PD-L1 variant immunomodulatory proteins and uses thereof

Abstract

The present invention relates to PD-L1 variant immunomodulatory proteins and uses thereof. Provided herein are immunomodulatory proteins comprising the variant PD-L1 and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

Description

[0001] This application is a divisional application. The application date of the original application is March 13, 2018, the application number is 201880031791.0 (PCT / US2018 / 022265), and the name of the invention is “PD-L1 variant immunomodulatory protein and its use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 472,554, entitled “PD-L1 Variant Immunomodulatory Proteins and Uses Thereof,” filed on March 16, 2017, U.S. Provisional Application No. 62 / 475,076, entitled “PD-L1 Ligand Variant Immunomodulatory Proteins and Uses Thereof,” filed on March 22, 2017, U.S. Provisional Application No. 62 / 537,923, entitled “PD-L1 Ligand Variant Immunomodulatory Proteins and Uses Thereof,” filed on July 27, 2017, and U.S. Provisional Application No. 62 / 582,249, entitled “PD-L1 Ligand Variant Immunomodulatory Proteins and Uses Thereof,” filed on November 6, 2017, the contents of each of which are incorporated by reference in their entirety.

[0004] Sequence Listing Incorporated by Reference

[0005] This application is submitted in electronic format along with a sequence listing. The sequence listing is provided as a file named 761612001040SeqList.TXT, created on February 24, 2018, and the file size is 3,329,416 bytes. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Technical Field

[0006] 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 PD-L1 that exhibit improved binding (such as improved binding affinity or selectivity) to one or more of the cognate binding partner proteins PD-1 and CD80. Background Art

[0007] Regulating immune responses by intervening in the process occurring in the immunological synapse (IS) formed by and between antigen presenting cells (APCs) or target cells and lymphocytes has increasing medical significance. Mechanistically, the cell surface proteins in the IS can involve the coordinated and usually simultaneous interaction of multiple protein targets with the single proteins to which they bind. The IS interaction occurs closely related to the engagement of two cells, and the single protein in this structure can interact with both proteins on the same cell (cis) and proteins on the associating cells (trans) at the same time. Although therapeutic agents that can regulate the IS are known, improved therapeutic agents are still needed. Immunomodulatory proteins that meet such needs are provided, including soluble proteins or transmembrane immunomodulatory proteins that can be expressed on cells. Summary of the Invention

[0008] Provided herein is a variant PD-L1 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications corresponding to positions in unmodified PD-L1 or a specific binding fragment thereof selected from the group consisting of: 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 9, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 213 or 221, which are numbered with reference to SEQ ID NO: 30 or 1728. In some cases, the amino acid modification is an amino acid substitution, insertion, or deletion.

[0009] In some embodiments, the unmodified PD-L1 is a mammalian PD-L1 or a specific binding fragment thereof. In some embodiments, the unmodified PD-L1 is a human PD-L1 or a specific binding fragment thereof. In some of any such embodiments, the unmodified PD-L1 comprises (i) the amino acid sequence set forth in SEQ ID NO: 30 or 1728, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30 or 1728; or (iii) a portion thereof comprising an IgV structure or an IgC domain or a specific binding fragment thereof, or both.

[0010] In some of any such embodiments, the specific binding fragment of the IgV domain or IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or the specific binding fragment of the IgV domain contains at least 80% of the length of the IgV domain listed as amino acids 24-130 of SEQ ID NO: 3; and / or the specific binding fragment of the IgC domain contains at least 80% of the length of the IgC domain listed as amino acids 133-225 of SEQ ID NO: 3. In some of any such embodiments, the variant PD-L1 contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, optionally amino acid substitutions, insertions and / or deletions. In some any such embodiments, the variant PD-L1 polypeptide contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30, 1728, or a specific binding fragment thereof.

[0011] In some of any such embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of PD-1 compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1. In some embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of CD80 compared to the binding of unmodified PD-L1 to the extracellular domain of CD80. In some of any such embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of PD-1 compared to unmodified PD-L1. In some embodiments, the altered binding is altered binding affinity and / or altered binding selectivity.

[0012] In some any such embodiments, one or more amino acid modifications are selected from P6S, Y10F, V11A, V11E, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I46V, I47T, F49 S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65L, R68L, K7 1E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, G101G-ins( G101GG), G102D, A103V, D104G, K106E, K106R, V110M, K111E, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T130 A. S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171 R, F173I, F173L, K173Y, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, T221L, or conservative amino acid substitutions thereof. In some of any such embodiments, one or more amino acid modifications are selected from K28N / M41V / N45T / H51N / K57E, I20L / I36T / N45D / I47T, I20L / M41K / K44E,P6S / N45T / N78I / I83T、N78I、M41K / N78I、N45T / N78I、I20L / N45T、N 45T、M41K、I20L / I36T / N45D、N17D / N47T / V50A / D72G、I20L / F49S、N 45T / V50A、I20L / N45T / N78I、I20L / N45T / V50A、M41V / N45T、M41K / N 45T、A33D / S75P / D85E、M18I / M41K / D43G / H51R / N78I、V11E / I20L / I3 6T / N45D / H60R / S75P、A33D / V50A、S16G / A33D / K71E / S75P、E27G / N4 5T / M97I、E27G / N45T / K57R、A33D / E53V、D43G / N45D / V58A、E40G / D43 V / N45T / V50A、Y14S / K28E / N45T、A33D / N78S、A33D / N78I、A33D / N45 T、A33D / N45T / N78I、E27G / N45T / V50A、N45T / V50A / N78S、I20L / N45T / V110M、I20L / I36T / N45T / V50A、N45T / L74P / S75P、N45T / S75P、S75 P / K106R、S75P、A33D / S75P、A33D / S75P / D104G、A33D / S75P、I20L / E2 7G / N45T / V50A、I20L / E27G / D43G / N45D / V58A / N78I、I20L / D43G / N4 5D / V58A / N78I、I20L / A33D / D43G / N45D / V58A / N78I、I20L / D43G / N45 D / N78I、E27G / N45T / V50A / N78I、N45T / V50A / N78I、V11A / I20L / E27 G / D43G / N45D / H51Y / S99G、I20L / E27G / D43G / N45T / V50A、I20L / K28E / D43G / N45D / V58A / Q89R、I20L / I36T / N45D、I20L / K28E / D43G / N45D / E53G / V58A / N78I、A33D / D43G / N45D / V58A / S75P、K23R / D43G / N45D、 I20L / D43G / N45D / V58A / N78I / D90G / G101D、D43G / N45D / L56Q / V58A / G101G-ins(G101GG) 、I20L / K23E / D43G / N45D / V58A / N78I、I20L / K23E / D43G / N45D / V50A / N78I、T19I / E27G / N45I / V50A / N78I / M97K、I20L / M41K / D43G / N45D、K23R / N45T / N78I、I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins(G101GG)、K57R / S99G、K57R / S99G / F189L、 M18V / M97L / F193S / R195G / E200K / H202Q、I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F 、C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L 、M18V / I98L / L124S / P198T / L206F、S99G / N117S / I148V / K171R / R180S、I36T / M97L / A103V / Q155H、K28I / S99G、R195S、A79T / S 99G / T185A / R195G / E200K / H202Q / L206F、K57R / S99G / L124S / K144Q、K57R / S99G / R195G、D55V / M97L / S99G、E27G / I36T / D55N / M97L / K111E、E54G / M97L / S99G、G15A / I36T / M97L / K111E / H202Q、G15A / I36T / V129D、G15A / I36T / V129D / R195G、G15A / V129D、 I36S / M97L、I36T / D55N / M97L / K111E / A204T、I36T / D55N / M97L / K111E / V129A / F173L、I36T / D55S / M97L / K111E / I148V / R180S 、I36T / G52R / M97L / V112A / K144E / V175A / P198T 、I36T / I46V / D55G / M97L / K106E / K144E / T185A / R195G 、I36T / I83T / M97L / K144E / P198T、I36T / M97L / K111E、I36T / M97L / K144E / P198T、I36T / M97L / Q155H / F193S / N201Y、I36 T / M97L / V129D、L35P / I36S / M97L / K111E、M18I / I36T / E53G / M97L / K144E / E199G / V207A、M18T / I36T / D55N / M97L / K111E、M18V / M97L / T176N / R195G、M97L / S99G、N17D / M97L / S99G、S99G / T185A / R195G / P198T、V129D / H202Q、V129D / P198T、V129D / T150A、V93E / V129D、Y10F / M18V / S99G / Q138R / T203A、N45D、K160M / R195G、N45D / K144E、N45D / P198S、N45D / P198T、N45D / R195G、N45D / R195S、N45D / S131F、N45D / V58D、V129D / R195S、I98T / F173Y / L196S、N45D / E134G / L213P、N45D / F173I / S177C、N45D / I148V / R195G、N45D / K111T / R195G、N45D / N113Y / R195S、N45D / N165Y / E170G、N45D / Q89R / I98V、N45D / S131F / P198S、N45D / S75P / P198S、N45D / V50A / R195T、E27D / N45D / T183A / I188V 、 F173Y / T183I / L196S / T203A 、K23N / N45D / S75P / N120S 、 N45D / G102D / R194W / R195G 、N45D / G52V / Q121L / P198S 、 N45D / I148V / R195G / N201D 、N45D / K111T / T183A / I188V 、 N45D / Q89R / F189S / P198S 、N45D / S99G / C137R / V207A 、 N45D / T163I / K167R / R195G 、N45D / T183A / T192S / R194G 、 N45D / V50A / I119T / K144E 、T19A / N45D / K144E / R195G 、 V11E / N45D / T130A / P198T 、V26A / N45D / T163I / T185A、 K23N / N45D / L124S / K167T / R195G、K23N / N45D / Q73R / T163I 、 K28E / N45D / W149R / S158G / P198T 、K28R / N45D / K57E / I98V / R195S、K28R / N45D / V129D / T163N / R195T、M41K / D43G / N45D / R64S / R195G、M41K / D43G / N45D / R64S / S99G、N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D, or N45D / V50A / L124S / K144E / L179P / R195G.

[0013] In some any such embodiments, the one or more amino acid modifications correspond to one or more positions selected from 20, 27, 28, 33, 36, 41, 43, 45, 50, 58, 71, 75, or 78. In some embodiments, the one or more amino acid substitutions are selected from I20L, E27G, K28E, A33D, I36T, M41K, D43G, N45D, N45T, V50A, V58A, K71E, S75P, N78I, or conservative amino acid substitutions thereof.

[0014] In some any such embodiments, the one or more amino acid modifications correspond to one or more positions selected from 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, or 195. In some embodiments, the one or more amino acid substitutions are selected from I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide contains amino acid modifications I20L / I36T, I20L / D43G, I20L / N45D, I20L / N45T, I20L / N45T, I20L / V50A, I20L / V58A, I20L / S75P, I20L / N78I, I36T / D43G, I36T / N45D, I36T / N45T, I36T / V50A, I36T / V58A, I36T / S75P, I36T / N78I, D43G / N45D, In some embodiments, the variant PD-L1 polypeptide contains the amino acid modification D43G / N45D / V58A. In some aspects, the variant PD-L1 polypeptide contains amino acid modifications D43G / N45D / L56Q / V58A / G101G-ins (G101GG) or I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins (G101GG).

[0015] In some of any such embodiments, the variant PD-L1 polypeptide includes a variant PD-L1 polypeptide comprising or consisting of a PD-L1 extracellular domain (ECD); and / or a variant PD-L1 polypeptide comprising or consisting of an IgV domain or a specific fragment thereof and an IgC domain or a specific fragment thereof. In some of any such embodiments, the variant PD-L1 polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008, or a specific binding fragment thereof, or an amino acid sequence or a specific binding fragment thereof having at least 95% sequence identity to any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 and containing one or more amino acid substitutions.

[0016] In some of any such embodiments, the variant PD-L1 polypeptide contains an IgV domain or a specific binding fragment thereof. In some of any such embodiments, the IgV domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide. In some embodiments, the IgC domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.

[0017] In some of any such embodiments, the variant PD-L1 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937, or a specific binding fragment thereof, an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937, and containing one or more amino acid substitutions, or a specific binding fragment thereof. In some embodiments, the IgC domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.

[0018] In some any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain of PD-1 or CD80 with increased affinity compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1 or CD80. In some any such embodiments, the variant PD-L1 polypeptide specifically binds to the extracellular domain of PD-1 with increased affinity compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1. In some any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain with increased affinity compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1 and the extracellular domain of CD80, respectively. In some any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain of PD-1 with increased affinity and binds to the same extracellular domain of CD80 with reduced affinity compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1 and the extracellular domain of CD80.

[0019] In some of any such embodiments, the increased affinity for the extracellular domain of PD-1 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to unmodified PD-L1. In some aspects, the increased affinity for the extracellular domain of CD80 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to unmodified PD-L1. In some aspects, the reduced affinity for the extracellular domain of CD80 is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to unmodified PD-L1.

[0020] In some of any such embodiments, the variant polypeptide specifically binds to the extracellular domain of PD-1 with increased selectivity compared to unmodified PD-L1. In some cases, the increased selectivity comprises a greater ratio of binding of the variant polypeptide to PD-1 versus the corresponding extracellular domain of CD80 compared to the ratio of binding of the unmodified PD-L1 polypeptide to PD-1 versus CD80. In some instances, the ratio is at least or about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more greater.

[0021] In some any such embodiments, PD-1 is human PD-1. In some any such embodiments, CD80 is human CD80.

[0022] In some any such embodiments, the binding activity is altered (increased or decreased) by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold compared to unmodified PD-L1.

[0023] In some of any such embodiments, the variant PD-L1 polypeptide is a soluble protein. In some of any such embodiments, the variant PD-L1 polypeptide lacks the PD-L1 transmembrane domain and the intracellular signaling domain; and / or the variant PD-L1 polypeptide is not capable of being expressed on the surface of a cell. In some of any such embodiments, the variant PD-L1 polypeptide is linked to a multimerization domain. In some of any such embodiments, the variant PD-L1 polypeptide is a multimeric polypeptide, optionally a dimeric polypeptide, comprising a first variant PD-L1 polypeptide linked to a multimerization domain and a second variant PD-L1 polypeptide linked to a multimerization domain. In some embodiments, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are the same or different.

[0024] In some any such embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some any such embodiments, the variant PD-L1 polypeptide is linked to a moiety that increases the biological half-life of the polypeptide. In some any such embodiments, the variant PD-L1 polypeptide is linked to an Fc domain or a variant thereof with reduced effector function.

[0025] In some any such embodiments, the Fc domain is a mammalian Fc domain, optionally a human Fc domain; or the variant Fc domain comprises one or more amino acid modifications compared to a mammalian (optionally human) unmodified Fc domain. In some any such embodiments, the Fc domain or its variant contains an amino acid sequence as set forth in SEQ ID NO: 187 or SEQ ID NO: 188, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 187 or SEQ ID NO: 188. In some embodiments, the Fc domain comprises one or more amino acid modifications selected from the group consisting of E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, R292C, V302C, and K447del, each of which is numbered by EU. In some embodiments, the Fc domain comprises an amino acid modification C220S by EU numbering. In some any such embodiments, the Fc domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940, or an amino acid sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940 and exhibits reduced effector function.

[0026] In some any such embodiments, the variant PD-L1 polypeptide is indirectly linked via a linker (optionally a G4S linker). In some any such embodiments, the variant PD-L1 polypeptide is a transmembrane immunomodulatory protein that further contains a transmembrane domain linked to the extracellular domain (ECD) of the variant PD-L1 polypeptide or a specific binding fragment thereof.

[0027] In some of any such embodiments, the transmembrane domain comprises an amino acid sequence set forth as residues 239-259 of SEQ ID NO: 3, or a functional variant thereof having at least 85% sequence identity to residues 239-259 of SEQ ID NO: 3. In some embodiments, the variant PD-L1 polypeptide further comprises a cytoplasmic signaling domain linked to the transmembrane domain. In some cases, the cytoplasmic signaling domain comprises an amino acid sequence set forth as residues 260-290 of SEQ ID NO: 3, or a functional variant thereof having at least 85% sequence identity to residues 260-290 of SEQ ID NO: 3.

[0028] In some any of the provided embodiments, the variant PD-L1 polypeptide modulates the response of immune cells (such as T cells). In some embodiments, the response (e.g., T cell response) is increased or decreased. In some any of these embodiments, in an in vitro T cell assay, the variant PD-L1 increases IFN-γ (interferon-γ) expression relative to unmodified PD-L1. In some any of these embodiments, in an in vitro T cell assay, the variant PD-L1 reduces IFN-γ (interferon-γ) expression relative to unmodified PD-L1.

[0029] In some of any such embodiments, the variant PD-L1 polypeptide is aglycosylated.

[0030] In some embodiments of any of the variant PD-L1 polypeptides described herein, the variant PD-L1 polypeptide increases T cell signaling relative to unmodified PD-L1, such as determined using a reporter gene assay involving T cells (e.g., Jurkat) engineered with a reporter gene (e.g., luciferase) operably linked to an IL-2 promoter. In some embodiments of any of the variant PD-L1 polypeptides described herein, the variant PD-L1 polypeptide reduces T cell signaling relative to unmodified PD-L1, such as determined using a reporter gene assay involving T cells (e.g., Jurkat) engineered with a reporter gene (e.g., luciferase) operably linked to an IL-2 promoter. In some any such embodiments, the variant PD-L1 polypeptide is provided in any of a variety of forms, such as soluble or fixed (e.g., plate-bound).

[0031] Also provided is an immunomodulatory polypeptide comprising a variant PD-L1 according to any of the provided embodiments, the variant PD-L1 being linked directly or indirectly via a linker to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain. In some cases, the IgSF domain is affinity-modified and exhibits altered binding to one or more of the same cognate binding partners compared to the binding of the unmodified or wild-type IgSF domain to one or more of its cognate binding partners. In some embodiments, the affinity-modified IgSF domain contains one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of an IgSF family member. In some cases, the IgSF domain exhibits increased binding to the same one or more cognate binding partners compared to the binding of the unmodified or wild-type IgSF domain of an IgSF family member to one or more of its cognate binding partners. In some examples, the IgSF domain is affinity-modified and exhibits altered binding to one or more of its cognate binding partners compared to binding of an unmodified or wild-type IgSF domain of an IgSF family member to one or more of its cognate binding partners. In some embodiments, the variant PD-L1 is a first PD-L1 variant and the IgSF domain of the second polypeptide is an IgSF domain from a second variant PD-L1, wherein the first PD-L1 variant and the second PD-L1 variant are the same or different.

[0032] In some embodiments, the variant PD-L1 polypeptide is capable of specifically binding to PD-1 or CD80, and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than the cognate binding partner specifically bound by the PD-L1 variant polypeptide. In some embodiments, the IgSF domain is from a member of the B7 family. In some cases, the IgSF domain is a tumor-localized portion that binds to a ligand expressed on a tumor, or an inflammatory-localized portion that binds to cells or tissues associated with an inflammatory environment. In some embodiments, the IgSF domain is a tumor-localized portion that binds to a ligand expressed on a tumor. In some cases, the ligand is B7H6. In some embodiments, the IgSF domain is from NKp30.

[0033] In some embodiments, the IgSF domain of the second or third polypeptide or its affinity-modified IgSF domain is or contains an IgV domain. In some embodiments, the variant PD-L1 polypeptide is or contains an IgV domain. In some embodiments, the immunomodulatory protein comprises a multimerization domain linked to one or both of the variant PD-L1 polypeptide or the IgSF domain. In some cases, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the immunomodulatory protein is a dimer. In some embodiments, the immunomodulatory protein is a homodimer. In some embodiments, the immunomodulatory protein is a heterodimer.

[0034] In some of any such embodiments, the IgSF domain of the second polypeptide is an IgSF domain of a ligand that binds to an inhibitory receptor or is an affinity-modified IgSF domain thereof. In some cases, the affinity-modified IgSF domain exhibits increased binding affinity and / or binding selectivity for the same inhibitory receptor compared to the binding of the unmodified IgSF domain to the inhibitory receptor. In some embodiments, the inhibitory receptor is TIGIT, PD-1, or CTLA-4; or the ligand of the inhibitory receptor is PD-L2, CD155, CD112, or CD80.

[0035] In some any such embodiments, the IgSF domain of the second polypeptide is an affinity-modified IgSF domain comprising: (i) wild-type CD155 comprising an IgSF listed in any one of SEQ ID NOs: 47, 310, or 353, or a variant CD155 polypeptide comprising an IgSF domain of any one of SEQ ID NOs listed in Table 5, optionally any one of SEQ ID NOs: 311-352, 354-665, 1505-1576, 1551-1714; (ii) wild-type CD112 comprising an IgSF domain listed in any one of SEQ ID NOs: 48, 666, or 761, or a variant CD112 polypeptide comprising an IgSF domain of any one of SEQ ID NOs listed in Table 4, optionally any one of SEQ ID NOs: 667-760, 762-931, 1433-1504; (iii) a variant CD112 polypeptide comprising an IgSF domain of any one of SEQ ID NOs listed in Table 4, optionally any one of SEQ ID NOs: (iv) a wild-type CD80 polypeptide comprising an IgSF domain as set forth in any one of SEQ ID NOs: 31, 1203, or 1263, or a variant PD-L2 polypeptide comprising an IgSF domain as set forth in any one of SEQ ID NOs: 1204-1254, 1256-1331, 1333-1407, 1409-1432; (v) a variant PD-L2 polypeptide comprising an IgSF domain as set forth in any one of SEQ ID NOs: 1204-1254, 1256-1331, 1333-1407, 1409-1432; An amino acid sequence having at least 95% sequence identity to any of NO and comprising amino acid substitutions; or (vi) a specific binding fragment of any of (i)-(v).

[0036] In some embodiments, the immunomodulatory protein further comprises a third polypeptide comprising an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, wherein the affinity-modified IgSF domain comprises one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of the IgSF family member. In some cases, the third polypeptide is identical to the first polypeptide and / or the second polypeptide, or the third polypeptide is different from the first polypeptide and / or the second polypeptide. In some examples, the third polypeptide is selected from (i) wild-type CD155 comprising an IgSF domain as set forth in any one of SEQ ID NOs: 47, 310, or 353, or a variant CD155 polypeptide comprising an IgSF domain as set forth in any one of SEQ ID NOs: 311-352, 354-665, 1505-1576, 1551-1714; (ii) wild-type CD112 comprising an IgSF domain as set forth in any one of SEQ ID NOs: 48, 666, or 761, or a variant CD112 polypeptide comprising an IgSF domain as set forth in any one of SEQ ID NOs: 667-760, 762-931, 1433-1504; (iii) wild-type CD80 comprising an IgSF domain as set forth in any one of SEQ ID NOs: 28, 1005, or 2030, or a variant CD80 comprising an IgSF domain as set forth in any one of SEQ ID NOs: (iv) a wild-type PD-L2 polypeptide comprising an IgSF domain set forth in any one of SEQ ID NOs: 31, 1203, or 1263, or a variant PD-L2 polypeptide comprising an IgSF domain set forth in any one of SEQ ID NOs: 1204-1254, 1256-1331, 1333-1407, 1409-1432; (v) an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs of (i) to (iv) and comprising an amino acid substitution; or (vi) a specific binding fragment of any one of (i) to (v). In some cases, optionally the IgSF domain of the second polypeptide or the third polypeptide, or its affinity-modified IgSF domain, is or contains an IgV domain. In some cases, the variant PD-L1 polypeptide is or contains an IgV domain.

[0037] In some embodiments, the immunomodulatory protein further comprises at least one additional polypeptide comprising an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, wherein the affinity-modified IgSF domain comprises one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member. In some embodiments, the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide or the second polypeptide. In some aspects, the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide, the second polypeptide, and / or the third polypeptide. In some cases, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the multimerization domain promotes heterodimer formation.

[0038] An immunomodulatory protein is provided, comprising a first variant PD-L1 polypeptide, wherein the multimerization domain is a first multimerization domain; and a second variant PD-L1 polypeptide, wherein the multimerization domain is a second multimerization domain, wherein the first multimerization domain and the second multimerization domain interact to form a multimer containing the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide, optionally wherein the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are identical. In some cases, the multimerization domain is the first multimerization domain and interacts with the second multimerization domain to form a multimer comprising the immunomodulatory protein. In some instances, the immunomodulatory protein is the first immunomodulatory protein and the second immunomodulatory protein is directly or indirectly linked to the second multimerization domain via a linker, wherein the multimer comprises the first immunomodulatory protein and the second immunomodulatory protein. In some embodiments, the second immunoglobulin regulatory protein is any of the immunoglobulin regulatory proteins described, and the multimerization structure is the second multimerization domain. In some cases, the multimer is a dimer. In some embodiments, the second polypeptide is a variant CD155 polypeptide, and the first and / or second immunomodulatory protein comprises a sequence as set forth in any one of SEQ ID NOs: 1716-1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1716-1721; or the second polypeptide is CD112 or CD155 and the third polypeptide is the other of CD112 or CD155, and the first and / or second immunomodulatory protein comprises a sequence as set forth in any one of SEQ ID NOs: 1722-1724, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1716-1721. Any of NOs: 1716-1721 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0039] In some cases, the immunomodulatory protein is a homodimer.In some aspects, the immunomodulatory protein is a heterodimer, optionally wherein the first multimerization domain and the second multimerization domain are different and / or capable of interacting to mediate heterodimer formation.

[0040] In some embodiments, the first and / or second multimerization domain is an Fc domain or a variant thereof with reduced effector function, optionally wherein the Fc domain is an Fc domain of a human immunoglobulin and / or the Fc region is a human Fc region, optionally wherein the Fc region is the Fc region of immunoglobulin G1 (IgG1) or immunoglobulin G2 (IgG2), optionally set forth in SEQ ID NO: 187 or SEQ ID NO: 188, optionally wherein the Fc region exhibits one or more effector functions; or the variant Fc domain comprises one or more amino acid substitutions in a wild-type Fc region, optionally wherein the reduced effector function is reduced compared to a wild-type Fc region, optionally wherein the wild-type human Fc is the Fc of human IgG1. In some cases, the first and second multimerization domains are the same or different.

[0041] In some embodiments, the variant Fc region contains the amino acid substitutions E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, wherein the residue numbering is according to the EU index as in Kabat; or the amino acid substitutions R292C / N297G / V302C or L234A / L235E / G237A, wherein the residue numbering is according to the EU index as in Kabat. In some instances, the Fc region or variant Fc region contains the amino acid substitution C220S, wherein the residue numbering is according to the EU index as in Kabat. In some cases, the Fc region or variant Fc region contains K447del, wherein the residue numbering is according to the EU index as in Kabat.

[0042] Also provided is a conjugate comprising a variant PD-L1 according to any provided embodiment or an immunomodulatory polypeptide according to any provided embodiment linked to a moiety. In some cases, the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell. In some aspects, the targeting moiety specifically binds to a molecule on the surface of an immune cell.

[0043] In some embodiments, the immune cell is an antigen presenting cell or a lymphocyte. In some cases, the targeting moiety is a tumor localizing moiety that binds to a molecule on the surface of the tumor. In some instances, 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 of any such embodiments, the conjugate is bivalent, tetravalent, hexavalent, or octavalent. In some aspects, the conjugate is a fusion protein.

[0044] Also provided is a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any provided embodiment, or an immunomodulatory polypeptide or conjugate according to any provided embodiment as a fusion protein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is a cDNA.

[0045] Also provided is a vector comprising a nucleic acid molecule according to any of the provided embodiments. In some cases, the vector is an expression vector. In some aspects, the vector is a mammalian expression vector or a viral vector.

[0046] Also provided is a cell comprising a vector according to any of the provided embodiments. In some cases, the cell is a mammalian cell. In some aspects, the cell is a human cell.

[0047] Also provided is a method for producing a variant PD-L1 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule according to any of the provided embodiments or a vector according to any of the provided embodiments into a host cell under conditions where the protein is expressed. In some cases, the method further comprises isolating or purifying the variant PD-L1 polypeptide or immunomodulatory protein from the cell. Also provided is a method for engineering a cell that expresses a variant PD-L1 variant polypeptide, comprising introducing a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any of the provided embodiments into the cell under conditions where the polypeptide is expressed in the host cell.

[0048] Also provided is an engineered cell that expresses a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, a conjugate according to any provided embodiment as a fusion protein, a nucleic acid molecule according to any provided embodiment, or a vector according to any provided embodiment. In some cases, the variant PD-L1 polypeptide or immunomodulatory protein comprises a signal peptide. In some cases, the variant PD-L1 polypeptide or immunomodulatory protein is encoded by a nucleic acid comprising a nucleotide sequence encoding a signal peptide. In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein does not contain a transmembrane domain and / or is not expressed on the cell surface.

[0049] In some embodiments of the engineered cells, the variant PD-L1 polypeptide or immunomodulatory protein is secreted or capable of being secreted from the engineered cells. In some embodiments, the engineered cells contain a variant PD-L1 polypeptide that contains a transmembrane domain and / or is a transmembrane immunomodulatory protein according to any of the provided embodiments. In some embodiments, the variant PD-L1 polypeptide is expressed on the cell surface.

[0050] In some embodiments of the engineered cell, the cell is an immune cell. In some cases, the immune cell is an antigen presenting cell (APC) or a lymphocyte. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the APC is an artificial APC. In some instances, the cell is a lymphocyte, and the lymphocyte is a T cell. In some aspects, the cell is an APC, and the APC is an artificial APC. In some any such embodiments, the engineered cell also contains a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0051] Also provided is an infectious agent containing a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any provided embodiment, a conjugate according to any provided embodiment as a fusion protein, or an immunomodulatory polypeptide according to any provided embodiment. In some cases, the encoded variant PD-L1 polypeptide or immunomodulatory polypeptide does not contain a transmembrane domain and / or is not expressed on the surface of the cell in which it is expressed. In some embodiments, the encoded variant PD-L1 polypeptide or immunomodulatory polypeptide is secreted or capable of being secreted from the cell in which it is expressed. In some cases, the encoded variant PD-L1 polypeptide contains a transmembrane domain. In some embodiments, the encoded variant PD-L1 polypeptide is expressed on the surface of the cell in which it is expressed.

[0052] In some any such embodiments, the infectious agent is a bacterium or virus. In some embodiments, the virus is a slow virus or retroviral construct or its heterocomplex. In some respects, the virus is an oncolytic virus. In some instances, the oncolytic virus is adenovirus, adeno-associated virus, herpes virus, herpes simplex virus, vesicular oral virus (VesticularStomatic virus), reovirus, Newcastle disease virus, parvovirus, measles virus, vesicular stomatitis virus (VSV), coxsackie virus or vaccinia virus. In some cases, the virus-specific targeting dendritic cells (DC) and / or is dendritic cell tropism. In some instances, the virus is a slow virus vector pseudotyped with modified Sindbis virus (Sindbis virus) envelope product. In some any such embodiments, the infectious agent also contains a nucleic acid molecule encoding another gene product, and the another gene product causes target cell death or can enhance or improve immune response. In some examples, the other gene product is selected from an anticancer agent, an antimetastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degradation gene, a gene for tissue regeneration or reprogramming human cells to pluripotency.

[0053] Also provided is a pharmaceutical composition containing a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, a conjugate according to any embodiment, an engineered cell according to any provided embodiment, or an infectious agent according to any provided embodiment. In some cases, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0054] Also provided is an article of manufacture containing a pharmaceutical composition according to any of the provided embodiments in a vial. In some cases, the vial is sealed.

[0055] Also provided is a kit comprising a pharmaceutical composition according to any of the provided embodiments and instructions for use. Also provided is a kit comprising an article of manufacture according to any of the provided embodiments and instructions for use.

[0056] Also provided is a method for regulating an immune response of a subject (such as increasing or decreasing an immune response), comprising administering to the subject a pharmaceutical composition according to any of the provided embodiments. In some embodiments, the method for regulating the immune response of a subject comprises administering to the subject an engineered cell according to any of the provided embodiments. In some cases, the engineered cell is autologous to the subject. In some cases, the engineered cell is allogeneic to the subject. In some embodiments, the method for regulating the immune response treats a disease or condition in the subject. In some embodiments, the method comprises administering to the subject a soluble variant PD-L1 polypeptide according to any one of the embodiments described herein, an immunomodulatory protein according to any one of the embodiments described herein, or a conjugate according to any one of the embodiments described herein. In some embodiments, the method comprises administering to the subject an infectious agent encoding a variant PD-L1 polypeptide according to any one of the embodiments described herein.

[0057] In some of any such embodiments, the immune response is increased. In some embodiments, a soluble variant PD-L1 polypeptide or immunomodulatory protein is administered to a subject. In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein is an Fc fusion protein. In some embodiments, a soluble variant PD-L1 polypeptide or immunomodulatory protein, optionally lacking the PD-L1 transmembrane domain and intracellular signaling domain, is administered to a subject. In some cases, the soluble immunomodulatory protein is an immunomodulatory Fc fusion protein. In some embodiments, a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, or a conjugate according to any provided embodiment is administered to a subject. In some embodiments, an engineered cell containing a secretable variant PD-L1 polypeptide is administered to a subject. In some of any such embodiments, an engineered cell according to any provided embodiment is administered to a subject. In some aspects, the infectious agent is optionally administered to a subject under conditions where the infectious agent encoding the variant PD-L1 polypeptide that secretes the immunomodulatory protein infects a tumor cell or an immune cell and the secretable immunomodulatory protein is secreted from the infected cell.

[0058] In some embodiments, the disease or condition is a tumor or cancer. In some instances, 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 cancers.

[0059] In some of any such embodiments, the immune response is reduced. In some embodiments, an immunomodulatory protein or conjugate containing a variant PD-L1 polypeptide linked to an IgSF domain or a portion thereof is administered to a subject, wherein the portion is localized to cells or tissues in an inflammatory environment. In some cases, the binding molecule comprises an antibody or antigen-binding fragment thereof or contains a wild-type IgSF domain or a variant thereof. In some embodiments, an immunomodulatory protein according to any provided embodiment or a conjugate according to any provided embodiment is administered to a subject.

[0060] In some embodiments, a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject. In some embodiments, an engineered cell containing a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein according to any of the provided embodiments is administered to a subject. In some cases, the infectious agent is optionally administered to a subject under conditions where the infectious agent encoding the variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein infects a tumor cell or an immune cell and the transmembrane immunomodulatory protein is expressed on the surface of the infected cell.

[0061] In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition. In some any such embodiments, the disease or condition is anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatism, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease or autoimmune blood disease. In some instances, the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease (Crohn's disease), ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis or psoriasis. In some any such embodiments, a variant PD-L1 in a form that reduces the immune response of the subject is administered. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figures 1A to 1C Depicted are different formats of the provided variant IgSF domain molecules. Figure 1A Soluble molecules are depicted, including: (1) a variant IgSF domain (vIgD) fused to an Fc chain; (2) a stacked molecule containing a first variant IgSF domain (first vIgD) and a second IgSF domain, such as a second variant IgSF domain (second vIgD); (3) a tumor-targeting IgSF molecule containing a first variant IgSF domain (vIgD) and an IgSF domain that targets a tumor antigen, such as an NKp30 IgSF domain; and (4) a variant IgSF domain (vIgD) linked to an antibody (V-mAb). Figure 1BDepicts a transmembrane immunomodulatory protein (TIP) containing a variant IgSF domain (vIgD) expressed on the cell surface. In an exemplary embodiment, the cognate binding partner of the transmembrane bound vIgD is an inhibitory receptor (e.g., PD-L1), and the TIP containing vIgD (e.g., PD-L1vIgD) antagonizes or blocks the negative signaling of the inhibitory receptor, thereby generating activated T cells or effector T cells. In some cases, if the clustering of inhibitory receptors (PD-1) is close to an activating receptor (e.g., CD28), then the active excitement performed by TIP can be achieved. Figure 1C Describe secreted immunomodulatory protein (SIP), wherein variant IgSF domain (vIgD) is secreted from a kind of cell, such as the first T cell (for example, CAR T cell).In an exemplary embodiment, the cognate binding partner of secreted vIgD is inhibitory receptor (for example, PD-1), which can be expressed by the first cell (for example, T cell, such as CAR T cell) and / or expressed on the second cell (for example T cell;Homologous or engineered cells, such as CAR T cells).When SIP is combined with its cognate binding partner, SIP antagonizes or blocks the negative signal conduction carried out via inhibitory receptors, thereby producing activated T cells or effector T cells.In all cases, vIgD can be any combination of the combination of only V- domains (IgV), V- domains (IgV) and C- domains (IgC) (including the entire extracellular domain (ECD)) or the Ig domains of IgSF superfamily members.

[0063] Figure 2 An exemplary schematic depicting the activity of a variant IgSF domain (vIgD) fused to Fc (vIgD-Fc), wherein vIgD is a variant of the IgSF domain of PD-L1. As shown, soluble vIgD of PD-L1 interacts with its cognate binding partner to block the interaction of PD-L1 or PD-L2 with PD-1, thereby blocking the PD-1 inhibitory receptor and, in some cases, allowing T cells to differentiate into an effector phenotype.

[0064] Figure 3An exemplary schematic diagram depicting a stacking molecule is a multi-target checkpoint antagonist containing a first variant IgSF domain (first vIgD) that is a PD-L1 or PD-L2 vIgD and a second IgSF domain (e.g., a second vIgD) that binds to a second inhibitory receptor. In the exemplary schematic diagram, the second IgSF domain (e.g., a second vIgD) is a CD112 or CD155 vIgD. As shown, the first vIgD and the second vIgD interact with their cognate binding partners to block the interaction of PD-L1 or PD-L2 with PD-1, respectively, and to block the interaction of CD155 or CD112 with TIGIT and / or CD112R, thereby blocking multiple inhibitory receptors.

[0065] Figure 4 Depicts an exemplary schematic diagram of a stacking molecule for localizing variant IgSF (vIgD) to tumor cells. In this form, the stacking molecule contains a first variant IgSF domain (a first vIgD) and a second IgSF domain (e.g., a second vIgD), wherein the second IgSF domain (e.g., a second vIgD) is a tumor-targeting IgSF domain that is bound to a tumor antigen. An exemplary tumor-targeting IgSF domain is an IgSF domain of NKp30 that is bound to the tumor antigen B7-H6. In this depiction, the variant IgSF domain (vIgD) is a variant of the IgSF domain of PD-L1. As shown in the figure, the tumor-targeting IgSF domain is bound to the surface of the tumor cell to locate the first variant IgSF domain on the surface of the tumor cell, wherein the first variant IgSF domain can interact with one or more cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells) to antagonize PD-1 inhibitory activity and promote T cell activation.

[0066] Figure 5A Various exemplary configurations of stacking molecules are depicted, the stacking molecules containing a first variant IgSF domain (a first vIgD) and a second IgSF domain, such as a second variant IgSF domain (a second vIgD). As shown, the first vIgD and the second IgSF domain are independently directly or indirectly connected to the N-terminus or C-terminus of the Fc region. In order to generate homodimeric Fc molecules, the Fc region is an Fc region that can form homodimers with the matching Fc region by co-expressing a single Fc region in a cell. In order to generate heterodimeric Fc molecules, a single Fc region contains a mutation (e.g., a "knob-into-hole" mutation in the CH3 domain) such that when a single Fc region is co-expressed in a cell, it prefers to form heterodimers compared to homodimers.

[0067] Figure 5BDescribe various exemplary configurations of stacking molecules, the stacking molecules containing a first variant IgSF domain (the first vIgD); a second IgSF domain, such as a second variant IgSF domain (the second vIgD); and a third IgSF domain, such as a third variant IgSF domain (the third vIgD). As shown, the first vIgD, the second IgSF, and the third IgSF domain are independently directly or indirectly connected to the N-terminus or C-terminus of the Fc region. In order to generate homodimeric Fc molecules, the Fc region is an Fc region that can form homodimers with matching Fc regions by co-expressing a single Fc region in a cell.

[0068] Figure 6 Describe the exemplary schematic diagram of the activity of the variant IgSF domain (vIgD) conjugated to the antibody (V-Mab), wherein the antibody (e.g., anti-HER2 antibody) is bound to the antigen on the tumor cell surface, so that vIgD is positioned to the cell. As shown in the figure, the antibody is bound to the tumor cell surface and vIgD is positioned on the tumor cell surface, wherein the vIgD can interact with one or more cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells) to excite or antagonize receptor signaling. In the exemplary embodiment as shown in the figure, the variant IgSF domain (vIgD) is a variant of the IgSF domain of the PD-L1 with respect to inhibitory receptor PD-1 (such as with increased affinity for the inhibitory receptor PD-1). The combination of PD-L1 vIgD and PD-1 inhibitory receptors antagonizes or blocks the negative signaling of inhibitory receptors, thereby producing activated T cells or effector T cells. In some cases, if an inhibitory receptor (PD-1) is clustered in proximity to an activating receptor (eg, CD28), then agonism of inhibitory receptor activity by TIP can be achieved.

[0069] 7A to 7C Various exemplary configurations of variant IgSF-antibody conjugates (V-Mab) are depicted. Figure 7A Various configurations are shown in which the variant IgSF domain is linked directly or indirectly to the N-terminus and / or C-terminus of the antibody light chain. Figure 7B Various configurations are shown in which the variant IgSF domain is linked directly or indirectly to the N-terminus and / or C-terminus of the antibody heavy chain. Figure 7C Describe when Figure 7A The light chain and Figure 7B The V-Mab configuration is obtained when the heavy chains of α-binding protein α and α-binding protein α are co-expressed in cells.

[0070] Figure 8 and Figure 9Figure 2 depicts the results of bioactivity of soluble variant PD-L1 IgV-Fc tested in a human mixed lymphocyte reaction (MLR). Approximately 10,000 mature DCs and 100,000 purified allogeneic CD3+ T cells were co-cultured with increasing concentrations of various variant PD-L1 IgV-Fc fusion proteins. Irrelevant human IgG or culture medium alone (indicated as "no supplement") served as negative controls. Control protein PDL1-Fc (full wild-type PD-L1 extracellular domain), wild-type PD-L1 IgV-Fc, and a positive control anti-PD-1 monoclonal antibody (nivolumab) were evaluated. Figure 8 and Figure 9 The calculated levels of IFN-γ (pg / mL) in culture supernatants at the indicated concentrations of variant IgV-Fc fusion molecules are listed.

[0071] Figure 10 Depicted are proliferation studies of T cells transduced with exemplary tested variant PD-L1 SIPs.

[0072] Figure 11 Depicted are the dose responses for binding of the indicated variant IgV-Fc fusion molecules, PD-L1 / CD155 stacked Fc fusion molecules, or PD-L1 / CD155 / CD112 stacked Fc fusion molecules to exhausted T cells.

[0073] Figure 12 Listed are the calculated levels of IFN-γ (pg / mL) in culture supernatants of exhausted T cells at the indicated concentrations of variant IgV-Fc fusion molecules, PD-L1 / CD155 stacked Fc fusion molecules, or antibody controls. DETAILED DESCRIPTION

[0074] Provided herein are immunomodulatory proteins that are or comprise variants or mutants of programmed cell death 1 ligand 1 or PD-L1 (also known as cluster of differentiation 274, CD274.B7 homolog 1, or B7-H1) or specific binding fragments thereof that exhibit binding activity to at least one target ligand cognate binding partner (also known as counter-structure protein). In some embodiments, the variant PD-L1 polypeptide contains one or more amino acid sequences (e.g., amino acid substitutions, deletions, or additions) compared to an unmodified or wild-type PD-L1 polypeptide. In some embodiments, one or more amino acid modifications (e.g., substitutions) are in the IgSF domain (e.g., IgV or ECD) of an unmodified or wild-type PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide and immunomodulatory protein exhibit altered (such as increased or decreased) binding activity or affinity for at least one cognate binding partner (such as at least one of PD-1 or CD80). In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is a transmembrane immunomodulatory protein capable of being expressed on the cell surface. In some embodiments, one or more other immunomodulatory proteins are also provided herein, which are conjugates or fusions containing a variant PD-L1 polypeptide provided herein and one or more other moieties or polypeptides.

[0075] In some embodiments, the variant PD-L1 polypeptides and immunomodulatory proteins modulate an immunological immune response, such as increasing or decreasing the immune response. In some embodiments, the variant PD-L1 polypeptides and immunomodulatory proteins provided herein can be used to treat diseases or conditions associated with a dysregulated immune response.

[0076] In some embodiments, the variant PD-L1 polypeptide provided regulates T cell activation via co-stimulatory and / or co-inhibitory signaling molecules. In general, antigen-specific T cell activation generally requires two different signals. The first signal is provided by the interaction of the T cell receptor (TCR) with the major histocompatibility complex (MHC)-associated antigen present on antigen presenting cells (APCs). The second signal is co-stimulatory for TCR engagement and is necessary for T cell proliferation, differentiation, and / or survival (including avoiding T cell apoptosis or incapacity in some cases).

[0077] In some embodiments, under normal physiological conditions, the T cell-mediated immune response begins with antigen recognition by T cell receptors (TCR) and is regulated by a balance of co-stimulatory signals and inhibitory signals (e.g., immune checkpoint proteins). The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and to prevent tissues from being excessively damaged during an immune response, such as during an attack against a pathogen infection. However, in some cases, these immunomodulatory proteins can be dysregulated in diseases and conditions (including tumors) as a mechanism to escape the immune system.

[0078] In some embodiments, known T cell co-stimulatory receptor is programmed cell death protein 1 or PD-1, which is a T cell co-stimulatory receptor of ligand PD-L1 (also referred to as differentiation cluster 274, CD274.B7 homolog 1 or B7-H1) and programmed cell death 1 ligand 2 or PD-L2 (also referred to as PDCD1L2, PDCD1LG2, differentiation cluster 273, CD273. or B7-DC). PD-L1 and PD-L2 are generally expressed on the surface of T cells, B cells and bone marrow cells. PD-L1 and PD-L2 are negative regulators of immune activation and can lower immune responses via interaction with programmed death 1 (PD-1) receptors. In some aspects, PD-1 is expressed on NK cells and T cells (including CD4+ and CD8+ T cells), whereby engagement of PD-1 can inhibit activation of activated cells, proliferation and / or amplification.

[0079] However, the PD-L1 ligand can also bind to cluster of differentiation 80 (also known as CD80 or B7-1). The binding of PD-L1 to CD80 can block the interaction between PD-L1 and PD-1, and thus strengthen or enhance the immune response. Therefore, in some cases, the interaction between PD-L1 and CD80 and between PD-L1 and PD-1 produces opposite effects in regulating the immune response. Therefore, PD-1 and CD80 can play opposite roles in the immune response to regulate pro-inflammatory or anti-inflammatory responses, which in some cases are associated with a variety of diseases and conditions.

[0080] In some embodiments, PD-1 and CD80 can play a complementary role in modeling immune responses. In some embodiments, the enhancement or inhibition of the activity of PD-1 receptors has clinical significance for treating inflammatory and autoimmune disorders, cancer and viral infections. However, in some cases, the therapy of intervening and changing the immunomodulatory effect of such receptors is limited to spatial orientation requirements and the size restrictions imposed by the boundaries of immunological synapses. In some respects, existing therapeutic drugs (including antibody drugs) may not be able to interact with the multiple target proteins involved in regulating these interactions at the same time. In addition, in some cases, existing therapeutic drugs may only have the ability to antagonize rather than excite immune responses. In addition, the pharmacokinetic differences between the drugs that independently target one of these receptors may produce difficulties in properly maintaining the required blood concentration of such drug combinations throughout the treatment process.

[0081] In some embodiments, the variant PD-L1 polypeptides or immunomodulatory proteins provided regulate (e.g., increase or decrease) the immune activity associated with PD-1. Therefore, in some embodiments, the polypeptides provided overcome these constraints by providing variant PD-L1 with altered (e.g., increased or decreased) binding affinity to PD-1, thereby exciting or antagonizing the effects of the receptor. In some embodiments, the polypeptides provided overcome these constraints by providing variant PD-L1 with altered (e.g., increased or decreased) binding affinity to CD80, thereby regulating the effects of the interaction between PD-1 and PD-L1. Methods for preparing and using these variant PD-L1 are also provided.

[0082] All publications (including patents, patent applications, scientific papers, and databases) mentioned in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication (including patents, patent applications, scientific papers, or databases) was specifically and individually indicated to be incorporated by reference. If the definitions listed herein are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions listed herein shall prevail over the definitions incorporated herein by reference.

[0083] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0084] I. Definition

[0085] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or nouns used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

[0086] Unless otherwise limited in specific circumstances, the terms used throughout this specification are defined as follows. As used in the specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Unless otherwise defined, all technical and scientific terms, abbreviations, and abbreviations used herein have the same meaning as those generally understood by those skilled in the art to which the invention belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names are according to IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values ​​defining the range and all integer values ​​therebetween.

[0087] The term "affinity modified" as used in the context of immunoglobulin superfamily domains means that a mammalian immunoglobulin superfamily (IgSF) domain has an altered amino acid sequence (relative to a corresponding wild-type parent or unmodified IgSF domain) such that it has increased or decreased binding affinity or avidity for at least one of its cognate binding partners (or "relative structure") compared to a parent wild-type or unmodified (i.e., non-affinity modified) IgSF control domain. Affinity-modified PD-L1 IgSF domains are included in this context. In some embodiments, the affinity-modified IgSF domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions, in a wild-type or unmodified IgSF domain. An increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). See also Linsley et al., Immunity, Vol. 1(9): 793-801 (1994). The increase in binding affinity or avidity of a protein for one or more of its cognate binding partners is at least 10% greater than a 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 a wild-type IgSF domain control value. The reduction in binding affinity or avidity of a protein for at least one cognate binding partner is no greater than 90% of a control but no less than 10% of a wild-type IgSF domain control value, and in some embodiments, no greater than 80%, 70%, 60%, 50%, 40%, 30%, or 20% but no less than 10% of a wild-type IgSF domain control value. Affinity-modified proteins are altered in their primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as any condition that utilizes any particular starting composition or method by which the affinity-modified IgSF domain is generated. Thus, the affinity-modified IgSF domains of the present invention are not limited to wild-type IgSF domains that are then converted into affinity-modified IgSF domains by any particular affinity modification method.Affinity-modified IgSF domain polypeptides can be generated, for example, starting with wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to their cognate binding partners, and finally recombinantly or chemically synthesized to produce the subject affinity-modified IgSF domain compositions. In just one alternative example, affinity-modified IgSF domains can be produced by site-directed mutagenesis of wild-type IgSF domains. Thus, affinity-modified IgSF domains represent a product and do not necessarily represent a product produced by any given method. A variety of techniques can be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0088] As used herein, the term "allogeneic" means that cells or tissues are removed from one organism and then infused or adoptively transferred into a genetically different organism of the same species. In some embodiments of the invention, the species is murine or human.

[0089] As used herein, the term "autologous" means that cells or tissues are taken out from an organism and then infused or adoptively transferred to the same organism. Autologous cells or tissues can be changed by, for example, recombinant DNA methods so that they are no longer genetically identical to the natural cells or natural tissues taken out from an organism. For example, natural autologous T cells can be genetically engineered by recombinant DNA technology to become self-engineered cells expressing transmembrane immunomodulatory proteins and / or chimeric antigen receptors (CARs), which in some cases involve engineering T cells or TILs (tumor infiltrating lymphocytes). The engineered cells are then infused into patients from whom natural T cells are separated. In some embodiments, the organism is a human or mouse.

[0090] As used herein, the terms "binding affinity" and "binding avidity" refer to the specific binding affinity and specific binding avidity of a protein for its relative structure under specific binding conditions, respectively. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of individual non-covalent binding interactions (such as between PD-L1 and its relative structure PD-1 and / or CD80). Therefore, avidity is different from the affinity that describes the strength of a single interaction. The increase or decrease in the binding affinity of a variant PD-L1 containing an affinity-modified PD-L1 IgSF domain to its relative structure is determined relative to the binding affinity of unmodified PD-L1, such as unmodified PD-L1 containing a native or wild-type IgSF domain (such as an IgV domain). Methods for determining binding affinity or avidity are known in the art. See, for example, Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). In some embodiments, the variant PD-L1 of the invention (i.e., a PD-L1 protein containing an affinity-modified IgSF domain) specifically binds to PD-1 and / or CD80 as measured by flow cytometry with a binding affinity that results in a mean fluorescence intensity (MFI) value in the binding assay that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than a wild-type PD-L1 control.

[0091] The term "biological half-life" refers to the amount of time it takes for a substance, such as an immunomodulatory polypeptide comprising a variant PD-L1 of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be achieved by elimination, excretion, degradation (e.g., enzymatic) of the substance in certain organs or tissues of the body, or absorption and concentration. In some embodiments, biological half-life can be assessed 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 and increase the biological half-life of the polypeptides of the present invention are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0092] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial (i.e., man-made) transmembrane protein comprising at least an extracellular domain, a transmembrane domain, and an intracellular domain (ectodomain) expressed on mammalian cells. Optionally, the CAR protein includes a "spacer" that covalently links the extracellular domain to the transmembrane domain. The spacer is typically a polypeptide that connects the extracellular domain to the transmembrane domain via a peptide bond. CAR is typically expressed on mammalian lymphocytes. In some embodiments, CAR is expressed on mammalian cells such as T cells or tumor infiltrating lymphocytes (TIL). CAR expressed on T cells is referred to herein as "CAR T cells" or "CAR-T". In some embodiments, CAR-T is a T helper cell, a cytotoxic T cell, a natural killer T cell, a memory T cell, a regulatory T cell, or a γδ T cell. When used clinically with, for example, adoptive cell transfer, CAR-T having antigen-binding specificity for the patient's tumor is typically engineered to express on T cells obtained from the patient. The engineered T cells expressing CAR are then infused back into the patient. Therefore, CAR-T is usually autologous CAR-T, although allogeneic CAR-T is included in the scope of the present invention. The extracellular domain of CAR includes an antigen binding region, such as an antibody or its antigen binding fragment (e.g., scFv), which specifically binds to a target antigen such as a tumor-specific antigen under physiological conditions. When specifically bound, the biochemical chain of events (i.e., signal transduction) causes the regulation of the immunological activity of CAR-T. Therefore, for example, when specifically bound to its target antigen by the antigen binding region of CAR-T, changes in the immunological activity of T cell activity can be caused, as reflected by changes in cytotoxicity, proliferation or cytokine production. In some embodiments, signal transduction during CAR-T activation is achieved by CD3-ζ chains ("CD3-z") that participate in signal transduction in natural mammalian T cells. CAR-T may also include a variety of signal transduction domains, such as CD28, 41BB or OX40, to further regulate the immunoregulatory response of T cells. CD3-z includes a conserved motif that participates in T cell receptor signal transduction, which is called an immunoreceptor tyrosine activation motif (ITAM).

[0093] The term "collectively" or "collective," when used with reference to cytokine production induced by the presence of two or more variant PD-L1 in an in vitro assay, means the overall cytokine expression level, regardless of the cytokine production induced by the individual variant PD-L1. In some embodiments, the cytokine being assayed is IFN-γ in an in vitro primary T cell assay.

[0094] The term "cognate binding partner" (used interchangeably with "relative structure") of a reference polypeptide, such as an IgSF domain of a reference variant PD-L1, refers to at least one molecule (typically a native mammalian protein) to which the reference polypeptide specifically binds under specific binding conditions. In some aspects, a variant PD-L1 containing an affinity-modified IgSF domain specifically binds to a relative structure corresponding to native or wild-type PD-L1, but with increased or decreased affinity. A ligand species that is recognized and that specifically binds to its cognate receptor under specific binding conditions is an example of a relative structure or cognate binding partner for that receptor. A "cognate cell surface binding partner" is a cognate binding partner expressed on the surface of a mammalian cell. A "cell surface molecule species" is a cognate binding partner of a ligand of an immunological synapse (IS) that is expressed on and by a cell (such as a mammalian cell), thereby forming an immunological synapse.

[0095] As used herein, "conjugate," "conjugate," or grammatical variations thereof refers to the joining or linking of two or more compounds together by any joining or linking method known in the art to form another compound. It may also refer to a compound generated by joining or linking two or more compounds together. For example, a variant PD-L1 polypeptide directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include fusion proteins, conjugates produced by chemical conjugation, and conjugates produced by any other method.

[0096] As used herein, the term "competitive binding" means that a protein is able to specifically bind to at least two cognate binding partners, but that specific binding of one cognate binding partner inhibits (such as prevents or blocks) simultaneous binding of a second cognate binding partner. Thus, in some cases, it is not possible for a protein to simultaneously bind to two cognate binding partners. Typically, competitive binders contain identical or overlapping binding sites for specific binding, but this is not required. In some embodiments, competitive binding results in measurable inhibition (partial or complete) of the specific binding of a protein to one of its cognate binding partners due to specific binding of a second cognate binding partner. Various methods are known to quantify competitive binding, such as ELISA (enzyme-linked immunosorbent assay) assays.

[0097] As used herein, the term "conservative amino acid substitution" refers to 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 amino acid groups with side chains of similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0098] The term "corresponding to" a position of a reference protein (such as a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (such as listed in the sequence listing)) refers to the nucleotide or amino acid position identified when aligned with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues can be determined by aligning a reference sequence with the sequence of wild-type PD-L1 set forth in SEQ ID NO: 30 or 1728 (ECD domain) or in SEQ ID NO: 55 or 309 (IgV domain) via a structural alignment method as described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0099] As used herein, the term "reduce" or "mitigate" or "inhibit" means a decrease by a statistically significant amount. The decrease can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0100] The term "derivative" or "derivation" refers to the modification of a protein by covalently linking it directly or indirectly to a composition in order to alter characteristics such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy while retaining or enhancing its therapeutic benefit. Derivatives of the immunomodulatory polypeptides of the invention are within the scope of the invention and can be prepared, for example, by glycosylation, PEGylation, lipidation, or Fc fusion.

[0101] As used herein, a domain (usually three or more, typically 5 or 7 or more amino acids, such as a sequence of 10 to 200 amino acid residues) refers to a portion of a molecule (such as a protein or encoding nucleic acid) that is structurally and / or functionally different from the other portions of the molecule and is identifiable. For example, a domain includes those portions of a polypeptide chain that can form an independent folding structure within a protein, the independent folding structure being composed of one or more structural motifs and / or being identified by means of a functional activity such as binding activity. A protein can have one or more different domains. For example, a domain can be identified, defined, or distinguished by homology to a primary sequence or structure with a related family member, such as homology to a motif. In another example, a domain can be distinguished by its function, such as its ability to interact with a biomolecule such as a cognate binding partner. A domain can independently exhibit a biological function or activity, such that a domain independently or fused to another molecule can perform an activity, such as binding. A domain can be a linear amino acid sequence or a nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For purposes of illustration herein, definitions are provided, but it will be appreciated that identifying specific domains by name is within the skill of the art. Where desired, domains can be identified using appropriate software.

[0102] As used herein, the term "extracellular domain" refers to a region of a membrane protein (such as a transmembrane protein) located outside the cytoplasmic membrane. The extracellular domain typically comprises a binding domain that specifically binds to a ligand or cell surface receptor, such as a binding domain that specifically binds to a ligand or cell surface receptor. The extracellular domain of a cell transmembrane protein is alternately referred to as an extracellular domain.

[0103] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition of the present invention (including a protein composition or a cell composition) that causes a statistically significant reduction in disease progression, for example by alleviating or eliminating the symptoms and / or causes of the disease, when administered ex vivo (by contacting with cells from the patient) or in vivo (by administering to the patient) alone (i.e., as a monotherapy) or in combination with an additional therapeutic agent. An effective amount can be an amount that alleviates, reduces or relieves at least one symptom or biological response or effect associated with a disease or condition, prevents the progression of a disease or condition, or improves the patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments, the patient is a mammal, such as a non-human primate or a human patient.

[0104] As used herein, the term "intracellular domain" refers to a region present in some membrane proteins (such as transmembrane proteins) that extends to the inner space defined by the cell surface membrane. In mammalian cells, the intracellular domain is the cytoplasmic region of a membrane protein. In cells, the intracellular domain interacts with intracellular components and can play a role in signal transduction, and therefore can be an intracellular signaling domain in some cases. The intracellular domain of a cell transmembrane protein is alternatively referred to as a cytoplasmic domain, which can be a cytoplasmic signaling domain in some cases.

[0105] As used herein, the term "enhanced" or "increased" in the context of increasing the immunological activity of mammalian lymphocytes means increasing one or more activities in the lymphocytes. The increased activity can be one or more of: increased cell survival, cell proliferation, cytokine production, or T cell cytotoxicity, such as an increase in a statistically significant amount. In some embodiments, reference to increased immunological activity means increasing interferon gamma (IFN-γ) production, such as an increase in a statistically significant amount. In some embodiments, immunological activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9): 846-56. Other methods for assessing lymphocyte activity are known in the art, including any assay as described herein. In some embodiments, the enhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

[0106] As used herein, the term "engineered cell" refers to a mammalian cell that has been genetically modified by human intervention, such as by recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., T cell, B cell, NK cell) or an antigen presenting cell (e.g., dendritic cell). The cell can be a primary cell from a patient or can be a cell line. In some embodiments, the engineered cell of the present invention comprises a variant PD-L1 of the present invention, which is engineered to modulate the immunological activity of T cells expressing PD-1 and / or CD80, to which the variant PD-L1 specifically binds. In some embodiments, the variant PD-L1 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") containing an extracellular domain or a portion thereof containing an IgV or ECD domain and optionally an intracellular signaling domain connected to a transmembrane domain (e.g., a PD-L1 transmembrane domain). In some cases, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or an intracellular domain. In some embodiments, the engineered cells are capable of expressing and secreting immunomodulatory proteins as described herein. The engineered cells provided also include cells further comprising an engineered T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0107] As used herein, the term "engineered T cells" refers to T cells that have been genetically modified by human intervention, such as by recombinant DNA methods or viral transduction methods, such as T helper cells, cytotoxic T cells (or, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells. The engineered T cells comprise a variant PD-L1 transmembrane immunomodulatory protein (TIP) or secreted immunomodulatory protein (SIP) of the present invention, which is expressed on the T cells and is engineered to modulate the immunological activity of the engineered T cells themselves or mammalian cells to which the variant PD-L1 expressed on the T cells specifically binds.

[0108] The term "engineered T cell receptor" or "engineered TCR" refers to a T cell receptor (TCR) that is engineered to specifically bind to a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity, which is selected, cloned, and / or subsequently introduced into a T cell population that is typically used for adoptive immunotherapy. Compared to engineered TCRs, CARs are engineered to bind to target antigens in an MHC-independent manner.

[0109] As used herein, the term "expressed on" is used with reference to a protein expressed on the surface of a cell, such as a mammalian cell. Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety, such as a drug or a detectable label. Proteins expressed on the cell surface can include cell surface proteins expressed on mammalian cells, such as cell surface receptors.

[0110] The term "half-life extending moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of a protein not so conjugated to the portion. In some embodiments, the half-life is extended by greater than or greater than about 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, or 6.0 times. In some embodiments, after in vivo administration, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week compared to a protein without the half-life extending moiety. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using an ELISA assay or activity assay. Exemplary half-life extending moieties include Fc domains, multimerization domains, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation) and poly Pro-Ala-Ser (PAS) and polyglutamic acid (glutamylation).

[0111] As used herein, the term "immunological synapse" or "immune synapse" means the interface between a mammalian cell expressing MHC I (major histocompatibility complex) or MHC II, such as an antigen-presenting cell or a tumor cell, and a mammalian lymphocyte, such as an effector T cell or a natural killer (NK) cell.

[0112] The Fc (fragment crystallizable) region or domain (also referred to as an Fc polypeptide) of an immunoglobulin molecule corresponds primarily to the constant region of an immunoglobulin heavy chain and is responsible for different functions, including one or more effector functions of the antibody. The Fc domain contains part or all of the hinge domain of an immunoglobulin molecule plus the CH2 and CH3 domains. The Fc domain can form a dimer of two polypeptides joined by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g., a reduction of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) activity to promote effector function. In some embodiments, unless described with reference to a specific SEQ ID NO, reference to amino acid substitutions in the Fc region is by the EU numbering system. EU numbering is known and is based on the most recently updated IMGT Scientific Icon ( , international ImMunoGeneTics information , http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (Created: May 17, 2001, Last Updated: January 10, 2013) and the EU index as reported in Kabat, EA et al. Sequences of Proteins of Immunological Interest. 5th ed. US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0113] Immunoglobulin Fc fusions ("Fc fusions") (such as immunomodulatory Fc fusion proteins) are molecules comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. An Fc fusion can comprise, for example, the Fc region of an antibody (which promotes effector function and pharmacokinetics) and a variant PD-L1. The immunoglobulin Fc region can be indirectly or directly linked to one or more variant PD-L1 or small molecules (fusion partners). Different linkers are known in the art and can optionally be used to link the Fc to the fusion partner to generate an Fc fusion. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or different species can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a mouse or human Fc.

[0114] 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 prokaryotic organism, such as Escherichia coli, or it can be a eukaryotic organism, such as a unicellular eukaryotic organism (for example, yeast or other fungi), a plant cell (for example, a tobacco or tomato plant cell), an animal cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell) or a hybridoma. The example of a host cell includes Chinese hamster ovary (CHO) cells or derivatives thereof such as Veggie CHO and the related cell lines grown in serum-free medium or the CHO strain DX-B11 lacking DHFR. Another example is human endothelial kidney 293 cells or derivatives thereof. In some embodiments, the host cell is a mammalian cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell).

[0115] As used herein, the term "immunoglobulin" (abbreviated as "Ig") refers to mammalian immunoglobulins, including any of the five human classes of antibodies: IgA (including the subclasses IgA1 and IgA2), IgD, IgE, IgG (including the subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes less than full-length immunoglobulins, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V-containing fragments, and fragments thereof. H and V L The variable fragment (Fv) contains V H and V L Single chain variable fragments (scFv), and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc and Fd polypeptide fragments. The term includes within its meaning bispecific antibodies, i.e., homologous bispecifics and heterologous bispecifics.

[0116] As used herein, the term "immunoglobulin superfamily" or "IgSF" refers to a group of cell surface and soluble proteins involved in the recognition, binding or adhesion processes of cells. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins (i.e., antibodies); they all have domains or folds called immunoglobulin domains. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular actin. They are generally associated with roles in the immune system. Proteins in immunological synapses are typically members of the IgSF. The IgSF is also classified into "subfamilies" based on shared characteristics such as function. Such subfamilies are typically composed of 4 to 30 IgSF members.

[0117] As used herein, the term "IgSF domain" or "immunoglobulin domain" or "Ig domain" refers to the domain of the IgSF protein. Ig domains are named after immunoglobulin molecules. They contain approximately 70-110 amino acids and are classified according to their size and function. The Ig domain has a characteristic Ig fold, which has a sandwich-like structure formed by two sheets of antiparallel β chains. The interaction between the hydrophobic amino acids on the inner side of the sandwich and the highly conserved disulfide bonds formed between the cysteine ​​residues of the B chain and the F chain stabilizes the Ig fold. One end of the Ig domain has a segment called the complementary determining region, which is important for the specificity of the antibody for its ligand. Ig-like domains can be classified into (the following categories): IgV, IgC (which can be IgC1 or IgC2) or IgI. Most Ig domains are variable (IgV) or constant (IgC). The IgV domain with 9 β chains is generally longer than the IgC domain with 7 β chains. Some members of the IgSF have Ig domains with amino acid sequences similar to IgV domains but similar in size to IgC domains. These domains are referred to as IgC2 domains, while the standard IgC domain is referred to as IgC1 domain. T cell receptor (TCR) chains contain two Ig domains in their extracellular portion: an IgV domain at the N-terminus and an IgC1 domain adjacent to the cell membrane. PD-L1 contains two Ig domains: an IgV domain and an IgC domain.

[0118] As used herein, the term "IgSF species" refers to all IgSF member proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines the unique identity of all IgSF species belonging to that IgSF member. Thus, each IgSF family member is unique relative to other IgSF family members, and thus each species of a particular IgSF family member is unique relative to the species of another IgSF family member. However, there can be variation between molecules of the same IgSF species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, minor sequence differences within a single IgSF species due to genetic polymorphisms constitute another form of variation within a single IgSF species, as do wild-type truncated forms of an IgSF species due to, for example, proteolytic cleavage. "Cell surface IgSF species" are IgSF species expressed on the surface of cells (typically mammalian cells).

[0119] As used herein in the context of mammalian lymphocytes such as T cells, the term "immunological activity" refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon-γ) or T cell cytotoxic activity. In some cases, immunological activity can refer to cellular expression of cytokines such as chemokines or interleukins. Assays for determining enhancement or inhibition of immunological activity include MLR (mixed lymphocyte reaction) assays (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9): 846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assays (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9): 846-56) and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med. 2010: 8: 104) measuring interferon-γ cytokine levels in culture supernatants. Since T cell activation is associated with the secretion of IFN-γ cytokines, these in vitro human T cell assays can be assayed using commercial ELISA kits for the detection of IFN-γ levels in culture supernatants (Wu et al., Immunol Lett 2008 Apr 15; 117(1): 57-62). The induction of an immune response results in an increase in immunological activity relative to resting lymphocytes. Immunomodulatory proteins (such as variant PD-L1 polypeptides) containing affinity-modified IgSF domains as provided herein can increase or, in alternative embodiments, decrease IFN-γ (interferon-γ) expression in primary T cell assays relative to wild-type IgSF members or IgSF domain controls. The skilled person will recognize that the format of the primary T cell assay for determining an increase in IFN-γ expression can be different from the assay for determining a decrease in IFN-γ expression. When determining the ability of the immunomodulatory proteins of the present invention or affinity-modified IgSF domains to alter IFN-γ expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used. Conveniently, in some cases, a soluble form of the affinity-modified IgSF domain of the present invention can be used to determine its ability to increase or decrease IFN-γ expression in the MLR. Alternatively, a co-fixation assay can be used. In a co-fixation assay, the T cell receptor signal provided by an anti-CD3 antibody in some embodiments is used in conjunction with a co-fixed affinity-modified IgSF domain (such as a variant PD-L1) to determine the ability to increase or decrease IFN-γ expression relative to a wild-type IgSF domain control.Methods for determining the immunological activity of engineered cells (including evaluating the activity of variant PD-L1 transmembrane immunomodulatory proteins) are known in the art and include, but are not limited to, the ability to expand T cells after antigen stimulation, the ability to maintain T cell expansion in the absence of restimulation, and anti-cancer activity in appropriate animal models. Assays also include assays to assess cytotoxicity, including standards. 51 Cr release assay (see, e.g., Milone et al., (2009) Molecular

[0120] 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).

[0121] "Immunomodulatory polypeptides" or "immunomodulatory proteins" are polypeptides or protein molecules that modulate immunological activity. "Modulating" an immune response means increasing or decreasing immunological activity. An immunomodulatory protein can be a single polypeptide chain or a multimer (dimer or higher multimer) of at least two polypeptide chains covalently bonded to each other, for example, by intrachain disulfide bonds. Thus, monomers, dimers, and higher multimer polypeptides are within the scope of the defined terms. Multimeric polypeptides can be homomultimers (same polypeptide chains) or heteromultimers (different polypeptide chains). The immunomodulatory proteins of the present invention include variant PD-L1.

[0122] As used herein, the term "increase" means an increase by a statistically significant amount. The increase can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100% or greater than a non-zero control value.

[0123] An "isoform" of PD-L1 is one of a variety of naturally occurring PD-L1 polypeptides that differ in their amino acid sequence. Isoforms can be the products of splice variants of RNA transcripts expressed from a single gene or the expression products of highly similar but distinct genes, resulting in functionally similar proteins, such as can be produced by gene duplication. As used herein, the term "isoform" of PD-L1 also refers to the products of different alleles of the PD-L1 gene.

[0124] As used herein, the term "lymphocyte" refers to any of the three subtypes of white blood cells in the mammalian immune system. They include natural killer cells (NK cells) (which play a role in cell-mediated cytotoxic innate immunity), T cells (for cell-mediated cytotoxic acquired immunity) and B cells (for humoral antibody-driven acquired immunity). T cells include: T helper cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Innate lymphocytes (ILCs) are also included in the definition of lymphocytes.

[0125] The term "mammal" or "patient" specifically includes reference to at least one of the following: human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0126] As used herein, the term "membrane protein" means a protein that is directly or indirectly attached to a lipid bilayer under physiological conditions. The lipid bilayer forming the membrane can be a biological membrane, such as a eukaryotic (e.g., mammalian) cell membrane or an artificial (i.e., man-made) membrane, such as a membrane present on a liposome. Membrane proteins can be attached to the lipid bilayer by covalent attachment or by non-covalent interactions, such as hydrophobic or electrostatic interactions. Membrane proteins can be integral membrane proteins or peripheral membrane proteins. Membrane proteins that are peripheral membrane proteins are non-covalently attached to the lipid bilayer or non-covalently attached to integral membrane proteins. Peripheral membrane proteins form a temporary attachment to the lipid bilayer so that, within the conditions of mammalian physiology, the peripheral membrane proteins can associate and / or dissociate with the lipid bilayer. Compared to peripheral membrane proteins, integral membrane proteins form a substantially permanent attachment to the membrane lipid bilayer so that, within the conditions of mammalian physiology, integral membrane proteins will not dissociate from the attachment to the lipid bilayer. Membrane proteins can be attached to the membrane through one layer of the lipid bilayer (monotopic) or through both layers of the membrane (polytopic). Integral membrane proteins that interact with only one lipid bilayer are "integral monotopic proteins." Integral membrane proteins that interact with both lipid bilayers are "integral polytopic proteins," or, as they are referred to herein, "transmembrane proteins."

[0127] As used herein in the context of an immune response (such as a mammalian immune response), the term "modulating" refers to any change, such as an increase or decrease, in an existing or potential immune response that occurs due to the administration of an immunomodulatory polypeptide comprising a variant PD-L1 of the present invention or due to the administration of an immunomodulatory protein expressing the present invention (such as a variant PD-L1 transmembrane immunomodulatory protein). Thus, it refers to a change, such as an increase or decrease, in an immune response that occurs or exists in the absence of an immunomodulatory protein comprising a variant PD-L1 or a cell expressing such an immunomodulatory polypeptide. This modulation includes any induction, activation, inhibition, or change in the degree or scope of the immunological activity of an immune cell. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs) and non-professional antigen-presenting cells, and inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change in an existing immune response, a developing immune response, a potential immune response, or the ability to induce, regulate, influence, or respond to an immune response. Regulation 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. Regulation of immune response or regulation of immunological activity include, for example, the following: eliminating, missing or sequestering immune cells; inducing or generating immune cells that can regulate the functional capabilities of other cells such as autoreactive lymphocytes, antigen presenting cells or inflammatory cells; inducing a non-responsive state (i.e., disability) of immune cells; enhancing or inhibiting the activity or function of immune cells, including but not limited to changing the pattern of proteins expressed by these cells. Examples include changes in the production and / or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, co-stimulatory molecules or other cell surface receptors or any combination of these regulatory events. Regulation can be assessed in primary T cell assays, for example, by changes in IFN-γ (interferon γ) expression relative to wild-type PD-L1 controls (see Zhao and Ji, Exp Cell Res. 2016 January 1; 340 (1): 132-138). Modulation can be, for example, by a change in the immunological activity of the engineered cells relative to cells engineered with wild-type PD-L1 transmembrane protein, such as a change in the cytotoxic activity of the engineered cells or a change in cytokine secretion by the engineered cells.

[0128] As used herein, the term "molecular species" means all proteins having the same or substantially the same primary amino acid sequence. Each member of the mammalian immunoglobulin superfamily (IgSF) defines a set of identical or substantially identical molecular species. Thus, for example, human PD-L1 is a member of the IgSF and each human PD-L1 molecule is a molecular species of PD-L1. There can be variation between molecules of the same molecular species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, minor sequence differences within a single molecular species due to genetic polymorphisms constitute another form of variation within a single molecular species, as do wild-type truncated forms of a single molecular species due to, for example, proteolytic cleavage. "Cell surface molecular species" are molecular species expressed on the surface of mammalian cells. Two or more different protein species, each uniquely present on one of the two mammalian cells forming an IS or uniquely present on the other mammalian cell (but not on both mammalian cells), are considered to be in a "cis" or "cis configuration" relative to each other. A first of two distinct protein species is present exclusively on one of the two mammalian cells forming the IS and a second is present exclusively on the second of the two mammalian cells forming the IS, said protein species being said to be in "trans" or "trans configuration." Two distinct protein species are each present on both of the two mammalian cells forming the IS, said protein species being in both cis and trans configurations on those cells.

[0129] The term "multimerization domain" refers to an amino acid sequence that promotes stable interactions between a polypeptide molecule and one or more additional polypeptide molecules, each of which contains a complementary multimerization domain (e.g., a first multimerization domain and a second multimerization domain), which may be identical or different multimerization domains. The interaction between the complementary multimerization domains, such as the interaction between the first multimerization domain and the second multimerization domain, forms a stable protein-protein interaction to produce a multimer of a polypeptide molecule and an additional polypeptide molecule. In some cases, the multimerization domain is identical and interacts with itself to form a stable protein-protein interaction between two polypeptide chains. Typically, a polypeptide is directly or indirectly attached 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 may for example be an immunoglobulin constant region or domain, such as, for example, an Fc domain or portion thereof from IgG (including IgG1, IgG2, IgG3 or IgG4 subtypes), IgA, IgE, IgD and IgM, and modified forms thereof.

[0130] The terms "nucleic acid" and "polynucleotide" are interchangeably used to indicate a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form. Unless otherwise specified, the terms encompass nucleic acids containing known analogs of natural nucleotides, and have similar binding properties to the natural nucleotides and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as sequences explicitly indicated ("reference sequences"). Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. The terms nucleic acid or polynucleotide encompass cDNA or mRNA encoded by a gene.

[0131] As used herein, the term "non-competitive binding" means the ability of a protein to specifically bind to at least two cognate binding partners simultaneously. Thus, a protein is capable of binding to at least two different cognate binding partners simultaneously, although the binding interactions need not last for the same duration, such that in some cases the protein specifically binds to only one cognate binding partner. In some embodiments, binding occurs under specific binding conditions. In some embodiments, simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding with a second cognate binding partner. In some embodiments, non-competitive binding means that the binding of the second cognate binding partner to its binding site on the protein does not displace the binding of the first cognate binding partner to its binding site on the protein. Methods for determining non-competitive binding are well known in the art, such as those described in Perez de La Lastra et al., Immunology, April 1999: 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, so that the binding of the second cognate binding partner does not directly interfere with the binding of the first cognate binding partner. Therefore, any effect of the binding of the second cognate binding partner on the binding of the cognate binding partner is by a mechanism other than the binding that directly interferes with the first cognate binding partner. For example, in the context of enzyme-substrate interactions, non-competitive inhibitors are bound to sites other than the enzyme active site. Non-competitive binding encompasses non-competitive binding interactions, wherein the second cognate binding partner specifically binds at an interaction site that does not overlap with the binding of the first cognate binding partner, but is only bound to the second interaction site when the first interaction site is occupied by the first cognate binding partner.

[0132] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (typically a human). A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant PD-L1 or an engineered cell expressing a variant PD-L1 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0133] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked by 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 term includes post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. The term also includes molecules that include one or more amino acid analogs or atypical or unnatural amino acids, as they may be synthesized or recombinantly expressed using known protein engineering techniques. In addition, proteins may be derivatized.

[0134] As used herein, the term "primary T cell assay" refers to an in vitro assay that measures interferon-γ ("IFN-γ") expression. A variety of such primary T cell assays are known in the art. In a preferred embodiment, the assay used is an anti-CD3 co-fixation assay. In this assay, primary T cells are stimulated by anti-CD3 fixed in the presence or absence of additional recombinant proteins. At certain time points, typically 24-72 hours, the culture supernatant is harvested. In another embodiment, the assay used is an MLR. In this assay, primary T cells are stimulated with allogeneic APCs. At certain time points, typically 24-72 hours, the culture supernatant is harvested. The human IFN-γ level in the culture supernatant is measured by standard ELISA technology. Commercial kits are purchased from suppliers and the assays are performed according to the manufacturer's recommendations.

[0135] The term "purified" as applied to nucleic acids (such as those encoding immunomodulatory proteins of the invention) generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or protein of the invention is at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or greater pure (e.g., by weight percentage or molar basis).

[0136] The term "recombinant" indicates that a material (e.g., nucleic acid or polypeptide) is artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material when the material is in its natural environment or state or when removed therefrom. For example, a "recombinant nucleic acid" is a nucleic acid prepared by recombining nucleic acid during, for example, 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 means of such molecular biology techniques. As used herein, the terms "recombinant protein" or "recombinant polypeptide" refer to protein molecules expressed using recombinant DNA molecules. A "recombinant host cell" is a cell that contains and / or expresses recombinant nucleic acid or is otherwise genetically engineered, such as by introducing a nucleic acid molecule encoding a recombinant protein (such as a transmembrane immunomodulatory protein provided herein) into the cell. Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers are composed of short DNA sequence arrays that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes from yeast, insect and mammalian cells, and viruses (autologous control elements, i.e., promoters are also found in prokaryotes). The choice of a particular promoter and enhancer depends on what cell type will be used to express the protein of interest. As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the connection of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule is produced that is capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0137] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence necessary for expressing the operably connected coding sequence in a specific host cell. The nucleic acid sequence necessary for expression in prokaryotes includes a promoter, an optional operator sequence, a ribosome binding site, and possible other sequences. Known eukaryotic cells utilize promoters, enhancers, and termination signals and polyadenylation signals. The secretion signal peptide sequence can also be optionally encoded by a recombinant expression vector that is operably connected to the coding sequence of a recombinant protein (such as a recombinant fusion protein) so that the expressed fusion protein can be secreted by the recombinant host cell to make the fusion protein easier to separate from the cell as needed. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of the host cell into which it has been introduced. Vectors are viral vectors, such as lentiviral vectors.

[0138] The term "selectivity" refers to the preference of a subject protein or polypeptide for specific binding to one substrate (such as one cognate binding partner) over specific binding to another substrate (such as a different cognate binding partner of the subject protein). Selectivity can be reflected in the binding activity (e.g., binding affinity) (e.g., K) of the subject protein and a first substrate (such as a first cognate binding partner). d1 ) binding activity (e.g., binding affinity) to the same subject protein and a second cognate binding partner (e.g., K d2 ) ratio.

[0139] As used herein, the term "sequence identity" refers to the sequence identity between a gene or protein at the nucleotide or amino acid level, respectively." sequence identity" is a measure of the identity between proteins at the amino acid level and the identity between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at the given position in each sequence when comparing sequences. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at the given position in each sequence when comparing sequences. Methods for comparing sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates sequence identity percentages and performs statistical analysis on the similarity between two sequences. The software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (NCBI) website.

[0140] As used herein with reference to a protein, the term "soluble" means that the protein is not a membrane protein. Generally speaking, a soluble protein contains only the extracellular domain of an IgSF family member receptor or a portion thereof containing one or more IgSF domains or specific binding fragments thereof, but does not contain a transmembrane domain. In some cases, the solubility of a protein can be improved by linking or attaching it directly or indirectly via a linker to an Fc domain, 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.

[0141] As used herein with respect to polypeptides or nucleic acids, the term "species" refers to all molecules having the same or substantially the same sequence. Variation can exist between polypeptides of the same species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated sequences of a polypeptide that differ from the full-length species by no more than 1, 2, or 3 amino acid residues at the amino or carboxyl termini are considered a single species. This type of microscopic heterogeneity is a common feature of manufactured proteins.

[0142] As used herein with reference to a full-length wild-type mammalian PD-L1 polypeptide or its IgV or IgC (e.g., IgC2) domain, the term "specific binding fragment" means a polypeptide having a subsequence of the full-length polypeptide or IgV and / or IgC domain and specifically binding to mammalian PD-1 and / or mammalian CD80 (such as human or murine PD-1 or CD80) in vitro and / or in vivo. In some embodiments, the specific binding fragment comprises a PD-L1 IgV or PD-L1 IgC2 subsequence that 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 sequence or its IgV or IgC (e.g., IgC2) sequence. The sequence of the specific binding fragment can be altered to form a variant PD-L1 of the present invention.

[0143] As used herein, the term "specific binding" means that a protein is bound to a target protein under specific binding conditions with an affinity or avidity as great as at least 5 times the average affinity or avidity of the same protein to a set of random peptides or polypeptides of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times, or even at least 1000 times the ability. Specific binding proteins do not need to be uniquely bound to a single target molecule, but can be specifically bound to non-target molecules due to the structural conformational similarity between target and non-target (e.g., horizontal homologues or straight homologues). Technicians will recognize that specific binding to molecules with the same function in different animal species (i.e., straight homologues) or non-target molecules with substantially similar epitopes to target molecules (e.g., horizontal homologues) is possible, and will not weaken the binding specificity determined relative to a statistically effective set (e.g., random polypeptides) of a unique non-target. Therefore, the polypeptide of the present invention can specifically bind to more than one different target molecule species due to cross-reactivity. Solid phase ELISA immunoassays, ForteBio Octet or Biacore measurements can be used to determine the specific binding between two proteins. Typically, the dissociation constant (K) of the interaction between two binding proteins is d ) is less than 1x10 -5 M, and often as low as 1x 10 -12 In certain embodiments of the present disclosure, the dissociation constant of the interaction between two binding proteins is less than or about 1×10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or 1x10 -11 M or smaller.

[0144] The term "surface expresses" or "surface expression" in reference to mammalian cells expressing a polypeptide means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0145] As used herein, "synthetic" with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0146] As used herein, the term "targeting moiety" refers to a composition that is covalently or non-covalently attached to a polypeptide comprising a variant PD-L1 of the present invention or physically encapsulates the polypeptide. The targeting moiety has a specific binding affinity for a desired relative structure such as a cell surface receptor (e.g., PD-1) or a tumor antigen such as a tumor specific antigen (TSA) or a tumor associated antigen (TAA) such as B7-H6. Typically, the desired relative structure is localized to a specific tissue or cell type. Targeting moieties include: antibodies, antigen binding fragments (Fab), V-containing H and V L The variable fragment (Fv) contains V H and V L Single chain variable fragments (scFv), and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecule (<500 Dalton) compositions (e.g., specific binding receptor compositions). The targeting moiety can also be covalently or non-covalently attached to the lipid membrane of the liposome, which encapsulates the polypeptide of the invention.

[0147] As used herein, the term "membrane-spanning protein" means substantially or completely across a lipid bilayer, such as the membrane proteins of those lipid bilayers present in biological membranes (such as mammalian cells) or artificial constructs (such as liposomes). A membrane-spanning protein comprises a membrane-spanning domain ("membrane-spanning domain"), which is integrated into a lipid bilayer by the membrane-spanning domain and is thermodynamically stable under physiological conditions by the integration carried out by the membrane-spanning domain. A membrane-spanning domain is generally predictable by its amino acid sequence based on its hydrophobicity relative to the protein region interacting with an aqueous environment (e.g., cytosol, cell efflux) via any number of commercially available bioinformatics software applications. A membrane-spanning domain is typically a hydrophobic alpha helix across a membrane. A membrane-spanning protein can pass through two layers of a lipid bilayer once or multiple times. A membrane-spanning protein includes a membrane-spanning immunomodulatory protein provided as described herein. In addition to the membrane-spanning domain, the membrane-spanning immunomodulatory protein of the present invention also comprises an extracellular domain and, in some embodiments, an intracellular domain.

[0148] As used herein, the term "treating / treatment / therapy" of a disease or condition means slowing, halting, or reversing the progression of a disease or condition by administering a therapeutic composition (e.g., containing an immunomodulatory protein or engineered cells), alone or in combination with another compound as described herein, as evidenced by a reduction, cessation, or elimination of clinical or diagnostic symptoms. "Treatment" also means a reduction in the severity of symptoms of an acute or chronic disease or condition, or a reduction in the recurrence rate, such as, for example, in the case of a relapsing or remitting autoimmune disease course, or a reduction in inflammation in the case of the inflammatory aspects of an autoimmune disease. As used herein in the context of cancer, the term "treatment" or "inhibit / inhibiting / inhibition" of cancer refers to at least one of the following: a statistically significant reduction in tumor growth rate, cessation of tumor growth, or a reduction in the size, mass, metabolic activity, or volume of a tumor, as measured by standard criteria, such as, but not limited to, a statistically significant increase in response evaluation criteria for solid tumors (RECIST) or progression-free survival (PFS) or overall survival (OS). As used in the context of the present invention, "prevention" refers to the administration of an immunomodulatory polypeptide or engineered cell of the invention, alone or in combination with another compound, to prevent the occurrence or onset of a disease or condition or some or all of the symptoms of a disease or condition or to reduce the likelihood of the onset of a disease or condition.

[0149] As used herein, the term "tumor-specific antigen" or "TSA" refers to a relative structure that is predominantly present on tumor cells in a mammalian subject, but is not generally found on normal cells in the mammalian subject. The tumor-specific antigen need not be exclusive to tumor cells, but the percentage of cells in a particular mammal that possess the tumor-specific antigen is sufficiently high, or the level of the tumor-specific antigen on the surface of the tumor is sufficiently high, that it can be targeted by an anti-tumor therapeutic agent, such as the immunomodulatory polypeptide of the present invention, and provide prophylaxis or treatment of the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal having a tumor, at least 50% of the cells that display the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that display the TSA are cancerous.

[0150] The term "variant" (also "modified" or "mutant") used with reference to variant PD-L1 means PD-L1 that has been formed through human intervention, such as mammalian (e.g., human or murine) PD-L1. A variant PD-L1 is a polypeptide having an altered amino acid sequence relative to unmodified or wild-type PD-L1. A variant PD-L1 is a polypeptide that differs from the wild-type PD-L1 isoform sequence by one or more amino acid substitutions, deletions, additions, or a combination thereof. For the purposes herein, a variant PD-L1 contains at least one affinity-modified domain, and thus has one or more amino acid differences in the IgSF domain (e.g., IgV domain or ECD). Variant PD-L1 may 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 PD-L1 polypeptides typically have at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to a corresponding wild-type or unmodified PD-L1, such as SEQ ID NO: 3, its mature sequence containing the extracellular domain or its IgSF domain (lacking the signal sequence), or a portion thereof. In some embodiments, the variant PD-L1 polypeptide has at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the corresponding wild-type or unmodified PD-L1 comprising the sequence set forth in SEQ ID NO: 30 or 1728 or SEQ ID NO: 55 or 309. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the permissible substitutions or additions. The variant PD-L1 is not limited to any particular method of preparation and includes, for example, de novo chemical synthesis, de novo recombinant DNA technology, or a combination thereof. The variant PD-L1 of the present invention specifically binds to at least one or more of PD-1 or CD80 of a mammalian species. In some embodiments, the altered amino acid sequence results in altered (i.e., increased or decreased) binding affinity or avidity for PD-1 and / or CD80 compared to wild-type or unmodified PD-L1 protein. The increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005).See also Linsley et al., Immunity, Vol. 1 (9): 793-801 (1994). The increase in the binding affinity or avidity of variant PD-L1 for PD-1 and / or CD80 is a value greater than at least 5% of the wild-type or unmodified PD-L1, and in some embodiments is a value greater than at least 10%, 15%, 20%, 30%, 40%, 50%, 100% of the wild-type or unmodified PD-L1 control value. The decrease in the binding affinity or avidity of PD-L1 for PD-1 and / or CD80 is a value less than 95% of the wild-type or unmodified control value, and in some embodiments is a value less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or undetectable binding affinity or avidity. Variant PD-L1 is altered in the primary amino acid sequence by substitution, addition or deletion of amino acid residues. The term "variant" in the context of variant PD-L1 should not be interpreted as any condition utilizing any particular starting composition or method by which the variant PD-L1 is produced. Variant PD-L1 can be generated, for example, starting with wild-type mammalian PD-L1 sequence information, then modeled in silico to bind to PD-1 and / or CD80, and finally recombinantly or chemically synthesized to produce the variant PD-L1 of the present invention. In just one alternative example, variant PD-L1 can be produced by site-directed mutagenesis of wild-type PD-L1. Thus, variant PD-L1 represents a composition and does not necessarily represent a product produced by any given method. A variety of techniques can be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0151] As used herein, the terms "wild type" or "natural" or "native" are used in connection with biological materials such as nucleic acid molecules, proteins (e.g., PD-L1), IgSF members, host cells, etc., to refer to those materials found in nature and that have not been modified by human intervention.

[0152] II. Variant PD-L1 Polypeptides

[0153] Provided herein are variant PD-L1 polypeptides that exhibit altered (increased or decreased) binding activity or affinity for one or more PD-L1 cognate binding partners. In some embodiments, the PD-L1 cognate binding partner is PD-1 or CD80. In some embodiments, the PD-L1 cognate binding partner is PD-1. In some embodiments, the variant PD-L1 polypeptide contains one or more amino acid modifications, such as one or more substitutions (alternatively, "mutations" or "substitutions"), deletions, or additions in the immunoglobulin superfamily (IgSF) domain (IgD), relative to a wild-type or unmodified PD-L1 polypeptide or a portion of a wild-type or unmodified PD-L1 containing IgD or a specific binding fragment thereof. Thus, the variant PD-L1 polypeptide provided is or comprises a variant IgD (hereinafter referred to as "vIgD"), wherein the one or more amino acid modifications (e.g., substitutions) are in IgD.

[0154] In some embodiments, IgD comprises an IgV domain or an IgC (e.g., IgC2) domain, or a specific binding fragment of an IgV domain or an IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, IgD can be any combination of IgV alone, a combination of IgV and IgC (including the entire extracellular domain (ECD)), or the Ig domain of PD-L1. Table 2 provides exemplary residues corresponding to the IgV or IgC region of PD-L1. In some embodiments, the variant PD-L1 polypeptide contains an IgV domain or an IgC domain or a specific binding fragment thereof, wherein at least one amino acid modification (e.g., substitution) is in the IgV domain or the IgC domain or a specific binding fragment thereof. In some embodiments, the variant PD-L1 polypeptide contains an IgV domain or a specific binding fragment thereof, wherein at least one amino acid modification (e.g., substitution) is in the IgV domain or the IgC domain or a specific binding fragment thereof. In some embodiments, the altered IgV domain or IgC (eg, IgC2) domain is an affinity-modified IgSF domain with altered binding activity or affinity.

[0155] In some embodiments, the variant is modified relative to the sequence of the unmodified PD-L1 sequence in one or more IgSF domains. In some embodiments, the unmodified PD-L1 sequence is wild-type PD-L1. In some embodiments, the unmodified or wild-type PD-L1 has the sequence of natural PD-L1 or its ortholog. In some embodiments, the unmodified PD-L1 is or comprises the extracellular domain (ECD) of PD-L1 or a portion thereof containing one or more IgSF domains (see Table 2). In some embodiments, the extracellular domain of the unmodified or wild-type PD-L1 polypeptide comprises an IgV domain and one or more IgC (e.g., IgC2) domains. However, the variant PD-L1 polypeptide does not need to comprise both the IgV domain and the one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises an IgV domain or a specific binding fragment thereof or consists essentially of it. In some embodiments, the variant PD-L1 polypeptide comprises one or both of an IgC (e.g., IgC2) domain or a specific binding fragment thereof, or consists essentially of the same. In some embodiments, the variant PD-L1 polypeptide comprises only one of an IgC (e.g., IgC2) domain or a specific binding fragment thereof, or consists essentially of the same. In some embodiments, the variant PD-L1 polypeptide comprises: an IgV domain or a specific binding fragment thereof; and a first and a second IgC (e.g., IgC2) domain or a specific binding fragment thereof. In some embodiments, the variant PD-L1 polypeptide is soluble and lacks a transmembrane domain. In some embodiments, the variant PD-L1 polypeptide further comprises a transmembrane domain, and in some cases, a cytoplasmic domain.

[0156] In some embodiments, the wild-type or unmodified PD-L1 sequence is a mammalian PD-L1 sequence. In some embodiments, the wild-type or unmodified PD-L1 sequence can be a mammalian PD-L1, including but not limited to human, mouse, cynomolgus monkey, or rat PD-L1. In some embodiments, the wild-type or unmodified PD-L1 sequence is a human sequence.

[0157] In some embodiments, the wild-type or unmodified PD-L1 sequence has (i) the amino acid sequence set forth in SEQ ID NO:3, or a mature form thereof lacking a signal sequence, (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO:3, or a mature form thereof, or (iii) is a portion of (i) or (ii) containing an IgV domain or an IgC (e.g., IgC2) domain, or a specific binding fragment thereof.

[0158] In some embodiments, the wild-type or unmodified PD-L1 sequence is or comprises the extracellular domain of PD-L1 or a portion thereof. In some embodiments, the unmodified or wild-type PD-L1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30 or 1728 or an ortholog thereof. In some cases, the unmodified or wild-type PD-L1 polypeptide may comprise (i) the amino acid sequence set forth in SEQ ID NO: 30 or 1728, (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: 30 or 1728, or (iii) a specific binding fragment of a sequence of (i) or (ii) comprising an IgV domain or an IgC (e.g., IgC2) domain.

[0159] In some embodiments, the wild-type or unmodified PD-L1 polypeptide comprises an IgV domain or one or more IgC (e.g., IgC2) domains or a specific binding fragment thereof. In some embodiments, the IgV domain of the wild-type or unmodified PD-L1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 55 or 309 (corresponding to amino acid residues 24-130 of SEQ ID NO: 3) or an ortholog thereof. For example, the IgV domain of the unmodified or wild-type PD-L1 polypeptide can contain (i) the amino acid sequence set forth in SEQ ID NO: 55 or 309, (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: 55 or 309, or (iii) a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain is capable of binding to one or more PD-L1 cognate binding proteins, such as one or more of PD-1 or CD80.

[0160] In some embodiments, the first IgC2 domain of a wild-type or unmodified PD-L1 polypeptide comprises the amino acid sequence set forth as residues 133-225 of SEQ ID NO: 3, or an ortholog thereof. For example, the IgC2 domain of an unmodified or wild-type PD-L1 polypeptide can contain (i) the amino acid sequence set forth as residues 133-225 of SEQ ID NO: 3, (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 residues 133-225 of SEQ ID NO: 3, or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgC domain is capable of binding to one or more PD-L1 cognate binding proteins.

[0161] In some embodiments, the wild-type or unmodified PD-L1 polypeptide contains a specific binding fragment of PD-L1, such as a specific binding fragment of an IgV domain or an IgC (e.g., IgC2) domain. In some embodiments, the specific binding fragment can bind to PD-1 and / or CD80. The amino acid length of the specific binding fragment can be at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 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%, or 99% of the length of the IgV domain listed as amino acids 24-130 of SEQ ID NO: 3. In some embodiments, the specific binding fragment of an IgC (e.g., IgC2) domain comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgC domain listed as amino acids 133-225 of SEQ ID NO:3.

[0162] In some embodiments, the variant PD-L1 polypeptide comprises an ECD domain or a portion thereof comprising one or more affinity-modified IgSF domains. In some embodiments, the variant PD-L1 polypeptide may comprise an IgV domain or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain, or a specific binding fragment of one or more IgC (e.g., IgC2) domains, wherein one or more of the IgSF domains (IgV or IgC) contain one or more amino acid modifications (e.g., substitutions). In some embodiments, the variant PD-L1 polypeptide may comprise an IgV domain and one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain and a specific binding fragment of one or more IgC (e.g., IgC2) domains, wherein at least one of the IgV or IgC domains contains one or more amino acid modifications (e.g., one or more substitutions). In some embodiments, the variant PD-L1 polypeptide comprises a full-length IgV domain. In some embodiments, the variant PD-L1 polypeptide comprises one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain. In some embodiments, the variant PD-L1 polypeptide comprises a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a full-length IgV domain and a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain and one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain and one or more specific binding fragments of an IgC (e.g., IgC2) domain.

[0163] In any such embodiments, the one or more amino acid modifications (e.g., substitutions) of the variant PD-L1 polypeptide may be located in any one or more of the IgSF domains of the PD-L1 polypeptide. For example, in some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the extracellular domain of the variant PD-L1 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgV domain or the specific binding fragment of the IgV domain. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgC (e.g., IgC2) domain or the specific binding fragment of the IgC (e.g., IgC2) domain.

[0164] In general, each of the various properties of the polypeptides is disclosed separately below (e.g., soluble and membrane-bound polypeptides, affinity of PD-L1 for PD-1 and CD80, number of variations per polypeptide chain, number of linked polypeptide chains, number and nature of amino acid changes per variant PD-L1, etc.). However, it will be clear to the skilled artisan that any particular polypeptide may comprise a combination of these individual properties. It should be understood that reference to amino acids used to describe the domain composition of the IgSF domain (including specific sequences listed as SEQ ID NOs) is for illustrative purposes and is not intended to limit the scope of the embodiments provided. It should be understood that the description of the polypeptides and their domains is theoretically derived based on homology analysis and comparison with similar molecules. Therefore, the precise loci may vary and are not necessarily the same for each protein. Thus, a specific IgSF domain (such as a specific IgV domain or IgC domain) may be several amino acids (such as one, two, three, or four) longer or shorter.

[0165] In addition, the various embodiments of the present invention, as discussed below, are frequently provided within the meaning of the defined terms as disclosed above. The embodiments described in the specific definitions should therefore be interpreted as being incorporated by reference when the defined terms are used to discuss the various aspects and attributes described herein. Therefore, the order of presentation of the various aspects and embodiments and the separate disclosure of each independent attribute are not intended to limit the scope of the present disclosure.

[0166] A. Exemplary Modifications

[0167] Provided herein are variant PD-L1 polypeptides comprising at least one affinity-modified IgSF domain (e.g., ECD, IgV, or IgC) or a specific binding fragment thereof relative to the IgSF domain contained in a wild-type or unmodified PD-L1 polypeptide, such that the variant PD-L1 polypeptide exhibits altered (increased or decreased) binding activity or affinity for one or more ligands, PD-1 or CD80, compared to the wild-type or unmodified PD-L1 polypeptide. In some embodiments, the binding affinity of the variant PD-L1 polypeptide for PD-1 and / or CD80 is different from that of a wild-type or unmodified PD-L1 polypeptide control sequence, as determined by, for example, solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet, or Biacore assay. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and / or CD80. In some embodiments, the variant PD-L1 polypeptide has decreased binding affinity for PD-1 and / or CD80 relative to the wild-type or unmodified PD-L1 polypeptide. PD-1 and / or CD80 may be mammalian proteins, such as human proteins or murine proteins.

[0168] The binding affinity for each cognate binding partner is independent; that is, in some embodiments, the variant PD-L1 polypeptide has increased binding affinity for one or both of PD-1 and / or CD80 and decreased binding affinity for one or both of PD-1 and CD80 relative to the wild-type or unmodified PD-L1 polypeptide.

[0169] In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has decreased binding affinity for PD-1 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has decreased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide.

[0170] In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and decreased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has decreased binding affinity for PD-1 and CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has decreased binding affinity for PD-1 and increased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide.

[0171] In some embodiments, the variant PD-L1 polypeptide having increased or greater binding affinity for PD-1 and / or CD80 has a binding affinity for PD-1 and / or CD80 that is increased by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50% relative to a wild-type or unmodified PD-L1 polypeptide control. In some embodiments, the binding affinity is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold relative to the wild-type or unmodified PD-L1 polypeptide. In such examples, the wild-type or unmodified PD-L1 polypeptide has the same sequence as the variant PD-L1 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0172] In some embodiments, the variant PD-L1 polypeptide with reduced binding affinity for PD-1 and / or CD80 has a binding affinity for PD-1 and / or CD80 that is reduced by at least 5% relative to a wild-type or unmodified PD-L1 polypeptide control, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the binding affinity relative to the wild-type or unmodified PD-L1 polypeptide is reduced by more than 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times or 50 times. In such examples, the wild-type or unmodified PD-L1 polypeptide has the same sequence as the variant PD-L1 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0173] In some embodiments, the equilibrium dissociation constant (K) of any of the foregoing embodiments with PD-1 and / or CD80 is d ) can be less than 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or 1x10 -11 M or 1x10 -12 M or smaller.

[0174] A wild-type or unmodified PD-L1 sequence does not necessarily have to be used as a starting composition for generating the variant PD-L1 polypeptides described herein. Thus, the use of the term "modification," such as "substitution," does not imply that the embodiments of the present invention are limited to a particular method for preparing the variant PD-L1 polypeptides. Variant PD-L1 polypeptides can be prepared, for example, by de novo peptide synthesis and therefore do not necessarily require modification in the sense of changing codons to encode the modification (e.g., substitution), such as "substitution." This theory also extends to the terms "addition" and "deletion" of amino acid residues, which likewise do not imply a particular method of preparation. The means of designing or generating variant PD-L1 polypeptides are not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified PD-L1 are mutagenized from wild-type or unmodified PD-L1 genetic material and screened for the desired specific binding affinity and / or induction of IFN-γ expression or other functional activity. In some embodiments, variant PD-L1 polypeptides are de novo synthesized using protein or nucleic acid sequences available at any number of publicly available databases and then subsequently screened. The National Center for Biotechnology Information provides such information and its website is publicly accessible via the Internet, as is the UniProtKB database discussed previously.

[0175] Unless otherwise stated, as indicated throughout this disclosure, one or more amino acid modifications are indicated by the amino acid position number corresponding to the position number of the unmodified ECD sequence set forth in SEQ ID NO:30 or 1728, or, where appropriate, the following unmodified IgV sequence set forth in SEQ ID NO:309 (containing residues 1-114 of SEQ ID NO:30, respectively):

[0176] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV NAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER(SEQ ID NO:30)

[0177] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV NAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT(SEQ ID NO:1728)

[0178] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQF VHGEEDLKVQHSSYRQRARLL KDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKVNA(SEQ ID NO:309)

[0179] The modifications provided herein can be in the unmodified PD-L1 polypeptide set forth in SEQ ID NO: 30, 309, or 1728. In some cases, the modifications can also be in the unmodified IgV set forth in SEQ ID NO: 55. In some embodiments, the unmodified PD-L1 polypeptide has 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30, 55, 309, or 1728.

[0180] PKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEE DLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKV(SEQ ID NO:55)

[0181] Identifying the corresponding position of a modification (e.g., amino acid substitution) in a PD-L1 polypeptide (including a portion thereof containing an IgSF domain (e.g., ECD or IgV)) is within the level of skill, such as by aligning a reference sequence with SEQ ID NO: 30 or SEQ ID NO: 309. In lists of modifications throughout this disclosure, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g., wild-type) amino acid listed before the number and the identified variant amino acid substitution listed after the number. If the modification is a deletion at a position, "del" is indicated, and if the modification is an insertion at a position, "ins" is indicated. In some cases, insertions are listed with the amino acid position indicated in the middle, with the corresponding unmodified (e.g., wild-type) amino acid listed before and after the number and the identified variant amino acid insertion listed after the unmodified (e.g., wild-type) amino acid.

[0182] In some embodiments, the variant PD-L1 polypeptide has one or more amino acid modifications (e.g., substitutions) in the wild-type or unmodified PD-L1 sequence. The one or more amino acid modifications (e.g., substitutions) may be in the extracellular domain (extracellular domain) of the wild-type or unmodified PD-L1 sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or its specific binding fragment. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the ECD domain or its specific binding fragment. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgC (e.g., IgC2) domain or its specific binding fragment. In some embodiments of the variant PD-L1 polypeptide, some of the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or its specific binding fragment, and some of the one or more amino acid modifications (e.g., substitutions) are in one or more IgC domains (e.g., IgC2) or its specific binding fragment.

[0183] In some embodiments, the variant PD-L1 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications (e.g., substitutions). The modifications (e.g., substitutions) can be in the IgV domain or one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 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-L1 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 one or more IgC (e.g., IgC2) domains or specific binding fragments thereof. In some embodiments, the variant PD-L1 polypeptide has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified PD-L1 polypeptide or specific binding fragment thereof, such as the amino acid sequence of SEQ ID NO: 30, 1728, 55, or 309.

[0184] 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 198, 199, 200, 201, 202, 203, 204, 206, 207, 213 or 221, which refers to the position listed in SEQ ID NO: 30 or 1728. In some embodiments, such variant PD-L1 polypeptides exhibit altered binding affinity for one or more of PD-1 and / or CD80 compared to a wild-type or unmodified PD-L1 polypeptide. For example, in some embodiments, the variant PD-L1 polypeptides exhibit increased binding affinity for PD-1 and / or CD80 compared to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptides exhibit decreased binding affinity for PD-1 or CD80 compared to a wild-type or unmodified PD-L1 polypeptide.

[0185] In some embodiments, the variant PD-L1 polypeptide has one or more amino acid modifications, e.g., amino acid substitutions, selected from the group consisting of P6S, Y10F, V11A, V11E, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I 46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G , D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65 L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q 89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, G101 G-ins(G101GG), G102D, A103V, D104G, K106E, K106R, V110M, K111E, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T 130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A , Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171 R, F173I, F173L, K173Y, V175A, T176N, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P or T221L, or conservative amino acid substitutions thereof. A conservative amino acid substitution is any amino acid that belongs to the same class as the substituted amino acid, other than the wild-type or unmodified 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).

[0186] In some embodiments, the variant PD-L1 polypeptide has two or more amino acid modifications, e.g., amino acid substitutions, selected from the group consisting of P6S, Y10F, V11A, V11E, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28N, K28R, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N 45T, I46V, I47T, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G, D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R 64S, Q65L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G 101D, G101G-ins(G101GG), G102D, A103V, D104G, K106E, K106R, V110M, K111E, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V1 29A, V129D, T130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A, Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K16 7T, E170G, K171R, F173I, F173L, K173Y, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, or T221L.

[0187] In some embodiments, amino acid modifications, such as amino acid substitutions, include K28N / M41V / N45T / H51N / K57E, I20L / I36T / N45D / I47T, I20L / M41K / K44E, P6S / N45T / N78I / I83T, N78I, M41K / N78I, N45T / N78I, I20L / N45T, N45T, M41K, I20L / I36T / N45D, N17D / N45T / V50A / D72G, I20L / F49S, N45T / V50A, I20L / N45T / N78I, I20 L / N45T / V50A, M41V / N45T, M41K / N45T, A33D / S75P / D85E, M18I / M41K / D43G / H51R / N78I, V11E / I20L / I36T / N45D / H60R / S75P, A33D / V50 A. S16G / A33D / K71E / S75P, E27G / N45T / M97I, E27G / N45T / K57R, A33D / E53V, D43G / N45D / V58A, E40G / D43V / N45T / V50A, Y14S / K28E / N45TA33D / N78S、A33D / N78I、A33D / N45T、A33D / N45T / N78I、E27G / N45T / V50A、N45T / V50A / N78S、I20L / N45T / V110M、I20L / I3 6T / N45T / V50A、N45T / L74P / S75P、N45T / S75P、S75P / K106R、 S75P、A33D / S75P、A33D / S75P / D104G、A33D / S75P、I20L / E27G / N45T / V50A、I20L / E27G / D43G / N45D / V58A / N78I、I20L / D43G / N45D / V58A / N78I、I20L / A33D / D43G / N45D / V58A / N78I、I20 L / D43G / N45D / N78I、E27G / N45T / V50A / N78I、N45T / V50A / N78I、V11A / I20L / E27G / D43G / N45D / H51Y / S99G、I20L / E27G / D4 3G / N45T / V50A、I20L / K28E / D43G / N45D / V58A / Q89R、I20L / I36T / N45D、I20L / K28E / D43G / N45D / E53G / V58A / N78I、A33D / D43G / N45D / V58A / S75P、K23R / D43G / N45D、I20L / D43G / N45D / V58A / N78I / D90G / G101D、D43G / N45D / L56Q / V58A / G101GG、I 20L / K23E / D43G / N45D / V58A / N78I、I20L / K23E / D43G / N45D / V50A / N78I、T19I / E27G / N45I / V50A / N78I / M97K、I20L / M41K / D43G / N45D、K23R / N45T / N78I、I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins(G101GG)、K57R / S99G、K57R / S99G / F189L、 M18V / M97L / F193S / R195G / E200K / H202Q、I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F 、C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L、M18V / I98L / L124S / P198T / L206F、S99G / N117S / I148V / K171R / R180S、I36T / M97L / A103V / Q155H、K28I / S99G、R195S、A79T / S99G / T185A / R195G / E200K / H202Q / L206F、K57R / S99G / L124S / K144Q、K57R / S99G / R195G、D55V / M97L / S99G、E27G / I36T / D55N / M97L / K111E、E54G / M97L / S99G、G15A / I36T / M97L / K111E / H202Q、G15A / I36T / V129D、G15A / I36T / V129D / R195G、G15A / V129D、I36S / M97L、I36T / D55N / M97L / K111E / A204T、I36T / D55N / M97L / K111E / V129A / F173L、I36T / D55S / M97L / K111E / I148V / R180S 、I36T / G52R / M97L / V112A / K144E / V175A / P198T 、I36T / I46V / D55G / M97L / K106E / K144E / T185A / R195G、I36T / I83T / M97L / K144E / P198T、I36T / M97L / K111E、I36T / M97L / K144E / P198T、I36T / M97L / Q155H / F193S / N201Y、I36T / M97L / V129D、L35P / I36S / M97L / K111E、M18I / I36T / E53G / M97L / K144E / E199G / V207A、M18T / I36T / D55N / M97L / K111E、M18V / M97L / T176N / R195G、M97L / S99G、N17D / M97L / S99G、S99G / T185A / R195G / P198T、V129D / H202Q、V129D / P198T、V129D / T150A、V93E / V129D、Y10F / M18V / S99G / Q138R / T203A、N45D、K160M / R195G、N45D / K144E、N45D / P198S、N45D / P198T、N45D / R195G、N45D / R195S、N45D / S131F、N45D / V58D、V129D / R195S、I98T / F173Y / L196S、N45D / E134G / L213P、N45D / F173I / S177C、N45D / I148V / R195G、N45D / K111T / R195G、N45D / N113Y / R195S、N45D / N165Y / E170G、N45D / Q89R / I98V、N45D / S131F / P198S、N45D / S75P / P198S、N45D / V50A / R195T、E27D / N45D / T183A / I188V 、 F173Y / T183I / L196S / T203A 、K23N / N45D / S75P / N120S 、 N45D / G102D / R194W / R195G 、N45D / G52V / Q121L / P198S 、 N45D / I148V / R195G / N201D 、N45D / K111T / T183A / I188V 、 N45D / Q89R / F189S / P198S 、N45D / S99G / C137R / V207A 、 N45D / T163I / K167R / R195G 、N45D / T183A / T192S / R194G 、 N45D / V50A / I119T / K144E 、T19A / N45D / K144E / R195G 、 V11E / N45D / T130A / P198T 、V26A / N45D / T163I / T185A、K23N / N45D / L124S / K167T / R195G, K23N / N45D / Q73R / T163I, K28E / N45D / W149R / S158G / P198T , K28R / N45D / K57E / I98V / R195S, K28R / N45D / V129D / T163N / R195T, M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D, or N45D / V50A / L124S / K144E / L179P / R195G.

[0188] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 20 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of I20L or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / E27G, I20L / A33D, I20L / I36T, I20L / D43G, I20L / N45D, I20L / N45T, I20L / V50A, I20L / V58A, I20L / S75P, I20L / N78I, I20L / M97L, I20L / S99G, I20L / R195G, I20L / P198S, or I20L / R198T.

[0189] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 27 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of E27G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / E27G, E27G / A33D, E27G / I36T, E27G / D43G, E27G / N45D, E27G / N45T, E27G / V50A, E27G / V58A, E27G / S75P, E27G / N78I, E27G / M97L, E27G / S99G, E27G / R195G, E27G / P198S, or E27G / R198T.

[0190] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 33 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution A33D or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 36, 43, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / A33D, E27G / A33D, A33D / I36T, A33D / D43G, A33D / N45D, A33D / N45T, A33D / V50A, A33D / V58A, A33D / S75P, A33D / N78I, A33D / M97L, A33D / S99G, A33D / R195G, A33D / P198S, or A33D / R198T.

[0191] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 36 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of I36T or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 43, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, E27G, A33D, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / I36T, E27G / I36T, A33D / I36T, I36T / D43G, I36T / N45D, I36T / N45T, A33D / V50A, I36T / V58A, I36T / S75P, I36T / N78I, I36T / M97L, I36T / S99G, I36T / R195G, I36T / P198S, or I36T / R198T.

[0192] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 43 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution D43G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / D43G, E27G / D43G, A33D / D43G, I36T / D43G, D43G / N45D, D43G / N45T, D43G / V50A, D43G / V58A, D43G / S75P, D43G / N78I, D43G / M97L, D43G / S99G, D43G / R195G, D43G / P198S, or D43G / R198T. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications D43G / N45D / V58A.

[0193] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 45 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution N45D or N45T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, D43G, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / N45D, E27G / N45D, A33D / N45D, I36T / N45D, D43G / N45D, N45D / V50A, N45D / V58A, N45D / S75P, N45D / S75P, N45D / N78I, N45D / M97L, N45D / S99G, N45D / R195G, N45D / P198S, or N45D / R198T. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / N45T, E27G / N45T, A33D / N45T, I36T / N45T, D43G / N45T, N45T / V50A, N45T / V58A, N45T / S75P, N45T / N78I, N45T / M97L, N45T / S99G, N45T / R195G, N45T / P198S, or N45T / R198T. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications D43G / N45D / V58A.

[0194] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 50 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of V50A or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, E27G, A33D, I36T, D43G, N45D, N45T, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / V50A, E27G / V50A, A33D / V50A, I36T / V50A, D43G / V50A, N45D / V50A, V50A / V58A, V50A / S75P, V50A / N78I, V50A / M97L, V50A / S99G, V50A / R195G, V50A / P198S, or V50A / R198T.

[0195] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 58 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of V58A or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / V58A, E27G / V58A, A33D / V58A, I36T / V58A, D43G / V58A, N45D / V58A, V50A / V58A, V58A / S75P, V58A / N78I, V58A / M97L, V58A / S99G, V58A / R195G, V58A / P198S, or V58A / R198T. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications D43G / N45D / V58A.

[0196] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 75 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution S75P or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, N78I, M97L, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / S75P, E27G / S75P, A33D / S75P, I36T / S75P, D43G / S75P, N45D / S75P, V50A / S75P, V58A / S75P, S75P / N78I, S75P / M97L, S75P / S99G, S75P / R195G, S75P / P198S, or S75P / R198T.

[0197] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 78 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution N78I or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 75, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, M97L, S99G, R195G, P198S, or P198T, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / N78I, E27G / N78I, A33D / N78I, I36T / N78I, D43G / N78I, N45D / N78I, V50A / N78I, V58A / N78I, S75P / N78I, N78I / M97L, N78I / S99G, N78I / R195G, N78I / P198S, or N78I / R198T.

[0198] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 97 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of M97L or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, S99G, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / M97L, E27G / M97L, A33D / M97L, I36T / M97L, D43G / M97L, N45D / M97L, V50A / M97L, V58A / M97L, S75P / M97L, N78I / M97L, M97L / S99G, M97L / R195G, M97L / P198S, or M97L / R198T.

[0199] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 99 in unmodified PD-L1 or a specific binding fragment thereof with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution of S99G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions of I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, R195G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / S99G, E27G / S99G, A33D / S99G, I36T / S99G, D43G / S99G, N45D / S99G, V50A / S99G, V58A / S99G, S75P / S99G, N78I / S99G, M97L / S99G, S99G / R195G, S99G / P198S, or S99G / R198T.

[0200] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 195 in unmodified PD-L1 or a specific binding fragment thereof with reference to the position numbering set forth in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution R195G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, P198S, or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / R195G, E27G / R195G, A33D / R195G, I36T / R195G, D43G / R195G, N45D / R195G, V50A / R195G, V58A / R195G, S75P / R195G, N78I / R195G, M97L / R195G, S99G / R195G, R195G / P198S, or R195G / R198T.

[0201] In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position corresponding to position 198 in unmodified PD-L1 or a specific binding fragment thereof with reference to the position numbering set forth in SEQ ID NO: 30. In some embodiments, the amino acid modification is an amino acid substitution P198S or P198T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further comprises one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, or 195. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, or R195G, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / P198S, E27G / P198S, A33D / P198S, I36T / P198S, D43G / P198S, N45D / P198S, V50A / P198S, V58A / P198S, S75P / P198S, N78I / P198S, M97L / P198S, S99G / P198S, or R195G / P198S. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L / P198T, E27G / P198T, A33D / P198T, I36T / P198T, D43G / P198T, N45D / P198T, V50A / P198T, V58A / P198T, S75P / P198T, N78I / P198T, M97L / P198T, S99G / P198T, or R195G / P198T.

[0202] In some embodiments, the variant PD-L1 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences of the extracellular domain (ECD) or IgV domain of wild-type PD-L1 or exemplary variant PD-L1 polypeptides by reference to SEQ ID NOs. As indicated, the precise locus or residue corresponding to a given domain may vary, such as depending on the method used to identify or classify the domain. In addition, in some cases, the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., ECD or IgV) may be included in the sequence of the variant IgSF polypeptide, such as to ensure proper folding of the domain upon expression. Therefore, it should be understood that the illustration of SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain (such as an ECD or IgV domain) of a variant PD-L1 polypeptide may be several amino acids longer or shorter than the amino acid sequence listed in the corresponding SEQ ID NO, such as 1-10, for example, 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0203] In some embodiments, the variant PD-L1 polypeptide comprises or consists of any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008). In some embodiments, the variant PD-L1 polypeptide comprises or consists of a polypeptide sequence that is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, such as at least 96% identical, 97% identical, 98% identical, or 99% identical to any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008), and contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1. In some embodiments, the variant PD-L1 polypeptide comprises or consists of a specific binding fragment of any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008) and contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1.

[0204] In some embodiments, the variant PD-L1 polypeptide comprises or consists of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937). In some embodiments, the variant PD-L1 polypeptide comprises or consists of a polypeptide sequence that is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, such as at least 96% identical, 97% identical, 98% identical, or 99% identical to any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937), and contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1. In some embodiments, the variant PD-L1 polypeptide comprises or consists of a specific binding fragment of any IgV sequence listed in Table 1 (i.e., any one of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937) and contains one or more amino acid modifications (e.g., one or more substitutions) not present in wild-type or unmodified PD-L1.

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of CD80 compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309). In some embodiments, the PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 and the extracellular domain of CD80 compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309).

[0211] In some embodiments, the variant PD-L1 polypeptide exhibits increased binding affinity to one of the extracellular domains of PD-1 or CD80 and exhibits decreased binding affinity to the other of the extracellular domains of PD-1 or CD80, compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 and decreased affinity for the extracellular domain of CD80, compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of CD80 and decreased affinity for the extracellular domain of PD-1 compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NO: 30, 1728, 55, or 309).

[0212] In some embodiments, the variant PD-L1 polypeptide exhibits increased selectivity for PD-1 versus CD80, such as indicated by a ratio of PD-1 binding to CD80 binding (PD-1:CD80 binding ratio) greater than 1, as compared to the binding ratio of an unmodified PD-L1 polypeptide (e.g., set forth in SEQ ID NO: 30, 55, or 309) binding to PD-1 versus CD80. In some embodiments, the variant PD-L1 polypeptide exhibits a ratio of binding to PD-1 to CD80 that is greater than or about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more.

[0213] III. Forms of variant polypeptides

[0214] The immunomodulatory polypeptides comprising the variant PD-L1 provided herein containing vIgD can be formatted in a variety of ways, including as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, a specific form can be selected for the desired therapeutic application. In some cases, the immunomodulatory polypeptides comprising the variant PD-L1 polypeptides are provided in a form to antagonize or block the activity of their cognate binding partners, such as PD-1. In some embodiments, the antagonism to PD-1 can be applicable to promoting immunity in oncology. In some cases, the immunomodulatory polypeptides comprising the variant PD-L1 polypeptides are provided in a form to excite or stimulate the activity of their cognate binding partners, such as PD-1. In some embodiments, the excitation to PD-1 can be applicable to treating inflammation or autoimmunity. Technicians can easily determine the activity of a specific form, such as for antagonizing or exciting one or more specific cognate binding partners. Exemplary methods for assessing such activity are provided herein (including in the examples).

[0215] In some aspects, there is provided an immunomodulatory protein comprising the vIgD of PD-L1, wherein such protein is soluble, for example, fused to an Fc chain. In some aspects, one or more additional IgSF domains (such as one or more additional vIgD) can be connected to the vIgD (hereinafter referred to as "stacked" or "stacked" immunomodulatory protein) of PD-L1 as provided herein. In some embodiments, the modular form of the immunomodulatory protein provided provides flexibility for engineering or generating immunomodulatory proteins for regulating the activity of multiple relative structures (multiple homologous binding partners). In some embodiments, such "stacked" molecules can be provided in soluble form, or in some cases can be provided in the form of membrane-bound or secreted proteins. In some embodiments, variant PD-L1 immunomodulatory proteins are provided in the form of a conjugate containing the vIgD of PD-L1, which is directly or indirectly connected to a targeting agent or portion (e.g., an antibody or other binding molecule), which specifically binds to a ligand (e.g., an antigen), such as when administered to a subject, for example, for targeting or positioning vIgD to a specific environment or cell. In some embodiments, the targeting agent (e.g., an antibody or other binding molecule) binds to a tumor antigen, thereby localizing the variant PD-L1 containing vIgD to the tumor microenvironment, for example, to modulate the activity of tumor-infiltrating lymphocytes (TILs) that are specific for the tumor microenvironment. In some embodiments, the targeting agent (e.g., an antibody or other binding molecule) binds to an antigen expressed on an antigen-presenting cell or normal tissue in an inflammatory environment, thereby localizing the variant PD-L1 containing vIgD to an area of ​​unwanted autoimmune inflammation, for example, to modulate the activity of T cells that target self-antigens.

[0216] In some embodiments, the immunomodulatory protein provided is expressed in a cell and provided as part of an engineered cell therapy (ECT). In some embodiments, the variant PD-L1 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"). In some embodiments, according to the cognate binding partner identified by TIP, the engineered cells expressing TIP can excite the cognate binding partner by providing a co-stimulatory signal that is positive or negative for other engineered cells and / or endogenous T cells. In some aspects, the variant PD-L1 polypeptide is expressed in a secretable form in cells such as immune cells (e.g., T cells or antigen-presenting cells), such that when cells are administered to a subject, a secreted or soluble form of the variant PD-L1 polypeptide (hereinafter also referred to as "SIP") is produced. In some aspects, SIP can antagonize the cognate binding partner in the environment (e.g., tumor microenvironment) in which it is secreted. In some embodiments, the variant PD-L1 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that is capable of infecting cells in vivo, such as immune cells (e.g., T cells or antigen-presenting cells), when administered to a subject for delivery or expression of the variant polypeptide as a TIP or SIP in the cell.

[0217] In some embodiments, soluble immunomodulatory polypeptides (such as variant PD-L1 containing vIgD) can be encapsulated in liposomes, which themselves can be conjugated to any one or any combination of the provided conjugates (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the invention are deglycosylated. In more specific embodiments, the variant PD-L1 sequence is deglycosylated. In even more specific embodiments, the IgV and / or one or more IgC (e.g., IgC2) domains of the variant PD-L1 are deglycosylated.

[0218] Non-limiting examples of the forms provided are described in Figures 1A to 1C and further described below.

[0219] A. Soluble protein

[0220] In some embodiments, the immunomodulatory protein containing the variant PD-L1 polypeptide is a soluble protein. It will be appreciated by those skilled in the art that cell surface proteins typically have intracellular, transmembrane, and extracellular domains (ECD), and that soluble forms of such proteins can be prepared using the extracellular domain or an immunologically active subsequence thereof. Therefore, in some embodiments, the immunomodulatory protein containing the variant PD-L1 polypeptide lacks a transmembrane domain or a portion of a transmembrane domain. In some embodiments, the immunomodulatory protein containing the variant PD-L1 lacks an intracellular (cytoplasmic) domain or a portion of an intracellular domain. In some embodiments, the immunomodulatory protein containing the variant PD-L1 polypeptide comprises only a vIgD portion containing an ECD domain or a portion thereof containing an IgV domain and / or one or more IgC (e.g., IgC2) domain or a specific binding fragment thereof containing one or more amino acid modifications.

[0221] In some embodiments, the immunomodulatory protein is or contains a variant PD-L1 polypeptide in monomeric form and / or exhibits monovalent binding to its binding partner. In some aspects, the variant PD-L1 polypeptide (such as a soluble and / or variant PD-L1 lacking a transmembrane domain and an intracellular signaling domain) is directly or indirectly linked to another part. In some embodiments, the other part is a protein, peptide, small molecule or nucleic acid. In some embodiments, the monovalent immunomodulatory protein is a fusion protein. In some embodiments, the part 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), C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin binding small molecules or combinations thereof.

[0222] In some embodiments, an immunomodulatory polypeptide comprising a variant PD-L1 can be linked to a portion comprising a conformationally disordered polypeptide sequence consisting of amino acids Pro, Ala, and Ser (see, e.g., WO2008 / 155134, SEQ ID NO: 2025). In some cases, the amino acid repeat sequence is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat sequence comprises Ala, Ser, and Pro residues. Thus, provided herein are immunomodulatory proteins that are PASylated proteins, wherein the variant PD-L1 polypeptide is linked directly or indirectly via a linker to Pro / Ala / Ser (PAS). In some embodiments, one or more additional linker structures may be used.

[0223] In some embodiments, the portion is conducive to detecting or purifying variant PD-L1 polypeptides. In some cases, the immunomodulatory polypeptide comprises a tag or fusion domain directly or indirectly connected to the N-terminus and / or C-terminus of the PD-L1 polypeptide, such as an affinity or purification tag. Various suitable polypeptide tags and / or fusion domains are known and include, but are not limited to, polyhistidine (His) tags, FLAG tags (SEQ ID NO: 2010), Myc tags, and fluorescent protein tags (e.g., EGFP, listed in SEQ ID NO: 2027-2029). In some cases, the immunomodulatory polypeptide comprising a variant PD-L1 comprises at least six histidine residues (listed in SEQ ID NO: 2011). In some cases, the immunomodulatory polypeptide comprising a variant PD-L1 further comprises different combinations of parts. For example, the immunomodulatory polypeptide comprising a variant PD-L1 further comprises one or more polyhistidine tags and FLAG tags.

[0224] In some embodiments, the PD-L1 polypeptide is linked to a modified immunoglobulin heavy chain constant region (Fc), which is maintained in a monovalent format, such as set forth in SEQ ID NO: 1187.

[0225] In some embodiments, the immunomodulatory protein contains a variant PD-L1 polypeptide directly or indirectly linked to a multimerization domain via a linker. In some aspects, the multimerization domain increases the half-life of the molecule. The interaction of two or more variant PD-L1 polypeptides can be facilitated by direct or indirect linkage with any portion or other polypeptide that can itself interact to form a stable structure. For example, separately encoded variant PD-L1 polypeptide chains can be linked by multimerization, thereby mediating the multimerization of the polypeptides through the multimerization domain. Typically, the multimerization domain provides for the formation of a stable protein-protein interaction between a first variant PD-L1 polypeptide and a second variant PD-L1 polypeptide.

[0226] Homomultimeric or heteromultimeric polypeptides can be generated by co-expression of separate variant PD-L1 polypeptides. The first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide can be the same or different. In specific embodiments, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are identical in the homodimer and are each linked to the same multimerization domain. In other embodiments, heterodimers can be formed by linking together different first variant PD-L1 polypeptides and second variant PD-L1 polypeptides. In such embodiments, in some aspects, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are linked to different multimerization domains that can promote heterodimer formation.

[0227] In some embodiments, the multimerization domain includes any domain capable of forming stable protein-protein interactions. The multimerization domain can be via the following interactions: immunoglobulin sequence (e.g., Fc domain; See, e.g., International Patent Publication No. WO 93 / 10151 and WO 2005 / 063816 US; U.S. Publication No. 2006 / 0024298; U.S. Patent No. 5,457,035); Leucine zipper (e.g., from nuclear transformation protein fos and jun or proto-oncogene c-myc or from general nitrogen control (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 region; Hydrophilic region; Or the free thiol forming intramolecular disulfide bond between the chimeric molecules of homodimers or heterodimers. In addition, the multimerization domain can include an amino acid sequence containing a protrusion complementary to the amino acid sequence comprising the hole, such as, for example, U.S. Patent No. 5,731,168; International Patent Publication Nos. WO 98 / 50431 and WO 2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9: 617-621. This multimerization region can be engineered so that steric interactions not only promote stable interactions, but also further promote heterodimers to be formed by a mixture of chimeric monomers over homodimers. Typically, the protrusion is constructed by replacing the small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the protrusion are optionally produced on the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0228] The variant PD-L1 polypeptide may be joined at any position, but typically via its N-terminus or C-terminus, to the N-terminus or C-terminus of the multimerization domain to form a chimeric polypeptide. The connection may be direct or indirect via a linker. The chimeric polypeptide may be a fusion protein or may be formed by chemical linkage, such as by covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, a nucleic acid encoding all or part of a PD-L1 polypeptide may be operably linked directly or indirectly, or optionally via a linker domain, to a nucleic acid encoding a multimerization domain sequence. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant PD-L1 polypeptide is joined to the N-terminus of the multimerization domain. In some cases, the construct may encode a chimeric protein in which the N-terminus of the variant PD-L1 polypeptide is joined to the C-terminus of the multimerization domain.

[0229] The polypeptide multimer contains multiple (such as two) chimeric proteins formed by directly or indirectly linking two identical or different variant PD-L1 polypeptides to a multimerization domain. In some examples, when the multimerization domain is a polypeptide, a gene fusion encoding a variant PD-L1 polypeptide and a multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with a recombinant expression vector and assembled into a multimer, wherein the multimerization domains interact to form a multivalent polypeptide. Chemical linkage of the multimerization domain to the variant PD-L1 polypeptide can be achieved using a heterologous bifunctional linker.

[0230] The resulting chimeric polypeptides (such as fusion proteins) and multimers formed therefrom can be purified by any suitable method, such as, for example, by affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, the formation of homodimers and heterodimers will occur. The conditions for expression can be adjusted so that heterodimer formation is favored over homodimer formation.

[0231] In some embodiments, the multimerization domain is an Fc domain or portion thereof from an immunoglobulin. In some embodiments, the immunomodulatory protein comprises a variant PD-L1 polypeptide attached to an immunoglobulin Fc (generating an "immunomodulatory Fc fusion," such as a "PD-L1-Fc variant fusion," also known as a PD-L1 vIgD-Fc fusion). In some embodiments, the attachment of the variant PD-L1 polypeptide is at the N-terminus of the Fc. In some embodiments, the attachment of the variant PD-L1 polypeptide is at the C-terminus of the Fc. In some embodiments, two or more PD-L1 variant polypeptides (the same or different) are independently attached at the N-terminus and the C-terminus.

[0232] In some embodiments, the Fc is a murine 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 set forth in SEQ ID NO: 187, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 187.

[0233] In some embodiments, the Fc region contains one or more modifications that change (e.g., reduce) one or more normal functions of the Fc region. Generally speaking, the Fc region is responsible for effector functions, such as complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), and the antigen binding ability of the primary function of immunoglobulin. In some cases, the effector functions in the Fc region may include programmed cell death and phagocytosis. In addition, the FcRn sequence present in the Fc region increases the half-life in vivo by being puted together with the FcRn receptor in vivo, thereby playing a role in regulating serum IgG levels. In some embodiments, such functions in Fc can be reduced or changed to be used together with the Fc fusion protein provided.

[0234] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the PD-L1-Fc variant fusions provided herein, thereby generating Fc region variants. In some embodiments, the Fc region variants have reduced effector function. There are many examples of changes or mutations in Fc sequences that can alter effector function. For example, WO 00 / 42072, WO2006019447, WO2012125850, WO2015 / 107026, US2016 / 0017041 and Shields et al. J Biol. Chem. 9 (2): 6591-6604 (2001) describe exemplary Fc variants with increased or decreased binding to FcR. The contents of these publications are expressly incorporated herein by reference.

[0235] In some embodiments, the variant PD-L1-Fc fusion provided comprises an Fc region that exhibits reduced effector function, which makes it a desirable candidate for applications in which the in vivo half-life of the PD-L1-Fc variant fusion is important, while certain effector functions (such as CDC and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the PD-L1-Fc variant fusion lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. For primary cells that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, Calif.); and CytoTox 96 TMNon-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 in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to confirm that the PD-L1-Fc variant fusion cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0236] PD-L1-Fc variant fusions with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (by EU numbering) ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 (by EU numbering), including the so-called "DANA" Fc mutant in which residues 265 and 297 are replaced with alanine ( U.S. Patent No. 7,332,581 ).

[0237] In some embodiments, the Fc region of the PD-L1-Fc variant fusion has an Fc region in which any one or more amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with a different amino acid compared to the native Fc region. Such alterations in the Fc region are not limited to those described above and include, for example, those described in Current Opinion in Biotechnology (2009) 20(6), 685-691, such as aglycosylated 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, I gG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A and IgG4-L236E; WO Alterations described in 2008 / 092117, such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R; amino acid insertions at positions 233, 234, 235, and 237 (indicated by EU numbering); and alterations at sites described in WO2000 / 042072.

[0238] Certain Fc variants with improved or reduced binding to FcRs have been described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, WO2006019447, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0239] In some embodiments, a PD-L1-Fc variant fusion comprising a variant Fc region is provided, wherein the variant Fc region comprises one or more amino acid substitutions that increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO2015107026. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (by EU numbering), e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0240] In some embodiments, the Fc region of the PD-L1-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L (by EU numbering). In some embodiments, the Fc region of the PD-L1-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S and / or C229S (by EU numbering). In some embodiments, the Fc region of the PD-L1 variant fusion comprises one or more amino acid substitutions R292C and V302C. See also Duncan and 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.

[0241] In some embodiments, alterations are made in the Fc region that result in reduced CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0242] In some embodiments, a PD-L1-Fc variant fusion comprising a variant Fc region is provided, wherein the variant Fc region comprises 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 set forth in SEQ ID NO: 187. In some embodiments, the Fc region comprises at least one amino acid substitution, which is N82G by numbering of SEQ ID NO: 187 (corresponding to N297G by EU numbering). In some embodiments, the Fc region further comprises at least one amino acid substitution, which is R77C or V87C by numbering of SEQ ID NO: 187 (corresponding to R292C or V302C by EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification by numbering of SEQ ID NO: 187 (corresponding to C220S by EU numbering). For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: V297G by EU numbering and one or more of the following amino acid modifications C220S, R292C, or V302C (corresponding to N82G with reference to SEQ ID NO: 187 and one or more of the following amino acid modifications C5S, R77C, or V87C), e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 1157. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E, or G237A, e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 1158. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 1159. In some embodiments, the variant Fc comprises one or more of amino acid modifications C220S, L234A, L235E, G237A, E356D, or M358L, eg, the Fc region comprises the sequence set forth in SEQ ID NO: 1155.

[0243] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc listed in SEQ ID NO: 187 (corresponding to K447del by EU numbering). In some aspects, this Fc region can further comprise one or more additional modifications, e.g., amino acid substitutions, such as any of those described. Examples of such Fc regions are listed in SEQ ID NO: 1938, 1939, 1940, or 1715.

[0244] In some embodiments, a PD-L1-Fc variant fusion is provided, comprising a variant Fc region, wherein the variant Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1155, 1157, 1158, 1159, 1715, 1938, 1939, or 1940, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any one of SEQ ID NOs: 1155, 1157, 1158, 1159, 1715, 1938, 1939, or 1940.

[0245] In some embodiments, the Fc is derived from IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 188, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity thereto.

[0246] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 1200, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 1200. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced by the CH3 domain of human IgG1 and exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced by the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which the arginine at position 409 indicated in the EU index as proposed by Kabat et al. of human IgG4 is substituted with lysine and exhibits inhibited aggregate formation (see, e.g., U.S. Patent No. 8,911,726. In some embodiments, the Fc is an IgG4 containing an S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab-arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8):767-71.) In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 1201 or a sequence identical to SEQ ID NO: 1202. NO:1201 has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

[0247] In some embodiments, the variant PD-L1 polypeptide is directly connected to the Fc sequence. In some embodiments, the variant PD-L1 polypeptide is indirectly connected to the Fc sequence, such as via a linker. In some embodiments, one or more "peptide linkers" connect the variant PD-L1 polypeptide to the Fc domain. In some embodiments, the length of the peptide linker can be a single amino acid residue or larger. In some embodiments, the peptide linker has at least one amino acid residue but no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues in length. In some embodiments, the linker is three alanines (AAA). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the form of a one-letter amino acid code): GGGGS ("4GS" or "G4S"; SEQ ID NO: 1942) or a multimer of a 4GS linker, such as 2, 3, 4, or 5 repeats of a 4GS linker, such as listed in SEQ ID NO: 240 (2xGGGGS) or SEQ ID NO: 239 (3xGGGGS). In some embodiments, the linker (in the form of a one-letter amino acid code) is GSGGGGS (SEQ ID NO: 1941). In some embodiments, the linker can also include 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 series is: 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an alpha helical linker. In some embodiments, the linker is (in the form of a one-letter amino acid code): EAAAK or a polymer of an EAAAK linker, such as a repeat of 2, 3, 4, or 5 EAAAK linkers, such as those listed in SEQ ID NO: 2022 (1xEAAAK), SEQ ID NO: 2023 (3xEAAAK), or SEQ ID NO: 2024 (5xEAAAK). In some embodiments, the linker may further comprise amino acids introduced by cloning and / or from restriction sites, for example, the linker may comprise the amino acid GS (in the form of a one-letter amino acid code), such as those introduced by using the restriction site BAMHI. For example, in some embodiments, the linker (in the form of a one-letter amino acid code) is GSGGGGS (SEQ ID NO: 1941), GS (G4S) 3 (SEQ ID NO: 2031), or GS (G4S) 5 (SEQ ID NO: 2032). In some instances, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 241).In some cases, immunomodulatory polypeptides comprising variant PD-L1 comprise different combinations of peptide linkers.

[0248] In some embodiments, the variant PD-L1-Fc fusion protein is a dimer formed by two variant PD-L1 Fc polypeptides linked to an Fc domain. In some embodiments, the dimer is a homodimer in which the two variant PD-L1 Fc polypeptides are identical. In some embodiments, the dimer is a heterodimer in which the two variant PD-L1 Fc polypeptides are different.

[0249] Also provided are nucleic acid molecules encoding variant PD-L1-Fc fusion proteins. In some embodiments, to produce an Fc fusion protein, a nucleic acid molecule encoding a variant PD-L1-Fc fusion protein is inserted into an appropriate expression vector. The resulting variant PD-L1-Fc fusion protein can be expressed in a host cell transformed with an expression vector, wherein assembly between the Fc domains occurs through interchain disulfide bonds formed between the Fc portions to produce a dimeric (such as a bivalent) variant PD-L1-Fc fusion protein.

[0250] The resulting Fc fusion protein can be readily purified by affinity chromatography on a Protein A or Protein G column. To generate heterodimers, additional purification steps may be necessary. For example, when two nucleic acids encoding different variant PD-L1 polypeptides are transformed into cells, heterodimer formation must be achieved biochemically because variant PD-L1 molecules carrying Fc domains will also be expressed as disulfide-linked homodimers. Therefore, homodimers can be reduced under conditions that favor the destruction of interchain disulfide bonds without affecting intrachain disulfide bonds. In some cases, different variant PD-L1 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 engineering and expressing Fc fusion molecules containing variant PD-L1 polypeptides using the ball-in-hole method described below.

[0251] B. Stacking molecules with additional IgSF domains

[0252] In some embodiments, the immunomodulatory protein may contain any of the variant PD-L1 provided herein, directly or indirectly linked to one or more other immunoglobulin superfamily (IgSF) domains ("stacked" immunomodulatory protein constructs and also referred to as "type II" immunomodulatory proteins). In some aspects, this can produce a unique multi-domain immunomodulatory protein that binds two or more (such as three or more) cognate binding partners, thereby providing multi-targeted modulation of the immune synapse.

[0253] In some embodiments, the immunomodulatory protein comprises a variant PD-L1 domain in combination with one or more other affinity-modified and / or non-affinity-modified IgSF domain sequences that are not present in a wild-type IgSF family member of another IgSF family member (e.g., a mammalian IgSF family member) (a "non-wild-type combination") and / or arrangement (a "non-wild-type arrangement" or "non-wild-type arrangement"). In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5, or 6 immunoglobulin superfamily (IgSF) domains, wherein at least one of the IgSF domains is a variant PD-L1 IgSF domain (vIgD of PD-L1) according to the description provided.

[0254] In some embodiments, the sequence of the additional IgSF domain can be a modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain can be non-affinity-modified (e.g., wild-type) or affinity-modified. In some embodiments, the unmodified or wild-type IgSF domain can be from mouse, rat, cynomolgus monkey, or human, or a combination thereof. In some embodiments, the additional IgSF domain can be an IgSF domain of an IgSF family member listed in Table 2. In some embodiments, the additional IgSF domain can be an affinity-modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to an IgSF domain contained in an IgSF family member listed in Table 2.

[0255] In some embodiments, the additional IgSF domain is an affinity or non-affinity modified IgSF domain contained by an IgSF family member selected from the following families: signal regulatory protein (SIRP) family, triggering receptor expressed on myeloid cells-like (TREML) family, carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, salicylic acid binding Ig-like lectin (SIGLEC) family, butyrophilin family, B7 family, CD28 family, V-type and immunoglobulin domain-containing (VSIG) family, V-type 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, triggering receptor of natural cytotoxicity (NCR) family, T cell immunoglobulin and mucin (TIM) family, or killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domains are 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, CTLA 4. PDCD1 (PD-1), ICOS, BTLA (CD272), CD4, CD8A (CD8-α), CD8B (CD8-β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226, CD2, CD160, CD200, CD200R1(CD200R), NCR3(NKp30) and VSIG8.

[0256] The first column of Table 2 provides the name of the specific IgSF member and, optionally, the names of some possible synonyms. The second column provides the protein identifier from the UniProtKB database, or in some cases, the GenBank number, which is a publicly available database accessible via the internet at uniprot.org. The Universal Protein Resource (UniProt) is a comprehensive resource of protein sequence and annotation data. The UniProt database includes the UniProt Knowledgebase (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Bioinformatics Institute, and the Protein Information Repository (PIR) and is primarily supported by funding from the U.S. National Institutes of Health (NIH). GenBank is the NIH gene sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). The third column provides the region in which the indicated IgSF domain is located. The region is designated as a range, wherein the domain includes the residues that define the range. Column 3 also indicates the IgSF domain class of a specific IgSF region. Column 4 provides the region where the indicated additional domains are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It should be understood that the description of the domains can vary depending on the method used to identify or classify the domains and can be identified differently from different sources. The description of the residues corresponding to the domains in Table 2 is for illustration only and can be longer or shorter by several amino acids (such as one, two, three, or four). Column 5 indicates some of the listed IgSF members and some of their cognate cell surface binding partners.

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264] In some embodiments, the immunomodulatory proteins provided contain at least 1, 2, 3, 4, 5, or 6 additional immunoglobulin superfamily (IgSF) domains, such as the IgD domains of the IgSF family members listed in Table 2, in addition to the variant PD-L1 polypeptide. In some embodiments, the immunomodulatory proteins provided contain at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the immunomodulatory proteins provided contain at least two additional IgSF domains (e.g., a second IgSF domain and a third IgSF domain). In some embodiments, the immunomodulatory proteins provided contain at least three additional IgSF domains (e.g., a second, third, and fourth). In some embodiments, the immunomodulatory proteins provided contain at least four additional IgSF domains (e.g., a second, third, fourth, and fifth). In some embodiments, the immunomodulatory proteins provided contain at least five additional IgSF domains (e.g., a second, third, fourth, fifth, and sixth). In some embodiments, the immunomodulatory proteins provided contain at least six additional IgSF domains (e.g., a second, third, fourth, fifth, sixth, and seventh). In some embodiments, each IgSF domain in the immunomodulatory protein is different. In some embodiments, at least one of the additional IgSF domains is identical to at least one other IgSF domain in the immunomodulatory protein. In some embodiments, each IgSF domain 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.

[0265] In some embodiments, the additional IgSF domain comprises an IgV domain or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain or one or more IgC (e.g., IgC2) domains. 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 one or more full-length IgC (e.g., IgC2) domains. 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 one or more IgC (e.g., IgC2) domains. In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (identical) IgSF member. For example, in some aspects, the immunomodulatory protein contains the ECD of an IgSF member or a portion thereof containing a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains or their specific binding fragments.

[0266] In some embodiments, provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second IgSF domain, or in some cases a third IgSF domain, etc.), wherein at least one additional, e.g., second or third IgSF domain is an IgSF domain listed among the wild-type or unmodified IgSF domains contained in the amino acid sequence listed in any one of SEQ ID NOs: 1-27 and 216, or a specific binding fragment thereof. 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.

[0267] In some embodiments, provided immunomodulatory proteins contain, in addition to a variant PD-L1 polypeptide, at least one additional affinity-modified IgSF domain (e.g., a second IgSF domain, or in some cases 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 a wild-type or unmodified IgSF domain (such as an IgSF domain in an IgSF family member listed 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 greater sequence identity with the wild-type or unmodified IgSF domain contained in the amino acid sequence set forth in any one of SEQ ID NOs: 1-27 and 216, or a specific binding fragment thereof. 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, the one or more additional IgSF domains are affinity-modified IgSF domains comprising an IgV domain and / or one or more IgC (e.g., IgC2) domains, or specific binding fragments of an IgV domain and / or specific binding fragments of one or more IgC (e.g., IgC2) domains, wherein the IgV and / or IgC domains comprise one or more amino acid modifications (e.g., substitutions). In some embodiments, the one or more additional affinity-modified IgSF domains comprise an IgV domain comprising one or more amino acid modifications (e.g., substitutions). In some embodiments, the 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, such as a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains, or specific binding fragments thereof, wherein one or both of the IgV and IgC domains comprise one or more amino acid modifications (e.g., substitutions).

[0268] In some embodiments, the immunomodulatory polypeptide comprising a variant PD-L1 may comprise one or more vIgDs of the PD-L1 provided herein. In some embodiments, the variant PD-L1 immunomodulatory proteins provided herein will comprise exactly 1, 2, 3, 4, 5, or more variant PD-L1 sequences. In some embodiments, at least two of the variant PD-L1 sequences are identical variant IgSF domains.

[0269] In some embodiments, the immunomodulatory polypeptide provided comprises two or more vIgD sequences of PD-L1. The multiple variant PD-L1s within a polypeptide chain may be identical to each other (i.e., the same species) or may be different (i.e., different species) variant PD-L1 sequences. In addition to the single polypeptide chain embodiment, in some embodiments, two, three, four or more polypeptides of the present invention may be covalently or non-covalently attached to each other. Thus, monomers, dimers, and higher-level (e.g., 3rd, 4th, 5th, or higher) multimeric proteins are provided herein. For example, in some embodiments, exactly two polypeptides of the present invention may be covalently or non-covalently attached to each other to form a dimer. In some embodiments, the attachment is via an intrachain cysteine ​​disulfide bond. A composition comprising two or more polypeptides of the present invention may be of the same or substantially the same species (e.g., homodimers) or of different species (e.g., heterodimers). As described above, a composition having a plurality of connected polypeptides of the present invention may have one or more identical or different variant PD-L1s of the present invention in each polypeptide chain. In some embodiments, PD-L1-Fc variant fusion polypeptides of the same or substantially the same species (allowing for 3 or fewer N-terminal or C-terminal amino acid sequence differences) will dimerize to produce homodimers. Alternatively, PD-L1-Fc variant fusion polypeptides of different species can be dimerized to produce heterodimers.

[0270] In some embodiments, provided immunomodulatory proteins contain at least one additional (e.g., or a second or, in some cases, a third IgSF domain, etc.) IgSF domain that is, in addition to PD-L1, a vIgD that contains one or more amino acid substitutions compared to an IgSF domain (e.g., IgV) of a wild-type or unmodified IgSF domain.

[0271] In some embodiments, one or more additional IgSF domains (e.g., a second or third IgSF) domain is an IgSF domain (e.g., ECD or IgV) of another IgSF family member that also binds to an inhibitory receptor. In some aspects, one or more additional IgSF domains (e.g., a second or third IgSF) domain is an affinity-modified IgSF domain that is a variant IgSF domain (vIgD) of an IgSF family member that binds to an inhibitory receptor and contains one or more amino acid substitutions in an IgSF domain (e.g., ECD or IgV), wherein in some cases one or more amino acid modifications cause increased binding to the inhibitory receptor. In some embodiments, vIgD contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in a wild-type or unmodified IgSF domain (e.g., ECD or IgV) of an IgSF family member that binds to an inhibitory receptor. In addition to PD-1, exemplary examples of such inhibitory receptors are CTLA-4, LAG3, TIGIT, TIM-3, or BTLA. In some embodiments, the one or more additional IgSF domains are from an IgSF family member selected from: CD155, CD112, PD-L2, CD80, or CEACAM 1. Thus, in some aspects, multi-target checkpoint antagonists are provided that target or block the activity of more than one inhibitory receptor.

[0272] In some embodiments, the immunomodulatory protein in the multi-target checkpoint antagonist targets or blocks the activity of at least two, three, four or more inhibitory receptors. In some embodiments, an immunomodulatory protein containing any one of the variant PD-L1 polypeptides and one or more IgSF domains of an inhibitory receptor (such as a wild-type or unmodified inhibitory receptor) is provided. In some embodiments, an immunomodulatory protein containing any one of the variant PD-L1 polypeptides and one or more IgSF domains of CD80 (e.g., wild-type or unmodified CD80) (such as an IgV domain listed in SEQ ID NO: 1005, 1079 or 2030 or an ECD listed in SEQ ID NO: 28 or a portion thereof (containing IgV and IgC domains or specific binding fragments thereof)) is provided. In some embodiments, an immunomodulatory protein is provided that contains any of the variant PD-L1 polypeptides and one or more IgSF domains of CD112 (e.g., wild-type or unmodified CD112), such as the IgV domain set forth in SEQ ID NO: 666 or 761, or the ECD set forth in SEQ ID NO: 48 or a portion thereof (containing IgV and IgC domains, or specific binding fragments thereof). In some embodiments, an immunomodulatory protein is provided that contains any of the variant PD-L1 polypeptides and one or more IgSF domains of PD-L2 (e.g., wild-type or unmodified PD-L2), such as the IgV domain set forth in SEQ ID NO: 1203 or 1263, or the ECD set forth in SEQ ID NO: 31 or a portion thereof (containing IgV and IgC domains, or specific binding fragments thereof). In some embodiments, an immunomodulatory protein is provided that contains any of the variant PD-L1 polypeptides and one or more IgSF domains of CD155 (e.g., wild-type or unmodified CD155), such as the IgV domain set forth in SEQ ID NO: 310 or 353, or the ECD set forth in SEQ ID NO: 47 or a portion thereof (containing IgV and IgC domains or specific binding fragments thereof).

[0273] In some embodiments, an immunomodulatory protein is provided that contains one or more additional IgSF domains (e.g., a second or third IgSF), which domain is a vIgD that binds to an IgSF family member of an inhibitory receptor, wherein the one or more amino acid modifications in the IgSF domain (e.g., IgV) result in an increase in the binding affinity of the vIgD or a fusion containing vIgD or immunomodulatory protein for the inhibitory receptor cognate binding partner compared to the unmodified IgSF domain, such as an increase in binding affinity of more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In some embodiments, the one or more amino acid modifications in the IgSF domain (e.g., IgV) result in an increase in the selectivity of the vIgD or a fusion containing vIgD or immunomodulatory protein for its inhibitory receptor compared to the unmodified IgSF domain. In some embodiments, the increased selectivity is a greater ratio of vIgD binding to an inhibitory receptor versus another cognate binding partner (such as a cognate binding partner of a non-inhibitory receptor) compared to the ratio of unmodified IgSF binding to the inhibitory receptor versus another cognate binding partner, In some embodiments, the ratio is at least or about 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 greater.

[0274] In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant CD80 polypeptide containing one or more amino acid modifications (e.g., substitutions, deletions, or additions) in an IgSF domain (e.g., IgV), wherein the CD80 is an IgSF family member that binds to the inhibitory receptor TIGIT. In some embodiments, at least one additional (e.g., second) vIgD contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in an IgSF domain (e.g., IgV) of CD80 that binds to an IgSF family member of the inhibitory receptor CTLA-4. Exemplary amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., ECD or IgV containing IgV and IgC) of a variant CD80 polypeptide are listed in Table 3. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD80 polypeptide containing an IgV domain comprising any of the amino acid modifications listed in Table 3, such as an IgV domain listed in any of SEQ ID NOs: 1006-1078, 1080-1112, 1114-1152, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs: 1006-1078, 1080-1112, 1114-1152 and containing one or more amino acid modifications. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD80 polypeptide containing an ECD, or portion thereof, containing an IgV and / or IgC domain, containing any of the amino acid modifications listed in Table 3, such as an ECD listed in any of SEQ ID NOs: 932-964, 966-1004, 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: 932-964, 966-1004 and containing one or more amino acid modifications.

[0275] In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) containing one or more amino acid modifications (e.g., substitutions, deletions or additions) of a variant CD155 polypeptide compared to unmodified or wild-type CD155, which in some aspects results in increased binding to inhibitory receptor TIGIT. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., IgV or an ECD containing IgV and IgC) of a variant CD155 polypeptide are listed in Table 5. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD155 polypeptide containing an IgV domain comprising any of the amino acid modifications listed in Table 5, such as an IgV domain listed in any of SEQ ID NOs: 332-352, 354-374, 472-665, 1505-1550, 1570-1714, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs: 332-352, 354-374, 472-665, 1505-1550, 1570-1714 and containing one or more amino acid modifications. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD155 polypeptide containing an ECD, or a portion thereof containing an IgV and / or IgC domain, containing any of the amino acid modifications listed in Table 5, such as an ECD listed in any of SEQ ID NOs: 311-331, 375-471, 1551-1622, 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: 311-331, 375-471, 1551-1622 and containing one or more amino acid modifications.

[0276] In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) containing one or more amino acid modifications (e.g., substitutions, deletions or additions) of a variant CD112 polypeptide compared to unmodified or wild-type CD112, which results in increased binding to inhibitory receptor TIGIT in some aspects. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., IgV or an ECD containing IgV and IgC) of a variant CD112 polypeptide are listed in Table 4. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD112 polypeptide containing an IgV domain comprising any of the amino acid modifications listed in Table 4, such as an IgV domain listed in any of SEQ ID NOs: 714-760, 762-808, 850-931, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and one or more amino acid modifications of any of SEQ ID NOs: 714-760, 762-808, 850-931. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD112 polypeptide containing an ECD, or a portion thereof containing an IgV and / or IgC domain, containing any of the amino acid modifications listed in Table 4, such as an ECD listed in any of SEQ ID NOs: 667-713, 809-849, 1433-1456, 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: 667-713, 809-849, 1433-1456 and containing one or more amino acid modifications.

[0277] In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant PD-L2 polypeptide containing one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., ECD or IgV) compared to unmodified or wild-type PD-L2, which in some aspects results in increased binding to the inhibitory receptor PD-1. Exemplary amino acid modifications (such as substitutions, deletions, or additions) in the IgSF domain (e.g., IgV or an ECD containing IgV and IgC) of the variant PD-L2 polypeptide are listed in Table 8. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant PD-L2 polypeptide containing an IgV domain comprising any of the amino acid modifications listed in Table 8, such as an IgV domain listed in any of SEQ ID NOs: 1281-1331, 1333-1407, 1409-1432, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and one or more amino acid modifications of any of SEQ ID NOs: 1281-1331, 1333-1407, 1409-1432. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant PD-L2 polypeptide containing an ECD, or portion thereof, containing an IgV and / or IgC domain, containing any of the amino acid modifications listed in Table 8, such as an ECD listed in any of SEQ ID NOs: 1204-1254, 1256-1280, 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: 1204-1254, 1256-1280 and containing one or more amino acid modifications.

[0278] In some embodiments, one or more additional IgSF domains (e.g., a second IgSF) domain is an IgSF domain (e.g., IgV) of another IgSF family member that binds to or recognizes a tumor antigen. In such embodiments, the IgSF family member serves as a tumor localization moiety, thereby bringing the vIgD of PD-L1 close to immune cells in the tumor microenvironment. In some embodiments, the additional IgSF domain (e.g., a second IgSF) domain is an IgSF domain of NKp30 that binds to or recognizes B7-H6 expressed on tumor cells. 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, one or more amino acid modifications increase binding affinity and / or selectivity for B7-H6 compared to an unmodified IgSF domain (e.g., NKp30), such as by at least or at least about 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. Exemplary amino acid modifications (such as substitutions, deletions, or additions) in the IgSF domain (e.g., IgC-like or full ECD) of variant NKp30 polypeptides are listed in Table 6. An exemplary polypeptide is a NKp30 variant containing the mutations L30V / A60V / S64P / S86G with reference to positions in the NKp30 extracellular domain corresponding to the positions listed in SEQ ID NO: 54. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant NKp30 polypeptide containing an IgC-like domain comprising any of the amino acid modifications listed in Table 6, such as an IgC-like domain listed in any of SEQ ID NOs: 1184-1188, or an IgC-like domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and one or more amino acid modifications of any of SEQ ID NOs: 1184-1188.In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant NKp30 polypeptide containing an ECD, or a portion thereof, containing one or more IgSF domains, containing any of the amino acid modifications listed in Table 6, such as an ECD listed in any of SEQ ID NOs: 1178-1182, 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: 1178-1182 and containing one or more amino acid modifications.

[0279] In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant CD86 polypeptide containing 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 CD86, which in some aspects results in increased binding to its cognate binding partner. Exemplary amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV or an ECD containing IgV and IgC) of the variant CD86 polypeptide are listed in Table 7. Exemplary polypeptides include CD86 variants containing the mutations Q35H / H90L / Q102H with reference to positions in the CD86 extracellular domain corresponding to the positions listed in SEQ ID NO: 29. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD86 polypeptide containing an IgV domain comprising any of the amino acid modifications listed in Table 7, such as an IgV domain listed in any of SEQ ID NOs: 1196-1199, or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and one or more amino acid modifications of any of SEQ ID NOs: 1196-1199. In some embodiments, an immunomodulatory protein is provided containing any of the provided variant PD-L1 polypeptides and a variant CD86 polypeptide containing an ECD, or portion thereof, containing an IgV and / or IgC domain, containing any of the amino acid modifications listed in Table 7, such as an ECD listed in any of SEQ ID NOs: 1191-1194, 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: 1191-1194 and containing one or more amino acid modifications.

[0280] Tables 3-8 provide exemplary polypeptides containing one or more affinity-modified IgSF domains that can be used in the stacking constructs provided herein.

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] The number of such non-affinity-modified or affinity-modified IgSF domains present in a "stacked" immunomodulatory protein construct (whether in a non-wild-type combination or non-wild-type arrangement) is at least 2, 3, 4 or 5, and in some embodiments, exactly 2, 3, 4 or 5 IgSF domains (the determination of the number of affinity-modified IgSF domains thus disregards any non-specific binding partial sequences thereof and / or substantially immunologically inactive partial sequences thereof).

[0296] In some embodiments of the stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least 2, wherein the number of affinity-modified IgSF domains and the number of non-affinity-modified IgSF domains are each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus, the number of affinity-modified IgSF domains and the number of non-affinity-modified IgSF domains (affinity-modified IgSF domains:non-affinity-modified IgSF domains) can be exactly or at least: 2:0 (affinity-modified:wild type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1, respectively.

[0297] In some embodiments of the stacked immunomodulatory proteins, at least two of the non-affinity-modified IgSF domains and / or affinity-modified IgSF domains are the same IgSF domain.

[0298] In some embodiments, the stacked immunomodulatory proteins provided herein comprise at least two affinity-modified IgSF domains and / or non-affinity-modified IgSF domains from a single IgSF member but in a non-wild-type arrangement (alternatively "arrangement"). An illustrative example of a non-wild-type arrangement or arrangement is an immunomodulatory protein comprising affinity-modified IgSF domains and / or non-affinity-modified IgSF domains in a non-wild-type order relative to those immunomodulatory proteins present in wild-type PD-L1 used as a source of variant IgSF domains as provided herein. Thus, in one example, an immunomodulatory protein may be comprised of IgV at the proximal end of the transmembrane domain and IgC at the distal end, although in a non-affinity-modified form and / or an affinity-modified form. The presence of non-wild-type combinations of non-affinity-modified IgSF domains and / or affinity-modified IgSF domains and non-wild-type arrangements in the immunomodulatory proteins provided herein is also within the scope of the provided subject matter.

[0299] In some embodiments of stacked immunomodulatory proteins, the non-affinity-modified IgSF domains and / or the affinity-modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity-modified IgSF domains specifically bind to different cognate binding partners under specific binding conditions and are "non-identical," regardless of whether the wild-type or unmodified IgSF domains from which they are engineered are the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein may comprise at least one IgSF domain sequence derived from and unique to one PD-L1 and at least one second IgSF domain derived from and unique to another non-PD-L1 IgSF family member, wherein the IgSF domains of the immunomodulatory protein are in a non-affinity-modified form and / or an affinity-modified form. However, in alternative embodiments, the two non-identical IgSF domains are derived from the same IgSF domain sequence, but at least one is affinity-modified such that they specifically bind to different cognate binding partners.

[0300] Multiple non-affinity-modified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be directly covalently linked to each other. In some embodiments, the intermediate span of one or more amino acid residues indirectly covalently binds the non-affinity-modified and / or affinity-modified IgSF domains to each other. The linkage can be via the N-terminal to C-terminal residues.

[0301] In some embodiments, two or more IgSF domains (including vIgD of PD-L1 and one or more additional IgSF domains (e.g., a second or third variant IgSF domain) from another IgSF family member are covalently or non-covalently linked together. In some embodiments, the two or more IgSF domains are linked directly or indirectly, such as via a linker. In some embodiments, the intermediate span of one or more amino acid residues covalently binds the IgSF domains to each other indirectly. The linkage can be via the N-terminal to C-terminal residues. In some embodiments, the linkage can be via the side chain of an amino acid residue that is not located at the N-terminus or C-terminus of the IgSF domain. Thus, the linkage can be via a terminal or internal amino acid residue, or a combination thereof.

[0302] 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 configurations are shown in Figure 5A and Figure 5B middle.

[0303] In some embodiments, one or more "peptide linkers" connect vIgD and one or more additional IgSF domains (e.g., a second or third variant IgSF domain) of PD-L1 together. In some embodiments, the length of the peptide linker can be a single amino acid residue or greater. In some embodiments, the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the form of a one-letter amino acid code): GGGGS ("4GS"; SEQ ID NO: 1942) or a polymer of a 4GS linker, such as 2, 3, 4, or 5 repeats of a 4GS linker. In some embodiments, the peptide linker is (GGGGS)2 (SEQ ID NO: 240) or (GGGGS)3 (SEQ ID NO: 239). In some embodiments, the linker (in the form of a one-letter amino acid code) is GSGGGGS (SEQ ID NO: 1941). In some embodiments, the linker can also include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or a polymer thereof). In some embodiments, the number of alanine residues in each series is: 2, 3, 4, 5 or 6 alanines. In some embodiments, the linker can also include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or a polymer thereof). In some embodiments, the number of alanine residues in each series is: 2, 3, 4, 5 or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an alpha helical linker. In some embodiments, the linker is (in the form of a one-letter amino acid code): EAAAK or a multimer of an EAAAK linker, such as a repeat of 2, 3, 4, or 5 EAAAK linkers, such as those listed in SEQ ID NO: 2022 (1xEAAAK), SEQ ID NO: 2023 (3xEAAAK), or SEQ ID NO: 2024 (5xEAAAK). 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 the form of a one-letter amino acid code), such as those introduced by using the restriction site BAMHI. For example, in some embodiments, the linker (in the form of a one-letter amino acid code) is GSGGGGS (SEQ ID NO: 1941), GS(G4S)3 (SEQ ID NO: 2031), or GS(G4S)5 (SEQ ID NO: 2032).In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 241).In some cases, various combinations of peptides are used as linkers.

[0304] In some embodiments, the non-affinity-modified and / or affinity-modified IgSF domains are connected by a "wild-type peptide linker" inserted at the N-terminus and / or C-terminus of the non-affinity-modified and / or affinity-modified IgSF domains. These linkers are also referred to as leader sequences (the N-terminus of the non-affinity-modified or affinity-modified IgSF domain) or tail sequences (the C-terminus of the non-affinity-modified or affinity-modified IgSF domain), and are sequences that exist in the wild-type protein that span outside the structural prediction of the Ig fold of IgSF. In some embodiments, a "wild-type linker" is an amino acid sequence that exists after the signal sequence but before the IgSF domain (such as a defined IgV domain) in the amino acid sequence of the wild-type protein. In some embodiments, a "wild-type" linker is an amino acid sequence that exists immediately after the IgSF domain (such as 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 facilitate proper folding and function of one or more adjacent IgSF domains. In some embodiments, there is a leader peptide sequence inserted at the N-terminus of a first IgSF domain and / or a tail sequence inserted at the C-terminus of a first non-affinity-modified and / or affinity-modified IgSF domain. In some embodiments, there is a second leader peptide sequence inserted at the N-terminus of a second IgSF domain and / or a second tail sequence inserted at the C-terminus of a second non-affinity-modified and / or affinity-modified IgSF domain. When the first and second non-affinity-modified and / or affinity-modified IgSF domains are derived from the same parent protein and linked in the same orientation, the wild-type peptide linker between the first and second non-affinity-modified and / or affinity-modified IgSF domains is not duplicated. For example, when the first tail wild-type peptide linker and the second leader wild-type peptide linker are identical, the type II immunomodulatory protein does not include the first tail wild-type peptide linker or the second leader wild-type peptide linker.

[0305] In some embodiments, the type II immunomodulatory protein comprises a first leader wild-type peptide linker inserted at the N-terminus of the first non-affinity-modified and / or affinity-modified IgSF domain, wherein the first leader wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the insertion sequence of the wild-type protein from which the first non-affinity-modified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain). In some embodiments, the first leader wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the first non-affinity-modified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain).

[0306] In some embodiments, the type II immunomodulatory protein further comprises a first tail wild-type peptide linker inserted at the C-terminus of the first non-affinity-modified and / or affinity-modified IgSF domain, wherein the first tail wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the insertion sequence of the wild-type protein from which the first non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain). In some embodiments, the first tail wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the first non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain).

[0307] In some embodiments, the type II immunomodulatory protein further comprises a second leader wild-type peptide linker inserted at the N-terminus of the second non-affinity-modified and / or affinity-modified IgSF domain, wherein the second leader wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the insertion sequence of the wild-type protein from which the second non-affinity-modified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain). In some embodiments, the second leader wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the second non-affinity-modified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain).

[0308] In some embodiments, the type II immunomodulatory protein further comprises a second tail wild-type peptide linker inserted at the C-terminus of the second non-affinity modified and / or affinity modified IgSF domain, wherein the second tail wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the insertion sequence of the wild-type protein from which the second non-affinity modified and / or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain). In some embodiments, the second tail wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the second non-affinity modified and / or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain).

[0309] In some embodiments, two or more IgSF domains (including vIgD of PD-L1 and one or more additional IgSF domains from another IgSF family member (e.g., a second and / or third variant IgSF domain)) are linked or attached to a multimerization domain, such as an Fc, to form an Fc fusion, which, when expressed in a cell, can produce a dimeric multidomain stacked immunomodulatory protein in some aspects. Thus, dimeric multidomain immunomodulatory proteins are also provided.

[0310] In some embodiments, the variant PD-L1 polypeptide and the one or more additional IgSF domains are independently directly or indirectly linked to the N-terminus or C-terminus of the multimerization domain, such as the Fc region. In some embodiments, at least one of the variant PD-L1 polypeptide and the one or more additional IgSF domains is directly or indirectly linked to the N-terminus or C-terminus of the multimerization domain, such as the Fc region, and one of the variant PD-L1 and one of the one or more additional IgSF domains is also directly or indirectly linked to the N-terminus or C-terminus of the multimerization domain, such as the Fc region. In some embodiments, the N-terminus or C-terminus of the multimerization domain, such as the Fc region, is linked to the variant PD-L1 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 PD-L1 variants or the other of the one or more additional IgSF domains. In some embodiments, the connection to the multimerization domain, such as to the Fc, is via a peptide linker, such as a peptide linker, such as described above. In some embodiments, the connection between the variant PD-L1 and the one or more additional IgSF domains is via a peptide linker, such as a peptide linker, such as described above. In some embodiments, the vIgD, one or more additional IgSF domains, and a multimerization domain such as an Fc domain of PD-L1 can be as described above. Figure 5A and Figure 5BAny of the many configurations depicted in FIG. 4 is connected together. Exemplary configurations are described in the Examples.

[0311] 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 one of the stacked immunomodulatory proteins. In some embodiments, the dimer multidomain stacked immunomodulatory protein can be produced in a cell by expressing or, in some cases, co-expressing the stacked immunomodulatory Fc fusion polypeptides (such as described above for generating a dimeric Fc fusion protein).

[0312] In some embodiments, the dimeric multi-domain stacked immunomodulatory protein is bivalent for each Fc region, monovalent for each subunit, or bivalent for one subunit and tetravalent for the other subunit.

[0313] In some embodiments, the dimeric multi-domain stacked immunomodulatory protein is a homodimeric multi-domain stacked Fc protein. In some embodiments, the dimeric multi-domain stacked immunomodulatory protein comprises a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are identical. In some embodiments, the multi-domain stacked molecule contains a first Fc fusion polypeptide comprising a variant PD-L1 and a second IgSF domain and a second Fc fusion polypeptide comprising a variant PD-L1 and a second IgSF domain. In some embodiments, the multi-domain stacked molecule contains a first Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain and a third IgSF domain and a second Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain and a third IgSF domain. In some embodiments, the Fc portion of the first and / or second fusion polypeptide can be any Fc as described above. In some embodiments, the Fc portion or region of the first and second fusion polypeptides is the same.

[0314] In some embodiments, the multi-domain stacking molecule is a heterologous dimer comprising two different Fc fusion polypeptides (e.g., a first and a second Fc fusion polypeptide), at least one of which is an Fc fusion polypeptide containing a variant PD-L1 polypeptide and / or at least one is an Fc fusion polypeptide containing a second IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the first or second Fc fusion polypeptide further contains a third IgSF domain (e.g., a third variant IgSF domain).

[0315] In some embodiments, the multi-domain stacking molecule contains a first Fc fusion polypeptide comprising a variant PD-L1 and a second Fc fusion polypeptide comprising a second IgSF domain, wherein in some cases the first or second Fc fusion polypeptide additionally contains a third IgSF domain. In some embodiments, the multi-domain stacking molecule contains a first Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain, and (in some cases) a third IgSF domain, and a second Fc fusion polypeptide that is not connected to a variant PD-L1 polypeptide or an additional IgSF domain. In some embodiments, the Fc portion or region of the first and second fusion polypeptides is the same. In some embodiments, the Fc portion or region of the first and second fusion polypeptides is different.

[0316] In some embodiments, the multi-domain stacked molecule contains a first Fc fusion polypeptide comprising 1, 2, 3, 4 or more variant PD-L1 polypeptides and 1, 2, 3, 4 or more additional IgSF domains, wherein the total number of IgSF domains in the first stacked Fc fusion polypeptide is greater than 2, 3, 4, 5, 6 or more. In one example of this embodiment, the second stacked Fc fusion polypeptide contains 1, 2, 3, 4 or more variant PD-L1 polypeptides and 1, 2, 3, 4 or more additional IgSF domains, wherein the total number of IgSF domains in the second stacked Fc fusion polypeptide is greater than 2, 3, 4, 5, 6 or more. In another example of this embodiment, the second Fc fusion polypeptide is not linked to a variant PD-L1 polypeptide or an additional IgSF domain.

[0317] In some embodiments, the heterodimeric stacking molecule contains a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are different. In some embodiments, the heterodimeric stacking molecule contains a first Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain) and a second polypeptide fusion comprising an Fc region and the other of the first variant PD-L1 polypeptide or the second IgSF domain. In some embodiments, the heterodimeric stacking molecule contains a first Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain) and a second Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain) but in a different orientation or configuration from the first Fc region. In some embodiments, the first and / or second Fc fusion polypeptide further contains a third IgSF domain (eg, a third variant IgSF domain).

[0318] In some embodiments, the Fc domains of one or both of the first and second stacked immunomodulatory Fc fusion polypeptides comprise modifications (e.g., substitutions) such that the interface of the Fc molecules is modified to facilitate and / or promote heterodimerization. In some embodiments, the modifications include introducing a protrusion (knob) into the first Fc polypeptide and introducing a cavity (hole) into the second Fc polypeptide such that the protrusion can be positioned in the cavity to promote complication of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create a protrusion or cavity in the polypeptide are typically interface amino acids that interact or contact one or more amino acids in the interface of the second polypeptide.

[0319] In some embodiments, the amino acid sequence is added in front of the Fc sequence of the construct, wherein the Fc sequence is the N-terminal portion of the sequence. In some cases, the amino acid sequence HMSSVSAQ (SEQ ID NO: 1156) is added immediately in front of the Fc sequence of the construct, wherein the Fc sequence is the N-terminal portion of the sequence. In some embodiments, the heterodimer stacking molecule contains the first Fc polypeptide fusions comprising an Fc region (globular protrusion) and a first variant polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain) and the second Fc polypeptide fusions comprising an Fc region (hole), comprising a stuffer sequence HMSSVSAQ (SEQ ID NO: 1156) immediately in front of the two Fc regions of the first and second Fc polypeptide fusions.

[0320] In some embodiments, the first polypeptide modified to contain a protrusion (hole) amino acid comprises replacing a natural or initial amino acid with an amino acid having at least one side chain, wherein the at least one side chain extends from the interface of the first polypeptide and can therefore be positioned in a complementary cavity (hole) in the adjacent interface of the second polypeptide. Most often, the replacement amino acid is an amino acid with a side chain volume larger than the initial amino acid residue. Those skilled in the art know how to determine and / or assess the properties of an amino acid residue to identify an ideal replacement amino acid, thereby generating a protrusion. In some embodiments, the replacement residue for forming a protrusion is a naturally occurring amino acid residue and includes, for example, arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W). In some instances, the initial residue identified for replacement is an amino acid residue with a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0321] In some embodiments, the second polypeptide modified to contain a cavity (hole) is a polypeptide comprising replacing a natural or initial amino acid with an amino acid having at least one side chain, wherein the at least one side chain is recessed from the interface of the second polypeptide and is therefore capable of accommodating a corresponding protrusion from the interface of the first polypeptide. Most often, the replacement amino acid is an amino acid having a side chain volume smaller than that of the initial amino acid residue. Those skilled in the art know how to determine and / or assess the properties of an amino acid residue to identify an ideal replacement residue for forming a cavity. Generally speaking, the replacement residue for forming a cavity is a naturally occurring amino acid, and includes, for example, alanine (A), serine (S), threonine (T) and valine (V). In some instances, the initial amino acid identified for replacement is an amino acid with a large side chain, such as, for example, tyrosine, arginine, phenylalanine or tryptophan.

[0322] The CH3 interface of human IgG1, for example, involves sixteen residues on each domain located on four antiparallel β-strands, which hide 1090 residues from each surface. (See, e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al. (1990) J Mol. Biol., 216, 965-973; Ridgway et al. (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modifications of the CH3 domain that create protrusions or cavities are described, e.g., in U.S. Patent No. 5,731,168; International Patent Applications WO 98 / 50431 and WO 2005 / 063816; and Ridgway et al. (1996) Prot. Engin., 9:617-621. In some examples, modifications of the CH3 domain that create protrusions or cavities are typically targeted to residues located on the two central antiparallel β strands. The goal is to minimize the risk that the resulting protrusion might be accommodated by protruding into the surrounding solvent rather than by a complementary cavity in the partner CH3 domain.

[0323] In some embodiments, the heterodimer molecule contains a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain". In some cases, additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing a Y349C mutation into the CH3 domain of the "knob" or "hole" chain and an E356C mutation or an S354C mutation into the CH3 domain of the other chain. In some embodiments, the heterodimer molecule contains an S354C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimer molecule comprises E356C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimer molecule comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimer molecule comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. Examples of other ball-in-hole technologies are known in the art, for example, as described in EP 1870459A1.

[0324] In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, such as any described above. In some embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function.

[0325] In some embodiments, the Fc variant containing a CH3 protrusion (knob) or cavity (hole) modification can be attached to any position of the stacked immunomodulatory polypeptide, but is typically attached to the N-terminus or C-terminus of the first and / or second stacked immunomodulatory polypeptide via its N-terminus or C-terminus, such as to form a fusion polypeptide. The connection can be direct or indirectly via a linker. Typically, the knob and hole molecules are generated by co-expression of a first stacked immunomodulatory polypeptide connected to an Fc variant containing one or more CH3 protrusion modifications and a second stacked immunomodulatory polypeptide connected to an Fc variant containing one or more CH3 cavity modifications.

[0326] Provided herein is a homodimeric multidomain stack molecule produced by stacking immunomodulatory Fc fusion polypeptides containing an IgSF domain (e.g., an IgV domain) of a variant PD-L1 polypeptide and a second IgSF domain (e.g., IgV) of a variant CD155 polypeptide. In some embodiments, the first and second immunomodulatory Fc fusion polypeptides of the multidomain stacking molecule have a sequence as set forth in any one of SEQ ID NOs: 1716, 1717, 1718, 1719, 1720, or 1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity thereto and containing one or more amino acid modifications in the variant PD-L1 and / or CD155 IgSF domains. In some embodiments, the resulting multidomain stacking molecule binds to both TIGIT and PD-1. In some aspects, the extent of binding to TIGIT is the same or similar, or in some cases increased, compared to binding to TIGIT with the corresponding IgSF domain of unmodified or wild-type PD-L1 or CD155. In some aspects, the extent of binding to PD-1 is the same or similar, or in some cases increased, compared to binding to PD-1 with the corresponding IgSF domain of unmodified or wild-type PD-L1. In some embodiments, the binding to TIGIT or PD-1 is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the binding of the non-stacked variant PD-L1 IgSF-Fc to TIGIT or PD-1. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the binding of TIGIT with the non-stacked variant CD155 IgSF-Fc. In some embodiments, the resulting multidomain stacked molecule increases T cell immune responses, such as determined in a reporter gene assay, compared to non-stacked variant PD-L1 IgSF-Fc and / or variant CD155-IgSF-Fc. In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or more.

[0327] Provided herein is a homodimeric multidomain stacked molecule produced by stacking immunomodulatory Fc fusion polypeptides containing an IgSF domain (e.g., an IgV domain) of a variant PD-L1 polypeptide, a second IgSF domain (e.g., IgV) of a variant CD155 polypeptide, and a third IgSF domain (e.g., IgV) of a variant CD112 polypeptide. In some embodiments, the first and second immunomodulatory Fc fusion polypeptides of the multidomain stacking molecule have a sequence as set forth in any one of SEQ ID NOs: 1722, 1723, and 1724, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity thereto and containing one or more amino acid modifications in the variant CD112, CD155, and / or PD-L1 IgSF domains. In some embodiments, the resulting multidomain stacking molecule binds to both TIGIT, CD112R, and PD-1. In some aspects, the degree of binding to TIGIT is the same or similar, or in some cases increased, compared to the binding to TIGIT of the corresponding IgSF domain of unmodified or wild-type CD112 or CD155. In some aspects, the degree of binding to CD112R is the same or similar, or in some cases increased, compared to the binding to CD112R of the corresponding IgSF domain of unmodified or wild-type CD112. In some aspects, the degree of binding to PD-1 is the same or similar, or in some cases increased, compared to the binding to PD-1 of the corresponding IgSF domain of unmodified or wild-type PD-L1. In some embodiments, the binding to TIGIT or CD112R is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the binding to TIGIT or CD112R of the non-stacked variant CD112IgSF-Fc. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the binding to TIGIT of the non-stacked variant CD155 IgSF-Fc. In some embodiments, the binding to PD-1 is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the binding to PD-1 of the non-stacked variant PD-1 IgSF-Fc. In some embodiments, the resulting multidomain stacked molecule increases T cell immune responses, such as determined in a reporter gene assay, compared to non-stacked variant CD112 IgSF-Fc, variant CD155-IgSF-Fc and / or variant PD-L1-IgSF-Fc.In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or more.

[0328] C. Conjugates and Fusions of Variant Polypeptides and Immunomodulatory Proteins

[0329] In some embodiments, the variant polypeptides provided herein are immunomodulatory proteins comprising a variant of an Ig domain of the IgSF family (vIgD), which can be conjugated or fused directly or indirectly to a moiety (such as an effector moiety, such as another protein) to form a conjugate ("IgSF conjugate"). In some embodiments, the attachment can be covalent or non-covalent, for example, via a biotin-streptavidin non-covalent interaction. In some embodiments, the moiety can be a targeting moiety, a small molecule drug (a non-polypeptide drug with a molar mass of less than 500 Daltons), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressant, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent.

[0330] In some embodiments, the effector moiety is a cytotoxic, cytostatic, or other therapeutic agent that provides some therapeutic benefit, such as a cancer therapeutic agent. In some embodiments, the effector moiety is a targeting moiety or targeting agent, such as an agent that targets a cell surface antigen (e.g., an antigen on the surface of a tumor cell). In some embodiments, the effector moiety is a label that can directly or indirectly generate a detectable signal. In some embodiments, the effector moiety is a toxin. In some embodiments, the effector moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.

[0331] In some embodiments, 1, 2, 3, 4, 5 or more effector moieties (which may be the same or different) are conjugated, linked or fused to a variant polypeptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties can be attached to a variant polypeptide or immunomodulatory protein using molecular biology or chemical conjugation and linking methods known in the art and described below. In some embodiments, a linker such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that facilitates a conjugation reaction can be used to link or conjugate the effector moiety to a variant polypeptide or immunomodulatory protein.

[0332] In some embodiments, the IgSF conjugate comprises the following components: (protein or polypeptide), (L) q and (effect part) mwherein the protein or polypeptide is any of the variant polypeptides or immunomodulatory proteins capable of binding to the one or more cognate opposing structural ligands; L is a linker that connects the protein or polypeptide to the moiety; m is at least 1; q is 0 or greater; and the resulting IgSF conjugate binds to the one or more opposing structural ligands. In specific embodiments, m is 1 to 4 and q is 0 to 8.

[0333] In some embodiments, an IgSF conjugate comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a targeting agent that binds to a cell surface molecule is provided, for example, for targeted delivery of the variant polypeptide or immunomodulatory protein to a specific cell. In some embodiments, the targeting agent is one or more molecules that have the ability to locate and bind to molecules present on normal cells / tissues and / or tumor cells / tumors of a subject. In other words, the IgSF conjugate comprising the targeting agent can bind to a ligand (directly or indirectly) present on a cell (such as a tumor cell). Targeting agents encompassed for use herein include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof that can bind to a component of a target cell or molecule.

[0334] In some embodiments, after administration to a subject, the targeting agent binds to the tumor cell or can bind to the vicinity of the tumor cell (e.g., tumor blood vessels or tumor microenvironment). The targeting agent can bind to a receptor or ligand on the surface of the cancer cell. In another aspect of the invention, a targeting agent is selected that is specific for non-cancerous cells or tissues. For example, the targeting agent can be specific for a molecule that normally exists on a particular cell or tissue. In addition, in some embodiments, the same molecule can be present on normal cells and cancer cells. Various cellular components and molecules are known. For example, if the targeting agent is specific for EGFR, the resulting IgSF conjugate can target cancer cells that express EGFR as well as normal skin epithelial cells that express EGFR. Therefore, in some embodiments, the IgSF conjugates of the present invention can act through two separate mechanisms (targeting cancer cells and non-cancerous cells).

[0335] In various aspects of the invention disclosed herein, the IgSF conjugates of the invention comprise a targeting agent that can bind to / target a cellular component, such as a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasma antigen, a fungal antigen, a prion antigen, or an antigen from a parasite. In some aspects, the cellular component, antigen, or molecule can each be used to designate a desired target for the targeting agent.For example, in various embodiments, the targeting agent is specific for or binds to a component including, but not limited to, epidermal growth factor receptor (EGFR, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family; platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (F GFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; ephrin (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family, such as ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L1, tumor glycosylated antigens, such as sTn or Tn, such as sTn of MUC1 Ag; LHR (LHCGR); phosphatidylserine, lectin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; transforming growth factor-α (TGF-α) receptor, TGF-β; cytokine receptors, class I (erythropoietin family) and class II (interferon / IL-10 family) receptors, tumor necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family; cancer-testis (CT) antigen, lineage-specific antigen, differentiation antigen, α-actinin-4, ARTCl, cleavage cluster region-Abelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (C ASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-I, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), such as the A domain of fibronectin (EDA), GPNMB, low-density lipid receptor / GDP-L trehalose:β-D-galactose 2-α-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2. Arginine to isoleucine transition at residue 170 of the α helix of the α2 domain of the HLA-A2 gene (HLA-A2). *201-R170I), HLA-A11, mutated heat shock protein 70-2 (HSP70-2M), K1AA0205, MART2, mutated melanoma ubiquitin 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, class I myosin, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase, BAGE, BAGK-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE, LAGE-I, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-I), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-AI l, MAGE-A12, MAGE-3, MAGE-Bl, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-Cl, MAGE-C2, mucin 1 (MUCl), MART-1 / Melan-A (MLANA), gplOO, gplOO / Pmell7 (SILV), tyrosinase (TYR), TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-1,2,3,4, TRP2-INT2, carcinoembryonic antigen (CEA), kallikrein 4, mammaglobin-A, OAl, prostate-specific antigen (PSA), TRP-1 / gp75, TRP-2, adipose differentiation-related protein, interferon-induced protein 2 absent in melanoma (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250), epidermal growth factor receptor (ERBB1), HER-2 / neu (ERBB2), interferon 13 receptor alpha 2 chain (IL13Rα2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCl, p53 (TP53), PBF, PRAME, PSMA , RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRC5), human telomerase reverse transcriptase (hTERT), telomerase, Wilms tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMAl, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotype, urinary Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD 19, CD33, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), beta-human chorionic gonadotropin, beta-2 microglobulin, squamous cell carcinoma antigen, neurospecific enol, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-I), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigen, other), Epstein-Barr virus (EBV) proteins (EBV latent membrane protein - LMP1, LMP2; other), hepatitis B or hepatitis C proteins, and HIV proteins.

[0336] In some embodiments, the IgSF conjugate will bind to tumor cells, tumor blood vessels, or cellular components of the tumor microenvironment through its targeting agent, thereby promoting the killing of targeted cells by modulating the immune response (e.g., by activating co-stimulatory molecules or inhibiting negative regulatory molecules that activate immune cells), inhibiting survival signals (e.g., growth factors or cytokines or hormone receptor antagonists), activating death signals, and / or immune-mediated cytotoxicity (such as through antibody-dependent cellular cytotoxicity). Such IgSF conjugates can act through several mechanisms to prevent, reduce, or eliminate tumor cells, such as to facilitate delivery of the conjugated effector moiety to the tumor target, such as through receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and / or activate immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells). In addition, in some cases, one or more of the aforementioned pathways can act upon administration of one or more IgSF conjugates of the present invention.

[0337] In some embodiments, the IgSF conjugate is positioned to (such as bound to) cellular components of tumor cells, tumor blood vessels, or the tumor microenvironment through its targeting agent, thereby regulating immune response cells near the tumor. In some embodiments, the targeting agent is conducive to delivering the conjugated IgSF (e.g., vIgD) to the tumor target, such as to interact with its cognate binding partner to change the signal transduction of immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells) carrying the cognate binding partner. In some embodiments, the positioned delivery mediates the antagonism or blocking activity of the PD-1 inhibitory receptor. In some embodiments, the positioned delivery excites the PD-1 inhibitory receptor, which can occur in some cases where there is a proximal clustering of activated receptors.

[0338] In some embodiments, the targeting agent is an immunoglobulin. As used herein, the term "immunoglobulin" includes natural or artificial monovalent or multivalent antibodies, including but not limited to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, single-chain Fv (scFv); anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the present invention) and epitope binding fragments of any of the above. As used herein, the term "antibody" refers to immunoglobulin molecules and immunoglobulin molecules. Immunologically active parts, such as molecules containing antigen-binding sites that immunospecifically bind to antigens. The immunoglobulin molecules of the present invention can have any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0339] In some embodiments, the IgSF conjugate will bind to tumor cells, tumor vasculature, or cellular components of the tumor microenvironment through its antibody targeting moiety, thereby promoting apoptosis of the targeted cells by modulating the immune response (e.g., by activating co-stimulatory molecules or inhibiting negative regulatory molecules that activate immune cells), inhibiting survival signals (e.g., growth factors or cytokines or hormone receptor antagonists), activating death signals, and / or immune-mediated cytotoxicity (e.g., through antibody-dependent cellular cytotoxicity). Such IgSF conjugates can act through several mechanisms to prevent, reduce, or eliminate tumor cells, such as to facilitate delivery of the conjugated effector moiety to the tumor target, such as through receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and / or activate immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells).

[0340] In some embodiments, the IgSF conjugate binds to tumor cells, tumor blood vessels, or cellular components of the tumor microenvironment through its antibody targeting portion, thereby modulating the immune response (e.g., by activating co-stimulatory molecules or inhibiting negative regulatory molecules that activate immune cells). In some embodiments, such conjugates can recognize, bind to, and / or modulate (e.g., inhibit or activate) immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells).

[0341] The antibody targeting moieties of the present invention include antibody fragments, including but not limited to Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv) and fragments comprising VL or VH domains. Antigen-binding antibody fragments including single-chain antibodies may include one or more variable regions alone or in combination with all or part of the following: hinge region, CH1 domain, CH2 domain and CH3 domain. The present invention also includes antigen-binding fragments that also include one or more variable regions and any combination of hinge region, CH1 domain, CH2 domain and CH3 domain. The present invention also includes Fc fragments, antigen-Fc fusion proteins and Fc targeting moiety conjugates or fusion products (Fc peptides, Fc aptamers). The antibody targeting moieties of the present invention may be from any animal origin, including birds and mammals. In one aspect, the antibody targeting moiety is human, mouse (e.g., mouse and rat), monkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. In addition, such antibodies may be humanized versions of animal antibodies. The antibody targeting moieties of the invention can be monospecific, bispecific, trispecific or have greater multispecificity.

[0342] In various embodiments, the antibody / targeting moiety recruits, binds and / or activates immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells) via interactions between Fc (in the antibody) and Fc receptors (on immune cells) and via the conjugated variant polypeptides or immunomodulatory proteins provided herein. In some embodiments, the antibody / targeting moiety recognizes or binds to the tumor agent via the conjugated variant polypeptides or immunomodulatory proteins provided herein and localizes to the tumor cells to facilitate modulation of immune cells near the tumor.

[0343] Examples of antibodies that can be incorporated into the IgSF conjugates include, but are not limited to, antibodies such as cetuximab (IMC-C225; ), trastuzumab Rituximab Bevacizumab Alemtuzumab Panitumumab (ABX-EGF...

Claims

1. A variant PD-L1 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications at one or more positions corresponding to positions in unmodified PD-L1 or a specific binding fragment thereof selected from the group consisting of: 45, 43, 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85 ,89,90,93,97,98,99,101,102,103,104,106,110,111,112,113,117,119,120,121,124,129,130,131,134,137,138,144,148,149,150,155,158,160,163 , 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 213 or 221, which refers to the numbering of SEQ ID NO:

30.

2. The variant PD-L1 polypeptide of claim 1, wherein the unmodified PD-L1 comprises (i) the amino acid sequence set forth in SEQ ID NO: 30, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30; or (iii) a portion thereof comprising an IgV structure or an IgC domain or a specific binding fragment thereof or both.

3. The variant PD-L1 polypeptide of claim 1 or claim 2, wherein the variant PD-L1 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.

4. The variant PD-L1 polypeptide of any one of claims 1 to 3, wherein the variant PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30 or 1728, or a specific binding fragment thereof.

5. The variant PD-L1 polypeptide of any one of claims 1 to 4, wherein the one or more amino acid modifications are selected from P6S, Y10F, V11A, V11E, Y14S, G15A, S16G, N17D, M18I, M18T, M18V, T19A, T19I, I20L, C22R, K23E, K23N, K23R, E26A, E27D, E27G, K28E, K28I, K28R, K28N, A33D, L35P, I36S, I36T, E40G, M41K, M41V, D43G, D43V, K44E, N45D, N45I, N45T, I 47T, I46V, F49S, V50A, H51N, H51R, H51Y, G52R, G52V, E53G, E53V, E54G , D55G, D55N, D55S, D55V, L56Q, K57E, K57R, V58A, V58D, H60R, R64S, Q65 L, R68L, K71E, D72G, Q73R, L74P, S75P, N78I, N78S, A79T, I83T, D85E, Q 89R, D90G, V93E, M97I, M97K, M97L, I98L, I98T, I98V, S99G, G101D, G101 G-ins(G101GG), G102D, A103V, D104G, K106E, K106R, V110M, K111E, K111T, V112A, N113Y, N117S, I119T, N120S, Q121L, L124S, V129A, V129D, T 130A, S131F, E134G, C137R, Q138R, K144E, K144Q, I148V, W149R, T150A , Q155H, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171 R, F173I, F173L, K173Y, V175A, T176N, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, A204T, L206F, V207A, L213P, T221L, or conservative amino acid substitutions thereof.

6. The variant PD-L1 polypeptide of any one of claims 1-5, wherein the one or more amino acid modifications are selected from K28N / M41V / N45T / H51N / K57E, I20L / I36T / N45D / I47T, I20L / M41K / K44E, P6S / N45T / N78I / I83T, N78I, M41K / N78I, N45T / N78I, I20L / N45T, N45T, M41K, I20L / I36T / N45D, N17D / N47T / V50A / D72G, I20L / F49S, N45T / V50A, I20L / N45 T / N78I, I20L / N45T / V50A, M41V / N45T, M41K / N45T, A33D / S75P / D85E, M18I / M41K / D43G / H51R / N78I, V11E / I20L / I36T / N45D / H60R / S75P, A33 D / V50A, S16G / A33D / K71E / S75P, E27G / N45T / M97I, E27G / N45T / K57R, A33D / E53V, D43G / N45D / V58A, E40G / D43V / N45T / V50A, Y14S / K28E / N45T A33D / N78S, A33D / N78I, A33D / N45T, A33D / N45T / N78I, E27G / N45T / V50A, N45T / V 50A / N78S, I20L / N45T / V110M, I20L / I36T / N45T / V50A, N45T / L74P / S75P, N45T / S 75P, S75P / K106R, S75P, A33D / S75P, A33D / S75P / D104G, A33D / S75P, I20L / E27G / N45T / V50A, I20L / E27G / D43G / N45D / V58A / N78I, I20L / D43G / N45D / V58A / N78I, I 20L / A33D / D43G / N45D / V58A / N78I, I20L / D43G / N45D / N78I, E27G / N45T / V50A / N7 8I, N45T / V50A / N78I, V11A / I20L / E27G / D43G / N45D / H51Y / S99G, I20L / E27G / D43 G / N45T / V50A, I20L / K28E / D43G / N45D / V58A / Q89R, I20L / I36T / N45D, I20L / K28E / D43G / N45D / E53G / V58A / N78I、A33D / D43G / N45D / V58A / S75P、K23R / D43G / N45D、I20L / D43G / N45D / V58A / N78I / D90G / G101D、D43G / N45D / L56Q / V58A / G101G-ins(G101GG)、I20L / K23E / D43G / N45D / V58A / N78I、I20L / K23E / D43G / N45D / V50A / N78I, T19I / E27G / N45I / V50A / N78I / M97K, I20L / M41K / D43G / N45D, K23R / N45T / N78I, I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins(G101GG) and K5 7R / S99G、K57R / S99G / F189L、M18V / M97L / F193S / R195G / E200K / H202Q、I36S / M41K / M97L / K144Q / R195G / E200K / H202Q / L206F、C22R / Q65L / L124S / K144Q / R195G / E200N / H202Q / T221L、M18V / I98L / L124S / P198T / L206F、S99G / N117S / I148V / K171R / R180S、I36T / M97L / A103V / Q155H、K28I / S99G、R195S、A79T / S 99G / T185A / R195G / E200K / H202Q / L206F、K57R / S99G / L124S / K144Q、K57R / S 99G / R195G、D55V / M97L / S99G、E27G / I36T / D55N / M97L / K111E、E54G / M97L / S 99G、G15A / I36T / M97L / K111E / H202Q、G15A / I36T / V129D、G15A / I36T / V129D / R195G、G15A / V129D、I36S / M97L、I36T / D55N / M97L / K111E / A204T、I36T / D55 N / M97L / K111E / V129A / F173L、I36T / D55S / M97L / K111E / I148V / R180S、I36T / G52R / M97L / V112A / K144E / V175A / P198T、I36T / I46V / D55G / M97L / K106E / K1 44E / T185A / R195G、I36T / I83T / M97L / K144E / P198T、I36T / M97L / K111E、I36 T / M97L / K144E / P198T、I36T / M97L / Q155H / F193S / N201Y、I36T / M97L / V129D、L35P / I36S / M97L / K111E、M18I / I36T / E53G / M97L / K144E / E199G / V207A、M18T / I36T / D55N / M97L / K111E、M18V / M97L / T176N / R195G、M97L / S99G、N17D / M97L / S99G、S99G / T185A / R195G / P198T、V129D / H202Q、V129D / P198T、V129D / T150A、V93E / V129D、Y10F / M18V / S99G / Q138R / T203A、N45D、K160M / R195G、N45D / K144E、N45D / P198S、N45D / P198T、N45D / R195G、N45D / R195S、N45D / S131F、N45D / V58D、V129D / R195S、I98T / F173Y / L196S、N45D / E134G / L213P、N45D / F173I / S177C、N45D / I148V / R195G、N45D / K111T / R195G、N45D / N113Y / R195S、N45D / N165Y / E170G、N45D / Q89R / I98V、N45D / S131F / P198S、N45D / S75P / P198S、N45D / V50A / R195T、E27D / N45D / T183A / I188V、F173Y / T183I / L196S / T203A、K23N / N45D / S75P / N120S、N45D / G102D / R194W / R195G、N45D / G52V / Q121L / P198S、N45D / I148V / R195G / N201D、N45D / K111T / T183A / I188V、N45D / Q89R / F189S / P198S、N45D / S99G / C137R / V207A、N45D / T163I / K167R / R195G、N45D / T183A / T192S / R194G、N45D / V50A / I119T / K144E、T19A / N45D / K144E / R195G、V11E / N45D / T130A / P198T、V26A / N45D / T163I / T185A、K23N / N45D / L124S / K167T / R195G、K23N / N45D / Q73R / T163I、K28E / N45D / W149R / S158G / P198T、K28R / N45D / K57E / I98V / R195S、K28R / N45D / V129D / T163N / R195T、M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D or N45D / V50A / L124S / K144E / L179P / R195G.

7. The variant PD-L1 polypeptide of any one of claims 1-6, wherein the one or more amino acid modifications are at one or more positions corresponding to one or more positions selected from 20, 27, 33, 36, 43, 45, 50, 58, 75 or 78.

8. The variant PD-L1 polypeptide of any one of claims 1 to 7, wherein the one or more amino acid modifications are selected from I20L, E27G, A33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, or conservative amino acid substitutions thereof.

9. A variant PD-L1 polypeptide as described in any of embodiments 1-8, wherein the variant PD-L1 polypeptide comprises amino acid modifications I20L / I36T, I20L / D43G, I20L / N45D, I20L / N45T, I20L / N45T, I20L / V50A, I20L / V58A, I20L / S75P, I20L / N78I, I36T / D43G, I36T / N45D, I36T / N45T, I36T / V50A, I36T / V58A, I36T / S75P, I36T / N , D43G / N45D, D43G / N45T, D43G / V50A, D43G / V58A, D43G / S75P, D43G / N78I, N45D / V50A, N45D / V58A, N45D / S75P, N45D / N78I, N45T / V50A, N45T / V58A, N45T / S75P, N45T / N78I, V50A / V58A, V50A / S75P, V50A / N78I, V58A / S75P, V58A / N78I, or S75P / N78I.

10. The variant PD-L1 polypeptide of any one of claims 1 to 9, wherein the variant PD-L1 polypeptide comprises amino acid modifications D43G / N45D / V58A.

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