CD80 variant immunomodulatory proteins and uses thereof

Abstract

To provide specific variants of CD80 that exhibit altered binding, such as binding affinity or selectivity, for a cognate binding partner, such as increased affinity for CTLA-4 and / or PD-L1 and / or decreased affinity for CD28.SOLUTION: Provided herein are variant CD80 polypeptides, immunomodulatory proteins comprising variant CD80 polypeptides, 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.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 472,558, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,569, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,554, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,572, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,573, filed March 17, 2017, and U.S. Provisional Patent Application No. 62 / 472,574, filed March 17, 2017. This application claims priority from U.S. Provisional Patent Application No. 62 / 475,204, filed March 22, 2017, U.S. Provisional Patent Application No. 62 / 537,939, filed July 27, 2017, U.S. Provisional Patent Application No. 62 / 574,165, filed October 18, 2017, and U.S. Provisional Patent Application No. 62 / 582,266, filed November 6, 2017, the contents of each of which are incorporated by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled 761612001640SeqList.TXT, created on March 12, 2018, and which is 4,888,576 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to therapeutic compositions for modulating immune responses in the treatment of cancer and immune disorders. In some aspects, the present disclosure relates to specific variants of CD80 that exhibit altered binding, such as binding affinity or selectivity for cognate binding partners, e.g., increased affinity for CTLA-4 and / or PD-L1, and / or decreased affinity for CD28. [Background technology]

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

[0005] overview In some embodiments, provided herein is a variant CD80 polypeptide containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant CD80 polypeptide contains one or more amino acid modifications at one or more positions of unmodified CD80 or a specific binding fragment thereof that correspond to positions 7, 23, 26, 30, 34, 35, 46, 51, 55, 57, 58, 65, 71, 73, 78, 79, 82, and / or 84 based on the numbering of SEQ ID NO:2. In some embodiments, the one or more amino acid modifications of unmodified CD80 or a specific binding fragment thereof correspond to positions 26, 35, 46, 57, and / or 71 based on the numbering of SEQ ID NO:2. In any embodiment, the amino acid modification is an amino acid substitution, insertion, or deletion.

[0006] In some embodiments, the variant CD80 polypeptide contains one or more amino acid substitutions selected from among E7D, E23D, E23G, A26E, A26P, A26S, A26T, I30F, I30T, I30V, K34E, E35D, E35G, D46E, D46V, P51A, N55D, N55I, T57A, T57I, I58V, L65P, A71D, A71G, R73H, R73S, G78A, T79A, T79I, T79L, T79M, T79P, C82R, V84A, and V84I at one or more positions of unmodified CD80 or a specific binding fragment thereof, wherein the position(s) of the amino acid modifications correspond to the numbering of the CD80 positions as set forth in SEQ ID NO:2.

[0007] In some embodiments, the variant CD80 polypeptides provided contain one or more additional modifications at one or more positions corresponding to positions 7, 12, 13, 15, 16, 18, 20, 22, 23, 24, 25, 26, 27, 30, 31, 33, 34, 35, 36, 38, 41, 42, 43, 44, 46, 47, 48, 51, 54, 55, 57, 58, 61, 62, 65, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, and / or 97 based on the numbering of SEQ ID NO:2. In some embodiments, further modifications include E7D, T13A, T13R, S15P, S15T, C16R, V20A, V20I, V22D, V22I, V22L, E23D, E23G, E24D, L25S, A26E, A26P, A26S, A26T, Q27H, Q27L, I30F, I30T, I30V, Y31S, Q33E, Q33K, Q33 L, Q33R, K34E, E35D, E35G, K36R, T41S, M42I, M42V, M43L, M43T, D46E, D46V, M47I, M47L, M47V, N4 8H, N48D, N48H, N48K, N48R, N48S, N48T, N48Y, P51A, Y53F, K54E, K54N, K54R, N55D, N55I, T57A, T5 7I, I58V, I61F, I61V, T62A, T62N, L65P, I67L, I67V, V68E, V68L, I69F, L70M, L70P, L70Q, A71D, A 71G, L72V, R73H, R73S, P74S, D76H, E77A, G78A, T79A, T79I, T79L, T79M, T79P, E81G, E81K, C82R, and one or more amino acid substitutions in CD80 or a specific binding fragment thereof selected from among V84A, V84I, L85E, L85M, L85Q, K86M, Y87C, Y87D, Y87H, E88V, F92S, F92V, R94Q, R94W, E95D, E95V, L97M, and L97Q, wherein the position(s) of the amino acid substitutions correspond to the numbering of the CD80 positions as set forth in SEQ ID NO:2.

[0008] In some embodiments, the one or more amino acid substitutions are TIFF2025076434000002.tif187166TIFF2025076434000003.tif231162TIFF2025076434000004.tif231164TIFF2025076434000005.tif231164TIFF2025076434000006.tif99165, wherein the position(s) of the amino acid substitution correspond to the position(s) of CD80 set forth in SEQ ID NO:2.

[0009] In some embodiments, provided herein is a variant CD80 polypeptide containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, which variant CD80 polypeptide is capable of binding to a non-modified CD80 or a specific binding fragment thereof, TIFF2025076434000007.tif63165, wherein the position(s) of the amino acid substitution(s) correspond to the numbering of the CD80 positions as set forth in SEQ ID NO:2.

[0010] In some embodiments, the variant CD80 polypeptides provided contain one or more additional modifications at one or more positions corresponding to positions 7, 12, 13, 15, 16, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 51, 53, 54, 55, 57, 58, 61, 62, 63, 65, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and / or 97 based on the numbering of SEQ ID NO:2. TIFF2025076434000008.tif92165, wherein the position(s) of the amino acid substitution(s) correspond to the numbering of the CD80 positions as set forth in SEQ ID NO:2.

[0011] In some embodiments, the one or more amino acid substitutions are TIFF2025076434000009.tif99163TIFF2025076434000010.tif231162TIFF2025076434000011.tif231162TIFF2025076434000012.tif231166TIFF2025076434000013.tif179163, wherein the position(s) of the amino acid substitution correspond to the position(s) of CD80 set forth in SEQ ID NO:2.

[0012] In some embodiments, the one or more amino acid substitutions are selected from among V20I, V22I, V22L, A26E, Q27H, Q33L, Q33R, E35D, E35G, T41S, M43L, D46E, D46V, M47I, M47L, M47V, N55D, T57I, I61V, L70M, A71D, A71G, L72V, and L85M, L85Q, R94W, and L97Q, and wherein the position(s) of the amino acid substitutions correspond to positions of CD80 as set forth in SEQ ID NO:2. In some embodiments, the one or more amino acid substitutions are selected from among V20I, V22L, A26E, Q27H, Q33L, Q33R, E35D, E35G, M47I, D46E, D46V, M47L, M47V, T57I, L70M, A71D, A71G, L72V, and L85M, L85Q, L97Q; or the one or more amino acid modifications are selected from among A26E, Q33L, E35D, M47I, M47L, M47V, T57I, L70M, A71D, A71G, and L85Q; or one or more or the one or more amino acid modifications comprise A26E; or the one or more amino acid modifications comprise E35D; or the one or more amino acid modifications comprise D46V; or the one or more amino acid modifications comprise M47L; or the one or more amino acid modifications comprise M47V; or the one or more amino acid modifications comprise A71G, wherein the position(s) of said amino acid substitutions correspond to the numbering of the CD80 positions in SEQ ID NO:2. In some embodiments, the one or more amino acid substitutions are selected from among A26E, Q33L, E35D, M47I, M47L, M47V, T57I, L70M, A71D, A71G, and L85Q, and the position(s) of the amino acid substitution correspond to a position of CD80 set forth in SEQ ID NO:2.

[0013] In some embodiments, provided herein is a variant CD80 polypeptide containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant CD80 polypeptide comprises an amino acid modification in unmodified CD80 or a specific binding fragment thereof, the amino acid modification being E35D / D46E, E35D / D46V, E35D / M47I, E35D / M47L, E35D / M47V, E35D / V68M, E35D / A71G or E35D / D90G; D46E / M47I, ... 46E / M47L, D46E / M47V, D46E / V68M, D46E / A71G or D46E / D90G; M47I / V68M, M47I / A71G, M48I / D90G; M47L / V68M, M47L / A71G, M47L / D90G; M47V / V68M, M47V / A71G, M47V / D90G; V68M / A71G or V68M / D90G; A71G / D90G; or E35D / M47I / V68M, E35D / M47L / V68M, E35D / M47V / V68M, wherein the position(s) of the amino acid modification(s) correspond to the numbering of the positions of CD80 as set forth in SEQ ID NO:2.

[0014] In some embodiments, provided herein is a variant CD80 polypeptide containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant CD80 polypeptide comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions contain at least the amino acid substitution L70P but not the amino acid substitutions V68M, L72P and / or K86E, and wherein the position(s) of the amino acid substitutions correspond to a position of CD80 set forth in SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions are selected from L70P, I30F / L70P, I30V / T57I / L70P / A71D / A91T, N55D / L70P / E77G, and L70P / F92S.

[0015] In some embodiments, the unmodified CD80 is a mammalian CD80. In some embodiments, the CD80 is human CD80.

[0016] In some embodiments, the variant CD80 polypeptide contains an IgV domain, or a specific binding fragment thereof, and an IgC domain, or a specific binding fragment thereof.

[0017] In some embodiments, the unmodified CD80 (i) contains a sequence of amino acids set forth in SEQ ID NO:2, (ii) contains a sequence of amino acids having at least 95% sequence identity to SEQ ID NO:2, or (iii) is an IgV domain or an IgC domain or a portion thereof that contains a specific binding fragment thereof. In some embodiments, the IgV domain or specific binding fragment of the 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 a length that is at least 80% of the length of the IgV domain set forth as amino acids 35-135, 35-138, 37-138 or 35-141 of SEQ ID NO:1, or the specific binding fragment of the IgC domain contains a length that is at least 80% of the length of the IgC domain set forth as amino acids 145-230, 154-232 or 142-232 of SEQ ID NO:1.

[0018] In some embodiments, the variant CD80 polypeptide 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 embodiments, the variant CD80 polypeptide contains a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, or a specific binding fragment thereof.

[0019] In some embodiments, the variant CD80 polypeptides exhibit altered binding to the ectodomain of CTLA-4, PD-L1 and / or CD28 compared to the binding of unmodified CD80 to the ectodomain of CTLA-4, PD-L1 and / or CD28, hi some embodiments, the altered binding is altered binding affinity and / or altered binding selectivity.

[0020] In some embodiments, the variant CD80 polypeptide contains an IgV domain, or a specific fragment thereof, and an IgC domain, or a specific fragment thereof. In some embodiments, the variant CD80 polypeptide contains or consists of a sequence of amino acids set forth in any of SEQ ID NOs:3-75, 2009-2104, 2297-2507, and 2930-2960, or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs:3-75, 2009-2104, 2297-2507, and 2930-2960 and contains one or more amino acid substitutions, or a specific binding fragment thereof.

[0021] In some embodiments, the variant CD80 polypeptide contains an IgV domain or a specific binding fragment thereof, hi some embodiments, the IgV domain or a specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide.

[0022] In some embodiments, the variant CD80 polypeptide contains a sequence of amino acids set forth in any of SEQ ID NOs:77-149, 151-223, 2105-2296, 2508-2929, and 2961-3022, or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs:77-149, 151-223, 2105-2296, 2508-2929, and 2961-3022, or a specific binding fragment thereof, and contains one or more amino acid substitutions.

[0023] In some embodiments, the IgC domain or a specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide.

[0024] In some embodiments, the variant CD80 exhibits altered binding affinity and / or altered binding selectivity for the ectodomain of CTLA4, PD-L1 or CD28 compared to the binding specificity of unmodified CD80 to the ectodomain of CTLA4, PD-L1 or CD28. In some embodiments, the CTLA-4 is human CTLA-4. In some embodiments, the CD28 is human CD28. In some embodiments, the PD-L1 is human PD-L1.

[0025] In some embodiments, the variant CD80 exhibits increased binding affinity to the ectodomain of CTLA4 compared to the binding affinity of unmodified CD80 to the ectodomain of CTLA4, in some embodiments, the increased affinity to the ectodomain of CTLA-4 is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold increase compared to the binding affinity of unmodified CD80 to the ectodomain of CTLA-4.

[0026] In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications to unmodified CD80 or a specific binding fragment thereof corresponding to positions 7, 23, 26, 30, 35, 46, 57, 58, 71, 73, 79 and / or 84 based on the numbering of SEQ ID NO:2. In some embodiments, the variant CD80 polypeptide is a variant of unmodified CD80 or a specific binding fragment thereof, comprising any of the following: E7D, T13A, T13R, S15T, C16R, V20I, V22D, V22L, E23D, E23G, E24D, A26E, A26P, A26S, A26T, Q27H, Q27L, I30V, Q33L, Q33R, E35D, E35G, T41S, M42V, M43L, M43T, D46E, D46V, M47I, M47L, M47V, N48D, N48H, N48K, N48R, N48S, N48T ... and L97Q, wherein the position(s) of the amino acid substitutions correspond to those of CD80 as set forth in SEQ ID NO:2.

[0027] In any of the embodiments provided, the CD80 polypeptide can exhibit increased binding affinity to the ectodomain of CD28 compared to the binding affinity of unmodified CD80 to the ectodomain of CD28. In some of such embodiments, the increased affinity to the ectodomain of CD28 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, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold compared to the binding affinity of unmodified CD80 to the ectodomain of CD28. In some of such embodiments, the CD80 polypeptide contains one or more amino acid modifications in unmodified CD80 or a specific binding fragment thereof corresponding to positions 23, 26, 35, 46, 55, 57, 58, 71, 79, and / or 84 based on the numbering of SEQ ID NO:2. In some such embodiments, the CD80 polypeptide comprises one or more of the following: T13R, S15T, V20I, V22D, V22L, E23D, E23G, E24D, A26E, A26P, A26S, A26T, Q27H, Q27L, Q33R, E35D, E35G, T41S, M42V, M43L, D46E, D46V, M47I, M47L, M47V, N48K, N48Y, Y53F, K5 and one or more amino acid modifications selected from among 4E, N55I, T57A, T57I, I58V, I61F, I61V, T62A, T62N, I67L, V68E, V68L, I69F, L70M, A71D, A71G, L72V, T79I, T79M, V84I, L85M, L85Q, Y87C, Y87D, E88V, R94Q, R94W, E95V, and L97Q, wherein the position(s) of the amino acid substitutions correspond to the positions of CD80 as set forth in SEQ ID NO:2.

[0028] In some embodiments, the variant CD80 polypeptides exhibit increased binding affinity for the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1. In some such embodiments, the increased affinity for the ectodomain of PD-L1 is an increase 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, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, or 450-fold compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1. In some of such embodiments, the CD80 polypeptide contains one or more amino acid modifications corresponding to positions 7, 23, 26, 30, 34, 35, 46, 51, 55, 57, 58, 65, 71, 73, 78, 79, 82, and / or 84 of unmodified CD80 or a specific binding fragment thereof based on the numbering of SEQ ID NO:2.In some embodiments, the CD80 polypeptide is selected from the group consisting of unmodified CD80 or a specific binding fragment thereof, E7D, T13A, T13R, S15T, C16R, V20A, V20I, V22D, V22I, V22L, E23D, E23G, E24D, L25S, A26E, A26P, A26S, A26T, Q27H, Q27L, I30T, I30V, Q33E, Q33K, Q33L, Q33R, K34E, E35D, K36R, T41S, M42I, M42V, M43L, M43T, D46E, D46V, M47I, M47L, M47V, N48D, N48H, N48K, N48R, N48S, N48T, N48Y, P51A, Y53F, K54R, N55D, N55I, T57I, I58V, I61F, I61V, T62A, T62N, L65P, I67L, V68L, I69F , L70M, A71D, A71G, L72V, R73S, P74S, D76H, G78A, T79A, T79I, T79L, T79M, T79P, E81G , E81K, C82R, V84A, V84I, L85E, L85M, L85Q, K86M, Y87C, Y87D, F92S, F92V, R94Q, R94W, E95D, E95V, L97M, and L97Q, wherein the position(s) of the amino acid substitutions correspond to the positions of CD80 as set forth in SEQ ID NO:2.

[0029] In some embodiments, the variant CD80 exhibits a reduced binding affinity for the ectodomain of CD28 compared to the binding affinity of unmodified CD80 to the ectodomain of CD28. In some embodiments, the reduced affinity for the ectodomain of CD28 is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to the binding affinity of unmodified CD80 to the ectodomain of CD28.

[0030] In some embodiments, the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with increased selectivity as compared to unmodified CD80 for the ectodomain of CTLA-4. In some such embodiments, the increased selectivity comprises a greater binding ratio of the variant polypeptide to CTLA-4 to CD28 as compared to the binding ratio of the unmodified CD80 polypeptide to CTLA-4 to CD28. In some such embodiments, the CTLA-4 to CD28 binding ratio is at least 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, or at least about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold or more.

[0031] In any of the embodiments provided, the variant CD80 polypeptide specifically binds to the ectodomain of PD-L1 with increased selectivity compared to the unmodified CD80 of the ectodomain of PD-L1. In some such embodiments, the increased selectivity comprises a greater binding ratio of the variant polypeptide to PD-L1 to CD28 compared to the binding ratio of the unmodified CD80 polypeptide to PD-L1 to CD28. In some such embodiments, the ratio is at least 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, or at least about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold or more.

[0032] In some embodiments, the variant CD80 polypeptide is a soluble protein. In some embodiments, the variant CD80 polypeptide is linked to a multimerization domain. In some embodiments, the variant CD80 polypeptide is a multimeric, optionally a dimeric, polypeptide containing a first variant CD80 polypeptide linked to a multimerization domain and a second variant CD80 polypeptide linked to a multimerization domain. In some embodiments, the first variant CD80 polypeptide and the second variant CD80 polypeptide are identical. In some embodiments, the first variant CD80 polypeptide and the second variant CD80 polypeptide are different.

[0033] In some embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the variant CD80 polypeptide is linked to a moiety that extends the biological half-life of the polypeptide. In some embodiments, the variant CD80 polypeptide is linked to an Fc domain or a variant thereof with reduced effector function.

[0034] In some embodiments, the Fc domain is mammalian, optionally human; or the variant Fc domain contains one or more amino acid modifications compared to an unmodified Fc domain, which is mammalian, optionally human. In some embodiments, the Fc domain or variant thereof contains an amino acid sequence set forth in SEQ ID NO:277, SEQ ID NO:359, or SEQ ID NO:1712, or an amino acid sequence exhibiting at least 85% sequence identity to SEQ ID NO:277, SEQ ID NO:359, or SEQ ID NO:1712. In some embodiments, the Fc domain contains one or more amino acid modifications selected from among E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G,V302C, and K447del (each according to EU numbering). In some embodiments, the Fc region is not a human IgG1 Fc containing the mutations R292C, N297G, and V302C (corresponding to R77C, N82G, and V87C based on the wild type human IgG1 Fc set forth in SEQ ID NO:277). In some embodiments, the Fc is not an Fc set forth in SEQ ID NO:356. In some embodiments, the Fc domain contains the amino acid modification C220S (according to EU numbering). In some embodiments, the Fc domain contains an amino acid sequence set forth in any of SEQ ID NOs:356-358, 376, and 1713-1715, or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOs:356-358, 376, and 1713-1715 and exhibits reduced effector function. In some embodiments, the variant CD80 polypeptide is 4 It is indirectly linked to the multimerization domain or Fc via an S linker (SEQ ID NO:1716). In some embodiments, the linker is not composed of three alanines (AAA).

[0035] In some embodiments, provided herein is an immunomodulatory protein comprising any of the variant CD80 polypeptides provided herein and a half-life extending moiety. In some embodiments, the half-life extending moiety comprises a multimerization domain, albumin, an albumin binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin binding small molecule, or a combination thereof. In some embodiments, the half-life extending moiety is or comprises Pro / Ala / Ser (PAS) and the variant CD80 polypeptide is PASylated. In some embodiments, the half-life extending moiety is or contains a multimerization domain. In some embodiments, the multimerization domain is selected from an immunoglobulin Fc region, a leucine zipper, an isoleucine zipper, or a zinc finger.

[0036] In some embodiments, the immunomodulatory protein is a multimer containing a first variant CD80 polypeptide linked to a first multimerization domain and a second variant CD80 polypeptide linked to a second multimerization domain, and the first and second multimerization domains interact to form a multimer containing the first and second variant CD80 polypeptides. In some embodiments, the multimer is a dimer. In some embodiments, the first variant CD80 polypeptide and the second variant CD80 polypeptide are identical. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0037] In some embodiments, the multimerization domain is or contains an Fc region of an immunoglobulin. In some embodiments, the Fc region is of an immunoglobulin G1 (IgG1) or Immunoglobulin G2 (IgG2) protein. In some embodiments, the immunoglobulin protein is human and / or the Fc region is human. In some embodiments, the Fc region contains an amino acid sequence set forth in SEQ ID NO:278, or a variant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:278. In some embodiments, the Fc region contains an amino acid sequence set forth in SEQ ID NO:277, or a variant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:277.

[0038] In some embodiments, the Fc region exhibits one or more effector functions. In some embodiments, the immunomodulatory protein exhibits Fc-dependent CD28 costimulation in a T cell stimulation assay, optionally in the presence of antigen presenting cells, and optionally the T cells comprise Jurkat cells expressing an IL-2 reporter. In some embodiments, the Fc region exhibits one or more reduced effector functions compared to a wild-type Fc region, and optionally the wild-type Fc region is a human Fc of human IgG1. In some embodiments, the one or more effector functions are selected from among antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity, programmed cell death, and cellular phagocytosis.

[0039] In some embodiments, the Fc region is a variant Fc region that contains one or more amino acid substitutions compared to the wild-type Fc region.In some of these embodiments, the one or more amino acid substitutions of the variant Fc region are selected from Fc N297G, R292C / N297G / V302C, E233P / L234V / L235A / G236del / S267K or L234A / L235E / G237A, the residues being numbered according to the EU index of Kabat.In some embodiments, the variant Fc region further comprises the amino acid substitution C220S, the residues being numbered according to the EU index of Kabat. In some embodiments, the Fc region contains a sequence of amino acids set forth in any of SEQ ID NOs:356-358, or a sequence of amino acids that exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:356-358, and contains an amino acid substitution. In some embodiments, the Fc region comprises K447del, the residues numbered according to the EU index of Kabat. In some embodiments, the Fc region contains a sequence of amino acids set forth in any of SEQ ID NOs:1713-1715, or a sequence of amino acids that exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:1713-1715, and contains an amino acid substitution.

[0040] In some embodiments, the immunomodulatory protein comprises any of the variant CD80 polypeptides provided herein that exhibit increased affinity for PD-L1.

[0041] In some embodiments, the immunomodulatory protein exhibits PD-L1-dependent CD28 costimulation in a T cell stimulation assay, optionally in the presence of antigen presenting cells expressing PD-L1, and optionally the T cells include Jurkat cells expressing an IL-2 reporter or primary human T cells producing inflammatory cytokines such as IL-2.

[0042] In some embodiments of the immunomodulatory protein, the variant CD80 polypeptide is linked to the multimerization domain directly or indirectly via a linker. In some such embodiments, the linker contains 1-10 amino acids. In some embodiments, the linker is AAA, G4S (SEQ ID NO:1717) or (G 4 S) 2 (SEQ ID NO:330).

[0043] In some embodiments, the variant CD80 polypeptide is a transmembrane immunomodulatory protein further comprising a transmembrane domain linked to the extracellular domain (ECD) or specific binding fragment thereof of the variant CD80 polypeptide. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth as residues 243-263 of SEQ ID NO:1, or a functional variant thereof that exhibits at least 85% sequence identity to residues 243-263 of SEQ ID NO:1. In some embodiments, the variant CD80 polypeptide further comprises a cytoplasmic signaling domain linked to the transmembrane domain. In some embodiments, the cytoplasmic signaling domain comprises the amino acid sequence set forth as residues 264-288 of SEQ ID NO:1, or a functional variant thereof that exhibits at least 85% sequence identity to residues 254-288 of SEQ ID NO:1.

[0044] In some of the embodiments provided, the variant CD80 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 embodiments, the variant CD80 increases IFN-γ (interferon-gamma) expression compared to unmodified CD80 in an in vitro primary T cell assay. In some embodiments, the variant CD80 decreases IFN-γ (interferon-gamma) expression compared to unmodified CD80 in an in vitro primary T cell assay. In some embodiments of any one of the variant CD80 polypeptides described herein, the variant CD80 polypeptide increases T cell signaling compared to unmodified CD80, as determined using a reporter assay comprising T cells (e.g., Jurkat) engineered with a reporter (e.g., luciferase) operably linked to an IL-2 promoter. In some embodiments of any one of the variant CD80 polypeptides described herein, the variant CD80 polypeptide reduces T cell signaling compared to unmodified CD80, as determined using a reporter assay comprising T cells (e.g., Jurkat) engineered with a reporter (e.g., luciferase) operably linked to an IL-2 promoter. In some of any such embodiments, the variant CD80 polypeptide is provided in any of a wide variety of formats, such as soluble or immobilized (e.g., plate-bound).

[0045] In some embodiments, the variant CD80 polypeptide is aglycosylated.

[0046] In some embodiments, provided herein are immunomodulatory proteins containing any of the provided variant CD80 polypeptides linked to a second polypeptide containing an immunoglobulin superfamily (IgSF) domain. In some embodiments, the IgSF domain is affinity modified and exhibits altered binding to one or more of its cognate binding partner(s) compared to an unmodified or wild-type IgSF domain. In some embodiments, the IgSF domain exhibits increased binding to one or more of its cognate binding partner(s) compared to an unmodified or wild-type IgSF domain.

[0047] In some embodiments, the variant CD80 is a first CD80 variant polypeptide and the IgSF domain of the second polypeptide is an IgSF domain derived from a second variant CD80 polypeptide that is any of the variant CD80 polypeptides provided herein, and the first and second CD80 variants are the same or different. In some embodiments, the variant CD80 polypeptide is capable of specifically binding to CTLA-4 and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than that specifically bound by the CD80 variant polypeptide. In some embodiments, the IgSF domain of the second polypeptide is a tumor-localizing moiety that binds to a ligand expressed on tumors. In some embodiments, the ligand expressed on tumors is B7H6.

[0048] In some embodiments, the IgSF domain is from NKp30.

[0049] In some embodiments, the IgSF domain of the second polypeptide is an IgSF domain of a ligand that binds to an inhibitory receptor, or an affinity-modified IgSF domain thereof. In some embodiments, the affinity-modified IgSF domain exhibits increased binding affinity and / or binding selectivity to the inhibitory receptor compared to the binding of the unmodified IgSF domain to the inhibitory receptor. In some embodiments, the inhibitory receptor is TIGIT or PD-1; or the ligand of the inhibitory receptor is CD155, CD112, PD-L1 or PD-L2.

[0050] In some embodiments, the IgSF domain of the second polypeptide is an affinity engineered IgSF domain comprising: (i) a wild type CD112 containing an IgSF domain as set forth in any of SEQ ID NOs: 269, 734 or 829, or a variant CD112 polypeptide comprising an IgSF domain as set forth in any of SEQ ID NOs: in Table 3, optionally any of SEQ ID NOs: 735-828, 830-917, 918-999, 1430-1501; (ii) a wild type CD155 containing an IgSF domain as set forth in any of SEQ ID NOs: 268, 378 or 421, or a variant CD155 polypeptide comprising an IgSF domain as set forth in any of SEQ ID NOs: in Table 4, optionally any of SEQ ID NOs: 379-420, 422-539, 540-733, 1502-1573, 1548-1711; (iv) a variant PD-L1 polypeptide comprising an IgSF domain as set forth in any of SEQ ID NOs: 252, 1197 or 1257, or any of SEQ ID NOs: set forth in Table 6, or optionally any of SEQ ID NOs: 1198-1248, 1250-1325, 1327-1401, 1403-1426, 1719, 1720, 1901-1930; (v) a variant PD-L2 polypeptide comprising an IgSF domain as set forth in any of SEQ ID NOs: (i)-(iv) or (vi) an amino acid sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity to any of NO: and contains amino acid substitutions; or (vi) a specific binding fragment of any of (i)-(v). In some embodiments, the IgSF domain is or contains an IgV domain. In some embodiments, the variant CD80 polypeptide is or contains an IgV domain.

[0051] In some embodiments, the immunomodulatory protein contains a variant CD80 polypeptide and a multimerization domain linked to one or both of the IgSF domains of a second polypeptide. In some embodiments, the multimerization domain is an Fc domain or a variant thereof having 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.

[0052] In some embodiments, provided herein is a conjugate comprising an immune modulating polypeptide provided herein linked to a variant CD80 polypeptide or moiety provided herein. In some embodiments, the moiety is a targeting moiety that specifically binds to a molecule on a cell surface. In some embodiments, the targeting moiety specifically binds to a molecule on an immune cell surface, optionally the immune cell is an antigen presenting cell or a lymphocyte. In some embodiments, the immune cell is an antigen presenting cell or a lymphocyte. In some embodiments, the targeting moiety is a tumor localizing moiety that binds to a molecule on a tumor surface. In some embodiments, the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle. In some embodiments, the moiety is an antibody or antigen binding fragment. In some embodiments, the conjugate is bivalent, tetravalent, hexavalent, or octavalent. Exemplary depictions of such conjugates are provided in Figures 6A and 6B. In some embodiments, the conjugate is a fusion protein.

[0053] In some embodiments, provided herein is a nucleic acid molecule(s) encoding a conjugate that is a fusion protein containing any of the variant CD80 polypeptides provided herein, the immunomodulatory polypeptides provided herein, or the variant CD80 polypeptides provided herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is a cDNA.

[0054] In some embodiments, provided herein is a vector containing the nucleic acid molecule provided herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector or a viral vector.

[0055] In some embodiments, provided herein is a cell containing a vector provided herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0056] In some embodiments, provided herein are methods of producing a variant CD80 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule provided herein or a vector provided herein into a host cell under conditions to express the protein in the cell. In some embodiments, the method further comprises isolating or purifying the variant CD80 polypeptide or immunomodulatory protein from the cell.

[0057] In some embodiments, provided herein are methods of engineering a cell to express a variant CD80 variant polypeptide comprising introducing into a host cell a nucleic acid molecule encoding a variant CD80 polypeptide provided herein under conditions whereby the polypeptide is expressed in the cell.

[0058] In some embodiments, provided herein are engineered cells that express a variant CD80 polypeptide provided herein, an immunomodulatory protein provided herein, a conjugate provided herein, a nucleic acid molecule provided herein, or a vector provided herein.

[0059] In some embodiments, the variant CD80 polypeptide or immunomodulatory protein contains a signal peptide. In some embodiments, the variant CD80 polypeptide or immunomodulatory protein does not contain a transmembrane domain and / or is not expressed on the surface of the cell. In some embodiments, the variant CD80 polypeptide or immunomodulatory protein is secreted from the engineered cell.

[0060] In some embodiments, the engineered cell contains a variant CD80 polypeptide that contains a transmembrane domain and / or is a transmembrane immunomodulatory protein provided herein. In some embodiments, the variant CD80 polypeptide is expressed on the surface of the cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the immune cell is an antigen presenting cell (APC) or a lymphocyte.

[0061] In some embodiments, the engineered cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a lymphocyte that is a T cell. In some embodiments, the cell is an APC that is an artificial APC. In some embodiments, the engineered cell further contains a chimeric antigen receptor (CAR) or an engineered T cell receptor.

[0062] In some embodiments, provided herein is an infectious agent containing a nucleic acid molecule encoding a variant CD80 polypeptide provided herein, an immunomodulatory polypeptide provided herein, or a variant CD80 fusion conjugate provided herein. In some embodiments, the encoded variant CD80 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 CD80 polypeptide, immunomodulatory polypeptide, or conjugate is secreted from the cell in which it is expressed.

[0063] In some embodiments, the encoded variant CD80 polypeptide contained within the infectious agent contains a transmembrane domain, hi some embodiments, the encoded variant CD80 polypeptide is expressed on the surface of the cell in which it is expressed.

[0064] In some embodiments, the infectious agent is a bacterium or a virus. In some embodiments, the virus is a lentivirus or a retrovirus construct or a hybrid thereof. In some embodiments, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is an adenovirus, an adeno-associated virus, a herpes virus, a herpes simplex virus, a reovirus, a Newcastle disease virus, a parvovirus, a measles virus, a vesicular stomatitis virus (VSV), a coxsackie virus or a vaccinia virus.

[0065] In some embodiments, the infectious agent is a virus that specifically targets dendritic cells (DCs) and / or is a dendritic cell-tropic virus, hi some embodiments, the virus is a lentiviral vector pseudotyped with a modified Sindbis virus envelope product.

[0066] In some embodiments, the infectious agent further contains a nucleic acid molecule encoding an additional gene product that can cause death of a target cell or enhance or potentiate an immune response, in some embodiments, the additional gene product is selected from anti-cancer agents, anti-metastatic agents, anti-angiogenic agents, immunomodulatory molecules, immune checkpoint inhibitors, antibodies, cytokines, growth factors, antigens, cytotoxic gene products, pro-apoptotic gene products, anti-apoptotic gene products, cell matrix degrading genes, tissue regeneration, and genes for reprogramming human somatic cells to pluripotency.

[0067] In some embodiments, provided herein is a pharmaceutical composition comprising a variant CD80 polypeptide provided herein, an immunomodulatory protein provided herein, a conjugate provided herein, an engineered cell provided herein, or an infectious agent provided herein. In some embodiments, the pharmaceutical composition comprises a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0068] In some embodiments, provided herein is an article of manufacture containing a pharmaceutical composition provided herein in a vial. In some embodiments, the vial is sealed.

[0069] In some embodiments, provided herein are kits containing the pharmaceutical compositions provided herein and instructions for use. In some embodiments, provided herein are kits containing the articles of manufacture provided herein and instructions for use.

[0070] In some embodiments, provided herein are methods of modulating an immune response in a subject, such as increasing or decreasing the immune response, comprising administering to the subject a pharmaceutical composition provided herein.

[0071] In some embodiments, provided herein is a method of modulating an immune response in a subject, comprising administering an immunomodulating protein provided herein, such as an immunomodulating protein that exhibits increased binding affinity to PD-L1. In some embodiments, the immune response is increased. In some embodiments, the immunomodulating protein is an immunomodulating protein provided herein that exhibits Fc-dependent CD28 costimulation. In some embodiments, the immunomodulating protein is an immunomodulating protein provided herein that exhibits PD-L1-dependent CD28 costimulation, and optionally the immunomodulating protein contains a variant CD80 polypeptide provided herein.

[0072] In some embodiments, provided herein is a method of modulating an immune response in a subject, comprising administering an engineered cell provided herein. In some embodiments, the engineered cell is autologous to the subject. In some embodiments, the engineered cell is allogeneic to the subject.

[0073] Also provided herein are methods of modulating an immune response in a subject, e.g., increasing or decreasing an immune response, comprising administering to a subject a variant CD80 polypeptide, or an immunomodulatory protein or conjugate containing a variant CD80 polypeptide, or an engineered cell, or an infectious agent that secretes or expresses a variant CD80 polypeptide, wherein the variant CD80 polypeptide binds to CTLA-4 with increased affinity or selectivity compared to the binding of unmodified CD80 to CTLA-4. In some embodiments, the variant CTLA-4 polypeptide contains one or more modifications described herein.

[0074] In some embodiments of this method, the variant CTLA-4 polypeptide contains one or more amino acid modifications at one or more positions of an unmodified CD80 polypeptide or a specific binding fragment thereof corresponding to a position selected from 12, 13, 16, 18, 20, 22, 23, 24, 25, 26, 27, 30, 31, 33, 34, 35, 36, 38, 41, 42, 43, 44, 46, 47, 48, 51, 54, 55, 57, 58, 61, 62, 65, 67, 68, 69, 70, 71, 72, 73, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 88, 90, 91, 92, 93, 94, and / or 95, based on the positions set forth in SEQ ID NO:2. In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications at one or more positions of unmodified CD80 or a specific binding fragment thereof corresponding to positions 23, 26, 30, 34, 35, 46, 51, 55, 57, 58, 65, 71, 73, 78, 79, 82 or 84 based on the numbering of SEQ ID NO:2.

[0075] In some embodiments, the one or more modifications are A12T, A12V, T13R, C16R, H18Y, V20I, V22I, V22L, E23D, E23G, E24D, L25S, A26E, A26P, A26S, A26T, Q27H, Q27L, 30F, I30T, I30V, Y31H, Q33E, K34E, E35D, E35G, K36R, M38V, T41A, T41S, M42I, M42V, M43I, M43L, S44P, D46E, D46V, M47I, M47L, M47T, M47V, N48H, P51A, K5 4E, N55D, N55I, T57A, T57I, I58V, I61V, T62N, L65P, I67L, V68A, V68L, V68M, I69F, I69T, L70M, L70P, L70Q, L70R, A71D, A71G, L72P, L72V, R73S, D76H, E77G, G78A, T79I, T79P, E81K, C82R, V83I, V84A, V84I, L85M, L85Q, K86M, E88D, E88V, D90N, A91S, A91T, F92S, K93R, R94W, E95K and E95V. In some embodiments, the variant CD80 polypeptide comprises one or more amino acid substitutions in unmodified CD80 or a specific binding fragment thereof selected from among E23D, E23G, A26E, A26P, A26S, A26T, I30F, I30T, I30V, K34E, E35D, E35G, D46E, D46V, P51A, N55D, N55I, T57A, T57I, I58V, L65P, A71D, A71G, R73S, G78A, T79I, T79P, C82R, V84A, V84I, wherein the position(s) of the amino acid substitutions correspond to the numbering of the CD80 positions as set forth in SEQ ID NO:2.

[0076] In some embodiments, the one or more modifications include: Selected from TIFF2025076434000014.tif128166.

[0077] In some embodiments, the method comprises administering to a subject a soluble variant CD80 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 a subject an infectious agent encoding a variant CD80 polypeptide according to any one of the embodiments described herein.

[0078] In some embodiments, the disease or condition of the subject is treated by modulating immune response. In some embodiments, the immune response is increased. Various formats of variant CD80 polypeptides are contemplated for administration to the subject to increase immune response, such as antagonistic formats of variant CD80. In some cases, such methods are performed under conditions in which signal transduction by inhibitory receptor CTLA-4 is blocked or attenuated by administration.

[0079] In some embodiments, methods of modulating an immune response are provided in which a soluble variant CD80 polypeptide or immunomodulatory protein is administered to a subject. In some embodiments, the soluble immunomodulatory protein is an immunomodulatory Fc fusion protein.

[0080] In some embodiments, the methods provided include administering to a subject a variant CD80 polypeptide provided herein, or an immunomodulatory protein provided herein. In some embodiments, engineered cells containing a secretable variant CD80 polypeptide provided herein are administered to the subject. In some embodiments, engineered cells provided herein are administered to the subject.

[0081] In some embodiments of the provided methods, an infectious agent encoding a variant CD80 polypeptide that is a secretable immunomodulatory protein is administered to a subject, optionally under conditions where the infectious agent infects tumor cells or immune cells and the secretable immunomodulatory protein is secreted from the infected cells.

[0082] In some embodiments of the provided methods, the disease or condition is a tumor or cancer. In some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colon cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer. In some of any such embodiments, the variant CD80 is administered in a manner that increases the immune response of the subject.

[0083] Various formats of variant CD80 polypeptides are contemplated for administration to a subject to reduce an immune response, including agonistic formats of variant CD80. In some cases, such methods are performed under conditions in which signaling through the inhibitory receptor CTLA-4 is activated or stimulated or induced by administration.

[0084] In some embodiments of the provided methods, the immune response is reduced. In some embodiments of the provided methods, an immunomodulatory protein or conjugate containing a variant CD80 polypeptide linked to a moiety that localizes to cells or tissues in an inflammatory environment is administered to a subject. In some embodiments, the binding molecule contains an antibody or an antigen-binding fragment thereof, or contains a second polypeptide containing a wild-type IgSF domain or a variant thereof.

[0085] In some embodiments of the provided method, the immunomodulatory protein provided herein or the conjugate provided herein is administered to the subject.In some embodiments of the provided method, a variant CD80 polypeptide that is a transmembrane immunomodulatory protein is administered to the subject.In some embodiments of the provided method, an engineered cell that contains a variant CD80 polypeptide that is a transmembrane immunomodulatory protein provided herein is administered to the subject.

[0086] In some embodiments of the provided methods, an infectious agent encoding a variant CD80 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject, optionally under conditions where the infectious agent infects tumor cells or immune cells and the transmembrane immunomodulatory protein is expressed on the surface of the infected cells.

[0087] In some embodiments of the provided method, the disease or condition is an inflammatory or autoimmune disease or condition. In some embodiments, the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. In some embodiments, the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. In some of any such embodiments, the variant CD80 is administered in a manner that reduces the immune response of the subject.

[0088] In some embodiments, provided herein is a method of treating a disease or condition comprising administering an immunomodulatory protein comprising a variant CD80 polypeptide containing one or more amino acid modifications at one or more positions in the IgV domain or IgC domain or specific binding fragment of unmodified CD80 or a specific binding fragment thereof, and exhibiting PD-L1-dependent CD28 costimulation. In some embodiments, the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1. In some embodiments, provided herein is a method of mediating CD28 agonism through PD-L1-dependent CD28 costimulation in a subject, the method comprising administering an immunomodulatory protein comprising a variant CD80 polypeptide, the variant CD80 polypeptide containing one or more amino acid modifications at one or more positions in the IgV domain or IgC domain or specific binding fragment of unmodified CD80 or a specific binding fragment thereof, and the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1. In any embodiment, the increased affinity for the ectodomain of PD-L1 is an increase 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, 50-fold, or 60-fold compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

[0089] In some embodiments, the method is for use in treating a disease or condition. In some embodiments, PD-L1-dependent CD28 costimulation is assessed in a T cell stimulation assay in the presence of antigen presenting cells expressing PD-L1, optionally the T cell stimulation assay is an in vitro assay, and optionally the T cells comprise Jurkat cells expressing an IL-2 reporter or primary human T cells that produce a proinflammatory cytokine such as IL-2.

[0090] In some embodiments of the provided methods, prior to administration, a subject is selected to have a tumor comprising cells that are positive for surface PD-L1, and optionally the cells are tumor cells or tumor-infiltrating immune cells; or the subject has been selected as having a tumor comprising cell surface positive for PD-L1, and optionally the cells are tumor cells or tumor-infiltrating immune cells.

[0091] In some embodiments, selecting the subject comprises: (a) contacting a tumor tissue sample from the subject with a binding reagent capable of specifically binding to the ectodomain of PD-L1; (b) detecting the presence of the binding reagent bound to or on cells of the tumor tissue sample, optionally where the cells are tumor cells or tumor-infiltrating immune cells; and (c) treating the subject if the tumor tissue sample contains detectable levels of cell surface positive for PD-L1.

[0092] In some embodiments of the provided methods, prior to administration, a subject is selected to be treated having a tumor comprising a cell surface positive for CD28, optionally the cells are tumor-infiltrating lymphocytes, optionally the lymphocytes are T cells, optionally CD8+ T cells; or the subject is selected as having a tumor comprising a cell surface positive for CD28, optionally the cells are tumor-infiltrating lymphocytes, optionally the lymphocytes are T cells, optionally CD8+ T cells.

[0093] In some embodiments, selecting the subject comprises: (a) contacting a tumor tissue sample from the subject with a binding reagent capable of specifically binding to the ectodomain of CD28; (b) detecting the presence of the binding reagent bound to or on cells of the tumor tissue sample, optionally where the cells are tumor-infiltrating lymphocytes, optionally where the lymphocytes are T cells, optionally where the lymphocytes are CD8+ T cells; and (c) selecting the subject for treatment if the tumor tissue sample contains detectable levels of cell surface positive for CD28.

[0094] In some embodiments, provided herein are methods of selecting a subject for treatment comprising: (a) contacting a tumor tissue sample from a subject with a binding reagent capable of specifically binding PD-L1; (b) detecting the presence of the binding reagent bound to or on cells of the tumor tissue sample, where optionally the cells are tumor cells or tumor infiltrating immune cells; and (c) if the tumor sample contains cell surface positive for detectable levels of PD-L1, selecting the subject for treatment with an immunomodulatory protein comprising a variant CD80 polypeptide, wherein the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or IgC domain, or a specific binding fragment thereof, of unmodified CD80, or a specific binding fragment thereof, and wherein the variant CD80 polypeptide exhibits increased binding affinity for the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

[0095] In some embodiments, the method further comprises contacting the tumor tissue sample with a binding reagent capable of specifically binding to CD28, and the subject is selected if the tumor tissue sample further comprises tumor-infiltrating lymphocytes positive for detectable levels of CD28, optionally the lymphocytes are T cells, optionally CD8+ T cells.

[0096] In some embodiments, the tumor tissue sample contains tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof.

[0097] In some embodiments, the binding reagent is an antibody or antigen-binding fragment, a protein ligand or binding partner, an aptamer, an affimer, a peptide, or a hapten. In some embodiments, the binding reagent is an anti-PD-L1 antibody or antigen-binding fragment. In some embodiments, the binding reagent is a variant CD80 polypeptide provided herein. In some embodiments, the variant CD80 polypeptide comprises an IgV domain or a specific binding fragment thereof. In some embodiments, the IgV domain or a specific binding fragment thereof is the only CD80 portion of the binding reagent.

[0098] In some embodiments, the variant CD80 polypeptides exhibit increased affinity for binding to PD-L1 compared to a wild-type or unmodified CD80 polypeptide.

[0099] In some embodiments, the binding reagent is directly or indirectly linked to a moiety that is a detectable moiety or a moiety that can be detected. In some embodiments, the moiety is an Fc region. In some embodiments, the Fc region is non-human, optionally mouse or rabbit.

[0100] In some embodiments, detecting the presence of bound binding reagent is by immunohistochemistry, pseudo-immunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting.

[0101] In some embodiments, the method further comprises administering an immunomodulatory protein to a subject. In some embodiments, the subject is a human subject.

[0102] In some embodiments, the immunomodulatory protein is a multimer comprising a first variant CD80 polypeptide linked to a first multimerization domain and a second variant CD80 polypeptide linked to a second multimerization domain, and the first and second multimerization domains interact to form a multimer comprising the first and second variant CD80 polypeptides. In some embodiments, the multimer is a dimer. In some embodiments, the first variant CD80 polypeptide and the second variant CD80 polypeptide are identical.

[0103] In some embodiments, the multimerization domain is or comprises an Fc region, optionally a variant Fc region containing one or more amino acid substitutions compared to a wild-type Fc region, and the Fc region exhibits one or more reduced effector functions compared to the wild-type Fc region, and optionally the wild-type Fc is human IgG1.

[0104] In some of such embodiments, the CD80 polypeptide contains one or more amino acid modifications corresponding to positions 7, 12, 13, 15, 16, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 51, 53, 54, 55, 57, 58, 61, 62, 63, 65, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and / or 97 based on the numbering of SEQ ID NO:2 in unmodified CD80 or a specific binding fragment thereof. In some such embodiments, the CD80 polypeptide is a polypeptide that is a nucleotide sequence ... TIFF2025076434000015.tif77165, wherein the position(s) of the amino acid substitution(s) correspond to the position(s) of CD80 set forth in SEQ ID NO:2.

[0105] In some embodiments, the variant CD80 polypeptide retains binding to CD28. In some embodiments, the variant CD80 polypeptide retains binding to CD28. In some embodiments, the variant CD80 polypeptide retains binding to CD28. %, 70%, 75%, 80%, 85%, 90% or 95%, or at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some embodiments, the variant CD80 polypeptide exhibits increased binding affinity for the ectodomain of CD28 compared to the binding affinity of unmodified CD80 to the ectodomain of CD28. In some of such embodiments, the increased affinity for the CD28 ectodomain 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 the binding affinity of unmodified CD80 to the CD28 ectodomain.

[0106] In some embodiments, the disease or condition of the subject is treated by increasing the immune response. In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colon cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer.

[0107] In some embodiments, provided herein are methods of detecting a CD80 binding partner in a biological sample comprising: (a) contacting the biological sample with a binding reagent comprising any of the variant CD80 polypeptides provided herein; and (b) detecting the presence of bound binding reagent in or on cells of the biological sample. In some embodiments, the binding partner is PD-L1, CD28, CTLA-4, or a combination thereof.

[0108] In some embodiments, the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or the IgC domain, or a specific binding fragment thereof, of unmodified CD80, or a specific binding fragment thereof, and the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

[0109] In some embodiments, the biological sample is or comprises a bodily fluid, cell or tissue sample, such as a bodily fluid that is serum, plasma or urine, or a tissue sample that is a tumor tissue sample, in some embodiments, the tumor tissue sample contains tumor infiltrating immune cells, tumor cells, stromal cells, or any combination thereof.

[0110] In some embodiments, the variant CD80 polypeptide comprises an IgV domain or a specific binding fragment thereof, hi some embodiments, the IgV domain or a specific binding fragment thereof is the only CD80 portion of the binding reagent.

[0111] In some embodiments, the binding reagent is directly or indirectly linked to a label or detectable moiety that is a detectable moiety. In some embodiments, the moiety is an Fc region, optionally non-human, such as a mouse or rabbit Fc region. In some embodiments, the detection of the presence of the bound binding reagent is by immunohistochemistry, pseudoimmunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting. [Brief description of the drawings]

[0112] [Figure 1A] Various formats of variant IgSF domain molecules are provided: (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 soluble molecule comprising a variant IgSF domain (vIgD) linked to an antibody (V-mAb). [Figure 1B] Various formats of variant IgSF domain molecules are provided. A transmembrane immunomodulatory protein (TIP) containing a variant IgSF domain (vIgD) expressed on the surface of a cell is shown. In an exemplary embodiment, the cognate binding partner of the transmembrane-bound vIgD is an inhibitory receptor (e.g., CTLA-4), and the TIP containing vIgD (e.g., CD80 vIgD) antagonizes or blocks the negative signaling of the inhibitory receptor, thereby obtaining activated T cells or effector T cells. In some cases, when the clustering of the inhibitory receptor (CTLA-4) is in close proximity to the activating receptor (e.g., CD28), the agonistic activity of the TIP can be achieved. [Figure 1C]Various formats of variant IgSF domain molecules are provided. A variant IgSF domain (vIgD) is a secreted immunomodulatory protein (SIP) secreted from a cell, such as a first T cell (e.g., a CAR T cell). In an exemplary embodiment, the cognate binding partner of the secreted vIgD is an inhibitory receptor (e.g., CTLA-4), which can be expressed by a first cell (e.g., a T cell, such as a CAR T cell) and / or a second cell (e.g., an endogenous or engineered T cell, such as a CAR T cell). Upon binding of the cognate binding partner to the SIP, the SIP antagonizes or blocks negative signaling through the inhibitory receptor, resulting in an activated or effector T cell. In all cases, the vIgD can be the V domain (IgV) alone, a combination of the V domain (IgV) and C domain (IgC) including the entire extracellular domain (ECD), or any combination of Ig domains of IgSF superfamily members. [Diagram 2] 1 shows an exemplary schematic diagram of the activity of a variant IgSF domain (vIgD) fused to Fc (vIgD-Fc), where the vIgD is a variant of the IgSF domain of CD80. As shown, soluble vIgD of CD80 interacts with its cognate binding partner and blocks the interaction of CD80 with CTLA-4, thereby blocking the CTLA-4 inhibitory receptor and potentially allowing T cells to differentiate towards an effector phenotype. [Figure 3A] 1 shows an exemplary schematic diagram of the activity of variant IgSF domain (vIgD) conjugated to Fc, where the CD80-Fc provides PD-L1-dependent CD28 agonist activity. As shown, binding of CD80-Fc to PD-L1 expressed on the surface of tumor cells can prevent the association of PD-L1 on tumor cells with the inhibitory PD-1 receptor expressed on the surface of T cells. In addition, CD80-Fc can be used to bind to the costimulatory CD28 receptor on the surface of T cells, thereby localizing T cells to tumors while promoting T cell activation via CD28 costimulation of TCR signals. [Figure 3B]1 shows an exemplary schematic diagram of the activity of CD80 variant IgSF domain (vIgD) conjugated to Fc, where the CD80-Fc blocks CTLA-4 inhibitory activity. As shown, binding of CD80 vIgD-Fc to CTLA-4 expressed on the surface of T cells (e.g., Treg and Teff cells) thereby antagonizes binding of CTLA-4 to its cognate binding partners CD80 (B7-1) and CD86 (B7-2) (denoted as B7), blocking CTLA-4 inhibitory signaling, lowering the threshold for TCR signaling, and promoting T cell activation. [Figure 4] 1 shows an exemplary schematic diagram of a stack molecule that 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., second vIgD) that binds to a second inhibitory receptor. In the exemplary schematic diagram, the second IgSF domain (e.g., second vIgD) is a CD80 vIgD. As shown, the first vIgD and second vIgD interact with their cognate binding partners and block the interaction of PD-L1 or PD-L2 with PD-1 and CD80 with the CTLA-4 inhibitory receptor, respectively. [Diagram 5]1 shows an exemplary schematic diagram of a stack molecule for localizing variant IgSF (vIgD) to tumor cells. In this format, the stack molecule comprises a first variant IgSF domain (first vIgD) and a second IgSF domain (e.g., second vIgD), where the second IgSF domain (e.g., second vIgD) is a tumor-targeting IgSF domain that binds to a tumor antigen. An exemplary tumor-targeting IgSF domain is the IgSF domain of NKp30, which binds to tumor antigen B7-H6. In this depiction, the first variant IgSF domain (vIgD) is a variant of the IgSF domain of CD80. As shown, binding of the tumor-targeting IgSF domain to the tumor cell surface localizes the first variant IgSF domain on the tumor cell surface where it can interact with one or more cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells) and antagonize the cognate inhibitory receptor CTLA-4. [Figure 6A] Various exemplary arrangements of stacked molecules containing a first variant IgSF domain (first vIgD) and a second IgSF domain, such as a second variant IgSF domain (second vIgD), are shown. As shown, the first vIgD and the second IgSF domain are independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. When generating homodimeric Fc molecules, the Fc region is capable of forming homodimers with a compatible Fc region upon co-expression of the individual Fc regions in a cell. When generating heterodimeric Fc molecules, the individual Fc regions contain mutations (e.g., "knob-into-hole" mutations in the CH3 domain) that favor the formation of heterodimers compared to homodimers when the individual Fc regions are co-expressed in a cell. [Figure 6B]Various exemplary configurations of stack molecules containing a first variant IgSF domain (first vIgD), a second IgSF domain such as a second variant IgSF domain (second vIgD), and a third IgSF domain such as a third variant IgSF domain (third vIgD) are shown. As shown, the first vIgD, the second IgSF domain, and the third IgSF domain are independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. When generating homodimeric Fc molecules, the Fc region is one that can form homodimers with a compatible Fc region by co-expression of the individual Fc regions in a cell. [Figure 7] An exemplary schematic diagram of the activity of a variant IgSF domain (vIgD) (V-Mab) conjugated to an antibody is shown, where the antibody (e.g., an anti-HER2 antibody) binds to an antigen on the surface of a tumor cell, localizing vIgD to the cell. As shown, binding of the antibody to the tumor cell surface localizes vIgD on the tumor cell surface, where it can interact with one or more of its cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells), activating or antagonizing receptor signaling. In the exemplary embodiment shown, the variant IgSF domain (vIgD) is a variant of the IgSF domain of CD80 that binds (e.g., has increased affinity) to the inhibitory receptor CTLA-4. Binding of CD80 vIgD to the CTLA-4 inhibitory receptor antagonizes or blocks the negative signaling of the inhibitory receptor, resulting in activated or effector T cells. In some cases, the clustering of inhibitory receptors (CTLA-4) in close proximity to activating receptors (eg, CD28) may result in the agonism of inhibitory receptor activity by TIP. [Figure 8A] 1 shows various exemplary configurations of variant IgSF antibody conjugates (V-Mabs), in which the variant IgSF domain is linked directly or indirectly to the N-terminus and / or C-terminus of the light chain of the antibody. [Figure 8B]1 shows various exemplary configurations of variant IgSF antibody conjugates (V-Mabs), in which the variant IgSF domain is linked directly or indirectly to the N-terminus and / or C-terminus of the heavy chain of the antibody. [Figure 8C] 8A and 8B show various exemplary configurations of variant IgSF antibody conjugates (V-Mab).The resulting V-Mab configurations are shown when the light chain in FIG. 8A and the heavy chain in FIG. 8B are co-expressed in a cell. [Figure 9A] Binding of exemplary CD80 IgV-Fc variants to cell surface expressed PD-L1, CD28 and CTL44 ligands. [Figure 9B] FIG. 1 shows dose-dependent PD-L1-dependent CD28 costimulation in Jurkat / IL-2 reporter lines induced by exemplary CD80 IgV-Fc variants. [Figure 9C] FIG. 1 shows human primary T cell cytokine production following PD-L1-dependent costimulation induced by exemplary CD80 IgV-Fc variants. [Figure 9D] Exemplary CD80 IgV-Fc candidates bind to PD-L1 and demonstrate the ability to block fluorescently conjugated PD-1 binding. [Figure 9E] 1 shows PD-1 / PD-L1 interaction and subsequent functional activity antagonistic activity of exemplary variant CD80-Fc variants. [Figure 10] 1 shows the in vivo anti-tumor activity of exemplary variant CD80 polypeptides fused to wild-type IgG1 Fc (WT Fc) or inactive IgG1 Fc (inactive Fc). [Figure 11] Median (left panel) and mean (right panel) tumor volumes of mouse models following treatment with 50 μg, 100 μg, and 500 μg of an exemplary variant CD80 IgV-Fc (inactive) and 100 μg of an anti-PD-L1 antibody (durvalumab) are shown. [Figure 12]Figure 2 shows the concentrations of IFNγ in hPD-L1 MC38 tumor lysates following in vivo treatment with 50μg, 100μg, and 500μg of an exemplary variant CD80 IgV-Fc (inactive) and 100μg of an anti-PD-L1 antibody (durvalumab). [Figure 13] Median (left panel) and mean (right panel) tumor volumes in mouse models following treatment with several exemplary CD80 IgV-Fc (inactive) variants and an anti-PD-L1 antibody (Durvalumab) are shown. [Figure 14] Shown are median (left panel) and mean (right panel) tumor volumes in mice following restimulation with huPD-L1 / MC38 tumor cells that were designated tumor-free following treatment with an exemplary CD80 IgV-Fc (inactive) variant and an anti-PD-L1 antibody (durvalumab). [Figure 15] Detection of bound negative control Fc, CD80 variant-Fc, and anti-PD-L1 antibody by flow cytometry in single cell suspensions of live CD45 negative (CD45-) tumor cells. [Figure 16] Shown are median (left panel) and mean (right panel) tumor volumes in mouse models following treatment with an exemplary variant CD80 IgV-Fc (inactive) and an anti-PD-L1 antibody (Durvalumab). [Figure 17] A and B show the percentage of CD8 cells detected by flow cytometry in tumor-draining lymph nodes (A) and tumors (B) of mice treated with negative control Fc, CD80 variant-Fc, and anti-PD-L1 antibody. C shows the percentage of anti-human Fc detection reagent on CD45-negative tumors treated in vivo with negative control Fc, CD80 IgV-Fc, and human anti-PD-L1 antibody. [Figure 18] Figure 2 shows the specific cytotoxic activity of CD80 IgV-Fc variants against huPD-L1 transduced MC38 tumor cells but not against the non-transduced parental MC38, demonstrating specific killing of huPDL1. [Figure 19A] Binding of CD80 IgV-Fc variants to primary human T cells. [Figure 19B] Binding of CD80 IgV-Fc variants to primary human monocytes. [Figure 20] FIG. 1 shows CD80 IgV-Fc variant antagonism of PD-L1-mediated SHP-2 recruitment to PD-1 using an enzyme complementation assay. [Figure 21] FIG. 1 shows CD80 IgV-Fc variant antagonism of CD80 / CTLA-4 binding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] Detailed Description Provided herein are immunomodulatory proteins that are or contain variants or mutants of CD80 and specific binding fragments thereof that exhibit altered binding activity or affinity for at least one target ligand cognate binding partner (also referred to as counter-structure ligand protein). In some embodiments, the variant CD80 polypeptides contain one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) compared to unmodified or wild-type CD80 polypeptides. In some embodiments, the variant CD80 polypeptides contain one or more amino acid modifications (e.g., substitutions) compared to unmodified or wild-type CD80 polypeptides. In some embodiments, the one or more amino acid modifications are in the IgSF domain (e.g., IgV) of unmodified or wild-type CD80 polypeptides.

[0114] In some embodiments, the altered binding activity, such as binding affinity and / or binding selectivity, e.g., increased or decreased binding affinity or selectivity, is for at least one binding partner protein CD28, PD-L1, or CTLA-4. In some embodiments, the variant CD80 polypeptide exhibits altered, such as increased or decreased binding activity or affinity, for one or more of CD28, PD-L1, or CTLA-4, compared to unmodified or wild-type CD80 that does not contain one or more modifications.

[0115] In some embodiments, the variant CD80 polypeptides exhibit increased binding affinity for CTLA-4 and / or PD-L1 compared to unmodified or wild-type CD80 that does not comprise one or more modifications. In some embodiments, the variant CD80 polypeptides exhibit decreased binding affinity for CD28 compared to unmodified or wild-type CD80 that does not comprise one or more modifications. In some embodiments, the variant CD80 polypeptides exhibit increased binding affinity for one or both of CTLA-4 and PD-L1, and decreased binding affinity for CD28 compared to unmodified or wild-type CD80 that does not comprise one or more modifications.

[0116] In some embodiments, the variant CD80 polypeptides provided herein exhibit increased selectivity for binding to CTLA-4 relative to CD28 compared to the selectivity for binding to CTLA-4 relative to CD28 of unmodified or wild-type CD80 that does not contain one or more modifications. Increased selectivity can be characterized as a greater ratio of binding to CTLA-4 to CD28, e.g., a greater ratio of binding affinity, of the variant CD80 polypeptide compared to the ratio of binding to CTLA-4 to binding to CD28, e.g., the ratio of binding affinity, of unmodified or wild-type CD80. In some embodiments, the ratio is increased by 1.2x, 1.5x, 2.0x, 3.0x, 4.0x, 5.0x, 6.0x, 7.0x, 8.0x, 9.0x, 10.0x, 15.0x, 20x, 30x, 40x, 50x, 100x or more, or by about 1.2x, about 1.5x, about 2.0x, about 3.0x, about 4.0x, about 5.0x, about 6.0x, about 7.0x, about 8.0x, about 9.0x, about 10.0x, about 15.0x, about 20x, about 30x, about 40x, about 50x, about 100x or more.

[0117] In some embodiments, the variant CD80 polypeptides provided herein exhibit increased selectivity for binding to PD-L1 relative to CD28 compared to the selectivity of binding to PD-L1 relative to CD28 of unmodified or wild-type CD80 that does not contain one or more modifications. Increased selectivity can be characterized as a greater ratio of binding to PD-L1 to CD28, e.g., a greater ratio of binding affinity, of the variant CD80 polypeptide compared to the ratio of binding to PD-L1 to CD28, e.g., the ratio of binding affinity, of unmodified or wild-type CD80. In some embodiments, the ratio is increased by 1.2x, 1.5x, 2.0x, 3.0x, 4.0x, 5.0x, 6.0x, 7.0x, 8.0x, 9.0x, 10.0x, 15.0x, 20x, 30x, 40x, 50x, 100x or more, or by about 1.2x, about 1.5x, about 2.0x, about 3.0x, about 4.0x, about 5.0x, about 6.0x, about 7.0x, about 8.0x, about 9.0x, about 10.0x, about 15.0x, about 20x, about 30x, about 40x, about 50x, about 100x or more.

[0118] In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is a transmembrane immunomodulatory protein that can be expressed on the surface of a cell. In some embodiments, the immunomodulatory protein is a secretable immunomodulatory protein that can be secreted from the cell in which it is expressed. In some embodiments, also provided herein is one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD80 polypeptide provided herein and one or more other moieties or polypeptides. In some aspects, engineered cells are provided that contain a transmembrane immunomodulatory protein or a secretable immunomodulatory protein. In some aspects, an infectious agent is provided that can deliver a transmembrane immunomodulatory protein or a secretable immunomodulatory protein to a cell that the infectious agent infects for expression. In some embodiments, also provided herein is one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD80 polypeptide provided herein and one or more other moieties or polypeptides.

[0119] In some embodiments, the variant CD80 polypeptides and immunomodulatory proteins modulate an immunological immune response, such as increasing or decreasing an immune response, hi some embodiments, the variant CD80 polypeptides and immunomodulatory proteins provided herein can be used to treat diseases or conditions associated with a dysregulated immune response.

[0120] In some embodiments, the provided variant CD80 polypeptides regulate T cell activation, expansion, differentiation, and survival through interaction with costimulatory signaling molecules. In general, two distinct signals are generally required for activation of antigen-specific T cells. The first signal is provided by the interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC)-associated antigens present on antigen-presenting cells (APCs). The second signal is a costimulatory signal for TCR engagement, e.g., a CD28 costimulatory signal, which is required to avoid apoptosis or anergy of the T cells.

[0121] In some embodiments, under normal physiological conditions, T cell-mediated immune responses are initiated by antigen recognition by the T cell receptor (TCR) and are regulated by a balance of costimulatory and inhibitory signals (e.g., immune checkpoint proteins). The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and protect tissues from excessive damage during an immune response, such as an attack against a pathogenic infection. However, in some cases, these immune-regulating proteins can be dysregulated in diseases and conditions, including tumors, as a mechanism to evade the immune system.

[0122] In some embodiments, among the known T cell costimulatory receptors is CD28, which is the T cell costimulatory receptor for the ligands B7-1 (CD80) and B7-2 (CD86), both of which are present on APCs. These same ligands can also bind to the inhibitory T cell receptor CTLA4 (cytotoxic T lymphocyte-associated protein 4) with higher affinity than CD28; binding to CTLA4 acts to downregulate the immune response.

[0123] In some embodiments, CD80 can bind to programmed death ligand 1 (PD-L1). CD80 has a similar affinity to PD-L1 for CD28. PD-L1 is one of two ligands of the inhibitory immune receptor, programmed death 1 (PD-1). The interaction of PD-L1 with PD-1 negatively regulates immune activity by promoting T cell inactivation and downregulating T cell activity. PD-1 expression on T cells can be induced after T cells are activated as a strategy to prevent T cell overactivity. Many tumor cells express PD-L1 on their surface, potentially resulting in the inhibition of PD-1 / PD-L1 interaction and T cell responses against tumors. The binding of CD80 to PD-L1 can block the interaction between PD-L1 and PD-1, thus preventing the inhibition of T cell responses, for example at tumor sites, effectively enhancing or enhancing immune responses. However, at the same time, CD80 can also bind to CD28 or CTLA4 receptors and potentially participate in the induction or inhibition of T cell responses. Thus, in some cases, the interactions of CD80 with PD-L1, CD28, and CTLA-4 can have overlapping and complementary effects. In some embodiments, CD28 and PD-L1 may play complementary roles in modeling the immune response.

[0124] In some embodiments, provided variant CD80 polypeptides or immunomodulatory proteins modulate (e.g., increase or decrease) immunological activity induced by or associated with the inhibitory receptor CTLA-4, the PD-L1 / PD-1 negative regulatory complex, and / or the costimulatory receptor CD28. For example, in some embodiments, provided CD80 polypeptides, such as soluble forms of variant CD80 polypeptides provided herein, bind to the CTLA-4 inhibitory receptor and block its interaction with CD80 expressed on APCs, thereby preventing negative regulatory signaling of the CD80-bound CTLA-4 receptor, as shown in Figure 2. In some embodiments, provided CD80 polypeptides, such as soluble forms of variant CD80 polypeptides provided herein, can bind to PD-L1 on tumor cells or APCs, thereby blocking the interaction of PD-L1 with the PD-1 inhibitory receptor, thereby preventing the negative regulatory signaling that would otherwise result from the PD-L1 / PD-1 interaction. In some embodiments, provided CD80 polypeptides, such as soluble forms of variant CD80 polypeptides provided herein, bind to and costimulate CD28 receptors on localized T cells while blocking PD-L1 / PD-1 interactions, thereby promoting an immune response (Figure 3A). In some embodiments, provided CD80 polypeptides, such as soluble forms of variant CD80 polypeptides provided herein, can antagonize B7 / CTLA-4 binding, preventing CTLA-4 inhibitory signaling and lowering the TCR signaling threshold, thereby promoting T cell activation and immune responses (Figure 3B). In some embodiments, provided CD80 variant polypeptides can be stacked or associated with other immune modulating polypeptides to further modulate immune activity (Figure 4), or can be bundled or conjugated to targeting molecules to localize immune activity (Figures 5 and 7).Thus, in some embodiments, the provided polypeptides overcome these limitations by providing variant CD80s that have independent binding affinities for both CTLA-4 and / or PD-L1, and optionally CD28, thereby agonizing or antagonizing the complementary effects of receptor costimulation. Methods of making and using these variant CD80s are also provided.

[0125] Also provided are various formats of the variant polypeptides provided. As shown herein, the different formats can facilitate the manipulation of immune responses and thus have therapeutic applications. The ability to format the variant polypeptides in various configurations and antagonize or agonize immune responses depending on the situation provides flexibility for therapeutic applications based on the same increased binding and activity of variant CD80 to binding partners. As an example, tethering a variant CD80 protein to a surface can deliver a localized costimulatory signal, while in other cases presenting CD80 in a non-localized soluble form confers antagonistic activity. For example, delivery of enhanced CD80 protein in a soluble format with increased affinity to CTLA-4 and / or PD-L1 can antagonize inhibitory receptor signaling, such as blocking intracellular inhibitory signals that can occur to reduce responses to activating stimuli, e.g., CD3 and / or CD28 costimulatory or mitogenic signals. In some cases, this can result in an increased immune response.

[0126] In addition, certain formats may also, in some cases, mediate CD28 agonism. In some cases, CD28 agonism is mediated by certain variant CD80 polypeptides that exhibit increased binding to PD-L1, thereby facilitating tethering or bridging of variant CD80 molecules to surfaces of the immune synapse for interaction with CD28, thereby providing a costimulatory signal, thereby promoting T cell activation. This activity, referred to herein as PD-L1-dependent CD28 costimulation, is due in some embodiments to the ability of the variant CD80 polypeptides to bind both PD-L1 and CD80 in a non-competitive manner and / or by providing a dimeric format of the variant CD80 polypeptide (see, e.g., FIG. 3). In some cases, such PD-L1-dependent costimulation does not require an Fc with effector function, and may be mediated by an Fc fusion protein containing an effector-less or inactive Fc molecule. In some embodiments, tethering or cross-linking can also be achieved via Fc receptors when the variant CD80 polypeptide is provided as a fusion protein with a wild-type Fc region of an immunoglobulin that retains or exhibits effector function, referred to herein as Fc receptor-dependent CD28 costimulation. In some embodiments, cross-linking of Fc receptors can initiate antibody-dependent cellular cytotoxicity (ADCC)-mediated effector function, which results in the activation of CTLA-4 expressing cells (e.g., CTLA-4 expressing T regulatory cells) or PD-L1 expressing cells (e.g., PD-L1 hi This results in depletion of target cells that express the cognate binding partner, such as tumor cells.

[0127] Enhancement or inhibition of the activity of these receptors has clinical implications in the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, therapies that intervene and alter the costimulatory effect of both receptors are constrained by size limitations in addition to spatial orientation requirements imposed by the extent of the immunological synapse. In some embodiments, existing therapeutics, including antibody drugs, may not be able to simultaneously interact with multiple target proteins involved in regulating these interactions. In addition, in some cases, existing therapeutics may only antagonize immune responses, but not have the ability to agonize them. In addition, pharmacokinetic differences between drugs that independently target one or the other of these two receptors may result in difficulties in adequately maintaining the desired blood levels of such drug combinations throughout the course of treatment. The variant CD80 polypeptides and immunomodulatory proteins provided, and other formats described, address such issues.

[0128] All publications, including patents, patent applications, scientific articles, and databases, referred to in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, including patent, patent application, scientific article, or database, was specifically and individually indicated to be incorporated by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0129] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0130] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as significantly different from those commonly understood in the art.

[0131] Terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms, acronyms and abbreviations used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names are in accordance with IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include not only the values ​​defining the range but also all integer values ​​therebetween.

[0132] The term "affinity modified" when used in the context of immunoglobulin superfamily domains means a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence (relative to a corresponding wild-type parent or unmodified IgSF domain) that has been altered to increase or decrease the binding affinity or avidity for at least one of its cognate binding partners (or "counterstructure") compared to a parent wild-type or unmodified (i.e., non-affinity modified) IgSF control domain. In this context, affinity modified CD80 IgSF domains are included. In some embodiments, affinity modified IgSF domains can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, e.g., amino acid substitutions, in a wild-type or unmodified IgSF domain. Increased or decreased 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 the protein to its cognate binding partner(s) will be at least 10% greater than a wild-type IgSF domain control, and in some embodiments will be at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value. The decrease in binding affinity or avidity of the protein to at least one of its cognate binding partners will be 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments will be 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less but 10% or more of the wild-type IgSF domain control value.Affinity-modified proteins have altered primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any condition on any particular starting composition or method by which the affinity-modified IgSF domain is made. Thus, the affinity-modified IgSF domain of the present invention is not limited to wild-type IgSF domains that have been converted into affinity-modified IgSF domains by any particular affinity modification process. Affinity-modified IgSF domain polypeptides can be generated, for example, starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its cognate binding partner, and finally recombinantly or chemically synthesized to obtain the subject affinity-modified IgSF domain composition. However, in another example, affinity-modified IgSF domains can be generated by site-directed mutagenesis of wild-type IgSF domains. Thus, affinity-modified IgSF domain refers to a product, but not necessarily the product produced by any given process. A wide variety of techniques may be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0133] The term "allogeneic" as used herein means cells or tissues that are removed from one organism and then injected or adoptively transferred into a genetically distinct organism of the same species. In some embodiments of the invention, the species is murine or human.

[0134] The term "autologous" as used herein refers to cells or tissues that are taken from the same organism and then injected or adoptively transferred into the organism. Autologous cells or tissues can be altered, for example, by recombinant DNA techniques, so that they are no longer genetically identical to the native cells or native tissues that are taken from the organism. For example, native autologous T cells can be genetically engineered by recombinant DNA techniques to become autologous engineered cells that express transmembrane immunomodulatory proteins and / or chimeric antigen receptors (CARs), which may involve engineering T cells or TILs (tumor infiltrating lymphocytes). The engineered cells are then infused into the patient from which the native T cells were isolated. In some embodiments, the organism is human or murine.

[0135] The terms "binding affinity" and "binding activity" as used herein refer to the specific binding affinity and specific binding activity of a protein to its counter-structure under specific binding conditions, respectively. In biochemical kinetics, binding activity refers to the cumulative strength of the affinities of multiple individual non-covalent interactions, such as between CD80 and its counter-structures PD-L1, CD28, and / or CTLA-4. Thus, binding activity differs from affinity, which represents the strength of a single interaction. The increased or weakened binding affinity of a variant CD80 containing an affinity-modified CD80 IgSF domain to its counter-structure is determined relative to the binding affinity of an unmodified CD80 (e.g., an unmodified CD80 containing a native or wild-type IgSF domain (e.g., an IgV domain)). Methods for determining binding affinity or binding activity 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 CD80 (e.g., containing an affinity-modified IgSF domain) specifically binds to CD28, PD-L1, and / or CTLA-4 with a binding affinity that results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than an unmodified CD80 control, as measured by flow cytometry, in the binding assay described in Example 6.

[0136] The term "biological half-life" refers to the time it takes for a substance (e.g., an immunomodulatory polypeptide containing a variant CD80 polypeptide of the invention) to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by the elimination, excretion, degradation (e.g., enzymatic), or absorption and concentration in specific organs or tissues of the body of the substance. 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 the polypeptides of the invention to extend their biological half-life are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0137] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial (i.e., man-made) transmembrane protein expressed on a mammalian cell, comprising at least an ectodomain, a transmembrane, and an endodomain. Optionally, the CAR protein comprises a "spacer" that covalently links the ectodomain to the transmembrane domain. The spacer is often a polypeptide that links the ectodomain to the transmembrane domain via a peptide bond. CARs are typically expressed on mammalian lymphocytes. In some embodiments, CARs are expressed on mammalian cells, such as T cells or tumor infiltrating lymphocytes (TILs). CARs expressed on T cells are referred to herein as "CAR T cells" or "CAR-Ts". In some embodiments, CAR-Ts are helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. When used clinically, for example in adoptive cell transfer, CAR-Ts with antigen-binding specificity for the patient's tumor are typically engineered to be expressed on natural T cells obtained from the patient. The engineered T cells expressing the CAR are then infused back into the patient. Thus, the CAR-T is often an autologous CAR-T, although allogeneic CAR-Ts are also included within the scope of the present invention. The ectodomain of the CAR contains an antigen-binding region (e.g., an antibody or an antigen-binding fragment thereof (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Upon specific binding, a series of biochemical events (i.e., signal transduction) results in the regulation of the immune activity of the CAR-T. Thus, for example, specific binding to its target antigen by the antigen-binding region of the CAR-T can lead to changes in the immune activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction by CAR-T activation is achieved by the CD3 zeta chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells.CAR-T can further contain multiple signaling domains (e.g., CD28, 41BB, or OX40) that further regulate the immunoregulatory response of T cells. CD3-z contains a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM) that is involved in T cell receptor signal transduction.

[0138] The terms "collectively" or "total," when used in reference to cytokine production induced by the presence of two or more variant CD80 polypeptides in an in vitro assay, refer to the overall cytokine expression level, regardless of cytokine production induced by individual variant CD80 polypeptides. In some embodiments, the cytokine assayed is IFN-γ, e.g., in an in vitro primary T cell assay as described in Example 7.

[0139] The term "cognate binding partner" (used interchangeably with "counter structure"), in reference to a polypeptide (e.g., an IgSF domain of a variant CD80), refers to at least one molecule (typically a native mammalian protein) to which the referenced polypeptide specifically binds under specific binding conditions. In some embodiments, a variant CD80 containing an affinity-modified IgSF domain specifically binds to the counter structure of the corresponding native or wild-type CD80, but with increased or attenuated affinity. A species of ligand that is recognized and specifically binds to its cognate receptor under specific binding conditions is an example of a counter structure or cognate binding partner of that receptor. A "cell surface cognate binding partner" is a cognate binding partner expressed on the mammalian cell surface. A "cell surface molecular species" is a cognate binding partner of a ligand of an immunological synapse (IS) that is expressed on and by cells (e.g., mammalian cells) that form the immunological synapse (IS).

[0140] As used herein, "conjugate", "conjugation" or grammatical variations thereof refers to the connection or linking of two or more compounds together by any connection or linking method known in the art, resulting in the formation of another compound. It can also refer to a compound produced by connecting or linking two or more compounds together. For example, a variant CD80 polypeptide directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include those produced by fusion proteins, chemical conjugates, and those produced by any other method.

[0141] The term "competitive binding" as used herein means that a protein can specifically bind to at least two cognate binding partners, but the specific binding of one cognate binding partner inhibits (e.g., prevents or hinders) the simultaneous binding of a second cognate binding partner. Thus, in some cases, a protein cannot simultaneously bind to two cognate binding partners. Generally, competitive binders contain the same or overlapping binding sites for specific binding, but this is not a requirement. In some embodiments, competitive binding causes a measurable (partial or complete) inhibition of the specific binding of a protein to one of its cognate binding partners due to the specific binding of the second cognate binding partner. A wide variety of methods are known for quantifying competitive binding, such as ELISA (enzyme-linked immunosorbent assay) assays.

[0142] The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0143] The term "corresponding" with respect to a protein position, e.g., a statement that a nucleotide or amino acid position "corresponds" to a nucleotide or amino acid position in a disclosed sequence, e.g., as set forth in the sequence listing, refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm (e.g., the GAP algorithm). For example, corresponding residues can be determined by alignment of a reference sequence with the sequence of wild-type CD80 as set forth in SEQ ID NO:2 (ECD domain) or as set forth in SEQ ID NO:76, 3030 or 3031 (IgV domain) according to the structural alignment methods described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, e.g., using conserved and identical amino acid residues as a reference.

[0144] The terms "reduce" or "attenuate" or "inhibit" as used herein refer to a statistically significant amount of reduction. The reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0145] The term "derivative" or "derivatized" refers to the modification of a protein by covalently linking the protein directly or indirectly to a composition to alter properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy while retaining or enhancing its therapeutic effect. Derivatives of the immune modulating polypeptides of the invention are within the scope of the invention and can be made, for example, by glycosylation, PEGylation, lipidation, or Fc fusion.

[0146] As used herein, detection includes the method that allows protein to be visualized (by eye or instrument).Protein can be visualized using an antibody specific to the protein.Protein detection can also be promoted by fusing the protein with a tag that contains detectable label, or by contacting the protein with a second reagent that is specific to the protein, such as a second antibody that contains detectable label.

[0147] As used herein, a domain (typically a sequence of 3 or more, generally 5 or 7 or more amino acids, e.g., 10-200 amino acid residues) refers to a portion of a molecule (e.g., a protein or coding nucleic acid) that is structurally and / or functionally distinct from and identifiable with other portions of the molecule. For example, a domain includes a portion of a polypeptide chain that can independently form a folded structure within a protein that is composed of one or more structural motifs and / or is recognized by a functional activity, such as a binding activity. A protein can have one, two or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology to related family members, e.g., homology to a motif. In another example, a domain can be distinguished by its function (e.g., ability to interact with a biomolecule, such as a cognate binding partner). A domain can exhibit an independent biological function or activity, such that it can act (e.g., bind) independently or fused to another molecule. A domain can be a linear or non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those of skill in the art. For purposes of illustration herein, definitions are provided, but it is understood that it is well within the skill of the art to recognize a particular domain by name. If necessary, appropriate software can be used to identify the domains.

[0148] The term "ectodomain" as used herein refers to a region of a membrane protein (e.g., a transmembrane protein) that is outside the vesicle membrane. Ectodomains often contain a binding domain that specifically binds to a ligand or cell surface receptor, e.g., via a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a cellular transmembrane protein is alternatively referred to as an extracellular domain.

[0149] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition (including a protein composition or a cell composition) of the present invention that, when administered ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient) either alone (i.e., as a monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant reduction in disease progression, for example by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the physical function of a patient. 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.

[0150] The term "endodomain" as used herein refers to a region found in some membrane proteins (e.g., transmembrane proteins) that extends into the internal space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. In cells, the endodomain can interact with intracellular components and play a role in signal transduction, and thus, in some cases, can be an intracellular signaling domain. The endodomain of a cellular transmembrane protein is alternatively referred to as a cytoplasmic domain, which in some cases can be a cytoplasmic signaling domain.

[0151] The term "enhanced" or "increased" as used herein in the context of increasing immune activity of a mammalian lymphocyte means an increase in one or more activities of the lymphocyte. The increase in activity can be an increase in one or more of cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity, such as, for example, in a statistically significant amount. In some embodiments, reference to increased immune activity means increasing interferon gamma (IFNγ) production, for example, in a statistically significant amount. In some embodiments, immune activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56. Other methods of assessing lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, an enhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

[0152] The term "engineered cell" as used herein refers to a mammalian cell that has been genetically engineered by human intervention (e.g., recombinant DNA or viral transduction). In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., T cell, B cell, NK cell) or an antigen-presenting cell (e.g., dendritic cell). The cell may be a primary cell from a patient or may be a cell line. In some embodiments, the engineered cell of the invention contains a variant CD80 of the invention engineered to modulate the immune activity of T cells expressing CD28, PD-L1, and / or CTLA-4, or APCs expressing PD-L1, to which the variant CD80 polypeptide specifically binds. In some embodiments, the variant CD80 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") that contains an extracellular domain or a portion thereof that contains an IgV domain linked to a transmembrane domain (e.g., a CD80 transmembrane domain), and optionally contains an intracellular signaling domain. In some cases, the TIP is formatted as a chimeric receptor that contains a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the engineered cells are capable of expressing and secreting an immunomodulatory protein as described herein.Some of the engineered cells provided further contain an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0153] The term "engineered T cells," as used herein, refers to T cells (e.g., helper T cells, cytotoxic T cells (or cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells) that have been genetically engineered by human intervention (e.g., recombinant DNA or viral transduction methods). Engineered T cells contain a variant CD80 transmembrane immunomodulatory protein (TIP) or secreted immunomodulatory protein (SIP) of the invention that is expressed on the T cell and is engineered to modulate the immune activity of the engineered T cell itself or of a mammalian cell to which the variant CD80 expressed on the T cell specifically binds.

[0154] The term "engineered T cell receptor" or "engineered TCR" refers to a T cell receptor (TCR) that has been selected, cloned, and / or subsequently introduced into a population of T cells (often used in adoptive immunotherapy) that has been engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen. In contrast to engineered TCRs, CARs are engineered to bind target antigens in an MHC-dependent manner.

[0155] The term "expressed on" as used herein is used in reference to a protein expressed on the surface of a cell (e.g., a mammalian cell). Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety (e.g., a drug or a detectable label). A protein expressed on the surface of a cell can include a cell surface protein (e.g., a cell surface receptor) expressed on a mammalian cell.

[0156] 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 moiety. In some embodiments, the half-life is extended by more than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or by more than about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or by more than about 6.0-fold. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to a protein without the half-life extending moiety. Half-life refers to the 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 an 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).

[0157] The term "immunological synapse" or "immune synapse," as used herein, refers to the interface between a mammalian cell (e.g., an antigen-presenting cell or a tumor cell) that expresses MHC I (major histocompatibility complex) or MHC II, and a mammalian lymphocyte (e.g., an effector T cell or a natural killer (NK) cell).

[0158] The Fc (fragment crystallizable) region or domain (also referred to as Fc polypeptide) of an immunoglobulin molecule corresponds primarily to the constant region of an immunoglobulin heavy chain and is involved in various functions, including the effector function(s) of an antibody. The Fc domain contains part or all of the hinge domain and the CH2 and CH3 domains of an immunoglobulin molecule. The Fc domain can form a dimer of two polypeptide chains connected by one or more disulfide bonds. Exemplary dimerization polypeptides are shown in Figures 6A and 6B. In some embodiments, the Fc is a variant Fc with reduced activity (e.g., reduced by more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more) in promoting effector function. In some embodiments, references to amino acid substitutions in the Fc region are according to the EU numbering system, unless otherwise noted based on a particular SEQ ID NO:. EU numbering is known and follows the EU index reported in the recently updated IMGT Scientific Chart (IMGT®, i.e., the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed. USDepartment of Health and Human Services, NIH publication No. 91-3242 (1991).

[0159] Immunoglobulin Fc fusions ("Fc fusions"), e.g., immunomodulatory Fc fusion proteins, are molecules that comprise one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. Fc fusions can comprise, for example, an Fc region of an antibody (to facilitate pharmacokinetics) and a variant CD80 polypeptide. The immunoglobulin Fc region can be indirectly or directly linked to one or more variant CD80 polypeptides or small molecules (fusion partners). A variety of linkers are known in the art and can optionally be used to link the Fc to the fusion partner to generate the Fc fusion. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or non-identical species can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, e.g., a murine, rabbit or human Fc.

[0160] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. A host cell can be a prokaryote, such as Escherichia coli (E. Coli), or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include Chinese Hamster Ovary (CHO) cells or their derivatives, such as Veggie CHO, DG44, Expi CHO, or CHOZN and related cell lines grown in serum-free medium, or the DHFR-deficient CHO line DX-B11. In some embodiments, the host cell can be a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell).

[0161] The term "immunoglobulin" (abbreviated as "Ig"), as used herein, refers to mammalian immunoglobulin proteins including any of the five human classes of antibodies, namely IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also refers to immunoglobulins that are less than full-length, whether fully or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V, and VF. H and V L variable fragments (Fv), which contain V H and V L Single chain variable fragments (scFv) containing the V region of an antibody, as well as other antibody V region fragments, such as Fab', F(ab) 2 , F(ab′) 2 , dsFv diabodies, Fc, and Fd polypeptide fragments. Homo-bispecific and hetero-bispecific bispecific antibodies are included within the meaning of the term.

[0162] The term "immunoglobulin superfamily" or "IgSF" as used herein refers to a group of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. Molecules are categorized as members of this superfamily based on structural features they share with immunoglobulins (i.e., antibodies), all of which possess domains known as immunoglobulin domains or folds. Members of 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, as well as intracellular muscle proteins. These are usually associated with a role in the immune system. Proteins in the immunological synapse are often members of IgSF. IgSF can also be divided into "subfamilies" based on shared properties such as function. Such subfamilies typically consist of 4-30 IgSF members.

[0163] The terms "IgSF domain" or "immunoglobulin domain" or "Ig domain" as used herein refer to a structural domain of an IgSF protein. Ig domains are named after immunoglobulin molecules. They contain about 70-110 amino acids and are classified according to their size and function. Ig domains possess a characteristic Ig fold with a sandwich-like structure formed by two sheets of antiparallel β-strands. Interactions between hydrophobic amino acids on the inside of the sandwich as well as highly conserved disulfide bonds formed between cysteine ​​residues in the B and F strands stabilize the Ig fold. One end of an Ig domain has a portion called the complementarity determining region that is important for the specificity of antibodies for their ligands. Ig-like domains can be classified (into classes) as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) or constant (IgC) domains. IgV domains, with nine β-strands, are generally longer than IgC domains, with seven β-strands. The Ig domains of some members of the IgSF resemble IgV domains in amino acid sequence but are similar in size to IgC domains. These are called IgC2 domains, whereas the canonical IgC domain is called IgC1 domain. T cell receptor (TCR) chains contain two Ig domains in their extracellular portions, one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane. CD80 contains two Ig domains, IgV and IgC.

[0164] The term "IgSF species" as used herein refers to a population of IgSF member proteins that have the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines a unique identity for all IgSF species that belong to that IgSF member. Thus, each IgSF family member is unique compared to other IgSF family members, and thus each species of a particular IgSF family member is unique compared to another IgSF family member species. Nevertheless, differences between molecules of the same IgSF species may arise due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, small sequence differences within a single IgSF species due to genetic polymorphisms, as well as wild-type truncated forms of an IgSF species due to, for example, proteolytic cleavage, make other forms of differences within a single IgSF species. A "cell surface IgSF species" is an IgSF species that is expressed on the surface of a cell (typically a mammalian cell).

[0165] The term "immune activity" as used herein in the context of mammalian lymphocytes such as T cells refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon-γ), or T cytotoxic activity. In some cases, immune activity can refer to their expression of cytokines such as chemokines or interleukins. Assays for determining enhanced or suppressed immune activity include MLR (mixed lymphocyte reaction) assays that measure interferon-γ cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assays (Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med. 2010:8:104). Since T cell activation is associated with the secretion of IFN-γ cytokines, detection of IFN-γ levels in culture supernatants from these in vitro human T cell assays can be assayed using commercially available ELISA kits (Wu et al, Immunol Lett 2008 Apr 15;117(1):57-62). Induction of an immune response results in increased immune activity compared to resting lymphocytes. Immunomodulatory proteins as provided herein (e.g., variant CD80 polypeptides containing affinity-modified IgSF domains) can increase, in some embodiments, or decrease, in other embodiments, IFN-γ (interferon-γ) expression in primary T cell assays compared to wild-type IgSF members or IgSF domain controls. One skilled in the art will understand that the format of the primary T cell assay used to determine increased IFN-γ expression will differ from the format used to assay for decreased IFN-γ expression.In assaying for the ability of the immunomodulating protein or affinity-modified IgSF domain of the invention to reduce IFN-γ expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used as described in Example 6. Conveniently, a soluble form of the affinity-modified IgSF domain of the invention can be used to determine its ability to reduce IFN-γ expression by antagonizing it in an MLR, also as described in Example 6. Alternatively, in assaying for the ability of the immunomodulating protein or affinity-modified IgSF domain of the invention to increase IFN-γ expression in primary T cell assays, a co-fixation assay can be used. In the co-fixation assay, a T cell receptor signal (provided in some embodiments by an anti-CD3 antibody) is used in combination with a co-fixed affinity-modified IgSF domain, such as a variant CD80, to determine the ability to increase IFN-γ expression compared to a wild-type IgSF domain control. Methods for assaying the immune activity of engineered cells, including assessing the activity of variant CD80 transmembrane immunomodulatory proteins, are known in the art and include, but are not limited to, the ability to proliferate T cells following antigen stimulation, the ability to sustain T cell proliferation in the absence of restimulation, and anti-cancer activity in appropriate animal models. Assays are also standard. 51 Assays to assess cytotoxicity include Cr release assays (see, e.g., Milone et al., (2009) Molecular Therapy 17;1453-1464) or flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

[0166] An "immunomodulating polypeptide" or "immunomodulating protein" is a polypeptide or protein molecule that modulates immune activity. "Modulation" or "modulating" an immune response means either increasing or decreasing immune activity. An immunomodulating protein can be a single polypeptide chain or a multimer (dimer or higher order multimer) of at least two polypeptide chains covalently linked to each other (e.g., by interchain disulfide bonds). Thus, monomeric, dimeric, and higher order multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimers (of identical polypeptide chains) or heteromultimers (of non-identical polypeptide chains). An immunomodulating protein of the present invention can include a variant CD80 polypeptide.

[0167] The term "increase" as used herein means to 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.

[0168] An "isoform" of CD80 is one of multiple naturally occurring CD80 polypeptides that differ in amino acid sequence.Isoforms can be the product of splice variants of the RNA transcript expressed by a single gene, or the expression product of highly similar but different genes that produce functionally similar proteins, such as can result from gene duplication.As used herein, the term "isoform" of CD80 also refers to the product of different alleles of the CD80 gene.

[0169] The term "label" refers to a compound or composition that can be directly or indirectly bound or linked to provide a detectable signal, or can interact with a second label to modify the detectable signal. A label can be directly or indirectly conjugated to a polypeptide to produce a labeled polypeptide. A label can be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze a chemical change in a substrate compound composition that is detectable. Non-limiting examples of labels include a fluorogenic moiety, green fluorescent protein, or luciferase.

[0170] The term "lymphocyte" as used herein means any of the three subtypes of white blood cells in the mammalian immune system. These include natural killer cells (NK cells) (which function in cell-mediated cytotoxic innate immunity), T cells (for cell-mediated cytotoxic adaptive immunity), and B cells (for humoral antibody-based adaptive immunity). T cells include helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Also included within the definition of lymphocytes are innate lymphocytes (ILCs).

[0171] The term "mammal" or "patient" specifically includes reference to at least one of a human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0172] The term "membrane protein" as used herein means a protein that directly or indirectly binds to a lipid bilayer under physiological conditions. The lipid bilayer that forms the membrane can be a biological membrane, such as a cell membrane of a eukaryotic organism (e.g., a mammalian organism) or an artificial (i.e., man-made) membrane, such as a membrane found on a liposome. The binding of the membrane protein to the lipid bilayer can be by covalent bonds or by non-covalent interactions (e.g., hydrophobic or electrostatic interactions). The membrane protein can be an integral membrane protein or a peripheral membrane protein. A membrane protein that is a peripheral membrane protein is non-covalently bound to the lipid bilayer or non-covalently bound to an integral membrane protein. The peripheral membrane protein forms a temporary binding to the lipid bilayer so that the peripheral membrane protein can associate with and / or dissociate from the lipid bilayer under physiological range conditions in a mammal. In contrast to peripheral membrane proteins, integral membrane proteins form a virtually permanent bond to the lipid bilayer of the membrane, such that integral membrane proteins do not dissociate from the lipid bilayer under physiological range conditions in mammals. A membrane protein can form a bond to the membrane through one layer of the lipid bilayer (monotopic) or through both layers of the membrane (polytopic). An integral membrane protein that only interacts with one lipid bilayer is an "integral monotopic protein". An integral membrane protein that interacts with both lipid bilayers is an "integral polytopic protein", or is referred to herein as a "transmembrane protein".

[0173] The term "modulating" or "modulating", as used herein in the context of an immune response (e.g., a mammalian immune response), refers to any change (e.g., increase or decrease) in an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant CD80 of the invention or as a result of administration of an engineered cell expressing an immunomodulatory protein of the invention (e.g., a variant CD80 transmembrane immunomodulatory protein). Modulation thus refers to a change (e.g., increase or decrease) in an immune response compared to an immune response that occurs or exists in the absence of administration of an immunomodulatory protein comprising a variant CD80. Such modulation includes any induction, activation, suppression, or change in the degree or extent of immune 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, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted to an existing, developing, or potential immune response, or to the ability to induce, modulate, affect, or respond to an immune response. Modulation includes any change in the expression and / or function of genes, proteins, and / or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immune activity includes, for example, the elimination, deletion, or sequestration of immune cells; the induction or generation of immune cells that can modulate the functional capabilities of other cells, such as autoreactive lymphocytes, antigen-presenting cells, or inflammatory cells; the induction of an unresponsive state (i.e., anergy) in immune cells; enhancing or suppressing 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 specific classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events.Modulation can be assessed, for example, by changes in IFN-γ (interferon gamma) expression compared to wild-type or unmodified CD80 controls in primary T cell assays (see Zhao and Ji, Exp Cell Res. 2016 Jan1;340(1):132-138). Modulation can be assessed, for example, by changes in immune activity of engineered cells, such as changes in cytotoxic activity of engineered cells or changes in cytokine secretion of engineered cells, compared to cells engineered with wild-type CD80 transmembrane protein.

[0174] The term "multimerization domain" refers to an amino acid sequence that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, containing complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), each of which can be the same or different multimerization domains. The interaction between the complementary multimerization domains, e.g., the interaction between the first multimerization domain and the second multimerization domain, forms a stable protein-protein interaction to produce a multimer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the multimerization domain is the same and interacts with itself to form a stable protein-protein interaction between the two polypeptide chains. Generally, the polypeptide is directly or indirectly connected to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain or portion thereof, from IgG (including IgG1, IgG2, IgG3 or IgG4 subtypes), IgA, IgE, IgD and IgM, and modifications thereof.

[0175] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise limited, the terms encompass nucleic acids containing known analogs of natural nucleotides and nucleic acids that have similar binding properties and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly shown ("reference sequence"), but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0176] The term "molecular species" as used herein refers to a population of proteins with identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of identical or substantially identical molecular species. Thus, for example, human CD80 is an IgSF member, and each human CD80 molecule is a molecular species of CD80. Differences between molecules of the same molecular species can occur due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, small sequence differences within a single molecular species due to genetic polymorphisms constitute another form of difference within a single molecular species, as do wild-type truncated forms of a single molecular species due to, for example, proteolytic cleavage. A "cell surface molecular species" is a molecular species that is expressed on the surface of a mammalian cell. Two or more different protein species, each present only in one or only in the other (but not both) of two mammalian cells forming an IS, are said to be in "cis" or in a "cis configuration" with respect to each other. Two distinct protein species, the first of which is present only in the first of two mammalian cells that form an IS and the second of which is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or in a "trans configuration." Two distinct protein species, each of which is present in both of two mammalian cells that form an IS, are in both cis and trans configurations on those cells.

[0177] The term "non-competitive binding" as used herein refers to the ability of a protein to specifically bind to at least two cognate binding partners simultaneously. Thus, a protein can simultaneously bind to at least two different cognate binding partners, but the binding interactions do not have to be of the same duration, so in some cases, the protein is specifically bound to only one of the cognate binding partners. In some embodiments, the binding occurs under certain binding conditions. In some embodiments, the simultaneous binding is such that the binding of one cognate binding partner does not substantially inhibit the simultaneous binding to the 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 assessing non-competitive binding are well known in the art, such as those described in Perez de La Lastra et al., Immunology, 1999 Apr:96(4):663-670. In some cases, in a non-competitive interaction, 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. Thus, any effect of the binding of the second cognate binding partner on the binding of the cognate binding partner is via a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in an enzyme-substrate interaction, a non-competitive inhibitor binds to a site other than the active site of the enzyme. Non-competitive binding encompasses a non-competitive binding interaction in which 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 only binds to the second interaction site when the first interaction site is occupied by the first cognate binding partner.

[0178] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant CD80 or an engineered cell expressing a variant CD80 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharma- ceutically acceptable carrier, excipient, or diluent, respectively.

[0179] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked via peptide bonds. The term does not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of a polypeptide. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. The term also includes molecules of one or more amino acid analogs or non-standard or non-natural amino acids, which can be synthesized or recombinantly expressed using known protein engineering techniques. In addition, proteins may be derivatized.

[0180] The term "primary T cell assay" as used herein refers to an in vitro assay for measuring interferon-gamma ("IFN-γ") expression. A wide variety of such primary T cell assays are known in the art, such as the assay described in Example 6. In a preferred embodiment, the assay used is an anti-CD3 co-fixation assay. In this assay, primary T cells are stimulated with fixed anti-CD3 with or without additional recombinant proteins. At a certain time point (usually 24-72 hours), the culture supernatant is collected. In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. At a certain time point (usually 24-72 hours), the culture supernatant is collected. Human IFN-γ levels in the culture supernatant are measured by standard ELISA techniques. Commercial kits are available from suppliers, and the assay is performed according to the manufacturer's recommendations.

[0181] The term "purified," as applied to nucleic acids (e.g., encoding an immunomodulatory protein of the invention), generally refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or protein of the invention is at least about 50% pure, typically at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 99% or more pure (e.g., on a weight percent or molar basis).

[0182] The term "recombinant" indicates that a substance (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on a substance in or removed from its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining a nucleic acid, e.g., during cloning, affinity engineering, DNA shuffling or other well-known molecular biological procedures. A "recombinant DNA molecule" is made up of fragments of DNA that are connected together by such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or is otherwise altered by genetic engineering (e.g., by introducing into the cell a nucleic acid molecule that encodes a recombinant protein (e.g., a transmembrane immunomodulatory protein provided herein). Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a wide variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, as well as viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. The terms "in operable combination," "in operable order," and "operably linked," as used herein, refer to the linking of nucleic acid sequences in a manner or orientation that produces a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0183] The term "recombinant expression vector" as used herein refers to a DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Optionally, a secretory signal peptide sequence can also be encoded by the recombinant expression vector operably linked to the coding sequence of the recombinant protein (e.g., recombinant fusion protein) so that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell. The term includes vectors as autonomously replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Among the vectors are viral vectors, such as lentiviral vectors.

[0184] The term "selectivity" refers to the preference of a protein or polypeptide of interest for specific binding to one substrate, such as a cognate binding partner, compared to the specific binding to another substrate, such as a different cognate binding partner of the protein of interest. Selectivity can be measured by the binding activity (e.g., binding affinity) (e.g., K d1 ) and the binding activity (e.g., binding affinity) (e.g., K d2 ) can be reflected as a ratio.

[0185] The term "sequence identity" as used herein refers to sequence identity at the nucleotide or amino acid level between genes or proteins, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. The sequence identity of proteins can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, the sequence identity of nucleic acids can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for alignment of sequences for comparison are well known in the art, and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates the percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website.

[0186] The term "soluble" as used herein with respect to a protein means that the protein is not a membrane protein. In general, a soluble protein contains only the extracellular domain or a portion thereof of an IgSF family member receptor that contains an IgSF domain(s) or a specific binding fragment thereof, but does not contain a transmembrane domain. In some cases, the solubility of the protein can be improved by linking or binding 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 embodiments, the soluble protein is an Fc fusion protein.

[0187] The term "species" as used herein with respect to a polypeptide or nucleic acid refers to a population of molecules having identical or substantially identical sequences. Differences between polypeptides of the same species may occur due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated polypeptide sequences that differ from the full-length species by (or code for) no more than one, two, or three amino acid residues at the amino or carboxy terminus are considered to be sequences of a single species. Such microheterogeneity is a common feature of manufactured proteins.

[0188] The term "specific binding fragment", as used herein with reference to a full-length wild-type mammalian CD80 polypeptide or an IgV or IgC domain thereof, refers to a polypeptide having a subsequence of the IgV and / or IgC domain and specifically binds to mammalian CD28, mammalian PD-L1 and / or mammalian CTLA-4 (e.g., human or murine CD28, PD-L1 and / or CTLA-4) in vitro and / or in vivo. In some embodiments, a specific binding fragment of CD80 IgV or CD80 IgC is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the full-length wild-type sequence. The sequence of the specific binding fragment can be altered to form a variant CD80.

[0189] The term "specifically binds" as used herein refers to the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or avidity is at least 5 times greater than the average affinity or avidity of the same protein for a collection of random peptides or polypeptides of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times greater, or even at least 1000 times greater. A specifically binding protein need not only bind to a single target molecule, but may specifically bind to a non-target molecule due to similarity in structural form between the target and the non-target (e.g., paralog or ortholog). Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or specific binding to non-target molecules with substantially similar epitopes to the target molecule (e.g., paralogs) is possible and does not impair the specificity of binding determined for a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, the polypeptides of the present invention may specifically bind to two or more distinct target molecule species due to cross-reactivity. Solid-phase ELISA immunoassays or surface plasmon resonance (e.g., Biacore) measurements can be used to determine the specific binding between two proteins. Generally, the interaction between two binding proteins is greater than 1×10 -5 Less than M, often 1×10 -12 Low dissociation constant (K d In certain embodiments of the present disclosure, the interaction between the two binding proteins has a molecular weight of about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 It has a dissociation constant of M.

[0190] With respect to mammalian cells expressing a polypeptide, the term "surface expressed" or "surface expression" means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0191] As used herein, "synthetic" refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant and / or chemical synthesis methods, for example, with respect to a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide.

[0192] The term "targeting moiety" as used herein refers to a composition that is covalently or non-covalently attached to or physically encapsulates a polypeptide comprising a variant CD80. A targeting moiety has specific binding affinity for a desired counter structure, such as a cell surface receptor (e.g., the B7 family member PD-L1) or a tumor antigen (e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), e.g., B7-H6). Typically, the desired counter structure is localized on a particular tissue or cell type. A targeting moiety can be an antibody, an antigen-binding fragment (Fab), a V-cell, or a fusion protein. H and V L variable fragments (Fv), which contain V H and V L Single chain variable fragments (scFv) containing the V region of an antibody, as well as other antibody V region fragments, such as Fab', F(ab) 2 , F(ab′) 2 , dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecule (less than 500 daltons) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or non-covalently attached to the lipid membrane of liposomes that encapsulate the polypeptides of the invention.

[0193] The term "transmembrane protein" as used herein means a membrane protein that substantially or completely spans a lipid bilayer, which is found, for example, in biological membranes, such as mammalian cells, or in artificial constructs, such as liposomes. A transmembrane protein comprises a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. A transmembrane domain is generally predictable from the amino acid sequence of the transmembrane domain through any number of commercially available bioinformatics software applications, based on its increased hydrophobicity compared to the region of the protein that interacts with the aqueous environment (e.g., cytosol, extracellular fluid). A transmembrane domain is often a hydrophobic alpha helix that spans the membrane. A transmembrane protein can span both layers of a lipid bilayer one or more times. The transmembrane immunomodulatory proteins provided herein are included in transmembrane proteins. In addition to a transmembrane domain, the transmembrane immunomodulatory proteins of the present invention further comprise an ectodomain, and in some embodiments, an endodomain.

[0194] The terms "treating," "treatment," or "therapy" of a disease or disorder, as used herein, refer to slowing, halting, or reversing the progression of a disease or disorder, as evidenced by the reduction, arrest, or elimination of any of the clinical or diagnostic symptoms by administering a therapeutic composition of the invention (e.g., containing an immunomodulatory protein or engineered cells), either alone or in combination with another compound described herein. "Treating," "treatment," or "therapy" also refers to reducing the severity of symptoms in an acute or chronic disease or disorder, or reducing the relapse rate (e.g., in cases of relapsing or remitting autoimmune disease processes), or reducing inflammation in the case of inflammatory aspects of an autoimmune disease. As used herein in the context of cancer, the terms "treat" cancer, "inhibit" cancer, "inhibiting" cancer, or "inhibition" of cancer refer to at least one of a statistically significant reduction in tumor growth rate, halt in tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, or a statistically significant improvement in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria, such as, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST). "Prevent" a disease or disorder, "prophylaxis" of a disease or disorder, or "prevention" of a disease or disorder, as used in the context of the present invention, refers to the administration of an immune-modulating polypeptide or engineered cell of the present invention, either alone or in combination with another compound, to prevent the appearance or development of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of the development of a disease or disorder.

[0195] The term "tumor specific antigen" or "TSA" as used herein refers to a counter structure that is present primarily on tumor cells of a mammalian subject, but is not generally found on normal cells of the mammalian subject. A tumor specific antigen need not be present exclusively on tumor cells, but rather the proportion of cells of a particular mammal that bear the tumor specific antigen is sufficiently high, or the level of the tumor specific antigen on the surface of the tumor is sufficiently high, so that it can be targeted with an anti-tumor therapeutic agent (e.g., an immunomodulatory polypeptide of the present invention) and provide for preventing or treating the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells that present the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that present the TSA are cancerous.

[0196] The term "variant" (also "modified" or "mutant"), when used in reference to variant CD80, refers to a CD80 created by human intervention, e.g., mammalian (e.g., human or murine) CD80. A variant CD80 is a polypeptide having an altered amino acid sequence compared to unmodified or wild-type CD80. A variant CD80 is a polypeptide that differs from the wild-type CD80 isoform sequence by one or more amino acid substitutions, deletions, additions, or a combination thereof. For purposes herein, a variant CD80 contains at least one affinity-modifying domain whereby one or more amino acid differences occur in the IgSF domain (e.g., the IgV domain). A variant CD80 can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, e.g., amino acid substitutions. A variant CD80 polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified CD80, e.g., the sequence of SEQ ID NO:1, the mature sequence thereof, or portions thereof containing the extracellular domain or the IgSF domain. In some embodiments, the variant CD80 polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified CD80, including the sequence set forth in SEQ ID NO:2, SEQ ID NO:76, or SEQ ID NO:150, SEQ ID NO:3030, or SEQ ID NO:3031.

[0197] Non-naturally occurring and naturally occurring amino acids are included within the scope of permissible substitutions or additions. The variant CD80 is not limited to any particular method of production, including, for example, de novo chemical synthesis, de novo recombinant DNA technology, or a combination thereof. The variant CD80 of the present invention specifically binds to at least one or more of CD28, PD-L1, and / or CTLA-4 of a mammalian species. In some embodiments, the altered amino acid sequence results in altered (i.e., increased or decreased) binding affinity or avidity to CD28, PD-L1, and / or CTLA-4 compared to the unmodified or wild-type CD80 protein. Increased or decreased 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 the variant CD80 to CD28, PD-L1 and / or CTLA-4 can be at least 5% greater than that of unmodified or wild-type CD80, and in some embodiments can be at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of an unmodified or wild-type CD80 control value. The decrease in binding affinity or avidity of CD80 to CD28, PD-L1 and / or CTLA-4 can be to a value that is 95% or less of that of an unmodified or wild-type CD80 control value, and in some embodiments, can be 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of that of an unmodified or wild-type CD80 control value, or to a value that is undetectable. A variant CD80 polypeptide has an alteration in the primary amino acid sequence due to the substitution, addition, or deletion of amino acid residues. The term "variant" in the context of a variant CD80 polypeptide should not be construed as imposing any requirement of any particular starting composition or method by which the variant CD80 is made.A variant CD80 can be generated, for example, starting from wild-type mammalian CD80 sequence information, then modeled in silico for binding to CD28, PD-L1 and / or CTLA-4, and finally recombinantly or chemically synthesized to obtain a variant CD80. However, in another example, a variant CD80 can be generated by site-directed mutagenesis of unmodified or wild-type CD80. Thus, a variant CD80 refers to a composition of matter, but not necessarily a product produced by any given process. A wide variety of techniques may be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0198] The terms "wild-type" or "native" or "naturally occurring" as used herein in reference to biological material, such as a nucleic acid molecule, a protein (e.g., CD80), an IgSF member, a host cell, etc., refer to that which is found in nature and has not been modified by human intervention.

[0199] II. Variant CD80 Polypeptides Provided herein are variant CD80 polypeptides that exhibit altered (increased or decreased) binding activity or affinity to one or more CD80 binding partners. In some embodiments, the CD80 binding partner is CD28, PD-L1, or CTLA-4. In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications, such as one or more substitutions (or alternatively, "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain (IgD) compared to a wild-type or unmodified CD80 polypeptide, or a portion of a wild-type or unmodified CD80 containing IgD, or a specific binding fragment thereof. Thus, the variant CD80 polypeptides provided are or include variant IgD (hereinafter referred to as "vIgD"), in which one or more amino acid modifications (e.g., substitutions) are in IgD.

[0200] In some embodiments, the IgD comprises an IgV or IgC (e.g., IgC2) domain, or a specific binding fragment of an IgV or IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, the IgD can be IgV only, a combination of IgV and IgC including the entire extracellular domain (ECD), or any combination of the Ig domains of CD80. Table 2 provides exemplary residues corresponding to the IgV or IgC regions of CD80. In some embodiments, the variant CD80 polypeptide contains an IgV or IgC domain or a specific binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV or IgC domain or a specific binding fragment thereof. In some embodiments, the variant CD80 polypeptide contains an IgV domain or a specific binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV domain or a specific binding fragment thereof. In some embodiments, the altered IgV or IgC domain is an affinity-engineered IgSF domain, due to its altered binding activity or affinity.

[0201] In some embodiments, the variant has one more IgSF domain modified compared to the sequence of the unmodified CD80 sequence. In some embodiments, the unmodified CD80 sequence is wild-type CD80. In some embodiments, the unmodified or wild-type CD80 has the sequence of a native CD80 or an orthologue thereof. In some embodiments, the unmodified CD80 is or comprises the extracellular domain (ECD) of CD80 or a portion thereof that contains one or more IgSF domains (see Table 2). For example, the unmodified CD80 polypeptide is or comprises the IgV domain set forth as amino acids 35-135 of SEQ ID NO:1, amino acids 35-138 of SEQ ID NO:1 (see SEQ ID NO:3030), or amino acids 35-141 of SEQ ID NO:1. In some embodiments, the unmodified CD80 polypeptide is or comprises an IgC domain set forth as amino acids 145-230 of SEQ ID NO:1 or amino acids 142-232 of SEQ ID NO:1. In some embodiments, the extracellular domain of an unmodified or wild-type CD80 polypeptide comprises an IgV domain and an IgC domain(s). However, a variant CD80 polypeptide need not comprise both an IgV domain and an IgC domain(s). In some embodiments, a variant CD80 polypeptide comprises or consists essentially of an IgV domain or a specific binding fragment thereof. In some embodiments, a variant CD80 polypeptide comprises or consists essentially of an IgC domain or a specific binding fragment thereof. In some embodiments, a variant CD80 is soluble and lacks a transmembrane domain. In some embodiments, a variant CD80 further comprises a transmembrane domain and optionally also a cytoplasmic domain.

[0202] In some embodiments, the wild-type or unmodified CD80 polypeptide is a mammalian CD80 polypeptide, such as, but not limited to, a human, mouse, cynomolgus monkey, or rat CD80 polypeptide. In some embodiments, the wild-type or unmodified CD80 sequence is human.

[0203] In some embodiments, a wild-type or unmodified CD80 polypeptide (i) has an amino acid sequence as set forth in SEQ ID NO:1 or a mature form thereof lacking a signal sequence, (ii) has an amino acid sequence or a mature form thereof that exhibits at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO:1, or (iii) is a portion of (i) or (ii) that contains an IgV domain or an IgC domain or a specific binding fragment thereof.

[0204] In some embodiments, the wild-type or unmodified CD80 polypeptide is or comprises the extracellular domain of CD80 or a portion thereof. For example, in some embodiments, the unmodified or wild-type CD80 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or an orthologue thereof. For example, the unmodified or wild-type CD80 polypeptide can (i) comprise the sequence of amino acids set forth in SEQ ID NO:2, (ii) comprise an amino acid sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO:2, or (iii) be a specific binding fragment of (i) or (ii) comprising the IgV domain or the IgC domain. In some embodiments, the extracellular domain of wild-type or unmodified CD80 can bind to one or more CD80 binding proteins, such as one or more of CTLA-4, PD-L1, or CD28.

[0205] In some embodiments, the wild-type or unmodified CD80 polypeptide contains an IgV domain or an IgC domain or a specific binding fragment thereof. In some embodiments, the IgV domain of the wild-type or unmodified CD80 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:76, 150, 3030 or 3031, or an orthologue thereof. For example, the IgV domain of an unmodified or wild-type CD80 polypeptide can (i) contain an amino acid sequence set forth in SEQ ID NO:76, 150, 3030 or 3031, or (ii) contain an amino acid sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO:76, 150, 3030 or 3031, or (iii) is a specific binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain can bind to one or more CD80 binding proteins, such as one or more of CTLA-4, PD-L1 or CD28.

[0206] In some embodiments, the IgC domain of a wild-type or unmodified CD80 polypeptide comprises the amino acid sequence set forth as residues 145-230, 154-232, or 142-232 of SEQ ID NO:1, or an orthologue thereof. For example, an IgC domain of an unmodified or wild-type CD80 polypeptide can (i) contain an amino acid sequence set forth as residues 145-230, 154-232, or 142-232 of SEQ ID NO:1, or (ii) contain an amino acid sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to residues 145-230, 154-232, or 142-232 of SEQ ID NO:1, or (iii) is a specific binding fragment of (i) or (ii). In some embodiments, a wild-type or unmodified IgC domain can bind to one or more CD80 binding proteins.

[0207] In some embodiments, the wild-type or unmodified CD80 polypeptide contains a specific binding fragment of CD80, such as a specific binding fragment of an IgV domain or an IgC domain. In some embodiments, the specific binding fragment can bind to CTLA-4, PD-L1 and / or CD28. The specific binding fragment can have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific binding fragment of an IgV domain contains an amino acid sequence that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% of the length of the IgV domain set forth as amino acids 35-135, 35-138, 37-138 or 35-141 of SEQ ID NO:1. In some embodiments, a specific binding fragment of an IgC domain comprises an amino acid sequence that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the length of an IgC domain set forth as amino acids 145-230, 154-232, or 142-232 of SEQ ID NO:1.

[0208] In some embodiments, the variant CD80 polypeptide comprises an ECD domain or a portion thereof that comprises one or more affinity modified IgSF domains. In some embodiments, the variant CD80 polypeptide can comprise an IgV domain or an IgC domain, or a specific binding fragment of an IgV domain or a specific binding fragment of an IgC domain, where at least one of the IgV domain or the IgC domain contains one or more amino acid modifications (e.g., substitutions). In some embodiments, the variant CD80 polypeptide can comprise an IgV domain and an IgC domain, or a specific binding fragment of an IgV domain and a specific binding fragment of an IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific binding fragment of an IgV domain. In some embodiments, the variant CD80 polypeptide comprises a specific binding fragment of an IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain and a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain and a specific binding fragment of the IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific binding fragment of the IgV domain and a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific binding fragment of the IgV domain and a specific binding fragment of the IgC domain.

[0209] In any of such embodiments, the one or more amino acid modifications (e.g., substitutions) of the variant CD80 polypeptide can be located in any one or more of the CD80 polypeptide domains. For example, in some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the extracellular domain of the variant CD80 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgV domain or a specific binding fragment of the IgV domain. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgC domain or a specific binding fragment of the IgC domain.

[0210] Generally, each of the various attributes of the polypeptides (e.g., soluble and membrane-bound polypeptides, affinity of CD80 for CTLA-4, PD-L1, and CD28, number of mutations per polypeptide chain, number of linked polypeptide chains, number and nature of amino acid changes per variant CD80, etc.) are disclosed individually below. However, as will be apparent to one of skill in the art, any particular polypeptide may include a combination of these independent attributes. It will be understood that references to amino acids, including references to specific sequences set forth as SEQ ID NO: used to describe the domain organization of the IgSF domains, are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It will be understood that descriptions of the polypeptides, and their domains, are theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus may vary and is not necessarily the same from protein to protein. Thus, a particular IgSF domain (e.g., a particular IgV or IgC domain) may be several (e.g., 1, 2, 3, or 4) amino acids longer or shorter.

[0211] Moreover, various embodiments of the present invention as discussed below are frequently provided within the meaning of the terms defined as disclosed above. Thus, the embodiments described in a particular definition should be construed as being incorporated by reference when the defined terms are utilized in the discussion of the various aspects and attributes described herein. Thus, the headings, the order of presentation of the various aspects and embodiments, and the separate disclosure of each independent attribute are not meant to limit the scope of the present disclosure.

[0212] A. Exemplary Modifications Provided herein are variant CD80 polypeptides that contain at least one affinity-modified IgSF domain (e.g., IgV or IgC) or a specific binding fragment thereof, relative to the IgSF domain contained in a wild-type or unmodified CD80 polypeptide, such that the variant CD80 polypeptide exhibits altered (increased or decreased) binding activity or affinity for one or more cognate binding partners CTLA-4, PD-L1 or CD28, relative to the wild-type or unmodified CD80 polypeptide. In some embodiments, the variant CD80 polypeptide has a binding affinity for CTLA-4, PD-L1 or CD28 that differs from that of a wild-type or unmodified CD80 polypeptide control sequence, e.g., as measured by solid-phase ELISA immunoassay, flow cytometry or surface plasmon resonance (Biacore) assay. In some embodiments, the variant CD80 polypeptide has an increased binding affinity for CTLA-4, PD-L1 and / or CD28. In some embodiments, the variant CD80 polypeptide has a decreased binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide. CD28, PD-L1 and / or CTLA-4 can be mammalian proteins, such as human proteins or murine proteins.

[0213] The altered (e.g., increased or decreased) binding activity or affinity for CTLA-4, PD-L1, and / or CD28 is conferred by one or more amino acid modifications in the IgSF domain of the wild-type or unmodified IgSF domain. A wild-type or unmodified CD80 sequence does not necessarily have to be used as the starting composition to generate the variant CD80 polypeptides described herein. Thus, the use of the term "substitution" does not imply that the provided embodiments are limited to a particular method of making the variant CD80 polypeptide. Variant CD80 polypeptides can be made, for example, by de novo peptide synthesis, and thus do not necessarily require a "substitution" in the sense of changing the codon that codes for the substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which also do not imply a particular method of making. The means by which variant CD80 polypeptides are designed or made is not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified CD80 are mutagenized from wild-type or unmodified CD80 genetic material according to the methods disclosed in the Examples or other methods known to those of skill in the art, and screened for the desired specific binding affinity and / or induction of IFN-γ expression or other functional activity. In some embodiments, variant CD80 polypeptides are synthesized de novo utilizing protein or nucleic acid sequences available in 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, as previously described.

[0214] Unless otherwise indicated, as indicated throughout this disclosure, the amino acid modification(s) are designated by amino acid position numbers that correspond to the numbering of the positions in the unmodified ECD sequence set forth in SEQ ID NO:2, or, where applicable, the unmodified IgV sequence set forth in SEQ ID NO:76, 150, 3030 or 3031, as follows: TIFF2025076434000016.tif90166

[0215] It is within the level of ordinary skill in the art to identify the corresponding position of a modification (e.g., amino acid substitution) in a CD80 polypeptide (including a portion thereof containing its IgSF domain (e.g., IgV)) by alignment of a reference sequence with, for example, SEQ ID NO:2 or SEQ ID NO:76 or SEQ ID NO:150 or SEQ ID NO:3030 or SEQ ID NO:3031. In describing modifications throughout this disclosure, the amino acid position is shown in the center, the corresponding unmodified (e.g., wild-type) amino acid is listed before the number, and the amino acid substitution of the identified variant is listed after the number. If the modification is a deletion at the position, it is indicated as "del" and if the modification is an insertion at the position, it is indicated as "ins." In some cases, an insertion is described with the amino acid position shown in the center, the corresponding unmodified (e.g., wild-type) amino acid is listed before and after the number, and the amino acid insertion of the identified variant is listed after the unmodified (e.g., wild-type) amino acid.

[0216] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) in a wild-type or unmodified CD80 sequence. The one or more amino acid modifications (e.g., substitutions) can be in an ectodomain (extracellular domain) of the wild-type or unmodified CD80 sequence, e.g., the extracellular domain. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in an IgV domain or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in an IgC domain or a specific binding fragment thereof. In some embodiments of the variant CD80 polypeptide, some of the one or more amino acid modifications (e.g., substitutions) are in an IgV domain or a specific binding fragment thereof and some of the one or more amino acid modifications (e.g., substitutions) are in an IgC domain or a specific binding fragment thereof.

[0217] In some embodiments, the variant CD80 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 the IgC domain. In some embodiments, the variant CD80 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, a variant CD80 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 IgC domain or specific binding fragment thereof. In some embodiments, a variant CD80 polypeptide has at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to a wild-type or unmodified CD80 polypeptide or specific binding fragment thereof (e.g., the amino acid sequence of SEQ ID NO:2, 76, 150, 3030 or 3031).

[0218] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) in unmodified CD80 or a specific binding fragment thereof corresponding to position(s) 7, 13, 15, 16, 20, 22, 23, 24, 25, 26, 27, 30, 31, 33, 34, 35, 36, 38, 41, 42, 43, 46, 47, 48, 51, 53, 54, 55, 57, 58, 61, 62, 65, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 84, 85, 86, 87, 88, 92, 94, 95 and / or 97 based on the numbering of SEQ ID NO:2. In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) in unmodified CD80 or a specific binding fragment thereof corresponding to positions 7, 23, 26, 30, 34, 35, 46, 51, 55, 57, 58, 65, 71, 73, 78, 79, 82, or 84 based on the numbering of SEQ ID NO:2. In some embodiments, the variant CD80 polypeptide has modifications, e.g., amino acid substitutions, at any two or more of the foregoing positions, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more.

[0219] In some embodiments, the variant CD80 polypeptide is TIFF2025076434000017.tif77166. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from E7D, E23D, E23G, A26E, A26P, A26S, A26T, I30F, I30T, I30V, K34E, E35D, E35G, D46E, D46V, P51A, N55D, N55I, T57A, T57I, I58V, L65P, A71D, A71G, R73S, G78A, T79A, T79I, T79L, T79P, C82R, V84A, V84I, L85Q, or conservative amino acid substitutions thereof. In some embodiments, the variant CD80 polypeptide comprises any two or more of the above-mentioned amino acid substitutions, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the variant CD80 polypeptide comprises only one amino acid difference compared to an unmodified or wild-type CD80 polypeptide that comprises only one of the above-mentioned amino acid substitutions.

[0220] In some embodiments, the variant CD80 polypeptide contains one or more additional amino acid modifications (e.g., substitutions) to unmodified CD80 or a specific binding fragment thereof corresponding to positions 12, 18, 29, 31, 37, 38, 41, 43, 44, 47, 61, 67, 68, 69, 70, 72, 77, 83, 88, 89, 90, 91 or 93 based on the numbering of SEQ ID NO:2. In some embodiments, the variant CD80 polypeptide has one or more additional amino acid substitutions selected from among A12T, A12V, H18L, H18Y, R29H, Y31H, K37E, M38T, T41A, M43I, S44P, M47L, M47T, I67T, V68A, V68M, I69T, L70P, L70R, L70Q, L72P, E77G, V83A, V83I, E88D, K89E, K89N, D90G, D90N, A91T, K93R.

[0221] A conservative amino acid substitution is any amino acid that belongs to the same class of amino acids 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). Thus, for example, conservative amino acid substitutions for the A26E substitution include A26D, A26N, and A26Q amino acid substitutions.

[0222] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 18 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution H18Y, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 26, 35, 46, 47, 68, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: A26E, E35D, D46E, D46V, M47I, M47L, V68M, A71G, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / A26E, H18Y / E35D, H18Y / D46E, H18Y / D46V, H18Y / M47I, H18Y / M47L, H18Y / V68M, H18Y / A71G, H18Y / L85Q, H18Y / D90G. The variant CD80 polypeptide can be provided with further amino acid modifications according to the embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0223] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 26 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution A26E, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 35, 46, 47, 68, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, E35D, D46E, D46V, M47I, M47L, V68M, A71G, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / A26E, A26E / E35D, A26E / D46E, A26E / D46V, A26E / M47I, A26E / M47L, A26E / V68M, A26E / A71G, A26E / L85Q, A26E / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein, according to the embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0224] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 35 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution E35D, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 46, 47, 68, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, D46E, D46V, M47I, M47L, V68M, A71G, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / E35D, A26E / E35D, E35D / D46E, E35D / D46V, E35D / M47I, E35D / M47L, E35D / V68M, E35D / A71G, E35D / L85Q, E35D / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein according to the embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides as described. In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 46 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution D46E or D46V, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 47, 68, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of H18Y, A26E, E35D, M47I, M47L, V68M, A71G, L85Q, or D90G amino acid substitutions, or conservative amino acid substitutions thereof.In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / D46E, A26E / D46E, E35D / D46E, D46E / M47I, D46E / M47L, D46E / V68M, D46E / A71G, D46E / L85Q, D46E / D90G. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / D46V, A26E / D46V, E35D / D46V, D46V / M47I, D46V / M47L, D46V / V68M, D46V / A71G, D46V / L85Q, D46V / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein according to the embodiments provided. Table 1 describes exemplary amino acid modified and variant CD80 polypeptides.

[0225] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 47 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution M47I or M47L, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 46, 68, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, E35D, D46E, D46V, V68M, A71G, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / M47I, A26E / M47I, E35D / M47I, M47I / D46E, M47I / D46V, M47I / V68M, M47I / A71G, M47I / L85Q, or M47I / D90G. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / M47L, A26E / M47L, E35D / M47L, M47L / D46E, M47L / D46V, M47L / V68M, M47L / A71G, M47L / L85Q, or M47L / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein according to the embodiments provided. Table 1 describes exemplary amino acid modified and variant CD80 polypeptides.

[0226] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 68 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution V68M, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 46, 47, 71, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, E35D, D46E, D46V, M47I, M47L, A71G, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, variant CD80 polypeptides comprise amino acid modifications H18Y / V68M, A26E / V68M, E35D / V68M, D46E / V68M, D46V / D68M, M47I / V68M, M47L / V68M, V68M / A71G, V68M / L85Q, V68M / D90G. Variant CD80 polypeptides can comprise additional amino acid modifications, such as those described herein, according to embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0227] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 71 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution A71G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 46, 47, 68, 85, or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, E35D, D46E, D46V, M47I, M47L, V68M, L85Q, or D90G, or a conservative amino acid substitution thereof. In some embodiments, variant CD80 polypeptides comprise amino acid modifications H18Y / A71G, A26E / A71G, E35D / A71G, D46E / A71G, D46V / D68M, M47I / A71G, M47L / A71G, V68M / A71G, A71G / L85Q, A71G / D90G. Variant CD80 polypeptides can comprise additional amino acid modifications, such as those described herein, according to embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0228] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 85 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution L85Q, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 46, 47, 68, 71 or 90. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, E35D, D46E, D46V, M47I, M47L, V68M, A71G, or D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / L85Q, A26E / L85Q, E35D / L85Q, D46E / L85Q, D46V / D68M, M47I / L85Q, M47L / L85Q, V68M / L85Q, A71G / L85Q, L85Q / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein, according to the embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0229] In some embodiments, the variant CD80 polypeptide comprises an amino acid modification of unmodified CD80 or a specific binding fragment thereof at a position corresponding to position 90 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the amino acid modification is the amino acid substitution D90G, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more of positions 18, 26, 35, 46, 47, 68, 71, or 85. In some embodiments, the one or more amino acid modifications are one or more of the following amino acid substitutions: H18Y, A26E, E35D, D46E, D46V, M47I, M47L, V68M, A71G, or L85Q, or a conservative amino acid substitution thereof. In some embodiments, the variant CD80 polypeptide comprises the amino acid modifications H18Y / D90G, A26E / D90G, E35D / D90G, D46E / D90G, D46V / D68M, M47I / D90G, M47L / D90G, V68M / D90G, A71G / D90G, L85Q / D90G. The variant CD80 polypeptide can comprise further amino acid modifications such as those described herein, according to the embodiments provided. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0230] In some embodiments, the variant CD80 polypeptide does not contain amino acid modifications to the unmodified CD80 polypeptide set forth in SEQ ID NOs:2, 76 or 150, and the amino acid modifications are only H18Y / M47I / T57I / A71G, H18Y / A26T / E35D / A71D / L85Q or H18Y / A71D / L72P / E88V. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NOs:41, 59, 66, 115, 133, 140, 189, 207 or 214.

[0231] In some embodiments, the variant CD80 polypeptide does not contain the amino acid modifications in the unmodified CD80 polypeptide set forth in SEQ ID NOs:2, 76 or 150, and the only amino acid modifications are A26E / E35D / M47L / L85Q. In some embodiments, the variant CD80 polypeptide is not the polypeptide set forth in SEQ ID NOs:73, 147 or 221.

[0232] In some embodiments, the variant CD80 polypeptide does not contain an amino acid modification to the unmodified CD80 polypeptide set forth in SEQ ID NO:2, 76, or 150, and the amino acid modifications are E35D / M47I / L65P / D90N, L25S / E35D / M47I / D90N, E35D / A71D, E35D / M47I, E35D / T57I / L70Q / A71D, E35D / A71D, E35D / I67L / A71D.E35D, E35D / M47I / L70M, E35D / A71D / L72V, E35D / M43L / L70M, A26P / E35D / M43I / L85Q / E88D, E35D / D46V / L85Q, Q2 7L / E35D / M47I / T57I / L70Q / E88D, E35D / T57A / A71D / L85Q, H18Y / A26T / E35D / A71D / L85Q, E35D / M47L, E35D / M43I / A71D, E23G / A26S / E35D / T62N / A71D / L 72V / L85M, A12T / E24D / E35D / D46V / I61V / L72P / E95V, V22L / E35D / M43L / A71G / D76H, A26E / E35D / M47L / L85Q, Y31H / E35D / T41S / V68L / K93R / R94W. In some embodiments, the variant CD80 polypeptide is selected from the group consisting of SEQ ID NO: 72V / L85M, A12T / E24D / E35D / D46V / I61V / L72P / E95V, V22L / E35D / M43L / A71G / D76H, A26E / E35D / M47L / L85Q, Y31H / E35D / T41S / V68L / K93R / R94W. NO:19, 20, 28, 29, 37, 46, 47, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 64, 68, 69, 70, 7 3, 75, 93, 94, 102, 103, 111, 120, 121, 124, 125, 126, 127, 128, 129, 130, 132, 133, 1

[0033] The polypeptide is not a polypeptide described in any one of claims 34, 138, 142, 143, 144, 147, 149, 167, 168, 176, 177, 185, 194, 195, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 212, 216, 217, 218, 221 or 223.

[0233] In some embodiments, the variant CD80 polypeptide does not contain amino acid modifications to the unmodified CD80 polypeptide set forth in SEQ ID NOs:2, 76 or 150, and the amino acid modifications are only E35D / D46V / L85Q, A12T / E24D / E35D / D46V / I61V / L72P / E95V or D46E / A71D. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NOs:55, 69, 74, 129, 143, 148, 203, 217 or 222.

[0234] In some embodiments, the variant CD80 polypeptide does not contain amino acid modifications to the unmodified CD80 polypeptide set forth in SEQ ID NO:2, 76, or 150, and the only amino acid modifications are E35D / M47I / L65P / D90N, L25S / E35D / M47I / D90N, E35D / M47I, M47L / V68A, M47I / E88D, H18Y / M47I / T57I / A71G, T13R / M42V / M47I / A71D, E35D / M47I / L70M, Q27L / E35D / M47I / T57I / L70Q / E88D, E35D / M47L, A26E / E35D / M47L / L85Q. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NOs:19, 20, 29, 33, 38, 41, 49, 51, 56, 60, 73, 93, 94, 103, 107, 112, 115, 123, 125, 130, 134, 147, 167, 168, 177, 181, 186, 189, 197, 199, 204, 208, 221.

[0235] In some embodiments, the variant CD80 polypeptide does not contain the amino acid modifications in the unmodified CD80 polypeptide set forth in SEQ ID NOs:2, 76 or 150, and the only amino acid modifications are A26E / E35D / M47L / L85Q. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NOs:62, 136, 210.

[0236] In some embodiments, the variant CD80 polypeptide does not contain amino acid modifications to the unmodified CD80 polypeptide set forth in SEQ ID NOs:2, 76 or 150, and the amino acid modifications are only H18Y / M47I / T57I / A71G or V22L / E35D / M43L / A71G / D76H. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NOs:41, 70, 115, 144, 189 or 218.

[0237] In some embodiments, the variant CD80 polypeptide does not contain an amino acid modification to the unmodified CD80 polypeptide set forth in SEQ ID NO:2, 76, or 150, and the amino acid modifications are only A26P / E35D / M43I / L85Q / E88D, E35D / D46V / L85Q, E35D / T57A / A71D / L85Q, H18Y / A26T / E35D / A71D / L85Q, or A26E / E35D / M47L / L85Q. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NO:54, 55, 58, 59, 73, 128, 129, 132, 133, 147, 202, 203, 206, 207, or 221.

[0238] In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, at positions corresponding to E35D and M47L. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, corresponding to E35D and M47L. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, corresponding to E35D and A71G. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, corresponding to E35D and M47V. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, corresponding to E35D and V68M. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications in unmodified CD80, or a specific binding fragment thereof, corresponding to H18Y and E35D.

[0239] In some embodiments, the variant CD80 polypeptide comprises at least three amino acid modifications, including modifications at three or more positions corresponding to positions 18, 26, 35, 46, 47, 68, 71, 85, or 90 based on the numbering of positions set forth in SEQ ID NO:2. In some embodiments, the at least three amino acid modifications comprise amino acid modifications corresponding to H18Y, A26E, E35D, D46E, D46V, M47I, M47L, V68M, A71G, L85Q, or D90G, or conservative amino acid substitutions thereof, in unmodified CD80 or a specific binding fragment thereof.

[0240] In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D / M47L / V68M in unmodified CD80 or a specific binding fragment thereof.

[0241] In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D / M47V / V68M in unmodified CD80 or a specific binding fragment thereof.

[0242] In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D / M47L / L85Q in unmodified CD80 or a specific binding fragment thereof.

[0243] In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to H18Y / E35D / M47I in unmodified CD80 or a specific binding fragment thereof.

[0244] In some embodiments, the variant CD80 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NO: for the extracellular domain (ECD) or IgV domain of wild-type CD80 or exemplary variant CD80 polypeptides. As indicated, the exact locus or residues corresponding to a given domain may vary depending, for example, on the method used to identify or classify the domain. Additionally, in some cases, adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., IgV) may also be included in the sequence of the variant IgSF polypeptide, for example, to ensure proper folding of the domain when expressed. Thus, it will be understood that the SEQ ID NO: examples in Table 1 should not be construed as limiting. For example, a particular domain (e.g., IgV domain) of a variant CD80 polypeptide may be several amino acids longer or shorter than the amino acid sequence set forth in the respective SEQ ID NO:, e.g., 1-10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0245] In some embodiments, the variant CD80 polypeptide comprises any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs:3-75, 2009-2104, 2297-2507, 2930-2960). In some embodiments, the variant CD80 polypeptide comprises a polypeptide sequence exhibiting at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs:3-75, 2009-2104, 2297-2507, 2930-2960), and contains an amino acid modification (e.g., substitution)(s) that are not present in wild-type or unmodified CD80. In some embodiments, a variant CD80 polypeptide comprises a specific binding fragment of any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 3-75, 2009-2104, 2297-2507, 2930-2960) and contains an amino acid modification (e.g., substitution)(s) that are not present in wild-type or unmodified CD80. In some embodiments, a variant CD80 polypeptide comprises any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 77-149, 151-223, 2105-2296, 2508-2929, 2961-3022).In some embodiments, the variant CD80 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs:77-149, 151-223, 2105-2296, 2508-2929, 2961-3022), and contains an amino acid modification (e.g., substitution)(s) that are not present in wild-type or unmodified CD80. In some embodiments, the variant CD80 polypeptide comprises a specific binding fragment of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs:77-149, 151-223, 2105-2296, 2508-2929, 2961-3022) and contains an amino acid modification (e.g., substitution)(s) that are not present in wild-type or unmodified CD80.

[0246] Table 1 also provides exemplary sequences by reference to SEQ ID NO: for the extracellular domain (ECD) or IgV domain of wild-type CD80 or exemplary variant CD80 polypeptides. As indicated, the exact locus or residues corresponding to a given domain may vary, depending, for example, on the method used to identify or classify the domain. Additionally, in some cases, the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., ECD) may also be included in the sequence of the variant IgSF polypeptide, for example, to ensure proper folding of the domain when expressed. Thus, it will be understood that the SEQ ID NO: examples in Table 1 should not be construed as limiting. For example, a particular domain (e.g., IgV domain) of a variant CD80 polypeptide may be several amino acids longer or shorter than the amino acid sequence set forth in the respective SEQ ID NO:, e.g., 1-10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0247] Table 1. Exemplary variant CD80 polypeptides TIFF2025076434000018.tif187169TIFF2025076434000019.tif230169TIFF2025076434000020.tif230169TIFF2025076434000021.tif230169 TIFF2025076434000022.tif230169TIFF2025076434000023.tif230169TIFF2025076434000024.tif230169TIFF2025076434000025.tif243169

[0248] In some embodiments, one or more amino acid modifications of the variant CD80 polypeptides provided herein produce at least one affinity-modified IgSF domain (such as, for example, IgV or IgC) or a specific binding fragment thereof, compared to the IgSF domain contained in the wild-type or unmodified CD80 polypeptide, such that the variant CD80 polypeptide exhibits altered (increased or decreased) binding activity or affinity for one or more binding partners, CTLA-4, PD-L1, or CD28, compared to the wild-type or unmodified CD80 polypeptide. In some embodiments, the variant CD80 polypeptide has a binding affinity for CTLA-4, PD-L1, or CD28 that differs from that of a wild-type or unmodified CD80 polypeptide control sequence, e.g., as measured by solid-phase ELISA immunoassays, flow cytometry, or surface plasmon resonance (Biacore) assays. In some embodiments, the variant CD80 polypeptide has an increased binding affinity for CTLA-4, PD-L1, and / or CD28. In some embodiments, the variant CD80 polypeptide has a decreased binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide. CD28, PD-L1 and / or CTLA-4 can be mammalian proteins, such as human proteins or murine proteins.

[0249] The binding affinity of each of the binding partners is independent, i.e., in some embodiments, a variant CD80 polypeptide has increased binding affinity for one, two, or three of CD28, PD-L1, and CTLA-4, and / or has decreased binding affinity for one, two, or three of CD28, PD-L1, and CTLA-4, compared to a wild-type or unmodified CD80 polypeptide.

[0250] In some embodiments, the variant CD80 polypeptide has increased binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has increased binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has increased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0251] In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4 and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4 and decreased binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4 and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4 and increased binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides.

[0252] In some embodiments, the variant CD80 polypeptide has increased binding affinity for CTLA-4 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has increased binding affinity for CTLA-4 and decreased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for CTLA-4 and CD28 compared to wild-type or unmodified CD80 polypeptides. In these embodiments,

[0253] In some embodiments, the variant CD80 polypeptides have increased binding affinity for PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have increased binding affinity for PD-L1 and decreased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for PD-L1 and increased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides.

[0254] In some embodiments, the variant CD80 polypeptide exhibits a binding affinity to the ectodomain of human CTLA-4 that is equal to or less than the binding affinity of unmodified or wild-type CD80 to the ectodomain of human CTLA-4.

[0255] In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4, PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4 and PD-L1 and decreased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4 and CD28 and decreased binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4 and PD-L1 and increased binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4 and increased binding affinity for PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have increased binding affinity for CTLA-4 and decreased binding affinity for PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4, PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have decreased binding affinity for CTLA-4 and increased binding affinity for PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides.

[0256] In some embodiments, variant CD80 polypeptides having increased or greater binding affinity to CD28, PD-L1, and / or CTLA-4 have an increase in binding affinity of at least about 5%, such as at least about 10%, about 15%, about 20%, about 25%, about 35%, or about 50%, to a CTLA-4, PD-L1, and / or CD28 binding partner(s) relative to a wild-type or unmodified CD80 polypeptide control. In some embodiments, the increase in binding affinity relative to a wild-type or unmodified CD80 polypeptide is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold or more. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (eg, substitutions).

[0257] In some embodiments, a variant CD80 polypeptide that has reduced or decreased binding affinity to CTLA-4, PD-L1, and / or CD28 has a reduction in binding affinity to CTLA-4, PD-L1, and / or CD28 of at least 5%, e.g., at least about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more, relative to a wild-type or unmodified CD80 polypeptide control. In some embodiments, the reduction in binding affinity relative to a wild-type or unmodified CD80 polypeptide is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0258] In some embodiments, the equilibrium dissociation constant (K d ) is at least 1 × 10-5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1×10 -12 It can be M.

[0259] In some embodiments, provided variant CD80 polypeptides containing at least one affinity modified IgSF domain (e.g., IgV or IgC, etc.) or specific binding fragments thereof compared to an IgSF domain contained in a wild-type or unmodified CD80 polypeptide exhibit altered (increased / stimulating or decreased / inhibited) signaling induced by one or more functional binding partners, such as CTLA-4 or CD28 expressed on the surface of a signaling competent cell, such as a T cell, capable of releasing cytokines in response to an intracellular signal, compared to a wild-type or unmodified CD80 polypeptide upon binding to the one or more binding partners. In some embodiments, the altered signaling is different from that provided by a wild-type or unmodified CD80 polypeptide control sequence in the same format, as determined by an assay that measures, for example, cytokine release (e.g., IL-2 release) after incubation with a particular variant and / or wild-type or unmodified CD80 polypeptide. Exemplary assays are described in Examples 8-10. In the exemplary assays, cytokine release is a function of the sum of the signaling activity of functional binding partners expressed on the surface of the cytokine-releasing cell. As described elsewhere herein, in some embodiments, the format of the variant CD80 polypeptide provided may affect the type of activity (eg, agonistic or antagonistic).

[0260] Because CTLA-4 induces inhibitory signaling, increased CTLA-4 signaling results in decreased cytokine release in some exemplary assays. Conversely, decreased CTLA-4 signaling decreases inhibitory signaling but not cytokine release, which may result in increased cytokine release in some assays. Because CD28 signaling stimulates cytokine release, increased CD28 signaling results in increased cytokine release in exemplary assays. Conversely, decreased CD28 signaling results in decreased cytokine release in exemplary assays.

[0261] In some embodiments, the variant CD80 polypeptides increase CTLA-4, PD-L1, and / or CD28 mediated signaling, hi some embodiments, the variant CD80 polypeptides decrease CD28, PD-L1, and / or CTLA-4 mediated signaling compared to a wild-type or unmodified CD80 polypeptide.

[0262] The binding affinity of each of the cognate binding partners is independent, and thus, in some embodiments, a variant CD80 polypeptide can increase signaling induced by one, two or three of CD28, PD-L1, and CTLA-4, and / or decrease signaling induced by one, two or three of CD28, PD-L1, and CTLA-4, compared to a wild-type or unmodified CD80 polypeptide.

[0263] In some embodiments, the variant CD80 polypeptides increase CTLA-4 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides increase PD-L1 / PD-1 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides increase CD28 induced signaling upon binding compared to wild-type or unmodified CD80 polypeptides. In some preferred embodiments, the variant CD80 polypeptides decrease CD28 induced signaling upon binding compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease PD-L1 / PD-1 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 induced signaling compared to wild-type or unmodified CD80 polypeptides.

[0264] In some embodiments, the variant CD80 polypeptides increase CTLA-4 and CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides increase CTLA-4 induced signaling and decrease CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 and CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides.

[0265] In some embodiments, the variant CD80 polypeptides increase CTLA-4 and CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides increase CTLA-4 induced signaling and decrease CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides increase CTLA-4 and CD28 induced signaling. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 induced signaling and increase CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 induced signaling and increase CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 induced signaling and increase CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides decrease CTLA-4 and CD28 induced signaling compared to wild-type or unmodified CD80 polypeptides.

[0266] In some embodiments, variant CD80 polypeptides that stimulate or increase inhibitory signaling induced by CTLA-4 generate a signal that is 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than the signal induced by a wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0267] In some embodiments, a variant CD80 polypeptide that stimulates or increases CD28-induced signaling generates a signal that is at least 105%, 110%, 120%, 150%, 200%, 300%, 400%, or 500%, or more, of the signal induced by a wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0268] In some embodiments, a variant CD80 polypeptide that inhibits or reduces inhibitory signaling induced by CTLA-4 generates a signal that is at least 105%, 110%, 120%, 150%, 200%, 300%, 400%, or 500%, or more, of the signal induced by a wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0269] In some embodiments, a variant CD80 polypeptide that inhibits or reduces CD28-induced inhibitory signaling produces a signal that is 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than the signal induced by a wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0270] In some embodiments, variant CD80 polypeptides that affect inhibitory signaling induced by CTLA-4 and / or affect signaling by CD28 provide a sum of CTLA-4 and CD28 signaling that is less than the sum of CTLA-4 and CD28 signaling provided by the corresponding wild-type or unmodified CD80 polypeptide. In such embodiments, the sum of CTLA-4 and CD28 signaling is 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the signal provided by the corresponding wild-type or unmodified CD80 polypeptide. In such examples, the corresponding wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0271] In some embodiments, a variant CD80 polypeptide that affects inhibitory signaling induced by CTLA-4 and / or affects signaling by CD28 results in a sum of CTLA-4 and CD28 signaling that is greater than the sum of CTLA-4 and CD28 signaling affected by a corresponding wild-type or unmodified CD80 polypeptide. In such embodiments, the sum of CTLA-4 and CD28 signaling is at least 105%, 110%, 120%, 150%, 200%, 300%, 400%, or 500% or more of the signal affected by the corresponding wild-type or unmodified CD80 polypeptide. In such examples, the corresponding wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0272] 1.CTLA4 In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide, such as a wild-type or unmodified CD80 polypeptide comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of CTLA-4 and decreased affinity for the ectodomain of CD28 compared to a wild-type or unmodified CD80 polypeptide comprising, for example, a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the increased affinity for the ectodomain of CTLA-4 is increased by greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to the binding affinity of unmodified CD80 to the ectodomain of CTLA-4.

[0273] In some of these embodiments, the variant CD80 polypeptides that exhibit increased binding affinity for CTLA-4 compared to a wild-type or unmodified CD80 polypeptide have one or more amino acid modifications (e.g., substitutions) corresponding to positions 7, 12, 13, 16, 18, 20, 22, 23, 24, 26, 27, 30, 33, 35, 37, 38, 41, 42, 43, 44, 46, 47, 48, 52, 53, 54, 57, 58, 61, 62, 63, 67, 68, 69, 70, 71, 72, 73, 74, 77, 79, 81, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, and / or 97 of SEQ ID NO:2, 76, 150, 3030, or 3031. In some of these embodiments, the variant CD80 polypeptide that exhibits increased binding affinity for CTLA-4 compared to a wild-type or unmodified CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 7, 23, 26, 30, 35, 46, 57, 58, 71, 73, 79 and / or 84 of SEQ ID NO:2, 76, 150, 3030, or 3031.

[0274] In some embodiments, the variant CD80 polypeptide is In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2025076434000027.tif55165.

[0275] In some embodiments, the one or more amino acid substitutions are TIFF2025076434000028.tif85160TIFF2025076434000029.tif231163TIFF2025076434000030.tif252164.

[0276] In some embodiments, the variant CD80 polypeptides exhibit increased selectivity for CTLA-4 to CD28, as indicated by a ratio of CTLA-4 binding to CD28 binding (CTLA4:CD28 binding ratio) of greater than 1, compared to the binding ratio of CTLA-4 binding to CD28 binding to an unmodified CD80 polypeptide (e.g., as set forth in SEQ ID NOs:2, 76, 150, 3030, or 3031). In some embodiments, the variant CD80 polypeptide is greater than or equal to about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more. or greater than 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. In some of these embodiments, the variant CD80 polypeptide has one or more amino acid modifications (eg, substitutions) corresponding to positions 30, 35, 57, 71, or 84 of SEQ ID NO:2, 76, 150, 3030, or 3031.In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: T13A, T13R, S15T, V22I, V22L, Q27H, I30V, Q33R, E35D, E35G, T41S, M47I, M47L, M47V, N48Y, Y53F, T57I, I61F, I61V, I67L, L70M, A71D, A71G, L72V, T79M, E81G, E81K, V84A, V84I, and L85M, Y87C, Y87D. In some embodiments, the one or more amino acid substitutions are: TIFF2025076434000031.tif106165TIFF2025076434000032.tif231160TIFF2025076434000033.tif62164.

[0277] 2.CD28 In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of CD28 compared to wild-type or unmodified CD80 polypeptides, e.g., comprising a sequence set forth in SEQ ID NOs:2, 76, 150, 3030, or 3031. In some embodiments, the increased affinity for the ectodomain of CD28 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, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold compared to the binding affinity of unmodified CD80 to the ectodomain of CD28.

[0278] In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the CD28 ectodomain and the CTLA-4 ectodomain compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the CD28 ectodomain, the PD-L1 ectodomain, and the CTLA-4 ectodomain compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the increased affinity for CD28 and one or both of the ectodomains of CTLA-4 and PD-L1 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, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, or 450-fold compared to the binding affinity of unmodified CD80 to the ectodomains of CTLA-4 or PD-L1.

[0279] In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the CD28 ectodomain and decreased affinity for the CTLA-4 ectodomain compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the CD28 ectodomain and the PD-L1 ectodomain, and decreased affinity for the CTLA-4 ectodomain compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the decreased affinity for the ectodomain of CTLA-4 is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold decreased compared to the binding affinity of unmodified CD80 to the ectodomain of CTLA-4.

[0280] In some of these embodiments, the variant CD80 polypeptides that exhibit increased binding affinity for CD28 compared to a wild-type or unmodified CD80 polypeptide have one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 13, 18, 20, 22, 23, 24, 26, 27, 31, 35, 41, 42, 43, 46, 47, 54, 55, 57, 58, 61, 62, 67, 68, 69, 70, 71, 72, 79, 83, 84, 85, 88, 90, 93, 94, and / or 95 of SEQ ID NO:2, 76, 150, 3030, or 3031. In some of these embodiments, the variant CD80 polypeptide that exhibits increased binding affinity for CD28 compared to a wild-type or unmodified CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 23, 26, 35, 46, 55, 57, 58, 71, 79 and / or 84 of SEQ ID NO:2, 76, 150, 3030, or 3031.

[0281] In some embodiments, the variant CD80 polypeptide is selected from the group consisting of A12T, T13R, S15T, H18A, H18C, H18F, H18I, H18T, H18V, H18Y, V20I, S21P, V22A, V22D, V22L, E23D, E23G, E24D, A26D, A26E, A26G, A26H, A26L ... 26K, A26N, A26P, A26Q, A26R, A26S, A26T, Q27H, Q27L, Q27R, Y31H, Q33R, E35D, E35G, K 37E, M38I, T41S, M42V, M43I, M43L, D46E, D46N, D46V, M47I, M47L, M47V, M47Y, N48K, N 48Y, Y53F, K54E, N55I, T57A, T57I, I58V, I61F, I61V, T62A, T62N, T62S, N64S, I67L, V 68E, V68I, V68L, V68M, I69F, L70M, L70Q, L70R, A71D, A71G, L72P, L72V, T79I, T79M, V and one or more amino acid substitutions selected from the group consisting of 83I, V84I, L85M, L85Q, Y87C, Y87D, Y87N, E88D, E88V, D90G, D90N, D90P, A91G, A91S, K93E, K93R, R94L, R94Q, R94W, E95K, E95V, and L97Q. In some embodiments, the variant CD80 polypeptide is selected from the group consisting of T13R, S15T, H18A, H18C, H18F, H18I, H18T, H18V, V20I, V22D, V22L, E23D, E23G, E24D, A26D, A26E, A26G, A26H, A26K, A26N, A26P, A26Q, A26R, A26S, A26T, Q27H, Q27L, Q33R, E35D, E35G, T41S, M42V, M43L, D46E, D46N, D46V, M47I, The compound has one or more amino acid substitutions selected from the group consisting of M47L, M47V, M47Y, N48K, N48Y, Y53F, K54E, N55I, T57A, T57I, I58V, I61F, I61V, T62A, T62N, I67L, V68E, V68I, V68L, I69F, L70M, A71D, A71G, L72V, T79I, T79M, V84I, L85M, L85Q, Y87C, Y87D, E88V, D90P, R94Q, R94W, E95V, and L97Q.

[0282] In some embodiments, the one or more amino acid substitutions are TIFF2025076434000034.tif194162TIFF2025076434000035.tif215163.

[0283] 3.PD-L1 In some embodiments, the variant CD80 polypeptides exhibit increased affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of PD-L1 and the ectodomain of CTLA-4 compared to wild-type or unmodified CD80 polypeptides comprising, for example, a sequence set forth in SEQ ID NOs:2, 76, 150, 3030, or 3031. In some embodiments, the increased affinity for the ectodomain of PD-L1 is an increase of greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, or 450-fold compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

[0284] In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of PD-L1 and decreased affinity for the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the variant CD80 polypeptides exhibit increased affinity for the ectodomain of PD-L1 and decreased affinity for the ectodomain of CD28 compared to a wild-type or unmodified CD80 polypeptide, e.g., comprising a sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the decreased affinity for the CTLA-4 or CD28 ectodomain is a decrease 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, 50-fold, or 60-fold compared to the binding affinity of unmodified CD80 to the CTLA-4 or CD28 ectodomain.

[0285] In some of these embodiments, the variant CD80 polypeptide that exhibits increased binding affinity for PD-L1 compared to a wild-type or unmodified CD80 polypeptide has SEQ ID NO: and / or 97 of NO:2, 76, 150, 3030, or 3031. In some of these embodiments, the variant CD80 polypeptide that exhibits increased binding affinity for PD-L1 compared to a wild-type or unmodified CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 7, 23, 26, 30, 34, 35, 46, 51, 55, 57, 58, 65, 71, 73, 78, 79, 82, and / or 84 of SEQ ID NO:2, 76, 150, 3030, or 3031.

[0286] In some embodiments, the variant CD80 polypeptide is In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2025076434000037.tif63166.

[0287] In some embodiments, the one or more amino acid substitutions are TIFF2025076434000038.tif70165TIFF2025076434000039.tif231166TIFF2025076434000040.tif231162TIFF2025076434000041.tif252164.

[0288] In some embodiments, variant CD80 polypeptides provided herein that exhibit increased affinity for the ectodomain of PD-L1 compared to wild-type or unmodified CD80 polypeptides may exhibit or provide PD-L1-dependent CD28 costimulatory activity. In some embodiments, the variant CD80 polypeptides mediate or provide PD-L1-dependent CD28 costimulatory activity, and the affinity of the variant CD80 polypeptide is increased by at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, or 450-fold compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

[0289] In some embodiments, variant CD80 polypeptides provided herein that exhibit, mediate, or result in PD-L1-dependent CD28 costimulatory activity retain binding to the ectodomain of CD28 compared to wild-type or unmodified CD80. For example, variant CD80 polypeptides exhibit at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 10 ... 5%, 80%, 85%, 90% or 95%, or at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% can be retained.

[0290] In some embodiments, variant CD80 polypeptides provided herein that exhibit, mediate, or result in PD-L1-dependent CD28 costimulatory activity exhibit increased affinity for the ectodomain of CD28 compared to the binding affinity of unmodified CD80 to the ectodomain of CD28. For example, variant CD80 polypeptides can exhibit increased affinity for the ectodomain of CD28 that 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, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold compared to the binding affinity of unmodified CD80 to the ectodomain of CD28.

[0291] III. Variant Polypeptide Formats The immune regulating polypeptides comprising variant CD80, including vIgD, provided herein can be formatted in a wide variety of ways, including as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, the particular format can be selected for the desired therapeutic use. In some cases, the immune regulating polypeptides comprising variant CD80 polypeptides are provided in a format that antagonizes or blocks the activity of its binding partners, such as CTLA-4, CD28, and / or PD-L1. In some embodiments, antagonism of CTLA-4 or PD-L1 / PD-1 can be useful in promoting immunity in oncology. In some cases, the immune regulating polypeptides comprising variant CD80 polypeptides are provided in a format that agonizes or stimulates the activity of its binding partners, such as CTLA-4 and / or CD28. In some embodiments, agonism of CD28 can be useful in promoting immunity in oncology. In some embodiments, agonism of CD28 can be dependent on or enhanced by PD-L1 binding to CD80. Such PD-L1-dependent agonism of CD28 may be useful in promoting immunity in oncology. In some embodiments, agonism of CTLA-4 may be useful in treating inflammation or autoimmunity. Those skilled in the art can easily determine the activity of a particular format, for example, to antagonize or agonize one or more specific cognate binding partners. Exemplary methods for assessing such activity are provided herein, including in the examples.

[0292] In some aspects, immunomodulatory proteins are provided that include the vIgD of CD80 that is soluble (e.g., fused to an Fc chain). In some aspects, one or more additional IgSF domains (e.g., one or more additional vIgDs) can be linked to the vIgD of CD80 as provided herein (hereinafter referred to as "stacked" or "stacked" immunomodulatory proteins). In some embodiments, the modular format of the provided immunomodulatory proteins provides flexibility to engineer or generate immunomodulatory proteins to modulate the activity of multiple counter structures (multiple cognate binding partners). In some embodiments, such "stacked" molecules can be provided in a soluble format, and in some cases may be provided as membrane-bound or secreted proteins. In some embodiments, variant CD80 immunomodulatory proteins are provided as conjugates that contain the vIgD of CD80 linked directly or indirectly to a targeting agent or moiety (e.g., an antibody or other binding molecule) that specifically binds to a ligand (e.g., an antigen), e.g., to target or localize the vIgD to a particular environment or cell when administered to a subject. In some embodiments, the targeting agent, e.g., an antibody or other binding molecule, binds to a tumor antigen, thereby localizing the vIgD-containing variant CD80 to the tumor microenvironment, e.g., modulating the activity of tumor-infiltrating lymphocytes (TILs) specific to the tumor microenvironment.

[0293] In some embodiments, the provided immunomodulatory proteins are expressed in cells and provided as part of engineered cell therapy (ECT). In some embodiments, the variant CD80 polypeptide is expressed in a cell, e.g., an immune cell (e.g., a T cell or an antigen presenting cell), in a membrane-bound form, thereby providing a transmembrane immunomodulatory protein (hereinafter also referred to as "TIP"). In some embodiments, depending on the cognate binding partner recognized by the TIP, the engineered cell expressing the TIP can agonize the cognate binding partner by providing either a positive or negative costimulatory signal to other engineered cells and / or endogenous T cells. In some aspects, the variant CD80 polypeptide is expressed in a secretable form in a cell, e.g., an immune cell (e.g., a T cell or an antigen presenting cell), thereby producing a secreted or soluble form of the variant CD80 polypeptide (hereinafter also referred to as "SIP"), e.g., when the cell is administered to a subject. In some aspects, the SIP can antagonize the cognate binding partner in the environment in which it is secreted (e.g., the tumor microenvironment). In some embodiments, the variant CD80 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that is capable of infecting a cell, e.g., an immune cell (e.g., a T cell or an antigen presenting cell), in vivo upon administration to a subject, for delivery or expression of the variant polypeptide as a TIP or SIP in the cell.

[0294] In some embodiments, a soluble immunomodulatory polypeptide, such as a variant CD80 containing vIgD, can be encapsulated within a liposome which can itself be conjugated to any one or any combination of the conjugates provided (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory polypeptide of the invention is deglycosylated. In more specific embodiments, the variant CD80 sequence is deglycosylated. In even more specific embodiments, the IgV and / or IgC (e.g., IgC2) domain(s) of the variant CD80 are deglycosylated.

[0295] Non-limiting examples of formats that can be provided are depicted in Figures 1A-1C and further described below.

[0296] A. Soluble Proteins In some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide is a soluble protein. One of skill in the art will recognize that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that the extracellular domain or an immunologically active sequence thereof can be used to create a soluble form of such a protein. Thus, in some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide lacks a transmembrane domain or a portion of a transmembrane domain. In some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide lacks an intracellular (cytoplasmic) domain or lacks a portion of an intracellular domain. In some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide contains only a vIgD portion that contains an ECD domain or a portion thereof that contains an IgV domain and / or an IgC (e.g., IgC2) domain(s) or a specific binding fragment thereof that contains an amino acid modification(s).

[0297] In some embodiments, an immune modulating polypeptide comprising a variant CD80 can comprise one or more variant CD80 polypeptides of the invention. In some embodiments, a polypeptide of the invention comprises exactly one, two, three, four, five variant CD80 sequences. In some embodiments, at least two of the variant CD80 sequences are the same variant CD80 sequence.

[0298] In some embodiments, the immunomodulatory polypeptides provided comprise two or more vIgD sequences of CD80. The multiple variant CD80 polypeptides within a polypeptide chain can be identical (i.e., homogeneous) or non-identical (i.e., heterogeneous) variant CD80 sequences to each other. In addition to the embodiment of a single polypeptide chain, in some embodiments, two, three, four, or more of the polypeptides of the invention may be covalently or non-covalently linked to each other. Thus, monomeric, dimeric, and higher order (e.g., 3, 4, 5, or higher) multimeric proteins are provided herein. For example, in some embodiments, exactly two polypeptides of the invention can be covalently or non-covalently linked to each other to form a dimer. In some embodiments, the linkage is via an interchain cysteine ​​disulfide bond. Compositions comprising two or more polypeptides of the invention can be of the same or substantially the same species of polypeptide (e.g., homodimers) or of non-the same species of polypeptide (e.g., heterodimers). A composition having multiple linked polypeptides of the invention can have one or more identical or non-identical variant CD80 polypeptides of the invention in each polypeptide chain, as described above.

[0299] In some embodiments, the immunomodulatory protein is or comprises a variant CD80 polypeptide that is in a monomeric form and / or exhibits monovalent binding to its binding partner. In some aspects, the described variant CD80 polypeptide, such as a variant CD80 that is soluble and / or lacks a transmembrane domain and an intracellular signaling domain, is directly or indirectly linked to an additional moiety. In some embodiments, the additional moiety is a protein, peptide, small molecule, or nucleic acid. In some embodiments, the monovalent immunomodulatory protein is a fusion protein. In some embodiments, the moiety is a half-life extending moiety. Examples of such half-life extending moieties include, but are not limited to, albumin, albumin binding polypeptides, Pro / Ala / Ser (PAS), C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), albumin binding small molecules, or combinations thereof.

[0300] In some embodiments, the immunomodulatory polypeptide comprising a variant CD80 can be linked to a moiety comprising a conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and Ser (see, e.g., WO2008 / 155134, SEQ ID NO:904). In some cases, the amino acid repeat 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, each repeat comprising Ala, Ser, and Pro residue(s). Thus, provided herein is an immunomodulatory protein that is a PASylated protein, wherein the variant CD80 polypeptide is linked to Pro / Ala / Ser (PAS) directly or indirectly via a linker. In some embodiments, one or more additional linker structures may be used.

[0301] In some embodiments, the moiety facilitates detection or purification of the variant CD80 polypeptide. Optionally, the immunomodulatory polypeptide comprises a tag or fusion domain, such as an affinity or purification tag, directly or indirectly linked to the N-terminus and / or C-terminus of the CD80 polypeptide. A variety of suitable polypeptide tags and / or fusion domains are known, including, but not limited to, polyhistidine (His) tags, FLAG tags (SEQ ID NO:3037), Myc tags, and fluorescent protein tags (e.g., EGFP as set forth in SEQ ID NOs:3033-3035). Optionally, the immunomodulatory polypeptide comprising a variant CD80 comprises at least six histidine residues (as set forth in SEQ ID NO:3038). Optionally, the immunomodulatory polypeptide comprising a variant CD80 further comprises various combinations of moieties. For example, the immunomodulatory polypeptide comprising a variant CD80 further comprises one or more polyhistidine tags and a FLAG tag.

[0302] In some embodiments, the CD80 polypeptide is linked to a modified immunoglobulin heavy chain constant region (Fc) that remains in a monovalent form as set forth in SEQ ID NO:374.

[0303] In some embodiments, the immunomodulatory protein contains a variant CD80 polypeptide linked directly or indirectly via a linker to a multimerization domain. In some aspects, the multimerization domain extends the half-life of the molecule. The interaction of two or more variant CD80 polypeptides can be facilitated by their direct or indirect linkage to any moiety or other polypeptide that can interact with itself to form a stable structure. For example, separate encoded variant CD80 polypeptide chains can be linked by multimerization, the multimerization of the polypeptides being mediated by the multimerization domain. Typically, the multimerization domain provides for the formation of a stable protein-protein interaction between a first variant CD80 polypeptide and a second variant CD80 polypeptide.

[0304] Homo- or heteromultimeric polypeptides can be generated from the co-expression of separate variant CD80 polypeptides. The first and second variant CD80 polypeptides can be the same or different. In certain embodiments, the first and second variant CD80 polypeptides are the same in a homodimer, each linked to the same multimerization domain. In other embodiments, heterodimers can be formed by linking different first and second variant CD80 polypeptides. In some such embodiments, the first and second variant CD80 polypeptides are linked to different multimerization domains that can promote heterodimer formation.

[0305] In some embodiments, the multimerization domain includes any that can form stable protein-protein interactions. The multimerization domain can interact through immunoglobulin sequences (e.g., Fc domains; see, e.g., International Patent Publications WO93 / 10151 and WO2005 / 063816 US; U.S. Patent No. 2006 / 0024298; U.S. Patent No. 5,457,035); leucine zippers (e.g., from nuclear transcription proteins fos and jun or from the proto-oncogene c-myc or from the general control of nitrogen (GCN4)) (see, e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz ​​et al., (1989) Science, 243:1695-1699); hydrophobic regions; hydrophilic regions; or free thiols that form intermolecular disulfide bonds between homo- or heteromultimeric chimeric molecules. In addition, the multimerization domain can include an amino acid sequence that includes a protuberance that is complementary to an amino acid sequence that includes a hole, as described, for example, in U.S. Pat. No. 5,731,168; International Patent Publications WO 98 / 50431 and WO 2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerization regions can be engineered so that steric interactions not only promote stable interactions, but also favor the formation of heterodimers rather than homodimers from a mixture of chimeric monomers. Generally, the protuberance is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protuberance are optionally created at the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0306] The variant CD80 polypeptide can be linked anywhere, but typically through its N-terminus or C-terminus, to the N-terminus or C-terminus of the multimerization domain to form a chimeric polypeptide. Linkage can be direct or indirect through a linker. The chimeric polypeptide can be a fusion protein or can 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 a portion of the variant CD80 polypeptide can be operably linked to a nucleic acid encoding a multimerization domain sequence, directly or indirectly, or optionally through a linker domain. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant CD80 polypeptide is linked to the N-terminus of the multimerization domain. In some cases, the construct can encode a chimeric protein in which the N-terminus of the variant CD80 polypeptide is linked to the C-terminus of the multimerization domain.

[0307] Polypeptide multimers contain multiple (e.g., two) chimeric proteins made by directly or indirectly linking two of the same or different variant CD80 polypeptides to a multimerization domain. In some examples, when the multimerization domain is a polypeptide, a gene fusion encoding the variant CD80 polypeptide and the multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with the recombinant expression vector and allowed to associate into multimers, where the multimerization domains interact to form a multivalent polypeptide. Chemical linkage of the multimerization domain to the variant CD80 polypeptide can be performed using a heterobifunctional linker.

[0308] The resulting chimeric polypeptide, such as a fusion protein, and multimers formed therefrom can be purified by any suitable method, such as, for example, affinity chromatography on a Protein A or Protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, homo- and heterodimer formation occurs. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0309] In some embodiments, the multimerization domain is an Fc domain or a portion thereof from an immunoglobulin. In some embodiments, the immunomodulatory protein comprises a variant CD80 polypeptide linked to an immunoglobulin Fc (to give an "immunomodulatory Fc fusion", such as a "variant CD80-Fc fusion", also called a CD80 vIgD-Fc fusion). In some embodiments, the variant CD80 polypeptide is linked to the N-terminus of the Fc. In some embodiments, the variant CD80 polypeptide is linked to the C-terminus of the Fc. In some embodiments, two or more CD80 variant polypeptides (same or different) are independently linked to the N-terminus and C-terminus.

[0310] 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 an IgG1, such as a human IgG1. In some embodiments, the Fc comprises an amino acid sequence set forth in SEQ ID NO:277, 359, or 1712, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:277, 359, or 1712.

[0311] In some embodiments, the Fc region contains another modification to change (e.g., reduce) one or more normal functions. In general, the Fc region is involved in effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen-binding ability, which is the main function of immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating IgG levels in serum by extending in vivo half-life through conjugation to the in vivo FcRn receptor. In some embodiments, such functions can be reduced or altered in the Fc for use with the provided Fc fusion proteins.

[0312] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the CD80-Fc variant fusions provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of modifications or mutations to Fc sequences that can alter effector function. For example, WO00 / 42072, WO2006019447, WO2012125850, WO2015 / 107026, US2016 / 0017041, and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcR. The contents of these publications are expressly incorporated herein by reference.

[0313] In some embodiments, the variant CD80-Fc fusions provided comprise an Fc region that exhibits reduced effector function, making them desirable candidates for applications where the half-life of the CD80-Fc variant fusion in vivo is important, but where certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the CD80-Fc variant fusion lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in an animal model as 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 CD80-Fc variant fusion cannot bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0314] CD80-Fc variant fusions with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0315] In some embodiments, the Fc region of a CD80-Fc variant fusion comprises an Fc region in which any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as indicated by EU numbering) have been substituted with a different amino acid compared to a native Fc region. Such changes in the Fc region are not limited to the above changes, and include, for example, deglycosylated chains (N297A and N297Q) described in Current Opinion in Biotechnology (2009) 20(6), 685-691, IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1- Changes such as E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E alterations such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (as indicated by EU numbering); and alterations at sites described in WO2000 / 042072.

[0316] Certain Fc variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO2004 / 056312, WO2006019447, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0317] In some embodiments, CD80-Fc variant fusions are provided that comprise a variant Fc region that comprises one or more amino acid substitutions that extend half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO2015107026. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of 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 according to EU numbering, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0318] In some embodiments, the Fc region of a CD80-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L (according to EU numbering). In some embodiments, the Fc region of a CD80-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S and / or C229S (according to EU numbering). In some embodiments, the Fc region of a CD80 variant fusion comprises one or more amino acid substitutions R292C and V302C. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0319] In some embodiments, changes are made in the Fc region that result in reduced C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0320] In some embodiments, a CD80-Fc variant fusion is provided that comprises a variant Fc region comprising one or more amino acid modifications, the variant Fc region being derived from an IgG1, e.g., human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:277. In some embodiments, the Fc contains at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO:277 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C according to the numbering of SEQ ID NO:277 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S (corresponding to C220S according to EU numbering) amino acid modification according to the numbering of SEQ ID NO:277, such as the Fc region set forth in SEQ ID NO:1429. For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: V297G and one or more of the following amino acid modifications C220S, R292C or V302C according to EU numbering (which corresponds to N82G and one or more of the following amino acid modifications C5S, R77C or V87C based on SEQ ID NO:277), e.g., the Fc region comprises the sequence set forth in SEQ ID NO:356. 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:357. 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:358. In some embodiments, the variant Fc comprises one or more of the amino acid modifications C220S, L234A, L235E, G237A, E356D or M358L, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:376.

[0321] In some embodiments, the CD80-Fc variant fusions provided herein comprise a variant CD80 polypeptide according to the description in Section II above. In some embodiments, a CD80-Fc variant fusion is provided comprising any one of the described variant CD80 polypeptides linked to a variant Fc region, where the variant Fc region is not a human IgG1 Fc containing the mutations R292C, N297G and V302C (corresponding to R77C, N82G and V87C based on the wild type human IgG1 Fc set forth in SEQ ID NO:xxx). In some embodiments, a CD80-Fc variant is provided comprising any one of the variant CD80 polypeptides linked to an Fc region or a variant Fc region, where the variant CD80 polypeptide is not linked to the Fc by a linker consisting of three alanines.

[0322] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO:277 (corresponding to K447del according to EU numbering). In some aspects, such an Fc region can further comprise one or more additional modifications, e.g., amino acid substitutions such as those described. Examples of such Fc regions are set forth in SEQ ID NOs:356-358, 376, or 1713-1715.

[0323] In some embodiments, a CD80-Fc variant fusion is provided comprising a variant Fc region, wherein the variant Fc comprises an amino acid sequence set forth in any of SEQ ID NOs: 376, 356, 357, 358, 1429, or 1713-1715, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 376, 356, 357, 358, 1429, or 1713-1715.

[0324] In some embodiments, the Fc is derived from an IgG2, such as a human IgG2. In some embodiments, the Fc comprises an amino acid sequence set forth in SEQ ID NO:278, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:278.

[0325] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 1427, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1427. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1 and exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which arginine at position 409 as indicated by the EU index proposed by Kabat et al. is replaced with lysine and exhibits inhibited aggregate formation (see, e.g., U.S. Pat. No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8)767-71). In some embodiments, the Fc comprises an amino acid sequence set forth in SEQ ID NO:1428, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1428.

[0326] In some embodiments, the variant CD80 polypeptide is indirectly linked to the Fc sequence, for example, via a linker. In some embodiments, one or more "peptide linkers" link the variant CD80 polypeptide and the Fc domain. In some embodiments, the peptide linker can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue, but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a linker that is (in the single letter amino acid code) GGGGS ("4GS" or "GGGGS"). 4 SEQ ID NO:1717) or multimers of 4GS linkers, such as those described in SEQ ID NO:330 (2xGGGGS; (G 4 S) 2 ) or as set forth in SEQ ID NO:329 (3xGGGGS;(G 4 S) 3) for example, two, three, four, or five repeats of the 4GS linker. In some embodiments, the linker can comprise a series of alanine residues alone or in addition to another peptide linker (such as a4GS linker or multimers 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 three alanines (AAA). In some embodiments, the variant CD80 polypeptide is indirectly linked to the Fc sequence via a linker, and the linker is not composed of three alanines (AAA). In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO:331). In some embodiments, the linker can further comprise amino acids introduced by cloning and / or from a restriction site, for example, the linker can comprise the amino acids GS (single letter amino acid code) introduced by use of the restriction site BAMHI. For example, in some embodiments, the linker (one letter amino acid code) is GSGGGGS (SEQ ID NO:1716), GS (G 4 S) 3 (SEQ ID NO:3028), or GS(G 4 S) 5 (SEQ ID NO:3029). 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 single letter amino acid code) EAAAK or a multimer of EAAAK linkers, such as (1xEAAAK) as described in SEQ ID NO:3026, (3xEAAAK) as described in SEQ ID NO:3027, or (5xEAAAK) as described in SEQ ID NO:3036, e.g., two, three, four, or five repeats of the EAAAK linker. In some cases, the immunomodulatory polypeptides comprising variant CD80 comprise various combinations of peptide linkers.

[0327] In some embodiments, the variant CD80 polypeptide is directly linked to an Fc sequence. In some embodiments, the variant CD80 polypeptide is directly linked to an Fc, such as an inactive Fc that further lacks all or part of the hinge region. An exemplary Fc lacking a portion of the hinge region (6 amino acids) is set forth in SEQ ID NO:3025. In some embodiments, when the CD80 polypeptide is directly linked to an Fc sequence, the CD80 polypeptide can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or more amino acids at the C-terminus. In some embodiments, the variant CD80 polypeptide is truncated to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids that connect the IgV region to the IgC region. For example, the variant CD80 polypeptide can contain modifications to the exemplary wild-type CD80 backbone set forth in SEQ ID NO:3030.

[0328] In some embodiments, the variant CD80-Fc fusion protein is a dimer formed by two variant CD80 Fc polypeptides linked to Fc domains. In some specific embodiments, identical or substantially identical species (allowing for no more than three N-terminal or C-terminal amino acid sequence differences) of CD80-Fc variant fusion polypeptides dimerize to create homodimers. In some embodiments, the dimer is a homodimer in which the two variant CD80 Fc polypeptides are identical. Alternatively, CD80-Fc variant fusion polypeptides of different species can be dimerized to obtain heterodimers. Thus, in some embodiments, the dimer is a heterodimer in which the two variant CD80 Fc polypeptides are different.

[0329] Also provided are nucleic acid molecules encoding variant CD80-Fc fusion proteins. In some embodiments, for production of Fc fusion proteins, the nucleic acid molecules encoding the variant CD80-Fc fusion proteins are inserted into a suitable expression vector. The resulting variant CD80-Fc fusion proteins can be expressed in transformed host cells with expression resulting in assembly between the Fc domains via interchain disulfide bonds formed between the Fc moieties to give rise to dimeric, e.g., bivalent, variant CD80-Fc fusion proteins.

[0330] The resulting Fc fusion protein can be easily purified by affinity chromatography on protein A or protein G columns. In the case of the generation of heterodimers, additional steps may be required for purification. For example, when two nucleic acids encoding different variant CD80 polypeptides are transformed into cells, the formation of heterodimers must be achieved biochemically, since variant CD80 molecules that retain the Fc domain are also expressed as disulfide-linked homodimers. Thus, homodimers can be reduced under conditions that favor the disruption of interchain disulfides but do not affect intrachain disulfides. In some cases, different variant CD80 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 CD80 polypeptides using the knob-into-hole method described below.

[0331] B. Stacked molecules with additional IgSF domains In some embodiments, the immunomodulatory protein can contain any of the variant CD80 polypeptides provided herein linked directly or indirectly to one or more other immunoglobulin superfamily (IgSF) domains ("stacked" immunomodulatory protein constructs, also referred to as "type II" immunomodulatory proteins). In some aspects, this can create unique multi-domain immunomodulatory proteins that provide multi-targeted modulation of the immune synapse by binding to two or more, e.g., three or more, cognate binding partners.

[0332] In some embodiments, the immunomodulatory protein comprises a combination ("non-wild-type combination") and / or arrangement ("non-wild-type arrangement" or "non-wild-type permutation") of a variant CD80 domain with one or more other affinity modified and / or non-affinity modified IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) that are not found in a wild-type IgSF family member. In some embodiments, the immunomodulatory protein contains two, three, four, five or six immunoglobulin superfamily (IgSF) domains, at least one of which is a variant CD80 IgSF domain (vIgD of CD80) according to the description provided.

[0333] 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 origin, 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.

[0334] In some embodiments, the additional IgSF domain is an affinity-modified or non-affinity-modified IgSF domain contained in an IgSF family member of a family selected from the following: the signal-regulatory protein (SIRP) family, the triggering receptor-like expressed on myeloid cells (TREML) family, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, the sialic acid-binding Ig-like lectin (SIGLEC) family, the butyrophilin family, the B7 family, the CD28 family, the V-set and immunoglobulin domain-containing (VSIG) family, the V-set transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signaling lymphocyte activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the natural cytotoxicity-inducing receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domains are independently selected from CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOSLG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3), VTCN1 (B7-H4), CD28, CTLA4, PDCD1 (PD-1), ICOS, BTLA (CD272), CD4, CD8A (CD8-α), CD8B (CD8-β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NC R3 (NKp30).

[0335] The first column of Table 2 provides the name and optionally some possible aliases for that particular IgSF member. The second column provides the protein identifier in the UniProtKB database, a publicly available database accessible via the Internet at uniprot.org, and in some cases, the GenBank number. The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The UniProt database includes the UniProt Knowledge Base (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Bioinformatics Institute, and the Protein Information Resource (PIR), and is supported primarily by a grant from the National Institutes of Health (NIH). GenBank is the NIH gene sequence database, which is a collection of all publicly available DNA sequences with annotations (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 identified as the range encompassing the residues that the domain defines. Column 3 also indicates the IgSF domain class of the particular IgSF region. Column 4 provides the region in which the indicated additional domains are located (S for signal peptide, E for extracellular domain, T for transmembrane domain, C for cytoplasmic domain). It is understood that the domain descriptions may vary depending on the method used to identify or classify the domain and may be identified differently from different sources. The descriptions of the residues corresponding to the domains in Table 2 are merely exemplary and may be several amino acids longer or shorter (e.g., 1, 2, 3, or 4). Column 5 indicates some of the IgSF members listed (some of their cognate cell surface binding partners).

[0336] Table 2: IgSF members according to the present disclosure TIFF2025076434000042.tif59167TIFF2025076434000043.tif224167TIFF2025076434000044.tif220167TIFF2025076434000045.tif156167

[0337] The number of such non-affinity modified or affinity modified IgSF domains (whether in non-wild-type combinations or non-wild-type arrangements) present in a "stacked" immunomodulatory protein construct is at least two, three, four, or five, and in some embodiments exactly two, three, four, or five IgSF domains (the determination of the number of affinity modified IgSF domains disregards any non-specific binding subsequences thereof and / or substantially immunologically inactive subsequences thereof).

[0338] In some embodiments of the stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least two, and 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 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 (affinity modified IgSF domains:non-affinity modified IgSF domains).

[0339] In some embodiments of the stacked immunomodulatory proteins, at least two of the non-affinity modified IgSF domains and / or the affinity modified IgSF domains are identical IgSF domains.

[0340] In some embodiments, the stacked immunomodulatory proteins provided herein comprise at least two affinity modified IgSF domains and / or non-affinity modified IgSF domains derived from a single IgSF member but in a non-wild-type arrangement (or "permutation"). One illustrative example of a non-wild-type arrangement or permutation is an immunomodulatory protein that comprises affinity modified IgSF domain sequences and / or non-affinity modified IgSF domain sequences in a non-wild-type order compared to those found in wild-type CD80, whose IgSF domain sequences serve as the source of the variant IgSF domains as provided herein. Thus, in one example, the immunomodulatory protein can comprise an IgV proximal to the transmembrane domain and an IgC distal to the transmembrane domain, regardless of the non-affinity modified and / or affinity modified form. It is also within the scope of the provided subject matter that both non-wild-type combinations and non-wild-type arrangements of non-affinity modified IgSF domains and / or affinity modified IgSF domains are present in the immunomodulatory proteins provided herein.

[0341] In some embodiments of stacked immunomodulatory proteins, the non-affinity modified IgSF domains and / or affinity modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity modified IgSF domains specifically bind to different cognate binding partners under specific binding conditions and are "non-identical" regardless of whether the wild-type or non-modified IgSF domains from which they are engineered were the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein can include at least one IgSF domain sequence whose origin is derived from and unique to one CD80, and at least one second IgSF domain sequence whose origin is derived from and unique to another IgSF family member that is not CD80, the IgSF domains of the immunomodulatory protein being in non-affinity modified and / or affinity modified form. However, in other embodiments, the two non-identical IgSF domains are derived from the same IgSF domain sequence, but at least one of the non-identical IgSF domains has been affinity engineered so that it specifically binds to a different cognate binding partner.

[0342] In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant CD80 polypeptide, also contain at least one, two, three, four, five, or six additional immunoglobulin superfamily (IgSF) domains, such as an IgD domain of an IgSF family member listed in Table 2. In some embodiments, the provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the provided immunomodulatory proteins contain at least two additional IgSF domains (e.g., a second and a third IgSF domain). In some embodiments, the provided immunomodulatory proteins contain at least three additional IgSF domains (e.g., a second, a third, and a fourth). In some embodiments, the provided immunomodulatory proteins contain at least four additional IgSF domains (e.g., a second, a third, a fourth, and a fifth). In some embodiments, the provided immunomodulatory proteins contain at least five additional IgSF domains (e.g., a second, a third, a fourth, a fifth, and a 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 of the IgSF domains of the immunomodulatory protein is different. In some embodiments, at least one of the additional IgSF domains is the same as at least one other IgSF domain of the immunomodulatory protein. In some embodiments, each of the IgSF domains is from or derived from a different IgSF family member. In some embodiments, at least two of the IgSF domains are from or derived from the same IgSF family member.

[0343] In some embodiments, the additional IgSF domain comprises an IgV domain or an IgC (e.g., IgC2) domain(s), or a specific binding fragment of an IgV domain or a specific binding fragment of an IgC (e.g., IgC2) domain(s). In some embodiments, the additional IgSF domain is or comprises a full-length IgV domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgC (e.g., IgC2) domain(s). In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of an IgV domain. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of an IgC (e.g., IgC2) domain(s). In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (same) IgSF member. For example, in some aspects, the immunomodulatory protein contains the ECD or a portion thereof of an IgSF member that contains a full-length IgV domain and a full-length IgC (e.g., IgC2) domain(s) or a specific binding fragment thereof.

[0344] In some embodiments, the immunomodulatory proteins provided contain at least one additional IgSF domain (e.g., a second or, optionally, a third IgSF domain), wherein the at least one additional or second IgSF domain is a wild-type or unmodified IgSF domain contained in an amino acid sequence set forth in any of SEQ ID NOs:224-249 and 306, 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.

[0345] In some embodiments, the immunomodulatory proteins provided, in addition to containing a variant CD80 polypeptide, also contain at least one additional affinity-modified IgSF domain (e.g., a second, or optionally, a third affinity-modified IgSF domain), where the at least one additional IgSF domain is vIgD that contains one or more amino acid modifications (e.g., substitutions, deletions, or mutations) relative to an IgSF domain in a wild-type or unmodified IgSF domain, e.g., an IgSF domain of an IgSF family member set forth in Table 2. In some embodiments, the additional, e.g., second or third affinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified IgSF domain contained in an amino acid sequence set forth in any of SEQ ID NOs:224-249 and 306, or a specific binding fragment thereof. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, e.g., an IgC1 or IgC2 domain. In some embodiments, the additional, e.g., second or third, IgSF domain is an affinity-modified IgV domain and / or an IgC domain. In some embodiments, the one or more additional IgSF domains are an IgV domain and / or an IgC (e.g., IgC2) domain(s), or an affinity-modified IgSF domain containing a specific binding fragment of an IgV domain and / or a specific binding fragment of an IgC (e.g., IgC2) domain(s), wherein the IgV domain and / or IgC domain contain(s) an amino acid modification (e.g., substitution). In some embodiments, the one or more additional affinity-modified IgSF domains contain an IgV domain containing(s) an amino acid modification (e.g., substitution).In some embodiments, the one or more additional affinity-modified IgSF domains comprise an IgSF domain present in the ECD or portion of the ECD of a corresponding unmodified IgSF family member, e.g., a full-length IgV domain and a full-length IgC (e.g., IgC2) domain(s) or specific binding fragments thereof, wherein one or both of the IgV and IgC contain amino acid modification (e.g., substitution)(s).

[0346] In some embodiments, the immunomodulatory proteins provided contain at least one additional or second IgSF domain that is vIgD that contains one or more amino acid substitutions compared to a wild-type or unmodified IgSF domain other than CD80 (e.g., IgV).

[0347] In some embodiments, the one or more additional IgSF domains (e.g., the second or third IgSF) domains are IgSF domains (e.g., IgV) of another IgSF family member that also binds to an inhibitory receptor. In some aspects, the one or more additional IgSF domains (e.g., the second or third IgSF) domains are affinity-modified IgSF domains that bind to an inhibitory receptor and are variant IgSF domains (vIgD) of an IgSF family member that contains one or more amino acid substitutions in the IgSF domain (e.g., IgV), and in some cases, the one or more amino acid modifications increase binding to the inhibitory receptor. In some embodiments, the vIgD contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the wild-type or unmodified IgSF domain (e.g., IgV) of an IgSF family member that binds to an inhibitory receptor. In addition to CTLA-4, examples of such inhibitory receptors are PD-1, 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-L1, PD-L2, or CEACAM1. Thus, in some aspects, a multi-target checkpoint antagonist is provided that targets or blocks the activity of two or more inhibitory receptors. 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.

[0348] In some embodiments, immunomodulatory proteins are provided that contain any one of the variant CD80 polypeptides and one or more IgSF domains of an inhibitory receptor, such as a wild-type or unmodified inhibitory receptor. In some embodiments, immunomodulatory proteins are provided that contain any one of the variant CD80 polypeptides and one or more IgSF domains of CD112, e.g., wild-type or unmodified CD112, e.g., an IgV domain set forth in SEQ ID NO:734 or 829, or an ECD or portion thereof set forth in SEQ ID NO:269 or a portion thereof (containing IgV and IgC domains or specific binding fragments thereof). In some embodiments, immunomodulatory proteins are provided that contain any one of the variant CD80 polypeptides and one or more IgSF domains of CD155, e.g., wild-type or unmodified CD155, e.g., an IgV domain set forth in SEQ ID NO:378 or 421, or an ECD or portion thereof set forth in SEQ ID NO:268 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 one of the variant CD80 polypeptides and one or more IgSF domains of PD-L1, such as a wild-type or unmodified PD-L1, such as an IgV domain set forth in SEQ ID NO:1000 or 1196, or an ECD or portion thereof set forth in SEQ ID NO:251 or 1721 or a portion thereof (containing the IgV and IgC domains or specific binding fragments thereof). In some embodiments, an immunomodulatory protein is provided that contains any one of the variant CD80 polypeptides and PD-L2, such as a wild-type or unmodified PD-L2, such as an IgV domain set forth in SEQ ID NO:1197 or 1257, or an ECD or portion thereof set forth in SEQ ID NO:252 or a portion thereof (containing the IgV and IgC domains or specific binding fragments thereof).

[0349] In some embodiments, immunomodulatory proteins are provided that contain one or more additional IgSF domains (e.g., a second or third IgSF) that are an IgSF family member vIgD that binds to an inhibitory receptor, and one or more amino acid modifications of the IgSF domain (e.g., IgV) increase the binding affinity of vIgD, or a fusion or immunomodulatory protein containing said vIgD, to its inhibitory receptor cognate binding partner, compared to the unmodified IgSF domain, e.g., 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. In some embodiments, one or more amino acid modifications of the IgSF domain (e.g., IgV) increase the selectivity of vIgD, or a fusion or immunomodulatory protein containing said vIgD, for its inhibitory receptor, compared to the unmodified IgSF domain. In some embodiments, the increase in selectivity is a ratio of vIgD binding to an inhibitory receptor to another cognate binding partner, such as a cognate binding partner that is not an inhibitory receptor, that is greater than the ratio of unmodified IgSF binding to an inhibitory receptor to another cognate binding partner, which in some embodiments is at least 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, or at least about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater.

[0350] In some embodiments, at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant CD112 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type CD112, and is an IgSF family member that binds to the inhibitory receptor TIGIT. Exemplary amino acid modifications, such as substitutions, deletions, or additions, in the IgSF domain (e.g., IgV, or ECD containing IgV and IgC) of a variant CD112 polypeptide are listed in Table 3. In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD80 polypeptides and an IgV domain that comprises any of the amino acid modifications set forth in Table 3, e.g., an IgV domain set forth in any of SEQ ID NOs:782-828, 830-876, 918-999, 1454-1501, or a variant CD112 polypeptide that contains an IgV domain that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to any of SEQ ID NOs:782-828, 830-876, 918-999, 1454-1501 and that comprises one or more amino acid modifications. In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD80 polypeptides and an ECD or portion thereof that contains an IgV domain and / or an IgC domain that includes any of the amino acid modifications listed in Table 3, e.g., an ECD set forth in any of SEQ ID NOs:735-781, 877-917, 1430-1453, or a variant CD112 polypeptide that contains an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs:735-781, 877-917, 1430-1453 and includes one or more amino acid modifications.

[0351] In some embodiments, at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant CD155 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type CD155, and is an IgSF family member that binds to the inhibitory receptor TIGIT. Exemplary amino acid modifications, such as substitutions, deletions, or additions, in the IgSF domain (e.g., IgV, or ECD containing IgV and IgC) of a variant CD155 polypeptide are listed in Table 4. In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD80 polypeptides and an IgV domain that comprises any of the amino acid modifications set forth in Table 4, e.g., an IgV domain set forth in any of SEQ ID NOs:400-420, 422-442, 540-733, 1502-1547, 1572, 1573, 1620-1711, or a variant CD155 polypeptide that contains an IgV domain that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to any of SEQ ID NOs:400-420, 422-442, 540-733, 1502-1547, 1572, 1573, 1620-1711 and that comprises one or more amino acid modifications. In some embodiments, immunomodulatory proteins are provided that contain any of the provided variant CD80 polypeptides and an ECD or portion thereof that contains an IgV domain and / or an IgC domain that contains any of the amino acid modifications listed in Table 4, such as an ECD set forth in any of SEQ ID NOs: 379-399, 443-539, 1548-1571, 1574-1619, or a variant CD155 polypeptide that contains an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs: 379-399, 443-539, 1548-1571, 1574-1619 and includes one or more amino acid modifications.

[0352] In some embodiments, at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g., IgV) of a variant PD-L1 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions or additions) in the IgSF domain (e.g., IgV or ECD) compared to unmodified or wild-type PD-L1, which in some aspects increases 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 ECD containing IgV and IgC) of a variant PD-L1 polypeptide are listed in Table 5. In some embodiments, immune modulating proteins are provided that contain any of the provided variant CD...

Claims

1. 1. A variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, comprising one or more amino acid modifications at one or more positions of unmodified CD80 or a specific binding fragment thereof corresponding to 35, 46, 71, 7, 23, 26, 30, 34, 51, 55, 57, 58, 65, 73, 78, 79, 82, or 84 based on the numbering of the positions set forth in SEQ ID NO:2; Contains up to 14 amino acid modifications; The variant CD80 polypeptide.

2. 1. A variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions of unmodified CD80 or a specific binding fragment thereof corresponding to 35, 46, 71, 7, 23, 26, 30, 34, 51, 55, 57, 58, 65, 73, 78, 79, 82, or 84 based on the numbering of positions set forth in SEQ ID NO:2; the IgV domain or specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide; The variant CD80 polypeptide.

3. 3. The variant CD80 polypeptide of claim 1 or claim 2, comprising one or more amino acid modifications corresponding to one or more of positions 26, 35, 46, 57, or 71 based on the numbering of SEQ ID NO:

2.

4. 1. A variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, The unmodified CD80 or a specific binding fragment thereof may be provided with a sequence similar to that described in SEQ ID NO:2, based on the numbering of the positions. and comprising one or more amino acid modifications corresponding to Contains up to 14 amino acid modifications; The variant CD80 polypeptide.

5. 1. A variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, The variant CD80 polypeptide is a variant of unmodified CD80 or a specific binding fragment thereof, based on the numbering of positions in SEQ ID NO:

2. and comprising an amino acid modification corresponding to the IgV domain or specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide; The variant CD80 polypeptide.

6. Amino acid modification A26E, Amino acid modification E35D, Amino acid modification D46E, the amino acid modification D46V, or Amino acid modification A71G A variant CD80 polypeptide according to any one of claims 1 to 5, comprising:

7. 6. The variant CD80 polypeptide of claim 4 or claim 5, comprising the amino acid modification M47L.

8. 1. A variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, Based on the numbering of positions described in SEQ ID NO:2 The amino acid modification is selected from The variant CD80 polypeptide.

9. Amino acid modification 9. The variant CD80 polypeptide of any one of claims 1 to 8, comprising:

10. 10. The variant CD80 polypeptide of any one of claims 4 to 9, comprising the amino acid modifications E35D / M47I, E35D / M47L, E35D / M47V, or E35D / V68M.

11. 11. The variant CD80 polypeptide of any one of claims 4 to 10, comprising the amino acid modifications E35D / M47L / V68M or E35D / M47V / V68M.

12. 12. The variant CD80 polypeptide of any one of claims 1 to 11, which exhibits increased binding affinity to the ectodomain of human PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of human PD-L1.

13. 13. The variant CD80 polypeptide of claim 12, wherein the 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, 50-fold, 60-fold, 80-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, or 450-fold compared to the binding affinity of the unmodified CD80 to the PD-L1 ectodomain.

14. Amino acid modifications based on the numbering of positions in SEQ ID NO:2 14. The variant CD80 polypeptide of any one of claims 4 to 13, comprising:

15. 14. The variant CD80 polypeptide of any one of claims 4 to 9 and 11 to 13, wherein said amino acid modifications comprise E35D / M47I / L70M.

16. 14. The variant CD80 polypeptide of any one of claims 4 to 13, wherein said amino acid modifications comprise E35D / M47V / N48K / V68M / K89N.

17. 14. The variant CD80 polypeptide of any one of claims 4 to 13, wherein said amino acid modifications comprise H18Y / A26E / E35D / M47L / V68M / A71G / D90G.

18. 14. The variant CD80 polypeptide of any one of claims 4 to 13, wherein said amino acid modifications comprise E35D / D46E / M47V / V68M / D90G / K93E.

19. 14. The variant CD80 polypeptide of any one of claims 4 to 13, wherein said amino acid modifications comprise E35D / D46V / M47L / V68M / L85Q / E88D.

20. 20. The variant CD80 polypeptide of any one of claims 1 to 19, wherein the unmodified CD80 (i) comprises a sequence of amino acids as set forth in SEQ ID NO:2, (ii) comprises a sequence of amino acids having at least 95% sequence identity to SEQ ID NO:2, or (iii) is a portion of (i) or (ii) that comprises an IgV domain or a specific binding fragment thereof.

21. 20. The variant CD80 polypeptide of any one of claims 1 to 19, which is or comprises the IgV domain or a specific binding fragment thereof.

22. 22. The variant CD80 polypeptide of any one of claims 1 to 21, wherein the IgV domain or a specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide.

23. 23. The variant CD80 polypeptide of any one of claims 1 to 22, comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid modifications.

24. A sequence of amino acids or a specific binding fragment thereof as set forth in any of SEQ ID NOs: 3-75, 2009-2104, 2297-2507, or 2930-2960, or a sequence of amino acids or a specific binding fragment thereof exhibiting at least 95% sequence identity to any of SEQ ID NOs: 3-75, 2009-2104, 2297-2507, or 2930-2960 and containing one or more amino acid modifications thereof; or A sequence of amino acids or a specific binding fragment thereof as set forth in any one of SEQ ID NOs: 77-149, 151-223, 2105-2296, 2508-2929, or 2961-3022, which shows at least 95% sequence identity to any one of SEQ ID NOs: 77-149, 151-223, 2105-2296, 2508-2929, or 2961-3022 and contains one or more amino acid modifications thereof.

24. The variant CD80 polypeptide of any one of claims 1 to 23, comprising:

25. 25. The variant CD80 polypeptide of any one of claims 1 to 24, comprising a sequence of amino acids set forth in any of SEQ ID NOs: 199, 208, 2250, 2276, 2280, or 2284.

26. lacking the CD80 transmembrane and intracellular signaling domains, and / or cannot be expressed on the cell surface, 26. A variant CD80 polypeptide according to any of embodiments 1 to 25.

27. 27. The variant CD80 polypeptide of any one of claims 1 to 26, linked to a moiety that increases the biological half-life of said polypeptide.

28. 28. The variant CD80 polypeptide of any one of claims 1 to 27, linked to a multimerization domain.

29. 29. The variant CD80 polypeptide of claim 28, wherein the multimerization domain is an Fc domain or a variant Fc domain having reduced effector function.

30. 30. The variant CD80 polypeptide of any one of claims 1 to 29, which is a transmembrane immunomodulatory protein, further comprising a transmembrane domain and / or a cytoplasmic signaling domain.

31. 30. An immunomodulatory protein comprising a first variant CD80 polypeptide of any one of claims 1 to 29 linked to a first multimerization domain and a second variant CD80 polypeptide of any one of claims 1 to 29 linked to a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first and second variant CD80 polypeptides.

32. 32. The immunomodulatory protein of claim 31 , wherein the multimer is a dimer, optionally a homodimer.

33. 33. The immunomodulatory protein of claim 31 or claim 32, wherein the first variant CD80 polypeptide and the second variant CD80 polypeptide are identical.

34. 34. The immunomodulatory protein of any one of claims 31 to 33, wherein the multimerization domain is or comprises an Fc region of an immunoglobulin, and optionally the immunoglobulin protein is human and / or the Fc region is human.

35. 35. The immunomodulatory protein of claim 34, wherein the Fc region is that of an immunoglobulin G1 (IgG1) or an immunoglobulin G2 (IgG2) protein.

36. 36. The immunomodulatory protein of claim 34 or claim 35, wherein the Fc region exhibits one or more effector functions.

37. The immunomodulatory protein of any one of claims 31 to 36, which exhibits Fc-dependent CD28 costimulation.

38. 35. The immunomodulatory protein of any one of claims 31 to 34, wherein the Fc region is a variant Fc region comprising one or more amino acid substitutions in a wild-type Fc region, the variant Fc region exhibiting one or more reduced effector functions compared to the wild-type Fc region, and optionally the wild-type human Fc is that of human IgG1.

39. 39. The immunomodulatory protein of claim 38, wherein the Fc region comprises the amino acid substitution N297G, said residues being numbered according to the EU index of Kabat.

40. 39. The immunomodulatory protein of claim 38, wherein the Fc region comprises the amino acid substitutions R292C / N297G / V302C, said residues being numbered according to the EU index of Kabat.

41. 39. The immunomodulatory protein of claim 38, wherein the Fc region comprises the amino acid substitutions L234A / L235E / G237A, said residues being numbered according to the EU index of Kabat.

42. 42. The immunomodulatory protein of any one of claims 39 to 41, wherein the variant Fc region further comprises the amino acid substitution C220S, said residues being numbered according to the EU index of Kabat.

43. 43. The immunomodulatory protein of any one of claims 38 to 42, wherein the Fc region comprises K447del, said residues being numbered according to the EU index of Kabat.

44. 44. An immunomodulatory protein according to any one of claims 38 to 43, comprising a variant CD80 polypeptide according to any one of claims 12 to 29.

45. The immunomodulatory protein of any one of claims 38 to 44, which exhibits PD-L1-dependent CD28 costimulation.

46. 30. An immunomodulatory protein comprising a variant CD80 polypeptide according to any one of claims 1 to 29 linked directly or indirectly via a linker to a second polypeptide comprising an IgSF domain of an immunoglobulin superfamily (IgSF) member.

47. 47. The immunomodulatory protein of claim 46, wherein the IgSF domain is an affinity modified IgSF domain, the affinity modified IgSF domain comprising one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of the IgSF family member.

48. 48. The immunomodulatory protein of claim 47, wherein the IgSF domain is an affinity-modified IgSF domain that exhibits increased binding to one or more of its cognate binding partner(s) compared to the binding of an unmodified or wild-type IgSF domain of the IgSF family member to the same cognate binding partner(s).

49. A conjugate comprising a variant CD80 polypeptide according to any one of claims 1 to 29 linked to a targeting moiety which specifically binds to a cell surface molecule.

50. 50. The conjugate of claim 49, wherein the cell is an immune cell or a tumor cell.

51. 51. The conjugate of claim 49 or claim 50, wherein the moiety is a protein, peptide, nucleic acid, small molecule or nanoparticle.

52. The conjugate of any one of claims 49 to 51, wherein the moiety is an antibody or an antigen-binding fragment.

53. The conjugate according to any one of claims 50 to 52, which is a fusion protein.

54. 54. A nucleic acid molecule encoding a variant CD80 polypeptide according to any one of claims 1 to 30, an immunomodulatory protein according to any one of claims 31 to 48, or a conjugate which is a fusion protein according to any one of claims 49 to 53.

55. A vector comprising the nucleic acid molecule of claim 54.

56. 56. The vector of claim 55 which is an expression vector.

57. A cell comprising the vector of claim 55 or claim 56.

58. 57. A method for producing a variant CD80 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule according to claim 54 or a vector according to claim 55 or claim 56 into a host cell under conditions to express said protein in said cell.

59. 59. The method of claim 58, further comprising isolating or purifying the variant CD80 polypeptide or immunomodulatory protein from the cells.

60. 31. A method of engineering a cell to express a variant CD80 variant polypeptide, comprising introducing into a host cell a nucleic acid molecule encoding a variant CD80 polypeptide of any of embodiments 1-30 under conditions whereby said polypeptide is expressed in said cell.

61. 57. An engineered cell comprising a variant CD80 polypeptide according to any one of claims 1 to 30, an immunomodulatory protein according to any one of claims 31 to 48, a conjugate which is a fusion protein according to any one of claims 49 to 53, a nucleic acid molecule according to claim 54, or a vector according to claim 55 or 56.

62. the variant CD80 polypeptide does not contain a transmembrane domain and / or is not expressed on the surface of the cell; and / or the variant CD80 polypeptide is capable of being secreted from the engineered cell upon expression; 62. The engineered cell of claim 61.

63. The variant CD80 polypeptide comprises a transmembrane domain or is a transmembrane immunomodulatory protein according to claim 29; and / or the variant CD80 polypeptide is expressed on the surface of the cell; 62. The engineered cell of claim 61.

64. 64. The engineered cell of any one of claims 61 to 63, wherein the cell is an immune cell, optionally an antigen presenting cell (APC) or a lymphocyte, optionally a T cell.

65. 65. The engineered cell of any one of claims 61-64, further comprising a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

66. 52. An infectious agent comprising a nucleic acid molecule encoding a variant CD80 polypeptide according to any of embodiments 1-30, an immunomodulatory protein according to any of embodiments 31-47, or a conjugate which is a fusion protein according to any of embodiments 48-52.

67. 67. The infectious agent of claim 66 which is a bacterium or a virus.

68. 68. The infectious agent of claim 67, wherein the infectious agent is a virus, and the virus is an oncolytic virus.

69. 13. A pharmaceutical composition comprising a variant CD80 polypeptide according to any one of claims 1 to 30, an immunomodulatory protein according to any one of claims 31 to 48, a conjugate according to any one of claims 49 to 53, an engineered cell according to any one of claims 61 to 65, or an infectious agent according to any one of claims 66 to 68.

70. 70. The pharmaceutical composition of claim 69, comprising a pharma- ceutically acceptable excipient.

71. 71. The pharmaceutical composition of claim 69 or claim 70, wherein the pharmaceutical composition is sterile.

72. An article of manufacture comprising the pharmaceutical composition of any one of claims 69 to 71 in a vial.

73. 73. The article of manufacture of claim 72, wherein the vial is sealed.

74. A kit comprising a pharmaceutical composition according to any one of claims 69 to 71, or an article of manufacture according to claim 72 or claim 73, and instructions for use.

75. A method of modulating an immune response in a subject comprising administering a pharmaceutical composition according to any one of claims 69 to 71.

76. A method of modulating an immune response in a subject comprising administering an immunomodulatory protein according to any one of claims 31 to 48.

77. The method of claim 76, wherein the immunomodulatory protein is the immunomodulatory protein of claim 37 that exhibits Fc-dependent CD28 costimulation.

78. 75. The method of claim 74, wherein said immunomodulatory protein is an immunomodulatory protein according to claim 44 that exhibits PD-L1 dependent CD28 costimulation, and optionally said immunomodulatory protein comprises a variant CD80 polypeptide according to any one of claims 12 to 29.

79. 66. A method of modulating an immune response in a subject comprising administering an engineered cell according to any one of claims 61 to 65.

80. 80. The method of claim 79, wherein the engineered cells are autologous to the subject.

81. The method of any one of claims 75 to 80, wherein the immune response is modulated to treat a disease or condition in the subject.

82. The method of any one of claims 75 to 81, wherein the immune response is increased.

83. 1. A method of increasing an immune response in a subject comprising administering an immunomodulating protein comprising a variant CD80 polypeptide, wherein the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or an IgC domain or a specific binding fragment thereof of unmodified CD80, or a specific binding fragment thereof, and wherein the immunomodulating protein exhibits PD-L1-dependent CD28 costimulation.

84. 84. The method of claim 83, wherein the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

85. 1. A method of mediating CD28 agonism through PD-L1-dependent CD28 costimulation in a subject comprising administering an immunomodulatory protein comprising a variant CD80 polypeptide, wherein the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or the IgC domain or a specific binding fragment thereof of unmodified CD80, or a specific binding fragment thereof, and wherein the variant CD80 polypeptide has a binding affinity to the ectodomain of PD-L1, compared to that of unmodified CD80: The method further comprises administering to the subject an antibody capable of binding to the ectodomain of PD-L1.

86. 86. The method of any one of claims 83 to 85, for use in the treatment of a disease or condition.

87. Prior to said administering, selecting a subject having a tumor comprising cells positive for surface PD-L1, and optionally, said cells are tumor cells or tumor-infiltrating immune cells; or The subject is selected as having a tumor comprising a cell surface positive for PD-L1, and optionally, the cell is a tumor cell or a tumor-infiltrating immune cell.

87. The method according to any one of claims 75 to 86.

88. The selection of the target, (a) contacting a tumor tissue sample from a subject with a binding reagent capable of specifically binding to the ectodomain of PD-L1; (b) detecting the presence of bound binding reagent in or on cells of said tumor tissue sample, optionally wherein said cells are tumor cells or tumor infiltrating immune cells; (c) selecting the subject for treatment if the tumor tissue sample contains a detectable level of cell surface positive for PD-L1; 88. The method of claim 87, comprising:

89. Prior to said administering, a subject is selected having a tumor comprising cell surface cells positive for CD28, optionally said cells are tumor-infiltrating lymphocytes, optionally said lymphocytes are T cells, optionally CD8+ T cells; or the subject is selected as having a tumor comprising a cell surface positive for CD28, optionally the cell is a tumor-infiltrating lymphocyte, optionally the lymphocyte is a T cell, optionally a CD8+ T cell; 89. The method according to any one of claims 75 to 88.

90. The selection of the object comprises: (a) contacting a tumor tissue sample from a subject with a binding reagent capable of specifically binding to the ectodomain of CD28; (b) detecting the presence of bound binding reagent in or on cells of said tumor tissue sample, optionally said cells being tumor infiltrating lymphocytes, optionally said lymphocytes being T cells, optionally CD8+ T cells; (c) selecting the subject for treatment if the tumor tissue sample contains a detectable level of CD28-positive cell surface.

90. The method of claim 89, comprising:

91. (a) contacting a tumor tissue sample from a subject with a binding reagent capable of specifically binding to PD-L1; (b) detecting the presence of bound binding reagent in or on cells of said tumor tissue sample, optionally wherein said cells are tumor cells or tumor infiltrating immune cells; (c) if the tumor sample contains cell surface positive for detectable levels of PD-L1, selecting the subject for treatment with an immunomodulatory protein comprising a variant CD80 polypeptide, wherein the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or the IgC domain or a specific binding fragment thereof of unmodified CD80, or a specific binding fragment thereof, and wherein the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1; A method for selecting a treatment target, comprising:

92. 92. The method of claim 91, comprising contacting the tumor tissue sample with a binding reagent capable of specifically binding to CD28, wherein the subject is selected if the tumor tissue sample further contains tumor-infiltrating lymphocytes that are positive for detectable levels of CD28, optionally wherein the lymphocytes are T cells, optionally CD8+ T cells.

93. 93. The method of any one of claims 88 and 90-92, wherein the tumor tissue sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof.

94. The method of claims 88 and 90-93, wherein the binding reagent is an antibody or antigen-binding fragment, a protein ligand or binding partner, an aptamer, an affimer, a peptide, or a hapten.

95. 95. The method of claim 88, 91, 93 or 94, wherein the binding reagent is an anti-PD-L1 antibody or antigen-binding fragment.

96. The method of any one of claims 88 to 94, wherein the binding reagent comprises a variant CD80 polypeptide according to any one of claims 1 to 30.

97. 97. The method of claim 96, wherein the variant CD80 polypeptide comprises the IgV domain or a specific binding fragment thereof.

98. 98. The method of claim 96 or 97, wherein the IgV domain or specific binding fragment thereof is the only CD80 portion of the binding reagent.

99. 99. The method of any one of claims 96-98, wherein the variant CD80 polypeptide exhibits increased affinity for binding to PD-L1 compared to the wild-type or unmodified CD80 polypeptide.

100. 100. The method of any one of claims 88 and 90 to 99, wherein the binding reagent is directly or indirectly linked to a moiety that is a detectable moiety or a moiety capable of being detected.

101. The method of claim 100, wherein the portion is an Fc region.

102. The method of claim 101, wherein the Fc region is non-human, optionally mouse or rabbit.

103. The method of any one of claims 88 and 90 to 102, wherein detecting the presence of bound binding reagent is by immunohistochemistry, pseudo-immunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting.

104. 104. The method of any one of claims 91-103, further comprising administering said immunomodulatory protein to said subject.

105. 105. The method of any one of claims 83-104, wherein the immunomodulatory protein is a multimer comprising a first variant CD80 polypeptide linked to a first multimerization domain and a second variant CD80 polypeptide linked to a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first and second variant CD80 polypeptides.

106. 106. The method of claim 105, wherein the multimer is a dimer.

107. 107. The method of claim 105 or claim 106, wherein the first variant CD80 polypeptide and the second variant CD80 polypeptide are identical.

108. The method of any one of claims 105 to 107, wherein the multimerization domain is or comprises an Fc region.

109. The method of claim 108, wherein the Fc region is a variant Fc region comprising one or more amino acid substitutions compared to a wild-type Fc region, and the Fc region exhibits one or more reduced effector functions compared to the wild-type Fc region, and optionally the wild-type Fc is a human IgG1.

110. The variant CD80 polypeptide is a polypeptide having a sequence similar to that of unmodified CD80 or a specific binding fragment thereof, as defined by SEQ ID NO:

110. The method of any one of claims 83 to 109, comprising one or more amino acid modifications corresponding to 7, 12, 13, 15, 16, 18, 21, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 51, 53, 54, 55, 57, 58, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and / or 97 based on the numbering of positions set forth in NO:

2.

111. The variant CD80 polypeptide is a variant of unmodified CD80 or a specific binding fragment thereof, based on the numbering of positions in SEQ ID NO:

2. The method of any one of claims 83 to 110, comprising one or more amino acid modifications corresponding to:

112. the variant CD80 polypeptide comprises one or more amino acid modifications corresponding to one or more positions 13, 18, 21, 22, 24, 26, 27, 33, 35, 37, 38, 41, 43, 46, 47, 48, 53, 61, 62, 68, 70, 71, 79, 85, 87, 88, 90, 91, 93, 94, 95, or 97, and optionally the one or more amino acid modifications are The method according to any one of claims 83 to 110, wherein the method is selected from

113. The one or more amino acid modifications are based on the numbering of positions in SEQ ID NO:

2. The method according to any one of claims 83 to 112, wherein the method is selected from the group consisting of

114. the variant CD80 polypeptide comprising: Amino acid modification H18Y, Amino acid modification A26E, Amino acid modification E35D, Amino acid modification D46E, Amino acid modification D46V, Amino acid modification M47I, Amino acid modification M47L, Amino acid modification V68M, Amino acid modification A71G, the amino acid modification L85Q, or Amino acid modification D90G The method according to any one of claims 83 to 113, comprising:

115. The variant CD80 polypeptide comprises an amino acid modification The method according to any one of claims 83 to 114, comprising:

116. 116. The method of any one of claims 83-115, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / M47I, E35D / M47L, E35D / M47V, or E35D / V68M.

117. 117. The method of any one of claims 83 to 116, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / M47L / V68M or E35D / M47V / V68M.

118. 117. The method of any one of claims 83 to 116, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / M47I / L70M.

119. 118. The method of any one of claims 83 to 117, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / M47V / N48K / V68M / K89N.

120. 118. The method of any one of claims 83 to 117, wherein the variant CD80 polypeptide comprises the amino acid modifications H18Y / A26E / E35D / M47L / V68M / A71G / D90G.

121. 118. The method of any one of claims 83 to 117, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / D46E / M47V / V68M / D90G / K93E.

122. 118. The method of any one of claims 83 to 117, wherein the variant CD80 polypeptide comprises the amino acid modifications E35D / D46V / M47L / V68M / L85Q / E88D.

123. 123. The method of any one of claims 83-122, wherein said immunomodulatory protein is soluble, and optionally said variant CD80 polypeptide lacks the CD80 transmembrane and intracellular signaling domains.

124. 124. The method of any one of claims 83 to 123, wherein the unmodified CD80 (i) comprises a sequence of amino acids as set forth in SEQ ID NO:2, (ii) comprises a sequence of amino acids having at least 95% sequence identity to SEQ ID NO:2, or (iii) is a portion of (i) or (ii) that comprises an IgV domain or a specific binding fragment thereof.

125. 125. The method of any one of claims 83 to 124, wherein the variant CD80 polypeptide is or comprises an IgV domain or a specific binding fragment thereof.

126. 126. The method of any one of claims 83 to 125, wherein said IgV domain or specific binding fragment thereof is the only CD80 portion of said immunomodulatory protein.

127. 127. The method of any one of claims 83-126, wherein the variant CD80 polypeptide 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.

128. The method of any one of claims 83 to 127, wherein the immunomodulatory protein is an Fc fusion protein.

129. 129. The method of any one of claims 81, 82, and 86-128, wherein the disease or condition is a tumor or cancer.

130. The method of any one of claims 75-129, wherein the subject has relapsed after remission, become refractory, or is a non-responder following treatment with a PD-1 / PD-L1 or PD-1 / PD-L2 antagonist.

131. 131. The method of claim 130, wherein the antagonist is an anti-PD-1 antibody, optionally nivolumab or pembrolizumab.

132. The method of any one of claims 75 to 81, wherein the immune response is reduced.

133. 133. The method of any one of claims 75 to 81 and 132, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

134. 1. A method for detecting a CD80 binding partner in a biological sample, comprising: (a) contacting a biological sample with a binding reagent comprising a variant CD80 polypeptide according to any one of claims 1 to 30; (b) detecting the presence of said binding reagent bound to or on cells of said biological sample; The method comprising:

135. The method of claim 134, wherein the binding partner is PD-L1, CD28, CTLA-4, or a combination thereof.

136. The method of claim 134 or claim 135, wherein the variant CD80 polypeptide comprises one or more amino acid modifications at one or more positions in the IgV domain or the IgC domain or a specific binding fragment thereof of unmodified CD80 or a specific binding fragment thereof, and wherein the variant CD80 polypeptide exhibits increased binding affinity to the ectodomain of PD-L1 compared to the binding affinity of unmodified CD80 to the ectodomain of PD-L1.

137. 137. The method of any one of claims 134 to 136, wherein the biological sample is or comprises a body fluid, cell, or tissue sample.

138. 138. The method of claim 137, wherein the bodily fluid is serum, plasma, or urine.

139. 138. The method of claim 137, wherein the tissue sample is a tumor tissue sample.

140. 140. The method of claim 139, wherein the tumor tissue sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof.

141. 141. The variant CD80 polypeptide of any one of claims 134 to 140, comprising the IgV domain or a specific binding fragment thereof.

142. 142. The method of claim 141, wherein the IgV domain or specific binding fragment thereof is the only CD80 portion of the binding reagent.

143. A method according to any one of claims 134 to 142, wherein the binding reagent is directly or indirectly linked to a label which is a detectable moiety or to a moiety which can be detected.

144. 144. The method of claim 143, wherein the portion is an Fc region.

145. The method of claim 144, wherein the Fc region is non-human, optionally mouse or rabbit.

146. The method of any one of claims 134 to 145, wherein detecting the presence of bound binding reagent is by immunohistochemistry, pseudo-immunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting.

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