant thereof that binds to the activating receptor. In some cases, the wild-type protein is a human protein. In some of any such embodiments, the binding partner of the activating receptor is a ICOSL polypeptide or a variant thereof and the ARBM contains (i) the sequence of amino acids set forth in SEQ ID NO:32, 2056, or 2244, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, 2056, or 2244; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the activating receptor is CD28 and the binding partner 2024204559   01 Jul 2024 of the activating receptor is a CD80 polypeptide or a variant thereof and the ARBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 28, 1005, 1079, 2615, 2654, or 3580, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28, 1005, 1079, 2615, 2654, or 3580; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the binding partner of the activating receptor is a CD86 polypeptide or a variant thereof and the ARBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 29 or 1195, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or 1195; or (iii) a specific binding fragment of (i) or (ii).

[0021] In some embodiments, the activating receptor is CD2. In some embodiments, the binding partner of the activating receptor is CD58 or is a variant thereof that binds to the activating receptor. In some cases, the wild-type protein is a human protein. In some of any such embodiments, the binding partner of the activating receptor is a CD58 polypeptide or a variant thereof and the ARBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 2946, 3239 or 3650, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2946, 3239 or 3650; or (iii) a specific binding fragment of (i) or (ii).

[0022] In some of any such embodiments, the binding partner is a variant of the activating receptor and the variant exhibits increased binding activity, optionally binding affinity, to the activating receptor compared to the binding of unmodified or wild-type binding partner to the same activating receptor. In some examples, the binding activity, optionally binding affinity, to the activating receptor is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold.

[0023] In some of any of the provided embodiments, the inhibitory receptor includes an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory receptor is PD-1, TIGIT or CTLA-4. In some embodiments, the IRBM is an antibody that specifically binds to the inhibitory receptor or is an antigen-binding antibody fragment. In some embodimetns, the IRBM contains at least one IgSF domain of a binding partner of the inhibitory receptor. In some examples, the binding partner of the inhibitory receptor is selected from a wild-type CD112, CD155, PD-L1 or PD-L2 or is a variant thereof that binds to the inhibitory receptor. In some instances, the wild-type protein is a human protein. 2024204559   01 Jul 2024

[0024] In some embodiments, the IRBM is not the full-length sequence of the binding partner; and / or the IRBM only contains the extracellular domain or an IgSF domain or specific binding fragment thereof of the binding partner, optionally wherein the IgSF domain is an IgV domain or an IgC domain. In some aspects, the at least one IgSF domain includes an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both. In some embodiments, the at least one IgSF domain consists of an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both.

[0025] In some of any such embodiments, the inhibitory receptor is a TIGIT. In some embodiments, the binding partner of the inhibitory receptor is a CD155 polypeptide or a variant thereof and the IRBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 47, 310 or 353, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47, 310 or 353; or (iii) a specific binding fragment of (i) or (ii). In some embodiments,the binding partner of the inhibitory receptor is a CD112 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 48, 666 or 761, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48, 666 or 761; or (iii) a specific binding fragment of (i) or (ii).

[0026] In some of any such embodiments, the inhibitory receptor is PD-1. In some embodiments, the binding partner of the inhibitory receptor is a PD-L1 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 30, 55, 309 or 1728, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 55, 309 or 1728; or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the binding partner of the inhibitory receptor is a PD-L2 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 31, 1203 or 1263, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31, 1203 or 1263; or (iii) a specific binding fragment of (i) or (ii). 2024204559   01 Jul 2024

[0027] In some of any of the provided embodiments, the IRBM contains at least one IgSF domain of a binding partner of the inhibitory receptor. Iln some of any of the provided embodiments, the IRBM is not the full-length sequence of the binding partner; and / or the IRBM only contains the extracellular domain or an IgSF domain or specific binding fragment thereof of the binding partner. In some embodiments, the IgSF domain is an IgV domain or an IgC domain. In some embodiments, the at least one IgSF domain contains an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both. In some embodimetns, the at least one IgSF domain consists of an IgV domain or specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both.

[0028] In some of any of the provided embodiments, the inhibitory is PD-1 and the binding partner of the inhibitory receptor is selected from a wild-type PD-L1 or PD-L2 or is a variant thereof that binds to the inhibitory receptor. In some cases, the wild-type protein is a human protein. In some embodiments, the binding partner of the inhibitory receptor is a PD-L1 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 30, 55, 309 or 1728, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 55, 309 or 1728; or (iii) a specific binding fragment of (i) or (ii); or the binding partner of the inhibitory receptor is a PD-L2 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 31, 1203 or 1263, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31, 1203 or 1263; or (iii) a specific binding fragment of (i) or (ii).

[0029] In some of any of the provided embodiments, the inhibitory is TIGIT and the binding partner of the inhibitory receptor is selected from a wild-type CD 155 or CD112 or is a variant thereof that binds to the inhibitory receptor. In some cases, the wild-type protein is a human protein. In some embodiments, the binding partner of the inhibitory receptor is a CD155 polypeptide or a variant thereof and the IRBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 47, 310 or 353, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47, 310 or 353; or (iii) a specific binding fragment of (i) or (ii). In some embodiments,the binding partner of the inhibitory receptor is a CD112 polypeptide or a variant thereof and the IRBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 48, 666 2024204559   01 Jul 2024 or 761, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48, 666 or 761; or (iii) a specific binding fragment of (i) or (ii).

[0030] In some of any such embodiments, the binding partner is a variant and the variant exhibits increased binding activity, optionally binding affinity, to the inhibitory receptor compared to the binding of unmodified or wild-type binding partner to the same inhibitory receptor. In some examples, the binding activity, optionally binding affinity, to the inhibitory receptor is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold.

[0031] In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD28 and the IRBM binds to an inhibitory receptor that is PD-1. In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD28 and the IRBM binds to an inhibitory receptor that is TIGIT.

[0032] In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD28 and the ARBM contains at least one IgSF domain of a binding partner of CD28.

[0033] In some of any such embodiments, the binding partner of CD28 is ICOSL or is a variant thereof that binds to CD28. In some aspects, the binding partner is a variant of ICOSL and the variant exhibits increased binding activity, optionally binding affinity, to CD28 compared to the binding of unmodified or wild-type ICOSL to CD28. In some embodiments, the binding activity, optionally binding affinity, to CD28 is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40fold, 50-fold or 60-fold. In some embodiments, the ARBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 32, 2056, or 2244, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, 2056, or 2244; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the ARBM is a variant ICOSL and the one or more amino acid modifications in a variant ICOSL can be any described herein, such as set forth in Section LB.La. In some embodiments, the one or more amino acid modifications include any set forth in Table 7. In some embodiments, the ARBM is a variant ICOSL and the variant ICOSL contains one or more amino acid modifications at one or more position corresponding to 52, 57, 100, 110, or 198 with reference to numbering set forth in SEQ ID NO: 32, 2056, or 2244. 2024204559   01 Jul 2024 In some cases, the variant ICOSL contains one or more amino acid modifications selected from N52H, N52D, N52S, N52K, S54A, S54P, N57Y, Q100P, Q100R, V110A, V110D, C198R, or a conservative amino acid substitution thereof, with reference to numbering set forth in SEQ ID NO: 32, 2056, or 2244. In some embodiments, the variant ICOSL contains one or more amino acid modifications selected from M10V, M10I, V11E, S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R, K42E, T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52L, N52K, N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y, S54A, S54P, N57A, N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94E, H94D, L96F, L96I, V97A, L98F, S99G, Q100A, Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100R, Q100P, Q100S, Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110D, VI10N, V110A, Ell Idel, T113E, H115R, H115Q, V116A, A117T, N119Q, F120I, F120S, S121G, V122A, V122M, S126T, S126R, H129P, S130G,S132F, Q133H, E135K, F138L, T139S, C140D, C140del, S142F,I143V, I143T, N144D, Y146C, V151A, Y152C, Y152H,W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M, L166Q, N168Q, F172S, L173S, M175T, T190S, T190A, S192G, V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T, I218N, E220G, R221G, R221I, I224V, T225A, N227K or a conservative amino acid modification thereof, with reference to numbering of reference to numbering of SEQ ID NO:32, 2056 or 2244.

[0034] In some of any such embodiments, the variant ICOSL polypeptide contains the amino acid modification(s) N52S, N52S, N52D, N52Y / N57Y / F138L / L203P, N52H / N57Y / Q100P, N52S / Y146C / Y152C, N52H / C198R, N52H / C140del / T225A, N52H / C198R / T225A, N52H / K92R, N52H / S99G, N57Y, N57Y / Q100P, N52S / S130G / Y152C, N52S / Y152C, N52S / C198R, N52Y / N57Y / Y152C, N52Y / N57Y / H129P / C198R, N52H / L161P / C198R, N52S / T113E, S54A, N52D / S54P, N52K / L208P, N52S / Y152H, N52H / I143T, N52S / L80P, N52S / D158G, N52D / Q133H, N52H / N57 Y / Q100R / V110D / C198R / S212G, N52H / N57 Y / Q100R / C198R, N52H / N57Y / L74Q / V110D / S192G, N52H / Q100R, N52H / S121G / C198R, A20V / N52H / N57Y / Q100R / S109G, N52H / N57Y / Q100P / C198R, N52H / N57Y / Q100R / V122A, N52H / N57Y / Q100R / F172S, N52H / N57Y / Q100R, N52S / F120S / N227K, N52S / N194D, N52S / F120S, N52S / G72R, N52S / A71T / A117T / T190A / C198R, 2024204559   01 Jul 2024 N52H / N57 Y / Q100R / V107I / V110D / S132F / I154F / C198R / R221G, E16V / N52H / N57 Y / Q 100R / V11OD / H115R / Y152C / K156M / C198R, N52H / N57Y / Q100R / V110D / C198R, F27S / N52H / N57Y / V1 ION, N52S / H94E / L96VS109N / L166Q, S18R / N52S / F93L / I143V / R221G, V11E / N30D / N52H / N57Y / H94E / L96VL98F / N194D / V210A / I218T, N52S / H94E / L96VV122M, N52H / N57Y / H94E / L96I / F120VS126T / W153R / I218N, M10V / S18R / N30D / N52S / S126R / T139S / L203F, S25G / N30D / N52S / F120S / N227K, N52H / N57 Y / Q 100R / V11OD / F172S / C198R, S25G / F27C / N52H / N57Y / Q100R / V110D / E135K / L173S / C198R, N52H / N57Y / V110A / C198R / R221I, N52H / N57Y / Q100R / L102R / V110D / H115R / C198R, N52H / N57Y / Q100R / V110D / N144D / F172S / C198R, N52S / H94E / L98F / Q100R, N52S / E90A, N52S / F120S / I143V / I224V, N52H / N57Y / Q100R / F172S / C198R, N52H / N57Y / Q100R / H115R / F172S / C198R, N52Y / N57Y / Q100P / F172S, E16V / N52H / N57Y / Q100R / V110D / H115R / Y152C / K156M / F172S / C198R, N52S / H115R / F120S / I143V / C198R, N52H / N57Y / Q100P / C198R, N52H / N57Y / Q100P / H115R / F172S / C198R, N52H / N57Y / Q100P / F172S / C198R, N52H / N57Y / Q100P / H115R, N52H / N57Y / Q100P / H115R / C198R, N52H / Q100R / C198R, N52H / Q100R / H115R / F172S, N52H / Q100R / H115X / F172S / C198R, N52H / Q100R / H115R / F172S / C198R, N52H / N57Y / Q100R / F172S / C198R, N52H / N57Y / Q100R / H115R / F172S / C198R, N52H / N57Y / Q100R / H115R / F172S, Q100R, N52Y / F138L / L203P, N57Y / Q100R / C198R, N57Y / F138L / L203, N52H, N57Y, N57Y / Q100P, Q1OOR / F138L, N52H / N57Y / Q100R / H115R, N52H / N57Y / Q100R / F172S, N52H / N57Y / Q100R / H115R / F172S / I224V, N52H / N57Y / Q100R / H115R / F172S, N52H / N57Y / Q100R / H115R / C198R, N52H / N57Y / Q100R / F172S / C198R, N52H / N57Y / Q100R / H115R / F172S / C198R, N52H / N57Y / Q100R / H115R / I143V / F172S / C198R, N52H / N57Y / Q100R / L102R / H115R / F172S / C198R, N52H / N57Y / Q100R / H115R / F172S / N194D, N52H / N57Y / H115R / F172S / C198R, N52H / N57Y / Q100R / H115R / C198R, N52H / N57Y / H115R, N52H / Q100R / H115R / I143T / F172S, N52H / N57Y / Q100P / H115R / F172S, E16V / N52H / N57Y / Q100R / V110D / H115R / C198R, N30D / K42E / N52S / H115R / C198R / R221I, N52S / E90A / H115R, N30D / K42E / N52S / H115R, N52S / H115R / F172S / C198R, N119Q, N207Q, N52Q / N207X, N168X / N207X, N52Q / N168Q, N84Q / N207Q, N119Q / N168Q, N119Q / N207Q, N119Q / N155X, N52Q / N119Q, 2024204559   01 Jul 2024 N52Q / N84Q / N207Q, N119Q / N155Q / N168Q, N52Q / N84Q / N155X / N168X, N52Q / N84Q / N119Q / N168Q , N52A / N57F / Q100S, N52A, / N57H / Q100S, N52A / N57Y / Q100A, N52D / N57A / Q100A, N52D / Q100S, N52G / Q100A, N52H / Q100A, N52M / N57H / Q100S, N52M / N57W / Q100P, N52Q / N57F, N52Q / N57S / Q100A, N52R / N57L / Q100A, N52R / N57Y / Q100P, N52R / N57Y / Q100S, N52S / N57A / Q100A, N52S / N57H / Q100E, N52S / N57L / Q100S, N52S / N57M / Q100S, N52S / N57Y / Q100S, N52S / N57Y / Q100M, N52S / N57Y / Q100V, N52T / N57H / Q100S, N52T / N57H / Q100A, N52T / N57Y / Q100A, N52V / N57L / Q100A, N52H / N57Y / Q100K, N52K / N57Y / Q100R, N52L / N57H / Q100R, N52R / N57F / Q100N, N52R / N57F / Q100P, N52R / N57F / Q100R, N52R / N57F / Q100T, N52R / N57L / Q100S, N52R / N57W / Q100K, N52R / N57W, N52G / N57V, N52L / N57V, N52S / N57L / Q100G, N52T / N57K / Q100P. In some cases, the variant ICOSL polypeptide contains the modification(s) N52H / N57Y / Q100R / F172S, N52H / N57Y / Q100R, N52H / N57Y / Q100R / V122A, N52H / Q100R, N52H / N57Y / Q100P, N52H / N57Y / V110A / C198R / R221I, N52Y / N57Y / I38L / L203P, N52S / N194D or N52D.

[0035] In some of any of the provided embodiments, the ARBM contains the sequence of amino acids set forth in any of SEQ ID NOs: 2022-2055, 2074, 2076-2121, 2137-2154, 21602197, 2200-2206, 2208-2243, 2299-2346, or a specific binding fragment thereof. In some of any of the provided embodiments, the ARBM includes the sequence of amino acids set forth in any of SEQ ID NOs: 2057-2073, 2075, 2122-2136, 2155-2159, 2189-2199, 2207, 2245-2298, 2347-2518, or a specific binding fragment thereof.

[0036] In some of any of the provided embodiments, the binding partner of CD28 is CD80 or CD86. In some embodiments, the ARBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 28, 1005, 1079, 2615, 2654 or 3580, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, 1005, 1079, 2615, 2654 or 3580; or (iii) a specific binding fragment of (i) or (ii). In some of any of the provided embodiments, the ARBM includes (i) the sequence of amino acids set forth in SEQ ID NO: 29 or 1195, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29 or 1195; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the ARBM is a variant CD80 and the one or more amino acid modifications can include any described herein, such as set forth in Section LB.l.b. In some embodiments, the one or more amino acid modifications include any 2024204559   01 Jul 2024 set forth in Table 8. In some of any such embodiments, the ARBM is a variant CD86 and the one or more amino acid modifications can be any described herein, such as set forth in Section I.B.l.c. In some embodiments, the one or more amino acid modifications include any set forth in Table 9.

[0037] In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD28 and the IRBM binds to an inhibitory receptor that is PD-1. In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD28 and the IRBM binds to an inhibitory receptor that is TIGIT.

[0038] In some of any of the provided embodiments, the ARBM binds to an activating receptor that is CD2 and the ARBM contains at least one IgSF domain of a binding partner of CD2. In some embodiments the binding partner of CD2 is CD58 or is a variant thereof.

[0039] In some of any of the provided embodiments, the ARBM comprises (i) the sequence of amino acids set forth in SEQ ID NO: 2946, 3239 or 3650, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2946, 3239 or 3650; or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the binding partner of CD2 is a wild-type human CD58. In some embodiments, the ARBM is set forth in SEQ ID NO:2946, 3239 or 3650 or is a specific binding fragment thereof that binds to CD2.

[0040] In some of any of the provided embodiments, the ARBM binds to a ligand of an activating receptor that is CD80 or CD86 and the IRBM binds to an inhibitory receptor that is PD-1. In some embodiments, the ARBM contains at least one IgSF domain of a binding partner of the ligand of the activating receptor. In some examples, the binding partner is CTLA-4 or is a variant thereof that binds to CD80 or CD86. In some of any such embodiments, the binding partner is a variant of CTLA-4 and the variant exhibits increased binding activity, optionally binding affinity, to CD80 or CD86 compared to the binding of unmodified or wild-type CTLA-4 to CD80 or CD86. In some cases, the binding activity, optionally binding affinity, to CD80 or CD86 is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold. In some embodiments, the ARBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 36, 2655 or 2947, (ii) a sequence of amino acids that has at least 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36, 2655 or 2947; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the ARBM is a 2024204559   01 Jul 2024 variant CTLA-4 and the one or more amino acid modifications can include any described herein, such as set forth in Section I.B.5.b. In some embodiments, the one or more amino acid modifications include any set forth in Table 10.In some embodiments, the variant CTLA-4 contains one or more modification at one or more position selected from 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 31, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67 69, 71, 72, 73, 75, 76, 82, 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117,118, 119, 120, 121 or 122 with reference to numbering set forth in SEQ ID NO:36 or 2655. In some embodiments, the variant CTLA-4 contains one or more amino acid modifications selected from A6T, V10A, L12F, L12H, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, A31Y, E33M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L63H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y105F, Y105L, L106E, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117M, I117T, I18T, or I18V, or a conservative amino acid substitution thereof, with reference to numbering set forth in SEQ ID NO: 36 or 2655.

[0041] In some of any such embodiments, the variant CTLA-4 contains the amino acid modification(s) A31Y / L106E, A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, L12F / K30R / S72G / Q82R / L98Q / M99L / Y105L, L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S, L12H / E33M / L98Q / Y105L, L12H / M55T / E59D / L63P / M99L, L12H / L63P / S72G / L98Q / Y105L, L12I / M5 5T / M5 6 V / I67T / M99L / L106R / 1108F, L12P / R16H / A26T / T61S / L63P / M87 V / L98Q / M99L / Y105L / L1061 / 1117L, L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y105L, L12P / A26T, L12P / A26T / L63P, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, L12P / G29W / L63P / S72G / L98Q / Y105L, L12P / G29W / L63P / S72G / L98Q / Y105L / L106I, L12P / A26T / L63P / L98Q / M99L / Y105L, L12P / A26T / L63P / L98Q / Y105L, L12P / A26T / L63P / L98Q / Y105L / L106I, L12P / G29W / D43N / N5 8S / L63P / L98Q / M99L / Y105L, 2024204559   01 Jul 2024 L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H, L12P / L63P / S72G / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105L / L106N, L12P / L63P / S72G / L98Q / M99L / Y105L / L106N / 1117L, S14N / R16C / I18T / M56K / T61A / L63P / A86T / M99L, S15P / I18V / M56T / L98Q / M99L / Y105L, R16C / G29W / E33V / M55T / L63P / L98Q / Y105L, I18A / L63P / S72G / L98Q / Y105L, I18F / L63P / L98Q / M99L / Y105L / P121S, I18N / A26T / L63H / T89A / L98Q / M99L / Y105L, I18N / L63P / S72T / M87T / L98Q / Y105L / N110S, I18T7A26S / M55T7M56V / L63P / S72G / L98Q / M99L / Y105L / I117K, I18T / A26T / L63P / S72G / L98Q / Y105L, I18T / A26T / L63P / Q82R / L98Q / Y105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y105L, I18T / G29W / L63P / L98Q / Y105L, I18T7E48R / L63P / T69S / L98Q / Y105L / N110Y, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, I18T / L63P / S72G / M87K / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / Y105L / I108V, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, I18V / G29W / L63P / S72G / L98Q / Y105L, A19V / G29W / R35K / L63P / L98Q / M99L / Y105L, S20N / A26T / L63P / L98Q / M99L / Y105L, V22A / L63P / L98Q / M99L / Y105L / P119H, V22I / L63P / L98Q / Y105L / I117M, E24Q / L63P / S72G / L98Q / M99L / Y105L, A26D / S72G / L98Q / M99L / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, A26T / V46E / L63P / D65G / L98Q, A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L, A26T / T53S / M56K / L63P / L98Q / Y105L, A26T / T53S / L63P / L98Q / Y105L / L106I / I117L, A26T / Y54F / M56K / M99L / Y105L, A26T / M55R / L98Q / M99L / Y105L, A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, A26T / M55T / L63P / L98Q / M99L / Y105L, A26T / L63P / D65G / L98Q / M99L / Y105L, A26T / L63P / M87V / N110K / I117E, A26T / L63P / S72G / L98Q / M99L / Y105L, A26T / L63P / S72G / L98Q / Y105L / L106PI117L, A26T / L63P / L98Q / M99L / Y105L, A26T / I67N / S72G / L98Q / M99L / Y105L, S27P / M56K / L63P / S72G / S73R / T89A / M99L / Y105L / I117M, P28L / E33V / L63P / S72G / L98Q / M99L / Y105L, P28L / E33V / L63P / S72G / L98R / M99L / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / T61N / L63P / L98Q / Y105L, G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L, G29W / T53S / M56K / L63P / L98Q / Y105L, G29W / T53S / L63P / S72G / L98Q / Y105L, G29W / M55V / E59G / L63P / L98Q / Y105L, G29W / M56T / L63P / L98Q / Y105L / L106PI117L, G29W / N58D / I67V / L98Q / M99L / Y105L, G29W / N58S / L63P / D64N / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, G29W / N58S / L63P / S72G / L98Q / Y105L, G29W / N58S / L63P / S72G / L98Q / Y105L / L106I, G29W / N58S / L63P / S72G / L98Q / Y105L / L106V, G29W / N58S / L63P / S72G / M87V / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, 2024204559   01 Jul 2024 G29W / N58S / L63P / M87T / L98Q / M99L / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / E59G / L63P / L98Q / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G29W / L63P / D65G / S72G / L98Q / Y105L, G29W / L63P / I67V / S72G / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / L106I, G29W / L63P / S72G / L98Q / Y105L / L106I / I117L, G29W / L63P / S72G / L98Q / Y105L / I117L, G29W / L63P / S72G / L98Q / Y105L / P121S,G29W / L63P / L98Q / M99L / Y105L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, G29W / M87K / I93V / L98Q / M99L / Y105L, G29W / L98Q / M99L / Y105L, E33M / A42T / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L / I108F, E33M / L63P / S72G / L98Q / Y105L / I117L, E33M / Q82H / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, T37S / M56V / L98Q / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, Q41L / Y54F / M56K / M99L / I108F, T53S / M56V / L98Q / Y105L, M55T / L63P / T71I / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L, M55T / E97Q / M99L / Y105F, M56K / L63P / N75D / V96I / M99L / Y105L / L106I, M56L / L63P / L98Q / Y105L / L106I / I117L, M56R / L63P / L98Q / M99L / Y105L, M56T / L91R / L98Q / Y105L, M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E, T61A / L63P / S72G / L98Q / M99L / Y105L, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, L63P / S72G / M87A / L98Q / Y105L, L63P / S72G / I93L / L98Q / M99L / Y105L, L63P / S72G / L98Q / M99L / Y105L, L63P / S72G / L98Q / M99L / Y105L / L106I / I117L, L63P / S72G / L98Q / Y105L, L63P / S72G / L98Q / Y105L / L106I / I117L, L63P / S72G / Y105L, L63P / M87K / M99L / L106R, L63P / Q82H / L98Q / M99L / Y105L, L63P / K95R, L63P / L98Q, L63P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L106I, L63P / L98Q / M99L / Y105L / I108V, L63P / L98Q / M99L / Y105L / I117M, L63P / L98Q / Y1O5L, L63P / L98Q / V116A, L63P / L98R / N110K, L63P / M99L / Y105L / I108F, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, S72G / L98Q / M99L / Y105L / I117T, L98Q / M99L / Y105L, L98Q / M99L / Y105L / L106I / I117T, L98Q / M99L / Y105L / L106I / Y115N, L98Q / Y105L, and L98R / N110K, with reference to numbering set forth in SEQ ID NO:36 or 2655. In some embodiments, the variant CTLA-4 polypeptide contains the modification(s) A31Y / L106E, with reference to numbering set forth in SEQ ID NO:36 or 2655.

[0042] In some of any of the provided embodiments, the ARBM includes the sequence of amino acids set forth in any of 2519, 2520, 2948-3041, 3043-3048, 3050-3099, 3230, 3231, or a specific binding fragment thereof. In some of any of the provided embodiments, the ARBM includes the sequence of amino acids set forth in any of SEQ ID NOs: 3100-3229, 3232 or 3233, or a specific binding fragment thereof. 2024204559   01 Jul 2024

[0043] In some of any of the provided embodiments, the IRBM contains at least one IgSF domain of a binding partner of PD-1. In some embodiments, the binding partner of PD-1 is PD-L1 or PD-L2 or is a variant of PD-L1 or PD-L2 that binds to PD-1. In some embodiments, the binding partner is a variant of PD-L1 or PD-L2 and the variant exhibits increased binding activity, optionally binding affinity, to PD-1 compared to the binding of unmodified or wild-type PD-L1 or PD-L2, respectively, to PD-1. In some embodiments, the binding activity, optionally binding affinity, to PD-1 is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold. In some embodiments, the IRBM contains (i) the sequence of amino acids set forth in SEQ ID NO: 30, 55, 309 or 1728, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 55, 309 or 1728; or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the IRBM includess (i) the sequence of amino acids set forth in SEQ ID NO: 31, 1203 or 1263, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31, 1203 or 1263; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the one or more amino acid modifications is a variant PD-L1 and can include any described herein, such as set forth in Section I.A.l.a. In some embodiments, IRBM is a variant PD-L1 and the one or more amino acid modifications include any set forth in Table 2. In some of any such embodiments, the IRBM is a variant PD-L2 and the one or more amino acid modifications can include any described herein, such as set forth in Section I.A.l.b. In some embodiments, the one or more amino acid modifications include any set forth in Table 3.

[0044] In some of any such embodiments, the IRBM is a variant of PD-L1 and the variant PD-L1 contains one or more amino acid modifications at one or more positions corresponding to 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 213, or 221, with reference to numbering set forth in SEQ ID NO: 30, 309 or 1728. In some of any such embodiments, the variant PD-L1 contains one or more amino acid modifications selected from P6S, Y10F, VI1A, VI IE, Y14S, G15A, S16G, N17D, M18I, 2024204559   01 Jul 2024 M18T, M18V, TWA, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, GlOlG-ins (G101GG), G102D, A103V, D104G, K106E, K106R, V110M,, KI HE, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171R, F173I, F173L, K173Y, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, or T221L or a conservative amino acid substitution thereof, with reference to numbering set forth in SEQ ID NO: 30, 309 or 1728.

[0045] In some embodiments, the variant PD-L1 polypeptide contains the amino acid modification(s) K28N / M41V / N45T / H51N / K57E, I20L / I36T / N45D / I47T, I20L / M41K / K44E, P6S / N45T / N78PI83T, N78I, M41K / N78I, N45T / N78I, I20L / N45T, N45T, M41K, I20L / I36T / N45D, N17D / N45T / V50A / D72G, I20L / F49S, N45T / V50A, I20L / N45T / N78I, I20L / N45T / V50A, M41V / N45T, M41K / N45T, A33D / S75P / D85E, M18VM41K / D43G / H51R / N78I, VI1E / I20L / I36T / N45D / H60R / S75P, A33D / V50A, S16G / A33D / K71E / S75P, E27G / N45T / M97I, E27G / N45T / K57R, A33D / E53V, D43G / N45D / V58A, E40G / D43V / N45T / V50A, Y14S / K28E / N45T A33D / N78S, A33D / N78I, A33D / N45T, A33D / N45T / N78I, E27G / N45T / V50A, N45T / V50A / N78S, I20L / N45T / V110M, I20L / I36T / N45T / V50A, N45T / L74P / S75P, N45T / S75P, S75P / K106R, S75P, A33D / S75P, A33D / S75P / D104G, A33D / S75P, I20L / E27G / N45T / V50A, I20L / E27G / D43G / N45D / V58A / N78I, I20L / D43G / N45D / V58A / N78I, I20L / A33D / D43G / N45D / V58A / N78I, I20L / D43G / N45D / N78I, E27G / N45T / V50A / N78I, N45T / V50A / N78I, VI1A / I20L / E27G / D43G / N45D / H51Y / S99G, I20L / E27G / D43G / N45T / V50A , I20L / K28E / D43G / N45D / V58A / Q89R , I20L / I36T / N45D , I20L / K28E / D43G / N45D / E53G / V58A / N78I, A33D / D43G / N45D / V58A / S75P, K23R / D43G / N45D, I20L / D43G / N45D / V58A / N78PD90G / G101D, D43G / N45D / L56Q / V58A / G101GG, I20L / K23E / D43G / N45D / V58A / N78I, 2024204559   01 Jul 2024 I20L / K23E / D43G / N45D / V50A / N78I, T19VE27G / N45PV50A / N78VM97K, I20L / M41K / D43G / N45D, K23R / N45T / N78I, I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins (G101GG), K57R / S99G, K57R / S99G / F189L, M18V / M97L / F193S / R195G / E200K / H202Q, I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F, C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L, M18V / I98L / L124S / P198T / L206F, S99G / N117S / I148V / K171R / R180S, I36T / M97L / A103V / Q155H, K28VS99G, R195S, A79T / S99G / T185A / R195G / E200K / H202Q / L206F, K57R / S99G / L124S / K144Q, K57R / S99G / R195G, D55V / M97L / S99G, E27G / I36T / D55N / M97L / K111E, E54G / M97L / S99G, G15A / I36T / M97L / K111E / H202Q, G15A / I36T / V129D, G15A / I36T / V129D / R195G, G15A / V129D, I36S / M97L, I36T / D55N / M97L / K111E / A204T, I36T / D55N / M97L / K111E / V129A / F173L, I36T / D55S / M97L / K111E / I148V / R180S, I36T / G52R / M97L / V112A / K144E / V175A / P198T, I36T / I46V / D55G / M97L / K106E / K144E / T185A / R195G, I36T / I83T / M97L / K144E / P198T, I36T / M97L / K11 IE, I36T / M97L / K144E / P198T, I36T / M97L / Q155H / F193S / N201Y, I36T / M97L / V129D, L35P / I36S / M97L / K11 IE, M18VI36T / E53G / M97L / K144E / E199G / V207A, M18T / I36T / D55N / M97L / K111E, M18V / M97L / T176N / R195G, M97L / S99G, N17D / M97L / S99G, S99G / T185A / R195G / P198T, V129D / H202Q, V129D / P198T, V129D / T150A, V93E / V129D, Y10F / M18V / S99G / Q138R / T203A, N45D, K160M / R195G, N45D / K144E, N45D / P198S, N45D / P198T, N45D / R195G, N45D / R195S, N45D / S131F, N45D / V58D, V129D / R195S, I98T / F173Y / L196S, N45D / E134G / L213P, N45D / F173VS177C, N45D / I148V / R195G, N45D / K111T / R195G, N45D / N113Y / R195S, N45D / N165Y / E17OG, N45D / Q89R / I98V, N45D / S131F / P198S, N45D / S75P / P198S, N45D / V50A / R195T, E27D / N45D / T183A / I188V, F173Y / T183VL196S / T203A, K23N / N45D / S75P / N120S, N45D / G102D / R194W / R195G, N45D / G52V / Q121L / P198S, N45D / I148V / R195G / N201D, N45D / K111T / T183A / I188V, N45D / Q89R / F189S / P198S, N45D / S99G / C137R / V207A, N45D / T163PK167R / R195G, N45D / T183A / T192S / R194G, N45D / V50A / I119T / K144E, T19A / N45D / K144E / R195G, V11E / N45D / T13OA / P198T, V26A / N45D / T163VT185A, K23N / N45D / L124S / K167T / R195G, K23N / N45D / Q73R / T163I, K28E / N45D / W149R / S158G / P198T, K28R / N45D / K57E / I98V / R195S, K28R / N45D / V129D / T163N / R195T, M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D, or 2024204559   01 Jul 2024 N45D / V50A / L124S / K144E / L179P / R195G, with reference to numbering set forth in SEQ ID NO: 30, 309 or 1728. In some embodiments, the variant PD-L1 contains the modification(s) D43 G / N45D / L56Q / V5 8 A / G101 G-ins.

[0046] In some of any of the provided embodiments, the IRBM contains the sequence of amino acids set forth in any of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 or a specific binding fragment thereof. In some of any of the provided embodiments, the IRBM contains the sequence of amino acids set forth in any of SEQ ID NOs: 121-185, 244-308, 17261727, 1908-1937.

[0047] In some of any of the provided embodiments, the IRBM is a variant of PD-L2 and the variant PD-L2 contains one or more amino acid modifications at one or more positions corresponding to 2, 12, 13, 15, 18, 20, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 82, 85, 86, 89, or 91, with reference to SEQ ID NO:31 and 1263. In some of any such embodiments, the variant PD-L2 contains one or more amino acid modifications selected from F2L, I12V, I13V, H15Q, N18D, T20A, N24S, C23S, G28V, N24D,V31A,V31M, N32D, L33P, ,L33H, L33F, I36V, T37A, S48C, S39I, E44D, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, R76G, D77N, Q82R, I85F, I86T, V89D, or W91R, with reference to numbering set forth in SEQ ID NO:31 and 1263. In some of any such embodiments, the variant PD-L2 contains the amino acid modification(s) H15Q, N24D, E44D, V89D, Q82R / V89D, E59G / Q82R, S39EV89D, S67L / V89D, S67L / I85F, S67L / I86T, H15Q / K65R, H15Q / Q72H / V89D, H15Q / S67L / R76G, H15Q / R76G / I85F, H15Q / T47A / Q82R, H15Q / Q82R / V89D, H15Q / C23S / I86T, H15Q / S39EI86T, E44D / V89D / W91R, I13V / S67L / V89D, H15Q / S67L / I86T, I13V / H15Q / S67L / I86T, I13V / H15Q / E44D / V89D, I13V / S39EE44D / Q82R / V89D, I13V / E44D / Q82R / V89D, I13V / Q72H / R76G / I86T, I13V / H15Q / R76G / I85F, H15Q / S39ER76G / V89D, H15Q / S67L / R76G / I85F, H15Q / T47A / Q72H / R76G / I86T, H15Q / T47A / Q72H / R76G, I13V / H15Q / T47A / Q72H / R76G, H15Q / E44D / R76G / I85F, H15Q / S39I / S67L / V89D, H15Q / N32D / S67L / V89D, N32D / S67L / V89D, H15Q / S67L / Q72H / R76G / V89D, H15Q / Q72H / Q74R / R76G / I86T, G28V / Q72H / R76G / I86T, I13V / H15Q / S39EE44D / S67L, E44D / S67L / Q72H / Q82R / V89D, H15Q / V89D, H15Q / T47A, I13V / H15Q / Q82R, I13V / H15Q / V89D, I13V / S67L / Q82R / V89D, I13V / H15Q / Q82R / V89D, H15Q / V31M / S67L / Q82R / V89D, I13V / H15Q / T47A / Q82R, I13V / H15Q / V31A / N45S / Q82R / V89D, H15Q / T47A / H69L / Q82R / V89D, 2024204559   01 Jul 2024 I13V / H15Q / T47A / H69L / R76G / V89D, I12V / I13V / H15Q / T47A / Q82R / V89D, I13V / H15Q / R76G / D77N / Q82R / V89D, I13V / H15Q / T47A / R76G / V89D, I13V / H15Q / T47A / Q82R / V89D, I13V / H15Q / N24D / Q82R / V89D, I13V / H15Q / I36V / T47A / S67L / V89D, H15Q / T47A / K65R / S67L / Q82R / V89D, H15Q / L33P / T47A / S67L / P71S / V89D, I13V / H15Q / Q72H / R76G / I86T, H15Q / T47A / S67L / Q82R / V89D, F2L / H15Q / D46E / T47A / Q72H / R76G / Q82R / V89D, I13V / H15Q / L33F / T47A / Q82R / V89D, I13V / H15Q / T47A / E58G / S67L / Q82R / V89D, H15Q / N24S / T47A / Q72H / R76G / V89D, I13V / H15Q / E44V / T47A / Q82R / V89D, H15Q / N18D / T47A / Q72H / V73A / R76G / I86T / V89D, I13V / H15Q / T37A / E44D / S48C / S67L / Q82R / V89D, H15Q / L33H / S67L / R76G / Q82R / V89D, I13V / H15Q / T47A / Q72H / R76G / I86T, H15Q / S39I / E44D / Q72H / V75G / R76G / Q82R / V89D, H15Q / T47A / S67L / R76G / Q82R / V89D, or I13V / H15Q / T47A / S67L / Q72H / R76G / Q82R / V89D, with reference to numbering set forth in SEQ ID NO:31 and 1263. In some embodiments, the variant PD-L2 contains the modification(s) H15Q / T47A / K65R / S67L / Q82R / V89D.

[0048] In some of any of the provided embodiments, the IRBM contains the sequence of amino acids set forth in any of SEQ ID NOs: 1204-1280 or a specific binding fragment thereof. In some of any of the provided embodiments, the IRBM contains the sequence of amino acids set forth in any of SEQ ID NOs: 1283-1331, 1333-1407, 1309-1432.

[0049] In some of any of the provided embodiments, the IRBM contains at least one IgSF domain of a binding partner of TIGIT. In some embodiments, the binding partner of TIGIT is CD 155 or CD 122 or is a variant of CD 155 or CD 122 that binds to PD-1. In some embodiments, the binding partner is a variant of CD 155 or CD112 and the variant exhibits increased binding activity, optionally binding affinity, to TIGIT compared to the binding of unmodified or wildtype CD155 or CD112, respectively, to TIGIT. In some embodiments, the binding activity, optionally binding affinity, to TIGIT is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold. In some of any of the provided embodiments, the IRBM comprises (i) the sequence of amino acids set forth in SEQ ID NO: 47, 310 or 353, (ii) a sequence of amino acids that has at least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47, 310 or 353; or (iii) a specific binding fragment of (i) or (ii). In some of any of the provided embodiments, the IRBM comprises (i) the sequence of amino acids set forth in SEQ ID NO: 48, 666 or 761, (ii) a sequence of amino acids that has at 2024204559   01 Jul 2024 least 85%, 86%, 87%, 88%%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48, 666 or 761; or (iii) a specific binding fragment of (i) or (ii). In some of any such embodiments, the IRBM is a variant CD155 and the one or more amino acid modification can be any described herein, such as set forth in Section I.A.2.a. In some embodiments, the IRBM is a variant CD155 and the one or more amino acid modifications include any set forth in Table 4. In some of any such embodiments, the IRBM is a variant CD112 and the one or more amino acid modification can be any described herein, such as set forth in Section I.A.2.b. In some embodiments, the one or more amino acid modifications include any set forth in Table 5.

[0050] In some of any of the provided embodiments, the variant CD155 comprises one or more amino acid modifications selected from P18F, P18S, S67A, L104M, L104Q, Gil ID or G111R. In some embodiments, the variant CD 155 polypeptide comprises the amino acid modification(s) P18F / T26M / L44V / Q62K / L79P / F91S, / L104M / Gl 1 ID, P18S / T45S / T61K / S65W / S67A / F91S / G111R, P18S / L79P / L104M / T107M, P18S / S65W / S67A / M90V / V95A / L104Q / G111R, P18S / S65W / S67A / L104Q / G111R.

[0051] In some of any of the provided embodiments, the IRBM comprises the sequence of amino acids set forth in any of SEQ ID NOs: 312-331, 375-471, 1551-1575, 1577-1622. In some of any of the provided embodiments, the IRBM comprises the sequence of amino acids set forth in any of SEQ ID NOs: 332-352, 354-374, 472-665, 1505-1550, 1575, 1576, 1623-1714.

[0052] In some of any of the provided embodiments, the ARBM or IRBM is a specific binding fragment of any of the above IgSF domain and has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids.

[0053] In some of any of the provided embodiments, the ARBM or IRBM is a variant of any of the above IgSF domains and the variant contains one or more amino acid modifications at one or more positions in an IgSF domain of an unmodified or wild-type IgSF domain, such as an IgSF domain of a binding partner of an activating receptor, a binding partner of a ligand of an activating receptor or a binding partner of an inhibitory receptor. In some embodiments, the one or more amino acid modification is an amino acid substitution, insertion or deletion. In some embodiments, the variant contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, optionally amino acid substitutions, insertions and / or deletions. 2024204559   01 Jul 2024

[0054] In some of any of the provided embodiments, the ARBM contains an IgV domain or a specific binding fragment thereof. In some embodiments, the at least one ARBM in a provided immunomodulatory protein contains only one ARBM.

[0055] In some of any such embodiments, the IRBM contains at least one IgSF domain that is an IgV domain or a specific binding fragment thereof. In some embodiments, the at least one IRBM in a provided immunomodulatory protein contains only one IRBM. In some embodiments, the at least one IRBM in a provided immunomodulatory protein contains 2, 3, 4, 5 or more IRBMs, optionally wherein each IRBM is the same. In some embodiments, each IRBM of a provided immunomodulatory protein is linked directly or indirectly via a linker, optionally wherein the linker is a peptide linker.

[0056] In some of any of the provided embodiments, the at least one IRBM and the at least one ARBM are linked direct or indirectly via a linker. In some instances, the embodiments is a peptide linker.

[0057] In some of any such embodiments, the peptide linker is selected from GSGGS (SEQ ID NO: 2523), GGGGS (G4S; SEQ ID NO: 1942), GSGGGGS (SEQ ID NO: 1941), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 240), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 239), GGGGSSA (SEQ ID NO: 2524) or combinations thereof.

[0058] In some embodiments, the at least one IRBM is amino-terminal to the at least one ARBM in the polypeptide. In some embodiments, the at least one IRBM is carboxy-terminal to the at least one ARBM in the polypeptide. In some embodiments, the immunomodulatory protein contains at least two IRBM. In some of any such embodiments, at least one IRBM is amino-terminal to the at least one ARBM in the polypeptide and at least one IRBM is carboxyterminal to the at least one ARBM in the polypeptide.

[0059] In some of any of the provided embodiments, the immunomodulatory protein is a multi-domain protein in which the ARBM contains at least one IgSF domain of CTLA-4 or a variant therof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of PD-L1 or a variant thereof, e.g affinity-modified domain thereof.

[0060] In some of any of the provided embodiments, the immunomodulatory protein a multi-domain protein in which the ARBM contains at least one IgSF domain of ICOS-L or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of PD-L1 or a variant thereof, e.g affinity-modified domain thereof. 2024204559   01 Jul 2024

[0061] In some of any of the provided embodiments, the immunomodulatory protein a multi-domain protein in which the ARBM contains at least one IgSF domain of ICOS-L or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of PD-L2 or a variant thereof, e.g affinity-modified domain thereof.

[0062] In some of any of the provided embodiments, the immunomodulatory protein is a multi-domain protein in which the ARBM contains at least one IgSF domain of CD58 or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of PD-L1 or a variant thereof, e.g affinity-modified domain thereof.

[0063] In some of any of the provided embodiments, the immunomodulatory protein is a multi-domain protein in which the ARBM contains at least one IgSF domain of CTLA-4 or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of CD155 or a variant thereof, e.g affinity-modified domain thereof.

[0064] In some of any of the provided embodiments, the immunomodulatory protein is a multi-domain protein in which the ARBM contains at least one IgSF domain of CD58 or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of CD155 or a variant thereof, e.g affinity-modified domain thereof.

[0065] In some of any of the provided embodiments, the immunomodulatory protein is a multi-domain protein in which the ARBM contain at least one IgSF domain of ICOSL or a variant thereof, e.g affinity-modified domain thereof, and the IRBM contains at least one IgSF domain of CD155 or a variant thereof, e.g affinity-modified domain thereof.

[0066] In some of any of the provided embodiments, the immunomodulatory protein is an immunomodulatory protein set forth in any of Tables E2, E3A, E3B, E4 or E5.

[0067] In some embodiments, the immunomodulatory protein is a monomer and / or contains a single polypeptide chain. In some embodiments, the immunomodulatory protein comprises the sequence of amino acids set forth in any of SEQ ID NOS: 2563, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605, 2607, 2609, 2612, 2614, 2619, 2621, 2623, 2625, 2627, 2629, 2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653, 3496, 3497, 3498, 3499, 3500, 3501, 3502, 3535, 3656, 3658, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID Nos: 2563, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581, 2583, 2585, 2587,2589,2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605, 2607, 2609, 2612, 2614, 2619, 2621, 2623, 2625, 2627, 2024204559   01 Jul 2024 2629, 2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653, 3496, 3497, 3498, 3499, 3500, 3501, 3502, 3535, 3656, 3658 and retains the same or similar activity.

[0068] In some of any such embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS:2541, 2543, 2563, 2567, 2569, 2579, 2583, 2585, 2587, 2589, 2591, 2595, 2597, 2599, 2601, 2603, 2605, 2607, 2609, 2631, 2643, 2651, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retains the same or similar activity.

[0069] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 2583, 2587, 2603, 2651, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retains the same or similar activity.

[0070] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 2541, 2533, 2651, 3522, 3523, 3664, 3666, 3668, 3674, 3766 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3522, 3523, 3664, 3666, 3668, 3674, 3766 and retains the same or similar activity. In some embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in SEQ ID NO:3522 or 3666 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3522 or 3566. In some embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in SEQ ID NO:3523 or 3668 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3523 or 3568. In some embodiments the immunomodulatory proteins binds CD80 or CD86 and binds PD-1.

[0071] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 2583, 2587, 2603, 3678 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 2583, 2587, 2603, 3678 and retains the same or similar activity. In some embodiments the immunomodulatory proteins binds CD28 and binds PD-1. 2024204559   01 Jul 2024

[0072] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 3680, 3682, 3684, 3686, 3688 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3680, 3682, 3684, 3686, 3688 and retains the same or similar activity. In some embodiments the immunomodulatory proteins binds CD2 and binds PD-1.

[0073] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 3652, 3654, or 3664 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3652, 3654, or 3664 and retains the same or similar activity. In some embodiments the immunomodulatory proteins binds CD80 or CD86 and binds TIGIT.

[0074] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 3656 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3656 and retains the same or similar activity. In some embodiments the immunomodulatory proteins binds CD28 and binds TIGIT.

[0075] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 3660 or 3662 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3660 or 3662 and retains the same or similar activity. In some embodiments the immunomodulatory proteins binds CD2 and binds TIGIT.

[0076] In some of any of the provided embodiments, the ARBM and IRBM are linked by a linker and the linker is or includes a multimerization domain. In some embodiments, the multimerization domain promotes dimerization, trimerization, tetramerization, or pentamerization. In some embodiments, the immunomodulatory protein is a dimer, trimer, tetramer, or a pentamer.

[0077] In some embodiments, the immunomodulatory protein is a dimer, optionally wherein each polypeptide of the dimer is linked to a multimerization domain. In some aspects, the multimerization domain is an Fc domain. In some of any such embodiments, the Fc domain is 2024204559   01 Jul 2024 an IgG, optionally an IgGl, IgG2 or IgG4, Fc domain. In some cases, the Fc domain is an IgGl Fc domain. In some embodiments, the Fc domain is a human Fc domain.

[0078] In some of any such embodiments, the Fc domain exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, optionally as compared to a native IgGl Fc domain. In some aspects, the Fc domain contains one or more amino acid substitution that reduces binding to an Fc receptor and or effector function, optionally wherein the one or more amino acid substitutions are in a native IgGl Fc domain. In some examples, the one or more amino acid substitutions in the Fc domain are selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C and K447del, each by EU numbering. In some embodiments, the Fc contains the amino acid substitutions L234A, L235E an G237A by EU numbering. In some embodiments, the Fc contains the amino acid substitutions R292C, N297G and V302C by EU numbering.

[0079] In some embodiments, the immunomodulatory protein contains a first polypeptide chain comprising the at least one ARBM, the at least one IRBM and a first multimerization domain, optionally wherein the first multimerization domain is a first Fc domain, and a second polypeptide chain comprising the at least one ARBM, the at least one IRBM and a second multimerization domain, optionally wherein the second multimerization domain is a second Fc domain, wherein the first and second multimerization domains. In some embodiments the first and second Fc domains are able to interact to form a dimer comprising the first and second polypeptides. In some embodiments, the at least one IRBM is amino-terminal to the at least one ARBM in the first and / or second polypeptide.

[0080] In some embodiments, the first and second multimerization domain, optionally the first and second Fc domain, are the same. In some embodiments, the immunomodulatory protein is a homodimer.

[0081] In some embodiments, the Fc domain contains the sequence of amino acids set forth in any of SEQ ID NOs:187 or 3538 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 187 or 3538. In some embodiments, the Fc domain contains the sequence of amino acids set forth in any of SEQ ID NOs: 1155, 1157, 1158, 1159, 1938, 1939 or 1940 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1155, 1157, 1158, 1159, 1938, 1939 or 1940. In some embodiments, the Fc domain contains the sequence of amino acids set forth in any of SEQ ID NOs: 1158, 1715 or 3579 or a sequence of 2024204559   01 Jul 2024 amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1158, 1715 or 3579.

[0082] In some of any such embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 2529, 2530, 2531, 2533, 2535, 2537, 2539, 2541, 2543, 3516, 3517, 3518, 3519, 3520, 3521, 3522, 3523, 3524, 3652, 3654, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674, 3676, 3680, or 3682 or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 2529, 2530, 2531, 2533, 2535, 2537, 2539, 2541, 2543, 3516, 3517, 3518, 3519, 3520, 3521, 3522, 3523, 3524, 3652, 3654, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674, 3676, 3680, or 3682 and retains the same activity.

[0083] In some embodiments, the immunomodulatory includes a first polypeptide chain containing (i) one of the at least one ARBM or the at least one IRBM and (ii) a first multimerization domain, optionally wherein the first multimerization domain is a first Fc domain and a second polypeptide chain comprising (i) the other of the at least one ARBM or the at least one IRBM and (b) a second multimerization domain, optionally wherein the second multimerization domain is a second Fc domain, wherein the first and second multimerization domains, optionally the first and second Fc domains, interact to form a dimer comprising the first and second polypeptides. In some embodiments, the first and second multimerization domains, optionally the first and second Fc domains, are different. In some of any of the provided embodiments, the immunomodulatory protein is a heterodimer.

[0084] In some embodiments, each polypeptide of the dimer is linked to an Fc domain and wherein the Fc domains contain a knob-into-hole modification or contain a charge mutation to reduce or prevent self-association due to charge repulsion.

[0085] In some embodiments, the Fc domain contains a knob-into-hole modification wherein one of the Fc domains contains a knob modification comprising the amino acid substitution T366W and the other one of the Fc domain contains a hole modification selected from T366S, L368A and / or Y407V, each by EU numbering. In some examples, the Fc domain containing the knob modification further contains the amino acid substitution S354C and the Fc domain comprising the hole modification further contains the amino acid substitution Y349C, each by EU numbering. In some embodiments, one of the Fc domains includes the sequence set forth in any of SEQ ID NO: 1153 or 2558 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO: 1153 or 2024204559   01 Jul 2024 2558 and contains the knob-into-hole modifications, optionally amino acid substitutions T366W and / or S354C, and the other Fc domain includes the sequence set forth in SEQ ID NO: 1154 or 2559 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1154 or 2559 and contains the knob-into-hole modifications, optionally Y349C, T366S, L368A and / or Y407V.

[0086] In some embodiments, the Fc domain includes a charge mutation wherein one of the Fc domains contains the amino acid substitution E356K, E357K and / or D399K and the other of the Fc domains contains the amino acid substitution K370D, K392D and / or K409D. In some cases, one of the Fc domains contains the sequence set forth in SEQ ID NO:2544 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2544 and includes the charge mutation, optionally the amino acid substitution E356K, E357K and / or D399K, and the other of the Fc domains includes the sequence set forth in SEQ ID NO:2544 or a sequence of amino acids that exhibits at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2544 and contains the charge mutation, optionally the amino acid substitution K370D, K392D and / or K409D.

[0087] In some of any such embodiments, the immunomodulatory protein contains a first and second polypeptide comprising the sequence of amino acids set forth in SEQ ID NOs: 2547 and 2549, SEQ ID Nos: 2547 and 2551, SEQ ID Nos: 2553 and 2549, SEQ ID Nos: 2553 and 2551, SEQ ID Nos: 2547 and 2555, SEQ ID Nos: 2547 and 2557, SEQ ID Nos: 2553 and 2555 and SEQ ID Nos: 2553 and 2557, SEQ ID Nos: 2526 and 2528, SEQ ID Nos: 2526 and 2561, SEQ ID Nos: 3513 and 3514 or SEQ ID Nos: 3515 and 3514, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to each of SEQ ID NOs: 2547 and 2549, SEQ ID Nos: 2547 and 2551, SEQ ID Nos: 2553 and 2549, SEQ ID Nos: 2553 and 2551, SEQ ID Nos: 2547 and 2555, SEQ ID Nos: 2547 and 2557, SEQ ID Nos: 2553 and 2555 and SEQ ID Nos: 2553 and 2557, SEQ ID Nos: 2526 and 2528, SEQ ID Nos: 2526 and 2561, SEQ ID Nos: 3513 and 3514 or SEQ ID Nos: 3515 and 3514 and retains the same activity.

[0088] In some embodiments, the ARBM and IRBM are linked by a multimerization domain that promotes dimerization, trimerization, tetramerization, or pentamerization. In some embodiments, the multimerization domain is a portion of a cartilage oligomeric protein (COMP), such as set forth in SEQ ID NO:3503. In some embodiments, the multimerization 2024204559   01 Jul 2024 domain is a vasodilatory-stimulated phosphoprotein (VASP) tetramerization domain, such as set forth in SEQ ID NO:3504. In some embodiments, the multimerization domain is a ZymoZipper (ZZ) domain, such as set forth in SEQ ID NO:3505.

[0089] In some of any of the provided embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS: 3507, 3509 or 3678, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3507, 3509 or 3678 and retains the same activity.

[0090] In some embodiments, the immunomodulatory protein contains the sequence of amino acids set forth in any of SEQ ID NOS:2541, 2533, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS:2541 or 2533 and retains the same activity.

[0091] Provided herein is an immunomodulatory protein that contains the sequence of amino acids set forth in any of SEQ ID NOS: 3506, 3508, 3510 or 3511, or a sequence that exhibits at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3506, 3508, 3510 or 3511 and retains the same activity.

[0092] In some of any of the provided embodiments, the immunomodulatory protein further contains a detectable moiety, optionally wherein the detectable moiety is a peptide capable of detection, optionally wherein the peptide capable of detection is a flag tag or a his tag.

[0093] In some of any of the provided embodiments, the IRBM has a dissociation constant for binding the inhibitory receptor of from 0.001 nM to 1000 nM, from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 2024204559   01 Jul 2024 50 nM, from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM.

[0094] In some of any of the provided embodiments, the ARBM has a dissociation constant for binding the activating receptor or a ligand of the activating receptor of from or from about 0.001 nM to 1000 nM, from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 50 nM, from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM.

[0095] In some of any of the provided embodiments, the immunomodulatory protein attenuates, decreases or reduces an immune response. In some embodimetns, the immune response is a T cell response. In some embodiments, a reduced, decreased or attenuated T cell response includes one or more of cell cycle inhibition, reduced cell survival, reduced cell proliferation, reduced cytokine production (e.g. IFN-gamma or IL-2), or reduced T-cell cytotoxicity. In some embodiments, the reduced activity is observed in vitro in a primary T cell activation assays, such as in a Jurkat reporter assay, SEB assay or mixed lymphocyte reaction (MLR) assay. In some embodiments, the activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the activity in the same assay in the absence of the immunomodulatory protein. In some embodiments, the activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the activity in the same assay presence of a control in which the control is a protein that is known or suspected not to modulate T cell activity, e.g. an Fc only control. In some embodiments, the activity is reduced to a level that is greater than the reduction observed by a reference immunomodulatory protein containing only the ARBM or containing only the IRBM, such as is reduced by greater 2024204559   01 Jul 2024 than or greater than about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold 5.0-fold or more compared to the reference immunomodulatory protein.

[0096] In some of any such embodiments, upon contact of the immunomodulatory protein with the immune cell, the immunomodulatory protein proximalizes association of the inhibitory receptor and activating receptor by the immune cell. In some cases, upon contact of the immunomodulatory protein with the immune cell, the immunomodulatory protein recruits a cytoplasmic protein tyrosine phosphatase to the intracellular region of the inhibitory receptor, optionally wherein the protein tyrosine phosphatase is a SHP-1 or SHP-2. In some of any such embodiments, the immune cell is a T cell. In some embodiments, contact of the immunomodulatory protein and immune cell is in vitro or in vivo.

[0097] In some of any of the provided embodiments, binding of the IRBM of the immunomodulatory protein to the inhibitory receptor on the immune cell blocks or antagonizes binding between the inhibitory receptor and its cognate ligand. In some such embodiments,the cognate ligand is expressed on an antigen presenting cell. In some such embodiments, the immune cell is a T cell.

[0098] In some of any of the provided embodiments, binding of the ARBM of the immunomodulatory protein to the activating receptor on the immune cell blocks or antagonizes binding between the activating receptor and its cognate ligand. In some such embodimetns,the cognate ligand is expressed on an antigen presenting cell. In some such embodiments, the immune cell is a T cell.

[0099] In some of any of the provided embodiments, binding of the ARBM of the immunomodulatory protein to the ligand of an activating receptor, in which said activating receptor is expressed on the immune cell, blocks or antagonizes binding between the activating receptor and the ligand. In some such embodimetns,the ligand is expressed on an antigen presenting cell. In some such embodiments, the immune cell is a T cell.

[0100] In some of any of the provided embodiments, the immunomodulatory protein increases an immune response. In some embodiments, the immune response is a T cell response. In some embodiments, an increased T cell response includes one or more of increased cell activation, increased cell differentiation, increased or greater cell survival, increased cell proliferation, increased cytokine production (e.g. IFN-gamma or IL-2), or increased T-cell cytotoxicity. In some embodiments, the increased activity is observed in vitro in a primary T cell activation assays, such as in a Jurkat reporter assay, SEB assay or mixed lymphocyte reaction 2024204559   01 Jul 2024 (MLR) assay. In some embodiments, the activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the activity in the same assay in the absence of the immunomodulatory protein. In some embodiments, the activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the activity in the same assay in the presence of a control in which the control is a protein that is known or suspected not to modulate T cell activity, e.g. an Fc only control. In some embodiments, the activity is increased to a level that is greater than the reduction observed by a reference immunomodulatory protein containing only the ARBM or containing only the IRBM, such as is increased by greater than or greater than about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold 5.0-fold or more compared to the reference immunomodulatory protein.

[0101] Provided are nucleic acid molecules encoding any of the provided immunomodulatory proteins. In some cases, the nucleic acid molecule is a synthetic nucleic acid. In some cases, the nucleic acid molecule is a cDNA. In some of any of the provided embodiments, the nucleic acid includes any having a DNA SEQ ID NO set forth in any of Tables E2, E3A, E3B, E4 or E5.

[0102] Provided are vectors containing any of the provided nucleic acid molecules. In some cases, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector or a viral vector.

[0103] Provided are cells containing any of the provided nucleic acid molecules. Provided are cells containing any of the provided vectors. In some cases, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0104] Provided are methods of producing an immunomodulatory protein including introducing any of the provided nucleic acid molecules or vectors into a host cell under conditions to express the protein in the cell. In some cases, the method further includes isolating or purifying the immunomodulatory protein from the cell.

[0105] Provided are methods of engineering a cell expressing an immunomodulatory protein including introducing a nucleic acid molecule encoding a polypeptide or polypeptides of any of the provided immunomodulatory proteins into a host cell under conditions in which the polypeptide is expressed in the cell.

[0106] Provided are engineered cells expressing any of the provided immunomodulatory proteins, nucleic acid molecules, or vectors. In some cases, the immunomodulatory protein is capable of being secreted from the engineered cell. In some embodiments, the 2024204559   01 Jul 2024 immunomodulatory protein does not contain a cytoplasmic signaling domain or transmembrane domain and / or is not capable of mediating or modulating an intracellular signal when secreted from a cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some examples, the T cell is a CD4+ and / or CD8+ T cell. In some examples, the T cell is a regulatory T cell (Treg). In some embodiments, the engineered cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the engineered cell further contains a chimeric antigen receptor (CAR). In some embodiments, the engineered cell further contains an engineered T-cell receptor (TCR).

[0107] Provided are infectious agents containing a nucleic acid molecule encoding any of the provided immunomodulatory proteins. In some embodiments, the infectious agent is a bacterium or a virus.

[0108] Also provided are pharmaceutical compositions containing any of the provided immunomodulatory proteins. Also provided are pharmaceutical compositions containing any of the provided engineered cells. Also provided are pharmaceutical compositions containing any of the provided infectious agents. In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0109] Provided are articles of manufacture containing any of the provided pharmaceutical compositions in a vial or container. In some cases, the vial or container is sealed.

[0110] Provided are kits containing any of the provided pharmaceutical compositions and instructions for use. Also provided are kits containing any of the provided articles of manufacture and instructions for use.

[0111] Provided are methods of modulating an immune response in a subject including administering any of the provided immunomodulatory proteins to the subject. Provided are methods of modulating an immune response in a subject including administering any of the provided pharmaceutical compositions to the subject. Also provided are methods of modulating an immune response in a subject, including administering any of the provided engineered cells. In some cases, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogenic to the subject.

[0112] In some of any of the provided embodiments, modulating the immune response treats a disease or condition in the subject. In some cases, the immune response is decreased. Also 2024204559   01 Jul 2024 provided are methods of treating a disease or condition in a subject including administering any of the provided immunomodulatory proteins to the subject. Also provided are methods of treating a disease or condition in a subject including administering any of the provided pharmaceutical compositions to the subject. In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition, or is a disease or condition associated with an overactive immune response. In some embodiments, the disease or condition is an Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, a thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, an autoimmune hematological disease, an autoimmune demyelinating disease, or an autoimmune disease involving a systemic autoimmune disorder.

[0113] In some examples, the disease or condition is selected from among inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, rheumatoid arthritis, psoriasis, lupus erythematosus, celiac disease, type I diabetes mellitus, Guillain-Barre syndrome, Chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, DeQuervains thyroiditis, myasthenia gravis, Vasculitis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic opthalmia, primary biliary cirrhosis, chronic aggressive hepatitis, membranous glomerulopathy, primary idiopathic myxedema, scleroderma, chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, autoimmune enteropathy syndrome, idiopathic thrombocytic purpura, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sensoneural hearing loss, Sjogren's syndrome, polymyositis, multiple sclerosis, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, and idiopathic facial paralysis. In some cases, the disease or condition is a vasculitis that is a giant cell arteritis (GCA).

[0114] In some embodiments, the immunomodulatory protein increases an immune response in the subject. In some embodiments, the disease or condition is a cancer. Brief Description of the Drawings 2024204559   01 Jul 2024

[0115] FIG. 1A depicts the proximalization of ITIM bearing inhibitory receptors with activating receptors on T cells or the inhibitory ligand and activating ligand of such receptors on APC to make possible attenuation of T cell activation and / or tolerance induction. FIG. IB depicts a cis binding strategy where the multi-domain immunomodulatory protein targets the inhibitory receptor (e.g. PD-1) and activating receptor (e.g. CD3, CD4 or CD28) on T cells, which, in some cases, proximalize or cluster the inhibitory receptor and the activating receptor on the surface of the same T cell and a trans binding strategy where the multi-domain immunomodulatory protein targets the inhibitory receptor (e.g. PD-1) on the T cell and a ligand of the activating receptor (e.g. CD80 or CD86) on an antigen-presenting cell (APC).

[0116] FIG. 2 depicts binding of the multi-domain immunomodulatory proteins in a cis binding strategy where an ARBM (e.g., ICOSL) binds the activating receptor (e.g. CD28) and an IRBM (e.g., PD-L1 or PD-L2) binds the inhibitory receptor (e.g., PD-1).

[0117] FIG. 3A depicts a reporter assay (Assay #1) used to assess activity of exemplary multi-domain immunomodulatory proteins where Jurkat reporter cells expressing an IL-2-luciferase reporter were incubated with artificial antigen presenting cells (aAPC) displaying cell surface anti-CD3 single chain Fv (OKT3) and CD80 (K562 / OKT3 / CD80 aAPC). FIG. 3B depicts a reporter assay (Assay #2) used to assess activity of exemplary multi-domain immunomodulatory proteins where Jurkat reporter cells were additionally transfected with PD-1 and incubated with the K562 / OKT3 / CD80 aAPCs. The two assays were used to distinguish between inhibitory activity due to blockade of CD28 signaling versus inhibitory activity via activity of the PD-1-binding IRBM.

[0118] FIGS. 4A-4B show results from assessing exemplary PD-L1 / PD-L2 and CTLA-4 multi-domain immunomodulatory proteins in the Jurkat / IL2 (Assay #1) and Jurkat / IL2 / PD-1 (Assay #2) reporter assays.

[0119] FIGS. 5A-5B and 6A-6B show results from assessing multi-domain immunomodulatory proteins containing a PD-L1 IgV or a PD-L2 IgV and ICOSL IgV, generated as monomeric proteins in the Jurkat / IL2 (Assay #1) and Jurkat / IL2 / PD-1 (Assay #2) reporter assays.

[0120] FIG. 7 depicts results from the Jurkat / IL2 / PD-1 reporter assay (Assay #2) carried out in the presence of a PD-lblocking antibody demonstrating PD-1 specificity for exemplary multidomain immunomodulatory molecules containing PD-L1 IgV / PD-L2 IgV and CTLA-4-ECD. 2024204559   01 Jul 2024

[0121] FIGS. 8A-8C show results from assessing multi-domain immunomodulatory proteins containing PD-L1 IgV / PD-L2 IgV and CD80 / CD86 IgV generated as monomeric proteins in the Jurkat / IL2 / PD-1 (Assay #2) reporter assay.

[0122] FIGS. 9A-9C show results from a PD-1 / CD28 proximalization assay using a betaGalactosidase (beta-Gal) Enzyme Fragment Complementation system to assess effects of exemplary multi-domain immunomodulatory protein containing PD-L1 / PD-L2 and ICOSL IgV upon binding to its targets.

[0123] FIGS. 10A-10B show results from a Jurkat PD-1 SHP2 Signaling Assay to assess the effect of the multi-domain immunomodulatory protein (PD-L1 303 - ICOSL 2264) to recruit the cytoplasmic protein tryrosine phosphatase, SHP-1 or SHP-2, to PD-1.

[0124] FIGS. 11A-11B show results from assessment of binding of immunomodulatory proteins containing PD-L1 IgV / PD-L2 IgV and CTLA-4 to binding partners PD-1 and / or CD80.

[0125] FIG. 12A-12B show results from assessing multi-domain immunomodulatory proteins containing a PD-L1 IgV or a PD-L2 IgV and CTLA-4 ECD, generated as multimeric homodimer immunomodulatory proteins in the Jurkat / IL2 (Assay #1) and Jurkat / IL2 / PD-1 (Assay #2) reporter assays.

[0126] FIG. 13A-13B show results from assessing multi-domain immunomodulatory proteins containing a PD-L1 IgV or a PD-L2 IgV and CTLA-4 ECD, generated as multimeric homodimer immunomodulatory proteins in the Jurkat / IL2 / PD-1 (Assay #2) reporter assay.

[0127] FIG. 14A-14B, 15A-15B, and 16A-16B show results from assessing multi-domain immunomodulatory proteins containing a PD-L1 IgV or a PD-L2 IgV and ICOSL IgV, generated as multimeric homodimer immunomodulatory proteins in the Jurkat / IL2 (Assay #1) and Jurkat / IL2 / PD-1 (Assay #2) reporter assays.

[0128] FIG. 17A-17C show results from a Jurkat PD-1 SHP2 Signaling Assay to assess the effect of monomeric or multimeric heterodimer PD-L1-ICOSL multi-domain immunomodulatory proteins to recruit the cytoplasmic protein tryrosine phosphatase, SHP-2, to PD-1.

[0129] FIG. 18 shows results from a Jurkat PD-1 SHP2 Signaling Assay to assess the effect of monomeric or multimeric heterodimer PD-L1-CTLA-4 multi-domain immunomodulatory proteins to recruit the cytoplasmic protein tryrosine phosphatase, SHP-2, to PD-1. 2024204559   01 Jul 2024

[0130] FIG. 19A-19B shows results from an assay using a beta-Galactosidase (beta-Gal) Enzyme Fragment Complementation system to assess the effect of immunomodulatory proteins on colocalization of CD28 and PD-1.

[0131] FIG. 20 shows results from a Staphylococcal enterotoxin B (SEB) assay assessing bioactivity of PD-L1, ICOSL and / or CTLA-4-containing multi-domain immunomodulatory proteins.

[0132] FIG. 21A-21E shows results from a Jurkat / IL and Jurkat / IL2 / PD1 stimulation assay using exemplary PD-L1-CTLA4 and PD-L1-ICOSL multidomain immunomodulatory proteins.

[0133] FIG. 22A-22E shows results from a Jurkat / IL2 and Jurkat / IL2 / PD1 assay using using exemplary CD58-PD-L1 and CD58-ICOSL multidomain innunomodulatory proteins.

[0134] FIG. 23A-23E shows results from a Jurkat / IL2 and Jurkat / IL2 / PD1 assay using exemplary CD155-CTLA-4, CD155-CD58, and CD155-ICOSL multidomain immunomodulatory proteins. Detailed Description

[0135] Provided herein are multi-domain immunomodulatory proteins that are capable of binding to two or more protein cell surface molecules to modulate, e.g. suppress, immunological immune responses. In some embodiments, the cell surface molecules are cell surface proteins expressed by immune cells, such as T lymphocytes, that engage with one or more other immune receptor or ligand, e.g. on antigen-presenting cells, to induce inhibitory or activating signals. For example, the interaction of certain receptors on lymphocytes with their cognate cell surface ligands to form an immunological synapse (IS) between antigen-presenting cells (APCs) or target cells and lymphocytes can provide costimulatory or inhibitory signals that can regulate the immune system. In some aspects, the multi-domain immunomodulatory proteins provided herein can alter the interaction of cell surface protein ligands with their receptors and / or alter the cell signal(s) induced in a cell to thereby modulate immune cells, such as T cell, activity. In some embodiments, the immunomodulatory proteins provided herein can be used for the treatment of diseases or conditions that are associated with a dysregulated immune response, such as autoimmune symptoms or an autoimmune disease.

[0136] In general, antigen specific T-cell activation generally requires two distinct signals. The first signal is provided by the interaction of the T-cell receptor (TCR) with major histocompatibility complex (MHC) associated antigens present on antigen presenting cells 2024204559   01 Jul 2024 (APCs). The second signal is costimulatory to TCR engagement and is necessary for T cell proliferation, differentiation and / or survival, including, in some cases, to avoid T-cell apoptosis or anergy. In some embodiments, under normal physiological conditions, the T cell-mediated immune response is initiated by antigen recognition by the T cell receptor (TCR) and is regulated by a balance of co-stimulatory activating signals and inhibitory signals (i.e., immune checkpoint proteins).

[0137] Among activating receptors on T cells are the costimulatory receptor CD28, which binds to costimulatory ligands CD80 (also called B7-1) and CD86 (also called B7-2), and promotes activation of naive T cells in the presence of a TCR signal. T cell activation, however, can be attenuated by engagement of inhibitory receptors, such as PD-1, CTLA-4 or TIGIT. For example, CTLA-4 competes with CD28 for binding of CD80 and CD86 to induce negative regulation of T cell activation. When CTLA-4 binds CD80 and / or CD86, and prevents CD28 from binding its cognate ligands, T cells do not effectively transmit the activating signaling cascade, and T cell activation and effector function can be eliminated or attenuated. In some cases, engagement of PD-1, expressed on NK cells and T cells, by its ligands PD-L1 and PD-L2, negatively regulate immune activation to inhibit activation, including by inhibiting cytolytic activity, proliferation and / or cytokine production. Similarly, TIGIT, which also can be expressed on NK cells and T cells, can suppress or inhibit the cytolytic activity of NK cells and T cells, T cell proliferation and / or proinflammatory cytokine production via engagement by its ligands CD112 or CD155. Inhibitory receptor ligands are, in some cases, expressed on antigen presenting cells, such that the ability to negatively regulate an immune response is often dependent on cells expressing activating or inhibitory receptors being present in the same space at the same time.

[0138] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self- tolerance) and to protect tissues from excessive damage during an immune response, for example during an attack against a pathogenic infection. In some cases, however, the immune system can become dysregulated and an abnormal immune response can be mounted against a normal body part or tissue, resulting in an autoimmune disease or condition or autoimmune symptoms. In other cases an unwanted immune response can be mounted to a foreign tissue, such as a transplant, resulting in transplant rejection. 2024204559   01 Jul 2024

[0139] In some aspects, immunotherapy that alters immune cell activity, such as T cell activity, can treat certain diseases and conditions in which the immune response is dysregulated. In particular, inhibition or attenuation of an immune response, such as a T cell response, could be desirable to reduce or prevent unwanted autoimmune symptoms and / or transplant rejection. Therapeutic approaches that seek to modulate interactions in the IS, however, are not entirely satisfactory. In some cases, therapies to intervene and alter the immunomodulatory effects of immune cell, e.g. T cell, activation are constrained by the spatial orientation requirements as well as size limitations imposed by the confines of the immunological synapse. In some aspects, existing therapeutic drugs, including antibody drugs, may not be able to interact simultaneously with the multiple target proteins involved in modulating these interactions. For example, soluble receptors and antibodies generally bind competitively (e.g., to no more than one target species at a time) and therefore lack the ability to simultaneously bind multiple targets. And while bispecific antibodies, as well as modalities comprising dual antigen binding regions, can bind to more than one target molecule simultaneously, the three-dimensional configuration typical of these modalities often precludes them intervening in key processes occurring in the IS in a manner consistent with their temporal and spatial requirements. Additionally, pharmacokinetic differences between drugs that independently target one of these receptors can create difficulties in properly maintaining a desired blood concentration of a drug combination targeting two different targets throughout the course of treatment.

[0140] Further, in some cases, existing therapeutic drugs may only have the ability to antagonize but not agonize an immune response. For example, the inhibitory receptor PD-1 has proven to be an effective inhibitory checkpoint receptor that regulates T cell activation. Therapeutic molecules that antagonize PD-1 / PD-L1 interactions have proven to be efficacious in stimulating patient immune responses towards tumors. Therapeutic molecules targeting PD-1 or its ligands, however, have not been demonstrated to do the reverse, such as to mediate an inhibitory signal intrinsically into the T cell to attenuate an immune response. Such a therapeutic molecule would be desirable for use in treating inflammatory or autoimmune diseases or conditions.

[0141] Thus, there is a need for therapeutic molecules that have the specificity and affinity of antibodies or soluble receptors but, in addition, attenuate immune responses intrinsically through an immune cell, such as a T cell. It is contemplated herein that the inability for molecules targeting inhibitory receptors, such as PD-1, to deliver negative signals into a cell is 2024204559   01 Jul 2024 due to the inability of such molecules to proximalize signaling of an inhibitory and activating receptor in the cell. Inhibitory receptors contain an immunoreceptor tyrosine-based inhibitory motif (ITIM), which when phosphorylated upon engagement of the inhibitor receptor by a ligand, can recruit phosphotyrosine phosphatases, such as a SHP-1 or SHP-2. Recruiting phosphatases can attenuate activating signaling cascades when the ITIM bearing receptors are in close proximity to the activating receptor (see FIG. 1A, IB, and 2). It is believed that, in some aspects, monoclonal antibodies may not be efficacious as PD-1 agonists because simply crosslinking the inhibitory receptor does not facilitate inhibitory activity.

[0142] The provided embodiments are based on findings that strategies that physically proximalize the inhibitory and the activating receptor of the surface of the same immune cell, such as a T cell, include the uses of reagents that can bind both the inhibitory and activating receptor. In some embodiments, binding of both the inhibitory and activating receptor can keep the ITIM and IT AM signaling components in close proximity, including, in some cases for extended periods of time, and thereby favor phosphatase dependent dephosphorylation of the activating receptor signaling cascades to attenuate immune responses.

[0143] Provided are therapeutic molecules that, in some embodiments, bind both T-cell activation signaling components, such as the activating receptor or its ligand, and inhibitory receptors. In some cases, the inhibitory receptor is an ITIM containing receptor and the activating receptor is a molecule that contains an IT AM and / or that associates or interacts with an adaptor protein involved in or regulating a tyrosine phosphorylation signaling cascade in the cell (e.g. T cell). In some embodiments, the provided immunomodulatory proteins contain at least two binding molecules: (1) an activating receptor binding molecule (ARBM) that is specific for an activating receptor or a ligand of the activating receptor and (2) an inhibitory receptor binding molecule (IRBM) that is specific for an inhibitory receptor. In some embodiments, the ARBM and IRBM are distinct or different polypeptides that independently bind different target cell surface molecules. In some aspects, the binding can occur simultaneously or in a non-competitive manner. In some embodiments, the activating receptor and inhibitory receptor are expressed on the same cell, e.g. a T cell, such that interactions of the ARBM and IRBM with their target cell surface molecules can physically proximalize the inhibitory receptor and activating receptor on the surface of the immune cells, e.g. T cell. In some cases, the close proximity of the inhibitory and activating receptors induced by binding of the immunomodulatory protein to its target cell surface molecules recruits a cytoplasmic protein 2024204559   01 Jul 2024 tyrosine phosphatase to the intracellular region of the inhibitory receptor and / or favors phosphatase dependent phosphorylation of the activating receptor signaling cascades. In some embodiments, binding of the immunomodulatory protein to a target cell expressing the activating receptor and inhibitor receptor, such as a T cells, can lead to attenuation of immune responses, such as attenuation of T cell responses.

[0144] In some embodiments, the provided multi-domain immunomodulatory proteins can interact with the activating receptor / ligand in cis or in trans (FIG. 1A and IB ). In some aspects, a cis strategy can be employed in which the IRBM binds to an inhibitory receptor (e.g. PD-1) and the ARBM binds to an activating receptor (e.g. CD3 / CD4 / CD28) on the surface of the same cell, e.g. same T cell. In some aspects, such embodiments benefit from the ability to bind multiple IS targets (e.g., an inhibitory and activating receptor) in a manner that is not dependent on the presence of other immune cells. In some aspects a trans strategy can be employed in which the IRBM binds an inhibitory receptor (e.g. PD-1) on a cell, e.g. T cell, that also expresses an activating receptor, and the ARBM binds to a ligand of the activating receptor found on another cell, such as an antigen presenting cell (APC). An exemplary immunomodulatory molecule that can act in a trans strategy includes one containing an IRBM that binds to an inhibitory receptor (e.g. PD-1) and an ARBM targeting a B7 costimulatory ligand (e.g. CD80 or CD86) on the APC to localize the CD28 costimulatory receptor and the inhibitory receptor to the immune synapse to attenuate the response. Similar trans strategies can use ARBM molecules targeting other activating ligands on an antigen presenting cell, such as an MHC molecule. In some cases, the trans strategy also may antagonize B7 / CD28 signaling, thereby regulating TCR and CD28 activating signaling cascades.

[0145] In particular aspects, the provided immunomodulatory proteins provide an immunotherapy platform using binding domains of wild-type or affinity modified native immune ligands or receptors as the IRBM and / or ARBM component. In some aspects, the binding domain is or includes an immunoglobulin superfamily (IgSF) domain of an IgSF family member. In some aspects, the IgSF domain excludes those found in antibodies (i.e., immunoglobulins), such that the provided embodiments include embodiments that relate to immunomodulatory proteins containing non-immunoglobulin (i.e., non-antibody) IgSF domains. Wild-type mammalian IgSF family members that are not immunoglobulins (i.e. antibodies) are known as are their nucleic and amino acid sequences. In some embodiments, the nonimmunoglobulin IgSF family members, and the corresponding IgSF domains present therein, are 2024204559   01 Jul 2024 of mouse, rat, cynomolgus monkey, or human origin. In some embodiments, the IgSF family members are members of an IgSF subfamily such as: Signal-Regulatory Protein (SIRP) Family, Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family, Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family, Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family, B7 family, CD28 family, V-set and Immunoglobulin Domain Containing (VSIG) family, V-set transmembrane Domain (VSTM) family, Major Histocompatibility Complex (MHC) family, Signaling lymphocytic activation molecule (SLAM) family, Leukocyte immunoglobulin-like receptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family, Poliovirus receptor related (PVR) family, Natural cytotoxicity triggering receptor (NCR) family, or Killer-cell immunoglobulin-like receptors (KIR) family. For purposes herein, the IgSF domain is one whose binding partner is an activating receptor or a ligand of an activating receptor or is an inhibitory receptor.

[0146] In some embodiments, non-immunoglobulin IgSF family members, and the corresponding IgSF domains present therein, of an immunomodulatory proteins provided herein, are affinity-modified compared to a mammalian IgSF member. The affinity-modified IgSF domains include IgSF domains that are modified, such as by one or more amino acid substitution, to bind with tunable affinities to one or more of their cognate binding partner. An IgSF domain can be affinity modified to independently increase or decrease specific binding affinity or avidity to each of the multiple cognate binding partners to which it binds. By this mechanism, specific binding to each of multiple cognate binding partners is independently tuned to a particular affinity or avidity. Exemplary affinity-modified or variant IgSF domains having altered, such as increased, binding activity to one or more cognate binding partners are known (e.g. International published PCT App. Nos. WO 2016 / 168771, WO 2017 / 181148 and WO 2017 / 181152) or are described.

[0147] In some aspects, the provided immunomodulatory proteins containing one or more IgSF domain based on an immune system molecule, such as human immune system molecule, themselves are more likely to retain their ability to normally assemble into key pathways of the immune synapse and maintain normal interactions and regulatory functions, in part, because they are based on natural components of the immune synapse. Further, the relatively small molecular weight of individual IgSF domain may be beneficial in bringing two receptors together in close enough proximity, such as to induce or mediate phosphtase regulatory activity. Such features may not be possible with antibodies, including next-generation bispecific regents, 2024204559   01 Jul 2024 due to the relatively large size of antibodies as well as from the fact that antibodies are not natural components of the immune synapse.

[0148] In some embodiments, the provided immunomodulatory proteins are single polypeptide fusions or monomers containing the IRBM or ARBM, which in some cases, can be tagged with a detectable moiety, such as a flag and / or his tag. In some aspects, such single polypeptide fusion or monomer formats minimize the size of the immunomodulatory protein and / or avoid steric issues that may result from other formats, including those involving larger molecules, such as dimeric molecules, e.g. molecules containing a multimerization domain, such as an Fc domain. In some cases, such polypeptide fusions can be made in various orientations, e.g. IRBM-ARBM or ARBM-IRBM, to create optimal binding pairs. Further, in some aspects, multiple IRBM and / or ARBM, such as independently two or more, such as 2, 3, 4, 5 or more IRBM or ARBM binding domain, e.g. IgSF domain, can be configured in a single polypeptide molecule to increase or improve binding affinity or avidity of one or both components, e.g. IRBM-IRBM-IRBM-ARBM.

[0149] In some aspects, the immunomodulatory proteins are provided as a dimeric protein. Generally, such formats are generated as a heterodimer, e.g. heterodimeric Fc fusion protein, such as to avoid dimeric binding of the activating receptor (e.g. CD28), which could result in unwanted firing of activating receptor. In some aspects, a heterodimeric immunomodulatory protein, while larger, may exhibit pharmacodynamics advantages, such as increased half-life.

[0150] In some aspects, the immunomodulatory proteins provided herein have therapeutic utility by attenuating immunological activity in a mammal with a disease or disorder in which modulation of the immune system response is beneficial. In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition.

[0151] All publications, including patents, patent applications scientific articles and databases, mentioned in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patent, patent application, scientific article or database, were specifically and individually indicated to be incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference. 2024204559   01 Jul 2024

[0152] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Further various embodiments of the invention as discussed below are frequently provided. Thus, the headings, the order of presentation of the various aspects and embodiments, and the separate disclosure of each independent attribute is not meant to be a limitation to the scope of the present disclosure. DEFINITIONS

[0153] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0154] Unless defined otherwise, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Unless indicated otherwise, abbreviations and symbols for chemical and biochemical names are per IUPAC-IUB nomenclature. Unless indicated otherwise, all numerical ranges are inclusive of the values defining the range as well as all integer values in-between.

[0155] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0156] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0157] As used herein, an “activating receptor” refers to a cell surface molecule in which engagement or ligation of the molecule results in the direct or indirect activation of one or more tyrosine kinases in an immune cell and / or culminates in the induction or potentiation of one or more effector cell functions in an immune cell in which it is expressed. An activating receptor generally contains an extracellular portion, a transmembrane domain and cytoplasmic region. In some embodiments, the cytoplasmic region contains an intracellular signaling domain that contains an immunoreceptor tyrosine-based activation motif (ITAM; defined by the sequence YXX(L / I)X6-8YXX(L / I)) or that otherwise is capable of interacting with or associating with 2024204559   01 Jul 2024 one or more accessory proteins, such as one or more adaptor proteins, involved in or regulating tyrosine phosphorylation in a signal transduction pathway. In some cases, an activating receptor interacts with or associates with an adaptor protein that contains an IT AM or an adaptor protein that contains one or more protein-binding domains, such as e.g., Src homology 2 (SH2) and SH3 domains, that bind specific amino acid sequences, e.g phosphotyrosine residues, within a protein in a signal transduction pathway. Examples of adaptor proteins include, but are not limited to, Lek, Fyn, ZAP70, SLP76, PI3K, Grb2, PKC0 and SHC1. Thus, it is understood that the activating receptor itself need not possess intrinsic enzymatic activity but may indirectly mediate enzymatic activities via accessory or adaptor proteins. In some embodiments, an activating receptor is a cell surface molecule on a T cell. Typically, engagement of an activating receptor initiates, mediates or potentiates activation of a cell resulting in a measurable morphological, phenotypic, and / or functional changes in the cell, such as a T cell, including cell proliferation, cytolytic activity, cytokine production or secretion, or expression of cell surface molecules such as receptors or adhesion molecules. In some embodiments, an activating receptor includes a T cell receptor (TCR), CD3, CD4, CD8, CD28, ICOS, or CD2.

[0158] The term “activating receptor binding molecule” or ARBM refers to a protein that specifically binds to an activating receptor or a ligand of an activating receptor.

[0159] The term “affinity-modified” as used in the context of an immunoglobulin superfamily domain, means a mammalian immunoglobulin superfamily (IgSF) domain having an altered amino acid sequence (relative to the corresponding wild-type parental or unmodified IgSF domain) such that it has an increased or decreased binding activity, such as binding affinity or avidity, to at least one of its binding partners (alternatively “counter-structures”) compared to the parental wild-type or unmodified (i.e., non-affinity modified) IgSF control domain. In some embodiments, the affinity-modified IgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions, in a wild-type or unmodified IgSF domain. An increase or decrease in binding activity, e.g. binding affinity or avidity, can be determined using well known binding assays, including flow cytometry. Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also, Linsley et al., Immunity, 1: 7930801 (1994). An increase in a protein’s binding activity, e.g. affinity or avidity, to its binding partner(s) is to a value at least 10% greater than that of the wild-type IgSF domain control and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 2024204559   01 Jul 2024 10000% greater than that of the wild-type IgSF domain control value. A decrease in a protein’s binding activity, e.g. affinity or avidity, to at least one of its binding partner is to a value no greater than 90% of the control but no less than 10% of the wild-type IgSF domain control value, and in some embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, or 20% but no less than 10% of the wild-type IgSF domain control value. An affinity-modified protein is altered in primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term “affinity-modified IgSF domain” is not be construed as imposing any condition for any particular starting composition or method by which the affinity-modified IgSF domain was created. Thus, the affinity-modified IgSF domains of the present invention are not limited to wild-type IgSF domains that are then transformed to an affinity-modified IgSF domain by any particular process of affinity modification. An affinity-modified IgSF domain polypeptide can, for example, be generated starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its binding partner, and finally recombinantly or chemically synthesized to yield the affinity-modified IgSF domain composition of matter. In but one alternative example, an affinity-modified IgSF domain can be created by site-directed mutagenesis of a wild-type IgSF domain. Thus, affinity modified IgSF domain denotes a product and not necessarily a product produced by any given process. A variety of techniques including recombinant methods, chemical synthesis, or combinations thereof, may be employed.

[0160] The term “allogeneic” as used herein means a cell or tissue that is removed from one organism and then infused or adoptively transferred into a genetically dissimilar organism of the same species.

[0161] The term “autologous” as used herein means a cell or tissue that is removed from the same organism to which it is later infused or adoptively transferred. An autologous cell or tissue can be altered by, for example, recombinant DNA methodologies, such that it is no longer genetically identical to the native cell or native tissue which is removed from the organism. For example, a native autologous T-cell can be genetically engineered by recombinant DNA techniques to become an autologous engineered cell expressing a immunomodulatory protein (which can be secreted from the engineered cell), which in some cases involves engineering a T-cell or TIL (tumor infiltrating lymphocyte). The engineered cell can then be infused into a patient from which the native T-cell was isolated. In some embodiments, the organism is human or murine. 2024204559   01 Jul 2024

[0162] As used herein, "bind," "bound" or grammatical variations thereof refers to the participation of a molecule in any attractive interaction with another molecule, resulting in a stable association in which the two molecules are in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds including drugs.

[0163] As used herein, binding activity refer to characteristics of a molecule, e.g. a polypeptide, relating to whether or not, and how, it binds one or more binding partners. A binding activity can include any measure of binding of one molecule for a binding partner. Binding activities include the ability to bind the binding partner(s), the affinity with which it binds to the binding partner (e.g. high affinity), the avidity with which it binds to the binding partner, the strength of the bond with the binding partner and / or specificity or selectivity for binding with the binding partner.

[0164] The term “binding affinity” as used herein means the specific binding affinity of a protein for its binding partner (i.e., its counter-structure) under specific binding conditions. The binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, typically the strength of the noncovalent interactions between two binding partners. An increase or attenuation in binding affinity of an affinity-modified IgSF domain, or an immunomodulatory protein containing an affinity-modified IgSF domain, to a binding partner is determined relative to the binding affinity of the unmodified IgSF domain (e.g., the native or wild-type IgSF domain). Methods for determining binding affinity, or relative binding affinity, are known in art, solid-phase ELISA immunoassays, ForteBio Octet, Biacore measurements or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1 (9): 793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, such as based on a Mean Fluorescence Intensity (MFI) in a binding assay.

[0165] The term “binding avidity” as used herein means the specific binding avidity, of a protein for its binding partner (i.e., its counter-structure) under specific binding conditions. In biochemical kinetics avidity refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between an IgSF domain and its binding 2024204559   01 Jul 2024 partner (i.e., its counter-structure). As such, avidity is distinct from affinity, which describes the strength of a single interaction.

[0166] The term “biological half-life” refers to the amount of time it takes for a substance, such as an immunomodulatory protein, to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic degradation / digestion) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”). Conjugates that can be used to derivatize and increase the biological half-life of a protein are known in the art and include, but are not limited to, multimerization domains (e.g. Fc), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see, WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0167] The term “cell surface counter-structure” (alternatively “cell surface binding partner”) as used herein is a counter-structure (alternatively is a binding partner) expressed on a mammalian cell. Typically, the cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor.

[0168] The terms “binding partner” or “counter-structure” in reference to a protein, such as an IgSF domain or an affinity-modified IgSF domain, refers to at least one molecule (typically a native mammalian protein) to which the referenced protein specifically binds under specific binding conditions. In some aspects, an affinity-modified IgSF domain, or an immunomodulatory protein containing an affinity-modified IgSF domain, specifically binds to the binding partner of the corresponding native or wild-type IgSF domain but with increased or attenuated affinity. A “cell surface binding partner” is a binding partner expressed on a mammalian cell. Typically, the cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor or a ligand of a receptor expressed on and by cells, such as mammalian cells, forming the immunological synapse, for example immune cells.

[0169] The term “cis” with reference to binding to cell surface molecules refers to binding to two or more different cell surface molecules, each of which is present on the surface of the same cell. In some embodiments, cis means that the two or more cell surface molecules are 2024204559   01 Jul 2024 exclusively on one or exclusively the other (but not both) of the two mammalian cells forming the IS.

[0170] The term “competitive binding” as used herein means that a protein is capable of specifically binding to at least two binding partners but that specific binding of one binding partner inhibits, such as prevents or precludes, simultaneous binding of the second binding partner. Thus, in some cases, it is not possible for a protein to bind the two binding partners at the same time. Generally, competitive binders contain the same or overlapping binding site for binding but this is not a requirement. In some embodiments, competitive binding causes a measurable inhibition (partial or complete) of specific binding of a protein to one of its binding partner due to specific binding of a second binding partner. A variety of methods are known to quantify competitive binding such as ELISA (enzyme linked immunosorbent assay) or Forte-Bio Octet experimental systems.

[0171] The term “conservative amino acid substitution” as used herein means an amino acid substitution in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic -hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0172] The term, “corresponding to” with reference to positions of a protein, such as recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0173] The term “cytokine” includes, e.g., but is not limited to, interleukins, interferons (IFN), chemokines, hematopoietic growth factors, tumor necrosis factors (TNF), and 2024204559   01 Jul 2024 transforming growth factors. In general, these are small molecular weight proteins that regulate maturation, activation, proliferation, and differentiation of cells of the immune system.

[0174] The terms “decreased,” “reduced,” “suppressed” or “inhibits,” which can be used interchangeably, as used herein in the context of an immunological activity of a mammalian lymphocyte in the presence of a provided immunomodulatory protein means to decrease one or more activities of the lymphocyte, as compared to a control, such as an untreated control or a control involving treatment with another molecule, such as a molecule containing an individual IgSF domain of an immunomodulatory protein or an alternative molecule, was employed under the same conditions. A decreased activity can refer to one or more of cell cycle inhibition, reduced cell survival, reduced cell proliferation, reduced cytokine production, or reduced T-cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to reduced immunological activity means to reduce interferon gamma (IFN-gamma) production compared to in the absence of treatment, such as by a statistically significant amount. In some embodiments, the immunological activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods of conducting MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. Other methods of assessing activities of lymphocytes are known in the art, including any assay as described herein. In some embodiments, a decrease can be by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, as compared to a control value, such as an untreated control value or a non-zero control value.

[0175] The terms “derivatives” or “derivatized” refer to modification of an immunomodulatory protein by covalently linking it, directly or indirectly, so as to alter such characteristics as half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy while retaining or enhancing its therapeutic benefit. Derivatives can be made by glycosylation, pegylation, lipidation, or Fc-fusion. In some embodiments, the immunomodulatory protein is not derivatized. In some embodiments, the immunomodulatory protein is not conjugated to a half-life extending moiety, such as an Fc domain.

[0176] As used herein, “domain” (typically a sequence of three or more, generally 5 or 7 or more amino acids, such as 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or encoding nucleic acid, that is structurally and / or functionally distinct from other portions of the molecule and is identifiable. For example, domains include those portions of a polypeptide chain that can form an independently folded structure within a protein made up of 2024204559   01 Jul 2024 one or more structural motifs and / or that is recognized by virtue of a functional activity, such as binding activity. A protein can have one, or more than one, distinct domains. For example, a domain can be identified, defined or distinguished by homology of the primary sequence or structure to related family members, such as homology to motifs. In another example, a domain can be distinguished by its function, such as an ability to interact with a biomolecule, such as a cognate binding partner. A domain independently can exhibit a biological function or activity such that the domain independently or fused to another molecule can perform an activity, such as, for example binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain a plurality of domains. Such domains are known, and can be identified by those of skill in the art. For exemplification herein, definitions are provided, but it is understood that it is well within the skill in the art to recognize particular domains by name. If needed appropriate software can be employed to identify domains. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization of an IgSF domain are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It is understood that polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar molecules. Also, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g. ECD or IgV) also can be included in a sequence of an IgSF domain, such as to ensure proper folding of the domain when expressed. Thus, the exact locus can vary, and is not necessarily the same for each protein. Hence, the specific IgSF domain, such as specific IgV domain or IgC domain, can be several amino acids (1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) longer or shorter.

[0177] The term “ectodomain,” “extracellular domain,” or “ECD,” which are used interchangeably herein, refers to the region of a membrane protein, such as a transmembrane protein, that lies outside the vesicular membrane (e.g., the space outside of a cell). Ectodomains often interact with specific ligands or specific cell surface receptors, such as via a binding domain that specifically binds to the ligand or cell surface receptor. Ectodomains of members of the immunoglobulin superfamily contain immunoglobulin domains.

[0178] The terms “effective amount” or “therapeutically effective amount” refer to a quantity and / or concentration of a therapeutic composition of the invention, such as containing an immunomodulatory protein or engineered cells expressing an immunomodulatory protein, that when administered ex vivo (by contact with a cell from a patient) or in vivo (by 2024204559   01 Jul 2024 administration into a patient) either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a statistically significant inhibition of disease progression as, for example, by ameliorating or eliminating symptoms and / or the cause of the disease. An effective amount for treating a disease or disorder, such as an immune system disease or disorder, may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with the disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient. In the case of cell therapy, the effective amount is an effective dose or number of cells administered to a patient. In some embodiments the patient is a human patient.

[0179] The term “endodomain” as used herein refers to the region found in some membrane proteins, such as transmembrane proteins, that extends into the interior space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. In cells, the endodomain interacts with intracellular constituents and can be play a role in signal transduction and thus, in some cases, can be an intracellular signaling domain. The endodomain of a cellular transmembrane protein is alternately referred to as a cytoplasmic domain, which, in some cases, can be a cytoplasmic signaling domain.

[0180] The term “enhanced” or “increased,” which can be used interchangeably, as used herein in the context of increasing immunological activity of a mammalian lymphocyte in the presence of a molecule means to increase one or more activities of the lymphocyte. An increased activity can be one or more of an increase cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to increased immunological activity means to increase interferon gamma (IFN-gamma) production, such as by a statistically significant amount. Typically, the increase is relative to or compared to a control, such as an untreated control or a control involving treatment with another molecule. Methods of assessing activities of lymphocytes are known in the art, including any assay as described herein. In some embodiments an enhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%,100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

[0181] The term “engineered cell” as used herein refers to a mammalian cell that has been genetically modified by human intervention such as by recombinant DNA methods or viral transduction. In some embodiments, the engineered cell is an immune cell, such as a lymphocyte (e.g. T cell, B cell, NK cell) or an antigen presenting cell (e.g. dendritic cell). The 2024204559   01 Jul 2024 cell can be a primary cell from a patient or can be a cell line. In some embodiments, an engineered cell is capable of expressing and secreting a immunomodulatory protein as described herein.

[0182] The term “engineered T-cell” as used herein refers to a T-cell such as a T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL), natural killer T-cell, regulatory T-cell, memory T-cell, or gamma delta T-cell, that has been genetically modified by human intervention such as by recombinant DNA methods. In some embodiments, an engineered T-cell is capable of expressing and secreting an immunomodulatory protein as described herein.

[0183] As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing a coding sequence for two or more proteins, in some cases 2, 3, 4, 5 or more protein, in which the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, the protein is produced containing the two or more proteins. Each of the two or more proteins can be adjacent to another protein in the construct or separated by a linker polypeptide that contains, 1, 2, 3, or more, but typically fewer than 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide.

[0184] The term “half-life extending moiety” refers to a moiety of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety. In some embodiments, half-life is extended by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold. In some embodiments, half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. The half-life refers to the amount of time it takes for the protein to lose half of its concentration, amount, or activity. Half-life can be determined for example, by using an ELISA assay or an activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see, WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

[0185] An Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also termed an Fc polypeptide) corresponds largely to the constant region of the 2024204559   01 Jul 2024 immunoglobulin heavy chain, and is responsible for various functions, including the antibody’s effector function(s). The Fc domain contains part or all of a hinge domain of an immunoglobulin molecule plus a CH2 and a CH3 domain. The Fc domain can form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g. reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) activity to facilitate an effector function. In some embodiments, reference to amino acid substitutions in an Fc region is by EU numbering system unless described with reference to a specific SEQ ID NO. EU numbering is known and is according to the most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013) and the EU index as reported in Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0186] An immunoglobulin Fc fusion (“Fc-fusion”), such as an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides operably linked to an Fc region of an immunoglobulin. An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) operably linked to an ARBM or IRBM of the provided immunomodulatory proteins. An immunoglobulin Fc region may be linked indirectly or directly to the ARBM and / or IRBM. Various linkers are known in the art and can optionally be used to link an Fc to a fusion partner to generate an Fc-fusion. Fc-fusions of identical species can be dimerized to form Fc-fusion homodimers, or using nonidentical species to form Fc-fusion heterodimers. In some embodiments, the Fc is a mammalian Fc such as a murine or human Fc.

[0187] The term “host cell” refers to any cell that can be used to express a protein encoded by a recombinant expression vector. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media or CHO strain DX-B11, which is deficient in DHFR.

[0188] The term “immunological synapse” or “immune synapse” (abbreviated “IS”) as used herein means the interface between a mammalian cell that expresses MHC I (major 2024204559   01 Jul 2024 histocompatibility complex) or MHC II, such as an antigen-presenting cell or tumor cell, and a mammalian lymphocyte such as an effector T cell or Natural Killer (NK) cell.

[0189] The term “immunoglobulin” (abbreviated “Ig”) as used herein is synonymous with the term “antibody” (abbreviated “Ab”) and refers to a mammalian immunoglobulin protein including any of the five human classes: IgA (which includes subclasses IgAl and IgA2), IgD, IgE, IgG (which includes subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. The term is also inclusive of immunoglobulins that are less than full-length, whether wholly or partially synthetic (e.g., recombinant or chemical synthesis) or naturally produced, including any fragment thereof containing at least a portion of the variable heavy (VH) chain and / or variable light (VL) chain region of the immunoglobulin molecule that is sufficient to form an antigen binding site and, when assembled, to specifically bind antigen. The antibody also can include all or a portion of the constant region. Such fragments include antigen binding fragment (Fab), variable fragment (Fv) containing VH and VL, the single chain variable fragment (scFv) containing VH and VL linked together in one chain, as well as other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabody, Fc, and Fd polypeptide fragments. Hence, it is understood that reference to an antibody herein includes full-length antibody and antigen-binding fragments. The term antibody also includes antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules. Bispecific antibodies, homobispecific and heterobispecific, are included within the meaning of the term. Antibodies include polyclonal antibodies or monoclonal antibodies. Antibody also includes synthetic antibodies or recombinantly produced antibodies. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0190] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. A full-length antibody is an antibody typically having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VLCL) and hinge regions, such as antibodies produced from mammalian species (e.g. human, mouse, rat, rabbit, non-human primate, etc.) by antibody secreting B cells and antibodies with the same domains that are produced synthetically. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native 2024204559   01 Jul 2024 sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0191] An “antibody fragment” comprises a portion of an intact antibody, the antigen binding and / or the variable region of the intact antibody. Antibody fragments, include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules, including single-chain Fvs (scFv) or single-chain Fabs (scFab); antigenbinding fragments of any of the above and multispecific antibodies from from antibody fragments.

[0192] “Fv” is composed of one heavy- and one light-chain variable region domain linked by non-covalent association. From the folding of these two domains emanate six complementarity determining regions (CDR) (3 in each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although, in some cases, at a lower affinity than the entire binding site.

[0193] “dsFv” refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.

[0194] An “Fd fragment” is a fragment of an antibody containing a variable domain (Vh) and one constant region domain (ChI) of an antibody heavy chain.

[0195] A “Fab fragment” is an antibody fragment that results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods. A Fab fragment contains a light chain (containing a Vl and Cl) and another chain containing a variable domain of a heavy chain (Vh) and one constant region domain of the heavy chain (ChI).

[0196] A “F(ab')2 fragment” is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods. The F(ab')2 fragment essentially contains two Fab fragments where each heavy chain portion contains an additional few amino acids, including cysteine residues that form disulfide linkages joining the two fragments. 2024204559   01 Jul 2024

[0197] A “Fab' fragment” is a fragment containing one half (one heavy chain and one light chain) of the F(ab')2 fragment.

[0198] An “Fd’ fragment” is a fragment of an antibody containing one heavy chain portion of a F(ab')2 fragment.

[0199] An “Fv’ fragment” is a fragment containing only the Vh and Vl domains of an antibody molecule.

[0200] An “scFv fragment” refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Exemplary linkers are (Gly-Ser)n residues with some Glu or Lys residues dispersed throughout to increase solubility.

[0201] “Diabodies” are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and preferentially dimerize.

[0202] The term “immunoglobulin superfamily” or “IgSF” as used herein means the group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Molecules are categorized as members of this superfamily based on shared structural features with immunoglobulins (i.e., antibodies); they all possess a domain known as an immunoglobulin domain or fold. Many “non-antibody IgSF” members include cell surface proteins or receptors that are not antibodies. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular muscle proteins. They are commonly associated with roles in the immune system. Proteins in the immunological synapse are often members of the IgSF. IgSF can also be classified into “subfamilies” based on shared properties such as function. Such subfamilies typically include from 4 to 30 IgSF members.

[0203] The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” or “IgD” as used herein refers to a structural domain or domains of IgSF proteins. Ig domains are named after the immunoglobulin molecules. They contain about 70-110 amino acids and are categorized according to their size and function. Ig-domains possess a characteristic Ig-fold, which has a sandwich-like structure formed by two sheets of antiparallel beta strands. Interactions between hydrophobic amino acids on the inner side of the sandwich and highly conserved disulfide bonds formed between cysteine residues in the B and F strands, stabilize the 2024204559   01 Jul 2024 Ig-fold. In some cases, one end of the Ig domain has a section called the complementarity determining region, which, in some aspects, is involved in the specificity of antibodies for their ligands. The Ig like domains can be classified (into classes) as: IgV, IgCl, IgC2, or Igl. Most Ig domains are either variable (IgV) or constant (IgC). IgV domains with 9 beta strands are generally longer than IgC domains with 7 beta strands. Ig domains of some members of the IgSF resemble IgV domains in the amino acid sequence, yet are similar in size to IgC domains. These are called IgC2 domains, while standard IgC domains are called IgCl domains. T-cell receptor (TCR) chains contain two Ig domains in the extracellular portion; one IgV domain at the N-terminus and one IgCl domain adjacent to the cell membrane. A “non-antibody IgSF domain” refers to IgSF domain or domains present in proteins other than antibodies, which typically are present in the extracellular portion or domain of certain cell surface proteins. Thus, the extracellular domain (ECD) of IgSF family members contains one or more Ig domains; hence, the term Ig domain is also used with reference to the ECD of such protein molecules.

[0204] The term “immunological activity” as used herein in the context of mammalian lymphocytes, such as T cells, refers to one or more of activation, cell survival, cell proliferation, cytokine production (e.g. interferon-gamma), or T-cell cytotoxicity activities. Assays for determining enhancement or suppression of immunological activity include MLR (mixed lymphocyte reaction) assays measuring interferon-gamma cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), SEB (staphylococcal enterotoxin B), T cell stimulation assays (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med. 2010: 8: 104). Since T cell activation is associated with secretion of IFN-gamma cytokine, detecting IFN-gamma levels in culture supernatants from these in vitro human T cell assays can be assayed using commercial ELISA kits (Wu et al, Immunol Lett 2008 Apr 15; 117(1): 57-62). Assays also include assays to assess cytotoxicity, including a standard 51Cr-release assay (see e.g. Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flow based cytotoxicity assays, or an impedance based cytotoxicity assay (Peper et al. (2014) Journal of Immunological Methods, 405:192-198). Assays to assess immunological activity of immunomodulatory proteins can be compared to control proteins with a known activity.

[0205] An “immunomodulatory protein” or “immunomodulatory polypeptide” is a protein that modulates immunological activity. By “modulation” or “modulating” an immune response is meant that immunological activity is either enhanced or suppressed. An immunomodulatory 2024204559   01 Jul 2024 protein can be a single polypeptide chain or a multimer (dimers or higher order multimers) of at least two polypeptide chains covalently bonded to each other by, for example, interchain disulfide bonds. Thus, monomeric, dimeric, and higher order multimeric proteins are within the scope of the defined term. Multimeric proteins can be homomultimeric (of identical polypeptide chains) or heteromultimeric (of different polypeptide chains). Secretable immunomodulatory proteins are a type of immunomodulatory protein.

[0206] As used herein, an “inhibitory receptor” refers to a cell surface molecule in which engagement of the molecule transmits a negative signal to an immune cell and / or downregulates or reduces activation of an immune cell. In some embodiments, transmission of an inhibitory signal to an immune cell can result in unresponsiveness or anergy or programmed cell death in the immune cell. An inhibitory receptor generally contains an extracellular portion, a transmembrane domain and a cytoplasmic region that directly or indirectly activates or recruits phosphatases. In some embodiments, the cytoplasmic region contains an immunoreceptor tyrosine-based inhibition motif (fTIM), which is a conserved sequence of amino acids (S / I / V / LxYxxI / V / L) that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. In some aspects, engagement of an ITIM-containing receptor, such as with a ligand, results in phosphorylation of the ITIM motif to recruit SH2-domain containing phosphatases, such as the phosphotyrosine phosphatases SHP-1 and SHP-2 or the inositolphosphatase called SHIP. In some aspects, the phosphatases can dephosphorylate kinases associated with IT AM-mediated cell activation, thereby attenuating signaling and effector functions mediated by an activating receptor, such as inhibition or reduction of proliferation, cytokine production or secretion or cytotoxic activity. In some embodiments, an inhibitory receptor includes PD-1, CTLA-4 or TIGIT.

[0207] The term “inhibitory receptor binding molecule” or IRBM refers to a protein that specifically binds to an inhibitory receptor.

[0208] The term “lymphocyte” as used herein means any of three subtypes of white blood cell in a mammalian immune system. They include natural killer cells (NK cells) (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). T cells include T helper cells, cytotoxic T-cells, natural killer T-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells. Innate lymphoid cells (ILC) are also included within the definition of lymphocyte.. 2024204559   01 Jul 2024

[0209] The term “lymphocyte” as used herein means any of three subtypes of white blood cell in a mammalian immune system. They include natural killer cells (NK cells) (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). T cells include: T helper cells, cytotoxic T-cells, natural killer T-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells. Innate lymphoid cells (ILC) are also included within the definition of lymphocyte.

[0210] The terms “mammal,” “subject,” or “patient” specifically includes reference to at least one of a: human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0211] The term “membrane protein” as used herein means a protein that, under physiological conditions, is attached directly or indirectly to a lipid bilayer. A lipid bilayer that forms a membrane can be a biological membrane such as a eukaryotic (e.g., mammalian) cell membrane or an artificial (i.e., man-made) membrane such as that found on a liposome. Attachment of a membrane protein to the lipid bilayer can be by way of covalent attachment, or by way of non-covalent interactions such as hydrophobic or electrostatic interactions. A membrane protein can be an integral membrane protein or a peripheral membrane protein. Membrane proteins that are peripheral membrane proteins are non-covalently attached to the lipid bilayer or non-covalently attached to an integral membrane protein. A peripheral membrane protein forms a temporary attachment to the lipid bilayer such that under the range of conditions that are physiological in a mammal, peripheral membrane protein can associate and / or disassociate from the lipid bilayer. In contrast to peripheral membrane proteins, integral membrane proteins form a substantially permanent attachment to the membrane's lipid bilayer such that under the range of conditions that are physiological in a mammal, integral membrane proteins do not disassociate from their attachment to the lipid bilayer. A membrane protein can form an attachment to the membrane by way of one layer of the lipid bilayer (monotopic), or attached by way of both layers of the membrane (polytopic). An integral membrane protein that interacts with only one lipid bilayer is an “integral monotopic protein”. An integral membrane protein that interacts with both lipid bilayers is an “integral polytopic protein” alternatively referred to herein as a “transmembrane protein”.

[0212] As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes 2024204559   01 Jul 2024 deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

[0213] The terms “modulating” or “modulate” as used herein in the context of an immune response, such as a mammalian immune response, refer to any alteration, such as an increase or decrease, of an existing or potential immune responses that occurs as a result of administration of an immunomodulatory protein or as a result of administration of engineered cells expressing an immunomodulatory protein, such as a secretable immunomodulatory protein of the present invention. Such modulation includes any induction, or alteration in degree or extent, or suppression of immunological activity of an immune cell. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs), and nonprofessional antigen-presenting cells, and inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration in the expression and / or function of genes, proteins and / or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immunological activity includes, for example, the following: elimination, deletion, or sequestration of immune cells; proliferation, induction, survival or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); enhancing or suppressing the activity or function of immune cells, including but not limited to altering the pattern of proteins expressed by these cells. Examples include altered production and / or secretion of certain classes of molecules such as cytokines, chemokines, perforins, granzymes, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors or any combination of these modulatory events. Modulation can be assessed, for example, by an alteration of an immunological activity.

[0214] The term, a “multimerization domain” refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each containing a complementary multimerization domain (e.g. a first multimerization domain and a second multimerization domain), which can be the same or a 2024204559   01 Jul 2024 different multimerization domain. The interactions between complementary multimerization domains, e.g. interaction between a first multimerication domain and a second multimerization domain, form a stable protein-protein interaction to produce a multimer of the polypeptide molecule with the additional polypeptide molecule. In some cases, the multimerization domain is the same and interacts with itself to form a stable proten-protein interaction between two polypeptide chains. Generally, a polypeptide is joined directly or indirectly to the multimerization domain. Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain, for example, can be an immunoglobulin constant region or domain, such as, for example, the Fc domain or portions thereof from IgG, including IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

[0215] The term “non-competitive binding” as used herein means the ability of a protein to specifically bind simultaneously to at least two binding partners. In some embodiments, the binding occurs under specific binding conditions. Thus, the protein is able to bind to at least two different binding partners at the same time although the binding interaction need not be for the same duration such that, in some cases, the protein is specifically bound to only one of the binding partners. In some embodiments, the simultaneous binding is such that binding of one binding partner does not substantially inhibit simultaneous binding to a second binding partner. In some embodiments, non-competitive binding means that binding a second binding partner to its binding site on the protein does not displace the binding of a first binding partner to its binding site on the protein. Methods of assessing non-competitive binding are well known in the art such as the method described in Perez de La Lastra et al., Immunology, 1999 Apr: 96(4): 663-670. In some cases, in non-competitive interactions, the first binding partner specifically binds at an interaction site that does not overlap with the interaction site of the second binding partner such that binding of the second binding partner does not directly interfere with the binding of the first binding partner. Thus, any effect on binding of the binding partner by the binding of the second binding partner is through a mechanism other than direct interference with the binding of the first binding partner. For example, in the context of enzyme-substrate interactions, a non-competitive inhibitor binds to a site other than the active site of the enzyme. Non-competitive binding encompasses uncompetitive binding interactions in which a second binding partner specifically binds at an interaction site that does not overlap with the binding of 2024204559   01 Jul 2024 the first binding partner but binds to the second interaction site only when the first interaction site is occupied by the first binding partner.

[0216] The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogues of natural nucleotides and that have similar binding properties to it and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0217] The terms “in operable combination,” “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner or orientation that the segments are arranged so that they function in concert for their intended purposes. In some embodiments, the term refers to linkage of nucleic acids to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or to produce a desired protein molecule that is functional. For example, segments of a DNA sequence, e.g. a coding sequence and a regulatory sequence(s), are linked in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence.

[0218] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory protein or engineered cells expressing and / or secreting an immunomodulatory protein of the present invention) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.

[0219] The terms “polypeptide” and “protein” are used interchangeably herein and refer to a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” and “oligopeptides,” are included 2024204559   01 Jul 2024 within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, proteins can be derivatized as described herein by well-known organic chemistry techniques.

[0220] The term “primary T-cell assay” as used herein refers to an in vitro assay to measure interferon-gamma (“IFN-gamma”) expression. A variety of such primary T-cell assays are known in the art. In some embodiments, the assay used is anti-CD3 coimmobilization assay. In this assay, primary T cells are stimulated by anti-CD3 immobilized with or without additional recombinant proteins. Culture supernatants are harvested at timepoints, usually 24-72 hours. In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are simulated with allogenic APC. Culture supernatants are harvested at timepoints, usually 24-72 hours. Human IFN-gamma levels are measured in culture supernatants by standard ELISA techniques. In some cases, commercial kits are available from vendors and the assay can be performed according to manufacturer’s recommendation.

[0221] The term “purified” as applied to nucleic acids, such as encoding immunomodulatory proteins, or proteins (e.g. immunomodulatory proteins) generally denotes a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.” A purified nucleic acid or protein is at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or on a molar basis).

[0222] The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, affinity modification, DNA shuffling or other well-known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant 2024204559   01 Jul 2024 protein” or “recombinant polypeptide” as used herein refers to a protein molecule (e.g., an immunomodulatory protein) which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and / or expresses a recombinant nucleic acid or that is otherwise altered by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, such as a immunomodulatory protein provided herein. Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.

[0223] The term “recombinant expression vector” as used herein refers to a DNA molecule containing a desired coding sequence (e.g., encoding an immunomodulatory protein) and appropriate nucleic acid sequences necessary for the expression of an operably linked coding sequence in a particular cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the recombinant expression vector, operably linked to the coding sequence so that the expressed protein can be secreted by the recombinant host cell, such as for its expression as a secretable protein or for more facile isolation or purification of the immunomodulatory protein from the cell, if desired. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Among the vectors are viral vectors, such as lentiviral vectors.

[0224] The term “selectivity” refers to the preference of a subject protein, or polypeptide, for specific binding of one substrate, such as one binding partner, compared to specific binding for another substrate, such as a different binding partner of the subject protein. Selectivity can be reflected as a ratio of the binding activity (e.g. binding affinity) of a subject protein and a first substrate, such as a first binding partner, (e.g., Kai) and the binding activity (e.g. binding affinity) of the same subject protein with a second binding partner (e.g., Kd2). 2024204559   01 Jul 2024

[0225] The term “sequence identity” as used herein refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. “Sequence identity” is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, FASTA and TFASTA. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. In some cases, a percent sequence identity can be determined as the percentage of amino acid residues (or nucleotide residues) in a candidate sequence that are identical with the amino acid residues (or nucleotide residues) in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0226] The term “soluble” as used herein in reference to proteins, means that the protein is not a membrane protein. In some cases, a soluble protein contains only an extracellular domain of an IgSF family member or members, or a portion thereof containing an IgSF domain or domains or specific-binding fragments thereof, but does not contain a transmembrane domain. In some cases, solubility of a protein can be improved by linkage or attachment, directly or indirectly via a linker, to an Fc domain, which, in some cases, also can improve the stability and / or half-life of the protein. In some aspects, a soluble protein is an Fc fusion protein.

[0227] The term “specifically binds” as used herein means the ability of a protein, under specific binding conditions, to bind to a target protein such that its affinity or avidity is at least 10 times as great, but optionally 50, 100, 250 or 500 times as great, or even at least 1000 times as great as the average affinity or avidity of the same protein to a collection of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind exclusively to a single target molecule but may specifically bind to more than one target 2024204559   01 Jul 2024 molecule. In some cases, a specifically binding protein may bind to a protein that has similarity in structural conformation with the target protein (e.g., paralogs or orthologs). Those of skill will recognize that specific binding to a molecule having the same function in a different species of animal (i.e., ortholog) or to a molecule having a substantially similar epitope as the target molecule (e.g., paralog) is possible and does not detract from the specificity of binding which is determined relative to a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, an immunomodulatory protein of the invention, or an ARBM or IRBM thereof, may specifically bind to more than one distinct species of target molecule due to crossreactivity. Solid-phase ELISA immunoassays, ForteBio Octet or Biacore measurements can be used to determine specific binding between two proteins. Generally, interactions between two binding proteins have dissociation constants (Kd) less than about IxlO'5 M, and often as low as -12 about 1 x 10' M. In certain aspects of the present disclosure, interactions between two binding proteins have dissociation constants of less than about IxlO'6 M, IxlO'7 M, 1x10'8M, IxlO'9 M, IxlO'10 M, or IxlO'11 M or less.

[0228] The term “specific binding fragment” or “fragment” as used herein in reference to a protein means a polypeptide that is shorter than a full-length protein or a specific domain or region thereof and that specifically binds in vitro and / or in vivo to a binding partner of the fulllength protein or of the specific domain or region. In some cases, a specific binding fragment is in reference to a fragment of a full-length IgSF family member or a full-length IgSF domain thereof (e.g. IgV or IgC), but that still binds to a binding partner of the IgSF family member or of an IgSF domain of an IgSF family member. In some embodiments, the specific binding fragment is at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% the sequence length of the full-length sequence or of a domain or region of an IgSF family member. In some embodiments, the specific binding fragment can have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. The specific binding fragment can be altered in sequence to form an affinity modified IgSF domain.

[0229] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

[0230] The terms “surface expresses” or “surface expression” in reference to a mammalian cell expressing a polypeptide means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein. 2024204559   01 Jul 2024

[0231] As used herein, “synthetic,” with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0232] The term “trans” with reference to binding to cell surface molecules refers to binding to two different cell surface molecules, each of which is present on the surface of a different cell. In some embodiments, trans means that with respect to two diffemt cell surface molecules, the first is exclusively present on one of the two mammalian cells forming the IS and the second is present exclusively on the second of the two mammalian cells forming the IS.

[0233] The term “transmembrane protein” as used herein means a membrane protein that substantially or completely spans a lipid bilayer such as those lipid bilayers found in a biological membrane such as a mammalian cell, or in an artificial construct such as a liposome. The transmembrane protein comprises a transmembrane domain (“transmembrane domain”) by which it is integrated into the lipid bilayer and by which the integration is thermodynamically stable under physiological conditions. Transmembrane domains are generally predictable from their amino acid sequence via any number of commercially available bioinformatics software applications on the basis of their elevated hydrophobicity relative to regions of the protein that interact with aqueous environments (e.g., cytosol, extracellular fluid). A transmembrane domain is often a hydrophobic alpha helix that spans the membrane. A transmembrane protein can pass through the both layers of the lipid bilayer once or multiple times.

[0234] The terms “treating,” “treatment,” or “therapy” of a disease or disorder as used herein mean slowing, stopping or reversing the disease or disorders progression, as evidenced by decreasing, cessation or elimination of either clinical or diagnostic symptoms, by administration of an immunomodulatory protein or engineered cells of the present invention either alone or in combination with another compound as described herein. “Treating,” “treatment,” or “therapy” also means a decrease in the severity of symptoms in an acute or chronic disease or disorder or a decrease in the relapse rate as for example in the case of a relapsing or remitting autoimmune disease course or a decrease in inflammation in the case of an inflammatory aspect of an autoimmune disease. “Preventing,” “prophylaxis,” or “prevention” of a disease or disorder as used in the context of this invention refers to the administration of an immunomodulatory protein or engineered cells expressing an immunomodulatory protein of the present invention, either alone or in combination with another compound, to prevent the occurrence or onset of a 2024204559   01 Jul 2024 disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.

[0235] The term “variant” (also “modified” or mutant,” which can be used interchangeably) as used in reference to a variant protein or polypeptide, such as a variant of an IgSF family member or IgSF domain thereof, means a protein, such as a mammalian (e.g., human or murine) protein created by human intervention. The variant is a polypeptide having an altered or modified amino acid sequence, such as by one or more amino acid substitutions, deletions, additions or combinations thereof, relative to an unmodified or wild-type protein or to a domain (e.g. IgSF domain, such as an IgV domain) thereof. A variant polypeptide can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions. A variant polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding form of a wildtype or unmodified protein (e.g. an IgSF family member), such as a mature sequence thereof (lacking the signal sequence) or a portion thereof containing the extracellular domain or an IgSF domain thereof. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. A variant protein is not limited to any particular method of making and includes, for example, chemical synthesis, recombinant DNA techniques, or combinations thereof. A variant protein, such as a variant IgSF domain, of the invention specifically binds to at least one or more binding partners. In some embodiments, the altered amino acid sequence results in an altered (i.e., increased or decreased) binding activity, such as binding affinity or avidity, to the one or more binding partners.

[0236] The term “wild-type” or “natural” or “native,” which are used interchangeably, as used herein is used in connection with biological materials such as nucleic acid molecules, proteins, IgSF members, host cells, and the like, refers to those which are found in nature and not modified by human intervention I. MULTI-DOMAIN IMMUNOMODULATORY PROTEINS

[0237] Provided herein are multi-domain immunomodulatory proteins that contain one or more inhibitory receptor binding molecule (IRBM) that binds to an inhibitory receptor and one or more activating receptor binding molecule (ARBM) that binds to an activating receptor or a 2024204559   01 Jul 2024 ligand of an activating receptor. In some embodiments, the activating receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM) or interacts with an adaptor protein involved in signal transduction pathways in an immune cell, such as a T cell, to transduce activation signals. In some embodiments, the inhibitory receptor comprises an immunoreceptor tyrosine-based inhibitory motif (fTIM). In some embodiments, the inhibitory receptor and the activating receptor are expressed on the same immune cell. In some embodiments, the activating receptor and inhibitory receptor are both expressed on a T cell.

[0238] In some embodiments, the provided multi-domain immunomodulatory proteins can be generated in a cis binding strategy to target an inhibitory receptor and activating receptor on the same immune cell, such as the same T cell, which, in some cases, proximalize or cluster the inhibitory receptor and the activating receptor on the surface of the same cell. In such embodiments, the IRBM binds to the inhibitory receptor and the ARBM binds to the activating receptor.

[0239] In other embodiments, the provided multi-domain immunomodulatory proteins can be generated in a trans binding strategy to target an inhibitory receptor and a ligand of an activating receptor, each present on different immune cells in an immune synapse. For example, in some embodiments, the IRBM binds to the inhibitory receptor on a T cell and the ARBM binds to a ligand of the activating receptor on an antigen-presenting cell (APC). In the trans strategy, localization of the ligand on the APC during immune synapse formation may be sufficient for signaling by its cognate activating receptor, such that the presence of the immunomodulatory protein could antagonize such as a signal and instead present an inhibitory ligand to recruit the inhibitory receptor to the immune synapse.

[0240] In some embodiments, the ARBM can be any binding molecule that binds to an activating receptor or a ligand thereof. In some embodiments, the IRBM can be any binding molecule that binds to an inhibitory receptor. In some embodiments, the one or more IRBM and / or ARBM independently include an antibody or an antigen-binding antibody fragment. In some aspects, the IRBM and / or ARBM can be a human antibody and / or an antibody that binds a human protein.

[0241] In some embodiments, at least one of the IRBM or ARBM is not an antibody or antigen-binding fragment. In some embodiments, at least one of the IRBM or ARBM is or contains a non-antibody immunoglobulin superfamily (IgSF) domain (IgD) of an IgSF member, or is a specific binding fragment of such an IgSF domain. In some embodiments, the at least 2024204559   01 Jul 2024 one of the IRBM or ARBM can be a variant IgD (hereinafter called “vlgD”) in which is contained one or more amino acid modifications (e.g. substitutions) in an IgD. In some aspects, the vlgD is an affinity-modified domain that exhibits increased binding activity, such as increased binding affinity, for the activating receptor (or ligand of the activating receptor) or inhibitory receptor compared to the binding activity of the unmodified or wild-type IgD for the same molecule. In some embodiment, both the IRBM and ARBM contain one or more IgD or vlgD of an IgSF member, in which, typically, the IgD or vlgD of the IRBM and ARBM are from, or derived from, a different IgSF member.

[0242] In some embodiments, the multi-domain immunomodulatory protein provided herein are soluble proteins and / or do not contain a portion that includes a transmembrane domain. Those of skill will appreciate that cell surface proteins, including proteins of the IgSF, typically have an intracellular domain, a transmembrane domain, and extracellular domain (ECD), and that a soluble form of such proteins can be made using the extracellular domain or an immunologically active subsequence thereof. Thus, in some embodiments, the ARBM or IRBM lacks a transmembrane domain or a portion of the transmembrane domain of an IgSF member. In some embodiments, the ARBM or IRBM lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain of an IgSF member. In some embodiments, the ARBM or IRBM only contains the ECD domain or a portion thereof containing an IgSF domain, such an IgV domain, or specific binding fragments thereof. In some cases, the ARBM and IRBM independently can include the extracellular domain of an IgSF family member or an IgSF domain or specific binding fragment thereof of an IgSF family member. In some aspects, the IgSF domain is an IgV domain or an IgC domain. In some aspects, the IgSF domain is an IgV domain or an IgC domain. In some aspects, the IRBM and / or ARBM is an IgSF domain of a IgSF family member that is a human protein and / or binds a human protein.

[0243] In some embodiments, the ARBM can bind to an activating receptor with at least a certain binding activity, such as binding affinity, as measured by any of a number of known methods. In some embodiments, the IRBM can bind to an inhibitory receptor with at least a certain binding activity, such as binding affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by an equilibrium dissociation constant (Kd) or is represented by EC50. A variety of assays are known for assessing binding activity, including binding affinity, and / or determining whether a binding molecule (e.g., an ARBM or IRBM) specifically binds to a particular binding partner. In some embodiments, a 2024204559   01 Jul 2024 BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detection of expressed nucleic acids or binding of proteins.

[0244] In some embodiments, the ARBM and IRBM independently exhibit a binding affinity for a binding partner with a Kd (i.e., an equilibrium dissociation constant of a particular binding interaction with units of M; equal to the ratio of the off-rate [kOff or kd] to the on-rate [kon or ka] for this association reaction, assuming bimolecular interaction) of equal to or less than 10’5 M. For example, the equilibrium dissociation constant KD ranges from 10’6 M to 10’12 M, such as 10'7 M to 10’11 M, 10’8 M to 10’10 M, or 10’9 M to 10’10 M. The on-rate (association rate constant; kon or ka; units of 1 / Ms) and the off-rate (dissociation rate constant; kOff or ka; units of 1 / s) can be determined using any of the assay methods known in the art, for example, surface plasmon resonance (SPR).

[0245] In some embodiments, the IRBM exhibits a binding affinity for an inhibitory receptor that is from or from about 0.001 nM to 1000 nM, such as from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 50 nM, 2024204559   01 Jul 2024 from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM. In certain embodiments, the binding affinity of the IRBM for the inhibitory receptor is at or less than or about 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less.

[0246] In some embodiments, the ARBM exhibits a binding affinity for an activating receptor or a ligand of an activating receptor that is from or from about 0.001 nM to about 1000 nM, such as from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 50 nM, from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM. In certain embodiments, the binding affinity of the ARBM for the activating receptor or a ligand of the activating receptor is at or less than or about 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less.

[0247] In some embodiments, the IRBM exhibits higher affinity for the inhibitory receptor than the ARBM exhibits for the activating receptor or a ligand of the activating receptor. For example, in some embodiments, the IRBM exhibits 1.2-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0 fold or higher affinity for the inhibitory receptor than the ARBM exhibits for the activating receptor or a ligand of the activating receptor.

[0248] In some embodiments, the provided multi-domain immunomodulatory proteins can include the ARBM and IRBM in various configurations or formats, including formats with one or more further moieties. In some embodiments, the provided immunomodulatory proteins 2024204559   01 Jul 2024 include polypeptides in which the one or more IRBM is N-terminal to the ARBM. In some embodiments, the one or more IRBM is C-terminal to the ARBM. The one or more ARBM and the one or more IRBM can be linked directly or indirectly, via a linker. In some embodiments, the immunomodulatory proteins can be formatted as multimeric molecules via fusion with a multimerization domain, such as an Fc protein. In some embodiments, the multi-domain immunomodulatory proteins can be formatted as multimeric molecules, e.g., dimeric, trimer, tetrameric, or pentameric molecules. In some embodiments, the immunomodulatory proteins are formatted as a monomeric molecules containing single polypeptide fusions of the one or more ARBM and the one or more IRBM. In some aspects, the configurations can be chosen to effect spatial proximalization of ITIM bearing inhibitory receptors with activating receptors on immune cells, such as T cells, to make possible attenuation of T cell activation and / or tolerance induction.

[0249] In the subsections below, exemplary ARBM and IRBM components of the provided multi-domain immunomodulatory protein are described, as are exemplary formats for such immunomodulatory proteins. A. Inhibitory Receptor Binding Molecule (IRBM)

[0250] In some embodiments, the provided immunomodulatory polypeptides contain an IRBM that binds to an inhibitory receptor. In some aspects, the inhibitory receptor includes an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory receptor target of the IRBM is CTLA-4, PD-1, or TIGIT, including any mammalian orthologs thereof. In some embodiments, the inhibitory receptor target is a human CTLA-4, human PD-1 or human TIGIT. In some embodiments, the inhibitory receptor is expressed on a T cell, such as a human T cell.

[0251] In some embodiments, the IRBM is an antibody or antigen-binding fragment that binds an inhibitory receptor. In some embodiments, the IRBM is an antibody or antigen-binding fragment that binds CTLA-4, PD-1 or TIGIT, such as a human CTLA-4, human PD-1 or human TIGIT.

[0252] In some embodiments, the IRBM is or contains a binding partner of an inhibitory receptor. For example, in some aspects, the IRBM is or contains an IgD of an IgSF family member that binds to an inhibitory receptor, such as a PD-1, TIGIT or CTLA-4 inhibitory receptor, or is a specific fragment or vlgD thereof that binds to the inhibitory receptor. Exemplary IgSF family members that are binding partners of or that bind to a PD-1 inhibitory 2024204559   01 Jul 2024 receptor include, for example, PD-L1 and PD-L2, such as human PD-L1 or human PD-L2. Exemplary IgSF family members that are binding partners of or that bind to a TIGIT inhibitory receptor include, for example, CD155 or CD112, such as human CD155 or human CD112. In some embodiments, the IgSF binding partner of an inhibitory receptor is a molecule set forth in Table 1. In some examples, the IRBM is or contains an IgD of a wild-type PD-L1 or PD-L2 or is or contains a vlgD thereof, wherein the IRBM specifically binds to PD-1. In other examples, the IRBM is or contains an IgD of CD155 or CD112 or is or contains a vlgD thereof, wherein the IRBM specifically binds to TIGIT.

[0253] The first column of Table 1 provides the name and, optionally, the name of some possible synonyms for that particular domain. The second column provides the protein identifier of the UniProtKB database, a publicly available database accessible via the internet at uniprot.org. The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The UniProt databases include the UniProt Knowledgebase (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics and the Protein Information Resource (PIR) and supported mainly by a grant from the U.S. National Institutes of Health (NIH). The third column provides the region where the indicated IgSF domain is located. The region is specified as a range where the domain is inclusive of the residues defining the range. Column 3 also indicates the domain class for the specified region. Column 4 provides the region where the indicated additional domains are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). Column 5 indicates for some of the listed IgSF members, some of its cognate cell surface binding partners. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization, such as of an IgSF domain, are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It is understood that polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar molecules. Thus, the exact locus can vary, and is not necessarily the same for each protein. Hence, the specific IgSF domain, such as specific IgV domain or IgC domain, can be several amino acids (such as one, two, three, four, five, six or more amino acids) longer or shorter. 2024204559   01 Jul 2024 TABLE 1. Exemplary IgSF as Inhibitory Receptor Binding Molecules (IRBM) Receptorbinding Domains (Synony ms) NCBI Protein Accession Number / UniProtKB Protein Identifier Region & Domain Class Other Domains Cognate Cell Surface Binding Partners IgSF Member Amino Acid Sequence (SEQ ID NO) Precursor (mature residues) Mature ECD CD274 (PD-L1, B7-H1) Q9NZQ7.1 24-130 IgV, 133-225 IgC2 S: 1-18, E: 19-238, T: 239-259, C: 260-290 PD-1, B7-1 SEQ ID NO: 3 (19-290) SEQ ID NO: 383 SEQ ID NO: 30 PDCD1L G2 (PD-L2, CD273) Q9BQ51.2 21-118 IgV, 122-203 IgC2 S: 1-19, E: 20-220, T: 221-241, C: 242-273 PD-1, RGMb SEQ ID NO: 4 (20-273) SEQ ID NO: 384 SEQ ID NO: 31 PVR (CD155) P15151.2 24-139 IgV, 145-237 IgC2, 244328 IgC2 S: 1-20, E: 21-343, T: 344-367, C: 368-417 TIGIT, CD226, CD96, poliovirus SEQ ID NO: 20 (21-417) SEQ ID NO: 208 SEQ ID NO: 47 PVRL2 (CD 112) Q92692.1 32-156 IgV, 162-256 IgC2, 261345 IgC2 S: 1-31, E: 32-360, T: 361-381, C: 382-538 TIGIT, CD226, CD112R SEQ ID NO: 21 (32-538) SEQ ID NO: 209 SEQ ID NO: 48

[0254] In some embodiments, the IRBM is or contains a wild-type or unmodified IgD of a binding partner of an inhibitory receptor, such as a sequence that is or contains an ECD or an IgD domain or domains of a native binding partner of an inhibitory receptor or an ortholog thereof. In some embodiments, the IRBM is or comprises the extracellular domain (ECD), or a portion thereof containing one or more IgSF domains, of an IgSF member set forth in Table 1, e.g. human PD-L1, human PD-L2, human CD 155 or human CD112. In some embodiments, the extracellular domain comprises an IgV domain or domains and, in some cases, an IgC (e.g. IgCl and / or IgC2) domain or domains. In some embodiments, the IRBM is less than the full length sequence of the IgSF binding partner of the inhibitory receptor. For example, in some aspects, the IRBM is or only contains the extracellular domain (ECD) or specific binding fragment thereof of the binding partner. In some embodiments, the IRBM is or only contains the IgV domain or the IgC domain or specific binding fragment of the IgV domain or the IgC domain, or combinations thereof. In some embodiments, the IRBM can be an IgV only, the combination of the IgV and IgC, including the entire extracellular domain (ECD), or any combination of Ig domains. In some embodiments, the IRBM consists or consists essentially of the ECD or an IgD domain or domain thereof of a binding partner of an inhibitory receptor, such as consists or 2024204559   01 Jul 2024 consists essentially of the ECD, IgV or IgC domain or domains. In some embodiments, the sequence of the IRBM containing an IgD of a binding partner of an inhibitory receptor is a mammalian sequence that includes, but is not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the sequence of IRBM containing an IgD is human. Table 1 provides exemplary residues that correspond to ECD, IgV, or IgC regions of various IgSF domains of binding partners of exemplary inhibitory receptors.

[0255] In some embodiments, the IRBM is or contains a vlgD that contains one or more amino acids modifications, such as one or more substitutions (alternatively, “mutations” or “replacements”), deletions or additions, in an IgD relative to a wild-type or unmodified IgD of a binding partner of the inhibitory receptor. In some aspects, the vlgD contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, such as amino acid substitutions, deletions or additions in an IgD domain of an IgSF binding partner of an inhibitory receptor, e.g. in an IgD domain of a binding partner set forth in Table 1. The modifications (e.g., substitutions) can be in the IgV domain or the IgC domain. In some embodiments, the vlgD has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgV domain or specific binding fragment thereof. In some embodiments, the vlgD has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgC domain or specific binding fragment thereof. In some embodiments, the vlgD has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type or unmodified IgD or specific binding fragment thereof.

[0256] In some embodiments, the IRBM is a variant of a binding partner of an inhibitory receptor (e.g. variant of a molecule set forth in Table 1, e.g. human PD-L1, human PD-L2, human CD155 or human CD112), in which the IRBM is or comprises an ECD, or portion thereof, containing one or more vlgD of an IgSF binding partner of an inhibitory receptor. In some embodiments, the IRBM can comprise an IgV domain or an IgC domain or domains, or a specific binding fragment of the IgV domain or a specific binding fragment of the IgC domain or domains in which one or more of the IgSF domains (IgV or IgC) contains the one or more amino acid modifications (e.g. substitutions). In some embodiments, the IRBM can comprise an IgV domain and an IgC domain or domains, or a specific binding fragment of the IgV domain and a specific binding fragment of the IgC domain or domains, in which at least one of the IgV or IgC domain contains the amino acid modifications (e.g. substitutions). In some embodiments, 2024204559   01 Jul 2024 the IRBM consists or consists essentially of the ECD containing a vlgD (e.g. IgV and / or IgC). In some embodiments, the IRBM contains only, such as consists or consists essentially of, an IgV domain or a specific binding fragment of the IgV domain, in which the one or more amino acid modifications (e.g. substitutions) are located in the IgV domain or specific binding fragment of the IgV domain. In some embodiments, the IRBM contains only, such as consists or consists essentially of, an IgC domain or a specific binding fragment of the IgC domain, in which the one or more amino acid modifications (e.g. substitutions) are located in an IgC domain or specific binding fragment of an IgC domain.

[0257] In some embodiments, the one or more amino acid modifications alter, such as increase, the binding activity, e.g. binding affinity, of the extracellular domain of the binding partner or an IgD domain thereof (e.g. IgV) for its cognate inhibitory receptor. In some embodiments, by virtue of the altered binding activity, such as binding affinity, the vlgD domain is an affinity-modified IgSF domain. Typically, the affinity-modified IgSF domain used in or as the IRBM is a human or murine affinity modified IgSF domain.

[0258] In some embodiments, an IRBM containing a vlgD has a binding activity, such as binding affinity, for the inhibitory receptor that is altered, e.g. increased, from that of an IRBM containing a wild-type or unmodified IgD sequence as determined by, for example, solid-phase ELISA immunoassays, flow cytometry or surface plasmon resonance (Biacore) assays. In some embodiments, the vlgD results in an increased binding activity, such as binding affinity, for the inhibitory receptor, relative to a wild-type or unmodified IgD. In some embodiments, the increase in binding activity, such as binding affinity, for the inhibitory receptor is at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, 40%, 50%, 60%, 70%, 90%, 100%, 200% or more. In some embodiments, the increase in binding activity, such as binding affinity, is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10fold, 20-fold, 30-fold 40-fold or 50-fold. In such examples, the wild-type or unmodified IgD has the same sequence as the vlgD except that it does not contain the one or more amino acid modifications (e.g. substitutions). In some embodiments, the equilibrium dissociation constant (Ka) of the IRBM to the inhibitory receptor can be less than IxlO-5 M, IxlO-6 M, IxlO-7 M, IxlO-8 M, IxlO-9 M, IxlO-10 M or lxl0-11M, or IxlO-12 M or less.

[0259] The use of the term “modification”, such as “substitution” does not imply that the present embodiments are limited to a particular method of making the immunomodulatory proteins. An IRBM that is or contains IgD or vlgDs can be made, for example, by de novo 2024204559   01 Jul 2024 peptide synthesis and thus does not necessarily require a modification, such as a “substitution” in the sense of altering a codon to encode for the modification, e.g. substitution. This principle also extends to the terms “addition” and “deletion” of an amino acid residue which likewise do not imply a particular method of making. The means by which the vlgDs are designed or created is not limited to any particular method. In some embodiments, however, a wild-type or unmodified IgD encoding nucleic acid is mutagenized from wild-type or unmodified IgD genetic material and screened for desired specific binding activity, such as binding affinity, and / or alteration of IFN-gamma expression or other functional activity. In some embodiments, a vlgD is synthesized de novo utilizing protein or nucleic acid sequences available at any number of publicly available databases and then subsequently screened. The National Center for Biotechnology Information provides such information and its website is publicly accessible via the internet as is the UniProtKB database.

[0260] In some embodiments, the IRBM has (i) the sequence of amino acids set forth in SEQ ID NO: 3, 4, 20, 21 or a mature form thereof lacking the signal sequence, (ii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3, 4, 20 or 21 or the mature form thereof, or (iii) a portion of (i) or (ii) containing the extracellular domain or an IgV domain or IgC domain or specific binding fragments thereof. In some embodiments, the IRBM has (i) the sequence of amino acids (i) set forth in SEQ ID NO: 208, 209, 383 or 384, (ii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 208, 209, 383 or 384; or (iii) a portion of (i) or (ii) containing the extracellular domain or an IgV domain or IgC domain or specific binding fragment thereof.

[0261] In some embodiments, the IRBM is or comprises an extracellular domain or a portion thereof of an IgSF member that is a binding partner of an inhibitory receptor (e.g. Table 1). In some embodiments, the IRBM has or comprises the amino acid sequence set forth in SEQ ID NO: 30, 31, 47, 48, or an ortholog thereof. In some cases, the IRBM has or comprises (i) the sequence of amino acids set forth in SEQ ID NO: 30, 31, 47, 48, (ii) a sequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30, 31, 47, 48, or (iii) is a specific binding fragment of the sequence of (i) or (ii) comprising an IgV domain or an IgC domain. 2024204559   01 Jul 2024

[0262] In some embodiments, the IRBM is or comprises an IgV domain or an IgC domain or domains, or a specific binding fragment thereof. In some embodiments, the IRBM has or comprises the amino acid sequence set forth in SEQ ID NO: 55, 309, 310, 353, 666, 761, 1203, 1263, or an ortholog thereof. For example, the IRBM has or comprises (i) the sequence of amino acids set forth in SEQ ID NO: 55, 309, 310, 353, 666, 761, 1203 or 1263, (ii) a sequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 55, 309, 310, 353, 666, 761, 1203 or 1263, or (iii) a specific binding fragment of the sequence of (i) or (ii). / . PD-1 Binding Molecule

[0263] Provided are immunomodulatory proteins containing an IRBM that is or contains a binding molecule that binds to PD-1, such as to human PD-1. Programmed death 1 (PD-1) receptor or PD-1 is expressed on NK cells and T cells, including CD4+ and CD8+ T cells, whereby engagement of PD-1 can inhibit activation cell activation, proliferation, and / or expansion. In some embodiments, the IRBM of the immunomodulatory protein binds to the ectodomain of PD-1. In some embodiments, the IRBM binds to PD-1 on the surface of a cell, such as on the surface of a T cell.

[0264] In some embodiments, the provided immunomodulatory protein contains an IRBM that is or contains an antibody that binds PD-1 or is an antigen-binding antibody fragment thereof (e.g. Fab or scFv). In some embodiments, the antibody or antigen-binding antibody fragment binds human PD-1. For example, in some embodiments, the antibody is nivolumab, pembrolizumab, pidilizumab or BMS-936559 or an antigen binding fragment containing a VH chain region and / or VL chain region of nivolumab, pembrolizumab, pidilizumab or BMS-936559. In some embodiments, the antibody is a single chain variable fragment (e.g. scFv) containing a VH and VL of nivolumab, pembrolizumab, pidilizumab or BMS-936559.

[0265] In some embodiments, the IRBM is or contains one or more IgD (e.g. IgV or IgC) or a specific binding fragment thereof, such as an unmodified or wild-type IgD or a vlgD or a specific binding fragment thereof, of an IgSF family member that binds PD-1. In some embodiments, the PD-1 is human PD-1. PD-1 is the T-cell costimulatory receptor for the ligands PD-L1 (also known as cluster of differentiation 274, CD274. B7 homolog 1 or B7-H1) and PD-L2 (also known as PDCD1L2, PDCD1LG2, cluster of differentiation 273, CD273. or B7-DC). PD-L1 and PD-L2 are normally expressed on the surface of T cells, B cells, and myeloid cells. 2024204559   01 Jul 2024 PD-Ll annd PD-L2 are negative regulators of immune activation and are capable of downmodulating the immune response via interactions with PD-1.

[0266] In some embodiment, the IRBM is or contains one or more IgD (e.g. IgV or IgC) that is an IgD of PD-Ll or PD-L2 polypeptide, such as a wild-type PD-Ll or PD-L2, e.g. a human PD-Ll or human PD-L2. In some aspects, the IRBM contains one or more IgD (e.g. IgV or IgC) that is an vlgD containing one or more amino acid modifications (e.g., substitutions, deletions or additions) compared to an IgD of a wild-type or unmodified PD-Ll or PD-L2, which, in some aspects, result in increased binding of the IRBM to PD-1. Exemplary IgDs or vlgDs of PD-Ll or PD-L2 binding partners for inclusion as an IRBM in the provided immunomodulatory proteins are described. In some embodiments, the IRBM is or contains a vlgD polypeptide that exhibit increased binding activity , such as binding affinity, for PD-1 compared to a corresponding wild-type or unmodified IgD. a. PD-Ll IgD or vlgD

[0267] Provided herein are immunomodulatory proteins containing an IRBM that is or contains one or more IgD, such as an unmodified or wild-type IgD or a vlgD, of PD-LL In some embodiments, the IRBM is or contains one or more IgD domain of a wild-type or unmodified PD-Ll, such as a mammalian PD-Ll, e.g. a human PD-LL In some embodiments, the IRBM is not the full length sequence of the PD-LL In some aspects, the IRBM is a soluble polypeptide, is not membrane-expressed and / or lacks the transmembrane and / or cytoplasmic domain of PD-LL In some embodiments, the IRBM only contains an extracellular domain (ECD) or a specific binding fragment thereof containing a IgD or vlgD, such as only contains an IgV domain or an IgC domain or specific binding fragment thereof, or combinations thereof.

[0268] In some embodiments, the IRBM is or contains the ECD sequence set forth in SEQ ID NO:30 or 1728 or is a specific binding fragment thereof. In some embodiments, the IRBM is or contains the IgV sequence set forth in SEQ ID NO: 309 (containing residues 1-114 of SEQ ID NO:30) or set forth in SEQ ID NO:55, or is a specific binding fragment thereof. FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHS SYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQ RILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTT TNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO:30) 2024204559   01 Jul 2024 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHS SYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQ RILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTT TNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT (SEQ ID NO: 1728) FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV HGEEDLKVQHSSYRQRARLL KDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKVNA (SEQ ID NO:309) PKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQR ARLL KDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV (SEQ ID NO:55)

[0269] In some embodiments, the immunomodulatory protein contains an IRBM that is or contains a vlgD containing one or more amino acid modifications, e.g. substitutions, in an IgD of a wild-type or unmodified PD-L1. In some embodiments, modifications provided herein can be in an IRBM containing an unmodified IgD set forth in SEQ ID NO:30, 55, 309 or 1728 or in a sequence that has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 30, 55, 309 or 1728. In some embodiments, an IRBM containing a vlgD of PD-L1 has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in any of SEQ ID NOs: 30, 1728, 55 or 309.

[0270] In some embodiments, the vlgD is an affinity-modified IgSF domain that has an increased binding activity, such as binding affinity, for PD-1 relative to the binding activity of the wild-type or unmodified IgD for PD-1. In some embodiments, the increase in binding activity, e.g. binding affinity, for PD-1 is increased at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 200% or more. In some embodiments, the increase in binding activity, e.g. binding affinity, is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold, or 50-fold. In such examples, the wild-type or unmodified IgD has the same sequence as the vlgD except that it does not contain the one or more amino acid modifications (e.g. substitutions). In some embodiments, the equilibrium dissociation constant (Ka) of the IRBM to PD-1 can be less than IxlO'5 M, IxlO'6 M, IxlO'7 M, IxlO'8 M, IxlO'9 M, IxlO'10 M or lxl0'nM, or IxlO12 M or less. 2024204559   01 Jul 2024

[0271] Unless stated otherwise, the amino acid modification(s) present in a vlgD of a PD-L1 ECD or an IgD (e.g. IgV) thereof are designated by amino acid position number corresponding to the numbering of positions of the unmodified ECD sequence set forth in SEQ ID NO:30 or 1728 or, where applicable, the unmodified IgV sequence set forth in SEQ ID NO: 309 (containing residues 1-114, respectively, of SEQ ID NO:30). It is within the level of a skilled artisan to identify the corresponding position of a modification, e.g. amino acid substitution, in an ECD or a portion thereof containing an IgSF domain (e.g. IgV) thereof, such as by alignment of a reference sequence with SEQ ID NOs: 30, 309 or 1728. In the listing of modifications throughout this disclosure, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution listed after the number. If the modification is a deletion of the position a “del” is indicated and if the modification is an insertion at the position an “ins” is indicated. In some cases, an insertion is listed with the amino acid position indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before and after the number and the identified variant amino acid insertion listed after the unmodified (e.g. wild-type) amino acid.

[0272] In some embodiments, the IRBM contains a vlgD that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions. The one or more amino acid modifications, e.g. substitutions, can be in the ectodomain (extracellular domain) of the wild-type or unmodified PD-L1. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the ECD domain of PD-L1 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of PD-L1 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in an IgC domain of PD-L1 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of PD-L1 or a specific binding fragment thereof and in an IgC domain or domains of PD-L1 or a specific binding fragment thereof.

[0273] In some embodiments, the IRBM is or contains a vlgD that has one or more amino acid modifications, e.g. substitutions, in an unmodified IgD of PD-L1 or a specific binding fragment thereof corresponding to position(s) 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 2024204559   01 Jul 2024 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 213, or 221, with reference to positions set forth in SEQ ID NO: 30 or 1728. In some embodiments, a polypeptide containing the vlgD exhibits altered, such as increased, binding activity, e.g. binding affinity, to PD-1 compared to a polypeptide containing the wild-type or unmodified PD-L1 IgD.

[0274] In some embodiments, the IRBM is or contains a vlgD of PD-L1 that has one or more amino acid modification selected from P6S, Y10F, Vil A, VI IE, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, GlOlG-ins (G101GG), G102D, A103V, D104G, K106E, K106R, V110M,, KI HE, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171R, F173I, F173L, K173Y, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, OR T221L or a conservative amino acid substitution thereof.

[0275] In some embodiments, the IRBM is or contains a vlgD that has two or more amino acid modifications selected from P6S, Y10F, Vil A, VI IE, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, TI9A. T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, GlOlG-ins (G101GG), G102D, A103V, D104G, K106E, K106R, V110M, KI HE, K111T, V112A, N113Y, N117S, I119T, 2024204559   01 Jul 2024 N120S, Q121L, L124S, V129A, V129D, T130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171R, F173I, F173L, K173Y, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, or T221L.

[0276] In some embodiments, the modification(s), e.g. substitutions(s), can be K28N / M41V / N45T / H51N / K57E, I20L / I36T / N45D / I47T, I20L / M41K / K44E, P6S / N45T / N78PI83T, N78I, M41K / N78I, N45T / N78I, I20L / N45T, N45T, M41K, I20L / I36T / N45D, N17D / N45T / V50A / D72G, I20L / F49S, N45T / V50A, I20L / N45T / N78I, I20L / N45T / V50A, M41V / N45T, M41K / N45T, A33D / S75P / D85E, M18I / M41K / D43G / H51R / N78I, V11E / I20L / I36T / N45D / H60R / S75P, A33D / V50A, S16G / A33D / K71E / S75P, E27G / N45T / M97I, E27G / N45T / K57R, A33D / E53V, D43G / N45D / V58A, E40G / D43V / N45T / V50A, Y14S / K28E / N45T A33D / N78S, A33D / N78I, A33D / N45T, A33D / N45T / N78I, E27G / N45T / V50A, N45T / V50A / N78S, I20L / N45T / V110M, I20L / I36T / N45T / V50A, N45T / L74P / S75P, N45T / S75P, S75P / K106R, S75P, A33D / S75P, A33D / S75P / D104G, A33D / S75P, I20L / E27G / N45T / V50A, I20L / E27G / D43G / N45D / V58A / N78I, I20L / D43G / N45D / V58A / N78I, I20L / A33D / D43G / N45D / V58A / N78I, I20L / D43G / N45D / N78I, E27G / N45T / V50A / N78I, N45T / V50A / N78I, VI1A / I20L / E27G / D43G / N45D / H51Y / S99G, I20L / E27G / D43G / N45T / V50A , I20L / K28E / D43G / N45D / V58A / Q89R, I20L / I36T / N45D , I20L / K28E / D43G / N45D / E53G / V58A / N78I, A33D / D43G / N45D / V58A / S75P, K23R / D43G / N45D, I20L / D43G / N45D / V58A / N78I / D90G / G101D, D43G / N45D / L56Q / V58A / G101GG, I20L / K23E / D43G / N45D / V58A / N78I, I20L / K23E / D43G / N45D / V50A / N78I, T19I / E27G / N45FV50A / N78VM97K, I20L / M41K / D43G / N45D, K23R / N45T / N78I, I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins (G101GG), K57R / S99G, K57R / S99G / F189L, M18 V / M97L / F193S / R195G / E200K / H202Q, I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F, C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L, M18V / I98L / L124S / P198T / L206F, S99G / N117S / I148V / K171R / R180S, I36T / M97L / A103V / Q155H, K28FS99G, R195S, A79T / S99G / T185A / Rl 95G / E200K / H202Q / L206F, K57R / S99G / L124S / K144Q, K57R / S99G / R195G, D55V / M97L / S99G, E27G / I36T / D55N / M97L / K111E, E54G / M97L / S99G, G15A / I36T / M97L / K111E / H202Q, G15A / I36T / V129D, G15A / I36T / V129D / R195G, G15A / V129D, I36S / M97L, I36T / D55N / M97L / K111E / A204T, I36T / D55N / M97L / K111E / V129A / F173L, I36T / D55S / M97L / K111E / I148V / R180S, I36T / G52R / M97L / V112A / K144E / V175A / P198T, I36T / I46V / D55G / M97L / K106E / K144E / T185A / R195G, I36T / I83T / M97L / K144E / P198T, I36T / M97L / K11 IE, I36T / M97L / K144E / P198T, I36T / M97L / Q155H / F193S / N201Y, I36T / M97L / V129D, L35P / I36S / M97L / K11 IE, Ml 8I / I36T / E53G / M97L / K144E / E199G / V207A, 2024204559   01 Jul 2024 M18T / I36T / D55N / M97L / K111E, M18V / M97L / T176N / R195G, M97L / S99G, N17D / M97L / S99G, S99G / T185A / R195G / P198T, V129D / H202Q, V129D / P198T, V129D / T150A, V93E / V129D, Y10F / M18V / S99G / Q138R / T203A, N45D, K160M / R195G, N45D / K144E, N45D / P198S, N45D / P198T, N45D / R195G, N45D / R195S, N45D / S131F, N45D / V58D, V129D / R195S, I98T / F173Y / L196S, N45D / E134G / L213P, N45D / F173PS177C, N45D / I148V / R195G, N45D / K111T / R195G, N45D / N113Y / R195S, N45D / N165Y / E170G, N45D / Q89R / I98V, N45D / S131F / P198S, N45D / S75P / P198S, N45D / V50A / R195T, E27D / N45D / T183A / I188V, F173Y / T183PL196S / T2O3A, K23N / N45D / S75P / N120S, N45D / G102D / R194W / R195G, N45D / G52V / Q121L / P198S, N45D / I148V / R195G / N201D, N45D / K111T / T183A / I188V, N45D / Q89R / F189S / P198S, N45D / S99G / C137R / V207A, N45D / T163I / K167R / R195G, N45D / T183 A / T192S / R194G, N45D / V50A / I119T / K144E, T19A / N45D / K144E / R195G, VI1E / N45D / T130A / P198T, V26A / N45D / T163PT185A, K23N / N45D / L124S / K167T / R195G, K23N / N45D / Q73R / T163I, K28E / N45D / W149R / S158G / P198T, K28R / N45D / K57E / I98V / R195S, K28R / N45D / V129D / T163N / R195T, M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D, or N45D / V50A / L124S / K144E / L179P / R195G.

[0277] In some embodiments, the IRBM contains a vlgD that has one or more amino acid modification, e.g. substitutions, in an unmodified PD-L1 or specific binding fragment thereof corresponding to position(s) 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, 195, and / or 198 with reference to positions set forth in SEQ ID NO:36 or 2655. In some emboidments, the IRBM is or contains a vlgD of PD-L1 that has one or more amino acid modifications selected from I20L, E27D, E27G, A33D, I36S, I36T, D43G, D43V, N45D, N45I, N45T, V50A, V58A, V58D, S75P, N78I, N78S, M97I, M97K, M97L, S99G, R195G, R195S, R195T, P198S, and / or P198T, or a conservative amino acid substitution thereof.

[0278] In some embodiments, the IRBM is or contains an IgD (e.g. IgV) of wild-type PD-L1 set forth in Table 2 or a vlgD thereof comprising any of the modifications (e.g. substitutionss) listed in Table 2. Table 2 also provides exemplary sequences by reference to SEQ ID NO for IRBMs containing an ECD or IgV domain of PD-L1. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g. ECD or IgV) also can be included in a sequence of an IRBM, such as to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplification of the SEQ ID NOSs in Table 2 is not to be construed as limiting. For example, the particular 2024204559   01 Jul 2024 domain, such as the ECD or IgV domain, can be several amino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO.

[0279] In some embodiments, the IRBM is or contains a wild-type PD-L1 ECD set forth in SEQ ID NO:30 or 1728 or a variant ECD sequence set forth in any one of SEQ ID NOS: 56120, 1725, 1729-1818, 1819-1907, 1943-2008. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the ECD sequences set forth in any one of SEQ ID NOS: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 and contains the amino acid modification(s), e.g. substitution(s), not present in the wild-type or unmodified PD-L1 ECD, e.g. not present in SEQ ID NO:30 or 1728. In some embodiments, the IRBM is or contains a specific binding fragment of any of the ECD sequences set forth in any one of SEQ ID NOS: 56120, 1725, 1729-1818, 1819-1907, 1943-2008 and contains the amino acid modification(s), e.g. substitution(s), not present in a wild-type or unmodified PD-L1 ECD, e.g. not present in SEQ ID NO: 30 or 1728.

[0280] In some embodiments, the IRBM is or contains a wild-type PD-L1 IgV set forth in SEQ ID NO: 55 or 309 or a variant IgV sequence set forth in any one of SEQ ID NOS: 121-185, 244-308, 1726-1727, 1908-1937. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences set forth in any one of SEQ ID NOS: 121-185, 244-308, 1726-1727, 1908-1937 and contains the amino acid modification(s), e.g. substitution(s), not present in the wild-type or unmodified PD-L1, e.g. not present in SEQ ID NO:55 or 309. In some embodiments, the IRBM is or contains a specific binding fragment of any of the IgV sequences set forth in any one of SEQ ID NOS: 121-185, 244-308, 1726-1727, 1908-1937 and that contains the amino acid modification(s), e.g. substitution(s), not present in a wild-type or unmodified PD-L1 IgV, e.g. not present in SEQ ID NO:55 or 309. TABLE 2: Exemplary PD-L1 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO Wild-type 30, 1728 55, 309 K28N / M41V / N45T / H51N / K57E 56, 1943 121, 244 2024204559   01 Jul 2024 TABLE 2: Exemplary PD-L1 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO I20L / I36T / N45D / I47T 57, 1944 122, 245 I20L / M41K / K44E 58, 1945 123, 246 P6S / N45T / N78I / I83T 59, 1946 124, 247 N78I 60, 1947 125, 248 M41K / N78I 61, 1948 126, 249 N45T / N78I 62, 1949 127, 250 I20L / N45T 63, 1950 128,251 N45T 64, 1951 129, 252 M41K 65, 1952 130, 253 I20L / I36T / N45D 66, 1953 131,254 N17D / N45T / V50A / D72G 67, 1954 132, 255 I20L / F49S 68, 1955 133, 256 N45T / V50A 69, 1956 134, 257 I20L / N45T / N78I 70, 1957 135, 258 I20L / N45T / V50A 71, 1958 136, 259 M41V / N45T 72, 1959 137, 260 M41K / N45T 73, 1960 138,261 A33D / S75P / D85E 74, 1961 139, 262 M18I / M41K / D43G / H51R / N78I 75, 1962 140, 263 VI1E / I20L / I36T / N45D / H60R / S75P 76, 1963 141, 264 A33D / V50A 77, 1964 142, 265 S16G / A33D / K71E / S75P 78, 1965 143, 266 E27G / N45T / M97I 79, 1966 144, 267 E27G / N45T / K57R 80, 1967 145, 268 A33D / E53V 81, 1968 146, 269 D43G / N45D / V58A 82, 1969 147, 270 E40G / D43V / N45T / V50A 83, 1970 148, 271 Y14S / K28E / N45T 84, 1971 149, 272 A33D / N78S 85, 1972 150, 272 A33D / N78I 86, 1973 151,274 A33D / N45T 87, 1974 152, 275 A33D / N45T / N78I 88, 1975 153, 276 E27G / N45T / V50A 89, 1976 154, 277 N45T / V50A / N78S 90, 1977 155, 278 I20L / N45T / V110M 91, 1978 156, 279 I20L / I36T / N45T / V50A 92, 1979 157, 280 N45T / L74P / S75P 93, 1980 158,281 N45T / S75P 94, 1981 159, 282 S75P / K106R 95, 1982 160, 283 S75P 96, 1983 161,284 A33D / S75P 97, 1984 162, 285 A33D / S75P / D104G 98, 1985 163, 286 A33D / S75P 99, 1986 164, 287 2024204559   01 Jul 2024 TABLE 2: Exemplary PD-L1 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO I20L / E27G / N45T / V50A 100, 1987 165, 288 I20L / E27G / D43G / N45D / V58A / N78I 101, 1988 166, 289 I20L / D43G / N45D / V58A / N78I 102, 1989 167, 290 I20L / A33D / D43G / N45D / V58A / N78I 103, 1990 168, 291 I20L / D43G / N45D / N78I 104, 1991 169, 292 E27G / N45T / V50A / N78I 105, 1992 170, 293 N45T / V50A / N78I 106, 1993 171,294 V11A / I20L / E27G / D43G / N45D / H51Y / S99G 107, 1994 172, 295 I20L / E27G / D43G / N45T / V50A 108, 1995 173, 296 I20L / K28E / D43G / N45D / V58A / Q89R 109, 1996 174, 297 I20L / I36T / N45D 110, 1997 175, 298 I20L / K28E / D43G / N45D / E53G / V58A / N78I 111, 1998 176, 299 A33D / D43G / N45D / V58A / S75P 112, 1999 177, 300 K23R / D43G / N45D 113,2000 178, 301 I20L / D43G / N45D / V58 A / N78I / D90G / G101D 114, 2001 179, 302 D43G / N45D / L56Q / V58A / GlOlG-ins (G101GG) 115, 2002 180, 303 I20L / K23E / D43G / N45D / V58A / N78I 116, 2003 181, 304 I20L / K23E / D43G / N45D / V50A / N78I 117, 2004 182, 305 T19I / E27G / N45I / V50A / N78I / M97K 118,2005 183, 306 I20L / M41K / D43G / N45D 119, 2006 184, 307 K23R / N45T / N78I 120, 2007 185, 308 I20L / K28E / D43G / N45D / V58A / Q89R / G101 G-ins (G101GG) 1725, 2008 1726, 1727 K57R / S99G 1729,1819 1908, 1923 K57R / S99G / F189L 1730, 1820 M18 V / M97L / F193S / R195G / E200K / H202Q 1731, 1821 I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F 1732, 1822 C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L 1733 M18V / I98L / L124S / P198T / L206F 1734, 1823 S99G / N117S / I148V / K171R / R180S 1735, 1824 I36T / M97L / A103V / Q155H 1736,1825 K28I / S99G 1737, 1826 1909, 1924 R195S 1738,1827 A79T / S99G / T185 A / Rl 95G / E200K / H202Q / L206F 1739, 1828 K57R / S99G / L124S / K144Q 1740, 1829 K57R / S99G / R195G 1741, 1830 D55V / M97L / S99G 1742, 1831 1910, 1925 E27G / I36T / D55N / M97L / K11 IE 1743,1832 1911, 1926 E54G / M97L / S99G 1744, 1833 1912, 1927 G15A / I36T / M97L / K111E / H202Q 1745,1834 G15A / I36T / V129D 1746,1835 2024204559   01 Jul 2024 TABLE 2: Exemplary PD-L1 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO G15 A / I36T / V129D / R195G 1747,1836 G15A / V129D 1748,1837 I36S / M97L 1749,1838 1913, 1928 I36T / D55N / M97L / K111E / A204T 1750, 1839 I36T / D55N / M97L / K111E / V129A / F173L 1751, 1840 I36T / D55S / M97L / K111E / I148V / R180S 1752, 1841 I36T / G52R / M97L / V112A / K144E / V175 A / P198T 1753, 1842 I36T / I46 V / D55G / M97L / K106E / K144E / T185 A / R195G 1754, 1843 I36T / I83T / M97L / K144E / P198T 1755, 1844 I36T / M97L / K11 IE 1756, 1845 1914, 1929 I36T / M97L / K144E / P198T 1757, 1846 I36T / M97L / Q155H / F193S / N201Y 1758, 1847 I36T / M97L / V129D 1759, 1848 L35P / I36S / M97L / K11 IE 1760, 1849 1915, 1930 M18I / I36T / E53G / M97L / K144E / E199G / V207A 1761, 1850 M18T / I36T / D55N / M97L / K11 IE 1762, 1851 1916,1931 M18 V / M97L / T176N / R195G 1763, 1852 M97L / S99G 1764, 1853 1917, 1932 N17D / M97L / S99G 1765, 1854 1918,1933 S99G / T185A / R195G / P198T 1766, 1855 V129D / H202Q 1767, 1856 V129D / P198T 1768, 1857 V129D / T150A 1769, 1858 V93E / V129D 1770, 1859 Y1OF / M18 V / S99G / Q138R / T203A 1771, 1860 N45D 1772, 1861 1919, 1934 K160M / R195G 1773, 1862 N45D / K144E 1774, 1863 N45D / P198S 1775, 1864 N45D / P198T 1776,1865 N45D / R195G 1777, 1866 N45D / R195S 1778, 1867 N45D / S131F 1779, 1868 N45D / V58D 1780, 1869 1920, 1935 V129D / R195S 1781, 1870 I98T / F173Y / L196S 1782, 1871 N45D / E134G / L213P 1783, 1872 N45D / F173I / S177C 1784, 1873 N45D / I148V / R195G 1785, 1874 N45D / K111T / R195G 1786,1875 N45D / N113Y / R195S 1787, 1876 N45D / N165Y / E170G 1788, 1877 N45D / Q89R / I98V 1789, 1878 1921, 1936 2024204559   01 Jul 2024 TABLE 2: Exemplary PD-L1 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO N45D / S131F / P198S 1790, 1879 N45D / S75P / P198S 1791, 1880 N45D / V50A / R195T 1792, 1881 E27D / N45D / T183A / I188V 1793, 1882 F173Y / T183I / L196S / T203A 1794, 1883 K23N / N45D / S75P / N120S 1795,1884 N45D / G102D / R194W / R195G 1796,1885 N45D / G52V / Q121L / P198S 1797, 1886 N45D / I148V / R195G / N201D 1798, 1887 N45D / K111T / T183A / I188V 1799, 1888 N45D / Q89R / F189S / P198S 1800, 1889 N45D / S99G / C137R / V207A 1801, 1890 N45D / T163I / K167R / R195G 1802, 1891 N45D / T183A / T192S / R194G 1803, 1892 N45D / V50A / I119T / K144E 1804, 1893 T19A / N45D / K144E / R195G 1805, 1894 V11E / N45D / T130A / P198T 1806, 1895 V26A / N45D / T163I / T185A 1807, 1896 K23N / N45D / L124S / K167T / R195G 1808,1897 K23N / N45D / Q73R / T163I 1809, 1898 K28E / N45D / W149R / S158G / P198T 1810, 1899 K28R / N45D / K57E / I98V / R195S 1811, 1900 K28R / N45D / V129D / T163N / R195T 1812, 1901 M41K / D43G / N45D / R64S / R195G 1813, 1902 M41K / D43G / N45D / R64S / S99G 1814, 1903 1922, 1937 N45D / R68L / F173L / D197G / P198S 1815, 1904 N45D / V50A / I148V / R195G / N201D 1816, 1905 M41K / D43G / K44E / N45D / R195G / N201D 1817, 1906 N45D / V50A / L124S / K144E / L179P / R195G 1818, 1907 b. PD-L2 IgD or vlgD

[0281] Provided herein are immunomodulatory proteins containing an IRBM that is or contains one or more IgD domain, such as an unmodified or wild-type IgD or a vlgD, of PD-L2. In some embodiments, the IRBM is or contains one or more IgD domain of a wild-type or unmodified PD-L2, such as a mammalian PD-L1, e.g. a human PD-L1. In some embodiments, the IRBM is not the full length sequence of the PD-L2. In some aspects, the IRBM is a soluble polypeptide, is not membrane-expressed and / or lacks the transmembrane and / or cytoplasmic domain of PD-L2. In some embodiments, the IRBM only contains an extracellular domain 2024204559   01 Jul 2024 (ECD) or a specific binding fragment thereof containing a IgD or vlgD, such as only contains an IgV domain or an IgC domain or specific binding fragment thereof, or combinations thereof.

[0282] In some embodiments, the IRBM is or contains the ECD sequence set forth in SEQ ID NO:31 or is a specific binding fragment thereof. In some embodiments, the IRBM is or contains the IgV sequence set forth in SEQ ID NO: 1203 or SEQ ID NO: 1263 (containing residues 1-98 or 1-102, respectively, of SEQ ID NO:31), or is a specific binding fragment thereof. LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPL GKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQ ATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLAS IDLQSQMEPRTHPT (SEQ ID NO:31) FTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLG KASFHIPQVQVRDEGQY QCIIIYGVAW DYKYLTLK (SEQ ID NO: 1203) LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPL GKASFHIPQVQVRDEGQY QCIIIYGVAW DYKYLTLKVKA (SEQ ID NO: 1263)

[0283] In some embodiments, the immunomodulatory protein contains an IRBM that is or contains a vlgD containing one or more amino acid modifications, e.g. substitutions, in an IgD of a wild-type or unmodified PD-L2. In some embodiments, modifications provided herein can be in an IRBM containing an unmodified IgD set forth in SEQ ID NO:31, 1203 or 1263 or in a sequence that has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 31, 1203 or 1263. In some embodiments, an IRBM containing a vlgD of PD-L2 has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in any of SEQ ID NOs: 31, 1203 or 1263.

[0284] In some embodiments, the vlgD is an affinity-modified IgSF domain that has an increased binding activity, such as binding affinity, for PD-1 relative to the binding activity of the wild-type or unmodified IgD for PD-1. In some embodiments, the increase in binding activity, e.g. binding affinity, for PD-1 is increased at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 200% or more. In some embodiments, the increase in binding activity, e.g. binding affinity, is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- 2024204559   01 Jul 2024 fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold, or 50-fold. In such examples, the wild-type or unmodified IgD has the same sequence as the vlgD except that it does not contain the one or more amino acid modifications (e.g. substitutions). In some embodiments, the equilibrium dissociation constant (Ka) of the IRBM to PD-1 can be less than IxlO'5 M, IxlO'6 M, IxlO'7 M, IxlO'8 M, IxlO'9 M, IxlO'10 M or lxlO'nM, or IxlO12 M or less.

[0285] Unless stated otherwise, the amino acid modification(s) present in a vlgD of a PD-L2 ECD or an IgD (e.g. IgV) thereof are designated by amino acid position number corresponding to the numbering of positions of the unmodified ECD sequence set forth in SEQ ID NO:31 or, where applicable, the unmodified IgV sequence set forth in SEQ ID NO: 1263. It is within the level of a skilled artisan to identify the corresponding position of a modification, e.g. amino acid substitution, in an ECD or a portion thereof containing an IgSF domain (e.g. IgV) thereof, such as by alignment of a reference sequence with SEQ ID NOs: 31 or 1263. In the listing of modifications throughout this disclosure, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution listed after the number. If the modification is a deletion of the position a “del” is indicated and if the modification is an insertion at the position an “ins” is indicated. In some cases, an insertion is listed with the amino acid position indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before and after the number and the identified variant amino acid insertion listed after the unmodified (e.g. wild-type) amino acid.

[0286] In some embodiments, the IRBM contains a vlgD that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions. The one or more amino acid modifications, e.g. substitutions, can be in the ectodomain (extracellular domain) of the wild-type or unmodified PD-L2. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the ECD domain of PD-L2 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of PD-L2 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in an IgC domain of PD-L2 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of PD-L2 or a specific binding fragment thereof and in an IgC domain or domains of PD-L2 or a specific binding fragment thereof. 2024204559   01 Jul 2024

[0287] In some embodiments, the IRBM is or contains a vlgD that has one or more amino acid modifications, e.g. substitutions in an unmodified IgD of PD-L2 or specific binding fragment thereof corresponding to position(s) 2, 12, 13, 15, 18, 20, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 82, 85, 86, 89, or 91 with reference to positions set forth in SEQ ID NO: 31. In some cases, a polypeptide containing the vlgD exhibits increased binding activity, e.g. binding affinity, to PD-1 compared to a polypeptide containing the wild-type or unmodified PD-L2 IgD.

[0288] In some embodiments, the IRBM is or contains a vlgD that has one or more amino acid modification, e.g. substitution, selected from F2L, I12V, I13V, H15Q, N18D, T20A, N24S, C23S, G28V, N24D,V31A,V31M, N32D, L33P, ,L33H, L33F, I36V, T37A, S48C, S39I, E44D, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, R76G, D77N, Q82R, I85F, I86T, V89D, or W91R , or a conservative amino acid substitution thereof.

[0289] In some embodiments, the IRBM is or contains a vlgD that has two or more amino acid modification, e.g. substitution, selected from F2L, I12V, I13V, H15Q, N18D, T20A, N24S, C23S, G28V, N24D,V31A,V31M, N32D, L33P, ,L33H, L33F, I36V, T37A, S48C, S39I, E44D, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, R76G, D77N, Q82R, I85F, I86T, V89D, W91R .

[0290] In some embodiments, the amino acid modification(s), e.g. substitution(s), is H15Q, N24D, E44D, V89D, Q82R / V89D, E59G / Q82R, S39PV89D, S67L / V89D, S67L / I85F, S67L / I86T, H15Q / K65R, H15Q / Q72H / V89D, H15Q / S67L / R76G, H15Q / R76G / I85F, H15Q / T47A / Q82R, H15Q / Q82R / V89D, H15Q / C23S / I86T, H15Q / S39I / I86T, E44D / V89D / W91R, I13V / S67L / V89D, H15Q / S67L / I86T, I13V / H15Q / S67L / I86T, I13V / H15Q / E44D / V89D, I13V / S39I / E44D / Q82R / V89D, I13V / E44D / Q82R / V89D, I13V / Q72H / R76G / I86T, I13V / H15Q / R76G / I85F, H15Q / S39I / R76G / V89D, H15Q / S67L / R76G / I85F, H15Q / T47A / Q72H / R76G / I86T, H15Q / T47A / Q72H / R76G, I13V / H15Q / T47A / Q72H / R76G, H15Q / E44D / R76G / I85F, H15Q / S39I / S67L / V89D, H15Q / N32D / S67L / V89D, N32D / S67L / V89D, H15Q / S67L / Q72H / R76G / V89D, H15Q / Q72H / Q74R / R76G / I86T, G28V / Q72H / R76G / I86T, I13V / H15Q / S39I / E44D / S67L, E44D / S67L / Q72H / Q82R / V89D, H15Q / V89D, H15Q / T47A, I13V / H15Q / Q82R, I13V / H15Q / V89D, I13V / S67L / Q82R / V89D, I13V / H15Q / Q82R / V89D, H15Q / V31M / S67L / Q82R / V89D, I13V / H15Q / T47A / Q82R, I13V / H15Q / V31A / N45S / Q82R / V89D, H15Q / T47A / H69L / Q82R / V89D, I13V / H15Q / T47A / H69L / R76G / V89D, I12V / I13V / H15Q / T47A / Q82R / V89D, I13V / H15Q / R76G / D77N / Q82R / V89D, I13V / H15Q / T47A / R76G / V89D, I13V / H15Q / T47A / Q82R / V89D, I13V / H15Q / N24D / Q82R / V89D, I13V / H15Q / I36V / T47A / S67L / V89D, H15Q / T47A / K65R / S67L / Q82R / V89D, H15Q / L33P / T47A / S67L / P71S / V89D, 2024204559   01 Jul 2024 I13V / H15Q / Q72H / R76G / I86T, H15Q / T47A / S67L / Q82R / V89D, F2L / H15Q / D46E / T47A / Q72H / R76G / Q82R / V89D, I13V / H15Q / L33F / T47A / Q82R / V89D, I13V / H15Q / T47A / E58G / S67L / Q82R / V89D, H15Q / N24S / T47A / Q72H / R76G / V89D, I13V / H15Q / E44V / T47A / Q82R / V89D, H15Q / N18D / T47A / Q72H / V73A / R76G / I86T / V89D, I13V / H15Q / T37A / E44D / S48C / S67L / Q82R / V89D, H15Q / L33H / S67L / R76G / Q82R / V89D, I13V / H15Q / T47A / Q72H / R76G / I86T, H15Q / S39I / E44D / Q72H / V75G / R76G / Q82R / V89D, H15Q / T47A / S67L / R76G / Q82R / V89D, or I13V / H15Q / T47A / S67L / Q72H / R76G / Q82R / V89D.

[0291] In some embodiments, the IRBM contains a vlgD that has one or more amino acid modification, e.g. substitutions, in an unmodified PD-L2 or specific binding fragment thereof corresponding to position(s) 13, 15, 47, 67, 72, 76, 82, 86, and / or 89 with reference to positions set forth in SEQ ID NO:36 or 2655. In some emboidments, the IRBM is or contains a vlgD of PD-L1 that has one or more amino acid modifications selected from I13V, H15Q, T47A, S67L, Q72H, R76G, Q82R, I86T, and / or V89D, or a conservative amino acid substitution thereof.

[0292] In some embodiments, the IRBM is or contains an IgD (e.g. IgV) of wild-type PD-L2 set forth in Table 3 or a vlgD comprising any of the modifications (e.g. substitutionss) listed in Table 3. Table 3 also provides exemplary sequences by reference to SEQ ID NO for IRBMs containing an ECD or IgV domain. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g. IgV) also can be included in an IRBM, such as to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplification of the SEQ ID NOSs in Table 3 is not to be construed as limiting. For example, the particular domain, such as the ECD or IgV domain, can be several amino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO.

[0293] In some embodiments, the IRBM is or contains a wild-type PD-L2 ECD set forth in SEQ ID NO:31 or a variant ECD sequence set forth in any one of SEQ ID NOS: 1204-1280. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the ECD sequences set forth in any one of SEQ ID NOS: 1204-1280 and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified PD-L2, e.g. not present in SEQ ID NO:31. In some embodiments, the IRBM is or contains a specific binding 2024204559   01 Jul 2024 fragment of any of the ECD set forth in any one of SEQ ID NOS: 1204-1280 and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified PD-L2 ECD, e.g. not present in SEQ ID NO:31.

[0294] In some embodiments, the IRBM is or contains a wild-type PD-L2 IgV set forth in SEQ ID NO: 1203 or 1263 or a variant IgV set forth in any one of SEQ ID NOS: 1283-1331, 1333-1407, 1309-1432. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences set forth in any one of SEQ ID NOS: 1283-1331, 1333-1407, 1309-1432 and contains the amino acid substitution(s) not present in the wild-type or unmodified PD-L2 IgV, e.g. not present in SEQ ID NO: 1203 or 1263. In some embodiments, the IRBM is or contains a specific binding fragment of any of the IgV sequences set forth in any one of SEQ ID NOS: 1283-1331, 1333-1407, 1309-1432 and that contains the amino acid modification(s), e.g., substitution(s) not present in the wild-type or unmodified PD-L2 IgV, e.g. not present in SEQ ID NO: 1203 or 1263. TABLE 3: Exemplary PD-L2 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO Wild-type 31 1203, 1263 H15Q 1204 1281, 1357 N24D 1205 1282, 1358 E44D 1206 1283, 1359 V89D 1207 1284, 1360 Q82R / V89D 1208 1285,1361 E59G / Q82R 1209 1286, 1362 S39I / V89D 1210 1287, 1363 S67L / V89D 1211 1288, 1364 S67L / I85F 1212 1289, 1365 S67L / I86T 1213 1290, 1366 H15Q / K65R 1214 1291, 1367 H15Q / Q72H / V89D 1215 1292, 1368 H15Q / S67L / R76G 1216 1293, 1369 H15Q / R76G / I85F 1217 1294, 1370 H15Q / T47A / Q82R 1218 1295,1371 H15Q / Q82R / V89D 1219 1296, 1372 H15Q / C23S / I86T 1220 1297, 1373 H15Q / S39I / I86T 1221 1298, 1374 H15Q / R76G / I85F 1222 1299, 1375 E44D / V89D / W91R 1223 1300, 1376 I13V / S67L / V89D 1224 1301, 1377 H15Q / S67L / I86T 1225 1302, 1378 I13V / H15Q / S67L / I86T 1226 1303, 1379 I13V / H15Q / E44D / V89D 1227 1304, 1380 2024204559   01 Jul 2024 TABLE 3: Exemplary PD-L2 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgVSEQ ID NO I13V / S39I / E44D / Q82R / V89D 1228 1305,1381 I13V / E44D / Q82R / V89D 1229 1306, 1382 I13V / Q72H / R76G / I86T 1230 1307, 1383 I13V / H15Q / R76G / I85F 1231 1308, 1384 H15Q / S39I / R76G / V89D 1232 1309, 1385 H15Q / S67L / R76G / I85F 1233 1310, 1386 H15Q / T47 A / Q72H / R76G / I86T 1234 1311, 1387 H15Q / T47 A / Q72H / R76G 1235 1312, 1388 113 V / H15Q / T47 A / Q72H / R76G 1236 1313, 1389 H15Q / E44D / R76G / I85F 1237 1314, 1390 H15Q / S39I / S67L / V89D 1238 1315,1391 H15Q / N32D / S67L / V89D 1239 1316, 1392 N32D / S67L / V89D 1240 1317, 1393 H15Q / S67L / Q72H / R76G / V89D 1241 1318, 1394 H15Q / Q72H / Q74R / R76G / I86T 1242 1319, 1395 G28V / Q72H / R76G / I86T 1243 1320, 1396 I13V / H15Q / S39I / E44D / S67L 1244 1321, 1397 E44D / S67L / Q72H / Q82R / V89D 1245 1322, 1398 H15Q / V89D 1246 1323, 1399 H15Q / T47A 1247 1324, 1400 I13V / H15Q / Q82R 1248 1325,1401 I13V / H15Q / V89D 1249 1326,1402 I13V / S67L / Q82R / V89D 1250 1327, 1403 I13V / H15Q / Q82R / V89D 1251 1328, 1404 H15Q / V31M / S67L / Q82R / V89D 1252 1329, 1405 113 V / H 15Q / T47 A / Q82R 1253 1330, 1406 I13V / H15Q / V31A / N45S / Q82R / V89D 1254 1331,1407 H15Q / T47A / H69L / Q82R / V89D 1256 1333, 1409 I13V / H15Q / T47A / H69L / R76G / V89D 1257 1334,1410 I12V / I13V / H15Q / T47A / Q82R / V89D 1258 1335,1411 I13V / H15Q / R76G / D77N / Q82R / V89D 1259 1336,1412 I13V / H15Q / T47A / R76G / V89D 1260 1337, 1413 I13V / H15Q / T47A / Q82R / V89D 1261 1338,1414 I13V / H15Q / N24D / Q82R / V89D 1262 1339, 1415 I13V / H15Q / I36V / T47A / S67L / V89D 1264 1340, 1416 H15Q / T47A / K65R / S67L / Q82R / V89D 1265 1341, 1417 H15Q / L33P / T47 A / S67L / P71S / V89D 1266 1342, 1418 I13V / H15Q / Q72H / R76G / I86T 1267 1343, 1419 H15Q / T47A / S67L / Q82R / V89D 1268 1344, 1420 F2L / H15Q / D46E / T47 A / Q72H / R76G / Q82R / V89D 1269 1345, 1421 I13V / H15Q / L33F / T47A / Q82R / V89D 1270 1346,1422 I13V / H15Q / T47A / E58G / S67L / Q82R / V89D 1271 1347, 1423 H15Q / N24S / T47A / Q72H / R76G / V89D 1272 1348, 1424 113 V / H 15Q / E44V / T47 A / Q82R / V89D 1273 1349, 1425 H15Q / N18D / T47A / Q72H / V73A / R76G / I86T / V89D 1274 1350, 1426 I13V / H15Q / T37A / E44D / S48C / S67L / Q82R / V89D 1275 1351, 1427 H15Q / L33H / S67L / R76G / Q82R / V89D 1276 1352, 1428 113 V / H 15Q / T47 A / Q72H / R76G / I86T 1277 1353, 1429 H15Q / S39I / E44D / Q72H / V75G / R76G / Q82R / V89D 1278 1354, 1430 H15Q / T47A / S67L / R76G / Q82R / V89D 1279 1355,1431 I13V / H15Q / T47A / S67L / Q72H / R76G / Q82R / V89D 1280 1356, 1432 2024204559   01 Jul 2024 2. T / G / T-Blndlng Molecules

[0295] Provided are immunomodulatory proteins containing an IRBM that is or contains a binding molecule that binds to TIGIT, such as to human TIGIT. Engagement of TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibits or down-modulate immune responses. TIGIT, which can be expressed on NK cells and T cells, can suppress or inhibit the cytolytic activity of NK cells, T cell proliferation and / or proinflammatory cytokine production. In some embodiments, the IRBM of the immunomodulatory protein binds to the ectodomain of TIGIT. In some embodiments, the IRBM binds to TIGIT on the surface of a cell, such as on the surface of a T cell.

[0296] In some embodiments, the provided immunomodulatory protein contains an IRBM that is or contains an antibody that binds TIGIT or is an antigen-binding antibody fragment thereof (e.g. Fab or scFv). In some embodiments, the antibody or antigen-binding antibody fragment binds human TIGIT. For example, in some embodiments, the antibody is BMS-986207, OMP-313M32, or RG6058 (MTIG7192A), or an antigen binding fragment containing a VH chain region and / or VL chain region of BMS-986207, OMP-313M32, or RG6058 (MTIG7192A). In some embodiments, the antibody is a single chain variable fragment (e.g. scFv) containing a VH and VL of BMS-986207, OMP-313M32, or RG6058 (MTIG7192A).

[0297] In some embodiments, the IRBM is or contains one or more IgD (e.g. IgV or IgC) or a specific binding fragment thereof, such as an unmodified or wild-type IgD or a vlgD or a specific binding fragment thereof, of an IgSF family member that binds TIGIT. In some embodiments, the TIGIT is human TIGIT. The ligands CD155 (also known as the poliovirus receptor, PVR) and CD112 (also known as Nectin-2), which are normally expressed on the surface of APCs (e.g. dendritic cells) can bind TIGIT to inhibit or down-modulate immune responses.

[0298] In some embodiments, the IRBM is or contains one or more IgD (e.g. IgV or IgC) that is an IgD of a CD112 or CD155 polypeptide, such as a wild-type CD112 or CD155, e.g. a human CD112 or a human CD155. In some aspects, the IRBM contains one or more IgD (e.g. IgV or IgC) that is a vlgD containing one or more amino acid modifications (e.g., substitutions, deletions or additions) compared to an IgD of a wild-type or unmodified CD112 or CD 155, which, in some aspects, result in increased binding to TIGIT. Exemplary IgDs or vlgDs of CD112 or CD155 binding partners for inclusion as an IRBM in the provided immunomodulatory proteins are described. In some embodiments, the IRBM is or contains a vlgD polypeptide that 2024204559   01 Jul 2024 exhibit increased binding activity or affinity for TIGIT compared to a corresponding wild-type or unmodified IgD.

[0299] In some aspects, a vlgD of CD112 or CD 155 may exhibit increased binding activity, e.g. binding affinity, to the activating receptor CD226. In such examples, such a vlgD may be used as an ARBM in the embodiments provided herein. In some embodiments, such an ARBM exhibits a greater binding activity, e.g. binding affinity, for CD226 than for TIGIT, such as a binding activity, e.g. binding affinity, that is at least 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold or 10.0-fold greater for CD226 than for TIGIT. a. CD155 IgD or vlgD

[0300] Provided herein are immunomodulatory proteins containing an IRBM that is or contains one or more IgD domain, such as an unmodified or wild-type IgD or a vlgD, of CD155. In some embodiments, the IRBM is or contains one or more IgD domain of a wild-type or unmodified CD155, such as a mammalian CD155, e.g. a human CD155. In some embodiments, the IRBM is not the full length sequence of the CD155. In some aspects, the IRBM is a soluble polypeptide, is not membrane-expressed and / or lacks the transmembrane and / or cytoplasmic domain of CD155. In some embodiments, the IRBM only contains an extracellular domain (ECD) or a specific binding fragment thereof containing a IgD or vlgD, such as only contains an IgV domain or an IgC domain or specific binding fragment thereof, or combinations thereof.

[0301] In some embodiments, the IRBM is or contains the ECD sequence set forth in SEQ ID NO:47 or is a specific binding fragment thereof. In some embodiments, the IRBM is or contains the IgV sequence set forth in SEQ ID NO: 310 or SEQ ID NO: 353, or is a specific binding fragment thereof. WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQ TQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVL AKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVT SLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLT CDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQ VKEGPPSEHSGISRN (SEQ ID NO:47) PGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQG PSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWL (SEQ ID NO:310) 2024204559   01 Jul 2024 WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQ TQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVL (SEQ ID NO:353)

[0302] In some embodiments, the immunomodulatory protein contains an IRBM that is or contains a vlgD containing one or more amino acid modifications, e.g. substitutions, in an IgD of a wild-type or unmodified CD155. In some embodiments, modifications provided herein can be in an IRBM containing an unmodified IgD set forth in SEQ ID NO:37, 310 or 353 or in a sequence that has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 37, 310 or 353. In some embodiments, an IRBM containing a vlgD of CD155 has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in any of SEQIDNOs: 37, 310 or 353.

[0303] In some embodiments, the vlgD is an affinity-modified IgSF domain that has an increased binding activity, such as binding affinity, for TIGIT relative to the binding affinity of the wild-type or unmodified IgD for TIGIT. In some embodiments, the increase in binding activity, e.g. binding affinity, for TIGIT is increased at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 200% or more. In some embodiments, the increase in binding activity, e.g. binding affinity, is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold, or 50-fold. In such examples, the wild-type or unmodified IgD has the same sequence as the vlgD except that it does not contain the one or more amino acid modifications (e.g. substitutions). In some embodiments, the equilibrium dissociation constant (Ka) of the IRBM to TIGIT can be less than IxlO'5 M, IxlO'6 M, IxlO'7 M, IxlO'8 M, IxlO'9 M, IxlO'10 M or lxl0'nM, or IxlO12 M or less.

[0304] In some aspects, a vlgD of CD155 may exhibit increased binding activity, e.g. binding affinity, to the activating receptor CD226. In some embodiments, the increase in binding activity, e.g. binding affinity, for TIGIT is increased at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 200% or more. In some embodiments, the increase in binding activity, e.g. binding affinity, is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold, or 50-fold. In such examples, such a vlgD may be used as an ARBM in the embodiments provided herein. In some embodiments, the equilibrium dissociation constant (Ka) of the ARBM to CD226 can be 2024204559   01 Jul 2024 less than 1x10’5M, IxlO-6 M, IxlO-7 M, IxlO-8 M, IxlO-9 M, IxlO-10 M or lxlO-11M, or IxlO12 M or less. In some embodiments, such an ARBM exhibits a greater binding activity, e.g. binding affinity, for CD226 than for TIGIT, such as a binding activity, e.g. binding affinity, that is at least 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold or 10.0-fold greater for CD226 than for TIGIT.

[0305] Unless stated otherwise, the amino acid modification(s) present in a vlgD of a CD155 ECD or an IgD (e.g. IgV) thereof are designated by amino acid position number corresponding to the numbering of positions of the unmodified ECD sequence set forth in SEQ ID NO:47 or, where applicable, the unmodified IgV sequence set forth in SEQ ID NO: 353. It is within the level of a skilled artisan to identify the corresponding position of a modification, e.g. amino acid substitution, in an ECD or a portion thereof containing an IgSF domain (e.g. IgV) thereof, such as by alignment of a reference sequence with SEQ ID NOs: 47 or 353. In the listing of modifications throughout this disclosure, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution listed after the number. If the modification is a deletion of the position a “del” is indicated and if the modification is an insertion at the position an “ins” is indicated. In some cases, an insertion is listed with the amino acid position indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before and after the number and the identified variant amino acid insertion listed after the unmodified (e.g. wild-type) amino acid.

[0306] In some embodiments, the IRBM contains a vlgD that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions. The one or more amino acid modifications, e.g. substitutions, can be in the ectodomain (extracellular domain) of the wild-type or unmodified CD 155. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the ECD domain of CD155 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of CD155 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in an IgC domain of CD 155 or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions, are in the IgV domain of CD155 or a specific binding fragment thereof and in an IgC domain or domains of CD 155 or a specific binding fragment thereof. 2024204559   01 Jul 2024

[0307] In some embodiments, the IRBM is or contains a vlgD that has one or more amino acid modification, e.g. substitutions, in an unmodified IgD of CD155 or specific binding fragment thereof corresponding to position(s) 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 67, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99, 100, 102, 104, 106, 107, 108, 110, 111, 112, 113, 114, 115, or 116 with reference to positions set forth in SEQ ID NO: 47. In some embodiments, a polypeptide containing the vlgD exhibits altered, such as increased, binding activity, e.g. binding affinity, to TIGIT compared to a polypeptide containing the wild-type or unmodified CD 155 IgD.

[0308] In some embodiments, the IRBM is or contains a vlgD of CD155 that has one or more amino acid modifications selected from G7E, D8G, V9A, V9D, V9I, V9L, V10F, V10G, V10I, VI1A, VI IE, VI IM, Q12H, Q12K, Q12L, AI3E. A13R, T15I, T15S, Q16H, P18C, P18F, P18H, P18L, P18S, P18T, P18Y, G19D, F20I, F20S, F20Y, L21S, L21M, G22S, D23A, D23G, D23N, D23Y, S24A, S24P, V25A, V25E, T26M, C29R, Y30C, Y30F, Y30H, Q32L, Q32R, V33M, P34S, N35D, N35F, N35S, M36I, M36R, M36T, E37G, E37P, E37S, E37V, V38A, V38G, T39A, T39S, H40Q, H40R, H40T, V41A, V41M, S42A, S42C, S42G, S42L, S42N, S42P, S42Q, S42T, S42V, S42W, L44P, L44V, T45A, T45G, T45I, T45S, T45Q, T45V, W46C, W46R, A47E, A47G, A47V, R48Q, H49L, H49Q, H49R, G50S, E51G, E51K, E51V, S52A, S52E, S52G, S52K, S52L, S52M, S52P, S52Q, S52R, S52T, S52W, G53R, S54C, S54G, S54H, S54N, S54R, M55I, M55L, M55V, A56V, V57A, V57L, V57T, F58L, F58Y, H59E, H59N, N59R, Q60H, Q60K, Q60P, Q60R, T61A, T61G, T61K, T61M, T61R, T61S, Q62F, Q62H, Q62K, Q62L, Q62M, Q62R, Q62Y, P64S, S65A, S65C, S65G, S65D, S65T, S65Y, S65H, S65N, S65T, S65W, S67A, S67E, S67G, S67H, S67L, S67T, S67V, S67W, E68G, S69L, S69P, K70E, K70R, K70Q, L72Q, E73D, E73G, E73R, V75A, V75L, A76E, A76G, A76T, A77T, A77V, R78G, R78K, R78S, L79P, L79Q, L79V, G80D, G80S, A81E, A81P, A81T, A81V, E82D, E82G, L83P, L83Q, R84W, N85D, N85Y, N87T, L88P, R89K, M90I, M90L, M90V, F91S, F91P, F91T, G92A, G92E, G92W, R94H, V95A, E96D, D97G, E98D, E98S, G99D, G99Y, N100Y, T102S, L104E, L104M, L104N, L104P, L104Q, L104T, L104Y, V106A, V106I, V106L, T107A, T107L, T107M, T107S, T107V, F108H, F108L, F108Y, Q110R, G111D, G111R, S1121, S112N, S112V, R113G, R113W, S114N, S114T, V115A, V115M, D116G, or D116N, or a conservative amino acid substitution thereof. 2024204559   01 Jul 2024

[0309] In some embodiments, the IRBM is or contains a vlgD that has two or more amino acid modifications selected from G7E, D8G, V9A, V9D, V9I, V9L, V10F, V10G, V10I, VI1A, V11E, V11M, Q12H, Q12K, Q12L, A13E, A13R, T15I, T15S, Q16H, P18C, P18F, P18H, P18L, P18S, P18T, P18Y, G19D, F20I, F20S, F20Y, L21S, L21M, G22S, D23A, D23G, D23N, D23Y, S24A, S24P, V25A, V25E, T26M, C29R, Y30C, Y30F, Y30H, Q32L, Q32R, V33M, P34S, N35D, N35F, N35S, M36I, M36R, M36T, E37G, E37P, E37S, E37V, V38A, V38G, T39A, T39S, H40Q, H40R, H40T, V41A, V41M, S42A, S42C, S42G, S42L, S42N, S42P, S42Q, S42T, S42V, S42W, L44P, L44V, T45A, T45G, T45I, T45S, T45Q, T45V, W46C, W46R, A47E, A47G, A47V, R48Q, H49L, H49Q, H49R, G50S, E51G, E51K, E51V, S52A, S52E, S52G, S52K, S52L, S52M, S52P, S52Q, S52R, S52T, S52W, G53R, S54C, S54G, S54H, S54N, S54R, M55I, M55L, M55V, A56V, V57A, V57L, V57T, F58L, F58Y, H59E, H59N, N59R, Q60H, Q60K, Q60P, Q60R, T61A, T61G, T61K, T61M, T61R, T61S, Q62F, Q62H, Q62K, Q62L, Q62M, Q62R, Q62Y, P64S, S65A, S65C, S65G, S65D, S65T, S65Y, S65H, S65N, S65T, S65W, S67A, S67E, S67G, S67H, S67L, S67T, S67V, S67W, E68D, E68G, S69L, S69P, K70E, K70R, K70Q, L72Q, E73D, E73G, E73R, V75A, V75L, A76E, A76G, A76T, A77T, A77V, R78G, R78K, R78S, L79P, L79Q, L79V, G80D, G80S, A81E, A81P, A81T, A81V, E82D, E82G, L83P, L83Q, R84W, N85D, N85Y, N87T, L88P, R89K, M90I, M90L, M90V, F91S, F91T, F91P, G92A, G92E, G92W, R94H, V95A, E96D, D97G, E98D, E98S, G99D, G99Y, N100Y, T102S, L104E, L104M, L104N, L104P, L104Q, L104T, L104Y, V106A, V106I, V106L, T107A, T107L, T107M, T107S, T107V, F108H, F108L, F108Y, Q110R, G111D, G111R, S1121, S112N, S112V, R113G, R113W, S114N, S114T, V115A, V115M, D116G, or D116N.

[0310] In some embodiments, the amino acid modification(s), e.g. substitutions(s), is P18S / P64S / F91S, P18S / F91S / L104P, P18L / L79V / F91S, P18S / F91S, P18T / F91S, P18T / S42P / F91S, G7E / P18T / Y30C / F91S, P18T / F91S / G111D, P18S / F91P, P18T / F91S / F108L, P18S / F91S, P18T / T45A / F91S, P18T / F91S / R94H, P18S / Y30C / F91S, A81V / L83P, A13E / P18S / A56V / F91S, P18T / F91S / V115A, P18T / Q60K, S52M, T45Q / S52L / L104E / G111R, S42G, Q62F, S52Q, S42A / L104Q / G111R, S42A / S52Q / L104Q / G111R, S52W / L104E, S42C, S52W, S52M / L104Q, S42L / S52L / Q62F / L104Q, S42W, S42Q, S52L, S52R, L104E, G111R, S52E, Q62Y, T45Q / S52M / L104E, S42N / L104Q / G111R, S52M / V57L, S42N / S52Q / Q62F, S42A / S52L / L104E / G111R, S42W / S52Q / V57L / Q62Y, L104Q, S42L / S52Q / L104E, S42C / S52L, S42W / S52R / Q62Y / L104Q, T45Q / S52R / L104E, S52R / Q62F / L104Q / G111R, T45Q / S52L / V57L / L104E, S52M / Q62Y, Q62F / L104E / G111R, T45Q / S52Q, S52L / L104E, S42V / S52E, T45Q / S52R / G111R, 2024204559   01 Jul 2024 S42G / S52Q / L104E / G111R, S42N / S52E / V57L / L104E, S42C / S52M / Q62F, S42L, S42A, S42G / S52L / Q62F / L104Q, S42N, P18T / S65A / S67V / F91S, P18F / T39A / T45Q / T61R / S65N / S67L / E73G / R78G, P18T / T45Q / T61R / S65N / S67L, P18F / S65A / S67V / F91S, P18F / T45Q / T61R / S65N / S67L / F91S / L104P, P18S / L79P / L104M, P18S / L104M, L79P / L104M, P18T / T45Q / L79P, P18T / T45Q / T61R / S65H / S67H, P18T / A81E, Pl8S / D23Y / E37P / S52G / Q62M / G80S / A81P / G99Y / S112N, A13R / D23Y / E37P / S42P / Q62Y / A8IE, A13R / D23Y / E37P / G99Y / S112N, A13R / D23Y / E37P / Q62M / A77V / G80S / A81P / G99Y, Pl8L / E37S / Q62M / G80S / A81P / G99Y / S112N, Pl8S / L104T, Pl8S / Q62H / L79Q / F91S, T45Q / S52K / Q62F / L104Q / G111R, T45Q / S52Q / Q62Y / L104Q / G1 HR, T45Q / S52Q / Q62Y / L104E / G111R, V57A / T61M / S65W / S67A / E96D / L104T, P18L / V57T / T61S / S65Y / S67A / L104T, P18T / T45Q, P18L / V57A / T61M / S65W / S67A / L104T, T61M / S65W / S67A / L104T, P18 S / V41A / S42G / T45 G / L104N, P18H / S42G / T45I / S52T / G53R / S54H / V57L / H59E / T61S / S65D / E68G / L104N, P18S / S42G / T45V / F58L / S67W / L104N, P18S / T45I / L104N, P18S / S42G / T45G / L104N / V106A, P18H / H40R / S42G / T45PS52T / G53R / S54H / V57L / H59E / T61S / S65D / E68G / L104Y / V106L / F108H, E37V / S42G / T45G / L104N, P18S / T45Q / L79P / L104T, P18L / Q62R, A13R / D23Y / E37P / S42L / S52G / Q62Y / A81E, P18L / H49R / L104T / D116N, A13R / D23Y / E37P / Q62M / G80S / A81P / L104T, S65T / L104T, A13R / D23Y / E37P / S52G / V57A / Q62M / K70E / L104T, P18L / A47V / Q62Y / E73D / L104T, H40T / V41M / A47V / S52Q / Q62L / S65T / E73R / D97G / E98S / L104T / D116N, P18L / S42P / T45Q / T61G / S65H / S67E / L104T / D116N, Pl 8S / H40T / V41M / A47V / S52Q / Q62L / S65T / E73R / L104M / V106 A, H40T / V41M / A47V / S52Q / Q62L / S65T / E68G / E73R / D97G / E98S / L104T, T45Q / S52E / L104E, T45Q / S52E / Q62F / L104E, P18F / T26M / L44V / Q62K / L79P / F91S / L104M / G11 ID, P18S / T45S / T61K / S65W / S67A / F91S / G111R, P18S / L79P / L104M / T107M, P18S / S65W / S67A / M90V / V95A / L104Q / G111R, P18S / A47G / L79P / F91S / L104M / T107A / R113W, P18T / D23G / S24A / N35D / H49L / L79P / F91S / L104M / G111R, V9L / P18S / Q60R / V75L / L79P / R89K / F91S / L104E / G111R, P18S / H49R / E73D / L79P / N85D / F91S / V95A / L104M / G111R, V11A / P18S / L79P / F91S / L104M / G111R, V11 A / Pl 8S / S54R / Q60P / Q62K / L79P / N85D / F91S / T107M, P18T / S52P / S65A / S67V / L79P / F91S / L104M / G111R, P18T / M36T / L79P / F91S / G111R, D8G / P18S / M36I / V38A / H49Q / A76E / F91S / L104M / T107A / R113W, P18S / S52P / S65A / S67V / L79P / F91S / L104M / T107S / R113W, T15I / P18T / L79P / F91S / L104M / G111R, P18F / T26M / L44V / Q62K / L79P / E82D / F91S / L104M / G11 ID, P18T / E37G / G53R / Q62K / L79P / F91S / E98D / L104M / T107M, P18L / K70E / L79P / F91S / V95A / G111R, 2024204559   01 Jul 2024 V9I / Q12K / P18F / S65A / S67V / L79P / L104T / G111R / S112I, P18F / S65A / S67V / F91S / L104M / G111R, V9I / V1OI / P18 S / F20S / T45 A / L79P / F91S / L104M / F108 Y / G111R / S112 V, V9L / P18L / L79P / M90PF91S / T102S / L104M / G111R, P18C / T26M / L44V / M55I / Q62K / L79P / F91S / L104M / T107M, V9I / P18T / D23G / L79P / F91S / G111R, P18F / L79P / M90L / F91S / V95A / L104M / G111R, P18T / M36T / S65A / S67E / L79Q / A81T / F91S / G111R, V9L / P18T / Q62R / L79P / F91S / L104M / G111R, P18S / S65W / S67A / L104Q / G111R, P18T / G19D / M36T / S54N / L79P / L83Q / F91S / T107M / F108Y,V9L / P18L / M55V / S69L / L79P / A81E / F91S / T 107M, Pl 8F / H40Q / T61K / Q62K / L79P / F91 S / L 104M / T107V, P18 S / Q32R / Q62K / R7 8G / L79P / F91 S / T 107 A / R113 W,Q 12H / P18T / L21S / G22S / V57 A / Q62R / L79P / F91S / T107M, V9I / P18S / S24P / H49Q / F5 8Y / Q60R / Q62K / L79P / F91 S / T 107M, P18T / W46C / H49R / S65A / S67V / A76T / L79P / S87T / L104M, P18S / S42T / E51G / L79P / F91S / G92W / T107M, V10F / T15S / P18L / R48Q / L79P / F91S / T107M / V115M, Pl 8S / L21M / Y30F / N35D / R84W / F91S / T107M / D116G, Pl 8F / E51V / S54G / Q60R / L79Q / E82G / S87T / M90I / F91S / G92R / T107M, Q16H / P18F / F91 S / T 107M, Pl 8T / D23G / Q60R / S67L / L79P / F91 S / T 107M / V115 A, D8G / V9I / V11A / P18T / T26M / S52P / L79P / F91S / G92A / T107L / V115A, V9I / P18F / A47E / G50S / E68G / L79P / F91S / T107M, P18S / M55I / Q62K / S69P / L79P / F91S / T107M, P18T / T39S / S52P / S54R / L79P / F91S / T107M, P18S / D23N / L79P / F91S / T107M / S114N, P18S / P34S / E51V / L79P / F91S / G111R, P18S / H59N / V75A / L79P / A81T / F91S / L104M / T107M, P18S / W46R / E68D / L79P / F91S / T107M / R113G,V9L / P18F / T45A / S65A / S67V / R78K / L79V / F91S / T107M / S114T, P18T / M55L / T61R / L79P / F91S / V106I / T107M, T15I / P18S / V33M / N35F / T39S / M55L / R78S / L79P / F91S / T107M, Pl8S / Q62K / K70E / L79P / F91S / G92E / R113W, Pl8F / F20I / T26M / A47V / E51K / L79P / F9IS, Pl 8T / D23 A / Q60H / L79P / M90V / F91 S / T 107M, P18S / D23G / C29R / N35D / E37G / M55I / Q62K / S65A / S67G / R78G / L79P / F91S / L104M / T107M / Q110R, A13E / P18S / M36R / Q62K / S67T / L79P / N85D / F91S / T107M, V9I / P18T / H49R / L79P / N85D / F91S / L104T / T107M, V9A / P18F / T61S / Q62L / L79P / F91S / G111R, D8E / P18T / T61A / L79P / F91S / T107M, P18S / V41A / H49R / S54C / L79S / N85Y / L88P / F91S / L104M / T107M, V11E / P18H / F20Y / V25E / N35S / H49R / L79P / F91S / T107M / G111R, V11A / P18F / D23A / L79P / G80D / V95A / T107M, P18S / K70R / L79P / F91S / G111R, V9L / V11M / P18S / N35S / S54G / Q62K / L79P / L104M / T107M / V115M, V9L / P18Y / V25A / V3 8G / M55V / A77T / L79P / M90I / F91 S / L 104M, VIOG / P18T / L72Q / L79P / F91 S / T 107M, P18S / H59R / A76G / R78S / L79P, V9A / P18S / M36T / S65G / L79P / F91S / L104T / G111R / S1121, Pl 8T / S52A / V57 A / Q60R / Q62K / S65C / L79P / F91T / N100Y / T107M, V11A / P18F / N35D / A47E / Q62K / L79P / F91S / G99D / T107M / S114N, V11A / P18T / N35S / L79P / S87T / F91S, 2024204559   01 Jul 2024 V9D / V11M / Q12L / P18S / E37V / M55I / Q60R / K70Q / L79P / F91S / L104M / T107M, or T15 S / P18 S / Y30H / Q32L / Q62R / L79P / F91S / T107M.

[0311] In some embodiments, the IRBM is or contains an IgD (e.g. IgV) of a wild-type CD 155 polypeptide set forth in Table 4 or a vlgD thereof comprising any of the modifications (e.g. substitutions) listed in Table 4. Table 4 also provides exemplary sequences by reference to SEQ ID NO for IRBMs containing an ECD or IgV domain of CD155. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g. ECD or IgV) also can be included in a sequence of an IRBM, such as to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplification of the SEQ ID NOSs in Table 4 is not to be construed as limiting. For example, the particular domain, such as the ECD or IgV domain, can be several amino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO.

[0312] In some embodiments, the IRBM is or contains a wild-type ECD set forth in SEQ ID NO: 47 or a variant ECD sequence set forth in any one of SEQ ID NOS: 311-331, 375-471, 1551-1622. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the ECD sequences set forth in any one of SEQ ID NOS: 311-331, 375-471, 1551-1622 and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified CD155 ECD, e.g. not present in SEQ ID NO:47. In some embodiments, the IRBM is or contains a specific binding fragment of any of the ECD sequences set forth in any one of SEQ ID NOS: 311-331, 375-471, 1551-1622 and that contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified CD 155 ECD, e.g. not present in SEQ ID NO:47.

[0313] In some embodiments, the IRBM is or contains a wild-type CD155 IgV set forth in SEQ ID NO: 310 or 353 or a variant IgV set forth in any one of SEQ ID NOS: 332-352, 354374, 472-665, 1505-1550, 1575-1576, 1623-1714. In some embodiments, the IRBM is or contains a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences set forth in any one of SEQ ID NOS: 332-352, 354-374, 472-665, 1505-1550, 1575-1576, 1623-1714 and 2024204559   01 Jul 2024 contains the amino acid modification(s), e.g. substitution(s), not present in the wild-type or unmodified CD155 IgV, e.g. not present in SEQ ID NO: 310 or 353. In some embodiments, the IRBM is or contains a specific binding fragment of any of the IgV sequences set forth in any one of SEQ ID NOS: 332-352, 354-374, 472-665, 1505-1550, 1575-1576, 1623-1714 and that contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified CD155 IgV, e.g. not present in SEQ ID NO: 310 or 353. TABLE 4: Exemplary CD155 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO Wild-type 47 310, 353 P18S, P64S, F91S 311 332, 354 P18S, F91S, L104P 312 333, 355 L44P 313 334, 356 A56V 314 335, 357 P18L, L79V, F91S 315 336, 358 P18S, F91S 316 337, 359 P18T, F91S 317 338, 360 P18T, S42P, F91S 318 339, 361 G7E, P18T, Y30C, F91S 319 340, 362 P18T,F91S,G111D 320 341, 363 P18S, F91P 321 342, 364 P18T, F91S, F108L 322 343, 365 P18T, T45A, F91S 323 344, 366 P18T, F91S, R94H 324 345, 367 P18S, Y30C, F91S 325 346, 368 A81V, L83P 326 347, 369 L88P 327 348, 370 R94H 328 349, 371 AI3E. P18S, A56V, F91S 329 350, 372 P18T, F91S, V115A 330 351, 373 P18T, Q60K 331 352, 374 S52M 375 472, 569 T45Q, S52L, L104E, G111R 376 473, 570 S42G 377 474, 571 Q62F 378 475, 572 S52Q 379 476, 573 S42A, L104Q, G111R 380 477, 574 S42A, S52Q, L104Q, G111R 381 478, 575 S52W, L104E 382 479, 576 S42C 383 480, 577 S52W 384 481,578 S52M, L104Q 385 482, 579 S42L, S52L, Q62F, L104Q 386 483, 580 2024204559   01 Jul 2024 TABLE 4: Exemplary CD155 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO S42W 387 484,581 S42Q 388 485, 582 S52L 389 486, 583 S52R 390 487, 584 L104E 391 488, 585 G111R 392 489, 586 S52E 393 490, 587 Q62Y 394 491,588 T45Q, S52M, L104E 395 492, 589 S42N, L104Q, G111R 396 493, 590 S52M, V57L 397 494, 591 S42N, S52Q, Q62F 398 495, 592 S42A, S52L, L104E, G111R 399 496, 593 S42W, S52Q, V57L, Q62Y 400 497, 594 L104Q 401 498, 595 S42L, S52Q, L104E 402 499, 596 S42C, S52L 403 500, 597 S42W, S52R, Q62Y, L104Q 404 501,598 T45Q, S52R, L104E 405 502, 599 S52R, Q62F, L104Q, G111R 406 503, 600 T45Q, S52L, V57L, L104E 407 504, 601 S52M, Q62Y 408 505, 602 Q62F, L104E, G111R 409 506, 603 T45Q, S52Q 410 507, 604 S52L, L104E 411 508, 605 S42V,S52E 412 509, 606 T45Q, S52R, G111R 413 510, 607 S42G, S52Q, L104E, G111R 414 511,608 S42N, S52E, V57L, L104E 415 512, 609 S42C, S52M, Q62F 416 513,610 S42L 417 514,611 S42A 418 515,612 S42G, S52L, Q62F, L104Q 419 516,613 S42N 420 517,614 P18T, S65A, S67V, F91S 421 518,615 P18F, T39A, T45Q, T61R, S65N, S67L, E73G, R78G 422 519,616 P18T, T45Q, T61R, S65N, S67L 423 520, 617 P18F, S65A, S67V, F91S 424 521,618 P18F, T45Q, T61R, S65N, S67L, F91S, L104P 425 522, 619 P18S, L79P, L104M 426 523, 620 P18S, L104M 427 524, 621 L79P, L104M 428 525, 622 P18T, T45Q, L79P 429 526, 623 2024204559   01 Jul 2024 TABLE 4: Exemplary CD155 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO P18T, T45Q, T61R, S65H, S67H 430 527, 624 P18T, A81E 431 528, 625 P18S, D23Y, E37P, S52G, Q62M, G80S, A81P, G99Y, S112N 432 529, 626 A13R, D23Y, E37P, S42P, Q62Y, A81E 433 530, 627 A13R, D23Y, E37P, G99Y, S112N 434 531,628 A13R, D23Y, E37P, Q62M, A77V, G80S, A81P, G99Y 435 532, 629 P18L, E37S, Q62M, G80S, A81P, G99Y, S112N 436 533, 630 P18S, L104T 437 534, 631 P18S, Q62H, L79Q, F91S 438 535, 632 T45Q, S52K, Q62F, L104Q, G111R 439 536, 633 T45Q, S52Q, Q62Y, L104Q, G111R 440 537, 634 T45Q, S52Q, Q62Y, L104E, G111R 441 538, 635 V57A, T61M, S65W, S67A, E96D, L104T 442 539, 636 P18L, V57T, T61S, S65Y, S67A, L104T 443 540, 637 P18T, T45Q 444 541,638 P18L, V57A, T61M, S65W, S67A, L104T 445 542, 639 T61M, S65W, S67A, L104T 446 543, 640 P18S, V41A, S42G, T45G, L104N 447 544, 641 P18H, S42G, T45I, S52T, G53R, S54H, V57L, H59E, T61S, S65D, E68G, L104N 448 545, 642 P18S, S42G, T45V, F58L, S67W, L104N 449 546, 643 P18S, T45I, L104N 450 547, 644 P18S, S42G, T45G, L104N, V106A 451 548, 645 P18H, H40R, S42G, T45I, S52T, G53R, S54H, V57L, H59E, T61S, S65D, E68G, L104Y, V106L, F108H 452 549, 646 E37V, S42G, T45G, L104N 453 550, 647 P18S, T45Q, L79P, L104T 454 551,648 P18L, Q62R 455 552, 649 A13R, D23Y, E37P, S42L, S52G, Q62Y, A81E 456 553, 650 P18L, H49R, L104T, D116N 457 554, 651 A13R, D23Y, E37P, Q62M, G80S, A81P, L104T 458 555, 652 S65T, L104T 459 556, 653 A13R, D23Y, E37P, S52G, V57A, Q62M, K70E, L104T 460 557, 654 P18L, A47V, Q62Y, E73D, L104T 461 558, 655 H40T, V41M, A47V, S52Q, Q62L, S65T, E73R, D97G, E98S, L104T, D116N 462 559, 656 P18L, S42P, T45Q, T61G, S65H, S67E, L104T, D116N 463 560, 657 P18S, H40T, V41M, A47V, S52Q, Q62L, S65T, E73R, L104M, V106A 464 561,658 H40T, V41M, A47V, S52Q, Q62L, S65T, E68G, E73R, D97G, E98S, L104T 465 562, 659 T45Q, S52E, L104E 466 563, 660 T45Q, S52E, Q62F, L104E 467 564, 661 P18F, T26M, L44V, Q62K, L79P, F91S, L104M, Gil ID 468 565, 662 P18S, T45S, T61K, S65W, S67A, F91S, G111R 469 566, 663 no 2024204559   01 Jul 2024 TABLE 4: Exemplary CD155 IRBMs containing an IgD or vlgD Mutation(s) ECD SEQ ID NO IgV SEQ ID NO P18S, L79P, L104M, T107M 470 567, 664 P18S, S65W, S67A, M90V, V95A, L104Q, G111R 471 568, 665 P18S, A47G, L79P, F91S, L104M, T107A, R113W 1551 1505, 1528 P18T, D23G, S24A, N35D, H49L, L79P, F91S, L104M, G111R 1552 1506, 1529 V9L, P18S, Q60R, V75L, L79P, R89K, F91S, L104E, G111R 1553 1507, 1530 P18S, H49R, E73D, L79P, N85D, F91S, V95A, L104M, G111R 1554 1508, 1531 Vil A, P18S, L79P, F91S, L104M, G111R 1555 1509, 1532 Vil A, P18S, S54R, Q60P, Q62K, L79P, N85D, F91S, T107M 1556 1510, 1533 P18T, S52P, S65A, S67V, L79P, F91S, L104M, G111R 1557 1511, 1534 P18T, M36T, L79P, F91S, G111R 1558 1512, 1535 D8G, P18S, M36I, V38A, H49Q, A76E, F91S, L104M, T107A, R113W 1559 1513, 1536 P18S, S52P, S65A, S67V, L79P, F91S, L104M, T107S, R113W 1560 1514, 1537 T15I, P18T, L79P, F91S, L104M, G111R 1561 1515, 1538 P18F, T26M, L44V, Q62K, L79P, E82D, F91S, L104M, Gil ID 1562 1516, 1539 P18T, E37G, G53R, Q62K, L79P, F91S, E98D, L104M, T107M 1563 1517, 1540 P18L, K70E, L79P, F91S, V95A, G111R 1564 1518, 1541 V9I, Q12K, P18F, S65A, S67V, L79P, L104T, G111R, SI 121 1565 1519, 1542 P18F, S65A, S67V, F91S, L104M, G111R 1566 1520, 1543 V9I, V10I, P18S, F20S, T45A, L79P, F91S, L104M, F108Y, G111R, SI 12V 1567 1521, 1544 V9L, P18L, L79P, M90I, F91S, T102S, L104...

Claims

WHAT IS CLAIMED:

1. An immunomodulatory protein comprising: (1) at least one inhibitory receptorbinding molecule (IRBM) that binds to an inhibitory receptor, wherein the inhibitory receptor is PD-1; and (2) at least one activating receptor binding molecule (ARBM) that binds to a ligand of an activating receptor,wherein the IRBM comprises a variant PD-L1 polypeptide comprising an N45D amino acid modification and one or more amino acid modifications selected from the group comprising I20L, K28E, D43G, L56Q, V58A, N78I, Q89R, and G101G-ins, with reference to numbering set forth in SEQ ID NO: 309, and wherein the variant PD-L1 polypeptide comprises a sequence of amino acids that is at least 90% identical to SEQ ID NO: 309, andwherein the ARBM comprises a variant CTLA-4 polypeptide comprising amino acid modification L63P and one or more amino acid modifications selected from the group consisting of G29W, T53S, M56K, N58S, Q82R, M87V, L98Q, and Y105L, with reference to numbering set forth in SEQ ID NO:36 or 2655, and wherein the variant CTLA-4 polypeptide comprises a sequence of amino acids that is at least 90% identical to SEQ ID NO: 36 or 2655.

2. The immunomodulatory protein of claim 1, wherein the ligand of the activatingreceptor is CD80, CD86, or ICOSL.

3. The immunomodulatory protein of claim 1 or 2, wherein the ARBM comprises atleast one IgSF domain of a binding partner of the ligand of the activating receptor, optionally wherein the ARBM only contains the extracellular domain of the IgSF domain or a specific binding fragment thereof of the binding partner of the ligand of the activating receptor.

4. The immunomodulatory protein of any one of claims 1-3, wherein the variantCTLA-4 polypeptide comprises (i) the sequence of amino acids set forth in SEQ ID NO: 36, 2655 or 2947, (ii) a sequence of amino acids that has at least 91%, 92%, 93%, 94%, 95%, 96%,2024204559   07 Aug 202697%, 98%, or 99% sequence identity to SEQ ID NO: 36, 2655 or 2947; or (iii) a specific binding fragment of (i) or (ii).

5. The immunomodulatory protein of claim 4, wherein the variant CTLA-4polypeptide further comprises one or more modifications at one or more positions selected from 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 31, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 64, 65, 67, 69, 71, 72, 73, 75, 76, 82, 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118, 119, 120, 121 and 122, with reference to numbering set forth in SEQ ID NO:36 or 2655.

6. The immunomodulatory protein of claim 4 or 5, wherein the variant CTLA-4polypeptide further comprises one or more amino acid modifications selected from A6T, V10A, L12F, L12H, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, A31Y, E33M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y105F, Y105L, L106E, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117M, I117T, I118T, and I118V, or a conservative amino acid substitution thereof, with reference to numbering set forth in SEQ ID NO: 36 or 2655.

7. The immunomodulatory protein of any one of claims 4-6, wherein the variantCTLA-4 polypeptide comprises amino acid modification(s) selected from L12P / A26T / L63P / L98Q / M99L / Y105L, L63P / S72G / L98Q / M99L / Y105L, S14N / R16C / I18T / M56K / T61A / L63P / A86T / M99L, S27P / M56K / L63P / S72G / S73R / T89A / M99L / Y105L / I117M, M56K / L63P / N75D / V96I / M99L / Y105L / L106I, L63P / S72G / Y105L,2024204559   07 Aug 2026A26T / L63P / S72G / L98Q / Y105L / L106I / I117L, L63P / L98Q / V116A, V22I / L63P / L98Q / Y105L / I117M, R16C / G29W / E33V / M55T / L63P / L98Q / Y105L, L12P / A26T / L63P / L98Q / Y105L / L106I, M56L / L63P / L98Q / Y105L / L106I / I117L, M55T / L63P / T71I / M99L / Y105L, A26T / T53S / M56K / L63P / L98Q / Y105L, I18T / A26T / L63P / Q82R / L98Q / Y105L, I18T / L63P / S72G / L98Q / Y105L / I108V, I18T / E48R / L63P / T69S / L98Q / Y105L / N110Y, I18N / L63P / S72T / M87T / L98Q / Y105L / N110S, G29W / M56T / L63P / L98Q / Y105L / L106I / I117L, L63P / L98Q, V10A / L63P / L98Q / Y105L, A26T / L63P / M87V / N110K / I117E, A26T / V46E / L63P / D65G / L98Q, G29W / E59G / L63P / L98Q / Y105L, L12H / L63P / S72G / L98Q / Y105L, L12P / G29W / D43N / N58S / L63P / L98Q / M99L / Y105L, E24Q / L63P / S72G / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L, E33M / L63P / S72G / L98Q / Y105L, L63P / S72G / M87A / L98Q / Y105L, V22A / L63P / L98Q / M99L / Y105L / P119H, I18A / L63P / S72G / L98Q / Y105L, G29W / M55V / E59G / L63P / L98Q / Y105L, L12P / G29W / L63P / S72G / L98Q / Y105L / L106I, G29W / L63P / S72G / L98Q / Y105L / L106I / I117L, G29W / N58S / L63P / S72G / L98Q / Y105L / L106I, G29W / T53S / L63P / S72G / L98Q / Y105L, N58S / L63P / T71A / S72G / L98Q / M99L / Y105L / D124I / S125P / D126T, R16G / E33M / N58S / E59G / L63P / L98Q / Y105L / E120D / C122P / D124P / S125I / D126P, or G29W / L63P / S72G / L98Q / Y105L / P121S / D126T with reference to numbering set forth in SEQ ID NO:36 or 2655.

8. The immunomodulatory protein of any one of claims 4-7, wherein the variantCTLA-4 polypeptide comprises the sequence of amino acids set forth in any of SEQ ID NOs: 2593, 2596, 2958, 2959, 2960, 2961, 2962, 2965, 2966, 2971, 2976, 2977, 2978, 2984, 2985,2986, 2988, 2995, 2996, 2997, 3003, 3007, 3009, 3011, 3013, 3014, 3018, 3023, 3025, 3029,3032, 3034, 3052, 3057, 3062, 3063, 3064, 3098, 3583, 3584, 3585, or a specific bindingfragment thereof.

9. The immunomodulatory protein of any one of claims 1-8, wherein the IRBMcomprises at least one IgSF domain of a binding partner of the inhibitory receptor, optionally wherein the IRBM only contains the extracellular domain of the IgSF domain or a specific binding fragment thereof of the binding partner of the inhibitory receptor, optionally wherein the 4012024204559   07 Aug 2026IgSF domain comprises an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both.

10. The immunomodulatory protein of any one of claims 1-9, wherein the variant PD-L1 polypeptide comprises (i) the sequence of amino acids set forth in SEQ ID NO: 30, 309 or 1728, (ii) a sequence of amino acids that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 309 or 1728; or (iii) a specific binding fragment of (i) or (ii).

11. The immunomodulatory protein of claim 10, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications at one or more positions selected from 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177,179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,206, 207, 213, or 221, with reference to numbering set forth in SEQ ID NO: 30, 309 and 1728.

12. The immunomodulatory protein of claim 10 or 11, wherein the variant PD-L1polypeptide comprises one or more amino acid modifications selected from P6S, Y10F, V11A, V11E, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, I46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, G101G-ins (G101GG), G102D, A103V, D104G, K106E, K106R, V110M, K111E, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171R, F173I, F173L, K173Y, V175A, S177C, L179P,2024204559   07 Aug 2026R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, and T221L, or a conservative amino acid substitution thereof, with reference to numbering set forth in SEQ ID NO: 30, 309 or 1728.

13. The immunomodulatory protein of any one of claims 10-12, wherein the variant PD-L1 polypeptide comprises amino acid modification(s) selected from I20L / I36T / N45D / I47T, V11E / I20L / I36T / N45D / H60R / S75P, I20L / K28E / D43G / N45D / E53G / V58A / N78I, I20L / D43G / N45D / V58A / N78I / D90G / G101D, I20L / M41K / D43G / N45D, N45D / Q89R / I98V, N45D / Q89R / F189S / P198S, M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, or M41K / D43G / K44E / N45D / R195G / N201D, with reference to numbering set forth in SEQ ID NO: 30, 309 or 1728.

14. The immunomodulatory protein of any one of claims 10-13, wherein the variant PD-L1 polypeptide comprises the sequence of amino acids set forth in SEQ ID NOs: 245, 264, 168, 299, 302, 307, 1936, 1937, 122, 131, 141, 147, 166, 167, 169, 172, 174, 175, 176, 177, 178, 179, 180, 181, 182, 184, 1726, 1921, 1889, 1992, 1906, 1800, 1813, 1817, or a specific binding fragment thereof.

15. The immunomodulatory protein of any one of claims 1-14, wherein the ARBM binds to a ligand of an activating receptor that is CD80, CD86, or ICOSL and the IRBM binds to an inhibitory receptor that is PD-1.

16. The immunomodulatory protein of any one of claims 1-15, wherein the at least one ARBM comprises only one ARBM, or wherein the at least one IRBM comprises only one IRBM.

17. The immunomodulatory protein of any one of claims 1-15, wherein the at least one ARBM comprises 2, 3, 4, 5 or more ARBMs, optionally wherein each ARBM is the same, 4032024204559   07 Aug 2026or wherein the at least one IRBM comprises 2, 3, 4, 5 or more IRBMs, optionally wherein each IRBM is the same.

18. The immunomodulatory protein of any one of claims 1-17, wherein the at least one IRBM and the at least one ARBM are linked directly or indirectly via a linker, optionally wherein the linker is a peptide linker.

19. The immunomodulatory protein of claim 18, wherein the peptide linker is selected from(i) [GxSy]z, where x is an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50,(ii) S(G4S)n, where n is an integer from 1 to 20,(iii) (GA)n, where n is an integer from 1 to 20,(iv) (A)n, where n is an integer from 1 to 20,(v) (EAAAK)n, where n is an integer from 1 to 20,(vi) GGGGS (SEQ ID NO: 1942), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 240), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 239), 4xGGGGS, 5xGGGGS, GSGGS (SEQ ID NO: 2523), GSGGGGS (SEQ ID NO: 1941), GGGGSSA (SEQ ID NO: 2524), GGGGSGGGGSAAA (SEQ ID NO:241), EAAAK (SEQ ID NO: 3240), 3xEAAAK (SEQ ID NO: 3241), 5xEAAAK (SEQ ID NO: 3242), and(vii) combinations thereof.

20. The immunomodulatory protein of any one of claims 1-19, wherein the at least one IRBM is amino-terminal to the at least one ARBM in the single polypeptide chain.

21. The immunomodulatory protein of any one of claims 1-19, wherein the at least one IRBM is carboxy-terminal to the at least one ARBM in the single polypeptide chain.2024204559   07 Aug 202622. The immunomodulatory protein of claim 20 or 21, wherein the single polypeptide chain comprises a multimerization domain, optionally wherein the multimerization domain is an Fc domain.

23. The immunomodulatory protein of claim 22, wherein the Fc domain is at least 85% identical to SEQ ID NO: 187, optionally wherein the one or more amino acid substitutions in the Fc domain are selected from C5S, L19A, L20E, G22A, E141D, M143L, and deletion of K232, with reference to numbering set forth in SEQ ID NO: 187.

24. The immunomodulatory protein of claim 22 or 23, wherein the Fc domain comprises the amino acid substitutions C5S, L19A, L20E, G22A, E141D, M143L and deletion of K232, optionally wherein the Fc region comprises the sequence of SEQ ID NO: 1715.

25. A nucleic acid molecule(s), encoding the immunomodulatory protein of any one of claims 1-24, optionally wherein the nucleic acid is a synthetic nucleic acid, optionally wherein the nucleic acid molecule is a cDNA.

26. A vector, comprising the nucleic acid molecule of claim 25, optionally wherein the vector is an expression vector, optionally wherein the vector is a mammalian expression vector or a viral vector.

27. A cell, comprising the nucleic acid of claim 25 or the vector of claim 26, optionally wherein the cell is a mammalian cell, optionally wherein the cell is a human cell.

28. A method of producing an immunomodulatory protein, comprising introducing the nucleic acid molecule of claim 25 or the vector of claim 26 into a host cell under conditions to express the protein in the cell, optionally wherein the method further comprises isolating or purifying the immunomodulatory protein from the cell.2024204559   07 Aug 202629. A pharmaceutical composition, comprising the immunomodulatory protein of any one of claims 1-24, optionally wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, optionally wherein the pharmaceutical composition is sterile.

30. An article of manufacture comprising the pharmaceutical composition of claim 29 in a vial or container, optionally wherein the vial or container is sealed.

31. A kit comprising the pharmaceutical composition of claim 29 or the article of manufacture of claim 30, and instructions for use.

32. A method of decreasing an immune response in a subject, comprising administering the immunomodulatory protein of any one of claims 1-24 or the pharmaceutical composition of claim 29 to the subject, optionally wherein modulating the immune response treats a disease or condition in the subject.

33. A method of treating a disease or condition in a subject, comprising administering the immunomodulatory protein of any one of claims 1-24 or the pharmaceutical composition of claim 29 to the subject, wherein the disease or condition is Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, a thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, an autoimmune hematological disease, an autoimmune demyelinating disease, an autoimmune disease involving a systemic autoimmune disorder, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, lupus erythematosus, celiac disease, Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Graves' disease, DeQuervains thyroiditis, Vasculitis, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis,2024204559   07 Aug 2026autoimmune thrombocytopenia, sympathetic opthalmia, chronic aggressive hepatitis, membranous glomerulopathy, primary idiopathic myxedema, chronic hepatitis, hypogonadism, pernicious anemia, autoimmune enteropathy syndrome, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sensoneural hearing loss, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, and idiopathic facial paralysis, giant cell arteritis (GCA), cancer, Addison’s Disease, Allergy, Alopecia Areata, Alzheimer’s, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), Arthritis, Atherosclerosis, Atherosclerotic plaque, autoimmune disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet’s Disease, Berger’s Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue / Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan’s Syndrome, Exopthalmos, Fibromyalgia, Goodpasture’s Syndrome, graft-related disease or disorder, GVHD, Hashimoto’s Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki’s Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, Lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere’s Disease, Miller Fish Syndrome / acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polychondritis, Polyglandular Syndromes (Whitaker’s syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis / Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud’s Phenomenon, Reiter’s Syndrome / Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt’s syndrome, Scleroderma, Sjorgen’s2024204559   07 Aug 2026Syndrome, Solid-organ transplant rejection, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis / Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Uveitis, Vitiligo, Wegener’s Granulomatosis, an immune response associated with rejection of a donor tissue, cell, graft, or organ transplant by a recipient subject, graft versus host disease (GVDH), or interstitial bowel disease.

34. Use of the immunomodulatory protein of any one of claims 1-24 or the pharmaceutical composition of claim 29 in the manufacture of a medicament for:decreasing an immune response in a subject; ortreating a disease or condition in a subject, wherein the disease or condition is Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, a thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, an autoimmune hematological disease, an autoimmune demyelinating disease, an autoimmune disease involving a systemic autoimmune disorder, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, lupus erythematosus, celiac disease, Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Graves' disease, DeQuervains thyroiditis, Vasculitis, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune thrombocytopenia, sympathetic opthalmia, chronic aggressive hepatitis, membranous glomerulopathy, primary idiopathic myxedema, chronic hepatitis, hypogonadism, pernicious anemia, autoimmune enteropathy syndrome, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sensoneural hearing loss, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, and idiopathic facial paralysis, giant cell arteritis (GCA), cancer, Addison’s Disease, Allergy, Alopecia Areata, Alzheimer’s, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), Arthritis, Atherosclerosis, Atherosclerotic plaque, autoimmune disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis,2024204559   07 Aug 2026Azoospermia, Behcet’s Disease, Berger’s Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue / Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan’s Syndrome, Exopthalmos, Fibromyalgia, Goodpasture’s Syndrome, graft-related disease or disorder, GVHD, Hashimoto’s Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki’s Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, Lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere’s Disease, Miller Fish Syndrome / acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polychondritis, Polyglandular Syndromes (Whitaker’s syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis / Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud’s Phenomenon, Reiter’s Syndrome / Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt’s syndrome, Scleroderma, Sjorgen’s Syndrome, Solid-organ transplant rejection, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis / Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Uveitis, Vitiligo, Wegener’s Granulomatosis, an immune response associated with rejection of a donor tissue, cell, graft, or organ transplant by a recipient subject, graft versus host disease (GVDH), or interstitial bowel disease.

Images