Immunomodulatory protein with tunable affinity

Abstract

To provide immunomodulatory proteins that provide therapeutic utility for a variety of immunological and oncological conditions.SOLUTION: There are provided immunomodulatory proteins that exhibit altered binding affinities to binding partners that are immune protein ligands involved in immunological responses, comprising at least one affinity modified non-immunoglobulin IgSF domain comprising one or more amino acid substitution(s) in a wild-type immunoglobulin superfamily (IgSF) domain.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 149,437, entitled "Immunomodulatory Proteins with Tunable Affinities," filed April 17, 2015, and U.S. Provisional Patent Application No. 62 / 218,534, entitled "Immunomodulatory Proteins with Tunable Affinities," filed September 14, 2015, the contents of each of which are incorporated by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format, which is provided as a 377 kilobyte file entitled 761612000140SEQLIST.TXT, created on April 15, 2016. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present invention relates to therapeutic proteins for modulating immune responses in the treatment of cancer and immune disorders. [Background technology]

[0004] background There is growing medical interest in modulating immune responses by intervening in processes occurring at the immune synapse (IS) formed between antigen-presenting cells (APCs) or target cells and lymphocytes. Currently, biologics used to enhance or suppress immune responses are generally limited to immunoglobulins (e.g., anti-PD-1 mAb) or soluble receptors (e.g., Fc-CTLA4). Soluble receptors suffer from numerous deficiencies. While useful for antagonizing protein-protein interactions, they often lack the ability to stimulate such interactions. Antibodies have proven less limited in this regard, and examples of both agonist and antagonist antibodies are known in the art. Nevertheless, both soluble receptors and antibodies lack key attributes essential for functioning at the IS. Mechanistically, cell surface proteins at the IS can participate in the cooperative and often simultaneous interactions between multiple protein targets and a single protein to which they bind. IS interactions occur in close association with the adhesion of two cells, and a single protein in this structure can interact (possibly simultaneously) with proteins on the same cell (cis) and with proteins on related cells (trans). Thus, there is a need for improved molecules for modulating immune responses. Embodiments that meet this need are provided. Summary of the Invention

[0005] overview Provided herein are immunomodulatory proteins that exhibit altered binding affinity to binding partners that are immune protein ligands involved in immune responses. In some embodiments, the provided immunomodulatory proteins can modulate (e.g., enhance or reduce) the activity of the immune protein ligand, thereby modulating the immune response. In some embodiments, methods and uses are also provided for modulating immune responses by contacting cells that express one or more immune protein ligands with the provided immunomodulatory proteins, for example, in immunotherapeutic treatment of diseases or conditions treatable by modulating the immune response.

[0006] In some embodiments, provided herein are immunomodulatory proteins comprising at least one affinity-modified non-immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type IgSF domain, wherein: the at least one affinity-modified IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type IgSF domain; and the at least one affinity-modified IgSF domain specifically binds to the at least two cognate binding partners in a non-competitive manner. In some embodiments, the at least two cognate binding partners are cell surface molecular species expressed on the surface of a mammalian cell. In some embodiments, the cell surface molecular species are expressed in a cis or trans configuration. In some embodiments, the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and each of the cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS. In some embodiments, at least one of the mammalian cells is a lymphocyte. In some embodiments, the lymphocyte is an NK cell or a T cell. In some embodiments, binding of the affinity-engineered IgSF domain modulates the immune activity of lymphocytes.

[0007] In some embodiments, the immunomodulating protein can confer improved immune activity compared to a wild-type protein comprising a wild-type IgSF domain. In some embodiments, the immunomodulating protein can confer reduced immune activity compared to a wild-type protein comprising a wild-type IgSF domain. In some embodiments, at least one of the mammalian cells is a tumor cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the affinity-engineered IgSF domain can specifically bind to two mammalian cells that form an IS.

[0008] In some embodiments of any one of the above immunomodulatory proteins, the wild-type IgSF domain is selected from the group consisting of the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling lymphocytic activation molecule (SLA) family, the IgSF domain, and the IgSF domain. It is derived from an IgSF family member of a family selected from the following: the SLAM family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor related (PVR) family, the natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the killer-cell immunoglobulin-like receptor (KIR) family.In some embodiments, the wild-type IgSF domain is derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30. In some embodiments, the wild-type IgSF domain is a human IgSF member.

[0009] In some embodiments of any one of the above immunomodulatory proteins, the wild-type IgSF domain is an IgV domain, an IgC1 domain, an IgC2 domain, or a specific-binding fragment thereof. In some embodiments, the affinity-modified IgSF domain is an affinity-modified IgV domain, an affinity-modified IgC1 domain, or an affinity-modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions.

[0010] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein comprises at least two affinity-modified non-immunoglobulin IgSF domains. In some embodiments, each of the affinity-modified non-immunoglobulin IgSF domains binds to a different cognate binding partner, wherein the two affinity-modified IgSF domains specifically bind to the at least two different cognate binding partners in a non-competitive manner. In some embodiments, the at least two affinity-modified non-immunoglobulin IgSF domains each comprise one or more different amino acid substitutions in the same wild-type IgSF domain. In some embodiments, the at least two affinity-modified non-immunoglobulin IgSF domains each comprise one or more amino acid substitutions in different wild-type IgSF domains. In some embodiments, the different wild-type IgSF domains are derived from different IgSF family members.

[0011] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein comprises only one affinity-engineered non-immunoglobulin IgSF domain.

[0012] In some embodiments of any one of the above immunomodulating proteins, the affinity-modified IgSF comprises at least 85% sequence identity to a wild-type IgSF domain, or a specific-binding fragment thereof, contained in the amino acid sequence set forth in any of SEQ ID NOs: 1-27. In some embodiments, the immunomodulating protein further comprises a second affinity-modified IgSF domain, wherein the second affinity-modified IgSF domain comprises at least 85% sequence identity to a wild-type IgSF domain, or a specific-binding fragment thereof, contained in the amino acid sequence set forth in any of SEQ ID NOs: 1-27.

[0013] In some embodiments of any one of the above immunomodulatory proteins, the wild-type IgSF domain is a member of the B7 family. In some embodiments, the wild-type IgSF domain is a domain of CD80, CD86, or ICOSLG. In some embodiments, the wild-type IgSF domain is a domain of CD80.

[0014] In some embodiments of any one of the above immunomodulatory proteins, provided herein are immunomodulatory proteins comprising at least one affinity-modified CD80 immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type CD80 IgSF domain, wherein the at least one affinity-modified CD80 IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type CD80 IgSF domain.

[0015] In some embodiments of any one of the above immunomodulatory proteins, the cognate binding partners are CD28 and PD-L1. In some embodiments, the wild-type IgSF domain is an IgV domain and / or the affinity-engineered CD80 domain is an affinity-engineered IgV domain. In some embodiments, the affinity-engineered domain comprises at least 85% sequence identity to the wild-type CD80 domain contained in the amino acid sequence set forth in SEQ ID NO:1, or a specific-binding fragment thereof.

[0016] In some embodiments of any one of the above immunomodulatory proteins, the at least one affinity-altered IgSF domain comprises between one and 20 amino acid substitutions in the wild-type IgSF domain. In some embodiments, the at least one affinity-altered IgSF domain comprises between one and 10 amino acid substitutions in the wild-type IgSF domain. In some embodiments, the at least one affinity-altered IgSF domain comprises between one and 5 amino acid substitutions in the wild-type IgSF domain.

[0017] In some embodiments of any one of the above immunomodulatory proteins, the affinity-modified IgSF domain has at least 120% of the binding affinity of its wild-type IgSF domain for each of at least two cognate binding partners.

[0018] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein further comprises an IgSF domain that has not been affinity modified.

[0019] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein lacks a transmembrane domain or a cytoplasmic domain. In some embodiments, the immunomodulatory protein comprises only an extracellular domain (ECD) or a specific binding fragment thereof, comprising an affinity-engineered IgSF domain.

[0020] In some embodiments of any one of the above immunomodulating proteins, the immunomodulating protein is glycosylated or pegylated.

[0021] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is linked to a multimerization domain. In some embodiments, the immunomodulatory protein is linked to an Fc domain or variant thereof with reduced effector function. In some embodiments, the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof with reduced effector function. In some embodiments, the Fc domain is mammalian (optionally human); or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified Fc domain that is mammalian (optionally human). In some embodiments, the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ ID NO:227.

[0022] In some embodiments of any one of the above immunomodulating proteins, the immunomodulating protein is indirectly linked via a linker.

[0023] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is a dimer.

[0024] In some embodiments of any one of the above immunomodulating proteins, the immunomodulating protein is bound to the liposome membrane.

[0025] Also provided are immunomodulatory proteins comprising at least two non-immunoglobulin immunoglobulin superfamily (IgSF) domains, wherein at least one is an affinity-modified IgSF domain, and the at least two non-immunoglobulin IgSF domains each independently bind to at least one different binding partner. In some embodiments, the at least two non-immunoglobulin IgSF domains bind non-competitively to the different binding partners. In some embodiments, the affinity-modified IgSF domain contains one or more amino acid substitutions in a first wild-type or unmodified IgSF domain. In some embodiments, the others of the IgSF domains are wild-type or unmodified IgSF domains. In some embodiments, the others of the IgSF domains are also affinity-modified IgSF domains.

[0026] In some embodiments of any of the above immunomodulatory proteins, the immunomodulatory protein contains at least two affinity-modified non-immunoglobulin IgSF domains (a first affinity-modified IgSF domain and a second affinity-modified IgSF domain), wherein the first modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a first wild-type IgSF domain and the second modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a second wild-type IgSF domain, and wherein the first and second modified non-immunoglobulin IgSF domains each specifically bind to at least one different cognate binding partner. In some embodiments, the at least two non-immunoglobulin IgSF domains bind non-competitively to the different binding partners. In some embodiments, the first and second modified non-immunoglobulin IgSF domains are affinity-modified so as to exhibit altered binding to their cognate binding partners. Thus, in some embodiments, the first and second modified non-immunoglobulin IgSF domains are affinity-modified IgSF domains. In some embodiments, the first modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the first wild-type IgSF domain; and the second modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the second wild-type IgSF domain. In some embodiments, the altered binding is independently either improved or decreased.

[0027] In some embodiments, the different cognate binding partners are cell surface molecular species expressed on the surface of a mammalian cell. In some embodiments, the different cell surface molecular species are expressed in cis or trans configuration. In some embodiments, the mammalian cell is one of two mammalian cells forming an immune synapse (IS), and the different cell surface molecular species is expressed on the surface of at least one of the two mammalian cells forming the IS. In some embodiments, at least one of the mammalian cells is a lymphocyte. In some embodiments, the lymphocyte is an NK cell or a T cell. In some embodiments, binding of the immunomodulatory protein to the cell regulates the immune activity of the lymphocyte. In some embodiments, the immunomodulatory protein can result in improved immune activity compared to a wild-type protein comprising a wild-type IgSF domain. In some embodiments, the immunomodulatory protein can result in reduced immune activity compared to a wild-type protein comprising a wild-type IgSF domain. In some embodiments, at least one of the mammalian cells is a tumor cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the immunomodulatory protein can specifically bind to two mammalian cells forming an IS.

[0028] In some embodiments of any one of the above immunomodulatory proteins, the first and second modified IgSF domains each comprise one or more amino acid substitutions in a different wild-type IgSF domain. In some embodiments, the different wild-type IgSF domains are derived from different IgSF family members. In some embodiments, the first and second modified IgSF domains are a non-wild-type combination.In some embodiments, the first wild-type IgSF domain and the second wild-type IgSF domain each individually are selected from the group consisting of the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling Lymphocyte Activation Molecule (SLA) family, the IgSF domain ... It is derived from an IgSF family member of a family selected from the following: the SLAM family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor related (PVR) family, the natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the killer-cell immunoglobulin-like receptor (KIR) family.In some embodiments, the first wild-type IgSF domain and the second wild-type IgSF domain are each individually derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

[0029] In some embodiments of any one of the above immunomodulatory proteins, the first modified IgSF domain and the second modified IgSF domain each individually comprise at least 85% sequence identity to a wild-type IgSF domain, or a specific-binding fragment thereof, contained in an amino acid sequence set forth in any of SEQ ID NOs:1-27.

[0030] In some embodiments of any one of the above immunomodulatory proteins, the first and second wild-type IgSF domains are each individually a member of the B7 family. In some embodiments, the first and second wild-type IgSF domains are each individually derived from CD80, CD86, or ICOSLG. In some embodiments, the first or second wild-type IgSF domain is derived from a member of the B7 family, and the other of the first or second wild-type IgSF domain is derived from another IgSF family member.

[0031] In some embodiments of any one of the above immunomodulatory proteins, the first and second wild-type IgSF domains are derived from ICOSLG and NKp30.

[0032] In some embodiments of any one of the above immunomodulatory proteins, the first and second wild-type IgSF domains are derived from CD80 and NKp30.

[0033] In some embodiments of any one of the above immunomodulatory proteins, the first and second wild-type IgSF domains are each individually a human IgSF member.

[0034] In some embodiments of any one of the above immunomodulatory proteins, the first and second wild-type IgSF domains are each independently an IgV domain and an IgC1 domain, an IgC2 domain, or a specific combination thereof. In some embodiments, the first modified non-immunoglobulin domain and the second modified non-immunoglobulin domain are each independently a modified IgV domain, a modified IgC1 domain, or a modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions. In some embodiments, at least one of the first modified non-immunoglobulin domain or the second modified non-immunoglobulin domain is a modified IgV domain. In some embodiments, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each independently comprise between one and 20 amino acid substitutions, inclusive. In some embodiments, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each independently comprise between one and 10 amino acid substitutions, inclusive. In some embodiments, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between one and five amino acid substitutions.

[0035] In some embodiments of any one of the above immunomodulatory proteins, at least one of the first or second modified non-immunoglobulin IgSF domains has 10% to 90% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners. In some embodiments, at least one of the first or second modified non-immunoglobulin IgSF domains has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

[0036] In some embodiments of any one of the above immunomodulatory proteins, the first and second modified non-immunoglobulin IgSF domains each individually have at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

[0037] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is soluble.

[0038] In some embodiments of any one of the above immunomodulating proteins, the immunomodulating protein is glycosylated or pegylated.

[0039] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is linked to a multimerization domain. In some embodiments, the immunomodulatory protein is linked to an Fc domain or variant thereof with reduced effector function. In some embodiments, the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof with reduced effector function. In some embodiments, the Fc domain is mammalian (optionally human); or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified Fc domain that is mammalian (optionally human). In some embodiments, the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ ID NO:227.

[0040] In some embodiments of any one of the above immunomodulatory proteins, the variant CD80 polypeptide is indirectly linked via a linker.

[0041] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein is a dimer.

[0042] In some embodiments of any one of the above immunomodulatory proteins, the immunomodulatory protein further comprises one or more additional non-immunoglobulin IgSF domains that are the same as or different from the first or second modified non-immunoglobulin IgSF domains, hi some embodiments, the one or more additional non-immunoglobulin IgSF domains are affinity-modified IgSF domains.

[0043] In some embodiments of any one of the above immunomodulating proteins, the immunomodulating protein is bound to the liposome membrane.

[0044] In some embodiments, provided herein is a nucleic acid molecule encoding an immunomodulatory polypeptide according to any one of the above embodiments. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is a cDNA.

[0045] In some embodiments, provided herein is a vector comprising the nucleic acid molecule according to any one of the above embodiments. In some embodiments, the vector is an expression vector.

[0046] In some embodiments, provided herein is a cell comprising a vector according to any one of the above embodiments. In some embodiments, the cell is a eukaryotic or prokaryotic cell.

[0047] In some embodiments, provided herein is a method for producing an immunomodulatory protein, the method comprising introducing a nucleic acid molecule according to any one of the above embodiments or a vector according to any one of the above embodiments into a host cell under conditions for expressing the protein in the cell. In some embodiments, the method further comprises isolating or purifying the immunomodulatory protein from the cell.

[0048] In some embodiments, provided herein is a pharmaceutical composition comprising an immunomodulatory protein according to any one of the above embodiments. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0049] In some embodiments, provided herein is an article of manufacture comprising a pharmaceutical composition according to any one of the above embodiments in a vial. In some embodiments, the vial is sealed.

[0050] In some embodiments, provided herein is a kit comprising a pharmaceutical composition according to any one of the above embodiments and instructions for use.

[0051] In some embodiments, provided herein is a kit comprising an article of manufacture according to any one of the above embodiments and instructions for use.

[0052] In some embodiments, provided herein are methods for modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an immunomodulatory protein according to any one of the above embodiments. In some embodiments, modulating the immune response treats a disease or condition in the subject. In some embodiments, the immune response is improved. In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, or hematological malignancies. In some embodiments, the immune response is reduced. In some embodiments, the disease or condition is an inflammatory disease or condition. In some embodiments, the disease or condition is selected from Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

[0053] In some embodiments, provided herein are methods for identifying immunomodulatory proteins with altered affinity, the methods comprising: a) contacting an altered protein comprising at least one altered non-immunoglobulin immunoglobulin superfamily (IgSF) domain or a specific-binding fragment thereof with at least two cognate binding partners under conditions capable of resulting in binding of the protein to the at least two cognate binding partners, wherein the at least one altered IgSF domain comprises one or more amino acid substitutions in the wild-type IgSF domain; b) identifying an altered protein comprising an altered IgSF domain that has improved binding to at least one of the two cognate binding partners compared to the protein comprising the wild-type IgSF domain; and c) selecting an altered protein comprising an altered IgSF domain that binds non-competitively to the at least two cognate binding partners, thereby identifying an immunomodulatory protein with altered affinity. In some embodiments, step b) comprises identifying an altered protein comprising an altered IgSF domain that has improved binding to each of the at least two cognate binding partners compared to the protein comprising the wild-type domain. In some embodiments, prior to step a), one or more amino acid substitutions are introduced into the wild-type IgSF domain, thereby generating an engineered protein comprising the engineered IgSF domain. In some embodiments, the engineered protein comprises at least two engineered IgSF domains or specific-binding fragments thereof, wherein a first IgSF domain comprises one or more amino acid substitutions in the first wild-type IgSF domain and a second affinity-engineered non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in the second wild-type IgSF domain. In some embodiments, the first and second affinity-engineered non-immunoglobulin IgSF domains each specifically bind to at least one different cognate binding partner.

[0054] In some embodiments, immunomodulatory proteins are also provided that comprise at least one affinity-engineered non-immunoglobulin immunoglobulin superfamily (IgSF) domain. In some embodiments, the affinity-engineered IgSF domain specifically binds to at least two cell surface molecular species in a non-competitive manner. In some embodiments, each of the molecular species is expressed on the surface of at least one of two mammalian cells that form an immune synapse (IS). In some embodiments, the molecular species are in a cis or trans configuration. In some embodiments, one of the mammalian cells is a lymphocyte, and binding of the affinity-engineered IgSF domain modulates the immune activity of the lymphocyte. In some embodiments, the affinity-engineered IgSF domain specifically binds to two mammalian cells that form an IS.

[0055] In some embodiments, the immunomodulatory protein comprises at least two affinity-engineered non-immunoglobulin IgSF domains, and the immunomodulatory protein specifically binds to two mammalian cells that form an IS. In some embodiments, the immunomodulatory protein comprises at least two affinity-engineered IgSF domains, wherein the affinity-engineered IgSF domains are not IgSF domains of the same species.

[0056] In some embodiments, one of the two mammalian cells is a tumor cell. In some embodiments, the lymphocyte is a NK cell or a T cell. In some embodiments, the mammalian cell is a mouse, rat, cynomolgus monkey, or human cell.

[0057] In some embodiments, the IgSF cell surface species is a human IgSF member.

[0058] In some embodiments, the immunomodulatory protein comprises an affinity-altered mammalian IgSF member. In some embodiments, the affinity-altered IgSF domain is an affinity-altered IgV, IgC1, or IgC2 domain. In some embodiments, the affinity-altered IgSF domain differs from its wild-type IgSF domain by between one and ten amino acid substitutions, inclusive. In some embodiments, the affinity-altered IgSF domain differs from its wild-type IgSF domain by between one and five amino acid substitutions, inclusive.

[0059] In some embodiments, the affinity-modified human IgSF member is at least one of CD80, PVR, ICOSLG, or HAVCR2. In some embodiments, the affinity-modified IgSF domain comprises at least one affinity-modified human CD80 domain. In some embodiments, the affinity-modified IgSF domain is a human CD80 IgSF domain.

[0060] In some embodiments, the immunomodulatory protein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof. In some embodiments, the immunomodulatory protein has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof. In some embodiments, the immunomodulatory protein has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof. In some embodiments, the immunomodulatory protein has at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof. In some embodiments, the immunomodulatory protein having at least 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof, further comprises a second immunomodulatory protein, wherein the second immunomodulatory protein has at least 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-27, or a fragment thereof.

[0061] In some embodiments, immune activity is enhanced. In other cases, it is suppressed.

[0062] In some embodiments, the affinity-modified IgSF domain has 10% to 90% of the binding affinity of the wild-type IgSF domain for at least one of two cell surface molecular species. In some embodiments, the affinity-modified IgSF domain specifically binds to exactly one IgSF member in a non-competitive manner. In some embodiments, the affinity-modified IgSF domain has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of two cell surface molecular species.

[0063] In some embodiments, the immunomodulatory protein is directly or indirectly covalently linked to the crystallizable fragment (Fc) of an antibody. In some embodiments, the immunomodulatory protein is glycosylated or pegylated. In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is embedded in a liposome membrane. In some embodiments, the immunomodulatory protein is dimerized by an intermolecular disulfide bond.

[0064] In some embodiments, the immunomodulatory protein is in a pharmaceutically acceptable carrier.

[0065] In another aspect, provided herein is an immunomodulatory protein comprising at least two affinity-modified non-immunoglobulin immunoglobulin superfamily (IgSF) domains. Each affinity-modified IgSF domain specifically binds to its own cell surface molecular species. Each molecular species is expressed on the surface of at least one of two mammalian cells forming an immune synapse (IS), and one of the mammalian cells is a lymphocyte. In some embodiments, the molecular species are in a cis or trans configuration. Binding of the affinity-modified IgSF domains regulates the immune activity of the lymphocyte. In some embodiments, at least one of the affinity-modified IgSF domains competitively binds. In some embodiments, the affinity-modified IgSF domains are not IgSF domains of the same species. In some embodiments, the affinity-modified IgSF domains are a non-wild-type combination. In some embodiments, the cell surface molecular species is a human IgSF member. In some embodiments, at least two affinity-engineered IgSF domains are derived from at least one of CD80, CD86, CD274, PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, NKp30, or CD200R1.

[0066] In some embodiments, the immunomodulatory protein comprises at least two affinity-engineered mammalian IgSF members. In some embodiments, the mammalian IgSF members are human IgSF members. In some embodiments, the mammalian IgSF members are at least two of CD80, CD86, CD274, PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, NKp30, or CD200R1. In some embodiments, immune activity is enhanced. In some embodiments, immune activity is suppressed. In some embodiments, one of the two mammalian cells is a tumor cell. In some embodiments, the lymphocyte is an NK cell or a T cell. In some embodiments, the mammalian cell is a mouse, rat, cynomolgus monkey, or human cell. In some embodiments, at least one of the two affinity-modified IgSF domains has 10% to 90% of the binding affinity of the wild-type IgSF domain for at least one of the cell surface molecular species. In some embodiments, at least one of the two affinity-modified IgSF domains specifically binds to exactly one cell surface molecular species. In some embodiments, at least one of the two affinity-modified IgSF domains has at least 120% of the binding affinity of its wild-type IgSF domain for at least one of the two cell surface molecular species. In some embodiments, the affinity-modified IgSF domain is at least one of an IgV, IgC1, or IgC2 domain. In some embodiments, each of the at least two affinity-modified IgSF domains differs from its wild-type IgSF domain by between one and ten amino acid substitutions. In some embodiments, each of the at least two affinity-modified IgSF domains differs from its wild-type IgSF domain by between one and five amino acid substitutions. In some embodiments, the immunomodulatory protein is covalently linked, directly or indirectly, to the crystallizable fragment (Fc) of the antibody.In some embodiments, the immunomodulatory protein is in a pharmaceutically acceptable carrier. In some embodiments, the immunomodulatory protein is glycosylated or pegylated. In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is embedded in a liposome membrane. In some embodiments, the immunomodulatory protein is dimerized by an intermolecular disulfide bond.

[0067] In another aspect, the present invention relates to recombinant nucleic acids encoding any of the immunomodulatory proteins summarized above.

[0068] In another aspect, the present invention relates to a recombinant expression vector comprising a nucleic acid encoding any of the immunomodulatory proteins summarized above.

[0069] In another aspect, the present invention relates to a recombinant host cell comprising an expression vector as summarized above.

[0070] In another aspect, the present invention relates to a method for producing any of the immunomodulatory proteins summarized above, the method comprising culturing recombinant host cells under immunomodulatory protein expression conditions, expressing in the cells an immunomodulatory protein encoded by a recombinant expression vector, and purifying the recombinant immunomodulatory protein thereby expressed.

[0071] In another aspect, the invention relates to a method of treating a mammalian patient in need of an enhanced or suppressed immune response by administering a therapeutically effective amount of an immunomodulatory protein of any of the above aspects. In some embodiments, the enhanced immune response treats melanoma, lung cancer, bladder cancer, or a hematological malignancy in the patient. In some embodiments, the suppressed immune response treats Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis in the patient. [Brief explanation of the drawings]

[0072] [Figure 1A] Figure 1 shows the results of a competitive binding assay for the binding of biotinylated recombinant CD28 Fc fusion protein (rCD28.Fc) to immobilized CD80 variant A91G ECD-Fc fusion molecules in the presence of unlabeled recombinant human PD-L1-his, human CTLA-4-his, or human PD-L2-Fc fusion proteins. [Figure 1B] Figure 1 shows the results of a competitive binding assay for the binding of biotinylated recombinant human PD-L1-his monomer protein to immobilized CD80 variant A91G ECD-Fc fusion molecules in the presence of unlabeled recombinant human rCD28.Fc, human CTLA-4.Fc, or human PD-L2.Fc.

[0073] INCORPORATION BY REFERENCE All publications (including patents, patent applications, scientific articles, and databases) mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication (including patents, patent applications, scientific articles, and databases) was specifically and individually indicated to be incorporated by reference. To the extent that a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference. DETAILED DESCRIPTION OF THE INVENTION

[0074] Detailed Description Provided herein are soluble immunomodulatory proteins that can bind to one or more (generally two or more) protein ligands to modulate (e.g., induce, enhance, or suppress) immunological immune responses. In some embodiments, the protein ligands are cell surface proteins expressed by immune cells that associate with one or more other immune receptors (e.g., on lymphocytes) to induce inhibitory or activating signals. For example, the interaction of certain receptors on lymphocytes with their cognate cell surface ligands to form an immune synapse (IS) between antigen-presenting cells (APCs) or target cells and lymphocytes can result in costimulatory or inhibitory signals that can control the immune system. In some aspects, the immunomodulatory proteins provided herein can alter the interaction between cell surface protein ligands and their receptors, thereby modulating the activity of immune cells (e.g., T cells).

[0075] In some embodiments, under normal physiological conditions, T cell-mediated immune responses are initiated by antigen recognition by T cell receptors (TCRs) and controlled by a balance of costimulatory and inhibitory signals (i.e., immune checkpoint proteins). The immune system relies on immune checkpoints to suppress autoimmunity (i.e., self-tolerance) and protect tissues from excessive damage during immune responses (e.g., during attacks against pathogen infections). However, in some cases, these immunomodulatory proteins may be deregulated as a mechanism to evade the immune system in diseases and conditions, including tumors.

[0076] Thus, in some respects, immunotherapies that alter immune cell activity, such as T cell activity, can treat certain diseases and conditions in which the immune response is deregulated. Therapeutic approaches that seek to modulate interactions at the IS would benefit from the ability to bind multiple IS targets simultaneously and in a manner that is sensitive to temporal and spatial orientation. Current therapeutic approaches fall short of this goal. Rather, soluble receptors and antibodies typically bind only one target protein at a time. This may be due to the lack of multiple target species. Furthermore, wild-type receptors and ligands possess low affinity for their cognate binding partners, which precludes their use as soluble therapeutics.

[0077] However, although not a major problem, soluble receptors and antibodies generally bind competitively (e.g., to only one target species at a time) and therefore lack the ability to simultaneously bind multiple targets. And, while bispecific antibodies and modalities containing two antigen-binding regions can simultaneously bind multiple target molecules, the three-dimensional configurations inherent to these modalities often prevent them from intervening in important processes occurring at the IS in a manner that meets their temporal and spatial requirements.

[0078] What is needed is an entirely new class of therapeutic molecules that possess the specificity and affinity of antibodies or soluble receptors, but that additionally maintain the size, volume, and spatial orientation constraints required at the IS and possess improved affinity for their cognate binding partners. Furthermore, such therapeutics would possess the ability to bind their targets noncompetitively and competitively. Molecules with these properties would therefore have novel functionality in their ability to integrate into multiprotein complexes at the IS and generate desired binding configurations and resulting biological activities.

[0079] To this end, emerging immuno-oncology therapeutics require the safe disruption of tumor-induced T cell tolerance. Current state-of-the-art immunotherapeutics block PD-1 or CTLA4, central inhibitory molecules of the B7 / CD28 family known to limit T cell effector function. Such single-target antagonist antibodies function to disrupt immune synapse checkpoint signaling complexes but are insufficient to simultaneously activate T cells. In contrast, bispecific antibody approaches activate T cells but are insufficient to simultaneously block the inhibitory ligands that regulate the induced signaling.

[0080] To address these shortcomings, in some embodiments, therapeutic molecules are provided that simultaneously stimulate T cell activation signaling and block inhibitory regulation. In some embodiments, the immunomodulatory proteins provided relate to the immunoglobulin superfamily (IgSF) component of the immune synapse, known for its dual role in both T cell activation and blocking inhibitory ligands. In certain aspects, the immunomodulatory proteins provided provide an immunotherapy platform using affinity-engineered natural immune ligands to generate immunotherapeutic biologics that bind with tunable affinity to one or more of their cognate immune receptors for the treatment of various oncological and immunological indications. In some aspects, IgSF-based therapeutics engineered from immune system ligands, such as human immune system ligands, are themselves more likely to retain their ability to normally assemble into critical pathways of the immune synapse and maintain normal interaction and regulatory functions in ways that antibodies or next-generation bispecific reagents cannot. This is due to the relatively large size of antibodies and the fact that they are not natural components of the immune synapse. These unique characteristics of human immune system ligands promise to provide new levels of immunotherapeutic efficacy and safety.

[0081] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0082] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or related terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as making a significant difference from what is commonly understood in the art.

[0083] Terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, 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 this invention belongs. Unless otherwise specified, abbreviations and symbols for chemical and biochemical names are in accordance with IUPAC-IUB nomenclature. Unless otherwise specified, all numerical ranges include not only the values ​​defining the range but also all integer values ​​therebetween.

[0084] The term "affinity-modified," when used in the context of an immunoglobulin superfamily domain, refers to a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence that has been altered such that it has improved or decreased binding affinity or avidity (relative to a wild-type control IgSF domain) for at least one of its cognate binding partners compared to a wild-type (i.e., non-affinity-modified) IgSF control domain. In some embodiments, the 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 (e.g., amino acid substitutions) in a wild-type or unmodified IgSF domain. An IgSF domain having an altered amino acid sequence that does not have an improved or reduced binding affinity or avidity (compared to a wild-type control IgSF domain) for at least one of its cognate binding partners compared to a wild-type control IgSF domain is an IgSF domain with unaltered affinity. The improved or reduced binding affinity or avidity can be determined using well-known binding assays, such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also Linsley et al., Immunity, 1: 7930801 (1994). The improvement in binding affinity or avidity of a protein for its cognate binding partner is an improvement that is at least 10% greater than the wild-type IgSF domain control, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value.The reduction in binding affinity or avidity of a protein for at least one of its cognate binding partners is 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type IgSF domain control value but 10% or more. Affinity-modified proteins have altered primary amino acid sequence due to substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any condition on any particular starting composition or method by which the affinity-modified IgSF domain is made. Thus, affinity-modified IgSF domains of the present invention are not limited to wild-type IgSF domains that are subsequently converted into affinity-modified IgSF domains by any particular affinity modification process. Affinity-modified IgSF domain polypeptides can be generated, for example, starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its cognate binding partner, and finally recombinantly or chemically synthesized to produce the subject affinity-modified IgSF domain composition. As just another example, affinity-modified IgSF domains can be generated by site-directed mutagenesis of wild-type IgSF domains. Thus, affinity-modified IgSF domains represent products, but not necessarily, produced by any given process. A variety of techniques may be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0085] The terms "binding affinity" and "binding avidity" as used herein refer to the specific binding affinity and specific binding avidity of a protein to its cognate binding partner under specific binding conditions, respectively. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of individual non-covalent interactions, for example, between an IgSF domain and its cognate binding partner. Thus, avidity differs from affinity, which represents the strength of a single interaction. Methods for determining binding affinity or avidity are known in the art. See, for example, Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005).

[0086] The term "biological half-life" refers to the length of time it takes for a substance (e.g., an immunomodulatory polypeptide of the invention) to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the substance's elimination, excretion, degradation (e.g., enzymatic), or absorption and concentration in certain organs or tissues in the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the substance's plasma concentration to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize the polypeptides of the invention to increase their biological half-life are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylated).

[0087] The term "cognate binding partner," with respect to a protein (e.g., an IgSF domain or affinity-engineered IgSF domain), refers to at least one molecule (typically a native mammalian protein) to which the reference protein specifically binds under specific binding conditions. A species of ligand that is recognized and specifically binds to its cognate receptor under specific binding conditions is an example of a cognate binding partner for that receptor. A "cognate cell surface binding partner" is a cognate binding partner that is expressed on the surface of a mammalian cell. In the present invention, a "cell surface molecular species" is a cognate binding partner of an immune synapse (IS) that is expressed on or by the surface of a cell (e.g., a mammalian cell) that forms the immune synapse.

[0088] The term "competitive binding," as used herein, means that a protein can specifically bind to at least two cognate binding partners, but the specific binding of one cognate binding partner inhibits (e.g., interferes with or prevents) the simultaneous binding of a second cognate binding partner. Thus, in some cases, a protein cannot simultaneously bind to two cognate binding partners. Generally, competitive binders contain identical or overlapping binding sites for specific binding, although this is not a requirement. In some embodiments, competitive binding causes measurable (partial or complete) inhibition of the specific binding of a protein to one of its cognate binding partners due to the specific binding of a second cognate binding partner. Various methods for quantifying competitive binding are known, such as ELISA (enzyme-linked immunosorbent assay) assays.

[0089] The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with 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 with 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.

[0090] The term "corresponding to" with respect to a protein position, e.g., a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (e.g., as shown in a sequence listing), refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm (e.g., the GAP algorithm). By aligning the sequences, one skilled in the art can identify corresponding residues, e.g., using conserved and identical amino acid residues as criteria.

[0091] The term "cytokine" includes, but is not limited to, for example, interleukins, interferons (IFNs), chemokines, hematopoietic growth factors, tumor necrosis factors (TNFs), and transforming growth factors. Generally, these are small molecular weight proteins that regulate the maturation, activation, proliferation, and differentiation of cells of the immune system.

[0092] The terms "reduce" or "alleviate" or "inhibit," as used herein, mean to decrease by a statistically significant amount. The decrease can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0093] The term "derivative" or "derivatized" refers to the modification of an immunomodulatory protein by direct or indirect covalent attachment to the protein to alter properties such as half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy while retaining or enhancing its therapeutic benefit. Derivatives can be produced by glycosylation, pegylation, lipidation, or Fc fusion.

[0094] As used herein, a domain (typically a sequence of three or more, generally five or seven or more amino acids, e.g., 10-200 amino acid residues) refers to a portion of a molecule (e.g., a protein or coding nucleic acid) that is structurally and / or functionally distinct from and identifiable with the rest of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independently folded structure within a protein composed of one or more structural motifs and / or is recognized by a functional activity, such as binding activity. A protein can have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology to related family members, e.g., homology to a motif. In another example, a domain can be distinguished by its function (e.g., ability to interact with a biomolecule, such as a cognate binding partner). A domain can exhibit an independent biological function or activity, such that it can perform an activity (e.g., binding) independently or fused to another molecule. A domain can be a linear or nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For illustrative purposes herein, definitions are provided, but it is understood that recognizing specific domains by name is well within the skill of the art. If necessary, appropriate software can be employed to identify domains. It is understood that reference to amino acids, including specific sequences designated as SEQ ID NOs, used to describe the domain organization of IgSF domains is for illustrative purposes and is not intended to limit the scope of the provided embodiments. It is understood that descriptions of polypeptides and their domains are theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus can vary and is not necessarily the same from protein to protein. Thus, a particular IgSF domain, e.g., a particular IgV or IgC domain, can be several amino acids (1, 2, 3, or 4) longer or shorter.

[0095] The term "ectodomain," as used herein, refers to the region of a membrane protein (e.g., a transmembrane protein) that is outside the vesicle membrane. Ectodomains often contain binding domains that specifically bind to ligands or cell surface receptors. The ectodomain of a transmembrane protein is alternatively referred to as the extracellular domain.

[0096] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition of the present invention that, when administered ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient), either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant inhibition of disease progression, such as by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount for treating a disease or disorder of the immune system can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with the disease or disorder, prevents the progression of the disease or disorder, or improves the patient's physical function. In some embodiments, the patient is a human patient.

[0097] As used herein in the context of improving the immune activity of mammalian lymphocytes, the term "enhanced" or "enhanced" means increasing interferon-γ (IFN-γ) production, for example, by a statistically significant amount. In some embodiments, immune activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing an MLR assay are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. In some embodiments, 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.

[0098] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. A host cell can be a prokaryote, such as Escherichia coli (E. coli), or it can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., 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 grown in serum-free medium or the CHO line DX-B11, which is DHFR-deficient.

[0099] The term "immune synapse," as used herein, refers to the interface between a mammalian cell (e.g., an antigen-presenting cell or tumor cell) that expresses MHC (major histocompatibility complex) I or MHC II, and a mammalian lymphocyte (e.g., an effector T cell or natural killer (NK) cell).

[0100] The term "immunoglobulin" (abbreviated "Ig"), as used herein, is synonymous with the term "antibody" (abbreviated "Ab") and refers to mammalian immunoglobulin proteins including any of the five human classes: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also refers to immunoglobulins that are less than full length, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V, and VI. H and V L variable fragments (Fv) containing V linked together in one chain; H and V LBispecific antibodies include single-chain variable fragments (scFv) containing the V region fragments (Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments). Homo- and hetero-bispecific bispecific antibodies are included within the scope of the term.

[0101] The Fc (fragment crystallizable) region or domain (also referred to as an Fc polypeptide) of an immunoglobulin molecule corresponds primarily to the constant region of the immunoglobulin heavy chain and is responsible for various functions, including antibody effector functions. An immunoglobulin Fc fusion ("Fc fusion") is a molecule comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. An Fc fusion may comprise, for example, the Fc region of an antibody (which enhances pharmacokinetics) and a wild-type or affinity-engineered immunoglobulin superfamily domain ("IgSF") domain, or the Fc region of another protein or fragment thereof. In some embodiments, the Fc further enhances effector function. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced) activity that enhances effector function. The IgSF domain mediates recognition of cognate binding partners (comparable to the recognition of the antibody variable region of an antibody for an antigen). The immunoglobulin Fc region can be indirectly or directly linked to one or more polypeptides or small molecules (fusion partners). Various linkers are known in the art and can be used to link the Fc to a fusion partner to generate an Fc fusion. The Fc fusion proteins of the present invention typically comprise an immunoglobulin Fc region covalently linked, directly or indirectly, to at least one affinity-modified IgSF domain. Fc fusions of the same species can dimerize to form Fc fusion homodimers, or non-identical species can be used to form Fc fusion heterodimers.

[0102] The term "immunoglobulin superfamily" or "IgSF" as used herein refers to a group of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins (i.e., antibodies); they all possess domains known as immunoglobulin domains or folds. IgSF members include cell surface antigen receptors, coreceptors, and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. These are typically associated with roles in the immune system. Proteins in the immune synapse are often members of IgSF. IgSF can also be divided into "subfamilies" based on shared characteristics, such as function. Such subfamilies typically consist of 4 to 30 IgSF members.

[0103] The terms "IgSF domain" or "immunoglobulin domain" or "Ig domain" as used herein refer to the structural domain of an IgSF protein. Ig domains are named after immunoglobulin molecules. They contain approximately 70 to 110 amino acids and are classified according to their size and function. Ig domains possess a characteristic Ig fold, a sandwich-like structure formed by two sheets of antiparallel β-strands. Interactions between hydrophobic amino acids within the sandwich and highly conserved disulfide bonds formed between cysteine ​​residues in the B and F strands stabilize the Ig fold. One end of an Ig domain contains a region called the complementarity-determining region, which is important for the specificity of antibodies for their ligands. Ig-like domains can be classified as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) or constant (IgC) domains. IgV domains, with nine β-strands, are generally longer than IgC domains, with seven β-strands. The Ig domains of some members of the IgSF resemble IgV domains in amino acid sequence, yet are similar in size to IgC domains. These are called IgC2 domains, while standard IgC domains are called IgC1 domains. T cell receptor (TCR) chains contain two Ig domains in their extracellular portions: one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane.

[0104] The term "IgSF species" as used herein refers to a group of IgSF member proteins that have identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a unique identity for all IgSF species belonging to that IgSF family. Thus, each IgSF family member is unique compared to other IgSF family members, and each species of a particular IgSF family member is therefore unique compared to other IgSF family member species. Nevertheless, differences between molecules of the same IgSF species can arise due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Furthermore, minor sequence differences within a single IgSF species due to genetic polymorphism, as well as wild-type truncated forms of an IgSF species due to, for example, proteolytic cleavage, also constitute differences between different forms within a single IgSF species. A "cell surface IgSF species" is an IgSF species expressed on the surface of a cell (generally a mammalian cell).

[0105] The term "immune activity," as used herein in the context of mammalian lymphocytes, refers to the expression of cytokines, such as chemokines or interleukins, in the lymphocytes. Assays for determining enhanced or suppressed immune activity include the MLR assay for interferon-γ (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), the SEB (Staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), and the anti-CD3 T cell stimulation assay (Li and Kurlander, J Transl Med. 2010: 8: 104). Induction of an immune response results in enhanced immune activity compared to resting lymphocytes. The immunomodulatory proteins or affinity-modified IgSF domains of the invention can increase, in some embodiments, or decrease, IFN-γ (interferon-γ) expression in primary T cell assays relative to a wild-type IgSF member or IgSF domain control. Those skilled in the art will recognize that the format of the primary T cell assay used to determine increased IFN-γ expression will differ from the format employed to assay for decreased IFN-γ expression. When assaying for the ability of the immunomodulatory proteins or affinity-modified IgSF domains of the invention to decrease IFN-γ expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used, as described in Example 6. Conveniently, a soluble form of the affinity-modified IgSF domain of the invention can be employed to determine its ability to decrease IFN-γ expression by antagonizing it in an MLR, as also described in Example 6. Alternatively, a co-immobilization assay can be used when assaying for the ability of the immunomodulatory proteins or affinity-modified IgSF domains of the invention to increase IFN-γ expression in primary T cell assays.In the co-immobilization assay, a T cell receptor signal (in some embodiments, provided by an anti-CD3 antibody) is used in combination with a co-immobilized affinity-engineered IgSF domain to determine its ability to increase IFN-γ expression relative to a wild-type IgSF domain control.

[0106] An "immunomodulating protein" is a protein that modulates immune activity. "Modulating" or "modulating" an immune response means that immune activity is either enhanced or suppressed. An immunomodulating protein can be a single polypeptide chain or a multimer (dimer or higher order multimer) of at least two polypeptide chains covalently linked to each other, for example, by interchain disulfide bonds. Thus, monomeric, dimeric, and higher order multimeric proteins are within the scope of the defined term. A multimeric protein can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains).

[0107] The term "improve" as used herein means to improve by a statistically significant amount. The improvement can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or more than a non-zero control value.

[0108] The term "lymphocyte," as used herein, refers to any of three subtypes of white blood cells of the mammalian immune system. These include natural killer cells (NK cells) (which function in cell-mediated cytotoxic innate immunity), T cells (which are involved in cell-mediated cytotoxic adaptive immunity), and B cells (which are involved in humoral antibody-driven adaptive immunity). T cells include helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Also included within the definition of lymphocytes are innate lymphocytes (ILCs).

[0109] The terms "mammal," "subject," or "patient" specifically include reference to at least one of a human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0110] The term "modulate," as used herein in the context of an immune response, such as a mammalian immune response, refers to any change (e.g., improvement or decrease) in an existing or potential immune response that occurs as a result of administration of an immunomodulatory protein of the invention. Thus, modulation refers to a change (e.g., improvement or decrease) in an immune response compared to an immune response that occurs or exists in the absence of administration of the immunomodulatory protein. Such modulation includes any induction, or change in the degree or extent, or suppression of immune activity of immune cells. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs), and non-professional antigen-presenting cells, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted to an existing, developing, or potential immune response, or the ability to induce, control, influence, or respond to an immune response. Modulation includes any change in the expression and / or function of genes, proteins, and / or other molecules in immune cells as part of an immune response. Modulation of immune response or immune activity includes, for example, the elimination, deletion, or sequestration of immune cells; the induction or generation of immune cells that can modulate the functional capacity of other cells, such as autoreactive lymphocytes, antigen-presenting cells, or inflammatory cells; the induction of an unresponsive state in immune cells (i.e., anergy); and the enhancement or suppression of immune cell activity or function (including, but not limited to, altering the protein patterns expressed by these cells). Examples include altered production and / or secretion of certain classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events. Modulation can be assessed, for example, by changes in IFN-γ (interferon-γ) expression relative to or compared with wild-type or unmodified IgSF domain controls in primary T cell assays (see Zhao and Ji, Exp Cell Res. 2016 Jan. 1; 340(1) 132-138).

[0111] The term "molecular species" as used herein refers to a population of proteins with identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of identical or substantially identical molecular species. Thus, for example, human CD80 is an IgSF member, and each human CD80 molecule is a species of CD80. Differences between molecules of the same molecular species can occur due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Furthermore, minor sequence differences within a single molecular species due to genetic polymorphism, as well as wild-type truncated forms of a single molecular species due to, for example, proteolytic cleavage, also constitute differences between other forms within a single molecular species. A "cell surface molecular species" is a molecular species expressed on the surface of a mammalian cell. Two or more different protein species, each present in only one or the other (but not both) of two mammalian cells forming an IS, are said to be in "cis" or "cis configuration" with respect to each other. Two different protein species, the first of which is present only in the first of two mammalian cells that form an IS and the second of which is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or in a "trans configuration." Two different protein species, each of which is present in both two mammalian cells that form an IS, are in both cis and trans configurations on those cells.

[0112] The term "non-competitive binding," as used herein, refers to the ability of a protein to simultaneously specifically bind to at least two cognate binding partners. In some embodiments, binding occurs under specific binding conditions. Thus, a protein can simultaneously bind to at least two different cognate binding partners, although the binding interactions need not be of the same duration; in some cases, the protein specifically binds to only one of the cognate binding partners. In some embodiments, simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding to a second cognate binding partner. In some embodiments, non-competitive binding means that binding of a second cognate binding partner to its binding site on the protein does not displace binding of a first cognate binding partner to its binding site on the protein. Methods for assessing non-competitive binding are well known in the art, such as those described in Perez de La Lastra et al., Immunology, 1999 Apr: 96(4): 663-670. In some cases, in a non-competitive interaction, a first cognate binding partner specifically binds at an interaction site that does not overlap with the interaction site of a second cognate binding partner, such that the binding of the second cognate binding partner does not directly interfere with the binding of the first cognate binding partner. Thus, any effect of the binding of the second cognate binding partner on the binding of the cognate binding partner is via a mechanism other than direct interference with the binding of the first cognate 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 non-competitive binding interactions in which a second cognate binding partner specifically binds at an interaction site that does not overlap with the binding of the first cognate binding partner, but only binds to the second interaction site when the first interaction site is occupied by the first cognate binding partner.

[0113] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise limited, the terms encompass nucleic acids containing known analogs of natural nucleotides, nucleic acids with similar binding properties, and nucleic acids that are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences. 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.

[0114] 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 of the invention) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0115] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked via peptide bonds. The term does not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The term includes post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules containing one or more amino acid analogs or non-standard or unnatural amino acids, which can be synthesized or recombinantly expressed using known protein modification techniques. In addition, proteins can be derivatized as described herein using well-known organic chemistry techniques.

[0116] The term "primary T cell assay," as used herein, refers to an in vitro assay for measuring interferon-γ ("IFN-γ") expression. Various such primary T cell assays are known in the art, such as the assay described in Example 6. In a preferred embodiment, the assay used is an anti-CD3 co-fixation assay. In this assay, primary T cells are stimulated with immobilized anti-CD3, with or without additional recombinant proteins. Culture supernatants are collected at a certain time point (usually 24-72 hours). In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatants are collected at a certain time point (usually 24-72 hours). Human IFN-γ levels in the culture supernatants are measured by standard ELISA techniques. Commercial kits are available from suppliers, and the assay is performed according to the manufacturer's recommendations.

[0117] The term "purified," as applied to nucleic acids or immunomodulating proteins of the invention, generally refers to a nucleic acid or polypeptide that is substantially free of 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, a chromatographic eluate, and / or a medium 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." Purified nucleic acids or immunomodulating proteins of the invention are at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a weight percent or molar basis).

[0118] The term "recombinant" indicates that a substance (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on a substance in or removed from its natural environment or condition. For example, a "recombinant nucleic acid" is one produced by recombining nucleic acids, e.g., during cloning, affinity engineering, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide," as used herein, refers to a protein molecule (e.g., an immunomodulatory protein) expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid. Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from various eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control elements, i.e., promoters, are also found in prokaryotes). The selection of a specific promoter and enhancer depends on what cell type is to be used to express the protein of interest. The terms "in functional combination," "in functional order," and "operably linked" as used herein refer to the linking of nucleic acid sequences in a manner or orientation that produces a nucleic acid molecule that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. The terms also refer to the linking of amino acid sequences in a manner that produces and / or transports a functional protein.

[0119] The term "recombinant expression vector," as used herein, refers to a DNA molecule containing a desired coding sequence (e.g., an immune modulatory nucleic acid) and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host 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. Optionally, a secretory signal peptide sequence can also be encoded by the recombinant expression vector operably linked to the coding sequence of the recombinant fusion protein of the invention so that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell.

[0120] 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 at the amino acid level between proteins and at the nucleotide level between nucleic acids. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates the percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website.

[0121] The term "soluble," as used herein with respect to a protein, means that the protein is not a membrane protein. Generally, a soluble protein contains only the extracellular domain of an IgSF family member receptor, or a portion thereof, containing one or more IgSF domains or specific binding fragments thereof.

[0122] The term "species," as used herein in the context of nucleic acid or polypeptide sequences, refers to an identical collection of such sequences. Slightly truncated sequences that differ from the full-length species by (or encode differences in) only one, two, or three amino acid residues at the amino or carboxy termini are considered to be a single species of sequence. Such microheterogeneity is a common feature of manufactured proteins.

[0123] The term "specifically binds," as used herein, refers to the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or avidity is at least 10 times greater, but optionally 50, 100, 250, or 500 times greater, or even at least 1000 times greater, than the average affinity or avidity of the same protein for a population of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind only to a single target molecule (e.g., its cognate binding partner), but may specifically bind to non-target molecules (e.g., paralogs or orthologs) due to the similarity in configuration between the target and non-target molecules. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or to non-target molecules (e.g., paralogs) with substantially similar epitopes to the target molecule is possible and does not impair the specificity of binding determined for a statistically valid population of unique non-targets (e.g., random polypeptides). Therefore, the affinity-modified polypeptide of the present invention can specifically bind to multiple distinct target molecular species due to cross-reactivity.In general, such off-target specific binding can be alleviated by reducing the affinity or avidity for undesired targets.Solid-phase ELISA immunoassay or Biacore measurement can be used to determine the specific binding between two proteins.Generally, the interaction between two binding proteins is 1 × 10 -5 Less than M, often 1 × 10 -12 In certain aspects of the disclosure, the interaction between two binding proteins has a dissociation constant (Kd) as low as 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 -11 It has a dissociation constant of M.

[0124] The term "specific binding fragment" or "fragment," as used herein with reference to a mature (i.e., lacking a signal peptide) wild-type IgSF domain, refers to a polypeptide that is shorter than the full-length mature IgSF domain and that specifically binds to the mature wild-type IgSF domain's natural cognate binding partner in vitro and / or in vivo. 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% of the sequence length of the full-length mature wild-type sequence. The specific binding fragment can be altered in sequence to form an affinity-modified IgSF domain of the invention. In some embodiments, the specific binding fragment modulates the immune activity of a lymphocyte.

[0125] The term "inhibited" or "reduced" as used herein means a statistically significant decrease. In some embodiments, the decrease can be at least 10%, and up to 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0126] The term "targeting moiety," as used herein, refers to a composition that covalently or noncovalently binds to or physically encapsulates a polypeptide comprising a wild-type and / or affinity-modified IgSF domain of the present invention. A targeting moiety has specific binding affinity for a desired cognate binding partner, such as a cell surface receptor or tumor antigen (e.g., tumor-specific antigen (TSA) or tumor-associated antigen (TAA)). Typically, the desired cognate binding partner is localized on a specific tissue or cell type. Targeting moieties include antibodies, antigen-binding fragments (Fab), V-cells, and the like. H and V L variable fragments (Fv) containing V linked together in one chain; H and V Land other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecule (<500 dalton) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or noncovalently attached to the lipid membrane of liposomes encapsulating the immunomodulatory polypeptides of the invention.

[0127] The term "treating" a disease or disorder or "treatment" of a disease or disorder, as used herein, means slowing, halting, or reversing the progression of the disease or disorder, as evidenced by any reduction, arrest, or elimination of clinical or diagnostic symptoms by administration of an immunomodulatory protein of the invention, either alone or in combination with another compound as described herein. "Treating" or "treatment" also means reducing the severity of symptoms in acute or chronic diseases or disorders, or reducing the rate of relapse (e.g., as in relapsing or remitting autoimmune disease processes), or reducing inflammation in the case of inflammatory aspects of autoimmune diseases. As used herein in the context of cancer, the terms "treating" cancer or "inhibiting" cancer or "inhibition" of cancer refer to at least one of a statistically significant reduction in tumor growth rate, halting tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, or a statistically significant improvement in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria such as, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST). "Preventing" a disease or disorder or "prevention" of a disease or disorder, as used in the context of the present invention, refers to the administration of an immunomodulatory protein of the present invention, either alone or in combination with another compound, to prevent the appearance or development of a disease or disorder or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[0128] The term "tumor-specific antigen" or "TSA," as used herein, refers to an antigen that is primarily present on tumor cells of a mammalian subject but is not generally found on normal cells of the mammalian subject. A tumor-specific antigen need not be present exclusively on tumor cells, but the proportion of cells in a particular mammal that have the tumor-specific antigen or the level of the tumor-specific antigen on the surface of the tumor must be sufficiently high so that it can be targeted with an anti-tumor therapeutic agent, such as the immunomodulatory polypeptide of the present invention, and provide protection or treatment for the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells that display the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that display the TSA are cancerous.

[0129] As used herein, "screening" refers to the identification or selection of a molecule or portion thereof from a collection or library of molecules and / or portions thereof based on determining an activity or property of the molecule or portion thereof. Screening can be carried out in any of a variety of ways, including, for example, by assays that assess direct binding (e.g., binding affinity) of the molecule to a target protein or by functional assays that assess modulation of the activity of a target protein.

[0130] The terms "wild-type" or "native" or "parent" as used herein in conjunction with biological materials such as nucleic acid molecules, proteins, IgSF members, host cells, etc., refer to those found in nature and unmodified by human intervention. A wild-type IgSF domain is a type of IgSF domain that has not been affinity-modified. In some embodiments of the immunomodulating proteins of the present invention, the affinity-unmodified IgSF domain is a wild-type IgSF domain.

[0131] II. Affinity-engineered immunomodulatory proteins The present invention provides immunomodulatory proteins that have therapeutic utility by modulating immune activity in mammals having diseases or disorders in which modulation of the immune system response is beneficial.

[0132] IgSF family members included within the scope of the immunomodulatory proteins of the present invention exclude antibodies (i.e., immunoglobulins), such as mammalian antibodies or antibodies that may be of mammalian origin. Thus, the present invention relates to non-immunoglobulin (i.e., non-antibody) IgSF domains. Wild-type mammalian IgSF family members that are not immunoglobulins (i.e., antibodies) are known in the art, as are their nucleic acid and amino acid sequences. All non-immunoglobulin mammalian IgSF family members are included within the scope of the present invention.

[0133] In some embodiments, the non-immunoglobulin IgSF family member and the corresponding IgSF domains present therein are of murine, rat, cynomolgus monkey, or human origin. In some embodiments, the IgSF family members are members of the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling lymphocytic activation molecule (SLAM) family, the Leukocyte Immunoglobulin-like Receptor (LGR) family, the IL-1 ... The members are from at least or exactly one, two, three, four, five or more IgSF subfamilies, such as the IgSF family (immunoglobulin-like receptor (LIR)), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor related (PVR) family, the natural cytotoxicity triggering receptor (NCR) family, or the killer-cell immunoglobulin-like receptor (KIR) family.

[0134] In some embodiments, the non-immunoglobulin IgSF family members of the immunomodulatory proteins of the present invention and the corresponding IgSF domains present therein have modified affinities compared to mammalian IgSF members. In some embodiments, the mammalian IgSF members are one of the IgSF members or comprise an IgSF domain derived from one of the IgSF members as shown in Table 1, including any mammalian orthologs thereof. Orthologs are genes from different species that have evolved from a common ancestral gene through speciation. Orthologs usually retain the same function during evolution.

[0135] The first column of Table 1 provides the name and, optionally, several possible aliases for that particular IgSF member. The second column provides the protein identifier from 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 database includes 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 is primarily supported by grants from the US National Institutes of Health (NIH). The third column provides the region in which the indicated IgSF domain is located. The region is identified as the range encompassing the residues that the domain defines. Column 3 also indicates the IgSF domain class of the identified IgSF region. Column 4 provides the region in which the indicated additional domains are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). Column 5 indicates some of the listed IgSF members, i.e., some of their cognate cell surface binding partners.

[0136] Typically, the affinity-altered IgSF domains of the provided embodiments are human or murine affinity-altered IgSF domains.

[0137] Table 1. IgSF members according to the present disclosure TIFF2025166053000002.tif86166TIFF2025166053000003.tif221166TIFF2025166053000004.tif222166TIFF2025166053000005.tif98166

[0138] In some embodiments, the immunomodulatory proteins of the invention comprise at least one affinity-engineered mammalian IgSF domain. The affinity-engineered IgSF domain can be affinity-engineered to specifically bind to one or multiple (two, three, four, or more) cognate binding partners (also called "counterstructure ligands"). The IgSF domain can be affinity-engineered to independently increase or decrease its specific binding affinity or avidity for each of multiple cognate binding partners to which it binds. By this mechanism, specific binding to each of multiple cognate binding partners can be independently tailored to a particular affinity or avidity.

[0139] In some embodiments, the cognate binding partner of an IgSF domain is at least one, and sometimes at least two or three, of the cognate binding partners of the wild-type IgSF domain, such as those listed in Table 1. The sequence of an IgSF domain, such as a mammalian IgSF domain, is affinity-modified by altering its sequence with at least one substitution, addition, or deletion. The sequence alteration can occur at the binding site or allosteric site of the cognate binding partner. In some embodiments, a nucleic acid encoding an IgSF domain, such as a mammalian IgSF domain, is affinity-modified by substitution, addition, deletion, or a combination thereof, at specific and predetermined nucleotide sites to generate a nucleic acid of the invention. In some contrasting embodiments, a nucleic acid encoding an IgSF domain, such as a mammalian IgSF domain, is affinity-modified by substitution, addition, deletion, or a combination thereof, at random sites within the nucleic acid. In some embodiments, a combination of the two approaches (predetermined and random) is utilized. In some embodiments, the design of affinity-modified IgSF domains of the invention is performed in silico.

[0140] In some embodiments, the affinity-modified IgSF domain contains one or more amino acid substitutions (or "mutations" or "exchanges") relative to a wild-type or unmodified polypeptide or portion thereof containing an immunoglobulin superfamily (IgSF) domain, such as an IgV domain or an IgC domain or a specific-binding fragment of an IgV domain or an IgC domain. In some embodiments, the immunomodulatory protein comprises an affinity-modified IgSF domain containing an IgV domain or an IgC domain or a specific-binding fragment thereof, wherein at least one of the amino acid substitutions is in the IgV domain or an IgC domain or a specific-binding fragment thereof. In some embodiments, the IgV domain or an IgC domain is an affinity-modified IgSF domain because of altered binding activity or affinity.

[0141] In some embodiments, an IgSF domain, such as a mammalian IgSF domain, is affinity-altered with at least one, but not more than a total of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In some embodiments, an IgSF domain, such as a mammalian IgSF domain, is affinity-altered with at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the substitutions are non-conservative. In some embodiments, the substitutions are a combination of conservative and non-conservative substitutions. In some embodiments, the sequence modifications are made in the binding site of the IgSF domain for its cognate binding partner.

[0142] In some embodiments, the wild-type or unmodified IgSF domain is a mammalian IgSF domain. In some embodiments, the wild-type or unmodified IgSF domain can be an IgSF domain, including but not limited to, a human, mouse, cynomolgus monkey, or rat IgSF domain. In some embodiments, the wild-type or unmodified IgSF domain is human.

[0143] In some embodiments, the wild-type or unmodified IgSF domain is an IgSF domain contained in the amino acid sequence set forth in any of SEQ ID NOs: 1-27 or a specific-binding fragment thereof, or a mature form thereof lacking a signal sequence (an amino acid sequence exhibiting 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: 1-27 or a mature form thereof), or is an IgV domain or an IgC domain or a portion thereof containing a specific-binding fragment thereof.

[0144] In some embodiments, the wild-type or unmodified IgSF domain is or comprises an extracellular domain or portion thereof of an IgSF family member containing an IgSF domain (e.g., an IgV domain or an IgC domain). In some embodiments, the unmodified or wild-type IgSF domain comprises the amino acid sequence set forth in any of SEQ ID NOs:28-54, or an ortholog thereof. For example, an unmodified or wild-type IgSF domain can comprise (i) an amino acid sequence set forth in any of SEQ ID NOs:28-54, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:28-54, or (iii) a specific-binding fragment of an amino acid sequence set forth in any of SEQ ID NOs:28-54 or an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:28-54, comprising an IgV domain or an IgC domain.

[0145] In some embodiments, the extracellular domain of an unmodified or wild-type IgSF domain can comprise multiple IgSF domains (e.g., an IgV domain and an IgC domain). However, an affinity-modified IgSF domain need not comprise both an IgV domain and an IgC domain. In some embodiments, an affinity-modified IgSF domain comprises or consists essentially of an IgV domain, or a specific binding fragment thereof. In some embodiments, an affinity-modified IgSF domain comprises or consists essentially of an IgC domain, or a specific binding fragment thereof. In some embodiments, an affinity-modified IgSF domain comprises an IgV domain, or a specific binding fragment thereof, and an IgC domain, or a specific binding fragment thereof.

[0146] In some embodiments, the one or more amino acid substitutions in the affinity-altered IgSF domain can be located in any one or more of the IgSF polypeptide domains. For example, in some embodiments, the one or more amino acid substitutions are located in the extracellular domain of the IgSF polypeptide. In some embodiments, the one or more amino acid substitutions are located in the IgV domain or a specific-binding fragment of the IgV domain. In some embodiments, the one or more amino acid substitutions are located in the IgC domain or a specific-binding fragment of the IgC domain.

[0147] In some embodiments, at least one IgSF domain, such as a mammalian IgSF domain, of the immunomodulatory proteins provided herein is independently affinity modified to have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, or 80% sequence identity to a wild-type or unmodified IgSF domain or a specific-binding fragment thereof contained in a wild-type or unmodified IgSF protein, such as, but not limited to, those disclosed in Table 1 as SEQ ID NOs: 1-27.

[0148] In some embodiments, the IgSF domain of the immunomodulatory proteins provided herein is a specific-binding fragment of a wild-type or unmodified IgSF domain contained in a wild-type or unmodified IgSF protein, such as, but not limited to, those disclosed in Table 1 at SEQ ID NOs: 1-27. In some embodiments, the specific-binding fragment can have an amino acid length of at least 50 amino acids, e.g., at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific-binding fragment of an IgV domain contains an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the wild-type or unmodified IgV domain. In some embodiments, a specific binding fragment of an IgC domain comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of a wild-type or unmodified IgC domain. In some embodiments, the specific binding fragment modulates immune activity. In more specific embodiments, the specific binding fragment of an IgSF domain enhances immune activity. In alternative embodiments, the specific binding fragment reduces immune activity.

[0149] To determine the percent identity of two nucleic acid sequences or two amino acids, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number (#) of identical positions / total number (#) of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences are the same length. The sequences can be aligned manually to count the number of identical nucleic acids or amino acids. Alternatively, the alignment of two sequences for determining percent identity can be achieved using a mathematical algorithm. Such algorithms are incorporated into the NBLAST and XBLAST programs. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches may be performed with the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules of the present invention, BLAST protein searches may be performed with the XBLAST program, score = 50, word length = 3. Gapped BLAST may be used to obtain gapped alignments for comparison purposes. Alternatively, PSI-Blast may be used to perform an iterated search that detects distant relationships between molecules. When using NBLAST, XBLAST, and Gapped BLAST programs, default parameters of the respective programs may be used, such as those available on the NCBI website. Alternatively, sequence identity may be calculated after aligning sequences in the NCBI database, for example, using the BLAST program. Generally, default settings, for example, for "scoring matrix" and "gap penalty," may be used for alignment.In the context of the present invention, the default settings of BLASTN and PSI BLAST NCBI may be employed.

[0150] In some embodiments, the immunomodulatory protein contains at least one affinity-modified IgSF domain. In some embodiments, the immunomodulatory protein further contains at least one affinity-modified domain and further contains at least one non-affinity-modified IgSF domain (e.g., an unmodified or wild-type IgSF domain). In some embodiments, the immunomodulatory protein contains at least two affinity-modified domains. In some embodiments, the immunomodulatory protein can contain multiple non-affinity-modified and / or affinity-modified IgSF domains, for example, 1, 2, 3, 4, 5, or 6 affinity-modified and / or affinity-modified IgSF domains.

[0151] In some embodiments, at least one unaffinity-modified IgSF domain and / or one affinity-modified IgSF domain present in the immunomodulating proteins provided herein specifically binds to at least one cell surface molecular species expressed on the surface of mammalian cells that form the immune synapse (IS). Of course, in some embodiments, the immunomodulating proteins provided herein comprise multiple unaffinity-modified IgSF domains and / or affinity-modified IgSF domains, e.g., 1, 2, 3, 4, 5, or 6 unaffinity-modified IgSF and / or affinity-modified IgSF domains. One or more of these unaffinity-modified IgSF domains and / or affinity-modified IgSF domains can independently specifically bind to either one or both of the mammalian cells that form the IS.

[0152] Often, the cell surface species to which the affinity-altered IgSF domain specifically binds will be a wild-type IgSF family member or a cognate binding partner of the wild-type IgSF domain to which affinity has been altered. In some embodiments, the cell surface species is a mammalian IgSF member. In some embodiments, the cell surface species is a human IgSF member. In some embodiments, the cell surface species will be a cognate cell surface binding partner as shown in Table 1. In some embodiments, the cell surface species will be a viral protein, e.g., a poliovirus protein, on the cell surface of a mammalian cell, such as a human cell.

[0153] In some embodiments, the affinity-unmodified and / or affinity-modified IgSF domain of at least one of the immunomodulatory proteins provided herein binds to at least two or three cell surface molecular species present on the mammalian cells that form the IS. The cell surface molecular species to which the affinity-unmodified and / or affinity-modified IgSF domain of the invention specifically binds can be present only on the surface of one or the other of the two mammalian cells that form the IS (i.e., in cis configuration), or alternatively, the cell surface molecular species can be present on both.

[0154] In some embodiments, the affinity-modified IgSF domain specifically binds to at least two cell surface molecular species, where one of the molecular species is present on one of the two mammalian cells forming the IS and the other molecular species is present on the second of the two mammalian cells forming the IS. In such embodiments, the cell surface molecular species need not necessarily be present solely on one or the other of the two mammalian cells forming the IS (i.e., in trans configuration), although in some embodiments they are. Thus, embodiments provided herein include those in which each cell surface molecular species is present on the surface of only one or the other of the mammalian cells forming the IS (cis configuration), as well as those in which the cell surface molecular species bound by each affinity-modified IgSF is present on the surface of both mammalian cells forming the IS (i.e., cis and trans configuration).

[0155] Those skilled in the art will recognize that antigen-presenting cells (APCs) and tumor cells form immune synapses with lymphocytes. Thus, in some embodiments, at least one affinity-unmodified IgSF domain and / or at least one affinity-modified IgSF domain of an immunomodulatory protein specifically binds only to a cell surface molecular species present on cancer cells, where the cancer cells associate with lymphocytes to form an IS. In other embodiments, at least one affinity-unmodified IgSF domain and / or at least one affinity-modified IgSF domain of an immunomodulatory protein specifically binds only to a cell surface molecular species present on lymphocytes, where the lymphocytes associate with APCs or tumor cells to form an IS. In some embodiments, the affinity-unmodified IgSF domain and / or affinity-modified IgSF domain binds to a cell surface molecular species present on both target cells (or APCs) and lymphocytes that form an IS.

[0156] Embodiments of the present invention include those in which the immunomodulating proteins provided herein comprise at least one affinity-modified IgSF domain having an amino acid sequence that differs from a wild-type or unmodified IgSF domain (e.g., a mammalian IgSF domain), such that under specific binding conditions, the binding affinity (or avidity, when in a multimeric or other related structure) of the immunomodulating protein provided herein for at least one of its cognate binding partners is either improved or decreased compared to an unaltered wild-type or unmodified IgSF domain control. In some embodiments, the affinity-modified IgSF domain has a binding affinity for its cognate binding partner that differs from that of the wild-type or unmodified IgSF control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. In some embodiments, the IgSF domain has improved binding affinity for one or more cognate binding partners. In some embodiments, the affinity-modified IgSF domain has decreased binding affinity for one or more cognate binding partners compared to a wild-type or unmodified IgSF domain. In some embodiments, the cognate binding partner can be a mammalian protein, such as a human protein or a murine protein.

[0157] The binding affinity for each of the cognate binding partners is independent; i.e., in some embodiments, the affinity-modified IgSF domain has improved binding affinity for one, two or three different cognate binding partners and decreased binding affinity for one, two or three of the different cognate binding partners compared to the wild-type or unmodified ICOSL polypeptide.

[0158] In some embodiments of the immunomodulatory proteins provided herein, the binding affinity or avidity of the affinity-modified IgSF domain is improved by at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 1000%, 5000%, or 10,000% compared to a wild-type or unmodified control IgSF domain. In some embodiments, the improvement in binding affinity compared to a wild-type or unmodified IgSF domain is greater 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.

[0159] In some embodiments, the binding affinity or avidity is reduced by at least 10%, up to 20%, 30%, 40%, 50%, 60%, 70%, 80%, or up to 90% compared to a wild-type or unmodified control IgSF domain, hi some embodiments, the reduction in binding affinity compared to a wild-type or unmodified IgSF domain is greater 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.

[0160] In some embodiments, the specific binding affinity of the affinity-engineered IgSF domain for its cognate binding partner is at least 1×10 -5 M, 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 - 11M, or 1 x 10 -12 It can be M.

[0161] In some embodiments, the provided immunomodulatory proteins comprise at least two IgSF domains, in which at least one of the IgSF domains is affinity-engineered, while in some embodiments both are affinity-engineered, with at least one of the affinity-engineered IgSF domains having improved affinity (or avidity) for its cognate binding partner and at least one affinity-engineered IgSF domain having reduced affinity (or avidity) for its cognate binding partner.

[0162] In some embodiments, an IgSF domain that otherwise binds to multiple cell surface species is affinity engineered such that it no longer specifically binds to one of its cognate cell surface species. Thus, in these embodiments, specific binding to one of its cognate cell surface species is reduced to no more than 10% of wild-type levels of specific binding, often no more than 7%, 5%, 3%, 1%, or even to undetectable or statistically insignificant specific binding.

[0163] In these embodiments, the specific binding site on the mammalian IgSF domain is inactivated or substantially inactivated with respect to at least one of the cell surface molecular species. Thus, for example, if a wild-type IgSF domain specifically binds to exactly two cell surface molecular species, in some embodiments, the domain is affinity-engineered to specifically bind to exactly one cell surface molecular species (wherein substantially immunologically inactive subsequences thereof are ignored in determining the number of affinity-engineered IgSF domains). And, if a wild-type IgSF domain specifically binds to exactly three cell surface molecular species, in some embodiments, the domain is affinity-engineered to specifically bind to exactly two cell surface molecular species. An IgSF domain that is affinity-engineered so that it no longer substantially specifically binds to one of its cognate cell surface molecular species can be an IgSF domain that otherwise specifically binds to that cell surface molecular species, either competitively or non-competitively. Those skilled in the art will recognize that a wild-type IgSF domain that competitively binds to two cognate binding partners can still be inactivated with respect to exactly one of them (e.g., if their binding sites are not exactly in the same area, but simply overlap such that the specific binding of one inhibits binding of the other cognate binding partner, and yet both competitive binding sites are distinct).

[0164] In some embodiments, the affinity-unmodified and / or affinity-modified IgSF domains of the immunomodulating proteins provided herein are capable of competitively specific binding to their cognate cell surface molecular species. In other embodiments, the affinity-unmodified and / or affinity-modified IgSF domains of the immunomodulating proteins provided herein are capable of non-competitively specific binding to their cognate cell surface molecular species. Any number of affinity-unmodified and / or affinity-modified IgSF domains present in the immunomodulating proteins provided herein are capable of competitively or non-competitively specific binding.

[0165] In some embodiments, the immunomodulatory proteins provided herein comprise at least two affinity-unmodified IgSF domains, or at least one affinity-unmodified IgSF domain and at least one affinity-modified IgSF domain, or at least two affinity-modified IgSF domains, where one IgSF domain specifically binds to its cognate cell surface molecular species in a competitive manner and the second IgSF domain binds non-competitively. More generally, the immunomodulatory proteins provided herein can comprise 1, 2, 3, 4, 5, or 6 competitively or 1, 2, 3, 4, 5, or 6 non-competitively binding affinity-unmodified IgSF and / or affinity-modified IgSF domains, or any combination thereof. Thus, the immunomodulatory proteins provided herein can have a number of non-competitively and competitively binding IgSF domains of: 0 and 1, 0 and 2, 0 and 3, 0 and 4, 1 and 0, 1 and 1, 1 and 2, 1 and 3, 2 and 0, 2 and 1, 2 and 2, 2 and 3, 3 and 0, 3 and 1, 3 and 2, 3 and 3, 4 and 0, 4 and 1, and 4 and 2, respectively.

[0166] The multiple affinity-unmodified and / or affinity-modified IgSF domains of the immunomodulatory proteins provided herein need not be directly covalently linked to each other. In some embodiments, an intervening stretch of one or more amino acid residues indirectly covalently links the affinity-unmodified and / or affinity-modified IgSF domains to each other. The linkage can be via N- to C-terminal residues.

[0167] In some embodiments, the linkage can be via the side chain of an amino acid residue that is not located at the N-terminus or C-terminus of the non-affinity-modified or affinity-modified IgSF domain, and thus the linkage can be via a terminal or internal amino acid residue or a combination thereof.

[0168] The "peptide linker" connecting the affinity-unmodified and / or affinity-modified IgSF domains can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue or less in length. In some embodiments, the linker is (in the single-letter amino acid code): GGGGS ("4GS") or a multimer of 4GS linkers, e.g., 2, 3, 4, or 5 repeats of the 4GS linker. In further optional embodiments, a series of alanine residues is interposed between the 4GS linker and the Fc to which the immunomodulatory protein is covalently linked. In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines.

[0169] A. Exemplary affinity-engineered IgSF domains In some embodiments, the affinity-modified IgSF domain has one or more amino acid substitutions in the IgSF domain of a wild-type or unmodified IgSF protein, such as those shown in Table 1 above. The one or more amino acid substitutions can be in the ectodomain (e.g., the extracellular domain) of the wild-type or unmodified IgSF domain. In some embodiments, the one or more amino acid substitutions are in the IgV domain or a specific-binding fragment thereof. In some embodiments, the one or more amino acid substitutions are in the IgC domain or a specific-binding fragment thereof. In some embodiments of the affinity-modified IgSF domain, some of the one or more amino acid substitutions are in the IgV domain or a specific-binding fragment thereof, and some of the one or more amino acid substitutions are in the IgC domain or a specific-binding fragment thereof.

[0170] In some embodiments, the affinity-modified IgSF domain 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 substitutions. The substitutions can be in the IgV domain or the IgC domain. In some embodiments, the affinity-modified IgSF domain 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 substitutions in the IgV domain or specific-binding fragment thereof. In some embodiments, the affinity-modified IgSF domain 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 substitutions in the IgC domain or specific-binding fragment thereof. In some embodiments, the affinity-modified IgSF domain has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified IgSF domain or specific-binding fragment thereof, such as the IgSF domain contained in an IgSF protein set forth in any of SEQ ID NOs: 1-27.

[0171] In some embodiments, the affinity-modified IgSF domain contains one or more amino acid substitutions in the wild-type or unmodified IgSF domain of a B7 IgSF family member. In some embodiments, the B7 IgSF family member is CD80, CD86, or ICOS ligand (ICOSL). In some embodiments, the affinity-modified IgSF domain has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified IgSF domain, or a specific-binding fragment thereof, such as the IgSF domain contained in the IgSF protein set forth in any of SEQ ID NOs: 1, 2, or 5. Exemplary affinity-modified IgSF domains for CD80 are shown in Table 2. Exemplary affinity-modified IgSF domains for ICOSL are shown in Table 3. Exemplary affinity-engineered IgSF domains of CD86 are shown in Table 4.

[0172] Table 2: Exemplary variant CD80 polypeptides TIFF2025166053000006.tif157168TIFF2025166053000007.tif171168

[0173] Table 3: Exemplary variant ICOSL polypeptides TIFF2025166053000008.tif198169

[0174] Table 4: Exemplary variant CD86 polypeptides TIFF2025166053000009.tif41169

[0175] In some embodiments, the affinity-modified IgSF domain contains one or more amino acid substitutions in the wild-type or unmodified IgSF domain of an NkP30 family member. In some embodiments, the affinity-modified IgSF domain has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the wild-type or unmodified IgSF domain, or a specific-binding fragment thereof, such as the IgSF domain contained in the IgSF protein set forth in SEQ ID NO:27. Table 5 provides exemplary affinity-modified NkP30 IgSF domains.

[0176] Table 5. Exemplary variant NKp30 polypeptides TIFF2025166053000010.tif46169

[0177] B. Types of affinity-modified immunomodulatory proteins 1. Dual-binding affinity-engineered domains In some embodiments, the immunomodulatory proteins provided herein can comprise the sequence of at least one IgSF domain of a wild-type mammalian non-immunoglobulin (i.e., non-antibody) IgSF family member, wherein at least one IgSF domain therein has been affinity-modified (a "type I" immunomodulatory protein). In some embodiments, at least one modified IgSF domain specifically binds to at least two cognate binding partners in a non-competitive manner.

[0178] In some embodiments, the immunomodulatory protein comprises at least one affinity-modified non-immunoglobulin immunoglobulin superfamily (IgSF) domain that specifically binds non-competitively to at least two cognate binding partners. In some embodiments, the affinity-modified domain exhibits improved binding to at least one of the cognate binding partners compared to a wild-type or unmodified IgSF domain. In some embodiments, the affinity-modified domain exhibits improved binding to at least two different cognate binding partners.

[0179] In some embodiments of the type I immunomodulatory proteins of the invention, the unmodified or wild-type IgSF member, e.g., a mammalian IgSF member, is one of the IgSF members or comprises an IgSF domain derived from one of the IgSF members as set forth in Table 1, including any mammalian orthologs thereof.

[0180] In some embodiments, the additional IgSF domains present in the type I immunomodulatory protein, e.g., at least 2, 3, 4, or 5 IgSF domains, and in some embodiments exactly 2, 3, 4, or 5 IgSF domains, can be non-affinity-engineered and / or affinity-engineered.

[0181] In some embodiments, the type I immunomodulatory proteins herein comprise at least one affinity-modified non-immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type or unmodified IgSF domain, wherein the affinity-modified IgSF domain 1) has altered (e.g., improved or decreased) binding to at least two cognate binding partners compared to the wild-type or unmodified IgSF domain; and 2) the at least one affinity-modified IgSF domain specifically binds to at least two cognate binding partners in a non-competitive manner. In some embodiments, the affinity-modified IgSF domain of a type I immunomodulatory protein has improved binding to at least two cognate binding partners. In some embodiments, the affinity-modified IgSF domain of a type I immunomodulatory protein has decreased binding to at least two cognate binding partners. In some embodiments, the affinity-engineered IgSF domain of a type I immunomodulatory protein has improved binding to at least one cognate binding partner and decreased binding to at least one other, different, cognate binding partner.

[0182] In some embodiments, the two cognate binding partners are expressed on the surface of at least two different cells, e.g., two different mammalian cells. For example, in some embodiments, one cognate binding partner is expressed on the surface of a lymphocyte, and another cognate binding partner is expressed on the surface of an antigen-presenting cell. In some embodiments, the two cognate binding partners are expressed on the surface of the same cell type (e.g., the same immune cell). In some embodiments, the type I immunomodulatory protein can regulate one or more immune activities of immune cells, such as the immune activity of lymphocytes, e.g., T cells. In some embodiments, the immune activity is improved. In some embodiments, the immune activity is reduced.

[0183] In some embodiments, the immunomodulatory protein comprises or consists essentially of a single affinity-engineered IgSF domain that non-competitively binds to at least two cognate binding partners. In some embodiments, the affinity-engineered domain is an affinity-engineered IgV domain. In some embodiments, the affinity-engineered domain is an affinity-engineered IgC domain.

[0184] In some embodiments, the type I immunomodulatory proteins provided herein comprise an affinity-modified CD80 IgSF domain that specifically binds to CD28 and PDL1 in a non-competitive manner. In some embodiments, the affinity-modified CD80 IgSF domain is an IgV domain. In some embodiments, the immunomodulatory protein further comprises an additional, non-affinity-modified IgSF domain.

[0185] 2. Stacked or multidomain immunomodulatory proteins In some embodiments of the present invention, the immunomodulatory proteins comprise combinations ("non-wild-type combinations") and / or arrangements ("non-wild-type arrangements" or "non-wild-type hypermutations") of affinity-modified and / or affinity-unmodified IgSF domain sequences not found in wild-type IgSF family members ("Type II" immunomodulatory proteins). The affinity-unmodified (e.g., wild-type) or affinity-modified IgSF domain sequences can be derived from mammalian, e.g., mouse, rat, cynomolgus monkey, or human origin, or a combination thereof. The number of such affinity-unmodified or affinity-modified IgSF domains present in these embodiments of the Type II immunomodulatory proteins (whether in non-wild-type combinations or non-wild-type arrangements) is at least 2, 3, 4, or 5, and in some embodiments, exactly 2, 3, 4, or 5 IgSF domains (wherein determining the number of affinity-modified IgSF domains disregards nonspecific binding and / or substantially immunologically inactive subsequences thereof).

[0186] In some embodiments, the type II immunomodulatory proteins of the invention comprise a non-wild-type combination of IgSF domains, where the IgSF domains can be IgSF domains of an IgSF family member from those listed in Table 1. Thus, in some embodiments, the immunomodulatory protein can contain first and second IgSF domains, each of which can be affinity-altered IgSF domains containing one or more amino acid substitutions compared to the IgSF domain contained in the IgSF family member shown in Table 1.

[0187] In some embodiments, the IgSF domains each independently belong to the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling lymphocytic activation molecule (SLAM) family, the Leukocyte Immunoglobulin-like Receptor (LGR) family, the IL-1 ... and affinity-engineered or unengineered IgSF domains contained in IgSF family members of a family selected from the group consisting of the immunoglobulin-like receptor (LIR), nectin (Nec) family, nectin-like (NECL) family, poliovirus receptor related (PVR) family, natural cytotoxicity triggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM) family, and killer-cell immunoglobulin-like receptor (KIR) family.In some embodiments, the IgSF domains are each independently selected from the group consisting of CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOSLG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3), VTCN1 (B7-H4), CD28, CTLA4, PDCD1 (PD-1), ICOS, BTLA (CD272), CD4, CD8A (CD8-α), CD8B (CD8-β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NC R3 (NKp30).

[0188] In some embodiments, the IgSF domain independently contains one or more amino acid substitutions compared to the IgSF domain in a wild-type or unmodified IgSF domain, such as the IgSF domains in the IgSF family members shown in Table 1. In some embodiments, the affinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified IgSF domain, or a specific-binding fragment thereof, contained in the amino acid sequence set forth in any of SEQ ID NOs: 1-27. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, e.g., an IgC1 or IgC2 domain. In some embodiments, the affinity-modified IgSF domain is an affinity-modified IgV domain or an IgC domain.

[0189] In some embodiments of the type II immunomodulatory proteins of the invention, the number of IgSF domains is at least two, and wherein the number of affinity-modified IgSF domains and the number of affinity-unmodified IgSF domains are each independently at least 0, 1, 2, 3, 4, 5, or 6. Thus, the ratio of the number of affinity-modified IgSF domains and the number of affinity-unmodified IgSF domains, respectively, can be exactly or at least 2:0 (affinity-modified:wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

[0190] In some embodiments of the type II immunomodulatory protein, at least two of the un-affinity-modified and / or affinity-modified IgSF domains are identical IgSF domains.

[0191] In some embodiments, the type II immunomodulatory proteins of the invention comprise at least two affinity-modified and / or affinity-unmodified IgSF domains derived from a single IgSF member but in a non-wild-type configuration (alternatively, "hypermutated"). One example of a non-wild-type configuration or hypermutation is an immunomodulatory protein of the invention comprising an affinity-modified and / or affinity-unmodified IgSF domain sequence of a non-wild-type series relative to that found in the wild-type mammalian IgSF family member that serves as the source of the affinity-unmodified and / or affinity-modified IgSF domain. Mammalian wild-type IgSF members in the preceding embodiments specifically include those listed in Table 1. Thus, in one example, if a wild-type family member comprises an IgC1 domain proximal to the transmembrane domain and an IgV domain distal to the transmembrane domain of a cell surface protein, an immunomodulatory protein of the invention, albeit in an affinity-unmodified and / or affinity-modified form, can comprise an IgV proximal to the transmembrane domain and an IgC1 domain distal to the transmembrane domain. The presence of both non-wild-type combinations and arrangements of affinity-unmodified and / or affinity-modified IgSF domains in the immunomodulatory proteins of the invention is also within the scope of the invention.

[0192] In some embodiments of type II immunomodulatory proteins, the affinity-unmodified and / or affinity-modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity-modified IgSF domains specifically bind to different cognate binding partners under specific binding conditions and are "non-identical" regardless of whether the wild-type IgSF domains being modified are the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein of the present invention can include at least one IgSF domain sequence that originates from one IgSF family member and is unique, and at least one second IgSF domain sequence that originates from another IgSF family member and is unique, wherein the IgSF domains of the immunomodulatory protein are in their affinity-unmodified and / or affinity-modified form. However, in alternative embodiments, the two non-identical IgSF domains originate from the same IgSF domain sequence, but at least one has been affinity-modified so that they specifically bind to different cognate binding partners.

[0193] In some embodiments, the number of non-identical affinity-unmodified and / or affinity-modified IgSF domains present in the immunomodulatory proteins of the invention is at least 2, 3, 4, or 5, and in some embodiments exactly 2, 3, 4, or 5 non-identical affinity-unmodified and / or affinity-modified IgSF domains. In some embodiments, the non-identical IgSF domains are a combination derived from at least two IgSF members shown in Table 1, and in some embodiments at least three or four IgSF members of Table 1.

[0194] In some specific embodiments, the type II immunomodulatory proteins of the present invention comprise an affinity-modified NKp30 IgSF domain and an affinity-modified ICOSLG IgSF domain, an affinity-modified CD80 IgSF domain, or an affinity-modified CD86 IgSF domain. In some embodiments, the type II immunomodulatory protein comprises affinity-modified IgSF domains derived from at least two B7 family members. In some embodiments, the immunomodulatory protein comprises at least two affinity-modified domains derived from an affinity-modified CD80 IgSF domain, an affinity-modified ICOSL IgSF domain, or an affinity-modified CD86 IgSF domain, or specific-binding fragments thereof. In some embodiments, the affinity-modified domains are linked via at least or exactly one, two, three, or four G4S domains.

[0195] The multiple affinity-unmodified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be directly covalently linked to one another. In some embodiments, an intervening stretch of one or more amino acid residues indirectly covalently links the affinity-unmodified and / or affinity-modified IgSF domains to one another. The linkage can be via N- to C-terminal residues.

[0196] In some embodiments, the linkage can be via the side chain of an amino acid residue that is not located at the N-terminus or C-terminus of the non-affinity-modified and / or affinity-modified IgSF domain. Thus, the linkage can be via a terminal or internal amino acid residue or a combination thereof.

[0197] In some embodiments, the "peptide linker" connecting the affinity-unmodified and / or affinity-modified IgSF domains can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue or less in length. In some embodiments, the linker is (in the single-letter amino acid code): GGGGS ("4GS") or a multimer of 4GS linkers, e.g., 2, 3, 4, or 5 repeats of the 4GS linker. In further optional embodiments, a series of alanine residues is interposed between the peptide linker (e.g., a 4GS linker or multimer thereof) and the Fc to which the immunomodulatory protein is covalently linked. In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines.

[0198] In some embodiments, the affinity-unmodified and / or affinity-modified IgSF domains are linked by a "wild-type peptide linker" inserted at the N-terminus and / or C-terminus of the first and / or second affinity-unmodified and / or affinity-modified IgSF domain. In some embodiments, there is a leading peptide linker inserted at the N-terminus of the first IgSF domain and / or a first trailing sequence inserted at the C-terminus of the first affinity-unmodified and / or affinity-modified IgSF domain. In some embodiments, there is a second leading peptide linker inserted at the N-terminus of the second IgSF domain and / or a second trailing sequence inserted at the C-terminus of the second affinity-unmodified and / or affinity-modified IgSF domain. When the first and second affinity-unmodified and / or affinity-modified IgSF domains are derived from the same parent protein and are connected in the same orientation, there is no repeated occurrence of a wild-type peptide linker between the first and second affinity-unmodified and / or affinity-modified IgSF domains. For example, when the first trailing wild-type peptide linker and the second leading wild-type peptide linker are the same, the type II immunomodulatory protein does not include either the first trailing wild-type peptide linker or the second leading wild-type peptide linker.

[0199] In some embodiments, the type II immunomodulatory protein comprises a first leading wild-type peptide linker inserted at the N-terminus of a first affinity-unmodified and / or affinity-modified IgSF domain, wherein the first leading wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids derived from an intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain). In some embodiments, the first leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain).

[0200] In some embodiments, the type II immunomodulatory protein further comprises a first trailing wild-type peptide linker inserted at the C-terminus of the first affinity-unmodified and / or affinity-modified IgSF domain, wherein the first trailing wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids derived from an intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain). In some embodiments, the first trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain).

[0201] In some embodiments, the type II immunomodulatory protein further comprises a second leading wild-type peptide linker inserted at the N-terminus of the second affinity-unmodified and / or affinity-modified IgSF domain, wherein the second leading wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids derived from an intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain). In some embodiments, the second leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain).

[0202] In some embodiments, the type II immunomodulatory protein further comprises a second trailing wild-type peptide linker inserted at the C-terminus of the second affinity-unmodified and / or affinity-modified IgSF domain, wherein the second trailing wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids derived from an intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., IgSF domain or transmembrane domain). In some embodiments, the second trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., IgSF domain or transmembrane domain).

[0203] Exemplary leading and trailing sequences for a type II protein containing a CD80 IgSF domain are set forth in SEQ ID NO:231 and SEQ ID NO:232. Exemplary leading and trailing sequences for a type II protein containing an ICOSL IgSF domain are set forth in SEQ ID NOs:233 and 234. Exemplary leading and trailing sequences for a type II protein containing a CD86 IgSF domain are set forth in any of SEQ ID NOs:236-238. An exemplary wild-type linker sequence for a type II protein containing an NKp30 IgSF domain is set forth in SEQ ID NO:235.

[0204] C. Affinity-engineered immunomodulatory protein formats In some embodiments, the immunomodulatory proteins provided herein are in soluble form. Those skilled in the art will recognize that cell surface proteins typically have intracellular, transmembrane, and extracellular domains (ECDs), and that such soluble forms of proteins can be produced using the extracellular domain or an immunologically active subsequence thereof. Thus, in some embodiments, the immunomodulatory protein containing an affinity-modified IgSF domain lacks the transmembrane domain or a portion of the transmembrane domain. In some embodiments, the immunomodulatory protein containing an affinity-modified IgSF domain lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the immunomodulatory protein contains an affinity-modified IgSF domain containing only an ECD domain or a portion thereof containing an IgV domain and / or an IgC domain or a specific binding fragment thereof.

[0205] In some embodiments, the soluble form of the immunomodulatory protein of the present invention is covalently linked directly or indirectly to an immunoglobulin Fc. Generally, the Fc is covalently linked to the amino terminus of the immunomodulatory protein. In some embodiments, the immunoglobulin Fc is a mammalian IgG class immunoglobulin, such as IgG1 or IgG2. In certain embodiments, the Fc will be a human IgG1 or IgG2 Fc. Those skilled in the art will recognize that minor changes, such as one, two, three, or four amino acid substitutions, deletions, additions, or combinations thereof, can be made to the Fc without substantially altering its pharmacokinetic properties. Such changes may be made, for example, to aid manufacturability or to enhance, suppress, or eliminate antibody-dependent cell-mediated cytotoxicity. The term "Fc" as used herein is intended to encompass such molecules.

[0206] In some embodiments, the Fc is a murine or human Fc. In some embodiments, the Fc is derived from an IgG1, such as a human IgG1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:226, or an amino acid 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:226. In some embodiments, the Fc is derived from an IgG2, such as a human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:227, or an amino acid 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:227.

[0207] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the IgSF-Fc variant fusions provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations to the Fc sequence that can alter effector function. For example, WO 00 / 42072, WO 2006019447, and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcR. The contents of these publications are specifically incorporated herein by reference.

[0208] In some embodiments, the Fc region possesses some, but not all, effector functions that make it a desirable candidate for applications in which the in vivo half-life of the Fc fusion is important, yet certain effector functions (e.g., CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the Fc-ICOSL variant fusion lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding activity. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., the ACTI™ Non-Radiotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and the CytoTox 96™ Non-Radiotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the Fc-ICOSL variant fusion is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0209] Fc fusions with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0210] Certain Fc variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; WO 2006019447; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0211] In some embodiments, the changes occur in the Fc region, resulting in reduced C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0212] In some embodiments, ICOSL-Fc variant fusions are provided that comprise a variant Fc region comprising one or more amino acid substitutions that increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues according to EU numbering: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0213] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0214] In some embodiments, the Fc is an IgG1 variant containing at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO:226 (corresponding to N297G according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification. For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: C5S and N82G.

[0215] In some embodiments, indirect covalent attachment of Fc to the immunomodulating proteins of the invention can be achieved, for example, via a single amino acid or a peptide (two or more amino acid residues long) linker. Furthermore, the single polypeptide chains of such Fc fusion molecules can dimerize through various means, including via inter-polypeptide chain disulfide bonds. Dimerized forms of the immunomodulating proteins of the invention can comprise two identical or substantially identical species of polypeptides of the invention (homodimers) or polypeptide chains of the invention of different species (heterodimers). It will be recognized that minor heterogeneity can exist even between polypeptide chains of the same species due to minor differences in amino- and carboxy-terminal residues resulting from minor differences in expression or proteolysis, or due to differences arising from post-translational modifications. Nevertheless, such substantially identical chains are considered homodimers. Derivatized immunomodulating proteins are within the scope of the invention and are often manufactured to provide, for example, altered physicochemical or pharmacokinetic properties.

[0216] In even more specific embodiments, the preceding specific embodiments are covalently linked to an Fc, such as a human IgG1 or IgG2 domain. In further specific embodiments, the Fc is linked to an immunomodulatory protein via one or more G4S domains (often having at least or exactly 1, 2, 3, 4, or 5 consecutive alanine residues directly linked to the Fc and the immunomodulatory protein).

[0217] In other embodiments, the immunomodulatory proteins provided herein are bound to the liposome membrane. Various methods for covalently or non-covalently binding proteins to the liposome surface (e.g., by amide conjugation or disulfide / thioether conjugation) are known in the art.

[0218] D. Functional Activity of Immunomodulatory Proteins In some embodiments, immunomodulatory proteins (full-length and / or specific-binding fragments or stack constructs or fusions thereof) containing affinity-engineered IgSF domains provided herein exhibit immunomodulatory activity that modulates T cell activation. Functionally, and regardless of whether they have increased or decreased specific binding to their cognate binding partners, the immunomodulatory proteins provided herein act to enhance or suppress immune activity of lymphocytes, e.g., in an MLR assay, relative to lymphocytes under appropriate assay controls. In some embodiments, the immunomodulatory proteins provided herein comprise at least two affinity-engineered IgSF domains, wherein at least one of the affinity-engineered IgSF domains acts to enhance immune activity and at least one affinity-engineered IgSF domain acts to suppress immune activity.

[0219] In some embodiments, the provided immunomodulatory proteins modulate IFN-γ expression in primary T cell assays compared to a wild-type or unmodified IgSF domain control. In some cases, modulating IFN-γ expression can increase or decrease IFN-γ expression compared to a control. Assays for determining specific binding and IFN-γ expression are well known in the art and include the MLR (mixed lymphocyte reaction) assay, which measures interferon-γ cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), the SEB (Staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), and the anti-CD3 T cell stimulation assay (Li and Kurlander, J Transl Med. 2010: 8: 104).

[0220] In some embodiments, immunomodulatory proteins containing affinity-engineered domains can increase, or in alternative embodiments decrease, IFN-γ (interferon-γ) expression in primary T cell assays relative to a wild-type IgSF domain control. In some embodiments of provided polypeptides containing affinity-engineered IgSF domains, the polypeptides can increase, or in alternative embodiments decrease, IFN-γ expression in primary T cell assays relative to a wild-type ICOSL control. In some embodiments of provided polypeptides containing multiple affinity-engineered IgSF domains, the polypeptides can increase, or in alternative embodiments decrease, IFN-γ expression in primary T cell assays relative to a wild-type IgSF domain control.

[0221] Those skilled in the art will recognize that the format of the primary T cell assay used to determine increased IFN-γ expression can differ from that employed to assay for decreased IFN-γ expression. When assaying for the ability of an immunomodulatory protein to decrease IFN-γ expression in a primary T cell assay, a mixed lymphocyte reaction (MLR) assay can be used, as described in Example 6. In some cases, employing a soluble form of the immunomodulatory protein, the ability of the affinity-engineered IgSF domain to decrease IFN-γ expression by antagonizing it can be determined in an MLR, also as described in Example 6.

[0222] Alternatively, when assaying for the ability of an immunomodulatory protein to increase IFN-γ expression in primary T cell assays, a co-fixation assay can be used, as described in Example 6. In the co-fixation assay, a TCR signal (provided in some embodiments by an anti-CD3 antibody) is combined with a co-fixed immunomodulatory protein containing an affinity-engineered IgSF domain to determine its ability to increase IFN-γ expression relative to an IgSF domain control. In some cases, the ability of the immunomodulatory protein to increase its expression by stimulating IFN-γ expression can be determined in an MLR, also as described in Example 6, employing a soluble form of the immunomodulatory protein that has been multimerized to provide multivalent binding.

[0223] The use of appropriate controls will be known to those of skill in the art, but in the foregoing embodiments, the control will typically involve the use of an unmodified IgSF domain, e.g., a wild-type naturally occurring IgSF isoform from the same mammalian species from which the IgSF domain is derived or generated. Regardless of whether it has improved or decreased binding affinity for one or both of its cognate binding partners, a particular immunomodulatory protein will in some embodiments increase IFN-γ expression, and in alternative embodiments decrease IFN-γ expression, in primary T cell assays relative to the wild-type IgSF domain control.

[0224] In some embodiments, the immunomodulatory protein (e.g., containing an affinity-engineered IgSF domain) increases IFN-γ expression (i.e., protein expression) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a wild-type or unmodified IgSF domain control. In other embodiments, the immunomodulatory protein (e.g., containing an affinity-engineered IgSF domain) decreases IFN-γ expression (i.e., protein expression) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a wild-type or unmodified IgSF domain control. In some embodiments, enhanced immune activity 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, for example, in an MLR assay. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. In some embodiments, suppressed immune activity can be a decrease of at least 10%, and up to 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0225] III. Methods for Producing Nucleic Acids and Proteins The present invention provides isolated or recombinant nucleic acids, collectively referred to as "nucleic acids of the invention," that encode any of the various embodiments of the immunomodulatory proteins (type I and type II) of the invention. Nucleic acids of the invention, including all of the following, are useful in the recombinant production (e.g., expression) of the polypeptides of the invention. Nucleic acids of the invention can be in the form of RNA or DNA and can include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. Nucleic acids of the invention are typically DNA molecules, and usually double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA / RNA nucleic acids, or combinations thereof, comprising any of the nucleotide sequences of the invention are also provided.

[0226] The present invention also relates to expression vectors and host cells useful for producing the immunomodulatory proteins of the present invention. The immunomodulatory proteins of the present invention can be produced in transformed host cells using recombinant DNA technology. To this end, recombinant DNA molecules encoding the immunomodulatory proteins are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequence encoding the peptide can be excised from DNA using a suitable restriction enzyme. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques such as the phosphoramidite method. A combination of these techniques can also be used. In some examples, recombinant or synthetic nucleic acids can be generated through polymerase chain reaction (PCR).

[0227] The present invention also includes expression vectors capable of expressing immunomodulatory proteins in suitable host cells under conditions suitable for their expression. Recombinant expression vectors contain a DNA molecule encoding an immunomodulatory protein operably linked to appropriate expression control sequences. Methods for making this functional linkage either before or after the DNA molecule is inserted into a vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, capping signals, polyadenylation signals, and other signals involved in the control of transcription or translation. The resulting recombinant expression vector carrying the DNA molecule thereon is used to transform an appropriate host. This transformation can be carried out using methods well known in the art. In some embodiments, the nucleic acids of the present invention further contain a nucleotide sequence encoding a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory protein of the present invention so that the immunomodulatory protein can be recovered from the culture medium, the host cell, or the host cell periplasm.

[0228] Any of a number of available and well-known host cells can be used in the practice of the present invention. The selection of a suitable host depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biological safety, and cost. These factors must be balanced, understanding that not all hosts are equally efficient at expressing a particular DNA sequence. Host cells can be various eukaryotic cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) or HEK293 cells. Host cells can also be prokaryotic cells, such as Escherichia coli (E. coli). The transformed host is cultured under conditions for immunomodulatory protein expression and then purified. The recombinant host cells can be cultured under conventional fermentation conditions to express the desired immunomodulatory protein. Such fermentation conditions are well known in the art. Finally, the immunomodulatory protein is recovered and purified from the recombinant cell culture by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphate cellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. If desired, a protein refolding step can be used to complete the configuration of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be employed for final purification. The immunomodulatory proteins of the present invention can also be produced synthetically. Solid-phase synthesis is the preferred technique for producing individual peptides because it is the most cost-effective method for producing small peptides. For example, well-known solid-phase synthesis techniques involve the use of protecting groups, linkers, and solid-phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis. The peptides can then be assembled into the immunomodulatory proteins of the present invention.

[0229] The means by which the immunomodulatory affinity-modified IgSF domains of the present invention are designed or generated are not limited to any particular method. However, in some embodiments, wild-type IgSF domains are mutagenized (site-directed, random, or a combination thereof) from wild-type IgSF genetic material and screened for altered binding properties according to the methods disclosed in the Examples. Methods for mutagenizing nucleic acids are known to those of skill in the art. In some embodiments, affinity-modified IgSF domains are synthesized de novo using protein or nucleic acid sequences available in numerous public databases and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database discussed above.

[0230] IV. Methods for screening or identifying affinity-engineered IgSF domains Provided herein are methods for identifying affinity-modified immunomodulatory proteins that can simultaneously or non-competitively bind to two or more cognate binding partners. In some embodiments, the methods include the following steps: a) contacting a modified protein comprising at least one modified non-immunoglobulin immunoglobulin superfamily (IgSF) domain or its specific-binding fragment with at least two cognate binding partners under conditions that can result in binding of the protein to the at least two cognate binding partners, wherein at least one modified IgSF domain comprises one or more amino acid substitutions in the wild-type IgSF domain; b) identifying modified proteins comprising modified IgSF domains that have improved binding to at least one of the two cognate binding partners compared to proteins comprising the wild-type IgSF domain; and c) selecting modified proteins comprising modified IgSF domains that bind non-competitively to at least two cognate binding partners, thereby identifying affinity-modified immunomodulatory proteins. In some embodiments, the selected affinity-modified proteins can simultaneously bind to both of the two cognate binding partners. It is within the level of ordinary skill in the art to assess or determine the existence of non-competitive binding interactions of a protein to two different ligands. An exemplary such method is described in Example 7.

[0231] In some embodiments, the IgSF domain is a non-immunoglobulin IgSF domain. In some embodiments, the modified or variant protein comprises one or more amino acid substitutions, deletions, or insertions in the IgSF domain of any of the non-immunoglobulin IgSF family members (e.g., any shown in Table 1). In some embodiments, the modified or variant IgSF domain, or modified protein containing a modified or variant IgSF domain, contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid changes, e.g., amino acid substitutions.

[0232] In some embodiments, a library of modified or variant proteins is generated by mutating any one or more amino acid residues of a protein known to contain a non-immunoglobulin IgSF domain, using any method generally known in the art. In generating the modified IgSF domains herein, any of the methods employed in the art for generating, modifying, or diversifying binding molecules can be used. Exemplary such methods for generating, modifying or diversifying binding molecules are described in U.S. Patent Nos. 5,223,409; 5,571,698; 5,750,373; 5,821,047; 5,837,500; 5,733,743; 5,871,907; 5,969,108; 6,040,136; 6,172,197; 6,291,159; 6,955,877; 6,979,538; 6,831,161; 7,063,943; 7,118,879; 7,208,293; 7,332,571; 7,385 ,028; 7,696,312; 7,638,299; 7,888,533; 7,642,044; U.S. Patent Application Nos. US20080300163; US20090208454; US20090155843; US20080113412; US20100035812; US20100093608; US20110015345; for example, in methods for diversifying or modifying biomolecules, IgSF-contiain can be used in the method instead of other binding molecules.

[0233] The methods for generating a library of binding molecules and the methods for creating diversity in the library are well known in the art, and can be used to generate a library of protein variants.The approach for generating diversity includes the targeted and non-targeted approaches well known in the art.For example, the known approaches for generating diverse nucleic acid and polypeptide libraries include but are not limited to error-prone PCR, cassette mutagenesis; mutual primer extension method; template-assisted ligation and extension method; codon cassette mutagenesis; oligonucleotide-directed mutagenesis; amplification using degenerate oligonucleotide primers, including overlap and two-step PCR; and combination approaches, such as combinatorial multiple cassette mutagenesis (CMCM) and related techniques.Those skilled in the art are familiar with these techniques.

[0234] Examples of methods for mutating proteins to generate libraries of candidate modified protein molecules include methods that result in random mutagenesis throughout the protein sequence or methods that result in mutagenesis of selected regions or domains of the protein. Mutations can be introduced randomly using methods that result in random mutagenesis of proteins, or more systematically using methods that specifically create single or multiple amino acid changes at targeted positions. Both random mutagenesis and systematic site-directed mutagenesis can be used to introduce one or more mutations into a protein. The variant protein can have one or more amino acid substitutions, insertions, or deletions internally compared to the wild-type or unmodified protein used as a scaffold to generate the library. The substitutions or insertions can be with any naturally occurring or non-naturally occurring amino acid.

[0235] In a method for identifying or generating variant or modified proteins containing a non-immunoglobulin IgSF domain according to the provided methods, one or more regions of a protein, such as one or more IgSF domains of a protein, can be modified using random mutagenesis of the region to generate one or more modified protein molecules. For example, a library of variants can be generated containing multiple modified molecules, each differing by at least one amino acid exchange (i.e., substitution), deletion, or insertion in the IgSF domain compared to the corresponding unmodified or wild-type protein containing the IgSF domain. The amino acid substitution can be a naturally occurring or non-naturally occurring amino acid substitution compared to the unmodified or wild-type protein. Generally, the libraries provided herein contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 10, 25, 30, 35, 40, 50, 60, 75, 80, 95, 100, 120, 140, 160, 180, 220, 240, 260, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 710, 720, 730, 74 2 , 10 3 , 10 4 , 2 × 10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 Libraries containing 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,

[0236] In some embodiments, prior to the step of selecting engineered proteins comprising engineered IgSF domains that bind non-competitively to at least two cognate binding partners, the method comprises combining two or more engineered IgSF domains or specific binding fragments thereof identified in step (b) to generate a stacked molecular construct containing a plurality of different engineered IgSF domains.

[0237] Thus, in some embodiments, provided herein is a method for identifying an immunomodulatory protein with modified affinity, the method comprising the steps of: a) contacting a modified protein comprising at least one modified non-immunoglobulin immunoglobulin superfamily (IgSF) domain or a specific-binding fragment thereof with at least two cognate binding partners under conditions capable of resulting in binding of the protein to the at least two cognate binding partners, wherein at least one modified IgSF domain comprises one or more amino acid substitutions in a wild-type IgSF domain; b) identifying a modified protein comprising a modified IgSF domain that has improved binding to at least one of the two cognate binding partners compared to a protein comprising the wild-type IgSF domain; c) combining two or more modified IgSF domains present in two or more identified proteins to generate a fusion (stacked) protein comprising a first modified IgSF domain linked to a second IgSF domain; and d) selecting a modified protein comprising a modified IgSF domain that binds non-competitively to at least two cognate binding partners, thereby identifying an immunomodulatory protein with modified affinity.

[0238] In some embodiments, at least two cognate binding partners are cell surface molecular species expressed on the surface of a mammalian cell. In some embodiments, the cell surface molecular species are expressed in a cis or trans configuration. In some embodiments, the mammalian cell is one of two mammalian cells forming an immune synapse (IS), and each of the cell surface molecular species is expressed on the surface of at least one of the two mammalian cells forming the IS. In some embodiments, at least one of the mammalian cells is a lymphocyte, which can be an NK cell or a T cell. In some embodiments, at least one of the mammalian cells is a tumor cell. In some embodiments, at least one of the mammalian cells is an antigen-presenting cell.

[0239] In some embodiments, two or more cognate binding partners are independently ligands of an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30. In some embodiments, two or more cognate binding partners are independently ligands of a B7 family member. In some embodiments, two or more cognate binding partners are selected from two or more of CD28, CTLA-4, ICOS, or PD-L1.

[0240] V. Pharmaceutical Compositions and Formulations Pharmaceutical compositions, including therapeutic compositions of the present invention, may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. The primary vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection or saline, possibly supplemented with other ingredients common to compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute thereof. In one embodiment of the present invention, the binder composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired purity with any compounding agents. Additionally, the binder product may be formulated as a lyophilizate using appropriate excipients, such as sucrose.

[0241] The formulation components are present in concentrations acceptable to the site of administration. For example, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. A particularly suitable vehicle for parenteral administration is sterile distilled water, in which the binder is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation involves the formulation of the desired molecule with agents such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes, which provide controlled or sustained release of the product, which can then be delivered via depot injection.

[0242] In another aspect, pharmaceutical preparations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additional pharmaceutical compositions, including formulations of binding agent molecules in sustained- or controlled-delivery formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Pharmaceutical compositions to be used for in vivo administration must typically be sterilized. This may be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method may be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Additionally, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0243] In some embodiments, pharmaceutical compositions are sterilized.Sterilization can be achieved by filtration through a sterile filtration membrane or irradiation.When the composition is lyophilized, sterilization using this method can be carried out before or after lyophilization and reconstitution.Compositions for parenteral administration can be stored in lyophilized form or in solution.In addition, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0244] Once the pharmaceutical composition is formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution (e.g., lyophilized) prior to administration. The effective amount of the pharmaceutical composition to be employed therapeutically will depend, for example, on the therapeutic context and purpose. Thus, those skilled in the art will recognize that appropriate dosage levels for therapy will depend in part on the molecule being delivered, the indication for which the binding agent molecule is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and condition (age and overall health). Thus, clinicians may titrate dosages and modify the route of administration to obtain optimal therapeutic effects. Therapeutic compositions of the present invention can be administered parenterally, subcutaneously, or intravenously, or as described elsewhere herein. Therapeutic compositions of the present invention may be administered in therapeutically effective amounts one, two, three, or four times per month, twice per week, every two weeks, or every two months. Administration may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more (e.g., 1, 2, 3, 4 years or more, including the lifetime of the subject).

[0245] Generally, the dosage and route of administration of pharmaceutical compositions are determined according to standard pharmaceutical practice, according to the size and condition of the subject. For example, therapeutically effective doses can be estimated first either through cell culture assays or animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can also be used to determine appropriate concentration ranges and administration routes. Such information can then be used to determine useful doses and administration routes in humans. The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide a sufficient level of the active compound or to maintain the desired effect. Factors that can be considered include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, the time and frequency of administration, drug combinations, reaction sensitivities, and response to treatment.

[0246] In some embodiments, the pharmaceutical composition is administered to the subject through any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, by direct application to a wound site, by application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intraventricularly, intra-articularly, intraocularly, or intrathecally.

[0247] In some embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses. In some embodiments, the dose is given to a subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, about one or more doses (e.g., about two or more, about three or more, about four or more, about five or more, about six or more, or about seven or more) are given per week. In some embodiments, multiple doses are given over the course of several days, weeks, months, or years. In some embodiments, a course of treatment is about one or more doses (e.g., about two or more doses, about three or more doses, about four or more doses, about five or more doses, about seven or more doses, about ten or more doses, about fifteen or more doses, about twenty-five or more doses, about forty or more doses, about fifty or more doses, or about one hundred or more doses).

[0248] In some embodiments, the administered dose of the pharmaceutical composition is about 1 μg or more of protein per kg of subject body weight (e.g., about 2 μg or more of protein per kg of subject body weight, about 5 μg or more of protein per kg of subject body weight, about 10 μg or more of protein per kg of subject body weight, about 25 μg or more of protein per kg of subject body weight, about 50 μg or more of protein per kg of subject body weight, about 100 μg or more of protein per kg of subject body weight, about 250 μg or more of protein per kg of subject body weight, about 500 μg or more of protein per kg of subject body weight, about 1 mg or more of protein per kg of subject body weight, about 2 mg or more of protein per kg of subject body weight, or about 5 mg or more of protein per kg of subject body weight).

[0249] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models may also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans. The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, time and frequency of administration, drug combinations, reaction sensitivities, and response to treatment. Depending on the half-life and clearance rate of the particular formulation, long-acting pharmaceutical compositions may be administered every 3 to 4 days, weekly, or biweekly. The dosing frequency will depend on the pharmacokinetic parameters of the molecule in the formulation used. Typically, the composition is administered until a dosage that achieves the desired effect is reached. Thus, the composition may be administered as a single dose or as multiple doses (at the same or different concentrations / dosages) over time, or as a continuous infusion. Further refinement of appropriate dosages is routine. Appropriate dosages may be ascertained through the use of appropriate dose-response data.

[0250] In some embodiments, one or more biomarkers or physiological markers of therapeutic efficacy can be monitored, including T cell activation or proliferation, cytokine synthesis or production (e.g., production of TNF-α, IFN-γ, IL-2), induction of various activation markers (e.g., CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serum levels of C-reactive protein, anti-collagen antibody production, and / or T cell-dependent antibody responses.

[0251] Injectable pharmaceutical compositions comprising a suitable pharmaceutically acceptable excipient or carrier (e.g., PBS) and an effective amount of a therapeutic composition of the present invention can be administered to a mammalian patient parenterally, intramuscularly, intraperitoneally, intravenously, subcutaneously, transdermally, subcutaneously, or intradermally. Administration can be facilitated via liposomes. Skin and muscle are generally preferred targets for administration of a therapeutic composition of the present invention by any suitable technique. Thus, delivery of a therapeutic composition of the present invention to or through the skin of a mammalian subject (e.g., a human) is a feature of the present invention. Such molecules of the present invention can be administered to or through the skin in a pharmaceutically acceptable injectable solution, for example, intramuscularly or intraperitoneally. Administration can also be achieved by transdermal devices, or more typically, by biolistic delivery of a therapeutic composition of the present invention to or through the skin of a subject or to exposed muscles of a mammalian subject.

[0252] Various means are known for determining whether administration of a therapeutic composition of the invention sufficiently modulates immune activity, such as by eliminating, sequestering, or inactivating immune cells that mediate or are capable of mediating an undesired immune response; inducing, generating, or stimulating immune cells that mediate or are capable of mediating a protective immune response; altering the physical or functional properties of immune cells; or a combination of these effects. Exemplary measures of modulation of immune activity include examining the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measuring the functional capabilities of immune cells, including their ability to proliferate or divide in response to a signal, or their resistance to proliferation or division (e.g., using T cell proliferation assays and pepscan analysis based on H-thymidine incorporation after stimulation with antigen-presenting cells loaded with anti-CD3 antibodies, anti-T cell receptor antibodies, anti-CD28 antibodies, calcium ionophore, PMA, peptide or protein antigens; B cell proliferation assays); measuring the ability to kill or lyse other cells (e.g., using cytotoxic T cell assays); measurement of cytokines, chemokines, cell surface molecules, antibodies, and other products of cells (e.g., by flow cytometry, enzyme-linked immunosorbent assay, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of immune cells or signaling pathways within immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine, or threonine phosphorylation, polypeptide cleavage, and protein complex formation or dissociation; protein array analysis; DNA transcription profiling using DNA arrays or subtractive hybridization); measurement of cell death by apoptosis, necrosis, or other mechanisms (e.g., Annexin V staining, TUNEL assay, gel electrophoresis measuring DNA laddering, histology; fluorogenic caspase assay, Western blot analysis of caspase substrates);Measurement of genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); and measurement of clinical outcomes such as clinical symptoms or improvement of autoimmune diseases, neurodegenerative diseases, and other diseases involving self-proteins or polypeptides (clinical scores, need for additional therapy, functional status, imaging studies), for example, by measuring relapse rate or disease severity in the case of multiple sclerosis (using clinical scores known to those of skill in the art), blood glucose in the case of type I diabetes, or joint inflammation in the case of rheumatoid arthritis.

[0253] Also provided herein is an article of manufacture comprising the pharmaceutical composition described herein in suitable packaging. Suitable packaging for the compositions (such as ophthalmic compositions) described herein is known in the art and includes, for example, vials (e.g., sealed vials), jars, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These articles of manufacture may be further sterilized and / or sealed.

[0254] Additionally, kits are provided that contain the pharmaceutical compositions (or articles of manufacture) described herein, which may further include instructions for using the compositions, such as for the uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing any of the methods described herein.

[0255] VI. Therapeutic applications The immunomodulatory proteins of the present invention are believed to be useful in a variety of applications, including, but not limited to, prophylactic or therapeutic methods for treating various immune system diseases or conditions in mammals in which modulation or control of the immune system and immune system responses is beneficial (collectively, the "therapeutic compositions of the present invention"). For example, suppressing the immune response can be beneficial in prophylactic and / or therapeutic methods for inhibiting the recipient's rejection of a tissue, cell, or organ transplant from a donor. In a therapeutic context, the mammalian subject typically has an immune system disease or condition, and administration is carried out to further prevent the progression of the disease or condition. For example, administration of a therapeutic composition of the present invention to a subject suffering from an immune system disease (e.g., an autoimmune disease) can result in the suppression or inhibition of such immune system attack or its associated biological responses. By suppressing this immune system attack against healthy body tissue, the resulting physical symptoms resulting from or associated with such attack against healthy tissue (e.g., pain, joint inflammation, joint swelling or tenderness) can be reduced or alleviated, and the biological and physical damage resulting from or associated with the immune system attack can be reduced, slowed, or stopped. In a prophylactic setting, a subject may be considered to have, be susceptible to, or exhibit an immune system disease, disorder, or condition, and administration is typically carried out to prevent the progression of the disease, disorder, or condition, inhibit or reduce symptoms, signs, or biological responses associated therewith, prevent physical damage potentially resulting therefrom, and / or maintain or improve the subject's bodily function.

[0256] Diseases or disorders of the patient's immune system include, but are not limited to, Addison's disease, allergies, alopecia areata, Alzheimer's disease, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, ankylosing spondylitis, antiphospholipid syndrome (Hughes syndrome), arthritis, asthma, atherosclerosis, atherosclerotic plaques, autoimmune diseases (e.g., lupus, RA, MS, Graves' disease, etc.), autoimmune hemolytic anemia, autoimmune hepatitis, inner ear autoimmune disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, azoospermia, Behcet's disease, Berger's disease, bullous pemphigoid, cardiomyopathy, cardiovascular disease, celiac disease / ciliary disease, chronic fatigue and immune deficiency syndrome (CFIDS), chronic idiopathic polyneuropathy, chronic inflammatory demyelinating polyradiculopathy, Chronic Relapsing Polyradicalneuropathy (CIPD), Chronic Relapsing Polyneuropathy (Guillain-Barré syndrome), Churg-Strauss syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Crohn's disease, Dermatitis Herpetiformis, Dermatomyositis, Diabetes, Discoid Lupus, Eczema, Epidermolysis Bullosa Acquisita, Essential Mixed Cryoglobulinemia, Evans syndrome, Exopthalmos, Fibromyalgia, Goodpasture's syndrome, Transplant-related Disease or Disorder, Graves' Disease, GVHD, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia IgA nephropathy, immunoproliferative diseases or disorders (e.g., psoriasis), inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM), interstitial lung disease, juvenile diabetes mellitus, juvenile arthritis, juvenile idiopathic arthritis (JIA), Kawasaki disease, Lambert-Eaton myasthenic syndrome, lichen planus, lupus, lupus nephritis, lymphocytic lymphophysiitis, Meniere's disease, Miller Fisher syndrome / acute disseminated cerebrospinal radiculopathyencephalomyeloradiculopathy), mixed connective tissue disease, multiple sclerosis (MS), muscular rheumatism, myalgic encephalomyelitis (ME), myasthenia gravis, eye inflammation, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome (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 diseases, rheumatoid arthritis, The present invention relates to a method for preventing or suppressing immune responses associated with the rejection of donor tissue, cell, graft, or organ transplants by a recipient subject, and may be or include diseases such as leukosis, Schmidt's syndrome, scleroderma, Sjogren's syndrome, solid organ transplant rejection (kidney, heart, liver, lung, etc.), stiff-man syndrome, systemic lupus erythematosus (SLE), systemic scleroderma, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thyroiditis, type 1 diabetes, type 2 diabetes, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis. Transplant-related diseases or disorders include graft-versus-host disease (GVDH) (e.g., associated with bone marrow transplantation) and immune disorders arising from or associated with the rejection of organ, tissue, or cell graft transplants (e.g., tissue or cell allografts or xenografts), including, for example, skin, muscle, nerve cells, pancreatic islets, organ transplants, liver parenchymal cells, etc. With respect to donor tissue, cell, graft, or solid organ transplantation in a recipient subject, it is believed that the therapeutic compositions of the invention disclosed herein may be effective in preventing acute rejection of such transplant in the recipient and / or for long-term maintenance therapy to prevent rejection of such transplant in the recipient (e.g., inhibiting rejection of insulin-producing pancreatic islet cell transplants from a donor in a subject recipient suffering from diabetes).

[0257] The therapeutic compositions of the present invention can also be used as monotherapy (i.e., as a single agent), in combination with at least one chemotherapeutic agent (i.e., combination therapy), in combination with a cancer vaccine, in combination with an immune checkpoint inhibitor, and / or in combination with radiation therapy to inhibit the growth of mammalian, particularly human, cancer cells. In some aspects of the present disclosure, the immune checkpoint inhibitor is nivolumab, tremelimumab, pembrolizumab, ipilimumab, or the like. An effective amount of the therapeutic composition is administered to inhibit, stop, or reverse the progression of cancers that are sensitive to modulation of immune activity by the immunomodulatory proteins of the present invention. Human cancer cells can be treated in vivo or ex vivo. In ex vivo treatment of human patients, tissues or body fluids containing cancer cells are treated outside the body, and then the tissues or body fluids are reintroduced into the patient. In some embodiments, cancer is treated in vivo in human patients by administering a therapeutic composition to the patient. Thus, the present invention provides ex vivo and in vivo methods that inhibit, halt, or reverse tumor progression, or otherwise result in a statistically significant improvement in progression-free survival (i.e., the length of time a patient lives during and after treatment without their cancer getting worse) or overall survival (also called "survival rate"; i.e., the percentage of people in a study or treatment group who survive for a certain period of time after being diagnosed with or treated for cancer) compared to control treatments. Cancers that can be treated by the methods of the present invention include, but are not limited to, melanoma, bladder cancer, hematologic malignancies (leukemia, lymphoma, myeloma), liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), spleen cancer, cancer of the thymus or blood cells (i.e., leukemia), prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, or Ewing's sarcoma.

[0258] VII. Illustrative Embodiments The following aspects are provided:

[0259] Embodiment 1. In some embodiments, an immunomodulatory protein is provided that comprises at least one affinity-modified non-immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type IgSF domain, wherein the at least one affinity-modified IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type IgSF domain; and wherein the at least one affinity-modified IgSF domain specifically binds to the at least two cognate binding partners in a non-competitive manner.

[0260] Embodiment 2. In some further embodiments of Embodiment 1, said at least two cognate binding partners are cell surface species expressed on the surface of mammalian cells.

[0261] Embodiment 3. In some further embodiments of Embodiment 2, the cell surface molecular species are expressed in a cis or trans configuration.

[0262] Embodiment 4. In some further embodiments of Embodiment 2 or Embodiment 3, the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and each of the cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS.

[0263] Embodiment 5. In some further embodiments of any one of Embodiments 2-4, at least one of the mammalian cells is a lymphocyte.

[0264] Embodiment 6. In some further embodiments of Embodiment 5, the lymphocytes are NK cells or T cells.

[0265] Embodiment 7. In some further embodiments of any one of Embodiments 5-6, binding of the affinity-engineered IgSF domain modulates immune activity of the lymphocyte.

[0266] Embodiment 8. In some further embodiments of Embodiment 7, the immunomodulatory protein is capable of providing enhanced immune activity compared to a wild-type protein that includes the wild-type IgSF domain.

[0267] Embodiment 9. In some further embodiments of Embodiment 7, the immunomodulatory protein is capable of providing reduced immune activity compared to a wild-type protein that comprises the wild-type IgSF domain.

[0268] Embodiment 10. In some further embodiments of any one of Embodiments 2-9, at least one of the mammalian cells is a tumor cell.

[0269] Embodiment 11. In some further embodiments of any one of Embodiments 2 to 10, the mammalian cell is a human cell.

[0270] Embodiment 12. In some further embodiments of any one of Embodiments 4 to 11, the affinity-engineered IgSF domain is capable of specifically binding to the two mammalian cells that form the IS.

[0271] Embodiment 13. In some further embodiments of any one of Embodiments 1 to 12, the wild-type IgSF domain is a member of the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling Lymphocyte Activation Molecule (SLA) family, the IgSF domain ... It is derived from an IgSF family member of a family selected from the following: the SLAM family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor related (PVR) family, the natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the killer-cell immunoglobulin-like receptor (KIR) family.

[0272] Embodiment 14. In some further embodiments of any one of Embodiments 1 to 13, the wild-type IgSF domain is derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

[0273] Embodiment 15. In some further embodiments of any one of Embodiments 1 to 14, the wild-type IgSF domain is a human IgSF member.

[0274] Embodiment 16. In some further embodiments of any one of Embodiments 1 to 15, the wild-type IgSF domain is an IgV domain, an IgC1 domain, an IgC2 domain, or a specific-binding fragment thereof.

[0275] Embodiment 17. In some further embodiments of any one of Embodiments 1 to 16, the affinity-modified IgSF domain is an affinity-modified IgV domain, an affinity-modified IgC1 domain, or an affinity-modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions.

[0276] Embodiment 18. In some further embodiments of any one of Embodiments 1 to 17, the immunomodulatory protein comprises at least two affinity-engineered non-immunoglobulin IgSF domains.

[0277] Embodiment 19. In some further embodiments of embodiment 18, each of said at least two affinity-altered non-immunoglobulin IgSF domains comprises one or more different amino acid substitutions in the same wild-type IgSF domain.

[0278] Embodiment 20. In some further embodiments of embodiment 19, each of said at least two affinity-altered non-immunoglobulin IgSF domains comprises one or more amino acid substitutions in a different wild-type IgSF domain.

[0279] Embodiment 21. In some further embodiments of Embodiment 20, said different wild-type IgSF domains are derived from different IgSF family members.

[0280] Embodiment 22. In some further embodiments of any one of Embodiments 1 to 17, the immunomodulatory protein comprises only one affinity-engineered non-immunoglobulin IgSF domain.

[0281] Embodiment 23. In some further embodiments of any one of Embodiments 1-22, the affinity-modified IgSF comprises at least 85% sequence identity to a wild-type IgSF domain, or a specific-binding fragment thereof, contained in an amino acid sequence set forth in any of SEQ ID NOs:1-27.

[0282] Embodiment 24. In some further embodiments of Embodiment 23, the immunomodulatory protein further comprises a second affinity-engineered IgSF domain comprising at least 85% sequence identity to a wild-type IgSF domain, or a specific-binding fragment thereof, contained in an amino acid sequence set forth in any of SEQ ID NOs:1-27.

[0283] Embodiment 25. In some further embodiments of any one of Embodiments 1 to 24, the wild-type IgSF domain is a member of the B7 family.

[0284] Embodiment 26. In some further embodiments of any one of Embodiments 1 to 25, the wild-type IgSF domain is a domain of CD80, CD86, or ICOSLG.

[0285] Embodiment 27. In some further embodiments of any one of Embodiments 1 to 26, the wild-type IgSF domain is a domain of CD80.

[0286] Embodiment 28. In some embodiments, there is provided an immunomodulatory protein comprising at least one affinity-modified CD80 immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type CD80 IgSF domain, wherein the at least one affinity-modified CD80 IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type CD80 IgSF domain.

[0287] Embodiment 29. In some further embodiments of Embodiment 27 or Embodiment 28, said cognate binding partners are CD28 and PD-L1.

[0288] Embodiment 30. In some further embodiments of any one of embodiments 27 to 29, the wild-type IgSF domain is an IgV domain and / or the affinity-engineered CD80 domain is an affinity-engineered IgV domain.

[0289] Embodiment 31. In some further embodiments of any one of Embodiments 27 to 30, the affinity-modified domain comprises at least 85% sequence identity to the wild-type CD80 domain contained in the amino acid sequence set forth in SEQ ID NO:1, or a specific-binding fragment thereof.

[0290] Embodiment 32. In some further embodiments of any one of Embodiments 1 to 31, the at least one affinity-altered IgSF domain comprises between 1 and 20 amino acid substitutions in the wild-type IgSF domain.

[0291] Embodiment 33. In some further embodiments of any one of Embodiments 1 to 32, the at least one affinity-altered IgSF domain comprises between 1 and 10 amino acid substitutions in the wild-type IgSF domain.

[0292] Embodiment 34. In some further embodiments of any one of Embodiments 1 to 33, the at least one affinity-altered IgSF domain comprises between one and five amino acid substitutions in the wild-type IgSF domain.

[0293] Embodiment 35. In some further embodiments of any one of Embodiments 1 to 34, the affinity-modified IgSF domain has at least 120% of the binding affinity of its wild-type IgSF domain for each of said at least two cognate binding partners.

[0294] Embodiment 36. In some further embodiments of any one of Embodiments 1 to 35, the immunomodulatory protein further comprises an IgSF domain that is not affinity modified.

[0295] Embodiment 37. In some further embodiments of any one of Embodiments 1 to 36, the immunomodulatory protein is soluble.

[0296] Embodiment 38 In some further embodiments of any one of Embodiments 1 to 37, the immunomodulatory protein lacks a transmembrane domain or a cytoplasmic domain.

[0297] Embodiment 39. In some further embodiments of any one of Embodiments 1 to 38, the immunomodulatory protein comprises only an extracellular domain (ECD) or a specific binding fragment thereof that comprises an affinity-engineered IgSF domain.

[0298] Embodiment 40 In some further embodiments of any one of Embodiments 1 to 39, the immunomodulatory protein is glycosylated or pegylated.

[0299] Embodiment 41 In some further embodiments of any one of Embodiments 1 to 40, the immunomodulatory protein is linked to a multimerization domain.

[0300] Embodiment 42. In some further embodiments of any one of Embodiments 1 to 41, the immunomodulatory protein is linked to an Fc domain or variant thereof with reduced effector function.

[0301] Embodiment 43 In some further embodiments of embodiment 42, the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof with reduced effector function.

[0302] Embodiment 44. In some further embodiments of any one of Embodiments 39 to 41, the Fc domain is mammalian, optionally human; or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified Fc domain that is mammalian, optionally human.

[0303] Embodiment 45. In some further embodiments of any one of embodiments 42 to 44, the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence exhibiting at least 85% sequence identity to SEQ ID NO:226 or SEQ ID NO:227.

[0304] Embodiment 46 In some further embodiments of any one of Embodiments 38 to 41, the immunomodulatory proteins are indirectly linked via a linker.

[0305] Embodiment 47. In some further embodiments of any one of Embodiments 41 to 46, the immunomodulatory protein is a dimer.

[0306] Embodiment 48. In some further embodiments of any one of Embodiments 1 to 47, the immunomodulatory protein is bound to a liposome membrane.

[0307] Embodiment 49. In some embodiments, an immunomodulatory protein is provided, comprising at least two non-immunoglobulin immunoglobulin superfamily (IgSF) domains, wherein at least one of the modified non-immunoglobulin IgSF domains is affinity modified to exhibit altered binding to its cognate binding partner; and wherein each of the at least two modified non-immunoglobulin IgSF domains independently specifically binds to at least one different cognate binding partner.

[0308] Embodiment 50. In some further embodiments of embodiment 49, each of the at least two non-immunoglobulin IgSF domains is an affinity-modified IgSF domain, wherein a first modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a first wild-type IgSF domain, and wherein the second modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a second wild-type IgSF domain.

[0309] Embodiment 51. In some further embodiments of embodiment 50, said first modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the first wild-type IgSF domain; and said second modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the second wild-type IgSF domain.

[0310] Embodiment 52. In some further embodiments of any one of Embodiments 49 to 51, the distinct cognate binding partner is a cell surface molecular species expressed on the surface of a mammalian cell.

[0311] Embodiment 53 In some further embodiments of Embodiment 52, the different cell surface molecular species are expressed in a cis or trans configuration.

[0312] Embodiment 54. In some further embodiments of embodiment 52 or embodiment 53, the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and the different cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS.

[0313] Embodiment 55. In some further embodiments of any one of Embodiments 52-54, at least one of the mammalian cells is a lymphocyte.

[0314] Embodiment 56. In some further embodiments of Embodiment 55, the lymphocytes are NK cells or T cells.

[0315] Embodiment 57 In some further embodiments of embodiment 55 or embodiment 56, binding of the immunomodulatory protein to the cell modulates the immune activity of the lymphocyte.

[0316] Embodiment 58. In some further embodiments of embodiment 57, the immunomodulatory protein is capable of providing enhanced immune activity as compared to a wild-type protein that includes the wild-type IgSF domain.

[0317] Embodiment 59. In some further embodiments of embodiment 57, the immunomodulatory protein is capable of providing reduced immune activity compared to a wild-type protein that comprises the wild-type IgSF domain.

[0318] Embodiment 60. In some further embodiments of any one of Embodiments 52-59, at least one of the mammalian cells is a tumor cell.

[0319] Embodiment 61. In some further embodiments of any one of Embodiments 52 to 60, the mammalian cell is a human cell.

[0320] Embodiment 62. In some further embodiments of any one of Embodiments 54 to 61, the immunomodulatory protein is capable of specifically binding to the two mammalian cells that form the IS.

[0321] Embodiment 63. In some further embodiments of any one of embodiments 49 to 62, the first and second modified IgSF domains each comprise one or more amino acid substitutions in a different wild-type IgSF domain.

[0322] Embodiment 64. In some further embodiments of Embodiment 63, the different wild-type IgSF domains are derived from different IgSF family members.

[0323] Embodiment 65. In some further embodiments of any one of Embodiments 49 to 64, the first and second modified IgSF domains are a non-wild-type combination.

[0324] Embodiment 66. In some further embodiments of any one of embodiments 49 to 65, the first wild-type IgSF domain and the second wild-type IgSF domain each individually are selected from the group consisting of a Signal-Regulatory Protein (SIRP) family, a Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, a Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, a Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, a Butyrophilin family, a B7 family, a CD28 family, a V-set and Immunoglobulin Domain Containing (VSIG) family, a V-set transmembrane Domain (VSTM) family, a Major Histocompatibility Complex (MAH) family, a VEGF ... It is derived from an IgSF family member of a family selected from the MHC family, the signaling lymphocytic activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor related (PVR) family, the natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the killer-cell immunoglobulin-like receptor (KIR) family.

[0325] Embodiment 67. In some further embodiments of any one of Embodiments 49 to 66, the first wild-type IgSF domain and the second wild-type IgSF domain are each individually derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

[0326] Embodiment 68. In some further embodiments of any one of Embodiments 49 to 67, the first modified IgSF domain and the second modified IgSF domain each individually comprise at least 85% sequence identity to a wild-type IgSF domain contained in the amino acid sequence set forth in any of SEQ ID NOs:1 to 27, or a specific-binding fragment thereof.

[0327] Embodiment 69. In some further embodiments of any one of Embodiments 49 to 68, each of the first and second wild-type IgSF domains individually is a member of the B7 family.

[0328] Embodiment 70. In some further embodiments of embodiment 69, each of the first and second wild-type IgSF domains is independently derived from CD80, CD86, or ICOSLG.

[0329] Embodiment 71. In some further embodiments of any one of embodiments 49 to 68, the first or second wild-type IgSF domain is derived from a member of the B7 family, and the other of the first or second wild-type IgSF domain is derived from another IgSF family member.

[0330] Embodiment 72. In some further embodiments of any one of embodiments 49 to 68 and 71, the first and second wild-type IgSF domains are derived from ICOSLG and NKp30.

[0331] Embodiment 73. In some further embodiments of any one of embodiments 49 to 68 and 71, the first and second wild-type IgSF domains are derived from CD80 and NKp30.

[0332] Embodiment 74. In some further embodiments of any one of Embodiments 49 to 73, each of the first and second wild-type IgSF domains individually is a human IgSF member.

[0333] Embodiment 75. In some further embodiments of any one of embodiments 49 to 74, each of the first and second wild-type IgSF domains is individually an IgV domain, and an IgC1 domain, an IgC2 domain, or a specific combination thereof.

[0334] Embodiment 76. In some further embodiments of any one of embodiments 49 to 75, each of the first modified non-immunoglobulin domain and the second modified non-immunoglobulin domain individually is a modified IgV domain, a modified IgC1 domain, or a modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions.

[0335] Embodiment 77. In some further embodiments of any one of embodiments 49 to 76, at least one of the first modified non-immunoglobulin domain or the second modified non-immunoglobulin domain is a modified IgV domain.

[0336] Embodiment 78. In some further embodiments of any one of embodiments 49 to 77, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between 1 and 20 amino acid substitutions.

[0337] Embodiment 79. In some further embodiments of any one of embodiments 49 to 78, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between 1 and 10 amino acid substitutions.

[0338] Embodiment 80. In some further embodiments of any one of embodiments 49 to 79, the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between one and five amino acid substitutions.

[0339] Embodiment 81. In some further embodiments of any one of embodiments 49 to 80, at least one of the first or second modified non-immunoglobulin IgSF domains has between 10% and 90% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

[0340] Embodiment 82. In some further embodiments of any one of embodiments 49 to 81, at least one of the first or second modified non-immunoglobulin IgSF domains has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

[0341] Embodiment 83. In some further embodiments of any one of embodiments 49 to 80 and 82, each of the first and second modified non-immunoglobulin IgSF domains individually has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

[0342] Embodiment 84 In some further embodiments of any one of Embodiments 49 to 83, the immunomodulatory protein is soluble.

[0343] Embodiment 85 In some further embodiments of any one of Embodiments 49 to 84, the immunomodulatory protein is glycosylated or pegylated.

[0344] Embodiment 86. In some further embodiments of any one of embodiments 49 to 85, the immunomodulatory protein is linked to a multimerization domain.

[0345] Embodiment 87. In some further embodiments of any one of embodiments 49 to 86, the immunomodulatory protein is linked to an Fc domain or variant thereof with reduced effector function.

[0346] Embodiment 88 In some further embodiments of embodiment 87, the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof with reduced effector function.

[0347] Embodiment 89. In some further embodiments of embodiment 87 or embodiment 88, the Fc domain is mammalian, optionally human; or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified Fc domain that is mammalian, optionally human.

[0348] Embodiment 90. In some further embodiments of any one of embodiments 87 to 89, the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ ID NO:227.

[0349] Embodiment 91. In some further embodiments of any one of Embodiments 86 to 90, the variant CD80 polypeptide is indirectly linked via a linker.

[0350] Embodiment 92. In some further embodiments of any one of Embodiments 86 to 91, the immunomodulatory protein is a dimer.

[0351] Embodiment 93. In some further embodiments of any one of embodiments 49 to 92, the immunomodulatory protein further comprises one or more additional non-immunoglobulin IgSF domains that are the same as or different from the first or second modified non-immunoglobulin IgSF domain.

[0352] Embodiment 94. In some further embodiments of embodiment 93, said one or more additional non-immunoglobulin IgSF domains are affinity-modified IgSF domains.

[0353] Embodiment 95. In some further embodiments of any one of Embodiments 49 to 94, the immunomodulatory protein is bound to a liposome membrane.

[0354] Embodiment 96. In some embodiments, a nucleic acid molecule is provided that encodes the immunomodulatory polypeptide of any one of embodiments 1 to 95.

[0355] Embodiment 97. In some further embodiments of Embodiment 96, the nucleic acid molecule is a synthetic nucleic acid.

[0356] Embodiment 98. In some further embodiments of embodiment 96 or embodiment 97, the nucleic acid molecule is cDNA.

[0357] Embodiment 99. In some embodiments, a vector is provided comprising the nucleic acid molecule of any one of embodiments 96-98.

[0358] Embodiment 100. In some further embodiments of Embodiment 99, the vector is an expression vector.

[0359] Embodiment 101. In some embodiments, a cell is provided comprising the vector of embodiment 99 or embodiment 100.

[0360] Embodiment 102. In some further embodiments of Embodiment 101, the cell is a eukaryotic cell or a prokaryotic cell.

[0361] Embodiment 103. In some embodiments, there is provided a method for producing an immunomodulatory protein, the method comprising introducing the nucleic acid molecule of any one of embodiments 96 to 98 or the vector of embodiment 99 or embodiment 100 into a host cell under conditions such that the protein is expressed in the cell.

[0362] Embodiment 104. In some further embodiments of Embodiment 103, the method further comprises isolating or purifying the immunomodulatory protein from the cells.

[0363] Embodiment 105. In some embodiments, a pharmaceutical composition is provided comprising an immunomodulatory protein of any one of embodiments 1 to 95.

[0364] Embodiment 106. In some further embodiments of embodiment 105, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0365] Embodiment 107. In some further embodiments of embodiment 105 or embodiment 106, the pharmaceutical composition is sterile.

[0366] Embodiment 108. In some embodiments, an article of manufacture is provided comprising the pharmaceutical composition of any one of embodiments 105-107 in a vial.

[0367] Embodiment 109. In some further embodiments of Embodiment 108, the vial is sealed.

[0368] Embodiment 110. In some embodiments, a kit is provided that includes the pharmaceutical composition of any one of embodiments 105-107 and instructions for use.

[0369] Embodiment 111. In some embodiments, a kit is provided that includes the article of manufacture of embodiment 108 or embodiment 109, and instructions for use.

[0370] Embodiment 112. In some embodiments, provided is a method of modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunomodulatory protein of any one of Embodiments 1-95.

[0371] Embodiment 113 In some further embodiments of Embodiment 112, modulating the immune response treats a disease or condition in the subject.

[0372] Embodiment 114 In some further embodiments of embodiment 112 or embodiment 113, the immune response is enhanced.

[0373] Embodiment 115. In some further embodiments of Embodiment 114, the disease or condition is a tumor or cancer.

[0374] Embodiment 116. In some further embodiments of Embodiment 114 or Embodiment 115, the disease or condition is selected from melanoma, lung cancer, bladder cancer, or hematological malignancies.

[0375] Embodiment 117 In some further embodiments of embodiment 112 or embodiment 113, the immune response is reduced.

[0376] Embodiment 118. In some further embodiments of Embodiment 117, the disease or condition is an inflammatory disease or condition.

[0377] Embodiment 119. In some further embodiments of embodiment 117 or embodiment 118, the disease or condition is selected from Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

[0378] Embodiment 120. In some embodiments, a method for identifying an affinity-modified immunomodulatory protein is provided, comprising the steps of: a) contacting an engineered protein comprising at least one engineered non-immunoglobulin immunoglobulin superfamily (IgSF) domain or a specific-binding fragment thereof with at least two cognate binding partners under conditions capable of resulting in binding of the protein to the at least two cognate binding partners, wherein the at least one engineered IgSF domain comprises one or more amino acid substitutions in a wild-type IgSF domain; b) identifying modified proteins comprising the modified IgSF domain that have improved binding to at least one of the two cognate binding partners compared to proteins comprising the wild-type IgSF domain; and c) selecting modified proteins comprising the modified IgSF domain that bind non-competitively to the at least two cognate binding partners, thereby identifying immunomodulatory proteins with modified affinity.

[0379] Embodiment 121. In some further embodiments of embodiment 120, step b) comprises identifying an engineered protein comprising an engineered IgSF domain that has improved binding to each of said at least two cognate binding partners compared to a protein comprising said wild-type domain.

[0380] Embodiment 122. In some further embodiments of embodiment 120 or embodiment 121, prior to step a), one or more amino acid substitutions are introduced into the wild-type IgSF domain, thereby generating a modified protein comprising the modified IgSF domain.

[0381] Embodiment 123. In some further embodiments of any one of embodiments 120 to 122, the engineered protein comprises at least two engineered IgSF domains or specific-binding fragments thereof, wherein a first IgSF domain comprises one or more amino acid substitutions in a first wild-type IgSF domain, and a second affinity-engineered non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a second wild-type IgSF domain.

[0382] Embodiment 124. In some further embodiments of embodiment 123, each of the first and second affinity-engineered non-immunoglobulin IgSF domains specifically binds to at least one different cognate binding partner.

[0383] Embodiment 125. In some embodiments, there is provided an immunomodulatory protein comprising at least one affinity-engineered non-immunoglobulin immunoglobulin superfamily (IgSF) domain, wherein the affinity-engineered IgSF domain specifically binds to at least two cell surface molecular species in a non-competitive manner, each of the molecular species being expressed on the surface of at least one of two mammalian cells that form an immune synapse (IS), one of the mammalian cells being a lymphocyte, and wherein binding of the affinity-engineered IgSF domain modulates immune activity of the lymphocyte.

[0384] Embodiment 126. In some further embodiments of Embodiment 125, the affinity-engineered IgSF domain specifically binds to the two mammalian cells that form the IS.

[0385] Embodiment 127. In some further embodiments of embodiment 125 or embodiment 126, the immunomodulatory protein comprises at least two affinity-engineered non-immunoglobulin IgSF domains, and the immunomodulatory protein specifically binds to the two mammalian cells that form the IS.

[0386] Embodiment 128. In some further embodiments of any one of Embodiments 125 to 127, the IgSF cell-surface species is a human IgSF member.

[0387] Embodiment 129. In some further embodiments of any one of embodiments 125 to 128, the affinity-engineered IgSF domain comprises at least one affinity-engineered human CD80 domain.

[0388] Embodiment 130. In some further embodiments of any one of embodiments 125 to 129, the immunomodulatory protein comprises an affinity-engineered mammalian IgSF member.

[0389] Embodiment 131. In some further embodiments of any one of embodiments 125 to 130, the affinity-modified mammalian IgSF member is at least one of CD80, PVR, ICOSLG, or HAVCR2.

[0390] Embodiment 132. In some further embodiments of any one of Embodiments 125-131, the immune activity is enhanced.

[0391] Embodiment 133. In some further embodiments of any one of Embodiments 125-132, immune activity is suppressed.

[0392] Embodiment 134. In some further embodiments of any one of Embodiments 125-133, one of the two mammalian cells is a tumor cell.

[0393] Embodiment 135. In some further embodiments of any one of Embodiments 125 to 134, the lymphocyte is an NK cell or a T cell.

[0394] Embodiment 136. In some further embodiments of any one of Embodiments 125-135, the mammalian cell is a mouse, rat, cynomolgus monkey, or human cell.

[0395] Embodiment 137. In some further embodiments of any one of embodiments 125 to 136, the affinity-modified IgSF domain has between 10% and 90% of the binding affinity of the wild-type IgSF domain for at least one of the two cell surface molecular species.

[0396] Embodiment 138. In some further embodiments of any one of embodiments 125 to 137, the affinity-modified IgSF domain specifically binds to exactly one IgSF member in a non-competitive manner.

[0397] Embodiment 139. In some further embodiments of any one of embodiments 125 to 138, the affinity-modified IgSF domain has at least 120% of the binding affinity of its wild-type IgSF domain for at least one of the two cell surface molecular species.

[0398] Embodiment 140. In some further embodiments of any one of embodiments 125 to 139, the affinity-modified IgSF domain is an affinity-modified IgV, IgC1, or IgC2 domain.

[0399] Embodiment 141. In some further embodiments of any one of embodiments 125 to 140, the affinity-altered IgSF domain differs from its wild-type IgSF domain by between 1 and 10 amino acid substitutions, inclusive.

[0400] Embodiment 142. In some further embodiments of any one of embodiments 125 to 141, the affinity-altered IgSF domain differs from its wild-type IgSF domain by between 1 and 5 amino acid substitutions, inclusive.

[0401] Embodiment 143. In some further embodiments of any one of Embodiments 125 to 142, the affinity-modified IgSF domain is a human CD80 IgSF domain.

[0402] Embodiment 144. In some further embodiments of any one of embodiments 125 to 143, the immunomodulatory protein comprises at least two affinity-engineered IgSF domains, wherein the affinity-engineered IgSF domains are not IgSF domains of the same species.

[0403] Embodiment 145. In some further embodiments of any one of embodiments 125 to 144, the immunomodulatory protein is covalently linked, directly or indirectly, to a crystallizable fragment (Fc) of an antibody.

[0404] Embodiment 146. In some further embodiments of any one of embodiments 125 to 145, the immunomodulatory protein is in a pharmaceutically acceptable carrier.

[0405] Embodiment 147. In some further embodiments of any one of embodiments 125 to 146, the immunomodulatory protein is glycosylated or pegylated.

[0406] Embodiment 148. In some further embodiments of any one of Embodiments 125 to 147, the immunomodulatory protein is soluble.

[0407] Embodiment 149. In some further embodiments of any one of embodiments 125 to 148, the immunomodulatory protein is bound to a liposome membrane.

[0408] Embodiment 150. In some further embodiments of any one of embodiments 125 to 149, the immunomodulating protein is dimerized by an intermolecular disulfide bond.

[0409] Embodiment 151. In some further embodiments of any one of Embodiments 125-150, the cell surface molecular species is expressed in a cis or trans configuration.

[0410] Embodiment 152. In some embodiments, there is provided an immunomodulatory protein comprising at least two affinity-engineered non-immunoglobulin immunoglobulin superfamily (IgSF) domains, each of the affinity-engineered IgSF domains specifically binding to a different cell surface molecular species, each of the molecular species being expressed on the surface of at least one of two mammalian cells that form an immune synapse (IS), one of the mammalian cells being a lymphocyte, and wherein binding of the affinity-engineered IgSF domains modulates immune activity of the lymphocyte.

[0411] Embodiment 153. In some further embodiments of any one of Embodiments 125 to 152, at least one of the affinity-modified IgSF domains binds competitively.

[0412] Embodiment 154. In some further embodiments of any one of Embodiments 125 to 153, the affinity-altered IgSF domains are not IgSF domains of the same species.

[0413] Embodiment 155. In some further embodiments of any one of Embodiments 125 to 154, the affinity-altered IgSF domains are a non-wild-type combination.

[0414] Embodiment 156. In some further embodiments of any one of Embodiments 125 to 155, the cell surface molecular species is a human IgSF member.

[0415] Embodiment 157. In some further embodiments of any one of embodiments 125 to 156, the at least two affinity-engineered IgSF domains are derived from at least one of CD80, CD86, CD274, PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, or CD200R1.

[0416] Embodiment 158. In some further embodiments of any one of embodiments 125 to 157, the immunomodulatory protein comprises at least two affinity-engineered mammalian IgSF members.

[0417] Embodiment 159. In some further embodiments of any one of embodiments 125 to 158, the mammalian IgSF member is a human IgSF member.

[0418] Embodiment 160. In some further embodiments of any one of embodiments 125 to 159, the mammalian IgSF member is at least two of CD80, CD86, CD274, PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, or CD200R1.

[0419] Embodiment 161. In some further embodiments of any one of embodiments 125-160, the immune activity is enhanced.

[0420] Embodiment 38. In some further embodiments of any one of Embodiments 125-1, immune activity is suppressed.

[0421] Embodiment 162. In some further embodiments of any one of Embodiments 125-161, one of the two mammalian cells is a tumor cell.

[0422] Embodiment 163. In some further embodiments of any one of Embodiments 125 to 162, the lymphocyte is an NK cell or a T cell.

[0423] Embodiment 164. In some further embodiments of any one of Embodiments 125 to 163, the mammalian cell is a mouse, rat, cynomolgus monkey, or human cell.

[0424] Embodiment 165. In some further embodiments of any one of embodiments 125 to 164, at least one of the two affinity-altered IgSF domains has between 10% and 90% of the binding affinity of the wild-type IgSF domain for at least one of the cell surface molecular species.

[0425] Embodiment 166. In some further embodiments of any one of embodiments 125 to 165, at least one of the two affinity-engineered IgSF domains specifically binds to exactly one cell surface molecular species.

[0426] Embodiment 167. In some further embodiments of any one of embodiments 125 to 166, at least one of the two affinity-modified IgSF domains has at least 120% of the binding affinity of its wild-type IgSF domain for at least one of the two cell surface molecular species.

[0427] Embodiment 168. In some further embodiments of any one of embodiments 125 to 167, the affinity-altered IgSF domain is at least one of an IgV, an IgC1, or an IgC2 domain.

[0428] Embodiment 169. In some further embodiments of any one of embodiments 125 to 168, each of the at least two affinity-altered IgSF domains differs from its wild-type IgSF domain by between 1 and 10 amino acid substitutions.

[0429] Embodiment 170. In some further embodiments of any one of embodiments 125 to 169, each of the at least two affinity-altered IgSF domains differs from its wild-type IgSF domain by between one and five amino acid substitutions.

[0430] Embodiment 171. In some further embodiments of any one of embodiments 125 to 170, the immunomodulatory protein is covalently linked, directly or indirectly, to a crystallizable fragment (Fc) of an antibody.

[0431] Embodiment 172. In some further embodiments of any one of embodiments 125 to 171, the immunomodulatory protein is in a pharmaceutically acceptable carrier.

[0432] Embodiment 173. In some further embodiments of any one of embodiments 125 to 172, the protein is glycosylated or pegylated.

[0433] Embodiment 174. In some further embodiments of any one of embodiments 125 to 173, the protein is soluble.

[0434] Embodiment 175. In some further embodiments of any one of embodiments 125 to 174, the protein is bound to a liposome membrane.

[0435] Embodiment 176. In some further embodiments of any one of embodiments 125 to 175, the protein is dimerized by an intermolecular disulfide bond.

[0436] Embodiment 177. In some further embodiments of any one of Embodiments 125-176, the cell surface molecular species is expressed in a cis or trans configuration.

[0437] Embodiment 178. In some further embodiments of any one of embodiments 125-177, the immunomodulatory protein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-26, a combination, or a fragment thereof.

[0438] Embodiment 179. In some further embodiments of any one of embodiments 125-178, the immunomodulatory protein has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, a combination, or a fragment thereof.

[0439] Embodiment 180. In some further embodiments of any one of embodiments 125-179, the immunomodulatory protein has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, a combination, or a fragment thereof.

[0440] Embodiment 181. In some further embodiments of any one of embodiments 125-180, the immunomodulatory protein has at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, a combination, or a fragment thereof.

[0441] Embodiment 182. In some further embodiments of any one of embodiments 125-181, the immunomodulatory protein further comprises a second immunomodulatory protein, wherein the second immunomodulatory protein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, or a fragment thereof.

[0442] Embodiment 183. In some further embodiments of any one of embodiments 125-182, the immunomodulatory protein further comprises a second immunomodulatory protein, wherein the second immunomodulatory protein has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, or a fragment thereof.

[0443] Embodiment 184. In some further embodiments of any one of embodiments 125 to 183, the immunomodulatory protein further comprises a second immunomodulatory protein, wherein the second immunomodulatory protein has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, or a fragment thereof.

[0444] Embodiment 185. In some further embodiments of any one of embodiments 125-184, the immunomodulatory protein further comprises a second immunomodulatory protein, wherein the second immunomodulatory protein has at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1-26, or a fragment thereof.

[0445] Embodiment 186. In some embodiments, a recombinant nucleic acid encoding any one of the immunomodulatory proteins of embodiments 125-185 is provided.

[0446] Embodiment 187. In some embodiments, a recombinant expression vector is provided that comprises the nucleic acid of embodiment 186.

[0447] Embodiment 188. In some embodiments, a recombinant host cell is provided comprising the expression vector of embodiment 187.

[0448] Embodiment 189. In some embodiments, there is provided a method for producing the immunomodulatory protein of any one of embodiments 125 to 185, the method comprising culturing recombinant host cells under immunomodulatory protein expression conditions, expressing in the cells the immunomodulatory protein encoded by a recombinant expression vector, and purifying the recombinant immunomodulatory protein thereby expressed.

[0449] Embodiment 190. In some embodiments, a method of treating a mammalian patient in need of an enhanced or suppressed immune response by administering a therapeutically effective amount of an immunomodulatory protein of any one of embodiments 125 to 185 is provided.

[0450] Embodiment 191. In some further embodiments of embodiment 190, the enhanced immune response treats melanoma, lung cancer, bladder cancer, or a hematological malignancy in the patient.

[0451] Embodiment 192. In some further embodiments of embodiment 190, the suppressed immune response treats Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis in the patient. [Example]

[0452] VIII. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0453] Examples 1-8 describe the design, production, and screening of affinity-engineered CD80 (B7-1), CD86 (B7-2), ICOSL, and NKp30 immunomodulatory proteins, components of the immune synapse (IS) with a demonstrated dual role in both immune activation and inhibition. These examples demonstrate that affinity engineering of IgSF domains generates proteins that can act to enhance and attenuate immune activity. This work also describes various combinations of these domains fused in pairs (i.e., stacked) to form type II immunomodulatory proteins to achieve immunomodulatory activity.

[0454] Example 1 Generation of mutant DNA constructs of IgSF domains Example 1 describes the generation of mutant DNA constructs of human CD80, CD86, ICOSL, and NKp30 IgSF domains for translation and expression on the surface of yeast as yeast display libraries.

[0455] A. Degenerate Libraries For libraries targeting specific residues of target proteins for full or partial randomization with degenerate codons, coding DNA for the extracellular domains (ECDs) of human CD80 (SEQ ID NO:28), ICOSL (SEQ ID NO:32), and NKp30 (SEQ ID NO:54) was ordered from Integrated DNA Technologies (Coralville, IA) as a series of overlapping oligonucleotides up to 80 base pairs (bp) in length. To generate libraries of diverse variants of each ECD, the oligonucleotides contained the desired degenerate codons at the desired amino acid positions. Degenerate codons were generated using the algorithm at URL: rosettadesign.med.unc.edu / SwiftLib / .

[0456] Generally, positions and degenerate codons to be mutated were selected as follows: Crystal structures (CD80, NKp30) or homology models (ICOSL) of the target-ligand pairs of interest were used to identify ligand contact residues and residues at the protein interaction interface. This analysis was performed using the structure viewer available at URL: spdbv.vital-it.ch. For example, the crystal structure of CD80 bound to CTLA4 is published at URL: www.rcsb.org / pdb / explore / explore.do?structureId=1I8L. A targeted library was designed based on the CD80:CTLA4 interface to select improved binders to CTLA4. However, there were no available CD80 structures with the ligands CD28 and PDL1. Therefore, the same library was also used to select for binders of CD28 (which binds to the same region on CD80 as CTLA4) and PDL1 (it is not known whether PDL1 binds to the same site as CTLA4).

[0457] The next step in library design was the alignment of human, mouse, rat, and monkey CD80, ICOSL, or NKp30 sequences to identify conserved residues. Based on this analysis, conserved target residues were mutated with degenerate codons, which only identified conservative amino acid changes plus the wild-type residue. Nonconserved residues were mutated more aggressively, but also included the wild-type residue. Degenerate codons that also encoded the wild-type residue were included to avoid excessive mutagenesis of the target protein. For the same reason, only a maximum of 20 positions could be targeted for mutagenesis at one time. These residues were a combination of contact and noncontact interface residues.

[0458] The oligonucleotides were dissolved in sterile water, mixed in equal molar ratios, heated to 95°C for 5 minutes, and then slowly cooled to room temperature for annealing. PCR products were then generated using ECD-specific oligonucleotide primers that anneal to the beginning and end of the ECD, respectively. A modified version of the pBYDS03 cloning vector (Life Technologies, USA) and ECD-specific oligonucleotides overlapping by 40–50 bp outside and within the BamH1 and Kpn1 cloning sites were then used to amplify 100 ng of the PCR product from the previous step, generating a total of 5 μg of DNA. Both PCRs were performed by polymerase chain reaction (PCR) using OneTaq 2x PCR master mix (New England Biolabs, USA). The second PCR product was purified using a PCR purification kit (Qiagen, Germany) and resuspended in sterile deionized water.

[0459] To prepare the library insert, the modified yeast display version of the vector pBYDS03 was digested with BamH1 and Kpn1 restriction enzymes (New England Biolabs, USA), and the large vector fragment was gel-purified and dissolved in sterile deionized water. Electroporatable DNA for the next step was generated by mixing 12 μg of library DNA and 4 μg of linearized vector in a total volume of 50 μl of deionized and sterile water. An alternative method for generating targeted libraries was to perform site-directed mutagenesis (Multisite kit, Agilent, USA) of the target ECD using oligonucleotides containing degenerate codons. This approach was used to generate sublibraries that targeted only specific stretches of the target protein for mutagenesis. In these cases, the sublibraries were mixed before proceeding to the selection step. Generally, library sizes were around 10 7 ~10 8 The range of clones was 10 4 ~10 5The range of expression was only 1. Large libraries were generated for CD80, ICOSL, CD86, and NKp30. Sub-libraries were generated for CD80, ICOSL, and NKp30.

[0460] B. Random Library Random libraries were also constructed to identify variants of the ECDs of CD80 (SEQ ID NO: 28), CD86 (SEQ ID NO: 29), ICOSL (SEQ ID NO: 32), and NKp30 (SEQ ID NO: 54). DNA encoding the wild-type ECD was cloned between the BamH1 and Kpn1 sites of the modified yeast display vector pBYDS03 and then excised using the same restriction enzymes. The excised DNA was then mutagenized using the Genemorph II kit (Agilent, USA) to generate an average of three to five amino acid changes per library variant. The mutagenized DNA was then amplified by two-step PCR and further processed as described above for the targeted library.

[0461] Example 2 Introduction of DNA libraries into yeast Example 2 describes the introduction of CD80, CD86, ICOSL, and NKp30 DNA libraries into yeast.

[0462] To introduce the degenerate and random library DNA into yeast, electroporation-competent cells of the yeast strain BJ5464 (ATCC.org; ATCC number 208288) were prepared and electroporated using the electroporatable DNA from the above step using a Gene Pulser II (Biorad, USA) essentially as described (Colby, DW et al. 2004 Methods Enzymology 388, 348-358). The only exception was that transformed cells were grown in non-inducing, minimally selective SCD-Leu medium to accommodate the LEU2 selection marker carried by the modified plasmid pBYDS03.

[0463] Library size was determined by plating dilutions of freshly recovered cells onto SCD-Leu agar plates and then estimating library size from the number of single colonies from platings that produced at least 50 colonies per plate. The remainder of the electroporated culture was grown to saturation, and cells from this culture were subcultured once more in the same medium to minimize the fraction of non-transformed cells. To maintain library diversity, this subculture step was performed using an inoculum that contained at least 10x more cells than the calculated library size. Cells from the second saturated culture were plated 10 times in fresh medium containing sterile 25% (wt / vol) glycerol. 10 The cells were resuspended to a density of 1000 / ml, frozen and stored at -80°C (frozen library stock).

[0464] One liter of SCD-Leu medium contains 14.7 grams of sodium citrate, 4.29 grams of citric acid monohydrate, 20 grams of dextrose, 6.7 grams of Difco brand nitrogen base for yeast, and 1.6 grams of leucine-free synthetic yeast dropout medium supplement. Sterile filter the medium before use using a 0.2 μM vacuum filtration device.

[0465] Library size was determined by plating dilutions of freshly harvested cells onto SCD-Leu agar plates and then estimating library size from the number of single colonies from platings that yielded at least 50 colonies per plate.

[0466] To isolate plasmids from cells containing two or more distinct library clones, a number of cells equivalent to 10 times the library size was taken from the overnight SCD-Leu culture, subcultured 1 / 100 into fresh SCD-Leu medium, and grown overnight. Cells from this overnight culture were diluted to 10% in sterile 25% (wt / vol) glycerol. 10The cells were resuspended to a density of 1000 / ml, frozen and stored at -80°C (frozen library stock).

[0467] Example 3 Yeast selection Example 3 describes the selection of yeast expressing affinity-engineered variants of CD80, CD86, ICOSL, and NKp30.

[0468] A number of cells equal to at least 10 times the library size was thawed from each library stock and cultured at 0.1 x 10 in uninducing SCD-Leu medium. 6 The next day, cells equal to 10 times the library size were centrifuged at 2000 RPM for 2 minutes and diluted to 0.5 x 10 cells / ml in inducing SCDG-Leu medium. 6 The cells were resuspended at 1000 cells / ml. One liter of SCDG-Leu induction medium consisted of 5.4 grams of NaHPO, 8.56 grams of NaHPO*H2O, 20 grams of galactose, 2.0 grams of dextrose, 6.7 grams of Difco Yeast Nitrogen Base, and 1.6 grams of Leucine-Free Synthetic Yeast Dropout Medium Supplement, dissolved in water and sterilized by passing through a 0.22 μm membrane filter. Cultures were grown at 20°C for 2 days to induce expression of the library proteins on the yeast cell surface.

[0469] Cells were treated with magnetic beads to reduce nonbinders and enrich for all CD80, CD86, ICOSL, or NKp30 variants capable of binding to exogenous recombinant counter-structure proteins. For example, yeast-displayed targeted or random CD80 libraries were selected separately against CD28, CTL-4, PD-L1, ICOS, and B7-H6. This was followed by two to three rounds of flow cytometry sorting using exogenous counter-structure protein stains to enrich for the fraction of yeast cells displaying improved binders. Magnetic bead enrichment and flow cytometry selection were essentially as described by Keith D. Miller,1 Noah B. Pefaur,2 and Cheryl L. Baird1 in Current Protocols in Cytometry 4.7.1-4.7.30, July 2008.

[0470] The CD80, CD86, ICOSL, and NKp30 libraries, along with target ligand proteins, were supplied by R&D Systems (USA): human rCD28.Fc (i.e., recombinant CD28-Fc fusion protein), rPDL1.Fc, rCTLA4.Fc, rICOS.Fc, and rB7H6.Fc. Magnetic streptavidin beads were obtained from New England Biolabs, USA. A biotinylation kit (cat# 21955, Life Technologies, USA) was used for biotinylation of counter-structural proteins. A Becton Dickinson FACS Aria II sorter was used for two-color flow cytometry sorting. CD80, CD86, ICOSL, or NKp30 display levels were monitored with an anti-hemagglutinin antibody labeled with Alexafluor 488 (Life Technologies, USA). Ligand-binding Fc fusion proteins rCD28.Fc, rCTLA4.Fc, rPDL1.Fc, rICOS.Fc, or rB7-H6.Fc were detected with PE-conjugated human Ig-specific goat Fab (Jackson ImmunoResearch, USA). Dual yeast were gated using forward scatter (FSC) / side scatter (SSC) parameters, and sort gates were based on higher ligand binding detected in FL4 and more limited tag expression binding in FL1.

[0471] The yeast output from flow cytometry sorting was assayed for higher specific binding affinity. The sort output yeast was grown and re-induced to express the specific IgSF affinity-altered domain variants they encoded. This population could then be compared by flow cytometry to the parent wild-type yeast strain or any other selected output, such as the bead output yeast population.

[0472] For ICOSL, the second sort output (F2) was compared to the parental ICOSL yeast for binding of each rICOS.Fc, rCD28.Fc, and rCTLA4.Fc by double staining each population with anti-HA (hemagglutinin) tag expression and anti-human Fc secondary to detect ligand binding.

[0473] For the ICOSL yeast variant selected for binding to ICOS, the F2 sort output gave a mean fluorescence intensity (MFI) value of 997 when stained with 5.6 nM rICOS.Fc, whereas the MFI of the parental ICOSL lineage was measured as 397 when stained with the same concentration of rICOS.Fc. This represents a roughly 3-fold average binding improvement for this F2-selected pool of clones, and individual clones from that pool are predicted to have much better improved MFI / affinity when tested individually.

[0474] For ICOSL yeast variants selected for binding to CD28, the F2 sort output gave an MFI value of 640 when stained with 100 nM rCD28.Fc, whereas the MFI of the parental ICOSL lineage was measured to be 29 when stained with the same concentration of rCD28.Fc (a 22-fold improvement). For ICOSL yeast variants selected for binding to CTLA4, the F2 sort output gave an MFI value of 949 when stained with 100 nM rCTLA4.Fc, whereas the MFI of the parental ICOSL lineage was measured to be 29 when stained with the same concentration of rCTLA4.Fc (a 32-fold improvement).

[0475] For the NKp30 yeast variant selected for binding to B7-H6, the F2 sort output gave an MFI value of 533 when stained with 16.6 nM rB7H6.Fc, whereas the MFI of the parental NKp30 lineage was measured as 90 when stained with the same concentration of rB7H6.Fc (a 6-fold improvement).

[0476] Importantly, the MFI of all the above F2 outputs, as measured in FL1 using an anti-HA tag antibody, was not increased and in some cases was decreased compared to the wild-type strain, indicating that the improved binding was not a function of increased expression of the selected variants on the yeast surface and validating the gating strategy of selecting only low expressers from among those with high ligand binding.

[0477] Example 4 Reorganization of selection outputs as Fc fusions and in various immunomodulatory protein types Example 4 describes the recombination of selected outputs as immunomodulatory proteins containing affinity-altered (variant) extracellular domains (ECDs) of CD80 or ICOSL fused to Fc molecules (variant ECD-Fc fusion molecules).

[0478] Output cells from the final flow cytometry CD80 and ICOSL sorts were grown to terminal density in SCD-Leu medium. Plasmid DNA from each output was isolated using a yeast plasmid DNA isolation kit (Zymoresearch, USA). For Fc fusions, the coding DNA for the mutant target ECD was batch-amplified from plasmid DNA preps using PCR primers that added restriction sites suitable for cloning into the selected Fc fusion vector. After restriction digestion, the PCR product was ligated into the appropriate Fc fusion vector and subsequently chemically transformed into XL1 Blue strain E. coli (Agilent, USA) or NEB5α (New England Biolabs) according to the supplier's instructions. An exemplary Fc fusion vector is pFUSE-hIgG1-Fc2 (Invivogen, USA).

[0479] Dilutions of the transformation reactions were plated on LB-agar containing 100 μg / ml carbenicillin (Teknova, USA) to generate single colonies. Up to 96 colonies from each transformation were then grown to saturation overnight at 37°C in LB-broth (Teknova cat. # L8112) in 96-well plates, and a small aliquot from each well was sent for DNA sequencing of the ECD insert to identify mutations in all clones. Sample preparation for DNA sequencing was performed using a protocol provided by the service provider (Genewiz; South Plainfield, NJ). After removing the sample for DNA sequencing, glycerol was then added to the remaining culture to a final glycerol content of 25%, and the plate was stored at -20°C as a master plate (see below) for later use. Alternatively, samples for DNA sequencing were generated by replica plating from grown liquid cultures onto solid agar plates using a disposable 96-well replicator (VWR, USA). These plates were incubated overnight to generate growth patches, and the plates were sent to Genewiz as specified by Genewiz.

[0480] After identifying clones of interest from analysis of the DNA sequencing data generated by Genewiz, clones of interest were recovered from the master plate and individually grown to density in 5 ml of liquid LB-broth containing 100 μg / ml carbenicillin (Teknova, USA). Two ml of each culture was then used to prepare approximately 10 μg of miniprep plasmid DNA for each clone using a standard kit, such as the Pureyield kit (Promega). Identification of clones of interest generally involved the following steps: First, DNA sequence data files were downloaded from the Genewiz website. All sequences were then manually processed to begin at the start of the ECD coding region. The processed sequences were then batch-translated using a suitable program available at: www.ebi.ac.uk / Tools / st / emboss_transeq / . The translated sequences were then aligned using a suitable program available at: multalin.toulouse.inra.fr / multalin / multalin.html.

[0481] Clones of interest were then identified using the following criteria: 1.) the same clone occurs at least twice in the alignment, and 2.) the mutation occurs at least twice in the alignment, preferably in separate clones. Clones that met at least one of these criteria were enriched by our sorting process due to improved binding.

[0482] To generate immunomodulatory proteins containing the ECD of CD80 or ICOSL with at least one affinity-engineered domain, coding nucleic acid molecules were generated that encode proteins designed as follows: a signal peptide, followed by a variant ECD, followed by a linker of three alanines (AAA), followed by a human IgG1 Fc containing the mutation N82G relative to the wild-type human IgG1 Fc shown in SEQ ID NO: 226. Because the construct does not contain an antibody light chain capable of forming a covalent bond with cysteine, the human IgG1 Fc also contains a substitution of a cysteine ​​residue at position 5 (C5S) with a serine residue compared to the wild-type or unmodified Fc shown in SEQ ID NO: 226.

[0483] Additionally, Example 8 below describes additional immunomodulatory proteins produced as stack constructs containing at least two different affinity-engineered domains from identified variant CD80, CD86, ICOSL, and NKp30 molecules linked together and fused to Fc.

[0484] Example 5 Expression and purification of Fc fusions Example 5 describes the high-throughput expression and purification of Fc-fusion proteins containing the variant ECDs CD80, CD86, ICOSL, and NKp30.

[0485] Recombinant variant Fc fusion proteins were produced using the Expi293 Expression System (Invitrogen, USA). Four micrograms of each plasmid DNA from the previous step was added to 200 μl of Opti-MEM (Invitrogen, USA). Simultaneously, 10.8 μl of ExpiFectamine was added separately to another 200 μl of Opti-MEM. After 5 minutes, 200 μl of plasmid DNA was mixed with 200 μl of ExpiFectamine, and the mixture was further incubated for an additional 20 minutes before adding to the cells. Ten million Expi293 cells were dispensed into separate wells of a 10-ml sterile, deep, conical-bottom, 24-well growth plate (Thomson Instrument Company, USA) in 3.4 ml of Expi293 medium (Invitrogen, USA). The plate was shaken at 120 RPM for 5 days in a mammalian cell culture incubator set at 95% humidity and 8% CO2. After 5 days of incubation, the cells were pelleted and the culture supernatant was removed.

[0486] Protein was purified from the supernatant using a high-throughput 96-well Protein A purification kit (Catalog number 45202, Life Technologies, USA) according to the manufacturer's protocol. The resulting elution fraction was buffer-exchanged into PBS using a Zeba 96-well spin desalting plate (Catalog number 89807, Life Technologies, USA) according to the manufacturer's protocol. Purified protein was quantified using absorbance at 280 nm measured with a Nanodrop instrument (Thermo Fisher Scientific, USA). Protein purity was assessed by loading 5 μg of protein onto a NUPAGE precast polyacrylamide gel (Life Technologies, USA) under denaturing and reducing conditions followed by gel electrophoresis. Proteins were visualized in the gel using standard Coomassie staining.

[0487] Example 6 Assessment of binding and activity of affinity-matured IgSF domain-containing molecules A. Binding to cell-expressed counterstructures This example describes Fc fusion binding studies demonstrating the specificity and affinity of CD80 and ICOSL domain variant immunomodulatory proteins for their cognate binding partners.

[0488] To produce cells expressing their cognate binding partners, full-length mammalian surface expression constructs for each of human CD28, CTLA4, PD-L1, ICOS, and B7-H6 were engineered in the pcDNA3.1 expression vector (Life Technologies), which was supplied by Genscript, USA. Binding studies were performed using the Expi293F transient transfection system (Life Technologies, USA) described above. The number of cells required for the experiment was determined, and appropriate 30 ml-scale transfections were performed using the manufacturer's recommended protocol. For each 30 ml transfection of CD28, CTLA-4, PD-L1, ICOS, B7-H6, or mock, 75 million Expi293F cells were incubated with 30 μg of expression construct DNA and 1.5 ml of diluted ExpiFectamine 293 reagent for 48 hours, at which point cells were harvested for staining.

[0489] For flow cytometry staining, 200,000 cells of the appropriate transient transfection or negative control were plated in a 96-well round-bottom plate. The cells were spun down and resuspended in staining buffer (PBS (phosphate-buffered saline), 1% BSA (bovine serum albumin), and 0.1% sodium azide) for 20 minutes to block nonspecific binding. The cells were then centrifuged again and resuspended in 50 μl of staining buffer containing 100 nM–1 nM of each candidate CD80 variant Fc, ICOSL variant Fc, or stacked IgSF variant Fc fusion protein at the appropriate concentration, depending on the experiment. Primary staining was performed on ice for 45 minutes, after which the cells were washed twice in staining buffer. PE-conjugated anti-human Fc (Jackson ImmunoResearch, USA) was diluted 1:150 in 50 μl of staining buffer and added to the cells, which were then incubated on ice for an additional 30 minutes. The secondary antibody was washed out twice, the cells were fixed in 4% formaldehyde / PBS, and the samples were analyzed on a FACScan flow cytometer (Becton Dickinson, USA).

[0490] The mean fluorescence intensity (MFI) was calculated for each transfectant and negative parental line using Cell Quest Pro software (Becton Dickinson, USA).

[0491] B. Characterization of Bioactivity This example further describes the characterization of the biological activity of the Fc fusion variant proteins in a human primary T cell in vitro assay.

[0492] 1. Mixed Lymphocyte Reaction (MLR) The biological activity of soluble rICOSL.Fc or rCD80.Fc was tested in a human mixed lymphocyte reaction (MLR). Human primary dendritic cells (DCs) were generated by culturing monocytes isolated from PBMCs (BenTech Bio, USA) in Ex-Vivo 15 medium (Lonza, Switzerland) with 500 U / ml rIL-4 (R&D Systems, USA) and 250 U / ml rGM-CSF (R&D Systems, USA) in vitro for 7 days. 10,000 mature DCs and 100,000 purified allogeneic CD4+ T cells (BenTech Bio, USA) were cocultured with ICOSL or CD80 variant Fc fusion proteins and controls in a final volume of 200 μl of Ex-Vivo 15 medium in 96-well round-bottom plates. IFN-γ secretion in culture supernatants was analyzed using a Human IFN-gamma Duoset ELISA kit (R&D Systems, USA) on day 5. Optical density was measured using a VMax ELISA Microplate Reader (Molecular Devices, USA) and quantified against a titrated rIFN-γ standard included in the IFN-gamma Duo-set kit (R&D Systems, USA).

[0493] 2. Anti-CD3 Co-fixation Assay The costimulatory bioactivity of ICOSL and CD80 Fc fusion variants was determined in an anti-CD3 co-fixation assay. Mouse anti-human CD3 (OKT3, Biolegends, USA) was diluted at 1 nM or 4 nM with rICOSL.Fc or rCD80.Fc variant protein at 1 nM–80 nM in PBS. This mixture was added overnight to a tissue-culture-treated, flat-bottom 96-well plate (Corning, USA) to promote binding of the stimulatory protein to the wells. The next day, unbound protein was washed from the plate, and 100,000 purified human pan T cells (BenTech Bio, US) or human T cell clone BC3 (Astarte Biologics, USA) were added to each well in a final volume of 200 μl of Ex-Vivo 15 medium (Lonza, Switzerland). After culturing the cells for 3 days, culture supernatants were collected, and human IFN-γ levels were measured using the Duoset ELISA kit (R&D Systems, USA) as described above.

[0494] C. Results The results of binding and activity studies for exemplary test variants are shown in Tables 6-8. In particular, Table 6 shows exemplary IgSF domain amino acid substitutions (exchanges) in the ECD of CD80 selected in affinity maturation screens against the respective cognate structure CD28. Table 7 shows exemplary IgSF domain amino acid substitutions (exchanges) in the ECD of CD80 selected in affinity maturation screens against the respective cognate structure PD-L1. Table 8 shows exemplary IgSF domain amino acid substitutions (exchanges) in the ECD of ICOSL selected in affinity maturation screens against the respective cognate structures ICOS and CD28. For each table, exemplary amino acid substitutions are indicated by the amino acid position number corresponding to the respective reference unmodified ECD sequence, as follows: For example, the reference unmodified ECD sequence in Tables 6 and 7 is the unmodified CD80 ECD sequence shown in SEQ ID NO:28, and the reference unmodified ECD sequence in Table 8 is the unmodified ICOSL ECD sequence (SEQ ID NO:32). The amino acid position is shown in the center, where the corresponding unaltered (e.g., wild-type) amino acid is listed before the number and the identified variant amino acid substitution is listed after the number. Column 2 shows the SEQ ID NO identifier for the variant ECD for each variant ECD-Fc fusion molecule.

[0495] Also shown is binding activity, as measured by the mean fluorescence intensity (MFI) value for binding of each variant Fc fusion molecule to cells engineered to express its cognate counterstructure ligand, compared to the ratio of MFI for binding of a corresponding unmodified ECD-Fc fusion molecule containing no amino acid substitution to the counterstructure ligand expressed by the same cells. Also shown is the functional activity of the variant Fc fusion molecules in modulating T cell activity, based on the calculated IFN-γ (pg / ml) in culture supernatants produced with either i) the indicated variant ECD-Fc fusion molecule co-immobilized with anti-CD3 or ii) the indicated variant ECD-Fc fusion molecule in an MLR assay. The table also shows the ratio of IFN-γ produced by each variant ECD-Fc compared to the corresponding unmodified ECD-Fc in both functional assays.

[0496] As shown, selection led to the identification of numerous affinity-engineered CD80 or ICOSL IgSF domain variants that exhibited improved binding to at least one, and in some cases multiple, cognate counterstructure ligands. Additionally, the results indicated that the affinity-engineered variant molecules also exhibited improved activity, both enhancing and attenuating immune activity, depending on the format of the molecule. For example, co-immobilization of ligands likely enhances T cell activation by providing multivalent interactions with cells to cluster them or by improving avidity favoring agonist activity compared to unmodified (e.g., wild-type) ECD-Fc molecules that do not contain amino acid exchanges. However, when the molecules were provided as bivalent Fc molecules in solution, the same IgSF domain variants exhibited antagonist activity, reducing T cell activation, compared to unmodified (e.g., wild-type) ECD-Fv molecules that do not contain amino acid exchanges.

[0497] Table 6: CD80 variants selected against CD28. Molecular sequences, binding data, and costimulatory bioactivity data. TIFF2025166053000011.tif195169TIFF2025166053000012.tif217169

[0498] Table 7. CD80 variants selected against PD-L1. Molecular sequences, binding data, and costimulatory bioactivity data. TIFF2025166053000013.tif136169TIFF2025166053000014.tif129169

[0499] Table 8. Selected ICOSL variants against CD28 or ICOS. Molecular sequences, binding data, and costimulatory bioactivity data. TIFF2025166053000015.tif241169TIFF2025166053000016.tif92169*: Ratio to parent calculated using 346 pg / ml IFN-γ for WT ICOSL

[0500] Example 7 Ligand binding competition assay As shown in Example 6, several CD80 variant molecules exhibited improved binding to one or both of CD28 and PD-L1. To further evaluate the binding activity of CD80 to its ligands CD28 and PD-L1, this example describes a ligand competition assay to assess the non-competitive ability of exemplary CD80 variants to bind to both CD28 and PD-L1.

[0501] To assess the ability of CD80 to simultaneously bind to CD28 and PD-L1, an ELISA-based binding assay incorporating plate-bound CD80 variant A91G ECD-Fc was set up. Maxisorp 96-well ELISA plates (Nunc, USA) were coated overnight with 100 nM human recombinant CD80 variant A91G ECD-Fc fusion protein in PBS. The following day, unbound protein was washed away, and the plates were blocked with 1% bovine serum albumin (Millipore, USA) in PBS for 1 hour at room temperature. This blocking reagent was then washed off three times with PBS / 0.05% Tween. Each wash included a 2-minute incubation on a platform shaker.

[0502] In one arm of the competition assay, CD80 was incubated with CD28, and then CD28-bound CD80 was assessed for competitive binding in the presence of other known CD80 ligand counterstructures, PD-L1 or CTLA-4, or the negative control ligand PD-L2. Specifically, biotinylated recombinant human CD28 Fc fusion protein (rCD28.Fc; R&D Systems) was dose-escalated in the wells, starting at 10 nM in a 25 μl volume and diluted 1:2 for eight points. Immediately following the addition of biotinylated rCD28.Fc, unlabeled competing binders, recombinant human PD-L1 monomeric his-tagged protein, recombinant human CTLA-4 monomeric his-tagged protein, or negative control human recombinant PD-L2 Fc fusion protein (R&D Systems), were added to the wells at 2000, 1000, and 500 nM in a 25 μl volume, respectively, in a final volume of 50 μl. The proteins were incubated together for 1 hour, after which the three washing steps as described above were repeated.

[0503] After washing, 2.5 ng of HRP-conjugated streptavidin (Jackson Immunoresearch, USA) diluted in 1% BSA / PBS was added per well to detect bound biotinylated rCD28.Fc. After 1 hour of incubation, the wells were washed again three times as described above. To detect the signal, following washing, 50 μl of TMB substrate (Pierce, USA) was added to the wells and incubated for 7 minutes before adding 50 μl of 2 M sulfuric acid stop solution. Optical density was determined using an Emax Plus microplate reader (Molecular Devices, USA). Optical density values ​​were graphed using Prism (Graphpad, USA).

[0504] The results are shown in Figure 1A. The results demonstrated decreased binding of biotinylated rCD28.Fc to the CD80 variant A91G ECD-Fc fusion protein with increasing doses of rCD28.Fc. When rCD28.Fc binding occurred in the presence of the non-competitive control protein rPDL2, there was no decrease in CD28 binding to CD80 (closed triangles). In contrast, the competitive control protein rCTLA-4, when incubated with CD28.Fc, resulted in decreased CD28 binding to CD80, as expected (x-ray). When recombinant PD-L1 was incubated with CD28.Fc, no decrease in CD28 binding to CD80 was observed, demonstrating that the epitopes of CD28 and PD-L1 for CD80 are non-competitive. Binding of the recombinant PD-L1 protein used in the CD28 competition assay to CD80 was confirmed by incubating biotinylated PD-L1 in the presence of non-biotinylated rCD28.Fc (squares).

[0505] Additionally, after incubating CD80 with PD-L1 in a reverse competition setup, PD-L1-bound CD80 was then assessed for competitive binding in the presence of either other known CD80 ligand counterstructures, CD28 or CTLA-4, or the negative control ligand, PD-L2. Specifically, the assay was performed by dose-titrating biotinylated recombinant human PD-L1-his monomer protein into wells containing recombinant CD80 variants. Due to weak binding to this ligand, dose escalation was performed using eight similar 1:2 dilutions, starting at 5000 nM in 25 μL. When rPD-L1-his was used to detect binding, competing ligand human rCD28.Fc, human rCTLA-4.Fc, or human rPD-L2.Fc controls were added at a final concentration of 2.5 nM in 25 μL in a total volume of 50 μL. Subsequent washes, detection, and OD measurements were the same as described above.

[0506] The results are shown in Figure 1B. Dose-escalating PD-L1-his binding alone confirmed PD-L1 binding to the CD80 variant A91G ECD-Fc fusion molecule immobilized on the plate (squares). When PD-L1-his binding occurred in the presence of the non-competitive control protein rPDL2, there was no reduction in PD-L1 binding to CD80 (triangles). The CD28-competitive control protein rCTLA-4, when incubated with PD-L1-his, did not result in reduced PD-L1 binding to CD80, despite CTLA-4 being competitive for CD28 (x-ray). This result further demonstrated the lack of competition between CD28 and PD-L1 for CD80 binding. Finally, when PD-L1-his was incubated with CD28.Fc, no reduction in PD-L1 binding to CD80 was observed, demonstrating that the epitopes of CD28 and PD-L1 for CD80 are non-competitive.

[0507] Thus, the results showed that CTLA-4, but not PD-L1 or the negative control PD-L2, competed for CD28 binding to CD80 (Figure 1A), and that CD28, CTLA-4, and PD-L2 did not compete for PD-L1 binding to CD80 (Figure 1B). Thus, these results demonstrated that CD28 and PD-L1 are noncompetitive binders of CD80, and that this noncompetitive binding can be demonstrated regardless of the ligand detected by ELISA.

[0508] Example 8 Generation and evaluation of stacked molecules containing different affinity-engineered domains The above selected variant molecules with engineered affinities for one or more counterstructure ligands were used to generate "stack" molecules (i.e., type II immunomodulatory proteins) containing two or more affinity-engineered IgSF domains. Stack constructs were obtained as gene blocks (Integrated DNA Technologies, Coralville, IA) encoding the stack in a format that allows its fusion to Fc by standard Gibson assembly using a Gibson assembly kit (New England Biolabs).

[0509] Coding nucleic acid molecules for all stacks encoding the designed proteins were generated as follows: a signal peptide, followed by a first variant IgV of interest, followed by a 15-amino acid linker composed of three GGGGS (G4S) motifs (SEQ ID NO: 228), followed by a second IgV of interest, followed by two GGGGS linkers (SEQ ID NO: 229), followed by three alanines (AAA), followed by human IgG1 Fc as described above. To maximize the likelihood of correct folding of the IgV domains in each stack, the first IgV is preceded by all residues that normally occur in the wild-type protein between this IgV and the signal peptide (leading sequence). Similarly, the first IgV is followed by all residues that normally connect the IgV to either the next Ig domain in the wild-type protein (typically the IgC domain) or, if such a second IgV domain is not present, to the transmembrane domain (trailing sequence). The same design principles were applied to the second IgV domain, except that if both IgV domains were derived from the same parent protein (e.g., a CD80 IgV stacked with another CD80 IgV), the linker between them was not repeated.

[0510] Table 9 shows the design of an exemplary stacked construct. The exemplary stacked molecule shown in Table 9 contains the IgV domains as indicated and further leading or trailing sequences as described above. The table shows the following components in the following order: signal peptide (SP; SEQ ID NO:225), IgV domain 1 (IgV1), trailing sequence 1 (TS1), linker 1 (LR1; SEQ ID NO:228), IgV domain 2 (IgV2), trailing sequence 2 (TS2), linker 2 (LR2; SEQ ID NO:230), and Fc domain (SEQ ID NO:226 containing C5S / N82G amino acid substitutions). In some cases, leading sequence 1 (LS1) is present between the signal peptide and IgV1, and in some cases, leading sequence 2 (LS2) is present between the linker and IgV2.

[0511] Table 9: Amino acid sequences (SEQ ID NOs) of components of exemplary stacked constructs TIFF2025166053000017.tif75169TIFF2025166053000018.tif234169TIFF2025166053000019.ti f210169TIFF2025166053000020.tif253169TIFF2025166053000021.tif217169TIFF20251660530 00022.tif233169TIFF2025166053000023.tif245169TIFF2025166053000024.tif229169TIFF202 5166053000025.tif230169TIFF2025166053000026.tif233169TIFF2025166053000027.tif99169

[0512] High-throughput expression and purification of variant IgV-stacked Fc fusion molecules containing various combinations of variant IgV domains from CD80, CD86, ICOSL, or Nkp30, each containing at least one affinity-engineered IgV domain, was performed as described in Example 5. The binding of the variant IgV-stacked Fc fusion molecules to their respective counterstructures and functional activity in an anti-CD3 co-immobilization assay were also evaluated as described in Example 6. For example, the costimulatory bioactivity of stacked IgSF Fc fusion proteins was determined in a similar immobilized anti-CD3 assay described above. In this case, 4 nM anti-CD3 (OKT3, Biolegend, USA) was co-immobilized overnight with 4 nM to 120 nM human rB7-H6.Fc (R&D Systems, USA) or human rPD-L1.Fc (R&D Systems, USA) on tissue culture-treated 96-well plates (Corning, USA). The next day, unbound proteins were washed away with PBS, and 100,000 purified pan T cells were added to each well in 100 μl of Ex-Vivo 15 medium (Lonza, Switzerland). Stacked IgSF domains were then added at concentrations ranging from 8 nM to 40 nM in a 100 μl volume, for a total volume of 200 μl. After culturing the cells for 3 days, the culture supernatants were collected and human IFN-γ levels were measured using the Duoset ELISA kit (R&D Systems, USA) as described above.

[0513] The results are shown in Tables 10-14. Specifically, Table 10 shows binding and functional activity results for variant IgV-stacked Fc fusion molecules containing an NKp30 IgV domain and an ICOSL IgV domain. Table 11 shows binding and functional activity results for variant IgV-stacked Fc fusion molecules containing an NKp30 IgV domain and a CD80 or CD86 IgV domain. Table 12 shows binding and functional activity results for variant IgV-stacked Fc fusion molecules containing a variant CD80 IgV domain and a CD80, CD86, or ICOSL IgV domain. Table 13 shows binding and functional activity results for variant IgV-stacked Fc fusion molecules containing two variant CD80 IgV domains. Table 14 shows results for variant IgV-stacked Fc fusion molecules containing a variant CD80 or CD86 IgV domain and a variant ICOSL IgV domain.

[0514] For each of Tables 10-14, column 1 shows the structural organization and orientation of the stacked, affinity-engineered, or wild-type (WT) domains, starting with the amino-terminal (N-terminal) domain, followed by the central WT or affinity-engineered domain before the C-terminal human IgG1 Fc domain. Column 2 shows the SEQ ID NO identifier for the sequence of each IgV domain contained in each "stack" molecule. Column 3 indicates the binding partner for which the affinity-engineered stacked domains listed in column 1 were selected.

[0515] Also shown is binding activity, as measured by the mean fluorescence intensity (MFI) value for binding of each stack molecule to cells engineered to express various counterstructure ligands, compared to the ratio of MFI for binding of a corresponding stack molecule containing an unmodified IgV domain without amino acid substitutions to the counterstructure ligand expressed by the same cells. Also shown is the functional activity of the variant stack molecules in modulating T cell activity, based on the calculated levels of IFN-γ (pg / ml) in culture supernatants produced with the indicated variant stack molecule in solution and the appropriate ligand co-immobilized with anti-CD3 as described in Example 6. The table also shows the ratio of IFN-γ produced by each variant stack molecule compared to the corresponding unmodified stack molecule in the co-immobilization assay.

[0516] As shown, the results demonstrated that it was possible to generate stacked molecules containing at least one variant IgSF domain that exhibited improved avidity with altered affinity for at least one cognate counterstructure ligand compared to corresponding stacked molecules containing the respective unmodified (e.g., wild-type) IgV domains. In some cases, stacked molecules derived from either one or a combination of both variant IgSF domains in the molecule exhibited improved binding to multiple cognate counterstructure ligands. The results also demonstrated that the order of the IgV domains in the stacked molecules can, in some cases, alter the degree of improved avidity. In some cases, functional T cell activity was also altered as assessed in a targeted co-immobilization assay.

[0517] Table 10. Stacked variant IgV Fc fusion proteins containing NKp30 IgV domain and ICOSL IgV domain TIFF2025166053000028.tif159170

[0518] Table 11. Stacked variant IgV Fc fusion proteins containing the NKp30 IgV domain and the CD80 or CD86 IgV domain TIFF2025166053000029.tif223170

[0519] Table 12. Stacked variant IgV Fc fusion proteins containing a CD80 IgV domain and a CD80, CD86, or ICOSL IgV domain TIFF2025166053000030.tif219170TIFF2025166053000031.tif235170TIFF2025166053000032.tif243170

[0520] Table 13. Stacked variant IgV Fc fusion proteins containing two CD80 IgV domains TIFF2025166053000033.tif215170TIFF2025166053000034.tif69170

[0521] Table 14. Stacked variant IgV Fc fusion proteins containing CD80 or CD86 IgV domains and ICOSL IgV domains TIFF2025166053000035.tif150170TIFF2025166053000036.tif235170TIFF2025166053000037.tif134170

[0522] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and compositions within the scope of these claims and their equivalents be covered thereby.

[0523] Array information SEQUENCE LISTING <110> Alpine Immune Sciences, Inc. <120> IMMUNOMODULATORY PROTEINS WITH TUNABLE AFFINITIES <150> US 62 / 149,437 <151> 2015-04-17 <150> US 62 / 218,534 <151> 2015-09-14 <160> 240 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 288 <212> PRT <213> Homo sapiens <220> <223> CD80(B7-1) <400> 1 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> 2 <211> 329 <212> PRT <213> Homo sapiens <220> <223> CD86(B7-2) <400> 2 Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe 20 25 30 Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln 35 40 45 Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val 50 55 60 Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser 65 70 75 80 Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg 85 90 95 Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile 100 105 110 His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser 115 120 125 Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135 140 Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145 150 155 160 His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys 165 170 175 Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp Asn 180 185 190 Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro 195 200 205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln 225 230 235 240 Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val 245 250 255 Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys 260 265 270 Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu 275 280 285 Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro 290 295 300 Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser 305 310 315 320 Ser Cys Asp Lys Ser Asp Thr Cys Phe 325 <210> 3 <211> 290 <212> PRT <213> Homo sapiens <220> <223> CD274 (PD-L1, B7-H1) <400> 3 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 4 <211> 273 <212> PRT <213> Homo sapiens <220> <223> PDCD1LG2(PD-L2, CD273) <400> 4 Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45 His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60 Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu 65 70 75 80 Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90 95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215 220 Ile Phe Ile Pro Phe Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val 225 230 235 240 Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265 270 Or <210> 5 <211> 302 <212> PRT <213> Homo sapiens <220> <223> ICOSLG(B7RP1, CD275, ICOSL, B7-H2) <400> 5 Met Arg Leu Gly Ser Pro Gly Leu Leu Phe Leu Leu Phe Ser Ser Leu 1 5 10 15 Arg Ala Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp 20 25 30 Val Glu Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn 35 40 45 Asp Val Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr 50 55 60 Tyr His Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr 65 70 75 80 Arg Asn Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe 85 90 95 Ser Leu Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His 100 105 110 Cys Leu Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val 115 120 125 Glu Val Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser 130 135 140 Ala Pro His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr Cys Thr Ser 145 150 155 160 Ile Asn Gly Tyr Pro Arg Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp 165 170 175 Asn Ser Leu Leu Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn 180 185 190 Met Arg Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr 195 200 205 Pro Ser Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln 210 215 220 Asn Leu Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg Asp 225 230 235 240 Lys Ile Thr Glu Asn Pro Val Ser Thr Gly Glu Lys Asn Ala Ala Thr 245 250 255 Trp Ser Ile Leu Ala Val Leu Cys Leu Leu Val Val Val Ala Val Ala 260 265 270 Ile Gly Trp Val Cys Arg Asp Arg Cys Leu Gln His Ser Tyr Ala Gly 275 280 285 Ala Trp Ala Val Ser Pro Glu Thr Glu Leu Thr Gly His Val 290 295 300 <210> 6 <211> 534 <212> PRT <213> Homo sapiens <220> <223> CD276(B7-H3) <400> 6 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln 225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln 305 310 315 320 Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330 335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val 340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met...

Claims

1. 1. An immunomodulatory protein comprising at least one affinity-altered non-immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in a wild-type IgSF domain, the at least one affinity-altered IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type IgSF domain; and the at least one affinity-engineered IgSF domain specifically binds to the at least two cognate binding partners in a non-competitive manner; The immunomodulatory protein.

2. The immunomodulatory protein of claim 1 , wherein the at least two cognate binding partners are cell surface molecular species expressed on the surface of mammalian cells.

3. The immunomodulatory protein of claim 2, wherein the cell surface molecular species is expressed in a cis or trans configuration.

4. The immunomodulatory protein of claim 2 or claim 3, wherein the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and each of the cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS.

5. The immunomodulatory protein of any one of claims 2 to 4, wherein at least one of the mammalian cells is a lymphocyte.

6. The immunomodulatory protein of claim 5, wherein the lymphocyte is a NK cell or a T cell.

7. The immunomodulatory protein of any one of claims 5 to 6, wherein binding of said affinity-engineered IgSF domain modulates the immune activity of said lymphocytes.

8. The immunomodulatory protein of claim 7, which is capable of providing improved immune activity compared to a wild-type protein comprising the wild-type IgSF domain.

9. The immunomodulatory protein of claim 7, which is capable of conferring reduced immune activity compared to a wild-type protein comprising the wild-type IgSF domain.

10. The immunomodulatory protein of any one of claims 2 to 9, wherein at least one of the mammalian cells is a tumor cell.

11. The immunomodulatory protein of any one of claims 2 to 10, wherein the mammalian cell is a human cell.

12. The immunomodulatory protein of any one of claims 4 to 11, wherein the affinity-engineered IgSF domain is capable of specifically binding to the two mammalian cells that form the IS.

13. The wild-type IgSF domain is a member of the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling Lymphocyte Activation Molecule (SLAM) family, the Leukocyte Immunoglobulin-like Receptor (LGR) family, the IL-1 ...

13. The immunomodulatory protein of any one of claims 1 to 12, which is derived from an IgSF family member of a family selected from the group consisting of the LIR (Like Receptor), the Nectin (Nec) family, the Nectin-like (NECL) family, the Poliovirus receptor related (PVR) family, the Natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the Killer-cell immunoglobulin-like receptor (KIR) family.

14. 14. The immunomodulatory protein of any one of claims 1 to 13, wherein the wild-type IgSF domain is derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-α, CD8-β, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

15. The immunomodulatory protein of any one of claims 1 to 14, wherein the wild-type IgSF domain is a human IgSF member.

16. The immunomodulatory protein of any one of claims 1 to 15, wherein the wild-type IgSF domain is an IgV domain, an IgC1 domain, an IgC2 domain, or a specific binding fragment thereof.

17. 17. The immunomodulatory protein of any one of claims 1 to 16, wherein the affinity-modified IgSF domain is an affinity-modified IgV domain, an affinity-modified IgC1 domain, or an affinity-modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions.

18. 18. The immunomodulatory protein of any one of claims 1 to 17, comprising at least two affinity-engineered non-immunoglobulin IgSF domains.

19. 20. The immunomodulatory protein of claim 18, wherein each of the at least two affinity-altered non-immunoglobulin IgSF domains comprises one or more different amino acid substitutions in the same wild-type IgSF domain.

20. 20. The immunomodulatory protein of claim 19, wherein each of the at least two affinity-altered non-immunoglobulin IgSF domains comprises one or more amino acid substitutions in a different wild-type IgSF domain.

21. The immunomodulatory protein of claim 20 , wherein the different wild-type IgSF domains are derived from different IgSF family members.

22. 18. The immunomodulatory protein of any one of claims 1 to 17, comprising only one affinity-engineered non-immunoglobulin IgSF domain.

23. 23. The immunomodulatory protein of any one of claims 1 to 22, wherein the affinity-modified IgSF comprises at least 85% sequence identity with a wild-type IgSF domain or a specific-binding fragment thereof contained in an amino acid sequence set forth in any of SEQ ID NOs: 1 to 27.

24. The immunomodulatory protein of claim 23, further comprising a second affinity-modified IgSF domain comprising at least 85% sequence identity to a wild-type IgSF domain or a specific-binding fragment thereof contained in an amino acid sequence set forth in any of SEQ ID NOs: 1-27.

25. The immunomodulatory protein of any one of claims 1 to 24, wherein the wild-type IgSF domain is a member of the B7 family.

26. The immunomodulatory protein of any one of claims 1 to 25, wherein the wild-type IgSF domain is a domain of CD80, CD86, or ICOSLG.

27. The immunomodulatory protein of any one of claims 1 to 26, wherein the wild-type IgSF domain is a domain of CD80.

28. 1. An immunomodulatory protein comprising at least one affinity-altered CD80 immunoglobulin superfamily (IgSF) domain comprising one or more amino acid substitutions in the wild-type CD80 IgSF domain, wherein the at least one affinity-altered CD80 IgSF domain has improved binding to at least two cognate binding partners compared to the wild-type CD80 IgSF domain.

29. 29. The immunomodulatory protein of claim 27 or claim 28, wherein the cognate binding partners are CD28 and PD-L1.

30. 30. The immunomodulatory protein of any one of claims 27 to 29, wherein the wild-type IgSF domain is an IgV domain and / or the affinity-modified CD80 domain is an affinity-modified IgV domain.

31. 31. The immunomodulatory protein of any one of claims 27 to 30, wherein the affinity-modified domain comprises at least 85% sequence identity with the wild-type CD80 domain contained in the amino acid sequence set forth in SEQ ID NO:1, or a specific-binding fragment thereof.

32. 32. The immunomodulatory protein of any one of claims 1 to 31, wherein the at least one affinity-altered IgSF domain comprises between 1 and 20 amino acid substitutions in the wild-type IgSF domain.

33. The immunomodulatory protein of any one of claims 1 to 32, wherein the at least one affinity-altered IgSF domain comprises between 1 and 10 amino acid substitutions in the wild-type IgSF domain.

34. 34. The immunomodulatory protein of any one of claims 1 to 33, wherein said at least one affinity-altered IgSF domain comprises between one and five amino acid substitutions in said wild-type IgSF domain.

35. 35. The immunomodulatory protein of any one of claims 1 to 34, wherein the affinity-modified IgSF domain has at least 120% of the binding affinity of its wild-type IgSF domain for each of the at least two cognate binding partners.

36. 36. The immunomodulatory protein of any one of claims 1 to 35, further comprising an IgSF domain that has not been affinity modified.

37. The immunomodulatory protein of any one of claims 1 to 36, which is soluble.

38. 38. The immunomodulatory protein of any one of claims 1 to 37, which lacks a transmembrane or cytoplasmic domain.

39. 39. The immunomodulatory protein of any one of claims 1 to 38, comprising only the extracellular domain (ECD) or a specific binding fragment thereof comprising an affinity-engineered IgSF domain.

40. 40. The immunomodulatory protein of any one of claims 1 to 39, which is glycosylated or pegylated.

41. 41. The immunomodulatory protein of any one of claims 1 to 40, which is linked to a multimerization domain.

42. 42. The immunomodulatory protein of any one of claims 1 to 41, which is linked to an Fc domain or variant thereof with reduced effector function.

43. 43. The immunomodulatory protein of claim 42, wherein the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof, with reduced effector function.

44. the Fc domain is mammalian, optionally human; or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified mammalian, optionally human, Fc domain; 42. The immunomodulatory protein of any one of claims 39 to 41.

45. 45. The immunomodulatory protein of any one of claims 42 to 44, wherein the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity with SEQ ID NO:226 or SEQ ID NO:

227.

46. 42. The immunomodulatory protein of any one of claims 38 to 41, which is indirectly linked via a linker.

47. 47. The immunomodulatory protein of any one of claims 41 to 46, which is a dimer.

48. 48. The immunomodulatory protein of any one of claims 1 to 47, which is bound to a liposome membrane.

49. 1. An immunomodulatory protein comprising at least two non-immunoglobulin immunoglobulin superfamily (IgSF) domains, at least one of the modified non-immunoglobulin IgSF domains is affinity modified to exhibit altered binding to its cognate binding partner; and each of the at least two modified non-immunoglobulin IgSF domains independently specifically binds to at least one different cognate binding partner; The immunomodulatory protein.

50. 50. The immunomodulatory protein of claim 49, wherein each of the at least two non-immunoglobulin IgSF domains is an affinity-modified IgSF domain, wherein a first modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a first wild-type IgSF domain, and a second modified non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a second wild-type IgSF domain.

51. the first modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the first wild-type IgSF domain; and the second modified non-immunoglobulin IgSF domain exhibits altered binding to at least one of its cognate binding partners compared to the second wild-type IgSF domain; 51. The immunomodulatory protein of claim 50.

52. 52. The immunomodulatory protein of any one of claims 49 to 51, wherein said distinct cognate binding partners are cell surface species expressed on the surface of mammalian cells.

53. 53. The immunomodulatory protein of claim 52, wherein the different cell surface molecular species are expressed in a cis or trans configuration.

54. The immunomodulatory protein of claim 52 or claim 53, wherein the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and the different cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS.

55. The immunomodulatory protein of any one of claims 52 to 54, wherein at least one of said mammalian cells is a lymphocyte.

56. 56. The immunomodulatory protein of claim 55, wherein the lymphocyte is a NK cell or a T cell.

57. 57. The immunomodulatory protein of claim 55 or claim 56, wherein binding of the immunomodulatory protein to the cell modulates the immune activity of the lymphocyte.

58. 58. The immunomodulatory protein of claim 57, which is capable of providing improved immune activity compared to a wild-type protein comprising the wild-type IgSF domain.

59. 58. The immunomodulatory protein of claim 57, which is capable of conferring reduced immune activity compared to a wild-type protein comprising the wild-type IgSF domain.

60. The immunomodulatory protein of any one of claims 52 to 59, wherein at least one of said mammalian cells is a tumor cell.

61. The immunomodulatory protein of any one of claims 52 to 60, wherein the mammalian cell is a human cell.

62. 62. The immunomodulatory protein of any one of claims 54 to 61, capable of specifically binding to the two mammalian cells that form the IS.

63. 63. The immunomodulatory protein of any one of claims 49 to 62, wherein each of the first and second modified IgSF domains comprises one or more amino acid substitutions in a different wild-type IgSF domain.

64. 64. The immunomodulatory protein of claim 63, wherein the different wild-type IgSF domains are derived from different IgSF family members.

65. 65. The immunomodulatory protein of any one of claims 49 to 64, wherein the first and second modified IgSF domains are a non-wild-type combination.

66. The first and second wild-type IgSF domains individually bind to the Signal-Regulatory Protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, the Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, the Butyrophilin family, the B7 family, the CD28 family, the V-set and Immunoglobulin Domain Containing (VSIG) family, the V-set transmembrane Domain (VSTM) family, the Major Histocompatibility Complex (MHC) family, the Signaling Lymphocyte Activation Molecule (SLA) family, the IgSF domains ...

66. The immunomodulatory protein of any one of claims 49-65, derived from an IgSF family member of a family selected from the SLAM family, the Leukocyte immunoglobulin-like receptor (LIR), the Nectin (Nec) family, the Nectin-like (NECL) family, the Poliovirus receptor related (PVR) family, the Natural cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the Killer-cell immunoglobulin-like receptor (KIR) family.

67. 67. The immunomodulatory protein of any one of claims 49-66, wherein each of the first wild-type IgSF domain and the second wild-type IgSF domain is individually derived from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

68. 68. The immunomodulatory protein of any one of claims 49 to 67, wherein each of the first modified IgSF domain and the second modified IgSF domain individually comprises at least 85% sequence identity to a wild-type IgSF domain, or a specific binding fragment thereof, contained in an amino acid sequence set forth in any of SEQ ID NOs: 1 to 27.

69. 69. The immunomodulatory protein of any one of claims 49 to 68, wherein each of the first and second wild-type IgSF domains individually is a member of the B7 family.

70. 70. The immunomodulatory protein of claim 69, wherein each of the first and second wild-type IgSF domains is independently derived from CD80, CD86, or ICOSLG.

71. 69. The immunomodulatory protein of any one of claims 49 to 68, wherein the first or second wild-type IgSF domain is derived from a member of the B7 family, and the other of the first or second wild-type IgSF domain is derived from another IgSF family member.

72. The immunomodulatory protein of any one of claims 49-68 and 71, wherein the first and second wild-type IgSF domains are derived from ICOSLG and NKp30.

73. The immunomodulatory protein of any one of claims 49-68 and 71, wherein the first and second wild-type IgSF domains are derived from CD80 and NKp30.

74. 74. The immunomodulatory protein of any one of claims 49 to 73, wherein each of the first and second wild-type IgSF domains individually is a human IgSF member.

75. The immunomodulatory protein of any one of claims 49 to 74, wherein each of the first and second wild-type IgSF domains is individually an IgV domain, and an IgC1 domain, an IgC2 domain, or a specific combination thereof.

76. 76. The immunomodulatory protein of any one of claims 49-75, wherein each of the first modified non-immunoglobulin domain and the second modified non-immunoglobulin domain is individually a modified IgV domain, a modified IgC1 domain, or a modified IgC2 domain, or a specific-binding fragment thereof, comprising one or more amino acid substitutions.

77. 77. The immunomodulatory protein of any one of claims 49-76, wherein at least one of the first modified non-immunoglobulin domain or the second modified non-immunoglobulin domain is a modified IgV domain.

78. 78. The immunomodulatory protein of any one of claims 49-77, wherein the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between 1 and 20 amino acid substitutions.

79. 79. The immunomodulatory protein of any one of claims 49-78, wherein the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between 1 and 10 amino acid substitutions.

80. 80. The immunomodulatory protein of any one of claims 49-79, wherein the first modified non-immunoglobulin IgSF domain and the second modified non-immunoglobulin IgSF domain each individually comprise between one and five amino acid substitutions.

81. 81. The immunomodulatory protein of any one of claims 49 to 80, wherein at least one of the first or second modified non-immunoglobulin IgSF domains has 10% to 90% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

82. 82. The immunomodulatory protein of any one of claims 49-81, wherein at least one of the first or second modified non-immunoglobulin IgSF domains has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

83. 83. The immunomodulatory protein of any one of claims 49-80 and 82, wherein each of the first and second modified non-immunoglobulin IgSF domains individually has at least 120% of the binding affinity of the wild-type IgSF domain for at least one of its cognate binding partners.

84. The immunomodulatory protein of any one of claims 49 to 83, which is soluble.

85. 85. The immunomodulatory protein of any one of claims 49-84, which is glycosylated or pegylated.

86. 86. The immunomodulatory protein of any one of claims 49 to 85, linked to a multimerization domain.

87. 87. The immunomodulatory protein of any one of claims 49 to 86, which is linked to an Fc domain or variant thereof with reduced effector function.

88. 88. The immunomodulatory protein of claim 87, wherein the Fc domain is an IgG1 domain, an IgG2 domain, or a variant thereof, with reduced effector function.

89. the Fc domain is mammalian, optionally human; or the variant Fc domain comprises one or more amino acid modifications compared to an unmodified mammalian, optionally human, Fc domain; 89. The immunomodulatory protein of claim 87 or claim 88.

90. 90. The immunomodulatory protein of any one of claims 87 to 89, wherein the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity with SEQ ID NO:226 or SEQ ID NO:

227.

91. 91. The immunomodulatory protein of any one of claims 86-90, wherein the variant CD80 polypeptide is indirectly linked via a linker.

92. 92. The immunomodulatory protein of any one of claims 86 to 91, which is a dimer.

93. 93. The immunomodulatory protein of any one of claims 49-92, further comprising one or more additional non-immunoglobulin IgSF domains that are the same as or different from the first or second modified non-immunoglobulin IgSF domain.

94. 94. The immunomodulatory protein of claim 93, wherein said one or more additional non-immunoglobulin IgSF domains are affinity-engineered IgSF domains.

95. 95. The immunomodulatory protein of any one of claims 49 to 94, which is bound to a liposome membrane.

96. A nucleic acid molecule encoding the immunomodulatory polypeptide of any one of claims 1 to 95.

97. 97. The nucleic acid molecule of claim 96, which is a synthetic nucleic acid.

98. 98. The nucleic acid molecule of claim 96 or claim 97 which is a cDNA.

99. A vector comprising the nucleic acid molecule of any one of claims 96 to 98.

100. 100. The vector of claim 99, which is an expression vector.

101. 101. A cell comprising the vector of claim 99 or claim 100.

102. 102. The cell of claim 101, which is a eukaryotic or prokaryotic cell.

103. 100. A method for producing an immunomodulatory protein, the method comprising introducing into a host cell a nucleic acid molecule of any one of claims 96 to 98 or a vector of claim 99 or claim 100 under conditions such that the protein is expressed in the cell.

104. 104. The method of claim 103, further comprising isolating or purifying said immunomodulatory protein from said cells.

105. 96. A pharmaceutical composition comprising the immunomodulatory protein of any one of claims 1 to 95.

106. 106. The pharmaceutical composition of claim 105, comprising a pharmaceutically acceptable excipient.

107. 107. The pharmaceutical composition of claim 105 or claim 106, which is sterile.

108. 108. An article of manufacture comprising the pharmaceutical composition of any one of claims 105-107 in a vial.

109. 109. The article of manufacture of claim 108, wherein the vial is sealed.

110. 108. A kit comprising the pharmaceutical composition of any one of claims 105 to 107 and instructions for use.

111. 110. A kit comprising the article of manufacture of claim 108 or claim 109 and instructions for use.

112. 96. A method of modulating an immune response in a subject, comprising administering to said subject a therapeutically effective amount of an immunomodulatory protein of any one of claims 1-95.

113. 113. The method of claim 112, wherein modulating the immune response treats a disease or condition in the subject.

114. 114. The method of claim 112 or claim 113, wherein the immune response is enhanced.

115. 115. The method of claim 114, wherein the disease or condition is a tumor or cancer.

116. 116. The method of claim 114 or claim 115, wherein the disease or condition is selected from melanoma, lung cancer, bladder cancer, or a hematological malignancy.

117. 114. The method of claim 112 or claim 113, wherein the immune response is reduced.

118. 118. The method of claim 117, wherein the disease or condition is an inflammatory disease or condition.

119. 119. The method of claim 117 or claim 118, wherein the disease or condition is selected from Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

120. 1. A method for identifying affinity-modified immunomodulatory proteins, comprising the steps of: a) contacting an engineered protein comprising at least one engineered non-immunoglobulin immunoglobulin superfamily (IgSF) domain or a specific binding fragment thereof with at least two cognate binding partners under conditions capable of resulting in binding of the protein to the at least two cognate binding partners, wherein the at least one engineered IgSF domain comprises one or more amino acid substitutions in a wild-type IgSF domain; b) identifying modified proteins comprising the modified IgSF domain that have improved binding to at least one of the two cognate binding partners compared to proteins comprising the wild-type IgSF domain; and c) selecting modified proteins comprising the modified IgSF domain that bind non-competitively to the at least two cognate binding partners, thereby identifying immunomodulatory proteins with modified affinity.

121. 121. The method of claim 120, wherein step b) comprises identifying a modified protein comprising a modified IgSF domain that has improved binding to each of said at least two cognate binding partners compared to a protein comprising said wild-type domain.

122. 122. The method of claim 120 or claim 121, wherein prior to step a), one or more amino acid substitutions are introduced into the wild-type IgSF domain, thereby generating a modified protein comprising the modified IgSF domain.

123. 123. The method of any one of claims 120-122, wherein the engineered protein comprises at least two engineered IgSF domains or specific-binding fragments thereof, wherein a first IgSF domain comprises one or more amino acid substitutions in a first wild-type IgSF domain, and a second affinity-engineered non-immunoglobulin IgSF domain comprises one or more amino acid substitutions in a second wild-type IgSF domain.

124. 124. The method of claim 123, wherein each of said first and second affinity-engineered non-immunoglobulin IgSF domains specifically binds to at least one different cognate binding partner.

125. 125. The method of any one of claims 120 to 124, further comprising, prior to said step of selecting modified proteins, combining two or more modified IgSF domains or specific binding fragments thereof identified in step b) to generate proteins containing two or more different modified IgSF domains.

126. 126. The method of any one of claims 120-125, wherein the affinity-modified protein is capable of binding to both of said cognate binding partners simultaneously.

127. 127. The method of any one of claims 120 to 126, wherein said at least two cognate binding partners are cell surface molecular species expressed on the surface of mammalian cells.

128. 128. The method of claim 127, wherein the cell surface molecular species is expressed in a cis or trans configuration.

129. The method of claim 127 or claim 128, wherein the mammalian cell is one of two mammalian cells that form an immune synapse (IS), and each of the cell surface molecular species is expressed on the surface of at least one of the two mammalian cells that form the IS.

130. 130. The method of any one of claims 127-129, wherein at least one of said mammalian cells is a lymphocyte.

131. The method of any one of claims 127-130, wherein said lymphocytes are NK cells or T cells.

132. 132. The method of any one of claims 127-131, wherein at least one of said mammalian cells is a tumor cell.

133. 133. The method of any one of claims 127 to 132, wherein at least one of said mammalian cells is an antigen-presenting cell.

134. The method of any one of claims 127-133, wherein said two or more cognate binding partners are independently a ligand of an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOS ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R, or Nkp30.

135. The method of any one of claims 127-134, wherein said two or more cognate binding partners are independently ligands of a B7 family member.

136. 136. The method of any one of claims 127-135, wherein said two or more cognate binding partners are selected from two or more of CD28, CTLA-4, ICOS, or PD-L1.

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