CD86 variant immune regulatory protein and its use

Abstract

Provided herein are variant CD86 polypeptides, immunomodulatory proteins comprising variant CD86 polypeptides, and nucleic acids encoding the proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for producing and using the proteins are provided. TIFF2022510276000050.tif71170

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 774,131, filed on November 30, 2018, entitled "CD86 Variant Immunomodulatory Proteins and Uses Thereof", and U.S. Provisional Patent Application No. 62 / 862,001, filed on June 14, 2019, entitled "CD86 Variant Immunomodulatory Proteins and Uses Thereof", the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of the sequence listing This application is filed together with a Sequence Listing in electronic format. The Sequence Listing 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 herein 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 its immunomodulatory proteins that exhibit altered binding affinities for cognate binding partners (e.g., enhanced affinity for CD28). Also provided are methods and uses of such immunomodulatory proteins.

Background Art

[0004] Background There is increasing medical interest in regulating the immune response by intervening in the processes that occur at the immunological synapse (IS) formed between antigen-presenting cells (APCs) or target cells and lymphocytes. Mechanistically, cell surface proteins within the IS can be involved in coordinated and often simultaneous interactions between multiple protein targets and a single protein to which they bind. Interactions in the IS occur in close association with the junction of the two cells, and a single protein within this structure can interact (presumably simultaneously) with both proteins on the same cell (cis) and proteins on the interacting cell (trans). Therapeutic agents capable of modulating the IS are known, but improved therapeutic agents are needed. Provided is an immunomodulatory protein comprising a soluble protein or a transmembrane immunomodulatory protein that can be expressed on a cell, which meets such needs.

Summary of the Invention

[0005] Summary In this specification, a variant CD86 polypeptide containing an extracellular domain or an IgV domain or a specific binding fragment thereof, wherein the variant CD86 polypeptide contains one or more amino acid modifications at positions corresponding to positions selected from 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, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224 based on the positions shown in SEQ ID NO:29 in an unmodified CD86 polypeptide or a specific binding fragment thereof is provided. 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 one or more amino acid modifications are in one or more residues of the extracellular domain of the unmodified CD86 polypeptide. In some embodiments, the unmodified CD86 polypeptide contains (i) the amino acid sequence shown 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 that contains an IgV domain or a portion containing a specific binding fragment of the IgV domain. In some embodiments, the unmodified CD86 contains the amino acid sequence shown in SEQ ID NO:29. In some embodiments, the portion includes amino acid residues 33 to 131 or 24 to 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 that contains an IgV domain or a portion containing a specific binding fragment of the IgV domain. In some embodiments, the unmodified CD86 contains 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 contains 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 amino acids, or more amino acids. In some embodiments, the specific binding fragment comprises at least 80% of the length of the IgV domain as shown by 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 of the amino acid modifications are substitutions. In some embodiments, one or more of the amino acid modifications are insertions. In some embodiments, one or more of the amino acid modifications are deletions. In some embodiments, one or more of the amino acid modifications are one or more amino acid substitutions selected from TIFF0007713886000001.tif47165, or conservative amino acid substitutions thereof.

[0009] In some embodiments, the variant CD86 polypeptide It contains one or more amino acid modifications selected from TIFF0007713886000002.tif107164. In some embodiments, the one or more amino acid modifications are at position 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 position 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 additional amino acid modifications. In some embodiments, the one or more amino acid modifications contain Q25L / H90Y or Q25L / H90L and It contains one or more amino acid modifications selected from TIFF0007713886000003.tif41163, or conservative amino acid substitutions thereof.

[0010] In some embodiments, the variant CD86 polypeptide It contains one or more amino acid modifications selected from TIFF0007713886000004.tif106169.

[0011] In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications A13V / Q25L / H90L. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications A13V / Q25L / H90L / S181P / L197M / S206T. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications Q25L / H90L / K93T / M97L. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications Q25L / H90L / K93T / M97L / T133A / S181P / D215V. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications Q25L / Q86R / H90L. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications Q25L / Q86R / H90L / N104S. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications I89V / H90L. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications I89V / H90L / I193V. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications M60K / H90L. In some embodiments, the variant CD86 polypeptide contains one or more amino acid modifications Q25L / F33I / H90L. In some embodiments, the 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 or a specific binding fragment thereof 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:29.

[0013] In some embodiments, the variant CD86 polypeptide specifically binds to the ectodomain of CD28 with an improved affinity as compared to the binding of non-modified CD86 to the same ectodomain. In some embodiments, the binding affinity is 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 or at least about 125-fold improved.

[0014] In some embodiments, the variant CD86 polypeptide specifically binds to the ectodomain of CTLA-4 with a reduced affinity as compared to the binding of non-modified CD86 to the same ectodomain. In 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 reduced. 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 non-modified CD86 to the same ectodomain, and optionally, the same or equivalent binding affinity is 90% - 120% or about 90% - about 120% of the binding affinity of non-modified 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 shown in any of SEQ ID NOs: 85 to 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 to 121 and contains one or more of the amino acid modifications of each of the sequence numbers shown in any of SEQ ID NOs: 85 to 121 or a specific binding fragment thereof. In some embodiments, the variant CD86 polypeptide contains the amino acid sequence shown in any of SEQ ID NOs: 141 to 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 to 177 and contains one or more of the amino acid modifications of each of the sequence numbers shown in any of SEQ ID NOs: 141 to 177 or a specific binding fragment thereof.

[0016] In some embodiments, CD28 is human CD28. In some embodiments, 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 domain and intracellular signaling domain of CD86; and / or, the variant CD86 polypeptide cannot be expressed on the surface of the cell. In some embodiments, the variant CD86 polypeptide is linked to a multimerization domain. In some embodiments, the multimerization domain is the Fc domain or a variant thereof with reduced effector function. In some embodiments, the variant CD86 polypeptide is linked to the 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 shown 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 the amino acid sequence shown in SEQ ID NO:229 or contains it.

[0018] In some embodiments, the Fc domain is a variant IgG1 Fc domain containing one or more amino acid modifications selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del (by 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 (by EU numbering). In some embodiments, the Fc domain contains the amino acid modification K447del (by EU numbering). In some embodiments, the Fc domain contains 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 contains one or more of each amino acid modification set forth in SEQ ID NO:230 as 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 a multimerization domain or Fc via a linker (optionally a G4S linker). In some embodiments, the variant CD86 polypeptide is a transmembrane immunomodulatory protein further containing a transmembrane domain, and optionally, the transmembrane domain is directly or indirectly linked 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 shown as residues 248 - 268 of SEQ ID NO:2, or contains 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, and optionally, the cytoplasmic domain is directly or indirectly linked to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains the native CD86 cytoplasmic domain. In some embodiments, the cytoplasmic domain contains the amino acid sequence shown as residues 269 - 329 of SEQ ID NO:2, or contains 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 cannot mediate or regulate intracellular signaling when expressed on the cell surface.

[0021] As used herein, there is provided 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 containing 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] As used herein, there is provided an immunomodulatory protein comprising any of the variant CD86 polypeptides described herein directly or indirectly linked 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, the affinity-modified IgSF domain containing one or more amino acid modifications as compared to an unmodified or wild-type IgSF domain of an IgSF family member. In some embodiments, the IgSF domain is an affinity-modified IgSF domain that exhibits altered binding properties as compared to the binding properties of an unmodified or wild-type IgSF domain of an IgSF family member to one or more of its cognate binding partners, for the same one or more cognate binding partners. In some embodiments, the IgSF domain exhibits improved binding properties as compared to the binding properties of an unmodified or wild-type IgSF domain of an IgSF family member to one or more of its cognate binding partners, for the same one or more cognate binding partners.

[0023] In some embodiments, the IgSF domain of the second polypeptide is a tumor-localizing moiety that binds to a ligand expressed on the tumor, 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 immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant CD86 polypeptide or the second polypeptide. In some embodiments, the immunomodulatory protein described herein further comprises a third polypeptide comprising an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, the affinity-modified IgSF domain comprising 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 a variant CD86 polypeptide, a second polypeptide, and / or a third polypeptide. In some embodiments, the multimerization domain is the Fc domain of an immunoglobulin, optionally, 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 an amino acid sequence shown in SEQ ID NO:229 or an amino acid sequence showing 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 shown in SEQ ID NO:229. In some embodiments, the Fc domain is a variant IgG1 containing one or more amino acid substitutions, and 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 by Kabat). In some embodiments, the Fc domain contains the amino acid substitution N297G, the amino acid substitution R292C / N297G / V302C, or the amino acid substitution 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, and the residue is numbered according to the EU index of Kabat.In some embodiments, the Fc region contains K447del, and the residue is numbered according to the Kabat EU index. The Fc region may also be referred to herein as the Fc domain.

[0025] In some embodiments, the Fc domain contains the amino acid sequence set forth in SEQ ID NO: 230, or shows 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 as 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] As used herein, an immunomodulatory protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one IgSF domain linked via a linker to a first Fc domain, the at least one IgSF domain comprising one or both of: a variant CD86 polypeptide which is any of the variant CD86 polypeptides provided herein, or an IgSF domain of a PD1 polypeptide or a variant thereof; the second polypeptide comprises at least one IgSF linked via a linker to a second Fc domain, the at least one IgSF domain comprising one or both of: a variant CD86 polypeptide which is any of the variant CD86 polypeptides provided herein, or an IgSF domain of a PD1 polypeptide or a variant thereof, and the immunomodulatory protein comprises at least one IgSF domain of CD86 and at least one IgSF domain of PD-1 or a variant thereof, there is provided an immunomodulatory protein.

[0027] In some parts of any of the provided aspects, 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 parts of any of the provided aspects, at least one IgSF domain of the second polypeptide comprises a variant PD1 polypeptide. In some parts of any of the provided aspects, at least one IgSF domain of the first polypeptide is a first IgSF domain, the first IgSF domain 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 parts of any of the provided aspects, the second IgSF domain of the first polypeptide comprises a variant PD1 polypeptide. In some parts of any of the provided aspects, at least one IgSF domain of the second polypeptide is a first IgSF domain, the first IgSF domain 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 parts of any of the provided aspects, 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 to the N-terminus or C-terminus of the first Fc domain via a linker; 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. 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 to the end to which the first IgSF domain is linked. 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 to the end to which the first IgSF domain is linked. In some of the provided embodiments, the linker independently comprises the sequence of SEQ ID NO: 222 or 224, and optionally, the linker comprises 1 to 4 repeat sequences of the sequence of SEQ ID NO: 222 or 224. In some 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 polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a homodimer. In some of the provided embodiments, the first and second polypeptides of the homodimer comprise, from left to right, a 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, optionally, the first and second Fc domains comprise a knob-into-hole mutation, 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 polypeptide and the second polypeptide 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, Fc-linker-variant CD86 polypeptide, or 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; the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO:351, 352, or 353.

[0033] Provided herein are conjugates comprising 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 part of any of the provided embodiments, the variant CD86 polypeptide is linked to the N-terminus or C-terminus of the V H or V L of an antibody. In some embodiments, the variant CD86 polypeptide linked to the N-terminus or C-terminus of the V H or V L of an antibody is any variant CD86 polypeptide provided herein. In some part of any of the provided embodiments, the antibody is an anti-HER2 antibody or an anti-EGFR antibody. In some part of any of the provided embodiments, the anti-HER2 antibody is pertuzumab. In some part of any of the provided embodiments, the variant CD86 polypeptide is linked to the N-terminus of the V H of pertuzumab, the C-terminus of the V H of pertuzumab, the N-terminus of the V L of pertuzumab, or the C-terminus of the V L of pertuzumab, optionally comprising the sequences of SEQ ID NO:342, 344, 343, or 345, respectively. In some part of any of the provided embodiments, the anti-EGFR antibody is panitumumab. In some part of any 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, optionally comprising the sequences of SEQ ID NO:348, 350, 349, or 351, respectively (or an anti-EGFR antibody).

[0035] Provided herein is a nucleic acid molecule encoding any of the conjugate that is a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a fusion protein described herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is cDNA.

[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 is a method for producing a protein containing a variant CD86 polypeptide, the method comprising introducing into a host cell the nucleic acid molecule described herein or the vector described herein 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 is a method for modifying a cell that expresses a variant CD86 polypeptide, the method comprising introducing into a host cell a nucleic acid molecule encoding a conjugate that is a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a fusion protein 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 the 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 can be secreted from the modified cell. In some embodiments, the protein contains neither a cytoplasmic signaling domain nor a transmembrane domain and / or is not expressed on the surface of the cell; and / or the protein, when expressed, can be secreted from the modified cell.

[0041] In some embodiments, the modified cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a CD4+ and / or CD8+ T cell. In some embodiments, the T cell is a regulatory T cell (Treg). In some embodiments, the modified cell is a primary cell. In some embodiments, the modified cell is a mammalian cell. In some embodiments, the modified cell is a human cell. In some embodiments, the modified cell further contains a chimeric antigen receptor (CAR). In some embodiments, the modified cell further contains a modified T cell receptor (TCR).

[0042] Provided herein are infectious substances 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 substance is a bacterium or a virus. In some embodiments, the infectious substance 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 substance described herein. In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterilized.

[0044] Provided herein are manufactured articles containing 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 a manufactured article described herein and instructions for use.

[0046] Provided herein is a method 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 substance described herein, or a pharmaceutical composition described herein.

[0047] Provided herein is a method of modulating an immune response in a subject, comprising the step of 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 is a method of treating a disease or condition in a subject in need thereof, comprising the step of administering a conjugate that is a variant CD86 polypeptide described herein, an immunomodulatory protein described herein, or a fusion protein described herein, a modified cell described herein, an infectious agent described herein, or a pharmaceutical composition described herein.

[0049] Provided herein is a method of treating a disease or condition in a subject in need thereof, comprising the step of 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 localization moiety is administered to the subject. In some embodiments, the tumor localization moiety is or contains a binding molecule that recognizes a tumor antigen. In some embodiments, the binding molecule contains an antibody or an antigen-binding fragment thereof, or contains a wild-type IgSF domain or a variant thereof.

[0051] In some embodiments, a pharmaceutical composition containing the immunomodulatory protein described herein or the conjugate described herein is administered to a subject. In some embodiments, a modified cell containing a variant CD86 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject, and optionally, the modified cell is 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. In 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, colorectal 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 variant CD86 polypeptide or immunomodulatory protein that is soluble 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 the variant CD86 polypeptide described herein or the immunomodulatory protein described herein is administered to a subject. In some embodiments, a modified cell containing a secretable variant CD86 polypeptide is administered to a subject, and optionally, the modified cell is any of the cells 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 an anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory nerve disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. In some embodiments, the disease or condition is selected from inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. [Inventive concept 1001] A variant CD86 polypeptide comprising an extracellular domain or an IgV domain or a specific binding fragment thereof, wherein the variant CD86 polypeptide comprises one or more amino acid modifications in a non - modified CD86 polypeptide or a specific binding fragment thereof corresponding to a position selected from positions 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, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224, based on the positions shown in SEQ ID NO:29 [Inventive concept 1002] The variant CD86 polypeptide of Inventive concept 1001, wherein the amino acid modification comprises an amino acid substitution, deletion, or insertion [Inventive concept 1003] The variant CD86 polypeptide of Inventive concept 1001 or Inventive concept 1002, wherein the non - modified CD86 polypeptide is a mammalian CD86 polypeptide or a specific binding fragment thereof [Inventive concept 1004] The variant CD86 polypeptide of Inventive concept 1003, wherein the non - modified CD86 polypeptide is a human CD86 polypeptide or a specific binding fragment thereof [Inventive concept 1005] The variant CD86 polypeptide according to any one of Inventive concepts 1001 - 1004, wherein the variant CD86 polypeptide comprises the extracellular domain of human CD86, and the one or more amino acid modifications are in one or more residues of the extracellular domain of the non - modified CD86 polypeptide [Inventive concept 1006] The non - modified CD86 polypeptide comprises (i) the amino acid sequence shown 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 that comprises an IgV domain or a specific binding fragment of the IgV domain The variant CD86 polypeptide according to any one of Inventive concepts 1001 - 1005 [Inventive concept 1007] The unmodified CD86 is a variant CD86 polypeptide of any one of inventions 1001 to 1006, comprising the amino acid sequence shown in SEQ ID NO: 29. [Invention 1008] The variant CD86 polypeptide of invention 1006, wherein the 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. [Invention 1009] The unmodified CD86 polypeptide is (i) the amino acid sequence shown 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 the IgV domain and is a variant CD86 polypeptide of any one of inventions 1001 to 1006 and invention 1008. [Invention 1010] The unmodified CD86 is a variant CD86 polypeptide of any one of inventions 1001 to 1006, comprising the amino acid sequence shown in SEQ ID NO: 123. [Invention 1011] The unmodified CD86 polypeptide is (i) the amino acid sequence shown 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 and is a variant CD86 polypeptide of any one of inventions 1001 to 1006, 1008, and 1009. [Invention 1012] The unmodified CD86 is a variant CD86 polypeptide of any one of inventions 1001 to 1006, 1008, 1009, and 1011, comprising the amino acid sequence shown in SEQ ID NO: 122. [Invention 1013] The specific binding fragment has a length of at least 50, 60, 70, 80, 90, 95 amino acids, or more; 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 and is a variant CD86 polypeptide of any one of inventions 1001 to 1012. [Invention 1014] A variant CD86 polypeptide of any of 1001 to 1013 of the present invention, 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. [1015 of the present invention] Said one or more amino acid modifications are TIFF0007713886000005.tif48164 One or more amino acid substitutions selected from, or conservative amino acid substitutions thereof, a variant CD86 polypeptide of any of 1001 to 1014 of the present invention. [1016 of the present invention] TIFF0007713886000006.tif106163 A variant CD86 polypeptide of any of 1001 to 1015 of the present invention, comprising one or more amino acid modifications selected from among [1017 of the present invention] Said one or more amino acid modifications are at position 25 and / or 90, a variant CD86 polypeptide of any of 1001 to 1014 of the present invention. [1018 of the present invention] Said one or more amino acid modifications include Q25L, H90Y, or H90L, a variant CD86 polypeptide of any of 1001 to 1014 and 1017 of the present invention. [1019 of the present invention] Said one or more amino acid modifications include modifications at position 25 and 90, a variant CD86 polypeptide of any of 1001 to 1014 and 1017 of the present invention. [1020 of the present invention] Said one or more amino acid modifications are selected from Q25L / H90Y or Q25L / H90L, the variant CD86 polypeptide of 1019 of the present invention. [1021 of the present invention] TIFF0007713886000007.tif106170 A variant CD86 polypeptide of any of 1001 to 1020 of the present invention, comprising one or more amino acid modifications selected from among [1022 of the present invention] A variant CD86 polypeptide of any of 1001 to 1021 of the present invention, comprising one or more amino acid modifications A13V / Q25L / H90L. [1023 of the present invention] A variant CD86 polypeptide of any of 1001 to 1022 of the present invention, comprising one or more amino acid modifications A13V / Q25L / H90L / S181P / L197M / S206T. [1024 of the present invention] A variant CD86 polypeptide of any of 1001 to 1021 of the present invention, comprising one or more amino acid modifications Q25L / H90L / K93T / M97L. [1025 of the present invention] A variant CD86 polypeptide of any of the present inventions 1001 to 1021 and 1024, comprising one or more amino acid modifications Q25L / H90L / K93T / M97L / T133A / S181P / D215V. [Present Invention 1026] A variant CD86 polypeptide of any of the present inventions 1001 to 1021 and 1024, comprising one or more amino acid modifications Q25L / Q86R / H90L. [Present Invention 1027] A variant CD86 polypeptide of any of the present inventions 1001 to 1021 and 1026, comprising one or more amino acid modifications Q25L / Q86R / H90L / N104S. [Present Invention 1028] A variant CD86 polypeptide of any of the present inventions 1001 to 1021, comprising one or more amino acid modifications I89V / H90L. [Present Invention 1029] A variant CD86 polypeptide of any of the present inventions 1001 to 1021 and 1028, comprising one or more amino acid modifications I89V / H90L / I193V. [Present Invention 1030] A variant CD86 polypeptide of any of the present inventions 1001 to 1021, comprising one or more amino acid modifications M60K / H90L. [Present Invention 1031] A variant CD86 polypeptide of any of the present inventions 1001 to 1021, comprising one or more amino acid modifications Q25L / F33I / H90L. [Present Invention 1032] A variant CD86 polypeptide of any of the present inventions 1001 to 1021, comprising one or more amino acid modifications Q25L / H90L / P185S. [Present Invention 1033] A variant CD86 polypeptide of any of the present inventions 1001 to 1032, comprising an amino acid sequence showing 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. [Present Invention 1034] A variant CD86 polypeptide of any of the present inventions 1001 to 1033, which specifically binds to the ectodomain of CD28 with improved affinity as compared to the binding of the unmodified CD86 to the same ectodomain. [Present Invention 1035] The variant CD86 polypeptide of the present invention 1034, wherein 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 or at least about 125-fold. [The present invention 1036] The variant CD86 polypeptide according to any one of the present inventions 1001 to 1035, which specifically binds to the extracellular domain of CTLA-4 with a reduced affinity as compared to the binding of the unmodified CD86 to the same extracellular domain. [The present invention 1037] The variant CD86 polypeptide of the present invention 1036, wherein the reduced binding affinity is reduced by 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] The variant CD86 polypeptide according to any one of the present inventions 1001 to 1037, wherein the variant CD86 polypeptide specifically binds to the extracellular domain of CTLA-4 with the same or equivalent binding affinity as the binding of the unmodified CD86 to the same extracellular domain, 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. [The present invention 1039] The variant CD86 polypeptide according to any one of the present inventions 1001 to 1038, which includes the entire extracellular domain. [The present invention 1040] An amino acid sequence shown in any of SEQ ID NOs: 85 to 121 or a specific binding fragment thereof, an amino acid sequence that shows at least 95% sequence identity to any of SEQ ID NOs: 85 to 121 and contains one or more of the amino acid modifications of each sequence number shown in any of SEQ ID NOs: 85 to 121, or a specific binding fragment thereof, a variant CD86 polypeptide of any of the present inventions 1001 to 1039. [The present invention 1041] An amino acid sequence shown in any of SEQ ID NOs: 141 to 177 or a specific binding fragment thereof, an amino acid sequence that shows at least 95% sequence identity to any of SEQ ID NOs: 141 to 177 and contains one or more of the amino acid modifications of each sequence number shown in any of SEQ ID NOs: 141 to 177, or a specific binding fragment thereof, a variant CD86 polypeptide of any of the present inventions 1001 to 1040. [The present invention 1042] The variant CD86 polypeptide of any of the present inventions 1034 to 1041, wherein the CD28 is human CD28. [The present invention 1043] The variant CD86 polypeptide of any of the present inventions 1034 to 1042, wherein the CTLA-4 is human CTLA-4. [The present invention 1044] The variant CD86 polypeptide of any of the present inventions 1001 to 1043, which is a soluble protein. [The present invention 1045] Lacking the CD86 transmembrane domain and intracellular signaling domain; and / or Unable to be expressed on the surface of cells, The variant CD86 polypeptide of any of the present inventions 1001 to 1044. [The present invention 1046] The variant CD86 polypeptide of any of the present inventions 1001 to 1045, which is linked to a multimerization domain. [The present invention 1047] The variant CD86 polypeptide of the present invention 1046, wherein the multimerization domain is an Fc domain or a variant thereof with reduced effector function. [The present invention 1048] The variant CD86 polypeptide of any of the present inventions 1001 to 1047, which is linked to an Fc domain or linked to a variant thereof with reduced effector function. [The present invention 1049] The variant CD86 polypeptide of the present invention 1047 or the present invention 1048, wherein the Fc domain is human IgG1 or a variant thereof with reduced effector function. [The present invention 1050] The variant CD86 polypeptide of any one of the present inventions 1047 to 1049, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 229 or an amino acid sequence showing 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 1051] The variant CD86 polypeptide of any one of the present inventions 1047 to 1050, wherein the Fc domain is the amino acid sequence shown in SEQ ID NO: 229 or comprises the same. [The present invention 1052] The variant CD86 polypeptide of any one of the present inventions 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 according to EU numbering. [The present invention 1053] The variant CD86 polypeptide of any one of the present inventions 1047 to 1050 and 1052, wherein the Fc domain comprises the amino acid modifications L234A / L235E / G237A. [The present invention 1054] The variant CD86 polypeptide of any one of the present inventions 1047 to 1050, 1052, and 1053, wherein the Fc domain comprises the amino acid modification C220S according to EU numbering. [The present invention 1055] The variant CD86 polypeptide of any one of the present inventions 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] The Fc domain of any of the variant CD86 polypeptides of aspects 1047-1050 and 1052-1055 of the present invention comprises the amino acid sequence shown in SEQ ID NO:230, or shows 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 comprises an amino acid sequence containing one or more of each amino acid modification shown in SEQ ID NO:230 as compared to human IgG1. [Aspect 1057] The Fc domain of any of the variant CD86 polypeptides of aspects 1047-1050 and 1052-1056 of the present invention is or comprises the amino acid sequence shown in SEQ ID NO:230. [Aspect 1058] Any of the variant CD86 polypeptides of aspects 1047-1057 of the present invention, wherein the multimerization domain or Fc is indirectly linked via a linker, optionally a G4S linker. [Aspect 1059] The variant CD86 polypeptide is a transmembrane immunomodulatory protein further comprising a transmembrane domain, optionally wherein the transmembrane domain is directly or indirectly linked to the extracellular domain (ECD) of the variant CD86 polypeptide or a specific binding fragment thereof, any of the variant CD86 polypeptides of aspects 1001-1043 of the present invention. [Aspect 1060] The transmembrane domain of the variant CD86 polypeptide of aspect 1059 comprises the amino acid sequence shown as residues 248-268 of SEQ ID NO:2, or comprises a functional variant thereof showing at least 85% sequence identity to residues 248-268 of SEQ ID NO:2. [Aspect 1061] The variant CD86 polypeptide further comprises a cytoplasmic domain, optionally wherein the cytoplasmic domain is directly or indirectly linked to the transmembrane domain, the variant CD86 polypeptide of aspect 1059 or aspect 1060 of the present invention. [Aspect 1062] The cytoplasmic domain of the variant CD86 polypeptide of aspect 1061 is or comprises the native CD86 cytoplasmic domain. [Aspect 1063] The variant CD86 polypeptide of the present invention 1061 or the present invention 1062, wherein the cytoplasmic domain comprises the amino acid sequence shown as residues 269 to 329 of SEQ ID NO:2, or a functional variant thereof showing at least 85% sequence identity to residues 269 to 329 of SEQ ID NO:2. [The present invention 1064] The variant CD86 polypeptide of the present invention 1061, wherein the cytoplasmic domain comprises an ITAM signaling motif and / or is or comprises the intracellular signaling domain of CD3ζ. [The present invention 1065] The variant CD86 polypeptide of the present invention 1059 or the present invention 1060, which does not contain a cytoplasmic signaling domain and / or cannot mediate or regulate an intracellular signal when expressed on the cell surface. [The present invention 1066] An immunomodulatory protein comprising a first variant CD86 polypeptide of any one of the present inventions 1001 to 1058 and a second variant CD86 polypeptide of any one of the present inventions 1001 to 1058. [The present invention 1067] The immunomodulatory protein of the present invention 1066, wherein the first and second variant CD86 polypeptides are indirectly linked via a linker. [The present invention 1068] The immunomodulatory protein of the present invention 1066 or the present 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. [The present invention 1069] The immunomodulatory protein of the present invention 1068, wherein the multimer is a dimer, optionally a homodimer. [The present invention 1070] The immunomodulatory protein of any one of the present inventions 1066 to 1069, wherein the first variant CD86 polypeptide and the second variant CD86 polypeptide are the same. [The present invention 1071] An immunomodulatory protein comprising a variant CD86 polypeptide of any one of the present inventions 1001 to 1058 directly or indirectly linked via a linker to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of an IgSF family member. [The present invention 1072] The immunomodulatory protein of the present invention 1071, wherein the IgSF domain is an affinity-modified IgSF domain, and the affinity-modified IgSF domain contains one or more amino acid modifications as compared with the unmodified or wild-type IgSF domain of an IgSF family member. [The present invention 1073] The immunomodulatory protein of the present invention 1072, wherein the IgSF domain is an affinity-modified IgSF domain, and the affinity-modified IgSF domain exhibits altered binding properties as compared with the binding properties of the unmodified or wild-type IgSF domain of the IgSF family member to one or more of its cognate binding partners. [The present invention 1074] The immunomodulatory protein of the present invention 1073, wherein the IgSF domain exhibits enhanced binding properties as compared with the binding properties of the unmodified or wild-type IgSF domain of the IgSF family member to one or more of its cognate binding partners. [The present invention 1075] The immunomodulatory protein according to any one of the present inventions 1071 to 1074, wherein the IgSF domain of the second polypeptide is a tumor localization moiety that binds to a ligand expressed on a tumor or a tumor localization moiety that binds to a ligand expressed on a tumor, or an inflammation localization moiety that binds to a cell or tissue associated with an inflammatory environment. [The present invention 1076] The immunomodulatory polypeptide of the present invention 1075, wherein the ligand is B7H6. [The present invention 1077] The immunomodulatory polypeptide of the present invention 1075 or the present invention 1076, wherein the IgSF domain is derived from NKp30. [The present invention 1078] The immunomodulatory protein according to any one of the present inventions 1071 to 1077, further comprising a multimerization domain linked to at least one of the variant CD86 polypeptide or the second polypeptide. [The present invention 1079] The immunomodulatory protein according to any one of the present inventions 1071 to 1078, further comprising a third polypeptide containing an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, and the affinity-modified IgSF domain contains one or more amino acid modifications as compared with the unmodified or wild-type IgSF domain of the IgSF family member. [The present invention 1080] Is the third polypeptide the same as the first and / or second polypeptide; or Is the third polypeptide different from the first and / or second polypeptide, The immunomodulatory protein of the present invention 1079. [The present invention 1081] The immunomodulatory protein of the present invention 1079 or the present invention 1080, further comprising a multimerization domain linked to at least one of the variant CD86 polypeptide, the second polypeptide, and / or the third polypeptide. [The present invention 1082] The immunomodulatory protein according to any one of the present invention 1068 to 1070, 1078, and 1081, wherein the multimerization domain is an Fc domain of an immunoglobulin, optionally the immunoglobulin protein is of human origin, and / or the Fc domain is of human origin. [The present invention 1083] The immunomodulatory protein of the present invention 1082, wherein the Fc domain is IgG1, IgG2, or IgG4, or a variant thereof with reduced effector function. [The present invention 1084] The immunomodulatory protein of the present invention 1083, wherein the Fc domain is an IgG1 Fc domain, optionally human IgG1, or a variant thereof with reduced effector function. [The present invention 1085] The immunomodulatory protein according to any one of the present invention 1082 to 1084, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 229, or an amino acid sequence showing 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 according to any one of the present invention 1082 to 1085, wherein the Fc domain is the amino acid sequence shown in SEQ ID NO: 229 or comprises it. [The present invention 1087] The immunomodulatory protein of the present invention 1084 or the present invention 1085, wherein the Fc domain is a variant IgG1 containing one or more amino acid substitutions, and 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 by Kabat. [The present invention 1088] The immunomodulatory protein of the present invention 1087, wherein the Fc domain comprises the amino acid substitution N297G, the amino acid substitution R292C / N297G / V302C, or the amino acid substitution L234A / L235E / G237A, each of which is numbered according to the Kabat EU index. [The present invention 1089] The immunomodulatory protein of the present invention 1087 or the present invention 1088, wherein the variant Fc domain further comprises the amino acid substitution C220S, and the residue is numbered according to the Kabat EU index. [The present invention 1090] The immunomodulatory protein of any one of the present inventions 1087 to 1089, wherein the Fc domain comprises K447del, and the residue is numbered according to the Kabat EU index. [The present invention 1091] The immunomodulatory protein of any one of the present inventions 1084, 1085 and 1087 to 1090, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 230, or shows 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 shown in SEQ ID NO: 230 compared to human IgG1. [The present invention 1092] The immunomodulatory protein of any one of the present inventions 1084, 1085 and 1087 to 1091, wherein the Fc domain is or comprises the amino acid sequence shown in SEQ ID NO: 230. [The present invention 1093] 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, and the at least one IgSF domain comprises one or both of the following: a variant CD86 polypeptide of any one of the present inventions 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 to a second Fc domain via a linker, and the at least one IgSF domain comprises one or both of the following: any variant CD86 polypeptide of the present invention from 1001 to 1046, or the 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. [Inventive item 1094] The immunomodulatory protein of Inventive item 1093, wherein at least one IgSF domain of the first polypeptide comprises any variant CD86 polypeptide of the present invention from 1001 to 1046. [Inventive item 1095] The immunomodulatory protein of Inventive item 1093 or Inventive item 1094, wherein at least one IgSF domain of the second polypeptide comprises a variant PD1 polypeptide. [Inventive item 1096] The immunomodulatory protein of any one of Inventive items 1093 to 1095, wherein at least one IgSF domain of the first polypeptide is a first IgSF domain, the first IgSF domain is any variant CD86 polypeptide of the present invention from 1001 to 1046, and the first polypeptide comprises a second IgSF domain linked to the first Fc domain via a linker. [Inventive item 1097] The immunomodulatory protein of Inventive item 1096, wherein the second IgSF domain of the first polypeptide comprises a variant PD1 polypeptide. [Inventive item 1098] The immunomodulatory protein of any one of Inventive items 1093 to 1097, wherein at least one IgSF domain of the second polypeptide is a first IgSF domain, the first IgSF domain is any variant CD86 polypeptide of the present invention from 1001 to 1046, and the second polypeptide comprises a second IgSF domain linked to the second Fc domain via a linker. [Inventive item 1099] The immunomodulatory protein of Inventive item 1098, wherein the second IgSF domain of the second polypeptide comprises a variant PD1 polypeptide. [Inventive item 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. The immunomodulatory protein according to any one of 1093 to 1099 of the present invention. [Inventive Item 1101] The immunomodulatory protein according to any one of 1096 to 1097 of the present invention, 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. [Inventive Item 1102] The immunomodulatory protein according to any one of 1098 to 1101 of the present invention, 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. [Inventive Item 1103] The immunomodulatory protein according to any one of 1093 to 1102 of the present invention, wherein the linker independently contains the sequence of SEQ ID NO: 222 or 224, and optionally, the linker contains 1 to 4 repeat sequences of the sequence of SEQ ID NO: 222 or 224. [Inventive Item 1104] The immunomodulatory protein according to any one of 1093 to 1103 of the present invention, wherein the first Fc domain and the second Fc domain are identical, and optionally, the first Fc domain and the second Fc domain contain the sequence of SEQ ID NO: 230. [Inventive Item 1105] The immunomodulatory protein according to any one of 1093 to 1104 of the present invention, wherein the first polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a homodimer. [Inventive Item 1106] The immunomodulatory protein according to any one of 1093 to 1104 and 1105 of the present invention, wherein the first and second polypeptides of the homodimer contain, from left to right, a variant PD1 polypeptide - linker - Fc - linker - variant CD86 polypeptide. [Inventive Item 1107] The immunomodulatory protein according to any one of 1093 to 1104 and 1105 to 1106 of the present invention, wherein the variant PD1 polypeptide contains the sequence of SEQ ID NO: 315. [Inventive Item 1108] The immunomodulatory protein according to any one of aspects 1093 to 1104 and 1105 to 1107 of the present invention, wherein the variant CD86 polypeptide comprises the sequence of SEQ ID NO: 94 or 150. [Aspect 1109] The immunomodulatory protein according to any one of aspects 1093 to 1104 and 1105 to 1108 of the present invention, wherein the first and second polypeptides of the homodimer each comprise the sequence of SEQ ID NO: 348 or 349. [Aspect 1110] The immunomodulatory protein according to any one of aspects 1093 to 1103 of the present invention, wherein the first Fc domain and the second Fc domain are different, optionally the first and second Fc domains comprise a knob-into-hole mutation, 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. [Aspect 1111] The immunomodulatory protein according to any one of aspects 1093 to 1103 and 1110 of the present invention, wherein the first polypeptide and the second polypeptide dimerize through the first and second Fc domains to form a heterodimer. [Aspect 1112] The immunomodulatory protein according to any one of aspects 1093 to 1103, 1110, and 1111 of the present invention, 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, Fc-linker-variant CD86 polypeptide, or variant PD1-linker-Fc-linker-variant CD86. [Aspect 1113] The immunomodulatory protein according to any one of aspects 1093 to 1103, 1110, and 1111 to 1112 of the present invention, wherein the variant PD1 polypeptide comprises the sequence of SEQ ID NO: 315. [Aspect 1114] The immunomodulatory protein according to any one of aspects 1093 to 1103, 1110, and 1111 to 1113 of the present invention, wherein the variant CD86 polypeptide comprises the sequence of SEQ ID NO: 94 or 150. [Aspect 1115] The first polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 350; and wherein the second polypeptide of the heterodimer comprises the sequence of SEQ ID NO: 351, 352, or 353 Any immunomodulatory protein of the present invention from 1093 to 1103, 1110, and 1111 to 1114 [Inventive item 1116] A conjugate comprising any variant CD86 polypeptide of the present invention from 1001 to 1065 linked to a targeting moiety that specifically binds to a molecule on the surface of a cell [Inventive item 1117] The conjugate of inventive item 1116, wherein the cell is an immune cell or a tumor cell [Inventive item 1118] The conjugate of inventive item 1116 or 1117, wherein the moiety is a protein, peptide, nucleic acid, small molecule or nanoparticle [Inventive item 1119] The conjugate of any of inventive items 1116 to 1118, wherein the moiety is an antibody or an antigen-binding fragment [Inventive item 1120] wherein the variant CD86 polypeptide is linked to the N-terminus or C-terminus of the V H or V L of the antibody, the conjugate of inventive item 1119 [Inventive item 1121] The conjugate of inventive item 1119, wherein the antibody is an anti-HER2 antibody or an anti-EGFR antibody [Inventive item 1122] The conjugate of inventive item 1121, wherein the anti-HER2 antibody is pertuzumab [Inventive item 1123] wherein the variant CD86 polypeptide is linked to the N-terminus of V H of pertuzumab, the C-terminus of V H of pertuzumab, the N-terminus of V L of pertuzumab, or the C-terminus of V L of pertuzumab, and optionally comprises the sequence of SEQ ID NO: 342, 344, 343, or 345 respectively, the conjugate of inventive item 1122 [Inventive item 1124] The conjugate of inventive item 1121, wherein the anti-EGFR antibody is panitumumab [Inventive item 1125] wherein the variant CD86 polypeptide is linked to the N-terminus of V H of panitumumab, the C-terminus of V H of panitumumab, the N-terminus of V L of panitumumab, or the C-terminus of V L of panitumumab, and optionally comprises the sequence of SEQ ID NO: 348, 350, 349, or 351 respectively, the conjugate of inventive item 1124. Alternatively, an anti-EGFR antibody [Inventive item 1126] The conjugate of any of inventive items 1116 to 1125, which is a fusion protein [Inventive item 1127] A nucleic acid molecule encoding a conjugate that is any variant CD86 polypeptide of the present invention from 1001 to 1065, any immunomodulatory protein of the present invention from 1066 to 1115, or any fusion protein of the present invention from 1116 to 1126. [The present invention 1128] A vector comprising the nucleic acid molecule of the present invention 1127. [The present invention 1129] A cell comprising the vector of the present invention 1128. [The present invention 1130] A method for producing a protein comprising a variant CD86 polypeptide, the method comprising introducing the nucleic acid molecule of the present invention 1127 or the vector of the present invention 1128 into a host cell under conditions such that the protein is expressed in the host cell. [The present invention 1131] The method of the present invention 1130, further comprising the step of isolating or purifying the protein from the cell. [The present invention 1132] A method for modifying a cell expressing a variant CD86 polypeptide, the method comprising introducing into a host cell a nucleic acid molecule encoding a conjugate that is any variant CD86 polypeptide of the present invention from 1001 to 1065, any immunomodulatory protein of the present invention from 1066 to 1115, or any fusion protein of the present invention from 1116 to 1126 under conditions such that the polypeptide is expressed in the host cell. [The present invention 1133] A modified cell comprising a conjugate that is any variant CD86 polypeptide of the present invention from 1001 to 1065, any immunomodulatory protein of the present invention from 1066 to 1115, or any fusion protein of the present invention from 1116 to 1126, the nucleic acid molecule of the present invention 1127, or the vector of the present invention 1128. [The present invention 1134] The variant CD86 polypeptide comprises a transmembrane domain or is any transmembrane immunomodulatory protein of the present invention from 1059 to 1065; and / or The protein comprising the variant CD86 polypeptide is expressed on the surface of the cell, The modified cell of the present invention 1133. [The present 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 can be secreted from the modified cell, The modified cell of the present invention 1133. [The present invention 1136] The modified cell according to any one of the present inventions 1133 to 1135, which is an immune cell. [The present invention 1137] The modified cell of the present invention 1136, wherein the immune cell is a lymphocyte, and optionally the lymphocyte is a T cell. [The present invention 1138] The modified cell according to any one of the present inventions 1133 to 1137, which is a primary cell. [The present invention 1139] The modified cell according to any one of the present inventions 1133 to 1138, further comprising a chimeric antigen receptor (CAR). [The present invention 1140] The modified cell according to any one of the present inventions 1133 to 1139, further comprising a modified T cell receptor (TCR). [The present invention 1141] An infectious substance comprising a conjugate which is any one of the variant CD86 polypeptides of the present inventions 1001 to 1065, any one of the immunomodulatory proteins of the present inventions 1066 to 1115, or any one of the fusion proteins of the present inventions 1116 to 1126, the nucleic acid molecule of the present invention 1127, or the vector of the present invention 1128. [The present invention 1142] The infectious substance of the present invention 1141, which is a bacterium or a virus. [The present invention 1143] The infectious substance of the present invention 1142, wherein the infectious substance is a virus and the virus is an oncolytic virus. [The present invention 1144] A pharmaceutical composition comprising any one of the variant CD86 polypeptides of the present inventions 1001 to 1065, any one of the immunomodulatory proteins of the present inventions 1066 to 1115, or any one of the fusion proteins of the present inventions 1116 to 1126, a conjugate, a modified cell according to any one of the present inventions 1133 to 1140, or an infectious substance according to any one of the present inventions 1141 to 1143. [The present invention 1145] The pharmaceutical composition of the present invention 1144, comprising a pharmaceutically acceptable excipient. [The present invention 1146] An article of manufacture comprising the pharmaceutical composition according to any one of the present inventions 1144 to 1145 in a vial or container. [The present invention 1147] A kit comprising the pharmaceutical composition according to any one of the present inventions 1144 to 1145 or the article of manufacture of the present invention 1146 and instructions for use. [The present invention 1148] A method for modulating an immune response in a subject, comprising administering a conjugate that is any one of the variant CD86 polypeptides of the present invention from 1001 to 1065, any one of the immunomodulatory proteins of the present invention from 1066 to 1115, or any one of the fusion proteins of the present invention from 1116 to 1126, any one of the modified cells of the present invention from 1133 to 1140, any one of the infectious agents of the present invention from 1141 to 1143, or any one of the pharmaceutical compositions of the present invention from 1144 to 1145. [The present invention 1149] A method for modulating an immune response in a subject, comprising administering any one of the modified cells of the present invention from 1133 to 1140. [The present invention 1150] The method of the present invention 1149, wherein the modified cell is autologous to the subject. [The present invention 1151] The method of the present invention 1149, wherein the modified cell is allogeneic to the subject. [The present invention 1152] Any one of the methods of the present invention from 1148 to 1151, wherein modulating the immune response treats a disease or condition in the subject. [The present invention 1153] A method for treating a disease or condition in a subject in need thereof, comprising administering a conjugate that is any one of the variant CD86 polypeptides of the present invention from 1001 to 1065, any one of the immunomodulatory proteins of the present invention from 1066 to 1115, or any one of the fusion proteins of the present invention from 1116 to 1126, any one of the modified cells of the present invention from 1133 to 1140, any one of the infectious agents of the present invention from 1141 to 1143, or any one of the pharmaceutical compositions of the present invention from 1144 to 1145. [The present invention 1154] A method for treating a disease or condition in a subject in need thereof, comprising administering any one of the modified cells of the present invention from 1133 to 1140. [The present invention 1155] The method of the present invention 1154, wherein the modified cell is autologous to the subject. [The present invention 1156] The method of the present invention 1154, wherein the modified cell is allogeneic to the subject. [The present invention 1157] Any one of the methods of the present invention from 1148 to 1156, wherein the immune response is enhanced in the subject. [The present invention 1158] Any one of the methods of the present invention 1148, 1152, 1153, and 1157, wherein an immunomodulatory protein or conjugate comprising a variant CD86 polypeptide linked to a tumor localization site is administered to the subject. [The present invention 1159] The method of the present invention 1158, wherein the tumor-localized portion is a binding molecule that recognizes a tumor antigen or comprises the same. [The present invention 1160] The binding molecule comprises an antibody or an antigen-binding fragment thereof, or a wild-type IgSF domain or a variant thereof, and optionally comprises an anti-HER2 antibody or an antigen-binding fragment thereof or an anti-EGFR antibody or an antigen-binding fragment thereof; 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 an IgSF domain of PD-1 or Nkp30. The method of the present invention 1159. [The present invention 1161] The method according to any one of the present inventions 1148 and 1152 to 1160, wherein a pharmaceutical composition comprising any one of the immunomodulatory proteins of the present inventions 1071 to 1115 or any one of the conjugates of the present inventions 1116 to 1126 is administered to the subject. [The present invention 1162] The method according to any one of the present inventions 1148 to 1160, wherein a modified cell comprising a variant CD86 polypeptide, which is a transmembrane immunomodulatory protein, is administered to the subject, and optionally, the modified cell is one of the present inventions 1133, 1134, and 1136 to 1140. [The present invention 1163] The method according to any one of the present inventions 1152 to 1162, wherein the disease or condition is a tumor or cancer. [The present invention 1164] The method according to any one of the present inventions 1152 to 1163, wherein the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancy, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, stomach cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer. [The present invention 1165] The method according to any one of the present inventions 1148 to 1156, wherein the immune response is reduced. [The present invention 1166] The method according to any one of the present inventions 1148, 1152, 1153, and 1165, wherein a soluble variant CD86 polypeptide or an immunomodulatory protein is administered to the subject. [The present invention 1167] The method of the present invention 1166, wherein the soluble polypeptide or immunomodulatory protein is an Fc fusion protein. [The present invention 1168] A method according to any one of the present invention 1148, 1152, 1153, and 1165 - 1167, wherein a pharmaceutical composition comprising a variant CD86 polypeptide of any one of the present invention 1001 - 1058, or an immunomodulatory protein of any one of the present invention 1066 - 1074 and 1078 - 1115 is administered to the subject. [The present invention 1169] A method according to any one of the present invention 1148, 1152, 1153, and 1165, wherein a modified cell comprising a secretable variant CD86 polypeptide is administered to the subject, and optionally, the modified cell is any one of the present invention 1133 and 1135 - 1140. [The present invention 1170] A method according to any one of the present invention 1148, 1152, 1153, and 1165 - 1169, wherein the disease or condition is an inflammatory or autoimmune disease or condition. [The present invention 1171] A method according to any one of the present invention 1148, 1152, 1153, and 1165 - 1169, wherein the disease or condition is anti - neutrophil cytoplasmic antibody (ANCA) - associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory digestive disease, inflammatory eye disease, inflammatory nerve disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. [The present invention 1172] A method according to the present invention 1170 or 1171, wherein the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

Brief Description of the Drawings

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

[0058] In some embodiments, the variant CD86 polypeptide exhibits increased binding affinity for CD28 compared to an unmodified or wild-type CD86 that does not contain one or more modifications. In some embodiments, the variant CD86 polypeptide exhibits at least increased binding affinity for CD28 compared to an unmodified or wild-type CD86 that does not contain one or more modifications. In some embodiments, the binding affinity is altered (e.g., increased) 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 greater compared to an 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 not greater than that of the 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 changed (e.g., reduced) 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 as compared to the unmodified or wild-type CD86 that does not contain one or more modifications.

[0060] In some embodiments, the variant CD86 polypeptide and the immunomodulatory protein modulate an immunological immune response, e.g., enhance or reduce the immune response. The particular modulation can be based on the format of the variant CD86 polypeptide and depends on whether a particular format provides antagonist or blocking activity or agonist activity. Also provided are various immunomodulatory protein formats of the variant polypeptide provided herein. As shown herein, the selectable formats can facilitate the manipulation of the immune response and thus therapeutic applications. The ability to format the variant polypeptide in various forms to antagonize (antagonize) or stimulate (agonize) the immune response depending on the situation provides flexibility in therapeutic applications based on the similarly improved binding and activity of the variant CD86 to its binding partner. As an example, tethering the variant CD86 protein to a surface can transmit a localized co-stimulatory signal, while in other cases, presenting CD86 in a non-localized soluble form can confer antagonist activity. In some embodiments, the variant CD86 polypeptides and immunomodulatory proteins provided herein can be used in the treatment of diseases or conditions associated with dysregulated immune responses.

[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 the variant CD86 polypeptide provided herein and one or more other moieties or polypeptides. In some aspects, modified cells containing a transmembrane immunomodulatory protein or a secretable immunomodulatory protein are provided. In some aspects, an infectious agent is provided that can deliver a transmembrane immunomodulatory protein or a secretable immunomodulatory protein to a cell (where 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 the variant CD86 polypeptide provided herein and one or more other moieties or polypeptides.

[0062] In some embodiments, the variant CD86 polypeptide is provided in a format that exhibits agonist activity for its cognate binding partner CD28 and / or stimulates or initiates costimulatory signaling via CD28. Among the immunomodulatory protein formats included in such formats are modified cells that express the variant CD86 polypeptide as a transmembrane immunomodulatory protein. In other cases, the immunomodulatory format can include a fusion with another molecule, for example, certain "stacked molecules" with other IgSF domains, including a tumor localization domain (e.g., the vCD86-NkP30 construct), and certain "stacked molecules" with an antibody conjugate format (e.g., the vCD86-anti-HER2 or vCD86-anti-HER1 construct). Such variant CD86 immunomodulatory proteins and their formats (e.g., modified cells or fusion constructs) can be used to treat cancer, viral infection, or bacterial infection. In some embodiments, the variant CD86 immunomodulatory proteins and their formats (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, for example, in a primary T cell assay. In some aspects, T cell activity can be evaluated by assessing the production of cytokines such as IL-2, IFN-γ, or TNFα.In some instances, the increase (e.g., an increase in IFN-γ, IL-2, or TNFα) is an increase of 1.1-fold or more, or about 1.1-fold or more, 1.2-fold or more, or about 1.2-fold or more, 1.3-fold or more, or about 1.3-fold or more, 1.4-fold or more, or about 1.4-fold or more, 1.5-fold or more, or about 1.5-fold or more, 1.6-fold or more, or about 1.6-fold or more, 1.7-fold or more, or about 1.7-fold or more, 1.8-fold or more, or about 1.8-fold or more, 1.9-fold or more, or about 1.9-fold or more, 2.0-fold or more, or about 2.0-fold or more, 2.5-fold or more, or about 2.5-fold or more, 3.0-fold or more, or about 3.0-fold or more, 3.5-fold or more, or about 3.5-fold or more, 4.0-fold or more, or about 4.0-fold or more, 5.0-fold or more, or about 5.0-fold or more, 6.0-fold or more, or about 6.0-fold or more, 7.0-fold or more, or about 7.0-fold or more, 8.0-fold or more, or about 8.0-fold or more, 9.0-fold or more, or about 9.0-fold or more, 10.0-fold or more, or about 10.0-fold or more, or greater as compared to unmodified or wild-type CD86 that does not contain one or more modifications.

[0063] In some embodiments, the variant CD86 polypeptide is provided in a format that exhibits antagonist activity to its cognate binding partner CD28 and / or blocks or inhibits co-stimulation signaling via CD28. Among those included in such immunomodulatory protein formats are variant CD86 polypeptides that are soluble (e.g., variant CD86-Fc fusion proteins). Such variant CD86 immunomodulatory proteins can be used to treat inflammatory or autoimmune disorders. In some embodiments, the variant CD86 immunomodulatory protein and its format (e.g., soluble variant CD86-Fc fusion protein) inhibit or block co-stimulation signaling, thereby resulting in decreased T cell activity (e.g., in vivo or in vitro) compared to wild-type or unmodified CD86 controls, for example, in a primary T cell assay. In some aspects, T cell activity can be evaluated by assessing the production of cytokines such as IL-2, IFN-γ or TNFα. In some aspects, the decrease, e.g., the decrease in IFN-γ, IL-2, TNFα, is 1.1-fold or more or about 1.1-fold or more, 1.2-fold or more or about 1.2-fold or more, 1.3-fold or more or about 1.3-fold or more, 1.4-fold or more or about 1.4-fold or more, 1.5-fold or more or about 1.5-fold or more, 1.6-fold or more or about 1.6-fold or more, 1.7-fold or more or about 1.7-fold or more, 1.8-fold or more or about 1.8-fold or more, 1.9-fold or more or about 1.9-fold or more, 2.0-fold or more or about 2.0-fold or more, 3.0-fold or more or about 3.0-fold or more, 4.0-fold or more or about 4.0-fold or more, 5.0-fold or more or about 5.0-fold or more, 6.0-fold or more or about 6.0-fold or more, 7.0-fold or more or about 7.0-fold or more, 8.0-fold or more or about 8.0-fold or more, 9.0-fold or more or about 9.0-fold or more, 10.0-fold or more or about 10.0-fold or more, or a greater decrease compared to unmodified or wild-type CD86 that does not contain one or more modifications.

[0064] In some embodiments, the provided variant CD86 polypeptides regulate T cell activation, expansion, differentiation, and survival through interaction with co-stimulatory signaling molecules. Generally, 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 co-stimulatory signal (e.g., CD28 co-stimulatory signal) for TCR engagement and is required to avoid T cell apoptosis or anergy.

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

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

[0067] Enhancement or suppression of the activity of the CD28 receptor and the CTLA-4 receptor has clinical significance for the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, treatments that intervene in and alter the co-stimulatory effect of both receptors are subject to the spatial localization requirements and size limitations imposed by the confinement of the immune synapse. In some aspects, existing therapeutic agents, including antibody drugs, cannot interact simultaneously with multiple target proteins involved in the regulation of these interactions. Additionally, in some cases, existing therapeutic agents only have the ability to antagonize the immune response and cannot have the ability to stimulate (agonize) the immune response. Additionally, differences in the pharmacokinetics between drugs that independently target one or the other of these two receptors can make it difficult to appropriately maintain the desired blood concentrations of such drug combinations throughout the treatment process. The provided variant CD86 polypeptides and immunomodulatory proteins, as well as the other formats described, address such problems. Also provided are methods for manufacturing and using these CD86 variant polypeptides and immunomodulatory proteins.

[0068] All publications (including patents, patent applications, scientific papers, and databases) referred to 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 papers, and databases) were specifically and individually indicated to be incorporated by reference. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control.

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

[0070] I.Definitions Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or related terms used in this specification 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 with commonly understood meanings are defined in this specification for clarity and / or for ready reference, and the inclusion of such definitions in this specification should not necessarily be construed as making a substantial difference from what is commonly understood in the art.

[0071] The terms used throughout this specification are defined as follows, unless otherwise limited in a particular instance. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Unless otherwise indicated, chemical and biochemical abbreviations and symbols are in accordance with the IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include not only the values defining the range but also all integer values therebetween.

[0072] The term "affinity-modified (affinity modification)" when used in the context of an immunoglobulin superfamily domain means a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence that has been changed such that the binding affinity or avidity for at least one of its cognate binding partners (or "counterstructures") is improved or decreased (compared to the corresponding wild-type or unmodified (i.e., non-affinity-modified) IgSF control domain). In this context, affinity-modified CD86 IgSF domains are included. In some embodiments, the affinity-modified IgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences (e.g., amino acid substitutions) from the 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 one of its cognate binding partners is a value that is at least 10% greater than the wild-type IgSF domain control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value. A decrease in the binding affinity or avidity of a protein for at least one of its cognate binding partners is a value that is 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type IgSF domain control value but 10% or more of the wild-type IgSF domain control value.A protein with modified affinity has an altered primary amino acid sequence due to substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain (affinity-modified IgSF domain)" should not be construed to impose any conditions of any particular starting composition or method by which the affinity-modified IgSF domain was made. Thus, the affinity-modified IgSF domains of the present invention are not limited to being converted from wild-type IgSF domains to affinity-modified IgSF domains by any particular affinity-modification process. Affinity-modified IgSF domain polypeptides can be generated, for example, starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its cognate binding partner, and finally recombinantly or chemically synthesized to generate the subject affinity-modified IgSF domain composition. As another example, an affinity-modified IgSF domain can be made by site-directed mutagenesis of a wild-type IgSF domain. Thus, an affinity-modified IgSF domain represents a product that can be produced by any given process but not necessarily by it. A variety of techniques may be used, including recombinant methods, chemical synthesis, or combinations thereof.

[0073] The term "allogeneic (of)" as used herein means a cell or tissue that is removed from one organism and then injected or transplanted into a genetically different organism of the same species. In some aspects of the present invention, the species is murine or human.

[0074] As used herein, the term "autologous (of)" means cells or tissues that are removed from the same organism and later injected or transplanted into said organism. Autologous cells or tissues can be modified, for example, by recombinant DNA methods, so that they are no longer genetically identical to the natural cells or natural tissues removed 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 includes modifying T cells or tumor-infiltrating lymphocytes (TILs). The modified cells are then injected into the patient from whom the natural T cells were isolated. In some embodiments, the organism is a human or a mouse.

[0075] As used herein, the terms "binding affinity" and "binding avidity" each refer to the specific binding affinity and specific binding avidity of a protein for its counterstructure under specific binding conditions. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of individual non-covalent interactions, such as between CD86 and its counterstructures CD28 and / or CTLA-4. Thus, avidity is different from affinity, which represents the strength of a single interaction. The improvement or decrease in the binding affinity of a variant CD86 containing an IgSF domain of modified affinity for its counterstructure is determined relative to the binding affinity of unmodified CD86 (e.g., unmodified CD86 containing a native or wild-type IgSF domain (e.g., 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, a variant CD86 (e.g., CD86 containing an affinity-modified IgSF domain) specifically binds to CD28 and / or CTLA-4 with a binding affinity that results in an average fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of the unmodified CD86 control in a binding assay when measured by flow cytometry. In some embodiments, a variant CD86 (e.g., one containing an affinity-modified IgSF domain) specifically binds to CD28 with a binding affinity that results in an average fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than that of the unmodified CD86 control in a binding assay when measured by flow cytometry, and exhibits a binding affinity for CTLA-4 that is unchanged or not greater compared to the unmodified CD86 control in the binding assay.In some embodiments, a variant CD86 (e.g., one containing an affinity-modified IgSF domain) specifically binds to CD28 with a binding affinity that results in an average fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of the unmodified CD86 control in a binding assay as measured by flow cytometry, and exhibits a reduced binding affinity for CTLA-4 as measured by flow cytometry, the binding affinity being at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than that of the unmodified CD86 control in the binding assay and resulting in an average fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than that of the unmodified CD86 control in the binding assay.

[0076] The term "biological half-life" refers to the time it takes 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. The biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic degradation) of the substance, or absorption and concentration in a particular organ or tissue in the body. In some embodiments, the biological half-life can be evaluated by determining the time it takes for the plasma concentration of the substance 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 peptide; 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 that contains 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 peptide bonds. 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). A CAR expressed on a T cell is referred to herein as a "CAR T cell" 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 clinically used in adoptive cell transfer, the CAR-T having antigen-binding specificity for a patient's tumor is typically modified to be expressed on natural T cells obtained from the patient. The modified T cells expressing the CAR are then returned to the patient by infusion. Thus, CAR-Ts are often autologous CAR-Ts, but allogeneic CAR-Ts are also included within the scope of the present invention. The ectodomain of the CAR contains an antigen-binding region (e.g., an antibody or an antigen-binding fragment thereof (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Specific binding results in a series of biochemical events (i.e., signal transduction) that lead to the modulation of the immunological activity of the CAR-T. Thus, for example, specific binding of the CAR-T's antigen-binding region to its target antigen can lead to changes in immunological activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction by CAR-T activation is achieved by the CD3ζ chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells. The CAR-T can further contain multiple signal transduction domains (e.g., CD28, 4-1BB, or OX40) that further regulate the immunomodulatory response of the T cell.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 term "collectively" or "collective" when used with respect to cytokine production induced by the presence of two or more variant CD86 polypeptides in an in vitro assay means the overall cytokine expression level regardless of cytokine production induced by an individual variant CD86 polypeptide. In some embodiments, the cytokine being 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 variant CD86) refers to at least one molecule (typically a native mammalian protein) to which the polypeptide being referred to 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 enhanced or reduced affinity. One type of ligand that is recognized under specific binding conditions and specifically binds to its cognate receptor is an example of a counterstructure or cognate binding partner of that receptor. A "cell surface cognate binding partner" is a cognate binding partner that is expressed on the surface of a mammalian cell. A "cell surface molecular species" is a cognate binding partner of a ligand of an immunological synapse (IS) that is expressed on or by a cell (e.g., a mammalian cell) that forms an immunological synapse.

[0080] As used herein, "conjugate", "conjugation" or grammatical variations thereof refer to connecting or linking two or more compounds together by any connection or linking method known in the art to result 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 bind to two cognate binding partners simultaneously. Generally, competitive binders contain the same or overlapping binding sites for specific binding, although this is not an essential requirement. In some embodiments, competitive binding causes a measurable (partial or complete) inhibition of the specific binding of the 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, such as ELISA (enzyme-linked immunosorbent assay), are known.

[0082] The term "conservative amino acid substitution" as used herein means an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids having 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. The groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0083] The term "corresponding" with respect to the position of a protein, e.g., a description that a nucleotide or amino acid position "corresponds" to a nucleotide or amino acid position in a disclosed sequence (e.g., as shown in a sequence listing), refers to the position of a nucleotide or amino acid identified by alignment with the disclosed sequence, based on a 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 by the structural alignment method described herein. By aligning the sequences, one of ordinary skill in the art can identify corresponding residues, e.g., using conserved amino acid residues and identical amino acid residues as a guide. 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 "decrease" or "attenuate" or "suppress", when used herein, mean a statistically significant amount of reduction or decrease. The reduction or decrease 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 covalently attaching the protein directly or indirectly to a composition to change 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 present invention are within the scope of the present invention and can be produced, for example, by glycosylation, pegylation, lipidation, or Fc fusion.

[0086] As used herein, detection includes methods that enable visualization of the protein (visually or by instrument). The protein can be visualized using an antibody specific for the protein. Also, detection of the protein can be facilitated by fusion of the protein with a tag that includes a detectable label or by contact of the protein with a second reagent (e.g., a secondary antibody) specific for the protein that includes a detectable label.

[0087] As used herein, a domain (typically, a sequence of 3 or more, generally 5 or 7 or more amino acids, e.g., 10 - 200 amino acid residues) refers to a portion of a molecule (e.g., a protein or a coding nucleic acid) that is structurally and / or functionally distinct from other parts of the molecule and is identifiable. For example, a domain can form a structure that folds independently within a protein composed of one or more structural motifs and / or includes a portion of a polypeptide chain that is recognized by a functional activity such as a 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., the ability to interact with a biomolecule such as a cognate binding partner). A domain can exhibit a biological function or activity independently so that it can perform an activity (e.g., binding) either independently or when fused to another molecule. A domain can be a linear amino acid sequence or a non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by one of ordinary skill in the art. Definitions are provided herein for purposes of illustration, 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 domains.

[0088] The term "ectodomain" as used herein refers to the region of a membrane protein (e.g., a transmembrane protein) that is outside of the vesicle membrane. The ectodomain often contains a binding domain that specifically binds to a ligand or a cell surface receptor, e.g., via a binding domain that specifically binds to the ligand or the cell surface receptor. The ectodomain of a transmembrane protein of a cell is alternatively referred to as the extracellular domain (ECD).

[0089] The terms "effective amount" or "therapeutically effective amount" refer to the amount and / or concentration of a therapeutic composition of the invention (including a protein composition or a cell composition) that, when administered ex vivo (by contact with cells derived from a patient) or in vivo (by administration to a patient), either alone (i.e., as monotherapy) or in combination with additional therapeutic agents, results in a statistically significant decrease in disease progression, for example, by ameliorating or eliminating the symptoms and / or etiology of a disease. An effective amount can be an amount that alleviates, reduces, or mitigates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the physical function of a patient. In the case of cell therapy, the effective amount is the effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments, the patient is a mammalian patient, such as a non-human primate or a human patient.

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

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

[0092] As used herein, the term "modified cell" refers to a mammalian cell that has been genetically modified by human intervention (e.g., recombinant DNA methods or viral transduction). In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., a T cell, B cell, NK cell) or an antigen-presenting cell (e.g., a 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 the variant CD86 of the invention that is modified to modulate the immune activity of a T cell expressing 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" herein) that contains an extracellular domain or a portion thereof containing an IgV domain linked to a transmembrane domain (e.g., the CD86 transmembrane domain), and optionally contains an intracellular signaling domain. In some cases, the TIP takes the form of a chimeric receptor containing a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the modified cell can express and secrete the immunomodulatory protein described herein. Among the modified cells provided, there are also cells that further contain a modified T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0093] As used herein, the term "modified T cell" refers to a T cell (e.g., a helper T cell, a cytotoxic T cell (or cytotoxic T lymphocyte or CTL), a natural killer T cell, a regulatory T cell, a memory T cell, or a γδ T cell) that has been genetically modified (altered) by human intervention (e.g., recombinant DNA methods or viral transduction). The modified T cell contains the variant CD86 transmembrane immunomodulatory protein (TIP) or secreted immunomodulatory protein (SIP) of the invention expressed on the T cell, and the TIP is modified to modulate the immune activity of the modified T cell itself or the immune activity 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 selected, cloned, and / or subsequently introduced into a population of T cells (which population is often used in adoptive immunotherapy) and modified to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen.

[0095] The term "expressed on" as used herein refers 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 protein being expressed is a transmembrane protein. In some embodiments, the protein is conjugated to a moiety (e.g., a drug or a detectable label). The protein expressed on the surface of the cell can include cell surface proteins (e.g., cell surface receptors) expressed on mammalian cells.

[0096] The term "half-life extension moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum as compared to the half-life of a protein not conjugated to such a portion. In some embodiments, the half-life is greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or is extended by 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, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 1 week after in vivo administration as compared to a protein having no half-life extension moiety. Half-life refers to the time it takes for a protein to lose half of its concentration, amount, or activity. The half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extension moieties include the 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 expressing MHC I (major histocompatibility complex) or MHC II (e.g., an antigen-presenting cell or tumor cell) 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 the Fc polypeptide) corresponds mainly to the constant region of the immunoglobulin heavy chain and is involved in various functions including the effector functions of antibodies. The Fc domain contains a part or all of the hinge domain of the immunoglobulin molecule and the CH2 and CH3 domains. 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 (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more reduced) activity to promote effector functions. In some embodiments, references to amino acid substitutions in the Fc region are by reference to the EU numbering system unless specified with reference to a particular 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, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0099] An immunoglobulin Fc fusion (an "Fc fusion"), for example an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. The Fc fusion may, for example, comprise the Fc region of an antibody (which in some cases promotes pharmacokinetics) and a variant CD86 polypeptide. The immunoglobulin Fc region may be linked indirectly or directly to one or more variant CD86 polypeptides or small molecules (fusion partners). A variety of linkers are known in the art and optionally these can be used to link the Fc to the fusion partner to generate an Fc fusion. The same type of Fc fusion can be dimerized to form an Fc fusion homodimer or non-identical types can be used to form an Fc fusion heterodimer. In some embodiments, the Fc is a mammalian Fc, such as 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. The host cell can be a prokaryote, such as, for example, Escherichia coli (E. coli), or the host cell can be a eukaryote, such as, for example, a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof, such as, for example, Veggie CHO, DG44, Expi CHO, or CHOZN, and related cell lines that grow in serum-free media, or a DHFR-deficient CHO line DX-B11. In some embodiments, the host cell may be a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell).

[0101] The term "immunoglobulin" (abbreviated as "Ig") as used herein refers to mammalian immunoglobulin proteins that include 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 fully or partially synthetic (e.g., recombinant or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V H and V L containing variable fragments (Fv), V H and V L containing single-chain variable fragments (scFv) linked together in one chain, as well as other antibody V-region fragments (Fab’, F(ab)2, F(ab’)2, dsFv diabodies, Fc, and Fd polypeptide fragments). Bispecific antibodies, both homodimeric and heterodimeric, are included within the scope of the term.

[0102] The term "immunoglobulin superfamily" or "IgSF" as used herein means a group of cell surface and soluble proteins that are involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features common to immunoglobulins (i.e., antibodies); all of these possess domains known as immunoglobulin domains or folds. Members of the IgSF 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 usually associated with roles in the immune system. Proteins in the immune synapse are often members of the IgSF. The IgSF can also be classified into "subfamilies" based on common properties such as function. Such subfamilies typically consist of 4 to 30 IgSF members.

[0103] The term "IgSF domain" or "immunoglobulin domain" or "Ig domain", as used herein, refers to the structural domain of an IgSF protein. Ig domains are named after the immunoglobulin molecule. They contain approximately 70-110 amino acids and are classified according to their size and function. Ig domains possess a characteristic Ig fold with a sandwich-like structure formed by two sheets of antiparallel β-strands. Interactions between the hydrophobic amino acids inside the sandwich and highly conserved disulfide bonds formed between cysteine residues in the B and F strands stabilize the Ig fold. One end of the Ig domain has a portion called the complementarity-determining region, which is important for the specificity of the antibody for its ligand. Ig-like domains can be classified (into classes) as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) domains or constant (IgC) domains. The IgV domain with nine β-strands is generally longer than the IgC domain with seven β-strands. The Ig domains of some members of the IgSF resemble the IgV domain in the amino acid sequence but still have a size similar to that of the IgC domain. These are called IgC2 domains, while the standard IgC domain is called the IgC1 domain. The T cell receptor (TCR) chain contains two Ig domains in the extracellular portion (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] As used herein, the term "IgSF species" means a population of IgSF member proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines an identity unique to all IgSF species to which that IgSF member belongs. 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 species of another IgSF family member. 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. In addition, small sequence differences within a single IgSF species due to genetic polymorphism also constitute differences in another form within a single IgSF species, similar to the wild-type truncated form of the IgSF species due to proteolytic cleavage, for example. "Cell surface IgSF species" are IgSF species that are expressed on the surface of cells (generally mammalian cells).

[0105] The term "immunoreactivity", 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 cell cytotoxic activity. In some cases, immunoreactivity can mean the expression of cytokines such as chemokines or interleukins. Assays for determining the enhancement or suppression of immunoreactivity include the MLR (mixed lymphocyte reaction) assay that measures cytokine levels such as interferon-γ or IL-2 in the culture supernatant (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). Since T cell activation is associated with the secretion of cytokines such as IFN-γ or IL-2 cytokines, the detection of such cytokine levels in the culture supernatant 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 the immune response results in enhanced immunoreactivity compared to resting lymphocytes. Immunomodulatory proteins as provided herein (e.g., variant CD86 polypeptides containing affinity-modified IgSF domains) can, in some embodiments, increase the expression of IFN-γ (interferon-γ) or IL-2 in primary T cell assays compared to wild-type IgSF members or IgSF domain controls, or, in alternative embodiments, decrease it. One of ordinary skill in the art will recognize that the format of the primary T cell assay used to determine an increase in the expression of IFN-γ or IL-2 is different from the format employed to assay for a decrease in the expression of IFN-γ or IL-2.When assaying the ability of the immunomodulatory proteins or affinity-modified IgSF domains of the present invention to reduce the expression of IFN-γ or IL-2 in a primary T cell assay, a mixed lymphocyte reaction (MLR) assay can be used as described in Example 6. Advantageously, the soluble form of the affinity-modified IgSF domain of the present invention can be employed to determine its ability to antagonize the expression of IFN-γ or IL-2 and thereby reduce its expression in the MLR. Alternatively, when assaying the ability of the immunomodulatory proteins or affinity-modified IgSF domains of the present invention to increase the expression of IFN-γ or IL-2 in a primary T cell assay, a co-stimulation assay can be used. In the co-stimulation assay, T cell receptor signals (in some embodiments provided by anti-CD3 antibodies) are combined with a co-stimulated affinity-modified IgSF domain, such as variant CD86, to determine the ability to increase the expression of IFN-γ or IL-2 compared to a wild-type IgSF domain control. Methods for assaying the immunological activity of modified cells, including evaluating 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 an appropriate animal model. Assays are also standard. 51 Assays for evaluating cytotoxicity, including 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 "immunomodulatory polypeptide" or "immunomodulatory protein" is a polypeptide or protein molecule that modulates immune activity. "Modulation" of an immune response means either enhancement or suppression of immune activity. An immunomodulatory 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 interchain disulfide bonds). Thus, monomeric, dimeric, and higher-order multimeric polypeptides are within the scope of the defined term. A multimeric polypeptide can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains). The immunomodulatory proteins herein include variant CD86 polypeptides.

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

[0108] An "isoform" of CD86 is one of a plurality of naturally occurring CD86 polypeptides that differ in amino acid sequence. An isoform can be the product of a splice variant of an RNA transcript expressed by a single gene or the product of highly similar but different genes that give rise to functionally similar proteins such as can occur 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 attached or linked directly or indirectly to generate a detectable signal, or that can interact with a second label to modify a detectable signal. A label can be conjugated directly or indirectly to a polypeptide to generate a labeled polypeptide. A label is either 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 a fluorogenic moiety, green fluorescent protein, or luciferase.

[0110] The term "lymphocyte", as used herein, means 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 relate to cell-mediated cytotoxic acquired immunity), and B cells (which relate to humoral antibody-mediated acquired immunity). T cells include helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Also included within the definition of lymphocytes are innate lymphoid cells (ILCs).

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

[0112] As used herein, the term "membrane protein" means a protein that attaches (binds) directly or indirectly to a lipid bilayer under physiological conditions. The lipid bilayer forming the membrane can be a biological membrane, such as the cell membrane of a eukaryote (e.g., a mammal), or an artificial (i.e., man-made) membrane, such as the membrane found on a liposome. The binding of a membrane protein to the lipid bilayer can be by covalent bonding or by non-covalent interactions (e.g., hydrophobic or electrostatic interactions). A 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 non-covalent interactions or is bound to an integral membrane protein by non-covalent interactions. A peripheral membrane protein forms a transient binding to the lipid bilayer such that under physiological range conditions in a mammal, the peripheral membrane protein can interact with and / or dissociate from the lipid bilayer. In contrast to a peripheral membrane protein, an integral membrane protein forms a substantially permanent binding to the lipid bilayer of the membrane such that under physiological range conditions in a mammal, the integral membrane protein does not dissociate from its binding to the lipid bilayer. A membrane protein can form a binding to the membrane by one layer of the lipid bilayer (monotopic type) or can be bound by both layers of the membrane (polytopic type). An integral membrane protein that interacts with only one lipid bilayer is an "integral monotopic protein". An integral membrane protein that interacts with both lipid bilayers is an "integral polytopic protein". Alternatively, it is referred to herein as a "transmembrane protein".

[0113] The term "modulate" or "modulating", when used in the context of an immune response (e.g., a mammalian immune response) in this specification, refers to any change (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 a modified cell 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 as compared to an immune response that occurs in the absence or presence 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 imparted to an existing immune response, an immune response in the developmental stage, a potential immune response, or the ability to induce, modulate, affect, 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: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells capable of modulating the functional capacity of other cells such as autoreactive lymphocytes, antigen-presenting cells, or inflammatory cells; induction of anergy (i.e., unresponsiveness) in immune cells; enhancing or suppressing the activity or function of immune cells (including, without limitation, changing the pattern of proteins expressed by these cells). Examples include changes in the production and / or secretion of specific molecular classes such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events.The modulation can be evaluated by, for example, changes in the expression of IFN-γ (interferon γ) or IL-2 compared to wild-type or unmodified CD86 controls in a primary T cell assay (see Zhao and Ji, Exp Cell Res. 2016 Jan 1; 340(1): 132-138). The modulation can be evaluated by, for example, changes in the immunological activity of the modified cells, such as changes in the cytotoxic activity of the modified cells or changes in the cytokine secretion of the modified cells, compared to cells modified with the wild-type CD86 transmembrane protein.

[0114] The term "multimerization domain" refers to an amino acid sequence that promotes the stable interaction of a polypeptide molecule containing complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), which can be the same or different multimerization domains, with one or more additional polypeptide molecules. The interaction between complementary multimerization domains, e.g., the interaction between a first multimerization domain and a second multimerization domain, forms a stable protein-protein interaction to produce a multimer of the polypeptide molecule and the additional polypeptide molecules. In some cases, the multimerization domains are the same and interact with each other to form a stable protein-protein interaction between 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 Fc domain or a portion thereof derived from an immunoglobulin constant region or domain, e.g., 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 polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing analogs of known natural nucleotides, nucleic acids having similar binding properties thereto, 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 explicitly shown sequence ("reference sequence") but also its conservatively modified variants (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 a mixture of bases and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0116] As used herein, the term "molecular species" means a population of proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines an aggregate of the same or substantially the same 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, small sequence differences within a single molecular species due to genetic polymorphism also constitute differences in another form within a single molecular species, similar to the wild-type truncated form of a single molecular species due to proteolytic cleavage, for example. A "cell surface molecular species" is a molecular species that is expressed on the surface of mammalian cells. Two or more different protein species that are present in only one (but not both) of two mammalian cells that each form an IS are said to be in "cis" or "cis configuration" with respect to each other. Two different protein species, where the first is present only in the first of two mammalian cells that form an IS and the second is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or "trans configuration". Two different protein species that are present in both of two mammalian cells that each form an IS are in both cis and trans configurations on these cells.

[0117] As used herein, the term "non-competitive binding" means the ability of a protein to specifically bind simultaneously to at least two cognate binding partners. Thus, a protein can bind simultaneously to at least two different cognate binding partners, although the binding interactions need not be during the same period, so in some cases the protein is specifically bound to only one of the cognate binding partners. In some embodiments, the binding occurs under specific binding conditions. In some embodiments, the co-binding is such that the binding of one cognate binding partner does not substantially inhibit the co-binding to a second cognate binding partner. In some embodiments, non-competitive binding means that the binding of a second cognate binding partner to its binding site on the protein does not replace the 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 the method described in Perez de La Lastra et al., Immunology, 1999 Apr: 96(4): 663-670. In some cases, in non-competitive interactions, the first cognate binding partner specifically binds at an interaction site that does not overlap with the interaction site of the 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 through a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in the context of an enzyme-substrate interaction, a non-competitive inhibitor binds at 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 binds to the second interaction site only 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 mammalian subjects, often humans. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant CD86 or a modified cell expressing a variant CD86 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is each typically a pharmaceutically acceptable carrier, excipient, or diluent.

[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 terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. The terms also include molecules that can be synthesized or recombinantly expressed using known protein modification techniques, where one or more amino acids are amino acid analogs or non-standard or non-natural amino acids. In addition, the protein may be derivatized.

[0120] As used herein, the term "primary T cell assay" refers to an in vitro assay for measuring T cell activity, e.g., cytokine production, e.g., the 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-stimulation assay. In this assay, primary T cells are stimulated with anti-CD3 that is immobilized with or without additional recombinant protein. Culture supernatant is collected at a certain point (usually 24 - 72 hours). In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatant is collected at a certain point (usually 24 - 72 hours). Cytokine levels in the culture supernatant, e.g., levels of IFN-γ, IL-2, or TNFα, are measured by standard ELISA techniques. Commercially available kits are available from suppliers and the assays are performed according to the manufacturer's recommendations.

[0121] When applied to a nucleic acid (e.g., a nucleic acid encoding an immunomodulatory protein of the invention), the term "purified" generally refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that yields essentially one band in an electrophoretic gel is "purified". A purified nucleic acid or protein of the invention is at least about 50% pure, usually 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. Such alteration can be carried out on a substance within or removed from its natural environment or state. For example, a "recombinant nucleic acid" is produced by recombining nucleic acids, e.g., during cloning, affinity modification, DNA shuffling, or other well-known molecular biology procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by such molecular biology techniques. The terms "recombinant protein" or "recombinant polypeptide", as used herein, refer to protein molecules expressed using recombinant DNA molecules. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or, alternatively, a cell that has 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, as well as viruses (similar control elements, i.e., promoters, are also found in prokaryotes). The choice of a particular promoter and enhancer depends on what cell type should be used to express the protein of interest. The terms "in functional combination", "in functional order", and "functionally linked", as used herein, refer to the linkage of nucleic acid sequences in a manner or orientation such that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced.

[0123] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence and the appropriate nucleic acid sequences necessary for expression in a particular host cell of a coding sequence operably linked thereto. 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, as well as termination and polyadenylation signals. Optionally, a secretory signal peptide sequence may also be encoded by a recombinant expression vector operably linked to the coding sequence of a 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. The term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of the host cell into which it has been introduced. Such vectors include viral vectors, such as lentiviral vectors.

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

[0125] As used herein, the term "sequence identity" refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at the nucleotide level. The sequence identity of a protein can be determined by comparing the amino acid sequences at a given position in each sequence when the sequences are aligned. Similarly, the sequence identity of a nucleic acid can be determined by comparing the nucleotide sequences 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] As used herein with respect to a protein, the term "soluble" 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 that contains an IgSF domain or a specific binding fragment thereof, but does not contain a transmembrane domain. In some cases, the solubility of a protein can be improved by directly or indirectly linking or conjugating it to an Fc domain, either directly or 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] As used herein with respect to polypeptides or nucleic acids, the term "species" means a population of molecules having the same or substantially the same sequence. 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. A polypeptide sequence that is only slightly shorter (or encodes a difference) with the full-length species differing from the amino terminus or carboxy terminus by only 1, 2, or 3 amino acid residues is considered to be a single species of sequence. Such minor heterogeneity is a common feature of manufactured proteins.

[0128] As used herein with respect to the full-length wild-type mammalian CD86 polypeptide or its ECD, IgV, or IgC domain, the term "specific binding fragment" means a polypeptide having a subsequence of the ECD, IgV, and / or IgC domain and that 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, the 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 varied to form variant CD86.

[0129] As used herein, the term "specifically binds" means 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-fold greater, and in some cases at least 10, 20, 30, 40, 50, 100, 250, or 500-fold greater, or even at least 1000-fold greater, than the average affinity or avidity of the same protein for a collection of random peptides or polypeptides of sufficient statistical size. A protein that specifically binds does not necessarily bind to only a single target molecule, but may specifically bind to non-target molecules due to conformational similarities between the target and non-target (e.g., paralogs or orthologs). One of ordinary skill in the art will recognize that specific binding to molecules having the same function in different animal species (i.e., orthologs) or to non-target molecules having epitopes substantially similar to the target molecule (e.g., paralogs) is possible and does not compromise the binding specificity determined against a statistically valid collection of native non-targets (e.g., random polypeptides). Thus, the polypeptides of the present invention may specifically bind to multiple distinct target molecule species due to cross-reactivity. Specific binding between two proteins can be determined using a solid-phase ELISA immunoassay or surface plasmon resonance (e.g., Biacore) measurements. Generally, the interaction between two binding proteins has a dissociation constant (Kd) as low as less than 1×10 -5 M, often as low as 1×10 -12 M. In certain embodiments of the present disclosure, the interaction between two binding proteins has a dissociation constant of 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, or 1×10 -11 M.

[0130] With respect to mammalian cells expressing a polypeptide, the terms "surface-expresses" or "surface expression" mean 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 nucleic acid molecules or polypeptide molecules produced by recombinant and / or chemical synthesis methods, for example, with respect to synthetic nucleic acid molecules or synthetic genes or synthetic peptides.

[0132] The term "targeting moiety", as used herein, refers to a composition that is covalently or non-covalently bound to a polypeptide comprising variant CD86 or physically encapsulates it. The targeting moiety has specific binding affinity for a desired counter structure such as a cell surface receptor (e.g., CD28), or a tumor antigen (e.g., tumor specific antigen (TSA) or tumor associated antigen (TAA), such as B7-H6). Typically, the desired counter structure is localized on a particular tissue or cell type. The targeting moiety includes antibodies, antigen-binding fragments (Fab), variable fragments (Fv) containing V H and V L , single-chain variable fragments (scFv) containing V H and V L linked together in one chain, as well as other antibody V region fragments, such as Fab’, F(ab)2, F(ab’)2, dsFv diabody, nanobody, soluble receptor, receptor ligand, affinity matured receptor or ligand, and small molecule (less than 500 daltons) compositions (e.g., specific binding receptor compositions). The targeting moiety can also be covalently or non-covalently bound (attached) to the lipid membrane of liposomes encapsulating the polypeptide of the invention.

[0133] As used herein, the term "transmembrane protein" means a membrane protein that substantially or completely traverses a lipid bilayer, which can be found, for example, in a biological membrane such as a mammalian cell or in an artificial construct such as a liposome. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. The transmembrane domain is generally predictable from the amino acid sequence of the transmembrane domain based on the fact that the hydrophobicity of the transmembrane domain is high compared to the regions that interact with the aqueous environment (e.g., the cytosol, extracellular fluid) in the protein, through several commercially available bioinformatics software applications. The transmembrane domain is often a hydrophobic α-helix that traverses the membrane. The transmembrane protein may traverse both layers of the lipid bilayer one or more times. The provided transmembrane immunomodulatory protein described herein is included in the transmembrane protein. The transmembrane immunomodulatory protein of the present invention further includes an ectodomain in addition to the transmembrane domain and, in some embodiments, further includes an endodomain.

[0134] As used herein, the terms "treatment" or "therapy" of a disease or disorder means delaying, interrupting, or reversing the progression of a disease or disorder, as demonstrated by reduction, arrest, or elimination of any clinical or diagnostic symptom by administration of a therapeutic composition of the invention (e.g., one containing an immunomodulatory protein or modified cell), either alone or in combination with another compound as described herein. "Treatment" or "therapy" also means reduction in the severity of symptoms in acute or chronic disease or disorder, or reduction in the recurrence rate (e.g., as in the case of recurrence or remission of an autoimmune disease course), or reduction of inflammation in the case of the inflammatory aspects of an autoimmune disease. As used herein in the context of cancer, the terms "treatment" or "inhibition" of cancer mean, but are not limited to, at least one of a statistically significant decrease in tumor growth rate, arrest of tumor growth, or 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 Response Evaluation Criteria for Solid Tumors (RECIST). "Prevention" of a disease or disorder, as used in the context of the present invention, means administration of an immunomodulatory polypeptide or modified cell of the invention, either alone or in combination with another compound, to prevent the appearance or onset of a disease or disorder or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of onset of a disease or disorder.

[0135] The term "tumor-specific antigen" or "TSA", as used herein, refers to a counterstructure that is predominantly present on the tumor cells of a mammalian subject but generally not found on the normal cells of the mammalian subject. A tumor-specific antigen need not be present only on tumor cells, but the proportion of specific mammalian cells having the tumor-specific antigen is sufficiently high or the level of the tumor-specific antigen on the surface of the tumor is sufficiently high such that it can be targeted by an anti-tumor therapeutic agent (e.g., the immunomodulatory polypeptide of the present invention) and can provide prophylaxis or treatment of the mammalian subject from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammalian subject having a tumor, at least 50% of the cells presenting the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells presenting the TSA are cancerous.

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

[0137] Both non-natural and natural amino acids are included within the scope of acceptable substitutions or additions. Variant CD86 is not limited to any particular method of manufacture and includes, for example, de novo chemical synthesis, de novo recombinant DNA technology, or combinations thereof. The variant CD86 of the present invention specifically binds to at least one or more of mammalian species CD28 and / or CTLA-4. In some embodiments, the amino acid sequence changes result in a change (i.e., an increase or decrease) in the binding affinity or avidity for CD28 and / or CTLA-4 as compared to the unmodified or wild-type CD86 protein. The increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. See 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 increase in the binding affinity or avidity of variant CD86 for CD28 and / or CTLA-4 can be an increase to a value that is at least 5% greater than the value of the unmodified or wild-type CD86, and in some embodiments, an increase to a value that is at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than the value of the unmodified or wild-type CD86 control value. A decrease in the binding affinity or avidity of CD86 for CD28 and / or CTLA-4 is a decrease to a value that is 95% or less of the unmodified or wild-type CD86 control value, and in some embodiments, a decrease to a value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less, or undetectable, of the binding affinity or avidity of the unmodified or wild-type CD86 control 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 the binding affinity or avidity of the unmodified or wild-type CD86.In some embodiments, the binding affinity or avidity for one homophilic binding partner can change, but not for the other homophilic binding partner. For example, variant CD86 that can exhibit improved binding affinity or avidity for CD28 exhibits no change in binding affinity or avidity for CTLA-4 as compared to the binding affinity or avidity of wild-type or unmodified CD86 molecules. In some embodiments, the binding affinity or avidity for both homophilic binding partners can change. In some embodiments, the changes are in the same direction (e.g., both increase or decrease). In some embodiments, the changes are in different directions (e.g., increase for one homophilic binding partner and decrease for the other homophilic binding partner). For example, variant CD86 that can exhibit improved binding affinity or avidity for CD28 exhibits decreased binding affinity or avidity for CTLA-4 as compared to the binding affinity or avidity of wild-type or unmodified CD86 molecules. In some embodiments, the CD86 variant or wild-type or unmodified polypeptide binds to the extracellular domain of CD28 and / or CTLA-4. Thus, in some embodiments, the affinity and avidity are determined based on the binding of the CD86 variant or wild-type or unmodified polypeptide to the extracellular domain of CD28 and / or CTLA-4. The variant CD86 polypeptide has a changed primary amino acid sequence due to amino acid residue substitution, addition, or deletion. The term "variant" in the context of the variant CD86 polypeptide should not be construed as imposing any conditions of any particular starting composition or method by which the variant CD86 is made. Variant CD86 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 synthesized or chemically synthesized to generate the variant CD86. As another example, variant CD86 can be made by site-directed mutagenesis of unmodified or wild-type CD86.Thus, variant CD86 represents a composition but is not necessarily a product produced by any given process. A wide variety of techniques may be employed, including recombinant methods, chemical synthesis, or combinations thereof.

[0138] The terms "wild-type" or "natural" or "native", as used herein, are used in connection with biological materials such as nucleic acid molecules, proteins (e.g., CD86), IgSF members, host cells, etc., and refer to those found in nature and not modified by human intervention.

[0139] II. Variant CD86 Polypeptide Provided herein are variant CD86 polypeptides in which the binding activity or affinity to one or more CD86 cognate binding partners is altered (increased or decreased). 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 polypeptide contains one or more amino acid modifications, such as one or more substitutions (or "mutations" or "exchanges"), deletions, or additions, compared to a wild-type or unmodified CD86 polypeptide, or a portion containing the IgD of wild-type or unmodified CD86, or a specific binding fragment thereof, in the immunoglobulin superfamily (IgSF) domain (IgD). Thus, the provided variant CD86 polypeptide is or includes a variant IgD (hereinafter referred to as "vIgD" herein) 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 compared to the sequence of the unmodified CD86 sequence. In some embodiments, the unmodified CD86 sequence is the wild-type CD86. In some embodiments, the unmodified or wild-type CD86 has the sequence of native CD86 or its ortholog. In some embodiments, the unmodified CD86 is the extracellular domain (ECD) of CD86 or a portion thereof that contains the IgV domain or includes it (see Table 2). In some embodiments, the variant CD86 is the extracellular domain (ECD) of CD86 or a portion thereof that contains the IgV domain or contains it. In some embodiments, the unmodified or wild-type CD86 polypeptide contains the IgV domain or a specific binding fragment thereof. In some embodiments, the variant CD86 polypeptide contains the 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 includes a transmembrane domain and, in some cases, also includes 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 shown in SEQ ID NO:2 or its mature form lacking the signal sequence, (ii) an amino acid sequence showing 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 its mature form, 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 the IgV of CD86 or a specific binding fragment thereof. 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, the wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 (corresponding to amino acid residues 24 to 247 of SEQ ID NO: 2), or an ortholog thereof. In some embodiments, the wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 122 (corresponding to amino acid residues 33 to 131 of SEQ ID NO: 2), or an ortholog thereof. In some embodiments, the wild-type or unmodified CD86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 123 (corresponding to amino acid residues 24 to 134 of SEQ ID NO: 2), or an ortholog thereof. In some embodiments, the wild-type or unmodified CD86 that contains the 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, the specific binding fragment of the 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%, 99% of the length of the IgV domain shown as amino acids 33-131 of SEQ ID NO:2.

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

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

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

[0148] A. Exemplary Modifications Provided herein are variant CD86 polypeptides containing at least one affinity-modified IgSF domain (e.g., IgV) or a specific binding fragment thereof, compared to the IgSF domain contained in the wild-type or unmodified CD86 polypeptide, which exhibit an altered (increased or decreased) binding activity or affinity for one or more ligand CD28 or CTLA-4, compared to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide has a binding affinity for CD28 and / or CTLA-4 that is different from that of the 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 polypeptide has an increased binding affinity for CD28, compared to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide has a decreased binding affinity for CTLA-4, compared to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide exhibits an unchanged binding affinity for CTLA-4, compared to the wild-type or unmodified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide exhibits a binding affinity for CTLA-4 that is not increased, compared to the wild-type or unmodified CD86 polypeptide. CD28 and / or CTLA-4 can be mammalian proteins such as human or murine proteins. In some embodiments, the variant, wild-type and unmodified CD86 polypeptides bind to the ectodomain of CD28 and / or CTLA-4. Thus, in some embodiments, the affinity or binding activity is determined with respect to the binding of the variant, wild-type and unmodified CD86 polypeptides to the ectodomain of CD28 and / or CTLA-4.

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

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

[0151] In some embodiments, a variant CD86 polypeptide with an improved or greater binding affinity for CD28 will have an improvement in binding affinity for CD28 of at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50%, compared to a wild-type or unmodified CD86 polypeptide control. In some embodiments, the improvement in binding affinity compared to the 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 one or more amino acid modifications (e.g., substitutions).

[0152] In some embodiments, variant CD86 polypeptides with reduced or decreased binding affinity for CTLA-4 will have a decrease in binding affinity for CTLA-4 of at least 5%, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, 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 shows no change in binding affinity for CTLA-4 compared to a wild-type or unmodified CD86 polypeptide control. In some embodiments, the variant CD86 polypeptide shows no improvement in binding affinity for CTLA-4 compared to a wild-type or unmodified CD86 polypeptide control. In such instances, 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 ) for any of the foregoing embodiments with respect to CD28 and / or CTLA-4 is less than 1×10 -5 M, less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M or less than 1×10 -11 M, or less than 1×10 -12 M or less, and can be less than that.

[0154] The wild-type or unmodified CD86 sequence does not necessarily have to be used as the starting composition to generate the variant CD86 polypeptides described herein. Therefore, the use of terms such as "modification" like "substitution" does not imply that this aspect is limited to a particular method of manufacturing the variant CD86 polypeptides. Variant CD86 polypeptides can be produced, for example, by de novo peptide synthesis and thus do not necessarily require modifications such as substitutions in the sense of changing codons to encode such modifications. This principle also extends to the terms "addition" and "deletion" of amino acid residues and also does not imply a particular method of manufacture. The means by which variant CD86 polypeptides are designed or made are not limited to any particular method. However, in some aspects, 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 activities. In some aspects, variant CD86 polypeptides are de novo synthesized using protein or nucleic acid sequences available in several publicly available databases and then subsequently screened. The National Center for Biotechnology Information provides such information and its website is publicly accessible via the Internet, like the UniProtKB database.

[0155] Unless otherwise noted, as shown throughout this disclosure, amino acid modifications are designated by amino acid position numbers corresponding to the numbering of positions in the unmodified ECD sequence shown in SEQ ID NO:29, as follows: TIFF0007713886000008.tif25161

[0156] The modifications provided herein can be in the wild-type or unmodified CD86 polypeptide shown 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 the IgV of CD86 as shown in SEQ ID NO:122. In some embodiments, the unmodified CD86 polypeptide may be several amino acids longer or shorter than the IgV sequence shown in SEQ ID NO:122, for example, 1 to 20, for example, 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 the sequence shown in any of SEQ ID NO:29, 122, and 123. TIFF0007713886000009.tif27161

[0157] Identifying the corresponding positions of modifications (e.g., amino acid substitutions) in a CD86 polypeptide (e.g., a portion thereof containing the IgV domain) is within the skill of one of ordinary skill in the art, for example, by alignment of a reference sequence with SEQ ID NO:29. An exemplary alignment of SEQ ID NO:29 containing residues 24 to 247 of wild-type CD86 with SEQ ID NO:122 containing residues 33 to 131 of wild-type CD86 is shown in Figure 3. In the listings of modifications throughout this disclosure, the amino acid position is shown in the middle, 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. When the modification is a deletion at that position, it is denoted as "del", and when the modification is an insertion at that position, it is denoted as "ins". In some cases, the insertion is listed together with the amino acid position shown in the middle, the corresponding unmodified (e.g., wild-type) amino acid is listed before and after the number, and the amino acid insertion of the identified 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 the wild-type or unmodified CD86 sequence. One or more amino acid modifications (e.g., substitutions) can be in the extracellular domain (ECD) of the wild-type or unmodified CD86 sequence. In some embodiments, one or more amino acid modifications (e.g., substitutions) are in the IgV domain or a specific binding fragment thereof. In some embodiments, one or more amino acid modifications (e.g., substitutions) are in the IgC domain or a specific binding fragment thereof. In some embodiments, 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 modification (e.g., substitution) can be in the IgV domain. In some embodiments, the modification is in the ECD. In some embodiments, the modification is in the ECD and the IgV domain. In some embodiments, the modification is 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 a 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 a 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 a specific binding fragment thereof, such as the amino acid sequences of SEQ ID NO:29, 122, or 123.

[0160] In some embodiments, the variant CD86 polypeptide has one or more amino acid modifications (e.g., substitutions) in the non-modified CD86 or its specific binding fragment corresponding to positions 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, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224 based on the positions shown in SEQ ID NO:29. In some embodiments, the modification at position 224 is a deletion. In some embodiments, such variant CD86 polypeptides exhibit an altered binding affinity for one or more of CD28 and / or CTLA-4 as compared to the wild-type or non-modified CD86 polypeptide. For example, in some embodiments, the variant CD86 polypeptide exhibits an improved binding affinity for CD28 as compared to the wild-type or non-modified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide exhibits a decreased binding affinity for CTLA-4 as compared to the wild-type or non-modified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide shows no change in binding affinity for CTLA-4 as compared to the wild-type or non-modified CD86 polypeptide. In some embodiments, the variant CD86 polypeptide does not show an improvement in binding affinity for CTLA-4 as compared to the wild-type or non-modified CD86 polypeptide.

[0161] In some embodiments, the variant CD86 polypeptide is TIFF0007713886000010.tif has one or more amino acid substitutions selected from 47165, or conservative amino acid substitutions thereof. Conservative amino acid substitutions are any amino acids other than the wild-type or unmodified amino acid that belong to the same class of amino acids as the substituted amino acid. The classes of amino acids 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 TIFF0007713886000011.tif has two or more amino acid substitutions selected from 47165, or conservative amino acid substitutions thereof.

[0163] In some embodiments, the variant CD86 polypeptide contains one or more modifications (e.g., amino acid substitutions) at positions corresponding to positions selected from 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, 175, 178, 180, 181, 183, 185, 192, 193, 196, 197, 198, 205, 206, 207, 212, 215, 216, 222, 223, or 224, based on the positions shown in SEQ ID NO:29. In some embodiments, the amino acid modification TIFF0007713886000012.tif is one or more amino acid substitutions selected from 48164, or conservative amino acid substitutions thereof.

[0164] In some embodiments, the variant CD86 polypeptide comprises one or more amino acid substitutions corresponding to TIFF0007713886000013.tif47165, or conservative substitutions thereof.

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

[0166] In some embodiments, the variant CD86 polypeptide comprises an amino acid substitution selected from TIFF0007713886000014.tif106164. In some embodiments, the variant CD86 polypeptide comprises an amino acid substitution selected from TIFF0007713886000015.tif106166. In some embodiments, the variant CD86 polypeptide comprises the amino acid substitution Q25L / H90Y or Q25L / H90L.

[0167] In some embodiments, any of the variant CD86 polypeptides provided further comprises one or more amino acid substitutions from TIFF0007713886000016.tif48161.

[0168] In some embodiments, among the variant CD86 polypeptides provided, there is a CD86 polypeptide having the amino acid substitution TIFF0007713886000017.tif106164. In some embodiments, among the variant CD86 polypeptides provided, there is an amino acid substitution There is a CD86 polypeptide having TIFF0007713886000018.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 shown in Table 1 is shorter than the ECD and thus may not include all amino acid substitutions as listed in Table 1, for example, amino acid substitutions outside the IgV domain. As shown, the exact locus or residue corresponding to a given domain may vary depending on, for example, the method used to identify or classify the domain. Also, in some cases, the adjacent N-terminal 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, for example, to ensure proper folding of the domain when expressed. Thus, it will be understood that the exemplification of 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 shown in the respective SEQ ID NO, for example, 1 to 20, for example, 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 shown in SEQ ID NOs: 85-121, 124-134, 141-221, and 314. In some embodiments, the variant CD86 polypeptide has at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the sequences shown in any one of SEQ ID NOs: 85-121, 124-134, 141-221, and 314, and contains therein an amino acid modification (e.g., substitution) that is not present in wild-type or unmodified CD86, and is a polypeptide sequence or comprises such a polypeptide sequence. In some embodiments, the 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 therein an amino acid modification (e.g., substitution) that is not present in wild-type or unmodified CD86. In some embodiments, the variant CD86 is or comprises the sequence shown by SEQ ID NO: 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 exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the sequences shown in any one of SEQ ID NO: 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 amino acid modifications (e.g., substitutions) that do not exist in wild-type or unmodified CD86, and is a polypeptide sequence or includes such a polypeptide sequence.

[0171] In some embodiments, the variant CD86 polypeptide is or includes a specific binding fragment of any one of SEQ ID NO: 85 - 121, 124 - 134, 141 - 221, or 314, and contains amino acid modifications (e.g., substitutions) that do not exist in wild-type or unmodified CD86. In some embodiments, the variant CD86 polypeptide is or includes a specific binding fragment of any one of SEQ ID NO: 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 amino acid modifications (e.g., substitutions) that do not exist in wild-type or unmodified CD86.

[0172] (Table 1) Exemplary variant CD86 polypeptides TIFF0007713886000019.tif93170TIFF0007713886000020.tif160170

[0173] In some embodiments, any of the provided CD86 variants can be included as a polypeptide that is shorter or longer than the amino acid sequence shown in Table 1, as described, 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, provided that the CD86 polypeptide binds to CD28 (including binding with improved affinity compared to the wild-type or unmodified CD86 polypeptide).

[0174] In some embodiments, the variant CD86 polypeptide exhibits improved affinity for the extracellular domain of CD28 compared to the 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 extracellular domain of CD28 and reduced binding affinity for binding to CTLA-4 compared to the 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 extracellular domain of CD28 and no change in affinity for the extracellular domain of CTLA-4 compared to the 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 binding to CTLA-4 of the unmodified CD86 polypeptide (e.g., as shown in SEQ ID NO:29, 122, or 123), as indicated by the ratio of binding to CD28 to binding to CTLA-4 (e.g., CD28 binding:CTLA-4 binding ratio). In some embodiments, the binding ratio is greater than 1. In some embodiments, the variant CD86 polypeptide exhibits a ratio of binding to CD28 to binding to CTLA-4 that is greater than 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 about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, 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 is 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. Format of Variant Polypeptide The immunomodulatory polypeptides provided herein that contain vIgD can be formatted in various ways, such as, for example, as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, a particular format can be selected according to the desired therapeutic application. In some cases, the immunomodulatory polypeptide containing the variant CD86 polypeptide is provided in a format that antagonizes or blocks the activity of its binding partner (e.g., CTLA-4 and / or CD28). In some cases, the immunomodulatory polypeptide containing the variant CD86 polypeptide is provided in a format that agonizes or stimulates the activity of its binding partner (e.g., CD28). In some embodiments, CD28 agonism (receptor activation) may be useful for promoting immunity in oncology. One of ordinary skill in the art can readily determine the activity of a particular format for antagonizing (antagonizing) or agonizing 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 provided immunomodulatory protein provides flexibility for the modification or generation of immunomodulatory proteins for modulating the activity of multiple counterstructures (multiple cognate binding partners).

[0178] In some aspects, there are provided immunomodulatory proteins that include the vIgD of CD86 and 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 "stack" immunomodulatory protein). In some embodiments, such "stacked" molecules can be provided in a soluble format or, in some cases, may be provided as a membrane-bound or secreted protein. In some embodiments, a variant CD86 immunomodulatory protein is provided as a conjugate that contains a vIgD of CD86 directly or indirectly linked to a targeting agent, e.g., a targeting moiety or portion 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., when administered 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 variant CD86 containing the vIgD to the tumor microenvironment and modulating, e.g., the activity of tumor-infiltrating lymphocytes (TILs) specific to the tumor microenvironment.

[0179] In some embodiments, the provided immunomodulatory protein is expressed in cells and provided as part of a modified cell therapy (ECT). In some embodiments, the variant CD86 polypeptide is expressed in a membrane-bound form in cells such as immune cells (e.g., T cells or antigen-presenting cells), thereby providing a transmembrane immunomodulatory protein (hereinafter also referred to as "TIP" herein). In some embodiments, depending on the cognate binding partner recognized by the TIP, the modified cells expressing the TIP can stimulate (agonize) the cognate binding partner by providing either a positive or negative costimulatory signal to other modified cells and / or endogenous T cells. In some embodiments, the modified cells expressing the TIP bind to cognate binding partners on different cells. In some embodiments, when the modified cells expressing the TIP bind to cognate binding partners on different cells, costimulation is referred to as trans-costimulation. In some embodiments, the modified cells expressing the TIP bind to the cognate binding partner of the cell itself, thereby inducing costimulation in itself. In some embodiments, when the TIP on the cell binds to the cognate binding partner of the cell itself, costimulation is referred to as cis-costimulation. In some aspects, the variant CD86 polypeptide is expressed in a secreted form in cells such as immune cells (e.g., T cells or antigen-presenting cells), thereby producing a secreted or soluble form of the variant CD86 polypeptide (hereinafter also referred to as "SIP" herein) when the cells are administered to a subject, for example. In some aspects, depending on the cognate binding partner recognized by the SIP, the modified cells expressing the SIP can antagonize or agonize the cognate binding partner in the environment in which it is secreted (e.g., the tumor microenvironment). In some embodiments, the variant CD86 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that is infectable in vivo in cells such as immune cells (e.g., T cells or antigen-presenting cells) by administration to a subject for delivery or expression of the variant polypeptide as a TIP or SIP to the cells.

[0180] In some embodiments, variant CD86 containing a soluble immunomodulatory polypeptide, such as vIgD, can be encapsulated within liposomes that can themselves be conjugated to any one or any combination (e.g., targeting moieties) of the provided conjugates. In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the 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 variant CD86 are deglycosylated.

[0181] Non-limiting examples of the formats provided are further described below.

[0182] B. Soluble Proteins In some embodiments, the immunomodulatory protein containing the variant CD86 polypeptide is a soluble protein. One of ordinary skill in the art will recognize that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that soluble forms of such proteins can be produced using the extracellular domain or an immunologically active sequence thereof. Thus, in some embodiments, the immunomodulatory protein containing the variant CD86 polypeptide lacks a transmembrane domain or a portion of the transmembrane domain. In some embodiments, the immunomodulatory protein containing variant CD86 lacks an intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the immunomodulatory protein containing the variant CD86 polypeptide contains only the vIgD portion containing an IgV domain and / or an IgC (e.g., IgC2) domain with amino acid modifications or a portion of the ECD domain containing a specific binding fragment thereof.

[0183] In some embodiments, an immunomodulatory polypeptide comprising variant CD86 can comprise one or more of the variant CD86 polypeptides of the invention. In some embodiments, the polypeptide of the invention comprises exactly 1, 2, 3, 4, 5 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 provided immunomodulatory polypeptide comprises two or more vIgD sequences of CD86. The plurality of variant CD86 polypeptides within the polypeptide chain can be the same (i.e., homologous) or non-identical (i.e., heterologous) variant CD86 sequences to each other. In addition to embodiments of a single polypeptide chain, in some embodiments, two, three, four, or more of the polypeptides of the invention can be bound to each other by covalent or non-covalent bonds. Thus, monomers, dimers, and higher order (e.g., 3, 4, 5, or higher order) multimeric proteins are provided herein. In some embodiments, for example, exactly two polypeptides of the invention can be bound to each other by covalent or non-covalent bonds to form a dimer. In some embodiments, the bond is made through an interchain cysteine disulfide bond. A composition comprising two or more polypeptides of the invention can be a composition of the same or substantially the same species of polypeptide (e.g., a homodimer) or a non-identical species of polypeptide (e.g., a heterodimer). A composition having a plurality of linked polypeptides of the invention can, as described above, have one or more identical or non-identical variant CD86 polypeptides of the invention on each polypeptide chain.

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

[0186] In some embodiments, an immunomodulatory polypeptide comprising a variant CD86 can be linked to a moiety comprising a conformationally disrupted 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. Accordingly, provided herein is an immunomodulatory protein that is a PASylated protein in which a variant CD86 polypeptide is directly or indirectly linked to PAS via a linker. In some embodiments, one or more additional linker structures can be used.

[0187] In some embodiments, the moiety facilitates the detection or purification of the variant CD86 polypeptide. In some cases, the immunomodulatory polypeptide comprises a tag or fusion domain, such as an affinity or purification tag, directly or indirectly linked to the N-terminus and / or C-terminus of the CD86 polypeptide. A variety of 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, shown in SEQ ID NO:244 - 246). In some cases, the immunomodulatory polypeptide comprising variant CD86 comprises at least six histidine residues (shown in SEQ ID NO:249). In some cases, the immunomodulatory polypeptide comprising variant CD86 further comprises various combinations of moieties. For example, the immunomodulatory polypeptide comprising variant CD86 further comprises one or more poly-histidine-tags and FLAG tags.

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

[0189] In some embodiments, the immunomodulatory protein comprises a variant CD86 polypeptide directly or indirectly linked via a linker to a multimerization domain. In some aspects, the multimerization domain increases the half-life of the molecule. 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 itself interact to form a stable structure. For example, separately encoded variant CD86 polypeptide chains can be joined by multimerization, and multimerization of the polypeptide is mediated by the multimerization domain. Typically, the multimerization domain provides for the formation of stable protein-protein interactions between a first variant CD86 polypeptide and a second variant CD86 polypeptide.

[0190] Homomeric or heteromeric polypeptides can be generated from the co-expression of distinct variant CD86 polypeptides. The first and second variant CD86 polypeptides may be the same or different. In certain embodiments, the first and second variant CD86 polypeptides are the same in a homodimer and each is 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 includes any that can form stable protein-protein interactions. The multimerization domain includes immunoglobulin sequences (e.g., Fc domain; see, e.g., International Patent Publication Nos. WO 93 / 10151 and WO 2005 / 063816 US; US Patent Publication No. 2006 / 0024298; US Patent No. 5,457,035); leucine zippers (e.g., from the nuclear transforming proteins fos and jun or the oncogene c-myc or from 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); hydrophobic regions; hydrophilic regions; or can interact via free thiols that form intermolecular disulfide bonds between homo- or hetero-multimeric chimeric molecules. In addition, the multimerization domain can include an amino acid sequence containing a protrusion complementary to an amino acid sequence containing a hole, as described, for example, in US 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 modified not only to promote stable interactions through steric interactions, but also to further promote 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). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory cavity of the same or similar size to the protrusion is optionally created at the interface of the second polypeptide. Exemplary multimerization domains are described below.

[0192] The variant CD86 polypeptide can be connected, via its N-terminus or C-terminus, typically, to the N-terminus or C-terminus of the multimerization domain to form a chimeric polypeptide, anywhere is fine. 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 via covalent or non-covalent interaction. For example, when preparing a chimeric polypeptide containing a multimerization domain, a nucleic acid encoding all or part of the variant CD86 polypeptide can be functionally linked directly or indirectly or optionally via a linker domain to a nucleic acid encoding the multimerization domain sequence. 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] The polypeptide multimer contains a plurality, for example two, chimeric proteins produced by directly or indirectly connecting two of the same or different variant CD86 polypeptides directly or indirectly to the multimerization domain. In some examples, when the multimerization domain is a polypeptide, a gene fusion encoding the variant CD86 polypeptide and the multimerization domain is inserted into a suitable expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with the recombinant expression vector and can assemble into a multimer, in which case the multimerization domains interact to form a multivalent polypeptide. Chemical linkage of the multimerization domain to the variant CD86 polypeptide can be performed using a heterobifunctional linker.

[0194] The obtained chimeric polypeptides such as fusion proteins, and multimers formed therefrom, can be purified by any suitable method, such as by affinity chromatography on a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, homodimer and heterodimer formation occurs. The conditions of expression can be adjusted so that heterodimer formation is preferred over homodimer formation.

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

[0196] In some embodiments, the Fc is murine Fc or 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 IgG1, such as human IgG1. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 229, 230, or 253, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 229, 230, or 253.

[0197] In some embodiments, the Fc region contains another modification that changes (e.g., reduces) 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 cell-mediated cytotoxicity (ADCC), in addition to the antigen-binding ability, which is the main function of immunoglobulins. Additionally, the FcRn sequence present in the Fc region plays a role in regulating IgG levels in serum by extending the in vivo half-life through conjugation to the FcRn receptor in vivo. In some embodiments, such functions can be reduced or modified in the Fc for the Fc fusion protein provided for use.

[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 Fc sequences 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 content of those publications is specifically incorporated herein by reference.

[0199] In some embodiments, the provided variant CD86-Fc fusion is a desirable candidate 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 detrimental, and includes an Fc region with reduced effector function. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay 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, which are primary cells for mediating ADCC, express only FcγRIII, while 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 for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, a non-radioactive assay method may be employed (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in an animal model such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Also, a C1q binding assay may 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. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). Also, FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0200] CD86-Fc variant fusions with reduced effector function include those having one or more substitutions among residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants having 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 the CD86-Fc variant fusion has an Fc region in which one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as designated by EU numbering) are substituted with amino acids different from those of 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-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 modifications such as IgG4-L236E as described in Current Opinion in Biotechnology (2009) 20 (6), 685-691; modifications such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R as described in WO 2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (as designated by EU numbering); and modifications at the sites described in WO 2000 / 042072.

[0202] Certain Fc variants with improved or reduced binding to FcR have been described (see, for example, 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, provided is a CD86-Fc variant fusion comprising a variant Fc polypeptide described herein and a variant Fc region comprising one or more amino acid substitutions that prolong the half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with an 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 therein one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having a substitution at Fc region residue: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 according to EU numbering, e.g., those having 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. Also, see 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 shown in SEQ ID NO:229 having an allotype (e.g., the f allotype) containing Glu (E) and Met (M) residues at positions 356 and 358 (by 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 reconstruct the residues of allotype G1 m1 (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 render the Fc inactive with respect to Fc effector functions. Exemplary effector function-deficient or inactivating 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 (by EU numbering). In some embodiments, the wild-type Fc is further modified by removal of one or more cysteine residues, such as by substitution of the cysteine residue at position 220 (C220S) by EU numbering with a serine residue. Exemplary inactive Fc regions with reduced effector function are shown in SEQ ID NO:333 or 256 and SEQ ID NO:258 or 230, respectively, which are based on the allotypes shown in SEQ ID NO:229 or SEQ ID NO: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), such as those described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

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

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

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

[0210] In some embodiments, a CD86-Fc variant fusion protein comprising a variant Fc region is provided, wherein the variant Fc comprises an amino acid sequence set forth in any of SEQ ID NO: 255, 258, 256, 257, 254 or 259-261, or an amino acid sequence having 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 NO: 255, 258, 256, 257, 254 or 259-261.

[0211] In some embodiments, the Fc is derived from IgG2, such as human IgG2. In some embodiments, the Fc comprises an amino acid sequence set forth in SEQ ID NO: 262, or an amino acid sequence having 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 having 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 arginine at position 409 shown by the EU index proposed by Kabat et al. of human IgG4 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 shown to prevent recombination between a therapeutic antibody 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 having 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 of greater length. In some embodiments, the peptide linker has at least one amino acid residue, but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length or less. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is GGGGS (one-letter amino acid code, "4GS" or "G4S"; SEQ ID NO:223) or a multimer of the 4GS linker, e.g., a repeat of 2, 3, 4, or 5 4GS linkers as shown in SEQ ID NO:225 (2×GGGGS; (G4S)2) or SEQ ID NO:224 (3×GGGGS; (G4S)3). In some embodiments, the linker can include a series of alanine residues alone or in addition to another peptide linker, such as a 4GS linker or its multimer. In some embodiments, the number of alanine residues in each stretch is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is three alanines (AAA). In some embodiments, the variant CD86 polypeptide is indirectly linked to the Fc sequence via a linker that is not composed of three alanines. In some examples, the linker is 2×GGGGS 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 restriction sites, e.g., the linker can include the amino acid GS (one-letter amino acid code) 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-helix linker. In some embodiments, the linker is (in single-letter amino acid code) EAAAK or a multimer of the EAAAK linker, e.g., a repeat of 2, 3, 4, or 5 EAAAK linkers as shown in SEQ ID NO:265 (1×EAAAK), SEQ ID NO:266 (3×EAAAK), or SEQ ID NO:247 (5×EAAAK). In some cases, the immunomodulatory polypeptide comprising the variant CD86 comprises various combinations of peptide linkers.

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

[0215] In some embodiments, when the CD86 polypeptide is directly linked to the Fc sequence, the CD86 polypeptide can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids at the C-terminus. 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 (tolerating 3 or fewer N-terminal or C-terminal amino acid sequence differences) are dimerized to produce homodimers. 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 heterodimers. 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 the variant CD86-Fc fusion protein. In some embodiments, for the production of the Fc fusion protein, the nucleic acid molecule encoding the variant CD86-Fc fusion protein is inserted into a suitable expression vector. The resulting variant CD86-Fc fusion protein can be expressed in host cells transformed with the expression vector, where assembly between the Fc domains occurs by intermolecular disulfide bonds formed between the Fc moieties to produce dimers such as bivalent variant CD86-Fc fusion proteins.

[0218] The resulting Fc fusion protein can be easily purified by affinity chromatography on a Protein A or Protein G column. Additional steps for purification may be required for the generation of heterodimers. For example, when cells are transformed with two nucleic acids encoding different variant CD86 polypeptides, the variant CD86 molecules carrying the Fc domain are expressed as homodimers that are also disulfide-linked, so the formation of heterodimers must be achieved biochemically. Thus, homodimers can be reduced under conditions where disruption of interchain disulfides works favorably, without affecting 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 that are directly or indirectly linked 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 multi-domain immunomodulatory proteins that provide multi-targeted regulation of the immune synapse by binding to two or more, such as 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 and one or more other IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) that are not found in wild-type IgSF family members, where the affinity of the one or more other IgSF domain sequences is modified and / or unmodified. 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 the variant CD86 IgSF domain (vIgD of CD86) according to the provided description.

[0221] In some embodiments, the sequence of the additional IgSF domain can be a modified IgSF domain that contains one or more amino acid modifications (e.g., substitutions) compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain may or may not have a modified affinity (e.g., wild-type). In some embodiments, the unmodified or wild-type IgSF domain can be of 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 that contains 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 IgSF domain, with or without modified affinity, contained within an IgSF family member of a family selected from the following: signal regulatory protein (SIRP) family, triggering receptor expressed on myeloid cells-like (TREML) family, carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, sialic acid-binding Ig-like lectin (SIGLEC) family, butyrophilin family, B7 family, CD28 family, V-set and immunoglobulin domain-containing (VSIG) family, V-set transmembrane domain (VSTM) family, major histocompatibility complex (MHC) family, signaling lymphocyte activation molecule (SLAM) family, leukocyte immunoglobulin-like receptor (LIR), nectin (Nec) family, nectin-like (NECL) family, poliovirus receptor-related (PVR) family, natural cytotoxicity triggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM) family, or killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domain is independently derived from an IgSF protein 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 NCR3 (NKp30).

[0223] The first column of Table 2 provides the name and, in some cases, several possible alternative names for that particular IgSF member. The second column provides the protein identifier of the UniProtKB database, a publicly available database accessible via the internet at uniprot.org, and in some cases, a GenBank number. The Universal Protein Resource (UniProt) is an inclusive resource for protein sequence and annotation data. The UniProt database includes the UniProt Knowledgebase (UniProtKB). UniProt is a joint organization between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics, and the Protein Information Resource (PIR), and is mainly supported by grants from the U.S. National Institutes of Health (NIH). GenBank is the NIH's gene sequence database, in which all publicly available DNA sequences are collected with annotations (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). The third column provides the region where the indicated IgSF domain is located. The region is specified as a range that includes the residues delimiting the domain. The third column also indicates the IgSF domain class of the specified IgSF region. The fourth column provides the region where additional domains of the indicated type are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It should be understood that the description of domains may vary depending on the method used for their identification or classification and that they may be identified separately from different sources. The description of residues corresponding to the domains in Table 2 is illustrative only and may be longer or shorter by a few amino acids (e.g., 1, 2, 3, or 4).The fifth column indicates some of the listed IgSF members (i.e., some of its cell surface homophilic binding partners).

[0224] (Table 2) IgSF members of the present disclosure TIFF0007713886000021.tif212168TIFF0007713886000022.tif202168TIFF0007713886000023.tif196168TIFF0007713886000024.tif206168TIFF0007713886000025.tif224168TIFF0007713886000026.tif94168

[0225] The number of such IgSF domains (regardless of non-wild type combinations or non-wild type arrangements) in the "stacked" immunomodulatory protein construct that have an unmodified or modified affinity is at least 2, 3, 4, or 5, and in some embodiments, exactly 2, 3, 4, or 5 IgSF domains (whereby determination of the number of affinity-modified IgSF domains ignores any of its non-specific binding cleavage sequences and / or its cleavage sequences that are substantially immunologically inert).

[0226] In some embodiments of the stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least 2, where the number of affinity-modified IgSF domains and the number of IgSF domains with unmodified affinity 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 IgSF domains with unmodified affinity can each be (affinity-modified IgSF domain: IgSF domain with unmodified affinity), exactly or at least 2:0 (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 IgSF domains with unmodified and / or modified affinity are the same IgSF domain.

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

[0229] In some embodiments of the stacked immunomodulatory proteins, the IgSF domains that have not been modified in affinity and / or that have been modified in affinity 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 they are modified from were 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 is derived from and unique to one CD86, and at least one second IgSF domain sequence that is derived from and unique to another IgSF family member that is not derived from CD86, where the IgSF domains of the immunomodulatory protein are in a form that has not been modified in affinity and / or in a form that has been modified in affinity. However, in an alternative embodiment, the two non-identical IgSF domains are derived from the same IgSF domain sequence, but at least one has been modified in affinity such that they specifically bind to different cognate binding partners.

[0230] In some embodiments, the provided immunomodulatory protein, in addition to containing a variant CD86 polypeptide, also contains at least 1, 2, 3, 4, 5, or 6 additional immunoglobulin superfamily (IgSF) domains, such as the IgD domain of the IgSF family members shown in Table 2. In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the provided immunomodulatory protein contains at least two additional IgSF domains (e.g., a second and a third IgSF domain). In some embodiments, the provided immunomodulatory protein contains at least three additional IgSF domains (e.g., a second, a third, and a fourth). In some embodiments, the provided immunomodulatory protein contains at least four additional IgSF domains (e.g., a second, a third, a fourth, and a fifth). In some embodiments, the provided immunomodulatory protein contains at least five additional IgSF domains (e.g., a second, a third, a fourth, a fifth, and a sixth). In some embodiments, the provided immunomodulatory protein contains at least six additional IgSF domains (e.g., a second, a third, a fourth, a fifth, a sixth, and a 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 is an 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 portion containing a specific binding fragment thereof.

[0232] In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g., a second, or in some cases, a third IgSF domain, etc.), wherein the at least one additional or second IgSF domain is an IgSF domain or a specific binding fragment thereof as shown in the wild-type or unmodified IgSF domain contained in the amino acid sequences shown 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, such as an IgC1 or IgC2 domain.

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

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

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

[0236] 1. PD-1 IgSF domain In some embodiments, at least one additional (e.g., second or third) vIgD is the 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 the IgV domain or a specific binding fragment of the IgV domain. In some embodiments, the IgD can be IgV only, include 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 is (i) its mature form lacking the amino acid sequence shown in SEQ ID NO:10 or the signal sequence, (ii) has an amino acid sequence or its mature form 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) is 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 is (i) the amino acid sequence shown in SEQ ID NO:37, (ii) has 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) is a portion of (i) or (ii) that contains the 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 the sequence shown in any of SEQ ID NO: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-modified IgSF domain (e.g., IgV or IgC) or a specific binding fragment thereof, compared to the IgSF domain contained in the wild-type or unmodified PD-1 polypeptide, and exhibits altered (increased or decreased) binding activity or affinity for PD-L1 or PD-L2 compared to the wild-type or unmodified PD-1 polypeptide. In some embodiments, it is a variant PD-1 polypeptide that contains at least one affinity-modified IgSF domain (e.g., IgV) or a specific binding fragment thereof, compared to the IgSF domain contained in the wild-type or unmodified PD-1 polypeptide, and exhibits altered (increased or decreased) binding activity or affinity for one or more ligand PD-L1 or PD-L2 compared 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 is different from that of the 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 decreased binding affinity for PD-L2 compared to the 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; that is, in some embodiments, the variant PD-1 polypeptide has an improved binding affinity for one or both of PD-L1 and / or PD-L2 and a decreased binding affinity for one or both of PD-L1 and PD-L2 as compared to the wild-type or unmodified PD-1 polypeptide.

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

[0240] In some embodiments, a variant PD-1 polypeptide having an improved or greater binding affinity for PD-L1 and / or PD-L2 will have at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50% improvement in binding affinity for PD-L1 and / or PD-L2 as compared to a wild-type or unmodified PD-1 polypeptide control. In some embodiments, the improvement in binding affinity as compared to the wild-type or unmodified PD-1 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, or 50-fold. In such instances, the wild-type or unmodified PD-1 polypeptide has the same sequence as the variant PD-1 polypeptide except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0241] In some embodiments, variant PD-1 polypeptides with reduced or decreased binding affinity for PD-L2 will have at least a 5% decrease in binding affinity for PD-L2, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to wild-type or unmodified PD-1 polypeptide controls. In some embodiments, the decrease in binding affinity compared to wild-type or unmodified PD-1 polypeptides is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. 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 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 ) for any of the foregoing embodiments with respect to PD-L1 and / or PD-L2 is less than 1×10 -5 M, less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M, or less than 1×10 -11 M, or less than 1×10 -12 M, or less than that.

[0244] The wild-type or unmodified PD-1 sequence is not necessarily used as the starting composition to generate the variant PD-1 polypeptides described herein. Therefore, the use of terms such as "modification" like "substitution" does not imply that this aspect is limited to a particular method of manufacturing the variant PD-1 polypeptides. Variant PD-1 polypeptides can be produced, for example, by de novo peptide synthesis and thus do not necessarily require a modification such as a substitution in the sense of changing codons to encode the modification. This principle also extends to the terms "addition" and "deletion" of amino acid residues and also does not imply a particular method of manufacture. The means by which the variant PD-1 polypeptides are designed or made are not limited to any particular method. However, in some aspects, a nucleic acid encoding wild-type or unmodified PD-1 is mutagenized from wild-type or unmodified PD-1 genetic material and screened for the induction of desired specific binding affinity and / or IFN-γ expression or other functional activities. In some aspects, the variant PD-1 polypeptides are de novo synthesized using protein or nucleic acid sequences available in several publicly available databases and then subsequently screened. As described above, the National Center for Biotechnology Information provides such information and its website is publicly accessible via the Internet, like the UniProtKB database.

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

[0246] The modifications provided herein can be in the wild-type or unmodified PD-1 polypeptide shown 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 shown in SEQ ID NO:335. In some embodiments, the unmodified PD-1 polypeptide can be several amino acids longer or shorter than the amino acid sequence shown by SEQ ID NO:335, for example, 1 to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids longer or shorter, and contains an IgV. 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 the sequence shown in any of SEQ ID NO:37. In some embodiments, the unmodified PD-1 polypeptide has the sequence shown by SEQ ID NO:335. In some embodiments, the unmodified PD-1 polypeptide has the sequence shown by SEQ ID NO:336. In some embodiments, the unmodified PD-1 polypeptide has the sequence shown by SEQ ID NO:337. In some embodiments, the unmodified PD-1 polypeptide has the sequence shown by SEQ ID NO:339.

[0247] Identifying the corresponding positions of modifications, such as amino acid substitutions, in a PD-1 polypeptide that includes a portion thereof containing its IgSF domain (e.g., IgV), for example, by alignment of a reference sequence with SEQ ID NO:37, is within the skill of those in the art. For example, according to the alignment, the 112th residue of SEQ ID NO:37 corresponds to the 107th residue 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 the wild-type or unmodified PD-1 sequence. 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, 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 modification (e.g., substitution) 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 a specific binding fragment thereof. In some embodiments, the variant PD-1 polypeptide has less than 100% sequence identity with the wild-type or unmodified PD-1 polypeptide or a specific binding fragment thereof and 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 with, for example, the amino acid sequences of SEQ ID NO:37, 335, 336, 337, or 339.

[0250] In some embodiments, the variant PD-1 polypeptide has one or more amino acid modifications (e.g., substitutions) at positions corresponding to positions 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, 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 of the unmodified PD-1 or its specific binding fragment. 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 the wild-type or unmodified PD-1 polypeptide. For example, in some embodiments, the variant PD-1 polypeptide exhibits improved binding affinity for PD-L1 and / or PD-L2 compared to the wild-type or unmodified PD-1 polypeptide. In some embodiments, the variant PD-1 polypeptide exhibits decreased binding affinity for PD-L1 or PD-L2 compared to the wild-type or unmodified PD-1 polypeptide.

[0251] In some embodiments, the variant PD-1 polypeptide is selected from one or more amino acid substitutions selected from 187165, or a conservative amino acid substitution thereof.

[0252] In some embodiments, the variant PD-1 is A variant PD-1 that contains one or more amino acid substitutions from 77165, 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 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: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 can be directly or indirectly linked to one or more other immunoglobulin superfamily (IgSF) domains as described.

[0253] As used herein, there is provided an immunomodulatory protein (CD86 / PD-1 immunomodulatory protein) comprising a variant CD86 polypeptide (e.g., any of those 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 comprises an extracellular domain of CD86 or its IgSF (e.g., IgV) domain or a specific binding fragment thereof that contains one or more modifications (e.g., substitutions) such as any of those described herein. In some embodiments, the variant PD-1 polypeptide is or comprises an extracellular domain of PD-1 or its IgSF (e.g., IgV) domain or a specific binding fragment thereof that contains one or more modifications (e.g., substitutions) such as any of those described herein. The CD86 / PD-1 immunomodulatory protein can be provided in various 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) are IgSF domains (e.g., IgV) of another IgSF family member that binds to or recognizes a tumor antigen. In such embodiments, the IgSF family member functions as a tumor localization moiety, thereby bringing the vIgD of CD86 into proximity 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 the binding affinity and / or selectivity for B7-H6 compared to the unmodified IgSF domain (e.g., NKp30) by, for example, at least 1.2-fold or at least about 1.2-fold, at least 1.5-fold or at least about 1.5-fold, at least 2-fold or at least about 2-fold, at least 3-fold or at least about 3-fold, at least 4-fold or at least about 4-fold, at least 5-fold or at least about 5-fold, at least 6-fold or at least about 6-fold, at least 7-fold or at least about 7-fold, at least 8-fold or at least about 8-fold, at least 9-fold or at least about 9-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, or at least 50-fold or at least about 50-fold. Exemplary amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgC-like or the entire ECD) of the variant NKp30 polypeptide are shown in Table 2. Among the exemplary polypeptides is an NKp30 variant containing the mutations L30V / A60V / S64P / S86G relative to the positions in the NKp30 extracellular domain corresponding to the positions shown in SEQ ID NO:54. In some embodiments, provided is an immunomodulatory protein containing any of the variant CD86 polypeptides provided, and an IgC-like domain containing any of the amino acid modifications shown in Table 3, e.g., an IgC-like domain shown in any of SEQ ID NOs:268 - 272, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any of SEQ ID NOs:268 - 272 and containing one or more amino acid modifications.In some embodiments, an immunomodulatory protein is provided that contains any of the provided variant CD86 polypeptides and an ECD containing an IgSF domain containing any of the amino acid modifications shown in Table 3, for example, an ECD shown in any of SEQ ID NOs: 273-277, or an ECD containing 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 containing one or more amino acid modifications, and contains a variant NKp30 polypeptide.

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

[0257] (Table 3) Exemplary variant NKp30 polypeptides TIFF0007713886000029.tif39166

[0258] As used herein, there is provided an immunomodulatory protein (CD86 / NkP30 immunomodulatory protein) comprising a variant CD86 polypeptide (e.g., any of those 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 comprises the extracellular domain of CD86 or its IgSF (e.g., IgV) domain or a specific binding fragment thereof that contains one or more modifications (e.g., substitutions) such as any of those described herein. In some embodiments, the variant NKp30 polypeptide is or comprises the extracellular domain of Nkp30 or its IgSF (e.g., IgV) domain or a specific binding fragment thereof that contains one or more modifications (e.g., substitutions) such as any of those described herein. The CD86 / Nkp30 immunomodulatory protein 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 shown in any of SEQ ID NO:135, 136, 137, 138, 139 or 140. In some embodiments, the variant CD86 / Nkp30 immunomodulatory protein has the sequence shown in SEQ ID NO:135, 136, 137, 138, 139 or 140.

[0259] 3. Construct 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 linked by covalent or non-covalent bonds. Multiple unmodified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be directly linked to each other by covalent bonds. In some embodiments, two or more IgSF domains are linked directly or indirectly, such as via a linker. In some embodiments, the intervening range of one or more amino acid residues covalently links the IgSF domains to each other indirectly. The linkage can be through residues from the N-terminus to the C-terminus. In some embodiments, the linkage can be made through the side chains of amino acid residues that are not located at the N-terminus or C-terminus of the IgSF domain. Thus, the linkage can be made through terminal or internal amino acid residues or combinations thereof.

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

[0261] In some embodiments, one or more "peptide linkers" link the vIgD of CD86 to one or more additional IgSF domains (e.g., the second or third variant IgSF domain). In some embodiments, the peptide linker can be a single amino acid residue or of greater length. In some embodiments, the peptide linker has at least one amino acid residue but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length or less. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is GGGGS ("4GS") in (one-letter amino acid code) or a multimer of the 4GS linker, e.g., a repeat of 2, 3, 4, or 5 4GS linkers. 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 contain a series of alanine residues 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 stretch is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker can also contain a series of alanine residues alone or in addition to another peptidyl linker (such as a 4GS linker or a multimer thereof). In some embodiments, the number of alanine residues in each stretch is 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an α-helix linker. In some embodiments, the linker is EAAAK in (one-letter amino acid code) or a multimer of the EAAAK linker, e.g., a repeat of 2, 3, 4, or 5 EAAAK linkers as shown in SEQ ID NO:265 (1×EAAAK), SEQ ID NO:266 (3×EAAAK), or SEQ ID NO:247 (5×EAAAK).In some embodiments, the linker can further comprise amino acids introduced by cloning and / or from restriction sites. For example, the linker can comprise the amino acid GS (in one-letter amino acid code) as introduced by the use of the restriction site BAMHI. For example, in some embodiments, the linker (in one-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 examples, the linker is 2×GGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO:226). In some cases, the immunomodulatory polypeptide comprising the variant CD86 comprises 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 shown by SEQ ID NO:135, 136, 137, 138, 139, or 140. In some embodiments, the immunomodulatory protein comprises or has the sequence shown by SEQ ID NO:135, 136, 137, 138, 139, or 140. In some embodiments, any of the foregoing sequences form a homodimer. In some embodiments, the homodimer is formed via a multimerization domain that is an Fc domain contained within the immunomodulatory protein. In some embodiments, the homodimer comprises 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 shown by SEQ ID NO: 316, 317, 318, 319, 320, 321, 322, or 323. In some embodiments, the immunomodulatory protein comprises or has the sequence shown by 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 shown by SEQ ID NO: 326 or 327. In some embodiments, the immunomodulatory protein comprises or has the sequence shown by SEQ ID NO: 326 or 327. In some embodiments, any of the foregoing sequences forms a homodimer. In some embodiments, the homodimer is formed via 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 the sequence set forth in SEQ ID NO: 328, 329, 330, or 331. In some embodiments, any of the foregoing sequences form a heterodimer. In some embodiments, the heterodimer is formed via a multimerization domain that is an Fc domain contained within 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 unmodified and / or affinity-modified IgSF domains are linked by a "wild-type peptide linker" inserted at the N-terminus and / or C-terminus of the unmodified and / or affinity-modified IgSF domains. These linkers are also referred to as the leading sequence (N-terminus of the unmodified or affinity-modified IgSF domain) or the trailing sequence (C-terminus of the unmodified or affinity-modified IgSF domain), and the sequence that exists in the wild type extending just outside the structural prediction of the Ig fold of the IgSF. In some embodiments, the "wild-type linker" is the amino acid sequence that exists after the signal sequence but before the IgSF domain (e.g., the defined IgV domain) in the amino acid sequence of the wild-type protein. In some embodiments, the "wild-type" linker is the amino acid sequence that exists immediately after the IgSF domain (e.g., immediately after the defined IgV domain) but before the IgC domain in the amino acid sequence of the wild-type protein. These linker sequences can contribute to the proper folding and function of the adjacent IgSF domains. In some embodiments, there is a leading peptide linker inserted at the N-terminus of the first IgSF domain, and / or a trailing sequence inserted at the C-terminus of the first unmodified and / or affinity-modified IgSF domain. In some embodiments, there is a second leading peptide linker inserted at the N-terminus of the second IgSF domain, and / or a second trailing sequence inserted at the C-terminus of the second unmodified and / or affinity-modified IgSF domain. When the first and second 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 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 includes a first leading wild-type peptide linker inserted at the N-terminus of an IgSF domain with an unmodified and / or modified affinity, wherein the first leading wild-type peptide linker comprises at least 5 (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the intervening sequence in the wild-type protein from which the IgSF domain with the unmodified and / or modified affinity is derived, between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or an 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 IgSF domain with the unmodified and / or modified affinity is derived, between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or an 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 an IgSF domain with an unmodified and / or modified affinity, wherein the first trailing wild-type peptide linker comprises at least 5 (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the intervening sequence in the wild-type protein from which the IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately following (e.g., an IgSF domain or a 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 IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately following (e.g., an IgSF domain or a 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 an IgSF domain with an unmodified and / or modified affinity, wherein the second leading wild-type peptide linker comprises at least 5 (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the intervening sequence in the wild-type protein from which the IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately preceding (e.g., a signal peptide or an 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 IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately preceding (e.g., a signal peptide or an 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 an IgSF domain with an unmodified and / or modified affinity, wherein the second trailing wild-type peptide linker comprises at least 5 (e.g., any of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the intervening sequence in the wild-type protein from which the IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately following (e.g., an IgSF domain or a 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 IgSF domain with an unmodified and / or modified affinity is derived, between the parental IgSF domain and the domain immediately following (e.g., an IgSF domain or a transmembrane domain).

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

[0271] In some embodiments, a variant CD86 polypeptide and one or more IgSF domains are each independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. In some embodiments, a variant CD86 polypeptide is directly or indirectly linked to at least one of one or more additional IgSF domains, and one of the variant CD86 and one of the one or more additional IgSF domains is directly or indirectly linked 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 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 linkage to Fc is via a peptide linker, such as the peptide linker as described above. In some embodiments, the linkage between the variant CD86 and one or more additional IgSF domains is via a peptide linker, such as the 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., FIGS. 14A-14D.

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

[0273] In some embodiments, the dimeric multi-dom...

Claims

1. A variant CD86 polypeptide comprising an extracellular domain or an IgV domain, wherein the variant CD86 polypeptide comprises the amino acid substitution Q25L in the unmodified CD86 polypeptide relative to the position shown in SEQ ID NO: 29, the unmodified CD86 polypeptide comprises (i) the amino acid sequence shown in SEQ ID NO: 29, or (ii) a portion of the amino acid sequence shown in SEQ ID NO: 29 that comprises the amino acid sequence of the IgV domain of the CD86 polypeptide, wherein the portion of the amino acid sequence of the IgV domain is shown as amino acids 33 to 131 or 24 to 134 of SEQ ID NO: 2, and the variant CD86 polypeptide comprises (i) the amino acid sequence shown in SEQ ID NO: 29, or, (ii) a portion of the amino acid sequence shown in SEQ ID NO: 29 that comprises the amino acid sequence of the IgV domain of the CD86 polypeptide, wherein the portion of the amino acid sequence of the IgV domain is shown as amino acids 33 to 131 or 24 to 134 of SEQ ID NO: 2 and shows at least 95% sequence identity to an amino acid sequence, and the variant CD86 polypeptide specifically binds to the extracellular domain of CD28 with improved affinity as compared to the binding of the unmodified CD86 to the same extracellular domain. A variant CD86 polypeptide.

2. The variant CD86 polypeptide according to claim 1, wherein the unmodified CD86 comprises amino acid residues 33 to 131 or 24 to 134 of the IgV domain of the CD86 polypeptide.

3. The variant CD86 polypeptide according to claim 1 or claim 2, wherein the variant CD86 polypeptide comprises an amino acid sequence showing at least 96%, 97%, 98%, or 99% sequence identity to the unmodified CD86 polypeptide. A variant CD86 polypeptide according to claim 1 or claim 2.

4. Q25L / T71A / H90Y, Q25L / D53G / E212V, Q25L / H90L, Q25L / H90Y, A13V / Q25L / H90L / S181P / L197M / S206T, Q25L / Q86R / H90L / K93T / L132M / V148D / S181P / P216H, Q25L / F33I / H90Y / V128A / P141A / E158G / S181P, Q25L / N39D / K80R / Q86R / I88F / H90L / K93T / N123D / N154D, Q25L / H90L / K93T / M97L / T133A / S181P / D215V, Q25L / Q86R / H90L / N104S, Q25L / L40M / H90L / L180S / S183P, Q18K / Q25L / F33I / L40S / H90L, Q25L / Q86K / H90L / I137T / S181P, Q25L / L77P / H90Y / K153R / V170D / S181P, Q25L / S28G / F33I / F52L / H90L / Q102H / I178T, Q25L / F33I / H90L / K144E / L180S, Q25L / F33I / H90L / K153E / E172G / T192N, Q25L / F33I / Q86R / H90Y / D175E / I196V / E198D, Q25L / V45I / D68N / H90L / S183P / L205S, Q25L / F33I / H90L, Q25L / F33I / Q86R / H90L / K93T, Q25L / H90L / P185S, Q25L / H90L / P185S / P224L, Q25L / H90L / S179R, Q25L / H90Y / S181P / I193V, Q25L / K82T / H90L / T152S / S207P, Q25L / Q86R / H90L / K93T, A13V / Q25L / H90L, Q25L / H90L / K93T / M97L, and Q25L / Q86R / H90L A variant CD86 polypeptide according to any one of claims 1 to 3, comprising one or more amino acid modifications selected from the group consisting of:

5. A variant CD86 polypeptide according to any one of claims 1 to 4, which specifically binds to the extracellular domain of CTLA-4 with a reduced affinity as compared to the binding of the unmodified CD86 to the same extracellular domain.

6. The variant CD86 polypeptide according to claim 5, wherein the CTLA-4 is human CTLA-4, or the CD28 is human CD28, or the CTLA-4 is human CTLA-4 and the CD28 is human CD28.

7. The variant CD86 polypeptide according to any one of claims 1 to 6, comprising an amino acid sequence shown in any of SEQ ID NOs: 85 to 87, 89 to 103, 112 to 120, 141 to 143, 145 to 159, or 168 to 176, or an amino acid sequence showing at least 99% sequence identity to any of SEQ ID NOs: 85 to 87, 89 to 103, 112 to 120, 141 to 143, 145 to 159, or 168 to 176.

8. It is a soluble protein; Lacking the CD86 transmembrane domain and intracellular signaling domain; and / or Unable to be expressed on the surface of cells, The variant CD86 polypeptide according to any one of claims 1 to 7.

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

10. The immunomodulatory protein according to claim 9, wherein the multimerization domain is an Fc domain or a variant thereof with reduced effector function.

11. The variant CD86 polypeptide according to any one of claims 1 to 7, which is a transmembrane immunomodulatory protein further comprising a transmembrane domain.

12. The variant CD86 polypeptide according to claim 11, wherein the transmembrane domain is directly or indirectly linked to the extracellular domain (ECD) of the variant CD86 polypeptide.

13. The variant CD86 polypeptide according to claim 11 or claim 12, further comprising a cytoplasmic domain.

14. The variant CD86 polypeptide according to claim 13, wherein the cytoplasmic domain is directly or indirectly linked to the transmembrane domain.

15. An immunomodulatory protein comprising a first variant CD86 polypeptide according to any one of claims 1 to 8 and a second variant CD86 polypeptide according to any one of claims 1 to 8.

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

17. The immunomodulatory protein according to claim 16, wherein the IgSF domain is an affinity-modified IgSF domain, and the affinity-modified IgSF domain contains one or more amino acid modifications as compared with an unmodified or wild-type IgSF domain of an IgSF family member.

18. The immunomodulatory protein according to claim 16 or 17, wherein the IgSF domain is an affinity-modified IgSF domain, and the affinity-modified IgSF domain exhibits altered binding properties as compared with the binding properties of the unmodified or wild-type IgSF domain of the IgSF family member to one or more of its cognate binding partners, with respect to the same one or more cognate binding partners.

19. The immunomodulatory protein according to any one of claims 16 to 18, wherein the IgSF domain of the second polypeptide is a tumor localization moiety that binds to a ligand expressed on a tumor or a tumor localization moiety that binds to a ligand expressed on a tumor, or an inflammation localization moiety that binds to a cell or tissue associated with an inflammatory environment.

20. The immunomodulatory protein according to claim 19, wherein the ligand is B7H6.

21. The immunomodulatory protein according to claim 19 or 20, wherein the IgSF domain is derived from NKp30.

22. The immunomodulatory protein according to claim 16 or 17, wherein the IgSF family member is selected from the group consisting of the signal regulatory protein (SIRP) family, the triggering receptor expressed on myeloid cells-like (TREML) family, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, the sialic acid-binding Ig-like lectin (SIGLEC) family, the butyrophilin family, the B7 family, the CD28 family, the V-set and immunoglobulin domain-containing (VSIG) family, the V-set transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signaling lymphocytic activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the natural cytotoxicity receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, and the killer cell immunoglobulin-like receptor (KIR) family.

23. The immunomodulatory protein according to any one of claims 16 to 22, further comprising a multimerization domain linked to at least one of the variant CD86 polypeptide or the second polypeptide.

24. A conjugate comprising a variant CD86 polypeptide according to any one of claims 1 to 8 or an immunomodulatory protein according to claim 9 or claim 10, linked to a targeting moiety that specifically binds to a molecule on the surface of a cell.

25. The conjugate according to claim 24, which is a fusion protein.

26. The conjugate according to claim 25, wherein the cell is an immune cell or a tumor cell.

27. The conjugate according to claim 25 or claim 26, wherein the moiety is an antibody or an antigen-binding fragment.

28. A nucleic acid molecule encoding a conjugate that is a variant CD86 polypeptide according to any one of claims 1 to 8 or 11 to 14, an immunomodulatory protein according to any one of claims 9 to 10 or 15 to 23, or a fusion protein according to any one of claims 24 to 27.

29. A vector comprising the nucleic acid molecule according to claim 28.

30. A cell comprising the vector according to claim 29.

31. A method for producing a variant CD86 polypeptide or an immunomodulatory protein, comprising introducing the nucleic acid molecule according to claim 28 or the vector according to claim 29 into a host cell under conditions such that the protein is expressed in the host cell.

32. The method according to claim 31, further comprising isolating or purifying the variant CD86 polypeptide or the immunomodulatory protein from the host cell.

33. A method for modifying a cell that expresses a variant CD86 polypeptide, comprising introducing into a host cell a nucleic acid molecule encoding a conjugate that is a variant CD86 polypeptide according to any one of claims 1 to 8 or 11 to 14, an immunomodulatory protein according to any one of claims 9 to 10 or 15 to 23, or a fusion protein according to any one of claims 24 to 27, under conditions such that the polypeptide, the immunomodulatory protein, or the conjugate is expressed in the host cell.

34. A modified cell comprising a variant CD86 polypeptide according to any one of claims 1 to 8 or 11 to 14, an immunomodulatory protein according to any one of claims 9 to 10 or 15 to 23, or a conjugate that is a fusion protein according to any one of claims 24 to 27, a nucleic acid molecule according to claim 28, or a vector according to claim 29.

35. The modified cell according to claim 34, wherein the variant CD86 polypeptide or the immunomodulatory protein can be secreted from the modified cell.

36. The modified cell according to claim 34 or claim 35, which is an immune cell.

37. The modified cell according to claim 36, wherein the immune cell is a lymphocyte.

38. The modified cell according to claim 37, wherein the lymphocyte is a T cell.

39. The modified cell according to any one of claims 34 to 38, further comprising a chimeric antigen receptor (CAR).

40. The modified cell according to any one of claims 34 to 39, further comprising a modified T cell receptor (TCR).

41. An infectious substance comprising a variant CD86 polypeptide according to any one of claims 1 to 8 or 11 to 14, an immunomodulatory protein according to any one of claims 9 to 10 or 15 to 23, or a conjugate that is a fusion protein according to any one of claims 24 to 27, a nucleic acid molecule according to claim 28, or a vector according to claim 29.

42. A pharmaceutical composition comprising a variant CD86 polypeptide according to any one of claims 1 to 8 or 11 to 14, an immunomodulatory protein according to any one of claims 9 to 10 or 15 to 23, or a conjugate that is a fusion protein according to any one of claims 24 to 27, a modified cell according to any one of claims 33 to 39, or an infectious substance according to claim 41 and a pharmaceutically acceptable excipient.

43. An article of manufacture or kit comprising the pharmaceutical composition according to claim 42 and instructions for use.

44. The pharmaceutical composition according to claim 42 for use in modulating an immune response in a subject.

45. The pharmaceutical composition according to claim 42 for use in a method of treating a disease or condition in a subject in need thereof.

46. Use of the pharmaceutical composition according to claim 42 in the manufacture of a medicament for modulating the immune response in a subject.

47. Use of the pharmaceutical composition according to claim 42 in the manufacture of a medicament for treating a disease or disorder in a subject.

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