CTLA-4 variant immunomodulatory proteins and their uses

Abstract

Provided herein are mutant CTLA-4 polypeptides and immunomodulatory proteins, as well as nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of disease indications, including the treatment of autoimmune or inflammatory conditions. Compositions and methods for producing and using such proteins are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit to U.S. Provisional Application No. 62 / 733,615, filed September 19, 2018, U.S. Provisional Application No. 62 / 613,379, filed January 3, 2018, and U.S. Provisional Application No. 62 / 570,619, filed October 10, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 761612002040SeqList.txt, created on October 8, 2018, which is 1,138,300 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to immunomodulatory proteins, including mutant CTLA-4, and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of disease indications, including the treatment of autoimmune or inflammatory conditions. Compositions and methods for making and using such proteins are provided. [Background technology]

[0004] background There is growing medical interest in modulating immune responses by interfering with processes occurring at the immune synapse (IS) formed by and between antigen-presenting cells (APCs) or target cells and lymphocytes. Mechanistically, cell surface proteins within the IS can involve the coordinated and often simultaneous interaction of multiple protein targets with a single protein to which they bind. IS interactions occur in close association with the junction of two cells, and a single protein in this structure can potentially interact simultaneously with both proteins on the same cell (cis) as well as proteins on related cells (trans). While therapeutic agents that can modulate the IS are known, improved therapeutic agents are needed. Immunomodulatory proteins that meet this need are provided. Summary of the Invention

[0005] overview Mutant CTLA-4 polypeptides are provided herein. In some embodiments, mutant CTLA-4 polypeptides are provided herein that contain an IgV domain or a specific-binding fragment thereof, wherein the mutant CTLA-4 polypeptide contains one or more amino acid modifications in an unmodified CTLA-4 polypeptide or a specific-binding fragment thereof, and wherein the mutant CTLA-4 polypeptide specifically binds to the ectodomain of ICOSL with increased affinity compared to unmodified CTLA-4.

[0006] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide comprises a mutated CTLA-4 polypeptide having SEQ ID NO: 1 in an unmodified CTLA-4 polypeptide or a specific binding fragment thereof. With respect to the positions set forth in NO:2, it contains one or more amino acid modifications corresponding to positions selected from among 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67, 69, 71, 72, 73, 75, 76, 82, 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118, 119, 120, 121, and 122.

[0007] In some of any of the provided embodiments, a mutant CTLA-4 polypeptide containing an IgV domain or a specific binding fragment thereof, wherein the mutant CTLA-4 polypeptide has at least one of the following amino acids in a non-modified CTLA-4 polypeptide or a specific binding fragment thereof, with respect to positions set forth in SEQ ID NO:2: 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67, 69, 71, 72, 73, 75, 76, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 202, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55 , 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118, 119, 120, 121, and 122. In some embodiments, the amino acid modification comprises an amino acid substitution, deletion, or insertion.

[0008] In some of any of the provided embodiments, the unmodified CTLA-4 polypeptide is a mammalian CTLA-4 polypeptide or a specific-binding fragment thereof. In some embodiments, the unmodified CTLA-4 polypeptide is a human CTLA-4 polypeptide or a specific-binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide contains the extracellular domain of human CTLA-4, and the one or more amino acid modifications are in one or more residues of the extracellular domain of the unmodified CTLA-4 polypeptide.

[0009] In some of any of the provided embodiments, the unmodified CTLA-4 polypeptide contains (i) the sequence of amino acids set forth in SEQ ID NO:2, (ii) a sequence of amino acids having at least 95% sequence identity to SEQ ID NO:2, or (iii) a portion thereof containing an IgV domain or a specific-binding fragment of an IgV domain. In some embodiments, the unmodified CTLA-4 contains the sequence of amino acids set forth in SEQ ID NO:2.

[0010] In some of any of the provided embodiments, the specific binding fragment of the IgV 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 comprises a length that is at least 80% of the length of the IgV domain set forth as residues 39-140 of SEQ ID NO:1.

[0011] In some of any of the provided embodiments, the mutant CTLA-4 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.

[0012] In some of any of the provided embodiments, the mutant CTLA-4 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.

[0013] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomain of ICOSL, CD80, and / or CD86 with increased affinity compared to the binding of an unmodified CTLA-4 polypeptide to the same ectodomain.

[0014] Provided herein are mutant CTLA-4 polypeptides containing one or more amino acid modifications in the extracellular domain of human CTLA-4 set forth in SEQ ID NO:2, wherein the mutant CTLA-4 polypeptides specifically bind to the ectodomains of human ICOSL, CD80, and / or CD86 with increased affinity compared to CTLA-4 containing the extracellular domain set forth in SEQ ID NO:2.

[0015] In some of any of the provided embodiments, the one or more amino acid modifications are A6T, V10A, L12F, L12H, L12I, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G 29R, G29W, K30R, E33M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T 53S, Y54F, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T6 1R, T61S, L63H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S7 2T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93 V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, P102L, Y105F, Y105L, L106I, L106N, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117M, I117T, and P121S, or conservative amino acid substitutions thereof.

[0016] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, L12F / K30R / S72 G / Q82R / L98Q / M99L / Y105L, L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L1 06I / P121S, L12H / E33M / L98Q / Y105L, L12H / M55T / E59D / L63P / M99L, L12H / L63P / S72G / L98Q / Y105L, L12I / M55T / M56V / I67T / M99L / L106R / I108F, L12 P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L, L12P / I1 8T / A26T / M55T / T69S / S72G / M99L / Y105L, L12P / A26T, L12P / A26T / L63P, L1 2P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, L12P / G29W / L63P / S72G / L9 8Q / Y105L, L12P / G29W / L63P / S72G / L98Q / Y105L / L106I, L12P / A26T / L63P / L98Q / M99L / Y105L, L12P / A26T / L63P / L98Q / Y105L, L12P / A26T / L63P / L98Q / Y105L / L106I, L12P / G29W / D43N / N58S / L63P / L98Q / M99L / Y105L, L12P / M56 V / L63P / V96I / L98Q / M99L / Y105L / Y115H, L12P / L63P / S72G / L98Q / M99L / Y10 5L, L12P / L63P / S72G / L98Q / M99L / Y105L / L106N, L12P / L63P / S72G / L98Q / M 99L / Y105L / L106N / I117L, S14N / R16C / I18T / M56K / T61A / L63P / A86T / M99L,S15P / I18V / M56T / L98Q / M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L98Q / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / L63P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63H / T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M87T / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26T / L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q82R / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M99L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T / E48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105L / I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105L、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S20N / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L98Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105L / I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A26D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / I67N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / L98Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、A26T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / M87V / N110K / I117E、A26T / L63P / S72G / L98Q / M99L / Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / I117L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67N / S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S72G / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / L63P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P / S72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29W / T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T53S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / S72G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q / Y105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I117L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W / N58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58S / L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q / Y105L / L106V、G29W / N58S / L63P / S72G / M87V / L98Q / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W / N58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98Q / Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y105L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / L63P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G / L98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y105L / L106I / I117L、G29W / L63P / S72G / L98Q / Y105L / I117L、G29W / L63P / L98Q / M99L / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L、G29W / M87K / I93V / L98Q / M99L / Y105L、G29W / L98Q / M99L / Y105L、E33M / A42T / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L / I108F、E33M / L63P / S72G / L98Q / Y105L / I117L、E33M / Q82H / L98Q / M99L / Y105L、E33V / A42S / M55T / L98Q / M99L / Y105L、T37S / M56V / L98Q / Y105L、V38I / L63P / S72G / L98Q / M99L / Y105L、Q41L / Y54F / M56K / M99L / I108F、T53S / M56V / L98Q / Y105L、M55T / L63P / T71I / M99L / Y105L、M55T / S72G / L98Q / M99L / Y105L、M55T / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M99L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L106I / I117L、M56R / L63P / L98Q / M99L / Y105L、M56T / L91R / L98Q / Y105L、M56V、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S72G / L98Q / M99L / Y105L、L63P、L63P / T69A / L98Q / M99L / Y105L / L106R / V116A、L63P / S72G / M87A / L98Q / Y105L、L63P / S72G / I93L / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L / L106I / I117L、L63P / S72G / L98Q / Y105L / L106I / I117L、L63P / S72G / Y105L、L63P / M87K / M99L / L106R、L63P / Q82H / L98Q / M99L / Y105L、L63P / K95R、L63P / L98Q、L63P / L98Q / M99L / Y105L、L63P / L98Q / M99L / Y105L / L106I、L63P / L98Q / M99L / Y105L / I108V、L63P / L98Q / M99L / Y105L / I117M、L63P / L98Q / Y105L、L63P / L98Q / V116A、L63P / L98R / N110K、L63P / M99L / Y105L / I108F、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y105L / L106I、S72G / L98Q / M99L / Y105L / I117T、L98Q / M99L / Y105L、L98Q / M99L / Y105L / L106I / I117T、L98Q / M99L / Y105L / L106I / Y115N、L98Q / Y105L、L98R / N110K、T89A / L98Q / M99L / Y105L / L106I / Y115N / E120D / C122P / D124P / S125I / D126P、N58S / L63P / T71A / S72G / L98Q / M99L / Y105L / D124I / S125P / D126T、R16G / E33M / N58S / E59G / L63P / L98Q / Y105L / E120D / C122P / D124P / S125I / D126P、G29W / L63P / S72G / L98Q / Y105L / P121S / D126T、L12H / E33M / L98Q / Y105L、T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N58S / L 63P / M87V / Y105L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / , M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M 87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / Y105L, E 33V / M99L / Y105L, E33V / L98Q / M99L, E33V / M99L, L12F / R16H / G29W / M56T / L98 Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / and I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L.

[0017] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide contains a sequence of amino acids set forth in any of SEQ ID NOs: 4-97, 99-104, 106-155, or 570-637, or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide contains a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs: 4-97, 99-104, 106-155, or 570-637, as compared to wild-type or unmodified CTLA-4, e.g., as set forth in SEQ ID NO:2, and contains one or more of the amino acid modifications of the respective SEQ ID NO:, or a specific binding fragment thereof.

[0018] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomain of ICOSL with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomain.

[0019] In some of any of the provided embodiments, the one or more amino acid modifications are at a position corresponding to a position selected from among 10, 12, 16, 18, 19, 26, 28, 29, 33, 35, 38, 42, 45, 47, 53, 55, 56, 58, 61, 63, 64, 65, 67, 69, 72, 76, 82, 85, 87, 89, 93, 97, 98, 99, 105, 106, 108, 110, 113, 116, 117, or 121 with respect to the positions set forth in SEQ ID NO:2. Regarding the positions listed in NO:2, V10A, L12F, L12I, L12P, R16H, I18F, I18N, I18T, I18V, A19V, A26T, P28L, G29W, E33M, E33V, R35K, V38I, A42V, Q45H, T47A, T53S, M55T, M56K, M56T, M56V, N58D, N58S, T61A, T61R, L63H, L63P, D64E, D64N, D64V 4V, D65G, I67N, I67T, I67V, T69A, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87T, M87V, T89A, T89S, I93L, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F, I108V, N110K, Q113H, V116A, I117L, or P121S.

[0020] In some of any of the provided embodiments, the one or more amino acid modifications are V10A, L12F, L12I, R16H, I18N, I18T, I18V, A19V, A26T, G29W, E33M, E33V, R35K, V38I, A42V, Q45H, T47A, T53S, M55T, M56K, M56V, N58D, N58S, T61A, T61R, L63H, L63P, D64E, D64N ... and P121S, or a conservative amino acid substitution thereof.

[0021] In some of any of the provided embodiments, the one or more amino acid modifications are selected from the group consisting of L63P / S72G / L98Q / M99L / Y105L / L106I / I117L, G29W / L98Q / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L, L63P / Q82H / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99L / Y105L, T61A / L63P / S72G / L98Q / M99L / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, L6 3P / S72G / I93L / L98Q / M99L / Y105L, L12I / M55T / M56V / I67T / M99L / L106R / I1 08F, I18N / A26T / L63H / T89A / L98Q / M99L / Y105L, G29W / N58S / L63P / M87T / L9 8Q / M99L / Y105L, G29W / N58S / L63P / D64N / L98Q / M99L / Y105L, I18T / L63P / S7 2G / M87K / L98Q / M99L / Y105L, L63P / M87K / M99L / L106R, L63P / M99L / Y105L / I 108F, G29W / L63P / L98Q / M99L / Y105L, A26T / L63P / D65G / L98Q / M99L / Y105L, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, I18V / A26T / L63P / D64E / L98Q / Y 105L / L106R / N110K, A19V / G29W / R35K / L63P / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, G29W / T53S / M56K / L63P / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / T53S / L63P / L98Q / Y105L / L106I / I11 7L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / N58D / I67V / L98Q / M 99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M 99L / Y105L / L106I, A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L,A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, G29W / M87K / I93V / L98Q / M99L / Y105L, G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L , L63P / L98Q / M99L / Y105L / I108V, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S72G / L98Q / Y105L / I108F, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y10 5L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / M87V / L98Q, T5 3S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, L12F / R16H / G29W / M56T / Y105L, or L12F / L98Q / Y105L. In some embodiments, the amino acid substitutions are G29W / L98Q / M99L / Y105L, L63P / M99L / Y105L / I108F, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, G29W / N58D / I67V / L98Q / M99L / Y105L,I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, G29W / M87K / I93V / L98Q / M99L / Y105L, G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L, A26T / A42V / Q45H / I67N / M87K / E9 7Q / M99L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, G29W / T53S / M56K / N58S / L63P / M87V / L9 8Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99 L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M56K / N58 S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, or L12F / L98Q / Y105L.

[0022] In some of any of the provided embodiments, the one or more amino acid modifications include one or more modifications at positions corresponding to positions 12, 26, 63, 98, or 105, with respect to the positions set forth in SEQ ID NO:2, and / or one or more amino acid modifications containing one or more modifications selected from L12P, L12F, A26T, L63P, L98Q, or Y105L.

[0023] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of L12P / A26T / L63P / L98Q / Y105L, A26T / L63P / S72G / L98Q / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L, L63P / Q82H / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99L / Y105L, T61A / L63P / S72G / L98Q / M99L / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, L63P / S72G / I93 L / L98Q / M99L / Y105L, L12I / M55T / M56V / I67T / M99L / L106R / I108F, I18N / A26T / L63H / T89A / L98Q / M99L / Y105L, G29W / N58S / L63P / M87T / L98Q / M99L / Y105L, G29W / N58S / L63P / D64N / L98Q / M99L / Y105L, I18T / L63P / S72G / M87K / L98Q / M99L / Y105L, L63P / M87K / M99L / L106R, L63P / M99L / Y105L / I108F, G29W / L63P / L98Q / M99L / Y105L, A26T / L63P / D65G / L98Q / M99L / Y105L, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, A19V / G29W / R35K / L63P / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / T53S / M56K / L63P / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / I117L, L63P / S72G / L98Q / Y105L / L10 6I / I117L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / T53S / L63P / L98 Q / Y105L / L106I / I117L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H,G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99 L / Y105L / L106I, A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L, A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, G29W / M87K / I93V / L98Q / M99L / Y105L, P28L / E33V / L63P / S72G / L98Q / M99L / Y105L, G29W / T5 3S / M56K / L63P / Q82H / L98Q / M99I / Y105L, I18F / L63P / L98Q / M99L / Y105L / P121S, L63P / L98Q / M99L / Y105L / I108V, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L9 8Q / M99L / Y105L / I108V / I117L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S72G / L98Q / Y105 and G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V.

[0024] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomain of CD80 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomain. In some embodiments, the one or more amino acid modifications are at a position corresponding to a position selected from among 10, 12, 16, 18, 26, 29, 42, 45, 53, 56, 58, 63, 67, 72, 82, 87, 97, 98, 99, 105, 108, or 121 with respect to the positions set forth in SEQ ID NO:2. In some embodiments, the one or more amino acid modifications are selected from V10A, L12F, R16H, I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, M56T, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, I108V, or a conservative amino acid substitution thereof.

[0025] In some of any of the provided embodiments, the one or more amino acid modifications are at a position corresponding to a position selected from among V10A, L12F, R16H, I18T, A26D, A26T, G29W, A42V, Q45H, T53S, M56K, M56T, N58S, L63P, I67N, S72G, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, I108V, or P121S, with respect to the position set forth in SEQ ID NO:2.

[0026] In some of any of the provided embodiments, the one or more amino acid modifications are, with respect to the positions set forth in SEQ ID NO:2, I18T / G29W / L63P / L98Q / Y105L, G29W / L63P / L98Q / M99L / Y105L, G29W / N58S / L63P / L98Q / Y105L, A26D / S72G / L98Q / M99L / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / A42V / Q45H / I67N / Selected from M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, and V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S.

[0027] In some of any of the provided embodiments, the one or more amino acid modifications are selected from the group consisting of I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, and / or L12F / R16H / G29W / M56T / L98Q / Y105L. 05L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56T / L98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L, or M56T / L98Q / Y105L.In some embodiments, the one or more amino acid modifications are I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, L98Q / M99L / Y105L, E33V / L98Q / Y105L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, and E33V / L98Q / M99L.

[0028] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomains of ICOSL and CD80 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomains. In some embodiments, the one or more amino acid modifications are selected from V10A, L12F, R16H, I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, I108V, or conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid modifications are selected from I18T, A26T, G29W, E33V, Q45H, T53S, M56K, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, I108V.

[0029] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomain of CD86 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomain.

[0030] In some of any of the provided embodiments, the one or more amino acid modifications are at a position corresponding to a position selected from among 10, 12, 16, 18, 20, 26, 28, 29, 30, 33, 42, 47, 53, 55, 56, 58, 59, 61, 63, 65, 67, 69, 72, 76, 82, 85, 87, 89, 93, 96, 98, 99, 102, 105, 106, 108, 113, 115, 116, 117, or 121 with respect to the positions set forth in SEQ ID NO:2. With respect to the positions described in NO:2, V10A, L12F, L12H, L12P, R16H, I18T, I18V, S20N, A26S, A26T, P28L, G29R, G29W, K30R, E33M, E33V, A42S, A42T, T47A, T53S, M55T, M56K, M56R, M56T, M56V, N58D, N58S, E59G, T61I, T61N, T61R, T61S, L63P, D65G, I67N, I67V, T69A, and at a position corresponding to a position selected from among T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89M, T89S, I93V, V96I, L98Q, L98R, M99L, P102L, Y105L, L106I, L106N, L106R, L106V, I108F, I108V, Q113H, Y115H, V116A, I117E, I117L, I117K, I117T, and P121S.

[0031] In some of any of the provided embodiments, the one or more amino acid modifications are L12F, L12H, L12P, R16H, I18T, I18V, S20N, A26T, G29R, G29W, E33M, E33V, A42S, A42V, T47A, T53S, M55T, M56K, M56R, M56T, M56V, N58D, N58S, T61I, T61N, T61R, T61S, L63P, D65G, I67N, I6 7V, T69A, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89M, T89S, V96I, L98Q, L98R, M99L, P102L, Y105L, L106I, L106N, L106R, L106V, I108F, I108V, Q113H, Y115H, V116A, I117L, I117T, and P121S, or conservative amino acid substitutions thereof.

[0032] In some of any of the provided embodiments, the one or more amino acid modifications are, with respect to the positions set forth in SEQ ID NO:2, L12P / A26T / L63P / L98Q / Y105L, I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, P28L / E33V / L63P / S72G / L98R / M99L / Y105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y105L, S72G / L98Q / M99L / Y105L / I117T, M56R / L63P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L106I, A26T / M55T / L63P / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L6 3P / Q82R / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L / I117L, A26T / I6 7N / S72G / L98Q / M99L / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, S20 N / A26T / L63P / L98Q / M99L / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L9 8Q / M99L / Y105L / L106N, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58S / L63P / S72G / L98Q / Y105L, G29W / L63P / D65G / S72G / L98Q / Y10 5L, T53S / M56V / L98Q / Y105L, L63P / S72G / L98Q / Y105L, G29W / T53S / M56 K / L63P / L98Q / Y105L, I18V / G29W / L63P / S72G / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / L106I, G29W / L63P / I67V / S72G / L98Q / Y105L, L63P / S72G / L98Q / Y105L / L106I / I117L, L12F / R16H / G29W / M56T / L98Q / Y105L,L12P / G29W / L63P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / M87V / L98Q / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / N58S / L63P / S72G / L98Q / Y105L / L106V、A26T / L63P / L98Q / M99L / Y105L、G29W / N58D / I67V / L98Q / M99L / Y105L、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y105L / L106I、L63P / L98Q / M99L / Y105L、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / Y105L、L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S、I18T / A26T / L63P / S72G / L98Q / Y105L、L12F / K30R / S72G / Q82R / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L / Y105L / L106N / I117L、G29W / M87K / I93V / L98Q / M99L / Y105L、P28L / E33V / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L、L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H、G29W / T53S / M56K / T61N / L63P / L98Q / Y105L、I18T / A26S / M55T / M56V / L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y105L / I108F、Selected from among L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117LG29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / L63P / S72G / L98Q / Y105L / P121S, and V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S.

[0033] In some of any of the provided embodiments, the one or more amino acid modifications are I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y105L, S72G / L98Q / M99L / Y105L / I117T, M56R / L63P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L106I, A26T / M55T / L63P / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L / I117L, A26 T / I67N / S72G / L98Q / M99L / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, S 20N / A26T / L63P / L98Q / M99L / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y1 05L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M 99L / Y105L / L106N, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58 S / L63P / S72G / L98Q / Y105L, G29W / L63P / D65G / S72G / L98Q / Y105L, T53S / M5 6V / L98Q / Y105L, L63P / S72G / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / L 106I, L12F / R16H / G29W / M56T / L98Q / Y105L, G29W / N58S / L63P / S72G / M87V / L98Q / Y105L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / N58S / L63 P / S72G / L98Q / Y105L / L106V, G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S 72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I,L63P / L98Q / M99L / Y105L, A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L, A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I1 17L, L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H, G29W / T53S / M56K / T61N / L63P / L 98Q / Y105L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y 105L, E33M / L63P / S72G / L98Q / Y105L / I108F, L12P / R16H / A26T / T61S / L63P / M87V / L98Q / Selected from M99L / Y105L / L106I / I117L, L98Q / M99L / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q, L12F / G29W / L98Q / Y105L, or L12F / L98Q / Y105L.

[0034] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomains of CD80 and CD86 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomains. In some embodiments, the one or more amino acid modifications are selected from I18T, A26T, G29W, E33V, A42V, T53S, N58S, L63P, I67N, Q82R, M87K, M87V, L98Q, M99L, Y105L, and I108V.

[0035] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomains of ICOSL and CD86 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomains.

[0036] In some of any of the provided embodiments, the one or more amino acid modifications are selected from L12F, R16H, I18T, I18V, A26T, G29W, E33M, E33V, A42V, T47A, T53S, M55T, M56K, M56V, N58D, N58S, T61R, L63P, D65G, I67N, I67V, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89S, L98Q, M99L, Y105L, L106I, L106R, I108F, I108V, Q113H, I117L, and P121S, or a conservative amino acid substitution thereof.

[0037] In some of any of the provided embodiments, the one or more amino acid modifications are selected from L12F, R16H, I18T, I18V, A26T, G29W, E33V, A42V, T47A, T53S, M55T, M56K, N58D, N58S, L63P, I67N, I67V, S72G, Q82H, Q82R, R85G, M87K, M87V, T89A, T89S, L98Q, M99L, Y105L, L106I, L106R, I108F, I108V, and I117L.

[0038] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomains of ICOSL, CD80, and CD86 with increased affinity compared to the binding of unmodified CTLA-4 to the same ectodomains.

[0039] In some of any of the provided embodiments, the one or more amino acid modifications are selected from L12F, R16H, I18T, A26T, G29W, E33V, A42V, T53S, M55T, M56K, N58S, L63P, I67N, Q82R, M87K, M87V, L98Q, M99L, Y105L, or I108V. In some embodiments, the one or more amino acid modifications are selected from A26T, G29W, T53S, M56K, N58S, L63P, L98Q, M99L, or Y105L. In some embodiments, the one or more amino acid modifications are selected from G29W, L63P, L98Q, M99L, or Y105L. In some embodiments, the one or more amino acid modifications comprise a modification selected from G29W / L63P, G29W / L98Q, G29W / M99L, G29W / Y105L, L63P / L98Q, L63P / M99L, L63P / Y105L, L98Q / M99L, L98Q / Y105L, or M99L / Y105L. In some embodiments, the amino acid modification is G29W / L98Q / Y105L. In some embodiments, the amino acid modification is G29W / N58S / L63P / Q82R / L98Q / Y105L. In some embodiments, the amino acid modification is L12P / G29W / L63P / S72G / L98Q / Y105L.

[0040] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide specifically binds to the ectodomain of ICOSL with increased affinity and specifically binds to one or more ectodomains of the other of CD80 or CD86 with decreased affinity compared to the binding of unmodified CTLA-4 to the same ectodomain.

[0041] In some of any of the provided embodiments, the increase in binding affinity for one or more ectodomains is independently 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, 100-fold or more.

[0042] In some of any of the provided embodiments, the reduction in binding affinity for one or more ectodomains is independently 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 or more.

[0043] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide contains an IgV domain or a specific-binding fragment thereof. In some embodiments, the IgV domain or a specific-binding fragment thereof is only the CTLA-4 portion of the mutant CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide contains a sequence of amino acids set forth in any of SEQ ID NOs: 156-285, 603-635, or 637, or a specific-binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide contains a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs: 156-285, 603-635, or 637, or a specific-binding fragment thereof, e.g., contains one or more of the amino acid modifications of the respective SEQ ID NOs compared to wild-type or unmodified CTLA-4 set forth in SEQ ID NOs: 156-285, 603-635, or 637. In some embodiments, the mutant CTLA-4 polypeptide comprises a sequence of amino acids of the extracellular domain set forth in any of SEQ ID NOs: 4-97, 99-104, 106-155, 569-602, or 636, or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs: 4-97, 99-104, 106-155, 569-602, or 63, and contains one or more of the amino acid modifications of the respective SEQ ID NOs: 4-97, 99-104, 106-155, 569-602, or 636.

[0044] In some of any of the provided embodiments, the ICOSL is human ICOSL. In some embodiments, the CD80 is human CD80. In some embodiments, the CD86 is human CD86.

[0045] In some embodiments, the mutant CTLA-4 polypeptide is a soluble protein. In some embodiments, the mutant CTLA-4 polypeptide lacks the CTLA-4 transmembrane domain and the intracellular signaling domain, and / or the mutant CTLA-4 polypeptide cannot be expressed on the surface of a cell.

[0046] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide is linked to a multimerization domain. In some embodiments, the multimerization domain is an Fc domain or variant thereof with reduced effector function. In some embodiments, the mutant CTLA-4 polypeptide is linked to an Fc domain or variant thereof with reduced effector function. In some embodiments, the Fc domain is mammalian, optionally human, or the variant Fc domain contains one or more amino acid modifications compared to an unmodified Fc domain that is mammalian, optionally human. In some embodiments, the Fc domain or variant thereof contains a sequence of amino acids set forth in any of SEQ ID NOs:438-442, or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOs:438-442. 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 domain comprises the amino acid modifications L234A / L235E / G237A. In some embodiments, the Fc domain contains the amino acid modification C220S according to EU numbering. In some embodiments, the mutant CTLA-4 polypeptide is indirectly linked to the multimerization domain or Fc via a linker, optionally a G4S (Gly4Ser) linker.

[0047] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide linked to the Fc domain contains a sequence of amino acids set forth in any of SEQ ID NOs: 286-379, 381-386, or 388-437, or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOs: 286-379, 381-386, or 388-437.

[0048] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide is a transmembrane immunomodulatory protein that further contains a transmembrane domain, optionally linked, directly or indirectly, to the extracellular domain (ECD) of the mutant CTLA-4 polypeptide or a specific-binding fragment thereof. In some embodiments, the transmembrane domain contains the sequence of amino acids set forth as residues 162-182 of SEQ ID NO:1, or a functional variant thereof, that exhibits at least 85% sequence identity with residues 162-182 of SEQ ID NO:1.

[0049] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide further contains a cytoplasmic domain, optionally linked directly or indirectly to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains a native CTLA-4 cytoplasmic domain, an intracellular signaling domain, and / or contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling motif. In some embodiments, the cytoplasmic domain contains the sequence of amino acids set forth as residues 183-223 of SEQ ID NO:1, or a functional variant thereof, that exhibits at least 85% sequence identity with residues 183-223 of SEQ ID NO:1. In some embodiments, the cytoplasmic domain contains an immunoreceptor tyrosine-based activation motif (ITAM) signaling motif and / or contains the intracellular signaling domain of CD3 zeta.

[0050] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide does not contain a cytoplasmic signaling domain and / or is unable to mediate or regulate intracellular signals when expressed on a cell.

[0051] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide reduces IFN-gamma (interferon gamma) expression relative to an unmodified CTLA-4 polypeptide in an in vitro primary T cell assay. In some embodiments, the mutant CTLA-4 polypeptide is deglycosylated.

[0052] In some of any of the provided embodiments, provided herein are immunomodulatory polypeptides containing any of the provided mutant CTLA-4s linked, directly or indirectly via a linker, to a second polypeptide containing an IgSF domain of an immunoglobulin superfamily (IgSF) member. In some embodiments, the IgSF domain is an affinity-modified IgSF domain, which contains one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of the IgSF family member. In some cases, the affinity-modified IgSF domain exhibits altered binding to one or more of its cognate binding partners compared to binding of an unmodified or wild-type IgSF domain of the IgSF family member to the same one or more cognate binding partners. In some embodiments, the IgSF domain exhibits increased binding to one or more of its cognate binding partners compared to binding of an unmodified or wild-type IgSF domain to the same one or more cognate binding partners.

[0053] In some of any of the provided embodiments, the mutant CTLA-4 is a first mutant CTLA-4 polypeptide, and the IgSF domain of the second polypeptide is an IgSF domain from a second mutant CTLA-4 polypeptide, and the first and second mutant CTLA-4s are the same or different. In some aspects, the immunomodulatory protein further contains a third polypeptide containing an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, and the affinity-modified IgSF domain contains one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member. In some examples, the third polypeptide is the same as the first and / or second polypeptide, or the third polypeptide is different from the first and / or second polypeptide.

[0054] In some of any of the provided embodiments, optionally the IgSF domain or affinity-modified IgSF domain of the second or third polypeptide is or includes an IgV domain. In some cases, the mutant CTLA-4 polypeptide is or contains an IgV domain.

[0055] In some of any of the provided embodiments, the immunomodulatory protein further contains a multimerization domain linked to at least one of the mutant CTLA-4 polypeptide or the second polypeptide. In some aspects, the immunomodulatory protein further comprises a multimerization domain linked to at least one of the mutant CTLA-4 polypeptide, the second polypeptide, and / or the third polypeptide. In some cases, the multimerization domain is an Fc domain or a variant thereof with reduced effector function.

[0056] In some of any of the provided embodiments, the multimerization domain promotes the formation of heterodimers. Provided herein is an immunomodulatory protein containing any of the provided immunomodulatory proteins, wherein the multimerization domain is a first multimerization domain and interacts with a second multimerization domain to form a multimer containing the immunomodulatory protein. In some cases, the immunomodulatory protein is a first immunomodulatory protein, and the second immunomodulatory protein is linked to the second multimerization domain, either directly or indirectly via a linker, and the multimer contains the first and second immunomodulatory proteins. In some embodiments, the second immunomodulatory protein is an immunomodulatory protein provided herein, and the multimerization domain is a second multimerization domain.

[0057] In some of any of the provided embodiments, provided herein is an immunomodulatory protein containing a first mutant CTLA-4 polypeptide, the first mutant CTLA-4 polypeptide having a multimerization domain that is a first multimerization domain, and a second mutant CTLA-4 polypeptide, the second mutant CTLA-4 polypeptide having a multimerization domain that is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer containing the first and second mutant CTLA-4 polypeptides. In some embodiments, the multimer is a dimer. In some embodiments, the immunomodulatory protein is a homodimer. In some embodiments, the immunomodulatory protein is a heterodimer. In some embodiments, the first and / or second multimerization domain is an Fc domain or variant thereof with reduced effector function. In some embodiments, the first and second multimerization domains are the same or different.

[0058] In some of any of the provided embodiments, provided herein is a conjugate containing a mutant CTLA-4 or immunomodulatory protein linked to a moiety. In some embodiments, the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell. In some embodiments, the targeting moiety specifically binds to a molecule on the surface of an immune cell. In some embodiments, the immune cell is an antigen-presenting cell or a lymphocyte. In some embodiments, the targeting moiety localizes to a cell or tissue in an inflammatory environment. 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.

[0059] In some of the embodiments provided herein, provided herein are nucleic acid molecules encoding the mutant CTLA-4 polypeptides provided herein or the immunomodulatory proteins provided herein. In some embodiments, the nucleic acid molecules are synthetic nucleic acids. In some embodiments, the nucleic acid molecules are cDNAs.

[0060] In some of the embodiments provided herein, a vector is provided containing any of the nucleic acid molecules 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.

[0061] In some of any of the embodiments provided, 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.

[0062] In some of any of the provided embodiments, provided herein are methods for producing a mutant CTLA-4 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule or 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 mutant CTLA-4 polypeptide or immunomodulatory protein from the cell.

[0063] In some of any of the provided embodiments, provided herein are methods for engineering cells to express a mutant CTLA-4 polypeptide, the methods comprising introducing a nucleic acid molecule encoding a mutant CTLA-4 polypeptide or an immunomodulatory protein provided herein into a host cell under conditions such that the polypeptide is expressed in the cell.

[0064] In some of any of the provided embodiments, provided herein are engineered cells that express a mutant CTLA-4 polypeptide, immunomodulatory protein, nucleic acid molecule, or vector provided herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a CD4+ and / or CD8+ T cell. In some embodiments, the T cell is a regulatory T cell (Treg). In some embodiments, the engineered cell is a primary cell. In some embodiments, the engineered cell is a mammalian cell. In some embodiments, the engineered cell is a human cell.

[0065] In some of any of the provided embodiments, the CTLA-4 polypeptide is expressed on the surface of a cell provided herein via a transmembrane domain. In some such embodiments, the CTLA-4 polypeptide contains a cytoplasmic domain, which optionally is linked, directly or indirectly, to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains a native CTLA-4 cytoplasmic domain, an intracellular signaling domain, and / or an ITIM signaling motif. In some embodiments, the cytoplasmic domain contains the sequence of amino acids set forth as residues 183-223 of SEQ ID NO:1, or a functional variant thereof, that exhibits at least 85% sequence identity with residues 183-223 of SEQ ID NO:1. In some embodiments, the cytoplasmic domain contains an ITAM signaling motif and / or is or contains the intracellular signaling domain of CD3 zeta. In some embodiments, the CTLA-4 polypeptide does not contain a cytoplasmic signaling domain and / or is unable to mediate or regulate intracellular signals when expressed on a cell.

[0066] In some of any of the provided embodiments, the engineered cells further contain a chimeric antigen receptor (CAR). In some embodiments, the engineered cells further contain an engineered T cell receptor (TCR).

[0067] In some of any of the provided embodiments, a nucleic acid molecule encoding a mutant CTLA-4 polypeptide provided herein or an immunomodulatory protein provided herein is included. Infectious substance In some embodiments, Infectious substance is a bacterium or a virus.

[0068] In some of any of the provided embodiments, a mutant CTLA-4 polypeptide provided herein, an immunomodulatory protein provided herein, a conjugate provided herein, an engineered cell provided herein, or a fusion protein provided herein is used. Infectious substance A pharmaceutical composition comprising Substance In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

[0069] In some of any of the embodiments provided, provided herein is an article of manufacture containing a pharmaceutical composition provided herein in a vial or container. In some embodiments, the vial or container is sealed.

[0070] In some of any of the embodiments provided, 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.

[0071] In some of any of the provided embodiments, provided herein are methods of modulating an immune response in a subject, comprising administering to the subject a pharmaceutical composition provided herein. In some embodiments, provided herein are methods of modulating an immune response in a subject, comprising administering to the subject an engineered cell provided herein. In some such embodiments, the engineered cell is autologous to the subject. In some embodiments, the engineered cell is allogeneic to the subject. In some embodiments, modulating the immune response treats a disease or condition in the subject.

[0072] In some of any of the provided embodiments, the immune response is reduced. In some embodiments of the provided methods, a mutant polypeptide provided herein, an immunomodulatory protein provided herein, or an engineered cell provided herein comprising a surface-expressed mutant CTLA-4 containing an inhibitory (e.g., ITIM-containing) cytoplasmic signaling domain is administered to a subject. In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition, or a disease or condition associated with an excessive immune response. In some embodiments, the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, an autoimmune skin disease, transplantation, a rheumatic disease, thyroiditis, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory lung disease, an inflammatory endocrine disease, an autoimmune blood disease, an autoimmune demyelinating disease, or an autoimmune disease involving a systemic autoimmune disorder. In some embodiments, the disease or condition is inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, rheumatoid arthritis, psoriasis, lupus erythematosus, celiac disease, type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, De Quervain's thyroiditis, myasthenia gravis, vasculitis, autoimmune hemolytic anemia, pernicious anemia autoimmune atrophic gastritis, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, primary biliary tract infections, and the like. The disease is selected from among idiopathic cirrhosis, chronic active hepatitis, membranous nephropathy, primary idiopathic myxedema, scleroderma, chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, autoimmune bowel disease syndrome, idiopathic thrombocytopenia, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antisperm antibodies, sensorineural hearing loss, Sjogren's syndrome, polymyositis, multiple sclerosis, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa, hemolytic anemia, glomerulonephritis, and idiopathic facial paralysis.

[0073] In some of the provided embodiments of any of the provided methods, an immune response is increased. In some of the provided methods, an engineered cell provided herein, such as a cell expressing a surface-expressed mutant CTLA-4 that lacks a cytoplasmic signaling domain or a surface-expressed mutant CTLA-4 that contains an activating (e.g., ITAM-containing) cytoplasmic signaling domain, is administered to a subject. In some embodiments, the provided method treats a disease or condition that is a tumor or cancer. In some of such embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, cancer of the musculoskeletal system, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer. [Brief explanation of the drawings]

[0074]

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[0075] Detailed Description Provided herein are immunomodulatory proteins that are or comprise mutants or variants of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as cluster of differentiation 152 or CD152), or specific-binding fragments thereof, that exhibit activity for binding to at least one target binding partner. In some embodiments, the mutant CTLA-4 polypeptide contains one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) compared to unmodified or wild-type CTLA-4 polypeptides. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the ECD of the unmodified or wild-type CTLA-4 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgSF domain (e.g., IgV) of the unmodified or wild-type CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptides and immunomodulatory proteins exhibit altered, e.g., increased, avidity or affinity for inducible T-cell costimulatory ligand (ICOSL, also known as B7-H2, CD275, and GL50). In some embodiments, the mutant CTLA-4 polypeptides and immunomodulatory proteins exhibit increased binding activity or affinity for ICOSL, CD80 (also known as B7-1), and / or CD86 (also known as B7-2).

[0076] In some embodiments, the mutant CTLA-4 polypeptide is a soluble immunomodulatory protein. Such molecules include CTLA-4 polypeptides that do not contain a transmembrane domain and / or are not membrane-anchored to cells or cannot be expressed on the surface of cells. In some embodiments, the mutant CTLA-4 protein can be provided as a transmembrane immunomodulatory protein that can be expressed on the surface of cells, or as a secretable immunomodulatory protein that can be secreted from cells. In some embodiments, one or more other immunomodulatory proteins are also provided herein, which are conjugates or fusions containing the mutant CTLA-4 polypeptides provided herein and one or more other moieties or polypeptides.

[0077] In some embodiments, the mutant CTLA-4 polypeptides and immunomodulatory proteins modulate an immunological immune response, such as increasing or decreasing an immune response. In some embodiments, the mutant CTLA-4 polypeptides and immunomodulatory proteins provided herein can be used for the treatment of a disease or condition associated with a dysregulated immune response, such as an autoimmune symptom or disease, or, in some cases, an oncological indication.

[0078] In general, antigen-specific T cell activation generally requires two distinct signals. The first signal is provided by the interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC)-associated antigens present on antigen-presenting cells (APCs). The second signal is costimulatory for TCR engagement and is necessary for T cell proliferation, differentiation, and / or survival, including, in some cases, avoiding T cell apoptosis or anergy. In some embodiments, under normal physiological conditions, a T cell-mediated immune response is initiated by antigen recognition by the T cell receptor (TCR) and regulated by a balance of costimulatory (e.g., T cell activation) and co-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, for example, during attack by a pathogenic infection. In some cases, however, the immune system can become dysregulated, and an aberrant immune response can be mounted against normal body sites or tissues, resulting in an autoimmune disease or condition or autoimmune symptoms. In other cases, an unwanted immune response can be mounted against foreign tissue, such as a transplant, which can lead to transplant rejection.

[0079] CTLA-4 is an inhibitory IgSF receptor that suppresses T cell responses through modulation of TCR / CD28 signaling. The costimulatory receptor CD28 binds to the costimulatory ligands CD80 (also known as B7-1) and CD86 (also known as B7-2) and promotes the activation of naive T cells in the presence of TCR signals. The co-inhibitory receptor CTLA-4 competes with CD28 for binding to CD80 and CD86, thereby inducing negative regulation of T cell activation (Figure 2). When CTLA-4 binds to CD80 and / or CD86 and prevents CD28 from its cognate ligand, T cells do not efficiently transduce the activation signaling cascade, eliminating or attenuating T cell activation and effector function. CTLA-4 also exerts its inhibitory effect by capturing and trans-endocytosing the costimulatory ligands CD80 and CD86 from opposing cells, such as APCs, thereby making these costimulatory ligands unavailable for binding to the CD28 costimulatory receptor (Figure 3). T cell receptor (TCR) engagement enhances acquisition of the costimulatory ligand CTLA-4 (Hou et al (2015) J. Immunol., 194(5):2148-59; Soskic et al. (2014) Adv. Immunol., 124:95-136; Qureshi et al. (2011), Science, 332(6029):600-603).

[0080] CTLA-4 has been exploited as a therapeutic agent for treating autoimmune diseases by attenuating T cell activation through modulation of CD80 and / or CD86 interactions. Specifically, abatacept and belatacept are FDA-approved therapeutic agents for use in rheumatoid arthritis and transplant settings, respectively. Abatacept is a wild-type CTLA-4 IgSF domain fused to the Fc portion of an antibody. Belatacept is an engineered variant of the CTLA-4 IgSF domain containing a substitution of tyrosine for alanine at position 31 and glutamic acid for leucine at position 106 (A31Y / L106E), corresponding to positions 31 and 106 of the wild-type reference CTLA-4 ECD sequence set forth in SEQ ID NO:2, to confer increased affinity for the CD80 and CD86 ligands (Kremer et al., N Engl J Med. 2003;349(20):1907-1915, Larsen et al., Am J Transplant. 2005;5(3):443-453).

[0081] In some embodiments, ICOSL (also known as B7-H2) has been reported to be a binding partner of CTLA-4 (Yao et al. (2011) Immunity, 34(5):729-740). ICOSL is a ligand for the costimulatory receptor ICOS (Figure 2). CD28 and CTLA-4 can also bind to ICOSL, although possibly with lower affinity than ICOS. CD80, CD86, and ICOSL are typically expressed on the surface of APCs (e.g., dendritic cells). ICOSL, CD80, and / or CD86 can also be expressed on T cells, including CD4+ and CD8+ T cells. Binding of CD80, CD86, and / or ICOSL to costimulatory receptors, CD28, and / or ICOS, enhances immune responses, which may involve T cell activation, T cell proliferation, and cytokine production, among other activities. For example, ICOSL binding to ICOS is involved in T helper cell and B cell differentiation.

[0082] A full-length CTLA-4 polypeptide contains a signaling sequence, an extracellular domain (ECD), a transmembrane region, and a cytoplasmic domain. The cytoplasmic domain contains the intracellular signaling domain. An exemplary human CTLA-4 sequence is set forth in SEQ ID NO:1. After removal of the signal sequence, the ECD contains amino acids corresponding to amino acids 36-161 of SEQ ID NO:1. An exemplary human CTLA-4 ECD amino acid sequence is set forth in SEQ ID NO:2 or 569. The ECD of CTLA-4 contains a single IgSF domain (i.e., a single IgV domain). In some cases, the IgV domain is or contains amino acids 39-140 of SEQ ID NO:1 (amino acids 4-105 of SEQ ID NO:2). In some cases, the IgV domain is or contains amino acids 39-152 of SEQ ID NO:1 (amino acids 4-117 of SEQ ID NO:2). Within the IgV domain are a CDR1-like loop (corresponding to amino acids 27-34 of SEQ ID NO:2), a CDR2-like loop (corresponding to amino acids 51-55 of SEQ ID NO:2), and a CDR3-like loop (corresponding to amino acids 97-104 of SEQ ID NO:2), which, in some embodiments, have been reported to play a role in the interaction of CTLA-4 with its CD80 and CD86 cognate ligands (Peach et al., J. Exp. Med. (1994), 180(6):2049-2058).

[0083] Provided herein are mutant CTLA-4 polypeptides that exhibit increased binding activity, such as increased binding affinity to CD80, CD86, and / or ICOSL. Some provided embodiments are directed to mutant CTLA-4 molecules that exhibit increased affinity for ICOSL, and in some cases, higher binding affinity, such as increased binding affinity to CD80 and / or CD86. In some embodiments, binding to CD80, CD86, and ICOSL is competitive, such that a single mutant CTLA-4 polypeptide does not bind to more than one ligand at a time. Thus, the provided mutant CTLA-4 polypeptides can bind to CD80, CD86, and / or ICOSL to modulate, e.g., antagonize, the normal function of these ligands at the binding-activating receptors CD28 and ICOS on T cells.

[0084] In some aspects, suppression or weakening of immune responses, such as T cell responses, may be desirable to reduce or prevent unwanted autoimmune symptoms and / or transplant rejection. Among the embodiments provided are methods for using mutant CTLA-4 polypeptides, such as soluble or cellularly expressed forms thereof, to treat autoimmune or inflammatory diseases or conditions. Thus, in some aspects, mutant CTLA-4 polypeptides can be delivered to patients experiencing unwanted autoimmunity, such as involving T cells, with the effect of reduced T cell activation, expansion, and / or effector function, weakening the autoimmune disorder. Methods of making and using these mutant CTLA-4 polypeptides are also provided.

[0085] In some cases, various forms of CTLA-4 polypeptides can be produced to promote or increase immune responses. For example, certain CTLA-4 switching receptors containing an activating cytoplasmic signaling domain or decoy receptor that competes for CTLA-4 binding to its cognate binding partner on effector cells can result in a promotion of immune responses, such as an increased immune response. Among the provided embodiments are methods for using mutant CTLA-4 polypeptides, such as specific cellular expression forms, that can induce activating signals and / or compete with inhibitory signals to treat cancer and oncological indications.

[0086] In some embodiments, the modulation of immune signaling achieved by the provided mutant CTLA-4 polypeptides and immunomodulatory polypeptides, conjugates, or engineered cells containing such mutant CTLA-4 polypeptides offers advantages over other therapies for the treatment of inflammatory and autoimmune disorders and other diseases and conditions. In some cases, therapies for interfering with and altering the immunomodulatory effects of such ligand / receptor interactions and subsequent signaling are limited by the spatial location requirements and size limitations imposed by the constraints of the immune synapse. In some aspects, existing therapeutic drugs, including antibody drugs, may not be able to simultaneously interact with multiple target proteins involved in regulating these interactions. Additionally, pharmacokinetic differences between drugs that independently target one of these ligand / receptor interactions can create difficulties in appropriately maintaining desired blood concentrations of such drug combinations throughout the course of treatment.

[0087] All publications, including patents, patent applications, scientific articles, and databases, mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patents, patent applications, scientific articles, or databases, were specifically and individually indicated to be incorporated by reference. To the extent that a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, application publication, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0088] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0089] 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 pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference beyond that commonly understood in the art.

[0090] Terms used throughout this specification are defined as follows, unless otherwise limited in specific examples. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references 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 pertains. Unless otherwise indicated, abbreviations and symbols for scientific and biochemical names are those of the IUPAC-IUB nomenclature system. Unless otherwise indicated, all numerical ranges include the values ​​defining the range and all integer values ​​therebetween.

[0091] The term "affinity modified," as used in the context of immunoglobulin superfamily domains, refers to a mammalian immunoglobulin superfamily (IgSF) domain having an altered amino acid sequence (relative to the corresponding wild-type parent or unmodified IgSF domain) such that it has increased or decreased binding affinity or avidity with at least one binding partner (alternatively, "counterstructure") compared to the parent wild-type or unmodified (i.e., modified non-affinity) IgSF regulatory domain. In this context, affinity-modified CTLA-4 IgSF domains are included. In some embodiments, affinity-modified IgSF domains may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, such as amino acid substitutions, in the wild-type or unmodified IgSF domain. Increases or decreases in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry (see also Larsen et al., Am J Transplant, 5(3):443-453 (2005); Linsley et al., Immunity, 1(9):793-801 (1994)). The increase in binding affinity or avidity of a protein to its binding partner is 10% greater than the control value for the wild-type IgSF domain, and in some embodiments is at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the control value for the wild-type IgSF domain. The reduction in binding affinity or activity of the protein to at least one of its binding partners is 90% or less of the control value but 10% or less of the control value for the wild-type IgSF domain, and in some embodiments is 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the control value for the wild-type IgSF domain but 10% or more thereof.

[0092] Affinity-modified proteins have been altered in primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any condition regarding any particular starting composition or method by which the affinity-modified IgSF domain is created. Thus, affinity-modified IgSF domains of the present invention are not limited to wild-type IgSF domains that are subsequently modified into affinity-modified IgSF domains by any particular process of affinity modification. Affinity-modified IgSF domain polypeptides can be created starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its binding partner, and ultimately recombinantly expressed or chemically synthesized to yield the affinity-modified IgSF domain composition of matter. In an alternative example, affinity-modified IgSF domains can be created by site-directed mutagenesis of wild-type IgSF domains. Thus, affinity-modified IgSF domains represent a product, not necessarily a product generated by any given process. A variety of techniques can be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0093] The terms "allogenic" or "allogeneic" are used interchangeably herein to refer to cells or tissues that are removed from one organism and then injected or adoptively transferred into a genetically different organism of the same species. In some embodiments of the invention, the species is murine or human.

[0094] As used herein, the term "autologous" refers to cells or tissues removed from the same organism that are subsequently injected or adoptively transferred. Autologous cells or tissues can be altered, for example, by recombinant DNA methodology, so that they are no longer genetically identical to the native cells or tissues removed from the organism. For example, native 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), possibly involving the manipulation of T cells or TILs (tumor-infiltrating lymphocytes). The engineered cells are then infused into the patient from whom the native T cells were isolated. In some embodiments, the organism is human or murine.

[0095] As used herein, the terms "binding affinity" and "binding activity" refer to the specific binding affinity and specific binding activity, respectively, of a protein for its counterstructure under specific binding conditions. In biochemical kinetics, binding activity refers to the cumulative strength of multiple affinities of individual non-covalent interactions, such as between CTLA-4 and its counterstructures ICOSL, CD80, and / or CD86. Thus, binding activity is distinct from affinity, which describes the strength of a single interaction. The increased or weakened binding affinity of a mutant CTLA-4 containing an affinity-modified CTLA-4 IgSF domain to its counterstructure is determined relative to the binding affinity of unmodified CTLA-4, such as unmodified CTLA-4 containing a native or wild-type IgSF domain, such as an IgV domain. Methods for determining binding affinity or binding activity are known in the art. See, for example, Larsen et al., Am J Transplant, 5(3):443-453 (2005). In some embodiments, the mutant CTLA-4 of the present invention (i.e., CTLA-4 proteins containing affinity-modified IgSF domains) specifically bind to ICOSL, CD80, and / or CD86, as measured by flow cytometry, with a binding affinity that produces a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than a wild-type CTLA-4 control in a binding assay.

[0096] The term "biological half-life" refers to the amount of time it takes for a substance, such as a mutant CTLA-4-containing cellular regulatory polypeptide of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic degradation / digestion), or absorption and concentration of the substance in a particular organ or tissue of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize and increase the biological half-life of the polypeptides of the invention are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), extended recombinant peptides sold under the trademark XTEN® (Amunix Operating, California, USA) (see WO 2013 / 130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

[0097] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial (i.e., man-made) transmembrane protein expressed on a mammalian cell, comprising at least an ectodomain, a transmembrane domain, and an ectodomain. 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 T helper cell, cytotoxic T cell, natural killer T cell, memory T cell, regulatory T cell, or gamma delta T cell. When used clinically, for example, in adoptive cell transfer, CAR-Ts with antigen-binding specificity for the patient's tumor or other tissue are typically engineered for expression on 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 within the scope of the present invention. The ectodomain (or ECD) of the CAR comprises an antigen-binding region, such as an antibody or antigen-binding fragment thereof (e.g., a single-chain variable fragment (scFv)), that specifically binds to a target antigen under physiological conditions, such as the ECD of an immunomodulatory peptide on a cell surface or tumor-specific antigen. Upon specific binding, a biochemical chain of events (i.e., signaling) 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 result in a change in the immune activity of T cell activity, as reflected by changes (increases or decreases) in cytotoxicity, proliferation, or cytokine production. Signaling upon CAR-T activation is, in some embodiments, achieved by the CD3-zeta chain ("CD3-z") ectodomain, which is involved in signaling in natural mammalian T cells.CAR-Ts may further contain multiple signaling domains, such as CD28, 4-1BB, or OX40, to 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 signaling.

[0098] The terms "collectively" or "collective," when used with respect to cytokines induced by the presence of two or more mutant CTLA-4s of the present invention in an in vitro assay, refer to overall cytokine expression, regardless of cytokine production induced by individual mutant CTLA-4 molecules. In some embodiments, the cytokine assayed is IFN-gamma, such as in an in vitro primary T cell assay.

[0099] The term "binding partner" (used interchangeably with "counterstructure") with respect to a polypeptide, such as with respect to the IgSF domain of a mutant CTLA-4, 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 mutant CTLA-4 containing an affinity-modified IgSF domain specifically binds to a corresponding binding partner of native or wild-type CTLA-4 with increased or weakened affinity. A "cell surface binding partner" is a binding partner expressed on the surface of a mammalian cell. Examples of binding partners for the mutant CTLA-4 molecules provided herein include CD80, CD86, and ICOSL, and specifically human CD80, human CD86, and human ICOSL.

[0100] As used herein, "conjugate," "conjugation," or grammatical variations thereof, refers to joining or linking two or more compounds together, resulting in the formation of another compound, by any joining or linking method known in the art. It may also refer to a compound created by joining or linking two or more compounds together. For example, a mutant CTLA-4 polypeptide directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include fusion proteins, conjugates produced by chemical conjugation, and conjugates produced by any other method.

[0101] As used herein, the term "competitive binding" means that a protein can specifically bind to at least two binding partners, but the specific binding of one binding partner inhibits, such as preventing or eliminating, the simultaneous binding of a second binding partner. Thus, in some cases, a protein cannot simultaneously bind to two 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 inhibition (partial or complete) of the specific binding of a protein to one of its binding partners due to the specific binding of a second binding partner. Various methods are known for quantifying competitive binding, such as ELISA (enzyme-linked immunosorbent assay) assays.

[0102] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which an 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 amino acid groups with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0103] The term "corresponding to" with respect to a protein position, such as a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence, such as in a sequence listing, refers to a nucleotide or amino acid position identified upon alignment with a disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues can be determined by alignment of a reference sequence with the sequence of wild-type CTLA-4 set forth in SEQ ID NO:2 or 569 (ECD) or set forth in SEQ ID NO:3 (IgV domain) by the structural alignment methods described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, using, for example, conserved and identical amino acid residues as a guide.

[0104] As used herein, the terms "reduce" or "attenuate" or "suppress" mean to decrease by a statistically significant amount. The decrease can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a control value, such as a non-zero control value.

[0105] As used herein in the context of reducing the immune activity of mammalian lymphocytes, the term "reduced" or "reduced" refers to a decrease in one or more activities of lymphocytes compared to a control, such as an untreated control or a control in which treatment using an unmodified or non-mutated control is used under the same conditions. Decreased activity may refer to one or more of cell cycle inhibition, reduced cell survival, reduced cell proliferation, reduced cytokine production, or reduced T-cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to reduced immune activity means that interferon gamma (IFN-gamma) production is reduced compared to the absence of treatment, such as 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) 2(9):846-56. Other methods for assessing lymphocyte activity, including the assays described herein, are known in the art. In some embodiments, the enhancement may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control value, such as an untreated control value or a non-zero control value.

[0106] The term "derivative" or "derivatized" refers to the modification of a protein by covalently linking the protein, directly or indirectly, to a composition to alter properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, efficacy, or potency, while retaining or enhancing the therapeutic utility of the protein. 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.

[0107] As used herein, a "domain" (typically a sequence of three or more amino acids, generally 5-7 or more amino acids, such as 10-200 amino acid residues) refers to a portion of a molecule, such as a protein, or a nucleic acid encoding molecule, that is structurally and / or functionally distinct and identifiable from the rest of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independent folding structure within a protein composed of one or more structural motifs and / or is recognized by a functional activity, such as binding activity. A protein can have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology with related family members, such as homology to a motif. In another example, a domain can be distinguished by its function, such as the ability to interact with a biomolecule, such as a binding partner. A domain can exhibit independent biological function or activity, such that a domain, either independently or fused to another molecule, can perform an activity, such as binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For purposes of illustration, definitions are provided herein, but it is understood that it is well within the skill of the art to recognize a particular domain by name. If necessary, appropriate software can be used to identify the domains.

[0108] As used herein, the term "ectodomain" refers to a region of a membrane protein, such as a transmembrane protein, that is outside the vesicle membrane. An ectodomain often includes a binding domain that specifically binds to a ligand or cell surface receptor, such as through a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a transmembrane protein is alternatively referred to as an extracellular domain.

[0109] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition of the invention, including a protein or cell composition, 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 additional therapeutic agents, results in a statistically significant reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or cause of the disease. An effective amount can be an amount that relieves, reduces, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the patient's physical function. In the case of cancer treatment, 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, such as a non-human primate or a human patient.

[0110] As used herein, the term "ectodomain" (also called "intracellular domain" or "cytoplasmic domain") refers to a region found in some proteins, such as transmembrane proteins, that extends into the interior space defined by the cell surface membrane. In mammalian cells, the ectodomain is the plasma membrane region of a membrane protein. In cells, the ectodomain may interact with intracellular components and play a role in signal transduction, and thus, in some cases, may be an intracellular signaling domain. The ectodomain of a transmembrane protein may alternatively be a cytoplasmic signaling domain, which in some cases mediates or plays a role in signal transduction, and is also referred to as a cytoplasmic domain. Thus, the terms intracellular signaling domain and cytoplasmic signaling domain are used interchangeably.

[0111] As used herein in the context of increasing the immune activity of mammalian lymphocytes, the terms "enhanced" or "increased" refer to increasing one or more activities of lymphocytes compared to a control, such as an untreated control or a control treated under the same conditions with an unmodified or unmutated control. Increased activity can be one or more of increased cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to increased immune activity refers to increased interferon gamma (IFN-gamma) production, such as by a statistically significant amount. In some embodiments, immune activity can be assessed in an MLR assay. Other methods of assessing lymphocyte activity, including the assays described herein, are known in the art. In some embodiments, enhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a control value, such as a non-zero control value.

[0112] As used herein, the term "engineered cell" refers to a mammalian cell that has been genetically modified by human intervention, such as by recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., a T cell, a B cell, an NK cell), or an antigen-presenting cell (e.g., a dendritic cell). The cell can be a primary cell from a patient or can be a cell line. In some embodiments, the engineered cell of the present invention comprises a mutant CTLA-4 provided herein. In some embodiments, the mutant CTLA-4 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") expressed on the engineered cell. In some embodiments, the TIP contains a transmembrane domain (e.g., a CTLA-4 transmembrane domain) and, optionally, an extracellular domain containing an IgV domain linked to an intracellular signaling domain, or a portion thereof. In some embodiments, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or ectodomain. In some embodiments, the engineered cell is capable of expressing and secreting the immunomodulatory protein described herein. Among the engineered cells provided are cells that further contain an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0113] As used herein, the term "engineered T cells" refers to T cells, such as T helper cells, cytotoxic T cells (alternatively, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or gamma delta T cells, that have been genetically modified by human intervention, such as recombinant DNA methods or viral signaling methods. The engineered T cells may comprise either the engineered T cells themselves, which are expressed on the T cells and to which mutant CTLA-4 expressed on the T cells specifically binds, or a mutant CTLA-4 transmembrane immunomodulatory protein (TIP) of the invention that is engineered to regulate the immune activity of mammalian cells. The engineered T cells may comprise either the engineered T cells themselves, which are expressed and / or secreted by the T cells and to which mutant CTLA-4 specifically binds when secreted by the T cells, or a mutant CTLA-4 secreted immunomodulatory protein (SIP) of the invention that is engineered to regulate the immune activity of mammalian cells.

[0114] The term "engineered T cell receptor" or "engineered TCR" refers to a T cell receptor (TCR) that has been engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen that is selected, cloned, and / or subsequently introduced into a population of T cells, often used for adoptive immunotherapy. In contrast to engineered TCRs, CARs are engineered to bind to target antigens in an MHC-independent manner.

[0115] As used herein, the term "expressed on" is used in reference to a protein expressed on the surface of a cell, such as a mammalian cell. Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety, such as a drug or a detectable label. A protein expressed on the surface of a cell may include a cell surface protein, such as a cell surface receptor expressed on a mammalian cell.

[0116] 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 blood serum compared to the half-life of the protein not conjugated to the moiety. In some embodiments, the half-life is extended by more than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 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 administration to an organism compared to the protein without the half-life extending moiety. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using an ELISA assay or activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), extended recombinant peptides sold under the trademark XTEN® (see WO 2013 / 130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

[0117] As used herein, the term "immune synapse" or "immune synapse" refers to the interface between a mammalian cell expressing MHC I (major histocompatibility complex class I) or MHC II, such as an antigen-presenting cell or tumor cell, and a mammalian lymphocyte, such as an effector T cell or natural killer (NK) cell.

[0118] The Fc (Fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as an Fc polypeptide) largely corresponds to the constant region of an immunoglobulin heavy chain and is involved in various functions, including antibody effector functions. The Fc domain contains part or all of the hinge domain plus the CH2 and CH3 domains of an immunoglobulin molecule. The Fc domain can form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced activity (e.g., reduced by more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) to enhance effector function. In some embodiments, reference to amino acid substitutions in the Fc region is according to the EU numbering system, unless otherwise noted with respect to a specific SEQ ID NO. EU numbering follows the EU index as known and most recently updated in the IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: May 17, 2001, last updated: June 8, 2016) and as reported in Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0119] Immunoglobulin Fc fusions ("Fc fusions"), such as immunomodulatory Fc fusion proteins, are molecules comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. An Fc fusion can comprise, for example, the Fc region of an antibody (to enhance effector function and pharmacokinetics) and a mutant CTLA-4. The immunoglobulin Fc region can be indirectly or directly linked to one or more mutant CTLA-4s or small molecules (fusion partners). Various linkers are known and can optionally be used to link the Fc to the fusion partner to create the Fc fusion. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or non-identical species can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a murine or human Fc.

[0120] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. Host cells can be prokaryotic, e.g., Escherichia coli (E. coli), or eukaryotic, e.g., unicellular eukaryotic cells (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human, monkey, hamster, rat, mouse, or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives, such as vegetable CHO and related cell lines grown in serum-free medium or the CHO strain DX-B11, which is deficient in dihydrofolate reductase (DHFR). Another example is human embryonic kidney 293 (HEK-293) cells or their derivatives. In some embodiments, the host cell is a mammalian cell (e.g., human, monkey, hamster, rat, mouse, or insect cell).

[0121] As used herein, the term "immunoglobulin" (abbreviated "Ig") 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 encompasses immunoglobulins that are less than full-length, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized), or naturally produced, such as antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL, single-chain variable fragments (scFv) containing VH and VL linked together in a single chain, and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments. Bispecific antibodies, homobispecifics, and heterobispecifics are included within the meaning of the term.

[0122] As used herein, the term "immunoglobulin superfamily" or "IgSF" 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 shared structural features shared by 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. They are generally associated with roles in the immune system. Proteins in the immune synapse are often members of IgSF. IgSF can also be classified into "subfamilies" based on shared characteristics, such as function. Such subfamilies typically consist of 4 to 30 IgSF members.

[0123] As used herein, the terms "IgSF domain" or "immunoglobulin domain" or "Ig domain" refer to a 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 on the interior of 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 has a section called the complementarity region, which determines the region important for antibody specificity for their ligands. Ig-like domains can be classified as IgV, IgC (which can be either IgC1 or IgC2), or IgI. Most Ig domains are either variable (IgV) or constant (IgC). IgV domains, which have nine beta chains, are generally longer than IgC domains, which have seven beta chains. 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. CTLA-4 contains one Ig domain: an IgV domain.

[0124] As used herein, the term "IgSF species" refers to an ensemble of IgSF member proteins with identical or nearly identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines the unique identity of 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, variation 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. Additionally, minor sequence differences within a single IgSF species due to genetic polymorphism constitute another form of variation within a single IgSF species, such as, for example, wild-type truncated versions of an IgSF species due to proteolytic cleavage. A "cell surface IgSF species" is an IgSF species expressed on the surface of a cell, typically a mammalian cell.

[0125] As used herein in the context of mammalian lymphocytes, such as T cells, the term "immune activity" refers to one or more of activation, cell survival, apoptosis, cell proliferation, cell cycle inhibition, cytokine production (e.g., interferon gamma), cytokine release, or T cell cytotoxic activity. In some cases, immune activity may refer to cellular 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 gamma cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 Sep:2(9):846-56), SEB (Staphylococcal enterotoxin B), T cell stimulation assays (Wang et al., Cancer Immunol Res, (2014) 2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med, (2010) 8:104). Because T cell activation is associated with the secretion of IFN-gamma cytokines, detection of IFN-gamma 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), 117(1):57-62). Eliciting an immune response results in increased immune activity relative to resting lymphocytes. Immunomodulatory proteins, such as mutant CTLA-4 polypeptides containing affinity-engineered IgSF domains as provided herein, can, in some embodiments, decrease, or in alternative embodiments, increase, IFN-gamma (interferon gamma) expression in primary T cell assays relative to wild-type IgSF members or IgSF domain controls. Those skilled in the art will recognize that the format of the primary T cell assay used to determine increased IFN-gamma expression can differ from the format used to assay for decreased IFN-gamma expression.

[0126] In assays for the ability of the immunomodulatory proteins or affinity-modified IgSF domains of the present invention to alter IFN-gamma expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used. Conveniently, in some cases, a soluble form of the affinity-modified IgSF domain of the present invention can be used to determine its ability to increase or decrease IFN-gamma expression in an MLR. Alternatively, a co-immobilization assay can be used. In a co-immobilization assay, a T cell receptor signal provided in some embodiments by an anti-CD3 antibody is used in conjunction with a co-immobilized affinity-modified IgSF domain, such as a mutant CTLA-4, to determine its ability to increase or decrease IFN-gamma expression relative to a wild-type IgSF domain control. Methods for assaying the immune activity of engineered cells, including assessing the activity of mutant CTLA-4 transmembrane immunomodulatory proteins, are known in the art and include, but are not limited to, assays for suppression or enhancement of cell expansion or proliferation after antigen stimulation, suppression or proliferation of primary and secondary allostimulatory T cells, and autoimmune activity, such as allograft survival assays and anti-donor antibody response assays in appropriate animal models. Assays also include assays for assessing cytotoxicity, including standard 51 chromium-release assays (see, e.g., Milone et al., Mol Ther (2009), 17(8):1453-1464) or flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014), J Immunol Methods, 405:192-198).

[0127] An "immunomodulating polypeptide" or "immunomodulating protein" is a polypeptide or protein molecule that modulates immune activity. By "modulate" or "modulating," it is meant that the immune response is either increased or decreased in immune activity. An immunomodulating protein can be, for example, a single-chain polypeptide or a multimer (dimer or higher multimer) of at least two polypeptide chains covalently linked to each other by interchain disulfide bonds. Thus, monomeric, dimeric, and higher 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 include mutant CTLA-4.

[0128] As used herein, the term "increase" means increase by a statistically significant amount. The increase may be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100% greater than a control value, such as a non-zero control value.

[0129] A "CTLA-4 isoform" is one of multiple naturally occurring CTLA-4 polypeptides that differ in amino acid sequence. Isoforms can be the product of a single gene-expressed RNA transcript that is a variant, or the expression product of a very similar but different gene that produces a functionally similar protein, which can result from gene duplication, etc. As used herein, the term "CTLA-4 isoform" also refers to the product of different alleles of the CTLA-4 gene.

[0130] As used herein, the term "lymphocyte" 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 (for cell-mediated cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). T cells include T helper cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or gamma delta T cells. Innate lymphoid cells (ILCs) are also included within the definition of lymphocytes.

[0131] 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.

[0132] As used herein, the term "membrane protein" refers to a protein that is directly or indirectly attached to a lipid bilayer under physiological conditions. The lipid bilayer that forms the membrane can be a biological membrane, such as a eukaryotic (e.g., mammalian) cell membrane, or an artificial (i.e., man-made) membrane, such as that found on a liposome. The attachment of a membrane protein to a lipid bilayer can be by covalent bonding or by non-covalent interactions, such as hydrophobic or electrostatic interactions. A membrane protein can be an integral membrane protein or a peripheral membrane protein. A membrane protein that is a peripheral membrane protein is non-covalently attached to a lipid bilayer or non-covalently attached to an integral membrane protein. A peripheral membrane protein forms a temporary attachment to a lipid bilayer such that the peripheral membrane protein can associate with and / or detach from the lipid bilayer under a range of conditions that are physiological in mammals. In contrast to peripheral membrane proteins, integral membrane proteins form substantially permanent attachments to the lipid bilayer of the membrane, such that under the range of conditions physiological in mammals, integral membrane proteins do not detach from their attachment to the lipid bilayer. Membrane proteins can form attachments to the membrane in a manner that involves one lipid bilayer (monotopic) or both membranes (multitopic). An integral membrane protein that interacts with only one lipid bilayer is an "integral monotopic protein." An integral membrane protein that interacts with both lipid bilayers is an "integral multitopic protein," alternatively referred to herein as a "transmembrane protein."

[0133] As used herein in the context of an immune response, such as a mammalian immune response, the term "modulating" or "modulating" refers to any change, such as an increase or decrease, in an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide, including a mutant CTLA-4 of the present invention, or as a result of administration of engineered cells expressing an immunomodulatory protein, such as a mutant CTLA-4 transmembrane immunomodulatory protein of the present invention. Thus, it refers to a change, such as an increase or decrease, in an immune response compared to an immune response that would occur or be present in the absence of administration of an immunomodulatory protein, including a mutant CTLA-4, or a cell expressing such an immunomodulatory polypeptide. 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, NKT cells, professional antigen-presenting cells (APCs), and non-professional antigen-presenting cells, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils).

[0134] Modulation includes any change imparted to an existing immune response, a developing immune response, a potential immune response, or the ability to induce, modulate, influence, or respond to an immune response. Modulation can be direct or indirect. 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 sequestration, induction, or creation of immune cells that may 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-gamma (interferon gamma) expression relative to wild-type CTLA-4 controls in primary T cell assays (see Zhao et al. (2016), Exp Cell Res, 340(1):132-138). Modulation can be assessed, for example, by changes in the immune activity of engineered cells, such as changes in the cytotoxic activity of engineered cells or changes in cytokine secretion of engineered cells, relative to cells engineered with wild-type CTLA-4 transmembrane proteins.

[0135] As used herein, the term "molecular species" refers to an ensemble of proteins with identical or nearly identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of identical or nearly identical molecular species. Thus, for example, human CTLA-4 is an IgSF member, and each human CTLA-4 molecule is a CTLA-4 molecular species. Variation between molecules of the same molecular species can arise due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Additionally, minor sequence differences within a single molecular species due to genetic polymorphism constitute another form of variation within a single molecular species, such as the wild-type and truncated forms of a single molecular species due to protein cleavage. A "cell surface molecular species" is a molecular species expressed on the surface of a mammalian cell. Two or more different species of proteins, each present exclusively on one or exclusively on the other of two mammalian cells forming an IS, are said to be "cis" or in "cis configuration" with each other. Two different species of proteins, one of which is present exclusively on one of the two mammalian cells that form the IS and the second of which is present exclusively on both mammalian cells that form the IS, are said to be in "trans" or "trans configuration." Two different species of proteins are present on both of the two mammalian cells that form the IS and are in both cis and trans configuration on these cells.

[0136] The term "multimerization domain" refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each of which contains complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), which may be the same or different. The interaction between the complementary multimerization domains, for example, the interaction between the first multimerization domain and the second multimerization domain, forms a stable protein-protein interaction to generate a multimer of the polypeptide molecule with the additional polypeptide molecule. In some cases, the multimerization domains are the same and interact with themselves to form a stable protein-protein interaction between the two polypeptide chains. Generally, the polypeptide is directly or indirectly joined to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain may be an immunoglobulin constant region or domain, such as, for example, the Fc domain or a portion thereof from IgG1, including, for example, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, and modified forms thereof.

[0137] The terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise specified, these terms encompass nucleic acids containing known analogs of natural nucleotides, which have similar binding properties and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as the explicitly indicated sequence ("reference sequence"). Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed group and / or a deoxyinosine residue. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0138] As used herein, the term "non-competitive binding" refers to the ability of a protein to specifically bind to at least two binding partners simultaneously. Thus, a protein can simultaneously bind to at least two different binding partners, although in some cases, the binding interactions need not be of equal duration, such that the protein specifically binds to only one of the binding partners. In some embodiments, binding occurs under specific binding conditions. In some embodiments, simultaneous binding is such that binding of one binding partner does not substantially inhibit simultaneous binding to a second binding partner. In some embodiments, non-competitive binding means that binding of a second binding partner to its binding site on the protein does not displace binding of a first 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. (1999), Immunology, 96(4):663-670. In some cases, in a non-competitive interaction, a first binding partner specifically binds to an interaction site that does not overlap with that of a second binding partner, such that the binding of the second binding partner does not directly interfere with the binding of the first binding partner. Thus, any effect of the binding of the second binding partner on the binding of the binding partner is through a mechanism other than directly interfering with the binding of the first binding partner. For example, in the context of enzyme-substrate interactions, a non-competitive inhibitor binds to a site other than the active site of the enzyme. Non-competitive binding encompasses non-competitive binding interactions in which a second binding partner specifically binds to an interaction site that does not overlap with the binding of the first binding partner, but only binds to the second interaction site when the first interaction site is presented by the first binding partner.

[0139] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. Pharmaceutical compositions typically contain an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a mutant CTLA-4, or an engineered cell expressing a mutant CTLA-4 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0140] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked through peptide bonds. These terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. These terms include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. These terms also include molecules containing one or more amino acid analogs or non-standard or unnatural amino acids, which can be synthesized using known protein engineering techniques or expressed recombinantly. In addition, proteins can be derivatized.

[0141] As used herein, the term "primary T cell assay" refers to an in vitro assay for measuring interferon gamma ("IFN-gamma") expression. Various such primary T cell assays are known in the art. In a preferred embodiment, the assay used is an anti-CD3 co-immobilization assay. In this assay, primary T cells are stimulated with immobilized anti-CD3, with or without additional recombinant proteins. Culture supernatants are typically collected at 24-72 hours. In another embodiment, the assay used is an MLR. In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatants are typically collected at 24-72 hours. Human IFN-gamma levels are measured in culture supernatants by standard ELISA techniques. Commercial kits are available from suppliers, and the assay is performed according to the manufacturer's recommendations.

[0142] The term "purified," as applied to nucleic acids, such as those encoding the 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 forms a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified." Purified nucleic acids or proteins of the invention are at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a weight percent or molar basis).

[0143] The term "recombinant" indicates that a material (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be made to or removed from the material in its natural environment or state. For example, a "recombinant nucleic acid" is a nucleic acid created by recombining nucleic acids, e.g., during cloning, affinity engineering, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by means of such molecular biological techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" 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 has otherwise been altered by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, such as the transmembrane immunomodulatory protein provided herein. Transcriptional control signals in eukaryotic cells include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on which cell type is to be used to express the protein of interest. As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the 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.

[0144] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing the appropriate desired coding sequence and nucleic acid sequences necessary for expression of an 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. A secretory signal peptide sequence may also be optionally encoded by the recombinant expression vector operably linked to the coding sequence for a recombinant protein, such as a recombinant fusion protein, so that the expressed fusion protein can be secreted by the recombinant host cell, if desired, for easier isolation of the fusion protein from the cell. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of the host cell into which they are introduced. Among these vectors are viral vectors, such as lentiviral vectors.

[0145] The term "selectivity" refers to the preference of a protein or polypeptide of interest for specific binding to one substrate, such as one binding partner, compared to specific binding to another substrate, such as a different binding partner of the protein of interest. Selectivity can be reflected as the ratio of the binding activity (e.g., binding affinity) of the protein of interest and a first substrate, such as a first binding partner (e.g., Kd1), to the binding activity (e.g., binding affinity) of the same protein of interest with a second binding partner (e.g., Kd2).

[0146] As used herein, the term "sequence identity" refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and 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 analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0147] As used herein with respect to a protein, the term "soluble" means that the protein is not a membrane protein. Generally, a soluble protein contains only the extracellular domain or a portion thereof of an IgSF family member receptor containing an IgSF domain or a specific binding fragment thereof, but does not contain a transmembrane domain and / or cannot be expressed on the surface of a cell. In some cases, the solubility of a protein can be improved by linking or attaching, directly or indirectly via a linker, to an Fc domain or other moiety, which can also improve the stability and / or half-life of the protein. In some embodiments, the soluble protein is an Fc fusion protein.

[0148] As used herein with respect to a polypeptide or nucleic acid, the term "species" refers to an ensemble of molecules having identical or nearly identical sequences. Variation 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 sequences of a polypeptide that differ from (or encode) the full-length species at the amino or carboxy terminus by no more than one, two, or three amino acid residues are considered to consist of a single species. Such microheterogeneity is a common feature of manufactured proteins.

[0149] As used herein with respect to a full-length wild-type mammalian CTLA-4 polypeptide or its IgV domain, the term "specific binding fragment" refers to a polypeptide having a subsequence of the full-length polypeptide or IgV domain and specifically binding to mammalian ICOSL, mammalian CD80, and / or mammalian CD86, such as human or murine ICOSL, CD80, or CD86, in vitro and / or in vivo. In some embodiments, a specific binding fragment comprises a CTLA-4 IgV subsequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the full-length wild-type sequence or its IgV sequence. Specific binding fragments can be altered in sequence to form mutant CTLA-4s of the present invention.

[0150] As used herein, the term "specifically binds" refers to the ability of a protein to bind to a target protein under specific binding conditions such that the affinity or avidity of the protein is at least 5-fold, and optionally at least 10, 20, 30, 40, 50, 100, 250, or 500-fold, or even at least 1000-fold, greater than the average affinity or avidity of the same protein to a collection of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind non-exclusively to a single target molecule, but may specifically bind to a non-target molecule due to similarities in structural conformation between the target and non-target (e.g., paralogs or orthologs). Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or non-target molecules with substantially similar epitopes as the target molecule (e.g., paralogs) is possible and does not compromise the specificity of binding determined against a statistically valid collection of unique non-targets (e.g., random polypeptides). Therefore, due to cross-reactivity, the polypeptides of the present invention may specifically bind to more than one distinct species of target molecule. Specific binding between two proteins can be determined using solid-phase ELISA immunoassays, ForteBio Octet®, or Biacore® measurements. Generally, the interaction between two binding proteins is greater than 1x10 -5 It has a dissociation constant (Kd) of less than 1 x 10 -12 In certain embodiments of the present disclosure, the interaction between two binding proteins is often as low as about 1x10 -6 Under M, 1x10 -7 Medium, 1x10 -8 Medium, 1x10 -9 Medium, 1x10 -10 Medium, or 1x10 -11 It has a dissociation constant of M or less.

[0151] With respect to a mammal expressing a polypeptide, the terms "surface expression," "surface expression," or "expressed on the surface" mean that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0152] As used herein, "synthetic," for example with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant and / or chemical synthesis methods.

[0153] As used herein, the term "targeting moiety" refers to a composition that is covalently or noncovalently attached to or physically encapsulates a polypeptide comprising a mutant CTLA-4 of the present invention. In some embodiments, the targeting moiety has specific binding affinity for a target molecule, such as a target molecule expressed on a cell. Typically, the target molecule is localized on a specific tissue or cell type. Targeting moieties include antibodies, antigen-binding fragments (Fab), variable fragments (Fv) containing a VH and a VL, single-chain variable fragments (scFv) containing a VH and a VL linked together in a single chain, 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, as well as small molecule (<500 dalton) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or noncovalently attached to the lipid membrane of liposomes encapsulating the polypeptides of the present invention.

[0154] As used herein, the term "transmembrane protein" refers to a membrane protein that substantially or completely spans a lipid bilayer, such as a lipid bilayer found in a biological membrane, such as a mammalian cell, or an artificial construct, such as a liposome. A transmembrane protein comprises a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. Transmembrane domains can generally be predicted from their amino acid sequences via any number of commercially available bioinformatics software applications based on their high hydrophobicity relative to regions of the protein that interact with the aqueous environment (e.g., cytosol, extracellular fluid). Transmembrane domains are often hydrophobic alpha helices that span the membrane. Transmembrane proteins may traverse both layers of the lipid bilayer one or more times. Transmembrane proteins include the provided transmembrane immunomodulatory proteins described herein. In addition to a transmembrane domain, the transmembrane immunomodulatory proteins of the present invention further comprise an ectodomain, and in some embodiments, an ectodomain.

[0155] As used herein, the terms "treating," "treatment," or "therapy" of a disease or disorder mean slowing, halting, or reversing the progression of the disease or disorder, as evidenced by the reduction, interruption, or elimination of any clinical or diagnostic symptoms, by administration of a therapeutic composition of the invention (e.g., containing an immunomodulatory protein or engineered cells), either alone or in combination with another compound as described herein. "Treating," "treatment," or "therapy" also means reducing the severity of symptoms in acute or chronic diseases or disorders, or reducing the relapse rate, for example, in the case of relapsing or ameliorating autoimmune disease processes, or reducing inflammation in the case of inflammatory aspects of autoimmune diseases. "Treating," "treatment," or "therapy" can also mean reducing inflammation and / or other symptoms associated with transplant rejection.

[0156] As used herein in the context of cancer, the terms "treat" or "inhibit," "inhibiting," or "inhibition" of cancer refer to at least one of a statistically significant decrease in tumor growth rate, a cessation of tumor growth, or a decrease in tumor size, mass, metabolic activity, or volume, as measured by standard criteria, including, but not limited to, Response Evaluation Criteria In Solid Tumors (RECIST), or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).

[0157] As used in the context of this invention, "preventing," "prophylaxis," or "prevention" of a disease or disorder 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 onset or onset of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[0158] As used herein, the term "tumor-specific antigen" or "TSA" refers to a counter structure that is primarily present on tumor cells of a mammalian subject but is generally not found on normal cells of the mammalian subject. A tumor-specific antigen need not be exclusive to tumor cells, but the proportion of cells in a particular mammal that have the tumor-specific antigen is sufficiently high, or the level of the tumor-specific antigen on the surface of the tumor is sufficiently high, that it can be targeted by an anti-tumor therapeutic agent, such as an immunomodulatory polypeptide of the present invention, and provide protection or treatment for the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells that display the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that display the TSA are cancerous.

[0159] The term "mutant" (also "modified" or "mutant") when used with reference to mutant CTLA-4 refers to CTLA-4, such as mammalian (e.g., human or murine) CTLA-4 created by human intervention. A mutant CTLA-4 is a polypeptide having an altered amino acid sequence relative to unmodified or wild-type CTLA-4. A mutant CTLA-4 is a polypeptide that differs from the wild-type CTLA-4 isoform sequence by one or more modifications, such as one or more amino acid substitutions, deletions, additions, or a combination thereof. For purposes herein, a mutant CTLA-4 contains at least one affinity-modified domain in which one or more of the amino acid differences occur in the IgSF domain (e.g., the IgV domain). Mutant CTLA-4 can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions. Mutant CTLA-4 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 with the corresponding wild-type or unmodified CTLA-4, such as the sequence of SEQ ID NO:1, its mature sequence (lacking the signal sequence), or a portion thereof, containing the extracellular domain or IgSF domain thereof. In some embodiments, the mutant CTLA-4 polypeptides 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 the corresponding wild-type or unmodified CTLA-4, including the sequence set forth in SEQ ID NO:2 or SEQ ID NO:3. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the range of permissible substitutions or additions. The mutant CTLA-4 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 mutant CTLA-4 of the present invention specifically binds to ICOSL, CD80, and / or CD86 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 ICOSL; CD80, CD86, CD80, and CD86; ICOSL and CD80; ICOSL and CD86; and / or ICOSL, CD80, and CD86, compared to wild-type or unmodified CTLA-4 protein. Increased or decreased binding affinity or avidity can be determined using well-known binding assays, such as flow cytometry. Such assays are described in Larsen et al., Am J Transplant, 5(3):443-453 (2005) and Linsley et al., Immunity, 1(9):793-801 (1994).

[0160] The increase in mutant CTLA-4 binding affinity or avidity to ICOSL, CD80, and / or CD86 is at least 5% greater than that of wild-type or unmodified CTLA-4, and in some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, or 100% greater than that of wild-type or unmodified CTLA-4 control values. The decrease in CTLA-4 binding affinity or avidity to ICOSL, CD80, and / or CD86 is 95% or less of the wild-type or unmodified control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the wild-type or unmodified control value, or undetectable binding affinity or avidity. Mutant CTLA-4 is altered in its primary amino acid sequence by substitution, addition, or deletion of amino acid residues.

[0161] The term "mutant" in the context of mutant CTLA-4 should not be construed as imposing any conditions on any particular starting composition or method by which the mutant CTLA-4 is made. Mutant CTLA-4 can be created, for example, starting from wild-type mammalian CTLA-4 sequence information, then modeled in silico for binding to ICOSL, CD80, and / or CD86, and finally recombinantly expressed or chemically synthesized to yield the mutant CTLA-4 of the present invention. In an alternative example, mutant CTLA-4 can be created by site-directed mutagenesis of wild-type CTLA-4. Thus, mutant CTLA-4 represents a composition, not necessarily a product, produced by any given process. A variety of techniques can be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0162] As used herein in connection with biological material, such as a nucleic acid molecule, a protein (e.g., CTLA-4), an IgSF member, or a host cell, the terms "wild-type" or "native" or "naturally occurring" refer to that which is found in nature and is not modified by human interference.

[0163] II. Mutant CTLA-4 Polypeptides Provided herein are mutant CTLA-4 polypeptides that exhibit altered (increased or decreased) binding activity or affinity to one or more of CTLA-4 binding partners. In some embodiments, the CTLA-4 binding partner is one or more of ICOSL, CD80, and / or CD86. In some embodiments, the CTLA-4 binding partner is ICOSL. In some embodiments, the one or more binding partners of CTLA-4 are ICOSL and CD80 or CD86. In some embodiments, the one or more binding partners of CTLA-4 are ICOSL, CD80, and CD86.

[0164] CTLA-4 is a member of the immunoglobulin superfamily of proteins, a family of proteins that all possess a domain known as an immunoglobulin domain or fold (hereafter "immunoglobulin superfamily domain" or IgSF domain). In some embodiments, other IgSF family members include members from the signal-regulating protein (SIRP) family, the Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family, the butyrophilin family, the B7 family, the CD28 family, the V-set and immunoglobulin domain-containing (VSIG) family, the V-set transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signaling lymphocyte activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the natural cytotoxicity-inducing receptor (NCR) family, the T-cell immunoglobulin and mucin (TIM) family, or the killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, other IgSF family members include CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOSLG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3), VTCN1 (B7-H4), CD28, PDCD1 (TIGI T), ICOS, BTLA (CD272), CD4, CD8A (CD8-alpha), CD8B (CD8-beta), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NCR3 (NKp30).

[0165] Table 1 summarizes exemplary members of the IgSF family. The first column of Table 1 provides the name and, optionally, several possible aliases for that particular IgSF member. The second column provides the protein identifier from the UniProtKB database, a publicly available database accessible via the internet at uniprot.org, or, in some cases, the GenBank number. The Universal Protein Source (UniProt) is a comprehensive source of protein sequence and annotation data. The UniProt database includes the UniProt Knowledge Base (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the Swiss Bioinformatics Institute (SIB), and the Protein Information Source (PIR), and is primarily supported by a grant from the National Institutes of Health (NIH). GenBank is the NIH gene sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). The third column provides the region in which the indicated IgSF domain is located. Regions are identified as ranges encompassing the residues that define the domain. Column 3 also indicates the IgSF domain class for the identified IgSF region. Column 4 provides the region in which the indicated additional domains are located (signal peptide, S, extracellular domain, E, transmembrane domain, T, cytoplasmic domain, C). It is understood that domain descriptions may vary depending on the method used to identify or classify domains and may be identified differently from different sources. The descriptions of residues corresponding to domains in Table 1 are for illustrative purposes only and may be several amino acids longer or shorter (e.g., one, two, three, or four). Column 5 indicates the binding partners of some of the listed IgSF members.

[0166] Table 1. IgSF members according to the present disclosure TIFF0007749319000001.tif67167TIFF0007749319000002.tif211167TIFF0007749319000003.tif211167 TIFF0007749319000004.tif205167TIFF0007749319000005.tif214167TIFF0007749319000006.tif88167

[0167] In some embodiments, mutant CTLA-4 polypeptides contain one or more amino acid modifications, such as one or more substitutions (alternatively, "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain (IgD) of a wild-type or unmodified CTLA-4 polypeptide, or a portion of wild-type or unmodified CTLA-4 containing IgD, or a specific-binding fragment thereof. Thus, the provided mutant CTLA-4 polypeptides are or include mutant IgDs (hereinafter referred to as "vIgDs") in which one or more amino acid modifications (e.g., substitutions) are within the IgD.

[0168] In some embodiments, the IgD comprises an IgV domain, a specific-binding fragment of an IgV domain, or a combination thereof. In some embodiments, the IgD can be only IgV or the entire extracellular domain (ECD) of CTLA-4. In some embodiments, the IgD comprises a specific-binding fragment of an ECD. Table 1 provides exemplary residues corresponding to the IgV region and ECD of CTLA-4. In some embodiments, a mutant CTLA-4 polypeptide contains an IgV domain or ECD, or a specific-binding fragment thereof, where at least one of the amino acid modifications (e.g., substitutions) is in the IgV domain or ECD, or a specific-binding fragment thereof. In some embodiments, a mutant CTLA-4 polypeptide contains an IgV domain, or a specific-binding fragment thereof, where at least one of the amino acid modifications (e.g., substitutions) is in the IgV domain, or a specific-binding fragment thereof. In some embodiments, a mutant CTLA-4 polypeptide contains an ECD, or a specific-binding fragment thereof, where at least one of the amino acid modifications (e.g., substitutions) is in the ECD, or a specific-binding fragment thereof. In some embodiments, the altered IgV domain or ECD is an affinity-engineered IgSF domain due to its altered avidity or affinity.

[0169] In some embodiments, the variant is a modification in another IgSF domain relative to the sequence of the unmodified CTLA-4 sequence. In some embodiments, the unmodified CTLA-4 sequence is wild-type CTLA-4. In some embodiments, the unmodified or wild-type CTLA-4 has the sequence of native CTLA-4, or an ortholog thereof. In some embodiments, the unmodified CTLA-4 is or comprises the extracellular domain (ECD) of CTLA-4, or a portion thereof, containing one or more IgSF domains (see Table 1). In some embodiments, the extracellular domain of the unmodified or wild-type CTLA-4 polypeptide comprises an IgV domain or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide comprises or consists essentially of an IgV domain or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide comprises or consists essentially of an ECD or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 is soluble and lacks a transmembrane domain. In some embodiments, the mutant CTLA-4 further comprises a transmembrane domain, which may contain an intracellular signaling domain, and optionally, a cytoplasmic domain.

[0170] In some embodiments, the wild-type or unmodified CTLA-4 sequence is a mammalian CTLA-4 sequence. In some embodiments, the wild-type or unmodified CTLA-4 sequence can be a mammalian CTLA-4, including, but not limited to, human, mouse, cynomolgus monkey, or rat CTLA-4. In some embodiments, the wild-type or unmodified CTLA-4 sequence is human.

[0171] In some embodiments, the wild-type or unmodified CTLA-4 sequence has (i) the sequence of amino acids set forth in SEQ ID NO:1 lacking the signal sequence, or a mature form thereof; (ii) 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:1, or a mature form thereof; or (iii) a portion of (i) or (ii) containing an IgV domain or ECD, or a specific-binding fragment thereof.

[0172] In some embodiments, the wild-type or unmodified CTLA-4 sequence is or comprises the extracellular domain (ECD) of CTLA-4 or a portion thereof. In some embodiments, the unmodified or wild-type CTLA-4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 (corresponding to amino acid residues 36-161 of SEQ ID NO:1), or an ortholog thereof. Optionally, the unmodified or wild-type CTLA-4 polypeptide can comprise (i) the sequence of amino acids set forth in SEQ ID NO:2, (ii) a sequence of amino acids having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2, or (iii) a specific-binding fragment of the sequence of (i) or (ii) comprising the IgV domain. In some embodiments, the wild-type or unmodified ECD is capable of binding to one or more CTLA-4 binding partners, such as ICOSL and / or one or more of CD80 and / or CD86.

[0173] In some embodiments, a wild-type or unmodified CTLA-4 polypeptide comprises an IgV domain, or a specific-binding fragment thereof. In some embodiments, the IgV domain of a wild-type or unmodified CTLA-4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 (corresponding to amino acid residues 39-140 of SEQ ID NO:1), or an ortholog thereof. For example, the IgV domain of an unmodified or wild-type CTLA-4 polypeptide can contain (i) the sequence of amino acids set forth in SEQ ID NO:3, (ii) a sequence of amino acids having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3, or (iii) a specific-binding fragment of the sequence of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain is capable of binding to one or more CTLA-4 binding partners, such as ICOSL and / or one or more of CD80 and / or CD86.

[0174] In some embodiments, wild-type or unmodified CTLA-4 polypeptides contain a specific binding fragment of CTLA-4, such as a specific binding fragment of the IgV domain or ECD. In some embodiments, the specific binding fragment may bind to ICOSL. In some embodiments, the specific fragment may bind to ICOSL and CD80 or CD86. In some embodiments, the specific binding fragment may bind to ICOSL, CD80, and CD86. The specific binding fragment may have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, or 100 amino acids. In some embodiments, the specific binding fragment of the IgV domain contains an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the IgV domain set forth as amino acids 39-140 of SEQ ID NO:1. In some embodiments, a specific binding fragment of an ECD comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the ECD set forth as amino acids 36-161 of SEQ ID NO:1.

[0175] In some embodiments, the mutant CTLA-4 polypeptide comprises an ECD domain or a portion thereof, comprising one or more affinity-modified IgSF domains. In some embodiments, the mutant CTLA-4 polypeptide may comprise an IgSF domain (IgV) or a specific-binding fragment of an IgV domain, or a specific-binding fragment of an ECD, wherein one or more of the IgV or ECD contain one or more amino acid modifications (e.g., substitutions). In some embodiments, the mutant CTLA-4 polypeptide comprises a full-length IgV domain. In some embodiments, the mutant CTLA-4 polypeptide comprises a full-length ECD. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific-binding fragment of an IgV domain. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific-binding fragment of an ECD.

[0176] In any of such embodiments, the one or more amino acid modifications (e.g., substitutions) of the mutant CTLA-4 polypeptide may be located within the CTLA-4 polypeptide ECD, such as within the IgSF domain. For example, in some embodiments, the one or more amino acid modifications (e.g., substitutions) are located within the extracellular domain of the mutant CTLA-4 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located within the IgV domain or a specific-binding fragment of the IgV domain.

[0177] Generally, each of the various attributes of the polypeptides is disclosed separately below (e.g., soluble and membrane-bound polypeptides, affinity of CTLA-4 for ICOSL, CD80, and / or CD86, number of variations per polypeptide chain, number of linked polypeptide chains, number and nature of amino acid changes per mutant CTLA-4, etc.). However, as will be apparent to those skilled in the art, any particular polypeptide may contain a combination of these independent attributes. It is understood that reference to amino acids, including specific sequences set forth as SEQ ID NOs, used to describe the domain organization of IgSF domains, is for illustrative purposes and is not intended to limit the scope of the provided embodiments. It is understood that the description of polypeptides and their domains is theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus may vary and is not necessarily the same for each protein. Thus, a particular IgSF domain, such as a particular IgV domain or ECD, may be several amino acids longer or shorter (e.g., one, two, three, or four).

[0178] Furthermore, the various embodiments of the present invention as discussed below are frequently provided within the meanings of the defined terms as disclosed above. Thus, embodiments described with specific definitions should be interpreted as if the defined terms were incorporated by reference when used in discussing the various aspects and attributes described herein. Thus, the headings, the order of presentation of the various aspects and embodiments, and the separate disclosure of each independent attribute are not meant to be limitations on the scope of the present disclosure.

[0179] A. Exemplary Modifications Provided herein are mutant CTLA-4 polypeptides, their IgSF domains, or specific-binding fragments thereof, containing modifications in the ECD relative to the IgSF or ECD contained in wild-type or unmodified CTLA-4 polypeptides. In some embodiments, at least one modification is in the IgSF domain (e.g., IgV) or specific-binding fragment thereof, such that the provided mutant CTLA-4 polypeptides contain at least one affinity-modified IgSF domain or specific-binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptides exhibit altered (increased or decreased) binding activity or affinity for ICOSL, CD80, and / or CD86 compared to wild-type or unmodified CTLA-4 polypeptides. ICOSL, CD80, and / or CD86 can be mammalian proteins, such as human or murine proteins.

[0180] In some embodiments, mutant CTLA-4 polypeptides exhibit altered (increased or decreased) binding activity and affinity for ICOSL, and optionally, one or more of the ligands CD80 and CD86, compared to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have binding affinity for ICOSL, and optionally, CD80 and / or CD86, that differs from the binding affinity of a wild-type or unmodified CTLA-4 polypeptide control (e.g., unmodified) sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet®, or Biacore assay. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL, and optionally, CD80 and / or CD86. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL, and optionally, decreased binding affinity for one or more of CD80 and / or CD86, relative to wild-type or unmodified CTLA-4 polypeptides.

[0181] In some embodiments, mutant CTLA-4 polypeptides exhibit altered (increased or decreased) binding activity or affinity for CD80, and optionally, one or more of the ligands ICOSL and CD86, compared to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have binding affinity for CD80, and optionally, ICOSL and / or CD86, that differs from the binding affinity of a wild-type or unmodified CTLA-4 polypeptide control (e.g., unmodified) sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet®, or Biacore assay. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD80, and optionally, ICOSL and / or CD86. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD80, and optionally, decreased binding affinity for one or more of ICOSL and / or CD86, relative to wild-type or unmodified CTLA-4 polypeptides.

[0182] In some embodiments, mutant CTLA-4 polypeptides exhibit altered (increased or decreased) binding activity or affinity for CD86, and optionally, one or more of the ligands ICOSL and CD80, relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have binding affinity for CD86, and optionally, ICOSL and / or CD80, that differs from the binding affinity of a wild-type or unmodified CTLA-4 polypeptide control (e.g., unmodified) sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet®, or Biacore assay. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD86, and optionally, ICOSL and / or CD80. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD86, and optionally, decreased binding affinity for one or more of ICOSL and / or CD80, relative to wild-type or unmodified CTLA-4 polypeptides.

[0183] The binding affinity for each of the binding partners is independent, i.e., in some embodiments, a mutant CTLA-4 polypeptide has increased binding affinity for one, two, or three of ICOSL, CD80, and / or CD86, and decreased binding affinity for one, two, or three of ICOSL, CD80, and / or CD86, relative to a wild-type or unmodified CTLA-4 polypeptide.

[0184] In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for CD80 and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, such mutant CTLA-4 polypeptides also have increased binding affinity for ICOSL relative to wild-type or unmodified CTLA-4 polypeptides.

[0185] In some embodiments, the mutant CTLA-4 polypeptide has increased binding affinity for ICOSL relative to wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide has increased or decreased binding affinity for ICOSL and increased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide has increased or decreased binding affinity for ICOSL and increased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide has decreased binding affinity for ICOSL relative to wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide has increased or decreased binding affinity for ICOSL and decreased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide has increased or decreased binding affinity for ICOSL and decreased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptide.

[0186] In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and CD80 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and decreased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and CD80 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and increased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptides.

[0187] In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and decreased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and increased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides.

[0188] In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL, CD80, and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and CD80 and decreased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and CD86 and decreased binding affinity for CD80 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and CD80 and increased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and CD80 and increased binding affinity for CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have increased binding affinity for ICOSL and decreased binding affinity for CD80 and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL, CD80, and CD86 relative to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, mutant CTLA-4 polypeptides have decreased binding affinity for ICOSL and increased binding affinity for CD80 and CD86 relative to wild-type or unmodified CTLA-4 polypeptides.

[0189] In some embodiments, a mutant CTLA-4 polypeptide with increased or greater binding affinity for ICOSL, CD80, and / or CD86 will have an increase in binding affinity for ICOSL, CD80, and / or CD86 relative to a wild-type or unmodified CTLA-4 polypeptide control of at least 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50%. In some embodiments, the increase in binding affinity relative to a wild-type or unmodified CTLA-4 polypeptide 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, or 50-fold. In such examples, the wild-type or unmodified CTLA-4 polypeptide has the same sequence as the mutant CTLA-4 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0190] In some embodiments, a mutant CTLA-4 polypeptide with reduced or decreased binding affinity for ICOSL, CD80, and / or CD86 will have a decrease in binding affinity relative to a wild-type or unmodified CTLA-4 polypeptide control of at least 5%, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for ICOSL, CD80, and / or CD86. In some embodiments, the decrease in binding affinity relative to a wild-type or unmodified CTLA-4 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 CTLA-4 polypeptide has the same sequence as the mutant CTLA-4 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0191] In some embodiments, the equilibrium dissociation constant (KD) of any of the foregoing embodiments for ICOSL, CD80, and / or CD86 is greater than or equal to 1x10 -5 Under M, 1x10 -6 Medium, 1x10 -7 Medium, 1x10 -8Medium, 1x10 -9 Medium, 1x10 -10 Medium, or 1x10 -11 Medium, or 1x10 -12 M, or even lower.

[0192] Wild-type or unmodified CTLA-4 sequences do not necessarily have to be used as starting materials for generating the mutant CTLA-4 polypeptides described herein. Thus, the use of the term "modification," such as "substitution," does not imply that embodiments of the present invention are limited to a particular method for generating the mutant CTLA-4 polypeptide. Mutant CTLA-4 polypeptides can be generated, for example, by de novo peptide synthesis and thus do not necessarily require a modification, such as a "substitution," in the sense of changing a codon to code for the substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which do not otherwise imply a particular method of generation. The means by which mutant CTLA-4 polypeptides are designed or generated are not limited to any particular method. However, in some embodiments, wild-type or unmodified CTLA-4-encoding nucleic acids are mutated from wild-type or unmodified CTLA-4 genetic material and screened for the desired specific binding affinity and / or suppression or reduction of IFN-gamma expression or other functional activity.

[0193] In some embodiments, mutant CTLA-4 polypeptides are synthesized de novo and subsequently screened using protein or nucleic acid sequences available in any number of publicly available databases. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, such as in the UniProtKB database discussed above.

[0194] Unless otherwise stated, as indicated throughout this disclosure, amino acid modifications are designated by amino acid position numbers that correspond to the numbering of positions in the unmodified ECD sequence set forth in SEQ ID NO:2, or, where applicable, the unmodified ECD sequence set forth in SEQ ID NO:569, as follows: TIFF0007749319000007.tif37165

[0195] In some embodiments, the mutant CTLA-4 polypeptide contains any one or more of the amino acid modifications, e.g., amino acid substitutions, provided relative to SEQ ID NO:2 or 569, or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide contains any one or more of the amino acid modifications provided in a polypeptide that is or comprises an IgV domain or a specific binding fragment thereof, such as the exemplary sequence set forth in SEQ ID NO:3. TIFF0007749319000008.tif9165

[0196] It is within the level of ordinary skill in the art to identify the corresponding position of a modification, e.g., an amino acid substitution, in a CTLA-4 polypeptide, including a portion thereof containing its IgSF domain (e.g., IgV), such as by aligning a reference sequence (e.g., SEQ ID NO:3) with SEQ ID NO:2 or SEQ ID NO:569. In listings of modifications throughout this disclosure, the amino acid position is shown in the middle, the corresponding unmodified (e.g., wild-type) amino acid is listed before the number, and the identified mutant amino acid substitution is listed after the number. If the modification is a deletion at the position, "del" is indicated; if the modification is an insertion at that position, "ins" is indicated. In some cases, insertions are listed with the amino acid position shown in the middle, the corresponding unmodified (e.g., wild-type) amino acid is listed before and after the number, and the identified mutant amino acid insertion is listed after the unmodified (e.g., wild-type) amino acid.

[0197] In some embodiments, the mutant CTLA-4 polypeptide has one or more amino acid modifications, e.g., substitutions, in a wild-type or unmodified CTLA-4 sequence. The one or more amino acid modifications, e.g., substitutions, can be in the ectodomain (extracellular domain) of the wild-type or unmodified CTLA-4 sequence. In some embodiments, the one or more amino acid modifications, e.g., substitutions, are in the IgV domain or a specific-binding fragment thereof.

[0198] In some embodiments, the mutant CTLA-4 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 substitutions may be in the IgV domain or ECD. In some embodiments, the mutant CTLA-4 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions, in the IgV domain or specific-binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions, in the ECD or specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified CTLA-4 polypeptide or specific binding fragment thereof, such as the amino acid sequence of SEQ ID NO:2 or 3.

[0199] In some embodiments, the mutant CTLA-4 polypeptides contain a nucleotide sequence similar to that of positions 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 20, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67, 6 , 75, 76, 82, 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118, 119, 120, 121, 122, 124, 125, and / or 126.

[0200] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 1 in unmodified CTLA-4 or a specific binding fragment thereof. With respect to the positions set forth in NO:2, having one or more amino acid modifications, e.g., substitutions, corresponding to positions 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 29, 30, 33, 35, 37, 38, 41, 42, 43, 45, 46, 47, 48, 53, 54, 55, 56, 58, 59, 61, 63, 64, 65, 67, 69, 71, 72, 73, 75, 76, 82, 85, 86, 87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118, 119, 120, 121, and / or 122.

[0201] In some embodiments, the mutant CTLA-4 polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to positions 12, 18, 26, 29, 33, 53, 55, 56, 58, 63, 72, 87, 98, 99, 105, 106, and / or 117 in unmodified CTLA-4 or a specific binding fragment thereof, with respect to the positions set forth in SEQ ID NO:2.

[0202] In some embodiments, the mutant CTLA-4 polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to positions 12, 18, 26, 29, 56, 63, 72, 98, 99, 105, 106, and / or 117 in unmodified CTLA-4 or a specific binding fragment thereof, with respect to the positions set forth in SEQ ID NO:2.

[0203] In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of A6T, V10A, L12F, L12H, L12I, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, E33M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D , N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L63H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I , T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89 S, L91R, I93L, I93V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, P102L, Y105F, Y105L, L106I, L106N, L106R, L106V, I and / or D126T, or a conservative amino acid substitution thereof.

[0204] In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of A6T, V10A, L12F, L12H, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, E33M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M55T, M5 5V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L63H, L63P , D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q 76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93V, K95R, V96 and / or I117T, or a conservative amino acid substitution thereof.

[0205] In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of L12F, L12H, L12I, L12P, I18A, I18F, I18N, I18T, I18V, A26D, A26S, A26T, G29R, G29W, E33M, E33V, T53S, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, L63H, L63P, S63H ... and / or I117T, or a conservative amino acid substitution thereof. In some embodiments, mutant CTLA-4 polypeptides have one or more amino acid modifications selected from I12F, L12P, I18T, A26T, G29W, T53S, M55T, M56K, M56T, N58S, S72G, M99L, L63P, L98Q, Y105L, L106I, and / or I117L, or conservative amino acid substitutions thereof, relative to the positions set forth in SEQ ID NO: 2. In some embodiments, mutant CTLA-4 polypeptides have one or more amino acid modifications selected from L12P, I18T, A26T, G29W, T53S, M55T, M56K, N58S, S72G, M99L, L63P, L98Q, Y105L, L106I, and / or I117L, or conservative amino acid substitutions thereof, relative to the positions set forth in SEQ ID NO: 2. In some embodiments, the mutant CTLA-4 polypeptide has one or more amino acid modifications selected from A26T, G29W, L63P, S72G, L98Q, M99L, Y105L, and / or L106I, or conservative amino acid substitutions thereof, with respect to the positions set forth in SEQ ID NO:2.

[0206] A conservative amino acid substitution is any amino acid that falls within the same class of amino acids as the substituting amino acid other than the wild-type or unmodified amino acid: aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl- or sulfur-containing (serine, cysteine, threonine, and methionine), cyclized (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).

[0207] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to A26T. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions G29W, T53S, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide comprises the amino acid substitutions A26T / G29W, A26T / T53S, A26T / L63P, A26T / S72G, A26T / L98Q, A26T / M99L, A26T / Y105L, A26T / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with the embodiments provided. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0208] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide corresponding to G29W, or a specific-binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, T53S, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide comprises the amino acid substitutions A26T / G29W, G29W / T53S, G29W / L63P, G29W / S72G, G29W / L98Q, G29W / M99L, G29W / Y105L, or G29W / L106I. The mutant CTLA-4 polypeptide, according to provided embodiments, may further comprise an amino acid modification (e.g., substitution), such as any described herein. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0209] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to T53S. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide contains the amino acid substitutions A26T / T53S, G29W / T53S, T53S / L63P, T53S / S72G, T53S / L98Q, T53S / M99L, T53S / Y105L, or T53S / L106I. The mutant CTLA-4 polypeptide, according to provided embodiments, may further comprise an amino acid modification (e.g., substitution), such as any described herein. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0210] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to L63P. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide contains the amino acid substitutions A26T / L63P, G29W / L63P, T53S / L63P, L63P / S72G, L63P / L98Q, L63P / M99L, L63P / Y105L, or L63P / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with the provided embodiments. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0211] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to S72G. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide comprises the amino acid substitutions A26T / S72G, G29W / S72G, T53S / S72G, L63P / S72G, S72G / L98Q, S72G / M99L, S72G / Y105L, or S72G / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with the embodiments provided. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0212] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to L98Q. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, M99L, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide comprises amino acid substitutions A26T / L98Q, G29W / L98Q, T53S / L98Q, L63P / L98Q, S72G / L98Q, L98Q / M99L, L98Q / Y105L, or L98Q / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with provided embodiments. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0213] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to M99L. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, Y105L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide contains amino acid substitutions A26T / M99L, G29W / M99L, T53S / M99L, L63P / M99L, S72G / M99L, L98Q / M99L, M99L / Y105L, or M99L / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with provided embodiments. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0214] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to Y105L. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L, and / or L106I. In some embodiments, the mutant CTLA-4 polypeptide contains amino acid substitutions A26T / Y105L, G29W / Y105L, T53S / Y105L, L63P / Y105L, S72G / Y105L, L98Q / Y105L, M99L / Y105L, or Y105L / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with provided embodiments. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0215] In some embodiments, the mutant CTLA-4 polypeptide comprises an amino acid substitution in an unmodified or wild-type CTLA-4 polypeptide or a specific-binding fragment thereof corresponding to L106I. In some embodiments, the mutant CTLA-4 polypeptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L, and / or Y105L. In some embodiments, the mutant CTLA-4 polypeptide contains amino acid substitutions A26T / L106I, G29W / L106I, T53S / L106I, L63P / L106I, S72G / L106I, L98Q / L106I, M99L / L106I, or Y105L / L106I. The mutant CTLA-4 polypeptide may further comprise an amino acid modification (e.g., substitution), such as any described herein, consistent with provided embodiments. Table 2 lists exemplary amino acid modifications (eg, substitutions) and mutant CTLA-4 polypeptides as described.

[0216] In some embodiments, the mutant CTLA-4 contains the amino acid substitution E33V / M99L. Mutant CTLA-4 polypeptides may further include amino acid modifications (e.g., substitutions), such as any described herein, consistent with the embodiments provided. Table 2 lists exemplary amino acid modifications (e.g., substitutions) and mutant CTLA-4 polypeptides, as described.

[0217] In some embodiments, the mutant CTLA-4 polypeptide comprises at least three amino acid modifications (e.g., substitutions), wherein the at least three modifications (e.g., substitutions) in unmodified or wild-type CTLA-4 or a specific-binding fragment thereof correspond to A26T, G29W, T53S, L63P, S72G, L98Q, M99L, Y105L, and / or L106I, or conservative amino acid substitutions thereof, with respect to the positions set forth in SEQ ID NO:2.

[0218] In some embodiments, the mutant CTLA-4 polypeptide comprises amino acid substitutions corresponding to G29W / L98Q / Y105L with respect to the positions set forth in SEQ ID NO:2 in unmodified or wild-type CTLA-4 or a specific binding fragment thereof.

[0219] In some embodiments, the mutant CTLA-4 polypeptide comprises amino acid substitutions corresponding to G29W / N58S / L63P / Q82R / L98Q / Y105L with respect to the positions set forth in SEQ ID NO:2 in unmodified or wild-type CTLA-4 or a specific-binding fragment thereof.

[0220] In some embodiments, the mutant CTLA-4 polypeptide comprises amino acid substitutions corresponding to L12F / R16H / G29W / M56T / L98Q / Y105L with respect to the positions set forth in SEQ ID NO:2 in unmodified or wild-type CTLA-4 or a specific-binding fragment thereof.

[0221] In some embodiments where the mutant CTLA-4 polypeptide has a single amino acid substitution in unmodified CTLA-4 or a specific-binding fragment thereof, the modification does not correspond to positions 27, 31, 32, 33, 35, 95, 98, 105, 106, or 107 with respect to positions set forth in SEQ ID NO:2. In some embodiments where the mutant CTLA-4 polypeptide has a single amino acid substitution in unmodified CTLA-4 or a specific-binding fragment thereof, the modification is not L106E. In some embodiments where the mutant CTLA-4 polypeptide has exactly two amino acid substitutions in unmodified CTLA-4 or a specific-binding fragment thereof, the modification is not A31Y and L106E (i.e., A31Y / L106E).

[0222] In some embodiments, the mutant CTLA-4 does not contain modifications corresponding to A26E, T32N, V34I, A52M, G57E, 167F, S66P, and / or S72F, with respect to the numbering set forth in SEQ ID NO: 2. In some embodiments, where the mutant CTLA-4 polypeptide has seven amino acid substitutions in unmodified CTLA-4 or a specific-binding fragment thereof, the modifications are not modifications corresponding to T32N, V34I, A52M, M56K, G57E, S66P, and S72F, with respect to the numbering set forth in SEQ ID NO: 2. In some embodiments in which the mutant CTLA-4 polypeptide has 10 amino acid substitutions in unmodified CTLA-4 or a specific-binding fragment thereof, the modifications are not those corresponding to A26E, T32N, V34I, A52M, M56K, N58D, S66P, I67S, S72F, and L106E, with respect to the numbering set forth in SEQ ID NO:2.

[0223] In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of A6T, V10A, L12F, L12H, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, E33 M, E33V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55 R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61 S, L63H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S7 2T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I9 In some embodiments, the two or more amino acid modifications are selected from among SEQ ID NO: 3L, I93V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y105F, Y105L, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117M, and I117T. ... With regard to the numbering listed in NO:2, A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98Q / Y105L, L12 F / R16H / G29W / M56T / L98Q / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, L12F / K30R / S7 2G / Q82R / L98Q / M99L / Y105L, L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S,L12H / E33M / L98Q / Y105L、L12H / M55T / E59D / L63P / M99L、L12H / L63P / S72G / L98Q / Y105L、L12I / M55T / M56V / I67T / M99L / L106R / I108F、L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L、L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y105L、L12P / A26T、L12P / A26T / L63P、L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L、L12P / G29W / L63P / S72G / L98Q / Y105L、L12P / G29W / L63P / S72G / L98Q / Y105L / L106I、L12P / A26T / L63P / L98Q / M99L / Y105L、L12P / A26T / L63P / L98Q / Y105L、L12P / A26T / L63P / L98Q / Y105L / L106I、L12P / G29W / D43N / N58S / L63P / L98Q / M99L / Y105L、L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H、L12P / L63P / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L / Y105L / L106N、L12P / L63P / S72G / L98Q / M99L / Y105L / L106N / I117L、S14N / R16C / I18T / M56K / T61A / L63P / A86T / M99L、S15P / I18V / M56T / L98Q / M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L98Q / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / L63P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63H / T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M87T / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26T / L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q82R / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M99L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T / E48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105L / I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105L、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S20N / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L98Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105L / I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A26D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / I67N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / L98Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、A26T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / M87V / N110K / I117E、A26T / L63P / S72G / L98Q / M99L / Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / I117L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67N / S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S72G / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / L63P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P / S72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29W / T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T53S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / S72G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q / Y105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I117L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W / N58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58S / L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q / Y105L / L106V、G29W / N58S / L63P / S72G / M87V / L98Q / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W / N58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98Q / Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y105L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / L63P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G / L98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y105L / L106I / I117L、<h2 style=";text-align:left;direction:ltr">G29W / L63P / S72G / L98Q / Y105L / I117L、G29W / L63P / S72G / L98Q / Y105L / P121 S,G29W / L63P / L98Q / M99L / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q1 13H、G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L、G29W / M87K / I93V / L98Q / M99L / Y105L、G29W / L98Q / M99L / Y105L、E33M / A42T / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L / I108F、E33M / L63P / S72G / L98Q / Y105L / I117L、E33M / Q82H / L98Q / M99L / Y105L、E33V / A42S / M5 5T / L98Q / M99L / Y105L、T37S / M56V / L98Q / Y105L、V38I / L63P / S72G / L98Q / M9 9L / Y105L、Q41L / Y54F / M56K / M99L / I108F、T53S / M56V / L98Q / Y105L、M55T / L 63P / T71I / M99L / Y105L、M55T / S72G / L98Q / M99L / Y105L、M55T / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M99L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L106I / I117L、M56R / L63P / L98Q / M99L / Y105L、M56T / L91R / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S7 2G / L98Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y105L / L106R / V116A、L63P / S72G / M87A / L98Q / Y105L、L63P / S72G / I93L / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L / L106I / I117L、L63P / S72 G / L98Q / Y105L、L63P / S72G / L98Q / Y105L / L106I / I117L、L63P / S72G / Y105L、L63P / M87K / M99L / L106R, L63P / Q82H / L98Q / M99L / Y105L, L63P / K95R, L63P / L98Q, L63P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L106I, L63P / L98Q / M99L / Y105L / I108V, L63P / L98Q / M99L / Y105L / I117M, L63P / L98Q / Y105L, L63P / L98Q / , V116A, L63P / L98R / N110K, L63P / M99L / Y105L / I108F, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, S72G / L98Q / M99L / Y105L / I117T, L98Q / M99L / Y105L, L98Q / M99L / Y105L / L106I / I117T, L98Q / M99L / Y105L / L106I / Y115N, L98Q / Y105L, and L98R / N110K.

[0224] In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of A6T, V10A, L12F, L12H, L12P, S14N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I18T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A26S, A26T, S27P, P28L, G29R, G29W, K30R, E33M, E33V, R35K, with respect to the numbering set forth in SEQ ID NO:2. , T37S, V38I, Q41L, A42S, A42T, A42V, D43N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M55T, M55V, M56K , M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L63H, L63P, D64E, D64N , D64V, D65G, I67N, I67T, I67V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q76R, Q82H, Q82R , R85G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93V, K95R, V96I, E97Q, L98Q, L98R , M99I, M99L, Y105F, Y105L, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117M, I117T, E120D, C122P, D124P, D124I, S125I, D126P, and D126T. In some embodiments, the two or more amino acid modifications are, with respect to the numbering set forth in SEQ ID NO:2, A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, L12F / K30R / S72G / Q82R / L98Q / M99L / Y105L,L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S、L12H / E33M / L98Q / Y105L、L12H / M55T / E59D / L63P / M99L、L12H / L63P / S72G / L98Q / Y105L、L12I / M55T / M56V / I67T / M99L / L106R / I108F、L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L、L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y105L、L12P / A26T、L12P / A26T / L63P、L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L、L12P / G29W / L63P / S72G / L98Q / Y105L、L12P / G29W / L63P / S72G / L98Q / Y105L / L106I、L12P / A26T / L63P / L98Q / M99L / Y105L、L12P / A26T / L63P / L98Q / Y105L、L12P / A26T / L63P / L98Q / Y105L / L106I、L12P / G29W / D43N / N58S / L63P / L98Q / M99L / Y105L、L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H、L12P / L63P / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L / Y105L / L106N、L12P / L63P / S72G / L98Q / M99L / Y105L / L106N / I117L、S14N / R16C / I18T / M56K / T61A / L63P / A86T / M99L、S15P / I18V / M56T / L98Q / M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L98Q / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / L63P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63H / T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M87T / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26T / L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q82R / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M99L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T / E48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105L / I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105L、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S20N / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L98Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105L / I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A26D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / I67N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / L98Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、A26T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / M87V / N110K / I117E、A26T / L63P / S72G / L98Q / M99L / Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / I117L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67N / S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S72G / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / L63P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P / S72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29W / T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T53S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / S72G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q / Y105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I117L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W / N58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58S / L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L98Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q / Y105L / L106V、G29W / N58S / L63P / S72G / M87V / L98Q / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W / N58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98Q / Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y105L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / L63P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G / L98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y105L / L106I / I117L、<h2 style=";text-align:left;direction:ltr">G29W / L63P / S72G / L98Q / Y105L / I117L、G29W / L63P / S72G / L98Q / Y105L / P121 S,G29W / L63P / L98Q / M99L / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q1 13H、G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L、G29W / M87K / I93V / L98Q / M99L / Y105L、G29W / L98Q / M99L / Y105L、E33M / A42T / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L / I108F、E33M / L63P / S72G / L98Q / Y105L / I117L、E33M / Q82H / L98Q / M99L / Y105L、E33V / A42S / M5 5T / L98Q / M99L / Y105L、T37S / M56V / L98Q / Y105L、V38I / L63P / S72G / L98Q / M9 9L / Y105L、Q41L / Y54F / M56K / M99L / I108F、T53S / M56V / L98Q / Y105L、M55T / L 63P / T71I / M99L / Y105L、M55T / S72G / L98Q / M99L / Y105L、M55T / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M99L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L106I / I117L、M56R / L63P / L98Q / M99L / Y105L、M56T / L91R / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S7 2G / L98Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y105L / L106R / V116A、L63P / S72G / M87A / L98Q / Y105L、L63P / S72G / I93L / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L / L106I / I117L、L63P / S72 G / L98Q / Y105L、L63P / S72G / L98Q / Y105L / L106I / I117L、L63P / S72G / Y105L、L63P / M87K / M99L / L106R、L63P / Q82H / L98Q / M99L / Y105L、L63P / K95R、L63P / L98Q、L63P / L98Q / M99L / Y105L、L63P / L98Q / M99L / Y105L / L106I、L63P / L98Q / M99L / Y105L / I108V、L63P / L98Q / M、 99L / Y105L / I117M、L63P / L98Q / Y105L、L63P / L98Q / V116A、L63P / L98R / N110K、L63P / M99L / Y105L / I108F、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y105L / L106I、S72G / L98Q / M99L / Y105L / I117T、L98Q / M99L / Y105L、L98Q / M99L / Y105L / L106I / I117T、L98Q / M99L / Y105L / L106I / Y115N、L98Q / Y105L、L98R / N110K、T89A / L98Q / M99L / Y105L / L106I / Y115N / E120D / C122P / D124P / S125I / D126P、N58S / L63P / T71A / S72G / L98Q / M99L / Y105L / D124I / S125P / D126T、R16G / E33M / N58S / E59G / L63P / L98Q / Y105L / E120D / C122P / D124P / S125I / D126P、G29W / L63P / S72G / L98Q / Y105L / P121S / D126T、L12H / E33M / L98Q / Y105L、T53S / M56K / N58S / L63P / M87V / L98Q / Y105L、I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L、I18T / A26T / M56K / L63P / L98Q / Y105L、T53S / L63P / L98Q、T53S / L63P / Y105L、T53S / M56K / N58S / L63P / M87V / L98Q、T53S / M56K / N58S / L63P / M87V / Y105L、T53S / M56K / N58S / L63P / L98Q / Y105L、T53S / M56K / N58S / M87V / L98Q / Y105L、T53S / M56K / L63P / M87V / L98Q / Y105L、T53S / N58S / L63P / M87V / L98Q / Y105L、M56K / N58S / L63P / M87V / L98Q / Y105L、E33V / L98Q / Y105L、E33V / M99L / Y105L、E33V / L98Q / M99L、E33V / M99L、L12F / R16H / G29W / M56T / L98Q、L12F / R16H / G29W / M56T / Y105L、L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56T / L98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L, M56T / L98Q / Y105L, L12F / R16H / G29W / M56T / S72G / L98Q / Y105L, and G29W / M56T / S72G / L98Q / Y105L.

[0225] In some embodiments, the mutant CTLA-4 polypeptide additionally comprises the amino acid modification C122S, relative to the position set forth in SEQ ID NO:2 or 569.

[0226] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the substitutions (mutations) listed in Table 2. Table 2 also provides exemplary sequences by reference to SEQ ID NOs for the extracellular domain (ECD) or IgV domain of wild-type CTLA-4 or exemplary mutant CTLA-4 polypeptides. Table 2 also provides exemplary sequences of mutant CTLA-4 polypeptides attached to immunoglobulin Fc (producing an "immunoregulatory Fc fusion," such as a "CTLA-4-Fc mutant fusion," also referred to as a CTLA-4 vIgD-Fc fusion). Such fusion polypeptides are further described below. As indicated, the exact locus or residues corresponding to a given domain may vary, depending on, for example, the method used to identify or classify the domain. Also, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g., IgV) may also be included in the mutant IgSF polypeptide to ensure correct folding of the domain when expressed. Therefore, it is understood that the exemplary SEQ ID NOs in Table 2 should not be construed as limiting. For example, a particular domain, such as the IgV domain of a mutant CTLA-4 polypeptide, may be several amino acids longer or shorter, such as 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids, than the sequence of amino acids set forth in the respective SEQ ID NO:

[0227] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the mutations listed in Table 2. In some embodiments, the mutant CTLA-4 polypeptide comprises any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 4-97, 99-104, or 106-155). In some embodiments, the mutant CTLA-4 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity, to any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 4-97, 99-104, or 106-155), and contains an amino acid modification, e.g., a substitution, of the respective SEQ ID NO that is not present in wild-type or unmodified CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific binding fragment of any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 4-97, 99-104, or 106-155) and contains one or more amino acid modifications, e.g., substitutions, of the respective SEQ ID NOs that are not present in wild-type or unmodified CTLA-4.

[0228] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the mutations listed in Table 2. In some embodiments, the mutant CTLA-4 polypeptide comprises any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs:4-97, 99-104, 106-155, 570-602, or 636). In some embodiments, the mutant CTLA-4 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity, to any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 4-97, 99-104, 106-155, 570-602, or 636), and contains an amino acid modification, e.g., a substitution, of the respective SEQ ID NO that is not present in wild-type or unmodified CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific binding fragment of any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 4-97, 99-104, 106-155, 570-602, or 636) and contains one or more amino acid modifications, e.g., substitutions, of the respective SEQ ID NOs that are not present in wild-type or unmodified CTLA-4.

[0229] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs:156-285). In some embodiments, the mutant CTLA-4 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity, to any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs:156-285), and contains an amino acid modification, e.g., a substitution, of the respective SEQ ID NO that is not present in wild-type or unmodified CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific binding fragment of any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs:156-285) and contains one or more amino acid modifications, e.g., substitutions, of the respective SEQ ID NOs that are not present in wild-type or unmodified CTLA-4.

[0230] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 156-285, 603-635, or 637). In some embodiments, the mutant CTLA-4 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity, to any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs: 156-285, 603-635, or 637), and contains an amino acid modification, e.g., a substitution, of the respective SEQ ID NO that is not present in wild-type or unmodified CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific binding fragment of any of the IgV sequences listed in Table 2 (i.e., any one of SEQ ID NOs:156-285, 603-635, or 637) and contains an amino acid modification, e.g., a substitution, of the respective SEQ ID NO that is not present in wild-type or unmodified CTLA-4.

[0231] Table 2: Exemplary mutant CTLA-4 polypeptides TIFF0007749319000009.tif64158TIFF0007749319000010.tif232158TIFF0007749319000011.tif232158 TIFF0007749319000012.tif232158TIFF0007749319000013.tif232158TIFF0007749319000014.tif29158

[0232] In some embodiments, the mutant CTLA-4 polypeptides exhibit increased binding affinity for the ectodomain of CD80 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the mutant CTLA-4 polypeptides exhibit increased binding affinity for the ectodomain of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 polypeptides exhibit increased affinity for the ectodomain of CD80 and the ectodomain of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3.

[0233] In some embodiments, the mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, such as those set forth in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 polypeptides exhibit increased affinity for the ectodomain of ICOSL and the ectodomain of CD80 compared to wild-type or unmodified CTLA-4, such as those set forth in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 polypeptides exhibit increased affinity for the ectodomain of ICOSL and the ectodomain of CD86 compared to wild-type or unmodified CTLA-4, such as those set forth in SEQ ID NO:2 or 3.

[0234] In some embodiments, the CTLA-4 polypeptide exhibits increased affinity for the ectodomain of ICOSL, the ectodomain of CD80, and the ectodomain of CD86 compared to wild-type or unmodified CTLA-4, such as those set forth in SEQ ID NO:2 or 3.

[0235] In some embodiments, mutant CTLA-4 polypeptides exhibit increased binding affinity for binding to the ectodomain of CD80 and decreased binding affinity for binding to the ectodomain of CD86 and / or ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of CD80 and the ectodomain of CD86 and decreased affinity for the ectodomain of ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of CD80 and the ectodomain of ICOSL and decreased affinity for the ectodomain of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the mutant CTLA-4 polypeptide exhibits increased affinity for the ectodomain of CD80 and decreased affinity for the ectodomain of CD86 and ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide, such as comprising the sequence set forth in SEQ ID NO:2 or 3.

[0236] In some embodiments, mutant CTLA-4 polypeptides exhibit increased binding affinity for binding to the ectodomain of CD86 and decreased binding affinity for binding to the ectodomain of CD80 and / or ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of CD86 and the ectodomain of CD80 and decreased affinity for the ectodomain of ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of CD86 and the ectodomain of ICOSL and decreased affinity for the ectodomain of CD80 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the mutant CTLA-4 polypeptide exhibits increased affinity for the ectodomain of CD86 and decreased affinity for the ectodomain of CD80 and ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide, such as comprising the sequence set forth in SEQ ID NO:2 or 3.

[0237] In some embodiments, mutant CTLA-4 polypeptides exhibit increased binding affinity for binding to the ectodomain of ICOSL and decreased binding affinity for binding to the ectodomain of CD80 and / or CD86 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of ICOSL and the ectodomain of CD80 and decreased affinity for the ectodomain of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, mutant CTLA-4 polypeptides exhibit increased affinity for the ectodomain of ICOSL and the ectodomain of CD86 and decreased affinity for the ectodomain of CD80 compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the mutant CTLA-4 polypeptide exhibits increased affinity for the ectodomain of ICOSL and decreased affinity for the ectodomain of CD80 and CD86 compared to a wild-type or unmodified CTLA-4 polypeptide, such as comprising the sequence set forth in SEQ ID NO:2 or 3.

[0238] In some embodiments, the mutant CTLA-4 polypeptide exhibits increased binding affinity for the ectodomain of CD80 compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising a sequence such as set forth in SEQ ID NO:2 or 3. In some embodiments, the increased affinity for the ectodomain of CD80 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 CTLA-4 for the ectodomain of CD80. In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased binding affinity to CD80 compared to wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions V10A, L12F, R16H, I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, M56T, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, I108V, and / or P121S in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain, with reference to the numbering of SEQ ID NO:2. In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased affinity for CD80 compared to wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M56K, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L, and / or I108V in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain, with reference to the numbering of SEQ ID NO:2.In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased binding affinity to CD80 compared to wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions I18T, A26T, G29W, A42V, Q45H, N58S, L63P, I67N, Q82R, M87K, M87V, L98Q, and / or Y105L in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain, with reference to the numbering of SEQ ID NO:2. In some of these embodiments, the mutant CTLA-4 polypeptide that exhibits increased binding affinity to CD80 compared to a wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain from among I18T, A26T, G29W, N58S, L63P, M87K, M87V, L98Q, and / or Y105L, with reference to the numbering of SEQ ID NO: 2. In some embodiments, the mutant CTLA-4 polypeptide has at least two, three, four, or five of such amino acid substitutions that exhibit increased binding to the ectodomain of CD80 compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 3.In some embodiments, the amino acid substitutions are I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, V10A / G29W / T53S / M56K / L63P / L 98Q / Y105L / P121S, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, T53S / M56K / N58S / L6 3P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98 Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56T / L98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L, or M56T / L98Q / Y105L.In some embodiments, the amino acid substitutions are I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, L98Q / M99L / Y105L, E33V / L98Q / Y105L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, or E33V / L98Q / M99L. In some embodiments, the amino acid substitutions are I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, or T53S / M56K / N58S / M87V / L98Q / Y105L. In some embodiments, any of the above substitutions is in the CTLA-4 extracellular domain set forth in SEQ ID NO:2, see e.g., the exemplary SEQ ID NOs set forth in Table 2. In some embodiments, any of the above substitutions is in the CTLA-4 extracellular domain set forth in SEQ ID NO:3, see e.g., the exemplary SEQ ID NOs set forth in Table 2.

[0239] In some embodiments, the mutant CTLA-4 polypeptide exhibits increased binding affinity for the ectodomain of CD86 compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising, for example, the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the increased affinity for the ectodomain of CD86 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 CTLA-4 for the ectodomain of CD86. In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased binding affinity for CD86 compared to a wild-type or unmodified CTLA-4 polypeptide comprises a sequence set forth in SEQ ID NO:3 in the extracellular domain of unmodified or wild-type CTLA-4 or in the IgV domain thereof. With respect to the numbering of NO:2, one or more amino acid substitutions L12F, L12H, L12P, R16H, I18T, I18V, S20N, A26T, G29R, G29W, E33M, E33V, A42S, A42V, T47A, T53S, M55T, M56K, M56R, M56T, M56V, N58D, N58S, T61I, T61N, T61R, T61S, L63P, D65G, I67N, I67N 7V, T69A, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89M, T89S, V96I, L98Q, L98R, M99L, P102L, Y105L, L106I, L106N, L106R, L106V, I108F, I108V, Q113H, Y115H, V116A, I117L, I117T, and / or P121S. In some of these embodiments, the mutant CTLA-4 polypeptide that exhibits increased binding affinity for CD86 compared to a wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions L12P, A26T, M55T, L63P, S72G, L98Q, M99L, and / or Y105L in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain, with reference to the numbering of SEQ ID NO:2.The CTLA-4 polypeptide has at least two, three, four, or five of such amino acid substitutions and exhibits increased binding to the ectodomain of CD86 compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the amino acid substitutions are I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y105L, S72G / L98Q / M99L / Y105L / I117T, M56R / L63P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L1 06I, A26T / M55T / L63P / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, E33M / L63P / S 72G / L98Q / Y105L / I117L, A26T / I67N / S72G / L98Q / M99L / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, S20N / A26T / L63P / L98Q / M99L / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105 L / L106N, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58S / L63P / S72G / L98Q / Y105L, G29W / L63P / D65G / S72G / L98Q / Y105L, G29 W / N58S / L63P / S72G / M87V / L98Q / Y105L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / N58S / L63P / S72G / L98Q / Y105L / L106V,G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M 99L / Y105L / L106I, L63P / L98Q / M99L / Y105L, A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L, A 26T / M55T / L63P / S72G / L98Q / M99L / Y105L, L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / Y115H, G29W / T53S / M56K / T61N / L63P / L98Q / Y105L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S72G / L98Q / Y105L / I108F, L12P / In some embodiments, the amino acid substitutions are R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L, L98Q / M99L / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q, L12F / G29W / L98Q / Y105L, or L12F / L98Q / Y105L. In some embodiments, the amino acid substitutions are A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, or L12P / L63P / S72G / L98Q / M99L / Y105L. In some embodiments, any of the above substitutions are in the CTLA-4 extracellular domain set forth in SEQ ID NO:2, see e.g., the exemplary SEQ ID NOs set forth in Table 2. In some embodiments, any of the above substitutions are in the CTLA-4 extracellular domain set forth in SEQ ID NO:3, see e.g., the exemplary SEQ ID NOs set forth in Table 2.

[0240] In some embodiments, the mutant CTLA-4 polypeptide exhibits increased binding affinity for the ectodomain of ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising the sequence set forth in SEQ ID NO:2 or 3. In some embodiments, the increased affinity for the ectodomain of ICOSL 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 CTLA-4 for the ectodomain of ICOSL. In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased binding affinity for ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide comprises a sequence set forth in SEQ ID NO:3 in the extracellular domain of unmodified or wild-type CTLA-4 or in the IgV domain thereof. With respect to the numbering of NO:2, one or more amino acid substitutions V10A, L12F, L12I, R16H, I18N, I18T, I18V, A19V, A26T, G29W, E33M, E33V, R35K, V38I, A42V, Q45H, T47A, T53S, M55T, M56K, M56V, N58D, N58S, T61A, T61R, L63H, L63P, D64E, D64N, D64R, and D65A. 4V, D65G, I67N, I67T, I67V, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87T, M87V, T89A, T89S, I93L, I93V, E97Q, L98Q, M99L, M99I, Y105L, L106I, L106R, I108F, I108V, N110K, Q113H, I117L, and / or P121S. In some of these embodiments, the mutant CTLA-4 polypeptide that exhibits increased binding affinity for ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide comprises one or more amino acid substitutions, with respect to the numbering of SEQ ID NO:2, in the extracellular domain of unmodified or wild-type CTLA-4 or in the IgV domain thereof: V10A, L12F, R16H, I18T, I18V, A19V, A26T, G29W, E33V, R35K, A42V, Q45H, T47A, T53S, M55T, M56K, M56T, N58D, N58S,In some of these embodiments, the mutant CTLA-4 polypeptide exhibiting increased binding affinity for ICOSL compared to wild-type or unmodified CTLA-4 polypeptides has the residues of SEQ ID NO: 1 in the extracellular domain of unmodified or wild-type CTLA-4 or in the IgV domain thereof. With respect to the numbering of NO:2, it has one or more amino acid substitutions L12F, I18T, I18V, A26T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, N58D, N58S, L63P, D64E, I67N, I67V, S72G, Q82H, R85G, M87K, M87V, T89A, T89S, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F, I108V, N110K, and / or N117L. In some of these embodiments, the mutant CTLA-4 polypeptide that exhibits increased binding affinity for ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide has one or more amino acid substitutions I18T, A26T, G29W, T53S, M55T, M56K, N58S, L63P, S72G, L98Q, M99L, Y105L, L106I, and / or N117L in the extracellular domain of unmodified or wild-type CTLA-4 or in its IgV domain, with reference to the numbering of SEQ ID NO: 2. In some embodiments, the mutant CTLA-4 polypeptide has at least two, three, four, or five of such amino acid substitutions and exhibits increased binding to the ectodomain of ICOSL compared to a wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 3. In some embodiments, the amino acid substitutions are L63P / S72G / L98Q / M99L / Y105L / L106I / I117L, G29W / L98Q / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L,<h2 style=";text-align:left;direction:ltr">L63P / Q82H / L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / M99L / Y105L、T61A / L63P / S72G / L98Q / M99L / Y105L、V38I / L63P / S72G / L98Q / M99L / Y105L、L63P / S 72G / I93L / L98Q / M99L / Y105L、L12I / M55T / M56V / I67T / M99L / L106R / I108F、I18N / A26T / L63H / T89A / L98Q / M99L / Y105L、G29W / N58S / L63P / M87T / L98Q / M9 9L / Y105L、G29W / N58S / L63P / D64N / L98Q / M99L / Y105L、I18T / L63P / S72G / M8 7K / L98Q / M99L / Y105L、L63P / M87K / M99L / L106R、L63P / M99L / Y105L / I108F、G 29W / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、V10A / L 63P / D64V / S72G / L98Q / M99L / Y105L、I18V / A26T / L63P / D64E / L98Q / Y105L / L1 06R / N110K、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、G29W / N58S / L63P / T 69I / L98Q / M99L / Y105L、G29W / T53S / M56K / L63P / L98Q / Y105L、L12F / R16H / G2 9W / M56T / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / N58D / I67V / L98Q / M99L / Y105L I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y105L / L106I、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / Y105L、G29W / M87K / I93V / L98Q / M99L / Y105L、G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L、L63P / L98Q / M99L / Y105L / I108V、A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105 L / I108V / I117L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S72G / L98Q / Y105L / I108F, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, T53S / M 56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E 33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, L12F / R16H / G29W / M56T / Y105L, or L12F / L98Q / Y105L. In some embodiments, the amino acid substitutions are G29W / L98Q / M99L / Y105L, L63P / M99L / Y105L / I108F, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, G29W / M87K / I93V / L98Q / M99L / Y105L,G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, G29W / T53S / M56K / N58S / L63 P / M87V / L98Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M 55T / M56K / L63P / L98Q / M99L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L 98Q, T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87 In some embodiments, any of the above substitutions is in the CTLA-4 extracellular domain set forth in SEQ ID NO:2, see e.g., the exemplary SEQ ID NOs: in Table 2. In some embodiments, any of the above substitutions is in the CTLA-4 extracellular domain set forth in SEQ ID NO:3, see e.g., the exemplary SEQ ID NOs: in Table 2.

[0241] In some embodiments, provided mutant CTLA-4 polypeptides containing an extracellular domain with at least one affinity-modified IgSF domain (e.g., IgV) or a specific-binding fragment thereof relative to the IgSF domain contained in a wild-type or unmodified CTLA-4 polypeptide exhibit altered signaling (e.g., reduced / inhibited signaling) by stimulatory receptors that can be engaged by one or more binding partners of CTLA-4, such as CD80, CD86, and / or ICOSL, compared to the wild-type or unmodified CTLA-4 polypeptide upon binding to the one or more binding partners. In some aspects, the stimulatory receptor is CD28 or ICOS, which are expressed on the surface of T cells and can release cytokines in response to intracellular signals. In some embodiments, the altered signaling differs from signaling affected by wild-type or unmodified CTLA-4 polypeptide regulatory sequences in the same format (e.g., Fc fusion protein), as determined, for example, by an assay that measures cytokine release (e.g., IL-2 release or IFN-gamma) after incubation with the identified mutant and / or wild-type or unmodified CTLA-4 polypeptide. Exemplary assays are described in the Examples. In some cases, the assay is a cell-based assay, such as a mixed lymphocyte reaction, and the resulting readout or function is the sum of the signaling activities of functional binding partners expressed on the surface of the cells in the assay. As discussed elsewhere herein, in some embodiments, the format of the provided mutant CTLA-4 polypeptide can affect the type of activity, for example, agonist or antagonist. In some embodiments, the mutant CTLA-4 polypeptide is a fusion protein of an extracellular domain or its specific binding fragment containing a multimerization domain, such as an IgV domain and an Fc domain, and the altered signaling results from an antagonist assay for inhibiting the activity of a stimulatory receptor. In other aspects, this format can result in increased or agonist activity of a stimulatory receptor.

[0242] III. Variant Polypeptide Formats The immunomodulatory polypeptides comprising mutant CTLA-4 provided herein can be formatted in a variety of ways, including as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, a particular format can be selected for a desired therapeutic application. In some cases, the immunomodulatory polypeptides comprising mutant CTLA-4 polypeptides are provided in a format that antagonizes or inhibits the activity of CD28 and / or ICOS by binding with their binding partners (e.g., ICOSL, CD80, and / or CD86), thereby preventing CD28 and / or ICOS costimulatory signaling. In some embodiments, CD28 and / or ICOSL antagonism can be useful for suppressing or suppressing immunity, for example, in autoimmunology. In some embodiments, CD28 and / or ICOSL antagonism can be useful for treating inflammation or autoimmunity. In some cases, the immunomodulatory polypeptide, including the mutant CTLA-4 polypeptide, is expressed on cells as a switch receptor, in which a CTLA-4 inhibitory receptor is converted into an activating receptor (e.g., by an ITAM signaling domain), and / or as a decoy receptor without an intracellular signaling domain. In some embodiments, the CTLA-4 activating receptor or decoy receptor may be useful for treating cancer. Those skilled in the art can easily determine the specific type of activity, such as to antagonize, compete with, and / or inhibit one or more specific binding partners, or to activate one or more downstream signaling pathways. Exemplary methods for assessing such activity, including examples, are provided herein.

[0243] In some aspects, immunomodulatory proteins are provided that include the vIgD of CTLA-4, e.g., fused to an Fc chain, where such proteins are soluble. In some aspects, one or more additional IgSF domains, such as one or more additional vIgDs, can be linked to the vIgD of CTLA-4 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 creating 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, mutant CTLA-4 immunomodulatory proteins are provided as conjugates that contain the vIgD of CTLA-4 linked, directly or indirectly, to a ligand, e.g., a targeting agent or moiety that specifically binds to an antigen, e.g., an antibody or other binding molecule, to target or localize the vIgD to a particular environment or cell, e.g., when administered to a subject. In some embodiments, the targeting agent, e.g., an antibody or other binding molecule, binds to an antigen on the surface of a leukocyte, lymphocyte, or lymphoid tissue, such as the spleen, tonsils, lymphatic vessels, lymph nodes, pharyngeal tonsils, and liver, thereby localizing the vIgD-containing mutant CTLA-4 to the immune system and, e.g., modulating the activity of leukocytes or lymphocytes within the immune system.

[0244] In some embodiments, the provided immunomodulatory proteins are expressed in or on cells and provided as part of engineered cell therapy (ECT). In some embodiments, the mutant CTLA-4 polypeptide is expressed in a membrane-bound form on a cell, such as an immune cell (e.g., a T cell or an antigen-presenting cell), thereby providing a transmembrane immunomodulatory protein (hereinafter also referred to as a "TIP"). In some aspects, the mutant CTLA-4 polypeptide is expressed in a cell, such as an immune cell (e.g., a T cell or an antigen-presenting cell), in a secretable form, thereby generating a secreted or soluble form of the mutant CTLA-4 polypeptide (hereinafter also referred to as a "SIP"), such as when the cell is administered to a subject. In some aspects, the SIP can antagonize a binding partner in the environment in which it is secreted (e.g., the immune microenvironment). In some embodiments, the mutant CTLA-4 polypeptide, upon administration to a subject, can affect a cell, such as an immune cell (e.g., a T cell or an antigen-presenting cell), in vivo due to delivery or expression of the mutant polypeptide as a TIP or SIP in the cell. Infectious substance (e.g., viral or bacterial pathogens).

[0245] In some embodiments, soluble immunomodulatory polypeptides, such as mutant CTLA-4 containing vIgD, can be encapsulated within liposomes that can themselves be conjugated to any one or any combination of the provided conjugates (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the invention are deglycosylated. In more specific embodiments, the mutant CTLA-4 sequence is deglycosylated. In even more specific embodiments, the IgV domain or ECD of the mutant CTLA-4 is deglycosylated.

[0246] A non-limiting example of the format provided is set forth in FIG. 1 and further described below.

[0247] A. Soluble Proteins In some embodiments, the immunomodulatory protein containing a mutant CTLA-4 polypeptide is a soluble protein. Those skilled in the art will understand that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that soluble forms of such proteins can be produced using the extracellular domain or an immunologically active subsequence thereof. Thus, in some embodiments, the immunomodulatory protein containing a mutant CTLA-4 polypeptide lacks the transmembrane domain or a portion thereof. In some embodiments, the immunomodulatory protein containing a mutant CTLA-4 polypeptide lacks the intracellular (cytoplasmic) domain or a portion thereof. In some embodiments, the immunomodulatory protein containing a mutant CTLA-4 polypeptide contains only an ECD domain or a portion thereof containing an IgV domain, or a vIgD portion containing a domain or specific-binding fragment thereof containing an amino acid modification. In some aspects, such soluble polypeptides can be used to inhibit and / or antagonize the activity of CD28 and ICOS, thereby suppressing or weakening immune responses. In some embodiments, soluble mutant CTLA-4 immunomodulatory proteins (eg, mutant CTLA-4-Fc) as provided can be used in methods for treating autoimmune or inflammatory conditions.

[0248] In some embodiments, an immunomodulatory polypeptide comprising a mutant CTLA-4 may comprise one or more mutant CTLA-4 polypeptides of the present invention. In some embodiments, a polypeptide of the present invention will comprise exactly one, two, three, four, or five mutant CTLA-4 sequences. In some embodiments, at least two of the mutant CTLA-4 sequences are the same mutant CTLA-4 sequence.

[0249] In some embodiments, the immunomodulatory polypeptides provided comprise two or more vIgD sequences of CTLA-4. Multiple mutant CTLA-4 polypeptides within a polypeptide chain can be identical (i.e., of the same species) or non-identical (i.e., of different species) mutant CTLA-4 sequences. In addition to single-chain polypeptide embodiments, in some embodiments, two, three, four, or more of the polypeptides of the invention can be covalently or non-covalently attached to each other. Thus, monomeric, dimeric, and higher order (e.g., three, four, five, or more) multimeric proteins are provided herein. For example, in some embodiments, exactly two polypeptides of the invention can be covalently or non-covalently attached to each other to form a dimer. In some embodiments, the attachment is via an interchain cysteine ​​disulfide bond. Compositions comprising two or more polypeptides of the invention can consist of polypeptides of the same or nearly the same species (e.g., homodimers) or polypeptides of different species (e.g., heterodimers). Compositions having multiple linked polypeptides of the invention can have one or more identical or non-identical mutant CTLA-4 polypeptides of the invention in the polypeptide chains, as described above.

[0250] In some embodiments, the immunomodulatory protein comprises a mutant CTLA-4 polypeptide attached to an immunoglobulin Fc (producing an "immunomodulatory Fc fusion," such as a "CTLA-4-Fc variant fusion," also referred to as a CTLA-4 vIgD-Fc fusion). In some embodiments, attachment of the mutant CTLA-4 polypeptide is at the N-terminus of the Fc. In some embodiments, attachment of the mutant CTLA-4 polypeptide is at the C-terminus of the Fc. In some embodiments, two or more CTLA-4 variant polypeptides (the same or different) are independently attached at the N-terminus and C-terminus.

[0251] In some embodiments, the Fc is a murine or human Fc. In some embodiments, the Fc is an Fc region of a mammalian or human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc is derived from an IgG1, such as human IgG1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:533, 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:533.

[0252] In some embodiments, the Fc region contains one or more modifications that alter (e.g., reduce) one or more of its normal functions. Generally, the Fc region is involved in effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen binding, which is the primary function of immunoglobulins. Additionally, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing in vivo half-life through conjugation to the FcRn receptor in vivo. In some embodiments, such functions can be reduced or altered in the Fc for use with the provided Fc fusion proteins.

[0253] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the CTLA-4-Fc variant fusions provided herein, thereby creating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations to the Fc sequence that can alter effector function. For example, WO2000 / 042072, WO2006 / 019447, WO2012 / 125850, WO2015 / 107026, US2016 / 0017041, and Shields et al., J. Biol. Chem., 276(9):6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcRs. The contents of these publications are specifically incorporated herein by reference.

[0254] In some embodiments, the provided variant CTLA-4-Fc fusions contain an Fc region that exhibits reduced effector function, making them desirable candidates for applications in which the in vivo half-life of the CTLA-4-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 ensure that the CTLA-4-Fc variant fusion lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., 9:457-492 (1991).

[0255] Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Natl. Acad. Sci. USA, 83(18):7059-7063 (1986)) and Hellstrom et al., Proc. Natl. Acad. Sci. USA, 82(5):1499-1502 (1985) and U.S. Pat. No. 5,821,337 (see, Bruggemann et al., J. Exp. Med. 166(5):1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA, 95(2):652-656 (1998).

[0256] C1q binding assay can also be carried out to confirm that CTLA-4-Fc mutant fusion cannot bind to C1q and therefore lacks CDC activity.For example, see the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402.To evaluate complement activation, CDC assay can be carried out (for example, see Gazzano-Santoro et al., J.Immunol.Methods,202:163(1997); Cragg et al., Blood,101(3):1045-1052(2003); and Cragg and Glennie, Blood,103(7):2738-2743(2004)). FcRn binding and in vivo clearance / half-life determinations can also be made using methods known in the art (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769 (2006)).

[0257] CTLA-4-Fc variant fusions with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions of two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant, which contains substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0258] In some embodiments, the Fc region of the CTLA-4-Fc variant fusion has an Fc region in which any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with a different amino acid compared to the native Fc region. Such alterations in the Fc region are not limited to the alterations described above, but include, for example, deglycosylated chains (N297A and N297Q) described in Stohl, Curr. Opin. Biotechnol., 20(6):685-691 (2009), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K. , changes such as IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E, changes such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO2008 / 092117, amino acid insertions at positions 233, 234, 235, and 237 (indicated by EU numbering), and changes at sites described in WO2000 / 042072.

[0259] Certain Fc mutants with improved or diminished binding have been described (see, e.g., U.S. Patent No. 6,737,056, WO2004 / 056312, WO2006019447, and Shields et al., J. Biol. Chem., 276(9):6591-6604 (2001)).

[0260] In some embodiments, CTLA-4-Fc variant fusions are provided that include a variant Fc region containing one or more amino acid substitutions that improve half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO2015107026. Such antibodies include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with a substitution of one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 according to EU numbering, e.g., a substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0261] In some embodiments, the Fc region of the CTLA-4-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L according to EU numbering. In some embodiments, the Fc region of the CTLA-4-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S, and / or C229S according to EU numbering. In some embodiments, the Fc region of the CTLA-4 variant fusion comprises one or more amino acid substitutions R292C and V302C according to EU numbering. See also Duncan & Winter, Nature, 332(6166):738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0262] In some embodiments, the wild-type IgG1 Fc can be the Fc set forth in SEQ ID NO:533, which has an allotype containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering (e.g., the f allotype). In other embodiments, the wild-type IgG1 Fc contains amino acids of the human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, as set forth in SEQ ID NO:638. Thus, in some cases, the Fc provided herein can contain amino acid substitutions E356D and M358L to reconstitute residues of allotype G1 m1 (e.g., the alpha allotype). In some aspects, the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or to render the Fc inactive with respect to Fc effector function. Exemplary effectorless or inactive mutations include those described herein. Among the effector-less mutations that may be included in the Fc of the constructs provided herein are L234A, L235E, and G237A according to EU numbering. In some embodiments, the wild-type Fc is further modified by removal of one or more cysteine ​​residues, such as by replacing the cysteine ​​residue with a serine residue at position 220 (C220S) according to EU numbering. Exemplary inactive Fc regions with reduced effector function are set forth in SEQ ID NO:526 and SEQ ID NO:438 or 439, which are based on the allotypes set forth in SEQ ID NO:533 or SEQ ID NO:638, respectively. In some embodiments, the Fc regions used in the constructs provided herein may further lack a C-terminal lysine residue.

[0263] In some embodiments, changes are made in the Fc region that result in reduced C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164(8):4178-4184 (2000).

[0264] In some embodiments, a CTLA-4-Fc variant fusion is provided comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from an IgG1, such as human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:533. In some embodiments, the Fc contains at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO:533 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C according to the numbering of SEQ ID NO:533 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification according to the numbering of SEQ ID NO:533 (corresponding to C220S according to EU numbering). For example, in some embodiments, the variant Fc region comprises the following amino acid modification:

[0265] and V297G according to EU numbering and one or more of the following amino acid modifications C220S, R292C, or V302C (which, relative to SEQ ID NO:533, corresponds to N82G and one or more of the following amino acid modifications C5S, R77C, or V87C), e.g., the Fc region comprises the sequence set forth in SEQ ID NO:440. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E, or G237A, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:441. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:442. In some embodiments, the variant Fc comprises one or more of the amino acid modifications C220S, L234A, L235E, G237A, E356D, or M358L, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:439.

[0266] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO:533 (corresponding to K447del according to EU numbering). In some aspects, such Fc regions may additionally include one or more additional modifications, e.g., amino acid substitutions, such as any of those described. Examples of such Fc regions are set forth in SEQ ID NOs:438, 526, 527, or 528.

[0267] In some embodiments, a CTLA-4-Fc variant fusion is provided that comprises a variant Fc region, wherein the variant Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs:438-442 or 526-528, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:438-442 or 526-528.

[0268] In some embodiments, the Fc is derived from an IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:529, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:529.

[0269] In some embodiments, the Fc comprises the amino acid sequence of human IgG4 set forth in SEQ ID NO: 530, 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: 530. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1 and exhibits reduced aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which the arginine at position 409 according to the EU index proposed by Kabat et al. of human IgG4 is replaced with lysine and exhibits reduced aggregate formation (see, e.g., U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between the therapeutic antibody and endogenous IgG4 by Fab arm exchange (see, e.g., Labrijin et al. (2009), Nat. Biotechnol., 27(8)767-71). In some embodiments, the Fc comprises the amino acid sequence set forth in human IgG4 with S228P set forth in SEQ ID NO:531, 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:531.

[0270] In some embodiments, the mutant CTLA-4 polypeptide is indirectly linked to the Fc sequence. In some embodiments, the mutant CTLA-4 polypeptide is indirectly linked to the Fc sequence, such as via a linker. In some embodiments, one or more "peptide linkers" link the mutant CTLA-4 polypeptide and the Fc domain. In some embodiments, the peptide linker can be a single amino acid residue or longer in length. In some embodiments, the peptide linker has at least one amino acid residue, but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is three alanines (AAA). In some embodiments, the linker is (in single letter amino acid code) GGGGS ("4GS", SEQ ID NO:535) or a multimer of a 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers, such as set forth in SEQ ID NO:537 (2xGGGGS) or SEQ ID NO:536 (3xGGGGS). In some embodiments, the linker (in single letter amino acid code) is GSGGGGS (SEQ ID NO:534). In some embodiments, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA, SEQ ID NO:538).

[0271] In some embodiments, the mutant CTLA-4 polypeptide comprises any of the CTLA-4-Fc sequences set forth in SEQ ID NOs: 286-379, 381-386, or 388-437. In some embodiments, the mutant CTLA-4 polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity, to any of the CTLA-4-Fc sequences set forth in SEQ ID NOs: 286-379, 381-386, or 388-437, and contains an amino acid modification, e.g., a substitution, that is not present in wild-type or unmodified CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific-binding fragment of any of the CTLA-Fc sequences set forth in SEQ ID NOs:286-379, 381-386, or 388-437, and contains an amino acid modification, e.g., a substitution, that is not present in wild-type or unmodified CTLA-4.

[0272] In some embodiments, the mutant CTLA-4-Fc fusion protein is a dimer formed by two mutant CTLA-4 Fc polypeptides linked to the Fc domain. In some embodiments, the dimer is a homodimer, in which the two mutant CTLA-4 Fc polypeptides are the same. In some embodiments, the dimer is a heterodimer, in which the two mutant CTLA-4 Fc polypeptides are different.

[0273] Nucleic acid molecules encoding mutant CTLA-4-Fc fusion proteins are also provided. In some embodiments, for production of Fc fusion proteins, nucleic acid molecules encoding the mutant CTLA-4-Fc fusion proteins are inserted into an appropriate expression vector. The resulting mutant CTLA-4-Fc fusion proteins can be expressed in host cells where assembly between the Fc domains occurs via interchain disulfide bonds formed between the Fc portions, resulting in dimeric, such as bivalent, mutant CTLA-4-Fc fusion proteins.

[0274] The resulting Fc fusion proteins can be easily purified by affinity chromatography over protein A or protein G columns. For heterodimer formation, additional purification steps may be required. For example, when two nucleic acids encoding different mutant CTLA-4 polypeptides are transformed into cells, heterodimer formation must be achieved biochemically, because mutant CTLA-4 molecules bearing the Fc domain are similarly expressed as disulfide-linked homodimers. Therefore, homodimer formation can be reduced under conditions that favor cleavage of interchain disulfides but do not affect intrachain disulfides. In some cases, different mutant CTLA-4 Fc monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing Fc fusion molecules containing mutant CTLA-4 polypeptides using the knob-into-hole method described below.

[0275] B. Stacked molecules with additional IgSF domains In some embodiments, the immunomodulatory protein may contain any of the mutant CTLA-4 polypeptides provided herein linked, directly or indirectly, to one or more other immunoglobulin superfamily (IgSF) domains (also referred to as "stacked" immunomodulatory protein constructs, and "type II" immunomodulatory proteins). In some aspects, this may create a unique multidomain immunomodulatory protein that binds to two or more cognate binding partners, such as three or more, thereby providing multi-targeted modulation of the immune synapse.

[0276] In some embodiments, the immunomodulatory protein comprises a combination ("non-wild-type combination") and / or sequence ("non-wild-type sequence" or "non-wild-type permutation") of a mutant CTLA-4 domain with one or more other affinity-modified and / or non-affinity-modified IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) that are not found in a wild-type IgSF family member. In some embodiments, the immunomodulatory protein contains two, three, four, five, or six immunoglobulin superfamily (IgSF) domains, and at least one of the IgSF domains is a mutant CTLA-4 IgSF domain (vIgD of CTLA-4) according to the description provided.

[0277] In some embodiments, the sequence of the additional IgSF domain may be a modified IgSF domain containing one or more amino acid modifications, e.g., substitutions, compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain may be non-affinity modified (e.g., wild-type) or affinity modified. In some embodiments, the unmodified or wild-type IgSF domain may be from mouse, rat, cynomolgus monkey, or human origin, or a combination thereof. In some embodiments, the additional IgSF domain may be an IgSF domain of an IgSF family member listed in Table 1. In some embodiments, the additional IgSF domain may be an affinity-modified IgSF domain containing one or more amino acid modifications, e.g., substitutions, compared to an IgSF domain contained in an IgSF family member listed in Table 1.

[0278] In some embodiments, the additional IgSF domain is a member of the signal-regulatory protein (SIRP) family, triggering receptor-like receptors expressed on myeloid cells. The affinity or non-affinity modified IgSF domains are contained in IgSF family members of a family selected from the following: the T-cell immunoglobulin-like (TREML) family, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family, the butyrophilin family, the B7 family, the CD28 family, the V-set and immunoglobulin domain-containing (VSIG) family, the V-set transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signaling lymphocyte activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the natural cytotoxicity-inducing receptor (NCR) family, the T-cell immunoglobulin and mucin (TIM) family, or the killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domains are independently selected from the group consisting of CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOSLG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3), VTCN1 (B7-H4), CD28, CTLA4, PDCD1 (TIGIT), ICOS, B Derived from an IgSF protein selected from the group consisting of TLA (CD272), CD4, CD8A (CD8-alpha), CD8B (CD8-beta), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NCR3 (NKp30).

[0279] The number of such non-affinity-modified or affinity-modified IgSF domains (whether in non-wild-type combinations or non-wild-type sequences) present in the "stacked" immunomodulatory protein construct is at least 2, 3, 4, or 5, and in some embodiments is exactly 2, 3, 4, or 5 IgSF domains (determining the number of affinity-modified IgSF domains disregards any non-specific binding fragment sequences thereof and / or substantially immunologically inactive fragment sequences thereof).

[0280] In some embodiments of the layered immunomodulatory proteins provided herein, the number of IgSF domains is at least 2, and the number of affinity modified and non-affinity modified IgSF domains is each independently at least 0, 1, 2, 3, 4, 5, or 6. Thus, the number of affinity modified IgSF domains and the number of non-affinity modified IgSF domains can be exactly or at least 2:0 (affinity modified:wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1, respectively (affinity modified IgSF domains:non-affinity modified IgSF domains).

[0281] In some embodiments of the stacked immunomodulatory protein, at least two of the non-affinity modified and / or affinity modified IgSF domains are identical IgSF domains.

[0282] In some embodiments of stacked immunomodulatory proteins, the non-affinity-modified and / or affinity-modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity-modified IgSF domains specifically bind to different cognate binding partners under specific binding conditions and are "non-identical" regardless of whether the engineered wild-type or non-modified IgSF domains are the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein may include at least one IgSF domain sequence that originates from and is unique to one CTLA-4 and at least one second IgSF domain sequence that originates from and is unique to another IgSF family member other than CTLA-4, where the IgSF domains of the immunomodulatory protein are non-affinity-modified and / or affinity-modified. However, in alternative embodiments, the two non-identical IgSF domains originate from the same IgSF domain sequence, but at least one is affinity-modified to specifically bind to a different cognate binding partner.

[0283] In some embodiments, the provided immunomodulatory proteins, in addition to containing a mutant CTLA-4 polypeptide, also contain at least one, two, three, four, five, or six additional immunoglobulin superfamily (IgSF) domains, such as the IgD domain of an IgSF family member listed in Table 1. In some embodiments, the provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the provided immunomodulatory proteins contain at least two additional IgSF domains (e.g., a second and third IgSF domains). In some embodiments, the provided immunomodulatory proteins contain at least three additional IgSF domains (e.g., a second, third, and fourth IgSF domains). In some embodiments, the provided immunomodulatory proteins contain at least four additional IgSF domains (e.g., a second, third, fourth, and fifth IgSF domains). In some embodiments, the provided immunomodulatory proteins contain at least five additional IgSF domains (e.g., a second, third, fourth, fifth, and sixth IgSF domains). In some embodiments, the provided immunomodulatory proteins contain at least six additional IgSF domains (e.g., the second, third, fourth, fifth, sixth, and seventh IgSF domains). In some embodiments, each of the IgSF domains in the immunomodulatory protein is different. In some embodiments, at least one of the additional IgSF domains is the same as at least one other IgSF domain in the immunomodulatory protein. In some embodiments, each of the IgSF domains is from or derived from a different IgSF family member. In some embodiments, at least two of the IgSF domains are from or derived from the same IgSF family member.

[0284] In some embodiments, the additional IgSF domain comprises an IgV domain or an IgC (e.g., IgC2) domain, or a specific-binding fragment of an IgV domain, or a specific-binding fragment of an IgC (e.g., IgC2) domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgV domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgC (e.g., IgC2) domain. In some embodiments, the additional IgSF domain is or comprises a specific-binding fragment of an IgV domain. In some embodiments, the additional IgSF domain is or comprises a specific-binding fragment of an IgC (e.g., IgC2) domain. In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (same) IgSF member. For example, in some aspects, the immunomodulatory protein contains the ECD or a portion thereof of an IgSF member containing a full-length IgV domain and a full-length IgC (e.g., IgC2) domain, or a specific-binding fragment thereof.

[0285] In some embodiments, provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second or, optionally, even a third IgSF domain), wherein the at least one additional, e.g., second or third, IgSF domain is an IgSF domain set forth in a wild-type or unmodified IgSF domain contained in the sequence of amino acids set forth in any of SEQ ID NOs: 1, 443-469, or a specific-binding fragment thereof. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain.

[0286] In some embodiments, the provided immunomodulatory proteins, in addition to containing a mutant CTLA-4 polypeptide, also contain at least one additional affinity-modified IgSF domain (e.g., a second, or optionally, a third affinity-modified IgSF domain), where the at least one additional IgSF domain is a vIgD that contains one or more amino acid modifications (e.g., substitutions, deletions, or mutations) compared to the IgSF domain in a wild-type or unmodified IgSF domain, such as the IgSF domain in an IgSF family member listed in Table 1. In some embodiments, the additional, e.g., second or third affinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified IgSF domain, or a specific-binding fragment thereof, contained in the sequence of amino acids set forth in any of SEQ ID NOs: 1, 443-469.

[0287] In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain. In some embodiments, an additional, e.g., second or third, IgSF domain is an affinity-modified IgV domain and / or an IgC domain. In some embodiments, one or more additional IgSF domains are affinity-modified IgSF domains containing an IgV domain and / or an IgC (e.g., IgC2) domain, or a specific-binding fragment of an IgV domain and / or a specific-binding fragment of an IgC (e.g., IgC2) domain, wherein the IgV and / or IgC domain contains an amino acid modification (e.g., substitution). In some embodiments, one or more additional affinity-modified IgSF domains contain an IgV domain containing an amino acid modification (e.g., substitution). In some embodiments, the one or more additional affinity-modified IgSF domains comprise an IgSF domain present in the ECD or portion of the ECD of a corresponding unmodified IgSF family member, such as a full-length IgV domain and a full-length IgC (e.g., IgC2) domain, or a specific-binding fragment thereof, wherein one or both of the IgV and IgC contain an amino acid modification (e.g., a substitution).

[0288] In some embodiments, the provided immunomodulatory proteins contain at least one additional (e.g., a second, or possibly a third, IgSF domain, etc.) IgSF domain that is vIgD that contains one or more amino acid substitutions compared to a wild-type or unmodified IgSF domain other than CTLA-4 (e.g., IgV).

[0289] In some embodiments, two or more IgSF domains, including the vIgD of CTLA-4 and one or more additional IgSF domains (e.g., a second mutant IgSF domain) from another IgSF family member, are covalently or non-covalently linked. Multiple non-affinity-modified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be directly covalently linked to each other. In some embodiments, two or more IgSF domains are linked directly or indirectly via a linker or the like. In some embodiments, an intervening range of one or more amino acid residues indirectly covalently links the IgSF domains to each other. Linkage can be via the N-terminus to the C-terminal residue. In some embodiments, linkage can be via the side chain of an amino acid residue that is not located at the N-terminus or C-terminus of the IgSF domain. Thus, linkage can be via terminal or internal amino acid residues, or a combination thereof.

[0290] In some embodiments, the immunomodulatory protein contains at least two IgSF domains, each linked directly or indirectly via a linker. In some embodiments, the immunomodulatory protein contains at least three immunomodulatory proteins, each linked directly or indirectly via a linker. Various configurations are shown in Figures 6A and 6B.

[0291] In some embodiments, one or more "peptide linkers" connect the vIgD of CTLA-4 and an additional IgSF domain (e.g., a second mutant IgSF domain). In some embodiments, the peptide linker can be longer than a single amino acid residue. In some embodiments, the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is (in the single letter amino acid code) GGGGS ("4GS", SEQ ID NO:535) or a multimer of a 4GS linker, such as two, three, four, or five repeats of the 4GS linker. In some embodiments, the peptide linker is (GGGGS)2 (SEQ ID NO:537) or (GGGGS)3 (SEQ ID NO:536). In some embodiments, the linker can also include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof, see e.g., SEQ ID NO:538). In some embodiments, the number of each consecutive alanine residue is 2, 3, 4, 5, or 6 alanines.

[0292] In some embodiments, the non-affinity-modified and / or affinity-modified IgSF domains are linked by a "wild-type peptide linker" inserted at the N-terminus and / or C-terminus of the second non-affinity-modified and / or affinity-modified IgSF domain. These linkers are also referred to as leading sequences (N-terminal to the non-affinity-modified or affinity-modified IgSF domain) or trailing sequences (C-terminal to the non-affinity-modified or affinity-modified IgSF domain), and sequences present in the wild-type protein that extend just outside the structural predictions of the Ig-fold structure of IgSF. In some embodiments, a "wild-type linker" is an amino acid sequence that occurs after the signal sequence but before the IgSF domain, such as a defined IgV domain, in the amino acid sequence of the wild-type protein. In some embodiments, a "wild-type" linker is an amino acid sequence that occurs immediately after the IgSF domain, such as immediately after the defined IgV domain, but before the IgC domain in the amino acid sequence of the wild-type protein. These linker sequences may contribute to the correct folding and function of adjacent IgSF domains.

[0293] In some embodiments, there is a leading peptide linker inserted at the N-terminus of a first IgSF domain and / or a trailing sequence inserted at the C-terminus of a first non-affinity-modified and / or affinity-modified IgSF domain. In some embodiments, there is a second leading peptide linker inserted at the N-terminus of a second IgSF domain and / or a second trailing sequence inserted at the C-terminus of a second non-affinity-modified and / or affinity-modified IgSF domain. When the first and second non-affinity-modified and / or affinity-modified IgSF domains are derived from the same parent protein and are connected in the same orientation, the wild-type peptide linkers between the first and second non-affinity-modified and / or affinity-modified IgSF domains do not overlap. For example, when the first trailing wild-type peptide linker and the second leading wild-type peptide linker are the same, the type II immunomodulatory protein does not include either the first trailing wild-type peptide linker or the second leading wild-type peptide linker.

[0294] In some embodiments, the type II immunomodulatory protein comprises a first leading wild-type peptide linker inserted at the N-terminus of the first non-affinity modified and / or affinity modified IgSF domain, wherein the first leading wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the interfering sequence in the wild-type protein from which the first non-affinity modified and / or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as the signal peptide or IgSF domain). In some embodiments, the first leading wild-type peptide linker comprises the entire interfering sequence in the wild-type protein from which the first non-affinity modified and / or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as the signal peptide or IgSF domain).

[0295] In some embodiments, the type II immunomodulatory protein further comprises a first trailing wild-type peptide linker inserted at the C-terminus of the first non-affinity-modified and / or affinity-modified IgSF domain, wherein the first trailing wild-type peptide linker comprises at least 5 (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from an interfering sequence in the wild-type protein where the first non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain). In some embodiments, the first trailing wild-type peptide linker comprises the entire interfering sequence in the wild-type protein where the first non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain).

[0296] In some embodiments, the type II immunomodulatory protein further comprises a second leading wild-type peptide linker inserted at the N-terminus of the second non-affinity-modified and / or affinity-modified IgSF domain, wherein the second leading wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from the interfering sequence in the wild-type protein from which the second non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as the signal peptide or IgSF domain). In some embodiments, the second leading wild-type peptide linker comprises the entire interfering sequence in the wild-type protein from which the second non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as the signal peptide or IgSF domain).

[0297] In some embodiments, the type II immunomodulatory protein further comprises a second trailing wild-type peptide linker inserted at the C-terminus of the second non-affinity-modified and / or affinity-modified IgSF domain, wherein the second trailing wild-type peptide linker comprises at least 5 (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from an interfering sequence in the wild-type protein where the second non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain). In some embodiments, the second trailing wild-type peptide linker comprises the entire interfering sequence in the wild-type protein where the second non-affinity-modified and / or affinity-modified IgSF domain is derived between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain).

[0298] In some embodiments, two or more IgSF domains, including the vIgD of CTLA-4 and one or more additional IgSF domains (e.g., second and / or third variant IgSF domains) from another IgSF family member, are linked or attached to an Fc to form an Fc fusion that, in some aspects, can generate a dimeric multidomain stacked immunomodulatory protein upon expression in a cell. Thus, dimeric multidomain immunomodulatory proteins are also provided.

[0299] In some embodiments, the mutant CTLA-4 polypeptide and one or more additional IgSF domains are independently linked, directly or indirectly, to the N-terminus or C-terminus of the Fc region. In some embodiments, the mutant CTLA-4 polypeptide and at least one of the one or more additional IgSF domains are linked, directly or indirectly, and one of the mutant CTLA-4 and one of the one or more additional IgSF domains are also linked, directly or indirectly, to the N-terminus or C-terminus of the Fc region. In some embodiments, the N-terminus or C-terminus of the Fc region is linked to the mutant CTLA-4 polypeptide or one or more additional IgSF domains, and the other of the N-terminus or C-terminus of the Fc region is linked to the other of the CTLA-4 variants or the other of the one or more additional IgSF domains. In some embodiments, the link to the Fc is via a peptide linker, e.g., a peptide linker as described above. In some embodiments, the link between the mutant CTLA-4 and the second IgSF domain is via a peptide linker, e.g., a peptide linker as described above. In some embodiments, the linkage between the mutant CTLA-4 and one or more additional IgSF domains is via a peptide linker, e.g., a peptide linker as described above. In some embodiments, the vIgD, one or more additional IgSF domains, and the Fc domain of CTLA-4 can be linked together in any of a number of configurations, as illustrated in Figures 6A and 6B. Exemplary configurations are described in the Examples.

[0300] In some embodiments, the stacked immunomodulatory protein is a dimer formed by two immunomodulatory Fc-fusion polypeptides. Nucleic acid molecules encoding any of the stacked immunomodulatory proteins are also provided. In some embodiments, the dimeric multidomain stacked immunomodulatory protein can be produced in a cell by expression, or in some cases co-expression, of stacked immunomodulatory Fc-region polypeptides, as described above, in conjunction with the creation of the dimeric Fc-fusion protein.

[0301] In some embodiments, the dimeric multidomain laminated immunomodulating protein is bivalent with respect to each Fc subunit, monovalent with respect to each subunit, or bivalent with respect to one subunit and tetravalent with respect to the other.

[0302] In some embodiments, the dimeric multidomain laminated immunomodulatory protein is a homodimeric multidomain laminated Fc protein. In some embodiments, the dimeric multidomain laminated immunomodulatory protein comprises a first laminated immunomodulatory Fc-fusion polypeptide and a second laminated immunomodulatory Fc-fusion polypeptide, wherein the first and second polypeptides are the same.

[0303] In some embodiments, the multidomain laminated molecule contains a first Fc fusion polypeptide containing a mutant CTLA-4 and a second IgSF domain, and a second Fc fusion polypeptide containing a mutant CTLA-4 and a second IgSF domain. In some embodiments, the multidomain laminated molecule contains a first Fc fusion polypeptide containing a mutant CTLA-4, a second IgSF domain, and a third IgSF domain, and a second Fc fusion polypeptide containing a mutant CTLA-4, a second IgSF domain, and a third IgSF domain. In some embodiments, the Fc portion of the first and / or second fusion polypeptide can be any Fc described above. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same.

[0304] In some embodiments, the multidomain laminate molecule is a heterodimer comprising two different Fc fusion polypeptides, e.g., a first and a second Fc polypeptide, at least one of which is an Fc fusion polypeptide containing at least one mutant CTLA-4 polypeptide and / or at least one of which is an Fc polypeptide containing a second IgSF domain (e.g., a second mutant IgSF domain). In some embodiments, the first or second Fc fusion polypeptide further contains a third IgSF domain (e.g., a third mutant IgSF domain). In some embodiments, the multidomain laminate molecule contains a first Fc fusion polypeptide containing a mutant CTLA-4 and a second Fc fusion polypeptide containing a second IgSF domain, and optionally, the first or second Fc fusion polypeptide additionally contains a third IgSF domain. In some embodiments, a multidomain laminate molecule contains a first Fc fusion polypeptide containing a mutant CTLA-4, a second IgSF domain, and optionally a third IgSF domain, and a second Fc fusion polypeptide that is not linked to either the mutant CTLA-4 polypeptide or an additional IgSF domain. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are different. In some embodiments, a multidomain laminate molecule contains a first fusion Fc polypeptide containing one, two, three, four, or more mutant CTLA-4 polypeptides and one, two, three, four, or more additional IgSF domains, and the total number of IgSF domains in the first laminated Fc fusion polypeptide is more than two, three, four, five, six, or more. In one example of such an embodiment, the second stacked Fc-fusion polypeptide contains one, two, three, four or more mutant CTLA-4 polypeptides and one, two, three, four or more additional IgSF domains, wherein the total number of IgSF domains in the second stacked Fc-fusion polypeptide is greater than two, three, four, five or six.In another example of such an embodiment, the second Fc polypeptide is not linked to either a mutant CTLA-4 polypeptide or an additional IgSF domain.

[0305] In some embodiments, the heterodimeric stack molecule contains a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are different. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first mutant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second mutant IgSF domain), and a second Fc polypeptide fusion containing an Fc region and the other of the first mutant CTLA-4 polypeptide or the second IgSF domain. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first mutant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second mutant IgSF domain), and a second Fc polypeptide fusion containing an Fc region and both a first mutant CTLA-4 polypeptide and a second IgSF domain (e.g., a second mutant IgSF domain), but in a different orientation or configuration than the first Fc region. In some embodiments, the first and / or second Fc fusion polypeptides also contain a third IgSF domain (e.g., a third mutant IgSF domain).

[0306] In some embodiments, the Fc domain of one or both of the first and second stacked immunomodulatory Fc fusion polypeptides comprises a modification (e.g., substitution) that alters the interface of the Fc molecule to facilitate and / or promote heterodimerization. In some embodiments, the modification comprises the introduction of a protrusion (convex) into the first Fc polypeptide and a cavity (concave) into the second Fc polypeptide, such that the protrusion is positionable within the cavity to promote complexation of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create the protrusion or cavity in the polypeptide are typically interface amino acids that interact or contact one or more amino acids at the interface of the second polypeptide.

[0307] In some embodiments, a sequence of amino acids is added before the Fc sequence for constructs in which the Fc sequence is the N-terminal portion of the sequence. Optionally, a sequence of amino acids HMSSVSAQ (SEQ ID NO:539) is added immediately before the Fc sequence for constructs in which the Fc sequence is the N-terminal portion of the sequence. In some embodiments, the heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region (convex) and a first mutant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second mutant IgSF domain), and a second Fc polypeptide fusion containing an Fc region (concave) and a stuffer sequence HMSSVSAQ (SEQ ID NO:539) added immediately before the Fc region of both the first and second Fc polypeptide fusions.

[0308] In some embodiments, a first polypeptide modified to contain a protruding (concave) amino acid comprises the replacement of a native or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide, and thus can locate in a compensatory cavity (concave) in the adjacent interface of the second polypeptide. Most often, the replacement amino acid is an amino acid with a larger side chain volume than the original amino acid residue. Those skilled in the art will know how to determine and / or evaluate the properties of amino acid residues to identify amino acids that are ideal replacement amino acids for creating protrusions. In some embodiments, replacement residues for forming protrusions are naturally occurring amino acid residues, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0309] In some embodiments, the second polypeptide modified to contain a cavity (concave) contains an amino acid with at least one side chain that is concave from the interface of the second polypeptide and a replacement of the native or original amino acid, thereby accommodating a corresponding protrusion from the interface of the first polypeptide. Most often, the replacement amino acid is an amino acid with a smaller side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify amino acids that are ideal replacement residues for cavity formation. Generally, replacement residues for cavity formation are naturally occurring amino acids, including, for example, alanine (A), serine, threonine (T), and valine (V). In some examples, the original amino acid identified for replacement is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0310] The CH3 interface of human IgG1, for example, contains 16 residues on each domain located on four antiparallel β-strands that bury 1090 Å from each surface (see, for example, Deisenhofer (1981), Biochemistry, 20(9):2361-2370; Miller (1990), J Mol. Biol., 216(4):965-973; Ridgway et al. (1996), Prot. Eng., 9(7):617-621; U.S. Pat. No. 5,731,168). Modifications of the CH3 domain to create protrusions or cavities are described, for example, in U.S. Pat. No. 5,731,168, International Patent Application Nos. WO98 / 50431 and WO2005 / 063816, and Ridgway et al. (1996), Prot. Eng., 9(7):617-621. In some instances, modifications of the CH3 domain to create protrusions or cavities are typically targeted to residues located on the two central antiparallel β-strands, with the goal of minimizing the risk that the resulting protrusions may be accommodated by protruding into the surrounding solvent rather than by a compensatory cavity in the partner CH3 domain.

[0311] In some embodiments, the heterodimeric molecule contains a T366W mutation in the CH3 domain of the "convex chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "concave chain". In some cases, additional interchain disulfide bridges between CH3 domains can also be used, for example, by introducing a Y349C mutation into the CH3 domain of the "convex" or "concave" chain, and an E356C or S354C mutation into the CH3 domain of the other chain (Merchant et al. (1998), Nature Biotech., 16(7):677-681). In some embodiments, the heterodimeric molecule contains an S354C, T366W mutation in one of the two CH3 domains, and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises an E356C, T366W mutation in one of the two CH3 domains and a Y349C, and a T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, T366W mutation in one of the two CH3 domains and an E356C, and a T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, T366W mutation in one of the two CH3 domains and an S354C, and a T366S, L368A, Y407V mutation in the other of the two CH3 domains. Other examples of knob-in-hole techniques are known in the art, for example, as described in EP1870459A1.

[0312] In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, such as any described above, hi some embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function.

[0313] In some embodiments, Fc variants containing CH3 protrusion (convex) or cavity (concave) modifications can be conjugated to stacked immunomodulatory polypeptides either at their N-terminus or C-terminus of the first and / or second stacked immunomodulatory polypeptides to form a fusion polypeptide. Linkage can be direct or indirect via a linker. Typically, convex and concave molecules are created by co-expression of a first stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 protrusion modifications with a second stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 cavity modifications.

[0314] C. Conjugates and Fusions of Mutant Polypeptides and Immunomodulatory Proteins In some embodiments, the variant polypeptides provided herein, which are immunomodulatory proteins comprising a variant of an Ig domain of the IgSF family (vIgD), can be conjugated or fused, directly or indirectly, to a moiety, such as an effector moiety, such as another protein, to form a conjugate (an "IgSF conjugate"). In some embodiments, attachment can be covalent or noncovalent, for example, via a biotin-streptavidin noncovalent interaction. In some embodiments, any one or combination of any two or more of the foregoing moieties can be attached to the Fc or the variant CTLA-4 polypeptide, or both. In some embodiments, the provided conjugates, such as fusion polypeptides, can be used in methods and uses for suppressing or attenuating immune responses, such as in connection with the treatment of autoimmune or inflammatory conditions.

[0315] In some embodiments, the moiety can be a targeting moiety, a small molecule drug (a non-polypeptide drug of less than 500 dalton molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressant, an antiproliferative agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent.

[0316] In some embodiments, the effector moiety is a therapeutic agent, such as a therapeutic immunosuppressant or anti-rejection agent, that provides some therapeutic benefit.

[0317] In some embodiments, the effector moiety is a targeting moiety or agent, such as an agent that targets a cell surface antigen, e.g., an antigen on the surface of an immune cell or activated immune cell. In some embodiments, the effector moiety is a label that can generate a detectable signal, either directly or indirectly. In some embodiments, the effector moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.

[0318] In some embodiments, one, two, three, four, five, or more effector moieties, which may be the same or different, may be conjugated, linked, or fused to a mutant polypeptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties may be attached to the mutant polypeptide or immunomodulatory protein using a variety of molecular biological or chemical conjugation and linking methods known in the art and described below. In some embodiments, the effector moieties may be linked or conjugated to the mutant polypeptide or immunomodulatory protein using a linker, such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that is conducive to the conjugation reaction.

[0319] In some embodiments, an IgSF conjugate comprises the following components: (protein or polypeptide), (L)q, and (effector moiety)m, where the protein or polypeptide is any of the described mutant polypeptides or immunomodulatory proteins capable of binding to one or more binding partners as described, L is a linker for joining the protein or polypeptide to the moiety, m is at least 1, and q is 0 or greater, and the resulting IgSF conjugate binds to one or more counter-structure ligands. In certain embodiments, m is 1-4 and q is 0-8.

[0320] In some embodiments, an IgSF conjugate is provided that includes a mutant polypeptide or immunomodulatory protein provided herein conjugated with a targeting agent that binds to a cell surface molecule, e.g., for targeted delivery of the mutant polypeptide or immunomodulatory protein to specific cells. In some embodiments, the targeting agent is a molecule capable of localizing to and binding to molecules present on normal cells / tissues, such as lymphoid tissue, immune cells, such as activated or non-activated immune cells, and / or tissues in an inflammatory environment in a subject. In other words, the IgSF conjugate includes a targeting agent and can bind (directly or indirectly) to a ligand present on cells, such as tumor cells. Targeting agents of the present invention contemplated for use include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind to components of target cells or molecules.

[0321] In some embodiments, the IgSF conjugate, via its targeting agent, will bind to or localize to cellular components of immune cells, tissues in an inflammatory environment, or lymphoid tissues such as the spleen, tonsils, lymphatic vessels, lymph nodes, adenoids, and / or liver tissue, thereby modulating the localized cellularity of the immune response. In some embodiments, the targeting agent facilitates delivery of the conjugated IgSF (e.g., vIgD) to immune cells so that it interacts with its binding partner to alter signaling in nearby immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells) that possess the binding partner and / or a costimulatory receptor for the binding partner. In some embodiments, the localized delivery mediates indirect antagonism or inhibitory activity of CD28 and / or ICOS costimulatory receptors.

[0322] In some embodiments, the targeting agent is an immunoglobulin. As used herein, the term "immunoglobulin" includes natural or artificial monovalent or polyvalent antibodies, including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, single-chain Fvs (scFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present invention), and epitope-binding fragments of any of the above. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules containing an antigen-binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Exemplary immunoglobulin molecules include immunoglobulin molecules directed against CD20, CD25, or CD3, such as anti-CD20, anti-CD25, or anti-CD3 monoclonal antibodies. Other exemplary immunoglobulin molecules include immunoglobulins directed against integrin molecules, such as alpha-4 integrin.

[0323] Antibody targeting moieties of the present invention include antibody fragments, including, but not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), and fragments comprising either the VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region alone or in combination with all or a portion of the foll...

Claims

1. (i) a CTLA-4 extracellular domain (ECD) comprising the amino acid modification L63P, and one or more amino acid modifications selected from the group consisting of G29W, T53S, M56K, N58S, Q82R, M87V, L98Q, and Y105L; CTLA-4 ECD, the amino acids of which are numbered with reference to SEQ ID NO:2; (ii) a first peptide linker; (iii) Fc region, (iv) a second peptide linker, and (v) a portion of a second immunoglobulin superfamily (IgSF) protein, wherein the second IgSF protein is PD-L1, and the portion comprises an IgV domain of PD-L1; Including, The orientation from N-terminus to C-terminus is (a) the CTLA-4 ECD, the first linker, the Fc region, the second linker, and the IgV domain of PD-L1; or (b) the Fc region, the first linker, the CTLA-4 ECD, the second linker, and the IgV domain of PD-L1. That is, Immunomodulatory Fc fusion polypeptides.

2. 2. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the amino acid modifications G29W / N58S / L63P / Q82R / L98Q / Y105L.

3. 2. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the amino acid modifications T53S / L63P / Y105L.

4. 2. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the amino acid modifications M56K / N58S / L63P / M87V / L98Q / Y105L.

5. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the sequence of SEQ ID NO:

93.

6. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the sequence of SEQ ID NO:

579.

7. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the sequence of SEQ ID NO:

586.

8. The polypeptide of claim 1, wherein the portion of PD-L1 does not include an IgC2 domain.

9. 9. The polypeptide of claim 8, wherein the portion of PD-L1 is at least 95% identical to amino acids 24 to 130 of SEQ ID NO:

445.

10. 2. The polypeptide of claim 1, wherein the first peptide linker is GGGGS (one 4GS linker; SEQ ID NO: 535), GSGGGGS (SEQ ID NO: 534), GGGGSGGGGS (two 4GS linker repeats; SEQ ID NO: 537), GGGGSGGGGSGGGGGS (three 4GS linker repeats; SEQ ID NO: 536), four 4GS linker repeats, five 4GS linker repeats, or a combination thereof.

11. 11. The polypeptide of claim 10, wherein the first peptide linker is SEQ ID NO:

534.

12. 2. The polypeptide of claim 1, wherein the second peptide linker is GGGGS (one 4GS linker; SEQ ID NO: 535), GSGGGGS (SEQ ID NO: 534), GGGGSGGGGS (two 4GS linker repeats; SEQ ID NO: 537), GGGGSGGGGSGGGGGS (three 4GS linker repeats; SEQ ID NO: 536), four 4GS linker repeats, five 4GS linker repeats, or a combination thereof.

13. 13. The method of claim 12, wherein the second peptide linker is SEQ ID NO:

536. The polypeptide described above.

14. 2. The polypeptide of claim 1, wherein the Fc region is at least 95% identical to SEQ ID NO:

533.

15. 15. The polypeptide of claim 14, wherein the Fc region comprises one or more amino acid modifications selected from the group consisting of C220S, L234A, L235E, G237A, E356D, M358L, and K447del, according to EU numbering.

16. 2. The polypeptide of claim 1, wherein the Fc region is at least 95% identical to any one of SEQ ID NOs: 438-442 or 526-528.

17. 17. The polypeptide of claim 16, wherein the Fc region is selected from the group consisting of SEQ ID NOs: 438-442, 526, 527, and 528.

18. 18. The polypeptide of claim 17, wherein the Fc region comprises the sequence of SEQ ID NO:

438.

19. 2. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the sequence of SEQ ID NO: 93, the first peptide linker comprises the sequence of SEQ ID NO: 534, the Fc region comprises the sequence of SEQ ID NO: 438, and the second peptide linker comprises the sequence of SEQ ID NO:

536.

20. 2. The polypeptide of claim 1, wherein the CTLA-4 ECD comprises the sequence of SEQ ID NO: 93, 579 or 586, the first peptide linker comprises the sequence of SEQ ID NO: 534, the Fc region comprises the sequence of SEQ ID NO: 438, and the second peptide linker comprises four 4GS linker repeats.

21. 2. The polypeptide of claim 1, wherein the Fc region comprises the sequence of SEQ ID NO: 438, the first peptide linker comprises four 4GS linker repeats, the CTLA-4 ECD comprises the sequence of SEQ ID NO: 93, 579 or 586, and the second peptide linker comprises the sequence of SEQ ID NO: 536.

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