CTLA-4 variant immunomodulatory proteins and uses thereof
Mutant CTLA-4 polypeptides with specific amino acid modifications improve immune regulation, addressing the limitations of existing agents in autoimmune and inflammatory conditions by enhancing binding affinity to ICOSL, CD80, and CD86, offering effective cancer treatment options.
Patent Information
- Application Number
- JP2025116732
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2018-09-19
- Filing Date
- 2025-07-10
- Publication Date
- 2025-10-07
AI Technical Summary
Existing therapeutic agents are inadequate in modulating immune responses effectively at the immune synapse, necessitating improved immunomodulatory proteins to treat autoimmune and inflammatory conditions, particularly in cancer treatment.
Development of mutant CTLA-4 polypeptides with specific amino acid modifications in the IgV domain or binding fragments, enhancing affinity for ICOSL, CD80, and CD86, to regulate immune responses with increased specificity and efficacy.
The mutant CTLA-4 polypeptides demonstrate enhanced binding affinity and immunomodulatory effects, providing therapeutic benefits for autoimmune and inflammatory conditions, including cancer treatment.
Smart Images
Figure 2025148471000001_ABST
Abstract
Description
[Technical Field]
[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of 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 Priority to U.S. Provisional Application No. 62 / 570,619, filed October 10, 2017 The benefit of each of the foregoing claims is hereby claimed, the contents of each of which are incorporated herein by reference in their entirety. do.
[0002] Incorporation by reference of sequence listing This application is being filed with a sequence listing in electronic format. Created on October 8, 2018, 76161200204, 0 bytes in size The electronic format of the sequence listing is provided as a file titled 0SeqList.txt. The information therein is incorporated by reference in its entirety.
[0003] Field The present disclosure relates to immunomodulatory proteins, including mutant CTLA-4, and methods for treating such proteins. Immunomodulatory proteins are used to treat autoimmune or inflammatory conditions. Such proteins provide therapeutic utility for a variety of disease indications, including for the treatment of cancer. Compositions and methods for making and using the materials are provided. [Background technology]
[0004] background Formed by and between antigen-presenting cells (APCs) or target cells and lymphocytes to regulate immune responses by interfering with processes occurring at the immune synapse (IS) where Mechanistically, cell surface proteins within the IS interact with multiple proteins. It involves the coordinated and often simultaneous interaction of protein targets with the single proteins to which they bind. The interaction of IS occurs in close association with the junction of two cells, and in this structure A single protein can interact with proteins on the same protein (cis), as well as with proteins on related cells. It can interact with both proteins (in trans), possibly simultaneously. Although therapeutic agents are known, improved therapeutic agents are needed to meet such needs. Immunomodulating proteins are provided. Summary of the Invention
[0005] overview Mutant CTLA-4 polypeptides are provided herein. In some embodiments, A mutant CTLA-4 polypeptide containing the gV domain or a specific binding fragment thereof. Thus, the mutant CTLA-4 polypeptide may be a non-modified CTLA-4 polypeptide or A mutant CTLA-4 polypeptide containing one or more amino acid modifications in a specific binding fragment. specific for the ectodomain of ICOSL with increased affinity compared to unmodified CTLA-4 Provided herein are mutant CTLA-4 polypeptides that bind to:
[0006] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide may be a peptide having SEQ ID NO: 1 in an unmodified CTLA-4 polypeptide or a specific binding fragment thereof. 6, 10, 12, 14, 15, 16, 18, 19 with respect to positions set forth in ID NO:2 , 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, 6 3, 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 contains one or more amino acid modifications corresponding to positions selected from among.
[0007] In some of any of the provided embodiments, a mutant CTLA-4 polypeptide containing an IgV domain or a specific binding fragment thereof, wherein the mutant CTLA -4 polypeptide has, in the unmodified CTLA-4 polypeptide or a specific binding fragment thereof, with respect to the positions set forth in SEQ ID NO:2, 6, 10, 12, 14, 15, 16, 1 8, 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, 8 6, 并含有87, 89, 91, 93, 95, 96, 97, 98, 99, 105, 106, 108 、110, 113, 115, 116, 117, 118, 119, 120, 121, and one or more amino acid modifications corresponding to positions selected from among 122, a mutant C TLA-4 polypeptide is provided herein. In some embodiments, the amino acid mod ifications include amino acid substitutions, deletions, or insertions.
[0008] In some of any of the provided embodiments, the unmodified CTLA-4 poly The peptide is a mammalian CTLA-4 polypeptide or a specific binding fragment thereof. In some embodiments, the unmodified CTLA-4 polypeptide is a human CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide is a polypeptide or a specific binding fragment thereof. The peptide contains the extracellular domain of human CTLA-4, and one or more amino acid modifications are In one or more residues of the extracellular domain of the unmodified CTLA-4 polypeptide.
[0009] In some of any of the provided embodiments, the unmodified CTLA-4 polynucleotide is The peptide comprises (i) the sequence of amino acids set forth in SEQ ID NO:2; (ii) SEQ a sequence of amino acids having at least 95% sequence identity with ID NO:2, or (ii) i) containing an IgV domain or a portion thereof containing a specific binding fragment of an IgV domain In some embodiments, the unmodified CTLA-4 has the sequence set forth in SEQ ID NO:2. It contains the amino acid sequence shown below.
[0010] In some of any of the provided embodiments, the specificity of the IgV domain The binding fragment may be at least 50, 60, 70, 80, 90, 100, 110 or more amino acids. The specific binding fragment of the IgV domain has the length of the residue of SEQ ID NO:1. A length that is at least 80% of the length of the IgV domain described as residues 39-140. include.
[0011] In some of any of the provided embodiments, the mutant CTLA-4 is Max 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 containing deletions.
[0012] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or a specific binding fragment thereof. .
[0013] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides were attached to the ectodomains of ICOSL, CD80, and / or CD86. and binding of the unmodified CTLA-4 polypeptide to the CTLA-4 polypeptide domain with increased affinity compared to unmodified CTLA-4 polypeptide binding to the unmodified CTLA-4 polypeptide domain. , specifically binds.
[0014] One or more amino acids in the extracellular domain of human CTLA-4 as set forth in SEQ ID NO:2 A mutant CTLA-4 polypeptide containing an amino acid modification, The polypeptide comprises a CTLA-4 polypeptide containing the extracellular domain set forth in SEQ ID NO:2. human ICOSL, CD80, and / or CD86 with increased affinity compared to Provided herein are mutant CTLA-4 polypeptides that specifically bind to the CTLA-4 region domain. do.
[0015] In some of any of the provided embodiments, one or more amino acid modifications 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, Y10 5F, Y105L, L106I, L106N, L106R, I108F, I108V, N 110K, N110S, N110Y, Y115N, V116A, I117E, I117L , I117M, I117T, and P121S, or their conservative amino acid substitutions be selected.
[0016] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides are A6T / A26T / M55T / M99L / Y105L and V10A / G29W. / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L6 <h2 style=";text-align:left;direction:ltr">3P / D64V / S72G / L98Q / M99L / Y105L、V10A / L63P / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L、L12F / R16H / G29W / M56T / L98Q / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、L12F / A26T / L63P / L98Q / Y105L / L106R、L12F / K3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0R / S72G / Q82R / L98Q / M99L / Y105L、L12H / I18V / A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 42T / M55T / N58D / L98R / Y105L / L106I / P121S、L12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H / E33M / L98Q / Y105L、L12H / M55T / E59D / L63P / M9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9L、L12H / L63P / S72G / L98Q / Y105L、L12I / M55T / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 56V / I67T / M99L / L106R / I108F、L12P / R16H / A26T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 17L、L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、L12P / A26T、L12P / A26T / L63P、L12P / A26T / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / T89M / L98Q / M99L / Y105L、L12P / G29W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / S72G / L98Q / Y105L、L12P / G29W / L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / Y105L / L106I、L12P / A26T / L63P / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、L12P / A26T / L63P / L98Q / Y105L、L12P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / A26T / L63P / L98Q / Y105L / L106I、L12P / G29W / D4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3N / N58S / L63P / L98Q / M99L / Y105L、L12P / M56V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / V96I / L98Q / M99L / Y105L / Y115H、L12P / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / L106N、L12P / L63P / S72G / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L / L106N / I117L、S14N / R16C / I18T / M56<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K / T61A / L63P / A86T / M99L、S15P / I18V / M56T / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H / T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7T / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T / L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2R / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / E48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1R / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">L、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0N / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I67N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A26T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M87V / N110K / I117E、A26T / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I117L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N / S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8S / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W / T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3S / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 53S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 117L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L / L106V、G29W / N58S / L63P / S72G / M87V / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">Q / Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L / L106I / I117L、G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I117L、G29W / L63P / L98Q / M99L / Y105L、G29W / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / M87<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K / T89S / L98Q / M99L / Y105L / I108V / I117L、G29W / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M87K / I93V / L98Q / M99L / Y105L、G29W / L98Q / M99L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、E33M / A42T / L98Q / Y105L、E33M / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I108F、E33M / L63P / S72G / L98Q / Y105L / I117L、E3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3M / Q82H / L98Q / M99L / Y105L、E33V / A42S / M55T / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L、T37S / M56V / L98Q / Y105L、V38I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / S72G / L98Q / M99L / Y105L、Q41L / Y54F / M56<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K / M99L / I108F、T53S / M56V / L98Q / Y105L、M55T / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / T71I / M99L / Y105L、M55T / S72G / L98Q / M99L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、M55T / E97Q / M99L / Y105F、M56K / L63P / N75<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D / V96I / M99L / Y105L / L106I、M56L / L63P / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L / L106I / I117L、M56R / L63P / L98Q / M99L / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L、M56T / L91R / L98Q / Y105L、M56V、M56V / E59G / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / S72G / M87K / I93V / L98Q / M99L / Y105L / I117E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T61A / L63P / S72G / L98Q / M99L / Y105L, L63P, L63<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> P / T69A / L98Q / M99L / Y105L / L106R / V116A、L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / M87A / L98Q / Y105L、L63P / S72G / I93L / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L、L63P / S72G / L98Q / M99L / Y105L、L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / M99L / Y105L / L106I / I117L、L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / Y105L / L106I / I117L、L63P / S72G / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、L63P / M87K / M99L / L106R、L63P / Q82H / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L、L63P / K95R、L63P / L98Q、L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L、L63P / L98Q / M99L / Y105L / L106I、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / L98Q / M99L / Y105L / I108V、L63P / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L / I117M、L63P / L98Q / Y105L、L63P / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V116A、L63P / L98R / N110K、L63P / M99L / Y105L / I1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 08F、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / R85G / L98Q / M99L / Y105L / L106I、S72G / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / I117T、L98Q / M99L / Y105L、L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">M99L / Y105L / L106I / I117T、L98Q / M99L / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106I / Y115N、L98Q / Y105L、L98R / N110K、T89A / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / M99L / Y105L / L106I / Y115N / E120D / C122P / D1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 24P / S125I / D126P、N58S / L63P / T71A / S72G / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L / D124I / S125P / D126T、R16G / E33M / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N58S / E59G / L63P / L98Q / Y105L / E120D / C122P / D1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 24P / S125I / D126P、G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / P121S / D126T、L12H / E33M / L98Q / Y105L、T53S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / N58S / L63P / M87V / L98Q / Y105L、I18T / A26T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M55T / M56K / L63P / L98Q / M99L / Y105L、I18T / A26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T / M56K / L63P / L98Q / Y105L、T53S / L63P / L98Q、T5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3S / L63P / Y105L、T53S / M56K / N58S / L63P / M87V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q、T53S / M56K / N58S / L63P / M87V / Y105L、T53S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / N58S / L63P / L98Q / Y105L、T53S / M56K / N58S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M87V / L98Q / Y105L、T53S / M56K / L63P / M87V / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、T53S / N58S / L63P / M87V / L98Q / Y105L、M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6K / N58S / L63P / M87V / L98Q / Y105L、E33V / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、E33V / M99L / Y105L、E33V / L98Q / M99L、E33V<h2 style=";text-align:left;direction:ltr"> / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y10 5L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56T / L 98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L, M 56T / L98Q / Y105L, L12F / R16H / G29W / M56T / S72G / L98Q / Y105L, G29W / M56T / S72G / L98Q / Y105L, and I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105 L contains one or more amino acid modifications selected from
[0017] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide is selected from the group consisting of SEQ ID NOs: 4-97, 99-104, 106-155, or 570 to 637, or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 polypeptide comprises, for example, an SE As compared to wild-type or unmodified CTLA-4 as set forth in SEQ ID NO:2 No.: 4-97, 99-104, 106-155, or 570-637 and each SEQ ID NO: A sequence of amino acids containing one or more of the amino acid modifications, or a specific binding fragment thereof, Contains.
[0018] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide was attached to the ectodomain of ICOSL and to the unmodified CTLA- 4 binds specifically with increased affinity compared to the binding of 4.
[0019] In some of any of the provided embodiments, one or more amino acid modifications 10, 12, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 6, 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, one or more The amino acid modifications are V10A, L12F, L16F, V17A, V18A, V19A, V20A, V21B, V22B, V23B, V24B, V25B, V26B, V27B, V28B, V29B, V30B, V31B, V32B, V33B, V34B, V35B, V36B, V37B, V3 12I, L12P, R16H, I18F, I18N, I18T, I18V, A19V, A 26T, P28L, G29W, E33M, E33V, R35K, V38I, A42V, Q 45H, T47A, T53S, M55T, M56K, M56T, M56V, N58D, N 58S, T61A, T61R, L63H, L63P, D64E, D64N, D64V, D 65G, I67N, I67T, I67V, T69A, T69I, S72G, Q76R, Q 82H, Q82R, R85G, M87K, M87T, M87V, T89A, T89S, I 93L, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I , L106R, I108F, I108V, N110K, Q113H, V116A, I11 7L or P121S.
[0020] In some of any of the provided embodiments, one or more amino acid modifications V10A, L12F, L12I, R16H, I18N, I18T, I18V, A19 V, A26T, G29W, E33M, E33V, R35K, V38I, A42V, Q45 H, T47A, T53S, M55T, M56K, M56V, N58D, N58S, T61 A, T61R, L63H, L63P, D64E, D64N, D64V, D65G, I67 N, I67T, I67V, T69I, S72G, Q76R, Q82H, Q82R, R85 G, M87K, M87T, M87V, T89A, T89S, I93L, I93V, E97 Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F , I108V, N110K, Q113H, I117L, and P121S, or Conservative amino acid substitutions are selected from:
[0021] In some of any of the provided embodiments, one or more amino acid modifications L63P / S72G / L98Q / M99L / Y105L / L106I / I117L, G29W / L98Q / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L, L63P / Q82H / L98Q / M99L / Y105L, I18T / L6 3P / S72G / L98Q / M99L / Y105L, T61A / L63P / S72G / L 98Q / M99L / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, L63P / S72G / I93L / L98Q / M99L / Y105L, L12 I / M55T / M56V / I67T / M99L / L106R / I108F, I18N / A 26T / L63H / T89A / L98Q / M99L / Y105L, G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W / N58S / L63P / D64N / L98Q / M99L / Y105L、I18T / L63P / S72G / M87 K / L98Q / M99L / Y105L、L63P / M87K / M99L / L106R、L 63P / M99L / Y105L / I108F、G29W / L63P / L98Q / M99L / Y105L、A26T / L63P / D65G / L98Q / M99L / Y105L、V1 0A / L63P / D64V / S72G / L98Q / M99L / Y105L、I18V / A 26T / L63P / D64E / L98Q / Y105L / L106R / N110K、A19 V / G29W / R35K / L63P / L98Q / M99L / Y105L、G29W / N5 8S / L63P / T69I / L98Q / M99L / Y105L、G29W / T53S / M 56K / L63P / L98Q / Y105L、L12F / R16H / G29W / M56T / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y105L / L10 6I / I117L、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / N58D / I67V / L98Q / M99L / Y105L、I6 7V / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R 85G / L98Q / M99L / Y105L / L106I、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / M55 T / L63P / S72G / L98Q / M99L / Y105L、G29W / M87K / I9 3V / L98Q / M99L / Y105L、G29W / T53S / M56K / L63P / Q <h2 style=";text-align:left;direction:ltr">82H / L98Q / M99I / Y105L、L63P / L98Q / M99L / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / I108V、A26T / A42V / Q45H / I67N / M87K / E97Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、E33M / L63P / S72G / L98Q / Y105L、G29W / M87K / T8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9S / L98Q / M99L / Y105L / I108V / I117L、I18T / T61R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / Y105L / I108F、G29W / T53S / M56K / N58S / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / M87V / L98Q / Y105L / P121S、G29W / T53S / M56K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / M87V / L98Q / Y105L / I108V、T53S / M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6K / N58S / L63P / M87V / L98Q / Y105L、I18T / A26T / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 55T / M56K / L63P / L98Q / M99L / Y105L、I18T / A26T / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / L63P / L98Q / Y105L、T53S / L63P / L98Q、T53S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / Y105L、T53S / M56K / N58S / L63P / M87V / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L、L98Q / M99L / Y105L、E33V / L98Q / Y105L、E33V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L、T53S / M56K / N58S / L63P / M87V / L98Q、T53S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / N58S / L63P / L98Q / Y105L、T53S / M56K / N58S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M87V / L98Q / Y105L、T53S / M56K / L63P / M87V / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / Y105L、T53S / N58S / L63P / M87V / L98Q / Y105L、M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 56K / N58S / L63P / M87V / L98Q / Y105L、E33V / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L、L12F / R16H / G29W / M56T / Y105L、またはL12F / L 98Q / Y105L. In some embodiments, the amino acid substitutions are G29W / L9 8Q / M99L / Y105L, L63P / M99L / Y105L / I108F, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, G 29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y105L / L106I, G29W / M87K / I93V / L98Q / M9 9L / Y105L, G29W / T53S / M56K / L63P / Q82H / L98Q / M 99I / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / M87K / T89S / L98Q / M99L / Y105L / I108 V / I117L, G29W / T53S / M56K / N58S / L63P / M87V / L9 8Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, I18T / A26T / M56K / L63P / L98Q / Y10 5L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M 56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L , E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58 S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L9 8Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, or L12F / L98Q / Y105L.
[0022] In some of any of the provided embodiments, one or more amino acid modifications is at positions 12, 26, 63, 98, if or one or more modifications at positions corresponding to 105, and / or L12P, L12F, A Contains one or more alterations selected from 26T, L63P, L98Q, or Y105L It contains one or more amino acid modifications that
[0023] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide has the sequence L12P / A26T / L6 with respect to the positions set forth in SEQ ID NO:2. 3P / L98Q / Y105L, A26T / L63P / S72G / L98Q / M99L / Y 105L, M55T / S72G / L98Q / M99L / Y105L, L63P / Q82H / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99 L / Y105L, T61A / L63P / S72G / L98Q / M99L / Y105L, V 38I / L63P / S72G / L98Q / M99L / Y105L, L63P / S72G / I93L / L98Q / M99L / Y105L, L12I / M55T / M56V / I67T / M99L / L106R / I108F, I18N / A26T / L63H / T89A / L9 8Q / M99L / Y105L, G29W / N58S / L63P / M87T / L98Q / M 99L / Y105L, G29W / N58S / L63P / D64N / L98Q / M99L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、I18T / L63P / S72G / M87K / L98Q / M99L / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、L63P / M87K / M99L / L106R、L63P / M99L / Y105L / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 108F、G29W / L63P / L98Q / M99L / Y105L、A26T / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / D65G / L98Q / M99L / Y105L、V10A / L63P / D64V / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / M99L / Y105L、I18V / A26T / L63P / D64E / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / Y105L / L106R / N110K、A19V / G29W / R35K / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / M99L / Y105L、G29W / N58S / L63P / T69I / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106R / V116A、G29W / T53S / M56K / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、G29W / L63P / S72G / L98Q / Y105L / I117L、L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / Y105L / L106I / I117L、L12F / R16H / G29<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W / M56T / L98Q / Y105L、A26T / T53S / L63P / L98Q / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L / L106I / I117L、G29W / S72G / Q76R / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / L106I / Q113H、G29W / N58D / I67V / L98Q / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、S72G / R85G / L98Q / M99L / Y105L / L106I、A26T / T4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / M55T / L63P / S72G / L98Q / M99L / Y105L、G29W / <h2 style=";text-align:left;direction:ltr"> M87K / I93V / L98Q / M99L / Y105L, P28L / E33V / L63P / S72G / L98Q / M99L / Y105L, G29W / T53S / M56K / L63 P / Q82H / L98Q / M99I / Y105L, I18F / L63P / L98Q / M9 9L / Y105L / P121S, L63P / L98Q / M99L / Y105L / I108 V, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, E33 M / L63P / S72G / L98Q / Y105L, G29W / M87K / T89S / L9 8Q / M99L / Y105L / I108V / I117L, I18T / T61R / L63P / S72G / L98Q / M99L / Y105L, E33M / L63P / S72G / L98 Q / Y105L / I108F, G29W / T53S / M56K / N58S / L63P / M 87V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, and G29W / T53S / M56K / Select from N58S / L63P / M87V / L98Q / Y105L / I108V Contains one or more amino acid modifications.
[0024] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. In some embodiments, one or more The above amino acid modifications are 10, 12, 16 with respect to the positions set forth in SEQ ID NO:2. , 18, 26, 29, 42, 45, 53, 56, 58, 63, 67, 72, 82, 87, A position corresponding to a position selected from among 97, 98, 99, 105, 108, or 121 In some embodiments, the one or more amino acid modifications are 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 warranty Conservative amino acid substitutions are selected from:
[0025] In some of any of the provided embodiments, one or more amino acid modifications V10A, L12F, R16H, I with respect to the positions set forth in SEQ ID NO:2 18T, A26D, A26T, G29W, A42V, Q45H, T53S, M56K, M 56T, N58S, L63P, I67N, S72G, Q82R, M87K, M87V, E Choose from 97Q, L98Q, M99L, Y105L, I108V, or P121S The position corresponds to the position where the
[0026] In some of any of the provided embodiments, one or more amino acid modifications is I18T / G29W / L63P / L with respect to the positions set forth in SEQ ID NO:2 98Q / Y105L, G29W / L63P / L98Q / M99L / Y105L, G29W / N58S / L63P / L98Q / Y105L, A26D / S72G / L98Q / M99 L / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, L 12F / R16H / G29W / M56T / 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 / T5 3S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, and V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P Selected from 121S.
[0027] In some of any of the provided embodiments, one or more amino acid modifications I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L6 3P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y 105L, L12F / R16H / G29W / M56T / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / P121S, G2 9W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G 29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, V10A / G29W / T53S / M56K / L63P / L98Q / Y105 L / P121S, T53S / M56K / N58S / L63P / M87V / L98Q / Y1 05L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y 105L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T5 3S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N 58S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105 L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L9 8Q / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R1 6H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y 105L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56T / L98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L9 8Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105L or M56T / L98Q / Y105L. , wherein the one or more amino acid modifications are I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, A26T / A42V / Q45H / I67N / M87 K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M87 V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L 63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, L98Q / M99L / Y10 5L, E33V / L98Q / Y105L, T53S / M56K / N58S / L63P / L 98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L , and E33V / L98Q / M99L.
[0028] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomains of ICOSL and CD80. In the form, one or more amino acid modifications are V10A, L12F, R16H, I18T, A2 6T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, N5 8S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M9 9L, Y105L, I108V, or a conservative amino acid substitution thereof. In some embodiments, the one or more amino acid modifications are I18T, A26T, G29W, E 33V, Q45H, T53S, M56K, N58S, L63P, I67N, Q82R, M Select from 87K, M87V, E97Q, L98Q, M99L, Y105L, I108V can be.
[0029] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomain of CD86.
[0030] In some of any of the provided embodiments, one or more amino acid modifications 10, 12, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 6, 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, Among 102, 105, 106, 108, 113, 115, 116, 117, or 121 In some embodiments, one or more amino The acid modifications are V10A, L12F, L12A, V10B, V10C, V10D, V10E, V10F, V10G, V10H ... H, L12P, R16H, I18T, I18V, S20N, A26S, A26T, P28 L, G29R, G29W, K30R, E33M, E33V, A42S, A42T, T47 A, T53S, M55T, M56K, M56R, M56T, M56V, N58D, N58 S, E59G, T61I, T61N, T61R, T61S, L63P, D65G, I67 N, I67V, T69A, T69I, S72G, Q76R, Q82H, Q82R, R85 G, M87K, M87V, T89A, T89M, T89S, I93V, V96I, L98 Q, L98R, M99L, P102L, Y105L, L106I, L106N, L106 R, L106V, I108F, I108V, Q113H, Y115H, V116A, I1 a position selected from 17E, I117L, I117K, I117T, and P121S; It is located in a position corresponding to the position.
[0031] In some of any of the provided embodiments, one or more amino acid modifications L12F, L12H, L12P, R16H, I18T, I18V, S20N, A26 T, G29R, G29W, E33M, E33V, A42S, A42V, T47A, T53 S, M55T, M56K, M56R, M56T, M56V, N58D, N58S, T61 I, T61N, T61R, T61S, L63P, D65G, I67N, I67V, T69 A, T69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87 V, T89A, T89M, T89S, V96I, L98Q, L98R, M99L, P10 2L, Y105L, L106I, L106N, L106R, L106V, I108F, I 108V, Q113H, Y115H, V116A, I117L, I117T, and P1 21S, or conservative amino acid substitutions thereof.
[0032] In some of any of the provided embodiments, one or more amino acid modifications is L12P / A26T / L63P / L with respect to the positions set forth in SEQ ID NO:2 98Q / Y105L, I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L, L12P / A26T / L63P / S72G / T89M / L98 Q / M99L / Y105L, P28L / E33V / L63P / S72G / L98R / M9 9L / Y105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y 105L, S72G / L98Q / M99L / Y105L / I117T, M56R / L63 P / L98Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L 106I, A26T / M55T / L63P / L98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63 P / Q82R / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y1 05L / I117L, A26T / I67N / S72G / L98Q / M99L / Y105L , L12F / A26T / L63P / L98Q / Y105L / L106R, S20N / A2 6T / L63P / L98Q / M99L / Y105L, G29W / T61I / L63P / S <h2 style=";text-align:left;direction:ltr">72G / L98Q / M99L / Y105L、G29W / N58S / L63P / T69I / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L / L106N、L63P / T69A / L98Q / M99L / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106R / V116A、G29W / N58S / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、G29W / L63P / D65G / S72G / L98Q / Y105L、T53S / M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6V / L98Q / Y105L、L63P / S72G / L98Q / Y105L、G29W / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T53S / M56K / L63P / L98Q / Y105L、I18V / G29W / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / Y105L、G29W / L63P / S72G / L98Q / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L / L106I、G29W / L63P / I67V / S72G / L98Q / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / Y105L / L106I / I117L、L12F / R1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6H / G29W / M56T / L98Q / Y105L、L12P / G29W / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / Y105L、G29W / N58S / L63P / S72G / M87V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6I / Q113H、G29W / N58S / L63P / S72G / L98Q / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L106V、A26T / L63P / L98Q / M99L / Y105L、G29W / N58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D / I67V / L98Q / M99L / Y105L、I67V / S72G / Q82H / T8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9A / L98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L / L106I、L63P / L98Q / M99L / Y105L、A26T / T47<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A / M56K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6T / M55T / L63P / S72G / L98Q / M99L / Y105L、L12H / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S、I18T / A26T / L63P / S72G / L98Q / Y105L、L12F / K3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0R / S72G / Q82R / L98Q / M99L / Y105L、L12P / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / M99L / Y105L / L106N / I117L、G29W / M87<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K / I93V / L98Q / M99L / Y105L、P28L / E33V / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、M56V / E59G / L63P / S72G / M87K / I93V / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L / I117E、G29W / M87K / T89S / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L / I108V / I117L、L12P / M56V / L63P / V96I / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L / Y115H、G29W / T53S / M56K / T61<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N / L63P / L98Q / Y105L、I18T / A26S / M55T / M56V / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / M99L / Y105L / I117K、I18T / T61R / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L / I108F、L12P / R16H / A26T / T61S / L63P / M87V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L / L106I / I117LG29W / T53S / M56K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / M87V / L98Q / Y105L / P121S、G29W / L6<h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / Y105L / P121S, and V10A / G29W / T5 Select from 3S / M56K / L63P / L98Q / Y105L / P121S.
[0033] In some of any of the provided embodiments, one or more amino acid modifications Fits I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y1 05L, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y 105L, I18T / G29R / L63P / S72G / L98Q / M99L / Y105L , S72G / L98Q / M99L / Y105L / I117T, M56R / L63P / L9 8Q / M99L / Y105L, L63P / L98Q / M99L / Y105L / L106I , A26T / M55T / L63P / L98Q / M99L / Y105L, E33V / A42 S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63P / Q8 2R / L98Q / Y105L, E33M / L63P / S72G / L98Q / Y105L / I117L, A26T / I67N / S72G / L98Q / M99L / Y105L, L12 F / A26T / L63P / L98Q / Y105L / L106R, S20N / A26T / L 63P / L98Q / M99L / Y105L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y10 5L / L106N, L63P / T69A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58S / L63P / S72G / L98Q / Y105L, G2 <h2 style=";text-align:left;direction:ltr">9W / L63P / D65G / S72G / L98Q / Y105L、T53S / M56V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L、L63P / S72G / L98Q / Y105L、G29W / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / Y105L / L106I、L12F / R16H / G29W / M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6T / L98Q / Y105L、G29W / N58S / L63P / S72G / M87V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L、G29W / S72G / Q76R / L98Q / Y105L / L106<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I / Q113H、G29W / N58S / L63P / S72G / L98Q / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106V、G29W / N58D / I67V / L98Q / M99L / Y105L、I67V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / Q82H / T89A / L98Q / M99L / Y105L、S72G / R85<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / M99L / Y105L / L106I、L63P / L98Q / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、L12H / I18V / A42T / M55T / N58D / L98R / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L / L106I / P121S、E33M / L63P / S72G / L98Q / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、G29W / M87K / T89S / L98Q / M99L / Y105L / I108V / I1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 17L、L12P / M56V / L63P / V96I / L98Q / M99L / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y115H、G29W / T53S / M56K / T61N / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、L1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2P / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / Y105L / I108F、L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117L, L 98Q / M99L / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q, L12F / G29W / L98Q / Y105L, or L12F / L98Q / Y105L.
[0034] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomains of CD80 and CD86. In some embodiments, the one or more amino acid modifications are I18T, A26T, G29W, E33V, A42 V, T53S, N58S, L63P, I67N, Q82R, M87K, M87V, L98 Choose from Q, M99L, Y105L, and I108V.
[0035] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomains of ICOSL and CD86.
[0036] In some of any of the provided embodiments, one or more amino acid modifications L12F, R16H, I18T, I18V, A26T, G29W, E33M, E33 V, A42V, T47A, T53S, M55T, M56K, M56V, N58D, N58 S, T61R, L63P, D65G, I67N, I67V, T69I, S72G, Q76 R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89S, L98 Q, M99L, Y105L, L106I, L106R, I108F, I108V, Q11 3H, I117L, and P121S, or conservative amino acid substitutions thereof; do.
[0037] In some of any of the provided embodiments, one or more amino acid modifications L12F, R16H, I18T, I18V, A26T, G29W, E33V, A42 V, T47A, T53S, M55T, M56K, N58D, N58S, L63P, I67 N, I67V, S72G, Q82H, Q82R, R85G, M87K, M87V, T89 A, T89S, L98Q, M99L, Y105L, L106I, L106R, I108F , I108V, and I117L.
[0038] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides showed increased binding of the parent CTLA-4 compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomains of ICOSL, CD80, and CD86.
[0039] In some of any of the provided embodiments, one or more amino acid modifications L12F, R16H, I18T, A26T, G29W, E33V, A42V, T53 S, M55T, M56K, N58S, L63P, I67N, Q82R, M87K, M87 V, L98Q, M99L, Y105L, or I108V. In embodiments, the one or more amino acid modifications are A26T, G29W, T53S, M56K, N Select from 58S, L63P, L98Q, M99L, or Y105L. In embodiments, the one or more amino acid modifications are G29W, L63P, L98Q, M99L, or Y105L. In some embodiments, the one or more amino acid modifications are selected from , G29W / L63P, G29W / L98Q, G29W / M99L, G29W / Y105 L, L63P / L98Q, L63P / M99L, L63P / Y105L, L98Q / M9 9L, L98Q / Y105L, or M99L / Y105L. In some embodiments, the amino acid modifications are G29W / L98Q / Y105L. In some embodiments, the amino acid modifications are G29W / N58S / L63P / Q82R / L 98Q / Y105L. In some embodiments, the amino acid modification is L12P / G2 9W / L63P / S72G / L98Q / Y105L.
[0040] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides increased binding compared to unmodified CTLA-4 to the same ectodomain. It specifically binds to the ectodomain of ICOSL with high affinity and also binds to CD80 with reduced affinity. or the other, one or more ectodomains of CD86.
[0041] In some of any of the provided embodiments, one or more external domains The increase in binding affinity for 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times or is more than that.
[0042] In some of any of the provided embodiments, one or more external domains The decrease in binding affinity for , 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 100x or It's more than that.
[0043] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide contains an IgV domain or a specific binding fragment thereof. In this case, the IgV domain or a specific binding fragment thereof is a mutant CTLA-4 polypeptide. In some embodiments, the mutant CTLA-4 polypeptide is The code is SEQ ID NO: 156-285, 603-635, or 637 The present invention relates to a method for producing a human immunodeficiency virus (HIV)-associated HIV-1 virus, comprising administering to a human anthracycline or a human anthracycline the ... or a specific binding fragment thereof. In embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NOs: 156-285. , 603-635, or 637. and the like, which contains the sequence of amino acids set forth in SEQ ID NO: :156-285, 603-635, or 637 of the wild-type or unmodified CTLA -4, each of which contains one or more of the amino acid modifications of the respective SEQ ID NO: In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NOs:4-9 Any of the following: 7, 99-104, 106-155, 569-602, or 636 or a specific binding fragment thereof, or SEQ ID NO: 4~97, 99~104, 106~155, 569~602, or 6 3 and shows at least 95% sequence identity with any of SEQ ID NOs: 4~97, 99~104, 106~155, 569~602, or 636 Amino acids containing one or more of the amino acid modifications of each of the SEQ ID NOs. It contains the sequence:
[0044] In some of any of the provided embodiments, the ICOSL is human IC In some embodiments, the CD80 is human CD80. In embodiments, the CD86 is human CD86.
[0045] In some embodiments, the mutant CTLA-4 polypeptide is a soluble protein. In some embodiments, the mutant CTLA-4 polypeptide is a CTLA-4 transmembrane polypeptide. domain and intracellular signaling domain and / or mutant CTLA The -4 polypeptide cannot be expressed on the surface of a cell.
[0046] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide is linked to a multimerization domain. In some embodiments, the multimerization domain The main one is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the mutant CTLA-4 polypeptide has a reduced effector function. In some embodiments, the Fc domain is linked to a nucleotide sequence of the Fc domain or a variant thereof. , mammalian, optionally human, or the variant Fc domain is mammalian, optionally human. Optionally, the Fc domain contains one or more amino acid modifications compared to an unmodified human Fc domain. In some embodiments, the Fc domain or variant thereof is selected from the group consisting of SEQ ID NO: 438 to 442, or the amino acid sequence set forth in any one of SEQ ID NOs. : An amino acid sequence showing at least 85% sequence identity with any of 438 to 442 In some embodiments, the Fc domains each contain a sequence of E233P, L234A, L234V, L235A, L235E, G236del, Choose from G237A, S267K, N297G, V302C, and K447del In some embodiments, the Fc domain contains one or more amino acid modifications In some embodiments, F comprises the amino acid modifications L234A / L235E / G237A. The c domain contains the amino acid modification C220S according to EU numbering. In this embodiment, the mutant CTLA-4 polypeptide is provided with a linker, optionally a G4S (Gl Indirectly linked to the multimerization domain or Fc via a (y4Ser) linker .
[0047] In some of any of the embodiments provided, The mutant CTLA-4 polypeptides are shown in SEQ ID NOs: 286-379, 38 a sequence of amino acids set forth in any one of 1 to 386 or 388 to 437, or SEQ ID NO: 286-379, 381-386, or 388-437 It contains an amino acid sequence that exhibits at least 85% sequence identity with any of the following:
[0048] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide is a transmembrane immunomodulatory protein that further contains a transmembrane domain, and optionally Alternatively, the transmembrane domain may directly or indirectly bind to the cellular domain of the mutant CTLA-4 polypeptide. linked to the extracellular domain (ECD) or a specific binding fragment thereof. In this embodiment, the transmembrane domain comprises residues 162-182 of SEQ ID NO:1 and at least It is set forth as residues 162-182 of SEQ ID NO:1, which shows 85% sequence identity. The amino acid sequence of the present invention is a sequence of amino acids selected from the group consisting of 2-amino-3-methyl-2-propanol, ...
[0049] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide further contains a cytoplasmic domain, and optionally, the cytoplasmic domain is a transmembrane domain. In some embodiments, the cytoplasmic domain is directly or indirectly linked to the cytoplasmic domain. The domain may be a native CTLA-4 cytoplasmic domain, an intracellular signaling domain, or or containing and / or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) In some embodiments, the cytoplasmic domain contains a signaling motif. SEQ ID NO: 1, which shows at least 85% sequence identity with residues 183-223 of SEQ ID NO: 1 The amino acid sequence set forth as residues 183-223 of ID NO:1 or its function In some embodiments, the cytoplasmic domain contains a target mutation of an immunoreceptor tyrosine. containing ITAM-based signaling activation motifs and / or CD It contains the intracellular signaling domain of 3 zeta.
[0050] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptide does not contain a cytoplasmic signaling domain and / or is expressed on cells Sometimes they are unable to mediate or regulate intracellular signals.
[0051] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide The peptides reacted with unmodified CTLA-4 polypeptide in an in vitro primary T cell assay. In some embodiments, the expression of IFN-gamma (interferon gamma) is reduced. In some embodiments, the mutant CTLA-4 polypeptide is deglycosylated.
[0052] In some of any of the embodiments provided, directly or via a linker Indirectly, the IgSF domain of immunoglobulin superfamily (IgSF) members The mutant CTLA-4 is provided linked to a second polypeptide containing an amino acid. Immunomodulating polypeptides containing any of the above are provided herein. In an embodiment, the IgSF domain is an affinity-modified IgSF domain, The IgSF domain is the unmodified or wild-type IgSF domain of an IgSF family member. Optionally, the affinity-modified IgS contains one or more amino acid modifications compared to the IgS. The F domain binds to one or more of its cognate binding partners. Unmodified or wild-type IgSF domains of IgSF family members with binding partners In some embodiments, the IgSF domain exhibits altered binding compared to its one or more of the cognate binding partners of the Increased binding is shown compared to binding of unmodified or wild-type IgSF domains.
[0053] In some of any of the provided embodiments, the mutant CTLA-4 is a first mutant CTLA-4 polypeptide and an IgSF domain of a second polypeptide; is an IgSF domain from a second mutant CTLA-4 polypeptide, The second mutant CTLA-4 is the same or different. Regulatory proteins are proteins that bind to the IgSF domain of an IgSF family member or its affinity modifier. and a third polypeptide containing a modified IgSF domain, The SF domain is identical to the unmodified or wild-type IgSF domain of the IgSF family members. In some examples, the third polypeptide contains one or more amino acid modifications compared to the first polypeptide. may be the same as the first and / or second polypeptide or may be a third polypeptide. The polypeptide is different from the first and / or second polypeptide.
[0054] In some of any of the embodiments provided, optionally, a second or the IgSF domain or affinity-modified IgSF domain of the third polypeptide is Optionally, the mutant CTLA-4 polypeptide is or comprises an IgV domain. The peptide is or contains an IgV domain.
[0055] In some of any of the provided embodiments, the immunomodulatory protein is at least one of the mutant CTLA-4 polypeptide or the second polypeptide In some embodiments, the immunomodulatory protein further comprises a linked multimerization domain. The protein comprises a mutant CTLA-4 polypeptide, a second polypeptide, and / or a third polypeptide. and further comprising a multimerization domain linked to at least one of the polypeptides of In some cases, the multimerization domain may be an Fc domain or a nucleotide sequence with reduced effector function. is a variant of it.
[0056] In some of any of the provided embodiments, the multimerization domain is a heme. Promotes the formation of heterodimers. wherein the multimerization domain is a first multimerization domain; and interacts with a second multimerization domain to form a multimer containing the immunomodulatory protein. Provided herein are immunomodulatory proteins that produce The protein is a first immunomodulatory protein, and the second immunomodulatory protein is a direct or reactive The first and second multimerization domains are indirectly linked to the second multimerization domain via a linker. In some embodiments, the second immunomodulatory protein The protein is an immunomodulatory protein provided herein, wherein the multimerization domain is a second multimer. It is a merization domain.
[0057] In some of any of the provided embodiments, the first mutant CTLA- 4 polypeptide, wherein the multimerization domain is a first multimerization domain. a mutant CTLA-4 polypeptide and a second mutant CTLA-4 polypeptide, a second mutant CTLA-4 polypeptide, wherein the multimerization domain is a second multimerization domain; and a peptide, wherein the first and second multimerization domains are The mutant CTLA-4 polypeptides interact with each other to form a multimeric complex containing the first and second mutant CTLA-4 polypeptides. Provided herein are immunomodulatory proteins that form the body. In some embodiments, The multimer is a dimer. In some embodiments, the immunomodulatory protein is a homodimer. In some embodiments, the immunomodulatory protein is a heterodimer. In some embodiments, the first and / or second multimerization domains may have an effector function. In some embodiments, the first and second Fc domains are reduced Fc domains or variants thereof. The two multimerization domains may be the same or different.
[0058] In some of the embodiments provided, a Conjugates containing mutant CTLA-4 or immunomodulatory proteins are described herein. In some embodiments, a moiety specifically binds to a molecule on the surface of a cell. In some embodiments, the targeting moiety is a targeting moiety that binds to the surface of an immune cell. In some embodiments, the immune cell is an antigen-presenting cell or In some embodiments, the targeting moiety is a cell or In some embodiments, the moiety is a protein, peptide, nucleic acid, or the like. In some embodiments, the moiety is an antibody or an anti- In some embodiments, the conjugate is a bivalent, tetravalent, hexavalent, or or octavalent.
[0059] In some of the embodiments provided, the variants provided herein may be Nucleic acid molecules encoding atypical CTLA-4 polypeptides or immunoglobulins provided herein. Regulatory proteins are provided herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is a cDNA.
[0060] In some of the embodiments provided, the nuclei provided herein Provided herein are vectors containing any of the acid molecules. In some embodiments, the vector is an expression vector. The vector is an expression vector or a viral vector.
[0061] In some of the embodiments provided, the bases provided herein Provided herein are cells containing a mammalian vector. In some embodiments, the cells are In some embodiments, the cell is a human cell.
[0062] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide A method for producing a peptide or immunomodulatory protein, comprising: Introducing the molecule or vector into a host cell under conditions that result in expression of the protein in the cell. In some embodiments, the method comprises: Isolating or purifying the LA-4 polypeptide or immunomodulatory protein from the cells. Further includes:
[0063] In some of any of the provided embodiments, the mutant CTLA-4 polypeptide A method of engineering cells to express a peptide comprising administering to a subject a mutant CTLA-1 polypeptide as provided herein. -4 A nucleic acid molecule encoding a polypeptide or an immunomodulatory protein, and introducing the vector into a host cell under conditions such that the vector is expressed in the host cell. .
[0064] In some of the embodiments provided, the variants provided herein may be expressing a variant CTLA-4 polypeptide, immunomodulatory protein, nucleic acid molecule, or vector In some embodiments, the cells are engineered to In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are CD4+ and In some embodiments, the T cells are regulatory T cells (T In some embodiments, the engineered cells are primary cells. In some embodiments, the engineered cells are mammalian cells. The engineered cells are human cells.
[0065] In some of any of the provided embodiments, the CTLA-4 polypeptide The domain is expressed on the surface of the cells provided herein via the transmembrane domain. In some such embodiments, the CTLA-4 polypeptide comprises a cytoplasmic domain. and optionally the cytoplasmic domain is linked, directly or indirectly, to the transmembrane domain. In some embodiments, the cytoplasmic domain is a naturally occurring CTLA-4 cytoplasmic domain. in the main, intracellular signaling domain, and / or ITIM signaling motif In some embodiments, the cytoplasmic domain is or contains SEQ SEQ ID NO: 1, which shows at least 85% sequence identity with residues 183-223 of SEQ ID NO: 1 The amino acid sequence set forth as residues 183-223 of ID NO:1 or its function In some embodiments, the cytoplasmic domain contains a specific variant of an ITAM signaling protein. Contains a targeting motif and / or the intracellular signaling domain of CD3 zeta In some embodiments, the CTLA-4 polypeptide is It does not contain a cytoplasmic signaling domain and / or does not produce an intracellular signal when expressed on a cell. It cannot mediate or regulate signals.
[0066] In some of any of the provided embodiments, the engineered cells are In some embodiments, the engineered cells further contain a CAR. , which further contain an engineered T cell receptor (TCR).
[0067] In some of the embodiments provided, the variants provided herein may be Nucleic acid molecules encoding atypical CTLA-4 polypeptides or immunoglobulins provided herein. Provided herein are infectious agents containing regulatory proteins. In some embodiments, In some cases, the infectious agent is a bacterium or a virus.
[0068] In some of the embodiments provided, the variants provided herein may be Atypical CTLA-4 polypeptides, immunomodulatory proteins provided herein, A provided conjugate, an engineered cell provided herein, or a method for producing a compound provided herein Provided herein are pharmaceutical compositions containing the infectious agent. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. The pharmaceutical composition is sterile.
[0069] In some of any of the provided embodiments, in a vial or container Provided herein are articles of manufacture containing the pharmaceutical compositions provided herein. In embodiments, the vial or container is sealed.
[0070] In some of the embodiments provided, the medical Kits containing the pharmaceutical composition and instructions for use are provided herein. In embodiments, a kit containing the articles of manufacture provided herein and instructions for use is provided by: Provided herein.
[0071] In some of any of the provided embodiments, the immune response in a subject is induced by A method of modulating the expression of steroid hormone, comprising administering to a subject a pharmaceutical composition provided herein. In some embodiments, methods for modulating an immune response in a subject are provided. The method comprises administering the engineered cells provided herein, In some of such embodiments, the engineered cells are In some embodiments, the engineered cells are allogeneic to the subject. In some embodiments, the modulation of the immune response is indicative of a disease or condition in a subject. Treat.
[0072] In some of any of the provided embodiments, the immune response is reduced. In some embodiments of the provided methods, suppressor (e.g., ITIM-containing) cells The present invention provides a method for the treatment of CTLA-4 comprising administering to a subject a therapeutic target, comprising administering to a subject a therapeutic target, a method for treating CTLA-4 ... a variant polypeptide provided herein, an immunomodulatory protein provided herein, or The engineered cells provided herein are administered to a subject. The disease or condition is an inflammatory or autoimmune disease or condition, or is caused by excessive immunity. In some embodiments, the disease or condition is a disease or condition associated with an immune response. , antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, Eumatic diseases, thyroiditis, inflammatory gastrointestinal diseases, inflammatory eye diseases, inflammatory neurological diseases, inflammatory lung diseases disease, inflammatory endocrine disease, autoimmune blood disease, autoimmune demyelinating disease, or systemic autoimmune In some embodiments, the disease or condition is an autoimmune disease involving an immune disorder. Inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, asthma, autoimmune asthma, rheumatoid arthritis , psoriasis, lupus erythematosus, celiac disease, type 1 diabetes, Guillain-Barré syndrome, chronic inflammation Demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, De Quervain's thyroiditis, myopathy Asthenia, vasculitis, autoimmune hemolytic anemia, pernicious anemia autoimmune atrophic gastritis, autoimmune Encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmopathy inflammation, primary biliary cirrhosis, chronic active hepatitis, membranous nephropathy, primary idiopathic myxedema, scleroderma, Chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, autoimmune Enteropathy syndrome, idiopathic thrombocytopenia, acquired splenic atrophy, idiopathic diabetes insipidus, and antisperm antibodies Infertility, sensorineural hearing loss, Sjogren's syndrome, polymyositis, multiple sclerosis, Myelitis dissecans, ataxic sclerosis, pemphigus, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa , hemolytic anemia, glomerulonephritis, and idiopathic facial paralysis.
[0073] In some of any of the provided embodiments of the provided methods, an immunoreaction In some embodiments of the provided methods, cytoplasmic signaling is increased. Surface-expressed mutant CTLA-4 lacking the I domain, or activating (e.g., I Surface-expressed mutant CTLA-containing (TAM-containing) cytoplasmic signaling domains The engineered cells provided herein, such as cells expressing 4, are administered to the subject. In some embodiments, the methods provided treat a disease or condition that is a tumor or cancer. In some such embodiments, the disease or condition is melanoma, lung cancer, bladder cancer, or the like. , hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer Adenocarcinoma, testicular cancer, ovarian cancer, uterine cancer, stomach cancer, cancer of the musculoskeletal system, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancers. [Brief explanation of the drawings]
[0074] [Figure 1] Illustrated are various soluble or cell-free expression formats of the provided mutant IgSF domain molecules, such as mutant CTLA-4 polypeptides comprising (1) a mutant IgSF domain (vIgD) fused to an Fc chain, (2) a mutant IgSF domain (vIgD) linked to an antibody (V-mAb). The vIgD may comprise an ECD containing the IgV or V domain (IgV) of an IgSF superfamily member of CTLA-4, or (3) a stacked molecule containing a second IgSF domain, such as a first mutant IgSF domain (first vIgD) and a second mutant IgSF domain (second vIgD). [Figure 2]The immunomodulatory activity of exemplary soluble CTLA-4 polypeptides is illustrated. In this exemplary schematic, T cell activation results from signaling through the T cell receptor (TCR) and CD28 and / or ICOS costimulatory receptors. Signaling, and thus T cell activation, is triggered by antigen-presenting cells (APCs) expressing MHC and the costimulatory ligands CD80 (B7-1), CD86 (B7-2), and / or ICOSL (see Figure 2, left). As shown, the provided mutant CTLA-4 polypeptides, as exemplified schematically by the IgSF domain (vIgD) of mutant CTLA-4 fused to Fc (vIgD-Fc of CTLA-4, see middle of Figure 2), interact with one or more binding partners ICOSL, CD80 (B7~1), and / or CD86 (B7~2) to inhibit binding of T cell CD28 and ICOS activating receptors to their costimulatory ligands, thereby suppressing T cell activation, proliferation, and effector function (see right of Figure 2). [Figure 3]Figures 3A and 3B illustrate an embodiment of the expression of CTLA-4, such as mutant CTLA-4, as a transmembrane immunoregulatory protein (TIP) on the surface of T cells. As shown in Figure 3A, in some cases, T cell activation is driven by CD28 / TCR signaling, which is triggered by engagement with CD80 (B7-1) and CD86 (B7-2) costimulatory molecules expressed on antigen-presenting cells (APCs). T cell-expressed CTLA-4, such as CTLA-4 expressed on activated T cells or T regulatory cells, is constantly recycled between the intracellular compartment and the cell surface through cellular internalization. CTLA-4 can also bind to CD80 (B7-1) and CD86 (B7-2) costimulatory molecules (and, in some cases, ICOSL). As shown in Figure 3B, in some cases, engagement of T cell-expressed CTLA-4 with its ligands, such as B7-1 and B7-2, allows the T cell to remove its cognate ligand from the surface of APCs in a process called trans-endocytosis. Functionally, this allows CTLA-4 to "shed" the ligand from the APC, thereby preventing the CD28 costimulatory ligand from subsequently activating additional T cells, thus limiting T cell activation. [Figure 4] 1 illustrates a secreted immunomodulatory protein (SIP) in which a mutant IgSF domain (vIgD) is secreted from a cell, such as a first T cell, e.g., a CAR (chimeric antigen receptor) T cell. In an exemplary embodiment, the SIP is a mutant CTLA-4 polypeptide, and the binding partner is a costimulatory ligand (e.g., CD80, CD86, and / or ICOSL) that can be expressed by an antigen-presenting cell. Upon binding of the SIP to its binding partner, the SIP inhibits binding of the costimulatory ligand to a costimulatory receptor (e.g., CD28), thereby inhibiting activation signals via the costimulatory receptor and attenuating T cell or effector T cell activation. [Figure 5A]Figures 5A-5C illustrate various exemplary configurations of mutant IgSF-antibody conjugates (V-Mabs). Figure 5A shows various configurations in which one or more mutant IgSF domains (vIgDs) are linked, directly or indirectly, to the amino and / or carboxyl termini of an antibody light chain. [Figure 5B] Figures 5A-5C illustrate various exemplary configurations of mutant IgSF-antibody conjugates (V-Mabs). Figure 5B shows various configurations in which one or more mutant IgSF domains are linked, directly or indirectly, to the amino and / or carboxyl termini of an antibody heavy chain. [Figure 5C] Figures 5A-5C illustrate various exemplary configurations of mutant IgSF-antibody conjugates (V-Mabs). Figure 5C illustrates the V-Mab configurations obtained when a light chain as illustrated in Figure 5A and a heavy chain as illustrated in Figure 5B are co-expressed in cells. [Figure 6A] Various exemplary configurations of stacked molecules containing a second IgSF domain, such as a first variant IgSF domain (first vIgD) and a second variant IgSF domain (second vIgD), are illustrated. As shown, the first vIgD and second IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of the Fc region. To create a homodimeric Fc molecule, the Fc region is a region capable of forming homodimers with matched Fc subunits by co-expression of the individual Fc regions in a cell. To create a heterodimeric Fc molecule, the individual Fc regions contain mutations (e.g., "knob-into-hole" mutations in the CH3 domain) that favor heterodimer formation compared to homodimers when the individual Fc regions are co-expressed in a cell. [Figure 6B]Various exemplary configurations of stacked molecules containing a first mutant IgSF domain (first vIgD), a second IgSF domain such as a second mutant IgSF domain (second vIgD), and a third IgSF domain such as a third mutant IgSF domain (third vIgD) are illustrated. As shown, the first vIgD, second IgSF, and third IgSF domains are independently linked, directly or indirectly, to the N- or C-terminus of the Fc region. To create a homodimeric Fc molecule, the Fc region is capable of forming a homodimer with a matching Fc region by co-expressing the individual Fc regions in a cell. [Figure 7] 1 illustrates collagen-induced arthritis (CIA) paw scores after treatment of mice in the CIA model with an exemplary CTLA-4 variant set forth in SEQ ID NO:93 (containing the mutations G29W / N58S / L63P / Q82R / L98Q / Y105L) fused to Fc, abatacept, or an Fc control. CIA paw scores were determined as the sum of each of the four paws per mouse and averaged across groups of 15 mice (mean CIA paw score). DETAILED DESCRIPTION OF THE INVENTION
[0075] Detailed Description Cytotoxic T lymphocytes exhibiting activity for binding to at least one target binding partner. CTL cell-associated protein 4 (also known as cluster of differentiation 152 or CD152) A-4) or a specific binding fragment thereof, or In some embodiments, the immunomodulatory protein comprises a variant of The CTLA-4 polypeptide has the following structure: , containing one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, one or more amino acid modifications (e.g., substitutions) are made to the unmodified or wild-type amino acid sequence. In some embodiments, one or more of the amino acid sequences are in the ECD of a CTLA-4 polypeptide. Amino acid modifications (e.g., substitutions) can be made to the IgS domain of unmodified or wild-type CTLA-4 polypeptides. In some embodiments, the mutant CTLA-4 Polypeptides and immunomodulatory proteins include inducible T cell costimulatory ligands (B7-H2, CD275, and GL50 (also known as ICOSL). In some embodiments, the mutant CTLA The B-4 polypeptide and immunomodulatory proteins are ICOSL, CD80 (also known as B7-1), and / or increased binding activity to CD86 (also known as B7-2) Or exhibits affinity.
[0076] In some embodiments, the mutant CTLA-4 polypeptide is an immunomodulatory Such molecules do not contain a transmembrane domain and / or are not transmembrane-localizing. CTLA-4 polypeptides that are not membrane-anchored or cannot be expressed on the surface of cells In some embodiments, the mutant CTLA-4 protein comprises a polypeptide. As a transmembrane immunomodulatory protein that can be expressed on the surface or secreted from the cell The antibody may be provided as a secretable immunomodulatory protein that can be administered intracellularly. In some embodiments, a mutant CTLA-4 polypeptide provided herein and one or more other one or more other immunosuppressants containing a conjugate or fusion containing the moiety or polypeptide Regulatory proteins are also provided herein.
[0077] In some embodiments, the mutant CTLA-4 polypeptide and the immunomodulatory protein The compound modulates an immunological immune response, such as increasing or decreasing an immune response. In the present invention, the mutant CTLA-4 polypeptides and immunomodulatory proteins provided may be an autoimmune condition or disease, or in some cases, an oncological indication , can be used for the treatment of diseases or conditions associated with a dysregulated immune response.
[0078] In general, antigen-specific T cell activation generally requires two distinct signals. Signal 1 is a major signaling pathway present on antigen-presenting cells (APCs) that activates the T cell receptor (TCR). The second signal is provided by interaction with histocompatibility complex (MHC)-associated antigens. , costimulatory for TCR engagement and, in some cases, induce T cell apoptosis or apoptosis required for T cell proliferation, differentiation, and / or survival, including evading vasculitis In some embodiments, under normal physiological conditions, a T cell-mediated immune response is initiated by T cell receptors. It is initiated by antigen recognition by the TCR and leads to costimulation (e.g., T cell activation) and Regulated by the balance of immune and co-inhibitory signals (e.g., immune checkpoint proteins) The immune system prevents autoimmunity (i.e., self-tolerance) and promotes immune responses, e.g., Immune checkpoints protect tissues from excessive damage during attacks by pathogenic infections In some cases, however, the immune system can become dysregulated. An abnormal immune response may be mounted against normal body parts or tissues, resulting in autoimmune disorders. It may result in an immune disease or condition or an autoimmune condition. Innate immune responses may be raised against foreign tissues, such as transplants, leading to transplant rejection. It can be achieved.
[0079] CTLA-4 suppresses T cell responses through regulation of TCR / CD28 signaling The costimulatory receptor CD28 is an inhibitory IgF receptor. The costimulatory receptor CD28 binds to the costimulatory ligand CD80 (B7 1) and CD86 (also known as B7-2), which mediates TCR signaling. In the presence of CD80, the co-inhibitory receptor CTLA-4 promotes the activation of naive T cells. and competes with CD28 for binding to CD86, inducing negative regulation of T cell activation CTLA-4 binds to CD80 and / or CD86 and binds to its cognate ligands (Figure 2). When CD28 is prevented from binding to T cells, T cells efficiently initiate activation signaling cascades. can eliminate or attenuate T cell activation and effector function CTLA-4 also binds to the costimulatory ligands CD80 and CD86, which act as antagonists to APCs. It exerts its inhibitory effect by trapping and trans-endocytosis from the target cell, which These costimulatory ligands are unavailable for binding to the CD28 costimulatory receptor by Engagement of the T cell receptor (TCR) triggers the release of the costimulatory ligand CTLA-4. Enhanced acquisition (Hou et al. (2015) J. Immunol., 194(5) :2148-59;Soskic et al.(2014)Adv.Immunol. ,124:95-136;Qureshi et al.(2011),Science ,332(6029):600-603).
[0080] CTLA-4 regulates T cell activity through regulation of CD80 and / or CD86 interactions It has been utilized as a therapeutic drug to treat autoimmune diseases by weakening the immune system. Specifically, abatacept and belatacept are used in the treatment of rheumatoid arthritis and Abatacept is an FDA-approved treatment for use in the transplant setting. Belatacept is a wild-type CTLA-4 IgSF domain fused to the CD4 domain. SEQ ID N to confer increased affinity for CD80 and CD86 ligands O: Corresponding to positions 31 and 106 of the wild-type reference CTLA-4 ECD sequence described in 2 , substitution of tyrosine for alanine at position 31, and guanylate for leucine at position 106 CTLA-4 IgSF domain containing glutamic acid substitutions (A31Y / L106E) It is a modified variant of (Kremer et al., N Engl J Med. 2003;349(20):1907-1915, Larsen et al.,Am J Transplant.2005;5(3):443-453).
[0081] In some embodiments, ICOSL (also known as B7-H2) inhibits the binding of CTLA-4. It has been reported that the partner (Yao et al. (2011) Immunit y,34(5):729-740). ICOSL is a ligand for the costimulatory receptor ICOS. CD28 and CTLA-4 can also bind to ICOSL, but CD80, CD86, and ICOSL may have lower affinity than ICOS. It is usually expressed on the surface of APCs (e.g., dendritic cells). ICOSL, CD80, and and / or CD86 are also expressed on T cells, including CD4+ and CD8+ T cells. CD80, CD86, and / or ICOSL co-stimulatory receptors, CD28, and Binding to IL-1 and / or ICOS mediates, among other activities, T cell activation, T cell proliferation, Enhances immune responses that may involve cytokine production. For example, ICOS binds to ICOS. L is involved in T helper cell and B cell differentiation.
[0082] The full-length CTLA-4 polypeptide contains the signaling sequence, the extracellular domain (ECD), and the membrane It contains a transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain acts as an intracellular signaling domain. An exemplary human CTLA-4 sequence is set forth in SEQ ID NO:1. After removal of the signal sequence, the ECD is located at amino acids 36 to 38 of SEQ ID NO:1. 161. Exemplary Human CTLA-4 ECD Amino Acid Sequence is set forth in SEQ ID NO: 2 or 569. The ECD of CTLA-4 is Contains a single IgSF domain (i.e., a single IgV domain). The IgV domain is amino acids 39 to 140 of SEQ ID NO:1 (SEQ ID NO: 0:2) or does not contain amino acids 4 to 105. The IgV domain is amino acids 39 to 152 of SEQ ID NO:1 (SEQ ID NO: O:2) or contains amino acids 4 to 117 within the IgV domain. , a CDR1-like loop (corresponding to amino acids 27 to 34 of SEQ ID NO:2), CD an R2-like loop (corresponding to amino acids 51-55 of SEQ ID NO:2), and a CD There is an R3-like loop (corresponding to amino acids 97-104 of SEQ ID NO:2) In some embodiments, the interaction of CTLA-4 with its CD80 and CD86 cognate ligands is It has been reported that it plays a role in the action of (1994), 180(6):2049-2058).
[0083] Increased binding affinity, such as increased binding affinity for CD80, CD86, and / or ICOSL Provided herein are mutant CTLA-4 polypeptides that exhibit increased binding activity. In some embodiments, the compounds exhibit increased affinity for ICOSL and, in some cases, CD They also exhibit higher binding affinity, including increased binding affinity to CD80 and / or CD86. In some embodiments, the present invention is directed to mutant CTLA-4 molecules. Binding to D80, CD86, and ICOSL is competitive, so the single mutant CTLA-4 polypeptides do not bind to more than one ligand at a time. The mutant CTLA-4 polypeptides to be used may be those that bind to CD80, CD86, and / or ICO. These bind to SL and bind to the activating receptors CD28 and ICOS on T cells. The normal function of the ligand of the agonist can be modulated, eg, antagonized.
[0084] In some embodiments, suppression or weakening of immune responses, such as T cell responses, is undesirable. May be desirable for reducing or preventing autoimmune conditions and / or transplant rejection Among the embodiments provided are those for treating an autoimmune or inflammatory disease or condition. Mutant CTLA-4 polypeptides, such as soluble or cell-expressed forms thereof, for use in therapy Thus, in some embodiments, methods are provided for using mutant CTLA-4 polypeptides. The peptides may induce decreased T cell activation, expansion, and / or effector function, autoimmunity, and Experiencing unwanted autoimmunity, such as involving T cells, which has the effect of weakening the immune system. Methods for making and using these mutant CTLA-4 polypeptides can be used to deliver the mutant CTLA-4 polypeptides to patients. It also provides the law.
[0085] In some cases, various forms of the CTLA-4 polypeptide are produced to stimulate an immune response. For example, binding to a cognate binding partner on an effector cell can be increased. Competing activation cytoplasmic signaling domains or decoy receptors for CTLA-4 Certain CTLA-4 switching receptors containing the body stimulate immune responses, including increased immune response. In some provided embodiments, the present invention may be used to treat cancer and oncological indications. In particular, specific proteins that can induce activating signals and / or compete with inhibitory signals. There are methods for using mutant CTLA-4 polypeptides, such as cellular expression forms of the polypeptides.
[0086] In some embodiments, mutant CTLA-4 polypeptides and the like are provided. Immunomodulating polypeptides and conjugates containing mutant CTLA-4 polypeptides The modulation of immune signaling achieved by these or engineered cells is comparable to other treatments. and provide benefits for the treatment of inflammatory and autoimmune disorders and other diseases and conditions. In some cases, such ligand / receptor interactions and subsequent signaling Therapies that interfere with and alter the immunomodulatory effects of immune signaling may contribute to the restriction of the immune synapse. Some aspects are limited by the spatial orientation requirements imposed by the Therefore, existing therapeutic drugs, including antibody drugs, target multiple proteins involved in regulating these interactions. In addition, these ligands may not be able to simultaneously interact with the protein. Pharmacokinetic differences between drugs that independently target one of the receptor interactions may be a therapeutic advantage. in adequately maintaining the desired blood concentration of such drug combinations throughout the course of This may cause difficulties.
[0087] All patents, patent applications, scientific papers, and databases mentioned herein All publications are based on the fact that each individual publication, including patents, patent applications, scientific papers, or databases, is a reference. for all purposes to the same extent as if specifically and individually indicated to be incorporated by reference. The definitions set forth herein are incorporated by reference in their entirety. Contrary to the definition set forth in any patent, application, published application, or other publication, all of which are incorporated herein by reference. In the event of any conflict or deviation, the definitions set forth herein shall prevail. take precedence over definitions incorporated into the document.
[0088] The section headings used herein are for organizational purposes and do not limit the scope of the subject matter described. It should not be construed as limiting the scope of the present disclosure.
[0089] I. Definition Unless otherwise defined, all technical terms, notations, and other terms used herein are Technical and scientific terms or terminology are understood to be understood by those skilled in the art to which the claimed subject matter pertains. In some cases, the term "common" is intended to have the same meaning as commonly understood by the public. Terms having commonly understood meanings are used herein for clarity and / or ease of reference. and the inclusion of such definitions herein does not necessarily imply that they are generally understood in the art. should not be construed as representing a substantial difference beyond that shown.
[0090] Terms used throughout this specification are intended to be illustrative unless otherwise limited in specific examples. As used in this specification and the appended claims, In this case, the singular forms "a", "an" and "the" are Plural references are included unless the context clearly indicates otherwise. All technical and scientific terms, acronyms and abbreviations used are those of the art to which this invention pertains. have the same meaning as commonly understood by those skilled in the art. Abbreviations and symbols for biochemical names are based on the IUPAC-IUB nomenclature system. Unless otherwise indicated, all numerical ranges include the values defining the range as well as any values therebetween. Includes all integer values.
[0091] The term "affinity modification" as used in the context of immunoglobulin superfamily domains The term refers to the parent wild-type or unmodified (i.e., modified non-tropism) IgSF regulatory domain. at least one binding partner (alternatively, a "counter structure") compared to the The amino acid sequence (e.g., α-glucan) may be altered to have increased or decreased binding affinity or activity. mammalian immunoglobulins having the corresponding wild-type parent or unmodified IgSF domains In this context, affinity-modified C In some embodiments, the affinity engineered IgS domain comprises a TLA-4 IgSF domain. The F domain may contain amino acid substitutions, such as amino acid substitutions, in the wild-type or unmodified IgSF domain. 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 The increase or decrease in binding affinity or avidity can be determined by flow cytometry. This can be determined using well-known binding assays such as ELISA (Larsen et al., Am J Transplant,5(3):443-453(2005 ), Linsley et al., Immunity, 1(9):793-801(1 See also 994). The binding affinity or binding of a protein to its binding partner The increase in activity was 10% over the wild-type IgSF domain control value, and several In some embodiments, the IgSF domain is at least 20%, 30%, or 40% greater than the wild-type IgSF domain control value. %, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000 %, or 10,000% greater. The decrease in binding affinity or activity to at least one of the target proteins is 90% or less of the control, but 10% or less of the control value of the wild-type IgSF domain, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 2% of the control value for the gSF domain It is below 0%, but above 10% of that.
[0092] Affinity-modified proteins are modified by substitution, addition, or deletion of amino acid residues to modify the primary amino acid sequence. The term "affinity-modified IgSF domain" refers to an affinity-modified IgSF domain. Any conditions regarding any particular starting composition or method by which the gSF domain was made Therefore, the affinity-engineered IgSF domains of the present invention should not be construed as imposing is then transformed into an affinity-engineered IgSF domain by any particular process of affinity engineering. The affinity-engineered IgSF domain polypeptides are not limited to wild-type IgSF domains. The domains are constructed starting from wild-type mammalian IgSF domain sequence information, and then the binding The affinity-modifying IgSF domain of the substance is modeled in silico for binding to the partner. Ultimately, the recombinant vector can be recombinantly expressed or chemically synthesized to produce a recombinant protein composition. In an alternative example, the affinity-engineered IgSF domain may be a site-specific IgSF domain. Thus, affinity-modified IgSF domains can be generated by selective mutagenesis. represents a product, not necessarily a product produced by any given process. It can be produced using a variety of techniques, including recombinant methods, chemical synthesis, or a combination of both. This can be done.
[0093] "Allogenic" or "allogeneic" The term refers to a gene that is removed from one organism and then injected or transferred into a genetically heterologous organism of the same species. are used interchangeably herein to refer to cells or tissues that are adoptively transferred. In some embodiments of the present invention, the species is murine or human.
[0094] As used herein, the term "autologous" refers to the same tissue that is subsequently injected or adoptively transferred. Autologous cells or tissues refer to cells or tissues that are removed from the same organism. The native cells or tissues may be modified so that they are no longer genetically identical to the native cells or tissues from which they were removed, e.g., They can be altered by recombinant DNA methodologies. For example, naturally occurring autologous T cells can optionally be These include transmembrane immunomodulatory proteins, which involve manipulation of T cells or TILs (tumor-infiltrating lymphocytes). The cells are then engineered to be autologous and express a target antigen and / or chimeric antigen receptor (CAR). The engineered cells can then be genetically engineered to resemble natural The isolated T cells are infused into the patient. In some embodiments, the organism is a human or a mouse. It's a smelt.
[0095] As used herein, the terms "binding affinity" and "binding activity" refer to a particular The specific binding affinity of each protein for its counter structure under bound conditions and specific avidity. In biochemical kinetics, avidity refers to the binding of CTLA-4 and its Counter structures such as ICOSL, CD80, and / or CD86 interact with individual non- Avidity refers to the cumulative strength of multiple affinities of covalent interactions. Affinity is distinct from affinity, which describes the strength of a single interaction. Binding affinity of mutant CTLA-4 containing the IgSF domain to its counter structure The increased or weakened IgSF domains include native or wild-type IgV domains. The binding affinity of the modified CTLA-4 is determined relative to the binding affinity of the unmodified CTLA-4, such as the unmodified CTLA-4 that binds to the modified CTLA-4. Methods for determining binding affinity or avidity are known in the art. , Larsen et al., Am J Transplant,5(3):443- 453 (2005). In some embodiments, the mutant CTLAs of the present invention -4 (i.e., a CTLA-4 protein containing an affinity-engineered IgSF domain) At least 10%, 20%, and 30% higher than wild-type CTLA-4 controls in binding assays , 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater mean fluorescence intensity Binding affinity, as measured by flow cytometry, yielding a molecular weight (MFI) value , ICOSL, CD80, and / or CD86.
[0096] The term "biological half-life" refers to the time period during which a substance loses half of its pharmacological or physiological activity or concentration. In order to eliminate the effect of the mutant CTLA-4 of the present invention, the mutant CTLA-4 is used as a regulatory polypeptide. The biological half-life refers to the amount of time it takes for a substance to dissipate in a particular organ or tissue of the body. Affected by elimination, excretion, degradation (e.g., enzymatic degradation / digestion), or absorption and concentration of In some embodiments, the biological half-life is the time at which the compound is released from its steady-state level. The time it takes for a substance's plasma concentration to reach half its normal concentration (its "plasma half-life") The biological half-life of the polypeptide of the present invention can be evaluated by determining the biological half-life of the polypeptide of the present invention. Conjugates that can be used to derivatize and increase are known in the art. It contains polyethylene glycol (PEG), hydroxyethyl starch (HES), WORK XTEN® (Amunix Operating, California) ,USA) (see WO2013 / 130683) Recombinant peptides, human serum albumin (HSA), bovine serum albumin (BSA), Lipids (acylated), poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation), but are not limited to these.
[0097] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that is a chimeric antigen receptor (CAR). expressed on mammalian cells containing at least an ectodomain, a transmembrane domain, and an ectodomain CAR protein refers to an artificial (i.e., man-made) transmembrane protein. Optionally, the CAR protein is It contains a "spacer" that covalently links the ectodomain to the transmembrane domain. It is a polypeptide that links the ectodomain to the transmembrane domain via a peptide bond. CARs are typically expressed on mammalian lymphocytes. In this setting, CARs are expressed on mammalian cells such as T cells or tumor-infiltrating lymphocytes (TILs). CARs expressed on T cells are called "CAR-T cells" or "CAR-T." In some embodiments, the CAR-T is a T helper cell, Cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or gastrointestinal When used clinically, for example in adoptive cell transfer, CAR-Ts with antigen-binding specificity for a patient's tumor or other tissue are typically The CAR is then engineered to be expressed on T cells obtained from the patient. The generated T cells are then infused back into the patient. Therefore, the CAR-T is considered to be autologous. Although allogeneic CAR-Ts are often used, allogeneic CAR-Ts are included within the scope of the present invention. (or ECD) refers to the ECD of an immunomodulatory peptide on a cell surface or tumor-specific antigen. , an antibody or antigen-binding fragment thereof (e.g., It contains an antigen-binding region such as a single-chain variable fragment (scFv). Upon specific binding, A biochemical chain of events (i.e., signal transduction) also regulates the immune activity of CAR-T. Therefore, for example, specific binding of CAR-T to its target antigen by the antigen-binding region Upon binding, a change (increase or decrease) in cytotoxicity, proliferation, or cytokine production This can result in changes in immune activity of T cell activity as reflected by CAR-T activity. Signal transduction during activation may, in some embodiments, be a signal transduction pathway in natural mammalian T cells. Reached by the CD3-zeta chain ("CD3-z") ectodomain, which is involved in signal transduction CAR-Ts are designed to target CD28, 4, and 6 to further modulate the immunoregulatory response of T cells. It may further comprise multiple signaling domains, such as 1BB, 1BB, or OX40. -z is an immunoreceptor tyrosine-based activation motif involved in T cell receptor signaling. It contains a conserved motif known as an ITAM.
[0098] In an in vitro assay, the presence of two or more mutant CTLA-4s of the present invention induces When used in reference to cytokines released, the term "collectively" or "collective" The term is used regardless of the cytokine production induced by each mutant CTLA-4 molecule. In some embodiments, the cytokines being assayed are The inhibitor is IFN-gamma, e.g., in an in vitro primary T cell assay.
[0099] With respect to a polypeptide, such as with respect to the IgSF domain of a mutant CTLA-4, "binding" refers to The term "partner" (used interchangeably with "counterstructure") refers to the specific binding conditions. At least one molecule (typically a polypeptide) to which the referenced polypeptide specifically binds, In some embodiments, affinity engineered IgSF domains are used. Mutant CTLA-4 containing CTLA-4 fragments exhibit the same binding patterns as the corresponding native or wild-type CTLA-4 fragments. Specific binding to the target with increased or weakened affinity. A "ner" is a binding partner that is expressed on the surface of a mammalian cell. Examples of binding partners for mutant CTLA-4 molecules include CD80, CD86, and IC and specifically human CD80, human CD86, and human ICOSL. It can be enjoyed.
[0100] As used herein, "conjugate," "conjugation," or the like These grammatical variations may be joined or concatenated by any method known in the art. Refers to the joining or linking of two or more compounds together resulting in the formation of another compound. It also includes compounds created by joining or linking two or more compounds together. For example, it may be linked directly or indirectly to one or more chemical moieties or polypeptides. A modified mutant CTLA-4 polypeptide is an exemplary conjugate. Conjugates include fusion proteins, conjugates produced by chemical conjugation, Conjugates produced by any other method are included.
[0101] As used herein, the term "competitive binding" refers to binding between two or more proteins in a single molecule. It can specifically bind to two binding partners, but the specificity of one binding partner It is meant that binding inhibits, i.e. prevents or eliminates, the simultaneous binding of a second binding partner. Thus, in some cases, a protein may bind to two binding partners simultaneously. Generally, competitive binding agents are those that have the same or similar affinity for specific binding. Although overlapping binding is included, this is not a requirement. The specific binding occurs due to the specific binding of a second binding partner to that binding part of the protein. This causes a measurable inhibition (partial or complete) of specific binding to one of the receptors. To quantify competitive binding, such as in ELISA (enzyme-linked immunosorbent assay) assays , various methods are known.
[0102] As used herein, the term "conservative amino acid substitution" refers to a substitution in which the amino acid residue is: Another amino acid with a side chain R group that has similar chemical properties (e.g., charge or hydrophobicity) It refers to an amino acid substitution in which an amino acid is replaced by a residue having a side chain with similar chemical properties. Examples of amino acid groups that may be used include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and and isoleucine, 2) aliphatic hydroxyl side chains: serine and threonine, 3) amide-containing 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 Conservative amino acid substitution groups include valine-leucine-isoleucine and methionine. phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine acid-aspartic acid, and asparagine-glutamine.
[0103] Nucleotide or amino acid positions in the disclosed sequences shown in the sequence listing, etc. References to protein positions, such as those that "correspond" to nucleotide or amino acid positions The term "corresponding to" as used herein refers to a sequence based on structural sequence alignment or GAP alignment. Alignment of sequences with the disclosed sequences can be performed using standard alignment algorithms, such as the Refers to the nucleotide or amino acid position identified during alignment. For example, The residues identified by the structural alignment methods described herein are those of SEQ ID NO: 2 or 569 (ECD), or SEQ ID NO:3 (IgV domain ) determined by alignment of the reference sequence with the sequence of wild-type CTLA-4 described in By aligning the sequences, the skilled artisan can use, as a guide, e.g. Conserved and identical amino acid residues can be used to identify corresponding residues.
[0104] As used herein, "reduce" or "weaken" or "suppress" The term "decreased" means decreased by a statistically significant amount. A decrease can be measured by comparing a non-zero control value, such as a non-zero control value. At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 4% of the control value 0%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9 It can be 0%, 95%, or 100%.
[0105] As used herein in the context of reducing the immune activity of mammalian lymphocytes, " The terms "decreased" or "reduced" refer to a condition that is compared to an untreated control or an unaltered or untreated control. Treatment with a mutant control compared with a control used under the same conditions This means that the activity of one or more cells in the body is reduced by a statistically significant amount. Inhibition of cell cycle, reduction of cell survival, reduction of cell proliferation, reduction of cytokine production, etc. or reduced T cell cytotoxicity. In this context, reference to a reduction in immune activity is compared to the absence of treatment, such as by a statistically significant amount. This means that interferon gamma (IFN-gamma) production is reduced. In some embodiments, immune activity is assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing MLR assays are known in the art. et al., Cancer Immunol Res.(2014)2(9):846 -56. Other methods for assessing lymphocyte activity, including the assays described herein, In some embodiments, the enhancement is greater than the untreated control value or a non-zero value, as known in the art. Compared to a control value, such as a control value, by at least 5%, 10%, 15%, 20%, 25%, 3 0%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 10 It can be reduced by 0%.
[0106] The term "derivative" or "derivatized" refers to a compound that retains the therapeutic utility of a protein. or enhancing biological half-life, bioavailability, immunogenicity, solubility, toxicity, efficacy The modification of proteins, directly or indirectly, to alter their properties, such as their activity or efficacy. The term "modification of a protein by covalently linking it to a polypeptide" refers to the modification of a protein by covalently linking it to a polypeptide of the present invention. Derivatives of tides are within the scope of the present invention, for example, glycosylated, pegylated, lipidated, or can be achieved by Fc fusion.
[0107] As used herein, a "domain" (typically a sequence of 10 to 200 amino acid residues) A sequence of three or more amino acids (typically 5-7 or more amino acids), such as a group, is structurally distinct from the rest of the molecule. and / or functionally distinct and distinguishable proteins, etc., or encoding nucleic acids For example, a domain is a molecule that consists of one or more structural motifs. It is possible to form an independent folding structure within the protein and / or to have functional properties such as binding activity. The protein contains a domain portion of the polypeptide chain that is recognized by the activity. or may have more than one distinct domain. For example, a domain may have homology to a motif Identified and defined by primary sequence or structural homology with related family members, such as sex In another example, a domain may be a molecule that interacts with a biomolecule, such as a binding partner. Domains can be distinguished by their functions, such as their ability to interact with one another. The domain, either isolated or fused to another molecule, can perform an activity, e.g., binding. Domains can independently exhibit biological functions or activities, such as: or may be a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. To exemplify, definitions are provided, but to recognize specific domains by name, It is understood that it is well within the skill of the art to identify domains, if necessary. , suitable software can be used.
[0108] As used herein, the term "ectodomain" refers to the domain that is located on the outside of the vesicle membrane. The ectodomain refers to a region of a membrane protein, such as a transmembrane protein. via a ligand or a binding domain that specifically binds to a cell surface receptor They often contain a binding domain that specifically binds to a receptor. The domain is alternatively referred to as the extracellular domain.
[0109] The terms "effective amount" or "therapeutically effective amount" include amounts administered ex vivo (by contact with cells from a patient). in vivo (by administration to a patient), alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, e.g., to improve symptoms and and / or a statistically significant reduction in disease progression by ameliorating or eliminating the cause of the disease. and / or An effective amount refers to a concentration or amount of a substance that reduces or eliminates at least one symptom or concentration associated with a disease or disorder. Attenuate, decrease, or alleviate a biological response or effect or slow the progression of a disease or disorder In the case of cancer treatment, an effective amount is an amount that prevents or improves the patient's physical function. , is the effective dose or number of cells administered to a patient by adoptive cell therapy. In embodiments, the patient is a mammal, such as a non-human primate or a human patient.
[0110] As used herein, an "ectodomain" (also referred to as an "intracellular domain" or "cytoplasmic domain") The term "intercellular matrix" (also called "matrix") refers to the membrane that extends into the interior space defined by the cell surface membrane. It refers to a region found in some proteins, such as transmembrane proteins, that binds to the nucleus. In cells, the ectodomain is the membrane region of a membrane protein. may interact with intracellular components and play a role in signal transduction, thus In some cases, it may be an intracellular signaling domain. The ectodomain may alternatively mediate signal transduction or It is also possible that the cytoplasmic domain plays a role in Therefore, the intracellular signaling domain and the cytoplasmic signaling domain The terms are used interchangeably.
[0111] As used herein in the context of increasing the immune activity of mammalian lymphocytes, "enhancement" refers to The terms "is increased" or "is increased" refer to an increase in a gene expression level relative to an untreated control or to an unaltered or unmutated A control treatment is used under the same conditions to compare one or more of the lymphocytes. Increased activity means increasing the activity of a compound by a statistically significant amount, etc. , increased cell survival, cell proliferation, cytokine production, or T cell cytotoxicity. In some embodiments, a reference to increased immune activity is a statistically significant amount. This means that it increases interferon gamma (IFN-gamma) production. In some embodiments, immune activity can be assessed in an MLR assay. Other methods for assessing lymphocyte activity, including the assays described herein, are known in the art. In some embodiments, the enhancement is less than a control value, such as a non-zero control value. At most 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300 %, 400%, or 500% greater increase.
[0112] As used herein, the term "engineered cells" refers to cells that have been engineered using recombinant DNA methods or refers to mammalian cells that have been genetically modified by human interference, such as by viral transduction. In some embodiments, the cells are lymphocytes (e.g., T cells, B cells, NK cells). or antigen-presenting cells (e.g., dendritic cells). The cells may be primary cells or may be cell lines. The engineered cells contain a mutant CTLA-4 provided herein. In this study, mutant CTLA-4 is a transmembrane immunoregulatory protein expressed on engineered cells. (hereinafter referred to as "TIP"). In some embodiments, the TIP is a transmembrane domain. a domain (e.g., a CTLA-4 transmembrane domain), and optionally, an intracellular signaling An extracellular domain or portion thereof containing an IgV domain linked to a transduction domain In some cases, TIPs contain heterologous cytoplasmic signaling domains or exons. In some embodiments, the engineered receptor is formatted as a chimeric receptor containing the engineered domain. The resulting cells are capable of expressing and secreting the immunomodulatory proteins described herein. Some of the engineered cells provided contain engineered T cell receptors (TCRs) or chimeric antibodies. Some cells also contain a primary receptor (CAR).
[0113] As used herein, the term "engineered T cells" refers to T cells engineered using recombinant DNA methods. or T helper cells that have been genetically modified by human intervention, such as viral signaling methods. , cytotoxic T cells (alternatively, cytotoxic T lymphocytes or CTLs), natural killer refers to T cells such as T cells, regulatory T cells, memory T cells, or gamma delta T cells. The engineered T cells are expressed on T cells and are capable of expressing mutant CTLs on T cells. A-4 specifically binds to engineered T cells or regulates the immune activity of mammalian cells Mutant CTLA-4 transmembrane immunomodulatory proteins (TIPs) of the present invention engineered to The engineered T cells may comprise a T cell-expressed and / or secreted T When secreted by the cells, the mutant CTLA-4 specifically binds to engineered T cells. The cells themselves, or variants of the invention engineered to modulate the immune activity of mammalian cells, It may comprise a variant CTLA-4 secreted immunomodulatory protein (SIP).
[0114] The term "engineered T cell receptor" or "engineered TCR" refers to a T cell receptor that is selected and cloned. T cells, which are often cloned and / or subsequently used for adoptive immunotherapy. Major histocompatibility complex (MHC) / peptide target antigens that are introduced into a population of cells with desired affinity This refers to a T cell receptor (TCR) that has been engineered to specifically bind to a specific antigen. In contrast, CARs are engineered to bind to target antigens in an MHC-independent manner.
[0115] As used herein, the term "expressed on" refers to a cell, such as a mammalian cell. It is used to refer to proteins that are expressed on the surface of a cell. Thus, proteins are membrane proteins. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is a drug or a detectable target. Proteins expressed on the surface of cells are conjugated to small molecule moieties such as ribonucleotides. It may include cell surface proteins such as cell surface receptors expressed on mammalian cells.
[0116] The term "half-life extending moiety" refers to a portion of a polypeptide fusion or chemical conjugate. minutes compared to the half-life of the protein not conjugated to the moiety. A steroid refers to a moiety that extends the half-life of a protein circulating in blood serum. In this state, the half-lives are approximately 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, or greater than 6.0 times. In some embodiments, the half-life is increased by a factor of 1 or greater than 6.0. Compared with proteins without the long portion, after administration to the body, Extended for more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than one week. Phase refers to the amount of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life is determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, a polyethylene glycol monoclonal antibody, and the like. Polyethylene glycol (PEG), Hydroxyethyl starch (HES), Mark XTEN (registered and the extended recombinant peptides sold under the trademarks (see WO2013 / 130683). (see reference), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamine Contains carboxylic acid (glutamyl).
[0117] As used herein, the term "immune synapse" or "immune synapse" refers to a MHC I (major histocompatibility complex class I) or M, such as antigen-presenting cells or tumor cells Mammalian cells expressing HC II and effector T cells or natural killer (N K) refers to the interface between mammalian lymphocytes such as lymphocytes.
[0118] The Fc (fragment crystallizable) region of an immunoglobulin molecule (also called an Fc polypeptide) The regions or domains correspond roughly to the constant regions of immunoglobulin heavy chains and are responsible for the expression of antibodies. The Fc domain is involved in various functions, including the vector function of immunoglobulin molecules. and the hinge domain plus some or all of the CH2 and CH3 domains. The Fc domain consists of two polypeptide chains joined by one or more disulfide bonds In some embodiments, the Fc can form a dimer of reduced (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%). In some embodiments, the variant Fc exhibits an activity that is reduced to 0% or more. Reference to amino acid substitutions in the Fc region refers to E unless otherwise specified in relation to a particular SEQ ID NO. The EU numbering system is the most recently updated and known IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system (registered trademark), http: / / www.imgt.org / IMGTSc ientificChart / Numbering / Hu_IGHGnber.html (Created: May 17, 2001, Last Updated: June 8, 2016), and Kabat ,EAet al.Sequences of Proteins of Immu nological interest.5th ed.US Department of Health and Human Services,NIH publica According to the EU guidelines as reported in 1991 No. 91-3242 There are.
[0119] Immunoglobulin Fc fusions ("Fc fusions"), such as immunomodulatory Fc fusion proteins, one or more polypeptides (or one or more polypeptides) operably linked to the Fc region of a globulin Fc fusions are molecules containing small molecules such as antibodies (effector functions and and the Fc region of a mutant CTLA-4. The Fc region indirectly binds one or more mutant CTLA-4 or small molecules (fusion partners). Alternatively, the Fc fusion may be directly linked. A variety of linkers are known, and may optionally be used to link the Fc fusion. To create a fusion partner, Fc can be used to link the Fc to a fusion partner of the same species. The Fc fusions can be dimerized to form Fc fusion homodimers or non-identical Fc fusions. In some embodiments, the species can be used to form Fc fusion heterodimers. , the Fc is a mammalian Fc, such as a murine or human Fc.
[0120] The term "host cell" refers to a cell in which a protein encoded by a recombinant expression vector is expressed. Host cells refer to cells that can be used to express the vector. Host cells are prokaryotes, such as Escherichia coli (E. coli), or a eukaryotic cell, such as a unicellular eukaryotic cell (e.g., yeast or 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), Examples of host cells include those that grow in serum-free medium, or in a dihydrofolate-reducing medium. Vegetable CHO and CHO grown in CHO strain DX-B11 lacking the DHFR enzyme. Chinese hamster ovary (CHO) cells or their derivatives, such as hamster ovary (HVO) cells and related cell lines Another example includes human embryonic kidney 293 (HEK-293) cells or derivatives thereof. In some embodiments, the host cell is a mammalian cell (e.g., a human cell, a mammalian ... The cells may be human cells, hamster cells, rat cells, mouse cells, or insect cells.
[0121] As used herein, the term "immunoglobulin" (abbreviated "Ig") refers to a Five human classes of antibodies: IgA (including subclasses IgA1 and IgA2), IgD , IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4) The term "immunoglobulin" refers to a mammalian immunoglobulin protein, including any of IgM, IgA, IgB, IgC, IgD, IgE, IgF, IgG, IgH, IgH, IgI, IgIgM, IgI ... The term also refers to antigen-binding fragments (Fab), variable fragments containing VH and VL (Fv), single-chain variable fragments (scFv) containing VH and VL linked together in one chain; and Fab′, F(ab)2, F(ab′)2, dsFv bispecific antibodies, Fc, and and other antibody V region fragments, such as Fd polypeptide fragments, which may be wholly or partially synthetic. whether produced by recombinant means (e.g., recombinantly or chemically synthesized) or naturally occurring. It includes immunoglobulins that are less than full-length. Bispecific antibodies, homobispecifics, and heterospecifics Hetero-bispecifics are included within the meaning of this term.
[0122] As used herein, the term "immunoglobulin superfamily" or "IgSF" refers to The term "cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes" refers to Immunoglobulins (i.e., antibodies) are a group of proteins that share a common structure. Based on structural features, they are all classified as members of this superfamily. Each protein contains a domain known as an immunoglobulin domain or fold. Members of the gSF are involved in cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, and lymphocytes. Molecules involved in antigen presentation to cytoplasmic cells, cell adhesion molecules, specific cytokine receptors, and They contain intramuscular proteins, which are commonly associated with a role in the immune system. Proteins in the sputum are often members of the IgSF. The IgSF also contains functional They can be classified into "subfamilies" based on shared characteristics. A family typically consists of 4-30 IgSF members.
[0123] As used herein, "IgSF domain" or "immunoglobulin domain" Alternatively, the term "Ig domain" refers to a structural domain of an IgSF protein. The g domains are named after immunoglobulin molecules. They consist of approximately 70-11 They contain 100 amino acids and are classified according to their size and function. The in has a sandwich-like structure formed by two sheets of antiparallel beta strands , possessing the characteristic Ig fold. interactions, and the highly conserved disulfides formed between cysteine residues in the B and F chains Binding stabilizes the Ig folded structure. One end of the Ig domains binds to the antigens of their ligands. It has a division called complementarity that determines the regions important for specificity. The antibodies can be IgV, IgC (which can be either IgC1 or IgC2), or IgI. Most Ig domains are variable (IgV) or The IgV domain, which has nine beta chains, is generally either constant (IgC) or constant (IgC). , longer than the IgC domain with seven beta chains. The Ig domain is similar to the IgV domain in amino acid sequence, but is different from the IgC domain. These are called IgC2 domains, but are not the same size as standard IgC domains. The main domain is called the IgC1 domain. The T cell receptor (TCR) chain is located in the extracellular portion. Two Ig domains in the nucleus: one IgV domain at the N-terminus and one adjacent to the cell membrane. CTLA-4 contains one Ig domain: the IgV domain. Contains.
[0124] As used herein, the term "IgSF species" refers to the same or nearly the same primary It refers to an ensemble of IgSF member proteins having the amino acid sequences The mammalian immunoglobulin superfamily (IgSF) members defines the unique identity of all IgSF species belonging to each member. IgSF family members are unique compared to other IgSF family members Therefore, each species of a particular IgSF family member may be associated with another IgSF family member. However, the molecules that are composed of the same IgSF species are unique compared to the species of the members. The variations between the two proteins are due to glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, and acetylation. This may arise due to differences in post-translational modifications such as methylation, methylation, and lipidation. Minor sequence differences within a single IgSF species due to polymorphisms can occur, e.g., due to protein cleavage. Other forms of variation within a single IgSF species can be constructed, such as wild-type truncated versions of the IgSF species. "Cell surface IgSF species" refers to IgSF expressed on the surface of a cell, typically a mammalian cell. It is a seed.
[0125] As used herein in the context of mammalian lymphocytes, such as T cells, the term "immune activity" refers to The terms used are activation, cell survival, apoptosis, cell proliferation, cell cycle inhibition, and cytokine production. cytotoxic activity (e.g., interferon gamma), cytokine release, or T-cell cytotoxic activity In some cases, immune activity refers to one or more of the following: chemokines or interleukins. To determine the enhancement or suppression of immune activity, The assay involves measuring interferon gamma cytokine levels in culture supernatants. MLR (mixed lymphocyte reaction) assay (Wang et al., Cancer Immunol. unol Res.2014 Sep:2(9):846-56), SEB (Staphylococcus aureus) enterotoxin B), T cell stimulation assay (Wang et al., Cancer Immunol Res, (2014) 2 (9): 846-56), and anti-CD3 T cells Cell stimulation assay (Li and Kurlander, J Transl Med, (2 010)8:104). T cell activation is mediated by the secretion of IFN-gamma cytokines. IF in culture supernatants from these in vitro human T cell assays is associated with Detection of N-gamma levels was performed using a commercially available ELISA kit (Wu et al., Immunol. ol Lett (2008), 117(1):57-62) The induction of an immune response results in an increase in immune activity associated with resting lymphocytes. Mutant CTLA-4 polypeptides containing affinity-engineered IgSF domains such as Which immunomodulatory proteins may be reduced in some embodiments or alternatively In one embodiment, primary T cell responses to wild-type IgSF members or IgSF domain controls are The present invention can increase IFN-gamma (interferon gamma) expression in a mouse model assay. If so, the primary T cell assay used to determine increased IFN-gamma expression The format may differ from that used in the assay for the reduction of IFN-gamma expression. You will recognize that.
[0126] Immunomodulation of the Invention to Alter IFN-gamma Expression in Primary T Cell Assays In assays for the ability of proteins or affinity-engineered IgSF domains to A lymphocyte response (MLR) assay may be used. A soluble form of the affinity-engineered IgSF domain of Alternatively, co-fixation can be used to determine its ability to increase or decrease the presence of In a co-immobilization assay, anti-CD3 antibodies are used to detect several In the embodiments provided, the T cell receptor signal is compared to a wild-type IgSF domain control. To determine their ability to increase or decrease N-gamma expression, mutant CTLA- 4. Mutant CT Immunotherapy of engineered cells, including assessing the activity of the LA-4 transmembrane immunomodulatory protein Methods for assaying activity are known in the art and include assays in suitable animal models. Cell expansion assays after antigen stimulation, such as allograft survival assays and anti-donor antibody response assays suppression or enhancement of proliferation or proliferation of primary and secondary allostimulatory T cells; Assays for immunosuppressive and autoimmune activities include, but are not limited to, standard 51-clonal assays. mAb-release assay (e.g., Milone et al., Mol Ther (2009) ), 17(8):1453-1464) or flow-based cytotoxicity cytotoxicity assay, or impedance-based cytotoxicity assay (Peper et al. l.(2014), J Immunol Methods,405:192-198) Assays for assessing cytotoxicity include:
[0127] An "immunomodulating polypeptide" or "immunomodulating protein" is a polypeptide that modulates immune activity. It is a peptide or protein molecule. By response is meant that immune activity is either increased or decreased. Immunomodulating proteins may be composed of, for example, small chains covalently linked to each other by interchain disulfide bonds. A single polypeptide or a multimer (dimer or higher order) of at least two polypeptide chains Thus, monomeric, dimeric, and higher multimeric polypeptides can be Multimeric polypeptides are within the scope of the defined term. The present invention may be a heteromultimer (of identical polypeptide chains) or a heteromultimer (of non-identical polypeptide chains). The immunomodulatory proteins include mutant CTLA-4.
[0128] As used herein, the term "increase" means to increase by a statistically significant amount. An increase means at least 5%, 10%, 20% above a control value, such as a non-zero control value. , 30%, 40%, 50%, 75%, 100% greater.
[0129] CTLA-4 "isoforms" are multiple naturally occurring proteins that differ in amino acid sequence. It is one of several CTLA-4 polypeptides that are expressed by a single gene. The product of a slice variant of an RNA transcript expressed by a gene, or resulting from gene duplication, etc. Expression of very similar but distinct genes that result in functionally similar proteins As used herein, the term "isoform" of CTLA-4 refers to a compound of the present invention. The term also refers to the products of different alleles of the CTLA-4 gene.
[0130] As used herein, the term "lymphocyte" refers to a white blood cell in a mammalian immune system. These are any of three subtypes of lymphocytes: natural killer cells ( NK cells) (function in cell-mediated cytotoxic innate immunity), T cells (cell-mediated cytotoxicity) B cells (for humoral, antibody-driven adaptive immunity) and B cells (for humoral, antibody-driven adaptive immunity). T cells include T helper cells, cytotoxic T cells, natural killer T cells, and memory T cells. -T cells, regulatory T cells, or gamma delta T cells. Innate lymphoid cells ( ILCs) are also included within the definition of lymphocytes.
[0131] The term "mammal" or "patient" specifically refers to a human, chimpanzee, lizard, or at least one reference to a monkey, a cynomolgus monkey, a dog, a cat, a mouse, or a rat Includes.
[0132] As used herein, the term "membrane protein" refers to a protein that is produced directly or indirectly under physiological conditions. It refers to proteins that are attached, or indirectly, to a lipid bilayer. are found on biological membranes, such as eukaryotic (e.g., mammalian) cell membranes, or on liposomes The membrane may be an artificial (i.e., man-made) membrane, such as one of the lipid bilayers. by means of covalent bonding or by non-covalent interactions such as hydrophobic or electrostatic interactions. The membrane protein may be an integral membrane protein or a peripheral membrane protein. Peripheral membrane proteins are membrane proteins that are non-covalently attached to the lipid bilayer. Peripheral membrane proteins are attached to the membrane or non-covalently to integral membrane proteins. The quality of the membrane is such that peripheral membrane proteins are able to form lipid bilayers under a range of conditions that are physiological in mammals. transient attachment to the lipid bilayer so that the In contrast to peripheral membrane proteins, integral membrane proteins form attachments in mammals. Integral membrane proteins are able to bind to the lipid bilayer under physiological conditions. They form a substantially permanent attachment to the lipid bilayer of the membrane, preventing it from detaching from the membrane. Proteins can attach to membranes by way of a single lipid bilayer (monotopic) or by way of both membranes (multitopic). ) can form attachments by the method of integral membrane proteins that interact with only one lipid bilayer. Proteins are "integral membrane proteins" that interact with both lipid bilayers. The protein is an "integral polytopic protein," alternatively referred to herein as a "transmembrane protein." It is also called "pak substance."
[0133] As used herein in the context of an immune response, such as a mammalian immune response, "modulating" refers to The terms "modulate" and "modulate" refer to the immunomodulatory polypeptides, including the mutant CTLA-4 of the present invention. or as a result of administration of a mutant CTLA-4 transmembrane immunomodulatory protein of the invention. Pre-existing diseases resulting from the administration of engineered cells expressing immunomodulatory proteins such as This refers to any change, such as an increase or decrease, in the immune system or potential immune response. This is an immunomodulatory protein containing mutant CTLA-4, or such an immunomodulatory polypeptide. compared to the immune response that occurs or is present in the absence of administration of cells expressing the peptide. Such modulation refers to changes in the immune activity of immune cells, such as an increase or decrease in the immune response. This includes any induction, activation, suppression, or alteration of the degree or extent of immune activity. cells, T cells, NK (natural killer) cells, NKT cells, professional antigen presenting cells Antigen-presenting cells (APCs), and non-professional antigen-presenting cells, as well as inflammatory cells (e.g., These include neutrophils, macrophages, monocytes, eosinophils, and basophils.
[0134] Modulation can involve the modulation of a pre-existing immune response, a developing immune response, a latent immune response, or an immune response that induces an immune response. the ability to emit, regulate, influence, or respond to Modulation can be direct or indirect. Modulation can be genetically Expression and function of molecules, proteins and / or other molecules in immune cells as part of an immune response Modulation of immune response or modulation of immune activity includes any change in immune function and / or function. For example: elimination, deletion, or sequestration of immune cells; autoreactive lymphocytes, antigen-presenting cells or the sequestration, induction, or activation of immune cells, which may modulate the functional capacity of other cells, such as inflammatory cells. or creating an unresponsive state (i.e., anergy) in immune cells; including, but not limited to, altering the pattern of proteins expressed by These include enhancing or suppressing the activity or function of immune cells. Examples include cytokines, chemokines, and kinases, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or Any combination of these regulatory events may contribute to the generation and / or management of specific classes of molecules. Modulation can include, for example, alterations in the secretion of wild-type CTLs in primary T cell assays. A-4 Assessed by changes in IFN-gamma (interferon gamma) expression relative to control (Zhao et al. (2016), Exp Cell Res, 340 ( 1):132-138). Regulation is achieved by the wild-type CTLA-4 transmembrane protein For example, to the engineered cells, a change in the cytotoxic activity of the engineered cells, or This is due to changes in the immune activity of the engineered cells, such as changes in cytokine secretion from the engineered cells. It can be evaluated as follows.
[0135] As used herein, the term "molecular species" refers to a group of molecules that are identical or nearly identical primary amino acids. Each mammalian immunoglobulin is an ensemble of proteins with a specific amino acid sequence. The immunoglobulin superfamily (IgSF) members represent a collection of identical or nearly identical molecular species. Thus, for example, human CTLA-4 is an IgSF member, and each These human CTLA-4 molecules are CTLA-4 molecular species. The changes in glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, and acetylation These differences may arise due to differences in post-translational modifications such as cleavage, ligation, and lipidation. The minute sequence differences within a single molecular species due to, for example, protein cleavage The wild-type and truncated forms of the nucleotides in the nucleotide sequence ... " is a molecular species expressed on the surface of mammalian cells. Two or more different species of proteins The plasm is composed of cells that are either exclusively or exclusively of one of the two mammalian cells that form the IS. They exist exclusively on one another and are said to be "cis" or in a "cis configuration" with one another. Proteins from different species are expressed in two mammalian cells, the first of which forms an IS. The second is present exclusively on the two mammalian cells that form the IS. These two different The protein of this species is present on both of the two mammalian cells that form the IS, and these It is in both cis and trans configurations on cells of
[0136] The term "multimerization domain" refers to a set of domains that can be used to form multimers, each of which can be the same or different. and complementary multimerization domains (e.g., a first multimerization domain and a and a second multimerization domain). It refers to a sequence of amino acids that promotes stable interactions between peptide molecules. Interactions between domains, e.g., between a first multimerization domain and a second multimerization domain Interactions between generate multimers of polypeptide molecules with additional polypeptide molecules In some cases, they form multimers. The covalent domains are identical and provide a stable protein-protein bond between the two polypeptide chains. Generally, a polypeptide interacts with itself directly or indirectly to form a complex interaction. or indirectly conjugated to a multimerization domain. Exemplary multimerization domains include immunoglobulins. globulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and and compatible protein-protein interaction domains. , e.g., IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, and modified forms thereof, e.g., the Fc domain from IgG1. or a portion thereof.
[0137] The terms "nucleic acid" and "polynucleotide" refer to either single-stranded or double-stranded forms. A polynucleotide of any nucleic acid residue (e.g., deoxyribonucleotide or ribonucleotide) Unless otherwise specified, these terms refer to naturally occurring nucleic acids. Nucleic acids containing known analogs of nucleotides have similar binding properties and are not naturally occurring. Unless otherwise specified, a particular nucleic acid sequence is Also implicitly included are conservatively modified variants thereof (e.g., degenerate codon substitutions) and complements thereof. The present invention encompasses the nucleotide sequence of a gene, as well as the sequence explicitly indicated (the "reference sequence"). Specifically, degenerate codon substitutions are those that substitute the third codon of one or more selected (or all) codons. Creating sequences where positions are substituted with mixed group and / or deoxyinosine residues The term nucleic acid or polynucleotide refers to a molecule encoded by a gene. This includes the cDNA or mRNA that is extracted.
[0138] As used herein, the term "non-competitive binding" refers to the simultaneous binding of at least two It refers to the ability of a protein to specifically bind to a binding partner. A protein can simultaneously bind to at least two different binding partners, In some cases, the protein is specifically bound to only one of the binding partners. As such, the binding interactions do not have to be of the same duration. In some embodiments, simultaneous binding occurs under specific binding conditions. such that binding of the partner does not substantially inhibit simultaneous binding to a second binding partner. In some embodiments, non-competitive binding is achieved by binding to a second binding partner on the protein. The binding of the toner to its binding site is linked to the binding site of the first binding partner on the protein. This means that the binding to the target molecule is not displaced. de la Lastra et al. (1999), Immunology, 96( 4):663-670. In some cases, in a non-competitive interaction, the first binding partner is selectively coupled to the second binding partner. of the second binding partner so that binding of the second binding partner does not directly interfere with binding of the first binding partner. Specifically binds to a non-overlapping interaction site. Any effect on binding of the binding partner due to binding of the first binding partner through mechanisms other than directly interfering with the binding of the hydroxyl group. For example, In this context, a non-competitive inhibitor binds to a site other than the active site of an enzyme. The two binding partners are specific for interaction sites that do not overlap with the binding of the first binding partner. binds to, but only when the first interaction site is presented by the first binding partner , which binds to a second interaction site, encompassing a non-competitive binding interaction.
[0139] The term "pharmaceutical composition" refers to a pharmaceutical composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically contains an effective amount of an active agent (e.g., a mutant C Immunomodulatory polypeptides, including CTLA-4, or mutant CTLA-4 transmembrane immunomodulatory proteins The carrier, excipient, or diluent may be a carrier, an engineered cell expressing the protein, or a carrier, excipient, or diluent. An excipient, or diluent, is typically a pharmaceutically acceptable carrier, excipient, or or a diluent.
[0140] The terms "polypeptide" and "protein" are used interchangeably herein; These terms refer to molecular chains of two or more amino acids linked through peptide bonds. The terms "peptide" and "oligonucleotide" do not refer to a specific length of the product. "Peptide" is included within the definition of polypeptide. These terms refer to polypeptides, e.g. This includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. These terms also refer to compounds containing one or more amino acid analogs or non-standard or unnatural amino acids. , may be synthesized using known protein engineering techniques or may be recombinantly expressed. In addition, proteins can be derivatized.
[0141] As used herein, the term "primary T cell assay" refers to an assay for interferon This refers to an in vitro assay for measuring IFN-gamma ("IFN-gamma") expression. Such primary T cell assays are known in the art. In a preferred embodiment The assay used is an anti-CD3 co-immobilization assay. T cells were incubated with immobilized anti-CD31 antibodies with or without additional recombinant proteins. The culture supernatant is usually harvested at 24-72 hours. In an embodiment, the assay used is an MLR. In this assay, primary T cells are The culture supernatant is typically collected at 24-72 hours. Human IFN-gamma levels were measured in culture supernatants by standard ELISA techniques. Commercial kits are available from suppliers and assays are This is done in accordance with the recommendations of
[0142] "Purified" as applied to nucleic acids, such as those encoding the immunomodulatory proteins of the present invention. The term "antibody" is generally determined by analytical techniques well known in the art. Other components such as purified polypeptides or polynucleotides may be removed by electrophoresis. in gels, chromatography eluates, and / or media subjected to density gradient centrifugation For example, a nucleic acid or polypeptide that is substantially free of a nucleic acid or polypeptide that forms a discrete band. A nucleic acid or polypeptide that gives rise to essentially one band in a running gel is "purified." A purified nucleic acid or protein of the invention is at least about 50% pure, usually at least Each has a purity of approximately 75%, 80%, 85%, 90%, 95%, 96%, 99% or more (e.g., on a weight percent or molar basis).
[0143] The term "recombinant" means that a material (e.g., a nucleic acid or polypeptide) has been modified without human intervention. The alteration indicates that the substance has been artificially (i.e., non-naturally) altered. may be performed on or removed from materials within a state. "Nucleic acids" may be prepared by, for example, cloning, affinity engineering, DNA shuffling, or other well-known methods. are nucleic acids produced by recombining nucleic acids during molecular biological procedures known "Recombinant DNA molecules" are molecules joined together by means of such molecular biological techniques. As used herein, "recombinant protein" refers to a protein that is composed of a segment of DNA that is expressed in a recombinant form. The term "protein" or "recombinant polypeptide" refers to a polypeptide that is developed using a recombinant DNA molecule. A "recombinant host cell" refers to a cell that contains and / or is capable of expressing a recombinant nucleic acid. or expressing a transmembrane immunomodulatory protein or a combination thereof, such as the transmembrane immunomodulatory protein provided herein. The protein can be differentiated by genetic engineering, such as by introducing into cells a nucleic acid molecule that encodes the recombinant protein. Transcriptional control signals in eukaryotic cells are called "promoters" and " Promoters and enhancers are cellular proteins involved in transcription. It consists of a short array of DNA sequences that interact specifically with proteins. Enhancer elements include genes in yeast, insect, and mammalian cells, and viruses. Genes from various eukaryotic sources, including ribosomal RNAs (similar regulatory elements, i.e., promoters, are also found in prokaryotes). The selection of specific promoters and enhancers is subject to It depends on which cell type is to be used to express the protein. When used, the terms "in operable combination," "in operable sequence," and "operable" are used interchangeably. The term "linked to" refers to the transcription of a given gene and / or a desired protein molecule. The linking of nucleic acid sequences in a manner or orientation that produces a nucleic acid molecule capable of directing the synthesis of Refers to the conclusion.
[0144] As used herein, the term "recombinant expression vector" refers to a vector that is capable of expressing a particular host cell. The appropriate desired coding sequence required for expression of the operably linked coding sequence in It refers to a DNA molecule containing a coding sequence and a nucleic acid sequence required for expression in prokaryotes. The essential nucleic acid sequences include a promoter, optionally an operator sequence, a ribosome binding site, Eukaryotic cells contain promoters, enhancers, and possibly other sequences. It is known that they utilize termination and polyadenylation signals. The peptide sequence may also optionally be used to facilitate the release of the fusion protein from the cell, if desired. The expressed fusion protein can be secreted by the recombinant host cells for easy isolation. The coding sequence for a recombinant protein, such as a recombinant fusion protein, may be manipulated to It can be encoded by a recombinant expression vector to which it is operably linked. Vectors as self-replicating nucleic acid structures and integrated into the genome of the host cell into which they are introduced Among the vectors are viral vectors such as lentiviral vectors. There is a tar.
[0145] The term "selectivity" refers to the ability to bind to different substrates, such as different binding partners of a protein of interest. specific binding of one substrate, such as one binding partner, compared to the specific binding of Selectivity refers to the preference for a target protein or polypeptide to be bound to a first binding partner. (e.g., Kd1) and the binding activity of the first substrate (e.g., binding parent affinity) and the binding activity of the same protein of interest with a second binding partner (e.g., Kd2) For example, it can be reflected as a ratio to the binding affinity.
[0146] As used herein, the term "sequence identity" refers to the difference between nucleotides or sequences, respectively. It refers to the sequence identity between genes or proteins at the amino acid level. " is a measure of identity between proteins at the amino acid level and at the nucleotide level. Protein sequence identity is a measure of identity between nucleic acids in a sequence. When the amino acid sequence is determined, it is possible to identify the amino acid sequence at a given position in each sequence. Similarly, nucleic acid sequence identity can be determined when sequences are aligned. can be determined by comparing the nucleotide sequence at a given position in each sequence. Methods for alignment of sequences for comparison are well known in the art and may be used interchangeably. Such methods include GAP, BESTFIT, BLAST, FASTA, and T The BLAST algorithm calculates percent sequence identity and BLAST is a statistical analysis of the similarity between two sequences. Through the website of the National Center for Biotechnology Information (NCBI) is publicly available.
[0147] As used herein with respect to proteins, the term "soluble" refers to a protein This means that the IgSF domain is not a membrane protein. The extracellular domain of an IgSF family member receptor containing a steroid or its specific binding fragment containing only the membrane domain or a portion thereof, but not the transmembrane domain, and / or In some cases, the solubility of the protein can be directly expressed on the surface of the cell. or indirectly via a linker, in some cases to improve protein stability and / or Linking or attaching to an Fc domain or other moiety can also improve half-life. In some embodiments, the soluble protein is an Fc fusion protein. is.
[0148] As used herein with respect to a polypeptide or nucleic acid, the term "species" refers to the same It refers to an ensemble of molecules that have identical or nearly identical sequences. The variations between peptides include glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, and acetylation. This may arise due to differences in post-translational modifications such as acetylation and lipidation. differs from the full-length species at the amino or carboxy terminus by no more than three or three amino acid residues A slightly truncated sequence of a polypeptide that encodes (or is encoding) a single species Such microheterogeneity is believed to be a common feature of the proteins produced. is.
[0149] The present invention relates to a full-length wild-type mammalian CTLA-4 polypeptide or its IgV domain. As used herein, the term "specific binding fragment" refers to a full-length polypeptide or an Ig V domain partial sequences and can be expressed in vitro and / or in vivo as human or murine mammalian ICOSL, mammalian CD80, such as ICOSL, CD80, or CD86; and / or a polypeptide that specifically binds to mammalian CD86. In this embodiment, the specific binding fragment comprises a full-length wild-type sequence or at least one of its IgV sequences. 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the CTLA-4 IgV partial sequence. , can be altered in sequence to form the mutant CTLA-4 of the present invention.
[0150] As used herein, the term "specifically binds" refers to the affinity of a protein. Or the binding activity is determined by random peptides of sufficient statistical size of the same protein or at least five times the average affinity or avidity for a collection of polypeptides, or Optionally, at least 10, 20, 30, 40, 50, 100, 250, or 50 0 fold or even at least 1000 fold under specific binding conditions, It refers to the ability of a protein to bind to a target protein. The binding site does not need to bind non-exclusively to a single target molecule, but rather to a structure between the target and non-target molecules. to non-target molecules due to similarities in target conformation (e.g., paralogs or orthologs). Those skilled in the art will be able to identify molecules ( i.e., orthologs), or non-target molecules with nearly identical epitopes to the target molecule (e.g., a statistically valid collection of unique non-targets capable of specific binding to a target molecule (e.g., a paralog) (e.g., random polypeptides) without compromising the specificity of binding determined Therefore, the polypeptides of the present invention may be used to identify targets due to cross-reactivity. can specifically bind to more than one distinct species of molecule. Solid-phase ELISA immunoassay, Fort Uses measurements from eBio Octet® or Biacore® The specific binding between two proteins can be determined by using the The protein-protein interaction has a dissociation constant (Kd) of less than 1x10-5 M and a dissociation constant of 1x10-1 In certain embodiments of the present disclosure, the interaction between two binding proteins is The interactions are approximately less than 1x10-6M, 1x10-7M, 1x10-8M, 1x10-9M, It has a dissociation constant of 1x10-10M or 1x10-11M or less.
[0151] With respect to a mammal that expresses a polypeptide, "surface expressed," "surface-expressed," or "expressed" The term "expressed on the surface of a polypeptide" refers to the expression of a polypeptide as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.
[0152] As used herein, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide. "Synthetic" with respect to a compound means that it is produced by recombinant and / or chemical synthesis methods. It refers to a nucleic acid molecule or a polypeptide molecule.
[0153] As used herein, the term "targeting moiety" refers to a mutant CTLA molecule of the invention. -4 or a polypeptide containing the covalently or non-covalently attached or physically attached to the polypeptide. In some embodiments, the targeting moiety is expressed on a cell. The target molecule has a specific binding affinity for a target molecule, such as a target molecule. Typically, the target molecule is Localized on specific tissues or cell types. Targeting moieties include antibodies, antigen-binding fragments (F ab), variable fragments (Fv) containing VH and VL, VH linked together in one chain and VL, as well as single-chain variable fragments (scFv), Fab′, F(ab)2, F (ab′)2, dsFv bispecific antibodies, nanobodies, soluble receptors, receptor ligands, Affinity matured receptors or other antibody V-region fragments such as ligands, as well as small molecules (<50 0 Daltons) compositions (e.g., specific binding receptor compositions). It is also covalently or non-covalently attached to the lipid membrane of the liposome that encapsulates the polypeptide of the present invention. It can be done.
[0154] As used herein, the term "transmembrane protein" refers to a protein that is present in a cell, such as a mammalian cell, Lipid bilayers, such as those found in biological membranes or artificial constructs such as liposomes A membrane protein is a protein that spans substantially or completely across a lipid bilayer. A transmembrane domain ("transmembrane domain") that is integrated into the target protein and whose integration is thermodynamically stable under physiological conditions. The transmembrane domain generally contains a transmembrane domain that is capable of transporting proteins into an aqueous environment (e.g., the cytosol, the cytosol, the Based on their high hydrophobicity relative to the regions of the protein that interact with the extracellular fluid (e.g., extracellular fluid), through any number of commercially available bioinformatics software applications. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Transmembrane proteins often pass through both layers of the lipid bilayer one or more times. The transmembrane protein may be any of the transmembrane immunomodulatory proteins provided herein. In addition to the transmembrane domain, the transmembrane immunomodulatory proteins of the present invention comprise an ectodomain. , and in some embodiments further comprises an ectodomain.
[0155] As used herein, "treating," "treatment," or "treatment of" a disease or disorder The term "therapy" refers to a compound, alone or in combination with another compound, as described herein. In any of the above, the therapeutic compositions of the present invention (e.g., immunomodulatory proteins or engineered administration of a therapeutic agent (containing a therapeutic agent containing ... Slowing or halting the progression of a disease or disorder, as evidenced by its progression or elimination "Treating," "treatment," or "therapy" means to improve or reverse a condition. may also be used to reduce the severity of symptoms in acute or chronic diseases or disorders, or to treat, for example, , a reduction in the recurrence rate in the case of relapse or remission of the autoimmune disease process, or "Treating," "treatment," or "therapeutic" refers to the reduction of inflammation in the case of inflammatory aspects of the disease. The method also aims to reduce inflammation and / or other symptoms associated with transplant rejection. You can taste it.
[0156] As used herein in the context of cancer, "treating" or "inhibiting" or "inhibiting" cancer The terms "resulting in" or "inhibition" are used in the Response Evaluation Criteria in Solid Tumors (RECIST) or a statistically significant increase in progression-free survival (PFS) or overall survival (OS), Statistically significant changes in tumor growth rate as measured by standard criteria, including but not limited to: a significant decrease, cessation of tumor growth, or reduction in tumor size, mass, metabolic activity, or volume refers to at least one of the following:
[0157] As used in the context of the present invention, "preventing," "prophylaxis," or "preventing" a disease or disorder refers to "Prevention" means the prevention of the onset or onset of a disease or disorder, or the alleviation of symptoms of a disease or disorder. Prevent some or all of the following: The present invention, either alone or in combination with another compound, This refers to administering immunomodulatory polypeptides or engineered cells.
[0158] As used herein, the term "tumor-specific antigen" or "TSA" refers primarily to , which are present on tumor cells of a mammalian subject, but are generally not found on normal cells of a mammalian subject. Tumor-specific antigens do not necessarily have to be exclusive to tumor cells. However, the proportion of cells in a particular mammal that have tumor-specific antigens is sufficiently high or The level of tumor-specific antigens on the surface of tumors can be used to administer anti-tumor agents such as the immunomodulatory polypeptides of the present invention. can be targeted by therapeutic agents and provide protection or treatment of mammals from the effects of tumors In some embodiments, the run-on of cells from a mammal having a tumor is high enough to allow for the production of a tumor-like substance. The undeniable statistics are that at least 50% of cells displaying TSA are cancerous. In other embodiments, at least 60% of the cells displaying the TSA 70%, 80%, 85%, 90%, 95%, or 99% are cancerous.
[0159] When used in reference to mutant CTLA-4, "mutated" (also "altered" or "mutated") The term "organism") refers to a mammal (e.g., a human or a mouse) created by human intervention. Mutant CTLA-4 refers to unmodified or wild-type CTLA-4. A polypeptide with an altered amino acid sequence relative to native CTLA-4. CTLA-4 may contain one or more amino acid substitutions, deletions, additions, or combinations thereof. A polypeptide that differs from the wild-type CTLA-4 isoform sequence by one or more modifications of For purposes herein, a mutant CTLA-4 is one having one or more of the amino acid differences. at least one affinity occurring in an IgSF domain (e.g., an IgV domain) Mutant CTLA-4 contains modified domains, such as 1, 2, 3, 4, or 5 amino acid substitutions. 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 acids Mutant CTLA-4 polypeptides generally contain differences in the extracellular domain or The sequence of SEQ ID NO: 1, which contains the IgSF domain of corresponding wild-type or unmodified CTLA- 4 and at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 In some embodiments, the mutant CTLA-4 polypeptide exhibits 9% or more sequence identity. The code is a corresponding code containing the sequence set forth in SEQ ID NO:2 or SEQ ID NO:3. wild-type or unmodified CTLA-4 by at least 50%, 60%, 70%, 80%, 85%, or %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more sequence identity. Acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. The mutant CTLA-4 is not limited to any particular method of production, for example, de novo chemical synthesis, The mutant CTLA of the present invention includes de novo recombinant DNA technology, or a combination thereof. -4 specifically binds to ICOSL, CD80, and / or CD86 in mammalian species. In some embodiments, the altered amino acid sequence is a wild-type or unmodified CTLA- Compared with 4 proteins, ICOSL; CD80, CD86, CD80, and CD86 ICOSL and CD80; ICOSL and CD86; and / or ICOSL Altered (i.e., increased or decreased) binding to CD80, and CD86 The increase or decrease in binding affinity or avidity is a function of the flow This can be determined using well-known binding assays such as cytometry. A suitable assay is described by Larsen et al., Am J Transplant, 5(3 ):443-453(2005) and Linsley et al., Immunit y,1(9):793-801(1994).
[0160] Mutant CTLA-4 binding affinity to ICOSL, CD80, and / or CD86 The increase in affinity or binding activity is at least 5% over that of wild-type or unmodified CTLA-4. % greater than, and in some embodiments, greater than, the wild-type or unmodified CTLA-4 control value. At least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than CTLA-4 binding affinity for ICOSL, CD80, and / or CD86 The reduction in binding activity was 95% or less of the wild-type or unmodified control value, and several In embodiments, the modified antibody concentration is 80%, 70%, 60%, 50%, 40%, or less than the wild-type or unmodified control value. Less than 30%, 20%, 10%, 5%, or undetectable binding affinity or avidity Mutant CTLA-4 has a primary amino acid sequence that is altered by substitution, addition, or deletion of amino acid residues. It is changed in a row.
[0161] The term "mutant" in the context of mutant CTLA-4 refers to the gene in which the mutant CTLA-4 was created. should not be construed as imposing any conditions on any particular starting composition or methodology used. Mutant CTLA-4 is not derived from, for example, wild-type mammalian CTLA-4 sequence information. can be generated by cleavage and then reacted with ICOSL, CD80, and / or CD86 for binding. These can be modeled in silico to ultimately yield the mutant CTLA-4 of the present invention. It can be recombinantly expressed or chemically synthesized. -4 can be generated by site-directed mutagenesis of wild-type CTLA-4. Mutant CTLA-4 represents a composition, not necessarily produced by any given process. It does not represent a product produced by recombinant methods, chemical synthesis, or a combination thereof. A variety of techniques can be used, including:
[0162] Nucleic acid molecules, proteins (e.g., CTLA-4), IgSF members, host cells, etc. As used herein in connection with biological material, "wild-type" or "native" or The term "native" refers to that found in nature and unaltered by human intervention .
[0163] II. Mutant CTLA-4 Polypeptides Altered (increased or decreased) activity against one or more of the CTLA-4 binding partners Provided herein are mutant CTLA-4 polypeptides that exhibit binding activity or affinity. In some embodiments, the CTLA-4 binding partner is ICOSL, CD80, and and / or CD86. In some embodiments, CTLA-4 In some embodiments, the binding partner is ICOSL. The binding partners are ICOSL and CD80 or CD86. In embodiments, the one or more binding partners of CTLA-4 include ICOSL, CD80, and and CD86.
[0164] CTLA-4 is a domain known as an immunoglobulin domain or fold. (hereafter referred to as "immunoglobulin superfamily domains" or IgSF domains) The immunoglobulin superfamily of proteins is a family of proteins that all humans possess. In some embodiments, the other IgSF family member is The signal-regulatory receptor (SIRP) family, a trigger receptor expressed on myeloid cells Body (Triggering Receptor Expressed On Myel T cell adhesion molecules related to carcinoembryonic antigen (TREML) family CEACAM family, sialic acid-binding immunoglobulin-like lectins (SIGLEC ) family, butyrophilin family, B7 family, CD28 family, V-cell V-set transmembrane domains and immunoglobulin domain-containing (VSIG) family VSTM family, major histocompatibility complex (MHC) family, signaling link lymphocyte activation molecule (SLAM) family, leukocyte immunoglobulin-like receptor (LIR), Nectin (Nec) family, nectin-like (NECL) family, poliovirus receptor Phenotype-related receptor (PVR) family, natural cytotoxicity-inducing receptor (NCR) family, T cells Immunoglobulin and mucin (TIM) family, or killer cell immunoglobulin-like In some embodiments, other Ig receptors include members from the KIR family. SF family members include CD80 (B7~1), CD86 (B7~2), and CD274. (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOS LG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3) , VTCN1(B7-H4), CD28, PDCD1(TIGIT), ICOS, BTL A (CD272), CD4, CD8A (CD8-alpha), CD8B (CD8-beta) ), LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(C D155), PVRL2 (CD112), CD226, CD2, CD160, CD200 , CD200R1 (CD200R), and NCR3 (NKp30).
[0165] Table 1 summarizes exemplary members of the IgSF family. The first column of Table 1 lists: Name, and optionally, some possible aliases for that particular IgSF member The second column provides the protein identifier from the UniProtKB database, uni Publicly available database accessible via the internet at prot.org Provide the source, or in some cases, the GenBank number. (UniProt) is a comprehensive source of protein sequence and annotation data. The UniProt database uses the UniProt Knowledge Base (UniProtKB) UniProt is a joint venture between the European Bioinformatics Institute (EMBL-EBI), SIB Swiss Institute of Bioinformatics and Protein Information Source (PIR) This is a collaboration between the National Institutes of Health (NIH) and the National Institutes of Health (NIH), and is supported primarily by a grant from the National Institutes of Health (NIH). GenBank is an annotated collection of all publicly available DNA sequences. The Nucleic Acids Research Database, which is part of the NIH gene sequence database, rch, 2013 Jan;41(D1):D36-42). The third column shows the Ig Provides the region in which the SF domain is located. The region encompasses the residues that define the domain. Column 3 also lists the IgSF domains for the identified IgSF regions. The main classes are shown. Column 4 indicates the region where the additional domains shown are located (signal peptides). The proteins provide the following: S, extracellular domain; E, transmembrane domain; T, cytoplasmic domain; and C. Domain descriptions vary depending on the method used to identify or classify the domain. It is understood that different sources may be identified differently. The corresponding residues are shown for illustrative purposes only and may be longer or shorter. Column 5 lists the IgSF amino acids (e.g., 1, 2, 3, or 4). Some of the members and some of their binding partners are shown.
[0166] Table 1. IgSF members according to the present disclosure [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]
[0167] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. CTLA-4 polypeptides, or wild-type or unmodified CTLA-4 containing IgD immunoglobulin superfamily (Ig) SF) domain (IgD) with one or more substitutions (alternatively, "mutations" or "exchanges" "), deletions, or additions. The mutant CTLA-4 polypeptides to be produced have one or more amino acid modifications (e.g., substitutions) that: Within IgD, it is a variant IgD (hereafter referred to as "vIgD"), or Includes.
[0168] In some embodiments, the IgD is an IgV domain or a specific In some embodiments, the IgD comprises an Ig binding fragment, an Ig binding fragment, or a combination thereof. It may be only V or the entire extracellular domain (ECD) of CTLA-4. In this form, IgD contains a specific binding fragment of the ECD. Table 1 shows the IgV domain of CTLA-4. Exemplary residues corresponding to the C region and ECD are provided. In some embodiments, the mutant C The TLA-4 polypeptide may have at least one amino acid modification (e.g., substitution) The IgV domain or ECD, or a specific binding fragment thereof, may also be present. or ECD, or a specific binding fragment thereof. The CTLA-4 polypeptide has at least one amino acid modification (e.g., substitution) , an IgV domain or its specific binding fragment, In some embodiments, the mutant CTLA-4 polypeptide comprises an adenosine triphosphate-binding fragment. At least one of the amino acid modifications (e.g., substitutions) is / are present in the ECD or a specific binding fragment thereof. In some embodiments, the modified ECD or a specific binding fragment thereof is present in a fragment. The altered IgV domain or ECD exhibits improved affinity due to the increased avidity or affinity. It is a variant IgSF domain.
[0169] In some embodiments, the variants are variants of the unmodified CTLA-4 sequence. In some embodiments, the modification is in one IgSF domain. The CTLA-4 sequence is wild-type CTLA-4. In some embodiments, the unmodified or wild-type CTLA-4 CTLA-4 has the sequence of native CTLA-4 or its ortholog. In embodiments, unmodified CTLA-4 is a CTLA containing one or more IgSF domains. -4 or a portion thereof (Table 1 In some embodiments, unmodified or wild-type CTLA-4 polypeptides are used. The extracellular domain of the IgV domain contains an IgV domain or a specific binding fragment thereof. In embodiments, the mutant CTLA-4 polypeptide is an IgV domain or its specific In some embodiments, the mutant CT comprises or consists essentially of a binding fragment. The LA-4 polypeptide comprises or essentially consists of an ECD or a specific binding fragment thereof. In some embodiments, the mutant CTLA-4 is soluble and transmembrane In some embodiments, the mutant CTLA-4 lacks an intracellular signaling domain. a transmembrane domain which may contain a transduction domain, and optionally a cytoplasmic domain. Further includes:
[0170] In some embodiments, the wild-type or unmodified CTLA-4 sequence is a mammalian CTLA In some embodiments, the wild-type or unmodified CTLA-4 sequence is a human CTLA-4 sequence. including, but not limited to, human, mouse, cynomolgus monkey, or rat CTLA-4. In some embodiments, the CTL may be a wild-type or unmodified CTL. The A-4 sequence is human.
[0171] In some embodiments, the wild-type or unmodified CTLA-4 sequence comprises (i) a signal sequence. the amino acid sequence set forth in SEQ ID NO:1, or its mature form, lacking the amino acid sequence , (ii) a sequence identical to SEQ ID NO: 1 by at least 85%, 86%, 87%, 88%, 89% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more sequence identity to the amino acid sequence of the mature form thereof, or ( iii) a portion of (i) or (ii) containing an IgV domain or ECD, or a specific binding fragment thereof.
[0172] In some embodiments, the wild-type or unmodified CTLA-4 sequence is a CTLA-4 cytosol. The extracellular domain (ECD) or a portion thereof may be or include the same. In one embodiment, the unmodified or wild-type CTLA-4 polypeptide is set forth in SEQ ID NO:2. The amino acid sequence shown (corresponding to amino acid residues 36 to 161 of SEQ ID NO:1) or an ortholog thereof. Optionally, the unmodified or wild-type CTLA-4 polypeptide The peptide comprises (i) the sequence of amino acids set forth in SEQ ID NO:2; (ii) SEQ ID NO:2 and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. or (iii) a sequence of amino acids comprising (i) or (ii) an IgV domain. In some embodiments, the wild-type or unmodified EC D is one or more of ICOSL and / or CD80 and / or CD86 The CTLA-4 binding partner may be capable of binding to one or more CTLA-4 binding partners, such as:
[0173] In some embodiments, the wild-type or unmodified CTLA-4 polypeptide is an IgV domain. In some embodiments, the wild-type or unmodified The IgV domain of the variant CTLA-4 polypeptide is the amino acid sequence set forth in SEQ ID NO:3. the amino acid sequence (corresponding to amino acid residues 39 to 140 of SEQ ID NO:1), or orthologs of the IgV domain of the unmodified or wild-type CTLA-4 polypeptide. The main sequence is (i) the amino acid sequence set forth in SEQ ID NO:3, (ii) SEQ ID NO:1 D NO:3 and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. or (iii) a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain may contain ICO one or more of SL and / or CD80 and / or CD86 can bind to the CTLA-4 binding partners listed above.
[0174] In some embodiments, the wild-type or unmodified CTLA-4 polypeptide is an IgV domain. It contains a specific binding fragment of CTLA-4, such as a specific binding fragment of the main or ECD. In some embodiments, the specific binding fragment is capable of binding to ICOSL. In some embodiments, the specific fragment may bind to ICOSL and CD80 or CD86. In some embodiments, the specific fragment can bind to ICOSL, CD80, and CD86. A specific binding fragment may be at least 60, 70, 80, 90, or 100 amino acids long. In some embodiments, the amino acid sequence may be at least 50 amino acids in length, such as: The specific binding fragment of the IgV domain is amino acids 39-140 of SEQ ID NO:1. at least about 85%, 86%, 87%, 88% of the length of the IgV domain as set forth above, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, In some embodiments, the specific binding fragment of the ECD comprises an amino acid sequence that is 99% or more of the amino acid sequence. The fragment is of the length of the ECD set forth as amino acids 36 to 161 of SEQ ID NO:1. At least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence.
[0175] In some embodiments, the mutant CTLA-4 polypeptide comprises one or more affinity modifications. In some embodiments, the ECD domain or a portion thereof includes an IgSF domain. In this study, the mutant CTLA-4 polypeptides were identified as having a specific IgSF domain (IgV) or ECD. IgV domains, one or more of which contain one or more amino acid modifications (e.g., substitutions). or an IgV domain-specific binding fragment, or a specific binding fragment of the ECD. In some embodiments, the mutant CTLA-4 polypeptide comprises a full-length IgV domain. In some embodiments, the mutant CTLA-4 polypeptide comprises a full-length ECD. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific IgV domain. In some embodiments, the mutant CTLA-4 polypeptide comprises a specific binding fragment of an EC D.
[0176] In any of such embodiments, one or more of the mutant CTLA-4 polypeptides The above amino acid modifications (e.g., substitutions) may be made in the CTLA-4 polypeptide, such as in the internal IgSF domain. It may be located within the peptide ECD. For example, in some embodiments, one or more amino acids The alterations (e.g., substitutions) are located within the extracellular domain of the mutant CTLA-4 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgV domain. It is located within the specific binding fragment of the IgV domain.
[0177] Generally, each of the various attributes of the polypeptides is disclosed separately below. (e.g., soluble and membrane-bound polypeptides, ICOSL, CD80 and / or is the affinity of CTLA-4 for CD86, the number of variables per polypeptide chain, the number of linked the number of polypeptide chains identified, the number and nature of amino acid changes per mutant CTLA-4, etc. However, as will be apparent to those skilled in the art, any particular polypeptide may It may contain a combination of independent attributes to describe the domain organization of the IgSF domain. Reference to amino acids, including specific sequences set forth as SEQ ID NOs, is for illustrative purposes only. It is understood that the following is for illustrative purposes only and is not meant to limit the scope of the embodiments provided. The description of the polypeptide and its domains is based on homology analysis and alignment with similar molecules. Therefore, it is understood that the exact locus is theoretically derived based on the can vary and is not necessarily the same for each protein. Certain IgSF domains, such as certain IgV domains or ECDs, are longer or It may be shorter, a few amino acids (such as 1, 2, 3, or 4).
[0178] Furthermore, various embodiments of the present invention as discussed below may be implemented using the methods disclosed above. It is frequently provided within the meaning of the defined term. Therefore, The embodiments described are intended to be illustrative and not restrictive, and are not intended to be limiting unless the defined terms are used to consider the various aspects and attributes described herein. When used in connection with the present application, the terms and conditions should be interpreted as if they were incorporated by reference. The headings, the order of presentation of the various aspects and embodiments, and the order of their respective independent attributes are Separate disclosures are not meant to be limitations on the scope of the present disclosure.
[0179] A. Exemplary Modifications against IgSF or ECD contained in wild-type or unmodified CTLA-4 polypeptides and mutant CTLA-4 polypeptides containing modifications in the ECD, their IgSF domains, In some embodiments, at least one of the nucleotides, or specific binding fragments thereof, is provided herein. At least one modification is in an IgSF domain (e.g., IgV) or a specific binding fragment thereof. and therefore, the provided mutant CTLA-4 polypeptides have a structure similar to that of at least one parent Some embodiments contain a hybrid-modified IgSF domain, or a specific-binding fragment thereof. In this context, a mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. Changes (increases) in ICOSL, CD80, and / or CD86 compared with controls or decreased) binding activity or affinity to ICOSL, CD80, and / or CD86 can be a mammalian protein, such as a human or mouse protein. do.
[0180] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. ICOSL, and optionally one or more linkers, as compared to a TLA-4 polypeptide. Altered (increased or decreased) binding activity to the ligands CD80 and CD86 and In some embodiments, the mutant CTLA-4 polypeptide exhibits affinity to, for example, Solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet ( trademark), or wild-type or unmodified as determined by Biacore assay The IC binding affinity of the mutated CTLA-4 polypeptide is different from that of a control (e.g., unmodified) sequence. and optionally, binding affinity to CD80 and / or CD86. In some embodiments, the mutant CTLA-4 polypeptide has a nucleotide sequence similar to that of ICOSL, and optionally, having increased binding affinity for CD80 and / or CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. Increased binding affinity for ICOSL relative to the mutated CTLA-4 polypeptide, and Optionally, a reduced binding affinity to one or more of CD80 and / or CD86 It has.
[0181] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. In comparison to the TLA-4 polypeptide, CD80, and optionally one or more ligases Altered (increased or decreased) binding activity to ICOSL and CD86 In some embodiments, the mutant CTLA-4 polypeptide exhibits affinity to, for example, Solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet ( Registered trademark), or wild-type or unmodified CT as determined by Biacore assay. CD80, which has a binding affinity different from that of a control (e.g., unmodified) sequence of the LA-4 polypeptide. and optionally having binding affinity for ICOSL and / or CD86 In some embodiments, the mutant CTLA-4 polypeptide is a CTLA-4 polypeptide that binds to CD80, as well as any Optionally, it has increased binding affinity for ICOSL and / or CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. For LA-4 polypeptides, increased binding affinity for CD80, as well as optional selectively, having reduced binding affinity for one or more of ICOSL and / or CD86 do.
[0182] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. In comparison to the TLA-4 polypeptide, CD86 and, optionally, one or more ligands Altered (increased or decreased) binding activity to ICOSL and CD80 In some embodiments, the mutant CTLA-4 polypeptide exhibits affinity to, for example, Solid-phase ELISA immunoassay, flow cytometry, ForteBio Octet ( Registered trademark), or wild-type or unmodified CT as determined by Biacore assay. CD86, which has a binding affinity different from that of a control (e.g., unmodified) sequence of the LA-4 polypeptide and optionally having binding affinity for ICOSL and / or CD80 In some embodiments, the mutant CTLA-4 polypeptide is a polypeptide that binds to CD86, optionally In addition, they have increased binding affinity for ICOSL and / or CD80. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 for the polypeptide, an increased binding affinity for CD86, optionally one or more have reduced binding affinity for ICOSL and / or CD80.
[0183] The binding affinity for each of the binding partners is independent. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTL A-4 polypeptide, ICOSL, CD80, and / or CD86 Increased binding affinity to one, two, or three of the following: ICOSL, CD80, and / or a reduced binding affinity to one, two, or three of CD86. Has.
[0184] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. TLA-4 polypeptides have increased binding affinity for CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA The -4 polypeptide has increased binding affinity to CD80. In embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. It has increased binding affinity to CD80 and CD86 relative to the polypeptide. In some embodiments, such mutant CTLA-4 polypeptides also refer to wild-type or exhibits increased binding affinity for ICOSL relative to unmodified CTLA-4 polypeptides. Has.
[0185] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. TLA-4 polypeptides have increased binding affinity for ICOSL. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTL Increased or decreased binding affinity for ICOSL to the A-4 polypeptide, and and has increased binding affinity to CD80. In some embodiments, the mutant CT The CTLA-4 polypeptide exhibits an IC Increased or decreased binding affinity for OSL and increased binding affinity for CD86 In some embodiments, the mutant CTLA-4 polypeptide has a wild-type or or reduced binding affinity for ICOSL relative to unmodified CTLA-4 polypeptides. In some embodiments, the mutant CTLA-4 polypeptide has a wild-type or Increased or decreased binding to ICOSL relative to unmodified CTLA-4 polypeptide In some embodiments, the antibody has a reduced binding affinity to CD80. The mutant CTLA-4 polypeptide may be a wild-type or unmodified CTLA-4 polypeptide. In contrast, increased or decreased binding affinity for ICOSL and decreased binding affinity for CD86 It has little binding affinity.
[0186] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. Increased binding affinity for ICOSL and CD80 relative to TLA-4 polypeptide In some embodiments, the mutant CTLA-4 polypeptide has the same properties as the wild-type or increased binding affinity for ICOSL relative to unmodified CTLA-4 polypeptide, and has a reduced binding affinity to CD80. In some embodiments, the mutant form The CTLA-4 polypeptide may be modified relative to a wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the antibody has a reduced binding affinity for ICOSL and CD80. The mutant CTLA-4 polypeptide may be a wild-type or unmodified CTLA-4 polypeptide. In contrast, the antibody showed a decreased binding affinity for ICOSL and an increased binding affinity for CD80. It has affinity for
[0187] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. Increased binding affinity for ICOSL and CD86 relative to TLA-4 polypeptide In some embodiments, the mutant CTLA-4 polypeptide has the same properties as the wild-type or increased binding affinity for ICOSL relative to unmodified CTLA-4 polypeptide, and has a reduced binding affinity to CD86. In some embodiments, the mutant form The CTLA-4 polypeptide may be modified relative to a wild-type or unmodified CTLA-4 polypeptide. In some embodiments, the antibody has a reduced binding affinity for ICOSL and CD86. The mutant CTLA-4 polypeptide may be a wild-type or unmodified CTLA-4 polypeptide. In contrast, the antibody showed a decreased binding affinity for ICOSL and an increased binding affinity for CD86. It has affinity for
[0188] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. Increased expression of TLA-4 polypeptide, ICOSL, CD80, and CD86 In some embodiments, the mutant CTLA-4 polypeptide has a binding affinity that is , wild-type or unmodified CTLA-4 polypeptide, ICOSL and CD80 It has increased binding affinity to CD86 and decreased binding affinity to CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTL increased binding affinity for ICOSL and CD86 relative to the A-4 polypeptide; and has a reduced binding affinity to CD80. The modified CTLA-4 polypeptide is a variant of the wild-type or unmodified CTLA-4 polypeptide. , decreased binding affinity for ICOSL and CD80, and increased binding affinity for CD86 In some embodiments, the mutant CTLA-4 polypeptide has increased binding affinity. reduced response to ICOSL relative to wild-type or unmodified CTLA-4 polypeptide It has good binding affinity to CD80 and CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. LA-4 polypeptide exhibits increased binding affinity to ICOSL, as well as CD In some embodiments, the variant has a reduced binding affinity to CD80 and CD86. A variant CTLA-4 polypeptide is a polypeptide that is a variant of a wild-type or unmodified CTLA-4 polypeptide. and have reduced binding affinity for ICOSL, CD80, and CD86. In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA -4 polypeptide, reduced binding affinity for ICOSL, as well as CD80 and has increased binding affinity to CD86.
[0189] In some embodiments, antibodies against ICOSL, CD80, and / or CD86 are Mutant CTLA-4 polypeptides with increased or greater binding affinity exhibit ICO activity. at least about 10%, 15%, 20% or more for SL, CD80, and / or CD86 At least 5% wild-type or unmodified CTLA, such as 25%, 35%, or 50% In some embodiments, the polypeptide will have increased binding affinity relative to the -4 polypeptide control. In this case, the increase in binding affinity to wild-type or unmodified CTLA-4 polypeptide is 1. More than 2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x , 30-fold, 40-fold, or 50-fold. A-4 polypeptides are intended to be useful in the art, except that they do not contain one or more amino acid modifications (e.g., substitutions). The mutant CTLA-4 polypeptide has the same sequence as the mutant CTLA-4 polypeptide.
[0190] In some embodiments, antibodies against ICSOL, CD80, and / or CD86 are Mutant CTLA-4 polypeptides with reduced or decreased binding affinity bind to ICOSL, CD 80, and / or at least about 10%, 15%, 20%, 30% for CD86; At least 5% wild-type or or will have a decreased binding affinity relative to an unmodified CTLA-4 polypeptide control. In some embodiments, the binding parent to a wild-type or unmodified CTLA-4 polypeptide The decrease in compatibility is 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times. , 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the wild-type Alternatively, the unmodified CTLA-4 polypeptide may contain one or more amino acid modifications (e.g., substitutions). It has the same sequence as the mutant CTLA-4 polypeptide, except that it does not contain the nucleotide sequence nucleotides ....
[0191] In some embodiments, antibodies against ICOSL, CD80, and / or CD86 are The equilibrium dissociation constant (KD) of any of the above-described embodiments is less than 1x10 M, less than 1x10 M, 0-6M, 1x10-7M, 1x10-8M, 1x10-9M, 1x10-10M, and can be 1x10-11M, or 1x10-12M, or even less.
[0192] A wild-type or unmodified CTLA-4 sequence does not necessarily correspond to a mutant CTLA-4 sequence described herein. It is not necessary that the -4 polypeptide be used as the starting material for generating the -4 polypeptide. The use of the term "modification," such as "substitution," indicates that embodiments of the invention are directed to a mutant CTLA- 4 is not intended to imply any limitation to any particular method for producing the polypeptide. A-4 polypeptides can be produced, for example, by de novo peptide synthesis, thus In the sense of modifying, for example, changing the codon to code for the substitution, This principle also does not necessarily require modifications such as "substitutions." This also extends to the terms "addition" and "deletion" of amino acid residues, which do not imply a specific mutation. The means by which the LA-4 polypeptide is designed or produced is not limited to any particular method. However, in some embodiments, the nucleic acid encoding the wild-type or unmodified CTL A-4 is mutated from wild-type or unmodified CTLA-4 genetic material to provide the desired specific binding to inhibit or reduce affinity and / or IFN-gamma expression or other functional activity are screened for.
[0193] In some embodiments, the mutant CTLA-4 polypeptide is available from any number of publicly available De novo synthesis using protein or nucleic acid sequences available in databases , followed by screening. National Center for Biotechnology Information provides such information and its website is part of the Uniform Resources and Information Act (UNRA) as discussed above. Publicly accessible via the internet, as in the ProtKB database be.
[0194] Unless otherwise stated, as set forth throughout this disclosure, amino acid modifications are those of SEQ The unmodified ECD sequence set forth in ID NO:2, or, where applicable, the SE as follows: Amino acids corresponding to the numbering of positions in the unmodified ECD sequence set forth in Q ID NO:569 It is specified by a position number. [ka]
[0195] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 569, any of the amino acid modifications, e.g., amino acid substitutions, provided In some embodiments, the variants include one or more of the following: CTLA-4 polypeptides may be used in conjunction with Ig polypeptides, such as the exemplary sequence set forth in SEQ ID NO:3. In a polypeptide which is or comprises a V domain or a specific binding fragment thereof The amino acid sequence may contain any one or more of the amino acid modifications provided above. [ka]
[0196] Reference sequences with SEQ ID NO:2 or SEQ ID NO:569 (e.g., SEQ ID NO: EQ ID NO:3), its IgSF domain (e.g. Modifications in the CTLA-4 polypeptide, including portions thereof containing the nucleotides ... Identifying the corresponding positions of amino acid substitutions is within the level of ordinary skill in the art. In the overall list of modifications, the amino acid positions are shown in the middle and the corresponding unmodified ( For example, wild-type amino acids are listed before a number, and identified mutant amino acid positions are listed before a number. The substitutions are listed after the number. If the modification is a deletion of the position, "del" is indicated. If the modification is an insertion at that position, "ins" is indicated. In the example, the amino acid position is shown in the middle, and the corresponding unmodified (e.g., wild-type) amino acid is shown in the middle. The amino acid sequence is listed before and after the number, and the identified mutant amino acid insertion is compared to the unmodified (e.g., wild-type) amino acid. The acid is listed in the state listed after the acid.
[0197] In some embodiments, the mutant CTLA-4 polypeptide is a wild-type or unmodified CTLA-4 polypeptide. One or more amino acid modifications, e.g., substitutions, in the TLA-4 sequence. Amino acid modifications, e.g., substitutions, can be made in the ectodomain (e.g., ectodomain) of the wild-type or unmodified CTLA-4 sequence. In some embodiments, one or more amino acid modifications, e.g., in the extracellular domain. For example, the substitution is in an IgV domain or a specific binding fragment thereof.
[0198] In some embodiments, the mutant CTLA-4 polypeptide comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions. The substitutions are in the IgV domain or E In some embodiments, the mutant CTLA-4 polypeptide may be in the Ig Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids In some embodiments, the mutant CTLA-4 polypeptide has an amino acid modification, e.g., a substitution. The tides may contain up to 1, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 In some embodiments, the mutant CTLA-4 polynucleotides have amino acid modifications, e.g., substitutions. The peptide may be a wild-type or unmodified peptide, such as the amino acid sequence of SEQ ID NO: 2 or 3. CTLA-4 polypeptide or a specific binding fragment thereof, and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 6%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 7%, 98%, or 99% sequence identity.
[0199] In some embodiments, the mutant CTLA-4 polypeptide is a variant of unmodified CTLA-4 or or a specific binding fragment thereof, with respect to the positions set forth in SEQ ID NO:2, at position 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 2 9, 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, 9 8, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118 , 119, 120, 121, 122, 124, 125, and / or 126 It has one or more amino acid modifications, eg, substitutions.
[0200] In some embodiments, the mutant CTLA-4 polypeptide is a variant of unmodified CTLA-4 or or a specific binding fragment thereof, with respect to the positions set forth in SEQ ID NO:2, at position 6, 10, 12, 14, 15, 16, 18, 19, 20, 22, 24, 26, 27, 28, 2 9, 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, 9 8, 99, 105, 106, 108, 110, 113, 115, 116, 117, 118 , 119, 120, 121, and / or 122; For example, with substitutions.
[0201] In some embodiments, the mutant CTLA-4 polypeptide is a variant of unmodified CTLA-4 or or a specific binding fragment thereof, at position 12 with respect to the positions set forth in SEQ ID NO:2 , 18, 26, 29, 33, 53, 55, 56, 58, 63, 72, 87, 98, 99, One or more amino acid modifications corresponding to 105, 106, and / or 117, e.g., substitutions It has exchange.
[0202] In some embodiments, the mutant CTLA-4 polypeptide is a variant of unmodified CTLA-4 or or a specific binding fragment thereof, at position 12 with respect to the positions set forth in SEQ ID NO:2 , 18, 26, 29, 56, 63, 72, 98, 99, 105, 106, and / or 117.
[0203] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: Regarding the positions described in 2, 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, L10 6V, I108F, I108V, N110K, N110S, N110Y, Q113H, Y 115H, Y115N, V116A, I117E, I117K, I117L, I117M , I117T, P119H, E120D, P121S, C122P, D124P, D12 4I, S125I, S125P, D126P, and / or D126T, or The amino acid sequence has one or more amino acid modifications selected from the following conservative amino acid substitutions:
[0204] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: With respect to the positions described in 2, 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, M87V, T89A, T89M, T89S, L91R, I93L, I93V , K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y105 F, Y105L, L106I, L106R, I108F, I108V, N110K, N1 10S, N110Y, Y115N, V116A, I117E, I117L, I117M, and / or I117T, or one or more conservative amino acid substitutions thereof The amino acid alterations are as follows:
[0205] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: With respect to the positions described in 2, L12F, L12H, L12I, L12P, I18A, I18 F, I18N, I18T, I18V, A26D, A26S, A26T, G29R, G29 W, E33M, E33V, T53S, M55R, M55T, M55V, M56K, M56 L, M56R, M56T, M56V, N58D, N58S, L63H, L63P, S72 G, S72T, M87A, M87K, M87T, M87V, L98Q, L98R, M99 I, M99L, Y105F, Y105L, L106I, L106N, L106R, L10 6V, I117E, I117K, I117L, I117M, and / or I117T or conservative amino acid substitutions thereof. In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NO:2 I12F, L12P, I18T, A26T, G29W, T53S for the positions listed in , M55T, M56K, M56T, N58S, S72G, M99L, L63P, L98Q , Y105L, L106I, and / or I117L, or conservative amino acids thereof In some embodiments, the mutation The CTLA-4 polypeptide has a sequence similar to that of SEQ ID NO:2, but differs from that of SEQ ID NO:3. P, I18T, A26T, G29W, T53S, M55T, M56K, N58S, S72 G, M99L, L63P, L98Q, Y105L, L106I, and / or I11 7L, or conservative amino acid substitutions thereof. In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NO: : Regarding the position described in 2, A26T, G29W, L63P, S72G, L98Q, M9 9L, Y105L, and / or L106I, or conservative amino acid substitutions thereof The amino acid sequence has one or more amino acid modifications selected from the following:
[0206] A conservative amino acid substitution is one that has the same amino acid sequence as the substituted amino acid other than the wild-type or unmodified amino acid. Any amino acid that fits within the amino acid class. The amino acid classes are aliphatic (glycosylated) and leucine, alanine, valine, leucine, and isoleucine), hydroxyl- or sulfur-containing Present (serine, cysteine, threonine, and methionine), cyclized (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine , and glutamine).
[0207] In some embodiments, the mutant CTLA-4 polypeptide is a non-mutated CTLA-4 polypeptide corresponding to A26T. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions G29W, T53S, L63P, S72G, L98Q, M99L, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide has the amino acid substitutions A26T / G29W, A26T / T53S, A26 T / L63P, A26T / S72G, A26T / L98Q, A26T / M99L, A26 The mutant CTLA-4 polypeptide contains T / Y105L, A26T / L106I. Consistent with the embodiments provided, amino acid modifications (such as any described herein) may be used. Table 2 provides exemplary amino acid modifications, such as: Modified (eg, substitution) and mutant CTLA-4 polypeptides are described.
[0208] In some embodiments, the mutant CTLA-4 polypeptide is a non-mutated CTLA-4 polypeptide corresponding to G29W. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions A26T, T53S, L63P, S72G, L98Q, M99L, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide has the amino acid substitutions A26T / G29W, G29W / T53S, G29 W / L63P, G29W / S72G, G29W / L98Q, G29W / M99L, G29 W / Y105L, or G29W / L106I. The peptide, according to provided embodiments, may have an amino acid modification, such as any of those described herein. Table 2 lists exemplary amino acids, as described. Acid-modified (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0209] In some embodiments, the mutant CTLA-4 polypeptide corresponds to T53S. The amino acid sequence of the unmodified or wild-type CTLA-4 polypeptide or its specific binding fragment In some embodiments, the mutant CTLA-4 polypeptide comprises one or more substitutions. Amino acid substitutions A26T, G29W, L63P, S72G, L98Q, M99L, Y105 In some embodiments, the mutant CT further contains L, and / or L106I. The LA-4 polypeptide contains the amino acid substitutions A26T / T53S, G29W / T53S, and T5 3S / L63P, T53S / S72G, T53S / L98Q, T53S / M99L, T5 Contains mutant CTLA-4 polypeptides with the mutations 3S / Y105L or T53S / L106I. The peptide, according to provided embodiments, may be an amino acid, such as any of those described herein. Table 2 shows exemplary amino acids, as described. Altered (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0210] In some embodiments, the mutant CTLA-4 polypeptide is a non-mutated CTLA-4 polypeptide corresponding to L63P. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions A26T, G29W, T53S, S72G, L98Q, M99L, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide contains the amino acid substitutions A26T / L63P, G29W / L63P, and T53 S / L63P, L63P / S72G, L63P / L98Q, L63P / M99L, L63 Mutant CTLA-4 polypeptides containing P / Y105L, or L63P / L106I. Consistent with the embodiments provided, the tide may be an amino acid, such as any described herein. Table 2 shows exemplary amino acids, as described. Altered (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0211] In some embodiments, the mutant CTLA-4 polypeptide comprises a non-mutated CTLA-4 polypeptide corresponding to S72G. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions A26T, G29W, T53S, L63P, L98Q, M99L, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide contains the amino acid substitutions A26T / S72G, G29W / S72G, T53 S / S72G, L63P / S72G, S72G / L98Q, S72G / M99L, S72 Mutant CTLA-4 polypeptides containing S72G / L106I, S72G / Y105L, or S72G / L106I. Consistent with the embodiments provided, the tide may be an amino acid, such as any described herein. Table 2 shows exemplary amino acids, as described. Altered (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0212] In some embodiments, the mutant CTLA-4 polypeptide is a non-mutated CTLA-4 polypeptide corresponding to L98Q. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions A26T, G29W, T53S, L63P, S72G, M99L, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide contains the amino acid substitutions A26T / L98Q, G29W / L98Q, T53 S / L98Q, L63P / L98Q, S72G / L98Q, L98Q / M99L, L98 Mutant CTLA-4 polypeptides containing Q / Y105L, or L98Q / L106I. Consistent with the embodiments provided, the tide may be an amino acid, such as any described herein. Table 2 shows exemplary amino acids, as described. Altered (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0213] In some embodiments, the mutant CTLA-4 polypeptide is a non-mutated CTLA-4 polypeptide corresponding to M99L. Amino acid substitutions in modified or wild-type CTLA-4 polypeptides or specific binding fragments thereof In some embodiments, the mutant CTLA-4 polypeptide comprises one or more amino acids. Amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, Y105L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide contains the amino acid substitutions A26T / M99L, G29W / M99L, T53 S / M99L, L63P / M99L, S72G / M99L, L98Q / M99L, M99 Mutant CTLA-4 polypeptides containing L / Y105L, or M99L / L106I. Consistent with the embodiments provided, the tide may be an amino acid, such as any described herein. Table 2 shows exemplary amino acids, as described. Altered (eg, substituted) and mutant CTLA-4 polypeptides are described.
[0214] In some embodiments, the mutant CTLA-4 polypeptide corresponds to Y105L The amino acid sequence of the unmodified or wild-type CTLA-4 polypeptide or its specific binding fragment In some embodiments, the mutant CTLA-4 polypeptide comprises one or more substitutions. Amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L and / or L106I. In some embodiments, the mutant CTL The A-4 polypeptide has the amino acid substitutions A26T / Y105L, G29W / Y105L, T 53S / Y105L, L63P / Y105L, S72G / Y105L, L98Q / Y10 5L, M99L / Y105L, or Y105L / L106I. The A-4 polypeptide may be any of the polypeptides described herein, consistent with the embodiments provided. Table 2 shows the amino acid sequences of the nucleotides as described above. , which describe exemplary amino acid modifications (e.g., substitutions) and mutant CTLA-4 polypeptides. do.
[0215] In some embodiments, the mutant CTLA-4 polypeptide corresponds to L106I. The amino acid sequence of the unmodified or wild-type CTLA-4 polypeptide or its specific binding fragment In some embodiments, the mutant CTLA-4 polypeptide comprises one or more substitutions. Amino acid substitutions A26T, G29W, T53S, L63P, S72G, L98Q, M99L and / or Y105L. The A-4 polypeptide contains the amino acid substitutions A26T / L106I, G29W / L106I, T 53S / L106I, L63P / L106I, S72G / L106I, L98Q / L10 6I, M99L / L106I, or Y105L / L106I. The A-4 polypeptide may be any of the polypeptides described herein, consistent with the embodiments provided. Table 2 shows the amino acid sequences of the nucleotides as described above. , which describe exemplary amino acid modifications (e.g., substitutions) and mutant CTLA-4 polypeptides. do.
[0216] In some embodiments, the mutant CTLA-4 comprises the amino acid substitutions E33V / M99L. The mutant CTLA-4 polypeptides may be used in accordance with the embodiments provided herein. The amino acid sequence may further include amino acid modifications (e.g., substitutions), such as any of those described in Table 2. Exemplary amino acid modifications (e.g., substitutions) and mutant CTLAs, as described in -4 polypeptides are described.
[0217] In some embodiments, the mutant CTLA-4 polypeptide comprises at least three amino acids. and unmodified or wild-type CTLA-4 or its specific At least three modifications (e.g., substitutions) in the binding fragment are as set forth in SEQ ID NO:2 Regarding the position of A26T, G29W, T53S, L63P, S72G, L98Q, M9 9L, Y105L, and / or L106I, or conservative amino acid substitutions thereof handle.
[0218] In some embodiments, the mutant CTLA-4 polypeptide is an unmodified or wild-type CTLA-4 polypeptide. CTLA-4 or a specific binding fragment thereof at the position set forth in SEQ ID NO:2 and contains amino acid substitutions corresponding to G29W / L98Q / Y105L.
[0219] In some embodiments, the mutant CTLA-4 polypeptide is an unmodified or wild-type CTLA-4 polypeptide. CTLA-4 or a specific binding fragment thereof at the position set forth in SEQ ID NO:2 And it is compatible with G29W / N58S / L63P / Q82R / L98Q / Y105L Contains acid substitutions.
[0220] In some embodiments, the mutant CTLA-4 polypeptide is an unmodified or wild-type CTLA-4 polypeptide. CTLA-4 or a specific binding fragment thereof at the position set forth in SEQ ID NO:2 And it is compatible with L12F / R16H / G29W / M56T / L98Q / Y105L Contains acid substitutions.
[0221] The mutant CTLA-4 polypeptide is a polypeptide that is a nucleotide sequence of unmodified CTLA-4 or a specific binding fragment thereof. In some embodiments, the modification has a single amino acid substitution in SEQ ID NO: :2, 27, 31, 32, 33, 35, 95, 98, 105, 10 6, or 107. 4 or a specific binding fragment thereof, The modification is not L106E. 4 or its specific binding fragments, some implementations have just two amino acid substitutions. In the form, the modifications are A31Y and L106E (i.e., A31Y / L106E). do not have.
[0222] In some embodiments, the mutant CTLA-4 is a mutant CTLA-4 having the sequence set forth in SEQ ID NO:2. Regarding designation, A26E, T32N, V34I, A52M, G57E, I67F, S6 6P, and / or S72F. The peptide has seven amino acid substitutions in unmodified CTLA-4 or a specific binding fragment thereof. In some embodiments, the modification is with respect to the numbering set forth in SEQ ID NO:2. for T32N, V34I, A52M, M56K, G57E, S66P, and S72F The mutant CTLA-4 polypeptide is not a corresponding modification. In some embodiments, the modified A26E, T32N, V34I with respect to the numbering set forth in SEQ ID NO:2 , A52M, M56K, N58D, S66P, I67S, S72F, and L106E It is not a corresponding modification.
[0223] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: Regarding the numbering in 2, A6T, V10A, L12F, L12H, L12P, S1 4N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I1 8T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A2 6S, A26T, S27P, P28L, G29R, G29W, K30R, E33M, E3 3V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D4 3N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M5 5T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N5 8S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L6 3H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I6 7V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S7 3R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M8 7K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I9 3V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y1 05F, Y105L, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117 and I117T. In embodiments, the two or more amino acid modifications are numbered according to the numbering set forth in SEQ ID NO:2. Regarding A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T 53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / Y105L、L12F / R16H / G29W / M56T / L98Q / Y105L、L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 12F / A26T / L63P / L98Q / Y105L / L106R、L12F / K30R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / Q82R / L98Q / M99L / Y105L、L12H / I18V / A42<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T / M55T / N58D / L98R / Y105L / L106I / P121S、L12H / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> E33M / L98Q / Y105L、L12H / M55T / E59D / L63P / M99L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、L12H / L63P / S72G / L98Q / Y105L、L12I / M55T / M56<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V / I67T / M99L / L106R / I108F、L12P / R16H / A26T / T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I117<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L、L12P / A26T、L12P / A26T / L63P、L12P / A26T / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / T89M / L98Q / M99L / Y105L、L12P / G29W / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / S72G / L98Q / Y105L、L12P / G29W / L63P / S72G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / Y105L / L106I、L12P / A26T / L63P / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、L12P / A26T / L63P / L98Q / Y105L、L12P / A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / L63P / L98Q / Y105L / L106I、L12P / G29W / D43N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / L98Q / M99L / Y105L、L12P / M56V / L63<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> P / V96I / L98Q / M99L / Y105L / Y115H、L12P / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">M99L / Y105L / L106N、L12P / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L / L106N / I117L、S14N / R16C / I18T / M56K / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T61A / L63P / A86T / M99L、S15P / I18V / M56T / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63H / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M87T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26T / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q82R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T / E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T61R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S20N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / I6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / M9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / M8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7V / N110K / I117E、A26T / L63P / S72G / L98Q / M99L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / I1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 17L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67N / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N58S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29W / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T53S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W / N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L / L106V、G29W / N58S / L63P / S72G / M87V / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、G2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W / N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / L106I / I117L、G29W / L63P / S72G / L98Q / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I117L、G29W / L63P / S72G / L98Q / Y105L / P121S,G2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9W / L63P / L98Q / M99L / Y105L、G29W / S72G / Q76R / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L / L106I / Q113H、G29W / M87K / T89S / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / I108V / I117L、G29W / M87K / I93V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L、G29W / L98Q / M99L / Y105L、E33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M / A42T / L98Q / Y105L、E33M / L63P / S72G / L98Q / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L、E33M / L63P / S72G / L98Q / Y105L / I108F、E33M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / S72G / L98Q / Y105L / I117L、E33M / Q82H / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / M99L / Y105L、E33V / A42S / M55T / L98Q / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L、T37S / M56V / L98Q / Y105L、V38I / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / M99L / Y105L、Q41L / Y54F / M56K / M99L / I10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8F、T53S / M56V / L98Q / Y105L、M55T / L63P / T71I / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、M55T / S72G / L98Q / M99L / Y105L、M55T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L106I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / I117L、M56R / L63P / L98Q / M99L / Y105L、M56T / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1R / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L / L106R / V116A、L63P / S72G / M87A / L98Q / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / I93L / L98Q / M99L / Y105L L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L / L106I / I117L、L63P / S72G / L98Q / Y105L、L63<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> P / S72G / L98Q / Y105L / L106I / I117L、L63P / S72G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、L63P / M87K / M99L / L106R、L63P / Q82H / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L、L63P / K95R、L63P / L98Q、L63P / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / M99L / Y105L、L63P / L98Q / M99L / Y105L / L106I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、L63P / L98Q / M99L / Y105L / I108V、L63P / L98Q / M9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 9L / Y105L / I117M、L63P / L98Q / Y105L、L63P / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V116A、L63P / L98R / N110K、L63P / M99L / Y105L / I1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 08F、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / R85G / L98Q / M99L / Y105L / L106I、S72G / L98<h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / I117T, L98Q / M99L / Y105L, L98Q / M99L / Y105L / L106I / I117T, L98Q / M99L / Y105L / L 106I / Y115N, L98Q / Y105L, and L98R / N110K.
[0224] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: Regarding the numbering in 2, A6T, V10A, L12F, L12H, L12P, S1 4N, S15P, R16C, R16G, R16H, I18A, I18F, I18N, I1 8T, I18V, A19V, S20N, V22A, V22I, E24Q, A26D, A2 6S, A26T, S27P, P28L, G29R, G29W, K30R, E33M, E3 3V, R35K, T37S, V38I, Q41L, A42S, A42T, A42V, D4 3N, Q45H, V46E, T47A, E48R, T53S, Y54F, M55R, M5 5T, M55V, M56K, M56L, M56R, M56T, M56V, N58D, N5 8S, E59D, E59G, T61A, T61I, T61N, T61R, T61S, L6 3H, L63P, D64E, D64N, D64V, D65G, I67N, I67T, I6 7V, T69A, T69I, T69S, T71A, T71I, S72G, S72T, S7 3R, N75D, Q76R, Q82H, Q82R, R85G, A86T, M87A, M8 7K, M87T, M87V, T89A, T89M, T89S, L91R, I93L, I9 3V, K95R, V96I, E97Q, L98Q, L98R, M99I, M99L, Y1 05F, Y105L, L106I, L106R, I108F, I108V, N110K, N110S, N110Y, Y115N, V116A, I117E, I117L, I117 M, I117T, E120D, C122P, D124P, D124I, S125I, D1 26P, and D126T. In some embodiments, the two or more amino acid modifications are selected from the numbered sequences set forth in SEQ ID NO:2. Regarding the A6T / A26T / M55T / M99L / Y105L, V10A / G29W / T53S / M56K / L63P / L98Q / Y105L / P121S, V10A / L6 3P / D64V / S72G / L98Q / M99L / Y105L, V10A / L63P / L 98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L , L12F / A26T / L63P / L98Q / Y105L / L106R, L12F / K3 0R / S72G / Q82R / L98Q / M99L / Y105L, L12H / I18V / A 42T / M55T / N58D / L98R / Y105L / L106I / P121S, L12 H / E33M / L98Q / Y105L, L12H / M55T / E59D / L63P / M9 9L, L12H / L63P / S72G / L98Q / Y105L, L12I / M55T / M 56V / I67T / M99L / L106R / I108F, L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M99L / Y105L / L106I / I1 17L, L12P / I18T / A26T / M55T / T69S / S72G / M99L / Y 105L, L12P / A26T, L12P / A26T / L63P, L12P / A26T / L63P / S72G / T89M / L98Q / M99L / Y105L, L12P / G29W / L63P / S72G / L98Q / Y105L, L12P / G29W / L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / L98Q / Y105L / L106I、L12P / A26T / L63P / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、L12P / A26T / L63P / L98Q / Y105L、L12P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / A26T / L63P / L98Q / Y105L / L106I、L12P / G29W / D4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3N / N58S / L63P / L98Q / M99L / Y105L、L12P / M56V / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / V96I / L98Q / M99L / Y105L / Y115H、L12P / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / L106N、L12P / L63P / S72G / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L / L106N / I117L、S14N / R16C / I18T / M56<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K / T61A / L63P / A86T / M99L、S15P / I18V / M56T / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L、R16C / G29W / E33V / M55T / L63P / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / Y105L、I18A / L63P / S72G / L98Q / Y105L、I18F / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / L98Q / M99L / Y105L / P121S、I18N / A26T / L63<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H / T89A / L98Q / M99L / Y105L、I18N / L63P / S72T / M8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7T / L98Q / Y105L / N110S、I18T / A26S / M55T / M56V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / M99L / Y105L / I117K、I18T / A26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T / L63P / S72G / L98Q / Y105L、I18T / A26T / L63P / Q8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2R / L98Q / Y105L、I18T / G29R / L63P / S72G / L98Q / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L、I18T / G29W / L63P / L98Q / Y105L、I18T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / E48R / L63P / T69S / L98Q / Y105L / N110Y、I18T / T6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1R / L63P / S72G / L98Q / M99L / Y105L、I18T / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / M87K / L98Q / M99L / Y105L、I18T / L63P / S72G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L、I18T / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I108V、I18V / A26T / L63P / D64E / L98Q / Y105L / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 106R / N110K、I18V / G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、A19V / G29W / R35K / L63P / L98Q / M99L / Y105L、S2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0N / A26T / L63P / L98Q / M99L / Y105L、V22A / L63P / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L / P119H、V22I / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I117M、E24Q / L63P / S72G / L98Q / M99L / Y105L、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26D / S72G / L98Q / M99L / Y105L、A26T / A42V / Q45H / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I67N / M87K / E97Q / M99L、A26T / V46E / L63P / D65G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q、A26T / T47A / M56K / L63P / S72G / Q82R / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L、A26T / T53S / M56K / L63P / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A26T / Y54F / M56K / M99L / Y105L、A26T / M55R / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L、A26T / M55T / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、A26T / M55T / L63P / L98Q / M99L / Y105L、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / L63P / D65G / L98Q / M99L / Y105L、A26T / L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M87V / N110K / I117E、A26T / L63P / S72G / L98Q / M99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / Y105L、A26T / L63P / S72G / L98Q / Y105L / L106I / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I117L、A26T / L63P / L98Q / M99L / Y105L、A26T / I67<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N / S72G / L98Q / M99L / Y105L、S27P / M56K / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / S73R / T89A / M99L / Y105L / I117M、P28L / E33V / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / S72G / L98Q / M99L / Y105L、P28L / E33V / L63P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / S72G / L98R / M99L / Y105L、G29W / T53S / M56K / N58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / L63P / M87V / L98Q / Y105L、G29W / T53S / M56K / N5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8S / L63P / M87V / L98Q / Y105L / I108V、G29W / T53S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M56K / N58S / L63P / M87V / L98Q / Y105L / P121S、G29<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W / T53S / M56K / T61N / L63P / L98Q / Y105L、G29W / T5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3S / M56K / L63P / Q82H / L98Q / M99I / Y105L、G29W / T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 53S / M56K / L63P / L98Q / Y105L、G29W / T53S / L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S72G / L98Q / Y105L、G29W / M55V / E59G / L63P / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、G29W / M56T / L63P / L98Q / Y105L / L106I / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 117L、G29W / N58D / I67V / L98Q / M99L / Y105L、G29W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / D64N / L98Q / M99L / Y105L、G29W / N58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / L63P / T69I / L98Q / M99L / Y105L、G29W / N58S / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">3P / S72G / L98Q / Y105L、G29W / N58S / L63P / S72G / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / Y105L / L106I、G29W / N58S / L63P / S72G / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L / L106V、G29W / N58S / L63P / S72G / M87V / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / Y105L、G29W / N58S / L63P / Q82R / L98Q / Y105L、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G29W / N58S / L63P / M87T / L98Q / M99L / Y105L、G29W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / N58S / L63P / L98Q / Y105L、G29W / E59G / L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / Y105L、G29W / T61I / L63P / S72G / L98Q / M99L / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L、G29W / L63P / D65G / S72G / L98Q / Y105L、G29W / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / I67V / S72G / L98Q / Y105L、G29W / L63P / S72G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L / L106I、G29W / L63P / S72G / L98Q / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L / L106I / I117L、G29W / L63P / S72G / L98Q / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L / I117L、G29W / L63P / S72G / L98Q / Y105L / P121S,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G29W / L63P / L98Q / M99L / Y105L、G29W / S72G / Q76R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L / L106I / Q113H、G29W / M87K / T89S / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L / I108V / I117L、G29W / M87K / I93<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V / L98Q / M99L / Y105L、G29W / L98Q / M99L / Y105L、E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 33M / A42T / L98Q / Y105L、E33M / L63P / S72G / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L、E33M / L63P / S72G / L98Q / Y105L / I108F、E3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3M / L63P / S72G / L98Q / Y105L / I117L、E33M / Q82H / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L、E33V / A42S / M55T / L98Q / M99L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / Y105L、T37S / M56V / L98Q / Y105L、V38I / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / M99L / Y105L、Q41L / Y54F / M56K / M99L / I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 108F、T53S / M56V / L98Q / Y105L、M55T / L63P / T71I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L、M55T / S72G / L98Q / M99L / Y105L、M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5T / E97Q / M99L / Y105F、M56K / L63P / N75D / V96I / M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L / L106I、M56L / L63P / L98Q / Y105L / L10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6I / I117L、M56R / L63P / L98Q / M99L / Y105L、M56T / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L91R / L98Q / Y105L、M56V / E59G / L63P / S72G / M87K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / I93V / L98Q / M99L / Y105L / I117E、T61A / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / M99L / Y105L、L63P / T69A / L98Q / M99L / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L / L106R / V116A、L63P / S72G / M87A / L98Q / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L、L63P / S72G / I93L / L98Q / M99L / Y105L、L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / M99L / Y105L、L63P / S72G / L98Q / M99L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y105L / L106I / I117L、L63P / S72G / L98Q / Y105L、L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / S72G / L98Q / Y105L / L106I / I117L、L63P / S72<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G / Y105L、L63P / M87K / M99L / L106R、L63P / Q82H / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L、L63P / K95R、L63P / L98Q、L63P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">L98Q / M99L / Y105L、L63P / L98Q / M99L / Y105L / L10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6I、L63P / L98Q / M99L / Y105L / I108V、L63P / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 99L / Y105L / I117M、L63P / L98Q / Y105L、L63P / L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / V116A、L63P / L98R / N110K、L63P / M99L / Y105L / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> I108F、I67V / S72G / Q82H / T89A / L98Q / M99L / Y105<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、S72G / R85G / L98Q / M99L / Y105L / L106I、S72G / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 98Q / M99L / Y105L / I117T、L98Q / M99L / Y105L、L98<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q / M99L / Y105L / L106I / I117T、L98Q / M99L / Y105L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L106I / Y115N、L98Q / Y105L、L98R / N110K、T89A / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L98Q / M99L / Y105L / L106I / Y115N / E120D / C122P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D124P / S125I / D126P、N58S / L63P / T71A / S72G / L9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8Q / M99L / Y105L / D124I / S125P / D126T、R16G / E33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M / N58S / E59G / L63P / L98Q / Y105L / E120D / C122P / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D124P / S125I / D126P、G29W / L63P / S72G / L98Q / Y1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 05L / P121S / D126T、L12H / E33M / L98Q / Y105L、T53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S / M56K / N58S / L63P / M87V / L98Q / Y105L、I18T / A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6T / M55T / M56K / L63P / L98Q / M99L / Y105L、I18T / A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / M56K / L63P / L98Q / Y105L、T53S / L63P / L98Q、<h2 style=";text-align:left;direction:ltr"> T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N58S / L63P / M87V / Y105L, T53 S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N5 8S / M87V / L98Q / Y105L, T53S / M56K / L63P / M87V / L 98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L , M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98 Q / Y105L, E33V / M99L / Y105L, E33V / L98Q / M99L, E 33V / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R 16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T / L98Q / Y105L, G29W / M56 T / L98Q / Y105L, L12F / G29W / L98Q / Y105L, L12F / L 98Q / Y105L, R16H / L98Q / Y105L, G29W / L98Q / Y105 L, M56T / L98Q / Y105L, L12F / R16H / G29W / M56T / S7 2G / L98Q / Y105L, and G29W / M56T / S72G / L98Q / Y10 It is 5L.
[0225] In some embodiments, the mutant CTLA-4 polypeptide additionally comprises SEQ ID NO: D NO: 2 or 569.
[0226] In some embodiments, the mutant CTLA-4 polypeptide has a substitution listed in Table 2. (mutations). Table 2 also includes wild-type CTLA-4 or exemplary mutations. against the extracellular domain (ECD) or IgV domain of the CTLA-4 polypeptide Exemplary sequences are provided by reference to SEQ ID NOs. Table 2 also provides immunoglobulins. Exemplary sequences of mutant CTLA-4 polypeptides attached to polypeptide Fc (CTLA-4 vI) "Immunomodulatory Fc fusions" such as "CTLA-4-Fc variant fusions" also known as gD-Fc fusions Such fusion polypeptides are further described below. The exact locus or residue corresponding to a given domain is used to identify the domain. or the method used to classify it. , the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., IgV) are Additionally, to ensure the correct folding of the domains when expressed, the mutant IgS The examples of SEQ ID NOs in Table 2 should not be construed as limiting. For example, it is understood that the IgV domain of a mutant CTLA-4 polypeptide should not be used. Certain domains, such as the nucleotide sequence of the nucleotides, may be more specific than the amino acid sequence set forth in the respective SEQ ID NOs. Longer or shorter, 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 The amino acid may be several amino acids, longer or shorter, such as 1 or 2 amino acids.
[0227] In some embodiments, the mutant CTLA-4 polypeptide has a mutation listed in Table 2. In some embodiments, the mutant CTLA-4 polypeptide comprises any of , any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., SEQ ID NO: 4~97, 99~104, or 106~155) In some embodiments, the mutant CTLA-4 polypeptide comprises any of the polypeptides listed in Table 2. Any of the extracellular domain (ECD) sequences of SEQ ID NO:4 ~97, 99–104, or 106–155) and at least 96 % identity, 97% identity, 98% identity, or 99% identity, etc. 90% identity, at least 91% identity, at least 92% identity, at least 9 Polypeptides showing 3% identity, at least 94% identity, and at least 95% identity The modified CTLA-4 fragments are also included in the modified CTLA-4 fragments. In some embodiments, the mutant CTLA-4 polypeptide contains an amino acid modification, e.g., a substitution. The polypeptide may be any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., i.e., any of SEQ ID NOs: 4-97, 99-104, or 106-155 and a specific binding fragment of either one of the following that is not present in wild-type or unmodified CTLA-4: Each SEQ ID NO contains one or more of the amino acid modifications, eg, substitutions.
[0228] In some embodiments, the mutant CTLA-4 polypeptide has a mutation listed in Table 2. In some embodiments, the mutant CTLA-4 polypeptide comprises any of , any of the extracellular domain (ECD) sequences listed in Table 2 (i.e., SEQ ID NO: 4-97, 99-104, 106-155, 570-602, or 63 In some embodiments, the mutant CTLA-4 polypeptide comprises The peptide may be any of the extracellular domain (ECD) sequences listed in Table 2 (i.e. , SEQ ID NO: 4-97, 99-104, 106-155, 570-602, or 636) at least 96% identity, 97% identity, 98% identity % identity, or 99% identity, such as at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% Identities, including polypeptide sequences that exhibit at least 95% identity, wild-type or unmodified The amino acid sequences of each SEQ ID NO: 1 contain amino acid modifications, e.g., substitutions, that are not present in CTLA-4. In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of those listed in Table 2. Any of the extracellular domain (ECD) sequences (i.e., SEQ ID NOs: 4-9 Any one of 7, 99-104, 106-155, 570-602, or 636 each of which contains a specific binding fragment that is not present in wild-type or unmodified CTLA-4 The amino acid sequence of SEQ ID NO: 1 contains one or more of the amino acid modifications, e.g., substitutions, of SEQ ID NO: 1.
[0229] In some embodiments, the mutant CTLA-4 polypeptide is an Ig polypeptide listed in Table 2. Any of the V sequences (i.e., any of SEQ ID NOs: 156-285) In some embodiments, the mutant CTLA-4 polypeptide comprises any one of the polypeptides listed in Table 2. Any of the IgV sequences listed in (i.e., SEQ ID NOs: 156-2 85) with at least 96% identity, 97% identity, 98% identity identity, or 99% identity, at least 90% identity, at least 91% identity, etc. Sex, at least 92% identity, at least 93% identity, at least 94% identity , comprising a polypeptide sequence that exhibits at least 95% identity, and A-4 contains amino acid modifications, e.g., substitutions, of the respective SEQ ID NOs. not present in A-4. In some embodiments, the mutant CTLA-4 polypeptide is an IgV sequence listed in Table 2. Any of the sequences (i.e., any of SEQ ID NOs: 156 to 285) 1) specific binding fragments of CTLA-4 that are not present in wild-type or unmodified CTLA-4, Each SEQ ID NO: contains one or more amino acid modifications, e.g., substitutions.
[0230] In some embodiments, the mutant CTLA-4 polypeptide is an Ig polypeptide listed in Table 2. Any of the V sequences (i.e., SEQ ID NOs: 156-285, 603-6 35, or 637). In some embodiments, the mutant C The TLA-4 polypeptide may be any of the IgV sequences listed in Table 2 (i.e., Any of SEQ ID NOs: 156-285, 603-635, or 637 at least 96% identity, 97% identity, 98% identity, or 99% identity with one of the Identity, etc., at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% containing polypeptide sequences that exhibit identity and are not present in wild-type or unmodified CTLA-4 Contains amino acid modifications, e.g., substitutions, of the respective SEQ ID NOs. The mutant CTLA-4 polypeptide may be any of the IgV sequences listed in Table 2 ( That is, among SEQ ID NOs: 156-285, 603-635, or 637 and (ii) a specific binding fragment of a CTLA-4 polypeptide that is present in wild-type or unmodified CTLA-4. Contains amino acid modifications, e.g., substitutions, that are not of the respective SEQ ID NO:
[0231] Table 2: Exemplary mutant CTLA-4 polypeptides [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]
[0232] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequences set forth in In some embodiments, the CD80 ectodomain exhibits increased binding affinity compared to the CD80 ectodomain. In some embodiments, the mutant CTLA-4 polypeptide is a polypeptide of the invention as set forth in SEQ ID NO: 2 or 3. of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, including, for example, In some embodiments, the CTLA- 4 Polypeptides include wild-type or nucleotide sequences, such as those set forth in SEQ ID NO: 2 or 3. The ectodomain of CD80 and the ectodomain of CD86 are significantly increased compared to unmodified CTLA-4. These results demonstrate increased affinity for cysteine.
[0233] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequences set forth in In some embodiments, the ectodomain of ICOSL is increased in affinity for the ectodomain of ICOSL compared to the ectodomain of ICOSL. In the present specification, the CTLA-4 polypeptide is a sequence such as that set forth in SEQ ID NO:2 or 3. compared to wild-type or unmodified CTLA-4, comprising the ectodomain of ICOSL and In some embodiments, CT LA-4 polypeptides include those having sequences such as those set forth in SEQ ID NO:2 or 3. Compared with live or unmodified CTLA-4, the ectodomain of ICOSL and CD86 Showing increased affinity for the ectodomain.
[0234] In some embodiments, the CTLA-4 polypeptide is SEQ ID NO:2 or 3, or a sequence similar to that described in Example 1. Increased for the ectodomain of CD80, CD80 ectodomain, and CD86 ectodomain It shows good affinity.
[0235] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequences set forth in It exhibits increased binding affinity to the ectodomain of CD80 compared to CD86 and / or exhibits reduced binding affinity for binding to the ectodomain of ICOSL. In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NO:2 or 3. and increased affinity for the ectodomain of CD80 and the ectodomain of CD86. and exhibits reduced affinity for the ectodomain of ICOSL. The mutant CTLA-4 polypeptide has the sequence set forth in SEQ ID NO: 2 or 3. and the like, which exhibits a reduced expression of CD80 compared to wild-type or unmodified CTLA-4 polypeptides, The CD86 ectodomain exhibits increased affinity for the CD86 ectodomain and ICOSL ectodomain. In some embodiments, the mutant CTLA- 4 Polypeptides include wild-type or nucleotide sequences, such as those set forth in SEQ ID NO: 2 or 3. or an increased binding to the ectodomain of CD80 compared to an unmodified CTLA-4 polypeptide. It showed a reduced affinity for the ectodomain of CD86 and the ectodomain of ICOSL. It shows little affinity.
[0236] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequences set forth in It exhibits increased binding affinity to the ectodomain of CD86 compared to CD80 and / or exhibits reduced binding affinity for binding to the ectodomain of ICOSL. In some embodiments, the mutant CTLA-4 polypeptide is selected from the group consisting of SEQ ID NO:2 or 3. and increased affinity for the ectodomain of CD86 and the ectodomain of CD80. and exhibits reduced affinity for the ectodomain of ICOSL. The mutant CTLA-4 polypeptide has the sequence set forth in SEQ ID NO: 2 or 3. and the like, which is a cytotoxic effect of CTLA-4 on the surface of CD86 compared to wild-type or unmodified CTLA-4 polypeptides, including The CD80 ectodomain exhibits increased affinity for the CD80 ectodomain and ICOSL ectodomain. In some embodiments, the mutant CTLA- 4 Polypeptides include wild-type or nucleotide sequences, such as those set forth in SEQ ID NO: 2 or 3. or an increased affinity to the ectodomain of CD86 compared to the unmodified CTLA-4 polypeptide. It showed a reduced affinity for the ectodomain of CD80 and the ectodomain of ICOSL. It shows little affinity.
[0237] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, compared to wild-type or unmodified CTLA-4 polypeptides, such as those comprising the sequences set forth in Compared to CD80, it exhibits increased binding affinity for the ectodomain of ICOSL. and / or exhibit reduced binding affinity for binding to the ectodomain of CD86. In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO:2 or or 3, or a wild-type or unmodified CTLA-4 polypeptide, and increased affinity for the ectodomain of ICOSL and the ectodomain of CD80. and exhibits reduced affinity for the ectodomain of CD86. The mutant CTLA-4 polypeptide may have a sequence such as that set forth in SEQ ID NO:2 or 3. the external appearance of ICOSL compared to wild-type or unmodified CTLA-4 polypeptides, including It exhibits increased affinity for the CD80 ectodomain and CD86 ectodomain. In some embodiments, the mutant CTLA-4 exhibits reduced affinity for the CTLA-4 domain. The polypeptide may be a wild-type or Increased ICOSL activity against the ectodomain compared to unmodified CTLA-4 polypeptide exhibiting similar affinity to the ectodomain of CD80 and the ectodomain of CD86. It shows good affinity.
[0238] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, such as a wild-type or unmodified CTLA-4 polypeptide. ectodomain of CD80 compared to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, the ectodomain of CD80 exhibits increased binding affinity to The increased affinity for the unmodified CTLA-4 to the ectodomain of CD80 is due to the increased affinity of the unmodified CTLA-4 to the ectodomain of CD80. Compared to the symbiotic sex, over 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold increases. In some embodiments, the modified CTLA-4 polypeptide is Thus, mutant CTLA-4 polypeptides that exhibit increased binding affinity to CD80 are non- In the extracellular domain of modified or wild-type CTLA-4 or in its IgV domain, there is With respect to the numbering of ID NO:2, one or more amino acid substitutions V10A, L12F, R 16H, I18T, A26T, G29W, E33V, A42V, Q45H, T53S, M 55T, M56K, M56T, N58S, L63P, I67N, Q82R, M87K, M 87V, E97Q, L98Q, M99L, Y105L, I108V, and / or P1 In some of these embodiments, wild-type or unmodified CTLA Mutant CTLA-4 polypeptides exhibit increased affinity for CD80 compared to the -4 polypeptide 4 polypeptide is the extracellular domain of unmodified or wild-type CTLA-4 or its IgV In the domain, one or more amino acid substitutions with respect to the numbering of SEQ ID NO:2 18T, A26T, G29W, E33V, A42V, Q45H, T53S, M56K, N 58S, L63P, I67N, Q82R, M87K, M87V, E97Q, L98Q, M 99L, Y105L, and / or I108V. Some have a reduced binding to CD80 compared to wild-type or unmodified CTLA-4 polypeptides. Mutant CTLA-4 polypeptides that exhibit increased binding affinity are comparable to unmodified or wild-type CTLA-4 polypeptides. In the extracellular domain of TLA-4 or in its IgV domain, Regarding numbering, one or more amino acid substitutions I18T, A26T, G29W, A42V, Q45H, N58S, L63P, I67N, Q82R, M87K, M87V, L98Q, and / or Y105L. In some of these embodiments, wild-type or or increased binding affinity to CD80 compared to unmodified CTLA-4 polypeptides. The mutant CTLA-4 polypeptides exhibiting the same extracellular domain as unmodified or wild-type CTLA-4. In the main or IgV domain, with respect to the numbering of SEQ ID NO:2, 18T, A26T, G29W, N58S, L63P, M87K, M87V, L98Q and and / or Y105L. The mutant CTLA-4 polypeptide has the sequence set forth in SEQ ID NO: 2 or 3. wild-type or unmodified CTL, such as a wild-type or unmodified CTLA-4 polypeptide comprising A-4 polypeptide, which exhibits increased binding to the ectodomain of CD80 compared to the A-4 polypeptide. Some embodiments have at least two, three, four, or five of such amino acid substitutions. In 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 / L98 Q / Y105L, A26T / A42V / Q45H / I67N / M87K / E97Q / M9 9L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y1 05L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, V10A / G29W / T53S / M56K / L6 3P / L98Q / Y105L / P121S, T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y105L, T53S / M56K / N58S / L63P / M87 V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L98Q / Y105L , T53S / M56K / N58S / M87V / L98Q / Y105L, T53S / M56 K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M8 7V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y 105L, E33V / L98Q / M99L, L12F / R16H / G29W / M56T / L98Q, L12F / R16H / G29W / M56T / Y105L, L12F / R16H / G29W / L98Q / Y105L, L12F / R16H / M56T / L98Q / Y10 5L, G29W / M56T / L98Q / Y105L, L12F / G29W / L98Q / Y 105L, L12F / L98Q / Y105L, R16H / L98Q / Y105L, G29 W / L98Q / Y105L, or M56T / L98Q / Y105L. In embodiments, the amino acid substitutions are I18T / G29W / L63P / L98Q / Y105L , G29W / N58S / L63P / L98Q / Y105L, G29W / N58S / L63 P / Q82R / L98Q / Y105L, A26T / A42V / Q45H / I67N / M8 7K / E97Q / M99L, G29W / T53S / M56K / N58S / L63P / M8 7V / L98Q / Y105L / P121S, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, L98Q / M99L / Y1 05L, E33V / L98Q / Y105L, T53S / M56K / N58S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105 In some embodiments, the amino acid substitutions are E33V / L98Q / M99L, or E33V / L98Q / M99L. Conversion is I18T / G29W / L63P / L98Q / Y105L, G29W / N58S / L 63P / 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 selected from the group consisting of C, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C39, C31, in the TLA-4 extracellular domain, see, e.g., exemplary SEQ ID NOs: in Table 2. In some embodiments, any of the above substitutions is selected from SEQ ID NO:3 and in the CTLA-4 extracellular domain set forth in, for example, exemplary SEQ ID NOs: Please refer to.
[0239] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3, such as a wild-type or unmodified CTLA-4 polypeptide. ectodomain of CD86, compared to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, the ectodomain of CD86 exhibits increased binding affinity to The increased affinity for the unmodified CTLA-4 to the ectodomain of CD86 is due to the increased affinity of the unmodified CTLA-4 to the ectodomain of CD86. Compared to the symbiotic sex, over 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold increases. In some of the forms, compared to wild-type or unmodified CTLA-4 polypeptide: The mutant CTLA-4 polypeptides that exhibit increased binding affinity to CD86 are or SEQ ID NO: 1 in the extracellular domain of wild-type CTLA-4 or in its IgV domain With respect to the numbering of D NO:2, one or more amino acid substitutions L12F, L12H, L12 P, R16H, I18T, I18V, S20N, A26T, G29R, G29W, E33 M, E33V, A42S, A42V, T47A, T53S, M55T, M56K, M56 R, M56T, M56V, N58D, N58S, T61I, T61N, T61R, T61 S, L63P, D65G, I67N, I67V, T69A, T69I, S72G, Q76 R, Q82H, Q82R, R85G, M87K, M87V, T89A, T89M, T89 S, V96I, L98Q, L98R, M99L, P102L, Y105L, L106I, L106N, L106R, L106V, I108F, I108V, Q113H, Y115 H, V116A, I117L, I117T, and / or P121S. In some embodiments, the modified CTLA-4 polypeptide is Thus, a mutant CTLA-4 polypeptide that exhibits increased binding affinity to CD86 is The extracellular domain of unmodified or wild-type CTLA-4 or its IgV domain contains SE With respect to the numbering of Q ID NO:2, one or more amino acid substitutions L12P, A26T, Possessing M55T, L63P, S72G, L98Q, M99L, and / or Y105L In some embodiments, the mutant CTLA-4 polypeptides include those amino acids and having at least two, three, four, or five of the substitutions, and wild-type or unmodified CTLA-4 polypeptide, such as a wild-type or unmodified CTLA-4 polypeptide comprising the sequence set forth in claim 3. The modified CTLA-4 polypeptide exhibits increased binding to the ectodomain of CD86 compared to the unmodified CTLA-4 polypeptide. In some embodiments, the amino acid substitutions are I18T / T61R / L63P / S72G / L98Q / M99L / P102L / Y105L, L12P / A26T / L63 P / S72G / T89M / L98Q / M99L / Y105L, I18T / G29R / L6 3P / S72G / L98Q / M99L / Y105L, S72G / L98Q / M99L / Y 105L / I117T, M56R / L63P / L98Q / M99L / Y105L, L63 P / L98Q / M99L / Y105L / L106I, A26T / M55T / L63P / L 98Q / M99L / Y105L, E33V / A42S / M55T / L98Q / M99L / Y105L, G29W / N58S / L63P / Q82R / L98Q / Y105L, E33 M / L63P / S72G / L98Q / Y105L / I117L, A26T / I67N / S 72G / L98Q / M99L / Y105L, L12F / A26T / L63P / L98Q / Y105L / L106R, S20N / A26T / L63P / L98Q / M99L / Y10 5L, G29W / T61I / L63P / S72G / L98Q / M99L / Y105L, G 29W / N58S / L63P / T69I / L98Q / M99L / Y105L, L12P / L63P / S72G / L98Q / M99L / Y105L / L106N, L63P / T69 A / L98Q / M99L / Y105L / L106R / V116A, G29W / N58S / L63P / S72G / L98Q / Y105L, G29W / L63P / D65G / S72G / L98Q / Y105L, T53S / M56V / L98Q / Y105L, L63P / S7 2G / L98Q / Y105L, G29W / L63P / S72G / L98Q / Y105L / L106I、L12F / R16H / G29W / M56T / L98Q / Y105L、G29 W / N58S / L63P / S72G / M87V / L98Q / Y105L、G29W / S7 2G / Q76R / L98Q / Y105L / L106I / Q113H、G29W / N58S / L63P / S72G / L98Q / Y105L / L106V、G29W / N58D / I6 7V / L98Q / M99L / Y105L、I67V / S72G / Q82H / T89A / L 98Q / M99L / Y105L、S72G / R85G / L98Q / M99L / Y105L / L106I、L63P / L98Q / M99L / Y105L、A26T / T47A / M5 6K / L63P / S72G / Q82R / L98Q / M99L / Y105L、A26T / M 55T / L63P / S72G / L98Q / M99L / Y105L、L12H / I18V / A42T / M55T / N58D / L98R / Y105L / L106I / P121S、E3 3M / L63P / S72G / L98Q / Y105L、G29W / M87K / T89S / L 98Q / M99L / Y105L / I108V / I117L、L12P / M56V / L63 P / V96I / L98Q / M99L / Y105L / Y115H、G29W / T53S / M 56K / T61N / L63P / L98Q / Y105L、I18T / T61R / L63P / S72G / L98Q / M99L / Y105L、L12P / L63P / S72G / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y105L / I1 08F、L12P / R16H / A26T / T61S / L63P / M87V / L98Q / M 99L / Y105L / L106I / I117L、L98Q / M99L / Y105L、T5 3S / M56K / N58S / M87V / L98Q / Y105L、L12F / R16H / G 29W / M56T / L98Q, L12F / G29W / L98Q / Y105L, or L1 2F / L98Q / Y105L. In some embodiments, the amino acid substitution is A26 T / M55T / L63P / S72G / L98Q / M99L / Y105L, or L12P / L63P / S72G / L98Q / M99L / Y105L. Any of the above substitutions may be used in the CTLA-4 extracellular domain of SEQ ID NO:2. domain, see, for example, the exemplary SEQ ID NOs listed in Table 2. In embodiments, any of the above substitutions is a CTLA as set forth in SEQ ID NO:3. -4 extracellular domain, see, for example, the exemplary SEQ ID NOs in Table 2.
[0240] In some embodiments, the mutant CTLA-4 polypeptide has SEQ ID NO: 2 or 3. The ectodomain of ICOSL is specifically targeted to the CTLA-4 polypeptide, as compared to the native or unmodified CTLA-4 polypeptide. In some embodiments, the ectodomain of ICOSL exhibits increased binding affinity to the The increased affinity for ICOSL is due to the binding of unmodified CTLA-4 to the ectodomain of ICOSL. 1.2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x compared to affinity , 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold increase. In some embodiments, compared to wild-type or unmodified CTLA-4 polypeptides The mutant CTLA-4 polypeptides exhibiting increased binding affinity for ICOSL are non- In the extracellular domain of modified or wild-type CTLA-4 or in its IgV domain, there is With respect to the numbering of ID NO:2, one or more amino acid substitutions V10A, L12F, L 12I, R16H, I18N, I18T, I18V, A19V, A26T, G29W, E 33M, E33V, R35K, V38I, A42V, Q45H, T47A, T53S, M 55T, M56K, M56V, N58D, N58S, T61A, T61R, L63H, L 63P, D64E, D64N, D64V, D65G, I67N, I67T, I67V, T 69I, S72G, Q76R, Q82H, Q82R, R85G, M87K, M87T, M 87V, T89A, T89S, I93L, I93V, E97Q, L98Q, M99L, M 99I, Y105L, L106I, L106R, I108F, I108V, N110K, Among these embodiments, the amino acid sequence has Q113H, I117L, and / or P121S. In some of the cases, ICOSL is more potent than wild-type or unmodified CTLA-4 polypeptides. Mutant CTLA-4 polypeptides that exhibit increased binding affinity to the unmodified or wild-type CTLA-4 polypeptides In the extracellular domain of native CTLA-4 or in its IgV domain, there is : With respect to numbering 2, one or more amino acid substitutions V10A, L12F, R16H, I1 8T, I18V, A19V, A26T, G29W, E33V, R35K, A42V, Q4 5H, T47A, T53S, M55T, M56K, M56T, N58D, N58S, L6 3P, D64E, D64V, I67N, I67V, S72G, Q82H, Q82R, R8 5G, M87K, M87T, M87V, T89A, T89S, I93V, E97Q, L9 8Q, M99L, Y105L, L106I, L106R, I108F, I108V, N1 In some of these embodiments, Increased response to ICOSL compared to wild-type or unmodified CTLA-4 polypeptide The mutant CTLA-4 polypeptides that exhibit binding affinity are similar to unmodified or wild-type CTLA-4. or in the IgV domain thereof, according to the numbering of SEQ ID NO:2 Regarding one or more amino acid substitutions L12F, I18T, I18V, A26T, G29W, E33V, A42V, Q45H, T53S, M55T, M56K, N58D, N58S, L63P, D64E, I67N, I67V, S72G, Q82H, R85G, M87K, M87V, T89A, T89S, I93V, E97Q, L98Q, M99I, M99L, Y105L, L106I, L106R, I108F, I108V, N110K, and / or or N117L. In some of these embodiments, the wild-type or unmodified exhibit increased binding affinity for ICOSL compared to mutant CTLA-4 polypeptides The mutant CTLA-4 polypeptides may be derived from the extracellular domain of unmodified or wild-type CTLA-4. or in its IgV domain, one or more with respect to the numbering of SEQ ID NO:2 Amino acid substitutions I18T, A26T, G29W, T53S, M55T, M56K, N58 S, L63P, S72G, L98Q, M99L, Y105L, L106I, and / or In some embodiments, the mutant CTLA-4 polypeptide has: having at least 2, 3, 4, or 5 of such amino acid substitutions, Wild-type or unmodified CTLA-4 polypeptides comprising the sequence set forth in NO:2 or 3 and the external domain of ICOSL, as compared to wild-type or unmodified CTLA-4 polypeptides. In some embodiments, the amino acid substitution is L63P / S 72G / L98Q / M99L / Y105L / L106I / I117L, G29W / L98 Q / M99L / Y105L, M55T / S72G / L98Q / M99L / Y105L, L 63P / Q82H / L98Q / M99L / Y105L, I18T / L63P / S72G / L98Q / M99L / Y105L, T61A / L63P / S72G / L98Q / M99L / Y105L, V38I / L63P / S72G / L98Q / M99L / Y105L, L6 3P / S72G / I93L / L98Q / M99L / Y105L, L12I / M55T / M 56V / I67T / M99L / L106R / I108F, I18N / A26T / L63H / T89A / L98Q / M99L / Y105L, G29W / N58S / L63P / M87 T / L98Q / M99L / Y105L, G29W / N58S / L63P / D64N / L9 8Q / M99L / Y105L, I18T / L63P / S72G / M87K / L98Q / M 99L / Y105L, L63P / M87K / M99L / L106R, L63P / M99L / Y105L / I108F, G29W / L63P / L98Q / M99L / Y105L, A 26T / L63P / D65G / L98Q / M99L / Y105L, V10A / L63P / D64V / S72G / L98Q / M99L / Y105L, I18V / A26T / L63P / D64E / L98Q / Y105L / L106R / N110K, A19V / G29W / R 35K / L63P / L98Q / M99L / Y105L, G29W / N58S / L63P / T69I / L98Q / M99L / Y105L, G29W / T53S / M56K / L63P / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5L、A26T / T53S / L63P / L98Q / Y105L / L106I / I117L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、G29W / S72G / Q76R / L98Q / Y105L / L106I / Q113H、G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 29W / N58D / I67V / L98Q / M99L / Y105L、I67V / S72G / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q82H / T89A / L98Q / M99L / Y105L、S72G / R85G / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99L / Y105L / L106I、A26T / T47A / M56K / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / Q82R / L98Q / M99L / Y105L、A26T / M55T / L63P / S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 72G / L98Q / M99L / Y105L、G29W / M87K / I93V / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L、G29W / T53S / M56K / L63P / Q82H / L98Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / M99I / Y105L、L63P / L98Q / M99L / Y105L / I108V、A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 26T / A42V / Q45H / I67N / M87K / E97Q / M99L、E33M / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 63P / S72G / L98Q / Y105L、G29W / M87K / T89S / L98Q / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M99L / Y105L / I108V / I117L、I18T / T61R / L63P / S7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2G / L98Q / M99L / Y105L、E33M / L63P / S72G / L98Q / Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 105L / I108F、G29W / T53S / M56K / N58S / L63P / M87V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L98Q / Y105L / P121S、G29W / T53S / M56K / N58S / L6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3P / M87V / L98Q / Y105L / I108V、T53S / M56K / N58S / <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L63P / M87V / L98Q / Y105L、I18T / A26T / M55T / M56K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / L63P / L98Q / M99L / Y105L、I18T / A26T / M56K / L63<h2 style=";text-align:left;direction:ltr"> P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y1 05L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L98Q / Y105L, E33V / M99L, T53 S / M56K / N58S / L63P / M87V / L98Q, T53S / M56K / N58 S / L63P / L98Q / Y105L, T53S / M56K / N58S / M87V / L9 8Q / Y105L, T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M87V / L98Q / Y105L, E33V / L98Q / M99L, L12 F / R16H / G29W / M56T / Y105L, or L12F / L98Q / Y105 In some embodiments, the amino acid substitutions are G29W / L98Q / M99L / Y105L, L63P / M99L / Y105L / I108F, I18V / A26T / L6 3P / D64E / L98Q / Y105L / L106R / N110K, G29W / N58D / I67V / L98Q / M99L / Y105L, I67V / S72G / Q82H / T89 A / L98Q / M99L / Y105L, S72G / R85G / L98Q / M99L / Y1 05L / L106I, G29W / M87K / I93V / L98Q / M99L / Y105L ,G29W / T53S / M56K / L63P / Q82H / L98Q / M99I / Y105 L, A26T / A42V / Q45H / I67N / M87K / E97Q / M99L, G29 W / M87K / T89S / L98Q / M99L / Y105L / I108V / I117L, G29W / T53S / M56K / N58S / L63P / M87V / L98Q / Y105L / I108V, T53S / M56K / N58S / L63P / M87V / L98Q / Y10 5L, I18T / A26T / M55T / M56K / L63P / L98Q / M99L / Y1 05L, I18T / A26T / M56K / L63P / L98Q / Y105L, T53S / L63P / L98Q, T53S / L63P / Y105L, T53S / M56K / N58S / L63P / M87V / Y105L, L98Q / M99L / Y105L, E33V / L9 8Q / Y105L, E33V / M99L, T53S / M56K / N58S / L63P / L 98Q / Y105L, T53S / M56K / N58S / M87V / L98Q / Y105L , T53S / M56K / L63P / M87V / L98Q / Y105L, T53S / N58 S / L63P / M87V / L98Q / Y105L, M56K / N58S / L63P / M8 7V / L98Q / Y105L, E33V / L98Q / M99L, or L12F / L98 Q / Y105L. In some embodiments, any of the above substitutions is SEQ in the CTLA-4 extracellular domain as set forth in ID NO:2, e.g., as set forth in Table 2 See exemplary SEQ ID NOs: 1 and 2. In some embodiments, any of the above substitutions may be is also in the CTLA-4 extracellular domain as set forth in SEQ ID NO:3, e.g., Table See exemplary SEQ ID NO:2.
[0241] In some embodiments, the CTLA-4 polypeptide may be modified to include a CTLA-4 polypeptide. at least one affinity-engineered IgSF domain (e.g., The provided mutants contain an extracellular domain having an IgV) or a specific binding fragment thereof. A CTLA-4 polypeptide is a wild-type or non-wild-type polypeptide when bound to one or more binding partners. CD80, CD86, and / or ICO compared to modified CTLA-4 polypeptides stimulatory receptors that can be engaged by one or more binding partners of CTLA-4, such as SL It exhibits altered signaling (eg, decreased / inhibited signaling) by the receptor. In some embodiments, the stimulatory receptor is expressed on the surface of a T cell and responds to intracellular signals. In response, they can release cytokines, such as CD28 or ICOS. In some embodiments, the altered signaling may be, for example, an identified variant and / or Cytokines were detected after incubation with wild-type or unmodified CTLA-4 polypeptides. Determined by assays measuring IL-2 release (e.g., IL-2 release or IFN-gamma) wild-type or unmodified in the same format (e.g., Fc fusion protein) as specified. Signal transduction affected by CTLA-4 polypeptide regulatory sequences is distinct. Typical assays are described in the Examples. In some cases, the assay is a method such as a mixed lymphocyte reaction. which cell-based assays, and the resulting readout or function is The signaling activity of the functional binding partners expressed on the surface of the cell is the sum of the signaling activities of the functional binding partners expressed on the surface of the cell. As discussed elsewhere in this document, in some embodiments, mutant CTLAs are provided. The form of the -4 polypeptide affects the type of activity, e.g., agonist or antagonist. In some embodiments, the mutant CTLA-4 polypeptide may affect IgV domains. an extracellular domain containing a multimerization domain such as an Fc domain or is a fusion protein of its specific binding fragment, and the altered signal transduction is In another embodiment, the present format is based on an antagonist assay for inhibiting the activity of the stimulator. This may result in increased or agonist activity of stimulatory receptors.
[0242] III. Variant Polypeptide Formats The immunomodulatory polypeptides comprising mutant CTLA-4 provided herein are soluble proteins. can be formulated in a variety of ways, including as proteins, membrane-bound proteins, or secreted proteins In some embodiments, a particular format may be selected for a desired therapeutic application. In some cases, immunomodulatory polypeptides, including mutant CTLA-4 polypeptides, inhibit CD28 and / or the activity of ICOS by its binding partners (e.g., ICOSL, CD80 , and / or CD86), thereby antagonizing or inhibiting CD2 8 and / or ICOS costimulatory signaling. In some embodiments, antagonism of CD28 and / or ICOSL is beneficial, e.g., in the prevention of autoimmunity. In epidemiology, it may be useful to suppress or dampen immunity. In embodiments, antagonism of CD28 and / or ICOSL is used to treat inflammation or autoimmunity. In some cases, the mutant CTLA-4 polypeptide may be useful for treating The immunomodulatory polypeptide converts the CTLA-4 inhibitory receptor into an activating receptor (e.g., , ITAM signaling domains) are expressed on cells as switch receptors, and and / or expressed on cells as decoy receptors without intracellular signaling domains In some embodiments, the CTLA-4 activating receptor or decoy receptor is used in the treatment of cancer. Those skilled in the art will appreciate that one or more specific binding partners may be used in combination to antagonize, compete, and and / or inhibit or activate one or more downstream signaling pathways. Therefore, the activity of a particular form can be easily determined. Exemplary methods for assessing the
[0243] In some embodiments, such proteins are soluble, e.g., fused to an Fc chain. In some embodiments, immunomodulatory proteins comprising the vIgD of CTLA-4 are provided. Alternatively, one or more additional IgSF domains, such as one or more additional vIgDs, may be used as described herein. The CTLA-4 vIgD can be linked to the CTLA-4 vIgD as provided herein (hereinafter referred to as "stacked" or "stacked"). In some embodiments, a modular approach is used to provide The immunomodulatory protein provided has multiple counter structures (multiple cognate binding partners). Providing flexibility to engineer or tailor immunomodulatory proteins to modulate their activity. In some embodiments, such "stacking" molecules are provided in a soluble format. It may be obtained as a protein or, in some cases, may be provided as a membrane-bound or secreted protein. In some embodiments, the mutant CTLA-4 immunomodulatory protein is administered to a subject, e.g., To target or localize vIgD to specific environments or cells, e.g., when In addition, a ligand, e.g., a targeting agent or moiety, e.g., an antibody or contains the vIgD of CTLA-4 linked directly or indirectly to other binding molecules. In some embodiments, the targeting agent, e.g., an antibody The body or other binding molecules are located in the spleen, spleen, tonsils, lymphatic vessels, lymph nodes, pharyngeal tonsils, and liver binds to antigens on the surface of white blood cells, lymphocytes, or lymphoid tissues, such as the liver, thereby The IgD-containing mutant CTLA-4 is localized to the immune system, e.g., white blood cells or or regulates lymphocyte activity.
[0244] In some embodiments, the immunomodulatory proteins provided are expressed in or on and provided as part of engineered cell therapy (ECT). In some embodiments, The mutant CTLA-4 polypeptide is expressed in a membrane-bound form by immune cells (e.g., T cells or antibodies). Transmembrane immunomodulatory proteins (hereafter, In some embodiments, mutant CTLA-4 polypeptides are provided. The peptide is delivered in a secretable form to cells such as immune cells (e.g., T cells or antigen-presenting cells). The nucleotides are expressed in the cells, thereby releasing them in secreted or soluble form, such as when the cells are administered to a subject. The mutant CTLA-4 polypeptide (hereinafter also referred to as "SIP") is produced. In some embodiments, SIPs bind to binding proteins in the environment in which they are secreted (e.g., the immune microenvironment). In some embodiments, the mutant CTLA-4 polypeptide can antagonize Upon administration to a subject, the mutant polypeptide is delivered as a TIP or SIP in a cell or is expressed in vivo in cells such as immune cells (e.g., T cells or antigen-presenting cells) expressed in infectious agents (e.g., viruses or bacterial pathogens) that can affect will be done.
[0245] In some embodiments, soluble immune responses, such as mutant CTLA-4 containing vIgD, The polypeptide may be any one of the conjugates provided (e.g., a targeting moiety). encapsulated within liposomes which themselves may be conjugated to In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the invention can be In a more specific embodiment, the mutant CTLA-4 sequence is deglycosylated. In an even more specific embodiment, the IgV domain of the mutant CTLA-4 is glycosylated. The lysin or ECD is deglycosylated.
[0246] A non-limiting example of the format provided is set forth in FIG. 1 and further described below.
[0247] A. Soluble Proteins In some embodiments, an immunomodulatory protein comprising a mutant CTLA-4 polypeptide Those skilled in the art will appreciate that cell surface proteins are typically , an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), Soluble forms of suitable proteins can be produced using the extracellular domain or immunologically active subsequences thereof. It will be appreciated that in some embodiments, the modified The immunomodulatory protein containing a variant CTLA-4 polypeptide has a transmembrane domain or In some embodiments, the mutant CTLA-4 lacks a portion of the transmembrane domain. The immunomodulatory protein contains the intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the mutant CTLA-4 polypeptide contains The immunomodulatory protein comprises an ECD domain or a portion thereof containing an IgV domain; or vIgD containing a domain or a specific binding fragment thereof containing an amino acid modification In some embodiments, such soluble polypeptides are used to Inhibiting and / or antagon...
Claims
[Claim 1] The invention described in the specification.