CD86 variant immunomodulatory proteins and uses thereof

Abstract

To provide variant CD86 polypeptides, immunomodulatory proteins comprising variant CD86 polypeptides, and nucleic acids encoding the proteins, having therapeutic utility for a variety of immunological and oncological conditions.SOLUTION: Provided is a variant CD86 polypeptide, comprising an extracellular domain or an IgV domain or specific binding fragment thereof, the variant CD86 polypeptide comprising one or more amino acid modifications among specific amino acid sequences.SELECTED DRAWING: Figure 24A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 774,131, entitled "CD86 VARIANT IMMUNOMODULATORY PROTEINS AND USES THEREOF," filed November 30, 2018, and U.S. Provisional Patent Application No. 62 / 862,001, entitled "CD86 VARIANT IMMUNOMODULATORY PROTEINS AND USES THEREOF," filed June 14, 2019, the contents of which are incorporated by reference in their entireties.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format, which is provided as a 599,034 byte file entitled 761612002840SeqList.txt, created on November 27, 2019. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to therapeutic compositions for modulating immune responses in the treatment of cancer and immunological diseases. In some aspects, the present disclosure relates to specific variants of CD86 and immunomodulatory proteins thereof that exhibit altered binding affinity to their cognate binding partners (e.g., improved affinity for CD28). Methods and uses of such immunomodulatory proteins are also provided. [Background technology]

[0004] background There is growing medical interest in modulating immune responses by intervening in processes occurring at the immune synapse (IS), the region formed between antigen-presenting cells (APCs) or target cells and lymphocytes. Mechanistically, cell surface proteins within the IS can engage in coordinated, often simultaneous, interactions between multiple protein targets and a single protein to which they bind. Interactions at the IS occur in close association with the junction of two cells, and a single protein within this structure can interact (possibly simultaneously) with proteins on both the same cell (cis) and interacting cells (trans). While therapeutic agents capable of modulating the IS are known, improved therapeutic agents are needed. We provide immunomodulatory proteins, including soluble or transmembrane immunomodulatory proteins that can be expressed on cells, that meet this need. Summary of the Invention

[0005] overview As used herein, a variant CD86 polypeptide containing the extracellular domain or IgV domain or a specific binding fragment thereof, wherein the variant CD86 polypeptide is selected from the group consisting of: 13, 18, 25, 28, 33, 38, 39, 40, 43, 45, 52, 53, 60, 68, 71, 77, 79, 80, 82, 86, 88, 89, 90, 92, 93, 97, 102, 104, 113, 114, 123, 128, 129, 132, 133, 137, 141, 143, 144, 148, 153, 154, 158, 170, 172, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

[0013] Variant CD86 polypeptides are provided that contain one or more amino acid modifications in an unmodified CD86 polypeptide or a specific-binding fragment thereof corresponding to a position selected from among: 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224. In some embodiments, the amino acid modification contains an amino acid substitution, deletion, or insertion. In some embodiments, the unmodified CD86 polypeptide is a mammalian CD86 polypeptide or a specific-binding fragment thereof. In some embodiments, the unmodified CD86 polypeptide is a human CD86 polypeptide or a specific-binding fragment thereof. In some embodiments, the variant CD86 polypeptide contains the extracellular domain of human CD86, wherein the one or more amino acid modifications are at one or more residues in the extracellular domain of the unmodified CD86 polypeptide. In some embodiments, the unmodified CD86 polypeptide comprises (i) the amino acid sequence set forth in SEQ ID NO:29; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:29; or (iii) a portion thereof, wherein the portion comprises an IgV domain or a specific-binding fragment of the IgV domain. In some embodiments, the unmodified CD86 comprises the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the portion comprises amino acid residues 33-131 or 24-134 of the IgV domain or a specific-binding fragment of the IgV domain.

[0006] In some embodiments, the unmodified CD86 polypeptide comprises (i) the amino acid sequence set forth in SEQ ID NO:123, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:123, or (iii) a portion thereof, which contains an IgV domain or a specific-binding fragment of an IgV domain. In some embodiments, the unmodified CD86 comprises the amino acid sequence set forth in SEQ ID NO:123.

[0007] In some embodiments, the unmodified CD86 polypeptide comprises (i) the amino acid sequence set forth in SEQ ID NO:122, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:122; or (iii) a specific-binding fragment thereof. In some embodiments, the unmodified CD86 comprises the amino acid sequence set forth in SEQ ID NO:122.

[0008] In some embodiments, the specific binding fragment has a length of at least 50, 60, 70, 80, 90, 95, or more amino acids. In some embodiments, the specific binding fragment comprises at least 80% of the length of the IgV domain set forth as residues 33-131 of SEQ ID NO:2. In some embodiments, the variant CD86 comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, optionally amino acid substitutions, insertions, and / or deletions. In some embodiments, one or more amino acid modifications are substitutions. In some embodiments, one or more amino acid modifications are insertions. In some embodiments, one or more amino acid modifications are deletions. In some embodiments, one or more amino acid modifications are TIFF2025060939000002.tif47165, or conservative amino acid substitutions thereof.

[0009] In some embodiments, the variant CD86 polypeptide is TIFF2025060939000003.tif107164. In some embodiments, the one or more amino acid modifications are at positions 25 and / or 90. In some embodiments, the one or more amino acid modifications contain Q25L, H90Y, or H90L. In some embodiments, the one or more amino acid modifications contain Q25L. In some embodiments, the one or more amino acid modifications contain H90Y. In some embodiments, the one or more amino acid modifications contain H90L. In some embodiments, the one or more amino acid modifications contain modifications at positions 25 and 90. In some embodiments, the one or more amino acid modifications are selected from Q25L / H90Y or Q25L / H90L. In some embodiments, the one or more amino acid modifications contain Q25L / H90Y or Q25L / H90L and an additional amino acid modification. In some embodiments, the one or more amino acid modifications are Q25L / H90Y or Q25L / H90L and TIFF2025060939000004.tif41163, or a conservative amino acid substitution thereof.

[0010] In some embodiments, the variant CD86 polypeptide is TIFF2025060939000005.tif106169.

[0011] In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications A13V / Q25L / H90L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications A13V / Q25L / H90L / S181P / L197M / S206T. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / H90L / K93T / M97L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / H90L / K93T / M97L / T133A / S181P / D215V. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / Q86R / H90L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / Q86R / H90L / N104S. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications I89V / H90L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications I89V / H90L / I193V. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications M60K / H90L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / F33I / H90L. In some embodiments, a variant CD86 polypeptide contains one or more amino acid modifications Q25L / H90L / P185S.

[0012] In some embodiments, the variant CD86 polypeptide comprises an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or a specific-binding fragment thereof.

[0013] In some embodiments, the variant CD86 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the binding of unmodified CD86 to the same ectodomain, hi some embodiments, the binding affinity is improved by at least 1.5-fold or at least about 1.5-fold, at least 2.0-fold or at least about 2.0-fold, at least 5.0-fold or at least about 5.0-fold, at least 10-fold or at least about 10-fold, at least 20-fold or at least about 20-fold, at least 30-fold or at least about 30-fold, at least 40-fold or at least about 40-fold, at least 50-fold or at least about 50-fold, at least 60-fold or at least about 60-fold, at least 70-fold or at least about 70-fold, at least 80-fold or at least about 80-fold, at least 90-fold or at least about 90-fold, at least 100-fold or at least about 100-fold, or at least 125-fold.

[0014] In some embodiments, the variant CD86 polypeptide specifically binds to the ectodomain of CTLA-4 with reduced affinity compared to the binding of unmodified CD86 to the same ectodomain, hi some embodiments, the reduced binding affinity is at least 1.2-fold or at least about 1.2-fold, at least 1.4-fold or at least about 1.4-fold, at least 1.5-fold or at least about 1.5-fold, at least 1.75-fold or at least about 1.75-fold, at least 2.0-fold or at least about 2.0-fold, at least 2.5-fold or at least about 2.5-fold, at least 3.0-fold or at least about 3.0-fold, at least 4.0-fold or at least about 4.0-fold, or at least 5.0-fold or at least about 5.0-fold. In some embodiments, the variant CD86 polypeptide specifically binds to the ectodomain of CTLA-4 with the same or equivalent binding affinity as the binding of unmodified CD86 to the same ectodomain, and optionally, the same or equivalent binding affinity is 90% to 120%, or about 90% to about 120%, of the binding affinity of unmodified CD86.

[0015] In some embodiments, the variant CD86 polypeptide contains the entire extracellular domain. In some embodiments, the variant CD86 polypeptide contains the amino acid sequence set forth in any of SEQ ID NOs:85-121, or a specific binding fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity to any of SEQ ID NOs:85-121 and contains one or more of the amino acid modifications of the respective SEQ ID NOs set forth in any of SEQ ID NOs:85-121, or a specific binding fragment thereof. In some embodiments, the variant CD86 polypeptide contains the amino acid sequence set forth in any of SEQ ID NOs:141-177, or a specific binding fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity to any of SEQ ID NOs:141-177 and contains one or more of the amino acid modifications of the respective SEQ ID NOs:141-177, or a specific binding fragment thereof.

[0016] In some embodiments, the CD28 is human CD28. In some embodiments, the CTLA-4 is human CTLA-4. In some embodiments, the variant CD86 polypeptide is a soluble protein.

[0017] In some embodiments, the variant CD86 polypeptide lacks the transmembrane and intracellular signaling domains of CD86; and / or the variant CD86 polypeptide cannot be expressed on the surface of a cell. In some embodiments, the variant CD86 polypeptide is linked to a multimerization domain. In some embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the variant CD86 polypeptide is linked to an Fc domain or a variant thereof with reduced effector function. In some embodiments, the Fc domain is human IgG1 or a variant thereof with reduced effector function. In some embodiments, the Fc domain contains the amino acid sequence set forth in SEQ ID NO:229 or contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:229. In some embodiments, the Fc domain is or contains the amino acid sequence set forth in SEQ ID NO:229.

[0018] In some embodiments, the Fc domain is a variant IgG1 Fc domain containing one or more amino acid modifications selected from among E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del (each according to EU numbering). In some embodiments, the Fc domain contains the amino acid modifications L234A / L235E / G237A. In some embodiments, the Fc domain contains the amino acid modification C220S (according to EU numbering). In some embodiments, the Fc domain contains the amino acid modification K447del (according to EU numbering). In some embodiments, the Fc domain contains the amino acid sequence set forth in SEQ ID NO:230, or contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:230 and contains one or more of each amino acid modification set forth in SEQ ID NO:230 compared to human IgG1. In some embodiments, the Fc domain is or contains the amino acid sequence set forth in SEQ ID NO:230.

[0019] In some embodiments, the variant CD86 polypeptide is indirectly linked to the multimerization domain or Fc via a linker (optionally a G4S linker). In some embodiments, the variant CD86 polypeptide is a transmembrane immunomodulatory protein that further contains a transmembrane domain, optionally linked directly or indirectly to the extracellular domain (ECD) of the variant CD86 polypeptide or a specific-binding fragment thereof. In some embodiments, the transmembrane domain contains the amino acid sequence set forth as residues 248-268 of SEQ ID NO:2, or a functional variant thereof that exhibits at least 85% sequence identity to residues 248-268 of SEQ ID NO:2. In some embodiments, the variant CD86 polypeptide further contains a cytoplasmic domain, optionally linked directly or indirectly to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains a native CD86 cytoplasmic domain. In some embodiments, the cytoplasmic domain contains the amino acid sequence set forth as residues 269-329 of SEQ ID NO:2, or a functional variant thereof that exhibits at least 85% sequence identity to residues 269-329 of SEQ ID NO:2. In some embodiments, the cytoplasmic domain contains an ITAM signaling motif and / or is or contains the intracellular signaling domain of CD3ζ.

[0020] In some embodiments, the variant CD86 polypeptide does not contain a cytoplasmic signaling domain and / or is unable to mediate or regulate intracellular signals when expressed on a cell.

[0021] Provided herein is an immunomodulatory protein comprising a first variant CD86 polypeptide of any of the variant CD86 polypeptides described herein and a second variant CD86 polypeptide of any of the variant CD86 polypeptides described herein. In some embodiments, the first and second variant CD86 polypeptides are indirectly linked via a linker. In some embodiments, the first and second variant CD86 polypeptides are each linked to a multimerization domain, and the immunomodulatory protein is a multimer comprising the first and second variant CD86 polypeptides. In some embodiments, the multimer is a dimer, optionally a homodimer. In some embodiments, the multimer is a homodimer. In some embodiments, the first variant CD86 polypeptide and the second variant CD86 polypeptide are the same.

[0022] Provided herein are immunomodulatory proteins comprising any of the variant CD86 polypeptides described herein linked, directly or indirectly via a linker, to a second polypeptide containing an immunoglobulin superfamily (IgSF) domain of an IgSF family member. In some embodiments, the IgSF domain is an affinity-modified IgSF domain, which contains one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member. In some embodiments, the IgSF domain is an affinity-modified IgSF domain that exhibits altered binding to one or more of its cognate binding partners compared to the binding of the unmodified or wild-type IgSF domain of the IgSF family member to the same cognate binding partners or partners. In some embodiments, the IgSF domain exhibits improved binding to one or more of its cognate binding partners compared to the binding of the unmodified or wild-type IgSF domain of the IgSF family member to the same cognate binding partners or partners.

[0023] In some embodiments, the IgSF domain of the second polypeptide is a tumor-localizing moiety that binds to a ligand expressed on tumors or an inflammation-localizing moiety that binds to cells or tissues associated with an inflammatory environment. In some embodiments, the ligand is B7H6. In some embodiments, the IgSF domain is derived from NKp30. In some embodiments, the immunomodulating protein further contains a multimerization domain linked to at least one of the variant CD86 polypeptide or the second polypeptide. In some embodiments, the immunomodulating protein described herein further contains a third polypeptide containing an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, wherein the affinity-modified IgSF domain contains one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member. In some embodiments, the third polypeptide is the same as the first and / or second polypeptide; or, the third polypeptide is different from the first and / or second polypeptide.

[0024] In some embodiments, the immunomodulatory protein further contains a multimerization domain linked to at least one of the variant CD86 polypeptide, the second polypeptide, and / or the third polypeptide. In some embodiments, the multimerization domain is an immunoglobulin Fc domain, optionally wherein the immunoglobulin protein is human and / or the Fc region is human. In some embodiments, the immunoglobulin protein is human and / or the Fc region is human. In some embodiments, the Fc domain is IgG1, IgG2, or IgG4, or a variant thereof with reduced effector function. In some embodiments, the Fc domain is an IgG1 Fc domain, optionally human IgG1, or a variant thereof with reduced effector function. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain contains the amino acid sequence set forth in SEQ ID NO:229 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:229. In some embodiments, the Fc domain is or contains the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, the Fc domain is a variant IgG1 containing one or more amino acid substitutions, wherein the one or more amino acid substitutions are selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G (each numbered according to the EU index as per Kabat). In some embodiments, the Fc domain contains the amino acid substitution N297G, the amino acid substitutions R292C / N297G / V302C, or the amino acid substitutions L234A / L235E / G237A (each numbered according to the EU index of Kabat). In some embodiments, the variant Fc region further contains the amino acid substitution C220S, where said residues are numbered according to the EU index of Kabat.In some embodiments, the Fc region contains K447del, where such residues are numbered according to the EU index of Kabat. An Fc region may also be referred to herein as an Fc domain.

[0025] In some embodiments, the Fc domain contains the amino acid sequence set forth in SEQ ID NO:230, or contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:230 and contains one or more of each amino acid modification set forth in SEQ ID NO:230 compared to human IgG1. In some embodiments, the Fc domain is or contains the amino acid sequence set forth in SEQ ID NO:230.

[0026] Provided herein is an immunomodulatory protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one IgSF domain linked to a first Fc domain via a linker, said at least one IgSF domain comprising one or both of the following: a variant CD86 polypeptide that is any of the variant CD86 polypeptides provided herein, or an IgSF domain of a PD1 polypeptide or a variant thereof; and the second polypeptide comprises at least one IgSF domain linked to a second Fc domain via a linker, said at least one IgSF domain comprising one or both of the following: a variant CD86 polypeptide that is any of the variant CD86 polypeptides provided herein, or an IgSF domain of a PD1 polypeptide or a variant thereof, wherein the immunomodulatory protein comprises at least one IgSF domain of CD86 and at least one IgSF domain of PD-1, or a variant thereof.

[0027] In some of the provided embodiments, at least one IgSF domain of the first polypeptide comprises a variant CD86 polypeptide that is any of the variant CD86 polypeptides provided herein. In some of the provided embodiments, at least one IgSF domain of the second polypeptide comprises a variant PD1 polypeptide. In some of the provided embodiments, at least one IgSF domain of the first polypeptide is a first IgSF domain, which is a variant CD86 polypeptide that is any of the variant CD86 polypeptides provided herein, and the first polypeptide comprises a second IgSF domain linked to a first Fc domain via a linker. In some of the provided embodiments, the second IgSF domain of the first polypeptide comprises a variant PD1 polypeptide. In some of the provided embodiments, at least one IgSF domain of the second polypeptide is a first IgSF domain, which is a variant CD86 polypeptide that is any of the variant CD86 polypeptides provided herein, and the second polypeptide comprises a second IgSF domain linked to a second Fc domain via a linker. In some of the provided embodiments, the second IgSF domain of the second polypeptide comprises a variant PD1 polypeptide.

[0028] In some of the provided embodiments, at least one IgSF domain of the first polypeptide is linked via a linker to the N-terminus or C-terminus of the first Fc domain; and at least one IgSF domain of the second polypeptide is linked via a linker to the N-terminus or C-terminus of the second Fc domain. In some of the provided embodiments, the second IgSF domain of the first polypeptide is linked to the end of the first Fc domain opposite the end linked to the first IgSF domain. In some of the provided embodiments, the second IgSF domain of the second polypeptide is linked to the end of the second Fc domain opposite the end linked to the first IgSF domain. In some of the provided embodiments, the linkers independently comprise the sequence of SEQ ID NO:222 or 224, and optionally, the linker comprises one to four repeats of the sequence of SEQ ID NO:222 or 224. In some of any of the provided embodiments, the first Fc domain and the second Fc domain are identical, and optionally, the first Fc domain and the second Fc domain comprise the sequence of SEQ ID NO:230.

[0029] In some of the provided embodiments, the first and second polypeptides dimerize through the first and second Fc domains to form a homodimer, and in some of the provided embodiments, the first and second polypeptides of the homodimer comprise, from left to right, variant PD1 polypeptide-linker-Fc-linker-variant CD86 polypeptide.

[0030] In some of the provided embodiments, the variant PD1 polypeptide comprises the sequence of SEQ ID NO: 315. In some of the provided embodiments, the variant CD86 polypeptide comprises the sequence of SEQ ID NO: 94 or 150. In some of the provided embodiments, the first Fc domain and the second Fc domain are different, and optionally, the first and second Fc domains comprise knob-into-hole mutations, and optionally, the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO: 346, and the other of the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO: 347.

[0031] In some of the provided embodiments, the first and second polypeptides dimerize through the first and second Fc domains to form a heterodimer. In some of the provided embodiments, the first polypeptide of the heterodimer comprises, from left to right, a variant PD1 polypeptide-linker-Fc, and the second polypeptide of the heterodimer comprises, from left to right, a variant CD86 polypeptide-linker-Fc, an Fc-linker-variant CD86 polypeptide, or a variant PD1-linker-Fc-linker-variant CD86.

[0032] In some of the provided embodiments, the variant PD1 polypeptide comprises the sequence of SEQ ID NO: 315. In some of the provided embodiments, the variant CD86 polypeptide comprises the sequence of SEQ ID NO: 94 or 150. In some of the provided embodiments, the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 350; and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 351, 352, or 353.

[0033] Provided herein are conjugates containing any of the variant CD86 polypeptides described herein linked to a targeting moiety that specifically binds to a molecule on the surface of a cell. In some embodiments, the cell is an immune cell or a tumor cell. In some embodiments, the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle. In some embodiments, the moiety is an antibody or antigen-binding fragment. In some embodiments, the conjugates described herein are fusion proteins.

[0034] In some of the provided embodiments, the variant CD86 polypeptide is a V H or V L In some embodiments, the V of the antibody is linked to the N-terminus or C-terminus of the V H or V L The variant CD86 polypeptide linked to the N-terminus or C-terminus of is any variant CD86 polypeptide provided herein. In some of the provided embodiments, the antibody is an anti-HER2 antibody or an anti-EGFR antibody. In some of the provided embodiments, the anti-HER2 antibody is pertuzumab. In some of the provided embodiments, the variant CD86 polypeptide is linked to the V of pertuzumab. H N-terminus of pertuzumab, V H C-terminus of pertuzumab L or the V of pertuzumab L and optionally comprising the sequence of SEQ ID NO:342, 344, 343, or 345, respectively. In some of the provided embodiments, the anti-EGFR antibody is panitumumab. In some of the provided embodiments, the variant CD86 polypeptide is linked to the N-terminus of the VH of panitumumab, the C-terminus of the VH of panitumumab, the N-terminus of the VL of panitumumab, or the C-terminus of the VL of panitumumab, and optionally comprising the sequence of SEQ ID NO:348, 350, 349, or 351, respectively (or an anti-EGFR antibody).

[0035] Provided herein are nucleic acid molecules encoding any of the variant CD86 polypeptides described herein, the immunomodulatory proteins described herein, or the conjugates that are fusion proteins described herein. In some embodiments, the nucleic acid molecules are synthetic nucleic acids. In some embodiments, the nucleic acid molecules are cDNAs.

[0036] Provided herein is a vector containing the nucleic acid molecule described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector or a viral vector.

[0037] Provided herein is a cell containing the vector described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0038] Provided herein are methods for producing a protein containing a variant CD86 polypeptide, comprising introducing a nucleic acid molecule described herein or a vector described herein into a host cell under conditions such that the protein is expressed in the host cell. In some embodiments, the method further comprises isolating or purifying the protein from the cell.

[0039] Provided herein are methods of modifying a cell to express a variant CD86 polypeptide, the method comprising introducing into a host cell a nucleic acid molecule encoding a variant CD86 polypeptide as described herein, an immunomodulatory protein as described herein, or a conjugate that is a fusion protein as described herein, under conditions such that the polypeptide is expressed in the host cell.

[0040] Provided herein are modified cells containing a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a conjugate that is a fusion protein described herein, a nucleic acid molecule described herein, or a vector described herein. In some embodiments, the variant CD86 polypeptide contains a transmembrane domain or is a transmembrane immunomodulatory protein described herein; and / or a protein containing the variant CD86 polypeptide is expressed on the surface of the cell. In some embodiments, the variant CD86 polypeptide does not contain a transmembrane domain and / or is not expressed on the surface of the cell; and / or the variant CD86 polypeptide is capable of being secreted from the modified cell. In some embodiments, the protein does not contain a cytoplasmic signaling domain or a transmembrane domain and / or is not expressed on the surface of the cell; and / or the protein, when expressed, is capable of being secreted from the modified cell.

[0041] In some embodiments, the modified cells are immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are CD4+ and / or CD8+ T cells. In some embodiments, the T cells are regulatory T cells (Tregs). In some embodiments, the modified cells are primary cells. In some embodiments, the modified cells are mammalian cells. In some embodiments, the modified cells are human cells. In some embodiments, the modified cells further comprise a chimeric antigen receptor (CAR). In some embodiments, the modified cells further comprise a modified T cell receptor (TCR).

[0042] Provided herein is an infectious agent containing a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a conjugate that is a fusion protein described herein, a nucleic acid molecule described herein, or a vector described herein. In some embodiments, the infectious agent is a bacterium or a virus. In some embodiments, the infectious agent is a virus, and the virus is an oncolytic virus.

[0043] Provided herein are pharmaceutical compositions containing a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a conjugate that is a fusion protein described herein, a modified cell described herein, or an infectious agent described herein. In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0044] Provided herein is an article of manufacture comprising a pharmaceutical composition described herein in a vial or container. In some embodiments, the vial or container is sealed.

[0045] Provided herein are kits containing a pharmaceutical composition described herein or an article of manufacture described herein and instructions for use.

[0046] Provided herein are methods of modulating an immune response in a subject, the method comprising administering a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a conjugate that is a fusion protein described herein, a modified cell described herein, an infectious agent described herein, or a pharmaceutical composition described herein.

[0047] Provided herein are methods for modulating an immune response in a subject, comprising administering the modified cells described herein. In some embodiments, the modified cells are autologous to the subject. In some embodiments, the modified cells are allogeneic to the subject. In some embodiments, modulating the immune response treats a disease or condition in the subject.

[0048] Provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a conjugate that is a fusion protein described herein, a modified cell described herein, an infectious agent described herein, or a pharmaceutical composition described herein.

[0049] Provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering the modified cells described herein. In some embodiments, the modified cells are autologous to the subject. In some embodiments, the modified cells are allogeneic to the subject.

[0050] In some embodiments, the immune response is enhanced in the subject. In some embodiments, an immunomodulatory protein or conjugate containing a variant CD86 polypeptide linked to a tumor-localizing moiety is administered to the subject. In some embodiments, the tumor-localizing moiety is or contains a binding molecule that recognizes a tumor antigen. In some embodiments, the binding molecule contains an antibody or antigen-binding fragment thereof, or contains a wild-type IgSF domain or a variant thereof.

[0051] In some embodiments, a pharmaceutical composition containing an immunomodulatory protein described herein or a conjugate described herein is administered to a subject. In some embodiments, modified cells containing a variant CD86 polypeptide that is a transmembrane immunomodulatory protein are administered to a subject, optionally, the modified cells are any of the cells described herein. In some embodiments, the transmembrane immunomodulatory protein is as described herein.

[0052] In some embodiments, the disease or condition is a tumor or cancer, hi some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colon cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer.

[0053] In some embodiments, the immune response is reduced. In some embodiments, a soluble variant CD86 polypeptide or immunomodulatory protein is administered to a subject. In some embodiments, the soluble polypeptide or immunomodulatory protein is an Fc fusion protein.

[0054] In some embodiments, a pharmaceutical composition containing a variant CD86 polypeptide described herein or an immunomodulatory protein described herein is administered to the subject. In some embodiments, modified cells containing a secretable variant CD86 polypeptide are administered to the subject, optionally, the modified cells are any described herein.

[0055] In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition. In some embodiments, the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. In some embodiments, the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. [The present invention 1001] 132, 133, 137, 141, 143, 144, 148, 153, 154, 158, 170, 171, 172, 173, 174, 175, 176, 177, 178, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 200, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, , 172, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224, [The present invention 1002] 1001. The variant CD86 polypeptide of the present invention, wherein said amino acid modification comprises an amino acid substitution, deletion, or insertion. [The present invention 1003] 1001 or 1002, wherein said unmodified CD86 polypeptide is a mammalian CD86 polypeptide or a specific binding fragment thereof. [The present invention 1004] 1003. The variant CD86 polypeptide of the present invention, wherein said unmodified CD86 polypeptide is a human CD86 polypeptide or a specific binding fragment thereof. [The present invention 1005] 1006. The variant CD86 polypeptide of any of claims 1001 to 1004, wherein said variant CD86 polypeptide comprises the extracellular domain of human CD86, and wherein said one or more amino acid modifications are at one or more residues in the extracellular domain of an unmodified CD86 polypeptide. [The present invention 1006] the unmodified CD86 polypeptide (i) the amino acid sequence set forth in SEQ ID NO:29, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:29; or (iii) a portion thereof, comprising an IgV domain or a specific binding fragment of an IgV domain. 1001 to 1005. A variant CD86 polypeptide according to any one of claims 1001 to 1005. [The present invention 1007] 1007. The variant CD86 polypeptide of any of claims 1001 to 1006, wherein said unmodified CD86 comprises the amino acid sequence shown in SEQ ID NO:29. [The present invention 1008] 1006. A variant CD86 polypeptide of the invention, wherein said portion comprises amino acid residues 33 to 131 or 24 to 134 of the IgV domain or a specific binding fragment of the IgV domain. [The present invention 1009] the unmodified CD86 polypeptide (i) the amino acid sequence set forth in SEQ ID NO:123, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:123; or (iii) a portion thereof, comprising an IgV domain or a specific binding fragment of an IgV domain. A variant CD86 polypeptide of any of claims 1001 to 1006 and claim 1008, comprising: [The present invention 1010] 1007. The variant CD86 polypeptide of any of claims 1001 to 1006, wherein said unmodified CD86 comprises the amino acid sequence shown in SEQ ID NO:123. [The present invention 1011] the unmodified CD86 polypeptide (i) the amino acid sequence set forth in SEQ ID NO:122, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:122; or (iii) a specific binding fragment thereof. A variant CD86 polypeptide of any of 1001 to 1006, 1008 and 1009 of the present invention, comprising: [The present invention 1012] 1009 and 1011. The variant CD86 polypeptide of any one of claims 1001 to 1006, 1008, 1009 and 1011, wherein the unmodified CD86 comprises the amino acid sequence shown in SEQ ID NO:122. [The present invention 1013] the specific binding fragment has a length of at least 50, 60, 70, 80, 90, 95, or more amino acids; or the specific binding fragment comprises at least 80% of the length of the IgV domain shown as residues 33 to 131 of SEQ ID NO:2; A variant CD86 polypeptide of any one of 1001 to 1012 of the present invention. [The present invention 1014] 10. The variant CD86 polypeptide of any of claims 1001 to 1013, comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, optionally amino acid substitutions, insertions, and / or deletions. [The present invention 1015] the one or more amino acid modifications are A variant CD86 polypeptide of any of claims 1001 to 1014, which has one or more amino acid substitutions selected from TIFF2025060939000006.tif48164, or conservative amino acid substitutions thereof. [The present invention 1016] A variant CD86 polypeptide of any of claims 1001 to 1015, comprising one or more amino acid modifications selected from among TIFF2025060939000007.tif106163. [The present invention 1017] 1001-1014. The variant CD86 polypeptide of any of claims 1001-1014, wherein said one or more amino acid modifications are at positions 25 and / or 90. [The present invention 1018] 1009. The variant CD86 polypeptide of any of claims 1001 to 1014 and 1017, wherein said one or more amino acid modifications comprise Q25L, H90Y, or H90L. [The present invention 1019] 10. The variant CD86 polypeptide of any of claims 1001 to 1014 and 1017, wherein said one or more amino acid modifications comprise modifications at positions 25 and 90. [The present invention 1020] 1019. The variant CD86 polypeptide of the invention, wherein said one or more amino acid modifications are selected from Q25L / H90Y or Q25L / H90L. [The present invention 1021] A variant CD86 polypeptide of any of claims 1001 to 1020, comprising one or more amino acid modifications selected from among TIFF2025060939000008.tif106170. [The present invention 1022] 1001-1021. A variant CD86 polypeptide of any of claims 1001-1021, comprising one or more amino acid modifications A13V / Q25L / H90L. [The present invention 1023] 10. The variant CD86 polypeptide of any of claims 1001 to 1022, comprising one or more amino acid modifications A13V / Q25L / H90L / S181P / L197M / S206T. [The present invention 1024] 1001-1021. A variant CD86 polypeptide of any of claims 1001-1021, comprising one or more amino acid modifications Q25L / H90L / K93T / M97L. [The present invention 1025] 10. A variant CD86 polypeptide of any of claims 1001 to 1021 and 1024 comprising one or more amino acid modifications Q25L / H90L / K93T / M97L / T133A / S181P / D215V. [The present invention 1026] A variant CD86 polypeptide of any of claims 1001 to 1021 and 1024 comprising one or more amino acid modifications Q25L / Q86R / H90L. [The present invention 1027] A variant CD86 polypeptide of any of claims 1001 to 1021 and 1026 comprising one or more amino acid modifications Q25L / Q86R / H90L / N104S. [The present invention 1028] 10. The variant CD86 polypeptide of any of claims 1001 to 1021, comprising one or more amino acid modifications I89V / H90L. [The present invention 1029] 1001-1021 and 1028. A variant CD86 polypeptide of any of claims 1001-1028, comprising one or more amino acid modifications I89V / H90L / I193V. [The present invention 1030] 10. The variant CD86 polypeptide of any of claims 1001 to 1021, comprising one or more amino acid modifications M60K / H90L. [The present invention 1031] 1001-1021. A variant CD86 polypeptide of any of claims 1001-1021, comprising one or more amino acid modifications Q25L / F33I / H90L. [The present invention 1032] 1001-1021. A variant CD86 polypeptide of any of claims 1001-1021, comprising one or more amino acid modifications Q25L / H90L / P185S. [The present invention 1033] A variant CD86 polypeptide of any of claims 1001 to 1032, comprising an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29, or a specific binding fragment thereof. [The present invention 1034] A variant CD86 polypeptide of any of claims 1001 to 1033, which specifically binds to the ectodomain of CD28 with improved affinity compared to the binding of said unmodified CD86 to the same ectodomain. [This invention 1035] 1034. A variant CD86 polypeptide of the invention, wherein the binding affinity is improved by at least 1.5 fold, at least 2.0 fold, at least 5.0 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, or at least 125 fold. [The present invention 1036] 1036. A variant CD86 polypeptide according to any of claims 1001 to 1035, which specifically binds to the ectodomain of CTLA-4 with reduced affinity compared to the binding of said unmodified CD86 to the same ectodomain. [This invention 1037] 1036. A variant CD86 polypeptide of the invention, wherein said reduced binding affinity is at least 1.2-fold or at least about 1.2-fold, at least 1.4-fold or at least about 1.4-fold, at least 1.5-fold or at least about 1.5-fold, at least 1.75-fold or at least about 1.75-fold, at least 2.0-fold or at least about 2.0-fold, at least 2.5-fold or at least about 2.5-fold, at least 3.0-fold or at least about 3.0-fold, at least 4.0-fold or at least about 4.0-fold, or at least 5.0-fold or at least about 5.0-fold. [The present invention 1038] 10. The variant CD86 polypeptide of any of claims 1001 to 1037, wherein the variant CD86 polypeptide specifically binds to the ectodomain of CTLA-4 with a binding affinity that is the same as or equivalent to the binding affinity of the unmodified CD86 for the same ectodomain, and optionally the same or equivalent binding affinity is 90% to 120%, or about 90% to about 120%, of the binding affinity of the unmodified CD86. [This invention 1039] 1001-1038. A variant CD86 polypeptide of any of claims 1001-1038, comprising the entire extracellular domain. [The present invention 1040] A variant CD86 polypeptide of any of claims 1001 to 1039, comprising an amino acid sequence as set forth in any of SEQ ID NOs: 85 to 121, or a specific binding fragment thereof, an amino acid sequence that exhibits at least 95% sequence identity to any of SEQ ID NOs: 85 to 121 and that contains one or more of the amino acid modifications of the respective SEQ ID NO: set forth in any of SEQ ID NOs: 85 to 121, or a specific binding fragment thereof. [This invention 1041] 10. A variant CD86 polypeptide according to any of claims 1001 to 1040, comprising an amino acid sequence as set forth in any of SEQ ID NOs: 141 to 177, or a specific binding fragment thereof, an amino acid sequence which exhibits at least 95% sequence identity to any of SEQ ID NOs: 141 to 177 and which contains one or more of the amino acid modifications of the respective SEQ ID NO: set forth in any of SEQ ID NOs: 141 to 177, or a specific binding fragment thereof. [The present invention 1042] The variant CD86 polypeptide of any of claims 1034 to 1041, wherein the CD28 is human CD28. [This invention 1043] The variant CD86 polypeptide of any of claims 1034 to 1042, wherein the CTLA-4 is human CTLA-4. [This invention 1044] A variant CD86 polypeptide of any one of claims 1001 to 1043, which is a soluble protein. [This invention 1045] lacks the CD86 transmembrane and intracellular signaling domains; and / or cannot be expressed on the surface of cells, A variant CD86 polypeptide of any one of 1001 to 1044 of the present invention. [The present invention 1046] 1001-1045. A variant CD86 polypeptide of any of claims 1001-1045, which is linked to a multimerization domain. [This invention 1047] 1046. The variant CD86 polypeptide of the invention, wherein said multimerization domain is an Fc domain or a variant thereof having reduced effector function. [This invention 1048] 10. The variant CD86 polypeptide of any of claims 1001 to 1047, which is linked to an Fc domain or a variant thereof having reduced effector function. [This invention 1049] 1047. The variant CD86 polypeptide of the invention 1048, wherein said Fc domain is human IgG1 or a variant thereof having reduced effector function. [The present invention 1050] 10. The variant CD86 polypeptide of any of claims 1047 to 1049, wherein the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:229 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:229. [This invention 1051] 1050. The variant CD86 polypeptide of any of claims 1047 to 1050, wherein the Fc domain is or comprises the amino acid sequence shown in SEQ ID NO:229. [This invention 1052] 10. The variant CD86 polypeptide of any of claims 1047 to 1050, wherein the Fc domain is a variant IgG1 Fc domain comprising one or more amino acid modifications selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del, each according to EU numbering. [This invention 1053] 1053. The variant CD86 polypeptide of any of claims 1047 to 1050 and 1052, wherein said Fc domain comprises the amino acid modifications L234A / L235E / G237A. [This invention 1054] The variant CD86 polypeptide of any of claims 1047 to 1050, 1052 and 1053, wherein the Fc domain comprises the amino acid modification C220S according to EU numbering. [This invention 1055] The variant CD86 polypeptide of any of claims 1047 to 1050 and 1052 to 1054, wherein the Fc domain comprises the amino acid modification K447del according to EU numbering. [The present invention 1056] 10. The variant CD86 polypeptide of any of claims 1047-1050 and 1052-1055, wherein the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:230 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:230 and includes one or more of the amino acid modifications set forth in SEQ ID NO:230 compared to human IgG1. [This invention 1057] A variant CD86 polypeptide of any of claims 1047 to 1050 and 1052 to 1056, wherein the Fc domain is or comprises the amino acid sequence shown in SEQ ID NO:230. [This invention 1058] 1047. The variant CD86 polypeptide of any of claims 1047 to 1057, which is indirectly linked to said multimerization domain or Fc via a linker, optionally a G4S linker. [This invention 1059] 10. The variant CD86 polypeptide of any of claims 1001 to 1043, wherein said variant CD86 polypeptide is a transmembrane immunomodulatory protein further comprising a transmembrane domain, and optionally said transmembrane domain is linked directly or indirectly to said extracellular domain (ECD) of said variant CD86 polypeptide or a specific binding fragment thereof. [The present invention 1060] 1059. A variant CD86 polypeptide of the invention, wherein the transmembrane domain comprises the amino acid sequence set forth as residues 248-268 of SEQ ID NO:2, or a functional variant thereof exhibiting at least 85% sequence identity to residues 248-268 of SEQ ID NO:2. [The present invention 1061] 1059. The variant CD86 polypeptide of claim 1060, wherein said variant CD86 polypeptide further comprises a cytoplasmic domain, optionally linked directly or indirectly to said transmembrane domain. [The present invention 1062] 1061. A variant CD86 polypeptide of the invention, wherein said cytoplasmic domain is or comprises a native CD86 cytoplasmic domain. [The present invention 1063] 1061 or 1062, a variant CD86 polypeptide of the invention, wherein the cytoplasmic domain comprises the amino acid sequence set forth as residues 269 to 329 of SEQ ID NO:2, or a functional variant thereof exhibiting at least 85% sequence identity to residues 269 to 329 of SEQ ID NO:2. [The present invention 1064] 1061. A variant CD86 polypeptide of the invention, wherein said cytoplasmic domain comprises an ITAM signaling motif and / or is or comprises the intracellular signaling domain of CD3ζ. [This invention 1065] A variant CD86 polypeptide of invention 1059 or invention 1060, which does not comprise a cytoplasmic signaling domain and / or is incapable of mediating or modulating an intracellular signal when expressed on a cell. [The present invention 1066] An immunomodulatory protein comprising a first variant CD86 polypeptide of any of claims 1001 to 1058 and a second variant CD86 polypeptide of any of claims 1001 to 1058. [This invention 1067] The immunomodulatory protein of the present invention 1066, wherein said first and second variant CD86 polypeptides are indirectly linked via a linker. [The present invention 1068] The immunomodulatory protein of invention 1066 or invention 1067, wherein the first and second variant CD86 polypeptides are each linked to a multimerization domain, and the immunomodulatory protein is a multimer comprising the first and second variant CD86 polypeptides. [This invention 1069] The immunomodulatory protein of the present invention 1068, wherein said multimer is a dimer, optionally a homodimer. [The present invention 1070] 1069. The immunomodulatory protein of any of claims 1066 to 1069, wherein said first variant CD86 polypeptide and said second variant CD86 polypeptide are the same. [This invention 1071] An immunomodulatory protein comprising a variant CD86 polypeptide of any of claims 1001 to 1058 linked directly, or indirectly via a linker, to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of an IgSF family member. [This invention 1072] 1071. The immunomodulatory protein of the present invention, wherein said IgSF domain is an affinity-modified IgSF domain, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of an IgSF family member. [This invention 1073] 1072. The immunomodulatory protein of the present invention, wherein said IgSF domain is an affinity-modified IgSF domain, and wherein said affinity-modified IgSF domain exhibits altered binding to one or more of its cognate binding partners compared to the binding of an unmodified or wild-type IgSF domain of said IgSF family member to the same cognate binding partner or partners. [This invention 1074] 1073. An immunomodulatory protein of the invention, wherein said IgSF domain exhibits improved binding to one or more of its cognate binding partners compared to the binding of an unmodified or wild-type IgSF domain of said IgSF family member to the same cognate binding partner or partners. [This invention 1075] The immunomodulatory protein of any of claims 1071 to 1074, wherein the IgSF domain of the second polypeptide is a tumor-localizing moiety that binds to a ligand expressed on a tumor or that binds to a ligand expressed on a tumor, or an inflammation-localizing moiety that binds to a cell or tissue associated with an inflammatory environment. [This invention 1076] The immunomodulatory polypeptide of the present invention 1075, wherein said ligand is B7H6. [This invention 1077] The immunomodulatory polypeptide of the present invention 1075 or 1076, wherein the IgSF domain is derived from NKp30. [This invention 1078] 1078. The immunomodulatory protein of any of claims 1071 to 1077, further comprising a multimerization domain linked to at least one of said variant CD86 polypeptide or said second polypeptide. [This invention 1079] The immunomodulatory protein of any of claims 1071 to 1078, wherein the immunomodulatory protein further comprises a third polypeptide comprising an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, wherein the affinity-modified IgSF domain comprises one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of the IgSF family member. [The present invention 1080] the third polypeptide is the same as the first and / or second polypeptide; or the third polypeptide is different from the first and / or second polypeptide; The immunomodulatory protein of the present invention. [This invention 1081] The immunomodulatory protein of claim 1079 or claim 1080, further comprising a multimerization domain linked to at least one of said variant CD86 polypeptide, said second polypeptide, and / or said third polypeptide. [This invention 1082] The immunomodulatory protein of any of claims 1068 to 1070, 1078 and 1081, wherein the multimerization domain is an immunoglobulin Fc domain, and optionally, the immunoglobulin protein is human and / or the Fc domain is human. [This invention 1083] 1082. The immunomodulatory protein of the present invention, wherein said Fc domain is IgG1, IgG2, or IgG4, or a variant thereof having reduced effector function. [This invention 1084] 1083. The immunomodulatory protein of the present invention, wherein said Fc domain is an IgG1 Fc domain, optionally human IgG1, or a variant thereof having reduced effector function. [This invention 1085] The immunomodulatory protein of any of the present inventions 1082 to 1084, wherein the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:229 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:229. [The present invention 1086] The immunomodulatory protein of any one of 1082 to 1085, wherein the Fc domain is or comprises the amino acid sequence shown in SEQ ID NO:229. [This invention 1087] 1084 or 1085. An immunomodulatory protein of the invention, wherein the Fc domain is a variant IgG1 comprising one or more amino acid substitutions, wherein the one or more amino acid substitutions are selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, each of which are numbered according to the EU index as per Kabat. [This invention 1088] 1087. An immunomodulatory protein of the invention, wherein the Fc domain comprises the amino acid substitution N297G, the amino acid substitutions R292C / N297G / V302C, or the amino acid substitutions L234A / L235E / G237A, each of which are numbered according to the EU index of Kabat. [This invention 1089] 1087. The immunomodulatory protein of claim 1088, wherein said variant Fc domain further comprises the amino acid substitution C220S, said residues being numbered according to the EU index of Kabat. [The present invention 1090] 1087-1089. The immunomodulatory protein of any one of claims 1087 to 1089, wherein the Fc domain comprises K447del, said residues being numbered according to the EU index of Kabat. [This invention 1091] The immunomodulatory protein of any of the present inventions 1084, 1085 and 1087 to 1090, wherein the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:230 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:230 and includes one or more of the amino acid modifications set forth in SEQ ID NO:230 compared to human IgG1. [This invention 1092] The immunomodulatory protein of any one of 1084, 1085 and 1087 to 1091, wherein the Fc domain is or comprises the amino acid sequence shown in SEQ ID NO:230. [This invention 1093] An immunomodulatory protein comprising a first polypeptide and a second polypeptide, the first polypeptide comprises at least one IgSF domain linked to a first Fc domain via a linker, and the at least one IgSF domain comprises one or both of the following: a variant CD86 polypeptide of any of claims 1001 to 1046, or an IgSF domain of a PD1 polypeptide or a variant thereof; and the second polypeptide comprises at least one IgSF linked via a linker to a second Fc domain, and the at least one IgSF domain comprises one or both of the following: a variant CD86 polypeptide of any of claims 1001 to 1046, or an IgSF domain of a PD1 polypeptide or a variant thereof; the immunomodulatory protein comprises at least one IgSF domain of CD86 and at least one IgSF domain of PD-1 or a variant thereof; Immunomodulatory protein. [This invention 1094] 1093. The immunomodulatory protein of the present invention, wherein at least one IgSF domain of said first polypeptide comprises a variant CD86 polypeptide of any of the present inventions 1001-1046. [This invention 1095] 1093. The immunomodulatory protein of claim 1094, wherein at least one IgSF domain of said second polypeptide comprises a variant PD1 polypeptide. [This invention 1096] 1093 to 1095. The immunomodulatory protein of any of claims 1093 to 1095, wherein at least one IgSF domain of said first polypeptide is a first IgSF domain, said first IgSF domain being a variant CD86 polypeptide of any of claims 1001 to 1046, and said first polypeptide comprising a second IgSF domain linked to said first Fc domain via a linker. [This invention 1097] 1096. The immunomodulatory protein of the present invention, wherein the second IgSF domain of said first polypeptide comprises a variant PD1 polypeptide. [This invention 1098] 1093 to 1097. The immunomodulatory protein of any of claims 1093 to 1097, wherein at least one IgSF domain of said second polypeptide is a first IgSF domain, said first IgSF domain being a variant CD86 polypeptide of any of claims 1001 to 1046, and said second polypeptide comprising a second IgSF domain linked to said second Fc domain via a linker. [This invention 1099] 1098. The immunomodulatory protein of the present invention, wherein the second IgSF domain of said second polypeptide comprises a variant PD1 polypeptide. [The present invention 1100] at least one IgSF domain of the first polypeptide is linked to the N-terminus or C-terminus of the first Fc domain via a linker; and At least one IgSF domain of the second polypeptide is linked to the N-terminus or C-terminus of the second Fc domain via a linker. An immunomodulatory protein according to any one of 1093 to 1099 of the present invention. [The present invention 1101] An immunomodulatory protein of any of claims 1096 to 1097, wherein the second IgSF domain of the first polypeptide is linked to the end of the first Fc domain opposite to the end linked to the first IgSF domain. [The present invention 1102] An immunomodulatory protein of any of the present inventions 1098 to 1101, wherein the second IgSF domain of the second polypeptide is linked to the end of the second Fc domain opposite to the end linked to the first IgSF domain. [The present invention 1103] The immunomodulatory protein of any of claims 1093 to 1102, wherein the linker independently comprises the sequence of SEQ ID NO: 222 or 224, and optionally the linker comprises 1 to 4 repeats of the sequence of SEQ ID NO: 222 or 224. [The present invention 1104] The immunomodulatory protein of any of claims 1093 to 1103, wherein the first Fc domain and the second Fc domain are identical, and optionally, the first Fc domain and the second Fc domain comprise the sequence of SEQ ID NO:230. [This invention 1105] The immunomodulatory protein of any of claims 1093 to 1104, wherein the first polypeptide and the second polypeptide dimerize via the first and second Fc domains to form a homodimer. [The present invention 1106] The immunomodulatory protein of any of claims 1093 to 1104 and 1105, wherein the first and second polypeptides of the homodimer comprise, from left to right, variant PD1 polypeptide-linker-Fc-linker-variant CD86 polypeptide. [This invention 1107] The immunomodulatory protein of any of claims 1093 to 1104 and 1105 to 1106, wherein the variant PD1 polypeptide comprises the sequence of SEQ ID NO:315. [This invention 1108] The immunomodulatory protein of any of claims 1093-1104 and 1105-1107, wherein the variant CD86 polypeptide comprises the sequence of SEQ ID NO:94 or 150. [This invention 1109] The immunomodulatory protein of any of claims 1093 to 1104 and 1105 to 1108, wherein the first and second polypeptides of the homodimer comprise the sequence of SEQ ID NO: 348 or 349, respectively. [The present invention 1110] The immunomodulatory protein of any of claims 1093 to 1103, wherein the first Fc domain and the second Fc domain are different, and optionally the first and second Fc domains comprise knob-into-hole mutations, and optionally the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO:346, and the other of the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO:347. [The present invention 1111] The immunomodulatory protein of any of 1093 to 1103 and 1110, wherein the first polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a heterodimer. [The present invention 1112] 1093-1103, 1110, and 1111. The immunomodulatory protein of any of claims 1093-1103, 1110, and 1111, wherein the first polypeptide of the heterodimer comprises, from left to right, a variant PD1 polypeptide-linker-Fc, and the second polypeptide of the heterodimer comprises, from left to right, a variant CD86 polypeptide-linker-Fc, an Fc-linker-variant CD86 polypeptide, or a variant PD1-linker-Fc-linker-variant CD86. [The present invention 1113] The immunomodulatory protein of any of claims 1093 to 1103, 1110, and 1111 to 1112, wherein the variant PD1 polypeptide comprises the sequence of SEQ ID NO:315. [This invention 1114] The immunomodulatory protein of any of claims 1093-1103, 1110, and 1111-1113, wherein the variant CD86 polypeptide comprises the sequence of SEQ ID NO:94 or 150. [This invention 1115] the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO:350; and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 351, 352, or 353; An immunomodulatory protein according to any one of 1093 to 1103, 1110, and 1111 to 1114 of the present invention. [The present invention 1116] A conjugate comprising a variant CD86 polypeptide of any of claims 1001 to 1065 linked to a targeting moiety that specifically binds to a molecule on the surface of a cell. [This invention 1117] The conjugate of the present invention 1116, wherein said cell is an immune cell or a tumor cell. [This invention 1118] The conjugate of claim 1116 or claim 1117, wherein said moiety is a protein, peptide, nucleic acid, small molecule or nanoparticle. [This invention 1119] 1118. The conjugate of any of claims 1116 to 1118, wherein said moiety is an antibody or an antigen-binding fragment. [The present invention 1120] The variant CD86 polypeptide is a V H or V L A conjugate of the present invention 1119, wherein the conjugate is linked to the N-terminus or C-terminus of [This invention 1121] The conjugate of the present invention 1119, wherein the antibody is an anti-HER2 antibody or an anti-EGFR antibody. [This invention 1122] The conjugate of the present invention 1121, wherein said anti-HER2 antibody is pertuzumab. [This invention 1123] The variant CD86 polypeptide is V of pertuzumab H N-terminus of pertuzumab, V H C-terminus of pertuzumab L or the V of pertuzumab L and optionally comprising the sequence of SEQ ID NO: 342, 344, 343, or 345, respectively. [This invention 1124] The conjugate of the present invention 1121, wherein the anti-EGFR antibody is panitumumab. [This invention 1125] The variant CD86 polypeptide is the V H N-terminus of panitumumab, V H C-terminus of panitumumab, V L or the V of panitumumab Land optionally comprising the sequence of SEQ ID NO: 348, 350, 349, or 351, respectively. Or an anti-EGFR antibody. [Invention 1126] A conjugate according to any one of claims 1116 to 1125, which is a fusion protein. [This invention 1127] A nucleic acid molecule encoding a conjugate which is a variant CD86 polypeptide of any of claims 1001 to 1065, an immunomodulatory protein of any of claims 1066 to 1115, or a fusion protein of any of claims 1116 to 1126. [This invention 1128] A vector comprising the nucleic acid molecule of the present invention. [This invention 1129] A cell comprising the vector of the present invention. [The present invention 1130] A method for producing a protein comprising a variant CD86 polypeptide, the method comprising the step of introducing a nucleic acid molecule of the invention 1127 or a vector of the invention 1128 into a host cell under conditions such that the protein is expressed in the host cell. [This invention 1131] 1130. The method of claim 1130, further comprising the step of isolating or purifying said protein from said cells. [This invention 1132] 1. A method for modifying a cell to express a variant CD86 polypeptide, the method comprising the step of introducing into a host cell a nucleic acid molecule encoding a conjugate that is a variant CD86 polypeptide of any of claims 1001 to 1065, an immunomodulatory protein of any of claims 1066 to 1115, or a fusion protein of any of claims 1116 to 1126, under conditions such that the polypeptide is expressed in the host cell. [This invention 1133] A modified cell comprising a variant CD86 polypeptide of any of claims 1001 to 1065, an immunomodulatory protein of any of claims 1066 to 1115, or a conjugate that is a fusion protein of any of claims 1116 to 1126, a nucleic acid molecule of claim 1127, or a vector of claim 1128. [This invention 1134] the variant CD86 polypeptide comprises a transmembrane domain or is a transmembrane immunomodulatory protein of any of claims 1059 to 1065 of the present invention; and / or a protein comprising the variant CD86 polypeptide is expressed on the surface of the cell. The modified cells of the present invention 1133. [This invention 1135] the variant CD86 polypeptide does not comprise a transmembrane domain and / or is not expressed on the surface of the cell; and / or the variant CD86 polypeptide is capable of being secreted from the modified cell; The modified cells of the present invention 1133. [This invention 1136] The modified cell of any one of 1133 to 1135 of the present invention, which is an immune cell. [This invention 1137] 1136. The modified cell of claim 1136, wherein said immune cell is a lymphocyte, and optionally said lymphocyte is a T cell. [This invention 1138] The modified cell of any one of 1133 to 1137 of the present invention, which is a primary cell. [This invention 1139] The modified cell of any of claims 1133 to 1138, further comprising a chimeric antigen receptor (CAR). [This invention 1140] The modified cell of any of claims 1133 to 1139, further comprising a modified T cell receptor (TCR). [This invention 1141] An infectious agent comprising a variant CD86 polypeptide of any one of claims 1001 to 1065, an immunomodulatory protein of any one of claims 1066 to 1115, or a conjugate which is a fusion protein of any one of claims 1116 to 1126, a nucleic acid molecule of claim 1127, or a vector of claim 1128. [This invention 1142] The infectious agent of this invention 1141 which is a bacterium or a virus. [This invention 1143] The infectious agent of claim 1142, wherein the infectious agent is a virus, and the virus is an oncolytic virus. [This invention 1144] A pharmaceutical composition comprising a variant CD86 polypeptide of any of claims 1001 to 1065, an immunomodulatory protein of any of claims 1066 to 1115, or a conjugate which is a fusion protein of any of claims 1116 to 1126, a modified cell of any of claims 1133 to 1140, or an infectious agent of any of claims 1141 to 1143. [Invention 1145] A pharmaceutical composition of the present invention 1144, comprising a pharmaceutically acceptable excipient. [Invention 1146] An article of manufacture comprising any one of the pharmaceutical compositions of the present inventions 1144 to 1145 in a vial or container. [This invention 1147] A kit comprising the pharmaceutical composition of any one of 1144 to 1145 or the article of manufacture of 1146 and instructions for use. [This invention 1148] A method of modulating an immune response in a subject, the method comprising administering a variant CD86 polypeptide of any of claims 1001 to 1065, an immunomodulatory protein of any of claims 1066 to 1115, or a conjugate which is a fusion protein of any of claims 1116 to 1126, a modified cell of any of claims 1133 to 1140, an infectious agent of any of claims 1141 to 1143, or a pharmaceutical composition of any of claims 1144 to 1145. [This invention 1149] A method for regulating an immune response in a subject, the method comprising the step of administering a modified cell of any of the present inventions 1133 to 1140. [This invention 1150] 1149. The method of claim 1149, wherein said modified cells are autologous to said subject. [This invention 1151] 1149. The method of claim 1149, wherein said modified cells are allogeneic to said subject. [This invention 1152] 1152. The method of any of claims 1148 to 1151, wherein modulating said immune response treats a disease or condition in said subject. [This invention 1153] A method of treating a disease or condition in a subject in need thereof, comprising the step of administering a variant CD86 polypeptide of any of inventions 1001-1065, an immunomodulatory protein of any of inventions 1066-1115, or a conjugate which is a fusion protein of any of inventions 1116-1126, a modified cell of any of inventions 1133-1140, an infectious agent of any of inventions 1141-1143, or a pharmaceutical composition of any of inventions 1144-1145. [This invention 1154] A method for treating a disease or condition in a subject in need thereof, the method comprising the step of administering a modified cell of any of the present inventions 1133 to 1140. [This invention 1155] 115. The method of claim 1154, wherein said modified cells are autologous to said subject. [Invention 1156] 115. The method of claim 1154, wherein said modified cells are allogeneic to said subject. [This invention 1157] The method of any of claims 1148 to 1156, wherein the immune response is enhanced in said subject. [This invention 1158] The method of any of claims 1148, 1152, 1153 and 1157, wherein an immunomodulatory protein or conjugate comprising a variant CD86 polypeptide linked to a tumor-localizing moiety is administered to said subject. [This invention 1159] 1158. The method of claim 1158, wherein said tumor-localizing moiety is or comprises a binding molecule that recognizes a tumor antigen. [The present invention 1160] the binding molecule comprises an antibody or antigen-binding fragment thereof or a wild-type IgSF domain or a variant thereof, optionally comprising an anti-HER2 antibody or antigen-binding fragment or an anti-EGFR antibody or antigen-binding fragment; or the binding molecule comprises an IgSF domain of an IgSF member that binds to a tumor antigen, or a specific binding fragment thereof, and optionally the IgSF domain is the IgSF domain of PD-1 or Nkp30. The method of the present invention 1159. [This invention 1161] The method of any of claims 1148 and 1152 to 1160, wherein a pharmaceutical composition comprising an immunomodulatory protein of any of claims 1071 to 1115 or a conjugate of any of claims 1116 to 1126 is administered to the subject. [This invention 1162] The method of any of claims 1148 to 1160, wherein modified cells comprising a variant CD86 polypeptide that is a transmembrane immunomodulatory protein are administered to said subject, and optionally the modified cells are those of claims 1133, 1134 and 1136 to 1140. [This invention 1163] The method according to any one of claims 1152 to 1162, wherein the disease or condition is a tumor or cancer. [Invention 1164] 1164. The method of any of claims 1152 to 1163, wherein said disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colon cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer. [Invention 1165] The method of any of claims 1148 to 1156, wherein the immune response is reduced. [Invention 1166] The method of any of claims 1148, 1152, 1153 and 1165, wherein a variant CD86 polypeptide or immunomodulatory protein that is soluble is administered to said subject. [This invention 1167] 1166. The method of claim 1166, wherein said soluble polypeptide or immunomodulatory protein is an Fc fusion protein. [Invention 1168] The method of any of claims 1148, 1152, 1153 and 1165-1167, wherein a pharmaceutical composition comprising a variant CD86 polypeptide of any of claims 1001-1058, or an immunomodulatory protein of any of claims 1066-1074 and 1078-1115, is administered to said subject. [This invention 1169] The method of any of claims 1148, 1152, 1153 and 1165, wherein modified cells comprising a secretable variant CD86 polypeptide are administered to the subject, and optionally the modified cells are any of claims 1133 and 1135-1140. [This invention 1170] The method according to any one of claims 1148, 1152, 1153 and 1165 to 1169, wherein said disease or condition is an inflammatory or autoimmune disease or condition. [This invention 1171] Any of the methods of the present invention 1148, 1152, 1153 and 1165 to 1169, wherein the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. [This invention 1172] 1172. The method of claim 1170 or 1171, wherein said disease or condition is selected from inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. [Brief explanation of the drawings]

[0056] [Figure 1-1] Figure 1A shows the release of IFN-γ (IFNγ; top left), IL2 (top right), and TNFα (bottom) in the supernatants from mock-transduced T cells and E6 TCR-transduced T cells expressing the TCR alone or coexpressing the indicated CD86 ECD TIP after 24 h of coculture with various numbers of HLA-A2+ HPV+ target cells (SCC152). [Figure 1-2] Figures 1B and 1C show the proliferation of CD4+ and CD8+ T cells, respectively, 3 days after the initiation of coculture of mock-transduced T cells or E6 TCR-transduced T cells expressing the TCR alone or co-expressing the indicated CD86 ECD TIP with various numbers of HLA-A2+ HPV+ target cells (SCC152). [Figure 1-3] Figure 1D shows the killing activity of mock-transduced T cells and E6 TCR-transduced T cells expressing the TCR alone or coexpressing the indicated CD86 ECD TIP after 4 days of coculture with HLA-A2+ HPV+ target cells (SCC152) at various effector:target ratios (E:T). [Figure 2-1] FIG. 2A illustrates HER2 expression levels on CEM-T2, SCC152, and NCI-N87 cell lines. [Figure 2-2]Figure 2B shows the killing activity of mock-transduced T cells and anti-HER2 CAR-transduced T cells expressing a CAR alone or co-expressing the indicated CD86 ECD TIP after 24 hours of coculture with NCI-N87 at various effector:target ratios (E:T). Figure 2C shows the killing activity of mock-transduced T cells and anti-HER2 CAR-transduced T cells expressing a CAR alone or co-expressing the indicated CD86 ECD TIP after 24 hours of coculture with SCC152 at various effector:target ratios (E:T). [Figure 3] Figure 3 illustrates an exemplary alignment of the wild-type CD86 extracellular domain (ECD) sequence shown in SEQ ID NO:29, containing residues 24-247 of CD86, designated "CD86(B7-2)" (SEQ ID NO:2), with the wild-type IgV sequence shown in SEQ ID NO:122, containing residues 33-131 of CD86, designated "CD86(B7-2)" (SEQ ID NO:2). The symbol "*" indicates that two aligned residues are identical. The absence of an "*" between two aligned residues indicates that the aligned amino acids are not identical. The symbol "-" indicates a gap in the alignment. Exemplary, non-limiting positions in SEQ ID NO:122 corresponding to the numbered positions shown in SEQ ID NO:29 are indicated by boxes. [Figure 4A] Figures 4A and 4B illustrate the binding of various concentrations (0.1 nM to 100 nM) of exemplary PD1-CD86 stack constructs to their cognate binding partner CTLA-4, as determined by mean fluorescence intensity (MFI) assessed by flow cytometry. [Figure 4B] See legend to Figure 4A. [Figure 5A] Figures 5A and 5B illustrate the binding of various concentrations (0.1 nM to 100 nM) of exemplary PD1-CD86 stack constructs to their cognate binding partner CD28, as determined by mean fluorescence intensity (MFI) assessed by flow cytometry. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figures 6A and 6B illustrate the binding of various concentrations (0.1 nM to 100 nM) of exemplary PD1-CD86 stack constructs to their cognate binding partner PD-L1, as determined by mean fluorescence intensity (MFI) assessed by flow cytometry. [Figure 6B] See legend to Figure 6A. [Figure 7A] Figure 7A illustrates the ability of exemplary variant PD1-CD86 stack constructs to deliver PD-L1-dependent CD28 costimulation, as measured by relative luminescence units (RLU) of IL-2 luminescence, using Jurkat / IL-2 reporter cells. [Figure 7B] Figure 7B illustrates the ability of exemplary variant PD1-CD86 stack constructs to deliver PD-L1-dependent CD28 costimulation using PD-L1-expressing Jurkat / IL-2 reporter cells, as measured by relative luminescence units (RLU) of IL-2. [Figure 8] Figure 8 illustrates cytokine concentrations (pg / mL) of T cell supernatants from a cytomegalovirus (CMV) antigen-specific functional assay. Supernatants were determined for IL-2 as assessed by ELISA. [Figure 9] Figure 9 illustrates cytokine concentrations (pg / mL) in T cell supernatants from a cytomegalovirus (CMV) antigen-specific functional assay. Supernatants were determined for IFNg as assessed by ELISA. [Figure 10] FIG. 10 illustrates the binding of exemplary NKp30-CD86 stack constructs to CD28 and CTLA-4 at various concentrations (100-100,000 pM), determined as the median hIgG PE. [Figure 11A] FIG. 11A illustrates the binding ability of exemplary NKp30-CD86 stack constructs to primary T cells as determined by mean fluorescence intensity (MFI) assessed by flow cytometry. [Figure 11B]FIG. 11B shows the rate of T cell proliferation assessed by flow cytometry using CFSE dye. [Figure 12] FIG. 12 illustrates the concentration (pg / mL) of IL-2 harvested from T cell supernatants as assessed by ELISA. [Figure 13] Figure 13 illustrates exemplary NKp30-CD86 stack construct costimulation in the presence (left) and absence (right) of B7H6. T cell proliferation rate assessed by flow cytometry. [Figure 14] 14A-14D illustrate the structure of an exemplary formatted stack construct. [Figure 15] Figures 15A and 15B illustrate the binding of exemplary formatted stack constructs at various concentrations (8 serial 1:4 dilutions of 100 nM) to their cognate binding partners PD-L1 (left) and CD28 (right) as assessed by flow cytometry and measured by mean fluorescence intensity (MFI). [Figure 16] 16A and 16B illustrate the costimulatory potential of exemplary formatted stack constructs tested in a luciferase reporter cell line and determined using relative luminescence units (RLU). [Figure 17A] Figure 17A illustrates the ability of exemplary CD86-PD-1 stack constructs to promote cytokine production in T cells, as measured by IFNg, IL2, and TNFa concentrations (pg / mL). [Figure 17B] Figure 17B illustrates the ability of exemplary CD86-PD-1 stack constructs to promote T cell cytotoxic activity against HLA-A2+ HPV+ target cells at 24, 48, and 72 hours after incubation, as assessed by relative luminescence units (RLU). [Figure 18-1] Figures 18A, 18B, and 18C illustrate exemplary geometries of conjugates of exemplary variant CD86 IgV molecules with antibodies targeting HER2 and EGFR. [Figure 18-2]See description of Figure 18-1. [Figure 19] Figure 19 illustrates the binding of an exemplary pertuzumab-CD86 conjugate to HER2 (Figure 19A) and an exemplary panitumumab-CD86 conjugate to EGFR (Figure 19B) as determined by mean fluorescence intensity. [Figure 20] Figure 20 illustrates the ability of pertuzumab-CD86 conjugates (Figure 20A) and exemplary panitumumab-CD86 conjugates (Figure 20B) to provide costimulation to T cells in an IL-2 luciferase reporter assay as measured in relative luminescence units (RLU). [Figure 21] Figure 21 illustrates the ability of an exemplary pertuzumab-CD86 conjugate (Figure 21A) and an exemplary panitumumab-CD86 conjugate (Figure 21B) to promote T cell cytotoxic activity when tested at various effector:target ratios (E:T) of primary human T cells, as measured by the killing rate of SCC-152 target cells. [Figure 22A] Figure 22A illustrates the ability of pertuzumab-CD86 conjugates to promote cytokine production in T cells by determining the concentrations of IFNg, IL2 and TNFa (nM protein) in cell supernatants. [Figure 22B] Figure 22B illustrates the ability of exemplary panitumumab-CD86 conjugates to promote cytokine production in T cells by determining the concentrations of IFNg, IL2, and TNFa (nM protein) in cell supernatants. [Figure 23A] FIG. 23A illustrates various exemplary shapes of stacked molecules containing a first variant IgSF domain (first vIgD) and a second IgSF domain, e.g., a second variant IgSF domain (second vIgD). [Figure 23B]Figure 23B illustrates various exemplary shapes of stacked molecules containing a first variant IgSF domain (first vIgD), a second IgSF domain, e.g., a second variant IgSF domain (second vIgD), and a third IgSF domain, e.g., a third variant IgSF domain (third vIgD). [Figure 24A] Figure 24 illustrates various formats of the variant IgSF domain molecules provided. Figure 24A illustrates a soluble molecule. [Figure 24B] FIG. 24B illustrates a transmembrane immunomodulatory protein (TIP) containing a variant IgSF domain (vIgD) expressed on the surface of a cell. [Figure 25] Figure 25 illustrates a secreted immunomodulatory protein (SIP) in which a variant IgSF domain (vIgD) is secreted from a cell, such as a first T cell (e.g., a CAR T cell). DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description Provided herein are immunomodulatory proteins that are or contain variants or mutants of CD86 and specific-binding fragments thereof, which exhibit altered binding activity or affinity for at least one target ligand cognate binding partner (also referred to as a counter-structure ligand protein). In some embodiments, the variant CD86 polypeptides contain one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) compared to unmodified or wild-type CD86 polypeptides. In some embodiments, the variant CD86 polypeptides contain one or more amino acid modifications (e.g., substitutions) compared to unmodified or wild-type CD86 polypeptides. In some embodiments, the one or more amino acid substitutions are in the extracellular domain of unmodified or wild-type CD86 polypeptides, for example, in the IgSF domain (e.g., IgV of IgC). In some embodiments, the variant CD86 polypeptides exhibit altered (e.g., improved or decreased) binding activity or affinity for one or more of CD28 or CTLA-4 compared to unmodified or wild-type CD86 that does not contain the one or more modifications.

[0058] In some embodiments, the variant CD86 polypeptides exhibit improved binding affinity to CD28 compared to unmodified or wild-type CD86 that does not contain one or more modifications. In some embodiments, the variant CD86 polypeptides exhibit at least improved binding affinity to CD28 compared to unmodified or wild-type CD86 that does not contain one or more modifications. In some embodiments, the binding affinity is altered (e.g., improved) by at least 1.2-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, 20.0-fold, 30.0-fold, 40.0-fold, 50.0-fold, 60.0-fold, 70.0-fold, 80.0-fold, 90.0-fold, 100.0-fold, 124.0-fold, or more, compared to unmodified or wild-type CD86 that does not contain one or more modifications.

[0059] In some embodiments, the variant CD86 polypeptide exhibits a binding affinity for CTLA-4 that is reduced, unchanged, or no greater than that of unmodified or wild-type CD86 that does not contain one or more modifications. In some embodiments, the binding affinity for CTLA-4 is reduced. In some embodiments, the binding affinity is altered (e.g., decreased) by at least 1.2-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, or more, compared to unmodified or wild-type CD86 that does not contain one or more modifications.

[0060] In some embodiments, variant CD86 polypeptides and immunomodulatory proteins modulate immunological immune responses, e.g., enhance or reduce immune responses. The specific modulation can be based on the format of the variant CD86 polypeptide, depending on whether the particular format provides antagonistic, blocking, or agonistic activity. Various immunomodulatory protein formats of the provided variant polypeptides are also provided. As demonstrated herein, selectable formats can facilitate manipulation of immune responses and, therefore, therapeutic applications. The ability to format variant polypeptides into various shapes to antagonize or agonize immune responses, depending on the situation, provides flexibility in therapeutic applications based on the similarly improved binding and activity of variant CD86 to binding partners. As one example, tethering a variant CD86 protein to a surface can deliver a localized costimulatory signal, while in other cases, presenting CD86 in a non-localized, soluble form can confer antagonistic activity. In some embodiments, the variant CD86 polypeptides and immunomodulatory proteins provided herein can be used to treat diseases or conditions associated with a dysregulated immune response.

[0061] In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is a transmembrane immunomodulatory protein that can be expressed on the surface of a cell. In some embodiments, the immunomodulatory protein is a secretable immunomodulatory protein that can be secreted from the cell that expresses it. In some embodiments, also provided herein are one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD86 polypeptide provided herein and one or more other moieties or polypeptides. In some aspects, modified cells are provided that contain a transmembrane immunomodulatory protein or a secretable immunomodulatory protein. In some aspects, infectious agents are provided that can deliver a transmembrane immunomodulatory protein or a secretable immunomodulatory protein to a cell (that the infectious agent infects) for expression. In some embodiments, also provided herein are one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD86 polypeptide provided herein and one or more other moieties or polypeptides.

[0062] In some embodiments, variant CD86 polypeptides are provided in formats that exhibit agonistic activity of their cognate binding partner, CD28, and / or stimulate or initiate costimulatory signaling through CD28. Included among such immunomodulatory protein formats are engineered cells that express the variant CD86 polypeptide as a transmembrane immunomodulatory protein. In other cases, immunomodulatory formats can include fusions with other molecules, such as those provided by certain "stack molecules" with other IgSF domains, including tumor-localization domains (e.g., vCD86-NkP30 constructs), as well as certain "stack molecules" with antibody conjugate formats (e.g., vCD86-anti-HER2 or vCD86-anti-HER1 constructs). Such variant CD86 immunomodulatory proteins and formats (e.g., engineered cells or fusion constructs) can be used to treat cancer, viral infections, or bacterial infections. In some embodiments, variant CD86 immunomodulatory proteins and formats thereof (e.g., modified cells or fusion constructs) exhibit enhanced costimulatory activity, thereby resulting in enhanced T cell activity (e.g., in vivo or in vitro) compared to wild-type or unmodified CD86 controls, e.g., in primary T cell assays. In some aspects, T cell activity can be assessed by assessing the production of cytokines such as IL-2, IFN-γ, or TNFα.In some aspects, the increase (e.g., increase in IFN-γ, IL-2, or TNFα) can be greater than or about 1.1 fold, greater than or about 1.1 fold, greater than or about 1.2 fold, greater than or about 1.2 fold, greater than or about 1.3 fold, greater than or about 1.3 fold, greater than or about 1.4 fold, greater than or about 1.4 fold, greater than or about 1.5 fold, greater than or about 1.5 fold, greater than or about 1.6 fold, greater than or about 1.6 fold, greater than or about 1.7 fold, greater than or about 1.7 fold, greater than or about 1.8 fold, greater than or about 1.8 fold, or even greater than 1.9 fold, as compared to unmodified or wild-type CD86 that does not contain one or more modifications. or an increase of about 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 5.0-fold or more, 6.0-fold or more, 7.0-fold or more, 8.0-fold or more, 9.0-fold or more, 10.0-fold or more, or more than 10.0-fold.

[0063] In some embodiments, variant CD86 polypeptides are provided in formats that exhibit antagonist activity of its cognate binding partner, CD28, and / or block or inhibit CD28-mediated costimulatory signaling. Among such immunomodulatory protein formats are soluble variant CD86 polypeptides (e.g., variant CD86-Fc fusion proteins). Such variant CD86 immunomodulatory proteins can be used to treat inflammatory or autoimmune disorders. In some embodiments, variant CD86 immunomodulatory proteins and formats thereof (e.g., soluble variant CD86-Fc fusion proteins) inhibit or block costimulatory signaling, thereby resulting in reduced T cell activity (e.g., in vivo or in vitro) compared to wild-type or unmodified CD86 controls, e.g., in primary T cell assays. In some aspects, T cell activity can be assessed by assessing the production of cytokines such as IL-2, IFN-γ, or TNFα. In some aspects, the decrease, e.g., decrease in IFN-γ, IL-2, TNFα, is greater than or about 1.1 fold, greater than or about 1.1 fold, greater than or about 1.2 fold, greater than or about 1.2 fold, greater than or about 1.3 fold, greater than or about 1.3 fold, greater than or about 1.4 fold, greater than or about 1.4 fold, greater than or about 1.5 fold, greater than or about 1.6 fold, greater than or about 1.6 fold, greater than or about 1.7 fold, greater than or about 1.7 fold, greater than or about 1.8 fold, or greater than or is a decrease of about 1.8 fold or more, 1.9 fold or more, 2.0 fold or more, 3.0 fold or more, 4.0 fold or more, 5.0 fold or more, 6.0 fold or more, 7.0 fold or more, 8.0 fold or more, 9.0 fold or more, 10.0 fold or more, or more than 10.0 fold.

[0064] In some embodiments, the provided variant CD86 polypeptides regulate T cell activation, expansion, differentiation, and survival through interaction with costimulatory signaling molecules. In general, antigen-specific T cell activation generally requires two distinct signals. The first signal is provided by the interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC)-associated antigens present on antigen-presenting cells (APCs). The second signal is a costimulatory signal for TCR engagement (e.g., a CD28 costimulatory signal) and is required to avoid T cell apoptosis or anergy.

[0065] In some embodiments, under normal physiological conditions, T cell-mediated immune response is initiated by antigen recognition by T cell receptor (TCR), and is regulated by the balance of costimulatory signal and inhibitory signal (e.g., immune checkpoint protein).The immune system relies on immune checkpoint to prevent autoimmunity (i.e., self-tolerance) and protect tissue from excessive damage during immune response (e.g., during attack against pathogenic infection).However, in some cases, these immune-regulating proteins may be dysregulated in diseases and pathologies, including tumors, as a mechanism to evade the immune system.

[0066] In some embodiments, among the known T cell costimulatory receptors is CD28, which is a T cell costimulatory receptor for the ligands B7-1 (CD80) and B7-2 (CD86), both of which are present on APCs. These same ligands can also bind to the inhibitory T cell receptor CTLA4 (cytotoxic T lymphocyte-associated protein 4) with higher affinity than to CD28; binding to CTLA-4 acts to downregulate the immune response.

[0067] Enhancement or suppression of the activity of CD28 receptor and CTLA-4 receptor has clinical significance for the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, therapies that intervene in and alter the costimulatory effect of both receptors are constrained by the spatial location requirements and size limitations imposed by the localization of the immune synapse. In some cases, existing therapeutic agents, including antibody drugs, cannot simultaneously interact with multiple target proteins involved in regulating these interactions. In addition, in some cases, existing therapeutic agents only have the ability to antagonize immune responses, but cannot stimulate immune responses. Additionally, the pharmacokinetic differences between drugs that independently target one or the other of these two receptors can make it difficult to adequately maintain the desired blood concentration of such drug combinations throughout the treatment process. The variant CD86 polypeptides and immunomodulatory proteins provided, as well as other formats described, address such problems. Also provided are methods of making and using these CD86 variant polypeptides and immunomodulatory proteins.

[0068] 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.

[0069] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0070] 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.

[0071] 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.

[0072] The term "affinity-modified (affinity-engineered)," when used in the context of immunoglobulin superfamily domains, refers to a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence that has been altered to improve or decrease (relative to the corresponding wild-type parent or unmodified IgSF domain) the binding affinity or avidity for at least one of its cognate binding partners (or "counterstructures") relative to a parent wild-type or unmodified (i.e., non-affinity-modified) IgSF control domain. Included in this context are affinity-modified CD86 IgSF domains. In some embodiments, an affinity-modified IgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences (e.g., amino acid substitutions) in a wild-type or unmodified IgSF domain. The improvement or decrease in 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, Vol 1(9): 793-801 (1994). An improvement in the 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, is 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 the protein for at least one of its cognate binding partners is a reduction of 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments, a reduction of 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 requirement on any particular starting composition or method by which the affinity-modified IgSF domain is generated. Thus, the affinity-modified IgSF domains of the present invention are not limited to converting a wild-type IgSF domain into an affinity-modified IgSF domain 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 their cognate binding partners, and finally recombinantly or chemically synthesized to generate the subject affinity-modified IgSF domain compositions. As 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, including recombinant methods, chemical synthesis, or a combination thereof, may be used.

[0073] The term "allogeneic," as used herein, refers to cells or tissues that are removed from one organism and then injected or adoptively transferred into a genetically different organism of the same species. In some embodiments of the invention, the species is murine or human.

[0074] The term "autologous" as used herein refers to cells or tissues that are extracted from the same organism and then injected or adoptively transferred into the organism. Autologous cells or tissues can be modified, for example, by recombinant DNA techniques, so that they are no longer genetically identical to the natural cells or natural tissues extracted from the organism. For example, natural autologous T cells can be genetically modified by recombinant DNA techniques to become autologous modified cells that express transmembrane immunomodulatory proteins and / or chimeric antigen receptors (CARs), which in some cases involves modifying T cells or TILs (tumor-infiltrating lymphocytes). The modified cells are then infused into the patient from which the natural T cells were isolated. In some embodiments, the organism is a human or a mouse.

[0075] 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 counterstructure under specific binding conditions, respectively. In biochemical kinetics, avidity refers to the cumulative strength of the affinities of multiple individual non-covalent interactions, such as between CD86 and its counterstructures CD28 and / or CTLA-4. Thus, avidity differs from affinity, which represents the strength of a single interaction. The improved or decreased binding affinity of an affinity-modified CD86 variant containing an IgSF domain to its counterstructure is determined compared to the binding affinity of an unmodified CD86 (e.g., an unmodified CD86 containing a native or wild-type IgSF domain (e.g., an IgV domain)). 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). In some embodiments, the variant CD86 (e.g., a CD86 containing an affinity-modified IgSF domain) specifically binds to CD28 and / or CTLA-4 with a binding affinity that results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater in a binding assay than an unmodified CD86 control, as measured by flow cytometry. In some embodiments, the variant CD86 (e.g., one containing an affinity-engineered IgSF domain) specifically binds to CD28 with a binding affinity that results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than an unmodified CD86 control in a binding assay, as measured by flow cytometry, and exhibits a binding affinity for CTLA-4 that is unchanged or no greater than an unmodified CD86 control in a binding assay.In some embodiments, the variant CD86 (e.g., one containing an affinity-modified IgSF domain) specifically binds to CD28 with a binding affinity that results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than an unmodified CD86 control in a binding assay, as measured by flow cytometry, and exhibits reduced binding affinity for CTLA-4 as measured by flow cytometry, wherein the binding affinity results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than the unmodified CD86 control in the binding assay compared to the unmodified CD86 control in the binding assay.

[0076] The term "biological half-life" refers to the time required for a substance (e.g., an immunomodulatory polypeptide containing a variant CD86 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 degradation), or absorption and concentration in specific organs or tissues in the body. In some embodiments, biological half-life can be assessed by determining the time required 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 extend 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 WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

[0077] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an artificial (i.e., man-made) transmembrane protein expressed on mammalian cells, containing at least an ectodomain, a transmembrane domain, and an endodomain. Optionally, the CAR protein includes a "spacer" that covalently links the ectodomain to the transmembrane domain. The spacer is often a polypeptide that links the ectodomain to the transmembrane domain via a peptide bond. CARs are typically expressed on mammalian lymphocytes. In some embodiments, the CAR is expressed on mammalian cells such as T cells or tumor-infiltrating lymphocytes (TILs). CARs expressed on T cells are referred to herein as "CAR T cells" or "CAR-T." In some embodiments, the CAR-T is a helper T cell, a cytotoxic T cell, a natural killer T cell, a memory T cell, a regulatory T cell, or a γδ T cell. For example, when used clinically in adoptive cell transfer, CAR-Ts with antigen-binding specificity for a patient's tumor are typically engineered to express on natural T cells obtained from the patient. The engineered T cells expressing the CAR are then infused back into the patient. Thus, while CAR-Ts are often autologous CAR-Ts, allogeneic CAR-Ts are also within the scope of the present invention. The ectodomain of the CAR contains an antigen-binding region (e.g., an antibody or its antigen-binding fragment (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Upon specific binding, a series of biochemical events (i.e., signal transduction) leads to modulation of the immune activity of the CAR-T. Thus, for example, specific binding of the antigen-binding region of the CAR-T to its target antigen can lead to changes in the immune activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction upon CAR-T activation is achieved by the CD3 zeta chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells. CAR-Ts can further contain multiple signaling domains (e.g., CD28, 4-1BB, or OX40) that further modulate the immunoregulatory response of T cells.CD3-z contains a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM) that is involved in T cell receptor signaling.

[0078] The terms "collectively" or "total," when used in reference to cytokine production induced by the presence of two or more variant CD86 polypeptides in an in vitro assay, refer to the overall cytokine expression level regardless of cytokine production induced by individual variant CD86 polypeptides. In some embodiments, the cytokine assayed is IFN-γ or IL-2 in an in vitro primary T cell assay.

[0079] The term "cognate binding partner" (used interchangeably with "counterstructure"), with respect to a polypeptide (e.g., an IgSF domain of a variant CD86), refers to at least one molecule (typically a native mammalian protein) to which the referenced polypeptide specifically binds under specific binding conditions. In some aspects, a variant CD86 containing an affinity-modified IgSF domain specifically binds to the counterstructure of the corresponding native or wild-type CD86 with improved or reduced affinity. A species of ligand that is recognized and specifically binds to its cognate receptor under specific binding conditions is an example of a counterstructure or cognate binding partner for that receptor. A "cell surface cognate binding partner" is a cognate binding partner expressed on the surface of a mammalian cell. A "cell surface molecular species" is a cognate binding partner of a ligand of the immune synapse (IS) that is expressed on or by cells (e.g., mammalian cells) that form the IS.

[0080] As used herein, "conjugate," "conjugation," or grammatical variations thereof, refers to the connection or linking of two or more compounds together by any connection or linking method known in the art, resulting in the formation of another compound. It can also refer to a compound produced by connecting or linking two or more compounds together. For example, a variant CD86 polypeptide directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by any other method.

[0081] 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 the same 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).

[0082] 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.

[0083] The term "corresponding" 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 the sequence listing), refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm (e.g., the GAP algorithm). For example, corresponding residues can be identified by alignment with a reference sequence having the sequence of wild-type CD86 (ECD domain) shown in SEQ ID NO:29 using the structural alignment methods described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as criteria. Figure 3 illustrates a sequence alignment with the reference sequence shown in SEQ ID NO:29 to identify corresponding residues. For example, in the exemplary alignment shown in Figure 3, the 13th residue of SEQ ID NO:29 corresponds to the 4th residue of SEQ ID NO:122.

[0084] The terms "reduce" or "reducing" or "attenuating" or "inhibiting," as used herein, refer to a statistically significant decrease or reduction. The decrease or reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction.

[0085] The term "derivative" or "derivatized" refers to the modification of a protein by directly or indirectly covalently attaching the protein to a composition to alter properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy, while retaining or enhancing its therapeutic benefit. Derivatives of the immunomodulatory polypeptides of the invention are within the scope of the invention and can be produced, for example, by glycosylation, pegylation, lipidation, or Fc fusion.

[0086] As used herein, detection includes methods that allow for visualization of a protein (visually or by instrumentation). Proteins can be visualized using antibodies specific to the protein. Protein detection can also be facilitated by fusing the protein with a tag containing a detectable label or by contacting the protein with a second reagent (e.g., a secondary antibody) specific to the protein and containing a detectable label.

[0087] 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 encoding 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 purposes of illustration herein, definitions are provided, but it is understood that recognizing a particular domain by name is well within the skill of the art. If necessary, appropriate software can be employed to identify the domains.

[0088] 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 a binding domain that specifically binds to a ligand or cell surface receptor, e.g., via a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a cellular transmembrane protein is alternatively referred to as the extracellular domain (ECD).

[0089] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition (including a protein or cell 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 reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of the disease or disorder, or improves the patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient via adoptive cell therapy. In some embodiments, the patient is a mammalian patient, e.g., a non-human primate or a human patient.

[0090] The term "endodomain," as used herein, refers to a region found in some membrane proteins (e.g., transmembrane proteins) that extends into the internal space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. Within the cell, the endodomain can interact with intracellular components and play a role in signal transduction, and thus, in some cases, can be an intracellular signaling domain. The endodomain of a cellular transmembrane protein is alternatively referred to as a cytoplasmic domain, which, in some cases, can be a cytoplasmic signaling domain.

[0091] The terms "enhanced," "increased," or "improved," when used herein in the context of enhancing the immune activity of mammalian lymphocytes, refer to enhancing one or more activities of the lymphocytes. Enhanced activity can be an enhancement (e.g., by a statistically significant amount) of one or more of cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity. In some embodiments, reference to enhanced immune activity refers to increasing (e.g., by a statistically significant amount) the production of interferon-γ (IFNγ), IL-2, or TNFα. In some embodiments, immune activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. Other methods for assessing lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, an enhancement can be an increase or improvement of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

[0092] The term "modified cell," as used herein, refers to a mammalian cell that has been genetically modified by human intervention (e.g., recombinant DNA or viral transduction). In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., T cell, B cell, NK cell) or an antigen-presenting cell (e.g., dendritic cell). The cell may be a primary cell derived from a patient or a cell line. In some embodiments, the modified cell of the invention contains a variant CD86 of the invention that has been modified to regulate the immune activity of a T cell that expresses CD28 or CTLA-4, to which the variant CD86 specifically binds. In some embodiments, the variant CD86 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") that contains an extracellular domain or portion thereof that contains an IgV domain linked to a transmembrane domain (e.g., a CD86 transmembrane domain), and optionally contains an intracellular signaling domain. In some cases, the TIP is configured as a chimeric receptor containing a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the modified cell is capable of expressing and secreting an immunomodulatory protein described herein. Some of the modified cells provided further contain a modified T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0093] The term "engineered T cells," as used herein, refers to T cells (e.g., helper T cells, cytotoxic T cells (or cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells) that have been genetically modified (engineered) by human intervention (e.g., recombinant DNA or viral transduction). The engineered T cells contain a variant CD86 transmembrane immunomodulatory protein (TIP) or secreted immunomodulatory protein (SIP) of the invention expressed on the T cell, which TIP has been engineered to modulate the immune activity of the engineered T cell itself or of a mammalian cell to which the variant CD86 expressed on the T cell specifically binds.

[0094] The term "modified T cell receptor" or "modified TCR" refers to a T cell receptor (TCR) that has been modified to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen that is selected, cloned, and / or subsequently introduced into a population of T cells (which populations of T cells are often used in adoptive immunotherapy).

[0095] The term "expressed on," as used herein, is used in reference to a protein expressed on the surface of a cell (e.g., a mammalian cell). Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety (e.g., a drug or a detectable label). A protein expressed on the surface of a cell can include a cell surface protein (e.g., a cell surface receptor) expressed on a mammalian cell.

[0096] The term "half-life extending moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of the protein not so conjugated to the moiety. In some embodiments, the half-life is extended by more than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or by more than about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or about 6.0-fold. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. Half-life refers to the time required for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using ELISA assay or activity assay. Exemplary half-life extending moieties include Fc domain, multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipid (acylation), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

[0097] 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 I (major histocompatibility complex) or MHC II, and a mammalian lymphocyte (e.g., an effector T cell or natural killer (NK) cell).

[0098] The Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as an Fc polypeptide) primarily corresponds to the constant region of the immunoglobulin heavy chain and is involved in various functions, including antibody effector functions. The Fc domain contains part or all of the hinge domain and the CH2 and CH3 domains of an immunoglobulin molecule. The Fc domain can form a dimer of two polypeptide chains connected by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc with reduced effector function-promoting activity (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction). In some embodiments, references to amino acid substitutions in the Fc region are in accordance with the EU numbering system, unless otherwise specified with reference to a specific SEQ ID NO. EU numbering is known and follows the EU index as reported in the recently updated IMGT Scientific Chart (IMGT®, i.e., international ImMunoGeneTics information system®) http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0099] Immunoglobulin Fc fusions ("Fc fusions"), e.g., immunomodulatory Fc fusion proteins, are molecules 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 in some cases enhances pharmacokinetics) and a variant CD86 polypeptide. The immunoglobulin Fc region may be indirectly or directly linked to one or more variant CD86 polypeptides or small molecules (fusion partners). Various linkers are known in the art and can optionally be used to link the Fc to the fusion partner to generate the Fc fusion. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or non-identical species can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, e.g., a murine, rabbit, or human Fc.

[0100] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. Host cells can be prokaryotic, e.g., Escherichia coli (E. coli), or they can be eukaryotic, e.g., unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives, e.g., Veggie CHO, DG44, Expi CHO, or CHOZN, and related cell lines grown in serum-free medium, or the DHFR-deficient CHO line DX-B11. In some embodiments, host cells can be mammalian cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells).

[0101] The term "immunoglobulin" (abbreviated "Ig"), as used herein, refers to mammalian immunoglobulin proteins, including any of the five human classes of antibodies: 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 L Bispecific 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.

[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 beta 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. CD86 contains two Ig domains: IgV and IgC.

[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 member. Thus, each IgSF family member is unique compared to other IgSF family members, and thus each species of a particular IgSF family member is 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 other 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 such as T cells, refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon-γ), or T-cytotoxic activity. In some cases, immune activity can refer to the expression of cytokines such as chemokines or interleukins. Assays for determining enhanced or suppressed immune activity include the MLR (mixed lymphocyte reaction) assay, which measures cytokine levels such as interferon-γ or IL-2 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). Because T cell activation is associated with the secretion of cytokines such as IFN-γ or IL-2 cytokines, detection of such cytokine levels in culture supernatants from these in vitro human T cell assays can be assayed using commercially available ELISA kits (Wu et al., Immunol Lett 2008 Apr 15; 117(1): 57-62). Induction of an immune response results in enhanced immune activity compared to resting lymphocytes. Immunomodulatory proteins (e.g., variant CD86 polypeptides containing affinity-engineered IgSF domains) as provided herein can, in some embodiments, increase, or in alternative embodiments, decrease, IFN-γ (interferon-γ) or IL-2 expression in primary T cell assays compared to wild-type IgSF members or IgSF domain controls. Those skilled in the art will recognize that the format of a primary T cell assay used to determine increased IFN-γ or IL-2 expression differs from the format employed to assay for decreased IFN-γ or IL-2 expression.When assaying for the ability of immunomodulating proteins or affinity-modified IgSF domains of the invention to reduce IFN-γ or IL-2 expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used, as described in Example 6. Conveniently, soluble forms of affinity-modified IgSF domains of the invention can be employed to determine their ability to antagonize and thereby reduce IFN-γ or IL-2 expression in an MLR. Alternatively, when assaying for the ability of immunomodulating proteins or affinity-modified IgSF domains of the invention to increase IFN-γ or IL-2 expression in primary T cell assays, a co-immobilization assay can be used. In the co-immobilization assay, a T cell receptor signal (provided, in some embodiments, by an anti-CD3 antibody) is used in combination with a co-immobilized affinity-modified IgSF domain, such as a variant CD86, to determine the ability to increase IFN-γ or IL-2 expression compared to a wild-type IgSF domain control. Methods for assaying the immune activity of modified cells, including assessing the activity of variant CD86 transmembrane immunomodulatory proteins, are known in the art and include, but are not limited to, the ability to proliferate T cells after antigen stimulation, the ability to sustain T cell proliferation in the absence of restimulation, and anti-cancer activity in appropriate animal models. Assays are also standard. 51 Assays to assess cytotoxicity include Cr release assays (see, e.g., Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

[0106] An "immunomodulating polypeptide" or "immunomodulating protein" is a polypeptide or protein molecule that modulates immune activity. "Modulating" an immune response means either enhancing or suppressing immune activity. 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 (e.g., by an interchain disulfide bond). Thus, monomeric, dimeric, and higher order multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimers (of the same polypeptide chain) or heteromultimers (of different polypeptide chains). As used herein, immunomodulating proteins include variant CD86 polypeptides.

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

[0108] An "isoform" of CD86 is one of multiple naturally occurring CD86 polypeptides that differ in amino acid sequence. Isoforms can be the product of splice variants of an RNA transcript expressed by a single gene, or the expression product of highly similar but different genes that produce functionally similar proteins, such as may result from gene duplication. As used herein, the term "isoform" of CD86 also refers to the products of different alleles of the CD86 gene.

[0109] The term "label" refers to a compound or composition that can be directly or indirectly attached or linked to generate a detectable signal, or that can interact with a second label to alter the detectable signal. A label can be directly or indirectly conjugated to a polypeptide to produce a labeled polypeptide. The label can be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical change in a substrate compound composition that is detectable. Non-limiting examples of labels include fluorogenic moieties, green fluorescent protein, or luciferase.

[0110] 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-mediated 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 lymphocyte are innate lymphocytes (ILCs).

[0111] The term "mammal" or "patient" specifically includes reference to at least one of a human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0112] The term "membrane protein," as used herein, refers to a protein that directly or indirectly attaches (binds) to a lipid bilayer under physiological conditions. The lipid bilayer that forms the membrane can be a biological membrane, such as a cell membrane of a eukaryote (e.g., a mammal), or an artificial (i.e., man-made) membrane, such as a membrane found on a liposome. The membrane protein can be bound to the lipid bilayer by a covalent bond or by a non-covalent interaction (e.g., a hydrophobic interaction or an electrostatic interaction). The membrane protein can be an integral membrane protein or a peripheral membrane protein. A membrane protein that is a peripheral membrane protein is bound to the lipid bilayer by a non-covalent interaction or to an integral membrane protein by a non-covalent interaction. The peripheral membrane protein forms a temporary bond to the lipid bilayer so that the peripheral membrane protein can interact with and / or dissociate from the lipid bilayer under physiological conditions in mammals. In contrast to peripheral membrane proteins, integral membrane proteins form a substantially permanent bond to the lipid bilayer of the membrane, so that they do not dissociate from the lipid bilayer under physiological conditions in mammals.Membrane proteins can form a bond to the membrane through one layer of the lipid bilayer (monotopic) or through both layers of the membrane (polytopic).Integral membrane proteins that interact with only one lipid bilayer are "integral monotopic proteins."Integral membrane proteins that interact with both lipid bilayers are "integral polytopic proteins."Alternatively, they are referred to herein as "transmembrane proteins."

[0113] The terms "modulation" or "modulating," as used herein in the context of an immune response (e.g., a mammalian immune response), refer to any alteration (e.g., enhancement or reduction) of an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant CD86 of the invention or as a result of administration of engineered cells expressing an immunomodulatory protein of the invention (e.g., a variant CD86 transmembrane immunomodulatory protein). Thus, modulation refers to a change (e.g., enhancement or reduction) of an immune response compared to an immune response that occurs or exists in the absence of administration of an immunomodulatory protein comprising a variant CD86. Such modulation includes any induction, activation, suppression, or change in the degree or extent 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), non-professional antigen-presenting cells, and inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change made to an existing, developing, or potential immune response, or to the ability to induce, regulate, 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 an immune response or modulation of 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 pattern of proteins expressed by these cells). Examples include alterations in the production and / or secretion of specific 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 gamma) or IL-2 expression compared to wild-type or unmodified CD86 controls in primary T cell assays (see Zhao and Ji, Exp Cell Res. 2016 Jan1;340(1):132-138). Modulation can be assessed, for example, by changes in the immune activity of the modified cells compared to cells modified with wild-type CD86 transmembrane protein, such as changes in the cytotoxic activity of the modified cells or changes in cytokine secretion of the modified cells.

[0114] The term "multimerization domain" refers to an amino acid sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, each containing complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), which can be the same or different multimerization domains. The interaction between the complementary multimerization domains, for example, the interaction between the first multimerization domain and the second multimerization domain, forms a stable protein-protein interaction to produce a multimer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the multimerization domains are the same and interact with each other to form a stable protein-protein interaction between the two polypeptide chains. Generally, the polypeptide is directly or indirectly connected to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain or portion thereof, from IgG (including IgG1, IgG2, IgG3 or IgG4 subtypes), IgA, IgE, IgD, IgM, and modified forms thereof.

[0115] 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 specified, the terms encompass nucleic acids containing analogs of known 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 set forth ("reference sequence"), 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.

[0116] 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 CD86 is an IgSF member, and each human CD86 molecule is a molecular species of CD86. 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.

[0117] 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. Thus, a protein can simultaneously bind to at least two different cognate binding partners, but the binding interactions do not necessarily have to be of the same duration; in some cases, the protein specifically binds to only one of the cognate binding partners. In some embodiments, binding occurs under specific binding conditions. 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.

[0118] 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 polypeptide comprising a variant CD86 or engineered cells expressing a variant CD86 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0119] 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 that can be synthesized or recombinantly expressed using known protein modification techniques, in which one or more amino acids are amino acid analogs or non-standard or non-natural amino acids. In addition, proteins may be derivatized.

[0120] The term "primary T cell assay," as used herein, refers to an in vitro assay for measuring T cell activity, e.g., cytokine production, e.g., expression of interferon-γ ("IFN-γ"), IL-2, or tumor necrosis factor α (TNFα). A variety of such primary T cell assays are known in the art. In some embodiments, 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. At a certain time point (usually 24-72 hours), the culture supernatant is collected. In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. At a certain time point (usually 24-72 hours), the culture supernatant is collected. Cytokine levels, e.g., IFN-γ, IL-2, or TNFα, in the culture supernatant are measured by standard ELISA techniques. Commercial kits are available from suppliers and the assays are performed according to the manufacturer's recommendations.

[0121] The term "purified," as applied to nucleic acids (e.g., nucleic acids encoding immunomodulatory proteins of the invention), generally refers to nucleic acids or polypeptides that are 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 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).

[0122] 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 state. 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 expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid, or a cell that has otherwise been genetically engineered (e.g., by introducing into the cell a nucleic acid molecule encoding a recombinant protein (e.g., a transmembrane immunomodulatory protein provided herein)). 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 a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. 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 capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0123] The term "recombinant expression vector," as used herein, refers to a DNA molecule containing a desired coding sequence 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. If desired, a secretory signal peptide sequence may also be encoded by the recombinant expression vector operably linked to the coding sequence of the recombinant protein (e.g., a recombinant fusion protein) so that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell. This term includes vectors as autonomously replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Such vectors include viral vectors, such as lentiviral vectors.

[0124] The term "selectivity" refers to the preference for specific binding of a protein or polypeptide of interest to one substrate (e.g., one cognate binding partner) compared to the specific binding of the protein of interest to another substrate (e.g., a different cognate binding partner). Selectivity refers to the binding activity (e.g., binding affinity) (e.g., K) of a protein of interest to a first substrate (e.g., a first cognate binding partner). d1 ) and the binding activity (e.g., binding affinity) (e.g., K d2 ) can be reflected as a ratio.

[0125] The term "sequence identity" as used herein refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. 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 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 website of the National Center for Biotechnology Information (NCBI).

[0126] The term "soluble" when used herein in reference to a protein means that the protein is not a membrane protein. Generally, a soluble protein contains only the extracellular domain or a portion thereof of an IgSF family member receptor containing an IgSF domain or its specific binding fragment, but does not contain a transmembrane domain. In some cases, the solubility of a protein can be improved by linking or binding to an Fc domain directly or indirectly via a linker, which in some cases can also improve the stability and / or half-life of the protein. In some aspects, the soluble protein is an Fc fusion protein.

[0127] The term "species," as used herein with respect to a polypeptide or nucleic acid, refers to a group of molecules having identical or substantially identical sequences. Differences between polypeptides of the same species can occur due to differences in post-translational modifications, such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated polypeptide sequences that differ from the full-length species by (or encode) only one, two, or three amino acid residues at the amino or carboxy terminus are considered to be sequences of a single species. Such microheterogeneity is a common feature of manufactured proteins.

[0128] The term "specific binding fragment," as used herein with reference to a full-length wild-type mammalian CD86 polypeptide or its ECD, IgV, or IgC domain, refers to a polypeptide that has a subsequence of the ECD, IgV, and / or IgC domain and specifically binds to mammalian CD28 and / or mammalian CTLA-4 (e.g., human or murine CD28 and / or CTLA-4) in vitro and / or in vivo. In some embodiments, a specific binding fragment of CD86 ECD, CD86 IgV, or CD86 IgC is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the full-length wild-type ECD, IgV, or IgC sequence. The sequence of the specific binding fragment can be altered to form a variant CD86.

[0129] 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 5 times greater than the average affinity or avidity of the same protein for a population of random peptides or polypeptides of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times greater, or even at least 1000 times greater. A specifically binding protein need not bind only to a single target molecule, but may specifically bind to a non-target molecule (e.g., a paralog or ortholog) due to the similarity in configuration between the target and the non-target. 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, due to cross-reactivity, the polypeptides of the present invention can specifically bind to multiple distinct target molecular species. Specific binding between two proteins can be determined using solid-phase ELISA immunoassays or surface plasmon resonance (e.g., Biacore) measurements. Generally, the interaction between two binding proteins is 1×10 -5 Less than M, often 1 × 10 -12 In certain embodiments of the present 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.

[0130] With respect to mammalian cells expressing a polypeptide, the term "surface-expressed" or "surface expression" means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0131] As used herein, "synthetic" refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant and / or chemical synthesis methods, e.g., with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide.

[0132] The term "targeting moiety," as used herein, refers to a composition that is covalently or non-covalently bound to or physically encapsulates a polypeptide comprising a variant CD86. A targeting moiety has specific binding affinity for a desired counterstructure, such as a cell surface receptor (e.g., CD28) or a tumor antigen (e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), e.g., B7-H6). Typically, the desired counterstructure is localized on a specific tissue or cell type. A targeting moiety can be an antibody, an antigen-binding fragment (Fab), a V-cell, or a mAb. H and V L variable fragments (Fv) containing V linked together in one chain; H and V L and 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 (less than 500 daltons) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or non-covalently bound (attached) to the lipid membrane of liposomes that encapsulate the polypeptides of the invention.

[0133] As used herein, the term "transmembrane protein" refers to a membrane protein that substantially or completely spans a lipid bilayer, which is found, for example, in biological membranes such as mammalian cells, or in artificial constructs such as liposomes. A transmembrane protein comprises a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. The transmembrane domain can generally be predicted from the amino acid sequence of the transmembrane domain through several commercially available bioinformatics software applications, based on the fact that the hydrophobicity of the transmembrane domain is higher than that of the region of the protein that interacts with the aqueous environment (e.g., cytosol, extracellular fluid). The transmembrane domain is often a hydrophobic α-helix that spans the membrane. A transmembrane protein may span both layers of the lipid bilayer once or multiple times. The transmembrane immunomodulatory proteins provided herein are included in transmembrane proteins. In addition to the transmembrane domain, the transmembrane immunomodulatory proteins of the present invention further comprise an ectodomain, and in some embodiments, an endodomain.

[0134] The term "treatment" or "therapy" of a disease or disorder, as used herein, means slowing, halting, or reversing the progression of the disease or disorder, as evidenced by the reduction, arrest, or elimination of any clinical or diagnostic symptoms by administering a therapeutic composition of the invention (e.g., containing an immunomodulatory protein or modified cells), either alone or in combination with another compound as described herein. "Treatment" or "therapy" 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 "treatment" 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). "Prevention" of a disease or disorder, as used in the context of the present invention, refers to the administration of an immunomodulatory polypeptide or modified cell 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.

[0135] The term "tumor-specific antigen" or "TSA" as used herein refers to a counter structure 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 (e.g., an 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.

[0136] The term "variant" (also "modified form" or "mutant"), when used in reference to variant CD86, refers to a CD86 created by human intervention, e.g., mammalian (e.g., human or murine) CD86. A variant CD86 is a polypeptide having an altered amino acid sequence compared to unmodified or wild-type CD86. A variant CD86 is a polypeptide that differs from the wild-type CD86 isoform sequence by one or more amino acid substitutions, deletions, additions, or a combination thereof. For purposes herein, a variant CD86 contains at least one affinity-altered domain whereby one or more of the amino acid differences occur in an IgSF domain (e.g., an IgV domain or an IgV domain). A variant CD86 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. A variant CD86 polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified CD86 (e.g., the sequence of SEQ ID NO:2), its mature sequence, or a portion thereof containing the extracellular domain or IgSF domain. In some embodiments, the variant CD86 polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the corresponding wild-type or unmodified CD86 comprising the sequence set forth in SEQ ID NO:2, SEQ ID NO:29, SEQ ID NO:122, or SEQ ID NO:123.

[0137] Both unnatural and natural amino acids are included within the scope of permissible substitutions or additions. The variant CD86 is not limited to any particular production method, including, for example, de novo chemical synthesis, de novo recombinant DNA technology, or a combination thereof. The variant CD86 of the present invention specifically binds to at least one or more of CD28 and / or CTLA-4 of a mammalian species. In some embodiments, the change in amino acid sequence results in a change (i.e., an improvement or decrease) in binding affinity or avidity to CD28 and / or CTLA-4 compared to the unmodified or wild-type CD86 protein. The improvement or decrease in 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, Vol. 1(9): 793-801 (1994). The improved binding affinity or avidity of variant CD86 for CD28 and / or CTLA-4 can be at least 5% greater than that of unmodified or wild-type CD86, and in some embodiments can be at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of an unmodified or wild-type CD86 control value. The decreased binding affinity or avidity of CD86 for CD28 and / or CTLA-4 is a decrease to no more than 95% of the unmodified or wild-type CD86 control value, and in some embodiments is a decrease to no more than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the binding affinity or avidity of the unmodified or wild-type CD86 control value, or to an undetectable value. In some embodiments, no change in binding affinity or avidity is considered to be no significant difference between the binding affinity or avidity of the variant CD86 and that of unmodified or wild-type CD86.In some embodiments, the binding affinity or avidity for one cognate binding partner may be altered but not for the other. For example, a variant CD86 that may exhibit improved binding affinity or avidity for CD28 exhibits unchanged binding affinity or avidity for CTLA-4 compared to the binding affinity or avidity of a wild-type or unmodified CD86 molecule. In some embodiments, the binding affinity or avidity for both cognate binding partners may be altered. In some embodiments, the changes are in the same direction (e.g., both are improved or decreased). In some embodiments, the changes are in different directions (e.g., improved for one cognate binding partner and decreased for the other cognate binding partner). For example, a variant CD86 that may exhibit improved binding affinity or avidity for CD28 exhibits decreased binding affinity or avidity for CTLA-4 compared to the binding affinity or avidity of a wild-type or unmodified CD86 molecule. In some embodiments, CD86 variants or wild-type or unmodified polypeptides bind to the ectodomains of CD28 and / or CTLA-4. Thus, in some embodiments, affinity and avidity are determined based on the binding of CD86 variants or wild-type or unmodified polypeptides to the ectodomains of CD28 and / or CTLA-4. Variant CD86 polypeptides have altered primary amino acid sequences due to substitution, addition, or deletion of amino acid residues. The term "variant" in the context of variant CD86 polypeptides should not be construed as imposing any conditions on any particular starting composition or method by which the variant CD86 is made. Variant CD86s can be generated, for example, starting from wild-type mammalian CD86 sequence information, then modeled in silico for binding to CD28 and / or CTLA-4, and finally recombinantly or chemically synthesized to produce the variant CD86. As another example, variant CD86s can be generated by site-directed mutagenesis of unmodified or wild-type CD86.Thus, a variant CD86 represents a composition, but not necessarily a product, produced by any given process. A wide variety of techniques may be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0138] The terms "wild-type" or "natural" or "native," as used herein in connection with biological materials such as nucleic acid molecules, proteins (e.g., CD86), IgSF members, host cells, etc., refer to those found in nature and unmodified by human intervention.

[0139] II. Variant CD86 Polypeptides Provided herein are variant CD86 polypeptides that have altered (enhanced or decreased) binding activity or affinity for one or more CD86 cognate binding partners. In some embodiments, the CD86 cognate binding partner is CD28 or CTLA-4. In some embodiments, the CD86 cognate binding partner is CD28. In some embodiments, the variant CD86 polypeptides contain one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain (IgD) compared to a wild-type or unmodified CD86 polypeptide, or an IgD-containing portion of a wild-type or unmodified CD86, or a specific-binding fragment thereof. Thus, the provided variant CD86 polypeptides are or comprise variant IgDs (hereinafter referred to as "vIgDs") in which one or more amino acid modifications (e.g., substitutions) are in the IgD.

[0140] In some embodiments, the variant has one or more IgSF domains modified relative to the sequence of the unmodified CD86 sequence. In some embodiments, the unmodified CD86 sequence is wild-type CD86. In some embodiments, the unmodified or wild-type CD86 has the sequence of a native CD86 or an orthologue thereof. In some embodiments, the unmodified CD86 is or comprises the extracellular domain (ECD) of CD86 or a portion thereof that contains an IgV domain (see Table 2). In some embodiments, the variant CD86 is or contains the extracellular domain (ECD) of CD86 or a portion thereof that contains an IgV domain. In some embodiments, the unmodified or wild-type CD86 polypeptide contains an IgV domain or a specific binding fragment thereof. In some embodiments, the variant CD86 polypeptide contains an IgV domain or a specific binding fragment thereof. In some embodiments, the variant CD86 is soluble and lacks a transmembrane domain. In some embodiments, the variant CD86 further comprises a transmembrane domain, and in some cases, a cytoplasmic domain.

[0141] In some embodiments, the wild-type or unmodified CD86 sequence is a mammalian CD86 sequence. In some embodiments, the wild-type or unmodified CD86 sequence can be a mammalian CD86, including but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the wild-type or unmodified CD86 sequence is human.

[0142] In some embodiments, the wild-type or unmodified CD86 sequence has (i) the amino acid sequence set forth in SEQ ID NO:2 or a mature form thereof lacking the signal sequence, (ii) 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:2 or a mature form thereof, or (iii) a portion of (i) or (ii) that contains the IgV domain or a specific binding fragment thereof.

[0143] In some embodiments, the wild-type or unmodified CD86 sequence is or comprises the extracellular domain of CD86 or a portion thereof that contains IgV or a specific-binding fragment thereof of CD86. In some embodiments, the unmodified or wild-type CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, 122, or 123, or an ortholog thereof. In some cases, the unmodified or wild-type CD86 polypeptide can comprise (i) the amino acid sequence set forth in SEQ ID NO:29, 122, or 123, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:29, 122, or 123, or (iii) a specific-binding fragment of the sequence of (i) or (ii). In some embodiments, a wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29 (corresponding to amino acid residues 24-247 of SEQ ID NO:2), or an orthologue thereof. In some embodiments, a wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:122 (corresponding to amino acid residues 33-131 of SEQ ID NO:2), or an orthologue thereof. In some embodiments, a wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:123 (corresponding to amino acid residues 24-134 of SEQ ID NO:2), or an orthologue thereof. In some embodiments, a wild-type or unmodified CD86 containing an IgV domain or a specific binding fragment thereof is capable of binding to one or more CD86 cognate binding proteins, such as one or more of CD28 or CTLA-4.

[0144] In some embodiments, the wild-type or unmodified CD86 polypeptide contains a specific binding fragment of CD86 (e.g., a specific binding fragment of the IgV domain). In some embodiments, the specific binding fragment can bind to CD28 and / or CTLA-4. In some embodiments, the specific binding fragment can bind to the ectodomain of CD28 and / or CTLA-4. The specific binding fragment can have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 amino acids. In some embodiments, a 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 IgV domain set forth as amino acids 33 to 131 of SEQ ID NO:2.

[0145] In some embodiments, a variant CD86 polypeptide comprises an extracellular domain or a portion thereof comprising one or more affinity-engineered IgSF domains. In some embodiments, a variant CD86 polypeptide can comprise an IgV domain, or a specific-binding fragment of an IgV domain, wherein the IgSF domain contains one or more amino acid modifications (e.g., substitutions). In some embodiments, a variant CD86 polypeptide comprises a full-length IgV domain. In some embodiments, a variant CD86 polypeptide comprises a specific-binding fragment of an IgV domain. In some embodiments, a variant CD86 polypeptide comprises a full-length extracellular domain (ECD). In some embodiments, a variant CD86 polypeptide comprises a specific-binding fragment of an ECD domain. In some embodiments, a variant CD86 polypeptide comprises a specific-binding fragment of an ECD domain comprising a full-length IgV domain. In some embodiments, a variant CD86 polypeptide comprises a specific-binding fragment of an ECD domain comprising a full-length IgV domain.

[0146] Generally, each of the various attributes of the polypeptides is disclosed separately below (e.g., soluble and membrane-bound polypeptides, affinity of CD86 for CD28 and CTLA-4, number of differences per polypeptide chain, number of linked polypeptide chains, number and nature of amino acid changes per variant CD86, etc.). However, as will be apparent to one of skill in the art, any particular polypeptide can include a combination of these independent attributes. It will be understood that references to amino acids, including references to specific sequences set forth as SEQ ID NOs, used to describe the domain organization of IgSF domains are for illustrative purposes and are not meant to limit the scope of the provided embodiments. It will be understood that descriptions of polypeptides and their domains are theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus may vary and is not necessarily the same from protein to protein. Thus, a particular IgSF domain, e.g., a particular IgV domain, may be several amino acids longer or shorter (e.g., 1, 2, 3, or 4).

[0147] Furthermore, various aspects of the present invention as discussed below are often provided within the meaning of defined terms as disclosed above. Therefore, aspects described in a particular definition should be construed as being incorporated by reference when the defined terms are utilized in the discussion of various aspects and attributes described herein. Thus, the headings, the order of presentation of various aspects and embodiments, and the separate disclosure of each independent attribute are not meant to limit the scope of the present disclosure.

[0148] A. Exemplary Modifications Provided herein are variant CD86 polypeptides that contain at least one affinity-modified IgSF domain (e.g., IgV) or a specific-binding fragment thereof relative to the IgSF domain contained in a wild-type or unmodified CD86 polypeptide, and that exhibit altered (enhanced or decreased) binding activity or affinity for one or more ligands, CD28 or CTLA-4, relative to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptides have binding affinity for CD28 and / or CTLA-4 that differs from that of a wild-type or unmodified CD86 polypeptide control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet, or Biacore assay. In some embodiments, the variant CD86 polypeptides have improved binding affinity for CD28 relative to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptides have decreased binding affinity for CTLA-4 relative to the wild-type or unmodified CD86 polypeptide. In some embodiments, variant CD86 polypeptides exhibit unchanged binding affinity for CTLA-4 compared to wild-type or unmodified CD86 polypeptides. In some embodiments, variant CD86 polypeptides exhibit unimproved binding affinity for CTLA-4 compared to wild-type or unmodified CD86 polypeptides. CD28 and / or CTLA-4 can be mammalian proteins, such as human or murine proteins. In some embodiments, variant, wild-type, and unmodified CD86 polypeptides bind to the ectodomain of CD28 and / or CTLA-4. Thus, in some embodiments, affinity or avidity is determined for binding of variant, wild-type, and unmodified CD86 polypeptides to the ectodomain of CD28 and / or CTLA-4.

[0149] The binding affinity for each of the cognate binding partners is independent; i.e., in some embodiments, the variant CD86 polypeptide has improved binding affinity for CD28 but not for CTLA-4 compared to wild-type or unmodified CD86 polypeptide.

[0150] In some embodiments, the variant CD86 polypeptide has improved binding affinity for CD28 compared to the wild-type or unmodified CD86 polypeptide and has reduced binding affinity for CTLA-4 compared to the wild-type or unmodified CD86 polypeptide, hi some embodiments, the variant CD86 polypeptide has improved binding affinity for CD28 compared to the wild-type or unmodified CD86 polypeptide and has unchanged binding affinity for CTLA-4 compared to the wild-type or unmodified CD86 polypeptide.

[0151] In some embodiments, a variant CD86 polypeptide with improved or greater binding affinity to CD28 will have at least about a 5%, e.g., at least about a 10%, 15%, 20%, 25%, 35%, or 50% improvement in binding affinity to CD28 relative to a wild-type or unmodified CD86 polypeptide control. In some embodiments, the improvement in binding affinity relative to a wild-type or unmodified CD86 polypeptide 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, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, or 400-fold. In such examples, the wild-type or unmodified CD86 polypeptide has the same sequence as the variant CD86 polypeptide, except that it does not contain the one or more amino acid modifications (eg, substitutions).

[0152] In some embodiments, a variant CD86 polypeptide with reduced or decreased binding affinity for CTLA-4 will have at least a 5%, e.g., at least about a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more decrease in binding affinity for CTLA-4 compared to a wild-type or unmodified CD86 polypeptide control. In some embodiments, the decrease in binding affinity compared to a wild-type or unmodified CD86 polypeptide is greater than 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In some embodiments, the variant CD86 polypeptide does not exhibit a change in binding affinity for CTLA-4 compared to a wild-type or unmodified CD86 polypeptide control. In some embodiments, the variant CD86 polypeptide does not exhibit improved binding affinity for CTLA-4 compared to a wild-type or unmodified CD86 polypeptide control. In such examples, the wild-type or unmodified CD86 polypeptide has the same sequence as the variant CD86 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0153] In some embodiments, the equilibrium dissociation constant (K d ) is 1×10 -5 Less than M, 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 M or 1×10 -11 Less than M or 1 x 10 -12 It can be less than M or less.

[0154] A wild-type or unmodified CD86 sequence need not necessarily be used as the starting composition to generate the variant CD86 polypeptides described herein. Therefore, the use of the term "modification," such as "substitution," does not imply that the present embodiment is limited to a particular method of producing the variant CD86 polypeptide. Variant CD86 polypeptides can be produced, for example, by de novo peptide synthesis and therefore do not necessarily require a modification, such as a "substitution," in the sense of altering a codon to encode the modification, e.g., substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which also do not imply a particular method of production. The means by which variant CD86 polypeptides are designed or generated are not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified CD86 are mutagenized from wild-type or unmodified CD86 genetic material and screened for the desired specific binding affinity and / or induction of IFN-γ expression or other functional activity. In some embodiments, variant CD86 polypeptides are synthesized de novo using protein or nucleic acid sequences available in a number of publicly available databases, which are then subsequently screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database.

[0155] Unless otherwise stated, as indicated throughout this disclosure, amino acid modifications are designated by amino position numbers that correspond to the numbering of positions in the unmodified ECD sequence shown in SEQ ID NO:29, as follows: TIFF2025060939000009.tif25161

[0156] The modifications provided herein can be in a wild-type or unmodified CD86 polypeptide set forth in SEQ ID NO:29, or in a portion thereof that contains the IgV domain or a specific-binding fragment thereof. In some embodiments, the wild-type or unmodified CD86 polypeptide contains an IgV of CD86 as set forth in SEQ ID NO:122. In some embodiments, the unmodified CD86 polypeptide contains an IgV that can be several amino acids longer or shorter than the IgV sequence set forth in SEQ ID NO:122, e.g., 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids longer or shorter. In some embodiments, the unmodified CD86 polypeptide has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:29, 122 or 123, or a specific binding fragment thereof. In some embodiments, the unmodified CD86 polypeptide has a sequence as set forth in any of SEQ ID NO:29, 122, and 123. TIFF2025060939000010.tif27161

[0157] It is within the skill of one in the art to identify the corresponding position of a modification (e.g., an amino acid substitution) in a CD86 polypeptide (e.g., a portion thereof containing an IgV domain), for example, by aligning a reference sequence with SEQ ID NO:29. An exemplary alignment of SEQ ID NO:29, containing residues 24-247 of wild-type CD86, with SEQ ID NO:122, containing residues 33-131 of wild-type CD86, is shown in Figure 3. In listings of modifications throughout this disclosure, the amino acid position is shown in the center, the corresponding unmodified (e.g., wild-type) amino acid is listed before the number, and the amino acid substitution of the identified variant is listed after the number. If the modification is a deletion at that position, it is indicated as "del," and if the modification is an insertion at that position, it is indicated as "ins." In some cases, the insertion is listed with the amino acid position indicated in the center, the corresponding unmodified (e.g., wild-type) amino acid is listed before and after the number, and the amino acid insertion of the specified variant is listed after the unmodified (e.g., wild-type) amino acid.

[0158] In some embodiments, the variant CD86 polypeptide has one or more amino acid modifications (e.g., substitutions) in a wild-type or unmodified CD86 sequence. The one or more amino acid modifications (e.g., substitutions) can be in the ectodomain (extracellular domain; ECD) of the wild-type or unmodified CD86 sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or a specific-binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgC domain or a specific-binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the ECD or a specific-binding fragment thereof.

[0159] In some embodiments, the variant CD86 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions). The modifications (e.g., substitutions) can be in the IgV domain. In some embodiments, the modifications are in the ECD. In some embodiments, the modifications are in the ECD and the IgV domain. In some embodiments, the modifications are in the IgV domain. In some embodiments, the variant CD86 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgV domain or specific-binding fragment thereof. In some embodiments, the variant CD86 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the ECD or specific-binding fragment thereof. In some embodiments, the variant CD86 polypeptide has less than 100% sequence identity and at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a wild-type or unmodified CD86 polypeptide or specific-binding fragment thereof, e.g., with the amino acid sequence of SEQ ID NO:29, 122, or 123.

[0160] In some embodiments, the variant CD86 polypeptide is selected from the group consisting of 13, 18, 25, 28, 33, 38, 39, 40, 43, 45, 52, 53, 60, 68, 71, 77, 79, 80, 82, 86, 88, 89, 90, 92, 93, 97, 102, 104, 113, 114, 123, 128, 129, 132, 133, 137, 141, 143, 144, 148, 153, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, , 154, 158, 170, 172, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224. In some embodiments, the modification at position 224 is a deletion. In some embodiments, such variant CD86 polypeptides exhibit altered binding affinity for one or more of CD28 and / or CTLA-4 compared to wild-type or unmodified CD86 polypeptides. For example, in some embodiments, variant CD86 polypeptides exhibit improved binding affinity for CD28 compared to wild-type or unmodified CD86 polypeptides. In some embodiments, the variant CD86 polypeptide exhibits reduced binding affinity for CTLA-4 compared to wild-type or unmodified CD86 polypeptides. In some embodiments, the variant CD86 polypeptide does not exhibit any change in binding affinity for CTLA-4 compared to wild-type or unmodified CD86 polypeptides. In some embodiments, the variant CD86 polypeptide does not exhibit improved binding affinity for CTLA-4 compared to wild-type or unmodified CD86 polypeptides.

[0161] In some embodiments, the variant CD86 polypeptide is TIFF2025060939000011.tif47165, or conservative amino acid substitutions thereof. A conservative amino acid substitution is any amino acid, other than a wild-type or unmodified amino acid, that belongs to the same class of amino acids as the substituted amino acid. The amino acid classes are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl- or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).

[0162] In some embodiments, the variant CD86 polypeptide is TIFF2025060939000012.tif47165, or a conservative amino acid substitution thereof.

[0163] In some embodiments, the variant CD86 polypeptide is selected from the group consisting of 13, 18, 25, 28, 33, 38, 39, 40, 43, 45, 52, 53, 60, 68, 71, 77, 79, 80, 82, 86, 88, 89, 90, 92, 93, 97, 102, 104, 113, 114, 123, 128, 129, 132, 133, 137, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, In some embodiments, the amino acid modification comprises one or more modifications (e.g., amino acid substitutions) at a position corresponding to a position selected from: 48, 153, 154, 158, 170, 172, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224. TIFF2025060939000013.tif48164, or a conservative amino acid substitution thereof.

[0164] In some embodiments, the variant CD86 polypeptide is It contains one or more amino acid substitutions corresponding to TIFF2025060939000014.tif47165, or conservative substitutions thereof.

[0165] In some embodiments, the variant CD86 polypeptide contains at least one alteration (e.g., substitution) at a position selected from 25 or 90. In some embodiments, the at least one amino acid substitution is Q25L, H90Y, or H90L. In some embodiments, the at least one amino acid substitution is Q25L. In some embodiments, the at least one amino acid substitution is H90Y or H90L.

[0166] In some embodiments, the variant CD86 polypeptide is TIFF2025060939000015.tif106164. In some embodiments, the variant CD86 polypeptide contains an amino acid substitution selected from: TIFF2025060939000016.tif106166. In some embodiments, the variant CD86 polypeptide contains the amino acid substitutions Q25L / H90Y or Q25L / H90L.

[0167] In some embodiments, any of the provided variant CD86 polypeptides further comprise: It may contain one or more amino acid substitutions from TIFF2025060939000017.tif48161.

[0168] In some embodiments, the variant CD86 polypeptides provided include amino acid substitutions TIFF2025060939000018.tif106164. In some embodiments, variant CD86 polypeptides are provided having an amino acid substitution There is a CD86 polypeptide with TIFF2025060939000019.tif107167.

[0169] In some embodiments, the variant CD86 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NOs for the extracellular domain (ECD) or IgV domain of wild-type CD86 or exemplary variant CD86 polypeptides. In some cases, the IgV as displayed in Table 1 is shorter than the ECD and therefore may not include all amino acid substitutions as listed in Table 1, e.g., amino acid substitutions outside the IgV domain. As indicated, the exact locus or residues corresponding to a given domain may vary, depending, for example, on the method used to identify or classify the domain. Also, in some cases, the adjacent N- and / or C-terminal amino acids of a given domain (e.g., ECD or IgV) can also be included in the sequence of the variant IgSF polypeptide, e.g., to ensure proper folding of the domain when expressed. Thus, it will be understood that the exemplary SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain, such as the ECD or IgV domain, of a variant CD86 polypeptide may be several amino acids longer or shorter than the amino acid sequence set forth in the respective SEQ ID NO, e.g., 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids longer or shorter.

[0170] In some embodiments, the variant CD86 polypeptide is or comprises any of the sequences set forth in SEQ ID NOs: 85-121, 124-134, 141-221, and 314. In some embodiments, the variant CD86 polypeptide is or comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the sequences set forth in any one of SEQ ID NOs: 85-121, 124-134, 141-221, and 314, and contains an amino acid modification (e.g., a substitution) therein that is not present in wild-type or unmodified CD86. In some embodiments, a variant CD86 polypeptide is or comprises any specific binding fragment of any one of SEQ ID NOs:85-121, 124-134, 314, and 141-221 and contains an amino acid modification (e.g., substitution) therein that is not present in wild-type or unmodified CD86. In some embodiments, a variant CD86 is or comprises the sequence set forth by SEQ ID NOs:89, 93, 94, 107, 111, 112, 115, 117, 124-134, 145, 149, 150, 163, 167, 168, 171, 173, 182, 186, 187, 200, 204, 205, 208, 210, 215-221, or 314.In some embodiments, the variant CD86 polypeptide has the sequence set forth in SEQ ID NO: or comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the sequences set forth in any one of SEQ ID NOs: 89, 93, 94, 107, 111, 112, 115, 117, 124-134, 145, 149, 150, 163, 167, 168, 171, 173, 182, 186, 187, 200, 204, 205, 208, 210, 215-221, or 314, and contains an amino acid modification (e.g., a substitution) therein that is not present in wild-type or unmodified CD86.

[0171] In some embodiments, a variant CD86 polypeptide is or comprises a specific binding fragment of any one of SEQ ID NOs:85-121, 124-134, 141-221, or 314, and contains an amino acid modification (e.g., substitution) therein that is not present in wild-type or unmodified CD86. In some embodiments, a variant CD86 polypeptide is or comprises a specific binding fragment of any one of SEQ ID NOs:89, 93, 94, 107, 111, 112, 115, 117, 124-134, 145, 149, 150, 163, 167, 168, 171, 173, 182, 186, 187, 200, 204, 205, 208, 210, 215-221, or 314, and contains an amino acid modification (e.g., substitution) therein that is not present in wild-type or unmodified CD86.

[0172] Table 1. Exemplary variant CD86 polypeptides TIFF2025060939000020.tif93170TIFF2025060939000021.tif160170

[0173] In some embodiments, any of the provided CD86 variants can include polypeptides that are shorter or longer than the amino acid sequences shown in Table 1, e.g., 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) longer or shorter, as described, provided that the CD86 polypeptide binds to CD28 (including with improved affinity compared to a wild-type or unmodified CD86 polypeptide).

[0174] In some embodiments, the variant CD86 polypeptide exhibits improved affinity for the ectodomain of CD28 compared to a wild-type or unmodified CD86 polypeptide (e.g., the sequence shown in SEQ ID NO:29, 122, or 123).

[0175] In some embodiments, the variant CD86 polypeptide exhibits improved binding affinity for binding to the ectodomain of CD28 and reduced binding affinity for binding to CTLA-4 compared to a wild-type or unmodified CD86 polypeptide (e.g., the sequence shown in SEQ ID NO:29, 122, or 123). In some embodiments, the variant CD86 polypeptide exhibits improved affinity for the ectodomain of CD28 and unchanged affinity for the ectodomain of CTLA-4 compared to a wild-type or unmodified CD86 polypeptide (e.g., the sequence shown in SEQ ID NO:29, 122, or 123).

[0176] In some embodiments, the variant CD86 polypeptide exhibits improved selectivity for CD28 over CTLA-4, as compared to the binding of an unmodified CD86 polypeptide (e.g., as shown in SEQ ID NO:29, 122, or 123) to CD28 relative to the binding of the unmodified CD86 polypeptide to CTLA-4 (e.g., as shown by the ratio of CD28 binding:CTLA-4 binding (CD28:CTLA-4 binding ratio)). In some embodiments, the binding ratio is greater than 1. In some embodiments, the variant CD86 polypeptide is greater than or equal to about 1.1, 1.2, 1.3, 1.4, 1.5, 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, 35, 40, 45, 50, 55, 60, 65, 70 or more. or greater than about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or more, or 1.1, 1.2, 1.3, 1.4, 1.5, 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, 35, 40, 45, 50, 55, 60, 65, 70 or more.

[0177] III. Variant Polypeptide Formats Immunomodulating polypeptides, including variant CD86s provided herein containing vIgD, can be formatted in various ways, e.g., as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, a particular format can be selected to suit the desired therapeutic application. In some cases, immunomodulating polypeptides, including variant CD86 polypeptides, are provided in a format that antagonizes or blocks the activity of their binding partners (e.g., CTLA-4 and / or CD28). In some cases, immunomodulating polypeptides, including variant CD86 polypeptides, are provided in a format that agonizes or stimulates the activity of their binding partners (e.g., CD28). In some embodiments, CD28 agonism can be useful for promoting immunity in oncology. One of skill in the art can readily determine the activity of a particular format, for example, to antagonize or agonize one or more specific binding partners. Exemplary methods for assessing such activity are provided herein, including in the Examples. In some embodiments, the modular format of the immunomodulating proteins provided provides flexibility for engineering or generating immunomodulating proteins to modulate the activity of multiple counter structures (multiple cognate binding partners).

[0178] In some aspects, immunomodulatory proteins comprising the vIgD of CD86 are provided, which are soluble, e.g., fused to an Fc chain. In some aspects, one or more additional IgSF domains, such as one or more additional vIgDs, may be linked to the vIgD of CD86 as provided herein (hereinafter referred to as a "stacked" or "stacked" immunomodulatory protein). In some embodiments, such "stacked" molecules can be provided in a soluble format, or in some cases, may be provided as membrane-bound or secreted proteins. In some embodiments, variant CD86 immunomodulatory proteins are provided as conjugates containing the vIgD of CD86 linked directly or indirectly to a ligand, e.g., a targeting agent or moiety that specifically binds to an antigen, e.g., an antibody or other binding molecule, to target or localize the vIgD to a particular environment or cell, e.g., upon administration to a subject. In some embodiments, the targeting agent, e.g., an antibody or other binding molecule, binds to a tumor antigen, thereby localizing the vIgD-containing variant CD86 to the tumor microenvironment and, e.g., modulating the activity of tumor-infiltrating lymphocytes (TILs) specific to the tumor microenvironment.

[0179] In some embodiments, the provided immunomodulatory proteins are expressed in cells and provided as part of engineered cell therapy (ECT). In some embodiments, variant CD86 polypeptides are expressed in a membrane-bound form in cells, such as immune cells (e.g., T cells or antigen-presenting cells), thereby providing transmembrane immunomodulatory proteins (also referred to hereinafter as "TIPs"). In some embodiments, depending on the cognate binding partner recognized by TIP, engineered cells expressing TIP can stimulate (agonize) the cognate binding partner by providing either a positive or negative costimulatory signal to other engineered cells and / or endogenous T cells. In some embodiments, engineered cells expressing TIP bind to their cognate binding partner on a different cell. In some embodiments, when engineered cells expressing TIP bind to their cognate binding partner on a different cell, the costimulation is referred to as trans costimulation. In some embodiments, engineered cells expressing TIP bind to their own cognate binding partner, thereby inducing costimulation in themselves. In some embodiments, when a TIP on a cell binds to the cell's own cognate binding partner, costimulation is referred to as cis costimulation. In some aspects, a variant CD86 polypeptide is expressed in a secreted form in a cell, such as an immune cell (e.g., a T cell or an antigen-presenting cell), thereby producing a secreted or soluble form of the variant CD86 polypeptide (hereinafter also referred to as a "SIP"), such as when the cell is administered to a subject. In some aspects, depending on the cognate binding partner recognized by the SIP, the engineered cell expressing the SIP can either antagonize or stimulate the cognate binding partner in the environment where it is secreted (e.g., the tumor microenvironment). In some embodiments, a variant CD86 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that is capable of infecting cells, such as immune cells (e.g., a T cell or an antigen-presenting cell) in vivo upon administration to a subject, for delivery or expression of the variant polypeptide to the cell as a TIP or SIP.

[0180] In some embodiments, a soluble immunomodulatory polypeptide, such as a variant CD86 containing vIgD, can be encapsulated in a liposome, which can itself be conjugated to any one or any combination of the provided conjugates (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the present invention are deglycosylated. In more specific embodiments, the variant CD86 sequence is deglycosylated. In even more specific embodiments, the IgV and / or IgC (e.g., IgC2) domains of the variant CD86 are deglycosylated.

[0181] Non-limiting examples of formats that may be provided are further described below.

[0182] B. Soluble Proteins In some embodiments, immunomodulatory proteins containing variant CD86 polypeptides are soluble proteins. Those skilled in the art will recognize that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that the extracellular domain or an immunologically active sequence thereof can be used to produce soluble forms of such proteins. Thus, in some embodiments, immunomodulatory proteins containing variant CD86 polypeptides lack the transmembrane domain or a portion of the transmembrane domain. In some embodiments, immunomodulatory proteins containing variant CD86 lack the intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, immunomodulatory proteins containing variant CD86 polypeptides contain only a vIgD portion containing an ECD domain or a portion thereof containing an IgV domain and / or an IgC (e.g., IgC2) domain or a specific-binding fragment thereof containing an amino acid modification.

[0183] In some embodiments, an immunomodulatory polypeptide comprising a variant CD86 can comprise one or more variant CD86 polypeptides of the invention. In some embodiments, a polypeptide of the invention comprises exactly one, two, three, four, or five variant CD86 sequences. In some embodiments, at least two of the variant CD86 sequences are the same variant CD86 sequence.

[0184] In some embodiments, the immunomodulatory polypeptides provided comprise two or more vIgD sequences of CD86. Multiple variant CD86 polypeptides within a polypeptide chain can be identical (i.e., homologous) or non-identical (i.e., heterologous) variant CD86 sequences. In addition to single polypeptide chain embodiments, in some embodiments, two, three, four, or more polypeptides of the invention may be covalently or non-covalently linked to one another. Thus, monomeric, dimeric, and higher (e.g., 3-, 4-, 5-, or higher) multimeric proteins are provided herein. In some embodiments, for example, exactly two polypeptides of the invention can be covalently or non-covalently linked to one another to form a dimer. In some embodiments, the linkage is via an interchain cysteine ​​disulfide bond. Compositions comprising two or more polypeptides of the invention can be compositions of polypeptides of the same or substantially the same species (e.g., homodimers) or polypeptides of different species (e.g., heterodimers). Compositions having multiple linked polypeptides of the invention can have one or more identical or non-identical variant CD86 polypeptides of the invention in each polypeptide chain, as described above.

[0185] In some embodiments, the immunomodulatory protein is or contains a variant CD86 polypeptide that is in a monomeric form and / or exhibits monovalent binding to its binding partner. In some aspects, a variant CD86 polypeptide as described, such as a soluble and / or variant CD86 lacking a transmembrane domain and an intracellular signaling domain, is directly or indirectly linked to an additional moiety. In some embodiments, the additional moiety is a protein, peptide, small molecule, or nucleic acid. In some embodiments, the monovalent immunomodulatory protein is a fusion protein. In some embodiments, the moiety is a half-life extension molecule. Examples of such half-life extension molecules include, but are not limited to, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding small molecule, or a combination thereof.

[0186] In some embodiments, an immunomodulatory polypeptide comprising a variant CD86 can be linked to a moiety comprising a conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and Ser (see, e.g., WO2008 / 155134, SEQ ID NO:242). In some cases, the amino acid repeat sequence is at least 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 residues, wherein each repeat sequence comprises Ala, Ser, and Pro residues. Thus, provided herein is an immunomodulatory protein that is a PAS-ylated protein in which a variant CD86 polypeptide is linked to Pro / Ala / Ser (PAS) directly or indirectly via a linker. In some embodiments, one or more additional linker structures may be used.

[0187] In some embodiments, the moiety facilitates detection or purification of the variant CD86 polypeptide. In some cases, the immunomodulatory polypeptide comprises a tag or fusion domain, e.g., an affinity or purification tag, linked directly or indirectly to the N-terminus and / or C-terminus of the CD86 polypeptide. Various suitable polypeptide tags and / or fusion domains are known and include, but are not limited to, poly-histidine (His) tags, FLAG-tags (SEQ ID NO:248), Myc-tags, and fluorescent protein-tags (e.g., EGFP, as shown in SEQ ID NOs:244-246). In some cases, the immunomodulatory polypeptide comprising a variant CD86 comprises at least six histidine residues (as shown in SEQ ID NO:249). In some cases, the immunomodulatory polypeptide comprising a variant CD86 further comprises various combinations of moieties. For example, the immunomodulatory polypeptide comprising a variant CD86 further comprises one or more polyhistidine-tags and FLAG-tags.

[0188] In some embodiments, the CD86 polypeptide is linked to a modified immunoglobulin heavy chain constant region (Fc) that remains in a monovalent form, such as that shown in SEQ ID NO:252.

[0189] In some embodiments, the immunomodulatory protein contains a variant CD86 polypeptide linked directly or indirectly via a linker to a multimerization domain. In some aspects, the multimerization domain increases the half-life of the molecule. The interaction of two or more variant CD86 polypeptides can be facilitated by their direct or indirect linkage to any moiety or other polypeptide that can interact with itself to form a stable structure. For example, separately encoded variant CD86 polypeptide chains can be connected by multimerization, where the multimerization of the polypeptides is mediated by the multimerization domain. Typically, the multimerization domain provides for the formation of a stable protein-protein interaction between a first variant CD86 polypeptide and a second variant CD86 polypeptide.

[0190] Homo- or heteromultimeric polypeptides can be generated from the co-expression of separate variant CD86 polypeptides. The first and second variant CD86 polypeptides can be the same or different. In certain embodiments, the first and second variant CD86 polypeptides are the same in a homodimer, each linked to the same multimerization domain. In other embodiments, heterodimers can be formed by linking different first and second variant CD86 polypeptides. In some such embodiments, the first and second variant CD86 polypeptides are linked to different multimerization domains capable of promoting heterodimer formation.

[0191] In some embodiments, the multimerization domain comprises any that can form stable protein-protein interactions. The multimerization domain can be an immunoglobulin sequence (e.g., an Fc domain; see, e.g., International Patent Publication Nos. WO 93 / 10151 and WO 2005 / 063816 US; U.S. Patent Publication No. 2006 / 0024298; U.S. Patent No. 5,457,035); a leucine zipper (e.g., derived from the nuclear transforming proteins fos and jun or the proto-oncogene c-myc or from the General Control of Nitrogen (GCN4)) (see, e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz ​​et al., (1989) Science, 243:1695-1699); a hydrophobic region; a hydrophilic region; or a free thiol that forms an intermolecular disulfide bond between homo- or heteromultimeric chimeric molecules. Additionally, the multimerization domain can include an amino acid sequence containing a protrusion complementary to the amino acid sequence containing the hole, as described, for example, in U.S. Patent No. 5,731,168; International Patent Publication Nos. WO 98 / 50431 and WO 2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerization regions can be engineered so that steric interactions not only promote stable interactions but also favor the formation of heterodimers over homodimers from a mixture of chimeric monomers. Generally, the protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Optionally, a compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0192] A variant CD86 polypeptide can be connected anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of a multimerization domain to form a chimeric polypeptide. The connection can be direct or indirect via a linker. The chimeric polypeptide can be a fusion protein or can be formed by chemical linkage, such as through covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, a nucleic acid encoding all or a portion of a variant CD86 polypeptide can be operably linked to a nucleic acid encoding the multimerization domain sequence, directly or indirectly, or optionally via a linker domain. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant CD86 polypeptide is connected to the N-terminus of the multimerization domain. In some situations, the construct can encode a chimeric protein in which the N-terminus of the variant CD86 polypeptide is connected to the C-terminus of the multimerization domain.

[0193] Polypeptide multimers contain multiple, for example, two chimeric proteins, which are created by directly or indirectly linking two of the same or different variant CD86 polypeptides to a multimerization domain. In some cases, when the multimerization domain is a polypeptide, a gene fusion encoding the variant CD86 polypeptide and the multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with a recombinant expression vector and allowed to assemble into a multimer, where the multimerization domains interact to form a multivalent polypeptide. Chemical linking of the multimerization domain to the variant CD86 polypeptide can be performed using a heterobifunctional linker.

[0194] The resulting chimeric polypeptide, such as a fusion protein, and the multimers formed therefrom can be purified by any suitable method, for example, by affinity chromatography on a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homo- and heterodimers are formed. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0195] In some embodiments, the multimerization domain is an Fc domain or portion thereof from an immunoglobulin. In some embodiments, the immunomodulatory protein comprises a variant CD86 polypeptide attached to an immunoglobulin Fc (resulting in an "immunomodulatory Fc fusion," such as a "variant CD86-Fc fusion," also referred to as a CD86 vIgD-Fc fusion). In some embodiments, attachment of the variant CD86 polypeptide is at the N-terminus of the Fc. In some embodiments, attachment of the variant CD86 polypeptide is at the C-terminus of the Fc. In some embodiments, two or more CD86 variant polypeptides (the same or different) are independently attached at the N-terminus and C-terminus. In some embodiments, the CD86-Fc variant fusions provided herein contain a variant CD86 polypeptide according to the description set forth in Section II above.

[0196] In some embodiments, the Fc is a murine Fc or a human Fc. In some embodiments, the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc is derived from an IgG1, e.g., a human IgG1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:229, 230, or 253, or 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 NO:229, 230, or 253.

[0197] In some embodiments, the Fc region contains additional modifications that alter (e.g., reduce) one or more of its normal functions. Generally, the Fc region is involved in effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), in addition to antigen binding, which is the primary function of immunoglobulins. Additionally, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by extending in vivo half-life through conjugation to the in vivo FcRn receptor. In some embodiments, such functions can be reduced or modified in the Fc for use with the provided Fc fusion proteins.

[0198] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the CD86-Fc variant fusion 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, WO 2012125850, WO 2015 / 107026, US 2016 / 0017041, 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.

[0199] In some embodiments, the provided variant CD86-Fc fusions comprise an Fc region with reduced effector function, making them desirable candidates for applications where the in vivo half-life of the CD86-Fc variant fusion is important but 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 CD86-Fc variant fusion lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. 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., ACTI™ Non-Radiotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and 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 CD86-Fc variant fusion is unable to bind C1q and thus 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)).

[0200] CD86-Fc variant fusions with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region 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).

[0201] In some embodiments, the Fc region of a CD86-Fc variant fusion comprises an Fc region in which any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as indicated by EU numbering) have been substituted with a different amino acid compared to the native Fc region. Such modifications of the Fc region are not limited to the above modifications, and include, for example, deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1- Modifications such as E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E; modifications such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO 2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (denoted by EU numbering); and modifications such as those described in WO 2008 / 092117. It includes modifications at the sites described in 2000 / 042072.

[0202] Certain Fc variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, WO 2006019447, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0203] In some embodiments, a CD86-Fc variant fusion is provided, comprising a variant Fc polypeptide described herein and a variant Fc region comprising one or more amino acid substitutions that extend half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO 2015107026. 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 residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0204] In some embodiments, the Fc region of the CD86-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L (according to EU numbering). In some embodiments, the Fc region of the CD86-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S, and / or C229S (according to EU numbering). In some embodiments, the Fc region of the CD86 variant fusion comprises one or more amino acid substitutions R292C and V302C. 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.

[0205] In some embodiments, the wild-type IgG1 Fc can be the Fc set forth in SEQ ID NO:229, which has an allotype (e.g., the f allotype) containing Glu (E) and Met (M) residues at positions 356 and 358 (according to EU numbering). In other embodiments, the wild-type IgG1 Fc contains amino acids of the human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, as shown, for example, in SEQ ID NO:332. Thus, in some cases, the Fc provided herein can contain the amino acid substitutions E356D and M358L to reconstitute residues of the G1 m1 allotype (e.g., the alpha allotype). In some aspects, the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or to render the Fc inactive for Fc effector function. Exemplary effector function-deficient or inactive mutations include those described herein. Among the effector function-deficient mutations that can be included in the Fc of the constructs provided herein are L234A, L235E, and G237A (according to EU numbering). In some embodiments, the wild-type Fc is further modified by removal of one or more cysteine ​​residues, such as by substituting a serine residue for the cysteine ​​residue at position 220 (C220S) according to EU numbering. Exemplary inactive Fc regions with reduced effector function are set forth in SEQ ID NOs:333 or 256 and 258 or 230, respectively, which are based on the allotypes set forth in SEQ ID NOs:229 or 332, respectively. In some embodiments, the Fc region used in the constructs provided herein can further lack a C-terminal lysine residue.

[0206] In some embodiments, alterations are made in the Fc region that result 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).

[0207] In some embodiments, a CD86-Fc variant fusion is provided comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from an IgG1, such as a human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, the Fc contains at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO:229 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C according to the numbering of SEQ ID NO:229 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification according to the numbering of SEQ ID NO:229 (corresponding to C220S according to EU numbering), such as the Fc region set forth in SEQ ID NO:254. For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: V297G according to EU numbering and one or more of the following amino acid modifications C220S, R292C or V302C (which corresponds to N82G and one or more of the following amino acid modifications C5S, R77C or V87C relative to SEQ ID NO:229), e.g., the Fc region comprises the sequence set forth in SEQ ID NO:255. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E or G237A, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:256. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 257. In some embodiments, the variant Fc comprises one or more of the amino acid modifications C220S, L234A, L235E, G237A, E356D, or M358L, e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 258.

[0208] In some embodiments, CD86-Fc variant fusions provided herein contain a variant CD86 polypeptide according to the description set forth in Section II, above. In some embodiments, CD86-Fc variant fusions are provided comprising any one of the described variant CD86 polypeptides linked to a variant Fc region, where the variant Fc region is not a human IgG1 Fc containing the mutations R292C, N297G, and V302C (corresponding to R77C, N82G, and V87C relative to the wild-type human IgG1 Fc set forth in SEQ ID NO:229). In some embodiments, CD86-Fc variant fusions are provided comprising any one of the variant CD86 polypeptides linked to an Fc region or a variant Fc region, where the variant CD86 polypeptide is not linked to the Fc by a linker consisting of three alanines.

[0209] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO:229 (corresponding to K447del according to EU numbering). In some aspects, such an Fc region can additionally comprise one or more additional modifications, e.g., amino acid substitutions (e.g., any as described). Examples of such Fc regions are set forth in SEQ ID NOs:255-257, 258, or 259-261.

[0210] In some embodiments, a CD86-Fc variant fusion is provided comprising a variant Fc region, wherein the variant Fc comprises an amino acid sequence set forth in any of SEQ ID NOs: 255, 258, 256, 257, 254, or 259-261, 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 any of SEQ ID NOs: 255, 258, 256, 257, 254, or 259-261.

[0211] In some embodiments, the Fc is derived from an IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:262, or 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 NO:262.

[0212] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 263, or 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 NO: 263. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1 and aggregate formation is inhibited, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which the arginine at position 409 of human IgG4 according to the EU index proposed by Kabat et al. is replaced with lysine and aggregate formation is inhibited (see, e.g., U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8):767-71). In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:264 or 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 NO:264.

[0213] In some embodiments, the variant CD86 polypeptide is indirectly linked to the Fc sequence, such as via a linker. In some embodiments, one or more "peptide linkers" link the variant CD86 polypeptide and the Fc domain. In some embodiments, the peptide linker can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the single-letter amino acid code) GGGGS ("4GS" or "G4S"; SEQ ID NO:223) or a multimer of 4GS linkers, e.g., 2, 3, 4, or 5 repeats of the 4GS linker as shown in SEQ ID NO:225 (2xGGGGS; (G4S)2) or SEQ ID NO:224 (3xGGGGS; (G4S)3). In some embodiments, the linker can comprise a series of alanine residues, alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is 3 alanines (AAA). In some embodiments, the variant CD86 polypeptide is indirectly linked to the Fc sequence via a linker, and the linker is not composed of 3 alanines. In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 226). In some embodiments, the linker can further include amino acids introduced by cloning and / or from a restriction site, for example, the linker can include the amino acid GS (in the single letter amino acid code) as introduced by use of the restriction site BAMHI.For example, in some embodiments, the linker (in single-letter amino acid code) is GSGGGGS (SEQ ID NO:222), GS(G4S)3 (SEQ ID NO:227), or GS(G4S)5 (SEQ ID NO:228). In some embodiments, the linker is a rigid linker. For example, the linker is an α-helical linker. In some embodiments, the linker is (in single-letter amino acid code) EAAAK or a multimer of EAAAK linkers, e.g., repeats of 2, 3, 4, or 5 EAAAK linkers as shown in SEQ ID NO:265 (1xEAAAK), SEQ ID NO:266 (3xEAAAK), or SEQ ID NO:247 (5xEAAAK). In some cases, immunomodulatory polypeptides comprising variant CD86 comprise various combinations of peptide linkers.

[0214] In some embodiments, the variant CD86 polypeptide is directly linked to an Fc sequence. In some embodiments, the variant CD86 polypeptide is directly linked to an Fc, such as an inactive Fc that additionally lacks all or a portion of the hinge region. An exemplary Fc lacking a portion of the hinge region (6 amino acids) is set forth in SEQ ID NO:267.

[0215] In some embodiments, when the CD86 polypeptide is directly linked to an Fc sequence, the CD86 polypeptide can be truncated at the C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids. In some embodiments, the variant CD86 polypeptide is truncated such that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids connecting the IgV region to the IgC region are removed.

[0216] In some embodiments, the variant CD86-Fc fusion protein is a dimer formed by two variant CD86 Fc polypeptides linked to the Fc domain. In some specific embodiments, CD86-Fc variant fusion polypeptides of the same or substantially the same species (allowing for three or fewer N- or C-terminal amino acid sequence differences) are dimerized to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two variant CD86 Fc polypeptides are the same. Alternatively, CD86-Fc variant fusion polypeptides of different species can be dimerized to produce a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two variant CD86 Fc polypeptides are different.

[0217] Also provided are nucleic acid molecules encoding variant CD86-Fc fusion proteins. In some embodiments, for production of the Fc fusion protein, the nucleic acid molecule encoding the variant CD86-Fc fusion protein is inserted into an appropriate expression vector. The resulting variant CD86-Fc fusion protein can be expressed in a host cell transformed with the expression vector, where assembly between the Fc domains occurs via interchain disulfide bonds formed between the Fc portions to generate a dimer, such as a bivalent variant CD86-Fc fusion protein.

[0218] The resulting Fc fusion protein can be easily purified by affinity chromatography on a Protein A or Protein G column. For heterodimer formation, additional purification steps may be required. For example, when cells are transformed with two nucleic acids encoding different variant CD86 polypeptides, the variant CD86 molecules bearing the Fc domain are also expressed as disulfide-linked homodimers, so heterodimer formation must be achieved biochemically. Thus, homodimer formation can be reduced under conditions that favor the disruption of interchain disulfides but do not affect the interchain disulfides. In some cases, different variant CD86 Fc monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically modifying and expressing Fc fusion molecules containing variant CD86 polypeptides using the knob-into-hole method described below.

[0219] C. Stacked molecules with additional IgSF domains In some embodiments, the immunomodulatory protein can contain any of the variant CD86 polypeptides provided herein linked, directly or indirectly, to one or more other immunoglobulin superfamily (IgSF) domains ("stacked" immunomodulatory protein constructs, also referred to as "type II" immunomodulatory proteins). In some aspects, this can create unique multidomain immunomodulatory proteins that provide multitargeted modulation of the immune synapse by binding to two or more, e.g., three or more, cognate binding partners.

[0220] In some embodiments, the immunomodulatory protein comprises a combination ("non-wild-type combination") and / or arrangement ("non-wild-type arrangement" or "non-wild-type permutation") of a variant CD86 domain with one or more other affinity-modified and / or affinity-unmodified IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) that is not found in a wild-type IgSF family member. In some embodiments, the immunomodulatory protein contains two, three, four, five, or six immunoglobulin superfamily (IgSF) domains, wherein at least one of the IgSF domains is a variant CD86 IgSF domain (vIgD of CD86) according to the description provided.

[0221] In some embodiments, the sequence of the additional IgSF domain can be a modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain can be affinity-unmodified (e.g., wild-type) or affinity-modified. In some embodiments, the unmodified or wild-type IgSF domain can be from mouse, rat, cynomolgus monkey, or human origin, or a combination thereof. In some embodiments, the additional IgSF domain can be an IgSF domain of an IgSF family member shown in Table 2. In some embodiments, the additional IgSF domain can be an affinity-modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to an IgSF domain contained in an IgSF family member shown in Table 2.

[0222] In some embodiments, the additional IgSF domain is an affinity-modified or unmodified IgSF domain contained in an IgSF family member of a family selected from the following: the signal-regulatory receptor (SIRP) family, the triggering receptor-like expressed on myeloid cells (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 (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the cytotoxicity-inducing receptor (NCR) family, the T-cell immunoglobulin and mucin (TIM) family, or the killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domains are 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 (CD8α), CD8B (CD8β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NC R3 (NKp30).

[0223] The first column of Table 2 provides the name and, in some cases, several possible aliases for that particular IgSF member. The second column provides the protein identifier from the UniProtKB database, a publicly accessible database via the Internet at uniprot.org, and in some cases, provides the GenBank number. 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). GenBank is the NIH's gene sequence database, a collection of all publicly available, annotated DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). Column 3 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). It should be understood that the domain descriptions may vary depending on the method used to identify or classify the domains and may be independently identified from different sources. The descriptions of residues corresponding to domains in Table 2 are exemplary only and may be several amino acids longer or shorter (e.g., 1, 2, 3, or 4).Column 5 indicates some of the IgSF members listed (ie, some of their cell surface cognate binding partners).

[0224] Table 2. IgSF members of the present disclosure TIFF2025060939000022.tif212168TIFF2025060939000023.tif202168TIFF2025060939000024.tif19616 8TIFF2025060939000025.tif206168TIFF2025060939000026.tif224168TIFF2025060939000027.tif94168

[0225] The number of such affinity-unmodified or affinity-modified IgSF domains (whether in non-wild-type combinations or non-wild-type arrangements) present in a "stacked" immunomodulatory protein construct is at least two, three, four, or five, and in some embodiments exactly two, three, four, or five IgSF domains (whereby the determination of the number of affinity-modified IgSF domains ignores any non-specific binding split sequences thereof and / or split sequences thereof that are substantially immunologically inactive).

[0226] In some embodiments of the stacked immunomodulatory proteins provided herein, 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 number of affinity-modified IgSF domains and the number of affinity-unmodified IgSF domains, respectively (affinity-modified IgSF domains:affinity-unmodified IgSF domains), 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.

[0227] In some embodiments of the stacked immunomodulatory proteins, at least two of the non-affinity-modified and / or affinity-modified IgSF domains are identical IgSF domains.

[0228] In some embodiments, the stacked immunomodulatory proteins provided herein comprise at least two affinity-modified and / or affinity-unmodified IgSF domains derived from a single IgSF member but in a non-wild-type arrangement (alternatively, "permutation"). One example of a non-wild-type arrangement or permutation is an immunomodulatory protein of the invention comprising affinity-modified and / or affinity-unmodified IgSF domain sequences of a non-wild-type series compared to those found in wild-type CD86, which IgSF domain sequence serves as the source of the variant IgSF domain as provided herein. Thus, in one example, the immunomodulatory protein can comprise an IgV proximal to the transmembrane domain and an IgC distal to the transmembrane domain, regardless of whether the affinity-unmodified and / or affinity-modified form is present. It is also within the scope of the provided subject matter that both non-wild-type combinations and non-wild-type arrangements of affinity-unmodified and / or affinity-modified IgSF domains are present in the immunomodulatory proteins provided herein.

[0229] In some embodiments of stacked 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 or unmodified IgSF domains to which they are modified are the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein can include at least one IgSF domain sequence that originates from and is unique to one CD86, and at least one second IgSF domain sequence that originates from and is unique to another IgSF family member other than CD86, where the IgSF domains of the immunomodulatory protein are in their affinity-unmodified and / or affinity-modified forms. 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 it specifically binds to a different cognate binding partner.

[0230] In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant CD86 polypeptide, also contain at least one, two, three, four, five, or six additional immunoglobulin superfamily (IgSF) domains, e.g., the IgD domain of an IgSF family member listed in Table 2. In some embodiments, the provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the provided immunomodulatory proteins contain at least two additional IgSF domains (e.g., a second and third IgSF domain). In some embodiments, the provided immunomodulatory proteins contain at least three additional IgSF domains (e.g., a second, third, and fourth). In some embodiments, the provided immunomodulatory proteins contain at least four additional IgSF domains (e.g., a second, third, fourth, and fifth). In some embodiments, the provided immunomodulatory proteins contain at least five additional IgSF domains (e.g., a second, third, fourth, fifth, and sixth). In some embodiments, the provided immunomodulatory proteins contain at least six additional IgSF domains (e.g., a second, third, fourth, fifth, sixth, and seventh). In some embodiments, each of the IgSF domains in the immunomodulatory protein is different. In some embodiments, at least one of the additional IgSF domains is the same as at least one other IgSF domain in the immunomodulatory protein. In some embodiments, each of the IgSF domains is from or derived from a different IgSF family member. In some embodiments, at least two of the IgSF domains are from or derived from the same IgSF family member.

[0231] In some embodiments, the additional IgSF domain comprises an IgV domain or an IgC (e.g., IgC2) domain, or a specific binding fragment of an IgV domain or a specific binding fragment of an IgC (e.g., IgC2) domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgV domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgC (e.g., IgC2) domain. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of an IgV domain. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of an IgC (e.g., IgC2) domain. In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (same) IgSF member. For example, in some aspects, the immunomodulatory protein comprises the ECD of an IgSF member, or a portion thereof, that contains a full-length IgV domain and a full-length IgC (e.g., IgC2) domain or a specific binding fragment thereof.

[0232] In some embodiments, the provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second, or in some cases, a third, etc. IgSF domain), wherein the at least one additional or second IgSF domain is an IgSF domain or a specific-binding fragment thereof set forth in a wild-type or unmodified IgSF domain contained in the amino acid sequence set forth in any of SEQ ID NOs:2-27 and 82. 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.

[0233] In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant CD86 polypeptide, also contain at least one additional affinity-modified IgSF domain (e.g., a second, or in some cases, a third affinity-modified IgSF domain), wherein the at least one additional IgSF domain is vIgD that contains one or more amino acid modifications (e.g., substitutions, deletions, or mutations) compared to the IgSF domain in a wild-type or unmodified IgSF domain, such as an IgSF domain in an IgSF family member shown in Table 2. In some embodiments, the additional, e.g., second or third, 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:2-27 and 82. 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 additional, e.g., second or third IgSF domain is an affinity-modified IgV domain and / or IgC domain. In some embodiments, the one or more additional IgSF domains are IgV and / or IgC (e.g., IgC2) domains, or affinity-modified IgSF domains containing a specific-binding fragment of an IgV domain and / or a specific-binding fragment of an IgC (e.g., IgC2) domain, wherein the IgV and / or IgC domains contain amino acid modifications (e.g., substitutions). In some embodiments, the one or more additional affinity-modified IgSF domains contain an IgV domain containing amino acid modifications (e.g., substitutions).In some embodiments, the one or more additional affinity-modified IgSF domains comprise IgSF domains present in the ECD or portion of the ECD of a corresponding unmodified IgSF family member, e.g., a full-length IgV domain and a full-length IgC (e.g., IgC2) domain, or specific-binding fragments thereof, wherein one or both of the IgV and IgC contain an amino acid modification (e.g., a substitution).

[0234] In some embodiments, the provided immunomodulatory proteins contain at least one additional or second IgSF domain that is vIgD containing one or more amino acid substitutions compared to a wild-type or unmodified IgSF domain other than CD86 (e.g., IgV).

[0235] Stacked molecule immunomodulatory proteins containing at least one IgSF domain of variant CD86 and one or more second or additional IgSF domains can be provided in a variety of construct formats, as described in Section III.C.3. Non-limiting examples of constructs are provided below.

[0236] 1. PD-1 IgSF domain In some embodiments, at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant PD-1 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type PD-1. In some embodiments, the IgSF domain of PD-1 comprises an IgV domain or a specific-binding fragment of an IgV domain. In some embodiments, the IgD can be only an IgV, the entire extracellular domain (ECD), or any combination of the Ig domains of PD-1. In some embodiments, the wild-type or unmodified PD-1 polypeptide has (i) the amino acid sequence set forth in SEQ ID NO:10 or a mature form thereof lacking the signal sequence, (ii) an amino acid sequence or mature form thereof that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:10, or (iii) a portion of (i) or (ii) that contains the IgV domain or a specific-binding fragment thereof. In some embodiments, the wild-type or unmodified PD-1 polypeptide has (i) the amino acid sequence set forth in SEQ ID NO:37, (ii) an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:37, or (iii) a portion of (i) or (ii) that contains an IgV domain or a specific binding fragment thereof. In some embodiments, the unmodified PD-1 polypeptide has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:37, 335, 336, or 337, or a specific binding fragment thereof. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in any of SEQ ID NOs:37, 335, 336, or 337.

[0237] In some embodiments, the IgSF domain of PD-1 is a variant PD-1 polypeptide that contains at least one affinity-engineered IgSF domain (e.g., IgV or IgC) or a specific-binding fragment thereof relative to the IgSF domain contained in the wild-type or unmodified PD-1 polypeptide, and that exhibits altered (enhanced or decreased) binding activity or affinity for PD-L1 or PD-L2 relative to the wild-type or unmodified PD-1 polypeptide. In some embodiments, the IgSF domain of PD-1 is a variant PD-1 polypeptide that contains at least one affinity-engineered IgSF domain (e.g., IgV) or a specific-binding fragment thereof relative to the IgSF domain contained in the wild-type or unmodified PD-1 polypeptide, and that exhibits altered (enhanced or decreased) binding activity or affinity for one or more ligands PD-L1 or PD-L2 relative to the wild-type or unmodified PD-1 polypeptide. In some embodiments, the variant PD-1 polypeptide has a binding affinity for PD-L1 and / or PD-L2 that differs from that of a wild-type or unmodified PD-1 polypeptide control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet, or Biacore assay. In some embodiments, the variant PD-1 polypeptide has an improved binding affinity for PD-L1 and / or PD-L2. In some embodiments, the variant PD-1 polypeptide has a reduced binding affinity for PD-L2 compared to a wild-type or unmodified PD-L1 polypeptide. PD-L1 and / or PD-L2 can be mammalian proteins, such as human or murine proteins.

[0238] The binding affinity for each of the cognate binding partners is independent; i.e., in some embodiments, the variant PD-1 polypeptide has improved binding affinity for one or both of PD-L1 and / or PD-L2 and decreased binding affinity for one or both of PD-L1 and PD-L2 compared to a wild-type or unmodified PD-1 polypeptide.

[0239] In some embodiments, the variant PD-1 polypeptide has improved binding affinity for PD-L1 compared to wild-type or unmodified PD-1 polypeptide. In some embodiments, the variant PD-1 polypeptide has improved or decreased binding affinity for PD-L2 compared to wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-1 polypeptide has improved binding affinity for PD-L1 compared to wild-type or unmodified PD-1 polypeptide, and decreased binding affinity for PD-L2 compared to wild-type or unmodified PD-1 polypeptide.

[0240] In some embodiments, a variant PD-1 polypeptide with improved or greater binding affinity to PD-L1 and / or PD-L2 will have at least about a 5%, e.g., at least about a 10%, 15%, 20%, 25%, 35%, or 50% improvement in binding affinity to PD-L1 and / or PD-L2 relative to a wild-type or unmodified PD-1 polypeptide control. In some embodiments, the improvement in binding affinity relative to a wild-type or unmodified PD-1 polypeptide is greater than 1.2-fold, greater than 1.5-fold, greater than 2-fold, greater than 3-fold, greater than 4-fold, greater than 5-fold, greater than 6-fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, greater than 10-fold, greater than 20-fold, greater than 30-fold, greater than 40-fold, or greater than 50-fold. In such examples, the wild-type or unmodified PD-1 polypeptide has the same sequence as the variant PD-1 polypeptide, except that it does not contain the one or more amino acid modifications (e.g., substitutions).

[0241] In some embodiments, a variant PD-1 polypeptide with reduced or decreased binding affinity for PD-L2 will have at least a 5%, e.g., at least about a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more decrease in binding affinity to PD-L2 compared to a wild-type or unmodified PD-1 polypeptide control. In some embodiments, the decrease in binding affinity compared to a wild-type or unmodified PD-1 polypeptide is greater than 1.2-fold, greater than 1.5-fold, greater than 2-fold, greater than 3-fold, greater than 4-fold, greater than 5-fold, greater than 6-fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, greater than 10-fold, greater than 20-fold, greater than 30-fold, greater than 40-fold, or greater than 50-fold. In such examples, the wild-type or unmodified PD-1 polypeptide has the same sequence as the variant PD-1 polypeptide, except that it does not contain the one or more amino acid modifications (e.g., substitutions).

[0242] PD-L1 and / or PD-L2 can be mammalian proteins, such as human or murine proteins. In some embodiments, PD-L1 is a human protein. In some embodiments, PD-L2 is a human protein.

[0243] In some embodiments, the equilibrium dissociation constant (K d ) is 1×10 -5 Less than M, 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M or 1 x 10 -11 Less than M or 1 x 10 -12 It can be less than M or less.

[0244] Wild-type or unmodified PD-1 sequences do not necessarily need to be used as the starting composition to generate the variant PD-1 polypeptides described herein. Therefore, the use of the term "modification," such as "substitution," does not imply that the present embodiment is limited to a particular method of producing the variant PD-1 polypeptide. Variant PD-1 polypeptides can be produced, for example, by de novo peptide synthesis and therefore do not necessarily require a modification, such as a "substitution," in the sense of altering a codon to encode the modification, e.g., substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which also do not imply a particular method of production. The means by which variant PD-1 polypeptides are designed or generated are not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified PD-1 are mutagenized from wild-type or unmodified PD-1 genetic material and screened for the desired specific binding affinity and / or induction of IFN-γ expression or other functional activity. In some embodiments, variant PD-1 polypeptides are synthesized de novo using protein or nucleic acid sequences available in a number of publicly available databases, which are then subsequently screened. As noted above, the National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database.

[0245] Unless otherwise noted, as indicated throughout this disclosure, amino acid substitutions are designated by amino position numbers that correspond to the numbering of positions in the unmodified ECD sequence shown in SEQ ID NO:37, or, where applicable, the unmodified IgV sequence containing residues 35-145 of SEQ ID NO:10.

[0246] The modifications provided herein can be in a wild-type or unmodified PD-1 polypeptide set forth in SEQ ID NO: 37, or in a portion thereof that contains the IgV domain or a specific-binding fragment thereof. In some embodiments, the wild-type or unmodified PD-1 polypeptide contains the IgV of PD-1 as set forth in SEQ ID NO: 335. In some embodiments, the unmodified PD-1 polypeptide contains an IgV that can be several amino acids longer or shorter than the amino acid sequence set forth by SEQ ID NO: 335, e.g., 1 to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids longer or shorter. In some embodiments, the unmodified PD-1 polypeptide has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:37, 335, 336, or 337. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in any of SEQ ID NO:37. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in SEQ ID NO:335. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in SEQ ID NO:336. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in SEQ ID NO:337. In some embodiments, the unmodified PD-1 polypeptide has a sequence set forth in SEQ ID NO:339.

[0247] It is within the skill of one in the art to identify the corresponding position of a modification, e.g., an amino acid substitution, in a PD-1 polypeptide, including a portion thereof containing its IgSF domain (e.g., IgV), by, for example, aligning a reference sequence with SEQ ID NO: 37. For example, according to the alignment, residue 112 of SEQ ID NO: 37 corresponds to residue 107 of SEQ ID NO: 336.

[0248] In some embodiments, the variant PD-1 polypeptide has one or more amino acid modifications (e.g., substitutions) in a wild-type or unmodified PD-1 sequence. The one or more amino acid modifications (e.g., substitutions) can be in the ectodomain (extracellular domain) of the wild-type or unmodified PD-1 sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or a specific-binding fragment thereof.

[0249] In some embodiments, the variant PD-1 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions). The modifications (e.g., substitutions) can be in the IgV domain. In some embodiments, the variant PD-1 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgV domain or specific-binding fragment thereof. In some embodiments, the variant PD-1 polypeptide has less than 100% sequence identity to a wild-type or unmodified PD-1 polypeptide or a specific-binding fragment thereof, e.g., to the amino acid sequence of SEQ ID NO:37, 335, 336, 337, or 339, and has at least about 85%, about 86%, about 86%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity.

[0250] In some embodiments, the variant PD-1 polypeptide is selected from the group consisting of 8, 9, 11, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 64, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, , 90, 91, 92, 93, 94, 95, 96, 100, 102, 104, 105, 107, 109, 111, 112, 113, 114, 115, 116, 119, 120, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144. In some embodiments, such variant PD-1 polypeptides exhibit altered binding affinity for one or more of PD-L1 and / or PD-L2 compared to wild-type or unmodified PD-1 polypeptides. For example, in some embodiments, variant PD-1 polypeptides exhibit improved binding affinity to PD-L1 and / or PD-L2 compared to wild-type or unmodified PD-1 polypeptides, hi some embodiments, variant PD-1 polypeptides exhibit decreased binding affinity to PD-L1 or PD-L2 compared to wild-type or unmodified PD-1 polypeptides.

[0251] In some embodiments, the variant PD-1 polypeptide is TIFF2025060939000028.tif187165, or a conservative amino acid substitution thereof.

[0252] In some embodiments, the variant PD-1 is TIFF2025060939000029.tif77165, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-1 polypeptide contains the amino acid substitutions S67N / C73R / F86Y / V91D / S107T / A112V / K115D / A120V. In some embodiments, the variant PD-1 polypeptide has the amino acid sequence set forth in SEQ ID NO:315, or 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 NO:315. In some embodiments, the variant PD-1 polypeptide contains the amino acid substitutions V44H / L45V / N46I / Y48H / M50E / N54G / K58T / L102V / A105V / A112I. In some embodiments, the variant PD-1 polypeptide has the amino acid sequence set forth in SEQ ID NO:334, 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:334. Such variant PD-1 polypeptides may be linked directly or indirectly to one or more other immunoglobulin superfamily (IgSF) domains as described.

[0253] Provided herein are immunomodulatory proteins (CD86 / PD-1 immunomodulatory proteins) that contain a variant CD86 polypeptide (e.g., any described in Section II) and an IgSF domain of a PD-1 polypeptide that binds to PD-L1 and / or PD-L2, or a variant thereof. In some embodiments, the variant CD86 polypeptide is or contains the extracellular domain of CD86 or its IgSF (e.g., IgV) domain, or a specific-binding fragment thereof, containing one or more modifications (e.g., substitutions), such as any as described herein. In some embodiments, the variant PD-1 polypeptide is or contains the extracellular domain of PD-1 or its IgSF (e.g., IgV) domain, or a specific-binding fragment thereof, containing one or more modifications (e.g., substitutions), such as any as described herein. CD86 / PD-1 immunomodulatory proteins can be provided in a variety of construct formats, as described in Section III.C.3.

[0254] 2. Tumor antigen-binding IgSF domain In some embodiments, one or more additional IgSF domains (e.g., a second or third IgSF domain) is an IgSF domain of another IgSF family member (e.g., IgV) that binds to or recognizes a tumor antigen. In such embodiments, the IgSF family member functions as a tumor-localizing moiety, thereby bringing vIgD of CD86 into access to immune cells in the tumor microenvironment. In some embodiments, the additional IgSF domain (e.g., a second IgSF) is an IgSF domain of NKp30 that binds to or recognizes B7-H6 expressed on tumor cells.

[0255] In some embodiments, at least one additional (e.g., second) IgSF domain (e.g., NKp30) is an affinity-modified IgSF domain or vIgD containing one or more amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the one or more amino acid modifications improve binding affinity and / or selectivity for B7-H6, e.g., by at least or at least about 1.2-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least about 40-fold, or at least 50-fold, compared to an unmodified IgSF domain (e.g., NKp30). Exemplary amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgC-like or entire ECD) of a variant NKp30 polypeptide are set forth in Table 2. Among exemplary polypeptides are NKp30 variants containing the mutations L30V / A60V / S64P / S86G relative to positions in the NKp30 extracellular domain corresponding to the positions set forth in SEQ ID NO: 54. In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD86 polypeptides and an IgC-like domain that includes any of the amino acid modifications set forth in Table 3, e.g., an IgC-like domain set forth in any of SEQ ID NOs:268-272, or a variant NKp30 polypeptide that contains an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to any of SEQ ID NOs:268-272 and contains one or more amino acid modifications.In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD86 polypeptides and an ECD or portion thereof containing an IgSF domain that contains any of the amino acid modifications set forth in Table 3, e.g., an ECD set forth in any of SEQ ID NOs:273-277, or a variant NKp30 polypeptide that contains an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs:273-277 and contains one or more amino acid modifications.

[0256] Table 3 provides exemplary polypeptides containing one or more affinity-engineered IgSF domains that can be used in the stack constructs provided herein.

[0257] Table 3: Exemplary variant NKp30 polypeptides TIFF2025060939000030.tif39166

[0258] Provided herein are immunomodulatory proteins (CD86 / NkP30 immunomodulatory proteins) containing a variant CD86 polypeptide (e.g., any described in Section II) and an NKp30 polypeptide or variant thereof that binds to B7-H6. In some embodiments, the variant CD86 polypeptide is or contains the extracellular domain of CD86 or its IgSF (e.g., IgV) domain or specific-binding fragment thereof, containing one or more modifications (e.g., substitutions), such as any as described herein. In some embodiments, the variant NKp30 polypeptide is or contains the extracellular domain of Nkp30 or its IgSF (e.g., IgV) domain or specific-binding fragment thereof, containing one or more modifications (e.g., substitutions), such as any as described herein. CD86 / Nkp30 immunomodulatory proteins can be provided in various construct formats, as described in Section III.C.3. In some embodiments, the CD86 / Nkp30 immunomodulatory protein exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any of SEQ ID NOs: 135, 136, 137, 138, 139 or 140. In some embodiments, the variant CD86 / Nkp30 immunomodulatory protein has the sequence set forth in SEQ ID NOs: 135, 136, 137, 138, 139 or 140.

[0259] 3. Constructs In some embodiments, two or more IgSF domains, including the vIgD of CD86, and one or more additional IgSF domains from another IgSF family member (e.g., a second or third variant IgSF domain) are covalently or non-covalently linked. Multiple unaffinity-modified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be directly covalently linked to each other. In some embodiments, two or more IgSF domains are linked directly or indirectly, such as via a linker. In some embodiments, an intervening stretch of one or more amino acid residues indirectly covalently links the IgSF domains to each other. Linkage can be via residues from the N-terminus to the C-terminus. In some embodiments, linkage can be via the side chain of an amino acid residue not located at the N-terminus or C-terminus of the IgSF domain. Thus, linkage can be via terminal or internal amino acid residues, or a combination thereof.

[0260] In some embodiments, the immunomodulatory protein contains at least two IgSF domains linked to each other either directly or indirectly via a linker. In some embodiments, the immunomodulatory protein contains at least three immunomodulatory proteins linked to each other either directly or indirectly via a linker. Various configurations are shown in Figures 23A and 23B.

[0261] In some embodiments, one or more "peptide linkers" connect the vIgD of CD86 to one or more additional IgSF domains (e.g., second or third variant IgSF domains). In some embodiments, the peptide linker can be a single amino acid residue or greater in length. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the single-letter amino acid code) GGGGS ("4GS") or a multimer of a 4GS linker, e.g., 2, 3, 4, or 5 repeats of the 4GS linker. In some embodiments, the peptide linker is (GGGGS)2 (SEQ ID NO:225) or (GGGGS)3 (SEQ ID NO:224). In some embodiments, the linker can also include a series of alanine residues, either alone or in addition to another peptide linker (such as a 4GS linker or a multimer thereof). In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker can also include a series of alanine residues, either alone or in addition to another peptilinker (such as a 4GS linker or a multimer thereof). In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an α-helical linker. In some embodiments, the linker is (in single letter amino acid code) EAAAK or a multimer of EAAAK linkers, e.g., repeats of 2, 3, 4 or 5 EAAAK linkers as shown in SEQ ID NO:265 (1xEAAAK), SEQ ID NO:266 (3xEAAAK) or SEQ ID NO:247 (5xEAAAK).In some embodiments, the linker can further comprise amino acids introduced by cloning and / or from a restriction site, e.g., the linker can comprise the amino acid GS (in the single-letter amino acid code) as introduced by use of the restriction site BAMHI. For example, in some embodiments, the linker (in the single-letter amino acid code) is GSGGGGS (SEQ ID NO:222), GS(GS)3 (SEQ ID NO:227), or GS(GS)5 (SEQ ID NO:228). In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO:226). In some cases, the immunomodulatory polypeptides comprising variant CD86 comprise various combinations of peptide linkers.

[0262] In some embodiments, the immunomodulatory protein comprises a variant CD86 molecule and a variant NKp30 molecule. In some embodiments, the immunomodulatory protein comprises or has a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence set forth in SEQ ID NO: 135, 136, 137, 138, 139, or 140. In some embodiments, the immunomodulatory protein comprises or has a sequence set forth in SEQ ID NO: 135, 136, 137, 138, 139, or 140. In some embodiments, any of the foregoing sequences form homodimers. In some embodiments, the homodimers are formed via a multimerization domain that is an Fc domain contained in the immunomodulatory protein. In some embodiments, the homodimers comprise the sequence of SEQ ID NO: 135. In some embodiments, the homodimer comprises the sequence of SEQ ID NO:136. In some embodiments, the homodimer comprises the sequence of SEQ ID NO:137. In some embodiments, the homodimer comprises the sequence of SEQ ID NO:138. In some embodiments, the homodimer comprises the sequence of SEQ ID NO:139. In some embodiments, the homodimer comprises the sequence of SEQ ID NO:140.

[0263] In some embodiments, the immunomodulatory protein comprises a variant CD86 molecule and a variant PD-1 molecule. In some embodiments, the immunomodulatory protein comprises or has a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence set forth in SEQ ID NO:316, 317, 318, 319, 320, 321, 322, or 323. In some embodiments, the immunomodulatory protein comprises or has a sequence set forth in SEQ ID NO:316, 317, 318, 319, 320, 321, 322, or 323. In some embodiments, the immunomodulatory protein comprises or has a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence set forth in SEQ ID NO:326 or 327. In some embodiments, the immunomodulatory protein comprises or has the sequence set forth in SEQ ID NO:326 or 327. In some embodiments, any of the foregoing sequences form homodimers. In some embodiments, the homodimers are formed through a multimerization domain that is an Fc domain contained in the immunomodulatory protein. In some embodiments, the homodimer comprises or has the sequence of SEQ ID NO:326. In some embodiments, the homodimer comprises or has the sequence of SEQ ID NO:327.

[0264] In some embodiments, the immunomodulatory protein comprises or has a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence set forth in SEQ ID NO:328, 329, 330, or 331. In some embodiments, the immunomodulatory protein comprises or has a sequence set forth in SEQ ID NO:328, 329, 330, or 331. In some embodiments, any of the foregoing sequences form heterodimers. In some embodiments, the heterodimer is formed through a multimerization domain that is an Fc domain contained in the immunomodulatory protein. In some embodiments, the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO:350, and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO:351. In some embodiments, the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 350 and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 352. In some embodiments, the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 350 and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 353.

[0265] In some embodiments, the affinity-unmodified and / or affinity-modified IgSF domains are linked by "wild-type peptide linkers" inserted at the N- and / or C-termini of the affinity-unmodified and / or affinity-modified IgSF domains. These linkers are also referred to as leading sequences (N-termini of the affinity-unmodified or affinity-modified IgSF domains) or trailing sequences (C-termini of the affinity-unmodified or affinity-modified IgSF domains), and sequences present in the wild-type that extend just outside the predicted structure of the IgSF Ig fold. In some embodiments, a "wild-type linker" is an amino acid sequence present in the amino acid sequence of a wild-type protein after the signal sequence but before the IgSF domain (e.g., a defined IgV domain). In some embodiments, a "wild-type" linker is an amino acid sequence present in the amino acid sequence of a wild-type protein immediately after the IgSF domain (e.g., immediately after the defined IgV domain) but before the IgC domain. These linker sequences can contribute to the proper folding and function of adjacent IgSF domains. In some embodiments, there is a leading peptide linker inserted at the N-terminus of a first IgSF domain and / or a trailing sequence inserted at the C-terminus of a 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 a second IgSF domain and / or a second trailing sequence inserted at the C-terminus of a 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, the wild-type peptide linkers between the first and second affinity-unmodified and / or affinity-modified IgSF domains do not overlap.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.

[0266] 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 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).

[0267] 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 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).

[0268] 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 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).

[0269] 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 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).

[0270] In some embodiments, two or more IgSF domains, including the vIgD of CD86 and one or more additional IgSF domains from another IgSF family member (e.g., a second and / or third variant IgSF domain), are linked or attached to Fc to form an Fc fusion, which, when expressed in a cell, can, in some aspects, produce a dimeric multidomain stack immunomodulatory protein. Thus, dimeric multidomain immunomodulatory proteins are also provided.

[0271] In some embodiments, the variant CD86 polypeptide and one or more IgSF domains are independently linked, directly or indirectly, to the N-terminus or C-terminus of the Fc region. In some embodiments, the variant CD86 polypeptide and at least one of the one or more additional IgSF domains are linked, directly or indirectly, and one of the variant CD86 and one or more additional IgSF domains is linked, directly or indirectly, to the N-terminus or C-terminus of the Fc region. In some embodiments, the N-terminus or C-terminus of the Fc region is linked to the variant CD86 polypeptide or to one or more additional IgSF domains, and the other of the N-terminus or C-terminus of the Fc region is linked to the other of the CD86 variant or another one or more additional IgSF domains. In some embodiments, the link to the Fc is via a peptide linker, e.g., a peptide linker as described above. In some embodiments, the link between the variant CD86 and one or more additional IgSF domains is via a peptide linker, e.g., a peptide linker as described above. In some embodiments, the vIgD of CD86, one or more additional IgSF domains, and the Fc domain can be linked together in any of a number of configurations. Exemplary configurations are described in the Examples. See, e.g., Figures 14A-14D.

[0272] In some embodiments, the stacked immunomodulatory protein is a dimer formed by two immunomodulatory Fc-fusion polypeptides. Nucleic acid molecules encoding any of the stacked immunomodulatory proteins are also provided. In some embodiments, the dimeric multidomain stacked immunomodulatory protein can be produced in a cell by expression, or in some cases co-expression, of stacked immunomodulatory Fc-fusion polypeptides as described above for the production of dimeric Fc-fusion proteins.

[0273] In some embodiments, the dimeric multidomain stack immunomodulatory protein is bivalent for each Fc region, monovalent for each subunit, or bivalent for one subunit and tetravalent for the other subunit.

[0274] In some embodiments, the dimeric multidomain stack immunomodulatory protein is a homodimeric multidomain stack Fc protein. In some embodiments, the dimeric multidomain stack immunomodulatory protein comprises a first stack immunomodulatory Fc fusion polypeptide and a second stack immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are the same. In some embodiments, the multidomain stack molecule comprises a first Fc fusion polypeptide containing a variant CD86 and a second IgSF domain, and a second Fc fusion polypeptide containing a variant CD86 and a second IgSF domain. In some embodiments, the multidomain stack molecule comprises a first Fc fusion polypeptide containing a variant CD86, a second IgSF domain, and a third IgSF domain, and a second Fc fusion polypeptide containing a variant CD86, a second IgSF domain, and a third IgSF domain. In some embodiments, the Fc portion of the first and / or second fusion polypeptide can be any Fc as described above. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same.

[0275] In some embodiments, the multidomain stack molecule is a heterodimer comprising two different Fc fusion polypeptides, e.g., a first and a second Fc fusion polypeptide, wherein at least one is an Fc fusion polypeptide containing at least one variant CD86 polypeptide and / or at least one is an Fc fusion polypeptide containing a second IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the first or second Fc fusion polypeptide further contains a third IgSF domain (e.g., a third variant IgSF domain). In some embodiments, the multidomain stack molecule contains a first Fc fusion polypeptide containing a variant CD86 and a second Fc fusion polypeptide containing a second IgSF domain, wherein in some cases the first or second Fc fusion polypeptide additionally contains a third IgSF domain. In some embodiments, the multidomain stack molecule contains a first Fc fusion polypeptide containing a variant CD86, a second IgSF domain, and in some cases a third IgSF domain, and a second Fc fusion polypeptide that is not linked to either the variant CD86 polypeptide or the additional IgSF domain. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are different.

[0276] In some embodiments, the multidomain stack molecule contains a first Fc fusion polypeptide containing one, two, three, four or more variant CD86 polypeptides and one, two, three, four or more additional IgSF domains, wherein the total number of IgSF domains in the first stacked Fc fusion polypeptide is greater than two, three, four, five, six or more. In one example of such an embodiment, the second stacked Fc fusion polypeptide contains one, two, three, four or more variant CD86 polypeptides and one, two, three, four or more additional IgSF domains, wherein the total number of IgSF domains in the first stacked Fc fusion polypeptide is greater than two, three, four, five, six or more. In another example of such an embodiment, the second Fc fusion polypeptide is not linked to either the variant CD86 polypeptide or the additional IgSF domains.

[0277] In some embodiments, the heterodimeric stack molecule contains a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are different. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first variant CD86 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and a second Fc polypeptide fusion containing an Fc region and the other of the first variant CD86 polypeptide or the second IgSF domain. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first variant CD86 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and a second Fc polypeptide fusion containing an Fc region and both the first variant CD86 polypeptide and a second IgSF domain (e.g., a second variant IgSF domain) in a different orientation or configuration than the first Fc region. In some embodiments, the first and / or second Fc fusion polypeptides also contain a third IgSF domain (e.g., a third variant IgSF domain).

[0278] In some embodiments, the Fc domains of one or both of the first and second stacked immunomodulatory Fc-fusion polypeptides comprise modifications (e.g., substitutions) such that the interface of the Fc molecules is altered to facilitate and / or promote heterodimerization. In some embodiments, the modifications comprise the introduction of a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide, thereby allowing the protrusion to be positioned within the cavity and facilitating the formation of a complex between the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create the protrusion or cavity in the polypeptide are typically interface amino acids that interact or contact with one or more amino acids at the interface of the second polypeptide.

[0279] In some embodiments, for constructs in which the Fc sequence is the N-terminal portion of the sequence, an amino acid sequence is added before the Fc sequence. In some cases, for constructs in which the Fc sequence is the N-terminal portion of the sequence, the amino acid sequence HMSSVSAQ (SEQ ID NO:279) is added immediately before the Fc sequence. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region (knob; e.g., the Fc sequence shown in SEQ ID NO:252 or 324) and a first variant polypeptide and / or a second IgSF domain (e.g., the second variant IgSF domain) and a second Fc polypeptide fusion containing an Fc region (hole; e.g., the Fc sequence shown in SEQ ID NO:280 or 325), and a stuffer sequence HMSSVSAQ (SEQ ID NO:279) is added immediately before the Fc region of both the first and second Fc polypeptide fusions.

[0280] In some embodiments, the first polypeptide modified to contain a knob amino acid comprises a replacement of a natural or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide and can therefore be positioned in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. In most cases, the replacement amino acid has a larger side chain volume than the or...

Claims

Claim 1: A variant CD86 polypeptide comprising an extracellular domain or an IgV domain, the variant CD86 polypeptide comprises the amino acid substitutions Q25L / Q86R / H90L / N104S relative to the positions shown in SEQ ID NO:29; the variant CD86 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the binding of the unmodified CD86 to the same ectodomain; Variant CD86 polypeptides.

2. A variant CD86 polypeptide comprising an extracellular domain or an IgV domain, the variant CD86 polypeptide comprises the amino acid substitutions A13V / Q25L / H90L / S181P / L197M / S206T relative to the positions shown in SEQ ID NO:29; the variant CD86 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the binding of the unmodified CD86 to the same ectodomain; Variant CD86 polypeptides.

3. A variant CD86 polypeptide comprising an extracellular domain or an IgV domain, the variant CD86 polypeptide comprises the amino acid substitutions Q25L / H90L / K93T / M97L / T133A / S181P / D215V relative to the positions shown in SEQ ID NO:29; the variant CD86 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the binding of the unmodified CD86 to the same ectodomain; Variant CD86 polypeptides.

4. The variant CD86 polypeptide of claim 1, (i) amino acid residues 24 to 247 of the CD86 extracellular domain, relative to the positions shown in SEQ ID NO: 2; (ii) amino acid residues 24 to 134 of the CD86 extracellular domain, relative to the positions set forth in SEQ ID NO:2; (iii) amino acid residues 33 to 131 of the CD86 extracellular domain, relative to the positions set forth in SEQ ID NO: 2; or (iv) an amino acid sequence comprising any one of (i) to (iii) above, including an IgV domain; 4. The variant CD86 polypeptide of any one of claims 1 to 3, comprising:

5. A variant CD86 polypeptide described in any one of claims 1 to 4, comprising the amino acid sequence shown in SEQ ID NO: 94 or 150.

6. A variant CD86 polypeptide according to claim 2 or 4, comprising the amino acid sequence shown in SEQ ID NO: 89 or 145.

7. A variant CD86 polypeptide according to claim 3 or 4, comprising the amino acid sequence shown in SEQ ID NO: 93 or 149.

8. (i) A soluble protein. (ii) lacks the CD86 transmembrane and intracellular signaling domains, and / or (iii) cannot be expressed on the surface of a cell; 8. The variant CD86 polypeptide of any one of claims 1 to 7.

9. An immunomodulatory protein comprising a variant CD86 polypeptide according to any one of claims 1 to 8 linked to a multimerization domain.

10. The immunomodulatory protein of claim 9, wherein the multimerization domain is an Fc domain, and optionally, the Fc domain comprises an amino acid sequence set forth in SEQ ID NO: 229 or an amino acid sequence exhibiting at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:

229. (i) the Fc domain is a variant IgG1 Fc domain comprising one or more amino acid modifications selected from C220S, E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C and K447del, each according to EU numbering; or (ii) the Fc domain is a variant IgG1 Fc domain comprising the amino acid modifications L234A / L235E / G237A, respectively according to EU numbering; and / or (iii) the Fc domain is or comprises the amino acid sequence set forth in SEQ ID NO: 230, or is or comprises an amino acid sequence that exhibits at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 230; The immunomodulatory protein of claim 10.

12. An immunomodulatory protein comprising a variant CD86 polypeptide according to any one of claims 1 to 8 linked directly or indirectly via a linker to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of an IgSF family member.

13. An immunomodulatory protein comprising a first polypeptide and a second polypeptide, the first polypeptide comprises at least one IgSF domain linked to a first Fc domain via a linker, the at least one IgSF domain comprising one or both of a variant CD86 polypeptide and an IgSF domain of a PD1 polypeptide or variant thereof, or is an IgSF domain of a PD1 polypeptide or variant thereof; and the second polypeptide comprises at least one IgSF domain linked via a linker to a second Fc domain, the at least one IgSF domain comprising one or both of a variant CD86 polypeptide and an IgSF domain of a PD1 polypeptide or variant thereof, or is an IgSF domain of a PD1 polypeptide or variant thereof; the immunomodulatory protein comprises at least one IgSF domain of CD86 and at least one IgSF domain of PD-1 or a variant thereof; the variant CD86 polypeptide comprises the amino acid substitution Q25L relative to the position shown in SEQ ID NO:29; The immunomodulatory protein.

14. The immunomodulatory protein described in claim 13, wherein the variant CD86 polypeptide comprises amino acids 33 to 131 or 24 to 134 of the CD86 extracellular domain based on the positions shown in SEQ ID NO:

2.

15. The variant CD86 polypeptide of claim 1, (i) the amino acid sequence set forth in SEQ ID NO: 29, or (ii) the amino acid sequence shown at positions 33 to 131 or 24 to 134 of SEQ ID NO: 2 15. The immunomodulatory protein of claim 13 or 14, comprising an amino acid sequence that exhibits at least 95% sequence identity to

16. An immunomodulatory protein described in any one of claims 13 to 15, wherein at least one IgSF domain of the first polypeptide comprises a variant CD86 polypeptide described in any one of claims 1 to 8.

17. An immunomodulatory protein described in any one of claims 13 to 16, wherein at least one IgSF domain of the second polypeptide comprises a variant PD1 polypeptide.

18. An immunomodulatory protein according to any one of claims 13 to 17, wherein at least one IgSF domain of the first polypeptide is a first IgSF domain, the first IgSF domain being a variant CD86 polypeptide according to any one of claims 1 to 8, and the first polypeptide comprising a second IgSF domain linked to the first Fc domain via a linker.

19. The immunomodulatory protein of claim 18, wherein the second IgSF domain of the first polypeptide comprises a variant PD1 polypeptide.

20. An immunomodulatory protein according to any one of claims 13 to 19, wherein at least one IgSF domain of the second polypeptide is a first IgSF domain, the first IgSF domain being a variant CD86 polypeptide according to any one of claims 1 to 8, and the second polypeptide comprising a second IgSF domain linked to the second Fc domain via a linker.

21. The immunomodulatory protein of claim 20, wherein the second IgSF domain of the second polypeptide comprises a variant PD1 polypeptide.

22. At least one IgSF domain of the first polypeptide is linked to the N-terminus or C-terminus of the first Fc domain via a linker; and At least one IgSF domain of the second polypeptide is linked to the N-terminus or C-terminus of the second Fc domain via a linker. An immunomodulatory protein according to any one of claims 13 to 21.

23. An immunomodulatory protein described in claim 18 or 19, wherein the second IgSF domain of the first polypeptide is linked to the end of the first Fc domain opposite the end linked to the first IgSF domain.

24. An immunomodulatory protein described in any one of claims 20 to 23, wherein the second IgSF domain of the second polypeptide is linked to the end of the second Fc domain opposite the end linked to the first IgSF domain.

25. An immunomodulatory protein described in any one of claims 13 to 24, wherein the linker independently comprises the sequence of SEQ ID NO: 222 or 224, and optionally the linker comprises 1 to 4 repeats of the sequence of SEQ ID NO: 222 or 224.

26. An immunomodulatory protein described in any one of claims 13 to 25, wherein the first Fc domain and the second Fc domain are identical, and optionally, the first Fc domain and the second Fc domain comprise the sequence of SEQ ID NO:

230.

27. The immunomodulatory protein of any one of claims 13 to 26, wherein the first polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a homodimer, and optionally, the first polypeptide and the second polypeptide comprise, from left to right, variant PD1 polypeptide-linker-Fc-linker-variant CD86 polypeptide.

28. An immunomodulatory protein described in any one of claims 13 to 27, wherein the variant PD1 polypeptide comprises the sequence of SEQ ID NO:

315.

29. An immunomodulatory protein described in any one of claims 13 to 28, wherein the variant CD86 polypeptide comprises the sequence of SEQ ID NO: 89, 93, 94, 145, 149, or 150.

30. An immunomodulatory protein described in any one of claims 13 to 29, wherein the first and second polypeptides comprise the sequence of SEQ ID NO: 326 or 327.

31. An immunomodulatory protein described in any one of claims 13 to 25, wherein the first Fc domain and the second Fc domain are different, and optionally the first and second Fc domains comprise knob-into-hole mutations, and optionally the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO: 324, and the other of the first Fc domain or the second Fc domain comprises the sequence of SEQ ID NO:

325.

32. An immunomodulatory protein described in any one of claims 13 to 25 and 31, wherein the first polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a heterodimer.

33. The immunomodulatory protein of any one of claims 13 to 25, 31, and 32, wherein the first polypeptide of the heterodimer comprises, from left to right, variant PD1 polypeptide-linker-Fc, and the second polypeptide of the heterodimer comprises, from left to right, variant CD86 polypeptide-linker-Fc, Fc-linker-variant CD86 polypeptide, or variant PD1-linker-Fc-linker-variant CD86.

34. An immunomodulatory protein described in any one of claims 13 to 25 and 31 to 33, wherein the variant PD1 polypeptide comprises the sequence of SEQ ID NO:

315.

35. 35. The immunomodulatory protein of any one of claims 13 to 25 and 31 to 34, wherein said variant CD86 polypeptide comprises the sequence of SEQ ID NO: 89, 93, 94, 145, 149, or 150.

36. The method of claim 36, wherein the first polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 328; and the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 329, 330, or 331; An immunomodulatory protein according to any one of claims 13 to 25 and 31 to 35.

37. A conjugate comprising a variant CD86 polypeptide according to any one of claims 1 to 8 linked to a targeting moiety that specifically binds to a molecule on the surface of a cell, optionally wherein the conjugate is a fusion protein.

38. The conjugate of claim 37, wherein the cell is an immune cell or a tumor cell.

39. The conjugate of claim 37 or 38, wherein the moiety is an antibody or antigen-binding fragment.

40. A nucleic acid molecule encoding a variant CD86 polypeptide according to any one of claims 1 to 8, an immunomodulatory protein according to any one of claims 9 to 36, or a conjugate according to any one of claims 37 to 39 that is a fusion protein.

41. A vector comprising the nucleic acid molecule of claim 40.

42. A cell comprising the vector described in claim 41.

43. A method for producing a variant CD86 polypeptide according to any one of claims 1 to 8, or an immunomodulatory protein according to any one of claims 9 to 36, comprising the step of introducing a nucleic acid molecule according to claim 40 or a vector according to claim 41 into a host cell under conditions in which the protein is expressed in the host cell, and optionally further comprising the step of isolating or purifying the variant CD86 polypeptide or immunomodulatory protein from the host cell.

44. A method of modifying a cell that expresses a variant CD86 polypeptide, the method comprising the step of introducing into a host cell a nucleic acid molecule encoding a variant CD86 polypeptide according to any one of claims 1 to 8, an immunomodulatory protein according to any one of claims 9 to 36, or a conjugate according to any one of claims 37 to 39 that is a fusion protein, under conditions such that the polypeptide, the immunomodulatory protein, or the conjugate is expressed in the cell.

45. A modified cell comprising a variant CD86 polypeptide according to any one of claims 1 to 8, an immunomodulatory protein according to any one of claims 9 to 36, a conjugate according to any one of claims 37 to 39 that is a fusion protein, a nucleic acid molecule according to claim 40, or a vector according to claim 41, wherein optionally the variant CD86 polypeptide or the immunomodulatory protein is capable of being secreted from the modified cell.

46. A modified cell described in claim 44 or 45, wherein the cell is an immune cell, optionally the immune cell is a lymphocyte, optionally the lymphocyte is a T cell.

47. A modified cell described in any one of claims 44 to 46, further comprising a chimeric antigen receptor (CAR) or a modified T cell receptor (TCR).

48. An infectious agent comprising a variant CD86 polypeptide of any one of claims 1 to 8, or an immunomodulatory protein of any one of claims 9 to 36, linked to a multimerization domain.

49. A pharmaceutical composition comprising a variant CD86 polypeptide according to any one of claims 1 to 8, an immunomodulatory protein according to any one of claims 9 to 36, a conjugate according to any one of claims 37 to 39 that is a fusion protein, a modified cell according to any one of claims 44 to 46, or an infectious agent according to claim 48, and a pharmaceutically acceptable excipient.

50. An article of manufacture comprising the pharmaceutical composition of claim 49 in a vial or container.

51. Use of the pharmaceutical composition of claim 49 in the manufacture of a medicament for modulating an immune response in a subject.

52. Use of the pharmaceutical composition of claim 49 in the manufacture of a medicament for the treatment of a disease or condition in a subject.

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