CD80 variant immunomodulatory protein and its use

Abstract

Variant CD80 polypeptides, immunomodulatory proteins comprising variant CD80 polypeptides, and nucleic acids encoding such proteins are provided. The immunomodulatory proteins provide therapeutic utility in a wide variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided. [Solution] Provided is 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, which comprises one or more amino acid modifications at one or more positions of unmodified CD80 or its specific-binding fragment that correspond to a specific sequence, and which comprises up to 14 amino acid modifications.

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, U.S. Provisional Patent Application No. 62 / 472,574, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,575, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,576, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,577, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,578, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,579, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,580, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,581, filed March 16, 2017, U.S. Provisional Patent Application No. 62 / 472,582, filed March 16, 2017, U.S. Provisional Patent Application No. 62 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 4,888,576 byte file entitled 761612001640SeqList.TXT, created on March 12, 2018. 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 disclosure relates to particular 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 between multiple protein targets and 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 potentially interact simultaneously with proteins on both the same cell (cis) and related cells (trans). While 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, are provided to meet this need. Summary of the Invention

[0005] overview In some embodiments, provided herein are variant CD80 polypeptides 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, provided variant CD80 polypeptides 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, additional 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 substitution(s) 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 TIFF0007702928000001.tif187166 TIFF0007702928000002.tif231162 TIFF0007702928000003.tif231164 TIFF0007702928000004.tif231164 TIFF0007702928000005.tif99165, wherein the position(s) of the amino acid substitution(s) correspond to the positions of CD80 set forth in SEQ ID NO:2.

[0009] In some embodiments, provided herein are variant CD80 polypeptides containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, which variant CD80 polypeptides bind to unmodified CD80 or a specific binding fragment thereof by: TIFF0007702928000006.tif63165, wherein the position(s) of the amino acid substitution(s) correspond to the numbering of the CD80 positions set forth in SEQ ID NO:2.

[0010] In some embodiments, provided variant CD80 polypeptides 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. TIFF0007702928000007.tif92165, wherein the position(s) of the amino acid substitution(s) correspond to the numbering of the positions of CD80 set forth in SEQ ID NO:2.

[0011] In some embodiments, the one or more amino acid substitutions are TIFF0007702928000008.tif99163 TIFF0007702928000009.tif231162 TIFF0007702928000010.tif231162 TIFF0007702928000011.tif231166 TIFF0007702928000012.tif179163, wherein the position(s) of the amino acid substitution(s) correspond to the positions 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 the position(s) of the amino acid substitution(s) correspond to positions in CD80 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 one or more amino acid modifications comprise D46V; or one or more amino acid modifications comprise M47L; or one or more amino acid modifications comprise M47V; or one or more amino acid modifications comprise A71G, wherein the position(s) of the amino acid substitutions correspond to the numbering of the positions of CD80 according to 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(s) correspond to positions in CD80 set forth in SEQ ID NO:2.

[0013] In some embodiments, provided herein are variant CD80 polypeptides 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 polypeptides comprise amino acid modifications in unmodified CD80 or a specific binding fragment thereof, the amino acid modifications being E35D / D46E, E35D / D46V, E35D / M47I, E35D / M47L, E35D / M47V, E35D / V68M, E35D / A71G, or E35D / D90G; D46E / M47I, D 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 modifications correspond to the numbering of the positions in 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 the position(s) of the amino acid substitution(s) corresponds 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 containing a specifically binding fragment thereof. In some embodiments, the IgV domain or a specifically 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 that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: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. In 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 or a specific-binding fragment thereof 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.

[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 comprises 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 specifically 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 specifically binding fragment thereof, and that contains one or more amino acid substitutions.

[0023] In some embodiments, the IgC domain or 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 for 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 for the CTLA4 ectodomain compared to the binding affinity of unmodified CD80 to the CTLA4 ectodomain, hi some embodiments, the increased affinity for the CTLA-4 ectodomain is greater than a 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 CTLA-4 ectodomain.

[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 polypeptides are modified to unmodified CD80 or specific binding fragments thereof with one or more 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 substitution(s) corresponds to a position in CD80 as set forth in SEQ ID NO:2.

[0027] In any of the provided embodiments, the CD80 polypeptide can exhibit 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 ectodomain of CD28 is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold increased 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: unmodified CD80 or a specific binding fragment thereof; 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 L97Q, wherein the position(s) of the amino acid substitution(s) corresponds to a position in CD80 as set forth in SEQ ID NO:2.

[0028] In some embodiments, the variant CD80 polypeptide exhibits increased binding affinity for the PD-L1 ectodomain compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain. In some such embodiments, the increased affinity for the PD-L1 ectodomain is greater than a 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 increase compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain. In some such embodiments, the CD80 polypeptide contains one or more amino acid modifications 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, and / or 84 based on the numbering of SEQ ID NO:2.In some embodiments, the CD80 polypeptide comprises one or more of the following: unmodified CD80 or a specific binding fragment thereof, including any of the following: 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 substitution(s) correspond to the positions of CD80 set forth in SEQ ID NO:2.

[0029] In some embodiments, the variant CD80 exhibits 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 CTLA-4 ectodomain with increased selectivity compared to unmodified CD80 for the CTLA-4 ectodomain. In some such embodiments, the increased selectivity comprises a greater binding ratio of the variant polypeptide to CTLA-4 to CD28 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 provided embodiments, the variant CD80 polypeptide specifically binds to the PD-L1 ectodomain with increased selectivity compared to unmodified CD80 for the PD-L1 ectodomain. 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 the same. 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 having 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 having 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 mammalian, optionally human Fc domain. In some embodiments, the Fc domain or variant thereof contains the amino acid sequence set forth in SEQ ID NO:277, SEQ ID NO:359, or SEQ ID NO:1712, or an amino acid sequence that exhibits 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 that exhibits reduced effector function. In some embodiments, the variant CD80 polypeptide is indirectly linked to the multimerization domain or Fc via a linker, optionally a GSG4S linker (SEQ ID NO:1716). In some embodiments, the linker is not composed of three alanines (AAA).

[0035] In some embodiments, provided herein are immunomodulatory proteins containing any of the variant CD80 polypeptides provided herein and a half-life extending moiety. In some embodiments, the half-life extending moiety includes 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 includes Pro / Ala / Ser (PAS), and the variant CD80 polypeptide is PAS-ylated. 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, wherein 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 immunoglobulin Fc region. 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 the 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 the 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 containing one or more amino acid substitutions compared to a wild-type Fc region. In some such embodiments, the one or more amino acid substitutions in the variant Fc region are selected from Fc N297G, R292C / N297G / V302C, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, where the residues are numbered according to the EU index of Kabat. In some embodiments, the variant Fc region further comprises the amino acid substitution C220S, where the residues are numbered according to the EU index of Kabat. In some embodiments, the Fc region contains the amino acid sequence set forth in any of SEQ ID NOs:356-358, or a sequence of amino acids exhibiting 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, wherein the residues are numbered according to the EU index of Kabat. In some embodiments, the Fc region contains the amino acid sequence set forth in any of SEQ ID NOs:1713-1715, or a sequence of amino acids exhibiting 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 that express PD-L1, and optionally the T cells comprise Jurkat cells that express an IL-2 reporter or primary human T cells that produce 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 to 10 amino acids. In some embodiments, the linker is selected from AAA, G4S (SEQ ID NO:1717), or (G4S)2 (SEQ ID NO:330).

[0043] In some embodiments, the variant CD80 polypeptide is a transmembrane immunomodulatory protein that further contains 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 contains 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 contains a cytoplasmic signaling domain linked to the transmembrane domain. In some embodiments, the cytoplasmic signaling domain contains 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 any of the provided embodiments, the variant CD80 polypeptide modulates immune cell responses, 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 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 the 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 the 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 for 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 containing: (i) a wild-type CD112 polypeptide containing an IgSF domain set forth in any of SEQ ID NOs: 269, 734 or 829, or a variant CD112 polypeptide comprising an IgSF domain of any of the SEQ ID NOs: set forth in Table 3, optionally any of SEQ ID NOs: 735-828, 830-917, 918-999, 1430-1501; (ii) a wild-type CD155 polypeptide containing an IgSF domain set forth in any of SEQ ID NOs: 268, 378 or 421, or a variant CD155 polypeptide comprising an IgSF domain of any of the SEQ ID NOs: set forth in Table 4, optionally any of SEQ ID NOs: 379-420, 422-539, 540-733, 1502-1573, 1548-1711; (iv) a wild-type PD-L1 polypeptide containing an IgSF domain as set forth in any of SEQ ID NOs: 251, 1000, 1721 or 1196, or any of SEQ ID NOs listed in Table 5, optionally any of SEQ ID NOs: 1001-1065, 1718-1900, 1931-1996; (iv) a wild-type PD-L2 polypeptide containing an IgSF domain as set forth in any of SEQ ID NOs: 252, 1197 or 1257, or any of SEQ ID NOs listed in Table 6, optionally any of SEQ ID NOs: 1198-1248, 1250-1325, 1327-1401, 1403-1426, 1719, 1720, 1901-1930; (v) a variant PD-L2 polypeptide containing an IgSF domain as set forth in any of SEQ ID NOs in (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 (i) to (v) and contains amino acid substitutions; or (vi) a specific-binding fragment of any of (i) to (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 are conjugates containing an immunomodulatory 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 cell surface molecule. In some embodiments, the targeting moiety specifically binds to a molecule on the surface of an immune cell, optionally an antigen-presenting cell or lymphocyte. In some embodiments, the immune cell is an antigen-presenting cell or lymphocyte. In some embodiments, the targeting moiety is a tumor-localizing moiety that binds to a molecule on the surface of a tumor. 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 variant CD80 polypeptide provided herein, an immunomodulatory polypeptide provided herein, or a conjugate that is a fusion protein containing any of 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 a 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 for expressing 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 cells 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 such that 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 are infectious agents 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 retrovirus construct or a hybrid thereof. In some embodiments, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is an adenovirus, adeno-associated virus, herpes virus, herpes simplex virus, reovirus, Newcastle disease virus, parvovirus, measles virus, vesicular stomatitis virus (VSV), coxsackievirus, or 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, hi some embodiments, the additional gene product is selected from an anti-cancer agent, an anti-metastatic agent, an anti-angiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degrading gene, a gene for tissue regeneration, and a gene for reprogramming human somatic cells to pluripotency.

[0067] In some embodiments, provided herein are pharmaceutical compositions containing 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 pharmaceutically 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 are methods of modulating an immune response in a subject, comprising administering an immunomodulatory protein provided herein, e.g., an immunomodulatory protein that exhibits increased binding affinity to PD-L1. In some embodiments, the immune response is increased. In some embodiments, the immunomodulatory protein is an immunomodulatory protein provided herein that exhibits Fc-dependent CD28 costimulation. In some embodiments, the immunomodulatory protein is an immunomodulatory protein provided herein that exhibits PD-L1-dependent CD28 costimulation, and optionally, the immunomodulatory protein contains a variant CD80 polypeptide provided herein.

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

[0073] Also provided herein are methods of modulating an immune response in a subject, e.g., increasing or decreasing the immune response, comprising administering to the 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 in 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 substitution(s) 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 are: Selected from TIFF0007702928000013.tif128166.

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

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

[0079] In some embodiments, provided are methods of modulating an immune response 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, provided methods 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 subject's immune response.

[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 by the inhibitory receptor CTLA-4 is activated, stimulated, or induced by the 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 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 methods, an immunomodulatory protein provided herein or a conjugate provided herein is administered to a subject. In some embodiments of the provided methods, a variant CD80 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject. In some embodiments of the provided methods, engineered cells containing a variant CD80 polypeptide that is a transmembrane immunomodulatory protein provided herein are administered to a 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 whereby 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 methods, 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 subject's immune response.

[0088] In some embodiments, provided herein are methods for 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 thereof, of unmodified CD80, or a specific binding fragment thereof, and which exhibits PD-L1-dependent CD28 costimulation. In some embodiments, the variant CD80 polypeptide exhibits increased binding affinity for the PD-L1 ectodomain compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain. In some embodiments, provided herein are methods for 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 comprising one or more amino acid modifications at one or more positions in the IgV domain or IgC domain, or specific binding fragment thereof, of unmodified CD80, or a specific binding fragment thereof, and the variant CD80 polypeptide exhibits increased binding affinity for the PD-L1 ectodomain compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain. 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 is selected as having a tumor comprising cell surface that is 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 wherein the cells are tumor cells or tumor-infiltrating immune cells; and (c) treating the subject if the tumor tissue sample contains cell surface positive for detectable levels of 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 wherein the cells are tumor-infiltrating lymphocytes, optionally wherein the lymphocytes are T cells, optionally 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 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 PD-L1 ectodomain compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain.

[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 that are positive for detectable levels of CD28, optionally wherein 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 polypeptide exhibits 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, wherein 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, wherein 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 such embodiments, the CD80 polypeptide contains one or more amino acid modifications in unmodified CD80 or a specific-binding fragment thereof 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 some such embodiments, the CD80 polypeptide is modified to unmodified CD80 or a specific binding fragment thereof. TIFF0007702928000014.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 exhibits at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, or 70% greater affinity for the ectodomain of CD28 compared to the binding affinity of the unmodified CD80 polypeptide for the ectodomain of CD28. %, 70%, 75%, 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% of the total binding affinity of the variant CD80 polypeptide to the ectodomain of CD28. In some embodiments, the variant CD80 polypeptide exhibits 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 for the CD28 ectodomain 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 CD28 ectodomain.

[0106] In some embodiments, the subject's disease or condition 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 the binding reagent bound to 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 IgC domain or specific binding fragment thereof of unmodified CD80 or a specific binding fragment thereof, and the variant CD80 polypeptide exhibits increased binding affinity to the PD-L1 ectodomain compared to the binding affinity of unmodified CD80 to the PD-L1 ectodomain.

[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, hi 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. In 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, detecting the presence of bound binding reagent is by immunohistochemistry, pseudoimmunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting. [The present invention 1001] 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, 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; containing up to 14 amino acid modifications, The variant CD80 polypeptide. [The present invention 1002] 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. [The present invention 1003] The variant CD80 polypeptide of invention 1001 or invention 1002, comprising one or more amino acid modifications corresponding to one or more positions 26, 35, 46, 57, or 71 based on the numbering of SEQ ID NO:2. [The present invention 1004] 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 its specific binding fragment may be selected from the group consisting of: TIFF0007702928000015.tif77165 and comprising one or more amino acid modifications corresponding to containing up to 14 amino acid modifications, The variant CD80 polypeptide. [The present invention 1005] 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 position numbering set forth in SEQ ID NO:2. TIFF0007702928000016.tif77166 and containing 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. [The present invention 1006] 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 1001 to 1005, [The present invention 1007] 1004 or 1005, a variant CD80 polypeptide of the invention comprising the amino acid modification M47L. [The present invention 1008] 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 in SEQ ID NO:2 TIFF0007702928000017.tif150166TIFF0007702928000018.tif216162TIFF0007702928000019.tif216162TIFF0007702928000020.tif216166TIFF0007702928000021.tif216163TIFF0007702928000022.tif77164 comprising an amino acid modification selected from The variant CD80 polypeptide. [The present invention 1009] Amino acid modification TIFF0007702928000023.tif41163 1009. A variant CD80 polypeptide according to any one of claims 1001 to 1008, comprising: [The present invention 1010] 1009. The variant CD80 polypeptide of any of claims 1004 to 1009, comprising the amino acid modifications E35D / M47I, E35D / M47L, E35D / M47V, or E35D / V68M. [The present invention 1011] 10. The variant CD80 polypeptide of any of claims 1004 to 1010, comprising the amino acid modifications E35D / M47L / V68M or E35D / M47V / V68M. [The present invention 1012] 10. The variant CD80 polypeptide of any of claims 1001 to 1011, 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. [The present invention 1013] 1012. A variant CD80 polypeptide of the invention, 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. [The present invention 1014] Amino acid modifications based on the numbering of positions in SEQ ID NO:2 TIFF0007702928000024.tif136166TIFF0007702928000025.tif216162TIFF0007702928000026.tif216162TIFF0007702928000027.tif218164 A variant CD80 polypeptide of any one of claims 1004 to 1013, comprising: [The present invention 1015] The variant CD80 polypeptide of any one of claims 1004 to 1009 and 1011 to 1013, wherein the amino acid modifications comprise E35D / M47I / L70M. [The present invention 1016] 1014. The variant CD80 polypeptide of any one of claims 1004 to 1013, wherein the amino acid modifications comprise E35D / M47V / N48K / V68M / K89N. [The present invention 1017] 1014. The variant CD80 polypeptide of any of claims 1004 to 1013, wherein the amino acid modifications comprise H18Y / A26E / E35D / M47L / V68M / A71G / D90G. [The present invention 1018] 1014. The variant CD80 polypeptide of any one of claims 1004 to 1013, wherein the amino acid modifications comprise E35D / D46E / M47V / V68M / D90G / K93E. [The present invention 1019] 1014. The variant CD80 polypeptide of any of claims 1004 to 1013, wherein the amino acid modifications comprise E35D / D46V / M47L / V68M / L85Q / E88D. [The present invention 1020] 10. The variant CD80 polypeptide of any of claims 1001 to 1019, wherein the unmodified CD80 (i) comprises the sequence of amino acids 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) comprising an IgV domain or a specific-binding fragment thereof. [The present invention 1021] A variant CD80 polypeptide of any of claims 1001 to 1019, which is or comprises the IgV domain or a specific-binding fragment thereof. [The present invention 1022] 1022. The variant CD80 polypeptide of any of claims 1001 to 1021, wherein said IgV domain or a specific binding fragment thereof is the only CD80 portion of said variant CD80 polypeptide. [The present invention 1023] 10. The variant CD80 polypeptide of any of claims 1001 to 1022, comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid alterations. [The present invention 1024] A sequence of amino acids or a specifically binding fragment thereof 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 specifically 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 set forth in any one of SEQ ID NOs: 77-149, 151-223, 2105-2296, 2508-2929, or 2961-3022, or a sequence of amino acids or a specific binding fragment thereof that exhibits 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. A variant CD80 polypeptide of any one of claims 1001 to 1023, comprising: [The present invention 1025] 1001-1024. A variant CD80 polypeptide of any of claims 1001-1024, comprising a sequence of amino acids set forth in any of SEQ ID NOs: 199, 208, 2250, 2276, 2280, or 2284. [The present invention 1026] lacking the CD80 transmembrane and intracellular signaling domains, and / or cannot be expressed on the cell surface, The variant CD80 polypeptide of any of embodiments 1001 to 1025. [The present invention 1027] 1027. A variant CD80 polypeptide of any of claims 1001 to 1026 linked to a moiety that increases the biological half-life of said polypeptide. [The present invention 1028] 10. The variant CD80 polypeptide of any of claims 1001 to 1027, linked to a multimerization domain. [The present invention 1029] 1028. A variant CD80 polypeptide of the invention, wherein said multimerization domain is an Fc domain or a variant Fc domain with reduced effector function. [The present invention 1030] A variant CD80 polypeptide of any of claims 1001 to 1029 of the present invention, which is a transmembrane immunomodulatory protein, further comprising a transmembrane domain and / or a cytoplasmic signaling domain. [The present invention 1031] 1. An immunomodulatory protein comprising a first variant CD80 polypeptide of any one of claims 1001 to 1029 linked to a first multimerization domain and a second variant CD80 polypeptide of any one of claims 1001 to 1029 linked to a second multimerization domain, wherein said first and second multimerization domains interact to form a multimer comprising the first and second variant CD80 polypeptides. [The present invention 1032] The immunomodulatory protein of the present invention 1031, wherein said multimer is a dimer, optionally a homodimer. [The present invention 1033] 103. The immunomodulatory protein of claim 1031 or 1032, wherein said first variant CD80 polypeptide and said second variant CD80 polypeptide are identical. [The present invention 1034] The immunomodulatory protein of any of claims 1031 to 1033, 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. [This invention 1035] The immunomodulatory protein of the present invention 1034, wherein said Fc region is that of an immunoglobulin G1 (IgG1) or immunoglobulin G2 (IgG2) protein. [The present invention 1036] The immunomodulatory protein of the present invention 1034 or 1035, wherein said Fc region exhibits one or more effector functions. [This invention 1037] The immunomodulatory protein of any of claims 1031 to 1036, which exhibits Fc-dependent CD28 costimulation. [The present invention 1038] The immunomodulatory protein of any of claims 1031 to 1034, wherein the Fc region is a variant Fc region comprising one or more amino acid substitutions in a wild-type Fc region, and the variant Fc region exhibits 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. [This invention 1039] 1038. An immunomodulatory protein of the invention, wherein said Fc region comprises the amino acid substitution N297G, said residues being numbered according to the EU index of Kabat. [The present invention 1040] 1038. An immunomodulatory protein of the invention, wherein said Fc region comprises the amino acid substitutions R292C / N297G / V302C, said residues being numbered according to the EU index of Kabat. [This invention 1041] 1038. An immunomodulatory protein of the invention, wherein said Fc region comprises the amino acid substitutions L234A / L235E / G237A, said residues being numbered according to the EU index of Kabat. [The present invention 1042] 1042. The immunomodulatory protein of any of claims 1039 to 1041, wherein said variant Fc region further comprises the amino acid substitution C220S, said residues being numbered according to the EU index of Kabat. [This invention 1043] 1042. The immunomodulatory protein of any of claims 1038 to 1042, wherein the Fc region comprises K447del, said residues being numbered according to the EU index of Kabat. [This invention 1044] An immunomodulatory protein according to any one of claims 1038 to 1043, comprising a variant CD80 polypeptide according to any one of claims 1012 to 1029. [This invention 1045] An immunomodulatory protein according to any one of claims 1038 to 1044 of the present invention, which exhibits PD-L1-dependent CD28 costimulation. [The present invention 1046] An immunomodulatory protein comprising a variant CD80 polypeptide of any of claims 1001 to 1029 linked directly or indirectly via a linker to a second polypeptide comprising an IgSF domain of an immunoglobulin superfamily (IgSF) member. [This invention 1047] The immunomodulatory protein of the present invention 1046, wherein said IgSF domain is an affinity-modified IgSF domain, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of said IgSF family member. [This invention 1048] 1047. The immunomodulatory protein of the present invention, wherein said 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 said IgSF family member to the same one or more cognate binding partner(s). [This invention 1049] A conjugate comprising a variant CD80 polypeptide of any of claims 1001 to 1029 linked to a targeting moiety that specifically binds to a cell surface molecule. [The present invention 1050] The conjugate of the present invention 1049, wherein said cell is an immune cell or a tumor cell. [This invention 1051] The conjugate of claim 1049 or claim 1050, wherein said moiety is a protein, peptide, nucleic acid, small molecule or nanoparticle. [This invention 1052] 1052. The conjugate of any of claims 1049 to 1051, wherein said moiety is an antibody or an antigen-binding fragment. [This invention 1053] The conjugate of any one of 1050 to 1052 of the present invention, which is a fusion protein. [This invention 1054] A nucleic acid molecule encoding a variant CD80 polypeptide of any one of claims 1001 to 1030, an immunomodulatory protein of any one of claims 1031 to 1048, or a conjugate which is a fusion protein of any one of claims 1049 to 1053. [This invention 1055] A vector comprising a nucleic acid molecule of the present invention. [This invention 1056] The vector of the present invention 1055, which is an expression vector. [This invention 1057] A cell comprising the vector of the present invention 1055 or 1056. [This invention 1058] 10. A method for producing a variant CD80 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule of claim 1054 or a vector of claim 1055 or a vector of claim 1056 into a host cell under conditions such that said protein is expressed in said cell. [This invention 1059] The method of claim 1058, further comprising isolating or purifying the variant CD80 polypeptide or immunomodulatory protein from said cells. [The present invention 1060] 10. A method of engineering a cell to express a variant CD80 variant polypeptide, the method comprising introducing into a host cell a nucleic acid molecule encoding the variant CD80 polypeptide of any of embodiments 1001-1030 under conditions such that said polypeptide is expressed in said cell. [This invention 1061] An engineered cell comprising a variant CD80 polypeptide of any of claims 1001 to 1030, an immunomodulatory protein of any of claims 1031 to 1048, a conjugate that is a fusion protein of any of claims 1049 to 1053, a nucleic acid molecule of claim 1054, or a vector of claim 1055 or 1056. [This invention 1062] 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; The engineered cells of the present invention 1061. [This invention 1063] the variant CD80 polypeptide comprises a transmembrane domain or is a transmembrane immunomodulatory protein of the invention; and / or the variant CD80 polypeptide is expressed on the surface of the cell. The engineered cells of the present invention 1061. [This invention 1064] The engineered cell of any of claims 1061 to 1063, wherein said cell is an immune cell, optionally an antigen-presenting cell (APC) or a lymphocyte, optionally a T cell. [This invention 1065] 1065. The engineered cell of any of claims 1061 to 1064, further comprising a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). [The present invention 1066] An infectious agent comprising a nucleic acid molecule encoding a variant CD80 polypeptide of any of embodiments 1001 to 1030, an immunomodulatory protein of any of embodiments 1031 to 1047, or a conjugate that is a fusion protein of any of embodiments 1048 to 1052. [This invention 1067] The infectious agent of the present invention 1066, which is a bacterium or a virus. [The present invention 1068] The infectious agent of claim 1067, wherein the infectious agent is a virus, and the virus is an oncolytic virus. [This invention 1069] A pharmaceutical composition comprising a variant CD80 polypeptide of any of claims 1001 to 1030, an immunomodulatory protein of any of claims 1031 to 1048, a conjugate of any of claims 1049 to 1053, an engineered cell of any of claims 1061 to 1065, or an infectious agent of any of claims 1066 to 1068. [The present invention 1070] A pharmaceutical composition of the present invention 1069 comprising a pharmaceutically acceptable excipient. [This invention 1071] The pharmaceutical composition of invention 1069 or invention 1070, wherein said pharmaceutical composition is sterile. [This invention 1072] An article of manufacture comprising any one of the pharmaceutical compositions of the present inventions 1069 to 1071 in a vial. [This invention 1073] 1072. The article of manufacture of claim 1072, wherein the vial is sealed. [This invention 1074] A kit comprising the pharmaceutical composition of any one of the present inventions 1069 to 1071, or the article of manufacture of the present invention 1072 or 1073, and instructions for use. [This invention 1075] A method for regulating an immune response in a subject, comprising administering any one of the pharmaceutical compositions of present inventions 1069 to 1071. [This invention 1076] A method for regulating an immune response in a subject, comprising administering the immunomodulatory protein of any of the present inventions 1031 to 1048. [This invention 1077] The method of claim 1076, wherein said immunomodulatory protein is an immunomodulatory protein of claim 1037 that exhibits Fc-dependent CD28 costimulation. [This invention 1078] The method of claim 1074, wherein said immunomodulatory protein is an immunomodulatory protein of claim 1044 that exhibits PD-L1-dependent CD28 costimulation, and optionally said immunomodulatory protein comprises a variant CD80 polypeptide of any of claims 1012 to 1029. [This invention 1079] A method for modulating an immune response in a subject, comprising administering an engineered cell of any of claims 1061 to 1065. [The present invention 1080] 1079. The method of claim 1079, wherein said engineered cells are autologous to said subject. [This invention 1081] The method of any of claims 1075 to 1080, wherein the disease or condition of the subject is treated by modulating the immune response. [This invention 1082] 1082. The method of any one of claims 1075 to 1081, wherein the immune response is increased. [This invention 1083] 1. A method of increasing an immune response 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 immunomodulatory protein exhibits PD-L1-dependent CD28 costimulation. [This invention 1084] 1083. The method of claim 1083, wherein said 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. [This invention 1085] 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, wherein the antibody exhibits increased binding affinity to the ectodomain of PD-L1. [The present invention 1086] 6. The method of any of claims 1083 to 1085, for use in treating a disease or condition. [This invention 1087] prior to said administering, selecting a subject having a tumor comprising cells positive for surface PD-L1, optionally wherein said cells are tumor cells or tumor-infiltrating immune cells; or the subject is selected as having a tumor comprising cell surface cells positive for PD-L1, and optionally, the cells are tumor cells or tumor-infiltrating immune cells; Any of methods 1075 to 1086 of the present invention. [This invention 1088] 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 the binding reagent bound to 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 detectable levels of cell surface positive PD-L1; The method of the present invention 1087, comprising: [This invention 1089] Prior to said administering, a subject is selected to be treated with a tumor comprising cell surface cells positive for CD28, optionally said cells being tumor-infiltrating lymphocytes, optionally said lymphocytes being T cells, optionally CD8+ T cells; or the subject is selected as having a tumor comprising cell surface cells positive for CD28, optionally wherein the cells are tumor-infiltrating lymphocytes, optionally wherein the lymphocytes are T cells, optionally CD8+ T cells; Any of methods 1075 to 1088 of the present invention. [The present invention 1090] said selecting a target (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 the binding reagent bound to or on cells of said tumor tissue sample, optionally wherein said cells are tumor-infiltrating lymphocytes, and optionally wherein said lymphocytes are T cells, optionally CD8+ T cells; (c) selecting the subject for treatment if the tumor tissue sample contains detectable levels of cell surface positive for CD28; The method of the present invention 1089, comprising: [This invention 1091] (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 the binding reagent bound to 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: [This invention 1092] 1091. The method of claim 1091, comprising contacting said tumor tissue sample with a binding reagent capable of specifically binding to CD28, wherein said subject is selected if said tumor tissue sample further comprises tumor-infiltrating lymphocytes that are positive for a detectable level of CD28, and optionally said lymphocytes are T cells, optionally CD8+ T cells. [This invention 1093] The method of any of claims 1088 and 1090 to 1092, wherein the tumor tissue sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. [This invention 1094] The method of any one of claims 1088 and 1090 to 1093, wherein said binding reagent is an antibody or antigen-binding fragment, a protein ligand or binding partner, an aptamer, an affimer, a peptide, or a hapten. [This invention 1095] 1095. The method of any one of claims 1088, 1091, 1093 or 1094, wherein the binding reagent is an anti-PD-L1 antibody or antigen-binding fragment. [This invention 1096] The method of any of claims 1088 to 1094, wherein said binding reagent comprises a variant CD80 polypeptide of any of claims 1001 to 1030. [This invention 1097] 1096. The method of claim 1096, wherein said variant CD80 polypeptide comprises said IgV domain or a specific binding fragment thereof. [This invention 1098] 1098. The method of claim 1096 or 1097, wherein said IgV domain or specific binding fragment thereof is the only CD80 portion of said binding reagent. [This invention 1099] 1098. The method of any of claims 1096 to 1098, wherein said variant CD80 polypeptide exhibits increased affinity for binding to PD-L1 compared to said wild-type or unmodified CD80 polypeptide. [The present invention 1100] 1099. The method of any of claims 1088 and 1090 to 1099, wherein said binding reagent is directly or indirectly linked to a moiety that is a detectable moiety or a moiety that can be detected. [The present invention 1101] 1100. The method of claim 1100, wherein said moiety is an Fc region. [The present invention 1102] 1102. The method of claim 1101, wherein said Fc region is non-human, optionally murine or rabbit. [The present invention 1103] The method of any of claims 1088 and 1090-1102, wherein detecting the presence of bound binding reagent is by immunohistochemistry, pseudo-immunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting. [The present invention 1104] The method of any of claims 1091 to 1103, further comprising administering said immunomodulatory protein to said subject. [This invention 1105] 1105. The method of any of claims 1083 to 1104, wherein said 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 said first and second multimerization domains interact to form a multimer comprising said first and second variant CD80 polypeptides. [The present invention 1106] 1105. The method of claim 1105, wherein the multimer is a dimer. [This invention 1107] 1107. The method of claim 1105 or claim 1106, wherein said first variant CD80 polypeptide and said second variant CD80 polypeptide are identical. [This invention 1108] The method of any of claims 1105 to 1107, wherein said multimerization domain is or comprises an Fc region. [This invention 1109] 1108. The method of claim 1108, wherein said Fc region is a variant Fc region comprising one or more amino acid substitutions compared to a wild-type Fc region, said Fc region exhibiting one or more reduced effector functions compared to said wild-type Fc region, and optionally said wild-type Fc is human IgG1. [The present invention 1110] The variant CD80 polypeptide may be a variant of unmodified CD80 or a specific binding fragment thereof, which has the sequence of SEQ ID NO: Any of the methods of 1083 to 1109 of the present invention, comprising one or more amino acid modifications corresponding to positions 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. [The present invention 1111] The variant CD80 polypeptide is a variant of unmodified CD80 or a specific binding fragment thereof, based on the position numbering set forth in SEQ ID NO:2. TIFF0007702928000028.tif99165 The method of any of claims 1083 to 1110, comprising one or more amino acid modifications corresponding to: [The present invention 1112] 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 based on the numbering of SEQ ID NO:2. TIFF0007702928000029.tif48166 Any of the methods of 1083 to 1110 of the present invention, selected from the group consisting of [The present invention 1113] The one or more amino acid modifications are based on the numbering of positions set forth in SEQ ID NO:2 TIFF0007702928000030.tif41163TIFF0007702928000031.tif216166TIFF0007702928000032.tif216162TIFF0007702928000033.tif216162TIFF0007702928000034.tif91164 Any of the methods of 1083 to 1112 of the present invention, selected from the following. [This invention 1114] the variant CD80 polypeptide is 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 Any of the methods of claims 1083 to 1113 of the present invention, comprising: [This invention 1115] The variant CD80 polypeptide comprises an amino acid modification TIFF0007702928000035.tif41165 Any of the methods of claims 1083 to 1114 of the present invention, comprising: [The present invention 1116] 1115. The method of any of claims 1083 to 1115, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / M47I, E35D / M47L, E35D / M47V, or E35D / V68M. [This invention 1117] 1116. The method of any of claims 1083 to 1116, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / M47L / V68M or E35D / M47V / V68M. [This invention 1118] 1116. The method of any of claims 1083 to 1116, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / M47I / L70M. [This invention 1119] 1117. The method of any of claims 1083 to 1117, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / M47V / N48K / V68M / K89N. [The present invention 1120] 1117. The method of any of claims 1083 to 1117, wherein said variant CD80 polypeptide comprises the amino acid modifications H18Y / A26E / E35D / M47L / V68M / A71G / D90G. [This invention 1121] 1117. The method of any of claims 1083 to 1117, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / D46E / M47V / V68M / D90G / K93E. [This invention 1122] 1117. The method of any of claims 1083 to 1117, wherein said variant CD80 polypeptide comprises the amino acid modifications E35D / D46V / M47L / V68M / L85Q / E88D. [This invention 1123] 13. The method of any of claims 1083 to 1122, wherein said immunomodulatory protein is soluble, and optionally said variant CD80 polypeptide lacks the CD80 transmembrane domain and the intracellular signaling domain. [This invention 1124] Any of the methods of claims 1083 to 1123, wherein the unmodified CD80 (i) comprises the sequence of amino acids 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) comprising an IgV domain or a specific-binding fragment thereof. [This invention 1125] 15. The method of any of claims 1083 to 1124, wherein said variant CD80 polypeptide is or comprises an IgV domain or a specific binding fragment thereof. [Invention 1126] 16. The method of any of claims 1083 to 1125, wherein said IgV domain or a specific binding fragment thereof is the only CD80 portion of said immunomodulatory protein. [This invention 1127] 1127. The method of any of claims 1083 to 1126, wherein said 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. [This invention 1128] The method of any of claims 1083 to 1127, wherein the immunomodulatory protein is an Fc fusion protein. [This invention 1129] The method of any one of claims 1081, 1082, and 1086 to 1128, wherein the disease or condition is a tumor or cancer. [The present invention 1130] Any of the methods of claims 1075 to 1129, 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. [This invention 1131] 1130. The method of claim 1130, wherein said antagonist is an anti-PD-1 antibody, optionally nivolumab or pembrolizumab. [This invention 1132] 1082. The method of any of claims 1075 to 1081, wherein the immune response is reduced. [This invention 1133] The method of any one of claims 1075 to 1081 and 1132, wherein said disease or condition is an inflammatory or autoimmune disease or condition. [This invention 1134] 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 of any of the inventions 1001 to 1030; (b) detecting the presence of said binding reagent bound to or on cells of said biological sample; The method comprising: [This invention 1135] 1135. The method of claim 1134, wherein said binding partner is PD-L1, CD28, CTLA-4 or a combination thereof. [This invention 1136] 1136. The method of claim 1134 or claim 1135, 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 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. [This invention 1137] 1137. The method of any of claims 1134 to 1136, wherein said biological sample is or comprises a body fluid, cell, or tissue sample. [This invention 1138] 1137. The method of claim 1137, wherein said body fluid is serum, plasma, or urine. [This invention 1139] 1137. The method of claim 1137, wherein said tissue sample is a tumor tissue sample. [This invention 1140] 1139. The method of claim 1139, wherein said tumor tissue sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. [This invention 1141] A variant CD80 polypeptide of any one of 1134 to 1140, comprising the IgV domain or a specific-binding fragment thereof. [This invention 1142] 1142. The method of claim 1141, wherein said IgV domain or specific binding fragment thereof is the only CD80 portion of said binding reagent. [This invention 1143] 1143. The method of any of claims 1134 to 1142, wherein said binding reagent is linked directly or indirectly to a label which is a detectable moiety or to a moiety which can be detected. [This invention 1144] 1143. The method of claim 1143, wherein said moiety is an Fc region. [Invention 1145] 114. The method of claim 1144, wherein said Fc region is non-human, optionally mouse or rabbit. [Invention 1146] 1146. The method of any of claims 1134 to 1145, wherein detecting the presence of bound binding reagent is by immunohistochemistry, pseudo-immunohistochemistry, immunofluorescence, flow cytometry, ELISA, or immunoblotting. [Brief explanation 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 (vIgD1) and a second IgSF domain, such as a second variant IgSF domain (vIgD2), (3) a tumor-targeting IgSF molecule containing a first variant IgSF domain (vIgD1) and an IgSF domain that targets a tumor antigen, such as an NKP30 IgSF domain, and (4) a soluble molecule containing a variant IgSF domain (vIgD) linked to an antibody (V-mAb). [Figure 1B] Illustrated are various formats of the variant IgSF domain molecules 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 a TIP containing vIgD (e.g., CD80 vIgD) antagonizes or blocks the negative signaling of the inhibitory receptor, thereby resulting in activated or effector T cells. In some cases, agonistic activity by the TIP can be achieved when the inhibitory receptor (CTLA-4) clusters in close proximity to an activating receptor (e.g., CD28). [Figure 1C]Various formats of variant IgSF domain molecules are provided. The variant IgSF domain (vIgD) represents 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 the 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 SIP to its cognate binding partner, the SIP antagonizes or blocks negative signaling via the inhibitory receptor, resulting in activated or effector T cells. 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 an IgSF superfamily member. [Figure 2]

[0023] Figure 1 shows an exemplary schematic 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 toward an effector phenotype. [Figure 3A] An exemplary schematic diagram of the activity of a variant IgSF domain (vIgD) conjugated to Fc is shown, in which the CD80-Fc confers PD-L1-dependent CD28 agonistic activity. As shown, binding of CD80-Fc to PD-L1 expressed on the surface of tumor cells can prevent the engagement 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]

[0023] Figure 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 the 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 stacked 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. [Figure 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 the 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 configurations 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 second IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of the Fc region. To generate homodimeric Fc molecules, the Fc regions are capable of forming homodimers with a compatible Fc region upon coexpression of the individual Fc regions in cells. To generate heterodimeric Fc molecules, the individual Fc regions contain mutations (e.g., "knob-into-hole" mutations in the CH3 domain) that favor heterodimer formation over homodimer formation when the individual Fc regions are coexpressed in cells. [Figure 6B]Various exemplary configurations of stacked 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 a homodimeric Fc molecule, the Fc region is one that can form a homodimer with a compatible Fc region by coexpression 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, in which 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 neighboring immune cells (e.g., T cells) and agonize or antagonize 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., with 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 antibody light chain. [Figure 8B]1 shows various exemplary configurations of variant IgSF antibody conjugates (V-Mabs), where 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-Mabs), and the resulting V-Mab configurations when the light chain in Figure 8A and the heavy chain in Figure 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 is shown. [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] Figure 1 shows the ability of exemplary CD80 IgV-Fc candidates to bind to PD-L1 and block fluorescently conjugated PD-1 binding. [Figure 9E] 1 shows the 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 1 shows the concentration of IFNγ in hPD-L1MC38 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] Median (left panel) and mean (right panel) tumor volumes in mice designated tumor-free after treatment with an exemplary CD80 IgV-Fc (inactive) variant and an anti-PD-L1 antibody (durvalumab) are shown after restimulation with huPD-L1 / MC38 tumor cells. [Figure 15] Figure 1 shows the 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] 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) are shown. [Figure 17] Panels 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. Panel 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 1 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] Figure 1 shows binding of CD80 IgV-Fc variants to primary human T cells. [Figure 19B] Figure 1 shows binding of CD80 IgV-Fc variants to primary human monocytes. [Figure 20] Figure 1 shows CD80 IgV-Fc variant antagonism of PD-L1-mediated SHP-2 recruitment to PD-1 using an enzyme complementation assay. [Figure 21] 1 shows CD80 IgV-Fc variant antagonism of CD80 / CTLA-4 binding. DETAILED DESCRIPTION OF THE INVENTION

[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 a 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 comprise 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 CD28, e.g., a ratio of binding affinity, of unmodified or wild-type CD80. In some embodiments, the ratio increases 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 for binding to PD-L1 relative to CD28 of unmodified or wild-type CD80 that does not comprise 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., a ratio of binding affinity, of unmodified or wild-type CD80. In some embodiments, the ratio increases 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 are 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 is capable of delivering a transmembrane immunomodulatory protein or a secretable immunomodulatory protein to a cell that it infects for expression. In some embodiments, also provided herein are 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. In 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, provided variant CD80 polypeptides regulate T cell activation, expansion, differentiation, and survival through interaction with costimulatory signaling molecules. Generally, two distinct signals are required for antigen-specific T cell activation. 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, costimulatory for TCR engagement, e.g., a CD28 costimulatory signal, is required to avoid T cell apoptosis or anergy.

[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 immunomodulatory proteins can become 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 similar affinity for CD28 as PD-L1. PD-L1 is one of two ligands for the inhibitory immune receptor, programmed death 1 (PD-1). The interaction between PD-L1 and 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 cell activation 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, thereby preventing the inhibition of T cell responses, for example, at tumor sites, effectively enhancing or boosting immune responses. However, at the same time, CD80 may also bind to CD28 or CTLA4 receptors and potentially be involved 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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 an immune response (Figure 3B). In some embodiments, provided CD80 variant polypeptides can be stacked or associated with other immunomodulatory 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] Various formats of the variant polypeptides are also provided. As demonstrated herein, different formats can facilitate manipulation of the immune response and thus have therapeutic applications. The ability to format the variant polypeptides in various configurations to antagonize or agonize the immune response, depending on the situation, provides flexibility in therapeutic applications based on the same increased binding and activity of the variant CD80 to its binding partner. As one example, tethering a variant CD80 protein to a surface can deliver a localized costimulatory signal, while in other cases, presenting CD80 in a delocalized, soluble form confers antagonistic activity. For example, delivery of enhanced CD80 protein in a soluble format with increased affinity for CTLA-4 and / or PD-L1 can antagonize inhibitory receptor signaling, such as blocking intracellular inhibitory signals that can diminish 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 tethering or bridging the variant CD80 molecule to the surface of the immune synapse for interaction with CD28, thereby providing a costimulatory signal and 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 polypeptide to bind to 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., Figure 3). In some cases, such PD-L1-dependent costimulation does not require an Fc with effector function and can be mediated by Fc fusion proteins containing effector-less or inactive Fc molecules. 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 in turn activates 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 the 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 for the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, therapies that intervene in and alter the costimulatory effects 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, not agonize them. In addition, pharmacokinetic differences between drugs that independently target one or the other of these two receptors can make it difficult to adequately maintain the desired blood levels of such drug combinations throughout the course of treatment. The provided variant CD80 polypeptides and immunomodulatory proteins, as well as 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 a patent, patent application, scientific article, or database, were 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 terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as making a significant difference from what is commonly understood in the art.

[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 one of ordinary skill 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 refers to 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") relative 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 for its cognate binding partner(s) will be at least 10% greater than the 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 for 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 due to substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any requirement on any particular starting composition or method by which the affinity-modified IgSF domain is produced. Thus, affinity-modified IgSF domains of the present invention are not limited to wild-type IgSF domains 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 their cognate binding partners, 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, refers to 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 removed 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 tissues from which they are removed from the organism. For example, naturally occurring autologous T cells can be genetically engineered by recombinant DNA technology to become autologous engineered cells that express transmembrane immunomodulatory proteins and / or chimeric antigen receptors (CARs), which in some cases involves 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 "avidity," as used herein, refer to the specific binding affinity and specific binding activity, respectively, of a protein to its counterstructure under specific binding conditions. In biochemical terms, avidity refers to the cumulative strength of the affinities of multiple individual noncovalent interactions, such as between CD80 and its counterstructures PD-L1, CD28, and / or CTLA-4. Therefore, avidity differs from affinity, which represents the strength of a single interaction. The increased or diminished binding affinity of a variant CD80 containing an affinity-modified CD80 IgSF domain to its counterstructure is determined relative to the binding affinity of unmodified CD80 (e.g., unmodified CD80 containing a native or wild-type IgSF domain (e.g., an IgV domain)). Methods for determining binding affinity or avidity are known in the art. See, e.g., 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 required 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 can be affected by the substance's elimination, excretion, degradation (e.g., enzymatic), or absorption and concentration in specific organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time required 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 (acylated), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylated).

[0137] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an artificial (i.e., man-made) transmembrane protein expressed on mammalian cells, comprising at least an ectodomain, a transmembrane, and an endodomain. Optionally, the CAR protein includes a "spacer" that covalently links the ectodomain to the transmembrane domain. The spacer is often a polypeptide that links the ectodomain to the transmembrane domain via a peptide bond. CARs are typically expressed on mammalian lymphocytes. In some embodiments, 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-T." In some embodiments, the CAR-T is a helper T cell, a cytotoxic T cell, a natural killer T cell, a memory T cell, a regulatory T cell, or a γδ T cell. When used clinically, for example, in adoptive cell transfer, CAR-Ts with antigen-binding specificity for a 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, while CAR-Ts are often autologous, allogeneic CAR-Ts are also within the scope of the present invention. The ectodomain of the CAR contains an antigen-binding region (e.g., an antibody or its antigen-binding fragment (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Upon specific binding, a series of biochemical events (i.e., signal transduction) results in modulation of the immune activity of the CAR-T. Thus, for example, specific binding of the antigen-binding region of the CAR-T to its target antigen can lead to changes in the immune activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction upon CAR-T activation is achieved by the CD3 zeta chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells.CAR-T can further contain multiple signaling domains (e.g., CD28, 41BB, or OX40) that further modulate the immunoregulatory response of T cells. CD3-z contains a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), which 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 the in vitro primary T cell assay described in Example 7.

[0139] The term "cognate binding partner" (used interchangeably with "counterstructure"), with respect 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-engineered IgSF domain specifically binds to the counterstructure of the corresponding native or wild-type CD80, but with increased or diminished affinity. A species of ligand that is recognized and specifically binds to its cognate receptor under specific binding conditions is an example of a counterstructure or cognate binding partner of that receptor. A "cell surface cognate binding partner" is a cognate binding partner expressed on the surface of a mammalian cell. A "cell surface molecular species" is a cognate binding partner of a ligand of an immunological synapse (IS) that is expressed on and by cells (e.g., mammalian cells) that form the IS.

[0140] As used herein, "conjugate," "conjugation," or grammatical variations thereof, refers to the joining or linking of two or more compounds together by any connecting or linking method known in the art, resulting in the formation of another compound. It can also refer to a compound produced by joining 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 fusion proteins, those produced by chemical conjugation, 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 identical or overlapping binding sites for specific binding, although this is not a requirement. In some embodiments, competitive binding causes measurable (partial or complete) inhibition of the specific binding of a protein to one of its cognate binding partners due to the specific binding of a second cognate binding partner. A wide variety of methods for quantifying competitive binding, such as ELISA (enzyme-linked immunosorbent assay) assays, are known.

[0142] The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. 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 the nucleotide or amino acid position as 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) by 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 decrease. The decrease 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 directly or indirectly covalently linking the protein to a composition to alter properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy, while retaining or enhancing its therapeutic effect. Derivatives of the immunomodulatory 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 the antibody specific to that 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 three or more, generally five or seven or more amino acids, e.g., 10-200 amino acid residues) refers to a portion of a molecule (e.g., a protein or coding nucleic acid) that is structurally and / or functionally distinct from and identifiable with the remainder of the molecule. For example, a domain includes a portion of a polypeptide chain that can independently fold within a protein, comprised of one or more structural motifs, and / or recognized by a functional activity, such as binding activity. A protein can have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology to related family members, e.g., homology to a motif. In another example, a domain can be distinguished by its function (e.g., ability to interact with a biomolecule, such as a cognate binding partner). A domain can exhibit an independent biological function or activity, such that it can function (e.g., bind) independently or fused to another molecule. A domain can be a linear or nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For purposes of illustration herein, definitions are provided, but it is understood that 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 domain.

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

[0149] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition (including a protein or cell composition) of the present invention that, when administered ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient), either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of the disease or disorder, or improves the physical function of the patient. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient via adoptive cell therapy. In some embodiments, the patient is a mammal, e.g., 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 terms "enhanced" or "increased (improved)," as used herein in the context of increasing the immune activity of mammalian lymphocytes, refer to an increase in one or more activities of the lymphocytes. An increase in activity can be an increase in one or more of cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity, e.g., by a statistically significant amount. In some embodiments, reference to increased immune activity refers to an increase in interferon-γ (IFNγ) production, e.g., by 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 for assessing lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, an enhancement can be an increase 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 modified by human intervention (e.g., recombinant DNA or viral transduction). In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., T cell, B cell, NK cell) or an antigen-presenting cell (e.g., dendritic cell). The cell may be a primary cell derived from a patient or a cell line. In some embodiments, the engineered cell of the invention contains a variant CD80 of the invention that has been 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 portion thereof containing an IgV domain linked to a transmembrane domain (e.g., a CD80 transmembrane domain), and optionally contains an intracellular signaling domain. In some embodiments, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the engineered cells are capable of expressing and secreting an immunomodulatory protein described herein. Some of the engineered cells provided further contain an engineered T cell receptor (TCR) or 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 modified by human intervention (e.g., recombinant DNA or viral transduction). Engineered T cells contain a variant CD80 transmembrane immunomodulatory protein (TIP) or secreted immunomodulatory protein (SIP) of the invention, which is expressed on the T cell and is engineered to regulate 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 engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen, which is then introduced into a population of T cells (often used in adoptive immunotherapy). 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 the protein not so conjugated to the moiety. In some embodiments, the half-life is extended by more than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or by more than about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or about 6.0-fold. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. Half-life refers to the time required for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using an ELISA assay or activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

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

[0158] The Fc (fragment crystallizable) region or domain (also referred to as an Fc polypeptide) of an immunoglobulin molecule primarily corresponds to the constant region of an immunoglobulin heavy chain and is responsible for various functions, including antibody effector function(s). 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 effector function-promoting 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 some embodiments, references to amino acid substitutions in the Fc region are by the EU numbering system, unless otherwise noted based on a specific SEQ ID NO: EU numbering is known and follows the EU index reported in the recently updated IMGT Scientific Chart (IMGT®, i.e., international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. USDapartment 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 comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. An Fc fusion can comprise, for example, the Fc region of an antibody (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 dimerize 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. Host cells can be prokaryotic, such as Escherichia coli (E. coli), or eukaryotic, 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, host cells can be mammalian cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells).

[0161] The term "immunoglobulin" (abbreviated "Ig"), as used herein, refers to mammalian immunoglobulin proteins including any of the five human classes of antibodies: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also refers to immunoglobulins that are less than full-length, whether fully or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V, and VI. H and V L variable fragments (Fv) containing V linked together in one chain; H and V L and other antibody V region fragments such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments. Homobispecific and heterobispecific 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 classified as members of this superfamily based on structural features shared with immunoglobulins (i.e., antibodies), all of which possess domains known as immunoglobulin domains or folds. IgSF members include cell surface antigen receptors, coreceptors, and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. These are typically associated with roles in the immune system. Proteins in the immunological synapse are often members of IgSF. IgSF can also be divided into "subfamilies" based on shared characteristics, such as function. Such subfamilies typically consist of 4 to 30 IgSF members.

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

[0164] The term "IgSF species," as used herein, refers to a group of IgSF member proteins that have identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a unique identity for all IgSF species belonging to that IgSF family. Therefore, each IgSF family member is unique compared to other IgSF family members, and thus each species of a particular IgSF family member is unique compared to other IgSF family member species. Nevertheless, differences between molecules of the same IgSF species can arise due to differences in post-translational modifications, such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, minor 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, can constitute distinct forms within a single IgSF species. A "cell surface IgSF species" is an IgSF species 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 the MLR (mixed lymphocyte reaction) assay, which measures interferon-γ cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56), the SEB (staphylococcal enterotoxin B) T-cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56), and the anti-CD3 T-cell stimulation assay (Li and Kurlander, J Transl Med. 2010:8:104). Because 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 (e.g., variant CD80 polypeptides containing affinity-engineered IgSF domains) as provided herein can increase, in some embodiments, or decrease, IFN-γ (interferon-γ) expression in primary T cell assays compared to wild-type IgSF members or IgSF domain controls. Those skilled in the art will understand that the format of a primary T cell assay used to determine increased IFN-γ expression differs from the format used to assay for decreased IFN-γ expression.When assaying for the ability of an immunomodulating protein or affinity-modified IgSF domain of the invention to reduce IFN-γ expression in a primary T cell assay, a mixed lymphocyte reaction (MLR) assay can be used, as described in Example 6. Conveniently, a soluble form of an affinity-modified IgSF domain of the invention can be used to determine its ability to antagonize and thereby reduce IFN-γ expression in an MLR, also as described in Example 6. Alternatively, when assaying for the ability of an immunomodulating protein or affinity-modified IgSF domain of the invention to increase IFN-γ expression in a primary T cell assay, a co-immobilization assay can be used, in which a T cell receptor signal (provided in some embodiments by an anti-CD3 antibody) is used in combination with a co-immobilized affinity-modified IgSF domain, such as a variant 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 expand T cells after antigen stimulation, the ability to sustain T cell proliferation in the absence of restimulation, and anti-cancer activity in appropriate animal models. Assays are also standard. 51 Assays to assess cytotoxicity include Cr release assays (see, e.g., Milone et al., (2009) Molecular Therapy 17;1453-1464) or flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

[0166] An "immunomodulating polypeptide" or "immunomodulating protein" is a polypeptide or protein molecule that modulates immune activity. "Modulation" or "modulating" an immune response refers to 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 an interchain disulfide bond). Thus, monomeric, dimeric, and higher order multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimers (of identical polypeptide chains) or heteromultimers (of non-identical polypeptide chains). The immunomodulating proteins of the present invention can include variant CD80 polypeptides.

[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 an RNA transcript expressed by a single gene, or the expression product of highly similar but different genes that produce functionally similar proteins, such as can result from gene duplication. As used herein, the term "isoform" of CD80 also refers to the products 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 that 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. The label can be detectable itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic 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, refers to any of 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, which are involved in cell-mediated cytotoxic adaptive immunity, and B cells, which are involved in humoral antibody-mediated adaptive immunity. T cells include helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Also included within the definition of lymphocyte are innate lymphoid cells (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, refers to 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 eukaryote (e.g., a mammal), or an artificial (i.e., man-made) membrane, such as a membrane found on a liposome. The binding of a membrane protein to a lipid bilayer can be by covalent bonding 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. A peripheral membrane protein forms a transient bond to the lipid bilayer so that the peripheral membrane protein can associate with and / or dissociate from the lipid bilayer under physiological conditions in mammals. In contrast to peripheral membrane proteins, integral membrane proteins form virtually permanent bonds to the lipid bilayer of the membrane, such that they do not dissociate from the lipid bilayer under physiological conditions in mammals. Membrane proteins can form bonds to the membrane through one layer of the lipid bilayer (monotopic) or through both layers of the membrane (polytopic). Integral membrane proteins that interact with only one lipid bilayer are "integral monotopic proteins." Integral membrane proteins that interact with both lipid bilayers are "integral polytopic proteins," or are referred to herein as "transmembrane proteins."

[0173] The terms "modulating" or "modulate," as used herein in the context of an immune response (e.g., a mammalian immune response), refer 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). Thus, modulation 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 immune cells. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs), and non-professional antigen-presenting cells, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted to an existing, developing, or potential immune response, or to the ability to induce, regulate, influence, or respond to an immune response. Modulation includes any change in the expression and / or function of genes, proteins, and / or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immune activity includes, for example, the elimination, deletion, or sequestration of immune cells; the induction or generation of immune cells that can modulate the functional capacity of other cells, such as autoreactive lymphocytes, antigen-presenting cells, or inflammatory cells; the induction of an unresponsive state (i.e., anergy) in immune cells; and the enhancement or suppression of immune cell activity or function (including, but not limited to, altering the pattern of proteins expressed by these cells). Examples include alterations in the production and / or secretion of specific classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events.Modulation can be assessed, for example, by changes in IFN-γ (interferon gamma) 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 the immune activity of the engineered cells compared to cells engineered with wild-type CD80 transmembrane protein, such as changes in the cytotoxic activity of the engineered cells or changes in cytokine secretion of the engineered cells.

[0174] The term "multimerization domain" refers to an amino acid sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, each of which contains 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 a first multimerization domain and a second multimerization domain, forms a stable protein-protein interaction to produce multimers 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, a polypeptide is connected directly or indirectly to a 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 modified forms 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- 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 set forth (the "reference sequence"), but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[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 other forms of differences 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 expressed on the surface of a mammalian cell. Two or more different protein species, each present in only one or only the other (but not both) of two mammalian cells forming an IS, are said to be in "cis" or "cis configuration" with each other. Two different protein species, the first of which is present only in the first of two mammalian cells that form an IS and the second of which is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or in a "trans configuration." Two different protein species, each of which is present in both 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, although the binding interactions need not be of the same duration; in some cases, the protein specifically binds to only one of the cognate binding partners. In some embodiments, binding occurs under specific binding conditions. In some embodiments, simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding to a second cognate binding partner. In some embodiments, non-competitive binding means that binding of a second cognate binding partner to its binding site on the protein does not displace binding of a first cognate binding partner to its binding site on the protein. Methods for assessing non-competitive binding are well known in the art, such as those described in Perez de La Lastra et al., Immunology, 1999 Apr:96(4):663-670. In some cases, in a non-competitive interaction, a first cognate binding partner specifically binds to a non-overlapping interaction site with a second cognate binding partner, such that the binding of the second cognate binding partner does not directly interfere with the binding of the first cognate binding partner. Thus, any effect of the binding of the second cognate binding partner on the binding of the cognate binding partner is via a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in 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 a second cognate binding partner specifically binds to a non-overlapping interaction site 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. Pharmaceutical compositions typically comprise an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant CD80 or engineered cells expressing a variant CD80 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically 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 polypeptide. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules of one or more amino acid analogs or non-standard or unnatural 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-γ ("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 immobilized anti-CD3, with or without additional recombinant proteins. Culture supernatants are collected at a time point (usually 24-72 hours). In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatants are collected at a time point (usually 24-72 hours). Human IFN-γ levels in the culture supernatants are measured by standard ELISA techniques. Commercial kits are available from suppliers, and the assay is performed according to the manufacturer's recommendations.

[0181] The term "purified," when applied to nucleic acids (e.g., encoding immunomodulatory proteins of the invention), generally refers to a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide will form 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." Purified nucleic acids or proteins of the invention are 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 condition. For example, a "recombinant nucleic acid" is one produced by recombining nucleic acids, e.g., during cloning, affinity engineering, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of fragments of DNA joined together by such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide," as used herein, refers to a protein molecule expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or is otherwise altered by genetic engineering (e.g., by introducing into the cell a nucleic acid molecule encoding a recombinant protein (e.g., a transmembrane immunomodulatory protein provided herein)). Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically 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, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type will 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 containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. If desired, a secretory signal peptide sequence can also be encoded by the recombinant expression vector operably linked to the coding sequence of the recombinant protein (e.g., a recombinant fusion protein) so that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell. This term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Among 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 of one substrate, such as a cognate binding partner, compared to specific binding of the protein of interest to another substrate, such as a different cognate binding partner. Selectivity can be measured by the binding activity (e.g., binding affinity) (e.g., K) of the protein of interest to a first substrate (e.g., a first cognate binding partner). d1 ) and the binding activity (e.g., binding affinity) (e.g., K d2 ) can be reflected as a ratio.

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

[0186] The term "soluble," as used herein with respect to a protein, means that the protein is not a membrane protein. Generally, a soluble protein contains only the extracellular domain or a portion thereof of an IgSF family member receptor containing 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 can arise due to differences in post-translational modifications, such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated polypeptide sequences that differ from the full-length species by (or encode differences in) 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 its IgV or IgC domain, refers to a polypeptide having a subsequence of the IgV and / or IgC domain and that 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-fold greater than the average affinity or avidity of the same protein for a population of random peptides or polypeptides of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times greater, or even at least 1000 times greater. A specifically binding protein need not bind only to a single target molecule; it may also specifically bind to non-target molecules (e.g., paralogs or orthologs) due to similarities in structural form between the target and non-target molecules. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or to non-target molecules (e.g., paralogs) with substantially similar epitopes to the target molecule is possible without compromising the specificity of binding determined for a statistically valid population 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 M has a low dissociation constant (K d In certain embodiments of the present disclosure, the interaction between the two binding proteins has a molecular weight of 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 -11 It has a dissociation constant of M.

[0190] The terms "surface-expressed" or "surface expression," with respect to a mammalian cell expressing a polypeptide, mean 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 polypeptide molecule that is produced by recombinant and / or chemical synthetic methods, e.g., with respect to a synthetic nucleic acid molecule or synthetic gene or 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 counterstructure, 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 counterstructure is localized on a specific tissue or cell type. A targeting moiety can be an antibody, an antigen-binding fragment (Fab), a V-cell, or a mAb. H and V L variable fragments (Fv) containing V linked together in one chain; H and V L and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecule (less than 500 daltons) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or non-covalently attached to the lipid membrane of liposomes that encapsulate the polypeptides of the invention.

[0193] The term "transmembrane protein" as used herein refers to a membrane protein that substantially or completely spans a lipid bilayer, such as those found in biological membranes, e.g., mammalian cells, or in artificial constructs, e.g., liposomes. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. Transmembrane domains can generally be predicted from the amino acid sequence of the transmembrane domain through any number of commercially available bioinformatics software applications based on their increased hydrophobicity compared to regions of the protein that interact with an aqueous environment (e.g., cytosol, extracellular fluid). Transmembrane domains are often hydrophobic α-helices that span 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 the transmembrane protein. In addition to the 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 the disease or disorder, as evidenced by the reduction, arrest, or elimination of any clinical or diagnostic symptoms by administering a therapeutic composition of the invention (e.g., containing an immunomodulatory protein or 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 acute or chronic diseases or disorders, or reducing the rate of relapse (e.g., in cases of relapsing or remitting autoimmune disease processes), or reducing inflammation in the case of inflammatory aspects of autoimmune diseases. As used herein in the context of cancer, the terms "treating" cancer, "inhibiting" cancer, "inhibiting" cancer, or "inhibition" of cancer refer to at least one of a statistically significant reduction in tumor growth rate, halting tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, or a statistically significant improvement in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria such as, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST). "Preventing" a disease or disorder, "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 immunomodulatory polypeptide or engineered cell of the invention, either alone or in combination with another compound, to prevent the appearance or development of a disease or disorder or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[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 in 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 invention) and provide for the prevention or treatment of the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells that display the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that display the TSA are cancerous.

[0196] The term "variant" (also "modified" or "mutant"), when used with 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. Variant CD80 polypeptides generally exhibit at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified CD80, e.g., the sequence of SEQ ID NO:1, its mature sequence, or a portion 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 the 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 the unmodified or wild-type CD80, and in some embodiments can be at least 10%, 15%, 20%, 30%, 40%, 50%, or 100% greater than that of the unmodified or wild-type CD80 control. The decrease in binding affinity or avidity of CD80 to CD28, PD-L1, and / or CTLA-4 can be to 95% or less of that of the unmodified or wild-type CD80 control, and in some embodiments, to 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the binding affinity or avidity of the unmodified or wild-type CD80 control, or to undetectable levels. Variant CD80 polypeptides have altered primary amino acid sequence due to substitution, addition, or deletion of amino acid residues. The term "variant" in the context of variant CD80 polypeptides should not be construed as imposing any requirement on 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 the variant CD80. However, in another example, a variant CD80 can be made 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 nucleic acid molecules, proteins (e.g., CD80), IgSF members, host cells, etc., refer to those found in nature and unmodified by human intervention.

[0199] II. Variant CD80 Polypeptides Provided herein are variant CD80 polypeptides that exhibit altered (increased or decreased) binding activity or affinity for 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, e.g., one or more substitutions (or "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain (IgD) compared to a wild-type or unmodified CD80 polypeptide, or a portion of a wild-type or unmodified CD80 containing IgD, or a specific-binding fragment thereof. Thus, the provided variant CD80 polypeptides are or comprise variant IgDs (hereinafter referred to as "vIgDs") in which one or more amino acid modifications (e.g., substitutions) are in the IgD.

[0200] In some embodiments, the IgD comprises an IgV domain or an IgC (e.g., IgC2) domain, or a specific-binding fragment of an IgV domain or an IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, 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, a variant CD80 polypeptide contains an IgV domain or an IgC domain or a specific-binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV domain or IgC domain or a specific-binding fragment thereof. In some embodiments, a variant CD80 polypeptide contains an IgV domain or an IgC domain or a specific-binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV domain or the specific-binding fragment thereof. In some embodiments, the IgV or IgC domain that has been altered by altered avidity or affinity is an affinity-engineered IgSF domain.

[0201] In some embodiments, the variant has one more IgSF domain modified relative 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 its ortholog. In some embodiments, the unmodified CD80 is or comprises the extracellular domain (ECD) of CD80 or a portion thereof containing 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 the 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, 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, the wild-type or unmodified CD80 polypeptide (i) has the amino acid sequence set forth in SEQ ID NO:1 or a mature form thereof lacking the signal sequence, or (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%, or 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, a wild-type or unmodified CD80 polypeptide is or comprises the extracellular domain of CD80 or a portion thereof. For example, in some embodiments, an unmodified or wild-type CD80 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or an ortholog thereof. For example, an unmodified or wild-type CD80 polypeptide can (i) comprise the sequence of amino acids set forth in SEQ ID NO:2, or (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%, or about 99% sequence identity to SEQ ID NO:2, or (iii) is 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 the 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 ortholog thereof. For example, the IgC domain of an unmodified or wild-type CD80 polypeptide can (i) contain the 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%, or 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, the wild-type or unmodified IgC domain is capable of binding to one or more CD80 binding proteins.

[0207] In some embodiments, a wild-type or unmodified CD80 polypeptide contains a specific-binding fragment of CD80, e.g., a specific-binding fragment of the IgV domain or the 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, e.g., at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific-binding fragment of the IgV domain contains an amino acid sequence that is at least about 85%, 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 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 the 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 comprising 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, wherein at least one of the IgV domain or 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 these 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 can include a combination of these independent attributes. It will be understood that references to amino acids, including references to specific sequences set forth as SEQ ID NO:, used to describe the domain organization of IgSF domains, are for illustrative purposes only and are not meant to limit the scope of the provided embodiments. It will be understood that descriptions of polypeptides and their domains are theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus may vary and is not necessarily the same from protein to protein. Thus, a particular IgSF domain (e.g., a particular IgV or IgC domain) may be several (e.g., 1, 2, 3, or 4) amino acids longer or shorter.

[0211] Furthermore, various embodiments of the present invention as discussed below are frequently provided within the meanings of the defined terms as disclosed above. Accordingly, embodiments described in a particular definition should be construed as being incorporated by reference when the defined terms are utilized in the discussion of various aspects and attributes described herein. Accordingly, 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, as measured, for example, by solid-phase ELISA immunoassay, flow cytometry, or surface plasmon resonance (Biacore) assay. In some embodiments, the variant CD80 polypeptide has increased binding affinity for CTLA-4, PD-L1, and / or CD28. In some embodiments, the variant CD80 polypeptide has a reduced 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, e.g., human or murine proteins.

[0213] 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 a wild-type or unmodified IgSF domain. A wild-type or unmodified CD80 sequence need not necessarily 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 a codon encoding 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 using 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 discussed above.

[0214] Unless otherwise indicated, as indicated throughout this disclosure, the amino acid modification(s) are designated by an amino acid position number that corresponds to the numbering of 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: TIFF0007702928000036.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 specified variant is listed after the number. If the modification is a deletion at that position, it is indicated as "del," and if the modification is an insertion at that position, it is indicated as "ins." In some cases, 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 specified 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 the 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 the IgV domain or a specifically binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgC domain or a specifically 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 the IgV domain or a specifically binding fragment thereof, and some of the one or more amino acid modifications (e.g., substitutions) are in the IgC domain or a specifically 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, 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 IgC domain or specific-binding fragment thereof. In some embodiments, the 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 TIFF0007702928000037.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 aforementioned 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 comprising only one of the aforementioned 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, other than the wild-type or unmodified amino acid, that belongs to the same class of amino acids as the substituted amino acid. The amino acid classes are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl- or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine). 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 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 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 additional amino acid modifications in accordance with provided embodiments. Table 1 lists 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, variant CD80 polypeptides comprise the amino acid modifications H18Y / A26E, A26E / E35D, A26E / D46E, A26E / D46V, A26E / M47I, A26E / M47L, A26E / V68M, A26E / A71G, A26E / L85Q, A26E / D90G. Variant CD80 polypeptides can comprise additional amino acid modifications, such as those described herein, in accordance with provided embodiments. Table 1 lists 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 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, or E35D / D90G. The variant CD80 polypeptide can comprise additional amino acid modifications, such as those described herein, according to provided embodiments. Table 1 describes exemplary amino acid modifications and variant CD80 polypeptides that are described. In some embodiments, the variant CD80 polypeptide comprises the 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 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, variant CD80 polypeptides comprise 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, variant CD80 polypeptides comprise the amino acid modifications H18Y / D46V, A26E / D46V, E35D / D46V, D46V / M47I, D46V / M47L, D46V / V68M, D46V / A71G, D46V / L85Q, D46V / D90G. Variant CD80 polypeptides can comprise additional amino acid modifications, such as those described herein, in accordance with provided embodiments. 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 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 additional amino acid modifications, such as those described herein, according to provided embodiments. 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 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 the 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, in accordance with provided embodiments. Table 1 lists 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 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 the 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, in accordance with provided embodiments. Table 1 lists 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 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 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 additional amino acid modifications, such as those described herein, in accordance with provided embodiments. 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 amino acid substitutions H18Y, A26E, E35D, D46E, D46V, M47I, M47L, V68M, A71G, or L85Q, or a conservative amino acid substitution thereof. In some embodiments, variant CD80 polypeptides comprise the amino acid modifications H18Y / D90G, A26E / D90G, E35D / D90G, D46E / D90G, D46V / D68M, M47I / D90G, M47L / D90G, V68M / D90G, A71G / D90G, L85Q / D90G. Variant CD80 polypeptides can comprise additional amino acid modifications, such as those described herein, in accordance with provided embodiments. Table 1 lists exemplary amino acid modifications and variant CD80 polypeptides that are described.

[0230] In some embodiments, the variant CD80 polypeptide does not contain the amino acid modifications in the unmodified CD80 polypeptide set forth in SEQ ID NO: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 NO: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 NO: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 NO: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 In some embodiments, the variant CD80 polypeptide is selected from the group consisting of SEQ ID NOs: 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 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 the amino acid modifications in the unmodified CD80 polypeptide set forth in SEQ ID NO: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 NO: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 A26E / E35D / M47L / L85Q in the unmodified CD80 polypeptide set forth in SEQ ID NO:2, 76, or 150. In some embodiments, the variant CD80 polypeptide is not a polypeptide set forth in SEQ ID NO:62, 136, 210.

[0236] In some embodiments, the variant CD80 polypeptide does not contain the amino acid modifications in the unmodified CD80 polypeptide set forth in SEQ ID NO: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 the polypeptide set forth in SEQ ID NO:41, 70, 115, 144, 189, or 218.

[0237] 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 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 corresponding to E35D and M47L in unmodified CD80, or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D and A71G in unmodified CD80, or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D and M47V in unmodified CD80, or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to E35D and V68M in unmodified CD80, or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide comprises amino acid modifications corresponding to H18Y and E35D in unmodified CD80, or a specific-binding fragment thereof.

[0239] In some embodiments, the variant CD80 polypeptide comprises at least three amino acid modifications, including modifications at three or more positions corresponding to 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, a variant CD80 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NOs: for the extracellular domain (ECD) or IgV domain of wild-type 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. Furthermore, in some cases, adjacent N- 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 is understood that the exemplary SEQ ID NOs 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 to 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, e.g., 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 amino acid modification(s) (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 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) that is not present in wild-type or unmodified CD80. In some embodiments, the 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, e.g., 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 amino acid modification(s) (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 amino acid modification(s) (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 NOs. 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. Furthermore, in some cases, the adjacent N- and / or C-terminal amino acids of a given domain (e.g., the ECD) may also be included in the sequence of the variant IgSF polypeptide, e.g., to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplary SEQ ID NOs. in Table 1 should not be construed as limiting. For example, a particular domain (e.g., the IgV domain) of a variant CD80 polypeptide may be several amino acids longer or shorter, e.g., 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter, than the amino acid sequence set forth in the respective SEQ ID NO.

[0247] Table 1. Exemplary variant CD80 polypeptides TIFF0007702928000038.tif187169TIFF0007702928000039.tif230169TIFF0007702928000040.tif230169TIFF0007702928000041.tif230169 TIFF0007702928000042.tif230169TIFF0007702928000043.tif230169TIFF0007702928000044.tif230169TIFF0007702928000045.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 (e.g., IgV or IgC, etc.) 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, as measured, for example, by solid-phase ELISA immunoassay, flow cytometry, or surface plasmon resonance (Biacore) assay. In some embodiments, the variant CD80 polypeptide has increased binding affinity for CTLA-4, PD-L1, and / or CD28. In some embodiments, the variant CD80 polypeptide has a reduced 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, e.g., human or murine proteins.

[0249] The binding affinity of each of the binding partners is independent, i.e., in some embodiments, the 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 polypeptide has increased binding affinity for CTLA-4 and PD-L1 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 PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for CTLA-4 and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has 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 polypeptide. 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 polypeptide. In some embodiments, the variant CD80 polypeptide has decreased binding affinity for CTLA-4 and CD28 compared to wild-type or unmodified CD80 polypeptide. 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 polypeptide has 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 polypeptide has decreased binding affinity for CTLA-4, PD-L1 and CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has 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 with increased or greater binding affinity for CD28, PD-L1, and / or CTLA-4 have an increase in binding affinity of at least about 5%, e.g., at least about 10%, about 15%, about 20%, about 25%, about 35%, or about 50%, for 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 the one or more amino acid modifications (eg, substitutions).

[0257] In some embodiments, variant CD80 polypeptides that have reduced or decreased binding affinity for CTLA-4, PD-L1, and / or CD28 have at least a 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 decrease in binding affinity to CTLA-4, PD-L1, and / or CD28 compared to a wild-type or unmodified CD80 polypeptide control. In some embodiments, the decrease in binding affinity compared 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 the 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 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 -11 M, or 1 x 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) or a specific-binding fragment thereof relative to the IgSF domain contained in wild-type or unmodified CD80 polypeptides exhibit altered (increased / stimulated or decreased / inhibited) signaling induced by one or more functional binding partners, such as CTLA-4 or CD28, expressed on the surface of signal-transducing cells, such as T cells, that are capable of releasing cytokines in response to intracellular signals, compared to wild-type or unmodified CD80 polypeptides upon binding to the one or more binding partners. In some embodiments, the altered signaling differs from that produced by wild-type or unmodified CD80 polypeptide control sequences in the same format, as determined, for example, by assays measuring 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 exemplary assays, cytokine release is a function of the sum of the signaling activity of functional binding partners expressed on the surface of cytokine-releasing cells. As described elsewhere herein, in some embodiments, the format of the variant CD80 polypeptide provided may affect the type of activity (e.g., 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 wild-type or unmodified CD80 polypeptides.

[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-induced signaling and increase CD28-induced signaling compared to wild-type or unmodified CD80 polypeptides.

[0266] In some embodiments, a variant CD80 polypeptide that stimulates or increases CTLA-4-induced inhibitory signaling generates 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 CTLA-4-induced inhibitory 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).

[0269] In some embodiments, a variant CD80 polypeptide that inhibits or reduces CD28-induced inhibitory signaling generates a signal that is 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less 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 the one or more amino acid modifications (e.g., substitutions).

[0270] In some embodiments, variant CD80 polypeptides that affect CTLA-4-induced inhibitory signaling and / or affect signaling by CD28 result in a combined CTLA-4 and CD28 signaling that is less than the combined CTLA-4 and CD28 signaling that is provided by a corresponding wild-type or unmodified CD80 polypeptide. In such embodiments, the combined 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 a 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 CTLA-4-induced inhibitory signaling and / or affects signaling by CD28 results in a combined CTLA-4 and CD28 signaling that is greater than the combined CTLA-4 and CD28 signaling affected by a corresponding wild-type or unmodified CD80 polypeptide. In such embodiments, the combined 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 polypeptide exhibits 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 the sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the variant CD80 polypeptide exhibits 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, e.g., comprising the sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the increased affinity for the CTLA-4 ectodomain 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 CTLA-4 ectodomain.

[0273] In some of these embodiments, the variant CD80 polypeptides that exhibit increased binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides 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: TIFF0007702928000047.tif55165.

[0275] In some embodiments, the one or more amino acid substitutions are TIFF0007702928000048.tif85160TIFF0007702928000049.tif231163TIFF0007702928000050.tif252164.

[0276] In some embodiments, the variant CD80 polypeptide exhibits increased selectivity for CTLA-4 versus 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 NO: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. TIFF0007702928000051.tif106165TIFF0007702928000052.tif231160TIFF0007702928000053.tif62164.

[0277] 2.CD28 In some embodiments, the variant CD80 polypeptide exhibits increased affinity for the ectodomain of CD28 compared to a wild-type or unmodified CD80 polypeptide. In some embodiments, the variant CD80 polypeptide exhibits increased 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 CD28 is greater than a 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 increase 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 independently 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 ectodomain 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 the 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 the sequence set forth in SEQ ID NO:2, 76, 150, 3030, or 3031. In some embodiments, the decreased affinity for the CTLA-4 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 ectodomain.

[0280] 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 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, A 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 TIFF0007702928000054.tif194162TIFF0007702928000055.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 PD-L1 ectodomain and the CTLA-4 ectodomain 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 PD-L1 ectodomain 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 PD-L1 ectodomain.

[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 the 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 the 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 is selected from the group consisting of 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: TIFF0007702928000057.tif63166.

[0287] In some embodiments, the one or more amino acid substitutions are TIFF0007702928000058.tif70165TIFF0007702928000059.tif231166TIFF0007702928000060.tif231162TIFF0007702928000061.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 can exhibit or result in PD-L1-dependent CD28 costimulation. In some embodiments, the variant CD80 polypeptides mediate or result in PD-L1-dependent CD28 costimulatory activity, wherein 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, the variant CD80 polypeptides may 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%, 105%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 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 Immune modulatory polypeptides, including variant CD80, including vIgD, provided herein can be formatted in a variety of ways, including as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, the particular format can be selected depending on the desired therapeutic use. In some cases, immune modulatory polypeptides, including variant CD80 polypeptides, are provided in a format that antagonizes or blocks the activity of their binding partners, such as CTLA-4, CD28, and / or PD-L1. In some embodiments, antagonism of CTLA-4 or PD-L1 / PD-1 may be useful in promoting immunity in oncology. In some cases, immune modulatory polypeptides, including variant CD80 polypeptides, are provided in a format that agonizes or stimulates the activity of their binding partners, such as CTLA-4 and / or CD28. In some embodiments, agonism of CD28 may be useful in promoting immunity in oncology. In some embodiments, agonism of CD28 may 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. One skilled in the art can readily 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 comprising the vIgD of CD80 that are soluble (e.g., fused to an Fc chain) are provided. 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 for engineering or generating 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, or in some cases, as membrane-bound or secreted proteins. In some embodiments, variant CD80 immunomodulatory proteins are provided as conjugates containing 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, variant CD80 polypeptides are expressed in cells, e.g., immune cells (e.g., T cells or antigen-presenting cells), in a membrane-bound form, thereby providing transmembrane immunomodulatory proteins (hereinafter also referred to as "TIPs"). In some embodiments, depending on the cognate binding partner recognized by TIP, engineered cells expressing TIPs 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, variant CD80 polypeptides are expressed in cells, e.g., immune cells (e.g., T cells or antigen-presenting cells), in a secretable form, thereby producing a secreted or soluble form of the variant CD80 polypeptide (hereinafter also referred to as "SIP"), e.g., when the cells are administered to a subject. In some aspects, the SIP can antagonize its cognate binding partner in the environment into 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, upon administration to a subject, is capable of infecting cells, e.g., immune cells (e.g., T cells or antigen-presenting cells), in vivo 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 provided conjugates (e.g., targeting moieties). In some embodiments, a 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 the formats provided are set forth in Figures 1A-1C and further described below.

[0296] A. Soluble Proteins In some embodiments, immunomodulatory proteins containing variant CD80 polypeptides are soluble proteins. Those skilled in the art will recognize that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that the extracellular domain, or an immunologically active sequence thereof, can be used to generate soluble forms of such proteins. Thus, in some embodiments, immunomodulatory proteins containing variant CD80 polypeptides lack a transmembrane domain or a portion of a transmembrane domain. In some embodiments, immunomodulatory proteins containing variant CD80 lack an intracellular (cytoplasmic) domain or a portion of an intracellular domain. In some embodiments, immunomodulatory proteins containing variant CD80 polypeptides contain only a vIgD portion containing an ECD domain or a portion thereof containing an IgV domain and / or IgC (e.g., IgC2) domain(s) or specific-binding fragments thereof containing an amino acid modification(s).

[0297] In some embodiments, an immunomodulatory 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, or 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, provided immunomodulatory polypeptides comprise two or more vIgD sequences of CD80. Multiple variant CD80 polypeptides within a polypeptide chain can be identical (i.e., homologous) or non-identical (i.e., heterologous) variant CD80 sequences. In addition to single polypeptide chain embodiments, in some embodiments, two, three, four, or more polypeptides of the invention can be covalently or non-covalently linked to each other. Thus, monomeric, dimeric, and higher (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 different species of polypeptide (e.g., heterodimers). Compositions 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 polypeptide, 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, an 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., WO 2008 / 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 are immunomodulatory proteins that are PAS-ylated proteins, 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, e.g., an affinity or purification tag, directly or indirectly linked to the N-terminus and / or C-terminus of the CD80 polypeptide. Various 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, separately encoded variant CD80 polypeptide chains can be linked by multimerization, where the multimerization of the polypeptides is mediated by a 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 coexpression 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 may be any that can form stable protein-protein interactions. Multimerization domains 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 the nuclear transcription proteins fos and jun or the proto-oncogene c-myc or from the general control of nitrogen (GCN4) gene) (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. Additionally, the multimerization domain can contain an amino acid sequence containing a protrusion complementary to an amino acid sequence containing a hole, as described, for example, in U.S. Patent 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 over homodimers from a mixture of chimeric monomers. Generally, the protrusion 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). A compensatory cavity of identical or similar size to the protrusion is 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 attached at any position, but typically via its N- or C-terminus, to the N- or C-terminus of the multimerization domain to form a chimeric polypeptide. Linkage can be direct or indirect via a linker. The chimeric polypeptide can be a fusion protein or can be formed by chemical linkage, such as 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, directly or indirectly, or optionally via a linker domain, to a nucleic acid encoding the multimerization domain sequence. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant 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 created 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 affinity chromatography on a Protein A or Protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, 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 portion thereof from an immunoglobulin. In some embodiments, the immunomodulatory protein comprises a variant CD80 polypeptide linked to an immunoglobulin Fc (yielding 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 (the same or different) are independently linked to the N-terminus and the 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 IgG1, e.g., human IgG1. In some embodiments, the Fc comprises the 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 alter (e.g., reduce) one or more of its normal functions. Generally, the Fc region is involved in effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen-binding ability, which is the primary function of immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by extending in vivo half-life through conjugation to the in vivo FcRn receptor. In some embodiments, such functions can be reduced or 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 a CD80-Fc variant fusion provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of modifications or mutations to the Fc sequence 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 FcRs. The contents of these publications are expressly incorporated herein by reference.

[0313] In some embodiments, provided variant CD80-Fc fusions comprise an Fc region that exhibits reduced effector function, making them desirable candidates for applications where in vivo half-life of the CD80-Fc variant fusion is important, but certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that CD80-Fc variant fusions lack FcγR binding (and thus likely lack ADCC activity) but retain 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 for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, 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 (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that a CD80-Fc variant fusion is unable to bind C1q and therefore lacks CDC activity. See, e.g., 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 of one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[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, ...

Claims

**Claim 1** A CD80-Fc fusion protein comprising a variant CD80 polypeptide operably linked to an Fc domain, wherein the variant CD80 polypeptide comprises a CD80 IgV domain, wherein the variant CD80 polypeptide comprises the amino acid substitution M47L based on the numbering of the positions set forth in SEQ ID NO: 2, wherein the variant CD80 polypeptide, (a) has the amino acid sequence set forth in SEQ ID NO: 2, or (b) has an amino acid sequence with at least 93% sequence identity to the IgV domain of CD80 having the amino acid sequence 35-141 of SEQ ID NO: 1 and wherein the variant CD80 polypeptide exhibits an increased binding affinity for the extracellular domain of human PD-L1 as compared to the binding affinity of wild-type human CD80 for the extracellular domain of human PD-L1, said CD80-Fc fusion protein. **Claim 2** The CD80-Fc fusion protein according to claim 1, wherein the affinity is increased by 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 more than 450-fold as compared to the binding affinity of the wild-type CD80 for the extracellular domain of human PD-L1. **Claim 3** The CD80-Fc fusion protein according to claim 1 or 2, wherein the variant CD80 polypeptide comprises the following amino acid substitutions based on the numbering of the positions set forth in SEQ ID NO: 2: D46V / M47L, E35D / M47L, A26E / E35D / M47L / L85Q, A26E / Q33R / E35D / M47L / L85Q / K86E, A26E / Q33R / E35D / M47L / L85Q, E35D / M47L / L85Q, A26E / Q33L / E35D / M47L / L85Q, A26E / Q33L / E35D / M47L, H18Y / A26E / Q33L / E35D / M47L / L85Q, H18Y / E35D / M47L, A26E / E35D / M43T / M47L / L85Q / R94Q, S15P / Q33L / E35D / M47L / L85Q, Y31S / E35D / M47L / T79L / E88G, H18L / V22A / E35D / M47L / N48T / L85Q, Q27H / E35D / M47L / L85Q / R94Q / E95K, A26E / E35D / M43I / M47L / L85Q / K86E / R94W, Q33L / E35D / M47L / A71G / F92S, Q33L / E35D / M47L / V68M / L85Q / E88D, E35D / M47L / A71G / L97Q, A26E / E35D / M47L / A71G, H18Y / A26E / E35D / M47L / L85Q / D90G, E35D / M47L / A71G / L85Q, V22D / E35D / M47L / L85Q, E35D / T41S / M43I / M47L / A71G, H18Y / A26E / E35D / M47L / V68M / A71G / D90G, Q27H / E35D / D46V / M47L / A71G, E35D / D46V / M47L / V68M / L85Q / E88D, E35D / M47L / A71G / L85M / F92Y, V22D / E35D / M47L / L70M / L97Q, E35D / M43I / M47L / L85M, H18Y / E35D / M47L / A71G / A91S, M43I / M47L / A71G, E35D / M47L / A71G / L85M, V22A / E35D / M47L / A71G, E35D / M47L / A71G, A26E / Q27R / E35D / M47L / N48Y / L85Q, E35D / D46E / M47L / V68M / L85Q / F92L, E35D / M47L / V68M / A71G / L85Q / D90G, E24D / E35D / M47L / V68M / E95V / L97Q,E35D / D46E / M47L / V68M / A71G / Y87C / K93R, E35D / D46E / M47L / V68M / T79M / L85M, E35D / D46E / M47L / V68M / T79M / L85M / L97Q, E35D / M43I / M47L / V68M, E35D / M47L / V68M / E95V / L97Q, E35D / M47L / Y53F / V68M / A71G / K93R / E95V, H18Y / E35D / M38I / M47L / V68M / L85M, H18Y / E35D / M47L / V68M / A71G / L85M, H18Y / E35D / M47L / V68M / A71G / L85M, H18Y / E35D / M47L / V68M / E95V / L97Q, H18Y / E35D / M47L / V68M / E95V / L97Q, H18Y / E35D / M47L / Y53F / V68M / A71G, H18Y / E35D / M47L / Y53F / V68M / A71G, H18Y / E35D / M47L / Y53F / V68M / A71G / K93R / E95V, H18Y / E35D / M47L / Y53F / V68M / A71G / K93R / E95V, Q33R / E35D / M38I / M47L / V68M, R29C / E35D / M47L / V68M / A71G / L85M, T13R / E35D / M47L / V68M, T13R / Q33L / E35D / M47L / V68M / L85M, T13R / Q33R / E35D / M38I / M47L / V68M, T13R / Q33R / E35D / M38I / M47L / V68M / E95V / L97Q, T13R / Q33R / E35D / M38I / M47L / V68M / L85M,T13R / Q33R / E35D / M38I / M47L / V68M / L85M / R94Q, T13R / Q33R / E35D / M47L / V68M, T13R / Q33R / E35D / M47L / V68M / L85M, V22D / E24D / E35D / M47L / V68M, V22D / E24D / E35D / M47L / V68M / L85M / D90G, H18C / A26P / E35D / M47L / V68M / A71G, H18T / A26N / E35D / M47L / V68M / A71G, H18V / A26K / E35D / M47L / V68M / A71G, H18V / A26P / E35D / M47L / V68M / A71G,H18V / A26R / E35D / M47L / V68M / A71G / D90G, H18A / A26P / E35D / M47L / V68M / A71G / D90G, H18A / A26N / E35D / M47L / V68M / A71G / D90G, H18F / A26H / E35D / M47L / V68M / A71G / D90G, or H18Y / A26E / E35D / M47L / V68M / A71G / D90G., **Claim 4** The CD80-Fc fusion protein according to any one of claims 1 to 3, wherein the variant CD80 polypeptide has at least 93% sequence identity to the IgV domain of CD80 having the amino acid sequence 35-141 of SEQ ID NO:

1. **Claim 5** The CD80-Fc fusion protein according to claim 4, wherein the variant CD80 polypeptide has at least 95% sequence identity to the IgV domain of CD80 having the amino acid sequence 35-141 of SEQ ID NO:

1. **Claim 6** The amino acid sequence as set forth in any one of SEQ ID NO: 60, 73, 2009 - 2014, 2019, 2026, 2030, 2035 - 2037, 2040, 2044, 2048, 2050, 2064, 2067, 2068, 2074, 2082, 2084, 2086, 2088, 2094, 2096, 2301, 2303, 2306 - 2310, 2323, 2324, 2328, 2334, 2338, 2339, 2343, 2346 - 2348, 2350, 2352, 2353, 2360, 2362 - 2365, 2373, 2374, 2377, 2380, 2382 - 2389, 2392, 2398, 2402, 2404, 2408, 2409, 2411, 2412, 2414, 2415, 2446 - 2448, 2450 - 2454, 2457 - 2459, 2461 - 2463, 2465 - 2470, 2473, 2474, 2478, 2480 - 2482, 2484 - 2486, 2490, 2492 - 2494, 2499, 2503, 2504, 2931, 2935, 2936, 2939, 2947, 2948, 2951, 2952, or 2954, or SEQ ID NO: 134, 147, 208, 221, 2105 - 2110, 2115, 2122, 2126, 2131 - 2133, 2136, 2140, 2144, 2146, 2160, 2163, 2164, 2170, 2178, 2180, 2182, 2184, 2190, 2192, 2201 - 2206, 2211, 2218, 2222, 2227 - 2229, 2232, 2236, 2240, 2242, 2256, 2259, 2260, 2266, 2274, 2276, 2278, 2280, 2286, 2288, 2512, 2514, 2517, 2519 - 2521, 2534, 2535, 2539, 2545, 2549, 2550, 2554, 2557 - 2559, 2561, 2563, 2564, 2571, 2573 - 2576, 2584, 2585, 2588, 2591, 2593 - 2600, 2603, 2609, 2613, 2615, 2619, 2620, 2622, 2623, 2625, 2626, 2657 - 2659, 2661 - 2665, 2668 - 2670, 2672 - 2674, 2676 - 2681, 2684, 2685, 2689, 2691 - 2693, 2695 - 2697, 2701, 2703 - 2705, 2710, 2714, 2715, 2723, 2725, 2728, 2730 - 2732, 2745, 2746, 2750, 2756, 2760, 2761, 2765, 2768 - 2770, 2772, 2774, 2775, 2795, 2796, 2799, 2802, 2804 - 2811, 2814, 2820, 2824, 2826, 2830, 2831, 2833, 2834, 2836, 2837, 2868 - 2870, 2872 - 2876, 2879 - 2881, 2883 - 2885, 2887 - 2892, 2895, 2896, 2900, 2902 - 2904, 2906 - 2908, 2912, 2914 - 2916, 2921, 2925, 2926, 2962, 2966, 2967, 2970, 2978, 2979, 2982, 2983, 2985,The amino acid sequence according to any one of 2993, 2997, 2998, 3001, 3009, 3010, 3013, or 3014, The CD80 - Fc fusion protein according to any one of claims 1 to 5, comprising **Claim 7** The CD80 - Fc fusion protein according to any one of claims 1 to 6, wherein the Fc domain is a variant Fc domain having reduced effector function. **Claim 8** The CD80 - Fc fusion protein according to claim 7, wherein the Fc domain is a variant human IgG1 Fc domain having reduced effector function. **Claim 9** The CD80 - Fc fusion protein according to any one of claims 1 to 8, wherein the Fc domain comprises one or more amino acid substitutions selected from E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del according to EU numbering. **Claim 10** The CD80 - Fc fusion protein according to any one of claims 1 to 9, wherein the Fc domain comprises the amino acid substitution N297G according to EU numbering. **Claim 11** The CD80-Fc fusion protein according to any one of claims 1 to 9, wherein the Fc domain contains amino acid substitutions R292C / N297G / V302C by EU numbering.

12. The CD80-Fc fusion protein according to any one of claims 1 to 9, wherein the Fc domain contains amino acid substitutions L234A / L235E / G237A by EU numbering.

13. The CD80-Fc fusion protein according to any one of claims 1 to 12, wherein the Fc domain contains amino acid substitution C220S by EU numbering and / or the Fc domain contains K447del by EU numbering.

14. The CD80-Fc fusion protein according to any one of claims 1 to 13, which exhibits PD-L1-dependent CD28 co-stimulation.

15. The CD80-Fc fusion protein according to any one of claims 1 to 14, wherein the Fc domain contains the amino acid sequence described in any of SEQ ID NOs: 356 to 358, 376, and 1712 to 1715, or shows at least 90% sequence identity to any of SEQ ID NOs: 356 to 358, 376, and 1712 to 1715, and has a reduced effector function.

16. The CD80-Fc fusion protein according to any one of claims 1 to 15, wherein the variant CD80 polypeptide is indirectly linked to the Fc domain via a linker.

17. The CD80-Fc fusion protein according to claim 16, wherein the linker has 1 to 20 amino acid residues.

18. The CD80-Fc fusion protein according to claim 16 or 17, wherein the linker is GGGGS (SEQ ID NO: 1717).

19. The CD80-Fc fusion protein according to any one of claims 16 to 18, wherein the linker is GSGGGS (SEQ ID NO: 1716).

20. An immunomodulatory protein which is a dimer containing two CD80-Fc fusion proteins according to any one of claims 1 to 19.

21. The immunomodulatory protein according to claim 20, wherein the dimer is a homodimer.

22. A purified CD80-Fc fusion protein according to any one of claims 1 to 19 or an immunomodulatory protein according to claim 20 or 21.

23. A conjugate, which is a fusion protein, comprising the CD80-Fc fusion protein according to any one of claims 1 to 19, linked to a targeting moiety that specifically binds to a molecule on the cell surface.

24. The conjugate according to claim 23, wherein the cell is an immune cell or a tumor cell.

25. The conjugate according to claim 23 or claim 24, wherein the moiety is a protein, a peptide, a nucleic acid, a small molecule, or a nanoparticle.

26. The conjugate according to any one of claims 23 to 25, wherein the moiety is an antibody or an antigen-binding fragment.

27. A nucleic acid molecule encoding the CD80-Fc fusion protein according to any one of claims 1 to 19, the immunomodulatory protein according to claim 20 or 21, or the conjugate which is a fusion protein according to any one of claims 23 to 26.

28. A vector comprising the nucleic acid molecule according to claim 27.

29. The vector according to claim 28, which is an expression vector.

30. An isolated cell comprising the vector according to claim 28 or claim 29.

31. An in vitro method for producing a CD80-Fc fusion protein or an immunomodulatory protein, comprising introducing the nucleic acid molecule according to claim 27 or the vector according to claim 28 or claim 29 into a host cell under conditions for expressing the protein in the host cell.

32. The method according to claim 31, further comprising isolating or purifying the CD80-Fc fusion protein or the immunomodulatory protein from the cell.

33. An isolated and engineered cell comprising the CD80-Fc fusion protein according to any one of claims 1 to 19, the immunomodulatory protein according to claim 20 or 21, the conjugate which is a fusion protein according to any one of claims 23 to 26, the nucleic acid molecule according to claim 27, or the vector according to claim 28.

34. The isolated and engineered cell according to claim 33, wherein the cell is an immune cell, optionally an antigen-presenting cell (APC) or a lymphocyte, optionally a T cell.

35. The isolated and engineered cell according to claim 33 or 34, further comprising a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

36. An infectious substance comprising a nucleic acid molecule encoding a CD80-Fc fusion protein according to any one of claims 1 to 19, an immunomodulatory protein according to claim 20 or 21, or a conjugate that is a fusion protein according to any one of claims 23 to 26.

37. The infectious substance according to claim 36, which is a bacterium or a virus.

38. The infectious substance according to claim 37, wherein the infectious substance is a virus and the virus is an oncolytic virus.

39. A pharmaceutical composition comprising a CD80-Fc fusion protein according to any one of claims 1 to 19, an immunomodulatory protein according to claim 20 or 21, a conjugate according to any one of claims 23 to 26, an isolated and engineered cell according to any one of claims 33 to 35, or an infectious substance according to any one of claims 36 to 38.

40. The pharmaceutical composition according to claim 39, comprising a pharmaceutically acceptable excipient.

41. Use of the pharmaceutical composition according to claim 39 or claim 40 in the manufacture of a medicament for treating a tumor or cancer in a human subject.

42. The pharmaceutical composition according to claim 39 or claim 40 for use in the treatment of a tumor or cancer in a human subject.

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