CTLA-4 variant immunomodulatory proteins and uses thereof
By modifying the amino acid composition of the CTLA-4 peptide, the binding affinity with ICOSL, CD80, and/or CD86 is enhanced, solving the problem of low efficiency of existing immunomodulators and achieving a more efficient immunomodulatory effect.
Patent Information
- Application Number
- CN202510950234.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2018-09-19
- Filing Date
- 2018-10-09
- Publication Date
- 2025-11-28
AI Technical Summary
Existing immunomodulators are inefficient and ineffective in regulating immune responses, especially at the immune synapses between antigen-presenting cells and lymphocytes. Improved therapeutics are needed to enhance immunomodulatory effects.
A variant CTLA-4 peptide is provided that enhances binding affinity to the extracellular domains of ICOSL, CD80, and/or CD86 by modifying specific amino acid positions, including amino acid substitution, deletion, or insertion, with the length and sequence identity of the specific binding fragment reaching at least 85%.
It improves the binding affinity of the variant CTLA-4 peptide to ICOSL, CD80 and/or CD86, enhances the immunomodulatory effect, and provides a more effective means of regulating the immune response.
Smart Images
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Abstract
Description
[0001] This application is a divisional of parent application number 201880077176.3, filed on October 9, 2018, with the title “CTLA-4 variant immunomodulatory proteins and uses thereof.”
[0002] Cross Reference to Related Applications
[0003] This application claims priority 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 hereby incorporated by reference in their entirety.
[0004] INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
[0005] This application is filed with an electronic format sequence listing. The sequence listing is provided in the file named 761612002040 SeqList.txt, created on October 8, 2018, having a size of 1,138,300 bytes. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. TECHNICAL FIELD
[0006] The present disclosure relates to immunomodulatory proteins including variant CTLA-4 and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of disease applications, including for treating autoimmune or inflammatory conditions. Compositions and methods of making and using such proteins are provided. BACKGROUND
[0007] Modulating immune responses by intervening in processes that occur in an immune synapse (IS) formed by or between an antigen presenting cell (APC) or target cell and a lymphocyte is of increasing medical interest. Mechanistically, cell surface proteins in the IS can be involved in coordinated and often simultaneous interactions of multiple protein targets with a single protein to which they bind. The occurrence of IS interactions is closely related to the connection of two cells, and a single protein in this structure can interact (possibly simultaneously) with both a protein on the same cell (cis) and a protein on the related cell (trans). Although therapeutic agents that can modulate the IS are known, there remains a need for improved therapeutic agents. Immunomodulatory proteins that meet such needs are provided. SUMMARY
[0008] Provided herein are variant CTLA-4 polypeptides. In some embodiments, provided herein is a variant CTLA-4 polypeptide comprising an IgV domain or specific binding fragment thereof, wherein the variant CTLA-4 polypeptide comprises one or more amino acid modifications in an unmodified CTLA-4 polypeptide or specific binding fragment thereof, wherein the variant CTLA-4 polypeptide specifically binds to the extracellular domain of ICOSL with increased affinity as compared to the unmodified CTLA-4.
[0009] In some embodiments of any of the provided embodiments, the variant CTLA-4 polypeptide comprises one or more amino acid modifications in the unmodified CTLA-4 polypeptide or specific binding fragment thereof, corresponding to one or more positions selected from: 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, with reference to positions set forth in SEQ ID NO: 2.
[0010] In some embodiments of any of the provided embodiments, provided herein is a variant CTLA-4 polypeptide comprising an IgV domain or specific binding fragment thereof, wherein the variant CTLA-4 polypeptide comprises one or more amino acid modifications in an unmodified CTLA-4 polypeptide or specific binding fragment thereof, corresponding to one or more positions selected from: 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, with reference to positions set forth in SEQ ID NO: 2. In some embodiments, the amino acid modifications comprise amino acid substitutions, deletions, or insertions.
[0011] In some of any of the provided embodiments, the unmodified CTLA-4 polypeptide is a mammalian CTLA-4 polypeptide or a specifically binding fragment thereof. In some embodiments, the unmodified CTLA-4 polypeptide is a human CTLA-4 polypeptide or a specifically binding fragment thereof. In some embodiments, the variant CTLA-4 polypeptide comprises an extracellular domain of human CTLA-4, wherein the one or more amino acid modifications are located in one or more residues of the extracellular domain of the unmodified CTLA-4 polypeptide.
[0012] In some of any of the provided embodiments, the unmodified CTLA-4 polypeptide comprises: (i) the amino acid sequence set forth in SEQ ID NO: 2; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2; or (iii) a portion thereof that comprises an IgV domain or a specifically binding fragment of the IgV domain. In some embodiments, the unmodified CTLA-4 comprises the amino acid sequence set forth in SEQ ID NO: 2.
[0013] In some of any of the provided embodiments, the specifically binding fragment of the IgV domain is at least 50, 60, 70, 80, 90, 100, 110, or more amino acids in length; or the specifically binding fragment of the IgV domain comprises at least 80% of the length of the IgV domain as set forth in residues 39-140 of SEQ ID NO: 1.
[0014] In some of any of the provided embodiments, the variant CTLA-4 comprises at most 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.
[0015] In some of any of the provided embodiments, the variant CTLA-4 polypeptide comprises an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 or a specifically binding fragment thereof.
[0016] In some of any of the provided embodiments, the variant CTLA-4 polypeptide specifically binds to the extracellular domain of ICOSL, CD80 and / or CD86 with increased affinity compared to binding of the unmodified CTLA-4 polypeptide to the same extracellular domain.
[0017] Provided herein are variant 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 variant CTLA-4 polypeptide specifically binds to the extracellular domain of human ICOSL, CD80 and / or CD86 with increased affinity compared to the CTLA-4 containing the extracellular domain set forth in SEQ ID NO: 2.
[0018] In some of any of the provided embodiments, the one or more amino acid modifications are selected from 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, T89S, L91R, I93L, I93V, 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.
[0019] In some of any of the provided embodiments, the variant CTLA-4 polypeptide contains one or more amino acid modifications 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 / 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 / T61 S / 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 / P121 S, 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 / M56 K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N5 8S / 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 / L9 8Q / 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 / G2 9W / L98Q / Y105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L, M56T / L98Q / Y105L, L12F / R16H / G29W / M56T / S72G / L98Q / Y105L, G29W / M56T / S72G / L98Q / Y105L and I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L.
[0020] In some embodiments of any of the provided embodiments, the variant CTLA-4 polypeptide contains the amino acid sequence shown in any one of SEQ ID NO:4-97, 99-104, 106-155, or 570-637 or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 polypeptide contains an amino acid sequence exhibiting at least 95% sequence identity with any one of SEQ ID NO:4-97, 99-104, 106-155, or 570-637 or a specific binding fragment thereof and containing one or more amino acid modifications of the corresponding SEQ ID NO compared to, for example, the wild-type or unmodified CTLA-4 shown in SEQ ID NO:2.
[0021] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds specifically to the same extracellular domain with increased affinity compared to the binding of the unmodified CTLA-4 to the extracellular domain of ICOSL.
[0022] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are located at positions corresponding to one or more of the following: 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. In some embodiments, referring to SEQ ID NO:2... At the position shown in NO:2, the one or more amino acid modifications are selected from 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, D6 4E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87T, M87V, T89A, T89 S, I93L, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F, I108V, N110K, Q113H, V116A, I117L or P121S.
[0023] In some embodiments of any of the provided examples, the one or more amino acid modifications are selected from 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, D64V, D65G, I67N, I67T, I67V, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87T, M87V, T89A, T89S, I93L, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F, I108V, N110K, Q113H, I117L and P121S or their conserved amino acid substitutions.
[0024] In some embodiments of any of the provided examples, the one or more amino acid modifications are 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, L63P / S72G / I93L / L98Q / M99L / Y105L, L12I / M55T / M56V / I67T / M99L / L106R / I108 F. 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 / I10 8F、G29W / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、V 10A / L63P / D64V / S72G / L98Q / M99L / Y105L, I18V / A26T / L63P / D64E / L98Q / Y1 05L / 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 / Y105 L、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 / Y105L / I108F, G29W / T53S / M56 K / N58S / L63P / M87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L 98Q / 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, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / M87V / L98 Q. T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L6 3P / 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 / 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 / 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 or L12F / L98Q / Y105L.
[0025] In some embodiments of any of the provided embodiments, the one or more amino acid modifications include one or more modifications located at positions corresponding to one or more positions 12, 26, 63, 98 or 105, and / or refer to the position shown in SEQ ID NO:2, the one or more amino acid modifications containing one or more modifications selected from L12P, L12F, A26T, L63P, L98Q or Y105L.
[0026] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide contains one or more amino acid modifications selected from the following: 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 / I93L / L98Q / M99L / Y105L; L12I / M55T / M56V / I67T / M 99L / L106R / I108F; I18N / A26T / L63H / T89A / L98Q / M99L / Y105L; G29W / N5 8S / L63P / M87T / L98Q / M99L / Y105L; G29W / N58S / L63P / D64N / L98Q / M99L / Y105L; I18T / L63P / S72G / M87K / L98Q / M99L / Y105L; L63P / M87K / M99L / L10 6R; L63P / M99L / Y105L / I108F; G29W / L63P / L98Q / M99L / Y105L; A26T / L63 P / D65G / L98Q / M99L / Y105L; V10A / L63P / D64V / S72G / L98Q / M99L / Y105L; I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K; A19V / G29W / R35K / L 63P / 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 / L106I / I117L; L12F / R16H / G29W / M56T / L98Q / Y105L; A26T / T53S / L63P / L 98Q / 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; P28L / E33V / L63P / S72G / L98Q / M99L / Y105 L; G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105L; I18F / L63P / L98Q / M99L / Y105L / P121S; L63P / L9 8Q / M99L / Y105L / I108V; A26T / A42V / Q45H / I67N / M87K / E97Q / M99L; E33M / L63P / S72G / L98Q / Y105L; G 29W / 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; and G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V.
[0027] In some embodiments of any of the provided embodiments, the variant CTLA-4 peptide binds specifically to the same extracellular domain with increased affinity compared to the binding of the unmodified CTLA-4 to the extracellular domain of CD80. In some embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are located at positions corresponding to one or more of the following: 10, 12, 16, 18, 26, 29, 42, 45, 53, 56, 58, 63, 67, 72, 82, 87, 97, 98, 99, 105, 108, or 121. 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 their conserved amino acid substitutions.
[0028] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are located at a position corresponding to one or more of the following: 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.
[0029] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are selected from 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 / 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.
[0030] In some embodiments of any of the provided embodiments, the one or more amino acid modifications are selected from 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 / Y 105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, V10A / G29W / T5 3S / 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 / N5 8S / 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 selected from 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.
[0031] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds with increased affinity to the extracellular domains of ICOSL and CD80 compared to the binding of the unmodified CTLA-4 to these domains. 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 their conserved amino acid substitutions. 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, and I108V.
[0032] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds specifically to the same extracellular domain with increased affinity compared to the binding of the unmodified CTLA-4 to the extracellular domain of CD86.
[0033] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are located at positions corresponding to one or more of the following: 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. In some embodiments, referring to SEQ ID NO:2 The position shown in NO:2, wherein the one or more amino acid modifications are located at positions corresponding to one or more of the following: 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, T6 1S, L63P, D65G, I67N, I67V, T69A, 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.
[0034] In some embodiments of any of the provided examples, the one or more amino acid modifications are selected from 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, I67V, 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 their conserved amino acid substitutions.
[0035] In some embodiments of any of the provided embodiments, referring to the position shown in SEQ ID NO:2, the one or more amino acid modifications are selected from 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; M56 R / L63P / L98Q / M99L / Y105L; L63P / L98Q / M99L / Y105L / L106I; A26T / M55T / L63P / L98Q / M99L / Y105L; E33V / A42S / M55T / L98Q / M99L / Y105L; G29W / N 58S / L63P / Q82R / L98Q / Y105L; E33M / L63P / S72G / L98Q / Y105L / I117L; A2 6T / I67N / S72G / L98Q / M99L / Y105L; L12F / A26T / L63P / L98Q / Y105L / L106 R;S20N / A26T / L63P / L98Q / M99L / Y105L;G29W / T61I / L63P / S72G / L98Q / M 99L / Y105L; G29W / N58S / L63P / T69I / L98Q / M99L / Y105L; L12P / L63P / S72 G / L98Q / M99L / Y105L / L106N; L63P / T69A / L98Q / M99L / Y105L / L106R / V11 6A; G29W / N58S / L63P / S72G / L98Q / Y105L; G29W / L63P / D65G / S72G / L98Q / Y105L; T53S / M56V / L98Q / Y105L; L63P / S72G / L98Q / Y105L; G29W / T53S / M 56K / L63P / L98Q / Y105L; I18V / G29W / L63P / S72G / L98Q / Y105L; G29W / L63 P / 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;L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S; G29W / L63P / S72G / L98Q / Y105L / P121S; and V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S.
[0036] In some embodiments of any of the provided examples, the one or more amino acid modifications are selected from 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 / L63 P / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63 P / Q82R / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L / I117L, A26T / I67N / S72 G / L98Q / M99L / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, S20N / A26T / L6 3P / L98Q / M99L / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G29W / N58 S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105L / L10 6N, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58S / L63P / S72G / L9 8Q / Y105L, G29W / L63P / D65G / S72G / L98Q / Y105L, T53S / M56V / L98Q / Y105L, L 63P / S72G / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / L106I, L12F / R16H / G 29W / M56T / L98Q / Y105L, G29W / N58S / L63P / S72G / M87V / L98Q / Y105L, G29W / S 72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / N58S / L63P / S72G / L98Q / Y105L / L106V, G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M 99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, L63P / L98Q / M99L / Y105L,A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L, A26T / M55T / L63P / S72G / L 98Q / 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 / Y 105L, E33M / L63P / S72G / L98Q / Y105L / I108F, L12P / R16H / A26T / T61S / L63P / M87V / L 98Q / 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.
[0037] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds with increased affinity to the extracellular domains of CD80 and CD86 compared to the binding of the unmodified CTLA-4 to these domains. 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.
[0038] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds with increased affinity to the extracellular domains of ICOSL and CD86 compared to the binding of the unmodified CTLA-4 to the extracellular domains of ICOSL and CD86.
[0039] In some embodiments of any of the provided examples, 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 their conserved amino acid substitutions.
[0040] In some embodiments of any of the provided examples, 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.
[0041] In some embodiments of any of the provided examples, the variant CTLA-4 peptide binds with increased affinity to the extracellular domains of ICOSL, CD80, and CD86 compared to the binding of the unmodified CTLA-4 to these extracellular domains.
[0042] In some embodiments 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 include modifications 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.
[0043] In some embodiments of any of the provided embodiments, compared to the binding of the unmodified CTLA-4 to the extracellular domain of ICOSL and one or more of the extracellular domains of another of CD80 or CD86, the variant CTLA-4 peptide binds specifically to the extracellular domain of ICOSL with increased affinity and specifically to the extracellular domains of one or more of the other of CD80 or CD86 with decreased affinity.
[0044] In some embodiments of any of the provided embodiments, the increase in binding affinity of the one or more extracellular domains is independently greater than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more.
[0045] In some embodiments of any of the provided embodiments, the reduction in binding affinity of the one or more extracellular domains is independently greater than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more times.
[0046] In some embodiments of any of the provided embodiments, the variant CTLA-4 polypeptide contains the IgV domain or a specific binding fragment thereof. In some embodiments, the IgV domain or a specific binding fragment thereof is the unique CTLA-4 portion of the variant CTLA-4 polypeptide. In some embodiments, the variant CTLA-4 polypeptide contains the amino acid sequence shown in any one of SEQ ID NO:156-285, 603-635, or 637 or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 polypeptide contains an amino acid sequence exhibiting at least 95% sequence identity with any one of SEQ ID NO:156-285, 603-635, or 637 or a specific binding fragment thereof and containing one or more amino acid modifications of the corresponding SEQ ID NO compared to, for example, the wild-type or unmodified CTLA-4 shown in SEQ ID NO:156-285, 603-635, or 637. In some embodiments, the variant CTLA-4 polypeptide contains the amino acid sequence of the extracellular domain shown in any one of SEQ ID NO:4-97, 99-104, 106-155, 569-602 or 636 or its specific binding fragment, or exhibits at least 95% sequence identity with any one of SEQ ID NO:4-97, 99-104, 106-155, 569-602 or 63 and contains one or more amino acid modified amino acid sequences of the corresponding SEQ ID NO shown in any one of SEQ ID NO:4-97, 99-104, 106-155, 569-602 or 636.
[0047] In some embodiments of any of the provided embodiments, the ICOSL is a human ICOSL. In some embodiments, the CD80 is a human CD80. In some embodiments, the CD86 is a human CD86.
[0048] In some embodiments, the variant CTLA-4 peptide is a soluble protein. In some embodiments, the variant CTLA-4 peptide lacks the CTLA-4 transmembrane domain and intracellular signal transduction domain; and / or the variant CTLA-4 peptide cannot be expressed on the cell surface.
[0049] In some embodiments of any of the provided embodiments, the variant CTLA-4 peptide is linked to a polymerizing domain. In some embodiments, the polymerizing domain is an Fc domain with reduced effector function or a variant thereof. In some embodiments, the variant CTLA-4 peptide is linked to an Fc domain with reduced effector function or a variant thereof. In some embodiments, the Fc domain is mammalian, optionally human; or the variant Fc domain contains one or more amino acid modifications compared to an unmodified Fc domain, which is mammalian, optionally human. In some embodiments, the Fc domain or a variant thereof contains an amino acid sequence shown in any one of SEQ ID NO:438-442 or an amino acid sequence exhibiting at least 85% sequence identity with any one of SEQ ID NO:438-442. In some embodiments, the Fc domain contains one or more amino acid modifications selected from the following, each numbered by an EU number: E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del. In some embodiments, the Fc domain includes the amino acid modification L234A / L235E / G237A. In some embodiments, the Fc domain contains the amino acid modification C220S, numbered by an EU number. In some embodiments, the variant CTLA-4 peptide is indirectly linked to the polymerized domain or Fc via a linker, optionally a G4S (Gly4Ser) linker.
[0050] In some embodiments of any of the provided embodiments, the variant CTLA-4 polypeptide linked to the Fc domain contains an amino acid sequence shown in any one of SEQ ID NO:286-379, 381-386 or 388-437 or an amino acid sequence showing at least 85% sequence identity with any one of SEQ ID NO:286-379, 381-386 or 388-437.
[0051] In some embodiments of any of the provided embodiments, the variant CTLA-4 peptide is a transmembrane immunomodulatory protein further comprising a transmembrane domain, optionally wherein the transmembrane domain is directly or indirectly linked to the extracellular domain (ECD) of the variant CTLA-4 peptide or a specific binding fragment thereof. In some embodiments, the transmembrane domain contains an amino acid sequence as shown in residues 162-182 of SEQ ID NO:1 or a functional variant thereof, the functional variant exhibiting at least 85% sequence identity with residues 162-182 of SEQ ID NO:1.
[0052] In some embodiments of any of the provided embodiments, the variant CTLA-4 polypeptide further comprises a cytoplasmic domain, optionally wherein the cytoplasmic domain is directly or indirectly linked to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains a native CTLA-4 cytoplasmic domain, an intracellular signal transduction domain, and / or contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) signal transduction motif. In some embodiments, the cytoplasmic domain contains an amino acid sequence as shown in residues 183-223 of SEQ ID NO:1 or a functional variant thereof, the functional variant exhibiting 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) signal transduction motif and / or contains an intracellular signal transduction domain containing CD3ζ.
[0053] In some embodiments of any of the provided examples, the variant CTLA-4 peptide does not contain a cytoplasmic signaling domain and / or cannot mediate or regulate intracellular signaling when expressed on cells.
[0054] In some embodiments of any of the provided examples, in an in vitro primary T cell assay, the variant CTLA-4 peptide reduces IFN-γ (interferon-γ) expression relative to the unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide is deglycosylated.
[0055] In some embodiments of any of the provided embodiments, an immunomodulatory polypeptide is provided comprising any variant CTLA-4 of the provided variant CTLA-4 that is directly linked to or indirectly linked via a linker to an IgSF domain containing an IgSF superfamily (IgSF) member. In some embodiments, the IgSF domain is an affinity-modified IgSF domain containing 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 homologous binding partners of the same one or more homologous binding partners compared to the binding of an unmodified or wild-type IgSF domain of the IgSF family member to one or more homologous binding partners of the affinity-modified IgSF domain. In some embodiments, the IgSF domain exhibits increased binding to the same one or more homologous binding partners compared to the binding of an unmodified or wild-type IgSF domain to one or more homologous binding partners of the IgSF domain.
[0056] In some embodiments of any of the provided examples, the variant CTLA-4 is a first variant CTLA-4 polypeptide, and the IgSF domain of the second polypeptide is an IgSF domain derived from the second variant CTLA-4 polypeptide, wherein the first variant CTLA-4 and the second variant CTLA-4 may be the same or different. In some aspects, the immunomodulatory protein further comprises a third polypeptide containing an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, wherein 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 instances, the third polypeptide is the same as the first polypeptide and / or the second polypeptide; or the third polypeptide is different from the first polypeptide and / or the second polypeptide.
[0057] In some embodiments of any of the provided embodiments, the IgSF domain or affinity-modified IgSF domain of the second or third polypeptide is or contains an IgV domain. In some cases, the variant CTLA-4 polypeptide is or contains an IgV domain.
[0058] In some embodiments of any of the provided embodiments, the immunomodulatory protein further comprises a polymerizing domain linked to at least one of the variant CTLA-4 peptide or the second peptide. In some aspects, the immunomodulatory protein further comprises a polymerizing domain linked to at least one of the variant CTLA-4 peptide, the second peptide, or the third peptide. In some cases, the polymerizing domain is an effector-reduced Fc domain or a variant thereof.
[0059] In some embodiments of any of the provided embodiments, the multimerizing domain promotes heterodimer formation. This document provides an immunomodulatory protein containing any of the provided immunomodulatory proteins, wherein the multimerizing domain is a first multimerizing domain and interacts with a second multimerizing domain to form a polymer containing the immunomodulatory protein. In some cases, the immunomodulatory protein is a first immunomodulatory protein, and a second immunomodulatory protein is directly linked to or indirectly linked to the second multimerizing domain via a linker, wherein the polymer contains both the first and second immunomodulatory proteins. In some embodiments, the second immunomodulatory protein is an immunomodulatory protein provided herein, wherein the multimerizing domain is the second multimerizing domain.
[0060] In some embodiments of any of the provided embodiments, this document provides an immunomodulatory protein comprising: a first variant CTLA-4 polypeptide, wherein the multimerizing domain is a first multimerizing domain; and a second variant CTLA-4 polypeptide, wherein the multimerizing domain is a second multimerizing domain, wherein the first and second multimerizing domains interact to form a multimer comprising the first variant CTLA-4 polypeptide and the second variant CTLA-4 polypeptide. 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 multimerizing domains are effector-reduced Fc domains or variants thereof. In some embodiments, the first and second multimerizing domains are the same or different.
[0061] In some embodiments of any of the provided embodiments, this document provides a conjugate containing a variant of CTLA-4 or an immunomodulatory protein linked to a portion. In some embodiments, the portion is a targeting portion that specifically binds to a molecule on the surface of a cell. In some embodiments, the targeting portion 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 portion is localized to cells or tissues in an inflammatory environment. In some embodiments, the portion is a protein, peptide, nucleic acid, small molecule, or nanoparticle. In some embodiments, the portion is an antibody or antigen-binding fragment. In some embodiments, the conjugate is bivalent, tetravalent, hexavalent, or octavalent.
[0062] In some embodiments of any of the provided examples, this document provides one or more nucleic acid molecules that encode the variant CTLA-4 polypeptide or the immunomodulatory protein provided herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is cDNA.
[0063] In some embodiments of any of the provided examples, this document provides a vector 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.
[0064] In some embodiments of any of the provided examples, this document provides a cell containing the vector provided herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.
[0065] In some embodiments of any of the provided examples, this document provides a method for generating a variant CTLA-4 peptide or immunomodulatory protein, the method comprising introducing a nucleic acid molecule or vector provided herein into the cells under conditions for expressing the protein in host cells. In some embodiments, the method further comprises isolating or purifying the variant CTLA-4 peptide or immunomodulatory protein from the cells.
[0066] In some embodiments of any of the provided examples, this document provides a method for engineering cells to express a variant CTLA-4 peptide, the method comprising introducing a nucleic acid molecule encoding the variant CTLA-4 peptide or an immunomodulatory protein provided herein into the cells under conditions in which the peptide is expressed in host cells.
[0067] In some embodiments of any of the provided examples, this document provides an engineered cell that expresses a variant of the CTLA-4 peptide, an immunomodulatory protein, a nucleic acid molecule, or a 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.
[0068] In some embodiments of any of the provided embodiments, the CTLA-4 peptide provided herein is expressed on the surface of the cell via a transmembrane domain. In some embodiments of such embodiments, the CTLA-4 peptide contains a cytoplasmic domain, optionally wherein the cytoplasmic domain is directly or indirectly linked to the transmembrane domain. In some embodiments, the cytoplasmic domain is or contains a native CTLA-4 cytoplasmic domain, an intracellular signal transduction domain, and / or an ITIM signal transduction motif. In some embodiments, the cytoplasmic domain contains an amino acid sequence as shown in residues 183-223 of SEQ ID NO:1 or a functional variant thereof, the functional variant exhibiting at least 85% sequence identity with residues 183-223 of SEQ ID NO:1. In some embodiments, the cytoplasmic domain contains an ITAM signal transduction motif and / or contains an intracellular signal transduction domain of CD3ζ. In some embodiments, the CTLA-4 peptide does not contain a cytoplasmic signal transduction domain and / or cannot mediate or regulate intracellular signaling when expressed on a cell.
[0069] In some embodiments 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).
[0070] In some embodiments of any of the provided examples, this document provides an infectious agent containing a nucleic acid molecule encoding a variant CTLA-4 polypeptide or an immunomodulatory protein provided herein. In some embodiments, the infectious agent is a bacterium or a virus.
[0071] In some embodiments of any of the provided examples, this document provides a pharmaceutical composition comprising a variant CTLA-4 peptide provided herein, an immunomodulatory protein provided herein, a conjugate provided herein, an engineered cell provided herein, or an infectious agent provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.
[0072] In some embodiments of any of the provided examples, this document provides an article of manufacture containing a pharmaceutical composition provided herein in a vial or container. In some embodiments, the vial or container is sealed.
[0073] In some embodiments of any of the provided examples, a kit is provided that contains the pharmaceutical composition provided herein and instructions for use. In some embodiments, a kit is provided that contains the article of manufacture provided herein and instructions for use.
[0074] In some embodiments of any of the provided embodiments, this document provides a method for modulating the immune response of a subject, the method comprising administering to the subject a pharmaceutical composition provided herein. In some embodiments, this document provides a method for modulating the immune response of a subject, the method comprising administering engineered cells provided herein. In some embodiments of such embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject. In some embodiments, the modulating of the immune response treats a disease or condition of the subject.
[0075] In some embodiments of any of the provided embodiments, the immune response is reduced. In some embodiments of the provided methods, the subject is administered a variant peptide, an immunomodulatory protein, or engineered cells having a variant CTLA-4 with a surface-expressed inhibitory (e.g., ITIM-containing) cytoplasmic signaling domain. In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition, or a disease or condition associated with an overactive immune response. In some embodiments, the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatism, thyroiditis, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, autoimmune blood disease, autoimmune demyelinating disease, or an autoimmune disease involving a systemic autoimmune condition. In some embodiments, the disease or symptom is selected from inflammatory bowel disease, grafts, 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, DeQuervains' thyroiditis, myasthenia gravis, vasculitis, autoimmune hemolytic anemia, autoimmune atrophic gastritis with pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease. Diseases including autoimmune thrombocytopenic purpura, sympathetic ophthalmia, primary biliary cirrhosis, chronic invasive hepatitis, membranous glomerulonephritis, primary idiopathic myxedema, scleroderma, chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, autoimmune enteropathy syndrome, idiopathic thrombocytopenic purpura, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antisperm antibodies, sensorineural hearing loss, Sjogren's syndrome, polymyositis, multiple sclerosis, transverse myelitis, ataxia-sclerosis, pemphigus, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa, hemolytic anemia, glomerulonephritis, and idiopathic facial paralysis.
[0076] In some embodiments of any of the provided embodiments, the immune response is enhanced. In some embodiments of the provided methods, the subject is administered engineered cells provided herein, such as engineered cells expressing a variant CTLA-4 lacking a cytoplasmic signaling domain or a variant CTLA-4 containing an activated (e.g., ITAM-containing) cytoplasmic signaling domain. In some embodiments, the provided methods treat a disease or condition that is a tumor or cancer. In some embodiments of such embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematologic malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer. Attached Figure Description
[0077] Figure 1 Various soluble or non-cellular expression formats of the provided variant IgSF domain molecules (such as variant CTLA-4 peptides) are described, including: (1) variant IgSF domains fused to the Fc chain (vIgD); (2) variant IgSF domains linked to an antibody (V-mAb) (vIgD). vIgD may contain an ECD containing an IgV domain or a V domain (IgV) of a member of the CTLA-4 IgSF superfamily; or (3) stacked molecules containing a first variant IgSF domain (first vIgD) and a second IgSF domain (such as a second variant IgSF domain (second vIgD)).
[0078] Figure 2 The immunomodulatory activity of an exemplary soluble CTLA-4 peptide is illustrated. In the exemplary schematic diagram, T cell activation is caused by signal transduction via the T cell receptor (TCR) and co-stimulatory receptors CD28 and / or ICOS. Signal transduction and T cell activation are induced by antigen-presenting cells (APCs) expressing MHC and co-stimulatory ligands CD80 (B7-1), CD86 (B7-2), and / or ICOS (see [link to diagram]). Figure 2 (Left side). As shown in the figure, the variant CTLA-4IgSF domain (vIgD) fused with Fc (CTLA-4vIgD-Fc; see also...) Figure 2 The variant CTLA-4 peptide illustrated in the middle of the text interacts with one or more binding mates ICOSL, CD80 (B7-1), and / or CD86 (B7-2) to block the binding of T cell CD28 and ICOS activation receptors to their co-stimulatory ligands, thereby inhibiting T cell activation, proliferation, and effector function (see [link to relevant documentation]). Figure 2 (Right side).
[0079] Figure 3A and Figure 3B Examples depicting CTLA-4 (such as variant CTLA-4) expressed as a transmembrane immune regulatory protein (TIP) on the surface of T cells are described. Figure 3A As shown, in some cases, T cell activation is driven by CD28 / TCR signaling, which is induced by binding to co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) expressed on antigen-presenting cells (APCs). CTLA-4 expressed on T cells (such as CTLA-4 expressed on activated T cells or regulatory T cells) circulates continuously between intracellular compartments and the cell surface via internalization. CTLA-4 can also bind to co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) (and in some cases, ICOSL). Figure 3B As shown, in some cases, the binding of CTLA-4 expressed by T cells to its ligands (such as B7-1 and B7-2) allows T cells to remove homologous ligands from the surface of APCs during a process called transcytosis. Functionally, this allows CTLA-4 to "strip" the ligand from the APC, thereby preventing the CD28 co-stimulatory ligand from subsequently activating other T cells and thus limiting T cell activation.
[0080] Figure 4 A secretory immunomodulatory protein (SIP) is described, wherein a variant IgSF domain (vIgD) is secreted from a cell (such as a first-order T cell, e.g., a CAR (chimeric antigen receptor) T cell). In one exemplary embodiment, the SIP is a variant CTLA-4 polypeptide, and its binding partner is a costimulatory ligand (e.g., CD80, CD86, and / or ICOSL), which may be expressed by antigen-presenting cells. When the SIP binds to its binding partner, the SIP blocks the binding of the costimulatory ligand to a costimulatory receptor (e.g., CD28), thereby blocking activation signals and attenuating T cell or effector T cell activation via the costimulatory receptor.
[0081] Figures 5A-5C Various exemplary configurations of the variant IgSF-antibody conjugate (V-Mab) are depicted. Figure 5A Various configurations are shown, in which one or more variant IgSF domains (vIgD) are directly or indirectly linked to the amino and / or carboxyl termini of the antibody's light chain. Figure 5B Various configurations are shown, in which one or more variant IgSF domains are directly or indirectly linked to the amino and / or carboxyl termini of the antibody's heavy chain. Figure 5C Describing when Figure 5A The described light chain and such Figure 5B The V-Mab configuration obtained when the described heavy chain is co-expressed in the cell.
[0082] Figure 6A Various exemplary configurations of stacked molecules containing a first variant IgSF domain (first vIgD) and a second IgSF domain (such as a second variant IgSF domain (second vIgD)) are depicted. As shown, the first vIgD and the second IgSF domain are independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. To generate homodimeric Fc molecules, the Fc region is an Fc region capable of forming a homodimer with a matching Fc subunit through co-expression of the individual Fc region in the cell. To generate heterodimeric Fc molecules, the individual Fc region contains a mutation (e.g., a "knob-into-hole" mutation in the CH3 domain) such that when the individual Fc region is co-expressed in the cell, homodimer formation is more favorable than heterodimer formation.
[0083] Figure 6B Various exemplary configurations of stacked molecules containing a first variant IgSF domain (first vIgD), a second IgSF domain (such as the second variant IgSF domain (second vIgD)), and a third IgSF domain (such as the third variant IgSF domain (third vIgD)) are depicted. As shown in the figure, the first vIgD, second IgSF domain, and third IgSF domain are independently directly or indirectly connected to the N-terminus or C-terminus of the Fc region. To generate homodimeric Fc molecules, the Fc region is an Fc region capable of forming a homodimer with a matching Fc region through co-expression of the individual Fc region in the cell.
[0084] Figure 7 The CIA paw scores in mice treated with an exemplary CTLA-4 variant fused to Fc as shown in SEQ ID NO:93 (containing the mutant G29W / N58S / L63P / Q82R / L98Q / Y105L), abatacept, or an Fc control in a collagen-induced arthritis (CIA) model were depicted. The CIA paw score was determined as the sum of the scores of each of the four paws of each mouse, and the average score was taken in a group of 15 mice (mean CIA paw score). Detailed Implementation
[0085] This document provides immunomodulatory proteins that are variants or mutants of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4; also known as differentiation cluster 152 or CD152) or a specific binding fragment thereof, exhibiting activity of binding to at least one target-binding mate. In some embodiments, the variant CTLA-4 peptide contains one or more amino acid modifications (e.g., amino acid substitution, deletion, or addition) compared to the unmodified or wild-type CTLA-4 peptide. In some embodiments, the one or more amino acid modifications (e.g., substitution) are located in the ECD of the unmodified or wild-type CTLA-4 peptide. In some embodiments, the one or more amino acid modifications (e.g., substitution) are located in the IgSF domain (e.g., IgV) of the unmodified or wild-type CTLA-4 peptide. In some embodiments, the binding activity or affinity of the variant CTLA-4 peptide and the immunomodulatory protein to inducible T-cell co-stimulatory molecular ligands (ICOSL; also known as B7-H2, CD275, and GL50) is altered, such as increased. In some embodiments, the variant CTLA-4 peptide and immunomodulatory protein exhibit increased binding activity or affinity for ICOSL, CD80 (also known as B7-1), and / or CD86 (also known as B7-2).
[0086] In some embodiments, the variant CTLA-4 peptide is a soluble immunomodulatory protein. Such molecules comprise a CTLA-4 peptide that lacks a transmembrane domain and / or is not membrane-anchored to the cell or cannot be expressed on the cell surface. In some embodiments, the variant CTLA-4 protein may be provided as a transmembrane immunomodulatory protein capable of being expressed on the cell surface or as a secretible immunomodulatory protein capable of being secreted from the cell. In some embodiments, one or more other immunomodulatory proteins are also provided herein as conjugates or fusions containing the variant CTLA-4 peptide provided herein and one or more other portions or peptides.
[0087] In some embodiments, the variant CTLA-4 peptide and immunomodulatory protein regulate immunological immune responses, such as increasing or decreasing them. In some embodiments, the variant CTLA-4 peptide and immunomodulatory protein provided herein can be used to treat diseases or conditions associated with immune response dysregulation, such as autoimmune symptoms or autoimmune diseases, or, in some cases, oncology indications.
[0088] Typically, antigen-specific T cell activation usually 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 co-stimulatory for TCR binding and is necessary for T cell proliferation, differentiation, and / or survival, including in some cases the necessity to avoid T cell apoptosis or unresponsiveness. In some embodiments, under normal physiological conditions, T cell-mediated immune responses are initiated by antigen recognition via the T cell receptor (TCR) and regulated by a balance of co-stimulatory (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 to protect tissues from excessive damage during immune responses, such as during an attack caused by pathogen infection. However, in some cases, the immune system may become dysregulated and may produce abnormal immune responses to normal body sites or tissues, leading to autoimmune diseases or conditions or symptoms of autoimmune disorders. In other cases, it may trigger a harmful immune response to foreign tissue (such as a graft), leading to graft rejection.
[0089] CTLA-4 is an inhibitory IgSF receptor that suppresses T cell responses by modulating TCR / CD28 signaling. The co-stimulatory receptor CD28 binds to its co-stimulatory ligands CD80 (also known as B7-1) and CD86 (also known as B7-2) and promotes the activation of primordial T cells in the presence of TCR signaling. The co-inhibitory receptor CTLA-4 competitively binds to CD80 and CD86 with CD28 to induce negative regulation of T cell activation. Figure 2 When CTLA-4 binds to CD80 and / or CD86 and prevents CD28 from binding to its homologous ligands, T cells cannot effectively transmit activation signaling cascades, and T cell activation and effector function may be eliminated or attenuated. CTLA-4 also exerts an inhibitory effect through the capture and transcytosis of costimulatory ligands CD80 and CD86 from opposing cells (such as APCs), thereby preventing these costimulatory ligands from binding to the CD28 costimulatory receptor (Figure 3). The binding of T cell receptor (TCR) enhances CTLA-4 acquisition of co-stimulatory ligands (Hou et al., (2015) Journal of Immunology, 194(5):2148-59; Soskic et al., (2014) Advances in Immunology, 124:95-136; Qureshi et al., (2011) Science, 332(6029):600-603).
[0090] CTLA-4 has been used as a therapeutic agent to treat autoimmune diseases by modulating the interaction between CD80 and / or CD86 to weaken T cell activation. Specifically, abatacept and belacept are FDA-approved therapeutic agents for rheumatoid arthritis and graft-related conditions, respectively. Abatacept is a wild-type CTLA-4 IgSF domain partially fused to the Fc region of an antibody. Berazip is a modified variant of the CTLA-4 IgSF domain containing a tyrosine substitution for alanine at position 31 and a glutamic acid substitution for leucine at position 106 (A31Y / L106E) (these positions correspond to positions 31 and 106 of the wild-type reference CTLA-4ECD sequence shown in SEQ ID NO:2) to increase affinity for CD80 and CD86 ligands (Kremer et al., New England Journal of Medicine 2003; 349(20):1907-1915; Larsen et al., American Journal of Transplantation 2005; 5(3):443-453).
[0091] In some respects, 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 of the co-stimulatory receptor ICOS ( Figure 2 CD28 and CTLA-4 can also bind to ICOSL, but with lower affinity than they bind to 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+ T cells and CD8+ T cells). The binding of CD80, CD86, and / or ICOSL to the co-stimulatory receptors CD28 and / or ICOS enhances the immune response, which may involve T cell activation, T cell proliferation, cytokine production, and other activities. For example, the binding of ICOSL to ICOS involves T helper cell and B cell differentiation.
[0092] The full-length CTLA-4 polypeptide contains a signal transduction sequence, an extracellular domain (ECD), a transmembrane region, and a cytoplasmic domain. The cytoplasmic domain contains an intracellular signal transduction domain. An exemplary human CTLA-4 sequence is shown in SEQ ID NO:1. After removing the signal sequence, the ECD contains amino acids 36-161 corresponding to amino acids in SEQ ID NO:1. An exemplary human CTLA-4 ECD amino acid sequence is shown in SEQ ID NO:2 or 569. The CTLA-4 ECD 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, there are CDR1-like rings (corresponding to amino acids 27-34 of SEQ ID NO:2), CDR2-like rings (corresponding to amino acids 51-55 of SEQ ID NO:2), and CDR3-like rings (corresponding to amino acids 97-104 of SEQ ID NO:2). In some respects, these rings have been reported to play a role in the interaction of CTLA-4 with its CD80 and CD86 homologous ligands (Peach et al., J. Exp. Med. (1994), 180(6):2049-2058).
[0093] This document provides variant CTLA-4 peptides with increased binding activity (e.g., increased binding affinity) to CD80, CD86, and / or ICOS. The provided examples include those involving variant CTLA-4 molecules that exhibit increased affinity for ICOS and, in some cases, also higher binding affinity for CD80 and / or CD86, such as increased binding affinity. In some embodiments, binding to CD80, CD86, and ICOS is competitive, such that a single variant CTLA-4 peptide will not bind more than one ligand at a time. Therefore, the provided variant CTLA-4 peptides are capable of binding to CD80, CD86, and / or ICOS to modulate, for example, antagonize, the normal function of these ligands in binding to the activating receptors CD28 and ICOS on T cells.
[0094] In some respects, it may be desirable to suppress or attenuate immune responses (such as T-cell responses) to reduce or prevent harmful autoimmune symptoms and / or graft rejection. The provided embodiments include methods for treating autoimmune or inflammatory diseases or conditions using variant CTLA-4 peptides (such as in their soluble or cellular expression forms). In this manner, in some respects, variant CTLA-4 peptides can be delivered to patients experiencing harmful autoimmunity (such as involving T cells) to produce effects that reduce T-cell activation, expansion, and / or effector function and attenuate autoimmune symptoms. Methods for preparing and using these variant CTLA-4 peptides are also provided.
[0095] In some cases, various formats of CTLA-4 peptides can be prepared to promote or enhance immune responses. For example, certain CTLA-4-switch receptors containing an activating cytoplasmic signaling domain or decoy receptors that compete with CTLA-4 for binding to homologous binding partners on effector cells can promote immune responses, such as enhancing them. The provided examples include methods for treating cancer and oncology indications using variant CTLA-4 peptides, such as certain cellular expression forms capable of inducing activating signals and / or competing with inhibitory signals.
[0096] In some embodiments, modulation of immune signaling via provided variant CTLA-4 peptides and immunomodulatory peptides, conjugates, or engineered cells containing such variant CTLA-4 peptides offers advantages for treating inflammatory and autoimmune diseases, as well as other diseases and conditions, compared to other treatments. In some cases, therapies for intervening in and altering such ligand / receptor interactions and subsequent signaling are constrained by spatial orientation requirements and size limitations imposed by the extent of immune synapses. In some aspects, existing therapeutic agents comprising antibody drugs may not simultaneously interact with multiple target proteins involved in regulating these interactions. Furthermore, pharmacokinetic differences between drugs that independently target one of these ligand / receptor interactions may make it difficult to properly maintain the desired blood concentrations of such drug combinations throughout treatment.
[0097] All publications mentioned in this specification, including patents, patent applications, scientific articles, and databases, are incorporated herein by reference in their entirety for all purposes, as if each individual publication, including a patent, patent application, scientific article, or database, were specifically and individually indicated to be incorporated herein by reference in general. If any definition set forth herein contradicts or otherwise is inconsistent with the definitions set forth in the patents, applications, publications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail over those incorporated herein by reference.
[0098] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.
[0099] I. Definition
[0100] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms having their commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from the meanings commonly understood in the art.
[0101] The terms used throughout this specification are defined as follows, unless otherwise specified in particular instances. The singular forms “a / an” and “described” as used in the specification and appended claims include plural indicators unless the context clearly indicates 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 stated, abbreviations and symbols for chemical and biochemical names are based on the IUPAC-IUB nomenclature. Unless otherwise stated, all numerical ranges include values within the defined range and all integer values between them.
[0102] In the context of immunoglobulin superfamily domains, the term "affinity-modified" means that a mammalian immunoglobulin superfamily (IgSF) domain has an altered amino acid sequence (relative to the corresponding wild-type parent or unmodified IgSF domain) such that its binding affinity or affinity for at least one binding partner (or "anti-structure") is increased or decreased compared to the parental wild-type or unmodified (i.e., unaffinity-modified) IgSF control domain. This context includes the affinity-modified CTLA-4IgSF domain. In some embodiments, the affinity-modified IgSF domain may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, thirty, or more amino acid differences, such as amino acid substitutions, from wild-type or unmodified IgSF domains. The increase or decrease in binding affinity or affinity can be determined using well-known binding assays such as flow cytometry (Larsen et al., *American Journal of Transplantation*, 5(3):443-453 (2005); see also Linsley et al., *Immunology*, 1(9):793-801 (1994)). The increase in binding affinity or affinity of the protein to one or more binding partners is an increase to a value at least 10% greater than that of the wild-type IgSF domain control, and in some embodiments, an increase to a value at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than that of the wild-type IgSF domain control. The decrease in binding affinity or affinity of the protein to at least one of its binding partners is a decrease to a value no greater than 90% of the control but not less than 10% of the wild-type IgSF domain control, and in some embodiments, a decrease to a value no greater than 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the wild-type IgSF domain control but not less than 10%.
[0103] Affinity-modified proteins undergo alterations in their primary amino acid sequence through the substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any conditions on any particular starting composition or method for generating affinity-modified IgSF domains. Therefore, the affinity-modified IgSF domains of this invention are not limited to wild-type IgSF domains subsequently converted to affinity-modified IgSF domains through any specific affinity modification process. For example, affinity-modified IgSF domain peptides can be generated starting from wild-type mammalian IgSF domain sequence information, followed by computer modeling of the binding of the affinity-modified IgSF domain peptide to its binding partner, and finally, recombinant expression or chemical synthesis of the affinity-modified IgSF domain peptide to produce an affinity-modified IgSF domain composition. In an alternative example, affinity-modified IgSF domains can be generated by site-directed mutagenesis of wild-type IgSF domains. Therefore, the affinity-modified IgSF domain represents the product, not necessarily the product produced by any given process. Various techniques can be employed, including recombination methods, chemical synthesis, or combinations thereof.
[0104] The term "allogenic" or "allogeneic" is used interchangeably herein to refer to cells or tissues taken from one organism and then injected or adopted into a different organism of the same species. In some embodiments of the invention, the species is a mouse or a human.
[0105] As used herein, the term "autologous" refers to cells or tissues taken from an organism and subsequently injected or adopted into the same organism. Autologous cells or tissues can be altered, for example, using recombinant DNA methods, so that they are no longer genetically identical to the natural cells or tissues taken from the organism. For example, natural autologous T cells can be genetically engineered using recombinant DNA technology to become autologous engineered cells expressing transmembrane immune regulatory proteins and / or chimeric antigen receptors (CARs). In some cases, this involves engineering T cells or TILs (tumor-infiltrating lymphocytes). The engineered cells are then injected into a patient from whom natural T cells were isolated. In some embodiments, the organism is a human or a mouse.
[0106] As used herein, the terms “binding affinity” and “binding affinity” refer to the specific binding affinity of a protein to its antistructure under specific binding conditions, respectively. In biochemical kinetics, affinity refers to the cumulative strength of multiple affinities between individual non-covalent binding interactions (such as between CTLA-4 and its antistructures ICOSL, CD80, and / or CD86). Thus, affinity differs from affinity, which describes the strength of a single interaction. An increase or decrease in the binding affinity of a variant of CTLA-4 containing an affinity-modified IgSF domain to its antistructure 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 affinity are known in the art. See, for example, Larsen et al., American Journal of Transplantation, 5(3):443-453 (2005). In some embodiments, the variant CTLA-4 of the present invention (i.e., the CTLA-4 protein containing an affinity-modified IgSF domain) binds specifically to ICOSL, CD80, and / or CD86 with a binding affinity at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of the wild-type CTLA-4 control in a binding assay, as measured by flow cytometry.
[0107] The term "biological half-life" refers to the amount of time it takes for a substance, such as an immunomodulatory polypeptide including the variant CTLA-4 of the present invention, to lose half of its pharmacological or physiological activity or concentration. Elimination, excretion, degradation (e.g., enzymatic degradation / digestion), or absorption and concentration in certain organs or tissues of the human body can affect the biological half-life. In some embodiments, the biological half-life can be assessed by determining the time it takes for a substance to reach half of its steady-state plasma concentration ("plasma half-life"). Peptides that can be used to derive the present invention and conjugates that increase their biological half-life are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), and other trademarked products. The products for sale include expanded recombinant peptides (Amunix Operating, California, USA) (see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).
[0108] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial (i.e., synthetic) transmembrane protein expressed on mammalian cells comprising at least an extracellular domain, a transmembrane domain, and an intracellular domain. Optionally, the CAR protein includes a "spacer region" that covalently links the extracellular domain to the transmembrane domain. The spacer region is typically a polypeptide that links the extracellular domain to the transmembrane domain via peptide bonds. 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, CAR-Ts are T helper cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. When used clinically, such as in adoptive cell transfer, CAR-Ts that have antigen-binding specificity to the patient's tumor or other tissues are typically engineered to be expressed on T cells obtained from the patient. The engineered T cells expressing the CAR are then injected back into the patient. CAR-Ts are therefore typically autologous CAR-Ts, but allogeneic CAR-Ts are included within the scope of this invention. The extracellular domain (or ECD) of a CAR includes an antigen-binding region, such as an antibody or an antigen-binding fragment thereof (e.g., a single-chain variable fragment (scFv)), which specifically binds to a target antigen (such as the ECD of an immunomodulatory peptide on the cell surface or a tumor-specific antigen) under physiological conditions. Following specific binding, a chain of biochemical events (i.e., signal transduction) modulates the immune activity of the CAR-T. Thus, for example, the specific binding of the antigen-binding region of a CAR-T to its target antigen can cause changes in the immune activity of T cells, reflected in alterations such as cytotoxicity, proliferation, or changes (increases or decreases) in cytokine production. In some embodiments, signal transduction during CAR-T activation is achieved through the intracellular domain of the CD3-ζ chain (“CD3-z”), which is involved in signal transduction in native mammalian T cells. CAR-Ts may further include multiple signal transduction domains, such as CD28, 4-1BB, or OX40, to further modulate the immune regulatory response of T cells. CD3-z includes a conserved motif called the immune receptor tyrosine-based activation motif (ITAM), which is involved in T cell receptor signal transduction.
[0109] When used in reference to cytokine production induced in in vitro assays due to the presence of two or more variants of CTLA-4 of the present invention, the term "uniformly" or "uniformly" means the overall cytokine expression level independent of cytokine production induced by a single variant CTLA-4 molecule. In some embodiments, the cytokine being measured is IFN-γ as in in vitro primary T cell assays.
[0110] The term "binding partner" (used interchangeably with "anti-structure") for a reference peptide, such as the IgSF domain of the reference variant CTLA-4, refers to at least one molecule (typically a native mammalian protein) to which the reference peptide specifically binds under specific binding conditions. In some respects, variant CTLA-4 containing an affinity-modified IgSF domain binds specifically to the binding partner of the corresponding native or wild-type CTLA-4, but with increased or decreased affinity. "Cell surface binding partner" refers to a binding partner expressed on the surface of mammalian cells. Examples of binding partners of variant CTLA-4 molecules provided herein include CD80, CD86, and ICOSL, specifically human CD80, human CD86, and human ICOSL.
[0111] As used herein, the terms "conjugate," "conjugation," or their grammatical variations refer to the linking of two or more compounds together by any joining or linking method known in the art to form another compound. It can also refer to compounds produced by linking two or more compounds together. For example, the variant CTLA-4 peptide directly or indirectly linked to one or more chemical moieties or peptides is an exemplary conjugate. Such conjugates include fusion proteins, conjugates produced by chemical conjugation, and conjugates produced by any other method.
[0112] As used herein, the term "competitive binding" means that a protein is capable of specifically binding to at least two binding partners, but the specific binding of one binding partner inhibits (e.g., prevents or blocks) the simultaneous binding of a second binding partner. Therefore, in some cases, it is impossible for a protein to bind to two binding partners simultaneously. Typically, competitive binders contain the same or overlapping binding sites for specific binding, but this is not necessary. In some embodiments, competitive binding causes a measurable inhibition (partial or complete) of the specific binding of a protein to one of its binding partners due to the specific binding of the second binding partner. Various methods for quantifying competitive binding are known, such as ELISA (enzyme-linked immunosorbent assay).
[0113] As used herein, the term "conservative amino acid substitution" refers to an amino acid residue in which one amino acid residue is replaced by another amino acid residue with a side-chain R group that has similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acid groups 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 substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.
[0114] The term "corresponds to" when referring to a location in a protein, such as when describing a nucleotide or amino acid position as "corresponding" to a nucleotide or amino acid position in a published sequence (as shown in the sequence listing), refers to the nucleotide or amino acid position identified when comparing the reference sequence to the published sequence based on structural sequence alignment or using a standard alignment algorithm (such as the GAP algorithm). For example, the corresponding residues can be determined by comparing the reference sequence with the wild-type CTLA-4 sequence shown in SEQ ID NO:2 or 569 (ECD) or SEQ ID NO:3 (IgV domain) using the structural alignment methods described herein. By aligning the sequences, those skilled in the art can, for example, use conserved and identical amino acid residues as guidance to identify the corresponding residues.
[0115] As used in this article, the terms “reduction,” “weakening,” or “inhibition” mean a reduction in a statistically significant amount. The reduction 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 the control value (e.g., a non-zero control).
[0116] The term “reduction” or “decreased” as used in the context of reducing the immune activity of mammalian lymphocytes herein means a reduction in one or more activities of lymphocytes compared to a control (such as an untreated control or a control treated under the same conditions using an unmodified or non-variant control). Decreased activity can refer to one or more of the following: cell cycle inhibition, decreased cell viability, decreased cell proliferation, decreased cytokine production, or decreased T-cell cytotoxicity, as in a statistically significant amount. In some embodiments, reference decreased immune activity means a statistically significant reduction in interferon-γ (IFN-γ) production compared to the absence of treatment. 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 are known in the art, including any of the assays described herein. In some embodiments, the enhancement may be a reduction of 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).
[0117] The term "derivative" or "derived" refers to protein modification that alters properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy by directly or indirectly covalently linking a protein to a composition, while retaining or enhancing its therapeutic benefits. Derivatives of the immunomodulatory peptides of this invention are within the scope of this invention and can be prepared by, for example, glycosylation, PEGylation, esterification, or Fc fusion.
[0118] As used herein, a “domain” (typically a sequence of three or more, usually five or seven or more amino acids (e.g., 10 to 200 amino acid residues) refers to a structurally and / or functionally distinct and identifiable portion of a molecule (such as a protein or encoding a nucleic acid). For example, a domain comprises those portions of a polypeptide chain that can form independently folded structures within a protein consisting of one or more structural motifs and / or are identified by functional activity (e.g., binding activity). Proteins may have one or more distinct domains. For example, domains can be identified, defined, or distinguished by homology of their primary sequence or structure with relevant family members, such as homology with motifs. In another instance, domains can be distinguished by their function (e.g., their ability to interact with biomolecules, such as binding couplers). A domain may independently exhibit a biological function or activity, such that the domain can perform an activity, such as binding, independently or in fusion with another molecule. A domain can be a linear or non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. Definitions are provided for illustrative purposes in this document; however, it should be understood that identifying a specific structural domain by name is entirely within the scope of the art. If necessary, appropriate software can be used to identify structural domains.
[0119] As used in this article, the term "extracellular domain" refers to the region of a membrane protein (transmembrane protein) located outside the membrane. Extracellular domains typically include binding domains that specifically bind to ligands or cell surface receptors, such as binding domains that bind specifically to ligands or cell surface receptors. The extracellular domain of a cellular transmembrane protein may alternatively be referred to as an extracellular structural domain.
[0120] The term "effective amount" or "therapeutic effective amount" refers to the amount and / or concentration of the therapeutic composition (comprising a protein composition or a cell composition) of the present invention, which, when administered ex vivo (by contact with the patient's cells) or in vivo (by administration to the patient) alone (i.e., as a monotherapy) or in combination with one or more other therapeutic agents, results in a statistically significant reduction in disease progression, for example, by alleviating or eliminating symptoms and / or the cause of the disease. An effective amount can be the amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with the disease or condition, prevents the progression of the disease or condition, or improves the patient's physical function. In the case of cell therapy, the effective amount is the effective dose or quantity of cells administered to the patient via adoptive cell therapy. In some embodiments, the patient is a mammal, such as a non-human primate, or a human patient.
[0121] As used herein, the term "intramocyte domain" (also known as "intracellular structural domain" or "cytoplasmic structural domain") refers to a region present in some membrane proteins (such as transmembrane proteins) that extends into the internal space defined by the cell surface membrane. In mammalian cells, an intracellular domain is a cytoplasmic region of a membrane protein. Within the cell, intracellular domains interact with intracellular components and can play a role in signal transduction, and therefore, in some cases, can be intracellular signal transduction domains. The intracellular domain of a transmembrane protein is alternatively referred to as a cytoplasmic structural domain, which in some cases can be a cytoplasmic signal transduction domain that mediates or plays a role in signal transduction. Therefore, the terms intracellular signal transduction domain and cytoplasmic signal transduction domain are used interchangeably.
[0122] The term “enhancement” or “increase” as used herein in the context of increasing the immune activity of mammalian lymphocytes means an increase in one or more activities of lymphocytes compared to a control (such as an untreated control or a control treated under the same conditions using an unmodified or non-variant control). An increase in activity can be one or more of the following: increased cell viability, increased cell proliferation, increased cytokine production, or increased T-cell cytotoxicity, if statistically significant. In some embodiments, reference increase in immune activity means an increase in interferon-γ (IFN-γ) production, if statistically significant. In some embodiments, immune activity can be assessed in an MLR assay. Other methods for assessing lymphocyte activity are known in the art, including any of the assays described herein. 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).
[0123] As used herein, the term "engineered cell" refers to a mammalian cell that has been genetically modified through human intervention, such as recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as lymphocytes (e.g., T cells, B cells, NK cells) or antigen-presenting cells (e.g., dendritic cells). The cell may be a primary cell derived from a patient or may be a cell line. In some embodiments, the engineered cells of the present invention comprise the variant CTLA-4 provided herein. In some embodiments, the variant CTLA-4 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") expressed on engineered cells. In some embodiments, the TIP contains an extracellular domain or a portion thereof comprising an IgV domain linked to a transmembrane domain (e.g., the CTLA-4 transmembrane domain) and optionally an intracellular signaling domain. In some cases, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or an intracellular domain. In some embodiments, the engineered cells are capable of expressing and secreting the immunomodulatory protein described herein. The engineered cells provided also include cells further comprising engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs).
[0124] As used herein, the term "engineered T cell" refers to a T cell that has been genetically modified through artificial intervention, such as recombinant DNA methods or viral transduction methods, including T helper cells, cytotoxic T cells (or alternatively, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells. Engineered T cells may include the variant CTLA-4 transmembrane immunomodulatory protein (TIP) of the present invention, expressed on T cells and engineered to regulate the immune activity of mammalian cells that specifically bind to the variant CTLA-4 expressed on T cells. Engineered T cells may also include the variant CTLA-4 secreted immunomodulatory protein (SIP) of the present invention, expressed and / or secreted by T cells and engineered to regulate the immune activity of mammalian cells that specifically bind to the variant CTLA-4 (when secreted by T cells).
[0125] The term "engineered T-cell receptor" or "engineered TCR" refers to a T-cell receptor (TCR) engineered to specifically bind with the major histocompatibility complex (MHC) / peptide target antigen in a selected, cloned, and / or subsequently introduced population of T cells commonly used for adoptive immunotherapy, with the desired affinity. In contrast to engineered TCRs, CARs are engineered to bind to the target antigen in an MHC-independent manner.
[0126] As used herein, the term "expressed on" refers to proteins expressed on the surface of cells, such as mammalian cells. 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 with a small molecule moiety, such as a drug or a detectable marker. Proteins expressed on the cell surface may include cell surface proteins expressed on mammalian cells, such as cell surface receptors.
[0127] The term "half-life extension" refers to a portion of the half-life of a protein circulating in mammalian serum that is extended by a polypeptide fusion or chemical conjugate, the extension being compared to the half-life of a protein not so conjugated to the portion. In some embodiments, the half-life extension is greater than or greater than about 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, or 6.0 times. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration, compared to a protein without a half-life extension portion. 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 an activity assay. An exemplary half-life extension portion comprises an Fc domain, a polymerizing domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), and other trademarked components. The extended recombinant peptides for sale (see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).
[0128] As used in this article, the term "immunological synapse" or "immunesynapse" refers to the interface between mammalian cells (such as antigen-presenting cells or tumor cells) expressing MHCI (major histocompatibility complex class I) or MHCII and mammalian lymphocytes (such as effector T cells or natural killer (NK) cells).
[0129] The Fc (crystallizable fragment) region or domain of an immunoglobulin molecule (also known as an Fc polypeptide) largely corresponds to the constant region of the immunoglobulin heavy chain and is responsible for a variety of functions, including one or more effector functions of an antibody. The Fc domain contains part or all of the hinge domain of the immunoglobulin molecule, plus CH2 and CH3 domains. The Fc domain can form a dimer of two polypeptide chains linked by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc with reduced activity (e.g., a reduction of more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) to promote effector function. In some embodiments, reference to amino acid substitutions in the Fc region is made using the EU numbering system unless described with reference to a specific SEQ ID NO. EU numbers are known and based on the most recently updated IMGT Scientific Chart (IMGTS Scientific Chart). theinternational ImMunoGeneTics information http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created on May 17, 2001, last updated June 8, 2016) and the EU index reported in the 5th edition of "Sequences of Proteins of Immunological Interest" by Kabat, EA et al. (US Department of Health and Human Services, NIH Publication No. 91-3242(1991)).
[0130] Immunoglobulin Fc fusions (“Fc fusions”), such as immunomodulatory Fc fusion proteins, are molecules comprising one or more polypeptides (or one or more small molecules) operatively linked to the Fc region of an immunoglobulin. Fc fusions may include, for example, an antibody (which promotes effector function and pharmacokinetics) and the Fc region of a variant CTLA-4. The immunoglobulin Fc region may be linked indirectly or directly to one or more variant CTLA-4 or small molecules (fusion partners). Various linkers are known in the art and may optionally be used to link Fc to a fusion partner to produce an Fc fusion. Fc fusions of the same species may be dimerized to form Fc fusion homodimers, or different species may be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a mouse or human Fc.
[0131] 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 prokaryotes, such as *Escherichia coli*, or eukaryotes, such as single-celled eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridoma cells. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO, grown in serum-free medium or in a dihydrofolate reductase (DHFR)-deficient CHO strain DX-B11. Another example is human embryonic kidney 293 (HEK-293) cells or derivatives thereof. In some embodiments, the host cell is a mammalian cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells).
[0132] As used herein, the term "immunoglobulin" (abbreviated as "Ig") refers to mammalian immunoglobulins, encompassing any one of the following five classes of human antibodies: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes immunoglobulins shorter than full length, whether wholly or partially synthetic (e.g., recombinant or chemically synthesized) or naturally occurring, such as antigen-binding fragments (Fab), containing V... H and V L The variable segment (Fv), containing V connected together to form a chain H and V L Single-chain variable fragments (scFv) and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv biantibodies, Fc polypeptide fragments, and Fd polypeptide fragments. The terminology includes bispecific antibodies (homological bispecific and heterospecific bispecific).
[0133] As used herein, the term "immunoglobulin superfamily" or "IgSF" refers to a group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins (i.e., antibodies); all of these molecules possess domains known as immunoglobulin domains or folds. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in lymphocyte antigen presentation, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. These members are generally associated with roles in the immune system. Proteins in immune synapses are typically members of the IgSF. The IgSF can also be classified into "subfamilies" based on shared properties, such as function. These subfamilies typically consist of 4 to 30 IgSF members.
[0134] The terms “IgSF domain”, “immunoglobulin domain”, or “Ig domain” as used herein refer to the domains of IgSF proteins. 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 distinctive Ig fold, which has a sandwich-like structure formed by two sheets of antiparallel β-chains. The interaction between the hydrophobic amino acids on the inner side of the sandwich and the highly conserved disulfide bonds formed between the cysteine residues in the B and F chains stabilizes the Ig fold. One end of the Ig domain has a region called the complementarity-determining region, which is important for the specificity of antibodies to their ligands. Ig-like domains can be classified as follows (into the following categories): IgV, IgC (which can be IgC1 or IgC2), or IgI. Most Ig domains are variable (IgV) or constant (IgC). The IgV domain, with nine β chains, is typically longer than the IgC domain, which has seven β chains. Some members of the IgSF have Ig domains that are amino acid sequence similar to the IgV domain but similar in size to the IgC domain. These Ig domains are referred to as the IgC2 domain, while the standard IgC domain is referred to as the IgC1 domain. The T cell receptor (TCR) chain contains two Ig domains in its extracellular portion; an IgV domain is located at the N-terminus, and an IgC1 domain is adjacent to the cell membrane. CTLA-4 contains one Ig domain: the IgV domain.
[0135] As used herein, the term "IgSF species" refers to all IgSF member proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines the unique identity of all IgSF species belonging to that IgSF member. Therefore, each IgSF family member is unique relative to other IgSF family members, and thus, each species of a particular IgSF family member is unique relative to the species of another IgSF family member. However, variations can exist between molecules belonging to the same IgSF species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosation, methylation, acetylation, and lipidation. Additionally, subtle sequence differences within a single IgSF species due to genetic polymorphism constitute another form of variation within a single IgSF species, as do wild-type truncated forms of IgSF species due to, for example, protein-lysing cleavage. "Cell surface IgSF species" are IgSF species expressed on the surface of cells (typically mammalian cells).
[0136] In this article, the term “immune activity” as used in the context of mammalian lymphocytes (such as T cells) refers to one or more of the following: activation, cell survival, apoptosis, cell proliferation, cell cycle inhibition, cytokine production (e.g., interferon-γ), cytokine release, or T cell cytotoxic activity. In some cases, immune activity can refer to cellular expression of cytokines (such as chemokines or interleukins). Assays used to determine enhancement or inhibition of immune activity include the MLR (mixed lymphocyte response) assay, which measures the level of interferon-γ cytokine in culture supernatant (Wang et al., Cancer Immunology Research, Sep 2014: 2(9): 846-56), the SEB (staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunology Research (2014) 2(9): 846-56), and the anti-CD3 T cell stimulation assay (Li and Kurlander, Journal of Translational Medicine (2010) 8: 104). Since T cell activation is associated with the secretion of the IFN-γ cytokine, commercial ELISA kits can be used to measure IFN-γ levels in culture supernatants from these in vitro human T cell assays (Wu et al., Immunology Letters (2008), 117(1):57-62). Induction of the immune response increases immunological activity relative to resting lymphocytes. In primary T cell assays, immunomodulatory proteins presented herein, such as the CTLA-4 peptide containing an affinity-modified IgSF domain, can reduce IFN-γ (interferon-γ) expression in some examples or increase it in alternative examples, relative to wild-type IgSF members or IgSF domain controls. Those skilled in the art will recognize that the format used to determine an increase in IFN-γ expression in primary T cell assays can differ from that used to determine a decrease in IFN-γ expression.
[0137] When determining the ability of the immunomodulatory protein of the present invention or the affinity-modified IgSF domain to alter IFN-γ expression in primary T cell assays, a mixed lymphocyte reaction (MLR) assay can be used. Conveniently, in some cases, the ability of the affinity-modified IgSF domain of the present invention to increase or decrease IFN-γ expression in the MLR can be determined using a soluble form. Alternatively, a co-immobilization assay can be used. In a co-immobilization assay, the ability to increase or decrease IFN-γ expression relative to a wild-type IgSF domain control is determined by using a T cell receptor signal provided by an anti-CD3 antibody in some examples, in conjunction with a co-immobilized affinity-modified IgSF domain (such as variant CTLA-4). Methods for determining the immune activity of engineered cells (including assessing the activity of the variant CTLA-4 transmembrane immunomodulatory protein) are known in the art and include, but are not limited to, inhibiting or enhancing T cell proliferation or proliferation following antigen stimulation, inhibiting the proliferation of primary and subcultured allogeneic stimulated T cells, and autoimmune activity, such as allogeneic graft survival assays and anti-donor antibody response assays, in appropriate animal models. Assays also include assays for assessing cytotoxicity, including standard... 51 Cr release assays (see, for example, Milone et al., Molecular Therapy (MolTher) (2009), 17(8):1453-1464) or flow-based cytotoxicity assays or impedance-based cytotoxicity assays (Peper et al., (2014), Journal of Immunology Methods, 405:192-198).
[0138] "Immunomotor polypeptide" or "immunomotor protein" is a polypeptide or protein molecule that regulates immune activity. "Modulation" of the immune response means an increase or decrease in immune activity. An immunomodulatory protein can be a single polypeptide chain or a polymer (dimer or higher-order polymer) of at least two polypeptide chains covalently bonded to each other, for example, by interchain disulfide bonds. Therefore, monomeric polypeptides, dimer polypeptides, and higher-order polymeric polypeptides are within the scope of the defined terminology. Polymeric polypeptides can be homopolymers (of the same polypeptide chain) or heteropolymers (of different polypeptide chains). The immunomodulatory protein of the present invention includes the variant CTLA-4.
[0139] As used in this article, the term "increase" means an increase that is statistically significant. An increase can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% greater than the control value (e.g., a non-zero control value).
[0140] A “type” of CTLA-4 is one of many naturally occurring CTLA-4 polypeptides with different amino acid sequences. An isotype can be the product of a splicing variant of an RNA transcript expressed by a single gene, or the expression product of highly similar but different genes that produce functionally similar proteins, such as those produced by gene duplication. As used herein, the term “type” of CTLA-4 also refers to the products of different alleles of the CTLA-4 gene.
[0141] As used in this article, the term "lymphocyte" refers to any of the three subtypes of white blood cells in the mammalian immune system. These subtypes include natural killer cells (NK cells) (which play a role in cell-mediated cytotoxic innate immunity), T cells (which are used in cell-mediated cytotoxic adaptive immunity), and B cells (which are used in humoral antibody-driven adaptive immunity). T cells include: helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. The definition of lymphocyte also includes innate lymphocytes (ILCs).
[0142] The term “mammal” or “patient” specifically includes reference to at least one of the following: human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.
[0143] As used herein, the term "membrane protein" refers to a protein that is directly or indirectly connected to the lipid bilayer under physiological conditions. The lipid bilayer forming the membrane can be a biological membrane, such as the cell membrane of a eukaryotic (e.g., mammalian) cell, or an artificial (i.e., synthetic) membrane, such as the membrane present on liposomes. The connection between membrane proteins and the lipid bilayer can be achieved through covalent linkage or through non-covalent interactions (such as hydrophobic or electrostatic interactions). Membrane proteins can be intrinsic or peripheral membrane proteins. Peripheral membrane proteins are non-covalently linked to the lipid bilayer or to intrinsic membrane proteins. Peripheral membrane proteins form temporary linkages with the lipid bilayer, allowing them to associate and / or dissociate within the physiological conditions of mammals. In contrast, intrinsic membrane proteins form substantially permanent linkages with the lipid bilayer, preventing dissociation within the physiological conditions of mammals. Membrane proteins can connect to the membrane via one layer of the lipid bilayer (monotopic) or via both layers (polytopic). Intrinsic membrane proteins that interact with only one lipid bilayer are called "intrinsic monotopic proteins." Intrinsic membrane proteins that interact with both lipid bilayers are called "intrinsic polytopic proteins," which may be alternatively referred to as "transmembrane proteins" in this paper.
[0144] The term "modulating / modulate" as used herein in the context of immune responses (such as mammalian immune responses) refers to any alteration, such as an increase or decrease, in an existing or potential immune response resulting from the administration of an immunomodulatory peptide comprising the variant CTLA-4 of the present invention or from the administration of engineered cells expressing an immunomodulatory protein, such as the transmembrane immunomodulatory protein variant CTLA-4 of the present invention. Therefore, the term refers to an alteration, such as an increase or decrease, in an immune response compared to an immune response that occurs or is present without the administration of an immunomodulatory protein comprising the variant CTLA-4 or cells expressing such immunomodulatory peptides. Such modulation includes any induction of immune cell activity, any activation of immune cells, any suppression of immune cell activity, or any alteration in the degree or extent of immune cell activity. 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).
[0145] Regulation encompasses any change that confers an existing immune response, an developing immune response, a potential immune response, or the ability to induce, regulate, influence, or respond to an immune response. Regulation can be direct or indirect. Regulation includes any alteration in the expression and / or function of genes, proteins, and / or other molecules in immune cells that are part of an immune response. Regulation of an immune response or regulation of immune activity includes, for example, eliminating, deleting, or isolating immune cells; inducing or generating immune cells capable of regulating the function of other cells (such as self-reactive lymphocytes, antigen-presenting cells, or inflammatory cells); inducing an unresponsive state (i.e., non-reactive) of immune cells; enhancing or inhibiting the activity or function of immune cells, including, but not limited to, altering the patterns of proteins expressed by these cells. Examples include alterations in the production and / or secretion of certain types of molecules (such as cytokines, chemokines, growth factors, transcription factors, kinases, co-stimulatory molecules, or other cell surface receptors) or any combination of these regulatory events. Regulation can be assessed, for example, by altering IFN-γ (interferon-γ) expression relative to wild-type CTLA-4 control in primary T cell assays (see Zhao et al., (2016), Exp CellRes, 340(1):132-138). Regulation can also be assessed, for example, by altering the immune activity of engineered cells (e.g., altering the cytotoxic activity or cytokine secretion of engineered cells) relative to cells engineered with wild-type CTLA-4 transmembrane protein.
[0146] As used herein, the term “molecular species” refers to all proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines a set of identical or substantially identical molecular species. Thus, for example, human CTLA-4 is an IgSF member, and each individual CTLA-4 molecule is a molecular species of CTLA-4. Variations can exist between molecules belonging to the same molecular species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosation, methylation, acetylation, and lipidation. Additionally, subtle sequence differences within a single molecular species due to genetic polymorphism constitute another form of variation within a single molecular species, as do wild-type truncated forms of a single molecular species due to, for example, protein-lysing cleavage. “Cell surface molecular species” are molecular species expressed on the surface of mammalian cells. Two or more different species of proteins—where each species is present only in one of the two mammalian cells forming an IS or only in the other mammalian cell (but not both mammalian cells)—are said to be “cis” or “cis-configured” to each other. Two proteins from different species—the first of which is present only in one of the two mammalian cells that form an isoform, and the second of which is present only in the second mammalian cell that forms an isoform—are described as having a “trans” or “trans” conformation. Two proteins from different species—each present in both mammalian cells that form an isoform—are present in both cis and trans conformations in those cells.
[0147] The term "multiplexing domain" refers to an amino acid sequence that promotes stable interactions between a polypeptide molecule and one or more other polypeptide molecules, each containing a complementary multiplexing domain (e.g., a first multiplexing domain and a second multiplexing domain), which may be the same or different. Interactions between complementary multiplexing domains, such as the interaction between a first and a second multiplexing domain, form stable protein-protein interactions to produce multimers of the polypeptide molecule with other polypeptide molecules. In some cases, the multiplexing domains are identical and interact with themselves to form stable protein-protein interactions between two polypeptide chains. Typically, the polypeptide is directly or indirectly linked to the multiplexing domain. Exemplary multiplexing domains include an immunoglobulin sequence or a portion thereof, a leucine zipper, a hydrophobic region, a hydrophilic region, and a compatible protein-protein interaction domain. The multiplying domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain or a portion thereof derived from IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes), IgA, IgE, IgD, and IgM and their modified forms.
[0148] The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form. Unless explicitly limited, the terms cover nucleic acids containing known analogs of natural nucleotides, having similar binding properties, and metabolizing in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, specific nucleic acid sequences also explicitly cover their conserved variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as explicitly specified sequences (“reference sequences”). Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. The terms nucleic acid or polynucleotide cover cDNA or mRNA encoded by genes.
[0149] As used herein, the term “non-competitive binding” refers to the ability of a protein to bind specifically to at least two binding partners simultaneously. Thus, a protein can bind to at least two different binding partners simultaneously, but the binding interactions do not need to last for the same duration, such that in some cases, the protein binds specifically 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 to one binding partner substantially does not inhibit simultaneous binding to a second binding partner. In some embodiments, non-competitive binding means that binding a second binding partner to its binding site on the protein does not replace binding of the 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 non-competitive interactions, the first binding partner binds specifically to an interaction site that does not overlap with the interaction site of the second binding partner, such that binding to the second binding partner does not directly interfere with binding to the first binding partner. Therefore, any effect of the binding of the second binding partner on the binding of the first binding partner is achieved through mechanisms other than directly interfering with the binding of the first binding partner. For example, in the case of enzyme-substrate interactions, non-competitive inhibitors bind to sites other than the enzyme's active site. Non-competitive binding encompasses non-competitive binding interactions in which the 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 if the first interaction site is occupied by the first binding partner.
[0150] The term "pharmaceutical composition" refers to a composition intended for pharmaceutical use in mammalian subjects, typically humans. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory peptide comprising a variant of CTLA-4 or engineered cells expressing a variant of the CTLA-4 transmembrane immunomodulatory protein) and a load, excipient, or diluent. The load, excipient, or diluent is typically a pharmaceutically acceptable load, excipient, or diluent, respectively.
[0151] The terms "peptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked by peptide bonds. The terms do not specify the exact length of the product. Therefore, the definition of a polypeptide includes both "peptide" and "oligopeptide." The term includes post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules containing one or more amino acid analogs or non-canonical or non-natural amino acids that can be synthesized or recombinantly expressed using known protein engineering techniques. Additionally, proteins can be derived.
[0152] As used herein, the term "primary T cell assay" refers to an in vitro assay for measuring interferon-γ ("IFN-γ") expression. Various such primary T cell assays are known in the art. In one preferred embodiment, the assay used is an anti-CD3 co-immobilization assay. In this assay, primary T cells are stimulated with anti-CD3, with or without additional recombinant protein immobilization. The culture supernatant is harvested at a time point typically 24–72 hours. In another embodiment, the assay used is an MLR. In this assay, primary T cells are stimulated with allogeneic APC. The culture supernatant is harvested at a time point typically 24–72 hours. Human IFN-γ levels are measured in the culture supernatant using standard ELISA techniques. Commercial kits are available from the vendor, and the assay should be performed according to the manufacturer's recommendations.
[0153] The term "purified" applied to nucleic acids encoding the immunomodulatory proteins of the present invention generally means nucleic acids or polypeptides that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified polypeptides or polynucleotides forming discrete bands in electrophoresis gels subjected to density gradient centrifugation, chromatographic eluents, and / or media). For example, nucleic acids or polypeptides that substantially produce one band in an electrophoresis gel are "purified". The purified nucleic acids or proteins of the present invention have a purity of at least about 50%, and a purity typically of at least about 75%, 80%, 85%, 90%, 95%, 96%, 99%, or higher (e.g., weight percentage or moles).
[0154] The term “recombinant” indicates that a material (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered through human intervention. Alterations can be performed on the material within its natural environment or state, or on material removed from its natural environment or state. For example, a “recombinant nucleic acid” is a nucleic acid prepared by recombining nucleic acids, for example, during cloning, affinity modification, DNA shuffling, or other well-known molecular biology procedures. A “recombinant DNA molecule” comprises DNA fragments linked together by such molecular biology techniques. The terms “recombinant protein” or “recombinant polypeptide” as used herein refer to protein molecules expressed using recombinant DNA molecules. A “recombinant host cell” is a cell containing and / or expressing recombinant nucleic acids or otherwise altered through genetic engineering, such as by introducing nucleic acid molecules encoding recombinant proteins (such as the transmembrane immune regulatory proteins described herein). Transcriptional control signals in eukaryotes include “promoter” and “enhancer” elements. Promoters and enhancers consist of arrays of short 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 from yeast, insects, mammalian cells, and viruses (similar control elements, i.e., promoters, have also been found in prokaryotes). The choice of a particular promoter and enhancer depends on the type of cell used to express the protein of interest. The terms “in an operable combination,” “in an operable sequence,” and “operably linked” as used herein refer to the manner or direction in which nucleic acid sequences are linked such that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of the desired protein molecule is produced.
[0155] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence required for expression in a specific host cell. The nucleic acid sequence required for expression in prokaryotes includes a promoter, optionally an operator gene sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Secretory signal peptide sequences may also optionally be encoded by a recombinant expression vector operatively linked to the coding sequence of a recombinant protein (such as a recombinant fusion protein), such that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell (if desired). The term encompasses vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of the host cell into which they have been introduced. Vectors include viral vectors, such as lentiviral vectors.
[0156] The term "selectivity" refers to the preferential and specific binding of the protein of the present invention to another substrate (such as another different binding partner) compared to the specific binding to one substrate (e.g., the binding partner of the protein or polypeptide of the present invention). Selectivity can be expressed as the binding activity (e.g., binding affinity) (e.g., Kconjugation) of the protein of the present invention and the first substrate (such as the first binding partner) to the first substrate (e.g., the Kconjugation). d1 ) and the binding activity (e.g., binding affinity) of the same protein of the present invention with a second binding partner (e.g., K) d2 The ratio of ).
[0157] 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 during amino acid sequence alignment. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence during nucleotide sequence alignment. Methods for sequence alignment used for comparison are well known in the art and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates the percentage of sequence identity and performs statistical analysis on the similarity between two sequences. Software used to perform BLAST analysis is publicly available from the website of the National Centre for Biotechnology Information (NCBI).
[0158] The term "soluble" as used in this article refers to proteins that are not membrane proteins. Typically, soluble proteins contain only the extracellular domain of an IgSF family member receptor or a portion thereof containing one or more IgSF domains or their specific binding fragments, but lack transmembrane domains and / or cannot be expressed on the cell surface. In some cases, protein solubility can be enhanced by direct or indirect linkage with an Fc domain or other portion, which in some cases can also improve protein stability and / or half-life. In some respects, soluble proteins are Fc fusion proteins.
[0159] In this article, the term "species" used for polypeptides or nucleic acids refers to all molecules having the same or substantially the same sequence. Variations may exist between polypeptides belonging to the same species due to differences in post-translational modifications (such as glycosylation, phosphorylation, ubiquitination, nitrosation, methylation, acetylation, and esterification). Slightly truncated sequences of polypeptides that differ from the full-length species by no more than one, two, or three amino acid residues at the amino or carboxyl terminus are considered to belong to a single species. This slight heterogeneity is a common characteristic of manufactured proteins.
[0160] The term "specifically binding fragment" as used herein with respect to the full-length wild-type mammalian CTLA-4 polypeptide or its IgV domain means a polypeptide having a subsequence of the full-length polypeptide or IgV domain that specifically binds to mammalian ICOSL, mammalian CD80, and / or mammalian CD86 (such as human or mouse ICOSL, CD80, or CD86) in vitro and / or in vivo. In some embodiments, the specific binding fragment comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the length of the full-length wild-type sequence or its IgV sequence. The sequence of the specific binding fragment may be modified to form variant CTLA-4 of the present invention.
[0161] As used herein, the term "specific binding" means the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or affinity is at least 5 times, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or affinity of the same protein for a set of random peptides or polypeptides of sufficient statistical size. Specific binding does not require binding to only a single target molecule, but can result in specific binding to non-target molecules due to the structural conformational similarity between the target molecule and non-target molecules (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 to non-target molecules with epitopes substantially similar to those of the target molecule (e.g., paralogous genes) is possible without diminishing the binding specificity determined relative to a statistically effective set of unique non-target molecules (e.g., random polypeptides). Therefore, due to cross-reactivity, the peptides of the present invention can specifically bind to target molecules from more than one different species. They can be used with solid-phase ELISA immunoassay, ForteBio... or Measurements can determine the specific binding between two proteins. Typically, the dissociation constant (K0) of the interaction between the two binding proteins is used to determine this. d Less than 1×10 -5M, and typically as low as 1×10 -12 M. In some embodiments of this disclosure, the dissociation constant of the interaction between the two binding proteins is less than or less than about 1 × 10⁻⁶. -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M or smaller.
[0162] The term "surface expression" or "expression on a surface" in reference to mammalian cells expressing peptides means that the peptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.
[0163] In this article, the term "synthetic" as used, for example, to refer to synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, refers to nucleic acid or polypeptide molecules produced by recombination and / or by chemical synthesis methods.
[0164] As used herein, the term "targeting portion" refers to a composition covalently or non-covalently linked to or physically encapsulating a peptide, including the variant CTLA-4 of the present invention. In some embodiments, the targeting portion has a specific binding affinity for a target molecule, such as a target molecule expressed on a cell. Typically, the target molecule is localized to a specific tissue or cell type. The targeting portion comprises: an antibody, an antigen-binding fragment (Fab), or a V-containing fragment. H and V L The variable segment (Fv) contains V connected together in a chain. H and V L The single-chain variable fragment (scFv) and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv biantibodies, nanobodies, soluble receptors, receptor ligands, affinity-matured receptors or ligands, and small molecule (<500 Daltons) compositions (e.g., specific receptor-binding compositions). The targeting portion can also be covalently or non-covalently linked to the lipid membrane of the liposome encapsulating the polypeptide of the present invention.
[0165] As used herein, the term "transmembrane protein" means a membrane protein that spans substantially or completely across a lipid bilayer, such as those present in biological membranes (e.g., mammalian cells) or artificial constructs (e.g., liposomes). Transmembrane proteins include transmembrane domains ("transmembrane domains") through which the protein integrates into the lipid bilayer and through which this integration is thermodynamically stable under physiological conditions. A transmembrane domain is generally predictable from its amino acid sequence using any number of commercially available bioinformatics software applications based on an increase in hydrophobicity relative to a protein region that interacts with an aqueous environment (e.g., cytosol, extracellular fluid). A transmembrane domain is typically a transmembrane hydrophobic α-helix. A transmembrane protein may cross both layers of the lipid bilayer once or multiple times. Transmembrane proteins include the transmembrane immunomodulatory proteins described herein. In addition to transmembrane domains, the transmembrane immunomodulatory proteins of the present invention further include extracellular domains and, in some embodiments, internal domains.
[0166] As used herein, the term "treating" means a disease or condition that slows, stops, or reverses the progression of the disease or condition by applying the therapeutic composition of the present invention (e.g., a therapeutic composition containing immunomodulatory proteins or engineered cells), alone or in combination with another compound described herein, as demonstrated by reducing, stopping, or eliminating clinical or diagnostic symptoms. "Treatment" also means a reduction in the severity of symptoms in an acute or chronic disease or condition, or, for example, a reduction in the relapse rate in the course of an autoimmune disease or in the inflammatory aspect of an autoimmune disease. "Treatment" can also mean a reduction in inflammation and / or other symptoms associated with graft rejection.
[0167] In the context of cancer, the terms “treatment” or “inhibit, inhibiting, or inhibition” used in this article refer to at least one of the following: a statistically significant reduction in tumor growth rate, cessation of tumor growth, or reduction in tumor size, mass, metabolic activity, or volume (as measured by normative criteria, such as, but not limited to, the Response Evaluation Criteria for Solid Tumors (RECIST)), or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).
[0168] As used in the context of this invention, “preventing (prophylaxis or prevention)” means, alone or in combination with another compound, the application of the immunomodulatory peptides or engineered cells of this invention to prevent the occurrence or onset of some or all of the symptoms of a disease or condition, or to reduce the likelihood of the onset of a disease or condition.
[0169] As used herein, the term "tumor-specific antigen" or "TSA" refers to a reverse structure that is primarily present on tumor cells of a mammalian subject but not typically on normal cells of the same mammalian subject. The tumor-specific antigen need not be unique to tumor cells, but the percentage of cells in a particular mammal possessing the tumor-specific antigen is sufficiently high, or the level of the tumor-specific antigen on the tumor surface is sufficiently high, such that the tumor-specific antigen can be targeted by antitumor therapeutic agents (such as the immunomodulatory peptides of this invention) and protect the mammal from the effects of the tumor or treat the tumor in the mammal. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells exhibiting TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells exhibiting TSA are cancerous.
[0170] The term "variant" (also known as "modified" or "mutant") used in this document to refer to variant CTLA-4 means CTLA-4 produced through artificial intervention (such as mammalian (e.g., human or mouse) CTLA-4). Variant CTLA-4 is a polypeptide whose amino acid sequence has been altered relative to unmodified or wild-type CTLA-4. Variant CTLA-4 is a polypeptide whose isotype of wild-type CTLA-4 differs from its wild-type counterpart by one or more modifications (such as one or more amino acid substitutions, deletions, additions, or combinations thereof). For the purposes of this document, variant CTLA-4 contains at least one affinity-modified domain, whereby one or more of the amino acid differences occur in the IgSF domain (e.g., the IgV domain). Variant 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. Variant CTLA-4 peptides typically 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 the portion of it containing the extracellular domain or its IgSF domain). In some embodiments, the variant CTLA-4 peptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the corresponding wild-type or unmodified CTLA-4 including the sequence shown in SEQ ID NO:2 or SEQ ID NO:3. Non-naturally occurring amino acids and naturally occurring amino acids are included within the permitted range of substitutions or additions. The variant CTLA-4 is not limited to any particular method of manufacture and includes, for example, de novo chemical synthesis, de novo recombinant DNA technology, or a combination thereof. The variant CTLA-4 of the present invention specifically binds to ICOSL, CD80, and / or CD86 in mammalian species. In some embodiments, changes in the amino acid sequence compared to wild-type or unmodified CTLA-4 protein result in alterations in binding affinity or affinity for the following: ICOSL; CD80; CD86; CD80 and CD86; ICOSL and CD80; ICOSL and CD86; and / or ICOSL, CD80 and CD86.Increases or decreases in binding affinity can be determined using well-known binding assays such as flow cytometry. Such assays are described in Larsen et al., American Journal of Transplantation, 5(3):443-453 (2005) and Linsley et al., Immunology, 1(9):793-801 (1994).
[0171] The binding affinity or affinity of variant CTLA-4 with ICOSL, CD80, and / or CD86 is increased by a value at least 5% greater than that of wild-type or unmodified CTLA-4, and in some embodiments, by at least 10%, 15%, 20%, 30%, 40%, 50%, or 100% greater than that of the wild-type or unmodified CTLA-4 control. The binding affinity or affinity of CTLA-4 with ICOSL, CD80, and / or CD86 is decreased by a value no greater than 95% of that of the wild-type or unmodified control, and in some embodiments, by no greater than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or no detectable binding affinity or affinity of the wild-type or unmodified control. Variant CTLA-4 is altered in its primary amino acid sequence through substitution, addition, or deletion of amino acid residues.
[0172] In the context of variant CTLA-4, the term "variant" should not be interpreted as imposing any conditions on any particular starting composition or method for producing variant CTLA-4. Variant CTLA-4 can be generated, for example, starting with wild-type mammalian CTLA-4 sequence information, followed by computer modeling of its binding to ICOSL, CD80, and / or CD86, and finally recombinant expression or chemical synthesis to produce the variant CTLA-4 of the present invention. In an alternative instance, variant CTLA-4 can be generated by site-directed mutagenesis of wild-type CTLA-4. Therefore, variant CTLA-4 refers to a composition, and not necessarily a product produced by any given process. Various techniques can be employed, including recombinant methods, chemical synthesis, or combinations thereof.
[0173] In this article, the terms “wild-type” or “natural” or “native” used in conjunction with biological materials (such as nucleic acid molecules, proteins (e.g., CTLA-4), IgSF members, host cells, etc.) refer to materials that exist in nature and have not been modified by human intervention.
[0174] II. Variant CTLA-4 peptide
[0175] This document provides variant CTLA-4 peptides that alter (increase or decrease) the binding activity or affinity of one or more 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.
[0176] CTLA-4 is a member of the immunoglobulin superfamily, which is a family of proteins that have an immunoglobulin domain or fold (hereinafter referred to as "immunoglobulin superfamily domain" or "IgSF domain"). In some embodiments, other IgSF family members include those from the following families: Signal-Regulatory Protein (SIRP) family, Triggering Receptor Expressed On Myeloid Cells Like (TREML) family, Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family, Sialic Acid Binding Ig-Like Lectin (SIGLEC) family, Butyrophilin family, B7 family, CD28 family, V-set and Immunoglobulin Domain Containing (VSIG) family, V-set Transmembrane Domain (VSTM) family, Major Histocompatibility Complex (MHC) family, and Signaling Lymphocytic Activating Molecule (SIL) family. The activation molecule (SLAM) family, leukocyte immunoglobulin-like receptor (LIR), nectin (Nec) family, nectin-like (NECL) family, poliovirus receptor related (PVR) family, natural cytotoxicity triggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM) family, or killer-cell immunoglobulin-like receptors (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 (TIGIT), ICOS, BTLA (CD272), CD4, CD8A (CD8-α), CD8B (CD8-β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NCR3 (NKp30).
[0177] Table 1 summarizes exemplary members of the IgSF family. The first column of Table 1 provides the name of a specific IgSF member and, optionally, some possible synonyms for that specific IgSF member. The second column provides the protein identifier for the UniProtKB database—a publicly available database accessible via the Internet at uniprot.org—or, in some cases, the GenBank number. The Universal Protein Resource (UniProt) is a comprehensive resource of protein sequence and annotation data. The UniProt database contains the UniProt Knowledgebase (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics, and the Protein Information Resource (PIR), and is primarily supported by grants 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, January 2013; 41(D1):D36-42). Column 3 provides the region where the indicated IgSF domain is located. The region is specified as a range, where the domain contains residues defining the range. Column 3 also indicates the IgSF domain category for the specified IgSF region. Column 4 provides the regions where the indicated additional domains are located (signal peptide – S; extracellular domain – E; transmembrane domain – T; cytoplasmic domain – C). It should be understood that the description of the domains can vary depending on the method used to identify or classify the domains, and can be identified differently from different sources. The descriptions of the residues corresponding to the domains in Table 1 are for illustrative purposes only and can be a few amino acids longer or shorter (e.g., one, two, three, or four). Column 5 indicates some of the binding couples of some of the listed IgSF members.
[0178]
[0179]
[0180]
[0181]
[0182] In some embodiments, relative to wild-type or unmodified CTLA-4 peptides or wild-type or unmodified CTLA-4 containing a portion of an immunoglobulin superfamily (IgSF) domain (IgD) or its specific binding fragment, the variant CTLA-4 peptide contains one or more amino acid modifications in the IgD, such as one or more substitutions (or alternatively, "mutations" or "substitutions"), deletions, or additions. Therefore, the provided variant CTLA-4 peptide is or includes a variant IgD (hereinafter referred to as "vIgD") having one or more amino acid modifications (e.g., substitutions) in the IgD.
[0183] In some embodiments, the IgD comprises an IgV domain or a specific binding fragment of the IgV domain, or a combination thereof. In some embodiments, the IgD may be simply the IgV of CTLA-4 or its entire extracellular domain (ECD). In some embodiments, the IgD comprises a specific binding fragment of the ECD. Table 1 provides exemplary residues corresponding to the IgV region and ECD of CTLA-4. In some embodiments, the variant CTLA-4 peptide contains at least one amino acid modification (e.g., substitution) located in the IgV domain or ECD or its specific binding fragment thereof. In some embodiments, the variant CTLA-4 peptide contains at least one amino acid modification (e.g., substitution) located in the IgV domain or its specific binding fragment thereof. In some embodiments, the variant CTLA-4 peptide contains at least one amino acid modification (e.g., substitution) located in the ECD or its specific binding fragment thereof. In some embodiments, the altered IgV domain or ECD is an affinity-modified IgSF domain due to changes in binding activity or affinity.
[0184] In some embodiments, the variant is modified in one or more IgSF domains 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 natural CTLA-4 or its ortholog. In some embodiments, the unmodified CTLA-4 is or includes the extracellular domain (ECD) of CTLA-4 or contains a portion of one or more IgSF domains (see Table 1). In some embodiments, the extracellular domain of the unmodified or wild-type CTLA-4 peptide includes an IgV domain or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 peptide includes or is substantially composed of an IgV domain or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 peptide includes or is substantially composed of an ECD or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 is soluble and lacks a transmembrane domain. In some embodiments, the variant CTLA-4 further includes a transmembrane domain and, in some cases, also includes a cytoplasmic domain, which may contain intracellular signal transduction domains.
[0185] 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 may be a mammalian CTLA-4, including but not limited to human CTLA-4, mouse CTLA-4, cynomolgus monkey CTLA-4, or rat CTLA-4. In some embodiments, the wild-type or unmodified CTLA-4 sequence is human.
[0186] In some embodiments, the wild-type or unmodified CTLA-4 sequence has (i) the amino acid sequence shown in SEQ ID NO:1 or its mature form lacking the signal sequence, (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:1 or its mature form, or (iii) a portion of (i) or (ii) containing an IgV domain or an ECD or a specific binding fragment thereof.
[0187] In some embodiments, the wild-type or unmodified CTLA-4 sequence is or includes 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 shown in SEQ ID NO:2 (corresponding to amino acid residues 36-161 of SEQ ID NO:1) or its orthologs. In some cases, the unmodified or wild-type CTLA-4 polypeptide may comprise (i) the amino acid sequence shown in SEQ ID NO:2, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:2, or (iii) a specific binding fragment including an IgV domain of the sequence of (i) or (ii). In some embodiments, wild-type or unmodified ECDs are capable of binding to one or more CTLA-4 binding partners, such as ICOSL and / or one or more of CD80 and / or CD86.
[0188] In some embodiments, the wild-type or unmodified CTLA-4 peptide includes an IgV domain or a specific binding fragment thereof. In some embodiments, the IgV domain of the wild-type or unmodified CTLA-4 peptide includes the amino acid sequence shown in SEQ ID NO:3 (corresponding to amino acid residues 39-140 of SEQ ID NO:1) or its orthologs. For example, the IgV domain of the unmodified or wild-type CTLA-4 peptide may contain (i) the amino acid sequence shown in SEQ ID NO:3, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 one or more CTLA-4 binding partners, such as ICOSL and / or one or more of CD80 and / or CD86.
[0189] In some embodiments, the wild-type or unmodified CTLA-4 peptide contains a CTLA-4-specific binding fragment, such as an IgV domain or an ECD-specific binding fragment. In some embodiments, the specific binding fragment may bind ICOSL. In some embodiments, the specific fragment may bind ICOSL and CD80 or CD86. In some embodiments, the specific fragment may bind ICOSL, CD80, and CD86. The amino acid length of the specific binding fragment may be at least 50 amino acids, such as at least 60, 70, 80, 90, or 100 amino acids. In some embodiments, the length of the amino acid sequence contained in the IgV domain-specific binding fragment 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 as shown in amino acids 39-140 of SEQ ID NO:1. In some embodiments, the length of the amino acid sequence comprising the specific binding fragment of ECD is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of ECD as shown in amino acids 36-161 of SEQ ID NO:1.
[0190] In some embodiments, the variant CTLA-4 peptide includes an ECD domain or a portion thereof comprising one or more affinity-modified IgSF domains. In some embodiments, the variant CTLA-4 peptide may include an IgV domain or a specific binding fragment of an IgV domain or an ECD, wherein one or more of the IgSF domain (IgV) or ECD contains one or more amino acid modifications (e.g., substitutions). In some embodiments, the variant CTLA-4 peptide includes a full-length IgV domain. In some embodiments, the variant CTLA-4 peptide includes a full-length ECD. In some embodiments, the variant CTLA-4 peptide includes a specific binding fragment of an IgV domain. In some embodiments, the variant CTLA-4 peptide ECD includes a specific binding fragment of an ECD.
[0191] In any of these embodiments, the one or more amino acid modifications (e.g., substitutions) of the variant CTLA-4 peptide may be located in the CTLA-4 peptide ECD, such as in the IgSF domain therein. For example, in some embodiments, one or more amino acid modifications (e.g., substitutions) are located in the extracellular domain of the variant CTLA-4 peptide. In some embodiments, one or more amino acid modifications (e.g., substitutions) are located in the IgV domain or in a specific binding fragment of the IgV domain.
[0192] In general, each of the various properties of the polypeptide is disclosed individually below (e.g., soluble and membrane-bound polypeptides, CTLA-4 affinity for ICOSL, CD80, and / or CD86, the number of variants per polypeptide chain, the number of linked polypeptide chains, the number and nature of amino acid changes in each variant of CTLA-4, etc.). However, it will be apparent to those skilled in the art that any particular polypeptide may include a combination of these individual properties. It should be understood that references to amino acids, including references to specific sequences as shown in SEQ ID NO for describing the organization of IgSF domains, are for illustrative purposes and do not imply limitation on the scope of the provided embodiments. It should be understood that the polypeptide and the description of its domains are theoretically derived based on homology analysis and comparison with similar molecules. Therefore, the exact loci may vary and are not necessarily the same for every protein. Thus, a particular IgSF domain, such as a particular IgV domain or ECD, may be a few amino acids longer or shorter (e.g., one, two, three, or four).
[0193] Furthermore, various embodiments of the invention discussed below are frequently provided within the meaning of the defined terms disclosed above. Therefore, when the defined terms are used in discussing the various aspects and properties described herein, embodiments described in a particular definition should be interpreted as incorporated by reference. Thus, the order in which the headings, aspects, and embodiments are presented, and the separate disclosure of each individual property, does not imply a limitation on the scope of this disclosure.
[0194] A. Exemplary Modification
[0195] This document provides a variant CTLA-4 peptide that, relative to the IgSF or ECD contained in the wild-type or unmodified CTLA-4 peptide, contains modifications in the ECD, its IgSF domain, or a specific binding fragment thereof. In some embodiments, at least one modification is located in the IgSF domain (e.g., IgV) or a specific binding fragment thereof, such that the provided variant CTLA-4 peptide contains at least one affinity-modified IgSF domain or a specific binding fragment thereof. In some embodiments, the variant CTLA-4 peptide exhibits altered (increased or decreased) binding activity or affinity for ICOSL, CD80, and / or CD86 compared to the wild-type or unmodified CTLA-4 peptide. ICOSL, CD80, and / or CD86 may be mammalian proteins, such as human or mouse proteins.
[0196] In some embodiments, the binding activity or affinity of the variant CTLA-4 peptide to ICOSL and optionally one or more ligands CD80 and CD86 is altered (increased or decreased) compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the binding affinity of the variant CTLA-4 peptide to ICOSL and optionally CD80 and / or CD86 differs from the binding affinity of the wild-type or unmodified CTLA-4 peptide control (e.g., unmodified) sequence, as determined by, for example, solid-phase ELISA immunoassay, flow cytometry, ForteBio... Or as determined by Biacore assays. In some embodiments, the binding affinity of the variant CTLA-4 peptide to ICOSL and optionally CD80 and / or CD86 is increased. In some embodiments, the variant CTLA-4 peptide has increased binding affinity to ICOSL relative to the wild-type or unmodified CTLA-4 peptide, and optionally decreased binding affinity to one or more CD80 and / or CD86.
[0197] In some embodiments, the binding activity or affinity of the variant CTLA-4 peptide for CD80 and optionally one or more ligands ICOSL and CD86 is altered (increased or decreased) compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the binding affinity of the variant CTLA-4 peptide for CD80 and optionally ICOSL and / or CD86 differs from the binding affinity of the wild-type or unmodified CTLA-4 peptide control (e.g., unmodified) sequence, as determined by, for example, solid-phase ELISA immunoassay, flow cytometry, ForteBio... Or as determined by Biacore assays. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD80 and optionally ICOSL and / or CD86. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD80 relative to the wild-type or unmodified CTLA-4 peptide, and optionally decreased binding affinity for one or more ICOSL and / or CD86.
[0198] In some embodiments, the binding activity or affinity of the variant CTLA-4 peptide for CD86 and optionally one or more ligands ICOSL and CD80 is altered (increased or decreased) compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the binding affinity of the variant CTLA-4 peptide for CD86 and optionally ICOSL and / or CD80 differs from the binding affinity of the wild-type or unmodified CTLA-4 peptide control (e.g., unmodified) sequence, as determined by, for example, solid-phase ELISA immunoassay, flow cytometry, ForteBio... Or as determined by Biacore assays. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD86 and optionally ICOSL and / or CD80. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD86 relative to the wild-type or unmodified CTLA-4 peptide, and optionally decreased binding affinity for one or more ICOSL and / or CD80.
[0199] The binding affinity of each binding partner in the binding partner is independent. That is, in some embodiments, the variant CTLA-4 peptide exhibits 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 the wild-type or unmodified CTLA-4 peptide.
[0200] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD86 relative to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for CD80 relative to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for both CD80 and CD86 relative to the wild-type or unmodified CTLA-4 peptide. In some embodiments, such variant CTLA-4 peptides also exhibit increased binding affinity for ICOSL relative to the wild-type or unmodified CTLA-4 peptide.
[0201] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased or decreased binding affinity for ICOSL and increased binding affinity for CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased or decreased binding affinity for ICOSL and increased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and decreased binding affinity for CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased or decreased binding affinity for ICOSL and decreased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide.
[0202] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for both ICOSL and CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL and decreased binding affinity for CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for both ICOSL and CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and increased binding affinity for CD80 compared to the wild-type or unmodified CTLA-4 peptide.
[0203] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for both ICOSL and CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL and decreased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for both ICOSL and CD86 relative to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and increased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide.
[0204] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL, CD80, and CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL and CD80 and decreased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL and CD86 and decreased binding affinity for CD80 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and CD80 and increased binding affinity for CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and increased binding affinity for CD80 and CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity for ICOSL and decreased binding affinity for CD80 and CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL, CD80, and CD86 compared to the wild-type or unmodified CTLA-4 peptide. In some embodiments, the variant CTLA-4 peptide exhibits decreased binding affinity for ICOSL and increased binding affinity for CD80 and CD86 compared to the wild-type or unmodified CTLA-4 peptide.
[0205] In some embodiments, variant CTLA-4 peptides exhibiting increased or greater binding affinity for ICOSL, CD80, and / or CD86 relative to wild-type or unmodified CTLA-4 peptide controls will show an increase of at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50%. In some embodiments, the increase in binding affinity relative to wild-type or unmodified CTLA-4 peptides 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 peptide has the same sequence as the variant CTLA-4 peptide, except that the wild-type or unmodified CTLA-4 peptide does not contain the one or more amino acid modifications (e.g., substitutions).
[0206] In some embodiments, variant CTLA-4 peptides exhibiting reduced or decreased binding affinity for ICSOL, CD80, and / or CD86 compared to wild-type or unmodified CTLA-4 peptide controls will show a reduction in binding affinity for ICSOL, CD80, and / or CD86 of at least 5%, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the reduction in binding affinity compared to wild-type or unmodified CTLA-4 peptides 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 peptide has the same sequence as the variant CTLA-4 peptide, except that the wild-type or unmodified CTLA-4 peptide does not contain the one or more amino acid modifications (e.g., substitutions).
[0207] In some embodiments, any of the foregoing embodiments relates to the equilibrium dissociation constant (K) of ICOSL, CD80, and / or CD86. D It can be less than 1×10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M or 1×10 -12 M or smaller.
[0208] Wild-type or unmodified CTLA-4 sequences are not necessarily required as starting compositions for generating the variant CTLA-4 peptides described herein. Therefore, the use of the term “modification” (e.g., “substitution”) does not imply that embodiments of the invention are limited to a specific method for preparing variant CTLA-4 peptides. Variant CTLA-4 peptides can be prepared, for example, by de novo peptide synthesis, and therefore modification (e.g., “substitution”) is not necessarily required in the sense of changing codons to encode a modification (e.g., substitution). This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of preparation. Methods for designing or generating variant CTLA-4 peptides are not limited to any particular method. However, in some embodiments, wild-type or unmodified CTLA-4 encoding nucleic acids is induced from wild-type or unmodified CTLA-4 genetic material, and screening is performed for desired specific binding affinity and / or inhibition or reduction of IFN-γ expression or other functional activities.
[0209] In some embodiments, variant CTLA-4 peptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases, followed by screening. Such information is provided by the National Center for Biotechnology Information (NCBI), and its website, like the UniProtKB database discussed earlier, is publicly accessible via the Internet.
[0210] Unless otherwise stated, as indicated throughout this disclosure, the one or more amino acid modifications are designated by amino acid position numbers corresponding to the positions of the unmodified ECD sequence shown in SEQ ID NO:2 or (where applicable) the unmodified ECD sequence shown in SEQ ID NO:569, as follows:
[0211] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI YVIDPEPCPDSD(SEQ ID NO:2)
[0212] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI YVIDPEPCPDSDQ(SEQ ID NO:569)
[0213] In some embodiments, the variant CTLA-4 polypeptide contains any one or more of the provided amino acid modifications (e.g., amino acid substitutions) of reference SEQ ID NO:2 or 569 or their specific binding fragment. In some embodiments, the variant CTLA-4 polypeptide contains any one or more of the provided amino acid modifications of a polypeptide that is or contains an IgV domain or its specific binding fragment (as shown in the exemplary sequence in SEQ ID NO:3).
[0214] HVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGN ELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY(SEQ ID NO:3)
[0215] Identifying the corresponding positions of modifications (e.g., amino acid substitutions) in the CTLA-4 peptide (including the portion containing its IgSF domain (e.g., IgV)) by comparing a reference sequence (e.g., SEQ ID NO:3) with SEQ ID NO:2 or SEQ ID NO:569 is within the skill of a person skilled in the art. Throughout the list of modifications, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g., wild-type) amino acid listed before the number, and the identified variant amino acid substitution listed after the number. If the modification is a positional omission, it is indicated as “del”, and if the modification is an insertion at the position, it is indicated as “ins”. In some cases, insertions are listed as follows: the amino acid position is indicated in the middle, the corresponding unmodified (e.g., wild-type) amino acid is listed before and after the number, and the identified variant amino acid insertion is listed after the unmodified (e.g., wild-type) amino acid.
[0216] In some embodiments, the variant CTLA-4 peptide has one or more amino acid modifications, such as substitutions, in the wild-type or unmodified CTLA-4 sequence. These modifications, such as substitutions, may be located in the extracellular domain (extracellular structural domain) of the wild-type or unmodified CTLA-4 sequence. In some embodiments, these modifications, such as substitutions, are located in the IgV domain or its specific binding fragment.
[0217] In some embodiments, the variant CTLA-4 peptide 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, such as substitutions. Substitutions may be located in the IgV domain or the ECD. In some embodiments, the variant CTLA-4 peptide 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, such as substitutions, in the IgV domain or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide 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, such as substitutions, in the ECD or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type or unmodified CTLA-4 peptide or its specific binding fragment (such as with the amino acid sequence of SEQ ID NO: 2 or 3).
[0218] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications corresponding to one or more of the following positions in the unmodified CTLA-4 or its specific binding fragment, such as substitutions: 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, 122, 124, 125 and / or 126.
[0219] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications corresponding to one or more of the following positions in the unmodified CTLA-4 or its specific binding fragment, such as substitutions: 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.
[0220] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide has one or more amino acid modifications corresponding to one or more of the following positions in the unmodified CTLA-4 or its specific binding fragment, such as substitutions for: 12, 18, 26, 29, 33, 53, 55, 56, 58, 63, 72, 87, 98, 99, 105, 106 and / or 117.
[0221] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide has one or more amino acid modifications corresponding to one or more of the following positions in the unmodified CTLA-4 or its specific binding fragment, such as substitutions: 12, 18, 26, 29, 56, 63, 72, 98, 99, 105, 106 and / or 117.
[0222] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: 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, M5 5T, 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, R85 G, A86T, M87A, M87K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I93V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, P 102L, Y105F, Y105L, L106I, L106N, L106R, L106V, I108F, I108V, N110K, N110S, N110Y, Q113H, Y115H, Y115N, V116A, I117E, I117K, I117L, I117M, I117T, P119H, E120D, P121S, C122P, D124P, D124I, S125I, S125P, D126P and / or D126T.
[0223] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: 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, 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, M 87V, 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 and / or I117T.
[0224] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: L12F, L12H, L12I, L12P, I18A, I18F, I18N, I18T, I18V, A26D, A26S, A26T, G29R, G29W, E33M, E33V, T53S, M55R, M55T, M55V, M56K, M5 6L, M56R, M56T, M56V, N58D, N58S, L63H, L63P, S72G, S72T, M87A, M87K, M87T, M87V, L98Q, L98R, M99I, M99L, Y105F, Y105L, L106I, L106N, L106R, L106V, I117E, I117K, I117L, I117M and / or I117T. In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: I12F, L12P, I18T, A26T, G29W, T53S, M55T, M56K, M56T, N58S, S72G, M99L, L63P, L98Q, Y105L, L106I and / or I117L. In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: L12P, I18T, A26T, G29W, T53S, M55T, M56K, N58S, S72G, M99L, L63P, L98Q, Y105L, L106I and / or I117L. In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide has one or more amino acid modifications selected from the following or their conserved amino acid substitutions: A26T, G29W, L63P, S72G, L98Q, M99L, Y105L and / or L106I.
[0225] Conservative amino acid substitutions are any amino acid that belongs to the same class as the substituted amino acid, except for wild-type or unmodified amino acids. Amino acids are categorized as aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, and tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).
[0226] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to A26T in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions of G29W, T53S, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions of A26T / G29W, A26T / T53S, A26T / L63P, A26T / S72G, A26T / L98Q, A26T / M99L, A26T / Y105L, and A26T / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0227] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to G29W in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, T53S, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / G29W, G29W / T53S, G29W / L63P, G29W / S72G, G29W / L98Q, G29W / M99L, G29W / Y105L, or G29W / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0228] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to T53S in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, L63P, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / T53S, G29W / T53S, T53S / L63P, T53S / S72G, T53S / L98Q, T53S / M99L, T53S / Y105L, or T53S / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0229] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to L63P in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, S72G, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / L63P, G29W / L63P, T53S / L63P, L63P / S72G, L63P / L98Q, L63P / M99L, L63P / Y105L, or L63P / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0230] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to S72G in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, L98Q, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / S72G, G29W / S72G, T53S / S72G, L63P / S72G, S72G / L98Q, S72G / M99L, S72G / Y105L, or S72G / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0231] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to L98Q in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, M99L, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / L98Q, G29W / L98Q, T53S / L98Q, L63P / L98Q, S72G / L98Q, L98Q / M99L, L98Q / Y105L, or L98Q / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0232] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to M99L in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, Y105L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / M99L, G29W / M99L, T53S / M99L, L63P / M99L, S72G / M99L, L98Q / M99L, M99L / Y105L, or M99L / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0233] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to Y105L in the unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L, and / or L106I. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / Y105L, G29W / Y105L, T53S / Y105L, L63P / Y105L, S72G / Y105L, L98Q / Y105L, M99L / Y105L, or Y105L / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0234] In some embodiments, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to L106I in an unmodified or wild-type CTLA-4 peptide or its specific binding fragment. In some embodiments, the variant CTLA-4 peptide further contains one or more amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L, and / or Y105L. In some embodiments, the variant CTLA-4 peptide contains amino acid substitutions A26T / L106I, G29W / L106I, T53S / L106I, L63P / L106I, S72G / L106I, L98Q / L106I, M99L / L106I, or Y105L / L106I. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0235] In some embodiments, the variant CTLA-4 contains the amino acid substitution E33V / M99L. The variant CTLA-4 peptide may contain additional amino acid modifications (e.g., substitutions) consistent with the provided embodiments, as described herein. Table 2 lists the described exemplary amino acid modifications (e.g., substitutions) and variant CTLA-4 peptides.
[0236] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide comprises at least three amino acid modifications (e.g., substitutions) or conserved amino acid substitutions of unmodified or wild-type CTLA-4 or its specific binding fragment corresponding to A26T, G29W, T53S, L63P, S72G, L98Q, M99L, Y105L and / or L106I, wherein the at least three amino acid modifications (e.g., substitutions) are described.
[0237] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide comprises an amino acid substitution corresponding to G29W / L98Q / Y105L in unmodified or wild-type CTLA-4 or its specific binding fragment.
[0238] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 peptide comprises an amino acid substitution corresponding to G29W / N58S / L63P / Q82R / L98Q / Y105L in unmodified or wild-type CTLA-4 or its specific binding fragment.
[0239] In some embodiments, referring to the position shown in SEQ ID NO:2, the variant CTLA-4 polypeptide comprises amino acid substitutions corresponding to L12F / R16H / G29W / M56T / L98Q / Y105L in unmodified or wild-type CTLA-4 or its specific binding fragment.
[0240] In some embodiments of the variant CTLA-4 peptide having a single amino acid substitution in unmodified CTLA-4 or its specific binding fragment, referring to the positions shown in SEQ ID NO:2, the modification does not correspond to positions 27, 31, 32, 33, 35, 95, 98, 105, 106, or 107. In some embodiments of the variant CTLA-4 peptide having a single amino acid substitution in unmodified CTLA-4 or its specific binding fragment, the modification is not L106E. In some embodiments of the variant CTLA-4 peptide having exactly two amino acid substitutions in unmodified CTLA-4 or its specific binding fragment, the modification is not A31Y and L106E (i.e., A31Y / L106E).
[0241] In some embodiments, referring to the numbers shown in SEQ ID NO:2, the variant CTLA-4 does not contain modifications corresponding to A26E, T32N, V34I, A52M, G57E, I67F, S66P, and / or S72F. In some embodiments where the variant CTLA-4 peptide has seven amino acid substitutions in unmodified CTLA-4 or its specific binding fragment, referring to the numbers shown in SEQ ID NO:2, the modifications are not corresponding to T32N, V34I, A52M, M56K, G57E, S66P, and S72F. In some embodiments where the variant CTLA-4 peptide has ten amino acid substitutions in unmodified CTLA-4 or its specific binding fragment, referring to the numbers shown in SEQ ID NO:2, the modifications are not corresponding to A26E, T32N, V34I, A52M, M56K, N58D, S66P, I67S, S72F, and L106E.
[0242] In some embodiments, referring to the numbers shown in SEQ ID NO:2, the variant CTLA-4 peptide has two or more amino acid modifications selected from the following: 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, V46 E, T47A, E48R, T53S, Y54F, M55R, M55T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N58S, E59D, E59 G, T61A, T61I, T61N, T61R, T61S, L63H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T69A, T6 9I, T69S, T71A, T71I, S72G, S72T, S73R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M87K, M87T, M 87V, 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, and I117T. In some embodiments, reference is made to SEQ ID. The two or more amino acid modifications shown in NO:2 are A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98Q / Y105L, and 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 / P121 S、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 / P121 S、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 / S72G / L98Q / Y105L / P121S, G29W / L63P / L98Q / M99L / Y105L, G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H, G29W / M87K / T89S / L98Q / M99L / Y 105L / I108V / I117L, G29W / M87K / I93V / L98Q / M99L / Y105L, G29W / L98Q / M99L / Y105L, E33M / A42T / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L, E33M / L6 3P / S72G / L98Q / Y105L / I108F, E33M / L63P / S72G / L98Q / Y105L / I117L, E33M / Q82H / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, T37S / M56 V / L98Q / Y105L、V38I / L63P / S72G / L98Q / M99L / Y105L、Q41L / Y54F / M56K / M9 9L / I108F、T53S / M56V / L98Q / Y105L、M55T / L63P / T71I / M99L / Y105L、M55T / S 72G / L98Q / M99L / Y105L、M55T / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M99L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L106I / I117L、M56R / L63P / L 98Q / M99L / Y105L、M56T / L91R / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S72G / L98Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y105L / L106R / V116A、L63P / S72G / M87A / L98Q / Y105L、L 63P / S72G / I93L / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L、L63P / S 72G / L98Q / M99L / Y105L / L106I / I117L、L63P / S72G / L98Q / Y105L、L63P / S72 G / 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 / L9 8Q / M99L / Y105L / I108V, L63P / L98Q / M99L / Y105L / I117M, L63P / L98Q / Y105L, L63P / L98Q / V116A, L63P / L98R / N110K, L63P / M9 9L / 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.
[0243] In some embodiments, referring to the numbers shown in SEQ ID NO:2, the variant CTLA-4 peptide has two or more amino acid modifications selected from the following: 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, 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, S72 T, 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, 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, referring to the numbers shown in SEQ ID NO:2, the two or more amino acid modifications are 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 / P121 S、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 / S72G / L98Q / Y105L / P121 S,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 / E59G / L63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S72G / 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 / S72G / 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、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, L 12F / R16H / G29W / M56T / L98Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T The following are listed: / 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.
[0244] In some embodiments, referring to the position shown in SEQ ID NO:2 or 569, the variant CTLA-4 polypeptide additionally comprises the amino acid modification C122S.
[0245] In some embodiments, the variant CTLA-4 polypeptide includes any of the substitutions (mutations) listed in Table 2. Table 2 also provides exemplary sequences by referring to the SEQ ID NO of the extracellular domain (ECD) or IgV domain of wild-type CTLA-4 or the exemplary variant CTLA-4 polypeptide. Table 2 also provides exemplary sequences of variant CTLA-4 polypeptides linked to immunoglobulin Fc (thus generating “immunomodulatory Fc fusions,” such as “CTLA-4-Fc variant fusions,” also known as CTLA-4vIgD-Fc fusions). Such fusion polypeptides are further described below. As indicated, the exact locus or residue corresponding to a given domain may vary, as depends on the method used to identify or classify the domain. Moreover, in some cases, the sequence of the variant IgSF polypeptide may also include adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., IgV) to ensure that the domain can fold properly upon expression. Therefore, it should be understood that the examples of SEQ ID NO in Table 2 should not be construed as limiting. For example, specific domains of the variant CTLA-4 peptide (such as the IgV domain) may be a few amino acids longer or shorter than the amino acid sequence shown in the corresponding SEQ ID NO, such as 1-10 amino acids longer or shorter, for example, 1, 2, 3, 4, 5, 6 or 7 amino acids.
[0246] In some embodiments, the variant CTLA-4 polypeptide comprises any mutation listed in Table 2. In some embodiments, the variant CTLA-4 polypeptide comprises any ECD sequence (i.e., any one of SEQ ID NO: 4-97, 99-104, or 106-155) of the extracellular domain (ECD) sequences listed in Table 2. In some embodiments, the variant CTLA-4 polypeptide comprises a polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, 97%, 98%, or 99% identity with any ECD sequence (i.e., any one of SEQ ID NO: 4-97, 99-104, or 106-155) of the extracellular domain (ECD) sequences listed in Table 2, and containing one or more amino acid modifications, such as one or more amino acid substitutions, that are not present in wild-type or unmodified CTLA-4 corresponding to the SEQ ID NO. In some embodiments, the variant 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 NO: 4-97, 99-104, or 106-155) and contains one or more amino acid modifications of the corresponding SEQ ID NO that are not present in wild-type or unmodified CTLA-4, such as one or more amino acid substitutions.
[0247] In some embodiments, the variant CTLA-4 peptide includes any mutation listed in Table 2. In some embodiments, the variant CTLA-4 peptide includes any ECD sequence (i.e., any one of SEQ ID NO: 4-97, 99-104, 106-155, 570-602, or 636) listed in Table 2. In some embodiments, the variant CTLA-4 polypeptide comprises a polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, 97%, 98%, or 99% identity with any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., any one of SEQ ID NO: 4-97, 99-104, 106-155, 570-602, or 636). In some embodiments, the variant 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 NO: 4-97, 99-104, 106-155, 570-602, or 636) and contains one or more amino acid modifications of the corresponding SEQ ID NO that are not present in wild-type or unmodified CTLA-4, such as one or more amino acid substitutions.
[0248] In some embodiments, the variant CTLA-4 polypeptide comprises any IgV sequence listed in Table 2 (i.e., any one of SEQ ID NO: 156-285). In some embodiments, the variant CTLA-4 polypeptide comprises a polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, 97%, 98%, or 99% identity with any IgV sequence listed in Table 2 (i.e., any one of SEQ ID NO: 156-285), and contains one or more amino acid modifications, such as one or more amino acid substitutions, that are not present in wild-type or unmodified CTLA-4 corresponding to the SEQ ID NO. In some embodiments, the variant CTLA-4 polypeptide comprises a specific binding fragment of any IgV sequence (i.e., any one of SEQ ID NO: 156-285) listed in Table 2, and contains one or more amino acid modifications of the corresponding SEQ ID NO that are not present in wild-type or unmodified CTLA-4, such as one or more amino acid substitutions.
[0249] In some embodiments, the variant CTLA-4 polypeptide comprises any IgV sequence listed in Table 2 (i.e., any one of SEQ ID NO: 156-285, 603-635, or 637). In some embodiments, the variant CTLA-4 polypeptide comprises a polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, 97%, 98%, or 99% identity with any IgV sequence listed in Table 2 (i.e., any one of SEQ ID NO: 156-285, 603-635, or 637), and contains one or more amino acid modifications, such as one or more amino acid substitutions, that are not present in wild-type or unmodified CTLA-4 according to the corresponding SEQ ID NO. In some embodiments, the variant CTLA-4 polypeptide comprises a specific binding fragment of any IgV sequence listed in Table 2 (i.e., any one of SEQ ID NO: 156-285, 603-635, or 637) and contains one or more amino acid modifications, such as one or more amino acid substitutions, that are not present in wild-type or unmodified CTLA-4 of the corresponding SEQ ID NO.
[0250]
[0251]
[0252]
[0253]
[0254]
[0255] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD80 compared to the wild-type or unmodified CTLA-4 peptide, as shown in SEQ ID NO:2 or 3. In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD86 compared to the wild-type or unmodified CTLA-4 peptide, as shown in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 peptide exhibits increased affinity to both the extracellular domains of CD80 and CD86 compared to the wild-type or unmodified CTLA-4 peptide, as shown in SEQ ID NO:2 or 3.
[0256] In some embodiments, the variant CTLA-4 peptide exhibits increased affinity for the extracellular domain of ICOSL compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 peptide exhibits increased affinity for both the extracellular domain of ICOSL and the extracellular domain of CD80 compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3. In some embodiments, the CTLA-4 peptide exhibits increased affinity for both the extracellular domain of ICOSL and the extracellular domain of CD86 compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3.
[0257] In some embodiments, the CTLA-4 peptide exhibits increased affinity for the extracellular domains of ICOSL, CD80, and CD86 compared to wild-type or unmodified CTLA-4, such as those comprising the sequence shown in SEQ ID NO:2 or 3.
[0258] In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD80 and decreased binding affinity to the extracellular domain of CD86 and / or ICOSL. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of CD80 and CD86 and decreased affinity to the extracellular domain of ICOSL. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of CD80 and ICOSL and decreased affinity to the extracellular domain of CD86. In some embodiments, the variant CTLA-4 peptide exhibits increased affinity for the extracellular domain of CD80 and decreased affinity for the extracellular domains of CD86 and ICOSL compared to the wild-type or unmodified CTLA-4 peptide, which includes the sequence shown in SEQ ID NO:2 or 3.
[0259] In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD86 and decreased binding affinity to the extracellular domain of CD80 and / or ICOSL. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of CD86 and CD80 and decreased affinity to the extracellular domain of ICOSL. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of CD86 and ICOSL and decreased affinity to the extracellular domain of CD80. In some embodiments, the variant CTLA-4 peptide exhibits increased affinity for the extracellular domain of CD86 and decreased affinity for the extracellular domains of CD80 and ICOSL compared to the wild-type or unmodified CTLA-4 peptide, which includes the sequence shown in SEQ ID NO:2 or 3.
[0260] In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of ICOSL and decreased binding affinity to the extracellular domains of CD80 and / or CD86. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of ICOSL and CD80 and decreased affinity to the extracellular domain of CD86. In some embodiments, compared to wild-type or unmodified CTLA-4 peptides including the sequence shown in SEQ ID NO:2 or 3, the variant CTLA-4 peptide exhibits increased affinity to the extracellular domains of ICOSL and CD86 and decreased affinity to the extracellular domain of CD80. In some embodiments, the variant CTLA-4 peptide exhibits increased affinity for the extracellular domain of ICOSL and decreased affinity for the extracellular domains of CD80 and CD86 compared to the wild-type or unmodified CTLA-4 peptide, which includes the sequence shown in SEQ ID NO:2 or 3.
[0261] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD80 compared to wild-type or unmodified CTLA-4 peptides, such as those comprising the sequence shown in SEQ ID NO:2 or 3. In some embodiments, the increased affinity to the extracellular domain of CD80 is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to the binding affinity of unmodified CTLA-4 to the extracellular domain of CD80. In some of these embodiments, referring to SEQ ID NO:2, a variant CTLA-4 peptide with increased binding affinity to CD80 compared to the wild-type or unmodified CTLA-4 peptide has one or more amino acid substitutions in the extracellular domain or IgV domain of the unmodified or wild-type CTLA-4, namely 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 some of these embodiments, referring to SEQ ID NO:2, a variant CTLA-4 peptide with increased binding affinity to CD80 compared to wild-type or unmodified CTLA-4 peptides has one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, namely I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M56K, N58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M99L, Y105L and / or I108V. In some of these embodiments, referring to SEQ ID NO:2, a variant CTLA-4 peptide with increased binding affinity to CD80 compared to wild-type or unmodified CTLA-4 peptides has one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, namely I18T, A26T, G29W, A42V, Q45H, N58S, L63P, I67N, Q82R, M87K, M87V, L98Q and / or Y105L.In some of these embodiments, referring to SEQ ID NO:2, the variant CTLA-4 peptide with increased binding affinity to CD80 compared to the wild-type or unmodified CTLA-4 peptide has one or more amino acid substitutions in the extracellular domain of the unmodified or wild-type CTLA-4 or its IgV domain, namely: I18T, A26T, G29W, N58S, L63P, M87K, M87V, L98Q, and / or Y105L. In some embodiments, the variant CTLA-4 peptide has at least two, three, four, or five of these amino acid substitutions and exhibits increased binding to the extracellular domain of CD80 compared to wild-type or unmodified CTLA-4 peptides, such as those including the sequence shown 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 / L 63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L 98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L 98Q / 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 / T53 S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, L98Q / M99L / Y105 L, 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 located in the CTLA-4 extracellular domain shown in SEQ ID NO:2, for example, see the exemplary SEQ ID NOs shown in Table 2. In some embodiments, any of the above substitutions is located in the CTLA-4 extracellular domain shown in SEQ ID NO:3, for example, see the exemplary SEQ ID NOs shown in Table 2.
[0262] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of CD86 compared to wild-type or unmodified CTLA-4 peptides, such as those comprising the sequence shown in SEQ ID NO:2 or 3. In some embodiments, the increased affinity to the extracellular domain of CD86 is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to the binding affinity of unmodified CTLA-4 to the extracellular domain of CD86. In some of these embodiments, reference is made to SEQ ID NO:2 or 3. IDNO:2 indicates a variant of CTLA-4 peptide with increased binding affinity to CD86 compared to wild-type or unmodified CTLA-4 peptides. This variant possesses one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, such as L12F, L12H, L12P, R16H, I18T, I18V, S20N, A26T, G29R, G29W, E33M, E33V, A42S, A42V, T47A, T53S, M55T, M56K, M56R, M56T, M56V, N58D, N... 58S, T61I, T61N, T61R, T61S, L63P, D65G, I67N, I67V, 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, referring to SEQ ID NO:2, a variant CTLA-4 peptide with increased binding affinity to CD86 compared to a wild-type or unmodified CTLA-4 peptide has one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, namely L12P, A26T, M55T, L63P, S72G, L98Q, M99L, and / or Y105L. In some embodiments, the variant CTLA-4 peptide has at least two, three, four, or five such amino acid substitutions and exhibits increased binding to the extracellular domain of CD86 compared to wild-type or unmodified CTLA-4 peptides such as those including sequences shown 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 / 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、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 / Y105L / L106N、L63P / T69A / L98Q / M99L / Y105L / L106R / V116A、G29W / N58S / L63P / S72G / L98Q / Y105L、G29W / L63P / D65G / S72G / L98Q / Y105L、T53S / M56V / L98Q / Y105L、L63P / S72G / L98Q / Y105L、G29W / L63P / S72G / L98Q / Y105L / L106I、L12F / R16H / G29W / M56T / L98Q / Y105L、G29W / 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 / 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 / L 98R / Y105L / L106I / P121S, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, L12P / M56V / L63P / V96I / L98Q / M99L / Y10 5L / Y115H, G29W / T53S / M56K / T61N / L63P / L98Q / Y105L, I18T / T61R / L63P / S7 2G / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S7 2G / L98Q / Y105L / I108F, L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y1 05L / 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 examples, the amino acid substitution is A26T / M55T / L63P / S72G / L98Q / M99L / Y105L or L12P / L63P / S72G / L98Q / M99L / Y105L. In some embodiments, any of the above substitutions is located in the extracellular domain of CTLA-4 as shown in SEQ ID NO:2, for example, see the exemplary SEQ ID NOs shown in Table 2. In some embodiments, any of the above substitutions are located in the extracellular domain of CTLA-4 as shown in SEQ ID NO:3, for example, see the exemplary SEQ ID NOs shown in Table 2.
[0263] In some embodiments, the variant CTLA-4 peptide exhibits increased binding affinity to the extracellular domain of ICOSL compared to wild-type or unmodified CTLA-4 peptides, such as those comprising the sequence shown in SEQ ID NO:2 or 3. In some embodiments, the increased affinity to the extracellular domain of ICOSL is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold compared to the binding affinity of unmodified CTLA-4 to the extracellular domain of ICOSL. In some of these embodiments, reference is made to SEQ ID NO:2 or 3. The NO:2 designation indicates a variant of CTLA-4 peptide with increased binding affinity to ICOSL compared to wild-type or unmodified CTLA-4 peptides. This variant possesses one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, such as V10A, L12F, L12I, R16H, I18N, I18T, I18V, A19V, A26T, G29W, E33M, E33V, R35K, V38I, A42V, Q45H, T47A, T53S, M55T, M56K, M56V, or N5. 8D, N58S, T61A, T61R, L63H, L63P, D64E, D64N, D64V, 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 embodiments of these examples, reference is made to SEQ ID. The NO:2 designation indicates a variant of CTLA-4 peptide with increased binding affinity to ICOSL compared to wild-type or unmodified CTLA-4 peptides. This variant possesses one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, such as V10A, L12F, R16H, I18T, I18V, A19V, A26T, G29W, E33V, R35K, A42V, and Q45H. , T47A, T53S, M55T, M56K, M56T, N58D, N58S, L63P, D64E, D64V, I67N, I67V, S72G, Q82H, Q82R, R8 5G, M87K, M87T, M87V, T89A, T89S, I93V, E97Q, L98Q, M99L, Y105L, L106I, L106R, I108F, I108V,N100K and / or I117L. In some of these embodiments, referring to SEQ ID NO:2, a variant CTLA-4 peptide with increased binding affinity to ICOSL compared to the wild-type or unmodified CTLA-4 peptide has one or more amino acid substitutions in the extracellular domain or IgV domain of the unmodified or wild-type CTLA-4, such as L12F, I18T, I18V, A26T, G29W, E33V, A42V, Q45H, T53S, M. 55T, 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, referring to SEQ ID NO:2, variant CTLA-4 peptides exhibiting increased binding affinity to ICOSL compared to wild-type or unmodified CTLA-4 peptides have one or more amino acid substitutions in the extracellular domain or IgV domain of unmodified or wild-type CTLA-4, namely I18T, A26T, G29W, T53S, M55T, M56K, N58S, L63P, S72G, L98Q, M99L, Y105L, L106I, and / or N117L. In some embodiments, the variant CTLA-4 peptide has at least two, three, four, or five of these amino acid substitutions and exhibits increased binding to the extracellular domain of ICOSL compared to wild-type or unmodified CTLA-4 peptides, such as those including the sequences shown 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, L63P / Q82H / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99L / Y105L, and T61A. / L63P / S72G / L98Q / M99L / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, L63P / S72G / I93L / L98Q / M99 L / 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 / M 99L / Y105L、I18T / L63P / S72G / M87K / L98Q / M99L / Y105L、L63P / M87K / M99L / L 106R、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、G29W / T53S / M56K / L63P / L98Q / Y105L、L12F / R16H / G29W / M56T / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G 29W / N58D / I67V / L98Q / M99L / Y105L、I67V / S72G / Q82H / T89A / L98Q / M99L / Y1 05L、S72G / R85G / L98Q / M99L / Y105L / L106I、A26T / T47A / M56K / L63P / S72G / Q 82R / L98Q / M99L / Y105L, A26T / M55T / L63P / S72G / L98Q / M99L / Y105L, G29W / M87K / I93V / L98Q / M99L / Y105L, G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y10 5L, L63P / L98Q / M99L / Y105L / I108V, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, E33M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L98Q / M99L / Y105L / I1 08V / I117L、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S72G / L 98Q / 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, I 18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T 53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E 33V / M99L, T53S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N58S / L63P / L98 Q / 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 / M87 K / E97Q / M99L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, T53S / M56K / N58S / L6 3P / 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 / L63 P / 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. In some embodiments, any of the above substitutions is located in the CTLA-4 extracellular domain shown in SEQ ID NO:2, for example, see the exemplary SEQ ID NOs shown in Table 2. In some embodiments, any of the above substitutions is located in the CTLA-4 extracellular domain shown in SEQ ID NO:3, for example, see the exemplary SEQ ID NOs shown in Table 2.
[0264] In some instances, variant CTLA-4 peptides, when binding to one or more binding partners of CTLA-4 (such as CD80, CD86, and / or ICOSL), contain an extracellular domain having at least one affinity-modified IgSF domain (e.g., IgV) or a specific binding fragment thereof, which, compared to wild-type or unmodified CTLA-4 peptides, alter signal transduction (e.g., reduce / inhibit signal transduction) by means of a stimulatory receptor capable of binding to said one or more binding partners. In some aspects, the stimulatory receptor is CD28 or ICOS, which in some respects is expressed on the surface of T cells and is capable of releasing cytokines in response to intracellular signals. In some embodiments, after incubation with a specified variant and / or wild-type or unmodified CTLA-4 peptide, the altered signaling differs from that achieved by a wild-type or unmodified CTLA-4 peptide control sequence of the same format (e.g., an Fc fusion protein), as determined by an assay, for example, measuring cytokine release (e.g., IL-2 release or IFN-γ). Exemplary assays are described in the examples. In some cases, the assay is cell-based, such as a mixed lymphocyte reaction, where the resulting readout or function is the sum of the signaling activities of functionally binding partner expressed on the surface of the cells in the assay. As discussed elsewhere herein, in some embodiments, the format of the provided variant CTLA-4 peptide may affect the type of activity, e.g., agonist or antagonist. In some embodiments, the variant CTLA-4 peptide is a fusion protein containing an extracellular domain containing an IgV domain and a polymerized domain (such as an Fc domain) or a specific binding fragment thereof, and the signaling is altered because the activity of the antagonist blocks the activity of the stimulating receptor. In other respects, the format may increase the activity of the stimulating receptor or produce agonist activity of the stimulating receptor.
[0265] III. Format of Variant Peptides
[0266] Immunomodulatory peptides including the variant CTLA-4 provided herein can be formatted in various ways, including as soluble proteins, membrane-bound proteins, or secreted proteins. In some embodiments, a specific format can be selected for a desired therapeutic application. In some cases, immunomodulatory peptides including the variant CTLA-4 peptide antagonize or block the activity of CD28 and / or ICOS by binding to their binding partners (e.g., ICOSL, CD80, and / or CD86), thereby preventing the delivery of a CD28 and / or ICOS co-stimulatory signal. In some embodiments, the antagonistic effect of CD28 and / or ICOSL can be used, for example, in autoimmunity to inhibit or suppress immunity. In some embodiments, the antagonistic effect of CD28 and / or ICOSL can be used to treat inflammation or autoimmunity. In some cases, immunomodulatory peptides including the variant CTLA-4 peptide are expressed on cells as a switch receptor that converts the CTLA-4 inhibitory receptor to an activating receptor (e.g., having 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 can be used to treat cancer. Those skilled in the art can readily determine the activity of a particular formulation, such as for antagonizing, competing with, and / or blocking one or more specific binding partners or activating one or more downstream signaling pathways. Exemplary methods for evaluating such activity are provided herein, included in the examples.
[0267] In some aspects, immunomodulatory proteins comprising vIgD of CTLA-4 are provided, wherein such proteins are soluble, for example, fused to an Fc chain. In some aspects, one or more additional IgSF domains (such as one or more additional vIgDs) may be linked to the vIgD of CTLA-4 provided herein (hereinafter referred to as “stacked” or “stacked” immunomodulatory proteins). In some embodiments, the modular format of the provided immunomodulatory proteins provides flexibility for engineering or generating immunomodulatory proteins for regulating the activity of multiple anti-structures (multiple homologous binding partners). In some embodiments, such “stacked” molecules may be provided in a soluble format, or in some cases, may be provided as membrane-bound or secreted proteins. In some embodiments, variant CTLA-4 immunomodulatory proteins are provided as conjugates containing vIgD of CTLA-4, which is directly or indirectly linked to a target agent or part of the conjugate (e.g., linked to an antibody or other binding molecule that specifically binds to a ligand (e.g., an antigen), for example, to target or localize the vIgD to a specific environment or cell, such as when administered to a subject. In some embodiments, the targeting agent (e.g., an antibody or other binding molecule) binds to antigens on the surface of leukocytes, lymphocytes, or lymphoid tissues (e.g., spleen, tonsils, lymphatic vessels, lymph nodes, adenoids, and liver), thereby targeting the vIgD-containing variant CTLA-4 to the immune system, for example, to modulate the activity of leukocytes or lymphocytes within the immune system.
[0268] In some embodiments, the provided immunomodulatory protein is expressed intracellularly or on cells and is provided as part of engineered cell therapy (ECT). In some embodiments, the variant CTLA-4 peptide is expressed on cells (such as immune cells, e.g., T cells or antigen-presenting cells) in a membrane-bound form, thereby providing a transmembrane immunomodulatory protein (hereinafter also referred to as "TIP"). In some aspects, the variant CTLA-4 peptide is expressed in cells (such as immune cells, e.g., T cells or antigen-presenting cells) in a secretible form, thereby producing a secretory or soluble form of the variant CTLA-4 peptide (hereinafter also referred to as "SIP"), as when the cells are administered to a subject. In some aspects, the SIP can antagonize binding partners in its secreted environment (e.g., the immune microenvironment). In some embodiments, the variant CTLA-4 peptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that, when administered to a subject, is capable of in vivo infecting cells (such as immune cells, e.g., T cells or antigen-presenting cells) to deliver or express the variant peptide in the cells as a TIP or SIP.
[0269] In some embodiments, a soluble immunomodulatory peptide (such as the vIgD-containing variant CTLA-4) can be encapsulated in liposomes, the liposomes themselves being conjugated to any or any combination of the provided conjugates (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory peptides of the present invention are deglycosylated. In more specific embodiments, the variant CTLA-4 sequence is deglycosylated. In even more specific embodiments, the IgV domain or ECD of the variant CTLA-4 is deglycosylated.
[0270] exist Figure 1 Furthermore, non-limiting examples of the provided format are described below.
[0271] A. Soluble proteins
[0272] In some embodiments, the immunomodulatory protein containing the variant CTLA-4 peptide 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 such proteins can be prepared in a soluble form using the extracellular domain or its immunomodulatory subsequence. Therefore, in some embodiments, the immunomodulatory protein containing the variant CTLA-4 peptide lacks a transmembrane domain or a portion thereof. In some embodiments, the immunomodulatory protein containing the variant CTLA-4 lacks an intracellular (cytoplasmic) domain or a portion thereof. In some embodiments, the immunomodulatory protein containing the variant CTLA-4 peptide comprises only a vIgD portion containing an ECD domain or a portion thereof containing an IgV domain, or a domain containing one or more amino acid modifications or a specific binding fragment thereof. In some aspects, such soluble peptides can be used to block and / or antagonize the activity of CD28 and ICOS, thereby inhibiting or attenuating the immune response. In some embodiments, the provided soluble variant CTLA-4 immunomodulatory protein (e.g., variant CTLA-4-Fc) can be used in methods for treating autoimmune or inflammatory indications.
[0273] In some embodiments, the immunomodulatory peptide including variant CTLA-4 may comprise one or more variant CTLA-4 peptides of the present invention. In some embodiments, the peptides of the present invention will comprise only one, two, three, four, or five variant CTLA-4 sequences. In some embodiments, at least two variant CTLA-4 sequences in the variant CTLA-4 sequences are the same variant CTLA-4 sequence.
[0274] In some embodiments, the provided immunomodulatory polypeptide comprises two or more vIgD sequences of CTLA-4. Multiple variant CTLA-4 polypeptides within a polypeptide chain may be identical to each other (i.e., of the same species) or may be different variant CTLA-4 sequences (i.e., of different species). In addition to the single polypeptide chain embodiments, in some embodiments, two, three, four, or more polypeptides of the present invention may be covalently or non-covalently linked to each other. Thus, monomeric proteins, dimer proteins, and higher-order (e.g., 3rd, 4th, 5th, or more) multimeric proteins are provided herein. For example, in some embodiments, only two polypeptides of the present invention may be covalently or non-covalently linked to each other to form a dimer. In some embodiments, the linkage is via interchain cysteine disulfide bonds. Compositions comprising two or more polypeptides of the present invention may belong to the same polypeptide species or substantially the same polypeptide species (e.g., homodimers) or different polypeptide species (e.g., heterodimers). As described above, compositions having multiple linked polypeptides of the present invention may have one or more identical or different CTLA-4 polypeptides of the present invention in each polypeptide chain.
[0275] In some embodiments, the immunomodulatory protein includes a variant CTLA-4 polypeptide linked to an immunoglobulin Fc (thus producing an "immunomodulatory Fc fusion," such as a "CTLA-4-Fc variant fusion," also known as a CTLA-4vIgD-Fc fusion). In some embodiments, the linking of the variant CTLA-4 polypeptide occurs at the N-terminus of the Fc. In some embodiments, the linking of the variant CTLA-4 polypeptide occurs at the C-terminus of the Fc. In some embodiments, two or more CTLA-4 variant polypeptides (identical or different) are independently linked at the N-terminus and C-terminus.
[0276] In some embodiments, Fc is mouse or human Fc. In some embodiments, Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, Fc is derived from IgG1, such as human IgG1. In some embodiments, Fc comprises the amino acid sequence shown in SEQ ID NO:533 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:533.
[0277] In some embodiments, the Fc region contains one or more modifications to alter (e.g., reduce) one or more of its normal functions. Typically, in addition to antigen-binding capacity (the primary function of immunoglobulins), the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). Additionally, the FcRn sequence present in the Fc region increases the in vivo half-life by conjugating to an in vivo FcRn receptor, thereby regulating serum IgG levels. In some embodiments, such functions in the Fc region can be reduced or altered for use with the provided Fc fusion protein.
[0278] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the CTLA-4-Fc variant fusions provided herein, thereby generating Fc region variants. In some embodiments, the effector function of the Fc region variants is reduced. There are many examples of Fc sequence changes or mutations that can alter effector function. For example, WO 2000 / 042072, WO2006 / 019447, WO2012 / 125850, WO2015 / 107026, US2016 / 0017041 and Shields et al., Journal of Biochemistry, 276(9):6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcR. These disclosures are incorporated herein by reference in full.
[0279] In some embodiments, the provided variant CTLA-4-Fc fusion includes an Fc region with reduced effector function, making it an ideal candidate for applications where 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 detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of 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 therefore may lack ADCC activity), but retains FcRn binding capacity. Primary cells used to mediate ADCC—NK cells—express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, *Annu. Rev. Immunol.*, 9:457-492 (1991), summarizes FcR expression on hematopoietic cells.
[0280] Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in the following literature: U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 83(18):7059-7063 (1986)) and Hellstrom et al., Proceedings of the National Academy of Sciences, 82(5):1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann et al., Journal of Experimental Medicine, 166(5):1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, California); and CytoTox 96 TM Non-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al., Proceedings of the National Academy of Sciences, 95(2):652-656 (1998).
[0281] C1q binding assays can also be performed to confirm that the CTLA-4-Fc variant fusion does not bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Journal of Immunological Methods, 202:163 (1997); Cragg et al., Blood, 101(3):1045-1052 (2003); and Cragg and Glennie et al., Blood, 103(7):2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova et al., International Immunology, 18(12):1759-1769 (2006)).
[0282] The CTLA-4-Fc variant fusion with reduced effector function comprises a CTLA-4-Fc variant fusion in which one or more Fc region residues substituted according to EU numbers 238, 265, 269, 270, 297, 327, and 329 are substituted (US Patent No. 6,737,056). Such Fc mutants comprise Fc mutants in which two or more amino acid positions 265, 269, 270, 297, and 327 according to EU numbers are substituted, including the so-called “DANA” Fc mutant in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).
[0283] In some embodiments, compared to the natural Fc region, the Fc region of the CTLA-4-Fc variant fusion has an Fc region in which any one or more amino acids (indicated by EU number) at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 are replaced by different amino acids. Such modifications to the Fc region are not limited to those described above and include, for example, the deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, I, as described in Stohl, *New Insights in Biotechnology*, 20(6):685-691 (2009). Changes in IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E, etc.; such as WO The changes described in 2008 / 092117, such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R; the amino acid insertions at positions 233, 234, 235, and 237 according to EU numbers; and the changes at the sites described in WO 2000 / 042072.
[0284] Certain Fc variants are described as having improved or reduced binding to FcR. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, WO 2006019447 and Shields et al., Journal of Biochemistry, 276(9):6591-6604(2001)).
[0285] In some embodiments, a CTLA-4-Fc variant fusion is provided, the CTLA-4-Fc variant fusion comprising a variant Fc region including one or more amino acid substitutions that increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO 2015107026. Those antibodies include an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more Fc region residues in the following Fc region numbers according to EU designations: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, for example, the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).
[0286] In some embodiments, the Fc region of the CTLA-4-Fc variant fusion comprises one or more amino acid substitutions according to EU designations E356D and M358L. In some embodiments, the Fc region of the CTLA-4-Fc variant fusion comprises one or more amino acid substitutions according to EU designations C220S, C226S, and / or C229S. In some embodiments, the Fc region of the CTLA-4 variant fusion comprises one or more amino acid substitutions according to EU designations R292C and V302C. For other examples of Fc region variants, see also Duncan and 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.
[0287] In some embodiments, the wild-type IgG1 Fc may be the Fc shown in SEQ ID NO:533, having an allotype (e.g., f allotype) containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbers. 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, for example, as shown in SEQ ID NO:638. Thus, in some cases, the Fc provided herein may contain amino acid substitutions for E356D and M358L to reconstruct residues of the allotype G1m1 (e.g., α 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 inert to Fc effector function. Exemplary effector-free or inert mutations include the mutations described herein. Effectorless mutations that may be included in the Fc of the constructs provided herein are L234A, L235E, and G237A according to EU numbers. In some embodiments, the wild-type Fc is further modified by removing one or more cysteine residues, such as replacing a cysteine residue with a serine residue at position 220 according to EU number (C220S). Exemplary inert Fc regions with reduced effector function are listed in SEQ ID NO:526 and SEQ ID NO:438 or 439, respectively, based on allotypes shown in SEQ ID NO:533 or SEQ ID NO:638. In some embodiments, the Fc region used in the constructs provided herein may be further lacking a C-terminal lysine residue.
[0288] In some embodiments, alterations are made in the Fc region that reduce C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., Journal of Immunology 164(8):4178-4184 (2000).
[0289] In some embodiments, a CTLA-4-Fc variant fusion is provided, the CTLA-4-Fc variant fusion comprising a variant Fc region having one or more amino acid modifications, wherein the variant Fc region is derived from IgG1, such as human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence shown in SEQ ID NO:533. In some embodiments, the Fc contains at least one amino acid substitution, said at least one amino acid substitution being N82G according to SEQ ID NO:533 (corresponding to N297G according to EU number). In some embodiments, the Fc further contains at least one amino acid substitution, said at least one amino acid substitution being R77C or V87C according to SEQ ID NO:533 (corresponding to R292C or V302C according to EU number). In some embodiments, the variant Fc region further includes a C5S amino acid modification according to SEQ ID NO:533 (corresponding to C220S according to EU number). For example, in some embodiments, the variant Fc region includes the following amino acid modifications:
[0290] According to EU designation V297G and one or more of the following amino acid modifications C220S, R292C, or V302C (corresponding to N82G of reference SEQ ID NO:533 and one or more of the following amino acid modifications C5S, R77C, or V87C), for example, the Fc region includes the sequence shown in SEQ ID NO:440. In some embodiments, the variant Fc region includes one or more amino acid modifications C220S, L234A, L235E, or G237A, for example, the Fc region includes the sequence shown in SEQ ID NO:441. In some embodiments, the variant Fc region includes one or more amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, for example, the Fc region includes the sequence shown in SEQ ID NO:442. In some embodiments, the variant Fc region includes one or more amino acid modifications selected from C220S, L234A, L235E, G237A, E356D, or M358L, for example, the Fc region includes the sequence shown in SEQ ID NO:439.
[0291] In some embodiments, the Fc region lacks the C-terminal lysine residue at position 232 of the wild-type or unmodified Fc shown in SEQ ID NO: 533 (corresponding to K447del according to EU designation). In some aspects, such Fc regions may additionally contain one or more additional modifications, such as amino acid substitutions, as described above. Examples of such Fc regions are listed in SEQ ID NO: 438, 526, 527, or 528.
[0292] In some embodiments, a CTLA-4-Fc variant fusion is provided, the CTLA-4-Fc variant fusion comprising a variant Fc region, wherein the variant Fc comprises an amino acid sequence shown in any one of SEQ ID NO:438-442 or 526-528 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO:438-442 or 526-528.
[0293] In some embodiments, Fc is derived from IgG2, such as human IgG2. In some embodiments, Fc comprises the amino acid sequence shown in SEQ ID NO:529 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:529.
[0294] In some embodiments, Fc comprises the human IgG4 amino acid sequence shown in SEQ ID NO:530 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:530. In some embodiments, IgG4Fc is a stable Fc in which the CH3 domain of human IgG4 is replaced by the CH3 domain of human IgG1 and exhibits inhibition of aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced by the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which the arginine at position 409 of human IgG4 indicated in the EU index proposed by Kabat et al. is replaced by a lysine and exhibits inhibition of aggregate formation (see, for example, U.S. Patent No. 8,911,726). In some embodiments, Fc is IgG4 containing the S228P mutation, which has been shown to prevent recombination between the therapeutic antibody and endogenous IgG4 via Fab arm exchange (see, for example, Labrijin et al. (2009), Nature Biotechnology, 27(8) 767-71). In some embodiments, Fc comprises the amino acid sequence shown in human IgG4 with S228P as shown in SEQ ID NO: 531 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 531.
[0295] In some embodiments, the variant CTLA-4 peptide is directly linked to the Fc sequence. In some embodiments, the variant CTLA-4 peptide is indirectly linked to the Fc sequence via a linker. In some embodiments, one or more "peptide linkers" connect the variant CTLA-4 peptide and the Fc domain. In some embodiments, the peptide linker can be a single amino acid residue or a longer amino acid residue. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the linker is three alanine residues (AAA). In some embodiments, the linker is (single-letter amino acid code): GGGGS (“4GS”; SEQ ID NO: 535) or a polymer of the 4GS linker, such as repeating sequences of 2, 3, 4, or 5 4GS linkers, as shown in SEQ ID NO: 537 (2xGGGGS) or SEQ ID NO: 536 (3xGGGGS). In some embodiments, the linker (single-letter amino acid code) is GSGGGGS (SEQ ID NO: 534). In some embodiments, the linker is 2xGGGGS plus three alanines (GGGGSGGGGSAAA; SEQ ID NO: 538).
[0296] In some embodiments, the variant CTLA-4 polypeptide comprises any CTLA-4-Fc sequence of the CTLA-4-Fc sequence shown in SEQ ID NO:286-379, 381-386, or 388-437. In some embodiments, the variant CTLA-4 polypeptide comprises a polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, 97%, 98%, or 99% identity with any CTLA-4-Fc sequence shown in SEQ ID NO:286-379, 381-386, or 388-437, and contains one or more amino acid modifications not present in wild-type or unmodified CTLA-4, for example, one or more amino acid substitutions. In some embodiments, the variant CTLA-4 polypeptide comprises a specific binding fragment of any CTLA-Fc sequence shown in SEQ ID NO:286-379, 381-386 or 388-437, and contains one or more amino acid modifications not present in wild-type or unmodified CTLA-4, such as one or more amino acid substitutions.
[0297] In some embodiments, the variant CTLA-4-Fc fusion protein is a dimer formed by two variant CTLA-4Fc peptides linked to the Fc domain. In some embodiments, the dimer is a homodimer of the two variant CTLA-4Fc peptides. In some embodiments, the dimer is a heterodimer of the two variant CTLA-4Fc peptides.
[0298] Nucleic acid molecules encoding variant CTLA-4-Fc fusion proteins are also provided. In some embodiments, to generate an Fc fusion protein, a nucleic acid molecule encoding a variant CTLA-4-Fc fusion protein is inserted into a suitable expression vector. The resulting variant CTLA-4-Fc fusion protein can be expressed in host cells transformed with assembly between Fc domains via interchain disulfide bonds formed between Fc moieties to generate a dimer variant CTLA-4-Fc fusion protein, such as the divalent variant CTLA-4-Fc fusion protein.
[0299] The resulting Fc fusion protein can be readily purified by affinity chromatography on a protein A or protein G column. Further purification steps may be required to generate heterodimers. For example, in the case of transforming two nucleic acids encoding different variant CTLA-4 peptides into cells, heterodimer formation must be achieved biochemically because the variant CTLA-4 molecule carrying the Fc domain will also be expressed as a disulfide-linked homodimer. Therefore, homodimers can be reduced under conditions that favor the disruption of inter-chain disulfide bonds without affecting intra-chain disulfide bonds. In some cases, different variant 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, this type of heterodimer formation can be promoted by genetically engineering and expressing Fc fusion molecules containing variant CTLA-4 peptides using the lumped-pore method described below.
[0300] B. Stacked molecules with additional IgSF domains
[0301] In some embodiments, the immunomodulatory protein may contain any variant CTLA-4 polypeptide that is directly or indirectly linked to one or more other immunoglobulin superfamily (IgSF) domains (“stacked” immunomodulatory protein constructs, also known as “type II” immunomodulatory proteins). In some aspects, this can produce a unique multidomain immunomodulatory protein that binds to two or more (e.g., three or more) homologous binding partners, thereby providing multi-targeted regulation of the immune synapse.
[0302] In some embodiments, the immunomodulatory protein comprises a combination (“non-wild-type combination”) and / or arrangement (“non-wild-type arrangement” or “non-wild-type sequence”) of a variant CTLA-4 domain with one or more other affinity-modified and / or unaffected IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) not found in wild-type IgSF family members. In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5, or 6 immunoglobulin superfamily (IgSF) domains, wherein at least one of the IgSF domains is a variant CTLA-4 IgSF domain (vIgD of CTLA-4) as described herein.
[0303] In some embodiments, the sequence of an additional IgSF domain, compared to a wild-type or unmodified IgSF domain, may be a modified IgSF domain containing one or more amino acid modifications (e.g., amino acid substitutions). In some embodiments, the IgSF domain may be unaffected (e.g., wild-type) or affinity-modified. In some embodiments, the unmodified or wild-type IgSF domain may be derived from mouse, rat, cynomolgus monkey, or human sources, or combinations thereof. In some embodiments, the additional IgSF domain may be an IgSF domain of a member of the IgSF family shown in Table 1. In some embodiments, the additional IgSF domain, compared to the IgSF domains contained in the IgSF family members shown in Table 1, may be an affinity-modified IgSF domain containing one or more amino acid modifications (e.g., amino acid substitutions).
[0304] In some embodiments, the additional IgSF domain is an affinity-modified or unaffected IgSF domain selected from members of the following IgSF family: signal regulatory protein (SIRP) family, myeloid cell expression trigger receptor-like (TREML) family, carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) family, sialic acid-binding Ig-like lectin (SIGLEC) family, lactolipin family, B7 family, CD28 family, and V-group and immunoglobulin domain-containing (VSIG) family. The family includes the V group transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signal transduction lymphocyte activation molecule (SLAM) family, leukocyte immunoglobulin-like receptor (LIR), the connexin (Nectin, Nec) family, the connexin-like (NECL) family, the poliovirus receptor-associated (PVR) family, the innate cytotoxicity triggering receptor (NCR) family, the T cell immunoglobulin and mucin (TIM) family, or the cytotoxic cell immunoglobulin-like receptor (KIR) family. In some embodiments, additional IgSF domains are independently derived from IgSF proteins 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, BTLA (CD272), CD4, CD8A (CD8-α), CD8B (CD8-β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NCR3 (NKp30).
[0305] The number of such unaffinity-modified or affinity-modified IgSF domains present in the “stacked” immunomodulatory protein construct (whether a non-wild-type combination or a non-wild-type arrangement) is at least 2, 3, 4 or 5 IgSF domains, and in some embodiments, only 2, 3, 4 or 5 IgSF domains (therefore, determining the number of affinity-modified IgSF domains ignores any non-specific binding portion sequences and / or its substantially non-immunogenic portion sequences).
[0306] In some embodiments of the stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least two, wherein the number of affinity-modified IgSF domains and the number of unaffected IgSF domains are each independently at least: 0, 1, 2, 3, 4, 5, or 6. Therefore, the number of affinity-modified IgSF domains and the number of unaffected IgSF domains (affinity-modified IgSF domains: unaffected IgSF domains) can be only 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.
[0307] In some embodiments of stacked immunomodulatory proteins, at least two IgSF domains in the unaffinity-modified and / or affinity-modified IgSF domains are the same IgSF domain.
[0308] In some embodiments of stacked immunomodulatory proteins, the unmodified and / or affinity-modified IgSF domains are non-identical (i.e., different) IgSF domains. Different affinity-modified IgSF domains specifically bind to different homologous binding partners under specific binding conditions and are "different" regardless of whether their engineered wild-type or unmodified IgSF domains are identical. Thus, for example, a non-wild-type combination of at least two different IgSF domains in an immunomodulatory protein may include at least one IgSF domain sequence derived from and specific to a CTLA-4 and at least one second IgSF domain sequence derived from and specific to another IgSF family member not of CTLA-4, wherein the IgSF domains of the immunomodulatory protein are in an unmodified and / or affinity-modified form. However, in an alternative embodiment, the two different IgSF domains originate from the same IgSF domain sequence, but at least one IgSF domain is affinity modified so that the two different IgSF domains specifically bind to different homologous binding partners.
[0309] In some embodiments, the provided immunomodulatory protein, in addition to containing the variant CTLA-4 polypeptide, contains at least one, two, three, four, five, or six additional immunoglobulin superfamily (IgSF) domains, such as the IgD domains of IgSF family members as shown in Table 1. In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, the provided immunomodulatory protein contains at least two additional IgSF domains (e.g., second and third IgSF domains). In some embodiments, the provided immunomodulatory protein contains at least three additional IgSF domains (e.g., second, third, and fourth IgSF domains). In some embodiments, the provided immunomodulatory protein contains at least four additional IgSF domains (e.g., second, third, fourth, and fifth IgSF domains). In some embodiments, the provided immunomodulatory protein contains at least five additional IgSF domains (e.g., second, third, fourth, fifth, and sixth IgSF domains). In some embodiments, the provided immunomodulatory protein contains at least six additional IgSF domains (e.g., second, third, fourth, fifth, sixth, and seventh IgSF domains). In some embodiments, each IgSF domain in the immunomodulatory protein is distinct. In some embodiments, at least one additional IgSF domain in the immunomodulatory protein is identical to at least one other IgSF domain. In some embodiments, each IgSF domain in the IgSF domains is derived from or originates from a different IgSF family member. In some embodiments, at least two IgSF domains in the IgSF domains are derived from or originate from the same IgSF family member.
[0310] In some embodiments, the additional IgSF domain includes an IgV domain or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of the IgV domain or a specific binding fragment of the one or more IgC (e.g., IgC2) domains. In some embodiments, the additional IgSF domain is or includes a full-length IgV domain. In some embodiments, the additional IgSF domain is or includes one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the additional IgSF domain is or includes a specific binding fragment of the IgV domain. In some embodiments, the additional IgSF domain is or includes a specific binding fragment of the one or more IgC (e.g., IgC2) domains. 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 an ECD of an IgSF member or a portion thereof, the ECD or a portion thereof containing a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains or specific binding fragments thereof.
[0311] In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g., a second IgSF domain, or in some cases a third IgSF domain), wherein at least one additional, such as a second or third IgSF domain, is a wild-type or unmodified IgSF domain contained in the amino acid sequence shown in any of SEQ ID NO:1, 443-469, or an IgSF domain shown in 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.
[0312] In some embodiments, the provided immunomodulatory protein contains, in addition to the variant CTLA-4 polypeptide, at least one additional affinity-modified IgSF domain (e.g., a second affinity-modified IgSF domain, or in some cases, a third affinity-modified IgSF domain, etc.), wherein the at least one additional IgSF domain is vIgD, which contains one or more amino acid modifications (e.g., substitution, deletion, or mutation) compared to the IgSF domain in the wild-type or unmodified IgSF domain (such as the IgSF domains in the IgSF family members shown in Table 1). In some embodiments, an additional, such as a second or third affinity-modified IgSF domain, has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the wild-type or unmodified IgSF domain or its specific binding fragment contained in the amino acid sequence shown in any of SEQ ID NO:1, 443-469.
[0313] 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, for example, second or third IgSF domain is an affinity-modified IgV domain and / or an IgC domain. In some embodiments, the one or more additional IgSF domains are affinity-modified IgSF domains containing an IgV domain and / or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of the IgV domain and / or a specific binding fragment of the one or more IgC (e.g., IgC2) domains, wherein the IgV and / or IgC domains contain one or more amino acid modifications (e.g., one or more amino acid substitutions). In some embodiments, the one or more additional affinity-modified IgSF domains contain an IgV domain, which contains one or more amino acid modifications (e.g., one or more amino acid substitutions). In some embodiments, the one or more additional affinity-modified IgSF domains comprise IgSF domains present in the ECD or a portion of the ECD of the corresponding unmodified IgSF family member, such as a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains or their specific binding fragments, wherein one or both of IgV and IgC contain one or more amino acid modifications (e.g., one or more amino acid substitutions).
[0314] In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g., a second IgSF domain, or in some cases a third IgSF domain, etc.), said IgSF domain being vIgD, which contains one or more amino acid substitutions compared to the IgSF domain of wild-type or unmodified IgSF domains other than CTLA-4 (e.g., IgV).
[0315] In some embodiments, two or more IgSF domains, including the vIgD of CTLA-4 and one or more additional IgSF domains from another IgSF family member (e.g., a second variant IgSF domain), are covalently or non-covalently linked. Multiple unaffinity-modified and / or affinity-modified IgSF domains in a stacked immunomodulatory protein polypeptide chain do not need to be directly covalently linked to each other. In some embodiments, the two or more IgSF domains are directly linked or indirectly linked, such as via a linker. In some embodiments, an intermediate span of one or more amino acid residues indirectly covalently links the IgSF domains to each other. Linkage can occur via N-terminal residues to C-terminal residues. In some embodiments, linking can occur via side chains of amino acid residues not located at the N-terminus or C-terminus of one or more IgSF domains. Therefore, linking can occur via terminal or internal amino acid residues or combinations thereof.
[0316] In some embodiments, the immunomodulatory protein contains at least two IgSF domains, each directly linked or indirectly linked via a adapter. In some embodiments, the immunomodulatory protein contains at least three immunomodulatory domains, each directly linked or indirectly linked via a adapter. Figure 6A and 6B Various configurations are shown in the figure.
[0317] In some embodiments, one or more "peptide linkers" connect the vIgD of CTLA-4 to an additional IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the peptide linker may be a single amino acid residue or a longer amino acid residue. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the linker is (single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO: 535) or a polymer of the 4GS linker, such as repeating sequences of 2, 3, 4, or 5 4GS linkers. 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 may also contain only a series of alanine residues or, in addition to another peptide linker (e.g., a 4GS linker or its multimer; see, for example, SEQ ID NO: 538), a series of alanine residues. In some embodiments, the number of alanine residues in each series is 2, 3, 4, 5, or 6 alanines.
[0318] In some embodiments, unaffinity-modified and / or affinity-modified IgSF domains are linked by “wild-type peptide linkers” inserted at the N-terminus and / or C-terminus of a second unaffinity-modified and / or affinity-modified IgSF domain. These linkers are also referred to as leader sequences (N-terminus to the unaffinity-modified or affinity-modified IgSF domain) or tail sequences (C-terminus to the unaffinity-modified or affinity-modified IgSF domain), and sequences present in wild-type proteins immediately outside the predicted structure spanning the Ig fold of the IgSF. In some embodiments, a “wild-type linker” is an amino acid sequence in the amino acid sequence of a wild-type protein that follows a signal sequence but precedes an IgSF domain (such as the defined IgV domain). In some embodiments, a “wild-type” linker is an amino acid sequence in the amino acid sequence of a wild-type protein that immediately follows an IgSF domain (such as immediately following the defined IgV domain) but precedes an IgC domain. These linker sequences can contribute to the proper folding and function of one or more adjacent IgSF domains.
[0319] In some embodiments, a leader peptide linker is present at the N-terminus of the first IgSF domain and / or a tailing sequence is present at the C-terminus of the first unaffinity-modified and / or affinity-modified IgSF domain. In some embodiments, a second leader peptide linker is present at the N-terminus of the second IgSF domain and / or a second tailing sequence is present at the C-terminus of the second unaffinity-modified and / or affinity-modified IgSF domain. When the first and second unaffinity-modified and / or affinity-modified IgSF domains originate from the same parent protein and are linked in the same orientation, the wild-type peptide linker between the first and second unaffinity-modified and / or affinity-modified IgSF domains will not be replicated. For example, when the first tailing wild-type peptide linker and the second leader wild-type peptide linker are the same, the type II immunomodulatory protein does not include the first tailing wild-type peptide linker or the second leader wild-type peptide linker.
[0320] In some embodiments, the type II immunomodulatory protein includes a first leader wild-type peptide linker inserted at the N-terminus of a first unaffinity-modified and / or affinity-modified IgSF domain, wherein the first leader wild-type peptide linker includes at least five (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from a wild-type protein derived from the first unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or IgSF domain). In some embodiments, the first leader wild-type peptide linker includes the entire intercalation sequence from a wild-type protein derived from the first unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or IgSF domain).
[0321] In some embodiments, the type II immunomodulatory protein includes a first tailed wild-type peptide linker inserted at the C-terminus of a first unaffinity-modified and / or affinity-modified IgSF domain, wherein the first tailed wild-type peptide linker includes at least five (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from a wild-type protein derived from the first unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately following domain (e.g., an IgSF domain or a transmembrane domain). In some embodiments, the first tailed wild-type peptide linker includes the entire intercalation sequence from a wild-type protein derived from the first unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately following domain (e.g., an IgSF domain or a transmembrane domain).
[0322] In some embodiments, the type II immunomodulatory protein further includes a second leader wild-type peptide linker inserted at the N-terminus of a second unaffinity-modified and / or affinity-modified IgSF domain, wherein the second leader wild-type peptide linker comprises at least five (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from a wild-type protein derived from the second unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or IgSF domain). In some embodiments, the second leader wild-type peptide linker comprises the entire intercalation sequence from a wild-type protein derived from the second unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately preceding domain (e.g., a signal peptide or IgSF domain).
[0323] In some embodiments, the type II immunomodulatory protein further includes a second tailed wild-type peptide linker inserted at the C-terminus of a second unaffinity-modified and / or affinity-modified IgSF domain, wherein the second tailed wild-type peptide linker comprises at least five (e.g., at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acids from a wild-type protein derived from the second unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately following domain (e.g., an IgSF domain or a transmembrane domain). In some embodiments, the second tailed wild-type peptide linker comprises the entire intercalation sequence from a wild-type protein derived from the second unaffinity-modified and / or affinity-modified IgSF domain, located between the parental IgSF domain and the immediately following domain (e.g., an IgSF domain or a transmembrane domain).
[0324] In some embodiments, two or more IgSF domains, including CTLA-4 vIgD and one or more additional IgSF domains from another IgSF family member (e.g., second and / or third variant IgSF domains), are linked or attached to an Fc to form an Fc fusion, which, when expressed in cells, can in some respects produce a dimer multidomain stacked immunomodulatory protein. Therefore, dimer multidomain immunomodulatory proteins are also provided.
[0325] In some embodiments, the variant CTLA-4 peptide 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 variant CTLA-4 peptide and at least one of the one or more additional IgSF domains are directly or indirectly linked, and one of the IgSF domains in the variant CTLA-4 and the one or more additional IgSF domains is also directly or indirectly linked to the N-terminus or C-terminus of the Fc region. In some embodiments, the N-terminus or C-terminus of the Fc region is linked to the variant CTLA-4 peptide or the one or more additional IgSF domains, and another end of the N-terminus or C-terminus of the Fc region is linked to another of the other IgSF domains in the variant CTLA-4 or the one or more additional IgSF domains. In some embodiments, the link to the Fc is performed via a peptide linker (e.g., a peptide linker as described above). In some embodiments, the link between the variant CTLA-4 and a second IgSF domain is performed via a peptide linker (e.g., a peptide linker as described above). In some embodiments, the connection between the variant CTLA-4 and the one or more additional IgSF domains is via a peptide linker (e.g., a peptide linker as described above). In some embodiments, the vIgD of CTLA-4, the one or more additional IgSF domains, and the Fc domain can be connected as follows: Figure 6A and 6B Any of the many configurations depicted are connected together. Exemplary configurations are described in the examples.
[0326] In some embodiments, the stacked immunomodulatory protein is a dimer formed from two immunomodulatory Fc fusion polypeptides. Nucleic acid molecules encoding any of the stacked immunomodulatory proteins are also provided. In some embodiments, the dimer multidomain stacked immunomodulatory protein can be generated in cells by expressing, or in some cases co-expressing, a stacked immunomodulatory Fc region polypeptide, as described above regarding the generation of the dimer Fc fusion protein.
[0327] In some embodiments, the dimer multidomain stacked immunomodulatory protein is divalent for each Fc subunit, monovalent for each subunit, or divalent for one subunit and tetravalent for another subunit.
[0328] In some embodiments, the dimer multidomain stacked immunomodulatory protein is a homodimeric multidomain stacked Fc protein. In some embodiments, the dimer multidomain stacked immunomodulatory protein includes a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are identical.
[0329] In some embodiments, the multi-domain stacked molecule contains a first Fc fusion peptide and a second Fc fusion peptide, wherein the first Fc fusion peptide contains a variant CTLA-4 and a second IgSF domain, and the second Fc fusion peptide contains a variant CTLA-4 and a second IgSF domain. In some embodiments, the multi-domain stacked molecule contains a first Fc fusion peptide and a second Fc fusion peptide, wherein the first Fc fusion peptide contains a variant CTLA-4, a second IgSF domain, and a third IgSF domain, and the second Fc fusion peptide contains a variant CTLA-4, a second IgSF domain, and a third IgSF domain. In some embodiments, the Fc portion of the first and / or second fusion peptide can be any Fc as described above. In some embodiments, the Fc portion or Fc region of the first and / or second fusion peptide is the same.
[0330] In some embodiments, the multi-domain stacked molecule is a heterodimer comprising two distinct Fc fusion peptides, such as first and second Fc peptides, wherein at least one Fc peptide is an Fc fusion peptide containing at least one variant CTLA-4 peptide and / or at least one Fc peptide is an Fc peptide containing a second IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the first or second Fc fusion peptide further contains a third IgSF domain (e.g., a third variant IgSF domain). In some embodiments, the multi-domain stacked molecule comprises a first Fc fusion peptide and a second Fc fusion peptide, the first Fc fusion peptide containing a variant CTLA-4, the second Fc fusion peptide containing a second IgSF domain, wherein in some cases, the first or second Fc fusion peptide additionally contains a third IgSF domain. In some embodiments, the multi-domain stacked molecule comprises a first Fc fusion peptide and a second Fc fusion peptide, the first Fc fusion peptide containing a variant CTLA-4, a second IgSF domain, and in some cases a third IgSF domain, wherein the second Fc fusion peptide is not linked to the variant CTLA-4 peptide or any other IgSF domain. In some embodiments, the Fc portions or Fc regions of the first and / or second fusion peptides are identical. In some embodiments, the Fc portions or Fc regions of the first and / or second fusion peptides are different. In some embodiments, the multi-domain stacked molecule contains a first fusion Fc peptide containing one, two, three, four or more variant CTLA-4 peptides and one, two, three, four or more additional IgSF domains, wherein the total number of IgSF domains in the first stacked Fc fusion peptide is greater than two, three, four, five, six or more. In one example of such embodiments, the second stacked Fc fusion peptide contains one, two, three, four or more variant CTLA-4 peptides and one, two, three, four or more additional IgSF domains, wherein the total number of IgSF domains in the second stacked Fc fusion peptide is greater than two, three, four, five, six or more. In another instance of such an embodiment, the second Fc polypeptide is not linked to the variant CTLA-4 polypeptide or an additional IgSF domain.
[0331] In some embodiments, the heterodimeric stacked 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 stacked molecule contains a first Fc polypeptide fusion and a second Fc polypeptide fusion, wherein the first Fc polypeptide fusion contains an Fc region and a first variant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and the second Fc polypeptide fusion contains an Fc region and another of the first variant CTLA-4 polypeptide or the second IgSF domain. In some embodiments, the heterodimeric stacked molecule contains a first Fc polypeptide fusion and a second Fc polypeptide fusion, wherein the first Fc polypeptide fusion contains an Fc region and a first variant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and the second Fc polypeptide fusion contains both an Fc region and the first variant CTLA-4 polypeptide and the second IgSF domain (e.g., a second variant IgSF domain), but in a different orientation or configuration than the first Fc region. In some embodiments, the first and / or second Fc fusion peptides further contain a third IgSF domain (e.g., a third variant IgSF domain).
[0332] In some embodiments, the Fc domain of one or both of the first and second stacked immunomodulatory Fc fusion peptides includes modifications (e.g., substitutions) such that the interface of the Fc molecule is modified to facilitate and / or promote heterodimerization. In some embodiments, the modification comprises introducing protrusions (bulges) into the first Fc peptide and introducing cavities (pores) into the second Fc peptide, such that the protrusions can be positioned within the cavities to promote the complexation of the first and second Fc-containing peptides. The amino acids used for substitution and / or modification to create protrusions or cavities in the peptide are typically interfacial amino acids that interact with or contact one or more amino acids at the interface of the second peptide.
[0333] In some embodiments, an amino acid sequence is added before the Fc sequence of the construct, wherein the Fc sequence is the N-terminal portion of the sequence. In some cases, the amino acid sequence HMSSVSAQ (SEQ ID NO: 539) is added immediately before the Fc sequence of the construct, wherein the Fc sequence is the N-terminal portion of the sequence. In some embodiments, the heterodimeric stacked molecule contains a first Fc polypeptide fusion and a second Fc polypeptide fusion, the first Fc polypeptide fusion containing an Fc region (bulge) and a first variant CTLA-4 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), the second Fc polypeptide fusion containing an Fc region (pore) and a filler sequence HMSSVSAQ (SEQ ID NO: 539) immediately before the two Fc regions of the first and second Fc polypeptide fusions.
[0334] In some embodiments, a first polypeptide modified to contain protruding (pore) amino acids comprises replacing a natural or original amino acid with an amino acid having at least one side chain, said at least one side chain protruding from the interface of the first polypeptide and thus being able to be positioned in a compensating cavity (pore) at an adjacent interface of a second polypeptide. Often, the substituted amino acid is an amino acid whose side chain volume is larger than that of 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 acid residues as ideal substitutes for generating protrusions. In some embodiments, the substituted residues for forming protrusions are naturally occurring amino acid residues and comprise, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some instances, the original residues identified for substitution are amino acid residues with small side chains, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
[0335] In some embodiments, the second polypeptide modified to contain a cavity (pore) is a polypeptide comprising replacing a natural or original amino acid with an amino acid having at least one side chain recessed from the interface of the second polypeptide and thus capable of accommodating a corresponding protrusion from the interface of the first polypeptide. Often, the substituted amino acid is an amino acid whose side chain volume is smaller than that of 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 acid residues as ideal substitutes for forming the cavity. Typically, the substitute residues for forming the cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T), and valine (V). In some instances, the original amino acid identified for substitution is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.
[0336] The CH3 interface of human IgG1, for example, involves embedding each surface onto each domain located on four antiparallel β chains. The 16 residues (see, for example, Deisenhofer (1981), Biochemistry, 20(9):2361-2370; Miller (1990), Journal of Molecular Biology, 216(4):965-973; Ridgway et al. (1996), Protein Engineering, 9(7):617-621; U.S. Patent No. 5,731,168). Modification of the CH3 domain to create protrusions or cavities is described in, for example, the following literature: U.S. Patent No. 5,731,168; International Patent Applications WO98 / 50431 and WO 2005 / 063816; and Ridgway et al. (1996), Protein Engineering, 9(7):617-621. In some instances, modifying the CH3 domain to create protrusions or cavities typically targets residues on two centrally antiparallel β chains. The aim is to minimize the risk that the resulting protrusions can be contained by protruding into the surrounding solvent, rather than by compensating cavities in the CH3 domain of the partner body.
[0337] In some embodiments, the heterodimer molecule contains the T366W mutation in the CH3 domain of the "lump chain" and the T366S, L368A, and Y407V mutations in the CH3 domain of the "pore chain". In some cases, additional interchain disulfide bridges between the CH3 domains can also be used (Merchant et al. (1998), Nature Biotechnology, 16(7):677-681), for example, by introducing the Y349C mutation into the CH3 domain of the "lump" or "pore" chain and introducing the E356C or S354C mutation into the CH3 domain of the other chain. In some embodiments, the heterodimer molecule contains the S354C and T366W mutations in one of the two CH3 domains and the Y349C, T366S, L368A, and Y407V mutations in the other CH3 domain. In some embodiments, the heterodimer molecule includes the E356C, T366W mutation in one of the two CH3 domains and the Y349C, T366S, L368A, Y407V mutation in the other CH3 domain. In some embodiments, the heterodimer molecule includes the Y349C, T366W mutation in one of the two CH3 domains and the E356C, T366S, L368A, Y407V mutation in the other CH3 domain. In some embodiments, the heterodimer molecule includes the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other CH3 domain. Other examples of protrusion-type entry hole techniques are known in the art, for example, as described in EP 1870459 A1.
[0338] In some embodiments, the Fc region of the heterodimer molecule may additionally contain one or more other Fc mutations, such as any of the mutations described above. In some embodiments, the heterodimer molecule contains an Fc region with a mutation that reduces effector function.
[0339] In some embodiments, Fc variants containing CH3 protrusions (knobs) or cavities (pores) can be linked anywhere to stacked immunomodulatory peptides, but typically via their N-terminus or C-terminus to the N-terminus or C-terminus of the first and / or second stacked immunomodulatory peptides, thus forming fusion peptides. The linkage can be direct or indirect, via a linker. Typically, a knob and hole molecule is generated by co-expressing a first stacked immunomodulatory peptide linked to one or more CH3 protrusion-modified Fc variants and a second stacked immunomodulatory peptide linked to one or more CH3 cavity-modified Fc variants.
[0340] C. Conjugates and fusions of variant peptides and immunomodulatory proteins
[0341] In some embodiments, the variant polypeptides provided herein, which are variants of the Ig domain of the IgSF family (vIgD), may be directly or indirectly conjugated or fused with portions such as effector portions (such as another protein) to form conjugates (“IgSF conjugates”). In some embodiments, the connection may be a covalent connection or a non-covalent connection, for example, through a biotin-streptavidin non-covalent interaction. In some embodiments, any one or any combination of any two or more of the foregoing portions may be linked to the Fc or variant CTLA-4 polypeptide or both. In some embodiments, the provided conjugates, such as fusion polypeptides, may be used in methods and uses for suppressing or attenuating immune responses, such as in combination with treatments for autoimmune or inflammatory indications.
[0342] In some embodiments, the portion may be a targeted portion, a small molecule drug (a non-peptide drug with a molar mass of less than 500 Daltons), a toxin, a cell growth inhibitor, a cytotoxic agent, an immunosuppressant, an antiproliferative agent, a radiopharmaceutical suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug activating enzyme, a drug that increases biological half-life, or a diagnostic or detectable drug.
[0343] In some embodiments, the effector portion is a therapeutic agent that provides a certain therapeutic benefit, such as a therapeutic immunosuppressant or an anti-rejection drug.
[0344] In some embodiments, the effector portion is a targeting portion 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 portion is a marker that can directly or indirectly generate a detectable signal. In some embodiments, the effector portion is a protein, peptide, nucleic acid, small molecule, or nanoparticle.
[0345] In some embodiments, one, two, three, four, five or more effector moieties, which may be identical or different, may be conjugated, linked, or fused with a variant peptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties may be linked to variant peptides or immunomodulatory proteins using various molecular biological or chemical conjugation and linking methods known in the art and described below. In some embodiments, effector moieties may be linked or conjugated to variant peptides or immunomodulatory proteins using adapters such as peptide adapters, cleavable adapters, non-cleavable adapters, or adapters that facilitate the conjugation reaction.
[0346] In some embodiments, the IgSF conjugate comprises the following components: (protein or polypeptide), (L... q and (effect component) m The protein or polypeptide is any of the described variant polypeptides or immunomodulatory proteins capable of binding one or more of the described binding couplers; L is a connector for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or greater; and the resulting IgSF conjugate binds to one or more anti-structural ligands. In a particular embodiment, m is 1 to 4, and q is 0 to 8.
[0347] In some embodiments, an IgSF conjugate is provided, comprising a variant polypeptide or immunomodulatory protein conjugated herein with a target agent, the target agent binding to a cell surface molecule, for example, to target and deliver the variant polypeptide or immunomodulatory protein to a specific cell. In some embodiments, the target agent is one or more molecules capable of localizing and binding to molecules present on normal cells / tissues (e.g., lymphoid tissue), immune cells (e.g., activated or inactivated immune cells), and / or tissues in an inflammatory environment of a subject. In other words, the IgSF conjugate comprising the target agent can (directly or indirectly) bind to a ligand present on cells such as tumor cells. It is contemplated that the target agents of the present invention for use comprise antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof capable of binding to components of target cells or molecules.
[0348] In some embodiments, via a targeting agent of an IgSF conjugate, the IgSF conjugate is localized (e.g., bound to) cellular components of immune cells, tissues in an inflammatory environment, or lymphoid tissues (e.g., spleen, tonsils, lymphatic vessels, lymph nodes, adenoids, and / or liver tissue), thereby modulating the targeted cells of the immune response. In some embodiments, the targeting agent promotes the delivery of the conjugated IgSF (e.g., vIgD) to immune cells, such as to interact with its binding partner to alter signaling in nearby immune cells carrying the binding partner and / or co-stimulatory receptors carrying the binding partner (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells). In some embodiments, local delivery mediates indirect antagonistic or blocking activity of one or more CD28 and / or ICOS co-stimulatory receptors.
[0349] In some embodiments, the target is an immunoglobulin. The term "immunoglobulin" as used herein includes natural or artificial monovalent or multivalent antibodies, including but not limited to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, single-chain Fv (scFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present invention), and epitope-binding fragments of any of the foregoing. The term "antibody" as used herein refers to an immunoglobulin molecule and an immunoglobulin molecule with immunological activity, such as a molecule containing an antigen-binding site that specifically binds to an antigen. The immunoglobulin ...
Claims
1. A variant CTLA-4 polypeptide comprising an IgV domain or a specific binding fragment thereof, wherein the variant CTLA-4 polypeptide comprises one or more amino acid modifications in an unmodified CTLA-4 polypeptide or a specific binding fragment thereof, wherein the variant CTLA-4 polypeptide binds specifically to the extracellular domain of ICOSL with increased affinity compared to the unmodified CTLA-4.
2. The variant CTLA-4 peptide of claim 1, wherein the variant CTLA-4 peptide comprises one or more amino acid modifications of the unmodified CTLA-4 peptide or its specific binding fragment, see SEQ ID. The positions shown in NO:2, wherein the one or more amino acid modifications correspond to one or more positions selected from the following: 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.
3. A variant CTLA-4 polypeptide comprising an IgV domain or a specific binding fragment thereof, wherein the variant CTLA-4 polypeptide comprises one or more amino acid modifications of an unmodified CTLA-4 polypeptide or a specific binding fragment thereof, see SEQ ID. The positions shown in NO:2, wherein the one or more amino acid modifications correspond to one or more positions selected from the following: 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.
4. The variant CTLA-4 polypeptide according to any one of claims 1 to 3, wherein the amino acid modification comprises amino acid substitution, deletion, or insertion.
5. The variant CTLA-4 polypeptide according to any one of claims 1 to 4, wherein the unmodified CTLA-4 polypeptide is a mammalian CTLA-4 polypeptide or a specific binding fragment thereof.
6. The variant CTLA-4 polypeptide of claim 5, wherein the unmodified CTLA-4 polypeptide is a human CTLA-4 polypeptide or a specific binding fragment thereof.
7. The variant CTLA-4 polypeptide according to any one of claims 1 to 6, wherein the variant CTLA-4 polypeptide comprises an extracellular domain of human CTLA-4, wherein the one or more amino acid modifications are located in one or more residues of the extracellular domain of the unmodified CTLA-4 polypeptide.
8. The variant CTLA-4 peptide according to any one of claims 1 to 7, wherein the unmodified CTLA-4 peptide comprises: (i) the amino acid sequence shown in SEQ ID NO:2; (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2; or (iii) a portion thereof comprising an IgV domain or a specific binding fragment of said IgV domain.
9. The variant CTLA-4 polypeptide according to any one of claims 1 to 8, wherein the unmodified CTLA-4 comprises the amino acid sequence shown in SEQ ID NO:
2.
10. The variant CTLA-4 peptide according to any one of claims 1 to 8, wherein: The length of the specific binding fragment of the IgV domain is at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or The length of the specific binding fragment of the IgV domain is at least 80% of the length of the IgV domain as shown by residues 39-140 of SEQ ID NO:1.
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