PD-L1 variant immunomodulatory proteins and uses thereof
阅读说明：本技术 Pd-l1变体免疫调节蛋白及其用途 (PD-L1 variant immunomodulatory proteins and uses thereof ) 是由 R·斯旺森 M·科纳克 M·F·莫伊雷尔 D·阿尔杜雷尔 D·W·德蒙泰 J·L·库伊普 于 2018-03-13 设计创作，主要内容包括：本文提供包含变体PD-L1的免疫调节蛋白和编码此类蛋白质的核酸。所述免疫调节蛋白提供用于多种免疫学和肿瘤学病状的治疗实用性。提供用于制备和使用此类蛋白质的组合物和方法。(Provided herein are immunomodulatory proteins comprising variant PD-L1 and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.)
1. A variant PD-L1 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications at one or more positions in the unmodified PD-L1 or a specific binding fragment thereof that correspond to a position selected from the group consisting of: 45. 43, 6, 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 206, 207, 221, 213, or 30, 199 ID of NO.
2. The variant PD-L1 polypeptide of claim 1, wherein the unmodified PD-L1 comprises (i) the amino acid sequence set forth in SEQ ID NO:30, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30; or (iii) it comprises a portion of an IgV structure or an IgC domain or a specific binding fragment thereof or both.
3. The variant PD-L1 polypeptide of claim 1 or claim 2, wherein the variant PD-L1 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, optionally amino acid substitutions, insertions, and/or deletions.
4. The variant PD-L1 polypeptide of any of claims 1-3, wherein the variant PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 30 or 1728, or a specific binding fragment thereof.
5. The variant PD-L polypeptide of any of claims 1-4, wherein the one or more amino acid modifications are selected from the group consisting of P6, Y10, V11, Y14, G15, S16, N17, M18, T19, I20, C22, K23, E26, E27, K28, A33, L35, I36, E40, M41, D43, K44, N45, I47, I46, F49, V50, H51, G52, E53, E54, D55, L56, K57, V58, H60, R64, Q65, R68, K72, D54, D55, L56, K57, V58, H60, R64, Q65, R68, Q72, K97, S101, V101, K78, V97, V101, V98, I98, V98, I101, K85, V97, I101, V98, I101, V95, V97, I101, V98, I101, I98, I7, K95, s131, E134, C137, Q138, K144, I148, W149, T150, Q155, S158, K160, T163, K163, N165, K167, E170, K171, F173, K173, V175, T176, S177, L179, R180, T183, T185, I188, F189, T192, F193, R194, R195, L196, D197, P198, E199, E200, N201, H202, T203, a204, L206, V207, L213, T221 or conservative amino acid substitutions thereof.
6. The variant PD-L polypeptide of any of claims 1 to 5, wherein the one or more amino acid modifications are selected from the group consisting of K28/M41/N45/H51/K57, I20/I36/N45/I47, I20/M41/K44, P6/N45/N78/I83, N78, M41/N78, N45/N78, I20/N45, M41, I20/I36/N45, N17/N47/V50/D72, I20/F49, N45/V50, I20/N45/N78, I20/N45/V50, M41/N45, A33/S75/D85, M18/M41/D43/H51/N78, V11/I20/I36/N45/H60/S75, A33/V50, S16/S33/S75, S33/S75, S33/, E27/N45/M97, E27/N45/K57, A33/E53, D43/N45/V58, E40/D43/N45/V50, Y14/K28/N45A 33/N78, A33/N45/N78, E27/N45/V50, N45/V50/N78, I20/N45/V110, I20/I36/N45/V50, N45/L74/S75, N45/S75, S75/K106, S75, A33/S75/D104, A33/S75, I20/E27/N45/V50, I20/E27/D43/N45/V58/N78, I20/A33/N45/N58/N78, I33/N45/N78, E27/N45/V50/N78, V11/I20/E27/D43/N45/H51/S99, I20/E27/D43/N45/V50, I20/K28/D43/N45/V58/Q89, I20/I36/N45, I20/K28/D43/N45/E53/V58/N78, A33/D43/N45/V58/S75, K23/D43/N45, I20/D43/N45/V58/N78/D90/G101, D43/N45/L56/V58/G101-ins (G101), I20/K23/D43/N45/V58/N78, I20/K23/D43/N45/V50/N78, T19/E27/N45/N50/N97/M45/M78, I20/M41/D43/N45, K23/N45/N78, I20/K28/D43/N45/V58/Q89/G101-ins (G101), K57/S99/F189, M18/M97/F193/R195/E200/H202, I36/M41/M97/K144/R195/E200/H202/L206, C22/Q65/L124/K144/R195/E200/H202/T221, M18/I98/L124/P198/L206, S99/N117/I148/K171/R180, I36/M97/A103/Q155, K28/S99, R195, A79/S99/T185/R195/E200/H202/L206, K57/S99/L124/K144, K99/S195/R, D55/M97/S99, E27/I36/D55/M97/K111, E54/M97/S99, G15/I36/M97/K111/H202, G15/I36/V129/R195, G15/V129, I36/M97, I36/D55/M97/K111/A204, I36/D55/M97/K111/V129/F173, I36/D55/M97/K111/I148/R180, I36/G52/M97/V112/K144/V175/P198, I36/I46/D55/M97/K106/K144/T185/R195, I36/I83/M97/K144/P198, I36/M97/K111, I36/M97/K144/P198, I36/M97/K144/M193/M155/F193/N195, I36T/M97L/V129D, L35P/I36S/M97L/K111E, M18I/I36T/E53G/M97L/K144E/E199G/V207A, M18T/I36T/D55N/M97L/K111E, M18V/M97L/T176L/R195L, M97L/S99L, N17/M97L/S L, S99L/T185/L/R195/P198L, V129L/H202L, V129L/P198L, V129L/T150, V93/V129L, Y10L/M18/S72/S99/S195/P198L, N129/N195/N72/N L/N198N L/N195/N72/N L/N198N 72/N L/N195/N72/N L/N72/N195/N72/N198N 72/N L/N198N 72/N L, N L/N198N L/N72/N L/N198N L/N195/N L/N72/N L/, N45D/N113Y/R195S, N45D/N165Y/E170G, N45D/Q89R/I98V, N45D/S131F/P198S, N45D/S75P/P198S, N45D/V50A/R195T, E27D/N45D/T183A/I188V, F173V/T183V/L196V/T203V, K23V/N45V/S75/N120V, N45V/G102/R194V/R195V, N45V/G52/Q V/P198, N45V/I148/R195/R72/N V/N72/N198/N72/N V/N183/N198/N72/N185/N72/N V/N198/N72/N V/N76/N72/N198/N72/N V/N72/N V/N72/N72/N72/N V/N72/N V/N72/N V/N72/N72/N V/N72/, K23/N45/L124/K167/R195, K23/N45/Q73/T163, K28/N45/W149/S158/P198, K28/N45/K57/I98/R195, K28/N45/V129/T163/R195, M41/D43/N45/R64/S99, N45/R68/F173/D197/P198, N45/V50/I148/R195/N201, M41/D43/K44/N45/R195/N201, or N45/V50/L124/K144/L179/R195.
7. The variant PD-L1 polypeptide of any of claims 1-6, wherein the one or more amino acid modifications are at one or more positions corresponding to one or more positions selected from 20, 27, 33, 36, 43, 45, 50, 58, 75, or 78.
8. The variant PD-L1 polypeptide of any one of claims 1-7, wherein the one or more amino acid modifications are selected from I20L, E27G, a33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, or conservative amino acid substitutions thereof.
9. The variant PD-L1 polypeptide of any one of embodiments 1 to 8, wherein the variant PD-L1 polypeptide comprises amino acid modifications I20L/I36T, I20L/D43G, I20L/N45D, I20L/N45T, I20L/N45T, I20L/V50A, I20L/V58A, I20L/S75P, I20L/N78I, I36T/D43G, I36T/N45D, I36T/N45T, I36T/V50A, I36A/V58A, I36A/S75A, I36A/N78A, D43A/N45A, D43A/N72/A, D A/V50, D43A/V A, D A/N72, N72/N A, N A/N72, N A/N72/N A, N72/N72, N72/N72, N72/N72.
10. The variant PD-L1 polypeptide of any one of claims 1-9, wherein the variant PD-L1 polypeptide comprises the amino acid modifications D43G/N45D/V58A.
11. The variant PD-L1 polypeptide of any one of claims 1-10, wherein the variant PD-L1 polypeptide comprises the amino acid modifications D43G/N45D/L56Q/V58A/G101G-I ns (G101GG) or I20L/K28E/D43G/N45D/V58A/Q89R/G101G-ins (G101 GG).
12. The variant PD-L1 polypeptide of any one of claims 1-11, wherein:
the variant PD-L1 polypeptide comprises a PD-L1 extracellular domain (ECD); and/or
The variant PD-L1 polypeptide comprises the IgV domain or a specific fragment thereof and the IgC domain or a specific fragment thereof.
13. The variant PD-L1 polypeptide of any of claims 1 to 12, which comprises the amino acid sequence as set forth in any of SEQ ID NOs 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 or a specific binding fragment thereof, or an amino acid sequence having at least 95% sequence identity with any of SEQ ID NOs 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 and comprising one or more of the amino acid modifications or a specific binding fragment thereof.
14. The variant PD-L1 polypeptide of any one of claims 1-19, wherein the variant PD-L1 polypeptide comprises the IgV domain or a specific binding fragment thereof.
15. The variant PD-L1 polypeptide of any one of claims 1-20, wherein the IgV domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.
16. The variant PD-L1 polypeptide of any of claims 1 to 15, which comprises an amino acid sequence as set forth in any of SEQ ID NOs 121-185, 244-308, 1726-1727, 1908-1937 or a specific binding fragment thereof, which has at least 95% sequence identity with any of SEQ ID NOs 121-185, 244-308, 1726-1727, 1908-1937 and which contains one or more of the amino acid modifications or a specific binding fragment thereof.
17. The variant PD-L1 polypeptide of any one of claims 1-16, wherein the variant PD-L1 polypeptide specifically binds to an extracellular domain of PD-1 with increased affinity compared to the binding of the unmodified PD-L1 to the extracellular domain of PD-1.
18. The variant PD-L1 polypeptide of any one of claims 1-17, wherein the variant PD-L1 polypeptide specifically binds with increased affinity to the extracellular domain of PD-1 and with decreased affinity to the extracellular domain of CD80, as compared to the binding of the unmodified PD-L1 to the extracellular domain of PD-1 and the extracellular domain of CD80.
19. The variant PD-L1 polypeptide of claim 17 or claim 18, wherein the increased affinity for the extracellular domain of PD-1 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold.
20. The variant PD-L1 polypeptide of claim 18 or claim 19, wherein the reduced affinity for the extracellular domain of CD80 is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold.
21. The variant PD-L1 polypeptide of any one of claims 1-20, wherein the variant polypeptide specifically binds to an extracellular domain of PD-1 with increased selectivity, including a greater ratio of binding of the variant polypeptide to the same extracellular domain of PD-1 versus CD80 as compared to the ratio of binding of the unmodified PD-L1 polypeptide to PD-1 versus CD80.
22. The variant PD-L1 polypeptide of claim 21, wherein the ratio is at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more greater.
23. The variant PD-L1 polypeptide of any one of claims 17-22, wherein the PD-1 is human PD-1.
24. The variant PD-L1 polypeptide of any one of claims 18-22, wherein the CD80 is human CD80.
25. The variant PD-L1 polypeptide of any one of claims 1-24, wherein:
the variant PD-L1 polypeptide is a soluble protein;
the variant PD-L1 polypeptide lacks a PD-L1 transmembrane domain and an intracellular signaling domain; and/or
The variant PD-L1 polypeptide is not expressed on the cell surface.
26. The variant PD-L1 polypeptide of any one of claims 1-25, wherein the variant PD-L1 polypeptide is linked to a moiety that increases the biological half-life of the polypeptide.
27. The variant PD-L1 polypeptide of any one of claims 1-26, wherein the variant PD-L1 polypeptide is linked to a multimerization domain.
28. The variant PD-L1 polypeptide of claim 27, wherein the multimerization domain is an Fc domain or a variant thereof having reduced effector function.
29. The variant PD-L1 polypeptide of any one of claims 1-24, which variant PD-L1 polypeptide is a transmembrane immunomodulatory protein, further comprising a transmembrane domain, optionally wherein the transmembrane domain is directly or indirectly linked to an extracellular domain (ECD) of the variant PD-L1 polypeptide or a specific binding fragment thereof.
30. The variant PD-L1 polypeptide of claim 29, further comprising a cytoplasmic signaling domain, optionally wherein the cytoplasmic signaling domain is directly or indirectly linked to the transmembrane domain.
31. An immunomodulatory protein comprising a first variant PD-L1 polypeptide of claim 27 or claim 28, wherein the multimerization domain is a first multimerization domain; and the second variant PD-L1 polypeptide of claim 27 or claim 28, wherein the multimerization domain is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide.
32. The immunomodulatory protein of claim 31, wherein the first variant PD-L2 polypeptide and the second variant PD-L2 polypeptide are the same.
33. The immunomodulatory protein of claim 31 or claim 32, wherein the multimer is a dimer.
34. The immunomodulatory protein of claim 33, which is a homodimer.
35. An immunomodulatory protein comprising the variant PD-L1 polypeptide of any one of claims 1-28, directly or indirectly linked via a linker to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of IgSF family members.
36. The immunomodulatory protein of claim 35, wherein the IgSF domain is an affinity modified IgSF domain comprising one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of the IgSF family member.
37. The immunomodulatory protein of claim 36, wherein the IgSF domain is an affinity modified IgSF domain and exhibits increased binding to the same one or more cognate binding partners as compared to binding of the unmodified or wild-type IgSF domain of the IgSF family member to one or more of its cognate binding partners.
38. The immunomodulatory protein of any of claims 35-37, wherein the variant PD-L1 polypeptide is capable of specifically binding to PD-1, and the IgSF domain of the second polypeptide is capable of binding to a homologous binding partner other than the homologous binding partner specifically bound by the PD-L1 variant polypeptide.
39. The immunomodulatory protein of any of claims 35-38, wherein the IgSF domain of the second polypeptide is an IgSF domain that binds to a ligand of an inhibitory receptor or an affinity modified IgSF domain thereof.
40. The immunomodulatory protein of claim 39, wherein:
the inhibitory receptor is TIGIT; or
Said ligand of said inhibitory receptor is CD155 or CD 112.
41. The immunomodulatory protein of any of claims 35-40, wherein the second polypeptide is selected from the group consisting of:
(i) a variant CD155 polypeptide comprising the IgSF domain as set forth in any one of SEQ ID Nos 311-352, 354-665, 1505-1576, 1551-1714; or
(ii) A variant CD112 polypeptide comprising an IgSF domain as set forth in any of SEQ ID Nos 667-760, 762-931, 1433-1504; (ii) a
(iii) (iii) an amino acid sequence having at least 95% sequence identity to any one of the SEQ ID NOs of (i) - (ii) and comprising amino acid modifications, optionally amino acid substitutions, insertions and/or deletions thereof; or
(vi) (iv) a specific binding fragment of any one of (i) - (iii).
42. The immunomodulatory protein of any of claims 35-41, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide or the second polypeptide.
43. An immunomodulatory protein comprising (1) the immunomodulatory protein of claim 42, wherein the multimerization domain is a first multimerization domain; and (2) a second multimerization domain, wherein the first multimerization domain interacts with the second multimerization domain to form a multimer comprising the immunomodulatory protein.
44. The immunomodulatory protein of claim 80, wherein the immunomodulatory protein is a first immunomodulatory protein and a second immunomodulatory protein of claim 42, wherein the multimerization domain is the second multimerization domain, wherein the multimerization domain comprises the first immunomodulatory protein and the second immunomodulatory protein.
45. The immunomodulatory protein of claim 43 or claim 44, wherein the multimer is a dimer.
46. The immunomodulatory protein of any of claims 43-45, being a homodimer.
47. The immunomodulatory protein of any of claims 43-46, wherein:
the second polypeptide is a variant CD155 polypeptide and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence set forth in any one of SEQ ID NO 1716-1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO 1716-1721; or
The second polypeptide is CD112 or CD155 and the third polypeptide is the other of CD112 or CD155 and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence as set forth in any one of SEQ ID NO 1722-1724 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO 1716-1721.
48. The immunomodulatory protein of any of claims 43-45, which is a heterodimer, optionally wherein the first and second multimerization domains are different and/or are capable of interacting to mediate heterodimer formation.
49. The immunomodulatory protein of any of claims 43-48, wherein the first and/or second multimerization domain is an Fc domain of an immunoglobulin, optionally wherein the immunoglobulin is a human immunoglobulin and/or the Fc region is a human Fc region.
50. The immunomodulatory protein of claim 49, wherein the Fc region is that of an immunoglobulin G1(IgG1) or immunoglobulin G2(IgG2) protein.
51. The immunomodulatory protein of claim 49 or claim 50, wherein the Fc region exhibits one or more effector functions.
52. The immunomodulatory protein of claim 49, wherein the Fc region is a variant Fc region comprising one or more amino acid substitutions in a wild-type Fc region, the variant Fc region exhibiting reduced one or more effector functions as compared to the wild-type Fc region, optionally wherein the wild-type Fc is an Fc of human IgG 1.
53. The immunomodulatory protein of claim 52, wherein the Fc region comprises the amino acid substitutions N292G, R292C/N297G/V302C, or L234A/L235E/G237A, wherein residue numbering is according to the EU index of Kabat.
54. The immunomodulatory protein of any of claims 49-53, wherein the Fc region comprises the amino acid substitution C220S, wherein the numbering of residues is according to the EU index of Kabat.
55. The immunomodulatory protein of any of claims 49-54, wherein the Fc region comprises K447del, wherein the numbering of residues is according to the EU index of Kabat.
56. A conjugate comprising the variant PD-L1 of any one of claims 1-30 or the immunomodulatory protein of any one of claims 31-55 linked to a moiety.
57. The conjugate of claim 56, wherein the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell.
58. The conjugate of claim 57, wherein the cell is an immune cell or a tumor cell.
59. The conjugate of any one of claims 56-58, wherein the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.
60. The conjugate of any one of claims 56-59, wherein the moiety is an antibody or antigen-binding fragment.
61. The conjugate of any one of claims 56-60, which is a fusion protein.
62. A nucleic acid molecule encoding the variant PD-L1 polypeptide of any one of claims 1-30, the immunomodulatory protein of any one of claims 31-55, or a conjugate that is the fusion protein of any one of claims 56-61.
63. A vector comprising the nucleic acid molecule of claim 62.
64. The vector of claim 63, which is an expression vector.
65. A cell comprising the vector of claim 63 or claim 64.
66. A method of producing a variant PD-L1 polypeptide or immunomodulatory protein, comprising introducing the nucleic acid molecule of claim 62 or the vector of claim 63 or claim 64 into a host cell under conditions in which the protein is expressed in the cell.
67. The method of claim 66, further comprising isolating or purifying the variant PD-L1 polypeptide or immunomodulatory protein from the cell.
68. A method of engineering a cell expressing a variant PD-L1 variant polypeptide, comprising introducing into the cell a nucleic acid molecule encoding the variant PD-L1 polypeptide of any one of claims 1-30, under conditions in which the polypeptide is expressed in the host cell.
69. An engineered cell expressing the variant PD-L1 polypeptide of any one of claims 1-30, the immunomodulatory protein of any one of claims 31-55, the nucleic acid molecule of claim 62, or the vector of claim 63 or claim 64.
70. The engineered cell of claim 69, wherein:
the variant PD-L1 polypeptide or immunomodulatory protein does not comprise a transmembrane domain and/or is not expressed on the surface of the cell; and/or
The variant PD-L1 polypeptide or immunomodulatory protein is secreted from or capable of being secreted from the engineered cell.
71. The engineered cell of claim 69, wherein:
the engineered cell comprises a variant PD-L1 polypeptide comprising a transmembrane domain and/or a transmembrane immunomodulatory protein of claim 29 or claim 30; and/or
The variant PD-L1 polypeptide is expressed on the surface of the cell.
72. The engineered cell of any one of claims 69-71, wherein the cell is an immune cell, optionally an Antigen Presenting Cell (APC) or a lymphocyte, optionally a T cell.
73. The engineered cell of any one of claims 69-72, wherein the cell further comprises a Chimeric Antigen Receptor (CAR) or an engineered T-cell receptor.
74. An infectious agent comprising a nucleic acid molecule encoding the variant PD-L1 polypeptide of any one of claims 1-30 or the immunomodulatory protein of any one of claims 51-55.
75. The infectious agent of claim 74, wherein the infectious agent is a bacterium or a virus.
76. The infectious agent of claim 75, wherein the infectious agent is a virus and the virus is an oncolytic virus.
77. A pharmaceutical composition comprising the variant PD-L1 polypeptide of any one of claims 1-30, the immunomodulatory protein of any one of claims 31-55, the conjugate of any one of claims 56-61, the engineered cell of any one of claims 69-73, or the infectious agent of any one of claims 74-76.
78. The pharmaceutical composition of claim 77, comprising a pharmaceutically acceptable excipient.
79. The pharmaceutical composition of claim 77 or claim 78, wherein the pharmaceutical composition is sterile.
80. An article of manufacture comprising the pharmaceutical composition of any one of claims 77-79 in a vial.
81. A kit comprising the pharmaceutical composition of any one of claims 77-79 or the article of manufacture of claim 80 and instructions for use.
82. A method of modulating an immune response in a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 77-79.
83. A method of modulating an immune response in a subject, comprising administering the engineered cell of any one of claims 69-73 to the subject.
84. The method of claim 83, wherein the engineered cells are autologous to the subject.
85. The method of any one of claims 82-84, wherein modulating the immune response treats a disease or condition in the subject.
86. The method of any one of claims 82-85, wherein the immune response is increased.
87. The method of any one of claims 82-86, wherein the pharmaceutical composition or engineered cell comprises a variant PD-L1 polypeptide in a form that is an antagonist and/or blocks the interaction of PD-L1 and PD-1 to attenuate negative signaling by PD-1.
88. The method of any one of claims 82 and 85-87, wherein the subject is administered a soluble variant PD-L1 polypeptide or immunomodulatory protein, optionally lacking a PD-L1 transmembrane domain and an intracellular signaling domain.
89. The method of any one of claims 82 and 85-88, wherein the variant PD-L1 polypeptide or immunomodulatory protein is an Fc fusion protein.
90. The method of any one of claims 82 and 85-89, wherein variant PD-L1 polypeptide is as set forth in any one of claims 1-28 or the immunomodulatory protein is as set forth in any one of claims 31-55.
91. The method of any one of claims 83-87, wherein the subject is administered an engineered cell comprising a secretable variant PD-L1 polypeptide.
92. The method of any one of claims 82-91, wherein the disease or condition is a tumor or cancer.
93. The method of any one of claims 82-92, wherein the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, stomach cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancers.
94. The method of any one of claims 82-85, wherein the immune response is reduced.
95. The method of any one of claims 82-85 and 94, wherein the pharmaceutical composition or engineered cell comprises a variant PD-L1 polypeptide in a form that is an agonist and/or is capable of stimulating inhibitory signaling through PD-1.
96. The method of any one of claims 82, 85, 94, and 95, wherein the subject is administered an immunomodulatory protein or conjugate comprising a variant PD-L1 polypeptide linked to an IgSF domain or a moiety that localizes to a cell or tissue of an inflammatory environment.
97. The method of any one of claims 83-85, 94, and 95, wherein the subject is administered an engineered cell comprising a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein.
98. The method of any one of claims 82-85 and 94-97, wherein the disease or condition is an inflammatory or autoimmune disease or condition.
99. The method of any one of claims 82-85 and 94-97, wherein the disease or condition is anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis, an autoimmune skin disease, transplantation, rheumatism, inflammatory gastrointestinal tract disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease.
100. The method of claim 98 or claim 99, wherein the disease or condition is selected from inflammatory bowel disease, transplantation, crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.
Technical Field
The present disclosure relates to therapeutic compositions for modulating immune responses in the treatment of cancer and immunological diseases. In some aspects, the disclosure relates to particular variants of PD-L1 that exhibit improved binding (such as improved binding affinity or selectivity) to one or more of the homologous binding partner proteins PD-1 and CD 80.
Background
Modulation of the immune response by intervening processes occurring in the Immunological Synapse (IS) formed by and between Antigen Presenting Cells (APC) or target cells and lymphocytes has increasing medical significance. Mechanistically, cell surface proteins in IS can involve coordinated and often simultaneous interactions of multiple protein targets with a single protein to which they bind. IS interactions occur in close association with the joining of two cells, and a single protein in this structure can interact with both proteins on the same cell (cis) and associated cells (trans) simultaneously. Although therapeutic agents that can modulate IS are known, there remains a need for improved therapeutic agents. Immunomodulatory proteins are provided that meet such needs, including soluble or transmembrane immunomodulatory proteins capable of being expressed on a cell.
Disclosure of Invention
Provided herein is a variant PD-L1 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications in an unmodified PD-L1 or a specific binding fragment thereof corresponding to a position selected from: 6. 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 87, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 203, 200, 201, 202, 203, 204, 206, 175, 176, 177, 213, or 1728 ID or NO. In some cases, the amino acid modification is an amino acid substitution, insertion, or deletion.
In some embodiments, unmodified PD-L1 is mammalian PD-L1 or a specific binding fragment thereof. In some embodiments, unmodified PD-L1 is human PD-L1 or a specific binding fragment thereof. In some of any such embodiments, unmodified PD-L1 contains (i) the amino acid sequence set forth in SEQ ID NO:30 or 1728, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:30 or 1728; or (iii) it comprises a portion of an IgV structure or an IgC domain or a specific binding fragment thereof or both.
In some any such embodiments, the IgV domain or specific binding fragment of an IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110, or more amino acids; or a specific binding fragment of an IgV domain having a length of at least 80% of the length of the IgV domain as set forth as amino acids 24-130 of SEQ ID NO. 3; and/or the specific binding fragment of the IgC domain has a length which is at least 80% of the length of the IgC domain as set forth as amino acids 133-225 of SEQ ID NO 3. In some any such embodiments, the variant PD-L1 contains up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, optionally amino acid substitutions, insertions, and/or deletions. In some of any such embodiments, the variant PD-L1 polypeptide contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs 30, 1728, or a specific binding fragment thereof.
In some of any such embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of PD-1 as compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1. In some embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of CD80 as compared to the binding of unmodified PD-L1 to the extracellular domain of CD 80. In some of any such embodiments, the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of PD-1 as compared to unmodified PD-L1. In some embodiments, the altered binding is altered binding affinity and/or altered binding selectivity.
In some such embodiments, the one or more amino acid modifications are selected from P6, Y10, V11, Y14, G15, S16, N17, M18, T19, I20, C22, K23, E26, E27, K28, a33, L35, I36, E40, M41, D43, K44, N45, I46, I47, F49, V50, H51, G52, E53, E54, D55, L56, K57, V58, H60, R64, Q65, R68, K71, D72, Q73, L74, S149, N55, D55, L56, K57, V58, N60, N64, Q65, R68, K71, Q72, Q73, Q74, S75, N85, N101, N98, K144, N101, M101, N99, N101, N95, M101, N99, M144, I101, N95, M101, N95, N101, N99, N95, M101, S158G, K160M, T163I, K163N, N165Y, K167R, K167T, E170G, K171R, F173I, K173I, V175I, S177I, L179I, R180I, T183I, T185I, I188I, F189I, F192I, F193I, R194I, R195I, L195I, D197I, P198I, E199I, E200I, N I, H202I, T36203 203, a204, L I, L36206, V36207, L I, T36221, T I, or conservative amino acid substitutions thereof. In some of any such embodiments, the one or more amino acid modifications are selected from K28/M41/N45/H51/K57, I20/I36/N45/I47, I20/M41/K44, P6/N45/N78/I83, N78, M41/N78, N45/N78, I20/N45, M41, I20/I36/N45, N17/N47/V50/D72, I20/F49, N45/V50, I20/N45/N78, I20/N45/V50, M41/N45, A33/S75/D85, M18/M41/D43/H51/N78, V11/I20/I36/N45/H60/S75, A33/V50, S16/A33/K71/S75, E27/N45/H51/N78, E11/I20/I36/N45/S75, E27/N45/K57/N57, E27/N45/N57, and E45/N78, 33/E53, D43/N45/V58, E40/D43/N45/V50, Y14/K28/N45, A33/N78, A33/N45/N78, E27/N45/V50, N45/V50/N78, I20/N45/V110, I20/I36/N45/V50, N45/L74/S75, N45/S75, S75/K106, S75, A33/S75/D104, A33/S75, I20/E27/N45/V50, I20/E27/D43/N45/V58/N78, I20/A33/D43/N45/V58/N78, I20/D43/N45/V58/N78, I45/N78, E45/N78, N45/V50/N78, V11/I20/E27/D43/N45/H51/S99, I20/E27/D43/N45/V50, I20/K28/D43/N45/V58/Q89, I20/I36/N45, I20/K28/D43/N45/E53/V58/N78, A33/D43/N45/V58/S75, K23/D43/N45, I20/D43/N45/V58/N78/D90/G101, D43/N45/L56/V58/G101-ins (G101), I20/K23/D43/N45/V58/N78, I20/K23/D43/N45/V50/N78, T19/E27/N45/V50/N78/M97, I20/M41/D43/N45, K23/N45/N78, I20/K28/D43/N45/V58/Q89/G101-ins (G101), K57/S99/F189, M18/M97/F193/R195/E200/H202, I36/M41/M97/K144/R195/E200/H202/L206, C22/Q65/L124/K144/R195/E200/H202/T221, M18/I98/L124/P198/L206, S99/N117/I148/K171/R180, I36/M97/A103/Q155, K28/S99, R195, A79/S99/T185/R195/E200/H202/L206, K57/S99/L124/K144, K99/S195/R, D55/M97/S99, E27/I36/D55/M97/K111, E54/M97/S99, G15/I36/M97/K111/H202, G15/I36/V129/R195, G15/V129, I36/M97, I36/D55/M97/K111/A204, I36/D55/M97/K111/V129/F173, I36/D55/M97/K111/I148/R180, I36/G52/M97/V112/K144/V175/P198, I36/I46/D55/M97/K106/K144/T185/R195, I36/I83/M97/K144/P198, I36/M97/K111, I36/M97/K144/P198, I36/M97/K144/M193/M155/F193/N195, I36T/M97L/V129D, L35P/I36S/M97L/K111E, M18I/I36T/E53G/M97L/K144E/E199G/V207A, M18T/I36T/D55N/M97L/K111E, M18V/M97L/T176L/R195L, M97L/S99L, N17/M97L/S L, S99L/T185/L/R195/P198L, V129L/H202L, V129L/P198L, V129L/T150, V93/V129L, Y10L/M18/S72/S99/S195/P198L, N129/N195/N72/N L/N198N L/N195/N72/N L/N198N 72/N L/N195/N72/N L/N72/N195/N72/N198N 72/N L/N198N 72/N L, N L/N198N L/N72/N L/N198N L/N195/N L/N72/N L/, N45D/N113Y/R195S, N45D/N165Y/E170G, N45D/Q89R/I98V, N45D/S131F/P198S, N45D/S75P/P198S, N45D/V50A/R195T, E27D/N45D/T183A/I188V, F173V/T183V/L196V/T203V, K23V/N45V/S75/N120V, N45V/G102/R194V/R195V, N45V/G52/Q V/P198, N45V/I148/R195/R72/N V/N72/N198/N72/N V/N183/N198/N72/N185/N72/N V/N198/N72/N V/N76/N72/N198/N72/N V/N72/N V/N72/N72/N72/N V/N72/N V/N72/N V/N72/N72/N V/N72/, K23/N45/L124/K167/R195, K23/N45/Q73/T163, K28/N45/W149/S158/P198, K28/N45/K57/I98/R195, K28/N45/V129/T163/R195, M41/D43/N45/R64/S99, N45/R68/F173/D197/P198, N45/V50/I148/R195/N201, M41/D43/K44/N45/R195/N201, or N45/V50/L124/K144/L179/R195.
In some any such embodiments, the one or more amino acid modifications correspond to one or more positions selected from 20, 27, 28, 33, 36, 41, 43, 45, 50, 58, 71, 75, or 78. In some embodiments, the one or more amino acid substitutions are selected from I20L, E27G, K28E, a33D, I36T, M41K, D43G, N45D, N45T, V50A, V58A, K71E, S75P, N78I, or conservative amino acid substitutions thereof.
In some any such embodiments, the one or more amino acid modifications correspond to one or more positions selected from 20, 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, or 195. In some embodiments, the one or more amino acid substitutions are selected from I20L, E27G, a33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L polypeptide contains amino acid modifications I20/I36, I20/D43, I20/N45, I20/V50, I20/V58, I20/S75, I20/N78, I36/D43, I36/N45, I36/V50, I36/V58, I36/S75, I36/N78, D43/N45, D43/V50, D43/V58, D43/S75, D43/N78, N45/V50, N45/V58, N45/S75, N45/N78, V50/V58, V50/S75, V50/N78, V58/S75, V78/V78, V78/V75, or N78/V75. In some embodiments, the variant PD-L1 polypeptide contains amino acid modifications D43G/N45D/V58A. In some aspects, variant PD-L1 polypeptides contain amino acid modifications D43G/N45D/L56Q/V58A/G101G-ins (G101GG) or I20L/K28E/D43G/N45D/V58A/Q89R/G101G-ins (G101 GG).
In some any such embodiments, the variant PD-L1 polypeptide includes a variant PD-L1 polypeptide that comprises or consists of a PD-L1 extracellular domain (ECD); and/or a variant PD-L1 polypeptide comprising or consisting of an IgV domain or a specific fragment thereof and an IgC domain or a specific fragment thereof. In some any such embodiments, the variant PD-L1 polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 or a specific binding fragment thereof, or an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs 56-120, 1725, 1729-1818, 1819-1907, 1943-2008 or a specific binding fragment thereof and containing one or more amino acid substitutions.
In some of any such embodiments, the variant PD-L1 polypeptide contains an IgV domain or a specific binding fragment thereof. In some of any such embodiments, the IgV domain or specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide. In some embodiments, the IgC domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.
In some any such embodiments, the variant PD-L1 polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID Nos. 121-185, 244-308, 1726-1727, 1908-1937 or a specific binding fragment thereof having at least 95% sequence identity to any one of SEQ ID Nos. 121-185, 244-308, 1726-1727, 1908-1937 or a specific binding fragment thereof and comprising one or more amino acid substitutions. In some embodiments, the IgC domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.
In some of any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain of PD-1 or CD80 with increased affinity as compared to the binding of unmodified PD-L1 to the same extracellular domain of PD-1 or CD 80. In some of any such embodiments, the variant PD-L1 polypeptide specifically binds to the extracellular domain of PD-1 with increased affinity as compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1. In some of any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain with increased affinity as compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1 and the extracellular domain of CD80, respectively. In some any such embodiments, the variant PD-L1 polypeptide specifically binds to the same extracellular domain of PD-1 with increased affinity and binds to the same extracellular domain of CD80 with decreased affinity as compared to the binding of unmodified PD-L1 to the extracellular domain of PD-1 and the extracellular domain of CD 80.
In some any such embodiments, the increased affinity for the extracellular domain of PD-1 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to unmodified PD-L1. In some aspects, the increased affinity for the extracellular domain of CD80 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to unmodified PD-L1. In some aspects, the reduced affinity for the extracellular domain of CD80 is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to unmodified PD-L1.
In some of any such embodiments, the variant polypeptide specifically binds to the extracellular domain of PD-1 with increased selectivity as compared to unmodified PD-L1. In some cases, the increased selectivity includes a greater ratio of binding of the variant polypeptide to the same extracellular domain of PD-1 versus CD80 as compared to the ratio of binding of the unmodified PD-L1 polypeptide to PD-1 versus CD 80. In some examples, the ratio is at least or at least about 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, or more times greater.
In some of any such embodiments, PD-1 is human PD-1. In some of any such embodiments, CD80 is human CD 80.
In some any such embodiments, the binding activity is altered (increased or decreased) by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold as compared to unmodified PD-L1.
In some of any such embodiments, the variant PD-L1 polypeptide is a soluble protein. In some any such embodiments, the variant PD-L1 polypeptide lacks a PD-L1 transmembrane domain and an intracellular signaling domain; and/or the variant PD-L1 polypeptide is not capable of being expressed on the surface of a cell. In some any such embodiments, the variant PD-L1 polypeptide is linked to a multimerization domain. In some any such embodiments, the variant PD-L1 polypeptide is a multimeric polypeptide comprising a first variant PD-L1 polypeptide linked to a multimerization domain and a second variant PD-L1 polypeptide linked to a multimerization domain, optionally a dimeric polypeptide. In some embodiments, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are the same or different.
In some of any such embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some of any such embodiments, the variant PD-L1 polypeptide is linked to a moiety that increases the biological half-life of the polypeptide. In some of any such embodiments, the variant PD-L1 polypeptide is linked to an Fc domain or a variant thereof having reduced effector function.
In some of any such embodiments, the Fc domain is a mammalian Fc domain, optionally a human Fc domain; or the variant Fc domain comprises one or more amino acid modifications as compared to a mammalian (optionally human) unmodified Fc domain. In some of any such embodiments, the Fc domain or variant thereof contains the amino acid sequence set forth in SEQ ID No. 187 or SEQ ID No. 188, or an amino acid sequence having at least 85% sequence identity to SEQ ID No. 187 or SEQ ID No. 188. In some embodiments, the Fc domain comprises one or more amino acid modifications selected from the group consisting of: E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, R292C, V302C, and K447del, each by EU numbering. In some embodiments, the Fc domain comprises the amino acid modification C220S by EU numbering. In some any such embodiments, the Fc domain comprises the amino acid sequence set forth in any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940 and exhibiting reduced effector function.
In some any such embodiments, the variant PD-L1 polypeptide is indirectly linked via a linker (optionally a G4S linker). In some any such embodiments, the variant PD-L1 polypeptide is a transmembrane immunomodulatory protein, further comprising a transmembrane domain linked to the extracellular domain (ECD) of the variant PD-L1 polypeptide, or a specific binding fragment thereof.
In some of any such embodiments, the transmembrane domain comprises the amino acid sequence set forth as residue 239-259 of SEQ ID NO. 3 or a functional variant thereof having at least 85% sequence identity to residue 239-259 of SEQ ID NO. 3. In some embodiments, the variant PD-L1 polypeptide further comprises a cytoplasmic signaling domain linked to the transmembrane domain. In some cases, the cytoplasmic signaling domain comprises the amino acid sequence set forth as residue 260-290 of SEQ ID NO. 3 or a functional variant thereof having at least 85% sequence identity to residue 260-290 of SEQ ID NO. 3.
In some of any of the provided embodiments, the variant PD-L1 polypeptide modulates a response of an immune cell (such as a T cell). In some embodiments, the response (e.g., T cell response) is increased or decreased. In some of any such embodiments, the variant PD-L1 increases IFN- γ (interferon- γ) expression relative to unmodified PD-L1 in an in vitro T cell assay. In some of any such embodiments, the variant PD-L1 reduces IFN- γ (interferon- γ) expression relative to unmodified PD-L1 in an in vitro T cell assay.
In some of any such embodiments, the variant PD-L1 polypeptide is deglycosylated.
In some embodiments of any of the variant PD-L1 polypeptides described herein, the variant PD-L1 polypeptide increases T cell signaling relative to unmodified PD-L1, such as determined using a reporter assay involving T cells (e.g., Jurkat) engineered with a reporter gene (e.g., luciferase) operably linked to an IL-2 promoter. In some embodiments of any of the variant PD-L1 polypeptides described herein, the variant PD-L1 polypeptide reduces T cell signaling relative to unmodified PD-L1, such as determined using a reporter assay involving T cells (e.g., Jurkat) engineered with a reporter gene (e.g., luciferase) operably linked to an IL-2 promoter. In some of any such embodiments, the variant PD-L1 polypeptide is provided in any of a variety of forms, such as soluble or immobilized (e.g., plate-bound).
Also provided is an immunomodulatory polypeptide comprising a variant PD-L1 according to any provided embodiment, said variant PD-L1 linked directly or indirectly via a linker to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain. In some cases, the IgSF domain is affinity modified and exhibits altered binding to the same one or more homologous binding partners as compared to binding of an unmodified or wild-type IgSF domain to one or more of its homologous binding partners. In some embodiments, the affinity modified IgSF domain contains one or more amino acid modifications compared to an unmodified or wild-type IgSF domain of an IgSF family member. In some cases, an IgSF domain exhibits increased binding to one or more of its homologous binding partners as compared to the binding of the unmodified or wild-type IgSF domain of an IgSF family member to one or more of its homologous binding partners. In some examples, the IgSF domain is affinity modified and exhibits altered binding to the same one or more homologous binding partners as compared to binding of an unmodified or wild-type IgSF domain of an IgSF family member to one or more of its homologous binding partners. In some embodiments, the variant PD-L1 is a first PD-L1 variant and the IgSF domain of the second polypeptide is an IgSF domain from a second variant PD-L1, wherein the first PD-L1 variant and the second PD-L1 variant are the same or different.
In some embodiments, the variant PD-L1 polypeptide is capable of specifically binding to PD-1 or CD80, and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than the cognate binding partner specifically bound by the PD-L1 variant polypeptide. In some embodiments, the IgSF domain is from a member of the B7 family. In some cases, the IgSF domain is a tumor-localized portion that binds to ligands expressed on tumors, or an inflammatory localized portion that binds to cells or tissues associated with an inflammatory environment. In some embodiments, the IgSF domain is a tumor localization moiety that binds to a ligand expressed on a tumor. In some cases, the ligand is
In some embodiments, optionally the IgSF domain of the second or third polypeptide or an affinity modified IgSF domain thereof is or comprises an IgV domain. In some embodiments, the variant PD-L1 polypeptide is or comprises an IgV domain. In some embodiments, the immunomodulatory protein comprises a multimerization domain linked to one or both of a variant PD-L1 polypeptide or an IgSF domain. In some cases, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the immunomodulatory protein is a dimer. In some embodiments, the immunomodulatory protein is a homodimer. In some embodiments, the immunomodulatory protein is a heterodimer.
In some of any such embodiments, the IgSF domain of the second polypeptide is an IgSF domain of a ligand that binds to an inhibitory receptor or an affinity modified IgSF domain thereof. In some cases, an affinity modified IgSF domain exhibits increased binding affinity and/or binding selectivity for an inhibitory receptor as compared to binding of an unmodified IgSF domain to the same inhibitory receptor. In some embodiments, the inhibitory receptor is TIGIT, PD-1, or CTLA-4; or the ligand of the inhibitory receptor is PD-L2, CD155, CD112 or CD 80.
In some of any such embodiments, the IgSF domain of the second polypeptide is an affinity modified IgSF domain comprising: (i) wild type CD155 comprising an IgSF as set out in any of SEQ ID NOs 47, 310 or 353, or a variant CD155 polypeptide comprising an IgSF domain as set out in any of SEQ ID NOs as set out in Table 5, optionally any of SEQ ID NOs 311-352, 354-665, 1505-1576, 1551-1714; (ii) wild type CD112 comprising an IgSF domain as set out in any of SEQ ID NOs 48, 666 or 761, or a variant CD112 polypeptide comprising an IgSF domain as set out in any of SEQ ID NOs, optionally any of SEQ ID NOs 667-760, 762-931, 1433-1504; (iii) wild-type CD80 comprising an IgSF domain as set out in any of SEQ ID NO 28, 1005 or 2030, or a variant CD80 polypeptide comprising an IgSF as set out in any of SEQ ID NO, optionally in any of SEQ ID NO 932-964, 966-1038, 1040-1078, 1080-1112, 1114-1152; (iv) a wild-type PD-L2 comprising an IgSF domain as set forth in any of SEQ ID NOs 31, 1203 or 1263, or a variant PD-L2 polypeptide comprising an IgSF domain as set forth in any of SEQ ID NOs as set forth in Table 3, optionally any of SEQ ID NOs 1204-1254, 1256-1331, 1333-1407, 1409-1432; (v) (iii) an amino acid sequence having at least 95% sequence identity to any one of the SEQ ID NOs of (i) - (iv) and comprising an amino acid substitution; or (vi) a specific binding fragment of any one of (i) - (v).
In some embodiments, the immunomodulatory protein further comprises a third polypeptide comprising an IgSF domain of an IgSF family member or an affinity modified IgSF domain thereof, the affinity modified IgSF domain comprising one or more amino acid modifications as compared to an unmodified or wild-type IgSF domain of the IgSF family member. In some cases, the third polypeptide is the same as the first polypeptide and/or the second polypeptide, or the third polypeptide is different from the first polypeptide and/or the second polypeptide. In some examples, the third polypeptide is selected from (i) wild-type CD155 comprising an IgSF listed in any of SEQ ID NOS: 47, 310 or 353, or a variant CD155 polypeptide comprising an IgSF domain listed in any of SEQ ID NOS: 311-352, 354-665, 1505-1576, 1551-1714; (ii) wild-type CD112 comprising an IgSF domain as set out in any of SEQ ID NO 48, 666 or 761, or a variant CD112 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 667-760, 762-931, 1433-1504; (iii) wild-type CD80 comprising an IgSF domain as set out in any of SEQ ID NO 28, 1005 or 2030, or a variant CD80 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 932-964, 966-1038, 1040-1078, 1080-1112, 1114-1152; (iv) wild-type PD-L2 comprising the IgSF domain as set forth in any of SEQ ID NOS 31, 1203 or 1263, or a variant PD-L2 polypeptide comprising the IgSF domain as set forth in any of SEQ ID NOS 1204-1254, 1256-1331, 1333-1407, 1409-1432; (v) (iii) an amino acid sequence having at least 95% sequence identity to any one of the SEQ ID NOs of (i) - (iv) and comprising an amino acid substitution; or (vi) a specific binding fragment of any one of (i) - (v). In some cases, optionally the IgSF domain of the second or third polypeptide or an affinity modified IgSF domain thereof is or comprises an IgV domain. In some cases, the variant PD-L1 polypeptide is or contains an IgV domain.
In some embodiments, the immunomodulatory protein further comprises at least one additional polypeptide comprising an IgSF domain of an IgSF family member or an affinity modified IgSF domain thereof, said affinity modified IgSF domain comprising one or more amino acid modifications as compared to an unmodified or wild-type IgSF domain of said IgSF family member. In some embodiments, the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide or the second polypeptide. In some aspects, the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide, the second polypeptide, and/or the third polypeptide. In some cases, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the multimerization domain promotes heterodimer formation.
Providing an immunomodulatory protein comprising a first variant PD-L1 polypeptide, wherein the multimerization domain is a first multimerization domain; and a second variant PD-L1 polypeptide, wherein the multimerization domain is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first and second variant PD-L1 and PD-L1 polypeptides, optionally wherein the first and second variant PD-L1 and PD-L1 polypeptides are identical. In some cases, the multimerization domain is a first multimerization domain and interacts with a second multimerization domain to form a multimer comprising the immunomodulatory protein. In some examples, the immunomodulatory protein is a first immunomodulatory protein and the second immunomodulatory protein is directly or indirectly linked to a second multimerization domain via a linker, wherein the multimer comprises the first immunomodulatory protein and the second immunomodulatory protein. In some embodiments, the second immunomodulating protein is any immunomodulating protein as described and the multimerization domain is a second multimerization domain. In some cases, the multimer is a dimer. In some embodiments, the second polypeptide is a variant CD155 polypeptide and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence set forth in any one of SEQ ID NO 1716-1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO 1716-1721; or the second polypeptide is CD112 or CD155 and the third polypeptide is the other of CD112 or CD155 and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence as set forth in any one of SEQ ID NO 1722-1724 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO 1716-1721.
In some cases, the immunomodulatory protein is a homodimer. In some aspects, the immunomodulatory protein is a heterodimer, optionally wherein the first and second multimerization domains are different and/or capable of interacting to mediate heterodimer formation.
In some embodiments, the first multimerization domain and/or the second multimerization domain is an Fc domain or a variant thereof having reduced effector function, optionally wherein the Fc domain is an Fc domain of a human immunoglobulin and/or the Fc region is a human Fc region, optionally wherein the Fc region is an Fc region of immunoglobulin G1(IgG1) or immunoglobulin G2(IgG2), optionally listed in SEQ ID NO:187 or SEQ ID NO:188, optionally wherein the Fc region exhibits one or more effector functions; or the variant Fc domain comprises one or more amino acid substitutions in a wild-type Fc region, optionally wherein the reduced effector function is reduced compared to the wild-type Fc region, optionally wherein the wild-type human Fc is an Fc of
In some embodiments, the variant Fc region contains the amino acid substitutions E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, wherein the residue numbering is according to the EU index of Kabat; or amino acid substitutions R292C/N297G/V302C or L234A/L235E/G237A, wherein the numbering of the residues is according to EU index of Kabat. In some examples, the Fc region or variant Fc region contains the amino acid substitution C220S, wherein the residue numbering is according to the EU index of Kabat. In some cases, the Fc region or variant Fc region comprises K447del, wherein the residue numbering is according to EU index of Kabat.
Also provided is a conjugate comprising a variant PD-L1 according to any provided embodiment or an immunomodulatory polypeptide according to any provided embodiment linked to a moiety. In some cases, the moiety is a targeting moiety that specifically binds to a molecule on the surface of the cell. In some aspects, the targeting moiety specifically binds to a molecule on the surface of an immune cell.
In some embodiments, the immune cell is an antigen presenting cell or a lymphocyte. In some cases, the targeting moiety is a tumor localization moiety that binds to a molecule on the surface of a tumor. In some examples, the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle. In some embodiments, the moiety is an antibody or antigen-binding fragment. In some of any such embodiments, the conjugate is bivalent, tetravalent, hexavalent, or octavalent. In some aspects, the conjugate is a fusion protein.
Also provided is a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any provided embodiment, or an immunomodulatory polypeptide or conjugate according to any provided embodiment as a fusion protein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is cDNA.
Also provided is a vector comprising a nucleic acid molecule according to any of the provided embodiments. In some cases, the vector is an expression vector. In some aspects, the vector is a mammalian expression vector or a viral vector.
Also provided is a cell comprising a vector according to any provided embodiment. In some cases, the cell is a mammalian cell. In some aspects, the cell is a human cell.
Also provided is a method of producing a variant PD-L1 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule according to any provided embodiment or a vector according to any provided embodiment into a host cell under conditions in which the protein is expressed in the cell. In some cases, the method further comprises isolating or purifying a variant PD-L1 polypeptide or immunomodulatory protein from the cell. Also provided is a method of engineering a cell expressing a variant PD-L1 variant polypeptide, comprising introducing into the cell a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any of the provided embodiments, under conditions in which the polypeptide is expressed in the host cell.
Also provided is an engineered cell expressing a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, a conjugate according to any provided embodiment as a fusion protein, a nucleic acid molecule according to any provided embodiment, or a vector according to any provided embodiment. In some cases, the variant PD-L1 polypeptide or immunomodulatory protein comprises a signal peptide. In some cases, the variant PD-L1 polypeptide or immunomodulatory protein is encoded by a nucleic acid comprising a nucleotide sequence encoding a signal peptide. In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein does not contain a transmembrane domain and/or is not expressed on the surface of a cell.
In some embodiments of the engineered cell, the variant PD-L1 polypeptide or immunomodulatory protein is secreted from or capable of being secreted from the engineered cell. In some embodiments, the engineered cell contains a variant PD-L1 polypeptide that contains a transmembrane domain and/or is a transmembrane immunomodulatory protein according to any provided embodiments. In some embodiments, the variant PD-L1 polypeptide is expressed on the surface of a cell.
In some embodiments of the engineered cell, the cell is an immune cell. In some cases, the immune cell is an Antigen Presenting Cell (APC) or a lymphocyte. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the APC is an artificial APC. In some examples, the cell is a lymphocyte, and the lymphocyte is a T cell. In some aspects, the cell is an APC, and the APC is an artificial APC. In some of any such embodiments, the engineered cell further contains a Chimeric Antigen Receptor (CAR) or an engineered T cell receptor.
Also provided is an infectious agent comprising a nucleic acid molecule encoding a variant PD-L1 polypeptide according to any provided embodiment, a conjugate according to any provided embodiment as a fusion protein, or an immunomodulatory polypeptide according to any provided embodiment. In some cases, the encoded variant PD-L1 polypeptide or immunomodulatory polypeptide does not contain a transmembrane domain and/or is not expressed on the surface of the cell in which it is expressed. In some embodiments, the encoded variant PD-L1 polypeptide or immunomodulatory polypeptide is secreted from, or capable of being secreted by, the cell in which it is expressed. In some cases, the encoded variant PD-L1 polypeptide contains a transmembrane domain. In some embodiments, the encoded variant PD-L1 polypeptide is expressed on the surface of a cell in which it is expressed.
In some of any such embodiments, the infectious agent is a bacterium or a virus. In some embodiments, the virus is a lentivirus or retrovirus construct or a hybrid thereof. In some aspects, the virus is an oncolytic virus. In some examples, the oncolytic virus is an adenovirus, an adeno-associated virus, a herpes simplex virus, a vesicular stomatitis virus (vesicular stomatic virus), a reovirus, a newcastle disease virus, a parvovirus, a measles virus, a Vesicular Stomatitis Virus (VSV), a coxsackie virus, or a vaccinia virus. In some cases, the virus specifically targets Dendritic Cells (DCs) and/or is dendritic cell tropic. In some examples, the virus is a lentiviral vector pseudotyped with a modified Sindbis virus (Sindbis virus) envelope product. In some of any such embodiments, the infectious agent further comprises a nucleic acid molecule encoding another gene product that causes death of the target cell or that can enhance or enhance the immune response. In some examples, the another gene product is selected from an anti-cancer agent, an anti-metastatic agent, an anti-angiogenic agent, an immune regulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degradation gene, a gene for tissue regeneration or reprogramming human cells to pluripotency.
Also provided is a pharmaceutical composition comprising a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, a conjugate according to any provided embodiment, an engineered cell according to any provided embodiment, or an infectious agent according to any provided embodiment. In some cases, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.
Also provided is an article of manufacture containing a pharmaceutical composition according to any provided embodiment in a vial. In some cases, the vial is sealed.
Also provided is a kit containing a pharmaceutical composition according to any of the provided embodiments and instructions for use. Also provided is a kit containing an article of manufacture according to any of the provided embodiments and instructions for use.
Also provided is a method of modulating an immune response (such as increasing or decreasing an immune response) in a subject, comprising administering to the subject a pharmaceutical composition according to any provided embodiment. In some embodiments, a method of modulating an immune response in a subject comprises administering to the subject an engineered cell according to any of the provided embodiments. In some cases, the engineered cells are autologous to the subject. In some cases, the engineered cell is allogeneic to the subject. In some embodiments, the method of modulating an immune response treats a disease or condition in a subject. In some embodiments, the method comprises administering to the subject a soluble variant PD-L1 polypeptide according to any one of the embodiments described herein, an immunomodulatory protein according to any one of the embodiments described herein, or a conjugate according to any one of the embodiments described herein. In some embodiments, the method comprises administering to the subject an infectious agent encoding a variant PD-L1 polypeptide according to any one of the embodiments described herein.
In some of any such embodiments, the immune response is increased. In some embodiments, a soluble variant PD-L1 polypeptide or immunomodulatory protein is administered to a subject. In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein is an Fc fusion protein. In some embodiments, a soluble variant PD-L1 polypeptide or immunomodulatory protein that optionally lacks a PD-L1 transmembrane domain and an intracellular signaling domain is administered to a subject. In some cases, the soluble immunomodulatory protein is an immunomodulatory Fc fusion protein. In some embodiments, a variant PD-L1 polypeptide according to any provided embodiment, an immunomodulatory protein according to any provided embodiment, or a conjugate according to any provided embodiment is administered to a subject. In some embodiments, the engineered cell containing the secretable variant PD-L1 polypeptide is administered to a subject. In some of any such embodiments, the engineered cells according to any provided embodiments are administered to a subject. In some aspects, the infectious agent is administered to the subject, optionally under conditions in which an infectious agent encoding a variant PD-L1 polypeptide that secretes an immunomodulatory protein infects tumor cells or immune cells and secretes the immunomodulatory protein from the infected cells.
In some embodiments, the disease or condition is a tumor or cancer. In some examples, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, stomach cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal tract cancer, germ cell cancer, or endocrine and neuroendocrine cancers.
In some of any such embodiments, the immune response is decreased. In some embodiments, an immunomodulatory protein or conjugate comprising a variant PD-L1 polypeptide linked to an IgSF domain or a moiety that localizes to a cell or tissue of an inflammatory environment is administered to a subject. In some cases, the binding molecule comprises an antibody or antigen-binding fragment thereof or comprises a wild-type IgSF domain or variant thereof. In some embodiments, an immunomodulatory protein according to any provided embodiments, or a conjugate according to any provided embodiments, is administered to a subject.
In some embodiments, a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject. In some embodiments, an engineered cell containing a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein according to any provided embodiment is administered to a subject. In some cases, the infectious agent is administered to the subject, optionally under conditions in which an infectious agent encoding a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein infects tumor cells or immune cells and the transmembrane immunomodulatory protein is expressed on the surface of the infected cells.
In some embodiments, the disease or condition is an inflammatory or autoimmune disease or condition. In some of any such embodiments, the disease or condition is an anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis, an autoimmune skin disease, transplantation, rheumatism, an inflammatory gastrointestinal tract disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory lung disease, an inflammatory endocrine disease, or an autoimmune hematologic disease. In some examples, the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. In some of any such embodiments, the variant PD-L1 is administered in a form that reduces the immune response in the subject.
Drawings
Fig. 1A-1C depict different forms of the variant IgSF domain molecules provided. Fig. 1A depicts a soluble molecule comprising: (1) a variant IgSF domain fused to an Fc chain (vigdd); (2) a stacked molecule comprising a first variant IgSF domain (first igd) and a second IgSF domain, such as a second variant IgSF domain (second igd); (3) tumor-targeting IgSF molecules comprising a first variant IgSF domain (vigdd) and an IgSF domain targeting a tumor antigen, such as NKp30 IgSF domain; and (4) a variant IgSF domain (vigdd) linked to an antibody (V-mAb). FIG. 1B depicts Transmembrane Immunomodulatory Protein (TIP) containing a variant IgSF domain (vIgD) expressed on the surface of a cell. In an exemplary embodiment, the cognate binding partner of the transmembrane-bound vigdd is an inhibitory receptor (e.g., PD-L1), and the TIP containing the vigdd (e.g., PD-L1 vigdd) antagonizes or blocks negative signaling of the inhibitory receptor, thereby producing an activated or effector T cell. In some cases, agonism of activity by TIP may be achieved if clustering of inhibitory receptors (PD-1) is in proximity to an activating receptor (e.g., CD 28). Figure 1C depicts Secreted Immunomodulatory Protein (SIP), wherein a variant IgSF domain (viggd) is secreted from a cell, such as a first T cell (e.g., CAR T cell). In an exemplary embodiment, the cognate binding partner of the secreted vigdd is an inhibitory receptor (e.g., PD-1), which can be expressed by a first cell (e.g., a T cell, such as a CAR T cell) and/or expressed on a second cell (e.g., a T cell; a cognate or engineered cell, such as a CAR T cell). Upon binding of SIP to its cognate binding partner, SIP antagonizes or blocks negative signaling through inhibitory receptors, thereby producing activated or effector T cells. In all cases, vigdd can be a combination of only V-domains (IgV), V-domains (IgV) and C-domains (IgC), including the entire extracellular domain (ECD), or any combination of Ig domains of IgSF superfamily members.
Fig. 2 depicts an exemplary schematic of the activity of a variant IgSF domain fused to Fc (vigdd-Fc), wherein vigdd is a variant of the IgSF domain of PD-L1. As shown, soluble vigdd of PD-L1 interacts with its cognate binding partner to block the interaction of PD-L1 or PD-L2 with PD-1, thereby blocking the PD-1 inhibitory receptor and, in some cases, differentiating T cells into an effector phenotype.
Fig. 3 depicts an exemplary schematic of a stacking molecule that is a multi-target checkpoint antagonist containing a first variant IgSF domain that is PD-L1 or PD-L2 igd (first igd) and a second IgSF domain that binds to a second inhibitory receptor (e.g., second igd). In an exemplary schematic, the second IgSF domain (e.g., second igd) is CD112 or CD155 igd. As shown, the first and second viggd interact with their cognate binding partners to block the interaction of PD-L1 or PD-L2 with PD-1 and to block the interaction of CD155 or CD112 with TIGIT and/or CD112R, respectively, thereby blocking multiple inhibitory receptors.
Fig. 4 depicts an exemplary schematic of a stacking molecule for localization of variant igsf (vigdd) to tumor cells. In this form, the stacking molecule contains a first variant IgSF domain (first igd) and a second IgSF domain (e.g., second igd), wherein the second IgSF domain (e.g., second igd) is a tumor-targeting IgSF domain that binds to a tumor antigen. An exemplary tumor-targeting IgSF domain is that of NKp30 that binds to tumor antigen B7-H6. In this depiction, the variant IgSF domain (viggd) is a variant of the IgSF domain of PD-L1. As shown, binding of the tumor-targeting IgSF domain to the surface of the tumor cell localizes a first variant IgSF domain on the surface of the tumor cell, wherein the first variant IgSF domain can interact with one or more homologous binding partners expressed on the surface of adjacent immune cells (e.g., T cells) to antagonize PD-1 inhibitory activity and promote T cell activation.
Fig. 5A depicts various exemplary configurations of a stacked molecule containing a first variant IgSF domain (first igd) and a second IgSF domain, such as a second variant IgSF domain (second igd). As shown, the first igd and second IgSF domains are independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. To generate a homodimeric Fc molecule, an Fc region is one that is capable of forming a homodimer with a matching Fc region by coexpression of a single Fc region in a cell. To generate heterodimeric Fc molecules, a single Fc region contains a mutation (e.g., a "knob-into-hole" mutation in the CH3 domain) such that the formation of heterodimers is favored when the single Fc region is co-expressed in a cell compared to homodimers.
Fig. 5B depicts various exemplary configurations of a stacked molecule containing a first variant IgSF domain (first igd); a second IgSF domain, such as a second variant IgSF domain (second igd); and a third IgSF domain, such as a third variant IgSF domain (a third igd). As shown, the first igd, second IgSF, and third IgSF domains are independently linked directly or indirectly to the N-terminus or C-terminus of the Fc region. To generate a homodimeric Fc molecule, an Fc region is one that is capable of forming a homodimer with a matching Fc region by coexpression of a single Fc region in a cell.
Fig. 6 depicts an exemplary schematic of the activity of a variant IgSF domain (vigdd) conjugated to an antibody (V-Mab), wherein the antibody (e.g., an anti-HER 2 antibody) binds to an antigen on the surface of a tumor cell to localize the vigdd to the cell. As shown, antibody binding to the surface of tumor cells localizes the igd on the surface of the tumor cells, where the igd can interact with one or more cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells) to agonize or antagonize receptor signaling. In the exemplary embodiments shown in the figures, a variant IgSF domain (viggd) is a variant of the IgSF domain of PD-L1 that binds to inhibitory receptor PD-1, such as having increased affinity for the inhibitory receptor PD-1. Binding of PD-L1 vIgD to a PD-1 inhibitory receptor antagonizes or blocks negative signaling of the inhibitory receptor, thereby producing an activated T cell or effector T cell. In some cases, agonism of inhibitory receptor activity by TIP may be achieved if clustering of inhibitory receptors (PD-1) is in proximity to an activating receptor (e.g., CD 28).
Fig. 7A-7C depict various exemplary configurations of variant IgSF-antibody conjugates (V-mabs). Fig. 7A shows various configurations in which variant IgSF domains are linked directly or indirectly to the N-terminus and/or C-terminus of the antibody light chain. Fig. 7B shows various configurations in which variant IgSF domains are linked directly or indirectly to the N-terminus and/or C-terminus of an antibody heavy chain. FIG. 7C depicts the resulting V-Mab configuration when the light chain of FIG. 7A and the heavy chain of FIG. 7B are co-expressed in a cell.
FIGS. 8 and 9 depict the results of the biological activity of the soluble variant PD-L1 IgV-Fc tested in human Mixed Lymphocyte Reaction (MLR). Approximately 10,000 mature DCs and 100,000 purified allogeneic CD3+ T cells were co-cultured with various increasing concentrations of the variant PD-L1 IgV-Fc fusion protein. Non-relevant human IgG or medium alone (indicated as "no addition") was used as a negative control. The control protein PDL1-Fc (whole wild-type PD-L1 extracellular domain), wild-type PD-L1 IgV-Fc and the positive control anti-PD-1 monoclonal antibody (nivolumab) were assessed. FIGS. 8 and 9 set forth the calculated levels of IFN-. gamma.in the culture supernatants (pg/mL) at the indicated concentrations of the variant IgV-Fc fusion molecules.
Figure 10 depicts a proliferation study of T cells transduced with the exemplary tested variant PD-L1 SIP.
Figure 11 depicts the dose response of indicated binding of variant IgV-Fc fusion molecules, PD-L1/CD155 stacked Fc fusion molecules, or PD-L1/CD155/CD112 stacked Fc fusion molecules to depleting T cells.
Figure 12 lists calculated levels of IFN- γ (pg/mL) in culture supernatants of depleted T cells under indicated concentrations of variant IgV-Fc fusion molecules, PD-L1/CD155 stacked Fc fusion molecules, or antibody controls.
Detailed Description
Provided herein are immunomodulatory proteins that are or comprise variants or mutants of
In some embodiments, the variant PD-L1 polypeptide and immunomodulatory protein modulate an immunological immune response, such as increasing or decreasing an immune response. In some embodiments, the variant PD-L1 polypeptides and immunomodulatory proteins provided herein may be used to treat diseases or conditions associated with a dysregulated immune response.
In some embodiments, provided variant PD-L1 polypeptides modulate T cell activation via co-stimulation and/or co-inhibition signaling molecules. In general, antigen-specific T cell activation typically requires two distinct signals. The first signal is provided by the interaction of the T Cell Receptor (TCR) with a Major Histocompatibility Complex (MHC) -associated antigen present on an Antigen Presenting Cell (APC). The second signal is co-stimulatory for TCR engagement and is necessary for T cell proliferation, differentiation and/or survival (including in some cases avoiding T cell apoptosis or disability).
In some embodiments, under normal physiological conditions, a T cell-mediated immune response is initiated by antigen recognition by the T Cell Receptor (TCR) and is modulated by a balance of costimulatory and inhibitory signals (e.g., immune checkpoint proteins). The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and to prevent tissues from being excessively damaged during immune responses, for example, during challenge against pathogen infection. However, in some cases, these immunomodulatory proteins may be dysregulated in diseases and conditions (including tumors) as a mechanism to evade the immune system.
In some embodiments, the known T cell co-stimulatory receptor is programmed
However, PD-L1 ligand may also bind to cluster of differentiation 80 (also known as CD80 or B7-1). Binding of PD-L1 to CD80 may block the interaction between PD-L1 and PD-1, and thus potentiate or enhance the immune response. Thus, in some cases, the interactions between PD-L1 and CD80 and PD-L1 and PD-1 produce opposite effects in modulating immune responses. Thus, PD-1 and CD80 may exert opposing effects in the immune response to modulate pro-inflammatory or anti-inflammatory responses, which in some cases are associated with a variety of diseases and conditions.
In some embodiments, PD-1 and CD80 may play complementary roles in modeling immune responses. In some embodiments, the enhancement or inhibition of the activity of the PD-1 receptor is of clinical interest for the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, therapies that intervene and alter the immunomodulatory effects of such receptors are limited by spatial orientation requirements and size limitations imposed by the boundaries of immunological synapses. In some aspects, existing therapeutic drugs (including antibody drugs) may not be able to interact simultaneously with multiple target proteins involved in modulating these interactions. Furthermore, in some cases, existing therapeutic drugs may only have the ability to antagonize, rather than agonize, the immune response. In addition, pharmacokinetic differences between drugs that target one of these receptors independently can create difficulties in properly maintaining the desired blood concentration of such drug combinations throughout the course of treatment.
In some embodiments, provided variant PD-L1 polypeptides or immunomodulatory proteins modulate (e.g., increase or decrease) PD-1 associated immune activity. Thus, in some embodiments, provided polypeptides overcome these constraints by providing variant PD-L1 with altered (e.g., increased or decreased) binding affinity for PD-1, thereby agonizing or antagonizing the effects of the receptor. In some embodiments, the provided polypeptides overcome these constraints by providing a variant PD-L1 that has altered (e.g., increased or decreased) binding affinity for CD80, thereby modulating the effects of the interaction between PD-1 and PD-L1. Methods of making and using these variants PD-L1 are also provided.
All publications (including patents, patent applications, scientific papers, and databases) mentioned in this specification are herein incorporated in their entirety by reference for all purposes to the same extent as if each individual publication (including patents, patent applications, scientific papers, or databases) was specifically and individually indicated to be incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with the definition set forth in the patents, applications, published applications and other publications that are incorporated by reference, the definition set forth herein controls over the definition that is incorporated by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. Definition of
Unless otherwise defined, all technical, scientific and other terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is commonly understood in the art.
Unless otherwise limited in specific instances, terms used throughout this specification are defined as follows. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms, acronyms, and abbreviations used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the invention belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names are according to the IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values defining the range and all integer values therebetween.
The term "affinity modified" as used in the context of immunoglobulin superfamily domains means that a mammalian immunoglobulin superfamily (IgSF) domain has an altered amino acid sequence (relative to the corresponding wild-type parent or unmodified IgSF domain) such that it has an increased or decreased binding affinity or avidity for at least one homologous binding partner (or "counterpart structure") thereof as compared to the parent wild-type or unmodified (i.e., non-affinity modified) IgSF control domain. In this context affinity modified PD-L1 IgSF domains are included. In some embodiments, the affinity modified IgSF domain can contain 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions, in the wild-type or unmodified IgSF domain. The increase or decrease in binding affinity or avidity can be determined using well known binding assays such as flow cytometry. Larsen et al, American Journal of Transplantation, Vol.5: 443-. See also Linsley et al, Immunity, Vol.1 (9): 793-. The increase in binding affinity or avidity of the protein for one or more of its cognate binding partners is a value that is at least 10% greater than the wild-type IgSF domain control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value. The reduction in binding affinity or avidity of a protein for its at least one cognate binding partner is a value no greater than 90% of the control but no less than 10% of the wild-type IgSF domain control value, and in some embodiments, no greater than 80%, 70%, 60%, 50%, 40%, 30%, or 20% but no less than 10% of the wild-type IgSF domain control value. Affinity modified proteins are altered in the primary amino acid sequence by substitution, addition or deletion of amino acid residues. The term "affinity modified IgSF domain" should not be construed as utilizing any conditions of any particular starting composition or method by which the affinity modified IgSF domain is produced. Thus, the affinity modified IgSF domains of the invention are not limited to wild-type IgSF domains, which are then converted to affinity modified IgSF domains by any particular affinity modification method. Affinity modified IgSF domain polypeptides can be generated, for example, starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for binding to its cognate binding partner, and finally recombined or chemically synthesized to produce the subject affinity modified IgSF domain compositions. In just one alternative example, affinity modified IgSF domains can be generated by site-directed mutagenesis of wild-type IgSF domains. Thus, an affinity modified IgSF domain represents one product and does not necessarily represent a product produced by any given method. Various techniques may be employed, including recombinant methods, chemical synthesis, or combinations thereof.
As used herein, the term "allogeneic" means that cells or tissue are removed from one organism and then infused or adoptively transferred into a genetically different organism of the same species. In some embodiments of the invention, the species is murine or human.
As used herein, the term "autologous" means that cells or tissue are removed from an organism and subsequently infused or adoptively transferred to the same organism. Autologous cells or tissues can be altered, for example, by recombinant DNA methods, such that it is no longer genetically identical to native cells or native tissues removed from an organism. For example, native autologous T cells can be genetically engineered by recombinant DNA techniques to become autologous engineered cells that express transmembrane immunomodulatory proteins and/or Chimeric Antigen Receptors (CARs), which in some cases involve engineering T cells or TILs (tumor infiltrating lymphocytes). The engineered cells are then infused into a patient from which native T cells are isolated. In some embodiments, the organism is a human or a mouse.
As used herein, the terms "binding affinity" and "binding affinity" refer to the specific binding affinity and specific binding affinity, respectively, of a protein to its relative structure under specific binding conditions. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of an individual's non-covalent binding interactions (such as between PD-L1 and its counterpart structures PD-1 and/or CD 80). Thus, affinity is different from affinity describing the strength of a single interaction. The increase or decrease in binding affinity of the variant PD-L1 containing an affinity modified PD-L1 IgSF domain to its relative structure is determined relative to the binding affinity of unmodified PD-L1, such as unmodified PD-L1 containing a native or wild-type IgSF domain, such as an IgV domain. Methods for determining binding affinity or avidity are known in the art. See, e.g., Larsen et al, American Journal of Transplantation, Vol.5: 443-. In some embodiments, a variant PD-L1 of the invention (i.e., a PD-L1 protein containing an affinity modified IgSF domain) specifically binds to PD-1 and/or CD80 as measured by flow cytometry with a binding affinity that produces a Mean Fluorescence Intensity (MFI) value in the binding assay that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than a wild-type PD-L1 control.
The term "biological half-life" refers to the amount of time it takes for a substance, such as an immunomodulatory polypeptide comprising the variant PD-L1 of the invention, to lose one-half of its pharmacological or physiological activity or concentration. Biological half-life can be achieved by elimination, excretion, degradation (e.g., enzymatic), or absorption and concentration of substances in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for a substance plasma concentration to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize and increase the biological half-life of the polypeptides of the invention are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO2013130683), Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), lipids (acylation), and poly Pro-Ala-ser (pas), poly glutamic acid (glutamylation).
As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial (i.e., artificial) transmembrane protein comprising at least an extracellular domain, a transmembrane, and an intracellular domain (ectodomain) that is expressed on mammalian cells. Optionally, the CAR protein comprises a "spacer" that covalently links the ectodomain to the transmembrane domain. Spacers are typically polypeptides that link the ectodomain to the transmembrane domain via a peptide bond. The CAR is typically expressed on mammalian lymphocytes. In some embodiments, the CAR is expressed on a mammalian cell, such as a T cell or a Tumor Infiltrating Lymphocyte (TIL). The CAR expressed on the T cell is referred to herein as a "CAR T cell" or "CAR-T". In some embodiments, the CAR-T is a T helper cell, a cytotoxic T cell, a natural killer T cell, a memory T cell, a regulatory T cell, or a γ δ T cell. When used clinically, e.g., with adoptive cell transfer, CAR-T having antigen binding specificity to a patient's tumor is typically engineered to be expressed on T cells obtained from the patient. The engineered T cells expressing the CAR are then infused back into the patient. Thus, the CAR-T is typically an autologous CAR-T, although an allogeneic CAR-T is included within the scope of the invention. The extracellular domain of the CAR comprises an antigen binding region, such as an antibody or antigen binding fragment thereof (e.g., scFv), that specifically binds to a target antigen, such as a tumor-specific antigen, under physiological conditions. Upon specific binding, the biochemical chain of events (i.e., signal transduction) causes modulation of the immunological activity of CAR-T. Thus, for example, upon specific binding to its target antigen by the antigen binding region of CAR-T, a change in immunological activity of the T cell activity may result, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signaling upon CAR-T activation is achieved through the CD 3-zeta chain ("CD 3-z") involved in signaling in native mammalian T cells. CAR-T may further comprise various signaling domains, such as CD28, 41BB, or OX40, to further modulate the immune regulatory response of T cells. CD3-z contains conserved motifs involved in T cell receptor signaling, called Immunoreceptor Tyrosine Activation Motifs (ITAMs).
The term "collective" or "collective" when used with reference to cytokine production induced by the presence of two or more variant PD-L1 in an in vitro assay means overall cytokine expression levels regardless of cytokine production induced by the individual variant PD-L1. In some embodiments, the cytokine being assayed is IFN- γ in an in vitro primary T cell assay.
The term "homologous binding partner" (used interchangeably with "relative structure") of a reference polypeptide, such as the IgSF domain of reference variant PD-L1, refers to at least one molecule (typically a native mammalian protein) to which the reference polypeptide specifically binds under specific binding conditions. In some aspects, a variant PD-L1 containing an affinity modified IgSF domain specifically binds to the opposite structure corresponding to native or wild-type PD-L1, but binds with increased or decreased affinity. The class of ligands that are recognized and that specifically bind to their cognate receptor under specific binding conditions are examples of the relative structure or cognate binding partner of this receptor. A "cognate cell surface binding partner" is a cognate binding partner expressed on the surface of a mammalian cell. A "cell surface molecular species" IS a cognate binding partner of a ligand of an Immunological Synapse (IS) which IS expressed on and by a cell, such as a mammalian cell, thereby forming the immunological synapse.
As used herein, "conjugate," "conjugation," or grammatical variations thereof refers to joining or linking two or more compounds together by any joining or linking method known in the art to form another compound. It may also refer to a compound produced by joining or linking two or more compounds together. For example, variant PD-L1 polypeptides linked directly or indirectly to one or more chemical moieties or polypeptides are exemplary conjugates. Such conjugates include fusion proteins, conjugates produced by chemical conjugation, and conjugates produced by any other method.
As used herein, the term "competitive binding" means that a protein is capable of specifically binding at least two homologous binding partners, but the specific binding of one homologous binding partner inhibits (such as prevents or prevents) the simultaneous binding of a second homologous binding partner. Thus, in some cases, it is not possible for a protein to bind to two homologous binding partners simultaneously. Typically, competitive binders contain the same or overlapping binding sites for specific binding, but this is not required. In some embodiments, competitive binding results in a measurable inhibition (partial or complete) of specific binding of the protein to one of its cognate binding partners due to specific binding of the second cognate binding partner. Various methods are known to quantify competitive binding, such as ELISA (enzyme linked immunosorbent assay) assays.
As used herein, the term "conservative amino acid substitution" means an amino acid substitution in which one amino acid residue is substituted with another amino acid residue having a side chain R group of similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acid groups having side chains of similar chemical character include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. Conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.
The term "corresponding to" (such as a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (such as listed in a sequence listing)) with reference to a position of a protein refers to a nucleotide or amino acid position that is identified when aligned with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, the corresponding residues can be determined by aligning the reference sequence with the sequence of wild-type PD-L1 as set forth in SEQ ID NO:30 or 1728(ECD domain) or as set forth in SEQ ID NO:55 or 309(IgV domain) via a structural alignment method as described herein. By aligning the sequences, one skilled in the art can, for example, use conserved and identical amino acid residues as guides to identify corresponding residues.
As used herein, the term "reduce" or "alleviate" or "inhibit" means reduce a statistically significant amount. The reduction may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
The term "derivative" or "derivatize" refers to a modification of a protein by covalently attaching it, directly or indirectly, to a composition in order to alter a characteristic such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy, while retaining or enhancing its therapeutic benefit. Derivatives of the immunomodulatory polypeptides of the invention are within the scope of the invention and may be prepared, for example, by glycosylation, pegylation, lipidation, or Fc fusion.
As used herein, a domain (typically a sequence of three or more, typically 5 or 7 or more amino acids, such as 10 to 200 amino acid residues) refers to a portion of a molecule (such as a protein or encoding nucleic acid) that is structurally and/or functionally different from other portions of the molecule and is identifiable. For example, domains include those portions of a polypeptide chain that can form an independent fold structure within a protein, which is made up of one or more structural motifs and/or is recognized by a functional activity, such as a binding activity. The protein may have one or more than one distinct domain. For example, domains may be identified, defined or distinguished by homology of the primary sequence or structure to the relevant family members, such as homology to motifs. In another example, domains may be distinguished by their function, such as their ability to interact with biomolecules such as homologous binding partners. The domains may independently exhibit a biological function or activity, such that the domains, independently or fused to another molecule, may perform an activity, such as binding, for example. The domains may be linear amino acid sequences or non-linear amino acid sequences. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For purposes of illustration herein, definitions are provided, but it is understood that identifying particular domains by name is within the skill of the art. Where desired, appropriate software can be used to identify the domains.
As used herein, the term "extracellular domain" refers to a region of a membrane protein (such as a transmembrane protein) that is located outside of the liquid cell membrane. The extracellular domain typically comprises a binding domain that specifically binds to a ligand or cell surface receptor, such as via specific binding to a ligand or cell surface receptor. The extracellular domain of a cellular transmembrane protein is alternatively referred to as the extracellular domain.
The term "effective amount" or "therapeutically effective amount" refers to the amount and/or concentration of a therapeutic composition (including a protein composition or a cell composition) of the invention that, when administered alone (i.e., as a monotherapy) or in combination with an additional therapeutic agent, ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient), causes a statistically significant reduction in the progression of the disease, e.g., by reducing or eliminating the symptoms and/or causes of the disease. An effective amount can be an amount that reduces, or alleviates at least one symptom or biological response or effect associated with a disease or condition, prevents the progression of a disease or condition, or improves a patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments, the patient is a mammal, such as a non-human primate or human patient.
As used herein, the term "endodomain" refers to a region present in some membrane proteins (such as transmembrane proteins) that extends into an interior space defined by a cell surface membrane. In mammalian cells, the intracellular domain is the cytoplasmic region of a membrane protein. In a cell, the endodomain interacts with intracellular components and may play a role in signal transduction, and thus may in some cases be an intracellular signaling domain. The intracellular domain of a cellular transmembrane protein is alternatively referred to as the cytoplasmic domain, which in some cases may be the cytoplasmic signaling domain.
The term "enhanced" or "increased" as used herein in the context of increasing the immunological activity of a mammalian lymphocyte means increasing one or more activities in the lymphocyte. The increased activity may be one or more of: increased cell survival, cell proliferation, cytokine production, or T cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to increased immunological activity means increased interferon gamma (IFN- γ) production, such as by a statistically significant amount. In some embodiments, immunological activity can be assessed in a Mixed Lymphocyte Reaction (MLR) assay. Methods of performing MLR assays are known in the art. Wang et al, Cancer Immunol Res.2014 9 months: 2(9): 846-56. Other methods of assessing lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, the enhancement may be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than the non-zero control value.
As used herein, the term "engineered cell" refers to a mammalian cell that has been genetically modified by human intervention, such as by recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., a T cell, B cell, NK cell) or an antigen presenting cell (e.g., a dendritic cell). The cells may be primary cells from a patient or may be cell lines. In some embodiments, the engineered cells of the invention comprise a variant PD-L1 of the invention engineered to modulate the immunological activity of a T cell expressing PD-1 and/or CD80, the variant PD-L1 specifically binding to said PD-1 and/or CD 80. In some embodiments, the variant PD-L1 is a transmembrane immunomodulatory protein (hereinafter "TIP") comprising an extracellular domain or a portion thereof comprising an IgV or ECD domain linked to a transmembrane domain (e.g., PD-L1 transmembrane domain) and optionally an intracellular signaling domain. In some cases, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the engineered cell is capable of expressing and secreting an immunomodulatory protein as described herein. Among the engineered cells provided are those further comprising an engineered T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).
As used herein, the term "engineered T cell" refers to a T cell that has been genetically modified by human intervention, such as by recombinant DNA methods or viral transduction methods, such as a T helper cell, a cytotoxic T cell (or, alternatively, a cytotoxic T lymphocyte or CTL), a natural killer T cell, a regulatory T cell, a memory T cell, or a γ δ T cell. The engineered T cells comprise a variant PD-L1 Transmembrane Immunomodulatory Protein (TIP) or Secreted Immunomodulatory Protein (SIP) of the invention expressed on T cells and engineered to modulate the immunological activity of mammalian cells to which the engineered T cells themselves or the variant PD-L1 expressed on T cells specifically bind.
The term "engineered T cell receptor" or "engineered TCR" refers to a T Cell Receptor (TCR) engineered to specifically bind to a Major Histocompatibility Complex (MHC)/peptide target antigen with a desired affinity, which is selected, cloned, and/or subsequently introduced into a population of T cells, which is typically used for adoptive immunotherapy. In contrast to engineered TCRs, CARs are engineered to bind a target antigen in an MHC independent manner.
As used herein, the term "express in.. the term" is used with reference to a protein expressed on the surface of a cell (such as a mammalian cell). Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety, such as a drug or a detectable label. Proteins expressed on the cell surface may include cell surface proteins expressed on mammalian cells, such as cell surface receptors.
The term "half-life extending moiety" refers to a moiety of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in the serum of a mammal compared to the half-life of a protein not so conjugated to the moiety. In some embodiments, the half-life is extended greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold. In some embodiments, the half-life is extended more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, amount, or activity. The half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extending moieties include Fc domains, multimerization domains, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO2013130683), Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), lipids (acylated) and poly Pro-Ala-ser (pas), and polyglutamic acid (glutamylated).
As used herein, the term "immunological synapse" or "immunological synapse" means the interface between a mammalian cell, such as an antigen presenting cell or a tumor cell, expressing MHC I (major histocompatibility complex) or MHC II and a mammalian lymphocyte, such as an effector T cell or a Natural Killer (NK) cell.
The Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as an Fc polypeptide) corresponds primarily to the constant region of an immunoglobulin heavy chain and is responsible for various functions, including one or more effector functions of an antibody. The Fc domain contains part or all of the hinge domain of an immunoglobulin molecule plus the CH2 and CH3 domains. The Fc domain may form a dimer of two polypeptides joined by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) activity to promote effector function. In some embodiments, reference to an amino acid substitution in the Fc region is by the EU numbering system unless described with reference to a particular SEQ ID NO. EU numbering is known and is based on the latest updated IMGT scientific icon (international ImMunoGeneTics informati on
An immunoglobulin Fc fusion ("Fc fusion"), such as an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. The Fc fusion may comprise, for example, the Fc region of an antibody (which promotes effector function and pharmacokinetics) and variant PD-L1. The immunoglobulin Fc region may be linked indirectly or directly to one or more variant PD-L1 or small molecules (fusion partners). Different linkers are known in the art and can optionally be used to link the Fc to a fusion partner to generate an Fc fusion. Fc fusions of the same species may be dimerized to form Fc fusion homodimers or different species may be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a murine or human Fc.
The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. The host cell may be a prokaryote, such as e.coli, or it may be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Examples of host cells include Chinese Hamster Ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines grown in serum-free media or the CHO line DX-B11 lacking DHFR. Another example is a human endothelial kidney 293 cell or derivative thereof. In some embodiments, the host cell is a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell).
As used herein, the term "immunoglobulin" (abbreviated "Ig") refers to a mammalian immunoglobulin, including any of the five human classes of antibodies: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. The term also includes less than full-length immunoglobulins, whether wholly or partially synthetic (e.g., recombinant or chemically synthesized) or naturally occurring, such as antigen binding fragments (fabs), V-containingHAnd VL(iv) variable fragment (Fv) comprising V linked together in one chainHAnd VLSingle chain variable fragments (scFv) of (A), and other antibody V region fragments, such as Fab', F (ab)2、F(ab′)2dsFv diabodies (diabodies), Fc, and Fd polypeptide fragments. Within the meaning of the term is included bispecific antibodies, i.e. both homologous and heterologous bispecific.
As used herein, the term "immunoglobulin superfamily" or "IgSF" means a group of cell surface and soluble proteins that are involved in the recognition, binding or adhesion processes of cells. Molecules are classified as members of this superfamily based on structural features common to immunoglobulins (i.e., antibodies); they all have domains or folds called immunoglobulin domains. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular myoproteins. They are often associated with roles in the immune system. Proteins in immunological synapses are typically members of IgSF. IgSF is also classified into "subfamilies" based on common properties such as function. Such subfamilies typically consist of 4 to 30 IgSF members.
As used herein, the term "IgSF domain" or "immunoglobulin domain" or "Ig domain" refers to the domain of an IgSF protein, Ig domains are named as immunoglobulin molecules, they contain about 70-110 amino acids and are classified according to their size and function, Ig domains have a characteristic Ig fold with a sandwich-like structure formed by two sheets of antiparallel β chains the interaction between hydrophobic amino acids on the inner side of the sandwich and highly conserved disulfide bonds formed between cysteine residues of B and F chains stabilizes the Ig fold, one end of the Ig domain has a segment called the complementarity determining region which is important for the specificity of the antibody for its ligand, Ig-like domains can be classified into (the following classes) IgV, IgC (which may be IgC1 or IgC2) or igi, most IgV domains are variable (IgV) or constant (IgV) or IgC, TCR domain with 9 IgV chains has a7 IgV domain which is generally longer than the IgV domain of IgC 9 chains, and a receptor domain similar to the IgC domain of these IgV domains is called the IgC 14, g domain which is located in the cell wall of a cell wall, the igg domain contains a similar to the IgC 3-g domain.
As used herein, the term "IgSF species" means all IgSF member proteins having the same or substantially the same primary amino acid sequence. Each member of the mammalian immunoglobulin superfamily (IgSF) defines the unique identity of all IgSF species belonging to that IgSF member. Thus, each IgSF family member is unique relative to other IgSF family members, and thus each species of a particular IgSF family member is unique relative to another species of IgSF family member. However, there may be variations between molecules of the same IgSF species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, minor sequence differences within a single IgSF species due to genetic polymorphisms constitute changes within another form of the single IgSF species, as do wild-type truncated forms of the IgSF species due to, for example, proteolytic cleavage. A "cell surface IgSF species" is an IgSF species expressed on the surface of a cell (typically a mammalian cell).
As used herein in the context of mammalian lymphocytes such as T cells, the term "immunological activity" refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon- γ), or T cell cytotoxic activity. In some cases, immunological activity may refer to cellular expression of cytokines such as chemokines or interleukins. Assays for determining enhancement or inhibition of immunological activity include MLR (mixed lymphocyte reaction) assays (Wang et al, Cancer Immunol Res.2014 9 months: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assays (Wang et al, Cancer Immunol Res.2014 9 months: 2(9):846-56), and anti-CD 3T cell stimulation assays (Li and Kurlander, J Transl Med.2010: 8: 104) which measure interferon- γ cytokine levels in culture supernatants. Since T cell activation is associated with secretion of IFN- γ cytokines, detection of IFN- γ levels in culture supernatants can be determined using a commercial ELISA kit for these in vitro human T cell assays (Wu et al, Immunol Lett 2008, 4/15 days; 117(1): 57-62). Induction of an immune response results in an increase in immunological activity relative to resting lymphocytes. Immunomodulatory proteins containing affinity-modified IgSF domains (such as variant PD-L1 polypeptides) as provided herein can be relative to wild-type IgSF member or IgSF domain control, in a primary T cell assay, increases in some embodiments or decreases in alternative embodiments IFN- γ (interferon- γ) expression. The skilled person will recognise that the format of the primary T cell assay used to determine the increase in IFN- γ expression may be different from the assay used to determine the decrease in IFN- γ expression. In assaying the ability of an immunomodulatory protein or affinity modified IgSF domain of the invention to alter IFN- γ expression in a primary T cell assay, a Mixed Lymphocyte Reaction (MLR) assay may be used. Conveniently, in some cases, soluble forms of the affinity modified IgSF domains of the invention can be used to determine their ability to increase or decrease IFN- γ expression in MLRs. Alternatively, a co-immobilization assay may be used. In a co-immobilization assay, the T cell receptor signal provided by the anti-CD 3 antibody is used in some embodiments in conjunction with a co-immobilized affinity modified IgSF domain (such as variant PD-L1) to determine the ability to increase or decrease IFN- γ expression relative to a wild-type IgSF domain control. Methods of determining the immunological activity of engineered cells, including assessing the activity of variant PD-L1 transmembrane immunomodulatory proteins, are known in the art and include, but are not limited to, the ability to expand T cells following antigen stimulation, the ability to maintain T cell expansion in the absence of restimulation, and anti-cancer activity in an appropriate animal model. Assays also include assays to assess cytotoxicity, including standards51Cr Release assay (see, e.g., Milone et al, (2009) Molecular
Therapy 17: 1453-.
An "immunomodulatory polypeptide" or "immunomodulatory protein" is a polypeptide or protein molecule that modulates immunological activity. By "modulating" an immune response is meant increasing or decreasing immunological activity. The immunomodulatory protein can be a single polypeptide chain or a multimer (dimer or higher multimer) of at least two polypeptide chains covalently bonded to each other, e.g., by intrachain disulfide bonds. Thus, monomeric, dimeric and higher multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimers (same polypeptide chain) or heteromultimers (different polypeptide chains). The immunomodulatory proteins of the invention comprise variant PD-L1.
As used herein, the term "increase" means an increase in a statistically significant amount. The increase can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or greater than a non-zero control value.
The "isoform" of PD-L1 is one of a plurality of naturally occurring PD-L1 polypeptides that differ in amino acid sequence. Isoforms may be the product of splice variants of an RNA transcript expressed from a single gene or the expression product of highly similar but different genes, thereby producing functionally similar proteins, such as may result from gene replication. The term "isoform" of PD-L1, as used herein, also refers to the product of different alleles of the PD-L1 gene.
As used herein, the term "lymphocyte" means any of the three subtypes of white blood cells in the mammalian immune system. They include natural killer cells (NK cells), which play a role in cell-mediated cytotoxic innate immunity, T cells (acquired immunity against cell-mediated cytotoxicity), and B cells (antibody-driven acquired immunity against humoral). The T cells include: t helper cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γ δ T cells. Innate Lymphocytes (ILCs) are also included within the definition of lymphocytes.
The term "mammal" or "patient" specifically includes reference to at least one of: human, chimpanzee, macaque, cynomolgus monkey, dog, cat, mouse or rat.
As used herein, the term "membrane protein" means a protein that is attached directly or indirectly to a lipid bilayer under physiological conditions. The membrane-forming lipid bilayer may be a biological membrane, such as a eukaryotic (e.g., mammalian) cell membrane or an artificial (i.e., man-made) membrane, such as a membrane present on a liposome. Attachment of the membrane protein to the lipid bilayer may be by covalent attachment 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. Membrane proteins that are peripheral membrane proteins are non-covalently attached to the lipid bilayer or non-covalently attached to integral membrane proteins. The peripheral membrane proteins form a temporary attachment to the lipid bilayer such that the peripheral membrane proteins can associate and/or disassociate with the lipid bilayer within the range of conditions of mammalian physiology. The integral membrane protein forms a substantially permanent attachment to the membrane lipid bilayer compared to the peripheral membrane protein, such that the integral membrane protein does not dissociate from the attachment to the lipid bilayer under a range of conditions in which the mammal is physiological. Membrane proteins can form attachments to the membrane through one layer of the lipid bilayer (unilamellar) or through two layers of the membrane (polytopic). The integral membrane protein that interacts with only one lipid bilayer is an "integral monoscopic protein". The integral membrane proteins that interact with both lipid bilayers are "integral multi-modal proteins," or "transmembrane proteins" as used herein.
The term "modulating" as used herein in the context of an immune response, such as a mammalian immune response, refers to any alteration, such as an increase or decrease, of an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant PD-L1 of the invention or as a result of administration of an immunomodulatory protein expressing an immunomodulatory protein of the invention, such as a variant PD-L1 transmembrane immunomodulatory protein. Thus, it refers to an alteration, such as an increase or decrease, in an immune response as compared to an immune response that occurs or is present in the absence of administration of an immunomodulatory protein comprising variant PD-L1 or a cell expressing such an immunomodulatory polypeptide. Such modulation includes any induction, activation, inhibition, or alteration of the degree or extent of immunological activity of the immune cell. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional Antigen Presenting Cells (APC) and non-professional antigen presenting cells, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils and basophils). Modulation includes any change imparted to an existing immune response, a developed immune response, a potential immune response, or the ability to induce, modulate, affect, or respond to an immune response. Modulation includes any change in the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response. Modulation of the immune response or modulation of immunological activity includes, for example, the following: elimination, deletion or sequestration of immune cells; inducing or generating immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; inducing a non-responsive state (i.e., incapacitating) of the immune cell; enhance or inhibit the activity or function of immune cells, including but not limited to altering the pattern of proteins expressed by these cells. Examples include alterations in the production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, co-stimulatory molecules or other cell surface receptors, or any combination of these regulatory events. Modulation can be assessed in primary T Cell assays, for example, by changes in IFN- γ (interferon γ) expression relative to wild-type PD-L1 controls (see Zhao and Ji, Exp Cell Res.2016, 1/1; 340(1): 132-. Modulation may be, for example, by alteration of the immunological activity of the engineered cell relative to a cell engineered with the wild-type PD-L1 transmembrane protein, such as alteration of the cytotoxic activity of the engineered cell or alteration of cytokine secretion of the engineered cell.
As used herein, the term "molecular species" means all proteins having the same or substantially the same primary amino acid sequence. Each member of the mammalian immunoglobulin superfamily (IgSF) defines a collection of identical or substantially identical molecular species. Thus, for example, human PD-L1 is a member of IgSF and each human PD-L1 molecule is a molecular species of PD-L1. There may be variations between molecules of the same molecular species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. In addition, minor sequence differences within a single molecular species due to genetic polymorphisms constitute changes within another form of a single molecular species, as do wild-type truncated forms of a single molecular species due to, for example, proteolytic cleavage. A "cell surface molecular species" is a molecular species expressed on the surface of a mammalian cell. Two or more different protein species, each present exclusively on one of the two mammalian cells forming the IS or exclusively on the other mammalian cell (but not on both mammalian cells), are said to be in "cis" or "cis configuration" relative to each other. The first of the two different protein species IS present exclusively on one of the two mammalian cells that form IS and the second IS present exclusively on the second of the two mammalian cells that form IS, said protein species being considered to be in "trans" or "trans configuration". Two different protein species are present on each of the two cells of the two mammalian cells forming the IS, said protein species being in cis and trans configurations on these cells.
The term "multimerization domain" refers to an amino acid sequence that facilitates stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each of which contains complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), which may be the same or different multimerization domains. Interactions between complementary multimerization domains, e.g., between a first multimerization domain and a second multimerization domain, form stable protein-protein interactions to generate multimers of polypeptide molecules and additional polypeptide molecules. In some cases, the multimerization domains are identical and interact with themselves to form stable protein-protein interactions between the two polypeptide chains. Typically, the polypeptide is directly or indirectly conjugated to a multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain may be, for example, an immunoglobulin constant region or domain, such as, for example, an Fc domain or portion thereof from IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes), IgA, IgE, IgD, and IgM, and modified forms thereof.
The terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides and which have similar binding properties as the natural nucleotides and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as the sequence explicitly indicated (the "reference sequence"). Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.
As used herein, the term "non-competitive binding" means the ability of a protein to specifically bind to at least two homologous binding partners simultaneously. Thus, the protein is capable of binding to at least two different cognate binding partners simultaneously, although the binding interactions need not be for the same duration, such that in some cases the protein specifically binds to only one cognate binding partner. In some embodiments, the binding occurs under specific binding conditions. In some embodiments, simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding to a second cognate binding partner. In some embodiments, noncompetitive binding means that binding of the second homologous binding partner to its binding site on the protein does not displace binding of the first homologous binding partner to its binding site on the protein. Methods for determining non-competitive binding are well known in the art, such as those described in Perez de La Lastra et al, Immunology,1999, month 4: 96(4): 663-. In some cases, in a non-competitive interaction, the first homologous binding partner specifically binds at an interaction site that does not overlap with the interaction site of the second homologous binding partner, such that binding of the second homologous binding partner does not directly interfere with binding of the first homologous binding partner. Thus, any effect of the binding of the second cognate binding partner on the binding of said cognate binding partner is by a mechanism other than directly interfering with the binding of the first cognate binding partner. For example, in the context of enzyme-substrate interactions, a non-competitive inhibitor binds to a site other than the enzyme active site. Non-competitive binding encompasses non-competitive binding interactions, wherein the second homologous binding partner specifically binds at an interaction site that does not overlap with the binding of the first homologous binding partner, but binds to the second interaction site only when the first interaction site is occupied by the first homologous binding partner.
The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (typically a human). Pharmaceutical compositions typically comprise an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising variant PD-L1 or an engineered cell expressing a variant PD-L1 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.
The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked by peptide bonds. The term does not relate to a specific length of the product. Thus, "peptides" and "oligopeptides" are included within the definition of polypeptide. The term includes post-translational modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation, and the like. The term also includes molecules in which one or more amino acid analogs, or atypical or unnatural amino acids, are included as a result of being synthesized or recombinantly expressed using known protein engineering techniques. In addition, the protein may be derivatized.
As used herein, the term "primary T cell assay" refers to an in vitro assay that measures expression of interferon-gamma ("IFN- γ"). A variety of such primary T cell assays are known in the art. In a preferred embodiment, the assay used is an anti-CD 3 co-immobilization assay. In this assay, primary T cells were stimulated by anti-CD 3 immobilized in the presence or absence of additional recombinant protein. At some time points, culture supernatants were harvested, typically 24-72 hours. In another embodiment, the assay used is MLR. In this assay, primary T cells are stimulated with allogeneic APCs. At some time points, culture supernatants were harvested, typically 24-72 hours. Human IFN- γ levels in culture supernatants were measured by standard ELISA techniques. Commercial kits were purchased from commercial suppliers and the assays were performed according to the manufacturer's recommendations.
The term "purified" as applied to a nucleic acid, such as an immunomodulatory protein-encoding of the invention, generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., the purified polypeptide or polynucleotide forms discrete bands in an electrophoresis gel, a chromatographic eluate, and/or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces a substantial band in an electrophoretic gel is "purified". The purified nucleic acids or proteins of the invention are at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a percent by weight or molar basis).
The term "recombinant" refers to an artificial (i.e., non-native) alteration of a material (e.g., a nucleic acid or polypeptide) by human intervention. The alteration may be performed on the material while it is in its natural environment or state, or while it is being removed therefrom. For example, a "recombinant nucleic acid" is a nucleic acid prepared by recombining a nucleic acid, e.g., during cloning, affinity modification, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by means of such molecular biological techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and/or expresses a recombinant nucleic acid or is otherwise altered by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein (such as a transmembrane immunomodulatory protein as provided herein). Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of an array of short DNA sequences that interact specifically with cellular proteins involved in transcription. Promoter and enhancer elements are isolated from a variety of eukaryotic sources, including yeast, insect and mammalian cells and genes of viruses (autologous control elements, i.e., promoters are also found in prokaryotes). The choice of a particular promoter and enhancer depends on what cell type will be used to express the protein of interest. As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the joining of nucleic acid sequences in a manner or orientation that results in the production of a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule.
The term "recombinant expression vector" as used herein refers to a DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include promoters, optional operator sequences, ribosome binding sites and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. The secretion signal peptide sequence may also optionally be encoded by a recombinant expression vector operably linked to the coding sequence of a recombinant protein (such as a recombinant fusion protein) so that the expressed fusion protein can be secreted by a recombinant host cell to make the fusion protein easier to isolate from the cell, if desired. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. The vector is a viral vector, such as a lentiviral vector.
The term "selective" refers to a preference of a subject protein or polypeptide for specific binding to one substrate (such as a cognate binding partner) as compared to specific binding to another substrate (such as a different cognate binding partner of the subject protein). Selectivity can be reflected as the binding activity (e.g., binding affinity) of the subject protein and a first substrate (such as a first homologous binding partner) (e.g., K)d1) Binding activity (e.g., binding affinity) to the same subject protein and a second homologous binding partner (e.g., K)d2) The ratio of (a) to (b).
As used herein, the term "sequence identity" refers to sequence identity at the nucleotide or amino acid level between genes or proteins, respectively. "sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at given positions in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art and include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLAST algorithm calculates the percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website.
The term "soluble" as used herein with reference to a protein means that the protein is not a membrane protein. In general, soluble proteins contain only the extracellular domain of an IgSF family member receptor or a portion thereof that contains one or more IgSF domains or specific binding fragments thereof, but no transmembrane domain. In some cases, the solubility of a protein may be improved by linking or attaching to an Fc domain, either directly or indirectly via a linker, which may also improve the stability and/or half-life of the protein in some cases. In some aspects, the soluble protein is an Fc fusion protein.
The term "species" as used herein with respect to a polypeptide or nucleic acid means all molecules having the same or substantially the same sequence. There may be variations between polypeptides of the same species due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated sequences of polypeptides that differ from the full-length species by no more than 1, 2 or 3 amino acid residues at the amino terminus or carboxy terminus are considered to be a single species. Such microscopic heterogeneity is a common feature of the proteins produced.
The term "specific binding fragment" as used herein with reference to a full-length wild-type mammalian PD-L1 polypeptide or an IgV or IgC (e.g., IgC2) domain thereof means a polypeptide having a subsequence of the full-length polypeptide or IgV and/or IgC domain and that specifically binds to mammalian PD-1 and/or mammalian CD80 (such as human or murine PD-1 or CD80) in vitro and/or in vivo. In some embodiments, a specific binding fragment comprises a PD-L1 IgV or PD-L1 IgC2 subsequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the full-length wild-type sequence or an IgV or IgC (e.g., IgC2) sequence thereof. The sequence of the specific binding fragment may be altered to form the variant PD-L1 of the present invention.
As used herein, the term "specific binding" means the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or avidity is at least 5-fold, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500-fold, or even at least 1000-fold greater than the average affinity or avidity of the same protein for a collection of random peptides or polypeptides of sufficient statistical size. A specific binding protein need not bind exclusively to a single target molecule, but may specifically bind to a non-target molecule due to structural conformational similarity between the target and the non-target (e.g., an ortholog or ortholog). The skilled artisan will recognize that specific binding to molecules having the same function in different animal species (i.e., orthologs) or non-target molecules having substantially similar epitopes to the target molecule (e.g., orthologs) is possible and does not diminish the binding specificity determined relative to a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, the polypeptides of the invention can specifically bind to more than one different target molecule species due to cross-reactivity. Solid phase ELISA immunoassays, ForteBio Octet or Biacore measurements can be used to determine specific binding between two proteins. In general, the dissociation constant (K) of the interaction between two binding proteinsd) Less than 1x10-5M, and is typically as low as 1x10-12And M. In certain embodiments of the present disclosure, the dissociation constant of the interaction between two binding proteins is less than or equal to about 1x10-6M、1x10- 7M、1x10-8M、1x10-9M、1x10-10M or 1x10-11M or less.
The term "surface expression" with reference to a mammalian cell expressing a polypeptide means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.
As used herein, reference to "synthesis" of, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule produced by recombinant methods and/or by chemical synthetic methods.
The term "targeting moiety" as used herein refers to a composition that is covalently or non-covalently attached to or physically encapsulates a polypeptide comprising the variant PD-L1 of the present invention. The targeting moiety has specific binding affinity for a desired relative structure such as a cell surface receptor (e.g., PD-1) or a tumor antigen such as a Tumor Specific Antigen (TSA) or a Tumor Associated Antigen (TAA) such as B7-H6. Typically, the desired relative structure is located on a particular tissue or cell type. The targeting moiety comprises: antibodies, antigen binding fragments (Fab), V-containingHAnd VL(iv) variable fragment (Fv) comprising V linked together in one chainHAnd VLSingle chain variable fragments (scFv) of (A), and other antibody V region fragments, such as Fab', F (ab)2、F(ab′)2dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecules (<500 daltons) composition (e.g., specifically binding receptor composition). The targeting moiety may also be covalently or non-covalently attached to the lipid membrane of the liposome, which encapsulates the polypeptide of the invention.
As used herein, the term "transmembrane protein" means a membrane protein that substantially or completely spans a lipid bilayer, such as those found in biological membranes (such as mammalian cells) or artificial constructs (such as liposomes), the transmembrane protein comprises a transmembrane domain ("transmembrane domain") through which the transmembrane protein integrates into the lipid bilayer and through which integration is thermodynamically stable under physiological conditions.
As used herein, the term "treating" of a disease or disorder means slowing, halting, or reversing the progression of the disease or disorder by administering a therapeutic composition (e.g., containing an immunomodulatory protein or engineered cells) alone or in combination with another compound as described herein, as evidenced by a reduction, cessation, or elimination of clinical or diagnostic symptoms. "treating" also means reducing the severity of symptoms of an acute or chronic disease or disorder, or reducing the rate of relapse as, for example, in the case of a relapsing or remitting autoimmune disease course, or reducing inflammation in the case of an inflammatory aspect of an autoimmune disease. As used herein in the context of cancer, the term "treatment" or "inhibition" of cancer refers to at least one of: a statistically significant decrease in tumor growth rate, tumor growth arrest, or a decrease in the size, mass, metabolic activity, or volume of a tumor, as measured by a normative standard such as, but not limited to, a solid tumor response assessment standard (RECIST) or a statistically significant increase in Progression Free Survival (PFS) or Overall Survival (OS). As used in the context of the present invention, "Preventing" refers to administering an immunomodulatory polypeptide or an engineered cell of the invention, alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to reduce the likelihood of the onset of a disease or disorder.
As used herein, the term "tumor-specific antigen" or "TSA" refers to a relative structure that is predominantly present on tumor cells of a mammalian subject, but is generally not visible on normal cells of the mammalian subject. The tumor-specific antigen need not be specific for a tumor cell, but the percentage of cells with tumor-specific antigen in a particular mammal is high enough, or the level of tumor-specific antigen on the surface of a tumor is high enough, so that it can be targeted by an anti-tumor therapeutic agent, such as the immunomodulatory polypeptides of the invention, and provide prophylaxis or treatment of protecting a mammal from the effects of a tumor. In some embodiments, at least 50% of the cells displaying TSA in a random statistical cell sample from a mammal with a tumor are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells displaying TSA are cancerous.
The term "variant" (also "modified" or "mutant") as used with reference to variant PD-L1 means PD-L1 formed by human intervention, such as mammalian (e.g., human or murine) PD-L1. Variant PD-L1 is a polypeptide having an altered amino acid sequence relative to unmodified or wild-type PD-L1. Variant PD-L1 is a polypeptide that differs from a wild-type PD-L1 isoform sequence by one or more amino acid substitutions, deletions, additions or a combination thereof. For purposes herein, variant PD-L1 contains at least one affinity modified domain, and thus one or more amino acid differences exist in the IgSF domain (e.g., IgV domain or ECD). Variant PD-L1 may contain 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions. Variant PD-L1 polypeptides typically have at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the corresponding wild-type or unmodified PD-L1, such as to the mature sequence of sequence SEQ ID NO:3, which contains the extracellular domain or IgSF domain thereof (lacking the signal sequence), or a portion thereof. In some embodiments, a variant PD-L1 polypeptide has at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified PD-L1 comprising a sequence set forth in SEQ ID NO 30 or 1728 or SEQ ID NO 55 or 309. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. Variant PD-L1 is not limited to any particular method of preparation and includes, for example, re-chemical synthesis, re-recombinant DNA techniques, or combinations thereof. The variant PD-L1 of the invention specifically binds to at least one or more of PD-1 or CD80 of a mammalian species. In some embodiments, the altered amino acid sequence results in altered (i.e., increased or decreased) binding affinity or avidity for PD-1 and/or CD80 as compared to the wild-type or unmodified PD-L1 protein. The increase or decrease in binding affinity or avidity can be determined using well known binding assays such as flow cytometry. Larsen et al, American journal transfer, Vol.5: 443-. See also Linsley et al, Immunity, Vol.1 (9): 793-. The increase in binding affinity or avidity of the variant PD-L1 for PD-1 and/or CD80 is a value that is at least 5% greater than that of wild-type or unmodified PD-L1, and in some embodiments is a value that is at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of wild-type or unmodified PD-L1 control. The reduction in binding affinity or avidity of PD-L1 for PD-1 and/or CD80 is a value no greater than 95% of the wild-type or unmodified control value, and in some embodiments is a value no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5% of the wild-type or unmodified control value or undetectable binding affinity or avidity. The variant PD-L1 is altered in the primary amino acid sequence by substitution, addition or deletion of an amino acid residue. The term "variant" in the context of variant PD-L1 should not be construed as utilizing any condition of any particular starting composition or method by which variant PD-L1 is produced. Variant PD-L1 can be generated, for example, starting from wild-type mammalian PD-L1 sequence information, then modeled in silico to bind to PD-1 and/or CD80, and finally synthesized recombinantly or chemically to produce variant PD-L1 of the invention. In but one alternative example, variant PD-L1 can be generated by site-directed mutagenesis of wild-type PD-L1. Thus, the variant PD-L1 represents a composition and does not necessarily represent a product produced by any given method. Various techniques may be employed, including recombinant methods, chemical synthesis, or combinations thereof.
As used herein, the terms "wild-type" or "native" are used in conjunction with biological materials such as nucleic acid molecules, proteins (e.g., PD-L1), IgSF members, host cells, and the like, and refer to those materials that are found in nature and that are not modified by human intervention.
Variant PD-L1 Polypeptides
Provided herein are variant PD-L1 polypeptides that exhibit altered (increased or decreased) binding activity or affinity for one or more PD-L1 cognate binding partners. In some embodiments, the PD-L1 homologous binding partner is PD-1 or CD 80. In some embodiments, the PD-L1 homologous binding partner is PD-1. In some embodiments, the variant PD-L1 polypeptide contains one or more amino acid modifications, such as one or more substitutions (alternatively, "mutations" or "substitutions"), deletions, or additions in the immunoglobulin superfamily (IgSF) domain (IgD) relative to the wild-type or unmodified PD-L1 polypeptide or the portion of wild-type or unmodified PD-L1 containing IgD or a specific binding fragment thereof. Thus, provided variant PD-L1 polypeptides are or comprise a variant IgD (hereinafter "vIgD"), wherein one or more amino acid modifications (e.g., substitutions) are in the IgD.
In some embodiments, the IgD comprises an IgV domain or an IgC (e.g., IgC2) domain, or a specific binding fragment of an IgV domain or an IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, IgD may be IgV alone, a combination of IgV and IgC, including the entire extracellular domain (ECD), or any combination of Ig domains of PD-L1. Exemplary residues corresponding to the IgV or IgC region of PD-L1 are provided in Table 2. In some embodiments, the variant PD-L1 polypeptide comprises an IgV domain or an IgC domain or a specific binding fragment thereof, wherein at least one amino acid modification (e.g., substitution) is in the IgV domain or the IgC domain or the specific binding fragment thereof. In some embodiments, the variant PD-L1 polypeptide comprises an IgV domain or a specific binding fragment thereof, wherein at least one amino acid modification (e.g., substitution) is in the IgV domain or the specific binding fragment thereof. In some embodiments, the altered IgV domain or IgC (e.g., IgC2) domain is an affinity modified IgSF domain by altered binding activity or affinity.
In some embodiments, the variant is modified in one or more IgSF domains relative to the sequence of the unmodified PD-L1 sequence. In some embodiments, the unmodified PD-L1 sequence is wild-type PD-L1. In some embodiments, unmodified or wild-type PD-L1 has the sequence of native PD-L1 or an ortholog thereof. In some embodiments, unmodified PD-L1 is or comprises the extracellular domain (ECD) of PD-L1 or a portion thereof containing one or more IgSF domains (see table 2). In some embodiments, the extracellular domain of an unmodified or wild-type PD-L1 polypeptide comprises an IgV domain and one or more IgC (e.g., IgC2) domains. However, a variant PD-L1 polypeptide need not comprise both the IgV domain and one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises or consists essentially of an IgV domain or a specific binding fragment thereof. In some embodiments, a variant PD-L1 polypeptide comprises or consists essentially of one or both of an IgC (e.g., IgC2) domain or a specific binding fragment thereof. In some embodiments, a variant PD-L1 polypeptide comprises or consists essentially of only one of an IgC (e.g., IgC2) domain or a specific binding fragment thereof. In some embodiments, the variant PD-L1 polypeptide comprises: an IgV domain or a specific binding fragment thereof; and first and second IgC (e.g., IgC2) domains or specific binding fragments thereof. In some embodiments, the variant PD-L1 polypeptide is soluble and lacks a transmembrane domain. In some embodiments, the variant PD-L1 polypeptide further comprises a transmembrane domain, and in some cases a cytoplasmic domain.
In some embodiments, the wild-type or unmodified PD-L1 sequence is a mammalian PD-L1 sequence. In some embodiments, the wild-type or unmodified PD-L1 sequence may be mammalian PD-L1, including but not limited to human, mouse, cynomolgus monkey or rat PD-L1. In some embodiments, the wild-type or unmodified PD-L1 sequence is a human sequence.
In some embodiments, the wild-type or unmodified PD-L1 sequence has (i) the amino acid sequence set forth in SEQ ID No. 3 or a mature form thereof lacking the signal sequence, (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 3 or a mature form thereof, or (iii) is a portion of (i) or (ii) that contains an IgV domain or an IgC (e.g., IgC2) domain or a specific binding fragment thereof.
In some embodiments, the wild-type or unmodified PD-L1 sequence is or comprises the extracellular domain of PD-L1 or a portion thereof. In some embodiments, the unmodified or wild-type PD-L1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30 or 1728, or an orthologue thereof. In some cases, an unmodified or wild-type PD-L1 polypeptide can comprise (i) the amino acid sequence set forth in SEQ ID NO:30 or 1728, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:30 or 1728, or (iii) a specific binding fragment of a sequence of (i) or (ii) that comprises an IgV domain or an IgC (e.g., IgC2) domain.
In some embodiments, the wild-type or unmodified PD-L1 polypeptide comprises an IgV domain or one or more IgC (e.g., IgC2) domains or specific binding fragments thereof. In some embodiments, the IgV domain of the wild-type or unmodified PD-L1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55 or 309 (corresponding to amino acid residues 24-130 of SEQ ID NO: 3) or an orthologue thereof. For example, the IgV domain of an unmodified or wild-type PD-L1 polypeptide may contain (i) the amino acid sequence set forth in SEQ ID NO:55 or 309, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:55 or 309, or (iii) a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain is capable of binding to one or more PD-L1 homologous binding proteins, such as one or more of PD-1 or CD 80.
In some embodiments, the first IgC2 domain of the wild-type or unmodified PD-L1 polypeptide comprises the amino acid sequence as set forth as residue 133-225 of SEQ ID NO. 3 or an orthologue thereof. For example, the IgC2 domain of an unmodified or wild-type PD-L1 polypeptide may contain (i) the amino acid sequence as set forth as residue 133-225 of SEQ ID NO:3, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to residue 133-225 of SEQ ID NO:3, or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgC domain is capable of binding to one or more PD-L1 homologous binding proteins.
In some embodiments, the wild-type or unmodified PD-L1 polypeptide contains a specific binding fragment of PD-L1, such as a specific binding fragment of an IgV domain or an IgC (e.g., IgC2) domain. In some embodiments, the specific binding fragment may bind to PD-1 and/or CD 80. The amino acid length of a specific binding fragment may be at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, a specific binding fragment of an IgV domain comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgV domain as set forth as amino acids 24-130 of SEQ ID NO 3. In some embodiments, a specific binding fragment of an IgC (e.g., IgC2) domain comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgC domain as set forth as amino acids 133-225 of SEQ ID NO: 3.
In some embodiments, the variant PD-L1 polypeptide comprises an ECD domain or portion thereof that comprises one or more affinity modified IgSF domains. In some embodiments, a variant PD-L1 polypeptide may comprise an IgV domain or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain, or a specific binding fragment of one or more IgC (e.g., IgC2) domains, wherein one or more of the IgSF domains (IgV or IgC) contain one or more amino acid modifications (e.g., substitutions). In some embodiments, a variant PD-L1 polypeptide may comprise an IgV domain and one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain and a specific binding fragment of one or more IgC (e.g., IgC2) domains, wherein at least one of the IgV or IgC domains contains one or more amino acid modifications (e.g., one or more substitutions). In some embodiments, the variant PD-L1 polypeptide comprises a full-length IgV domain. In some embodiments, a variant PD-L1 polypeptide comprises one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain. In some embodiments, a variant PD-L1 polypeptide comprises a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, a variant PD-L1 polypeptide comprises a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains. In some embodiments, a variant PD-L1 polypeptide comprises a full-length IgV domain and a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, a variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain and one or more full-length IgC (e.g., IgC2) domains. In some embodiments, a variant PD-L1 polypeptide comprises a specific binding fragment of an IgV domain and a specific binding fragment of one or more IgC (e.g., IgC2) domains.
In any such embodiment, one or more amino acid modifications (e.g., substitutions) of a variant PD-L1 polypeptide can be located in any one or more of the IgSF domains of the PD-L1 polypeptide. For example, in some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the extracellular domain of the variant PD-L1 polypeptide. In some embodiments, one or more amino acid modifications (e.g., substitutions) are located in the IgV domain or a specific binding fragment of the IgV domain. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in an IgC (e.g., IgC2) domain or a specific binding fragment of an IgC (e.g., IgC2) domain.
Generally, each of the various attributes of the polypeptide (e.g., soluble and membrane-bound polypeptide, PD-L1 affinity for PD-1 and CD80, varying number of polypeptide chains per polypeptide chain, number of polypeptide chains linked, number and nature of amino acid changes per variant PD-L1, etc.) are disclosed separately below. However, it will be clear to the skilled person that any particular polypeptide may comprise a combination of these independent attributes. It is to be understood that reference to amino acids (including the specific sequences listed as seq id NOs) used to describe the domain composition of IgSF domains is for illustrative purposes and is not intended to limit the scope of the embodiments provided. It will be appreciated that the description of polypeptides and their domains is theoretically deduced based on homology analysis and alignment with similar molecules. Thus, the precise locus may vary and is not necessarily the same for each protein. Thus, a specific IgSF domain (such as a specific IgV domain or IgC domain) may be several amino acids (such as one, two, three or four) longer or shorter.
In addition, various embodiments of the invention as discussed below are frequently provided within the meaning of the defined terms as disclosed above. The embodiments described in a particular definition should therefore be construed as being incorporated by reference when the defined terms are used to discuss the various aspects and attributes described herein. Thus, the order of presentation of the various aspects and embodiments, and the separate disclosure of the various independent attributes, is not intended to limit the scope of the present disclosure.
A. Exemplary modifications
Provided herein are the following variant PD-L1 polypeptides: the IgSF domain (e.g., ECD, IgV, or IgC) or specific binding fragment thereof containing at least one affinity modification relative to the IgSF domain contained in a wild-type or unmodified PD-L1 polypeptide, such that the variant PD-L1 polypeptide exhibits altered (increased or decreased) binding activity or affinity for one or more ligands PD-1 or CD80 as compared to the wild-type or unmodified PD-L1 polypeptide. In some embodiments, the binding affinity of the variant PD-L1 polypeptide to PD-1 and/or CD80 differs from a wild-type or unmodified PD-L1 polypeptide control sequence, as determined by, for example, a solid phase ELISA immunoassay, flow cytometry, ForteBio Octet, or Biacore assay. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and/or CD 80. In some embodiments, the variant PD-L1 polypeptide has reduced binding affinity for PD-1 and/or CD80 relative to a wild-type or unmodified PD-L1 polypeptide. PD-1 and/or CD80 may be a mammalian protein, such as a human protein or a murine protein.
The binding affinity for each cognate binding partner is independent; that is, in some embodiments, the variant PD-L1 polypeptide has increased binding affinity for one or both of PD-1 and/or CD80 and decreased binding affinity for one or both of PD-1 and CD80 relative to a wild-type or unmodified PD-L1 polypeptide.
In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has reduced binding affinity for PD-1 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has reduced binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide.
In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has increased binding affinity for PD-1 and decreased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has reduced binding affinity for PD-1 and CD80 relative to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide has reduced binding affinity for PD-1 and increased binding affinity for CD80 relative to a wild-type or unmodified PD-L1 polypeptide.
In some embodiments, a variant PD-L1 polypeptide having increased or greater binding affinity for PD-1 and/or CD80 has at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50% increased binding affinity for PD-1 and/or CD80 relative to a wild-type or unmodified PD-L1 polypeptide control. In some embodiments, the binding affinity relative to a wild-type or unmodified PD-L1 polypeptide is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In such examples, the wild-type or unmodified PD-L1 polypeptide has the same sequence as the variant PD-L1 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).
In some embodiments, a variant PD-L1 polypeptide having reduced binding affinity for PD-1 and/or CD80 has at least 5% reduced binding affinity for PD-1 and/or CD80 relative to a wild-type or unmodified PD-L1 polypeptide control, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the binding affinity relative to a wild-type or unmodified PD-L1 polypeptide is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In such examples, the wild-type or unmodified PD-L1 polypeptide has the same sequence as the variant PD-L1 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).
In some embodiments, the equilibrium dissociation constant (K) of any of the preceding embodiments to PD-1 and/or CD80d) May be less than 1x10-5M、1x10-6M、1x10-7M、1x10-8M、1x10-9M、1x10-10M or 1x10-11M or 1x10-12M orAnd is smaller.
The wild-type or unmodified PD-L1 sequence need not necessarily be used as a starting composition for the generation of the variant PD-L1 polypeptides described herein. Thus, use of the term "modification," such as "substitution," does not imply that embodiments of the present invention are limited to a particular method of making a variant PD-L1 polypeptide. Variant PD-L1 polypeptides can be prepared, for example, by de novo peptide synthesis and thus do not necessarily require modifications, such as "substitutions," in the sense of altering codons to encode the modifications (e.g., substitutions). This theory also extends to the terms "addition" and "deletion" of amino acid residues, which likewise does not imply a particular preparation method. The means of designing or producing a variant PD-L1 polypeptide is not limited to any particular method. However, in some embodiments, the nucleic acid encoding wild-type or unmodified PD-L1 is mutagenized by wild-type or unmodified PD-L1 genetic material and screened for the induction of a desired specific binding affinity and/or IFN- γ expression or other functional activity. In some embodiments, variant PD-L1 polypeptides are resynthesized using protein or nucleic acid sequences available at any number of publicly available databases and then subsequently screened. The National Center for Biotechnology Information provides such Information and its web site is publicly accessible via the internet, as with the UniProtKB database discussed previously.
Unless otherwise indicated, as indicated throughout this disclosure, one or more amino acid modifications are indicated by the amino acid position numbers corresponding to the position numbers of the unmodified ECD sequences listed in SEQ ID NO:30 or 1728, or the following unmodified IgV sequences (containing residues 1-114 of SEQ ID NO:30, respectively) listed in SEQ ID NO:309, as appropriate:
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER(SEQ ID NO:30)
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT(SEQ ID NO:1728)
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLL KDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKVNA(SEQ ID NO:309)
the modifications provided herein may be in the unmodified PD-L1 polypeptide set forth in SEQ ID NO 30, 309 or 1728. In some cases, the modification may also be in the unmodified IgV as set forth in SEQ ID NO: 55. In some embodiments, the unmodified PD-L1 polypeptide has 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID No. 30, 55, 309 or 1728.
PKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKV(SEQ ID NO:55)
It is within the level of the skilled person to identify the corresponding positions of modifications (e.g. amino acid substitutions) in a PD-L1 polypeptide, including portions thereof comprising an IgSF domain (e.g. ECD or IgV), such as by aligning the reference sequence with SEQ ID NO:30 or SEQ ID NO: 309. In the list of modifications throughout the present disclosure, amino acid positions are indicated in the middle, with corresponding unmodified (e.g., wild-type) amino acids listed before the numbering and identified variant amino acid substitutions listed after the numbering. "del" is indicated if the modification is a deletion of a position, and "ins" is indicated if the modification is an insertion at a position. In some cases, insertions are listed with amino acid positions in the middle indication, with corresponding unmodified (e.g., wild-type) amino acids listed before and after the numbering and identified variant amino acid insertions listed after unmodified (e.g., wild-type) amino acids.
In some embodiments, the variant PD-L1 polypeptide has one or more amino acid modifications (e.g., substitutions) in the wild-type or unmodified PD-L1 sequence. The one or more amino acid modifications (e.g., substitutions) can be in the extracellular domain (extracellular domain) of the wild-type or unmodified PD-L1 sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in an IgV domain or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the ECD domain or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in an IgC (e.g., IgC2) domain or a specific binding fragment thereof. In some embodiments of variant PD-L1 polypeptides, some of the one or more amino acid modifications (e.g., substitutions) are in an IgV domain or a specific binding fragment thereof, and some of the one or more amino acid modifications (e.g., substitutions) are in one or more IgC domains (e.g., IgC2) or a specific binding fragment thereof.
In some embodiments, a variant PD-L1 polypeptide has up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions). Modifications (e.g., substitutions) can be in an IgV domain or one or more IgC (e.g., IgC2) domains. In some embodiments, the variant PD-L1 polypeptide has up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgV domain or a specific binding fragment thereof. In some embodiments, a variant PD-L1 polypeptide has up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in one or more IgC (e.g., IgC2) domains or specific binding fragments thereof. In some embodiments, a variant PD-L1 polypeptide has at least about 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified PD-L1 polypeptide, or a specific binding fragment thereof, such as amino acid sequence SEQ ID No. 30, 1728, 55, or 309.
In some embodiments, the variant PD-L1 polypeptide has one or more amino acid modifications, e.g., substitutions, in unmodified PD-L1 or a specific binding fragment thereof corresponding to the following positions: 6. 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, SEQ, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 206, 207, 221, 175, 1728, or 1728: o for a reference to a position listed therein. In some embodiments, such variant PD-L1 polypeptides exhibit altered binding affinity for one or more of PD-1 and/or CD80 as compared to a wild-type or unmodified PD-L1 polypeptide. For example, in some embodiments, the variant PD-L1 polypeptide exhibits increased binding affinity for PD-1 and/or CD80 as compared to a wild-type or unmodified PD-L1 polypeptide. In some embodiments, the variant PD-L1 polypeptide exhibits reduced binding affinity for PD-1 or CD80 as compared to a wild-type or unmodified PD-L1 polypeptide.
In some embodiments, the variant PD-L1 polypeptide has one or more amino acid modifications, e.g., amino acid substitutions, selected from the group consisting of: p6, Y10, V11, Y14, G15, S16, N17, M18, T19, I20, C22, K23, E26, E27, K28, A33, L35, I36, E40, M41, D43, K44, N45, I46, I47, F49, V50, H51, G52, E53, E54, D55, L56, K57, V58, H60, R64, Q65, R68, K71, D72, Q73, L74, S75, N78, A79, I83, D149, Q85, V7, K160, K97, K170, K173, K170, K173, K170, K173, K95, K, V175A, T176N, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, a204T, L206F, V207A, L213P or T221L or conservative amino acid substitutions thereof. A conservative amino acid substitution is any amino acid, other than the wild-type or unmodified amino acid, that belongs to the same class as the substituted amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine and isoleucine), hydroxyl-or sulfur-containing (serine, cysteine, threonine and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine and arginine) and acidic/amide (aspartic acid, glutamic acid, asparagine and glutamine).
In some embodiments, the variant PD-L1 polypeptide has two or more amino acid modifications, e.g., amino acid substitutions, selected from the group consisting of: p6, Y10, V11, Y14, G15, S16, N17, M18, T19, I20, C22, K23, E26, E27, K28, A33, L35, I36, E40, M41, D43, K44, N45, I46, I47, F49, V50, H51, G52, E53, E54, D55, L56, K57, V58, H60, R64, Q65, R68, K71, D72, Q73, L74, S75, N78, A79, I83, D149, Q85, V7, K160, K97, K170, K173, K170, K173, K170, K173, K95, K, V175A, S177C, L179P, R180S, T183A, T183I, T185A, I188V, F189L, F189S, T192S, F193S, R194G, R194W, R195G, R195S, R195T, L196S, D197G, P198S, P198T, E199G, E200K, E200N, N201D, N201Y, H202Q, T203A, a204T, L206F, V207A, L213P or T221L.
In some embodiments, amino acid modifications, e.g., amino acid substitutions, include K28N/M41V/N45T/H51N/K57E, I20L/I36T/N45D/I47T, I20L/M41K/K44E, P6S/N45T/N78I/I83T, N78I, M41K/N78I, N45T/N78I, I20L/N45T, N45T, M41K, I20L/I36T/N45D, N17D/N45/N D/V50/D D, I20D/F49D, N45/V50D, I20D/N45/N D/N3678/N D/N72/D N72/N72/D/N72/D/N72/N72N, 33/E53, D43/N45/V58, E40/D43/N45/V50, Y14/K28/N45A 33/N78, A33/N45/N78, E27/N45/V50, N45/V50/N78, I20/N45/V110, I20/I36/N45/V50, N45/L74/S75, N45/S75, S75/K106, S75, A33/S75/D104, A33/S75, I20/E27/N45/V50, I20/E27/D43/N45/V58/N78, I20/A33/D43/N45/V58/N78, I20/D43/N45/V58/N78, I45/N78, E45/N78, N45/V50/N78, V11/I20/E27/D43/N45/H51/S99, I20/E27/D43/N45/V50, I20/K28/D43/N45/V58/Q89, I20/I36/N45, I20/K28/D43/N45/E53/V58/N78, A33/D43/N45/V58/S75, K23/D43/N45, I20/D43/N45/V58/N78/D90/G101, D43/N45/L56/V58/G101, I20/K23/D43/N45/V58/N78, I20/K23/D43/N45/V50/N78, T19/E27/N45/V50/N78/M97, I20/M41/N43/N45/N78, K23/N45/N78, I20/K28/D43/N45/V58/Q89/G101-ins (G101), K57/S99/F189, M18/M97/F193/R195/E200/H202, I36/M41/M97/K144/R195/E200/H202/L206, C22/Q65/L124/K144/R195/E200/H202/T221, M18/I98/L124/P198/L206, S99/N117/I148/K171/R180, I36/M97/A103/Q155, K28/S99, R195, A79/S99/T185/R195/E200/H202/L206, K57/S99/L124/K144, K57/S99/R195, D55/M97/S99, E27/I36/D55/M97/K111, E54/M97/S99, G15/I36/M97/K111/H202, G15/I36/V129/R195, G15/V129, I36/M97, I36/D55/M97/K111/A204, I36/D55/M97/K111/V129/F173, I36/D55/M97/K111/I148/R180, I36/G52/M97/V112/K144/V175/P198, I36/I46/D55/M97/K106/K144/T185/R195, I36/I83/M97/K144/P198, I36/M97/K111, I36/M97/K144/P198, I36/M97/K111/M97/K144/P198, I36/M97/Q155/N193F 201/M97/F193, I36T/M97L/V129D, L35P/I36S/M97L/K111E, M18I/I36T/E53G/M97L/K144E/E199G/V207A, M18T/I36T/D55N/M97L/K111E, M18V/M97L/T176L/R195L, M97L/S99L, N17/M97L/S L, S99L/T185/L/R195/P198L, V129L/H202L, V129L/P198L, V129L/T150, V93/V129L, Y10L/M18/S72/S99/S195/P198L, N129/N195/N72/N L/N198N L/N195/N72/N L/N198N 72/N L/N195/N72/N L/N72/N195/N72/N198N 72/N L/N198N 72/N L, N L/N198N L/N72/N L/N198N L/N195/N L/N72/N L/, N45D/N113Y/R195S, N45D/N165Y/E170G, N45D/Q89R/I98V, N45D/S131F/P198S, N45D/S75P/P198S, N45D/V50A/R195T, E27D/N45D/T183A/I188V, F173V/T183V/L196V/T203V, K23V/N45V/S75/N120V, N45V/G102/R194V/R195V, N45V/G52/Q V/P198, N45V/I148/R195/R72/N V/N72/N198/N72/N V/N183/N198/N72/N185/N72/N V/N198/N72/N V/N76/N72/N198/N72/N V/N72/N V/N72/N72/N72/N V/N72/N V/N72/N V/N72/N72/N V/N72/, K23/N45/L124/K167/R195, K23/N45/Q73/T163, K28/N45/W149/S158/P198, K28/N45/K57/I98/R195, K28/N45/V129/T163/R195, M41/D43/N45/R64/S99, N45/R68/F173/D197/P198, N45/V50/I148/R195/N201, M41/D43/K44/N45/R195/N201, or N45/V50/L124/K144/L179/R195.
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 20 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution I20L or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or more positions 27, 33, 36, 43, 45, 50, 58, 75, 78, 97, 99, 195, or 198. In some embodiments, the one or more amino acid modifications are one or more amino acid substitutions E27G, a33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, M97L, S99G, R195G, P198S, or P198T or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide comprises amino acid modifications I20L/E27G, I20L/a33D, I20L/I36T, I20L/D43G, I20L/N45D, I20L/N45T, I20L/V50A, I20L/V58A, I20L/S75P, I20L/N78I, I20L/M97L, I20L/S99G, I20L/R195G, I20L/P198S, or I20L/R198T.
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 27 with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is the amino acid substitution E27G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 33 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution a33D or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 36 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is amino acid substitution I36T or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 43 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution D43G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification at a position in unmodified PD-L1 or a specific binding fragment thereof that corresponds to position 45 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is an amino acid substitution N45D or N45T or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 50 referenced to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution V50A or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 58 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution V58A or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 75 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is amino acid substitution S75P or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 78 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution N78I or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 97 with reference to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is the amino acid substitution M97L or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 99 referenced to the numbering of the positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is amino acid substitution S99G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 195 with reference to the numbering of the positions listed in SEQ ID NO: 30. In some embodiments, the amino acid modification is the amino acid substitution R195G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises an amino acid modification in unmodified PD-L1 or a specific binding fragment thereof at a position corresponding to position 198 referenced to the numbering of positions listed in SEQ ID No. 30. In some embodiments, the amino acid modification is an amino acid substitution P198S or P198T or conservative amino acid substitutions thereof. In some embodiments, the variant PD-L1 polypeptide further contains one or more amino acid modifications, e.g., amino acid substitutions, at one or
In some embodiments, the variant PD-L1 polypeptide comprises any substitution (mutation) listed in table 1. Table 1 also provides exemplary sequences of extracellular domains (ECDs) or IgV domains of wild-type PD-L1 or exemplary variant PD-L1 polypeptides by reference to SEQ ID NOs. As indicated, the precise locus or residue corresponding to a given domain may vary, such as according to the method used to identify or classify the domain. In addition, in some cases, adjacent N-terminal and/or C-terminal amino acids of a given domain (e.g., ECD or IgV) may be included in the sequence of a variant IgSF polypeptide, such as to ensure proper folding of the domain upon expression. Thus, it is to be understood that the illustration of SEQ ID NOs in table 1 should not be construed as limiting. For example, a particular domain (such as an ECD or IgV domain) of a variant PD-L1 polypeptide may be several amino acids longer or shorter than the amino acid sequence set forth in the corresponding SEQ ID NO, such as 1-10, e.g., 1, 2, 3, 4, 5,6, or 7, amino acids longer or shorter.
In some embodiments, the variant PD-L1 polypeptide comprises or consists of any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008). In some embodiments, a variant PD-L1 polypeptide comprises or consists of a polypeptide sequence that is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, such as at least 96% identical, 97% identical, 98% identical, or 99% identical to any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008), and that contains or consists of one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1. In some embodiments, the variant PD-L1 polypeptide comprises or consists of a specific binding fragment of any one of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 56-120, 1725, 1729-1818, 1819-1907, 1943-2008) and contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1.
In some embodiments, the variant PD-L1 polypeptide comprises or consists of any of the IgV sequences listed in Table 1 (i.e., any of SEQ ID NOs: 121-185, 244-308, 1726-1727, 1908-1937). In some embodiments, the variant PD-L1 polypeptide comprises or consists of a polypeptide sequence that is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, such as at least 96% identical, 97% identical, 98% identical, or 99% identical to any of the IgV sequences listed in Table 1 (i.e., any of SEQ ID NOS: 121-185, 244-308, 1726-1727, 1908-1937), and that contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1. In some embodiments, the variant PD-L1 polypeptide comprises or consists of a specific binding fragment of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 121-185, 244-308, 1726-1727, 1908-1937) and contains one or more amino acid modifications (e.g., one or more substitutions) that are not present in wild-type or unmodified PD-L1.
In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 as compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NOs 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of CD80 as compared to wild-type or unmodified PD-L1 (such as comprising the sequence set forth in SEQ ID NOs: 30, 1728, 55, or 309). In some embodiments, the PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 and the extracellular domain of CD80 as compared to wild-type or unmodified PD-L1 (such as comprising the sequences set forth in SEQ ID NOs: 30, 1728, 55, or 309).
In some embodiments, the variant PD-L1 polypeptide exhibits increased binding affinity to one of the extracellular domains of PD-1 or CD80 and exhibits decreased binding affinity to the other of the extracellular domains of PD-1 or CD80, as compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NOs: 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of PD-1 and decreased affinity for the extracellular domain of CD80, as compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NOs: 30, 1728, 55, or 309). In some embodiments, the variant PD-L1 polypeptide exhibits increased affinity for the extracellular domain of CD80 and decreased affinity for the extracellular domain of PD-1, as compared to a wild-type or unmodified PD-L1 polypeptide (such as comprising the sequence set forth in SEQ ID NOs: 30, 1728, 55, or 309).
In some embodiments, the variant PD-L1 polypeptide exhibits increased selectivity for PD-1 versus CD80, such as indicated by a ratio of PD-1 binding to CD80 binding greater than 1 (PD-1: CD80 binding ratio), as compared to the binding ratio of an unmodified PD-L1 polypeptide (e.g., as set forth in SEQ ID NOs: 30, 55, or 309) to PD-1 versus CD 80. In some embodiments, the variant PD-L1 polypeptide exhibits a ratio of bound PD-1 to CD80 that is greater than or equal to about or 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 or greater.
Forms of variant polypeptides
Immunomodulatory polypeptides comprising variant PD-L1 provided herein that comprise a vigdd can be formatted in a variety of ways, including as soluble, membrane-bound, or secreted proteins. In some embodiments, the particular form may be selected for a desired therapeutic application. In some cases, an immunomodulatory polypeptide comprising a variant PD-L1 polypeptide is provided in a form to antagonize or block the activity of its cognate binding partner, e.g., PD-1. In some embodiments, antagonism of PD-1 may be useful in promoting oncological immunity. In some cases, an immunomodulatory polypeptide comprising a variant PD-L1 polypeptide is provided in a form to agonize or stimulate the activity of its cognate binding partner, e.g., PD-1. In some embodiments, agonism of PD-1 may be useful in the treatment of inflammation or autoimmunity. The skilled person can readily determine a particular form of activity, such as for antagonizing or agonizing one or more specific cognate binding partners. Exemplary methods for assessing such activity are provided herein (including in the examples).
In some aspects, immunomodulatory proteins comprising vigdd of PD-L1 are provided, wherein such proteins are soluble, e.g., fused to an Fc chain. In some aspects, one or more additional IgSF domains (such as one or more additional vigds) can be linked to a vigdd of PD-L1 as provided herein (hereinafter referred to as a "stacked" or "stacked" immunomodulatory protein). In some embodiments, the provided modular form of an immunomodulatory protein provides flexibility for engineering or generating immunomodulatory proteins to modulate the activity of multiple relative structures (multiple cognate binding partners). In some embodiments, such "stacked" molecules may be provided in soluble form, or in some cases, in the form of a membrane-bound or secreted protein. In some embodiments, the variant PD-L1 immunomodulatory protein is provided in the form of a conjugate comprising vigdd of PD-L1, directly or indirectly linked to a targeting agent or moiety (e.g., an antibody or other binding molecule) that specifically binds to a ligand (e.g., an antigen), such as when administered to a subject, for example, for targeting or localizing the vigdd to a particular environment or cell. In some embodiments, a targeting agent (e.g., an antibody or other binding molecule) binds to a tumor antigen, thereby localizing variant PD-L1 containing vigdd to the tumor microenvironment, e.g., to modulate the activity of Tumor Infiltrating Lymphocytes (TILs) specific for the tumor microenvironment. In some embodiments, the targeting agent (e.g., an antibody or other binding molecule) binds to an antigen expressed on antigen presenting cells or normal tissues in an inflammatory environment, thereby localizing the variant PD-L1 containing vigdd to areas of unwanted autoimmune inflammation, e.g., to modulate the activity of T cells targeting the autoantigen.
In some embodiments, the immunomodulatory proteins provided are expressed in a cell and provided as part of an Engineered Cell Therapy (ECT). In some embodiments, the variant PD-L1 polypeptide is expressed in a membrane-bound form in a cell, such as an immune cell (e.g., a T cell or antigen presenting cell), thereby providing a transmembrane immunomodulatory protein (hereinafter also referred to as a "TIP"). In some embodiments, depending on the cognate binding partner recognized by the TIP, the engineered cell expressing the TIP may agonize the cognate binding partner by providing a costimulatory signal positive or negative for other engineered cells and/or endogenous T cells. In some aspects, the variant PD-L1 polypeptide is expressed in a secreted form in a cell, such as an immune cell (e.g., a T cell or an antigen presenting cell), thereby producing the variant PD-L1 polypeptide (hereinafter also referred to as "SIP") in a secreted or soluble form, such as when the cell is administered to a subject. In some aspects, SIP can antagonize a cognate binding partner in the environment in which it is secreted (e.g., the tumor microenvironment). In some embodiments, the variant PD-L1 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that, when administered to a subject, is capable of infecting cells in vivo, such as immune cells (e.g., T cells or antigen presenting cells), for delivery or expression of the variant polypeptide as a TIP or SIP in the cells.
In some embodiments, a soluble immunomodulatory polypeptide (such as a variant PD-L1 containing vigdd) may be encapsulated within a liposome, which itself may be conjugated to any one of the provided conjugates (e.g., targeting moieties) or any combination thereof. In some embodiments, a soluble or membrane-bound immunomodulatory polypeptide of the invention is deglycosylated. In a more specific embodiment, the variant PD-L1 sequence is deglycosylated. In even more particular embodiments, the IgV and/or one or more IgC (e.g., IgC2) domains of variant PD-L1 are deglycosylated.
Non-limiting examples of the forms provided are described in fig. 1A-1C and further described below.
A. Soluble proteins
In some embodiments, the immunomodulatory protein comprising a variant PD-L1 polypeptide is a soluble protein. The skilled person will appreciate that cell surface proteins typically have an intracellular, transmembrane and extracellular domain (ECD) and that soluble forms of such proteins can be prepared using the extracellular domain or immunologically active subsequences thereof. Thus, in some embodiments, an immunomodulatory protein comprising a variant PD-L1 polypeptide lacks a transmembrane domain or a portion of a transmembrane domain. In some embodiments, the immunomodulatory protein comprising variant PD-L1 lacks an intracellular (cytoplasmic) domain or a portion of an intracellular domain. In some embodiments, an immunomodulatory protein comprising a variant PD-L1 polypeptide comprises only a viggd portion comprising an ECD domain or a portion thereof comprising an IgV domain and/or one or more IgC (e.g., IgC2) domain or a specific binding fragment thereof comprising one or more amino acid modifications.
In some embodiments, the immunomodulatory protein is or comprises a monomeric form and/or exhibits monovalent binding to its binding partner a variant PD-L1 polypeptide (such as a variant PD-L1 that is soluble and/or lacks a transmembrane domain and an intracellular signaling domain) is linked directly or indirectly to another moiety in some embodiments, another moiety is a protein, peptide, small molecule, or nucleic acid in some embodiments, a monovalent immunomodulatory protein is a fusion protein in some embodiments, a moiety is a half-life extending molecule.
In some embodiments, an immunomodulatory polypeptide comprising variant PD-L1 can be linked to a portion comprising a conformationally disordered polypeptide sequence consisting of amino acids Pro, Ala, and Ser (see, e.g., WO2008/155134, SEQ ID NO: 2025). In some cases, the amino acid repeat is at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat comprises Ala, Ser, and Pro residues. Accordingly, provided herein are immunomodulatory proteins that are PAS-based proteins, wherein a variant PD-L1 polypeptide is linked directly or indirectly via a linker to Pro/Ala/ser (PAS). In some embodiments, one or more additional joint structures may be used.
In some embodiments, the moiety facilitates detection or purification of a variant PD-L1 polypeptide. In some cases, the immunomodulatory polypeptide comprises a label or fusion domain, e.g., an affinity or purification label, directly or indirectly linked to the N-terminus and/or c-terminus of the PD-L1 polypeptide. Various suitable polypeptide tags and/or fusion domains are known and include, but are not limited to, a polyhistidine (His) tag, a FLAG tag (SEQ ID NO:2010), a Myc tag, and a fluorescent protein tag (e.g., EGFP, listed in SEQ ID NO: 2027-2029). In some cases, an immunomodulatory polypeptide comprising variant PD-L1 comprises at least six histidine residues (listed in SEQ ID NO: 2011). In some cases, an immunomodulatory polypeptide comprising a variant PD-L1 further comprises different combinations of the portions. For example, an immunomodulatory polypeptide comprising variant PD-L1 further comprises one or more of a polyhistidine tag and a FLAG tag.
In some embodiments, the PD-L1 polypeptide is linked to a modified immunoglobulin heavy chain constant region (Fc), which is maintained in a monovalent form, such as set forth in SEQ ID NO: 1187.
In some embodiments, the immunomodulatory protein comprises a variant PD-L1 polypeptide linked directly or indirectly via a linker to a multimerization domain. In some aspects, the multimerization domain increases the half-life of the molecule. The interaction of two or more variant PD-L1 polypeptides may be facilitated by their direct or indirect linkage to any moiety or other polypeptide that is capable of interacting with itself to form a stable structure. For example, an individually encoded variant PD-L1 polypeptide chain may be linked by multimerization, thereby mediating multimerization of the polypeptide by a multimerization domain. In general, the multimerization domain provides for the formation of stable protein-protein interactions between the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide.
Homo-or heteromultimeric polypeptides can be produced by co-expression of individual variant PD-L1 polypeptides. The first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide may be the same or different. In particular embodiments, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are identical in a homodimer and are each linked to the same multimerization domain. In other embodiments, a heterodimer may be formed by joining together different first and second variant PD-L1 and PD-L1 polypeptides. In such embodiments, in some aspects, the first variant PD-L1 polypeptide and the second variant PD-L1 polypeptide are linked to different multimerization domains capable of promoting heterodimer formation.
In some embodiments, the multimerization domain includes any domain capable of forming a stable protein-protein interaction. The multimerization domains may interact via: immunoglobulin sequences (e.g., Fc domains; see, e.g., International patent publication Nos. WO 93/10151 and WO 2005/063816 US; U.S. publication No. 2006/0024298; U.S. patent No. 5,457,035); leucine zippers (e.g., from the nuclear transformation proteins fos and jun or the proto-oncogene c-myc or from General Nitrogen Control (General Control of Nitrogen, GCN4)) (see, e.g., Busch and Sassone-Corsi (1990) Trends Genetics,6: 36-40; Gentz et al, (1989) Science,243: 1695-; a hydrophobic region; a hydrophilic region; or free thiols that form intramolecular disulfide bonds between chimeric molecules of homodimers or heterodimers. In addition, the multimerization domain may comprise an amino acid sequence comprising a protrusion that is complementary to an amino acid sequence comprising a pore, such as, for example, U.S. Pat. nos. 5,731,168; international patent publication nos. WO 98/50431 and WO 2005/063816; ridgway et al (1996) Protein Engineering,9: 617-621. This multimerization region may be engineered such that steric interactions not only promote stable interactions, but further promote the formation of heterodimers over homodimers from a mixture of chimeric monomers. Typically, the projections are constructed by replacing small amino acid side chains of the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities with the same or similar dimensions as the protrusions are optionally created on the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). Exemplary multimerization domains are described below.
The variant PD-L1 polypeptide can be joined to the N-terminus or C-terminus of the multimerization domain at any position, but typically via its N-terminus or C-terminus to form a chimeric polypeptide. The attachment may be directly or indirectly via a linker. Chimeric polypeptides may be fusion proteins or may be formed by chemical linkage, such as by covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide comprising a multimerization domain, a nucleic acid encoding all or a portion of the PD-L1 polypeptide may be operably linked, directly or indirectly or optionally via a linker domain, to a nucleic acid encoding a multimerization domain sequence. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant PD-L1 polypeptide is joined to the N-terminus of the multimerization domain. In some cases, the construct may encode a chimeric protein in which the N-terminus of the variant PD-L1 polypeptide is joined to the C-terminus of the multimerization domain.
Polypeptide multimers contain multiple (such as two) chimeric proteins formed by directly or indirectly linking two identical or different variant PD-L1 polypeptides to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, a gene fusion encoding the variant PD-L1 polypeptide and the multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion proteins can be expressed in host cells transformed with recombinant expression vectors and allowed to assemble into multimers, where the multimerization domains interact to form multivalent polypeptides. Chemical linkage of the multimerization domain to the variant PD-L1 polypeptide may be achieved using a heterobifunctional linker.
The resulting chimeric polypeptide (such as a fusion protein) and multimers formed therefrom may be purified by any suitable method, such as, for example, by affinity chromatography on a protein a or protein G column. Upon transformation of two nucleic acid molecules encoding different polypeptides into a cell, the formation of homodimers and heterodimers will occur. The conditions for expression may be adjusted so that heterodimer formation is favored over homodimer formation.
In some embodiments, the multimerization domain is an Fc domain from an immunoglobulin or a portion thereof. In some embodiments, the immunomodulatory protein comprises a variant PD-L1 polypeptide attached to an immunoglobulin Fc (resulting in an "immunomodulatory Fc fusion," such as a "PD-L1-Fc variant fusion," also known as a PD-L1 vIgD-Fc fusion). In some embodiments, the attachment of the variant PD-L1 polypeptide is at the N-terminus of the Fc. In some embodiments, the attachment of the variant PD-L1 polypeptide is at the C-terminus of the Fc. In some embodiments, two or more PD-L1 variant polypeptides (the same or different) are independently attached at the N-terminus and the C-terminus.
In some embodiments, the Fc is a murine or human Fc. In some embodiments, the Fc is a mammalian or human IgGl, lgG2, lgG3, or lgG4 Fc region. In some embodiments, the Fc is derived from IgG1, such as
In some embodiments, the Fc region contains one or more modifications that alter (e.g., reduce) one or more of its normal functions. In general, the Fc region is responsible for effector functions such as Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicity (ADCC), as well as antigen binding ability which is the primary function of an immunoglobulin. In some cases, effector functions of the Fc region may include programmed cell death and phagocytosis. In addition, the FcRn sequence present in the Fc region serves to modulate serum IgG levels by increasing half-life in vivo via conjugation to the FcRn receptor in vivo. In some embodiments, such functions may be reduced or altered in the Fc for use with provided Fc fusion proteins.
In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the PD-L1-Fc variant fusions provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations in the Fc sequence that can alter effector function. For example, WO 00/42072, WO2006019447, WO2012125850, WO2015/107026, US2016/0017041, and Shields et al Jbiol. chem.9(2):6591-6604(2001) describe exemplary Fc variants with increased or decreased binding to FcRs. The contents of these publications are expressly incorporated herein by reference.
In some embodiments, provided variant PD-L1-Fc fusions comprise Fc regions that exhibit reduced effector function, making them desirable candidates for applications where the in vivo half-life of the PD-L1-Fc variant fusion is important and where certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/absence of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that a PD-L1-Fc variant fusion lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells used to mediate ADCC, NK cells express Fc γ RIII only, while monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is found in ravatch and Kinet, annu.Immunol.9:457-492(1991) is summarized in Table 2 on page 464. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al Proc. nat 'l Acad. Sci. USA83:7059-7063(1986)) and Hellstrom, I. et al, Proc. nat' l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed, (see, e.g., ACTI for flow cytometryTMNon-radioactive cytotoxicity assay (cell technology company Mountain View, Calif.); and Cytotox96TMNon-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al Proc. nat' l Acad. Sci. USA 95: 652-. A C1q binding assay may also be performed to confirm that the PD-L1-Fc variant fusion is unable to bind C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg, M.S. et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12):1759-1769 (2006)).
PD-L1-Fc variant fusions with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (by EU numbering) (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327 (by EU numbering), including so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (U.S. Pat. No. 7,332,581).
In some embodiments, the Fc region of the PD-L1-Fc variant fusion has an Fc region in which any one or more amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with a different amino acid as compared to the native Fc region. Such changes in the Fc region are not limited to those described above, and include, for example, changes described in Current Opinion in Biotechnology (2009)20(6), 685-; changes described in WO 2008/092117, such as G236R/L328R, L235G/G236R, N325A/L328R and N325LL 328R; amino acid insertions at positions 233, 234, 235 and 237 (indicated by EU numbering); and alterations at the sites described in WO 2000/042072.
Certain Fc variants with improved or reduced binding to FcR have been described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, WO2006019447, and Shields et al, J.biol. chem.9(2):6591-6604 (2001))
In some embodiments, a PD-L1-Fc variant fusion is provided that comprises a variant Fc region comprising one or more amino acid substitutions that increase half-life and/or improve binding to a neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005/0014934a1(Hinton et al) or WO 2015107026. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (by EU numbering), for example a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826).
In some embodiments, the Fc region of the PD-L1-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L (by EU numbering). In some embodiments, the Fc region of the PD-L1-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S, and/or C229S (by EU numbering). In some embodiments, the Fc region of the PD-L1 variant fusion comprises one or more amino acid substitutions R292C and V302C. See also Duncan and Winter, Nature322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351 for other examples of variants of the Fc region.
In some embodiments, the alteration that causes a reduction in C1q binding and/or Complement Dependent Cytotoxicity (CDC) is made in the Fc region, e.g., as described in U.S. Pat. nos. 6,194,551, WO 99/51642, and Idusogie et al, j.immunol.164: 4178 (2000).
In some embodiments, there is provided a PD-L1-Fc variant fusion comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from IgG1, such as
In some embodiments, the Fc region lacks a C-terminal lysine corresponding to position 232 (corresponding to K447del by EU numbering) of the wild-type or unmodified Fc listed in SEQ ID NO: 187. In some aspects, this Fc region may further comprise one or more additional modifications, e.g., amino acid substitutions, such as any of those described. Examples of such Fc regions are set forth in SEQ ID NO:1938, 1939, 1940 or 1715.
In some embodiments, a PD-L1-Fc variant fusion is provided that comprises a variant Fc region, wherein the variant Fc comprises an amino acid sequence set forth in any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, or 1940, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, or 1940.
In some embodiments, the Fc is derived from IgG2, such as human IgG 2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID No. 188 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 188.
In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID No. 1200 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 1200. In some embodiments, IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is substituted with the CH3 domain of human IgG1 and exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are substituted with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which arginine at position 409 indicated in the EU index proposed by Kabat et al of human IgG4 is substituted with lysine and exhibits inhibited aggregate formation (see, e.g., U.S. patent No. 8,911,726. in some embodiments, Fc 4 contains a S228P mutation that has been shown to prevent recombination between a therapeutic antibody and endogenous IgG4 by Fab-arm exchange (see, e.g., Labrijin et al (2009) nat. biotechnol, 27(8): 1201-71.) in some embodiments, the Fc comprises an amino acid sequence in SEQ ID No. or has at least 85 ID No.: 7685: SEQ ID No. 1201: 1201, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
In some embodiments, the variant PD-L1 polypeptide is directly linked to an Fc sequence. In some embodiments, the variant PD-L1 polypeptide is indirectly linked to the Fc sequence, such as via a linker. In some embodiments, one or more "peptide linkers" link the variant PD-L1 polypeptide with an Fc domain. In some embodiments, the length of the peptide linker may be a single amino acid residue or greater. In some embodiments, the peptide linker has at least one amino acid residue but no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7,6, 5,4, 3,2, or1 amino acid residue in length. In some embodiments, the linker is three alanines (AAA). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the form of a one letter amino acid code): GGGGS (' 4GS ' or ' G)4S "; SEQ ID NO:1942) or multimers of 4GS linkers, such as2, 3,4 or 5 repeats of a 4GS linker, such as listed in SEQ ID NO:240 (2xGGGGS) or in SEQ ID NO:239 (3 xGGGGS). In some embodiments, the linker (in the form of a one letter amino acid code) is GSGGGGS (SEQ ID NO: 1941). In some embodiments, the linker may also include a series of alanine residues, alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each series is: 2. 3,4,5 or 6 alanines.In some embodiments, the linker is (in the form of an alpha amino acid code) EAAAK or a multimer of EAAAK linkers, such as a repeat sequence of 2,3,4, or 5 EAAAK linkers, such as (1xEAAAK) set forth in SEQ ID NO:2022, (3xEAAAK) set forth in SEQ ID NO:2023, or (5xEAAAK) set forth in SEQ ID NO:20244S)3(SEQ ID NO:2031) or GS (G)4S)5(SEQ ID NO: 2032). In some examples, the linker is 2XGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 241). In some cases, an immunomodulatory polypeptide comprising variant PD-L1 comprises a different combination of peptide linkers.
In some embodiments, the variant PD-L1-Fc fusion protein is a dimer formed by two variant PD-L1 Fc polypeptides linked to an Fc domain. In some embodiments, the dimer is a homodimer in which the two variant PD-L1 Fc polypeptides are the same. In some embodiments, the dimer is a heterodimer where the two variant PD-L1 Fc polypeptides are different heterodimers.
Also provided are nucleic acid molecules encoding the variant PD-L1-Fc fusion proteins. In some embodiments, to produce the Fc fusion protein, a nucleic acid molecule encoding the variant PD-L1-Fc fusion protein is inserted into an appropriate expression vector. The resulting variant PD-L1-Fc fusion protein can be expressed in a host cell transformed with expression, in which assembly between Fc domains occurs through interchain disulfide bonds formed between Fc portions to produce a dimeric (such as bivalent) variant PD-L1-Fc fusion protein.
The resulting Fc fusion protein can be easily purified by affinity chromatography on a protein a or protein G column. To generate the heterodimer, additional steps for purification may be necessary. For example, when two nucleic acids encoding different variant PD-L1 polypeptides are transformed into a cell, heterodimer formation must be achieved biochemically, as the variant PD-L1 molecule carrying the Fc domain will also be expressed as a disulfide-linked homodimer. Thus, homodimers can be reduced under conditions favorable to disrupting interchain disulfide bonds without affecting intrachain disulfide bonds. In some cases, different variant PD-L1 Fc monomers were mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing an Fc fusion molecule containing a variant PD-L1 polypeptide using the globular-well approach described below.
B. Stacking molecules with additional IgSF domains
In some embodiments, an immunomodulatory protein may contain any of the variant PD-L1 provided herein, linked directly or indirectly to one or more other immunoglobulin superfamily (IgSF) domains (a "stacked" immunomodulatory protein construct and also referred to as a "type II" immunomodulatory protein). In some aspects, this may result in a unique multidomain immunomodulatory protein that binds two or more (such as three or more) homologous binding partners, thereby providing multi-targeted modulation of the immunological synapse.
In some embodiments, an immunomodulatory protein comprises a variant PD-L1 domain in combination with (a "non-wild-type combination") and/or arrangement of (a "non-wild-type arrangement" or a "non-wild-type arrangement") one or more other affinity-modified and/or non-affinity-modified IgSF domain sequences not present in a wild-type IgSF family member of another IgSF family member (e.g., a mammalian IgSF family member). In some embodiments, the immunomodulatory protein contains 2,3,4,5, or 6 immunoglobulin superfamily (IgSF) domains, wherein at least one of the IgSF domains is a variant PD-L1IgSF domain (vigdd of PD-L1) according to the description provided.
In some embodiments, the sequence of the additional IgSF domain can be a modified IgSF domain comprising one or more amino acid modifications (e.g., substitutions) as compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain may be non-affinity modified (e.g., wild-type) or affinity modified. In some embodiments, the unmodified or wild-type IgSF domain may be from mouse, rat, cynomolgus monkey or human origin or combinations thereof. In some embodiments, the additional IgSF domain can be an IgSF domain of an IgSF family member listed in table 2. In some embodiments, the additional IgSF domain can be an affinity modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) as compared to the IgSF domain contained by an IgSF family member listed in table 2.
In some embodiments, the additional IgSF domain is an affinity or non-affinity modified IgSF domain contained by a member of the IgSF family selected from the group consisting of the family of signal-regulating proteins (SIRP), the family of trigger receptor-like (TREML) expressed on bone marrow cells, the family of carcinoembryonic antigen-associated cell adhesion molecules (CEACAM), the family of salicylic acid-binding Ig-like lectin (SIGLEC), the family of lactophagins, the family B, the family of CD, the family of V-type and immunoglobulin-containing domains (VSIG), the family of V-type transmembrane domains (VSTM), the family of Major Histocompatibility Complexes (MHC), the family of Signaling Lymphocyte Activating Molecules (SLAM), the family of leukocyte immunoglobulin-like receptors (LIR), the family of bindin (Nec), the family of binding to (CL), the family of poliovirus receptor-related (VSTM), the family of natural cytotoxicity trigger receptors (NCR), the family of T-cell immunoglobulin and mucin (TIM) or the family of killer cell immunoglobulin-like receptors (KIR), the family of CD 150 CD-G-CD-200, CD-150, CD-150, CD-150, CD-150, CD.
The first column of table 2 provides the names of the particular IgSF member and optionally the names of some possible synonyms. The second column provides protein identification or in some cases GenBank numbering of the UniProtKB database, which is a publicly available database accessible at uniprot. The universal protein resource (UniProt) is a comprehensive resource of protein sequences and annotation data. The UniProt database includes the UniProt knowledge base (UniProtKB). UniProt is a collaboration between the european bioinformatics institute (EMBL-EBI), SIB switzerland bioinformatics institute and the protein information base (PIR) and is largely supported by the U.S. national health institute (NIH) funding. GenBank is the NIH gene sequence database, which is an annotated set of all publicly available DNA sequences (Nucleic acids research, 1 month 2013; 41(D1): D36-42). The third column provides the region in which the indicated IgSF domain is located. The region is designated as a range, wherein the domain includes residues that define the range. Column 3 also indicates the IgSF domain class of the particular IgSF region. Column 4 provides the region in which the indicated additional domain is located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It is understood that the description of a domain may vary depending on the method used to identify or classify the domain, and may be identified differently from different sources. The depiction of residues corresponding to the domains in table 2 is for illustration only and may be several amino acids (such as one, two, three, or four) longer or shorter. Column 5 indicates some of the listed IgSF members, some of their cognate cell surface binding partners.
In some embodiments, provided immunomodulatory proteins comprise, in addition to a variant PD-L1 polypeptide, at least 1,2,3,4,5, or 6 additional immunoglobulin superfamily (IgSF) domains, such as IgD domains of IgSF family members listed in table 2. In some embodiments, provided immunomodulatory proteins comprise at least one additional IgSF domain (e.g., a second IgSF domain). In some embodiments, provided immunomodulatory proteins comprise at least two additional IgSF domains (e.g., a second IgSF domain and a third IgSF domain). In some embodiments, provided immunomodulatory proteins comprise at least three additional IgSF domains (e.g., second, third, and fourth). In some embodiments, provided immunomodulatory proteins comprise at least four additional IgSF domains (e.g., second, third, fourth, and fifth). In some embodiments, provided immunomodulatory proteins comprise at least five additional IgSF domains (e.g., second, third, fourth, fifth, and sixth). In some embodiments, provided immunomodulatory proteins comprise at least six additional IgSF domains (e.g., second, third, fourth, fifth, sixth, and seventh). In some embodiments, each IgSF domain in the immunomodulatory protein is different. In some embodiments, at least one of the additional IgSF domains is identical to at least one other IgSF domain in the immunomodulatory protein. In some embodiments, each IgSF domain is from or derived from a different IgSF family member. In some embodiments, at least two of the IgSF domains are from or derived from the same IgSF family member.
In some embodiments, the additional IgSF domain comprises an IgV domain or one or more IgC (e.g., IgC2) domains, or a specific binding fragment of an IgV domain or a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, the additional IgSF domain is or comprises a full-length IgV domain. In some embodiments, the additional IgSF domain is or comprises one or more full-length IgC (e.g., IgC2) domains. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of an IgV domain. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of one or more IgC (e.g., IgC2) domains. In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (same) IgSF member. For example, in some aspects, an immunomodulatory protein comprises an ECD of an IgSF member or portion thereof comprising a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domains or specific binding fragments thereof.
In some embodiments, immunomodulatory proteins are provided that comprise at least one additional IgSF domain (e.g., a second IgSF domain, or in some cases a third IgSF domain, etc.), wherein the at least one additional, e.g., second or third IgSF domain is a IgSF domain listed as a wild-type or unmodified IgSF domain contained in an amino acid sequence listed in any one of SEQ ID NOs 1-27 and 216, or a specific binding fragment thereof. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain.
In some embodiments, immunomodulatory proteins are provided that comprise, in addition to a variant PD-L1 polypeptide, at least one additional affinity modified IgSF domain (e.g., a second IgSF domain, or in some cases a third affinity modified IgSF domain, etc.), wherein the at least one additional IgSF domain is a viggd that comprises one or more amino acid modifications (e.g., substitutions, deletions, or mutations) as compared to a wild-type or unmodified IgSF domain, such as an IgSF domain in an IgSF family member listed in table 2. In some embodiments, the additional (e.g., second or third) affinity modified IgSF domain has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified IgSF domain or specific binding fragment thereof contained in an amino acid sequence set forth in any of SEQ ID NOs 1-27 and 216. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain. In some embodiments, the additional (e.g., or second or third) IgSF domain is an affinity modified IgV domain and/or an IgC domain. In some embodiments, the one or more additional IgSF domains are affinity modified IgSF domains comprising an IgV domain and/or one or more IgC (e.g., IgC2) domains or specific binding fragments of an IgV domain and/or specific binding fragments of one or more IgC (e.g., IgC2) domains, wherein the IgV and/or IgC domains comprise one or more amino acid modifications (e.g., substitutions). In some embodiments, the one or more additional affinity modified IgSF domains comprise an IgV domain comprising one or more amino acid modifications (e.g., substitutions). In some embodiments, the one or more additional affinity modified IgSF domains include an IgSF domain present in an ECD or a portion of an ECD corresponding to an unmodified IgSF family member, such as a full-length IgV domain and one or more full-length IgC (e.g., IgC2) domain or a specific binding fragment thereof, wherein one or both of IgV and IgC contain one or more amino acid modifications (e.g., substitutions).
In some embodiments, an immunomodulatory polypeptide comprising variant PD-L1 may comprise one or more vigds of PD-L1 provided herein. In some embodiments, a variant PD-L1 immunomodulatory protein provided herein will comprise exactly 1,2,3,4,5, or more variant PD-L1 sequences. In some embodiments, at least two of the variant PD-L1 sequences are the same variant IgSF domain.
In some embodiments, provided immunomodulatory polypeptides comprise two or more vigdd sequences of PD-L1. The plurality of variant PD-L1 within a polypeptide chain can be identical (i.e., the same species) to each other or can be different (i.e., different species) of the variant PD-L1 sequence. In addition to single polypeptide chain embodiments, in some embodiments, two, three, four, or more of the polypeptides of the invention can be covalently or non-covalently attached to each other. Thus, provided herein are monomeric, dimeric, and higher order (e.g., 3,4,5, or higher order) multimeric proteins. For example, in some embodiments, exactly two polypeptides of the invention may be covalently or non-covalently attached to each other to form a dimer. In some embodiments, attachment is via an intrachain cysteine disulfide bond. A composition comprising two or more polypeptides of the invention can be the same species or substantially the same species of polypeptide (e.g., homodimers) or a different species of polypeptide (e.g., heterodimers). As described above, a composition having a plurality of linked polypeptides of the invention can have one or more of the same or different variants of PD-L1 of the invention in each polypeptide chain. In some embodiments, PD-L1-Fc variant fusion polypeptides of the same or substantially the same species (allowing for 3 or fewer N-or C-terminal amino acid sequence differences) will be dimerized to produce homodimers. Alternatively, different species of PD-L1-Fc variant fusion polypeptides may be dimerized to produce heterodimers.
In some embodiments, provided immunomodulatory proteins comprise at least one additional (e.g., or second or in some cases third IgSF domain, etc.) IgSF domain that is a IgSF domain other than PD-L1 that comprises one or more amino acid substitutions compared to the IgSF domain (e.g., IgV) of a wild-type or unmodified IgSF domain.
In some embodiments, one or more additional IgSF domain (e.g., second or third IgSF) domain is an IgSF domain (e.g., ECD or IgV) of another IgSF family member that also binds itself to an inhibitory receptor. In some aspects, the one or more additional IgSF domain (e.g., second or third IgSF) domain is an affinity modified IgSF domain that is a variant IgSF domain (viggd) of an IgSF family member that binds to an inhibitory receptor and contains one or more amino acid substitutions in the IgSF domain (e.g., ECD or IgV), wherein in some cases the one or more amino acid modifications result in increased binding to the inhibitory receptor. In some embodiments, a vigdd contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in a wild-type or unmodified IgSF domain (e.g., ECD or IgV) of an IgSF family member that binds to an inhibitory receptor. Illustrative examples of such inhibitory receptors, other than PD-1, are CTLA-4, LAG3, TIGIT, TIM-3 or BTLA. In some embodiments, the one or more additional IgSF domains are from an IgSF family member selected from the group consisting of: CD155, CD112, PD-L2, CD80 or
In some embodiments, the immunomodulatory protein in the multi-target checkpoint antagonist targets or blocks the activity of at least two, three, four, or more inhibitory receptors. In some embodiments, an immunomodulatory protein comprising any one of the variant PD-L1 polypeptides and one or more IgSF domains of an inhibitory receptor (such as a wild-type or unmodified inhibitory receptor) is provided. In some embodiments, an immunomodulatory protein comprising any one of the variant PD-L1 polypeptides and one or more IgSF domains of CD80 (e.g., wild-type or unmodified CD80), such as the IgV domains set forth in SEQ ID NO:1005, 1079, or 2030 or the ECD or portion thereof (comprising IgV and IgC domains or specific binding fragments thereof) set forth in SEQ ID NO:28 or a portion thereof is provided. In some embodiments, an immunomodulatory protein comprising any one of the variant PD-L1 polypeptides and one or more IgSF domains of CD112 (e.g., wild-type or unmodified CD112), such as the IgV domain set forth in SEQ ID NO:666 or 761 or the ECD or portion thereof set forth in SEQ ID NO:48 or a portion thereof (comprising IgV and IgC domains or specific binding fragments thereof), is provided. In some embodiments, an immunomodulatory protein comprising any one of the variant PD-L1 polypeptides and one or more IgSF domains of PD-L2 (e.g., wild-type or unmodified PD-L2), such as the IgV domains set forth in SEQ ID NO:1203 or 1263 or the ECD or portion thereof set forth in SEQ ID NO:31 or portion thereof (comprising IgV and IgC domains or specific binding fragments thereof), is provided. In some embodiments, an immunomodulatory protein comprising any one of the variant PD-L1 polypeptides and one or more IgSF domains of CD155 (e.g., wild-type or unmodified CD155), such as the IgV domains listed in SEQ ID NO:310 or 353 or the ECD or portion thereof (comprising IgV and IgC domains or specific binding fragments thereof) listed in SEQ ID NO:47 or a portion thereof, is provided.
In some embodiments, an immunomodulatory protein is provided that comprises one or more additional IgSF domains (e.g., a second or third IgSF) that is a vgdd that binds to an IgSF family member of an inhibitory receptor, wherein one or more amino acid modifications in the IgSF domain (e.g., IgV) result in an increase in binding affinity of the vgdd or a fusion or immunomodulatory protein comprising the vgdd to an inhibitory receptor cognate binding partner, such as an increase in binding affinity of more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold, as compared to the unmodified IgSF domain. In some embodiments, one or more amino acid modifications in an IgSF domain (e.g., IgV) result in increased selectivity of igd or a fusion or immunomodulatory protein comprising igd for its inhibitory receptor as compared to an unmodified IgSF domain. In some embodiments, the increased selectivity is a greater binding ratio of vgdd to inhibitory receptor to another homologous binding partner than to the binding ratio of unmodified IgSF to inhibitory receptor to another homologous binding partner (such as a homologous binding partner of a non-inhibitory receptor). In some embodiments, the ratio is at least or at least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater.
In some embodiments, the at least one additional (e.g., second or third) igd is an IgSF domain (e.g., IgV) of a variant CD80 polypeptide comprising one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV), the CD80 being a member of the IgSF family that binds to the inhibitory receptor TIGIT. In some embodiments, at least one additional (e.g., second) igd contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) of CD80 that binds to an IgSF family member of the inhibitory receptor CTLA-4. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., ECD or IgV containing IgV and IgC) of a variant CD80 polypeptide are listed in table 3. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD80 polypeptide, the variant CD80 polypeptide comprising an IgV domain comprising any of the amino acid modifications listed in Table 3, such as an IgV domain listed in any of SEQ ID NOs 1006-1078, 1080-1112, 1114-1152 or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs 1006-1078, 1080-1112, 1114-1152 and comprising one or more amino acid modifications. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD80 polypeptide, the variant CD80 polypeptide comprising an ECD or a portion thereof comprising an IgV and/or IgC domain comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of the amino acid modifications listed in Table 3, such as the ECD listed in any of SEQ ID NO:932-964, 966-1004 or the ECD comprising any of SEQ ID NO:932-964, 966-1004 and comprising one or more amino acid modifications.
In some embodiments, the at least one additional (e.g., second or third) igd is an IgSF domain (e.g., IgV) of a variant CD155 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) as compared to unmodified or wild-type CD155, which in some aspects results in increased binding to the inhibitory receptor TIGIT. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain of a variant CD155 polypeptide (e.g., IgV or an ECD comprising IgV and IgC) are listed in table 5. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD155 polypeptide, the variant CD155 polypeptide comprising an IgV domain comprising any of the amino acid modifications listed in Table 5, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the IgV domain listed in any of SEQ ID NOs 332-. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD155 polypeptide comprising an ECD or a portion thereof comprising an IgV and/or IgC domain comprising any of the amino acid modifications listed in Table 5, such as an ECD listed in any of SEQ ID NO: 311-1622, 375-channel 471, 1551-channel 1622 or an ECD comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NO: 311-channel 331, 375-channel 471, 1551-channel 1622 and comprising one or more amino acid modifications.
In some embodiments, the at least one additional (e.g., second or third) igd is an IgSF domain (e.g., IgV) of a variant CD112 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) as compared to unmodified or wild-type CD112, which in some aspects results in increased binding to the inhibitory receptor TIGIT. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain of a variant CD112 polypeptide (e.g., IgV or an ECD comprising IgV and IgC) are listed in table 4. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD112 polypeptide, the variant CD112 polypeptide comprising an IgV domain comprising any of the amino acid modifications listed in Table 4, such as an IgV domain listed in any of SEQ ID NOs 714-760, 762-808, 850-931 or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs 714-760, 762-808, 850-931 and comprising one more amino acid modification. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD112 polypeptide comprising an ECD or a portion thereof comprising an IgV and/or IgC domain comprising any one of the amino acid modifications listed in Table 4, such as an ECD listed in any of SEQ ID NO 667-I713, 809-849, 1433-I1456 or an ECD comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NO 667-I713, 809-I849, 1433-I1456 and comprising one or more amino acid modifications.
In some embodiments, the at least one additional (e.g., second or third) igd is an IgSF domain (e.g., IgV) of a variant PD-L2 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., ECD or IgV) as compared to unmodified or wild-type PD-L2, which in some aspects results in increased binding to the inhibitory receptor PD-1. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., IgV or an ECD comprising IgV and IgC) of a variant PD-L2 polypeptide are listed in table 8. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant PD-L2 polypeptide, wherein the variant PD-L2 polypeptide comprises an IgV domain comprising any of the amino acid modifications listed in Table 8, such as an IgV domain listed in any of SEQ ID NOS 1281-1331, 1333-1407, 1409-1432 or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOS 1281-1401, 1333-1407, 1409-1432 and comprising one or more amino acid modifications. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant PD-L2 polypeptide, the variant PD-L2 polypeptide comprising an ECD or a portion thereof comprising an IgV and/or IgC domain comprising any one of the amino acid modifications listed in Table 8, such as an ECD listed in any of SEQ ID Nos 1204-1254, 1256-minus 1280 or an ECD comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID Nos 1204-1254, 1256-minus 1280 and comprising one or more amino acid modifications.
In some embodiments, the one or more additional IgSF domain (e.g., second IgSF) domain is an IgSF domain (e.g., IgV) of another IgSF family member that binds or recognizes a tumor antigen. In such embodiments, IgSF family members serve as tumor localization sites, thereby bringing the vigdd of PD-L1 close to immune cells in the tumor microenvironment. In some embodiments, the additional IgSF domain (e.g., the second IgSF) domain is an IgSF domain that binds to or recognizes NKp30 of B7-H6 expressed on tumor cells. In some embodiments, at least one additional (e.g., second) IgSF domain (e.g., NKp30) is an affinity modified IgSF domain or viggd containing one or more amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the one or more amino acid modifications increase binding affinity and/or selectivity for B7-H6, such as by at least or at least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold, as compared to an unmodified IgSF domain (e.g., NKp 30). Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., IgC-like or whole ECD) of a variant NKp30 polypeptide are listed in table 6. An exemplary polypeptide is a NKp30 variant containing mutations L30V/a60V/S64P/S86G with reference to positions in the NKp30 extracellular domain corresponding to the positions listed in SEQ ID NO: 54. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant NKp30 polypeptide, the variant NKp30 polypeptide comprising an IgC-like domain comprising any of the amino acid modifications listed in table 6, such as an IgC-like domain listed in any of SEQ ID NOs 1184-1188 or an IgC-like domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NOs 1184-1188 and comprising one or more amino acid modifications. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant NKp30 polypeptide, the variant NKp30 polypeptide comprising ECD or a portion thereof comprising one or more IgSF domains comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of the amino acid modifications listed in Table 6, such as ECD listed in any of SEQ ID NO:1178-1182 or ECD comprising any of SEQ ID NO:1178-1182 and comprising one or more amino acid modifications.
In some embodiments, the at least one additional (e.g., second or third) igd is an IgSF domain (e.g., IgV) of a variant CD86 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions, or additions) in the IgSF domain (e.g., IgV) as compared to unmodified or wild-type CD86, which in some aspects results in increased binding to its cognate binding partner. Exemplary amino acid modifications (such as substitutions, deletions or additions) in the IgSF domain (e.g., IgV or an ECD comprising IgV and IgC) of a variant CD86 polypeptide are listed in table 7. Exemplary polypeptides include CD86 variants comprising mutations Q35H/H90L/Q102H with reference to positions in the CD86 extracellular domain corresponding to the positions set forth in SEQ ID NO: 29. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD86 polypeptide, the variant CD86 polypeptide comprising an IgV domain comprising any of the amino acid modifications listed in Table 7, such as the IgV domain listed in any of SEQ ID NO:1196-1199 or an IgV domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of SEQ ID NO:1196-1199 and comprising one more amino acid modification. In some embodiments, an immunomodulatory protein is provided comprising any of the provided variant PD-L1 polypeptides and a variant CD86 polypeptide, said variant CD86 polypeptide comprising ECD or a portion thereof comprising an IgV and/or IgC domain comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of the amino acid modifications listed in Table 7, such as ECD listed in any of SEQ ID NO:1191-1194 or ECD comprising any of SEQ ID NO:1191-1194 and comprising one or more amino acid modifications.
Tables 3-8 provide exemplary polypeptides comprising IgSF domains that can be used for one or more affinity modifications in the stacked constructs provided herein.
The number of such non-affinity modified or affinity modified IgSF domains present in a "stacked" immunomodulatory protein construct (whether in non-wild-type combination or non-wild-type arrangement) is at least 2,3,4, or 5 and in some embodiments exactly 2,3,4, or 5 IgSF domains (thus determination of the number of affinity modified IgSF domains disregards any non-specific binding partial sequence thereof and/or substantially immunologically active partial sequence thereof).
In some embodiments of stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least 2, wherein the number of affinity modified IgSF domains and the number of non-affinity modified IgSF domains are each independently at least: 0. 1,2,3,4,5 or 6. Thus, the number of affinity modified IgSF domains and the number of non-affinity modified IgSF domains (affinity modified IgSF domains: non-affinity modified IgSF domains) may be exactly or at least: 2:0 (affinity modification: wild type), 0:2, 2:1, 1:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5 or 5: 1.
In some embodiments of stacked immunomodulatory proteins, the at least two non-affinity modified IgSF domains and/or affinity modified IgSF domains are the same IgSF domain.
In some embodiments, stacked immunomodulatory proteins provided herein comprise at least two affinity modified IgSF domains and/or non-affinity modified IgSF domains from a single IgSF member but in a non-wild type arrangement (alternatively "aligned"). An illustrative example of a non-wild-type arrangement or permutation are those immunomodulatory proteins present in wild-type PD-L1 used as a source of variant IgSF domains as provided herein, immunomodulatory proteins comprising affinity modified IgSF domains and/or non-affinity modified IgSF domains of the non-wild-type sequence, relative to the IgSF domain sequence. Thus, in one example, an immunomodulatory protein may comprise IgV proximal to a transmembrane domain and IgC distal, albeit in a non-affinity modified form and/or an affinity modified form. The presence of non-wild-type combinations and non-wild-type arrangements of non-affinity modified IgSF domains and/or affinity modified IgSF domains in the immunomodulatory proteins provided herein is also within the provided subject matter.
In some embodiments of the stacked immunomodulatory proteins, the non-affinity modified IgSF domain and/or the affinity modified IgSF domain are non-identical (i.e., different) IgSF domains. Non-identical affinity modified IgSF domains specifically bind to different homologous binding partners under specific binding conditions and are "non-identical" regardless of whether the wild-type or unmodified IgSF domains from which they are engineered are identical. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein may comprise at least one IgSF domain sequence derived from and unique to one PD-L1 and at least one second IgSF domain derived from and unique to another IgSF family member other than PD-L1, wherein the IgSF domains of the immunomodulatory protein are in a non-affinity modified and/or affinity modified form. However, in alternative embodiments, two non-identical IgSF domains are derived from the same IgSF domain sequence, but at least one is affinity modified such that they specifically bind to different cognate binding partners.
The plurality of non-affinity modified and/or affinity modified IgSF domains in the stacked immunomodulatory protein polypeptide chains need not be directly covalently linked to each other. In some embodiments, the intermediate spans of one or more amino acid residues are indirectly covalently bound to each other with non-affinity modified and/or affinity modified IgSF domains. The linkage may be via the N-terminal to the C-terminal residue.
In some embodiments, two or more IgSF domains (including a vigdd of PD-L1 and one or more additional IgSF domains from another IgSF family member (e.g., a second or third variant IgSF domain) are linked together covalently or non-covalently.
In some embodiments, the immunomodulatory protein comprises at least two IgSF domains each linked directly or indirectly via a linker. In some embodiments, the immunomodulatory protein comprises at least three immunomodulatory proteins each linked directly or indirectly via a linker. Various configurations are shown in fig. 5A and 5B.
In some embodiments, one or more "peptide linkers" link the viggd of PD-L1 and one or more additional IgSF domains (e.g., second or third variant IgSF domains). In some embodiments, the length of the peptide linker may be a single amino acid residue or greater. In some embodiments, the peptide linker has at least one amino acid residue but no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7,6, 5,4, 3,2, or1 amino acid residue in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in the form of a one letter amino acid code): GGGGS ("4 GS"; SEQ ID NO:1942) or multimers of 4GS linkers, such as2, 3,4, or 5 repeats of a 4GS linker. In some embodiments, the peptide linker is (GGGGS)2(SEQ ID NO:240) or (GGGGS)3(SEQ ID NO: 239). in some embodiments, the linker (in the form of one letter amino acid code) is GSGGGGS (SEQ ID NO: 1941). in some embodiments, the linker may also include a series of alanine residues, either alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). in some embodiments, the number of alanine residues in each series is 2,3,4,5, or 6 alanine.in some embodiments, the linker is a rigid linker.e.g., the linker is an α helical linkerRepeated sequences, such as those listed in SEQ ID NO:2022 (1xEAAAK), SEQ ID NO:2023 (3xEAAAK) or SEQ ID NO:2024 (5 xEAAAK). In some embodiments, the linker may further comprise amino acids introduced by cloning and/or from restriction sites, e.g. the linker may comprise the amino acids GS (in the form of one letter amino acid code) as introduced by using the restriction site BAMHI. For example, in some embodiments, the linker (in the form of one letter amino acid code) is GSGGGGS (SEQ ID NO:1941), GS (G)4S)3(SEQ ID NO:2031) or GS (G)4S)5(SEQ ID NO: 2032). In some examples, the linker is 2XGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 241). In some cases, various combinations of peptides are used as linkers.
In some embodiments, the non-affinity modified and/or affinity modified IgSF domains are linked by a "wild-type peptide linker" inserted at the N-terminus and/or C-terminus of the non-affinity modified and/or affinity modified IgSF domains. These linkers are also referred to as leader sequences (N-terminal of the non-affinity modified or affinity modified IgSF domain) or trailer sequences (C-terminal of the non-affinity modified or affinity modified IgSF domain), as well as sequences present in the wild-type protein that span outside of the structural prediction of the Ig folding of the IgSF. In some embodiments, a "wild-type linker" is an amino acid sequence that is present in the amino acid sequence of the wild-type protein after the signal sequence but before the IgSF domain (such as the defined IgV domain). In some embodiments, a "wild-type" linker is an amino acid sequence that is present immediately after an IgSF domain (such as immediately after a defined IgV domain) but before an IgC domain in the amino acid sequence of a wild-type protein. These linker sequences may facilitate proper folding and function of the adjacent one or more IgSF domains. In some embodiments, there is a leader peptide sequence inserted at the N-terminus of the first IgSF domain and/or a tail sequence inserted at the C-terminus of the first non-affinity modified and/or affinity modified IgSF domain. In some embodiments, there is a second leader peptide sequence inserted at the N-terminus of the second IgSF domain and/or a second tail sequence inserted at the C-terminus of the second non-affinity modified and/or affinity modified IgSF domain. When the first and second non-affinity modified and/or affinity modified IgSF domains are derived from the same parent protein and are linked in the same orientation, the wild-type peptide linker between the first and second non-affinity modified and/or affinity modified IgSF domains is not duplicated. For example, when the first tail wild-type peptide linker and the second leader wild-type peptide linker are the same, the type II immunomodulatory protein does not comprise the first tail wild-type peptide linker or the second leader wild-type peptide linker.
In some embodiments, the type II immunomodulatory protein comprises a first leader wild-type peptide linker inserted at the N-terminus of the first non-affinity and/or affinity modified IgSF domain, wherein the first leader wild-type peptide linker comprises at least 5 (such as at least about any of 6,7,8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the inserted sequence of the wild-type protein from which the first non-affinity and/or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain). In some embodiments, the first lead wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the first non-affinity modified and/or affinity modified IgSF domain is derived between the parent IgSF domain and an immediately preceding domain (such as a signal peptide or an IgSF domain).
In some embodiments, the type II immunomodulatory protein further comprises a first tail wild-type peptide linker inserted at the C-terminus of the first non-affinity and/or affinity modified IgSF domain, wherein the first tail wild-type peptide linker comprises at least 5 (such as at least about any of 6,7,8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the inserted sequence of the wild-type protein from which the first non-affinity and/or affinity modified IgSF domain is derived, between the parent IgSF domain and an immediately following domain (such as an IgSF domain or transmembrane domain). In some embodiments, the first tail wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the first non-affinity and/or affinity modified IgSF domain is derived between the parent IgSF domain and an immediately following domain (such as an IgSF domain or transmembrane domain).
In some embodiments, the type II immunomodulatory protein further comprises a second leader wild-type peptide linker inserted at the N-terminus of a second non-affinity modified and/or affinity modified IgSF domain, wherein the second leader wild-type peptide linker comprises at least 5 (such as at least about any of 6,7,8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the inserted sequence of the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain). In some embodiments, the second lead wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parent IgSF domain and the immediately preceding domain (such as a signal peptide or IgSF domain).
In some embodiments, the type II immunomodulatory protein further comprises a second tail wild-type peptide linker inserted at the C-terminus of a second non-affinity and/or affinity modified IgSF domain, wherein the second tail wild-type peptide linker comprises at least 5 (such as at least about any of 6,7,8, 9, 10, 11, 12, 13, 14, 15 or more) contiguous amino acids from the inserted sequence of the wild-type protein from which the second non-affinity and/or affinity modified IgSF domain is derived, between the parent IgSF domain and an immediately following domain (such as an IgSF domain or transmembrane domain). In some embodiments, the second tail wild-type peptide linker comprises the entire insertion sequence of the wild-type protein from which the second non-affinity and/or affinity modified IgSF domain is derived between the parent IgSF domain and an immediately following domain (such as an IgSF domain or transmembrane domain).
In some embodiments, two or more IgSF domains, including a vigdd of PD-L1 and one or more additional IgSF domains from another IgSF family member (e.g., second and/or third variant IgSF domains), are linked or attached to a multimerization domain, such as to an Fc, to form an Fc fusion that, when expressed in a cell, can in some aspects produce a dimeric multidomain stacking immunomodulatory protein. Thus, dimeric multidomain immunomodulatory proteins are also provided.
In some embodiments, the variant PD-L1 polypeptide and the one or more additional IgSF domains are independently linked directly or indirectly to the N-terminus or C-terminus of a multimerization domain, such as an Fc region. In some embodiments, at least one of the variant PD-L1 polypeptide and the one or more additional IgSF domains is directly or indirectly linked to the N-terminus or C-terminus of a multimerization domain, such as an Fc region, and one of the variant PD-L1 and one of the one or more additional IgSF domains is also directly or indirectly linked to the N-terminus or C-terminus of a multimerization domain, such as an Fc region. In some embodiments, the multimerization domain, such as the N-terminus or C-terminus of the Fc region, is linked to the variant PD-L1 polypeptide or the one or more additional IgSF domains, and the other of the N-terminus or C-terminus of the Fc region is linked to the other of the PD-L1 variant or the other of the one or more additional IgSF domains. In some embodiments, the linkage to the multimerization domain, such as to the Fc, is via a peptide linker, e.g., a peptide linker, such as described above. In some embodiments, the linkage between the variant PD-L1 and the one or more additional IgSF domains is via a peptide linker, e.g., a peptide linker, such as described above. In some embodiments, the vigdd, one or more additional IgSF domains, and a multimerization domain, such as an Fc domain, of PD-L1 may be linked together in any of a number of configurations as depicted in fig. 5A and 5B. Exemplary configurations are described in the examples.
In some embodiments, the stacked immunomodulatory protein is a dimer formed by two immunomodulatory Fc fusion polypeptides. Also provided are nucleic acid molecules encoding any of the stacked immunomodulatory proteins. In some embodiments, a dimeric multidomain stacked immunomodulatory protein may be produced in a cell by expressing or, in some cases, co-expressing a stacked immunomodulatory Fc fusion polypeptide (such as described above with respect to the production of a dimeric Fc fusion protein).
In some embodiments, the dimeric multidomain stacking immunomodulatory protein is bivalent for each Fc region, monovalent for each subunit, or bivalent for one subunit and tetravalent for another subunit.
In some embodiments, the dimeric multidomain stacking immunomodulatory protein is a homodimeric multidomain stacking Fc protein. In some embodiments, the dimeric multidomain stacked immunomodulatory protein comprises a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are the same. In some embodiments, the multidomain stacking molecule contains a first Fc fusion polypeptide comprising a variant PD-L1 and a second IgSF domain and a second Fc fusion polypeptide comprising a variant PD-L1 and a second IgSF domain. In some embodiments, the multidomain stacking molecule contains a first Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain, and a third IgSF domain and a second Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain, and a third IgSF domain. In some embodiments, the Fc portion of the first and/or second fusion polypeptide can be any Fc as described above. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same.
In some embodiments, the multidomain stacking molecule is a heterodimer comprising two different Fc fusion polypeptides (e.g., a first and a second Fc fusion polypeptide), at least one of which is an Fc fusion polypeptide comprising a variant PD-L1 polypeptide and/or at least one of which is an Fc fusion polypeptide comprising a second IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the first or second Fc fusion polypeptide further comprises a third IgSF domain (e.g., a third variant IgSF domain).
In some embodiments, the multidomain stacking molecule comprises a first Fc fusion polypeptide comprising variant PD-L1 and a second Fc fusion polypeptide comprising a second IgSF domain, wherein in some cases the first or second Fc fusion polypeptide additionally comprises a third IgSF domain. In some embodiments, the multidomain stacking molecule contains a first Fc fusion polypeptide comprising a variant PD-L1, a second IgSF domain, and (in some cases) a third IgSF domain, and a second Fc fusion polypeptide not linked to a variant PD-L1 polypeptide or an additional IgSF domain. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are the same. In some embodiments, the Fc portions or regions of the first and second fusion polypeptides are different.
In some embodiments, the multidomain stacking molecule contains a first Fc fusion polypeptide comprising 1,2,3,4, or more variant PD-L1 polypeptides and 1,2,3,4, or more additional IgSF domains, wherein the total number of IgSF domains in the first stacked Fc fusion polypeptide is greater than 2,3,4,5,6, or greater. In one example of such an embodiment, the second stacked Fc fusion polypeptide contains 1,2,3,4, or more variant PD-L1 polypeptides and 1,2,3,4, or more additional IgSF domains, wherein the total number of IgSF domains in the second stacked Fc fusion polypeptide is greater than 2,3,4,5,6, or greater. In another example of such an embodiment, the second Fc fusion polypeptide is not linked to a variant PD-L1 polypeptide or an additional IgSF domain.
In some embodiments, the heterodimeric stacking molecule comprises a first stacking immunomodulatory Fc fusion polypeptide and a second stacking immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are different. In some embodiments, the heterodimer stacking molecule comprises a first Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and/or a second IgSF domain (e.g., a second variant IgSF domain) and a second polypeptide fusion comprising an Fc region and the other of the first variant PD-L1 polypeptide or the second IgSF domain. In some embodiments, the heterodimer stacking molecule comprises a first Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and/or a second IgSF domain (e.g., a second variant IgSF domain) and a second Fc polypeptide fusion comprising an Fc region and a first variant PD-L1 polypeptide and a second IgSF domain (e.g., a second variant IgSF domain) but in a different orientation or configuration than the first Fc region. In some embodiments, the first and/or second Fc fusion polypeptide further comprises a third IgSF domain (e.g., a third variant IgSF domain).
In some embodiments, the Fc domain of one or both of the first and second stacked immunomodulatory Fc fusion polypeptides comprises a modification (e.g., a substitution) such that the interface of the Fc molecule is modified to facilitate and/or promote heterodimerization. In some embodiments, the modification comprises introducing a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide such that the protrusion can be positioned in the cavity to facilitate the complication of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and/or modification to create a protrusion or cavity in a polypeptide are typically interfacial amino acids that interact or contact one or more amino acids in the interface of a second polypeptide.
In some embodiments, the amino acid sequence is added before the Fc sequence of the construct, wherein the Fc sequence is the N-terminal portion of the sequence. In some cases, the amino acid sequence HMSSVSAQ (SEQ ID NO:1156) is added immediately before the Fc sequence of the construct, where the Fc sequence is the N-terminal portion of the sequence. In some embodiments, the heterodimer stacking molecule comprises a first Fc-polypeptide fusion comprising an Fc region (knob) and a first variant polypeptide and/or a second IgSF domain (e.g., a second variant IgSF domain) and a second Fc-polypeptide fusion comprising an Fc region (pore), comprising the stuffer sequence HMSSVSAQ (SEQ ID NO:1156) immediately preceding both Fc regions of the first and second Fc-polypeptide fusions.
In some embodiments, a first polypeptide modified to contain a tab (pore) amino acid comprises replacing a native or original amino acid with an amino acid having at least one side chain that extends from an interface of the first polypeptide and is thus positionable in a complementary cavity (pore) in an adjacent interface of a second polypeptide. Most often, the replacement amino acid is an amino acid with a larger side chain volume than the original amino acid residue. One skilled in the art would know how to determine and/or assess the identity of amino acid residues to identify the desired substituted amino acid to generate a protrusion. In some embodiments, the replacement residues used to form the projections are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the initial residue identified for substitution is an amino acid residue with a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
In some embodiments, the second polypeptide modified to contain a cavity (pore) is a polypeptide comprising the replacement of a native or original amino acid with an amino acid having at least one side chain that is recessed from the interface of the second polypeptide and is thus capable of accommodating a corresponding protrusion from the interface of the first polypeptide. Most often, the replacement amino acid is an amino acid with a smaller side chain volume than the original amino acid residue. Those skilled in the art know how to determine and/or assess the identity of amino acid residues to identify ideal replacement residues for cavity formation. Generally, the replacement residues for forming the cavity are naturally occurring amino acids and include, for example, alanine (a), serine (S), threonine (T), and valine (V). In some examples, the initial amino acid identified for substitution is an amino acid with a large side chain, such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.
The CH3 interface of human IgG1, for example, involves sixteen residues located on each domain on four antiparallel β chains, which are hidden from each surface
In some embodiments, the heterodimeric molecule contains a T366W mutation in the CH3 domain of the "globular projection chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "pore chain". In some cases, additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, a.m. et al, Nature biotech.16(1998) 677-. In some embodiments, the heterodimeric molecule contains an S354C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises an E356C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, a T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, a T366W mutation in one of the two CH3 domains and a S354C, a T366S, an L368A, a Y407V mutation in the other of the two CH3 domains. Examples of other ball-in-hole techniques are known in the art, for example, as described by EP 1870459 a 1.
In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, such as any of those described above. In some embodiments, the heterodimeric molecule contains a mutated Fc region with reduced effector function.
In some embodiments, Fc variants containing CH3 tab (knob) or cavity (hole) modifications can be ligated to any position of the stacked immunomodulatory polypeptides, but are typically ligated via their N-or C-termini to the N-or C-termini of the first and/or second stacked immunomodulatory polypeptides, such as to form a fusion polypeptide. The attachment may be directly or indirectly via a linker. Typically, the knob and hole molecules are generated by co-expressing a first stacked immunomodulatory polypeptide linked to an Fc variant comprising one or more CH3 tab modifications and a second stacked immunomodulatory polypeptide linked to an Fc variant comprising one or more CH3 cavity modifications.
Provided herein is a homodimeric multidomain stacked molecule produced from a stacked immunomodulatory Fc fusion polypeptide comprising an IgSF domain (e.g., IgV domain) of a variant PD-L1 polypeptide and a second IgSF domain (e.g., IgV) of a variant CD155 polypeptide. In some embodiments, the first and second immunomodulatory Fc fusion polypeptides of the multidomain stacking molecule have a sequence set forth in any one of SEQ ID NOs 1716, 1717, 1718, 1719, 1720, or 1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any one of SEQ ID NOs 1716, 1717, 1718, 1719, 1720, or 1721 and containing one or more amino acid modifications in a variant PD-L1 and/or CD155 IgSF domain. In some embodiments, the resulting multidomain stacked molecule binds to both TIGIT and PD-1. In some aspects, the degree of binding to TIGIT is the same or similar, or in some cases increased, as compared to binding to TIGIT of the corresponding IgSF domain of unmodified or wild-type PD-L1 or CD 155. In some aspects, the degree of binding to PD-1 is the same or similar, or in some cases increased, as compared to the binding to PD-1 of the corresponding IgSF domain of unmodified or wild-type PD-L1. In some embodiments, the variant PD-L1 IgSF-Fc that binds to TIGIT or PD-1 in a non-stacked form is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the binding to TIGIT or PD-1. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the binding to TIGIT of variant CD155 IgSF-Fc in non-stacked form. In some embodiments, the resulting multidomain stacked molecule increases a T cell immune response, such as determined in a reporter gene assay, as compared to a non-stacked variant PD-L1 IgSF-Fc and/or variant CD 155-IgSF-Fc. In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or more.
Provided herein is a homodimeric multidomain stacked molecule produced from a stacked immunomodulatory Fc fusion polypeptide comprising an IgSF domain (e.g., IgV domain) of a variant PD-L1 polypeptide, a second IgSF domain (e.g., IgV) of a variant CD155 polypeptide, and a third IgSF domain (e.g., IgV) of a variant CD112 polypeptide. In some embodiments, the first and second immunomodulatory Fc fusion polypeptides of the multidomain stacking molecule have a sequence set forth in any one of SEQ ID NOs 1722, 1723, and 1724 or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs 1722, 1723, and 1724 and contain an amino acid sequence that is one or more amino acid modifications of a variant CD112, CD155, and/or PD-L1IgSF domain. In some embodiments, the resulting multidomain stacked molecule binds to both TIGIT, CD112R, and PD-1. In some aspects, the degree of binding to TIGIT is the same or similar, or in some cases increased, as compared to binding to TIGIT of the corresponding IgSF domain of unmodified or wild-type CD112 or CD 155. In some aspects, the degree of binding to CD112R is the same or similar, or in some cases increased, as compared to binding to CD112R of the corresponding IgSF domain of unmodified or wild-type CD 112. In some aspects, the degree of binding to PD-1 is the same or similar, or in some cases increased, as compared to the binding to PD-1 of the corresponding IgSF domain of unmodified or wild-type PD-L1. In some embodiments, the binding to TIGIT or CD112R is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the binding to TIGIT or CD112R of the variant CD112IgSF-Fc in non-stacked form. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the binding to TIGIT of variant CD155 IgSF-Fc in non-stacked form. In some embodiments, the binding to PD-1 is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the binding to PD-1 of the variant PD-1 IgSF-Fc in non-stacked form. In some embodiments, the resulting multidomain stacked molecule increases a T cell immune response, such as determined in a reporter assay, as compared to a non-stacked variant CD112IgSF-Fc, variant CD155-IgSF-Fc, and/or variant PD-L1-IgSF-Fc. In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or more.
C. Conjugates and fusions of variant polypeptides and immunomodulatory proteins
In some embodiments, a variant polypeptide provided herein is an immunomodulatory protein comprising a variant of an Ig domain of the IgSF family (viggd), which variant polypeptide may be conjugated or fused, directly or indirectly, to a moiety, such as an effector moiety, such as another protein, to form a conjugate ("IgSF conjugate"). In some embodiments, the attachment may be covalent or non-covalent, e.g., via biotin-streptavidin non-covalent interactions. In some embodiments, the moiety can be a targeting moiety, a small molecule drug (non-polypeptide drug of less than 500 dalton molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug activating enzyme, an agent that increases biological half-life or a diagnostic or detectable agent.
In some embodiments, the effector moiety is a therapeutic agent that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit, such as a cancer therapeutic agent. In some embodiments, the effector moiety is a targeting moiety or targeting agent, such as an agent that targets a cell surface antigen (e.g., an antigen on the surface of a tumor cell). In some embodiments, the effector moiety is a tag, which can generate a detectable signal, either directly or indirectly. In some embodiments, the effector moiety is a toxin. In some embodiments, the effector moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.
In some embodiments, 1,2,3,4,5 or more effector moieties (which may be the same or different) are conjugated, linked or fused to a variant polypeptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties may be attached to variant polypeptides or immunomodulatory proteins using molecular biological or chemical conjugation and attachment methods known in the art and described below. In some embodiments, a linker, such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that facilitates the conjugation reaction, may be used to link or conjugate the effector moiety to the variant polypeptide or the immunomodulatory protein.
In some embodiments, the IgSF conjugate comprises the following components: (protein or polypeptide), (L)qAnd (Effect part)mWherein the protein or polypeptide is any of the variant polypeptide or immunomodulatory protein capable of binding to the one or more cognate relative structural ligands; l is a linker attaching the protein or polypeptide to the moiety; m is at least 1; q is 0 or greater; and the resulting IgSF conjugate binds to one or more opposing structural ligands. In particular embodiments, m is 1 to 4 and q is 0 to 8.
In some embodiments, an IgSF conjugate is provided comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a targeting agent that binds a cell surface molecule, e.g., for targeted delivery of the variant polypeptide or immunomodulatory protein to a specific cell. In some embodiments, the targeting agent is one or more molecules that have the ability to localize and bind to molecules present on normal cells/tissues and/or tumor cells/tumors of the subject. In other words, an IgSF conjugate comprising a targeting agent may bind to a ligand (directly or indirectly) present on a cell, such as a tumor cell. Targeting agents contemplated for use with the present invention include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof that can bind to a component of a target cell or molecule.
In some embodiments, the targeting agent binds to the tumor cell or can bind in the vicinity of the tumor cell (e.g., tumor vasculature or tumor microenvironment) after administration to the subject. The targeting agent can bind to a receptor or ligand on the surface of the cancer cell. In another aspect of the invention, a targeting agent is selected that is specific for a non-cancerous cell or tissue. For example, the targeting agent may be specific for a molecule normally present on a particular cell or tissue. Furthermore, in some embodiments, the same molecule may be present on both normal and cancer cells. Various cellular components and molecules are known. For example, if the targeting agent is specific for EGFR, the resulting IgSF conjugate can target EGFR-expressing cancer cells as well as EGFR-expressing normal skin epithelial cells. Thus, in some embodiments, the IgSF conjugates of the present invention can function by two separate mechanisms (targeting cancer cells and non-cancer cells).
In various aspects of the invention disclosed herein, the IgSF conjugates of the invention comprise a targeting agent that can bind to/target a cellular component, such as a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasma antigen, a fungal antigen, a prion antigen, an antigen from a parasitic organism in some aspects the cellular component, antigen, or molecule can each be used to refer to a desired target for the targeting agent, for example, in various embodiments, the targeting agent is specific for or binds to a component including, but not limited to, epidermal growth factor receptor (EGFR, ErbB-1, HERl), 2(HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family, insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family, platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family, Fibroblast Growth Factor Receptor (FGFR) family, FGFR ligand family, VEGF receptor (VEGF receptor) family, VEGF receptor family such as the TGF receptor family, VEGF receptor family, TGF receptor family, such as the TGF receptor family 11 receptor family, the TGF receptor family, the receptor family of the TGF receptor family of the orphan receptor of the TGF receptorReceptors, receptors of class I (erythropoietin family) and class II (interferon/IL-10 family), Tumor Necrosis Factor (TNF) receptor superfamily (TNFRSF), death receptor family; cancer-testis (CT) antigen, lineage specific antigen, differentiation antigen, α -actinin-4, ARTCl, cleavage cluster region-Abelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β -catenin (CTNNBl), cell division cycle 27(CDC27), cyclin-dependent kinase 4(CDK4), CDKN2A, COA-I, dek-can fusion protein, EFTUD-2, elongation factor 2(ELF2), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETC 4-1) fusion protein, Fibronectin (FN) domain such as the fucose-A-2-serine-alpha-serine-transferase domain, low density domain of fucose-serine transferase (HLA-serine transferase) for example, low density domain of fucose-serine transferase (HLA-serine transferase) 2, low density domain of fucose-serine transferase (HLA-637), low density transferase (HLA-serine transferase) for example, low density of fucose-serine transferase (HLA-serine transferase) for example, low density of fucose-serine transferase (HLA-serine transferase) for example, low density protein, low density of fucose-serine transferase (HLA-serine transferase (CDK-5), and*201-R170I), HLA-Al 1, mutated heat shock protein 70-2(HSP70-2M), K1AA0205, MART2, mutated melanoma ubiquitin 1,2, 3(MUM-I, 2, 3), Prostatic Acid Phosphatase (PAP), neo-PAP, class I sarcoplasmic globulin, NFYC, OGT, OS-9, MART 28-RAR 9 fusion protein, PRDX 2, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPDl, SYT-SSXl or-SSX 2 fusion protein, triose phosphate isomerase, BAGE, BlBAGK-1, MAGE-2, 3, GE 4,5, GAGE-1,2,3,4,5,6,7,8, T-V (abnormal N-acetyl transferase), MAGHESIL-N-I-4, MAG-K-1, MAG-2, MAGE-4, MAG-III, MAG-I-4, MAG-III, MAG-I-III, MAG-I-III, MAG-I-III, MAG-ING-4, CPSF, cyclin Dl, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250(gp250), EGFR (ERBBl), HER-2/neu (ERBB2), interferon 13 receptor α 2 chain (IL13R α), IL-6 receptor, Intestinal Carboxyesterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCl, p53(TP53), PBF, PRAME, PSMA, RAGE-RAGE I, RNF43, RU2AS, SOXlO, STEAPl, survivin (BIRC5), human telomerase reverse transcriptase (hTERT), telomerase, Wilms tumor gene (WTl), SYCPl, BR36707, SPANX, XAGE, ADAM2, EPGE-5, LIPL, CTAGE-I, PIGE, PIECE, BOAL, CAL, CAR-15, HCP-15, antigen, HCP-15, HCP receptor antigen (HCEP-15), antigen receptor antigen of human choriocarcinoma, HCP 15, HCEP 15, HCP receptor antigen, HCP 15, HCP 27, HCP 15, HCP 15, HCP 15, HCP 27, HCP 15, HCP 15, HCP 15, HCP 27, HCP 15, HCP 15, HCP 27, HCP 15, HCP 27, HCP 15, HCP 27.
In some embodiments, the IgSF conjugate will bind through its targeting agent to a tumor cell, tumor vasculature, or cellular component of the tumor microenvironment, thereby promoting targeted cell killing via modulation of the immune response (e.g., by activating a costimulatory molecule or a negative regulator molecule that inhibits immune cell activation), inhibition of survival signaling (e.g., a growth factor or cytokine or hormone receptor antagonist), activation of death signaling, and/or immune-mediated cytotoxicity (such as by antibody-dependent cytotoxicity). Such IgSF conjugates can function by several mechanisms to prevent, reduce, or eliminate tumor cells, such as to facilitate delivery of conjugated effector moieties to tumor targets, such as by receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and/or activate immune cells (e.g., NK cells, monocytes/macrophages, dendritic cells, T cells, B cells). Furthermore, in some cases, one or more of the aforementioned routes may function following administration of one or more of the IgSF conjugates of the invention.
In some embodiments, the IgSF conjugate will localize to (such as bind to) a tumor cell, tumor vessel, or cellular component of the tumor microenvironment via its targeting agent, thereby modulating immune response cells in the vicinity of the tumor. In some embodiments, targeting agents facilitate delivery of conjugated IgSF (e.g., vigdd) to a tumor target, such as to interact with its cognate binding partner to alter signaling of immune cells (e.g., NK cells, monocytes/macrophages, dendritic cells, T cells, B cells) bearing the cognate binding partner. In some embodiments, the localized delivery mediates antagonism or blockade of the activity of the PD-1 inhibitory receptor. In some embodiments, localized delivery agonizes a PD-1 inhibitory receptor, which may occur in some cases where there is proximal clustering of activated receptors.
In some embodiments, the targeting agent is an immunoglobulin. As used herein, the term "immunoglobulin" includes natural or artificial monovalent or multivalent antibodies, including but not limited to polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, single chain fv (scfv); anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention) and epitope-binding fragments of any of the above. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.
In some embodiments, the IgSF conjugates will bind, through its antibody targeting moiety, to a tumor cell, tumor vasculature, or a cellular component of the tumor microenvironment, thereby promoting apoptosis of the targeted cell via modulation of the immune response (e.g., by activating a costimulatory molecule or a negative regulator molecule that inhibits immune cell activation), inhibition of survival signaling (e.g., a growth factor or cytokine or hormone receptor antagonist), activation of death signaling, and/or immune-mediated cytotoxicity (such as by antibody-dependent cytotoxicity). Such IgSF conjugates can function by several mechanisms to prevent, reduce, or eliminate tumor cells, such as to facilitate delivery of conjugated effector moieties to tumor targets, such as by receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and/or activate immune cells (e.g., NK cells, monocytes/macrophages, dendritic cells, T cells, B cells).
In some embodiments, the IgSF conjugate, through its antibody targeting moiety, will bind to a tumor cell, tumor vessel, or cellular component of the tumor microenvironment, thereby modulating the immune response (e.g., by activating a costimulatory molecule or a negative regulatory molecule that inhibits immune cell activation). In some embodiments, such conjugates can recognize, bind to, and/or modulate (e.g., inhibit or activate) immune cells (e.g., NK cells, monocytes/macrophages, dendritic cells, T cells, B cells).
Antibody targeting moieties of the invention include antibody fragments, including but not limited to Fab, Fab 'and F (ab')2, Fd, single chain fv (scFv), single chain antibodies, disulfide linked fv (sdFv), and fragments comprising a VL or VH domain. Antigen-binding antibody fragments, including single chain antibodies, may include one or more variable regions, alone or in combination with all or a portion of: a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain. The invention also includes antigen binding fragments that also comprise any combination of one or more variable regions and the hinge, CH1, CH2, and CH3 domains. The invention also includes Fc fragments, antigen-Fc fusion proteins and Fc targeting moiety conjugates or fusion products (Fc peptides, Fc aptamers). The antibody targeting moiety of the invention may be from any animal source, including birds and mammals. In one aspect, the antibody targeting moiety is human, murine (e.g., mouse and rat), monkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Additionally, such antibodies can be humanized versions of animal antibodies. The antibody targeting moieties of the invention can be monospecific, bispecific, trispecific, or more multispecific.
In various embodiments, the antibody/targeting moiety recruits, binds and/or activates immune cells (e.g., NK cells, monocytes/macrophages, dendritic cells) via the interaction between Fc (in the antibody) and Fc receptors (on immune cells) and via conjugated variant polypeptides or immunomodulatory proteins provided herein. In some embodiments, the antibody/targeting moiety recognizes or binds to the tumor agent and localizes to the tumor cell via the conjugated variant polypeptides or immunomodulatory proteins provided herein, so as to modulate immune cells in the vicinity of the tumor.
Examples of antibodies that can be incorporated into IgSF conjugates include, but are not limited to, antibodies such as: cetuximab (IMC-C225;
In some embodiments, the antibody targeting moiety is a full length antibody or antigen binding fragment thereof comprising an Fc domain. In some embodiments, the variant polypeptide or immunomodulatory protein is conjugated to the Fc portion of an antibody targeting moiety, such as by conjugation to the N-terminus of the Fc portion of an antibody.
In some embodiments, the vigdd is directly or indirectly linked to the N-terminus or C-terminus of the light chain and/or heavy chain of the antibody. In some embodiments, the linking may be via a peptide linker, such as any of the linkers described above. Various configurations may be constructed. Fig. 7A-7C depict exemplary configurations. In some embodiments, the antibody conjugate can be produced by co-expressing the heavy and light chains of the antibody in a cell.
In one aspect of the invention, the targeting agent is an aptamer molecule. For example, in some embodiments, the aptamer comprises a nucleic acid that serves as a targeting agent. In various embodiments, the IgSF conjugates of the present invention comprise aptamers specific for molecules on tumor cells, tumor vessels, and/or tumor microenvironment. In some embodiments, the aptamer itself can comprise a biologically active sequence as well as a targeting module (sequence), wherein the biologically active sequence can induce an immune response to a target cell. In other words, such aptamer molecules are dual-use agents. In some embodiments, the IgSF conjugates of the present invention comprise conjugation of an aptamer to an antibody, wherein the aptamer and antibody specifically bind to separate molecules on tumor cells, tumor vessels, tumor microenvironment, and/or immune cells.
The term "aptamer" includes DNA, RNA or peptides selected based on their specific binding properties to a particular molecule. For example, one or more aptamers may be selected for binding to a particular gene or gene product in a tumor cell, tumor vasculature, tumor microenvironment, and/or immune cell, as disclosed herein, wherein selection is performed by methods known in the art and familiar to those skilled in the art.
Thus, such IgSF conjugates of the invention comprise a peptide targeting agent that binds to a tumor cell, a cellular component of a tumor blood vessel, and/or a component of the tumor microenvironment.
In one embodiment, the targeting agent is Vv β 3. integrin Vv β 3 is expressed on a variety of cells and has been shown to mediate several biologically related processes, including adhesion of osteoclasts to bone matrix, migration of vascular smooth muscle cells, and angiogenesis suitable targeting molecules for integrins include RGD peptides or peptide mimetics of other integrins such as V4.β i (VLA-4), V4-P7 (see, e.g., U.S. Pat. No. 6,365,619; Chang et al, Bioorganic & Medicinal Chem Lett,12:159-163 (2002); Lin et al, Bioorganic & Medicinal Chem Lett,12:133-136(2002)), as well as non-RGD peptides or peptide mimetics (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426), and the like.
In some embodiments, an IgSF conjugate is provided comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a therapeutic agent. In some embodiments, therapeutic agents include, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al, Cancer Immunol.Immunother.21:183-187, 1986). In some embodiments, the therapeutic agent has intracellular activity. In some embodiments, the IgSF conjugate is internalized and the therapeutic agent is a cytotoxin that blocks protein synthesis of the cell, where cell death results. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome inactivating activity, including, for example, gelonin, trigonal ribosome inactivating protein (bouganin), saporin, ricin a chain, bryodin, diphtheria toxin, restrictocin, pseudomonas exotoxin a, and variants thereof. In some embodiments, where the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome inactivating activity, the IgSF conjugate must internalize upon binding to the target cell in order for the protein to be cytotoxic to the cell.
In some embodiments, an IgSF conjugate is provided comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a toxin. In some embodiments, toxins include, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al, J.Nat. Cancer Inst.92(19):1573-1581 (2000); Mandler et al, Bioorganic & Med.Chem.letters 10:1025-1028 (2000); Mandler et al, Bioconjugate chem.13:786-791(2002)), maytansinoids (EP 1391213; Liu et al, Proc.Natl.Acad.Sci.USA 93:8618-8623(1996)), and calicheamicin (calicheamicin) (Lode et al, Cancer Res.58:2928 (1998); Hinman et al, Cancer Res.53:3336-3342 (1993)). Toxins may exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
In some embodiments, an IgSF conjugate is provided comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a tag that can indirectly or directly generate a detectable signal, such as for detecting cancer in vivo the tag is preferably capable of directly or indirectly generating a detectable signal, for example, the tag may be a radio-opaque or radioactive isotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I, a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or fluorescein, an enzyme, such as alkaline phosphatase, β -galactosidase or horseradish peroxidase, an imaging agent, or a metal ion in some embodiments, the tag is a radioactive atom for scintigraphic studies, such as 99Tc or 123I, or a spin tag for imaging (also referred to as magnetic resonance imaging, MRI) such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-19, 201113-carbon, 201113-manganese, or gadolinium-iron-manganese conjugates, for example, and can be used in some embodiments for the detection of an indirectly complexed metal antibody, e.g., a detectable magnetic resonance imaging ligand, e.g., a PET conjugate, a PET-sf-containing a detectable ligand.
IgSF conjugates can be prepared using any method known in the art. See also WO 2009/067800, WO2011/133886, and U.S. patent application publication No. 2014322129, which are hereby incorporated by reference in their entirety.
The variant polypeptide or immunomodulatory protein of an IgSF conjugate can be "attached" to an effector moiety by any means by which the variant polypeptide or immunomodulatory protein can be associated with or linked to the effector moiety. For example, a variant polypeptide or immunomodulatory protein of an IgSF conjugate can be chemically or recombinantly attached to an effector moiety. The chemistry used to prepare fusions or conjugates is known in the art and can be used to prepare IgSF conjugates. The method for conjugating a variant polypeptide or immunomodulatory protein to an effector moiety must be capable of conjugating the variant polypeptide or immunomodulatory protein to the effector moiety without interfering with the ability of the variant polypeptide or immunomodulatory protein to bind to one or more of its opposing structural ligands.
The variant polypeptide or immunomodulatory protein of the IgSF conjugate can be indirectly linked to an effector moiety. For example, the variant polypeptide or immunomodulatory protein of an IgSF conjugate can be directly linked to a liposome containing one of several types of effector moieties. One or more effector moiety and/or variant polypeptide or immunomodulatory protein may also be bound to a solid surface.
In some embodiments, the variant polypeptide of the IgSF conjugate or the immunomodulatory protein and the effector moiety are both proteins and can be conjugated using techniques well known in the art. There are hundreds of cross-linking agents that can conjugate two proteins. (see, e.g., "Chemistry of Protein Conjugation and Crosslinking," 1991, ShansWong, CRC Press, Ann Arbor). The cross-linking agent is typically selected based on reactive functional groups available on or inserted onto the variant polypeptide or immunomodulatory protein and/or effector moiety. In addition, if no reactive groups are present, a photoactivatable crosslinker may be used. In some cases, it may be desirable to include a spacer between the variant polypeptide or immunomodulatory protein and the effector moiety. Crosslinking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipimide ester, and bis (diazabenzidine); and a heterobifunctional agent: m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.
In some embodiments, variant polypeptides or immunomodulatory proteins of IgSF conjugates can be engineered using specific residues for chemical attachment of effector moieties. Specific residues for chemical attachment of molecules known in the art include lysine and cysteine. The cross-linking agent is selected based on the reactive functional groups available on the insert or effector moiety on the variant polypeptide or immunomodulatory protein.
IgSF conjugates can also be prepared using recombinant DNA techniques. In this case, the DNA sequence encoding the variant polypeptide or the immunomodulatory protein is fused to a DNA sequence encoding the effector moiety, thereby producing a chimeric DNA molecule. The chimeric DNA sequence is transfected into a host cell expressing the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.
Examples of attaching an effector moiety, which is a tag, to a variant polypeptide or immunomodulatory protein include the methods described in: hunter et al, Nature 144:945 (1962); david et al, Biochemistry 13:1014 (1974); pain et al, J.Immunol.meth.40:219 (1981); nygren, J.Histochem.and Cytochem.30:407 (1982); wensel And meases, radioimmunoasting And Radioimmunotherapy, Elsevier, n.y. (1983); and Colcher et al, "Use Of Monoclonal Antibodies As radiopharmaceuticals for The Localization Of Human cardio catalysts In analytical Rice", meth.Enzymol.,121:802-16 (1986).
Radioactive or other labels can be incorporated into the conjugate in known manner. For example, the peptide may be biosynthetic or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors comprising, for example, fluorine-19 in place of hydrogen. Tags such as 99Tc or 123I, 186Re, 188Re and 111In may be attached via cysteine residues In the peptide. Yttrium-90 may be attached via a lysine residue. The IODOGEN method (Fraker et al, biochem. Biophys. Res. Commun.80:49-57(1978)) can be used to incorporate iodine-123. Other methods are described in detail by "monoclonal antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).
A variety of bifunctional protein coupling agents can be used to generate conjugates of variant polypeptides or immunomodulatory proteins and cytotoxic agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-p-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See, for example, WO 94/11026. The linker may be a "cleavable linker" to facilitate release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, cancer research 52: 127-.
The IgSF conjugates of the present invention specifically encompass, but are not limited to, drug conjugates prepared with the following cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thioEMCS, thioGMBS, thioKMUS, thioMBS, thioSIAB, thioSMCC, and thioSMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Rockford, IL, U.S.A.). See pages 467 & 498, 2003 & 2004 application manual and catalog.
D. Transmembrane and secretable immunomodulatory proteins and engineered cells
Provided herein are engineered cells (alternatively, "engineered cells") that express an immunomodulatory variant PD-L1 polypeptide. In some embodiments, the expressed immunomodulatory variant PD-L1 polypeptide is a transmembrane protein and is expressed on a surface. In some embodiments, the expressed immunomodulatory variant PD-L1 polypeptide is expressed and secreted from a cell.
1. Transmembrane immunomodulatory proteins
In some embodiments, the immunomodulatory polypeptide comprising variant PD-L1 may be a membrane-bound protein. As described in more detail below, the immunomodulatory polypeptide can be a transmembrane immunomodulatory polypeptide comprising a variant PD-L1 comprising in the variant PD-L1: an extracellular domain comprising at least one affinity modified IgSF domain (IgV or IgC), a transmembrane domain, and optionally a cytoplasmic domain. In some embodiments, the immunomodulatory protein can be expressed on the surface of an immune cell (such as a mammalian cell), including on the surface of a lymphocyte (e.g., a T cell or NK cell) or an antigen presenting cell. In some embodiments, the transmembrane immunomodulatory protein is expressed on the surface of mammalian T cells, including T cells such as: t helper cells, cytotoxic T cells (alternatively, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γ δ T cells. In some embodiments, the mammalian cell is an Antigen Presenting Cell (APC). Typically, but not exclusively, the extracellular domain (alternatively, the "extracellular domain") comprises one or more amino acid changes (e.g., amino acid substitutions) of the variant PD-L1 of the invention. Thus, for example, in some embodiments, a transmembrane protein will comprise an extracellular domain comprising one or more amino acid substitutions of variant PD-L1 of the invention.
In some embodiments, the engineered cell expresses a variant PD-L1 polypeptide that is a Transmembrane Immunomodulatory Polypeptide (TIP), which may be a membrane protein such as a transmembrane protein. In typical embodiments, the extracellular domain of a membrane protein comprises the extracellular domain of variant PD-L1 provided herein or an IgSF domain thereof, wherein the at least one IgSF domain contains one or more amino acid substitutions. The transmembrane immunomodulatory proteins provided herein also contain a transmembrane domain linked to an extracellular domain. In some embodiments, the transmembrane domain produces an encoded protein that is expressed on the cell surface. In some embodiments, the transmembrane domain is directly linked to the extracellular domain. In some embodiments, the transmembrane domain is indirectly linked to the extracellular domain via one or more linkers or spacers. In some embodiments, the transmembrane domain contains predominantly hydrophobic amino acid residues, such as leucine and valine.
In some embodiments, a full-length transmembrane anchor domain may be used to ensure that the TIP will be expressed on the surface of an engineered cell, such as an engineered T cell. Conveniently, this may be from a particular native protein that is affinity modified (e.g., PD-L1 or other native IgSF protein) and simply fused to the sequence of the first membrane proximal domain in a manner similar to a native IgSF protein (e.g., PD-L1). In some embodiments, the transmembrane immunomodulatory protein comprises a transmembrane domain corresponding to a wild-type or unmodified member of an IgSF, such as the transmembrane domain contained in the amino acid sequence set forth in SEQ ID NO:3 (Table 2). In some embodiments, the membrane bound form comprises a transmembrane domain corresponding to a wild-type or unmodified polypeptide, such as corresponding to residues 239-259 of SEQ ID NO 3.
In some embodiments, the transmembrane domain is a non-native transmembrane domain that is not a transmembrane domain of native PD-L1. In some embodiments, the transmembrane domain is derived from a transmembrane domain from another non-PD-L1 family member polypeptide, which is membrane-bound or is a transmembrane protein. In some embodiments, a transmembrane anchor domain from another protein on a T cell may be used. In some embodiments, the transmembrane domain is derived from CD 8. In some embodiments, the transmembrane domain may further comprise the extracellular portion of CD8 that serves as a spacer domain. Exemplary CD 8-derived transmembrane domains are set forth in SEQ ID NO:242 or 1164 or portions thereof containing the CD8 transmembrane domain. In some embodiments, the transmembrane domain is a synthetic transmembrane domain.
In some embodiments, the transmembrane immunomodulatory protein further comprises an endodomain linked to the transmembrane domain, such as a cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain induces cell signaling. In some embodiments, the intracellular domain of the transmembrane immunomodulatory protein comprises a cytoplasmic domain corresponding to a wild-type or unmodified polypeptide, such as the cytoplasmic domain contained in the amino acid sequence set forth in SEQ ID NO:3 (see Table 2).
In some embodiments, provided transmembrane immunomodulatory proteins that are or comprise variant PD-L1 comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to seq id No. 191 and comprise an extracellular domain comprising at least one affinity modified PD-L1IgSF domain and a transmembrane domain as described. In some embodiments, an immunomodulatory protein comprises any one or more amino acid substitutions in the IgSF domain (e.g., an IgV domain), including any of the substitutions listed in table 1. In some embodiments, the transmembrane immunomodulatory protein may further comprise the cytoplasmic domain. In some embodiments, the transmembrane immunomodulatory protein may further comprise a signal peptide. In some embodiments, the signal peptide is a native signal peptide of a member of the wild-type IgSF, such as the signal peptide contained within the amino acid sequence set forth in SEQ ID NO:3 (see, e.g., Table 2).
Also provided is a nucleic acid molecule encoding such a transmembrane immunomodulatory protein. In some embodiments, a nucleic acid molecule encoding a transmembrane immunomodulatory protein comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 191, and comprises an extracellular domain comprising at least one affinity modified IgSF domain as described, a transmembrane domain, and optionally a cytoplasmic domain. In some embodiments, the nucleic acid molecule may further comprise a nucleotide sequence encoding a signal peptide. In some embodiments, the signal peptide is a native signal peptide corresponding to a member of a wild-type IgSF (see, e.g., table 2).
In some embodiments, a CAR-associated transmembrane immunomodulatory protein is provided, wherein the intracellular domain of the transmembrane immunomodulatory protein comprises a cytoplasmic signaling domain comprising at least one signaling domain comprising ITAM (immunoreceptor tyrosine activation motif). ITAMs are conserved motifs found in many protein signaling domains involved in signal transduction by immune cells, including in the CD 3-zeta chain ("CD 3-z") involved in T cell receptor signaling. In some embodiments, the endodomain comprises a CD 3-zeta signaling domain. In some embodiments, the CD 3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 243 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of SEQ ID No. 243 and retains T cell signaling activity. In some embodiments, the endodomain of the CAR-associated transmembrane immunomodulatory protein may further comprise a costimulatory signaling domain to further modulate an immunomodulatory response of the T cell. In some embodiments, the co-stimulatory signaling domain is CD28, ICOS, 41BB, or
In some embodiments, the transmembrane immunomodulatory protein does not contain an endodomain capable of mediating cytoplasmic signaling. In some embodiments, the transmembrane immunomodulatory protein lacks the signaling mechanism of the wild-type or unmodified polypeptide and therefore does not itself induce cell signaling. In some embodiments, the transmembrane immunomodulatory protein lacks an intracellular (cytoplasmic) domain or a portion of an intracellular domain corresponding to a wild-type or unmodified polypeptide, such as the cytoplasmic signaling domain contained within the amino acid sequence set forth in SEQ ID NO:3 (see Table 2). In some embodiments, the transmembrane immunomodulatory protein does not contain an ITIM (immunoreceptor tyrosine activation motif), such as that contained in certain inhibitory receptors, including inhibitory receptors of the IgSF family (e.g., PD-1 or TIGIT). Thus, in some embodiments, the transmembrane immunomodulatory protein contains only an extracellular domain and a transmembrane domain, such as any such domains described.
2. Secreted immunomodulatory proteins and engineered cells
In some embodiments, a PD-L1 variant immunomodulatory polypeptide containing any one or more of the amino acid mutations described herein is secretable, such as when expressed by a cell. This variant PD-L1 immunomodulatory protein does not comprise a transmembrane domain. In some embodiments, the variant PD-L1 immunomodulatory protein is not conjugated to a half-life extending moiety (such as an Fc domain or multimerization domain). In some embodiments, the variant PD-L1 immunomodulatory protein comprises a signal peptide, such as an antibody signal peptide or other effective signal sequence, to obtain a domain outside the cell. When the immunomodulatory protein comprises a signal peptide and is expressed by the engineered cell, the signal peptide causes the immunomodulatory protein to be secreted by the engineered cell. Typically, the signal peptide or a portion of the signal peptide is cleaved from the immunomodulatory protein upon secretion. The immunomodulatory protein may be encoded by a nucleic acid (which may be part of an expression vector). In some embodiments, the immunomodulatory protein is expressed and secreted by a cell (such as an immune cell, e.g., a primary immune cell).
Thus, in some embodiments, variant PD-L1 immunomodulatory proteins are provided that further comprise a signal peptide. In some embodiments, provided herein are nucleic acid molecules encoding a variant PD-L1 immunomodulatory protein operably linked to a secretory sequence encoding a signal peptide.
The signal peptide is a sequence that sends a signal for secretion of the immunomodulatory protein from cells on the N-terminus of the immunomodulatory protein. In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).
In some embodiments, the signal peptide is a native signal peptide from the corresponding wild-type PD-L1 (see table 2). In some embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding wild-type PD-L1, and may comprise one or more (such as2, 3,4,5,6,7,8, 9, or 10 or more) substitutions, insertions, or deletions. In some embodiments, the non-native signal peptide is a signal peptide from a family member of the IgSF family that is the same as the wild-type IgSF family member, or a mutant thereof. In some embodiments, the non-native signal peptide is a signal peptide from an IgSF family member of an IgSF family different from the wild-type IgSF family member, or a mutant thereof. In some embodiments, the signal peptide is a signal peptide from the non-IgSF protein family or a mutant thereof, such as a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interferon-2 (IL-2) or CD33), serum albumin (e.g., HSA or albumin), a human disintegrin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently secreting a protein from a cell. Exemplary signal peptides include any of the signal peptides described in table 9.
In some embodiments of the secretable variant PD-L1 immunomodulatory protein, the immunomodulatory protein comprises a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein upon secretion.
In some embodiments, the engineered cell expresses a variant PD-L1 polypeptide, which is secreted from the cell. In some embodiments, such a variant PD-L1 polypeptide is encoded by a nucleic acid molecule that encodes an immunomodulatory protein under the operable control of a signal sequence for secretion. In some embodiments, the encoded immunomodulatory protein is secreted after expression by the cell. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not comprise a transmembrane domain. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not comprise a half-life extending moiety (such as an Fc domain or multimerization domain). In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule comprises a signal peptide. In some embodiments, the nucleic acids of the invention further comprise a nucleotide sequence encoding a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory protein, thereby allowing secretion of the immunomodulatory protein.
3. Cells and engineered cells
Provided herein are engineered cells expressing any one of the provided immunomodulatory polypeptides. In some embodiments, the engineered cell expresses any of the provided transmembrane immunomodulatory polypeptides on its surface. In some embodiments, the engineered cell expresses an immunomodulatory protein and is capable of or is capable of secreting an immunomodulatory protein from the cell under conditions suitable for secretion of the protein. In some embodiments, the immunomodulatory protein is expressed on lymphocytes, such as Tumor Infiltrating Lymphocytes (TILs), T cells, or NK cells, or on bone marrow cells. In some embodiments, the engineered cell is an Antigen Presenting Cell (APC). In some embodiments, the engineered cell is an engineered mammalian T cell or an engineered mammalian Antigen Presenting Cell (APC). In some embodiments, the engineered T cell or APC is a human cell or a murine cell.
In some embodiments, the engineered T cells include, but are not limited to, T helper cells, cytotoxic T cells (alternatively, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γ δ T cells. In some embodiments, the engineered T cell is CD4+ or CD8 +.
In some embodiments, engineered APCs include, for example, MHC II expressing APCs, such as macrophages, B cells, and dendritic cells, and artificial APCs (aapcs), including cellular and non-cellular (e.g., biodegradable polymer microparticles) aapcs. Artificial APCs (aapcs) are synthetic versions of APCs that can function in a manner similar to APCs in that they present antigens to T cells and activate them. Antigen presentation is by MHC (class I or class II). In some embodiments, in an engineered APC such as an aAPC, the antigen loaded onto the MHC is in some embodiments a tumor-specific antigen or a tumor-associated antigen. The antigen loaded onto the MHC is recognized by the T Cell Receptor (TCR) of a T cell, which in some cases may express PD-1 or CD80 or other molecules recognized by the variant PD-L1 polypeptides provided herein. Materials that can be used to engineer aapcs include: poly (glycolic acid), poly (lactic-co-glycolic acid), iron oxide, liposomes, lipid bilayers, agarose, and polystyrene.
In some embodiments, the cellular aapcs can be engineered to contain TIPs and TCR agonists, which are used in adoptive cell therapy. In some embodiments, the cellular aapcs can be engineered to contain TIPs and TCR agonists for use in expanding human T cells ex vivo, such as prior to administration, e.g., for reintroduction into a patient. In some aspects, aapcs can include clones expressing at least one anti-CD 3 antibody, such as, for example, OKT3 and/or
In some embodiments, an immunomodulatory protein provided herein, such as a transmembrane immunomodulatory protein or a secretable immunomodulatory protein, is co-expressed or engineered into a cell that expresses an antigen-binding receptor, such as a recombinant receptor, such as a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). In some embodiments, engineered cells, such as engineered T cells, recognize a desired antigen associated with cancer, inflammatory and autoimmune disorders, or viral infections. In particular embodiments, the antigen-binding receptor comprises an antigen-binding portion that specifically binds to a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the engineered T cell is a CAR (chimeric antigen receptor) T cell that contains an antigen binding domain (e.g., scFv) that specifically binds to an antigen, such as a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the TIP protein is expressed in an engineered T cell receptor cell or and an engineered chimeric antigen receptor cell. In such embodiments, the engineered cell co-expresses the TIP and the CAR or TCR. In some embodiments, the SIP protein is expressed in an engineered T cell receptor cell or an engineered chimeric antigen receptor cell. In such embodiments, the engineered cell co-expresses SIP and CAR or TCR.
Chimeric Antigen Receptors (CARs) are recombinant receptors comprising an antigen binding domain (extracellular domain), a transmembrane domain, and an intracellular signaling region (endodomain) capable of inducing or mediating an activation signal on T cells upon antigen binding. In some examples, the CAR-expressing cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety that comprises an activation domain and, in some cases, a co-stimulatory domain. The costimulatory domain can be derived from, for example, CD28, OX-40, 4-1BB/CD137, induced T cell costimulator (ICOS). The activation domain may be derived, for example, from CD3, such as CD3 ζ, epsilon, delta, gamma, and the like. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. CAR scFv can be designed to target antigens expressed on cells associated with a disease or condition, e.g., tumor antigens such as, for example, CD19, which are transmembrane proteins expressed by cells in the B cell lineage, including ALL normal B cell and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL. Exemplary CAR + T cell therapies and constructs are described in U.S. patent publication nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, and these references are incorporated by reference in their entirety.
In some aspects, the antigen binding domain is an antibody or antigen binding fragment thereof, such as a single chain fragment (scFv). In some embodiments, the antigen is expressed on a tumor or cancer cell. An example of an antigen is CD 19. An example of a CAR is an anti-CD 19CAR, such as a CAR comprising the anti-CD 19 scFv listed in SEQ ID NO:1163 or SEQ ID NO: 1174. In some embodiments, the CAR further contains a spacer, a transmembrane domain, and an intracellular signaling domain or region comprising an ITAM signaling domain, such as a CD3 zeta signaling domain. In some embodiments, the CAR further comprises a costimulatory signaling domain.
In some embodiments, the spacers and transmembrane domains are hinge and transmembrane domains derived from CD8, such as amino acid sequences having the exemplary sequences set forth in SEQ ID NOs 242, 1164, 2014 or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NOs 242, 1164, or 2014. In some embodiments, the endodomain comprises a CD 3-zeta signaling domain. In some embodiments, the CD 3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 243 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID No. 243 and retaining T cell signaling activity. In some embodiments, the endodomain of the CAR may further comprise a costimulatory signaling region to further modulate the immune modulatory response of the T cell. In some embodiments, the co-stimulatory signaling domain is or comprises, or is derived from, a co-stimulatory region of CD28, ICOS, 41BB, or
In some embodiments, the construct encoding the CAR further encodes a second protein, such as a label, e.g., a detectable protein, that is separable from the CAR by the self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is a F2A, T2A, E2A, or P2A self-cleaving peptide. Exemplary sequences of T2A self-cleaving peptides are set forth in any one of SEQ ID NOs 1167, 1177, or 2021 or in an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NOs 1167, 1177, or 2021. In some embodiments, T2A is encoded by the nucleotide sequence set forth in SEQ ID NO:1176 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO: 1176. Exemplary sequences of P2A self-cleaving peptides are set forth in SEQ ID No. 2026 or in amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID No. 2026. In some cases, the nucleic acid construct encodes more than one P2A self-cleaving peptide (such as P2a1 and P2a2), where the nucleotide sequences P2a1 and P2a2 each encode P2A listed in SEQ ID NO:2026, and the nucleotide sequences may be different to avoid recombination between the sequences.
In some embodiments, the label is a detectable protein, such as a fluorescent protein, e.g., Green Fluorescent Protein (GFP) or Blue Fluorescent Protein (BFP). Exemplary sequences of fluorescent protein markers are set forth in SEQ ID NO 1170, 2020, 2027-2029 or in amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO 1170 or 2020.
In some embodiments, the CAR has an amino acid sequence set forth in any one of SEQ ID NOs 1160, 1171, 1172, 1173, 2015, 2016, 2018, or 2019, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to any one of SEQ ID NOs 1160, 1171, 1172, 1173, 2015, 2016, 2018, or 2019. In some embodiments, the CAR is encoded by a nucleotide sequence set forth in SEQ ID No. 1175 or 2017 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID No. 1175 or 2017.
In another embodiment, the engineered T cell has a TCR, including a recombinant or engineered TCR-in some embodiments, the TCR may be a native TCR-those skilled in the art will recognize that, in general, native mammalian T cell receptors comprise α and β chains (or γ or δ chains) involved in antigen-specific recognition and binding.
In some embodiments, an immunomodulatory polypeptide, such as a transmembrane immunomodulatory polypeptide or a secretable immunomodulatory polypeptide, can be incorporated into an engineered cell, such as an engineered T cell or an engineered APC, through a variety of strategies, such as those employed for recombinant host cells. Many methods of introducing DNA constructs into primary T cells are known in the art. In some embodiments, viral transduction or plasmid electroporation is employed. In typical embodiments, the nucleic acid molecule or expression vector encoding the immunomodulatory protein comprises a signal peptide that localizes the expressed transmembrane immunomodulatory protein to the cell membrane or for secretion. In some embodiments, a nucleic acid encoding a transmembrane immunomodulatory protein of the invention is subcloned into a viral vector, such as a retroviral vector, that is permissive for expression in a host mammalian cell. The expression vector may be introduced into a mammalian host cell and the immunomodulatory protein expressed on the surface or secreted under host cell culture conditions.
In one illustrative example, primary T cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with an activation protocol consisting of various TCR/CD28 agonists, such as anti-CD 3/anti-CD 28 coated beads. After a 2-or 3-day activation process, recombinant expression vectors containing immunomodulatory polypeptides can be stably introduced into primary T cells by standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies known in the art. The expression of a cell's immunomodulatory polypeptide can be monitored, for example, by flow cytometry using anti-epitope tags or antibodies that cross-react with the native parent molecule and the polypeptide comprising the variant PD-L1. T cells expressing an immunomodulatory polypeptide can be enriched by sorting using anti-epitope tag antibodies or high or low expressing T cells depending on the application.
In immunomodulating polypeptide expression, the appropriate function of the engineered T cell can be assessed in various ways. Engineered CAR or TCR co-expression can be validated to show that this portion of the engineered T cells is not significantly affected by the expression of the immunomodulatory protein. Once validated, the engineered T cells can be assessed for function using standard in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN- γ expression). Exemplary standard endpoints are percent lysis of tumor lines, proliferation of engineered T cells, or IFN- γ protein expression in culture supernatants. The engineered construct may be selected to cause a statistically significant increase in lysis of tumor lines, an increase in proliferation of engineered T cells, or an increase in IFN- γ expression relative to a control construct. In addition, non-engineered (such as native) primary or endogenous T cells can also be incorporated into the same in vitro assay to measure the ability of an immunomodulatory polypeptide construct expressed on an engineered cell (such as an engineered T cell) to modulate activity (including in some cases activation and generation of effector functions in a bystander native T cell). Increased expression of activation markers such as CD69, CD44, or CD62L on endogenous T cells can be monitored, and increased proliferation and/or cytokine production can indicate a desired activity of the immunomodulatory protein expressed on engineered T cells.
In some embodiments, a similar assay can be used to compare the function of engineered T cells containing CARs or TCRs alone to T cells containing CARs or TCRs and TIP constructs. Typically, these in vitro assays are performed by inoculating different ratios of engineered T cells containing homologous CAR or TCR antigens with "tumor" cell lines in culture. The standard endpoints are percent lysis of tumor lines, proliferation of engineered T cells, or IFN- γ production in culture supernatants. The engineered immunomodulatory protein can be selected to cause a statistically significant increase in lysis of tumor lines, an increase in proliferation of engineered T cells, or an increase in IFN- γ production relative to the same TCR or CAR construct alone. Engineered human T cells can be analyzed in immunocompromised mice (e.g., NSG strains) lacking mouse T cells, NK cells, and B cells. Engineered human T cells in which the CAR or TCR binds to a target counterpart structure on the xenograft and is co-expressed with the TIP affinity modified IgSF domain can be adoptively transferred in vivo at different cell numbers and ratios compared to xenografts. For example, transplantation of a luciferase/GFP vector-containing CD19+ leukemic tumor line can be monitored by bioluminescence or ex vivo by flow cytometry. In one general embodiment, the xenograft is introduced into a murine model, followed by several days later by the introduction of engineered T cells. Increased survival, tumor clearance, or expanded number of engineered T cells containing an immunomodulatory protein can be assessed relative to engineered T cells containing CARs or TCRs alone. As in vitro assays, native (i.e., un-engineered) human T cells can co-adoptively metastasize to see successful epitope spreading in this population, resulting in better survival or tumor clearance.
E. Infectious agents expressing variant polypeptides and immunomodulatory proteins
Also provided are infectious agents comprising a nucleic acid encoding any of the variant polypeptides, such as PD-L1vIgD polypeptides, including the secretable or transmembrane immunomodulatory proteins described herein. In some embodiments, such infectious agents can deliver a nucleic acid encoding a variant immunomodulatory polypeptide described herein, such as a PD-L1 vgld polypeptide, to a target cell of a subject, e.g., an immune cell and/or an Antigen Presenting Cell (APC) or a tumor cell of a subject. Also provided are nucleic acids contained in such infectious agents and/or nucleic acids used to generate or modify such infectious agents such as vectors and/or plasmids, and compositions containing such infectious agents.
In some embodiments, the infectious agent is a microorganism (microorganism/microbe). In some embodiments, the infectious agent is a virus or a bacterium. In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is a bacterium. In some embodiments, such infectious agents may deliver a nucleic acid sequence encoding any of the variant polypeptides, such as PD-L1 vgld polypeptides, including the secretable or transmembrane immunomodulatory proteins described herein. Thus, in some embodiments, cells of a subject infected or contacted by an infectious agent may be caused to express or secrete the variant immunomodulatory polypeptide on the cell surface. In some embodiments, the infectious agent may also deliver one or more additional therapeutic agents or nucleic acids encoding additional therapeutic agents to cells and/or the environment within the subject. In some embodiments, other therapeutic agents that may be delivered by the infectious agent include cytokines or other immune modulatory molecules.
In some embodiments, the infectious agent (e.g., a virus or a bacterium) contains a nucleic acid sequence encoding any of the variant polypeptides described herein, such as a PD-L1 vigdd polypeptide, including a secretable or transmembrane immunomodulatory protein, and upon contacting and/or infecting cells of a subject that express the variant polypeptide encoded by the nucleic acid sequence contained in the infectious agent, such as a PD-L1 vigdd polypeptide, including a secretable or transmembrane immunomodulatory protein. In some embodiments, an infectious agent may be administered to a subject. In some embodiments, the infectious agent may be contacted with cells from the subject ex vivo.
In some embodiments, the variant polypeptide expressed by the cell infected with the infectious agent, such as a PD-L1 vigdd polypeptide, including transmembrane immunomodulatory proteins, is a transmembrane protein and is expressed on the surface. In some embodiments, variant polypeptides expressed by cells infected with an infectious agent, such as PD-L1 vigdd polypeptides, including secretable immunomodulatory proteins, are expressed and secreted from the cells. The transmembrane immunomodulatory protein or secreted immunomodulatory protein may be any protein described herein.
In some embodiments, the cells targeted by the infectious agent in the subject include tumor cells, immune cells, and/or Antigen Presenting Cells (APCs). In some embodiments, the infectious agent targets cells in the Tumor Microenvironment (TME). In some embodiments, the infectious agent delivers a nucleic acid encoding a variant polypeptide, such as a PD-L1 vigdd polypeptide, including a secretable or transmembrane immunomodulatory protein, to an appropriate cell (e.g., an APC, such as a cell displaying a peptide/MHC complex on its cell surface, such as a dendritic cell) or tissue (e.g., a lymphocyte) that will induce and/or enhance a desired effect, e.g., an immunomodulatory and/or specific cell-mediated immune response, e.g., a CD4 and/or CD 8T cell response, which may include a cytotoxic T Cell (CTL) response. In some embodiments, the infectious agent targets an APC, such as a Dendritic Cell (DC). In some embodiments, the nucleic acid molecule delivered by an infectious agent described herein comprises the appropriate nucleic acid sequence (e.g., a regulatory element, such as a promoter) necessary for expression of an operably linked coding sequence encoding a variant immunomodulatory polypeptide in a particular target cell.
In some embodiments, an infectious agent comprising a nucleic acid sequence encoding an immunomodulatory polypeptide may also comprise a nucleic acid sequence encoding one or more additional gene products (e.g., a cytokine, a prodrug converting enzyme, a cytotoxin, and/or a detectable gene product). For example, in some embodiments, the infectious agent is an oncolytic virus and the virus may comprise a nucleic acid sequence encoding an additional therapeutic gene product (see, e.g., Kirn et al, (2009) Nat Rev Cancer9: 64-71; Garcia-aragancinillo et al, (2010) Curr Opin Mol Ther12:403-, a cytokine). Exemplary gene products also include anti-cancer agents, anti-metastatic agents, anti-angiogenic agents, immune regulatory molecules, immune checkpoint inhibitors, antibodies, cytokines, growth factors, antigens, cytotoxic gene products, pro-apoptotic gene products, anti-apoptotic gene products, cell matrix degradation genes, genes for tissue regeneration or reprogramming of human cells to pluripotency, and other genes described herein or known to those skilled in the art. In some embodiments, the additional gene product is granulocyte-macrophage colony stimulating factor (GM-CSF).
1. Virus
In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is an oncolytic virus or a virus that targets a particular cell (e.g., an immune cell). In some embodiments, the infectious agent targets tumor cells and/or cancer cells in the subject. In some embodiments, the infectious agent targets an immune cell or an Antigen Presenting Cell (APC).
In some embodiments, the infectious agent is an oncolytic virus. Oncolytic viruses are viruses that accumulate within tumor cells and replicate within the tumor cells. By virtue of replicating and optionally delivering nucleic acids encoding the variant PD-L1 polypeptides or immunomodulatory polypeptides described herein in cells, tumor cells are lysed and the tumor is reduced and can be eliminated. Oncolytic viruses can also have a wide range of hosts and cell types. For example, oncolytic viruses can accumulate in immune-privileged cells or immune-privileged tissues (including tumors and/or metastatic cancers, and also including injured tissues and cells), thus allowing the delivery and expression of nucleic acids encoding the variant immunomodulatory polypeptides described herein in a wide range of cell types. Oncolytic viruses can also replicate in a tumor cell-specific manner, resulting in tumor cell lysis and efficient tumor regression.
Exemplary oncolytic viruses include adenovirus, adeno-associated virus, herpes simplex virus, vesicular stomatitis virus, reovirus, newcastle disease virus, parvovirus, measles virus, Vesicular Stomatitis Virus (VSV), coxsackie virus, and vaccinia virus. In some embodiments, the oncolytic virus can specifically colonize solid tumors without infecting other organs, and can be used as an infectious agent to deliver nucleic acids encoding the variant immunomodulatory polypeptides described herein to such solid tumors.
Oncolytic viruses for delivery of nucleic acids encoding the variant PD-L1 polypeptides or immunomodulatory polypeptides described herein can be any of those known to those of skill in the art and include, for example, vesicular stomatitis virus, see, e.g., U.S. patent nos. 7,731,974, 7,153,510, 6,653,103 and U.S. patent publication nos. 2010/0178684, 2010/0172877, 2010/0113567, 2007/0098743, 20050260601, 20050220818 and european patent nos. 1385466, 1606411 and 1520175; herpes simplex viruses, see, e.g., U.S. patent nos. 7,897,146, 7,731,952, 7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. patent publication nos. 2014/0154216, 2011/0177032, 2011/0158948, 2010/0092515, 2009/0274728, 2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720, 2006/0039894, 2004/0009604, 2004/0063094, international patent publication nos. WO 2007/052029, WO 1999/038955; retroviruses, see, e.g., U.S. patent nos. 6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. patent publication No. 20110212530; vaccinia virus, see, e.g., 2016/0339066; and adeno-associated viruses, see, e.g., U.S. patent nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and 6,632,670.
Oncolytic viruses also include viruses that have been genetically altered to reduce their virulence, to improve their safety profile, to enhance their tumor specificity, and they have been equipped with additional genes, such as cytotoxins, cytokines, prodrug converting enzymes, to improve the overall efficacy of the virus (see, e.g., Kirn et al, (2009) Nat Rev Cancer9: 64-71; Garcia-aragannecillo et al, (2010) Curr Opin Mol Ther12: 403-. In some embodiments, the oncolytic viruses may be those that have been modified such that they selectively replicate in cancer cells and are therefore oncolytic. For example, oncolytic viruses are adenoviruses that have been engineered to have a modified tropism for tumor therapy and are also used as gene therapy vectors. Examples of such adenoviruses are ONYX-015, H101 and Ad5 Δ CR (Hallden and Portella (2012) Expert Opin Ther Targets,16:945-58) and TNFade (McLoughlin et al (2005) Ann. Surg. Oncol.,12:825-30) or conditionally replication competent adenoviruses
In some embodiments, the infectious agent is a modified herpes simplex virus. In some embodiments, the infectious agent is a modified version of taliomogene laherparvec (also referred to as T-Vec, Imlygic, or OncoVex GM-CSF) modified to contain a nucleic acid encoding any of the variant immunomodulatory polypeptides described herein, such as the variant PD-L1 polypeptide described herein. In some embodiments, the infectious agent is a modified herpes simplex virus described in WO 2007/052029, WO 1999/038955, US 2004/0063094, US 2014/0154216, or a variant thereof.
In some embodiments, the infectious agent is a virus that targets a particular type of cell of a subject to which the virus is administered, e.g., a virus that targets immune cells or Antigen Presenting Cells (APCs). Dendritic Cells (DCs) are essential APCs for initiating and controlling immune responses. DCs can capture and process antigens, migrate from peripheral organs to lymphoid organs, and present antigens to resting T cells in a Major Histocompatibility Complex (MHC) restricted manner. In some embodiments, the infectious agent is a virus that can be specifically targeted to a DC to deliver a nucleic acid encoding a variant PD-L1 polypeptide or an immunomodulatory polypeptide for expression in the DC. In some embodiments, the virus is a lentivirus or a variant or derivative thereof, such as an integration-defective lentivirus vector. In some embodiments, the virus is a lentivirus that is pseudotyped to efficiently bind to and efficiently infect cells, such as DCs, that express the cell surface marker dendritic cell-specific intracellular adhesion molecule-3-capture non-integrin (DC-SIGN). In some embodiments, the virus is a lentivirus pseudotyped with sindbis virus E2 glycoprotein or modified forms thereof, such as those described in WO 2013/149167. In some embodiments, the virus allows for the delivery and expression of a sequence of interest (e.g., a nucleic acid encoding any of the variant PD-L1 polypeptides or immunomodulatory polypeptides described herein) to a DC. In some embodiments, the virus comprises WO 2008/011636 or US 2011/0064763; those described by Tareen et al (2014) mol. ther.,22: 575-. An example of a dendritic cell tropism vector platform is ZVexTM。
2. Bacteria
In some embodiments, the infectious agent is a bacterium. For example, in some embodiments, the bacterium can deliver a nucleic acid encoding any of the variant immunomodulatory polypeptides described herein to a target cell (such as a tumor cell, an immune cell, an antigen presenting cell, and/or a phagocytic cell) of the subject. In some embodiments, the bacteria may preferably target a particular environment within the subject, such as a Tumor Microenvironment (TME), for expression and/or secretion of the variant immunomodulatory polypeptide, and/or target a particular target cell in the environment to express the variant immunomodulatory polypeptide.
In some embodiments, the bacteria deliver the nucleic acid to the cell via bacterially mediated transfer of plasmid DNA to a mammalian cell (also known as "bacterial infection"). For example, in some embodiments, delivery of genetic material is achieved by allowing the entire bacterium to enter a target cell. In some embodiments, spontaneous or induced bacterial lysis may result in plasmid release for subsequent eukaryotic expression. In some embodiments, the bacteria can deliver the nucleic acid to non-phagocytic mammalian cells (e.g., tumor cells) and/or phagocytic cells, such as certain immune cells and/or APCs. In some embodiments, nucleic acids delivered by bacteria may be transferred to the nucleus of a subject for expression. In some embodiments, the nucleic acid also includes the appropriate nucleic acid sequences (e.g., regulatory elements such as promoters or enhancers) necessary for expression of the operably linked sequence encoding the variant immunomodulatory polypeptide in a particular host cell. In some embodiments, an infectious agent that is a bacterium can deliver nucleic acid encoding an immunomodulatory protein in the form of RNA, such as pre-made translatable RNA that is delivered to the cytoplasm of a target cell for translation by the machinery of the target cell.
In some embodiments, the bacteria can replicate and lyse a target cell, such as a tumor cell. In some embodiments, the bacteria may contain and/or release nucleic acid sequences and/or gene products in the cytoplasm of the target cell, thereby killing the target cell, e.g., a tumor cell. In some embodiments, the infectious agent is a bacterium that can specifically replicate in a particular environment of the subject (e.g., a Tumor Microenvironment (TME)). For example, in some embodiments, the bacteria can specifically replicate in an anaerobic or hypoxic microenvironment. In some embodiments, conditions or factors present in a particular environment (e.g., aspartic acid, serine, citric acid, ribose, or galactose produced by cells in the TME) may act as chemical attractants to attract bacteria to the environment. In some embodiments, the bacteria can express and/or secrete an immunomodulatory protein described herein in the environment (e.g., TME).
In some embodiments, the infectious agent is a bacterium that is a listeria species, a bifidobacterium species, an escherichia species, a clostridium species, a salmonella species, a shigella species, a vibrio species, or a yersinia species. In some embodiments, the bacteria are selected from one or more of: listeria monocytogenes (Listeria monocytogenes), Salmonella typhimurium (Salmonella typhimurium), Salmonella cholera (Salmonella choleraesuis), Escherichia coli (Escherichia coli), Vibrio cholerae (Vibrio cholera), Clostridium perfringens (Clostridium perfringens), Clostridium butyricum (Clostridium butyricum), Clostridium norbomiae (Clostridium novyi), Clostridium acetobutylicum (Clostridium acetobutylicum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), and Bifidobacterium adolescentis (Bifidobacterium adolescentis). In some embodiments, the bacterium is an engineered bacterium. In some embodiments, the bacteria are engineered bacteria, such as those described below: such as show and Wood (2009) Molecular Therapy 17(5): 767-777; baban et al (2010) Bioengineered bubbles 1:6, 385-394; patylar et al (2010) J Biomed Sci17: 21; tangney et al (2010) Bioengineered bubbles 1:4, 284-287; van Pijkeren et al (2010) Hum Gene ther.21(4): 405-416; WO 2012/149364; WO 2014/198002; US 9103831; US 9453227; US 2014/0186401; US 2004/0146488; US 2011/0293705; US 2015/0359909 and EP 3020816. The bacteria may be modified to deliver nucleic acid sequences encoding any of the variant immunomodulatory polypeptides, conjugates, and/or fusions provided herein, and/or to express such variant immunomodulatory polypeptides in a subject.
F. Nucleic acids, vectors and methods for producing polypeptides or cells
Provided herein are isolated or recombinant nucleic acids, collectively referred to as "nucleic acids," encoding any of the various provided embodiments of the variant PD-L1 polypeptides or immunomodulatory polypeptides provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) are suitable for recombinant production (e.g., expression) of a variant PD-L1 polypeptide or immunomodulatory polypeptide provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) are suitable for expressing a variant PD-L1 polypeptide or immunomodulatory polypeptide provided herein in a cell, such as an engineered cell, e.g., an immune cell, or an infectious agent cell. Nucleic acids provided herein can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are typically DNA molecules, and typically double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleic acids, or combinations thereof, comprising any of the nucleotide sequences of the present invention are also provided.
Also provided herein are recombinant expression vectors and recombinant host cells suitable for producing the variant PD-L1 polypeptides or immunomodulatory polypeptides provided herein.
Also provided herein are engineered cells, such as engineered immune cells, containing any of the provided nucleic acids or encoded variant PD-L1 polypeptides or immunomodulatory polypeptides (such as any of transmembrane immunomodulatory polypeptides or secretable immunomodulatory polypeptides).
Also provided herein are infectious agents, such as bacterial or viral cells, containing any of the provided nucleic acids or encoded variant PD-L1 polypeptides or immunomodulatory polypeptides (such as any of transmembrane immunomodulatory polypeptides or secretable immunomodulatory polypeptides).
In any of the embodiments provided above, the nucleic acid encoding an immunomodulatory polypeptide provided herein can be introduced into a cell using recombinant DNA and cloning techniques. To achieve this, recombinant DNA molecules encoding immunomodulatory polypeptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequence encoding the peptide may be excised from the DNA using a suitable restriction enzyme. Alternatively, the DNA molecule may be synthesized using chemical synthesis techniques, such as the phosphoramidite method. Combinations of these techniques may also be used. In some cases, recombinant or synthetic nucleic acids can be generated by Polymerase Chain Reaction (PCR). In some embodiments, DNA inserts may be generated that encode one or more variant PD-L1 polypeptides that contain at least one affinity modified IgSF domain and, in some embodiments, a signal peptide, transmembrane domain, and/or endodomain according to the description provided. This DNA insert can be cloned into an appropriate transduction/transfection vector as known to those skilled in the art. Expression vectors containing the nucleic acid molecules are also provided.
In some embodiments, the expression vector is capable of expressing the immunomodulatory protein in an appropriate cell under conditions suitable for expression of the protein. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding an immunomodulatory protein operably linked to appropriate expression control sequences. Methods for performing this operative ligation either before or after inserting the DNA molecule into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in transcriptional or translational control.
In some embodiments, expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate expression. The promoter is operably linked to a portion of the nucleic acid molecule encoding the variant polypeptide or the immunomodulatory protein. In some embodiments, the promoter is a constitutively active promoter (such as a tissue-specific constitutively active promoter or other constitutive promoter). In some embodiments, the promoter is an inducible promoter, which may be responsive to an inducing agent (such as a T cell activation signal).
Exemplary constitutive promoters include the simian vacuolar virus 40(Sv40) promoter, the Cytomegalovirus (CMV) promoter, the ubiquitin c (ubc) promoter, and the EF-1 α (EF1a) promoter.
In some embodiments, the inducible promoter is operably linked to a nucleic acid molecule encoding a variant polypeptide or an immunomodulatory protein such that expression of the nucleic acid can be controlled by controlling the presence or absence of an appropriate transcription inducer. For example, the promoter may be a regulated promoter and transcription factor expression system, such as the published tetracycline regulated system or other regulatory systems (see, e.g., published International PCT application No. WO 01/30843), to allow for regulation of expression of the encoded polypeptide. An exemplary adjustable start subsystem is the Tet-On (and Tet-Off) system available from, for example, Clontech (Palo Alto, Calif.). This promoter system allows the regulation of the expression of transgenes controlled by tetracycline or tetracycline derivatives such as doxycycline. Other regulatable promoter systems are known (see, e.g., published U.S. application No. 2002-.
In some embodiments, the promoter is responsive to elements that signal transduction in response to T cell activation. By way of example only, in some embodiments, the engineered T cell comprises an expression vector encoding an immunomodulatory protein and a promoter operably linked to control expression of the immunomodulatory protein. Engineered T cells can be activated, for example, by signaling through an engineered T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR) and thus triggering expression and secretion of immunomodulatory proteins by responsive promoters.
In some embodiments, the inducible promoter is operably linked to a nucleic acid molecule encoding an immunomodulatory protein such that the immunomodulatory protein is expressed in response to nuclear factor for activated T cells (NFAT) or nuclear factor kappa light chain enhancer for activated B cells (NF-kappa B). For example, in some embodiments, an inducible promoter comprises a binding site for NFAT or NF-. kappa.B. For example, in some embodiments, the promoter is an NFAT or NF-. kappa.B promoter or a functional variant thereof. Thus, in some embodiments, the nucleic acid allows for control of expression of the immunomodulatory protein while also reducing or eliminating toxicity of the immunomodulatory protein. Specifically, engineered immune cells comprising the nucleic acids of the invention express and secrete immunomodulatory proteins only when the cell (e.g., a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR) expressed by the cell) is specifically stimulated by an antigen and/or the cell (e.g., the calcium signaling pathway of the cell) is non-specifically stimulated by, for example, Phorbol Myristate Acetate (PMA)/ionomycin. Thus, expression and in some cases secretion of the immunomodulatory proteins can be controlled to occur only when needed and at a desired site (e.g., in the presence of infectious disease initiators, in cancer, or at a tumor site), which can reduce or avoid unwanted immunomodulatory protein interactions.
In some embodiments, a nucleic acid encoding an immunomodulatory protein described herein comprises a suitable nucleotide sequence encoding an NFAT promoter, an NF- κ B promoter, or a functional variant thereof. As used herein, "NFAT promoter" means one or more NFAT responsive elements linked to a minimal promoter. "NF-. kappa.B promoter" refers to one or more NF-. kappa.B responsive elements linked to a minimal promoter. In some embodiments, the minimal promoter of the gene is the minimal human IL-2 promoter or the CMV promoter. The NFAT responsive element may include, for example, NFAT1, NFAT2, NFAT3, and/or NFAT4 responsive elements. The NFAT promoter, NF- κ B promoter, or functional variant thereof may comprise any number of binding motifs, for example at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs.
The resulting recombinant expression vector with the DNA molecule thereon is used to transform a suitable host. This transformation can be performed using methods well known in the art. In some embodiments, the nucleic acids provided herein further comprise a nucleotide sequence encoding a secretory peptide or a signal peptide operably linked to the nucleic acid encoding the immunomodulatory polypeptide such that the resulting soluble immunomodulatory polypeptide is recovered from the culture medium, the host cell, or the periplasm of the host cell. In other embodiments, appropriate expression control signals are selected to allow membrane expression of the immunomodulatory polypeptide. In addition, commercially available kits and contracts are also used by manufacturing companies to prepare engineered or recombinant host cells as provided herein.
In some embodiments, the resulting expression vector with the DNA molecule thereon is used to transform (such as transduce) an appropriate cell. Introduction may be carried out using methods well known in the art. Exemplary methods include methods for transferring nucleic acids encoding a receptor, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into the cell by a lentiviral or retroviral transduction method.
Any of a number of publicly available and well known mammalian host cells, including mammalian T cells or APCs, can be used to produce the polypeptide or engineered cell. The choice of cells depends on a number of factors recognized in the art. Such factors include, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of peptide recovery, expression characteristics, biosafety, and cost. These factors must be balanced against one another with the understanding that not all cells can express a particular DNA sequence as efficiently.
In some embodiments, the host cell may be a variety of eukaryotic cells, such as in a yeast cell, or a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell or a HEK293 cell, is used. In some embodiments, the host cell is a suspension cell and the polypeptide is engineered or produced in a cultured suspension, such as in cultured suspension CHO cells, e.g., CHO-S cells. In some examples, the cell line is a CHO cell line lacking DHFR (DHFR-) (such as DG44 and DUXB 11). In some embodiments, the cell lacks Glutamate Synthase (GS), such as CHO-S cells, CHOK1 SV cells, and CHOZN ((R)) GS-/-cells. In some embodiments, the CHO cells (such as suspension CHO cells) may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpicHO-S cells.
In some embodiments, the host cell may also be a prokaryotic cell, such as using E.coli. The transformed recombinant host is cultured under polypeptide expression conditions and then purified to obtain a soluble protein. The recombinant host cell may be cultured under conventional fermentation conditions in order to express the desired polypeptide. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. Protein refolding steps can be used as needed to complete the configuration of the mature protein. Finally, High Performance Liquid Chromatography (HPLC) can be used for the final purification step.
In some embodiments, the cell is an immune cell, such as any of the immune cells described above in connection with the preparation of the engineered cell. In some embodiments, such engineered cells are primary cells. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cell is allogeneic to the subject. In some embodiments, the engineered cells are obtained from a subject, such as by leukapheresis, and transformed ex vivo for expression of an immunomodulatory polypeptide, e.g., a transmembrane immunomodulatory polypeptide or a secretable immunomodulatory polypeptide.
Also provided are nucleic acids encoding any of the variant immunomodulatory polypeptides contained in the infectious agents described herein. In some embodiments, the infectious agent delivers the nucleic acid to a cell of the subject, and/or allows expression of the encoded variant polypeptide in the cell. Nucleic acids for generating, producing or modifying such infectious agents are also provided. For example, in some embodiments, vectors and/or plasmids containing nucleic acids encoding variant immunomodulatory polypeptides are provided for use in generating an infectious agent, delivering to cells of a subject, and/or expressing a variant immunomodulatory polypeptide in cells of a subject.
In some embodiments, the nucleic acid provided is a recombinant viral or bacterial vector comprising a nucleic acid sequence encoding a variant immunomodulatory polypeptide. In some embodiments, the recombinant vector may be used to produce an infectious agent comprising a nucleic acid sequence encoding a variant immunomodulatory polypeptide and/or to be delivered into a target cell of a subject for expression by the target cell. In some embodiments, the recombinant vector is an expression vector. In some embodiments, the recombinant vector includes the appropriate sequences necessary for the production and/or production of the infectious agent and expression in the target cell.
In some embodiments, the recombinant vector is a plasmid or cosmid. Plasmids or cosmids containing nucleic acid sequences encoding variant immunomodulatory polypeptides as described herein are readily constructed using standard techniques well known in the art. To produce infectious agents, vectors or genomes may be constructed in the form of plasmids, which may then be transfected into packaging or producer cell lines or host bacteria. Recombinant vectors may be generated using any of the recombinant techniques known in the art. In some embodiments, the vector may include a prokaryotic origin of replication and/or a gene whose expression confers a detectable or selectable marker, such as drug resistance for transmission and/or selection in prokaryotic systems.
In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include lentiviral vector genomes, poxvirus vector genomes, vaccinia virus vector genomes, adenoviral vector genomes, adeno-associated viral vector genomes, herpesvirus vector genomes, and alphavirus vector genomes. The viral vector may be live, attenuated, replication-conditional or replication-defective, non-pathogenic (defective), capable of replication, and/or modified to express a heterologous gene product, such as a variant immunomodulatory polypeptide provided herein. The vector used to generate the virus may be modified to alter the attenuation of the virus, including any method that increases or decreases the transcriptional or translational load.
Exemplary viral vectors that can be used include modified vaccinia virus vectors (see, e.g., Guerra et al, J.Virol.80:985-98 (2006); Tartaglia et al, AIDS Research and Human Retroviruses 8:1445-47 (1992); Gheradi et al, J.Gen.Virol.86:2925-36 (2005); Mayr et al, Infectin 3:6-14 (1975); Hu et al, J.Virol.75: 10300-308 (2001); U.S. Pat. Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615, and 7,368,116); adenoviral or adeno-associated viral vectors (see, e.g., Molin et al, J.Virol.72:8358-61 (1998); Narumi et al, Am J.Respir.cell mol.biol.19:936-41 (1998); Mercier et al, Proc.Natl.Acad.Sci.USA 101:6188-93 (2004); U.S. Pat. Nos. 6,143,290, 6,596,535, 6,855,317, 6,936,257, 7,125,717, 7,378,087, 7,550,296); retroviral vectors, including those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retrovirus, Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J.Virol.66:2731-39 (1992); Johann et al, J.Virol.66: 1635-40 (1992); Sommerfelt et al, Virology 176:58-59 (1990); Wilson et al, J.Virol.63:2374-78 (1989); Miller et al, J.Virol.65:2220-24 (1991); Miller et al, mol.cell biol.10:4239 (1990); Kolberg, NIH Res.4: 431992; Cornet et al, Hum.1992: 1991); lentiviral vectors, including those based on Human immunodeficiency virus (HIV-1), HIV-2, Feline Immunodeficiency Virus (FIV), equine infectious anemia virus, Simian Immunodeficiency Virus (SIV), and Maedi/visna virus (maedi/visna virus) (see, e.g., Pfeifer et al, Annu. Rev. genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al, J.Virol.72: 9873,1998; Miyoshi et al, J.Virol.72:8150,1998; Philpott and Thrasher, Human Gene Therapy 18:483,2007; Engelman et al, J.Virol. 69: 2729,1995; Nigeingale et al, mol.rapy, 13:1121,2006; Brown et al, J.Virol.73: 9011; WO 3578; WO 5635; Virol. J.3578; see, Virol et al, see, for any of the vectors described in Pfei, Annu. In some embodiments, the recombinant vector may comprise regulatory sequences, such as promoter or enhancer sequences, that can regulate expression of the viral genome (such as in the case of RNA viruses) in a packaging cell line (see, e.g., U.S. patent nos. 5,385,839 and 5,168,062).
In some embodiments, the recombinant vector is an expression vector, e.g., an expression vector that allows expression of the encoded gene product when delivered into a target cell, e.g., a cell of a subject, e.g., a tumor cell, an immune cell, and/or an APC. In some embodiments, the recombinant expression vector contained in the infectious agent is capable of expressing the immunomodulatory protein in a target cell of a subject under conditions suitable for expression of the protein.
In some aspects, the nucleic acid or expression vector comprises a nucleic acid sequence encoding an immunomodulatory protein operably linked to appropriate expression control sequences. Methods for performing such operative ligation either before or after inserting the nucleic acid sequence encoding the immunomodulatory protein into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in transcriptional or translational control. The promoter may be operably linked to a portion of the nucleic acid sequence encoding the immunomodulatory protein. In some embodiments, the promoter is a constitutively active promoter (such as a tissue-specific constitutively active promoter or other constitutive promoter) in the target cell. For example, the recombinant expression vector can also include a lymphoid tissue-specific Transcriptional Regulatory Element (TRE), such as a B lymphocyte, T lymphocyte, or dendritic cell-specific TRE. Lymphoid tissue-specific TREs are known in the art (see, e.g., Thompson et al, mol.cell.biol.12:1043-53 (1992); Todd et al, J.exp.Med.177:1663-74 (1993); Penix et al, J.exp.Med.178:1483-96 (1993)). In some embodiments, the promoter is an inducible promoter, which may be responsive to an inducing agent (such as a T cell activation signal). In some embodiments, a nucleic acid delivered to a target cell (e.g., a tumor cell, an immune cell, and/or an APC) of a subject can be operably linked to any of the regulatory elements described above.
In some embodiments, the vector is a bacterial vector, such as a bacterial plasmid or cosmid. In some embodiments, the bacterial vector is delivered to a target cell, such as a tumor cell, immune cell, and/or APC, via bacterial-mediated transfer of plasmid DNA to a mammalian cell (also referred to as "bacterial infection"). In some embodiments, the delivered bacterial vector also contains appropriate expression control sequences for expression in the target cell, such as promoter sequences and/or enhancer sequences or any of the regulatory or control sequences described above. In some embodiments, the bacterial vector contains appropriate expression control sequences for expression and/or secretion of the encoded variant polypeptide in an infectious agent (e.g., a bacterium).
In some embodiments, the polypeptides provided herein can also be prepared by synthetic methods. Solid phase synthesis is the preferred technique for preparing individual peptides, as it is the most cost-effective method for preparing small peptides. For example, well-known solid phase synthesis techniques include the use of protecting groups, linkers, and solid supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid phase peptide synthesis. The peptides can then be assembled into polypeptides as provided herein.
Methods of assessing active immunomodulation of variant PD-L1 polypeptides and immunomodulatory proteins
In some embodiments, a variant PD-L1 polypeptide provided herein (e.g., a full-length and/or specific binding fragment or conjugate, a stacked construct or fusion thereof, an engineered cell, or an infectious agent) exhibits immunomodulatory activity to modulate T cell activation. In some embodiments, the PD-L1 polypeptide modulates IFN- γ expression in a T cell assay relative to a wild-type or unmodified PD-L1 control. In some cases, modulation of IFN- γ expression can increase or decrease IFN- γ expression relative to a control. Assays to determine specific binding and IFN- γ expression are well known in the art and include MLR (mixed lymphocyte reaction) assays to measure interferon- γ cytokine levels in culture supernatants (Wang et al, Cancer Immunol Res.2014 9 months: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assays (Wang et al, Cancer Immunol Res.2014 9 months: 2(9):846-56), and anti-CD 3T cell stimulation assays (Li and Kurlander, J Transl Med.2010: 8: 104).
In some embodiments, a variant PD-L1 polypeptide may increase or in alternative embodiments decrease IFN- γ (interferon- γ) expression in a primary T cell assay relative to a wild-type PD-L1 control. In some embodiments, such activity may be provided in dependence on whether the variant PD-L1 polypeptide is in the form of antagonist activity or in the form of agonist activity. In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein is an antagonist of an inhibitory receptor in a cell, such as an inhibitory signal that a block may appear to reduce response to an activation stimulus (e.g., CD3 and/or CD28 co-stimulatory signals or mitogenic signals). The skilled person will recognise that there are different forms of primary T cell assays for determining an increase or decrease in INF-gamma expression.
When determining the ability of variant PD-L1 to increase or decrease IFN- γ expression in a primary T cell assay, a Mixed Lymphocyte Reaction (MLR) assay may be used. In some embodiments, a variant PD-L1 polypeptide or immunomodulatory protein (e.g., a variant PD-L1-Fc or secretable immunomodulatory protein) provided in an antagonist form (such as a soluble form) blocks the activity of the PD-1 inhibitory receptor and thus increases MLR activity in an assay, such as observed by an increase in IFN- γ production in an assay. In some embodiments, variant PD-L1 polypeptides or immunomodulatory proteins provided in agonist form (such as localized vigdd stacks or conjugates containing tumor localization moieties or engineered cells expressing transmembrane immunomodulatory proteins provided) can stimulate the activity of PD-1 inhibitory receptors and thus reduce MLR activity, such as evidenced by reduced IFN- γ production. In some embodiments, variant PD-L1 polypeptides or immunomodulatory proteins provided in agonist form (such as localized vigdd stacks or conjugates containing tumor localization moieties or engineered cells expressing transmembrane immunomodulatory proteins provided) can block the activity of PD-1 inhibitory receptors and thus increase MLR activity, such as increased IFN- γ production.
Alternatively, a co-immobilization assay may be used when determining the ability of variant PD-L1 to modulate an increase or decrease in IFN- γ expression in a primary T cell assay. In a co-immobilization assay, the TCR signal provided by the anti-CD 3 antibody is used in some embodiments in conjunction with a co-immobilized variant PD-L1 to determine the ability to increase or decrease IFN- γ expression relative to a PD-L1 unmodified or wild-type control. In some embodiments, a variant PD-L1 polypeptide or immunomodulatory protein (e.g., a co-immobilized variant PD-L1 (e.g., PD-L1-Fc)) reduces IFN- γ production in a co-immobilization assay.
In some embodiments, in determining the ability of variant PD-L1 to modulate an increase or decrease in IFN- γ expression, a T cell reporter gene assay may be used. In some embodiments, the T cell is or is derived from a Jurkat T cell line. In reporter assays, reporter cell lines (e.g., Jurkat reporter cells) are also generated to overexpress inhibitory receptors, which are homologous binding partners for variant IgSF domain polypeptides. For example, in the case of variant PD-L1, a reporter cell line (e.g., Jurkat reporter cell) is generated to overexpress PD-1. In some embodiments, the reporter T cell further comprises a reporter construct comprising an inducible promoter operably linked to the reporter in response to T cell activation. In some embodiments, the reporter gene is a fluorescent or luminescent reporter gene. In some embodiments, the reporter gene is luciferase. In some embodiments, the promoter is responsive to CD3 signaling. In some embodiments, the promoter is an NFAT promoter. In some embodiments, the promoter is responsive to costimulatory signaling, e.g., CD28 costimulatory signaling. In some embodiments, the promoter is an IL-2 promoter.
In aspects of reporter assays, the reporter cell line is stimulated, such as by co-incubation with Antigen Presenting Cells (APCs) that express a wild-type ligand of an inhibitory receptor, e.g., PD-L1. In some embodiments, the APC is an artificial APC. Artificial APCs are well known to the skilled artisan. In some embodiments, the artificial APC is derived from one or more mammalian cell lines, such as K562, CHO, or 293 cells.
In some embodiments, the Jurkat reporter cell is incubated with an artificial APC overexpressing an inhibitory ligand in the presence of a variant IgSF domain molecule or an immunomodulatory protein (e.g., a variant PD-L1 polypeptide or an immunomodulatory protein). In some embodiments, reporter gene expression is monitored, such as by determining the luminescence or fluorescence of the cell. In some embodiments, normal interaction between the inhibitory receptor and the ligand causes a regression or reduction in reporter signal, such as compared to a control, e.g., by co-incubation with control T cells and APCs in which there is no interaction of the inhibitory receptor and the ligand (e.g., APCs that do not overexpress PD-L1). In some embodiments, a variant PD-L1 polypeptide or immunomodulatory protein provided herein antagonizes the interaction such that reporter gene signal is increased compared to the absence of the variant PD-L1 polypeptide or immunomodulatory protein, e.g., when provided in soluble form as variant PD-L1-Fc or when expressed by an APC as a secretable immunomodulatory protein. In some cases, certain forms of the variant PD-L1 polypeptides or immunomodulatory proteins provided herein can provide agonist activity, thereby reducing reporter gene expression compared to when the variant PD-L1 polypeptide or immunomodulatory protein is absent.
The use of appropriate controls is known to those skilled in the art, however, in the above embodiments, control typically involves the use of unmodified PD-L1 (such as the wild-type or native PD-L1 isoform) from the same mammalian species from which the variant PD-L1 was derived or developed. In some embodiments, the wild-type or native PD-L1 has the same form or a corresponding form as the variant. For example, if the variant PD-L1 is a soluble form of a variant ECD containing a fusion to an Fc protein, then the control is a soluble form of a wild-type or native ECD containing PD-L1 fused to an Fc protein. Regardless of whether the binding affinity and/or selectivity to one or more of PD-1 and CD80 is increased or decreased, the variant PD-L1 will increase IFN- γ expression in some embodiments and decrease IFN- γ expression in alternative embodiments relative to a wild-type PD-L1 control in a T cell assay.
In some embodiments, the variant PD-L1 polypeptide or immunomodulatory protein increases IFN- γ expression (i.e., protein expression) relative to a wild-type or unmodified PD-L1 control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher. In other embodiments, the variant PD-L1 or immunomodulatory protein reduces IFN- γ expression (i.e., protein expression) relative to a wild-type or unmodified PD-L1 control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher. In some embodiments, the wild-type PD-L1 control is murine PD-L1, such as variant PD-L1 that would typically be used for sequence changes from the wild-type murine PD-L1 sequence. In some embodiments, wild-type PD-L1 control is human PD-L1, such as variant PD-L1 that would normally be used for alterations in sequence by wild-type human PD-L1 sequence, such as a PD-L1 sequence comprising amino acid sequence SEQ ID NO:30 or 1728 or SEQ ID NO:55 or 309.
V. pharmaceutical preparation
Provided herein are compositions containing any of the variant PD-L1 polypeptides, immunomodulatory proteins, conjugates, engineered cells, or infectious agents described herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. For example, the pharmaceutical composition may contain one or more excipients for adjusting, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or permeation of the composition. In some aspects, the skilled artisan understands that a cell-containing pharmaceutical composition can differ from a protein-containing pharmaceutical composition.
In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition may be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.
In some embodiments, the pharmaceutical composition is a liquid, e.g., a variant PD-L1 polypeptide dissolved in an aqueous solution, such as physiological saline or Ringer's solution. In some embodiments, the pH of the pharmaceutical composition is between about 4.0 and about 8.5 (such as between about 4.0 and about 5.0, between about 4.5 and about 5.5, between about 5.0 and about 6.0, between about 5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5 and about 7.5, between about 7.0 and about 8.0, or between about 7.5 and about 8.5).
In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, a binder, a coating agent, a preservative, a lubricant, a flavoring agent, a sweetener, a coloring agent, a solvent, a buffering agent, a chelating agent, or a stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coating agents include Hydroxypropylmethylcellulose (HPMC), shellac, zein, zeatin or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethyl cellulose or sodium carboxymethyl starch. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically acceptable preservatives include methyl paraben, ethyl paraben, propyl paraben, benzoic acid or sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates or tartrates.
In some embodiments, the pharmaceutical composition further comprises an agent for controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes.
In some embodiments, the pharmaceutical composition is sterile. Sterilization may be achieved by filtration through sterile filtration membranes or irradiation. In lyophilizing the composition, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution form. In addition, parenteral compositions are typically placed in containers having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
In some embodiments, pharmaceutical compositions containing transmembrane immunomodulatory proteins are provided, including engineered cells expressing such transmembrane immunomodulatory proteins. In some embodiments, the pharmaceutical compositions and formulations comprise one or more optional pharmaceutically acceptable carriers or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; sugars such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.
Such a formulation may, for example, be in a form suitable for intravenous infusion. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable substance, composition, or vehicle involved in carrying or transporting cells of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any body tissue, organ or part it may encounter, which means that it must not carry the risk of toxicity, irritation, allergic response, immunogenicity, or any other complications that outweigh their therapeutic benefits.
Article and kit
Also provided herein are articles of manufacture comprising the pharmaceutical compositions described herein in a suitable package. Suitable packaging for the compositions described herein (such as ophthalmic compositions) are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed mylar or plastic bags), and the like. These articles may be further sterilized and/or sealed.
Also provided are kits comprising a pharmaceutical composition (or article of manufacture) described herein, which may further comprise instructions for methods of using the composition, such as the uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein.
Therapeutic applications
Provided herein are methods for modulating an immune response (including with respect to treating a disease or condition) in a subject, such as a human patient, using provided pharmaceutical compositions containing a variant PD-L1 polypeptide immunomodulatory protein, conjugate, engineered cell, or infectious agent described herein. The pharmaceutical compositions described herein (including pharmaceutical compositions comprising the variant PD-L1 polypeptides, immunomodulatory proteins, conjugates, engineered cells, and infectious agents described herein) can be used in a variety of therapeutic applications, such as treating diseases. For example, in some embodiments, the pharmaceutical composition is used to treat an inflammatory or autoimmune disorder, cancer, organ transplantation, viral infection, and/or bacterial infection in a mammal. The pharmaceutical composition may modulate (e.g., increase or decrease) the immune response to treat a disease.
In some embodiments, the provided methods are suitable for therapeutic administration of the variant PD-L1 polypeptides, immunomodulatory proteins, conjugates, engineered cells, and infectious agents described herein. It is within the level of the skilled person to select the form for indication according to the type of modulation (e.g. increase or decrease required) of the immune response in light of the disclosure provided.
In some embodiments, the pharmaceutical compositions provided herein that stimulate an immune response are administered, which may be useful, for example, in the treatment of cancer, viral infections, or bacterial infections. In some embodiments, the pharmaceutical composition contains a form of the variant PD-L1 polypeptide that exhibits antagonist activity of its cognate binding partner PD-1 and/or inhibits co-stimulatory signaling via PD-1. Exemplary forms of PD-L1 polypeptides for use in binding to such therapeutic applications include, for example, a soluble variant PD-L1 polypeptide (e.g., a variant PD-L1-Fc fusion protein), an immunomodulatory protein or a "stack" of a variant PD-L1 polypeptide and another IgSF domain (including soluble forms of Fc fusions thereof), an engineered cell expressing a secretable immunomodulatory protein, or an infectious agent comprising a nucleic acid molecule encoding a secretable immunomodulatory protein, such as for expression and secretion of a secretable immunomodulatory protein in an infected cell (e.g., a tumor cell or APC, e.g., a dendritic cell). Such methods are those performed by delivering a soluble form of the variant PD-L1 polypeptide. Exemplary soluble forms are described herein, including forms in which the extracellular portion of a variant PD-L1 polypeptide comprising an affinity modified IgSF domain (e.g., IgV) is linked directly or indirectly to a multimerization domain (e.g., an Fc domain or region). In some embodiments, such a therapeutic agent is a variant PD-L1-Fc fusion protein.
The provided methods of modulating immune responses may be used to treat diseases or conditions, such as tumors or cancers. In some embodiments, the pharmaceutical composition can be used to inhibit the growth of mammalian cancer cells (such as human cancer cells). A method of treating cancer may comprise administering to a subject having cancer an effective amount of any of the pharmaceutical compositions described herein. An effective amount of the pharmaceutical composition can be administered to inhibit, halt, or reverse the progression of cancer, including cancers that are susceptible to modulation of immune activity, such as by provided variants or immunomodulatory proteins. Human cancer cells can be treated in vivo or ex vivo. In ex vivo treatment of human patients, tissues or fluids containing cancer cells are treated outside the body and then reintroduced back into the patient. In some embodiments, the cancer is treated in vivo in a human patient by administering a therapeutic composition to the patient. Thus, the present invention provides ex vivo and in vivo methods of inhibiting, halting or reversing the progression of a tumor or otherwise causing a statistically significant increase in progression free survival (i.e., the length of time during and after treatment in which patients with cancer without worsening survive) or overall survival (also referred to as "survival," i.e., the percentage of people in a study or treatment group that survive a period of time after people have been diagnosed with or treated for cancer) relative to treatment with a control.
Cancers that can be treated by the pharmaceutical compositions and methods of treatment described herein include, but are not limited to, melanoma, bladder cancer, hematological malignancies (white blood cells, lymphomas, myelomas), liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer (adenocarcinomas), colorectal cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), spleen cancer, thymus or blood cell cancer (i.e., leukemia), prostate cancer, testicular cancer, ovarian cancer, uterine cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal tract cancer, germ cell cancer, or endocrine and neuroendocrine cancers. In some embodiments, the cancer is ewing's sarcoma. In some embodiments, the cancer is selected from melanoma, lung cancer, bladder cancer, and hematologic malignancies. In some embodiments, the cancer is lymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer, neuroblastoma, or multiple myeloma.
In some embodiments, the pharmaceutical composition is administered as a monotherapy (i.e., as a single agent) or as a combination therapy (i.e., in combination with one or more additional anti-cancer agents, such as a chemotherapeutic drug, a cancer vaccine, or an immune checkpoint inhibitor). In some embodiments, the pharmaceutical composition may also be administered with radiation therapy. In some aspects of the disclosure, the immune checkpoint inhibitor is nivolumab, tremelimumab, pembrolizumab, Yipimema, or the like.
In some embodiments, the pharmaceutical composition inhibits an immune response, which may be useful for treating an inflammatory or autoimmune disorder or organ transplantation. In some embodiments, the pharmaceutical composition contains a form of the variant PD-L1 polypeptide that exhibits agonist activity of its cognate binding partner PD-1 and/or stimulates inhibitory signaling via PD-1. In some aspects, the variant PD-L1 polypeptide stimulates inhibitory signals, such as by CD80 expressed on lymphocytes or APCs. Exemplary forms of PD-L1 polypeptides for use in conjunction with such therapeutic applications include, for example, a "stack" of an immunomodulatory protein or a variant PD-L1 polypeptide and an IgSF domain or variant thereof localized to a cell or tissue of an inflammatory environment, a conjugate containing a variant PD-L1 polypeptide linked to a portion of a cell or tissue localized to an inflammatory environment, an engineered cell expressing a transmembrane immunomodulatory protein, or an infectious agent comprising a nucleic acid molecule encoding a transmembrane immunomodulatory protein, such as for expression of a transmembrane immunomodulatory protein in an infected cell.
The provided methods of modulating immune responses may be used to treat diseases or conditions, such as inflammatory or autoimmune disorders. In some embodiments, the inflammatory or autoimmune disorder is anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis, an autoimmune skin disease, transplantation, rheumatism, an inflammatory gastrointestinal tract disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory lung disease, an inflammatory endocrine disease, or an autoimmune blood disease.
In some embodiments, the inflammatory and autoimmune disorders that can be treated by the pharmaceutical compositions described herein are Addison's Disease, allergy, alopecia areata, alzheimer's Disease, anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, ankylosing spondylitis, antiphospholipid Syndrome (Hughes Syndrome), asthma, atherosclerosis, rheumatoid arthritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear Disease, autoimmune lymphoproliferative Syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune deltoid, azoospermia, Behcet's Disease, Berger's Disease, bullous pemphigoid, cardiomyopathy, cardiovascular Disease, alzheimer's Disease, diabetes mellitus, radiation, diabetes mellitus, radiation Syndrome, radiation therapy, radiation therapy for cancer, cancer,Sprue/chylemia, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic idiopathic polyneuritis, chronic inflammatory demyelination, polyneuropathies (CIDP), chronic recurrent polyneuropathy (Guillain-barre Syndrome), allergic granulomatous vasculitis (Churg-Strauss Syndrome, CSS), cicatricial pemphigoid, Cold Agglutinin Disease (CAD), COPD (chronic obstructive pulmonary Disease), CREST Syndrome, Crohn's Disease, dermatitis, papuloid dermatitis, dermatomyositis, diabetes, discoid lupus, eczema, epidermolysis bullosa acquisita, primary cryoglobulinemia, Evan's Syndrome, herniated nodules, fibromyalgia, Goodpasture's Syndrome, leiomyelitis's Syndrome (Graves ' Syndrome), and leishmaniasis, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, an immunoproliferative Disease or disorder, Inflammatory Bowel Disease (IBD), interstitial lung Disease, juvenile arthritis, Juvenile Idiopathic Arthritis (JIA), Kawasaki's Disease, leber-Eaton myasthenia Syndrome (Lambert-Eaton myasthetheic Syndrome), purpura, lupus nephritis, lymphocytic hypophysitis, meniere's Disease, Miller gill é Syndrome (Miller Fish Syndrome)/acute disseminated myeloneuropathy, mixed connective tissue Disease, Multiple Sclerosis (MS), myasthenia gravis, Myalgic Encephalomyelitis (ME), myasthenia gravis, ocular inflammation, pemphigus vulgaris, pernicious anemia, polyarteritis destruens, Multiple Sclerosis (MS), myasthenia gravis, nodular myasthenia gravis, ocular inflammation, pemphigus vulgaris, pernicious anemia, polyarteritis destruens, and multiple sclerosis, Polyadenylic Syndrome (Whitaker's Syndrome), polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy, psoriasis, psoriatic arthritis, Raynaud's Phenomenon (Raynaud's Phenomenon), Reiter's Syndrome/reactive arthritis, restenosis, rheumatic fever, rheumatic diseases, sarcoidosis, Schmidt's Syndrome, scleroderma, Sjogren's Syndrome
In some embodiments, the pharmaceutical composition is administered to modulate an autoimmune disorder. For example, suppressing an immune response may be advantageous for use in a method of suppressing rejection of a tissue, cell, or organ transplant from a donor by a recipient. Thus, in some embodiments, the pharmaceutical compositions described herein are used to limit or prevent graft-related or implant-related diseases or disorders, such as graft-versus-host disease (GVHD). In some embodiments, the pharmaceutical composition is for inhibiting autoimmune rejection of implanted or transplanted bone marrow, organs, skin, muscle, neurons, pancreatic islets, or parenchymal cells.
The pharmaceutical compositions and methods comprising engineered cells described herein can be used in adoptive cell transfer applications. In some embodiments, the cellular compositions comprising the engineered cells may be used in related methods, e.g., for modulating immunological activity in an immunotherapy regimen, to treat, for example, mammalian cancer or (in other embodiments) to treat an autoimmune disorder. The methods employed generally include methods of contacting a TIP of the invention with mammalian cells under conditions that allow specific binding of affinity-modified IgSF domains and modulation of the immunological activity of the mammalian cells. In some embodiments, immune cells, such as Tumor Infiltrating Lymphocytes (TILs), T cells (including CD8+ or CD4+ T cells), or APCs, are engineered to be expressed as membrane proteins and/or soluble variant PD-L1 polypeptides, immunomodulatory proteins, or conjugates as described herein. The engineered cell may then be contacted with a mammalian cell, such as an APC, a second lymphocyte, or a tumor cell, wherein modulation of immunological activity is desired under conditions that allow the affinity modified IgSF domain to specifically bind to a relative structure on the mammalian cell, such that the immunological activity may be modulated in the mammalian cell. The cells may be contacted in vivo or ex vivo.
In some embodiments, the engineered cell is an autologous cell. In other embodiments, the cells are allogeneic. In some embodiments, the cell is an autologous engineered cell that is reinfused into the mammal from which it was isolated. In some embodiments, the cells are alloengineered cells infused into a mammal. In some embodiments, the cells are harvested from the patient's blood or tumor, engineered to express a polypeptide (such as a variant PD-L1 polypeptide, immunomodulatory protein, or conjugate described herein), expanded in an in vitro culture system (e.g., by stimulating the cells), and reinfused into the patient to mediate tumor destruction. In some embodiments, the method is performed by adoptive cell transfer, wherein cells (e.g., T cells) expressing TIP are infused back into the patient. In some embodiments, the therapeutic compositions and methods of the invention are used to treat cancer, such as lymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer, neuroblastoma, or multiple myeloma in a mammalian patient.
In some embodiments, the pharmaceutical composition is administered to a subject. In some embodiments, the subject is a human patient. In general, the dosage and route of administration of the pharmaceutical composition is determined according to the size and condition of the subject, according to standard pharmaceutical practice. For example, a therapeutically effective dose can be initially evaluated in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration for the human. The precise dosage will be determined by factors associated with the subject in need of treatment. The dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, the time and frequency of administration, drug combination, response sensitivity and response to therapy.
The long-acting pharmaceutical composition may be administered every 3 to 4 days, weekly or biweekly depending on the half-life and clearance of the particular formulation the frequency of administration will depend on the pharmacokinetic parameters of the molecules in the formulation used in general, the composition is administered until a dose is reached that achieves the desired effect the composition may thus be administered in a single dose or over time in multiple doses (at the same or different concentrations/doses) or in continuous infusion.
In some embodiments, the pharmaceutical composition is administered to the subject by any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly to a wound site, to a surgical site, intraabdominally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intraspinally.
In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeat dose. In some embodiments, the subject is administered a dose once a day, twice a day, three times a day, or four or more times a day. In some embodiments, about 1 or more (such as about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, or about 7 or more) doses are administered within a week. In some embodiments, multiple doses are administered over the course of days, weeks, months, or years. In some embodiments, the course of treatment is about 1 or more doses (such as about 2 or more doses, about 3 or more doses, about 4 or more doses, about 5 or more doses, about 7 or more doses, about 10 or more doses, about 15 or more doses, about 25 or more doses, about 40 or more doses, about 50 or more doses, or about 100 or more doses).
In some embodiments, the dosage of the pharmaceutical composition administered is about 1 μ g protein/kg subject weight or greater (such as about 2 μ g protein/kg subject weight or greater, about 5 μ g protein/kg subject weight or greater, about 10 μ g protein/kg subject weight or greater, about 25 μ g protein/kg subject weight or greater, about 50 μ g protein/kg subject weight or greater, about 100 μ g protein/kg subject weight or greater, about 250 μ g protein/kg subject weight or greater, about 500 μ g protein/kg subject weight or greater, about 1mg protein/kg subject weight or greater, about 2mg protein/kg subject weight or greater, or about 5mg protein/kg subject weight or greater).
In some embodiments, a therapeutic amount of the cell composition is administered. In general, the precise amount of the composition of the invention to be administered can be determined by a physician considering the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). In general, a pharmaceutical composition comprising an engineered cell (e.g., a T cell) as described herein can be 104To 109Individual cells/kg body weight, such as 105To 106Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. Engineered cell compositions (such as T cell compositions) can also be administered multiple times at these doses. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319: 1676,1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
In some embodiments, the pharmaceutical composition containsAn infectious agent comprising a nucleic acid sequence encoding an immunomodulatory variant polypeptide. In some embodiments, the pharmaceutical composition contains one dose of an infectious agent suitable for administration to a subject suitable for treatment. In some embodiments, the pharmaceutical composition contains an infectious agent that is a virus in the following single or multiple dose amounts: about 1X105Each and about 1 × 10121X10 between plaque-Forming units (pfu)6And 1X1010Between pfu, or 1X107And 1X1010Each of which includes endpoints between, such as at least or at least about or about 1 × 106、1×107、1×108、1×109、2×109、3×109、4×109、5×109pfu or about 1X1010pfu. In some embodiments, the pharmaceutical composition contains the following virus concentrations: from or about 105To about 1010pfu/mL,
Various means are known for determining whether administration of the therapeutic compositions of the present invention will result in the elimination, sequestration, or inactivation of immune cells that mediate or are capable of mediating an undesired immune response; inducing, generating or initiating immune cells that mediate or are capable of mediating a protective immune response; altering a physical or functional characteristic of an immune cell; or a combination of these effects to substantially modulate immunological activity. Examples of measurements of modulation of immunological activity include, but are not limited to, examining the presence or absence of a population of immune cells (using flow cytometry, immunohistochemistry, histology, electron microscopy, Polymerase Chain Reaction (PCR)); measuring the functional capacity of immune cells, including the ability or resistance to proliferate or divide in response to a signal (such as using a T cell proliferation assay and peptide scanning analysis based on the 3H-thymine incorporation method after stimulation with anti-CD 3 antibody, anti-T cell receptor antibody, anti-CD 28 antibody, calcium ionophore, PMA (phorbol 12-myristate 13-acetate) antigen presenting cells loaded with peptide or protein antigens; B cell proliferation assay); measuring the ability to kill or lyse other cells (such as cytotoxic T cell assays); measuring cytokines, chemokines, cell surface molecules, antibodies, and other cell products (e.g., by flow cytometry, enzyme-linked immunosorbent assays, western blot analysis, protein microarray analysis, immunoprecipitation analysis); biochemical markers that measure activation of immune cells or signaling pathways within immune cells (e.g., western blot and immunoprecipitation assays for tyrosine, serine, or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array assays; DNA transcription profiles using DNA arrays or subtractive hybridization); measuring cell death by apoptosis, necrosis or other mechanisms (e.g., annexin V staining, TUNEL assay, gel electrophoresis to measure DNA gradients, histology; fluorescent caspase assay, western blot analysis of caspase substrates); measuring genes, proteins, and other molecules produced by immune cells (e.g., northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, 2-dimensional gel electrophoresis, western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); and measuring clinical symptoms or outcomes, such as improvement in autoimmunity, neurodegeneration, and other diseases involving self-proteins or self-polypeptides (clinical score, need for additional therapy, functional status, imaging studies), for example by measuring relapse rate or disease severity in the case of multiple sclerosis (using a clinical score known to the skilled artisan), measuring blood glucose in the case of type I diabetes, or measuring joint inflammation in the case of rheumatoid arthritis.
Exemplary embodiments
The embodiments provided are:
1. a variant PD-L1 polypeptide comprising an ECD or IgV domain or specific binding fragment thereof, an IgC domain or specific binding fragment thereof, or both, wherein the variant PD-L1 polypeptide comprises one or more amino acid modifications in an unmodified PD-L1 or specific binding fragment thereof corresponding to a position selected from: 6. 10, 11, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 33, 35, 36, 40, 41, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64, 65, 68, 71, 72, 73, 74, 75, 78, 79, 83, 85, 89, 90, 93, 97, 98, 99, 101, 102, 103, 104, 106, 110, 111, 112, 113, 117, 119, 120, 121, 124, 129, 130, 131, 134, 137, 138, 144, 148, 149, 150, 155, 158, 160, 163, 165, 167, 170, 171, 173, 175, 176, 177, 179, 180, 183, 185, 188, 189, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 206, 207, 221, 213, or 1728 ID or 199 of NO.
2. The variant PD-L1 polypeptide of
3. The variant PD-L1 polypeptide of
4. The variant PD-L1 polypeptide of embodiment 3, wherein the unmodified PD-L1 is human PD-L1 or a specific binding fragment thereof.
5. The variant PD-L1 polypeptide of any one of embodiments 1-4, wherein the unmodified PD-L1 comprises (i) the amino acid sequence set forth in SEQ ID NO:30 or 1728, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:30 or 1728; or (iii) it comprises a portion of an IgV structure or an IgC domain or a specific binding fragment thereof or both.
6. The variant PD-L1 polypeptide of any one of embodiments 1-5, wherein:
the IgV domain or a specific binding fragment of an IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or
The specific binding fragment of an IgV domain comprises a length that is at least 80% of the length of the IgV domain listed as amino acids 24-130 of SEQ ID NO 3; and/or the specific binding fragment of the IgC domain comprises a length which is at least 80% of the length of the IgC domain as set forth as amino acids 133-225 of SEQ ID NO 3.
7. The variant PD-L1 polypeptide of any one of embodiments 1-6, wherein:
the variant PD-L1 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, optionally amino acid substitutions, insertions, and/or deletions; or
The variant PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 30 or 1728, or a specific binding fragment thereof.
8. The variant PD-L1 polypeptide of any one of embodiments 1-7, wherein the variant PD-L1 polypeptide exhibits altered binding to an extracellular domain of PD-1 or CD80, as compared to the unmodified PD-L1.
9. The variant PD-L1 polypeptide of any one of embodiments 1-8, wherein:
the variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of PD-1 as compared to the binding of the unmodified PD-L1 to the extracellular domain of PD-1; and/or
The variant PD-L1 polypeptide exhibits altered binding to the extracellular domain of CD80 as compared to the binding of the unmodified PD-L1 to the extracellular domain of CD80.
10. The variant PD-L1 polypeptide of embodiment 8 or embodiment 9, wherein the altered binding is altered binding affinity and/or altered binding selectivity.
11. The variant PD-L polypeptide of any one of embodiments 1-10, wherein the one or more amino acid modifications are selected from P6, Y10, V11, Y14, G15, S16, N17, M18, T19, I20, C22, K23, E26, E27, K28, a33, L35, I36, E40, M41, D43, K44, N45, I47, I46, F49, V50, H51, G52, E53, E54, D55, L56, K57, V58, H60, S75, R64, Q65, R68, K72, E54, D55, L56, K57, V58, V60, S75, R64, Q65, R72, K97, N101, N99, N101, N97, N98, N97, N98, N101, N98, N97, N98, I9, N7, N101, N7, N95, N7, N95, N7, N95, t130, S131, E134, C137, Q138, K144, I148, W149, T150, Q155, S158, K160, T163, K163, N165, K167, E170, K171, F173, K173, V175, T176, S177, L179, R180, T183, T185, I188, F189, T192, F193, R194, R195, L196, D197, P198, E199, E200, N201, H202, T203, a204, L206, V207, L213, T221 or conservative amino acid substitutions thereof.
12. The variant PD-L polypeptide of any of embodiments 1 to 11, wherein the one or more amino acid modifications are selected from the group consisting of K28/M41/N45/H51/K57, I20/I36/N45/I47, I20/M41/K44, P6/N45/N78/I83, N78, M41/N78, N45/N78, I20/N45, M41, I20/I36/N45, N17/N47/V50/D72, I20/F49, N45/V50, I20/N45/N78, I20/N45/V50, M41/N45, A33/S75/D85, M18/M41/D43/H51/N78, V11/I20/I36/N45/H60/S75, A33/V50, S16/S33/S75, S33/S75, S33/, E27/N45/M97, E27/N45/K57, A33/E53, D43/N45/V58, E40/D43/N45/V50, Y14/K28/N45A 33/N78, A33/N45/N78, E27/N45/V50, N45/V50/N78, I20/N45/V110, I20/I36/N45/V50, N45/L74/S75, N45/S75, S75/K106, S75, A33/S75/D104, A33/S75, I20/E27/N45/V50, I20/E27/D43/N45/V58/N78, I20/A33/N45/N58/N78, I33/N45/N78, E27/N45/V50/N78, V11/I20/E27/D43/N45/H51/S99, I20/E27/D43/N45/V50, I20/K28/D43/N45/V58/Q89, I20/I36/N45, I20/K28/D43/N45/E53/V58/N78, A33/D43/N45/V58/S75, K23/D43/N45, I20/D43/N45/V58/N78/D90/G101, D43/N45/L56/V58/G101-ins (G101), I20/K23/D43/N45/V58/N78, I20/K23/D43/N45/V50/N78, T19/E27/N45/N50/N97/M45/M78, I20/M41/D43/N45, K23/N45/N78, I20/K28/D43/N45/V58/Q89/G101-ins (G101), K57/S99/F189, M18/M97/F193/R195/E200/H202, I36/M41/M97/K144/R195/E200/H202/L206, C22/Q65/L124/K144/R195/E200/H202/T221, M18/I98/L124/P198/L206, S99/N117/I148/K171/R180, I36/M97/A103/Q155, K28/S99, R195, A79/S99/T185/R195/E200/H202/L206, K57/S99/L124/K144, K99/S195/R, D55/M97/S99, E27/I36/D55/M97/K111, E54/M97/S99, G15/I36/M97/K111/H202, G15/I36/V129/R195, G15/V129, I36/M97, I36/D55/M97/K111/A204, I36/D55/M97/K111/V129/F173, I36/D55/M97/K111/I148/R180, I36/G52/M97/V112/K144/V175/P198, I36/I46/D55/M97/K106/K144/T185/R195, I36/I83/M97/K144/P198, I36/M97/K111, I36/M97/K144/P198, I36/M97/K144/M193/M155/F193/N195, I36T/M97L/V129D, L35P/I36S/M97L/K111E, M18I/I36T/E53G/M97L/K144E/E199G/V207A, M18T/I36T/D55N/M97L/K111E, M18V/M97L/T176L/R195L, M97L/S99L, N17/M97L/S L, S99L/T185/L/R195/P198L, V129L/H202L, V129L/P198L, V129L/T150, V93/V129L, Y10L/M18/S72/S99/S195/P198L, N129/N195/N72/N L/N198N L/N195/N72/N L/N198N 72/N L/N195/N72/N L/N72/N195/N72/N198N 72/N L/N198N 72/N L, N L/N198N L/N72/N L/N198N L/N195/N L/N72/N L/, N45D/N113Y/R195S, N45D/N165Y/E170G, N45D/Q89R/I98V, N45D/S131F/P198S, N45D/S75P/P198S, N45D/V50A/R195T, E27D/N45D/T183A/I188V, F173V/T183V/L196V/T203V, K23V/N45V/S75/N120V, N45V/G102/R194V/R195V, N45V/G52/Q V/P198, N45V/I148/R195/R72/N V/N72/N198/N72/N V/N183/N198/N72/N185/N72/N V/N198/N72/N V/N76/N72/N198/N72/N V/N72/N V/N72/N72/N72/N V/N72/N V/N72/N V/N72/N72/N V/N72/, K23/N45/L124/K167/R195, K23/N45/Q73/T163, K28/N45/W149/S158/P198, K28/N45/K57/I98/R195, K28/N45/V129/T163/R195, M41/D43/N45/R64/S99, N45/R68/F173/D197/P198, N45/V50/I148/R195/N201, M41/D43/K44/N45/R195/N201, or N45/V50/L124/K144/L179/R195.
13. The variant PD-L1 polypeptide of any one of embodiments 1-13, wherein the one or more amino acid modifications correspond to one or more positions selected from: 20. 27, 28, 33, 36, 41, 43, 45, 50, 58, 71, 75, 78, 97, 99 or 195, optionally wherein the one or more amino acid substitutions is selected from I20L, E27G, K28E, a33D, I36T, M41K, D43G, N45D, N45T, V50A, V58A, K71E, S75P, N78I, M97L, S99G or R195G or conservative amino acid substitutions thereof.
14. The variant PD-L1 polypeptide of any one of embodiments 1-13, wherein the one or more amino acid modifications correspond to one or more positions selected from: 20. 27, 33, 36, 43, 45, 50, 58, 75, or 78, optionally wherein the one or more amino acid substitutions is selected from I20L, E27G, a33D, I36T, D43G, N45D, N45T, V50A, V58A, S75P, N78I, or conservative amino acid substitutions thereof.
15. The variant PD-L1 polypeptide of any one of
16. The variant PD-L1 polypeptide of any one of claims 1-15, wherein the variant PD-L1 polypeptide comprises the amino acid modifications D43G/N45D/V58A.
17. The variant PD-L1 polypeptide of any one of claims 1-16, wherein the variant PD-L1 polypeptide comprises the amino acid modifications D43G/N45D/L56Q/V58A/G101G-ins (G101GG) or I20L/K28E/D43G/N45D/V58A/Q89R/G101G-ins (G101 GG).
18. The variant PD-L1 polypeptide of any one of embodiments 1-17, wherein:
the variant PD-L1 polypeptide comprises or consists of an extracellular domain (ECD); and/or
The variant PD-L1 polypeptide comprises or consists of the IgV domain or a specific fragment thereof and the IgC domain or a specific fragment thereof.
19. The variant PD-L1 polypeptide of any one of
20. The variant PD-L1 polypeptide of any one of embodiments 1-19, wherein the variant PD-L1 polypeptide comprises or consists of the ECD or IgV domain or a specific binding fragment thereof.
21. The variant PD-L1 polypeptide of any one of embodiments 1-20, wherein the IgV domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.
22. The variant PD-L1 polypeptide of any of
23. The variant PD-L1 polypeptide of any one of embodiments 1-17, wherein the IgC domain or a specific binding fragment thereof is the only PD-L1 portion of the variant PD-L1 polypeptide.
24. The variant PD-L1 polypeptide of any one of embodiments 1-23, wherein the variant PD-L1 polypeptide specifically binds to an extracellular domain of PD-1 or CD80 with increased affinity as compared to the unmodified PD-L1.
25. The variant PD-L1 polypeptide of any one of embodiments 1-24, wherein the variant PD-L1 polypeptide specifically binds to the extracellular domain of PD-1 with increased affinity as compared to the unmodified PD-L1.
26. The variant PD-L1 polypeptide of any one of embodiments 1-25, wherein the variant PD-L1 polypeptide specifically binds to the extracellular domain of PD-1 and the extracellular domain of CD80, respectively, with increased affinity as compared to the unmodified PD-L1.
27. The variant PD-L1 polypeptide of any one of embodiments 1-24, wherein the variant PD-L1 polypeptide specifically binds to the extracellular domain of PD-1 with increased affinity and specifically binds to the extracellular domain of CD80 with decreased affinity as compared to the unmodified PD-L1.
28. The variant PD-L1 polypeptide of any one of embodiments 24-27, wherein the increased affinity for the extracellular domain of PD-1 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to the unmodified PD-L1.
29. The variant PD-L1 polypeptide of embodiment 24 or embodiment 26, wherein the increased affinity for the extracellular domain of CD80 is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to the unmodified PD-L1.
30. The variant PD-L1 polypeptide of embodiment 27, wherein the reduced affinity for the extracellular domain of CD80 is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold as compared to the unmodified PD-L1.
31. The variant PD-L1 polypeptide of any one of embodiments 1-30, wherein the variant polypeptide specifically binds to the extracellular domain of PD-1 with increased selectivity as compared to the unmodified PD-L1.
32. The variant PD-L1 polypeptide of embodiment 31, wherein the increased selectivity comprises a greater ratio of binding of the variant polypeptide to PD-1 versus CD80 as compared to the ratio of binding of the unmodified PD-L1 polypeptide to PD-1 versus CD80.
33. The variant PD-L1 polypeptide of embodiment 32, wherein the ratio is at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more greater.
34. The variant PD-L1 polypeptide of any one of embodiments 9-33, wherein the PD-1 is human PD-1.
35. The variant PD-L1 polypeptide of any one of embodiments 9-34, wherein the CD80 is human CD80.
36. The variant PD-L1 polypeptide of any one of embodiments 1-35, wherein the binding activity is altered (increased or decreased) by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold as compared to the unmodified PD-L1.
37. The variant PD-L1 polypeptide of any one of embodiments 1-36, which is a soluble protein.
38. The variant PD-L1 polypeptide of any one of embodiments 1-37, wherein the variant PD-L1 polypeptide is linked to a multimerization domain.
39. The variant PD-L1 polypeptide of embodiment 38, wherein the multimerization domain is an Fc domain or a variant thereof having reduced effector function.
40. The variant PD-L1 polypeptide of any one of embodiments 1-39, wherein the variant PD-L1 polypeptide is linked to a moiety that increases the biological half-life of the polypeptide.
41. The variant PD-L1 polypeptide of any one of embodiments 1-40, wherein the variant PD-L1 polypeptide is linked to an Fc domain or a variant thereof having reduced effector function.
42. The variant PD-L1 polypeptide of any one of embodiments 39-41, wherein:
the Fc domain is a mammalian Fc domain, optionally a human Fc domain; or
The variant Fc domain comprises one or more amino acid modifications as compared to a mammalian, optionally human, unmodified Fc domain.
43. The variant PD-L1 polypeptide of any one of embodiments 39, 41 and 42, wherein the Fc domain or variant thereof comprises the amino acid sequence set forth in SEQ ID No. 187 or SEQ ID No. 188, or an amino acid sequence having at least 85% sequence identity to SEQ ID No. 187 or SEQ ID No. 188.
44. The variant PD-L1 polypeptide of any one of embodiments 39 and 41-43, wherein the Fc domain comprises one or more amino acid modifications selected from: E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, R292C, V302C, and K447del, each by EU numbering.
45. The variant PD-L1 polypeptide of any one of embodiments 39 and 41-44, wherein the Fc domain comprises the amino acid modification C220S by EU numbering.
46. The variant PD-L1 polypeptide of any one of embodiments 39 and 41-45, wherein the Fc domain comprises the amino acid sequence set forth in any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs 1155, 1157, 1158, 1159, 1715, 1938, 1939, and 1940 and exhibiting reduced effector function.
47. The variant PD-L1 polypeptide of any one of embodiments 38-46, wherein the variant PD-L1 polypeptide is indirectly linked via a linker, optionally a G4S linker.
48. The variant PD-L1 polypeptide of any one of embodiments 1-47, which variant PD-L1 polypeptide is a transmembrane immunomodulatory protein, further comprising a transmembrane domain linked to the extracellular domain (ECD) of the variant PD-L1 polypeptide or a specific binding fragment thereof.
49. The variant PD-L1 polypeptide of embodiment 48, wherein the transmembrane domain comprises the amino acid sequence as set forth in residues 239-259 of SEQ ID NO:3 or a functional variant thereof having at least 85% sequence identity with residues 239-259 of SEQ ID NO: 3.
50. The variant PD-L1 polypeptide of embodiment 48 or embodiment 49, further comprising a cytoplasmic signaling domain linked to the transmembrane domain.
51. The variant PD-L1 polypeptide of embodiment 50, wherein the cytoplasmic signaling domain comprises the amino acid sequence as set forth in residues 260-290 of SEQ ID NO. 3 or a functional variant thereof having at least 85% sequence identity to residues 260-290 of SEQ ID NO. 3.
52. The variant PD-L1 polypeptide of any one of embodiments 1-51, wherein the variant PD-L1 increases IFN- γ (interferon- γ) expression relative to the unmodified PD-L1 in an in vitro T cell assay.
53. The variant PD-L1 polypeptide of any one of embodiments 1-52, wherein the variant PD-L1 reduces IFN- γ (interferon- γ) expression relative to the unmodified PD-L1 in an in vitro T cell assay.
54. The variant PD-L1 polypeptide of any one of embodiments 1-55, which is deglycosylated.
55. An immunomodulatory protein comprising the variant PD-L1 polypeptide of any one of embodiments 1-54 and a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of IgSF family members or an affinity modified IgSF domain thereof, the affinity modified IgSF domain comprising one or more amino acid modifications as compared to an unmodified or wild-type IgSF domain of the IgSF family member.
56. The immunomodulatory protein of embodiment 55, wherein the variant CD112 polypeptide and the second polypeptide are directly linked or indirectly linked via a linker.
57. The immunomodulatory protein of embodiment 55 or embodiment 56, wherein the IgSF domain is affinity modified and exhibits altered binding to one or more of its cognate binding partners compared to the unmodified or wild-type IgSF domain of the IgSF family member.
58. The immunomodulatory protein of embodiment 57, wherein the IgSF domain exhibits increased binding to one or more of its cognate binding partners as compared to the unmodified or wild-type IgSF domain of the IgSF family member.
59. The immunomodulatory protein of any of embodiments 58-58, wherein the variant PD-L1 polypeptide is a first PD-L1 variant and the IgSF domain of the second polypeptide is an IgSF domain from a second variant PD-L1 polypeptide of any of embodiments 1-58, wherein the first PD-L1 variant polypeptide and the second PD-L1 variant polypeptide are the same or different.
60. The immunomodulatory protein of any of embodiments 58-59, wherein the variant PD-L1 polypeptide is capable of specifically binding to PD-1 or CD80, and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than the cognate binding partner specifically bound by the PD-L1 variant polypeptide.
61. The immunomodulatory protein of any of embodiments 58-60, wherein the IgSF domain is from a member of the B7 family.
62. The immunomodulatory protein of any of embodiments 58-61, wherein the IgSF domain is bound to a tumor-localization moiety of a ligand expressed on a tumor or to an inflammatory-localization moiety of a cell or tissue associated with an inflammatory environment.
63. The immunomodulatory protein of embodiment 62, wherein the ligand is B7H6.
64. The immunomodulatory protein of embodiment 62 or embodiment 63, wherein the IgSF domain is from NKp 30.
65. The immunomodulatory protein of any of embodiments 55-64, wherein the IgSF domain of the second polypeptide is an IgSF domain that binds to a ligand of an inhibitory receptor or is an affinity modified IgSF domain thereof, optionally wherein the affinity modified IgSF domain exhibits increased binding affinity and/or binding selectivity for the inhibitory receptor as compared to binding of the unmodified IgSF domain to the inhibitory receptor.
66. The immunomodulatory protein of embodiment 65, wherein:
the inhibitory receptor is TIGIT, PD-1 or CTLA-4; or
The ligand of the inhibitory receptor is PD-L2, CD155, CD112 or CD80.
67. The immunomodulatory protein of any of embodiments 55-66, wherein the second polypeptide is selected from the group consisting of:
(i) wild type CD155 comprising an IgSF as set out in any of SEQ ID NOs 47, 310 or 353, or a variant CD155 polypeptide comprising an IgSF domain as set out in any of SEQ ID NOs 311-352, 354-665, 1505-1576, 1551-1714;
(ii) wild-type CD112 comprising an IgSF domain as set out in any of SEQ ID NO 48, 666 or 761, or a variant CD112 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 667-760, 762-931, 1433-1504;
(iii) wild-type CD80 comprising an IgSF domain as set out in any of SEQ ID NO 28, 1005 or 2030, or a variant CD80 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 932-964, 966-1038, 1040-1078, 1080-1112, 1114-1152;
(iv) wild-type PD-L2 comprising the IgSF domain as set forth in any of SEQ ID NOS 31, 1203 or 1263, or a variant PD-L2 polypeptide comprising the IgSF domain as set forth in any of SEQ ID NOS 1204-1254, 1256-1331, 1333-1407, 1409-1432;
(v) (iii) an amino acid sequence having at least 95% sequence identity to any one of the SEQ ID NOs of (i) - (iv) and comprising an amino acid substitution; or
(vi) (vi) a specific binding fragment of any one of (i) - (v).
68. The immunomodulatory protein of any of embodiments 55-67, further comprising a third polypeptide comprising an IgSF domain of an IgSF family member or an affinity modified IgSF domain thereof, said affinity modified IgSF domain comprising one or more amino acid modifications as compared to the unmodified or wild-type IgSF domain of the IgSF family member, wherein:
the third polypeptide is identical to the first polypeptide and/or the second polypeptide; or
The third polypeptide is different from the first polypeptide and/or the second polypeptide.
69. The immunomodulatory protein of embodiment 68, wherein the third polypeptide is selected from the group consisting of:
(i) wild type CD155 comprising an IgSF as set out in any of SEQ ID NOs 47, 310 or 353, or a variant CD155 polypeptide comprising an IgSF domain as set out in any of SEQ ID NOs 311-352, 354-665, 1505-1576, 1551-1714;
(ii) wild-type CD112 comprising an IgSF domain as set out in any of SEQ ID NO 48, 666 or 761, or a variant CD112 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 667-760, 762-931, 1433-1504;
(iii) wild-type CD80 comprising an IgSF domain as set out in any of SEQ ID NO 28, 1005 or 2030, or a variant CD80 polypeptide comprising an IgSF domain as set out in any of SEQ ID NO 932-964, 966-1038, 1040-1078, 1080-1112, 1114-1152;
(iv) wild-type PD-L2 comprising the IgSF domain as set forth in any of SEQ ID NOS 31, 1203 or 1263, or a variant PD-L2 polypeptide comprising the IgSF domain as set forth in any of SEQ ID NOS 1204-1254, 1256-1331, 1333-1407, 1409-1432;
(v) (iii) an amino acid sequence having at least 95% sequence identity to any one of the SEQ ID NOs of (i) - (iv) and comprising an amino acid substitution; or
(vi) (vi) a specific binding fragment of any one of (i) - (v).
70. The immunomodulatory protein of any of embodiments 55-69, wherein optionally the IgSF domain of the second or third polypeptide, or an affinity modified IgSF domain thereof, is or comprises an IgV domain.
71. The immunomodulatory protein of any of embodiments 55-70, wherein the variant PD-L1 polypeptide is or comprises an IgV domain.
72. The immunomodulatory protein of any of embodiments 55-71, further comprising at least one additional polypeptide comprising an IgSF domain of an IgSF family member or an affinity modified IgSF domain thereof, said affinity modified IgSF domain comprising one or more amino acid modifications as compared to the unmodified or wild-type IgSF domain of the IgSF family member.
73. The immunomodulatory protein of any of embodiments 55-67, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide or the second polypeptide.
74. The immunomodulatory protein of any of embodiments 55-73, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L1 polypeptide, the second polypeptide, and/or the third polypeptide, optionally wherein the multimerization domain is an Fc domain or a variant thereof having reduced effector function.
75. The immunomodulatory protein of any of embodiments 68-75, wherein the multimerization domain promotes heterodimer formation.
76. An immunomodulatory protein comprising a first variant PD-L1 polypeptide of any one of embodiments 38-47, wherein the multimerization domain is a first multimerization domain; and a second variant PD-L1 polypeptide of any one of embodiments 38-47, wherein a multimerization domain is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first and second variant PD-L1 and PD-L1 polypeptides, optionally wherein the first and second variant PD-L1 and PD-L1 polypeptides are identical.
77. An immunomodulatory protein comprising the immunomodulatory protein of any of embodiments 73-75, wherein the multimerization domain is a first multimerization domain and interacts with a second multimerization domain to form a multimer comprising the immunomodulatory protein.
78. The immunomodulatory protein of embodiment 77, wherein the immunomodulatory protein is a first immunomodulatory protein and a second immunomodulatory protein is directly or indirectly linked to the second multimerization domain via a linker, wherein the multimer comprises the first immunomodulatory protein and the second immunomodulatory protein, optionally wherein the second immunomodulatory protein is the immunomodulatory protein of any of claims 68-70, wherein the multimerization domain is the second multimerization domain.
79. The immunomodulatory protein of any of embodiments 76-78, wherein the multimer is a dimer.
80. The immunomodulatory protein of any of embodiments 76-79, the immunomodulatory protein being a homodimer, optionally wherein the first and second multimerization domains are the same.
81. The immunomodulatory protein of any of claims 77-80, wherein:
the second polypeptide is a variant CD155 polypeptide and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence set forth in any one of SEQ ID NO 1716-1721, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO 1716-1721; or
Or the second polypeptide is CD112 or CD155 and the third polypeptide is the other of CD112 or CD155 and the first immunomodulatory protein and/or the second immunomodulatory protein comprises a sequence as set forth in any one of SEQ ID NO 1722-1724 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO 1716-1721.
82. The immunomodulatory protein of any of claims 76-79, being a heterodimer, optionally wherein the first and second multimerization domains are different and/or are capable of interacting to mediate heterodimer formation.
83. The immunomodulatory protein of any of embodiments 76-82, wherein the first and/or second multimerization domain is an Fc domain or a variant thereof with reduced effector function, optionally wherein:
the Fc domain is an Fc domain of a human immunoglobulin and/or the Fc region is a human Fc region, optionally wherein the Fc region is an Fc region of immunoglobulin G1(IgG1) or immunoglobulin G2(IgG2), optionally listed in SEQ ID NO:187 or SEQ ID NO:188, optionally wherein the Fc region exhibits one or more effector functions; or
The variant Fc domain comprises one or more amino acid substitutions in a wild-type Fc region, optionally wherein the reduced effector function is reduced as compared to the wild-type Fc region, optionally wherein the wild-type human Fc is an Fc of
84. The immunomodulatory protein of any of claims 76-83, wherein the first multimerization domain and the second multimerization domain are the same or different.
85. The immunomodulatory protein of any of embodiments 74, 75, 83, and 84, wherein the variant Fc region comprises:
amino acid substitutions E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, wherein the residue numbering is according to the EU index of Kabat; or
Amino acid substitutions R292C/N297G/V302C or L234A/L235E/G237A, wherein the numbering of the residues is according to EU index of Kabat.
86. The immunomodulatory protein of any of embodiments 74, 75, and 83-85, wherein the Fc region or variant Fc region comprises the amino acid substitution C220S, wherein the numbering of residues is according to the EU index of Kabat.
87. The immunomodulatory protein of any of embodiments 74, 75, and 83-86, wherein the Fc region or variant Fc region comprises K447del, wherein the numbering of residues is according to the EU index of Kabat.
88. A conjugate comprising the variant PD-L1 of any one of embodiments 1-54 or the immunomodulatory protein of any one of embodiments 55-87 linked to a moiety.
89. The conjugate of embodiment 88, wherein the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell.
90. The conjugate of embodiment 89, wherein the targeting moiety specifically binds to a molecule on the surface of an immune cell, optionally wherein the immune cell is an antigen presenting cell or a lymphocyte.
91. The conjugate of embodiment 89, wherein the targeting moiety is a tumor localization moiety that binds to a molecule on the surface of a tumor.
92. The conjugate of any one of embodiments 88-91, wherein the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.
93. The conjugate of any one of embodiments 88-93, wherein the moiety is an antibody or antigen-binding fragment.
94. The conjugate of any one of embodiments 88-94, wherein the conjugate is bivalent, tetravalent, hexavalent, or octavalent.
95. The conjugate of any one of embodiments 88-94, which is a fusion protein.
96. A nucleic acid molecule encoding the variant PD-L1 polypeptide of any one of embodiments 1-54, the immunomodulatory protein of any one of embodiments 55-87, or a conjugate that is a fusion protein of any one of embodiments 88-95.
97. The nucleic acid molecule of embodiment 96, which is a synthetic nucleic acid.
98. The nucleic acid molecule of embodiment 96 or embodiment 97, which is a cDNA.
99. A vector comprising the nucleic acid molecule of any one of embodiments 96-98.
100. The vector of embodiment 99, which is an expression vector.
101. The vector of embodiment 99 or
102. A cell comprising the vector of
103. The cell of embodiment 102, which is a mammalian cell.
104. The cell of embodiment 102 or embodiment 103, which is a human cell.
105. A method of producing a variant PD-L1 polypeptide or immunomodulatory protein, comprising introducing a nucleic acid molecule according to any one of embodiments 96-98 or a vector according to any one of embodiments 99-101 into a host cell under conditions in which the protein is expressed in the cell.
106. The method of embodiment 105, further comprising isolating or purifying the variant PD-L1 polypeptide or immunomodulatory protein from the cell.
107. A method of engineering a cell expressing a variant PD-L1 variant polypeptide, comprising introducing into the cell a nucleic acid molecule encoding the variant PD-L1 polypeptide of any one of embodiments 1-54 under conditions in which the polypeptide is expressed in the host cell.
108. An engineered cell that expresses the variant PD-L1 polypeptide of any one of embodiments 1-54, the immunomodulatory protein of any one of embodiments 55-87, a conjugate that is the fusion protein of any one of claims 88-95, the nucleic acid molecule of any one of embodiments 96-98, or the vector of any one of embodiments 99-101.
109. The engineered cell of embodiment 108, wherein the variant PD-L1 polypeptide or immunomodulatory protein is encoded by a nucleic acid comprising a nucleotide sequence encoding a signal peptide.
110. The engineered cell of embodiment 108 or embodiment 109, wherein the variant PD-L1 polypeptide or immunomodulatory protein does not comprise a transmembrane domain and/or is not expressed on the surface of the cell.
111. The engineered cell of any one of embodiments 108-110, wherein the variant PD-L1 polypeptide or immunomodulatory protein is secreted from the engineered cell.
112. The engineered cell of embodiment 108 or embodiment 110, wherein the engineered cell comprises a variant PD-L1 polypeptide that comprises a transmembrane domain and/or is a transmembrane immunomodulatory protein of any one of embodiments 48-54.
113. The engineered cell of any one of embodiments 108, 110, and 112, wherein the variant PD-L1 polypeptide is expressed on the surface of the cell.
114. The engineered cell of any one of embodiments 108-113, wherein the cell is an immune cell.
115. The engineered cell of embodiment 114, wherein the immune cell is an Antigen Presenting Cell (APC) or a lymphocyte.
116. The engineered cell of any one of embodiments 108-115, which is a primary cell.
117. The engineered cell of any one of embodiments 108-116, wherein the cell is a mammalian cell.
118. The engineered cell of any one of embodiments 108-117, wherein the cell is a human cell.
119. The engineered cell of any one of embodiments 108-118, wherein the cell is a lymphocyte and the lymphocyte is a T cell.
120. The engineered cell of embodiment 115, wherein the cell is an APC and the APC is an artificial APC.
121. The engineered cell of any one of embodiments 108-120, further comprising a Chimeric Antigen Receptor (CAR) or an engineered T cell receptor.
122. An infectious agent comprising a nucleic acid molecule encoding a variant PD-L1 polypeptide of any one of embodiments 1-54, or an immunomodulatory protein of any one of embodiments 55-84, or a conjugate that is a fusion protein of any one of claims 88-95.
123. The infectious agent of embodiment 122, wherein the encoded variant PD-L1 polypeptide or immunomodulatory protein does not comprise a transmembrane domain and/or is not expressed on the surface of a cell in which it is expressed.
124. The infectious agent of embodiment 122 or embodiment 123, wherein the encoded variant PD-L1 polypeptide or immunomodulatory protein is secreted from the cells in which it is expressed.
125. The infectious agent of embodiment 122, wherein the encoded variant PD-L1 polypeptide comprises a transmembrane domain.
126. The infectious agent of embodiment 122 or embodiment 125, wherein the encoded variant PD-L1 polypeptide is expressed on the surface of a cell in which it is expressed.
127. The infectious agent of any one of embodiments 122-126, wherein the infectious agent is a bacterium or a virus.
128. The infectious agent of embodiment 127, wherein said infectious agent is a virus and said virus is an oncolytic virus.
129. The infectious agent of embodiment 128, wherein the oncolytic virus is an adenovirus, an adeno-associated virus, a herpesvirus, a herpes simplex virus, a vesicular stomatitis virus, a reovirus, a newcastle disease virus, a parvovirus, a measles virus, a Vesicular Stomatitis Virus (VSV), a coxsackie virus, or a vaccinia virus.
130. The infectious agent of embodiment 129, wherein the virus specifically targets Dendritic Cells (DCs) and/or is dendritic cell tropic.
131. The infectious agent of embodiment 130, wherein the virus is a lentiviral vector pseudotyped with a modified sindbis virus envelope product.
132. The infectious agent of any one of embodiments 122-131, further comprising a nucleic acid molecule encoding another gene product that causes death of the target cell or is capable of enhancing or increasing an immune response.
133. The infectious agent of embodiment 132, wherein the another gene product is selected from the group consisting of an anti-cancer agent, an anti-metastatic agent, an anti-angiogenic agent, an immune modulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cellular matrix degradation gene, a gene for tissue regeneration or reprogramming of human cells to pluripotency.
134. A pharmaceutical composition comprising a variant PD-L1 polypeptide according to any one of embodiments 1-47 and 48-54, an immunomodulatory protein according to any one of embodiments 55-87, a conjugate according to any one of embodiments 88-95, or an engineered cell according to any one of embodiments 108-121 or an infectious agent according to any one of embodiments 122-133.
135. The pharmaceutical composition of embodiment 134, comprising a pharmaceutically acceptable excipient.
136. The pharmaceutical composition of embodiment 134 or embodiment 135, wherein the pharmaceutical composition is sterile.
137. An article of manufacture comprising a pharmaceutical composition as in any one of embodiments 134 and 136 in a vial.
138. The article of embodiment 137, wherein the vial is sealed.
139. A kit comprising a pharmaceutical composition as described in any one of embodiments 134-136 and instructions for use.
140. A kit comprising the article of manufacture of embodiment 137 or embodiment 138 and instructions for use.
141. A method of modulating an immune response in a subject comprising administering to the subject a pharmaceutical composition according to any one of embodiments 123-125.
142. A method of modulating an immune response in a subject comprising administering to the subject an engineered cell as described in any one of embodiments 108-121.
143. The method of embodiment 142, wherein the engineered cells are autologous to the subject.
144. The method of embodiment 142, wherein the engineered cells are allogeneic to the subject.
145. The method of any one of embodiments 141-144, wherein modulating the immune response treats the disease or condition in the subject.
146. The method of any one of embodiments 141-145, wherein the immune response is increased.
147. The method of any one of embodiments 141-146, wherein the subject is administered a soluble variant PD-L1 polypeptide or an immunomodulatory protein.
148. The method of any one of embodiments 141-147, wherein the variant PD-L1 polypeptide or immunomodulatory protein is an Fc fusion protein.
149. The method of any one of embodiments 142-148, wherein the variant PD-L1 polypeptide of any one of embodiments 1-47 and 52-54, the immunomodulatory protein of any one of embodiments 55-87, or the conjugate of 88-95 is administered to the subject.
150. The method of any one of embodiments 142-146, wherein the subject is administered an engineered cell comprising a secretable variant PD-L1 polypeptide.
151. The method of any one of embodiments 142-146 and 150, wherein the engineered cell of any one of embodiments 97-100 and 103-110 is administered to the subject.
152. The method of any one of embodiments 141, 145, and 146, wherein the infectious agent is administered to the subject, optionally under conditions in which an infectious agent encoding a variant PD-L1 polypeptide that is a secretable immunomodulatory protein infects tumor cells or immune cells, and the secretable immunomodulatory protein is secreted from the infected cells.
153. The method of any one of embodiments 141-152, wherein the disease or condition is a tumor or cancer.
154. The method of any one of embodiments 141-153, wherein the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancers.
155. The method of any one of embodiments 141-145, wherein the immune response is reduced.
156. The method of any one of embodiments 141, 145, and 155, wherein the subject is administered an immunomodulatory protein or conjugate comprising a variant PD-L1 polypeptide linked to an IgSF domain or a moiety that localizes to a cell or tissue of an inflammatory environment.
157. The method of embodiment 156, wherein the binding molecule comprises an antibody or antigen-binding fragment thereof or comprises a wild-type IgSF domain or variant thereof.
158. The method of any one of embodiments 141-145 and 155-157, wherein the immunomodulatory protein of any one of embodiments 57-76 or the conjugate of any one of embodiments 77-84 is administered to the subject.
159. The method of any one of embodiments 141-145 and 155, wherein the subject is administered a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein.
160. The method of any one of embodiments 142-145 and 155-159, wherein the engineered cell comprising a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein as described in any one of embodiments 48-87 is administered to the subject.
161. The method of any one of embodiments 141, 145, and 155, wherein the infectious agent is administered to the subject, optionally under conditions in which an infectious agent encoding a variant PD-L1 polypeptide that is a transmembrane immunomodulatory protein infects tumor cells or immune cells and the transmembrane immunomodulatory protein is expressed on the surface of the infected cells.
162. The method of any one of embodiments 141-145 and 155-161, wherein the disease or condition is an inflammatory or autoimmune disease or condition.
163. The method of any one of embodiments 141-145 and 155-162, wherein the disease or condition is anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, an autoimmune skin disease, transplantation, rheumatism, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory lung disease, an inflammatory endocrine disease, or an autoimmune hematological disease.
164. The method of embodiment 162 or embodiment 163, wherein the disease or condition is selected from inflammatory bowel disease, transplantation, crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.
IX. example
The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：在抗体制造期间控制粉红色形成的方法