anti-TREM 2 antibodies and methods of use thereof
阅读说明：本技术 抗trem2抗体及其使用方法 (anti-TREM 2 antibodies and methods of use thereof ) 是由 马克·S·丹尼斯 雪莉·邓肯 凯瑟琳·利辛戈 凯瑟琳·M·梦露 约书亚·I·帕克 瑞秋·普 于 2020-02-20 设计创作，主要内容包括：在一个方面,提供了特异性结合到人类髓系细胞表达的触发受体2(TREM2)蛋白的抗体。在一些实施方案中,所述抗体降低可溶性TREM2(sTREM2)的水平。在一些实施方案中,所述抗体增强TREM2活性。(In one aspect, antibodies are provided that specifically bind to a trigger receptor 2(TREM2) protein expressed by human myeloid lineage cells. In some embodiments, the antibody reduces the level of soluble TREM2(sTREM 2). In some embodiments, the antibody enhances TREM2 activity.)
1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof comprises:
(a) CDR-H1 sequence comprising the sequence G-F-T-F-T-alpha6-F-Y-M-S (SEQ ID NO:48), wherein alpha6Is D or N;
(b) CDR-H2 sequence comprising the sequence V-I-R-N-beta5-β6-N-β8-Y-T-β11-β12-Y-N-P-S-V-K-G (SEQ ID NO:49), wherein5Is K or R; beta is a6Is A or P; beta is a8Is G or A; beta is a11Is A or T; and beta12Is G or D;
(c) CDR-H3 sequence comprising the sequence gamma1-R-L-γ4-Y-G-F-D-Y (SEQ ID NO:50), wherein γ1Is A or T; and gamma is4Is T or S;
(d) CDR-L1 sequence comprising the sequence Q-S-S-K-S-L-L-H-S-delta10-G-K-T-Y-L-N (SEQ ID NO:51), wherein delta10Is N or T;
(e) a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and
(f) CDR-L3 sequence comprising the sequence Q-Q-F-L-E-phi6-P-F-T (SEQ ID NO:52), wherein phi6Is Y or F.
2. The isolated antibody or antigen-binding fragment of claim 1, wherein the CDR-H1 sequence is selected from any one of SEQ ID NOs 4 and 12.
3. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the CDR-H2 sequence is selected from any one of SEQ ID NOs 5, 13, and 25.
4. The isolated antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the CDR-H3 sequence is selected from any one of SEQ ID NOs 6, 14, and 17.
5. The isolated antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the CDR-L1 sequence is selected from any one of SEQ ID NOs 7 and 23.
6. The isolated antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the CDR-L3 sequence is selected from any one of SEQ ID NOs 9 and 18.
7. The isolated antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(e) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 6, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(f) CDR-H1 comprising the amino acid sequence SEQ ID NO. 12, CDR-H2 comprising the amino acid sequence SEQ ID NO. 13, CDR-H3 comprising the amino acid sequence SEQ ID NO. 14, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(g) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9.
8. The isolated antibody or antigen-binding fragment of any one of claims 1 to 7, comprising a V having at least 85% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, and 79HAnd (4) sequencing.
9. The isolated antibody or antigen-binding fragment of claim 8, wherein the VHThe sequence has at least 90% sequence identity with SEQ ID NO. 15.
10. The isolated antibody or antigen-binding fragment of claim 9, wherein the VHThe sequence has at least 95% sequence identity with SEQ ID NO. 15.
11. The isolated antibody or antigen-binding fragment of claim 10, wherein the VHThe sequence comprises SEQ ID NO 15.
12. The isolated antibody or antigen-binding fragment of claim 8, wherein the VHThe sequence has at least 90% sequence identity with SEQ ID NO. 24.
13. The isolated antibody or antigen-binding fragment of claim 12, wherein the VHThe sequence has at least 95% sequence identity with SEQ ID NO. 24.
14. The isolated antibody or antigen-binding fragment of claim 13, wherein the VHThe sequence comprises SEQ ID NO 24.
15. The isolated antibody or antigen-binding fragment of claim 8, wherein the VHThe sequence has at least 90% sequence identity with SEQ ID NO. 79.
16. The isolated antibody or antigen-binding fragment of claim 15, wherein the VHThe sequence has at least 95% sequence identity with SEQ ID NO. 79.
17. The isolated antibody or antigen-binding fragment of claim 16, wherein the VHThe sequence comprises SEQ ID NO 79.
18. The isolated antibody or antigen-binding fragment of any one of claims 1 to 17, comprising a V having at least 85% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, and 68LAnd (4) sequencing.
19. The isolated antibody or antigen-binding fragment of claim 18, wherein the VLThe sequence has at least 90% sequence identity with SEQ ID NO 16.
20. The isolated antibody or antigen-binding fragment of claim 19, wherein the VLThe sequence has at least 95% sequence identity with SEQ ID NO 16.
21. The isolated antibody or antigen-binding fragment of claim 20, wherein the VLThe sequence comprises SEQ ID NO 16.
22. The isolated antibody or antigen-binding fragment of claim 18, wherein the VLThe sequence has at least 90% sequence identity with SEQ ID NO. 22.
23. The isolated antibody or antigen-binding fragment of claim 22, wherein the VLThe sequence has at least 95% sequence identity with SEQ ID NO. 22.
24. The isolated antibody or antigen-binding fragment of claim 23, wherein the VLThe sequence comprises SEQ ID NO 22.
25. The isolated antibody of claim 18Or an antigen binding fragment, wherein said VLThe sequence has at least 90% sequence identity with SEQ ID NO 68.
26. The isolated antibody or antigen-binding fragment of claim 25, wherein the VLThe sequence has at least 95% sequence identity with SEQ ID NO 68.
27. The isolated antibody or antigen-binding fragment of claim 26, wherein the VLThe sequence comprises SEQ ID NO 68.
28. The isolated antibody or antigen-binding fragment of any one of claims 1 to 27, wherein the antibody or antigen-binding fragment comprises:
(a) v comprising SEQ ID NO 15HSequences and V comprising SEQ ID NO 16LA sequence; or
(b) V comprising SEQ ID NO 19HSequences and V comprising SEQ ID NO 20LA sequence; or
(c) V comprising SEQ ID NO 21HSequences and V comprising SEQ ID NO 20LA sequence; or
(d) V comprising SEQ ID NO 19HSequences and V comprising SEQ ID NO 22LA sequence; or
(e) V comprising SEQ ID NO 79HSequences and V comprising SEQ ID NO 22LA sequence; or
(f) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 20LA sequence; or
(g) V comprising SEQ ID NO 26HSequences and V comprising SEQ ID NO 20LA sequence; or
(h) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 22LA sequence; or
(i) V comprising SEQ ID NO 26HSequences and V comprising SEQ ID NO 22LA sequence; or
(j) V comprising SEQ ID NO 2HSequences and V comprising SEQ ID NO 3LA sequence; or
(k) V comprising SEQ ID NO 10HSequences and V comprising SEQ ID NO 11LA sequence; or
(l) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 68LAnd (4) sequencing.
29. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof comprises:
(a) a CDR-H1 sequence comprising sequence GFSIEDFYIH (SEQ ID NO: 29);
(b) CDR-H2 sequence comprising the sequence W-I-D-P-E-beta6-G-β8-S-K-Y-A-P-K-F-Q-G (SEQ ID NO:47), wherein6Is N or Q and beta8Is D or E;
(c) a CDR-H3 sequence comprising sequence HADHGNYGSTMDY (SEQ ID NO: 31);
(d) a CDR-L1 sequence comprising sequence HASQHINVWLS (SEQ ID NO: 32);
(e) a CDR-L2 sequence comprising the sequence KASNLHT (SEQ ID NO: 33); and
(f) a CDR-L3 sequence comprising sequence QQGQTYPRT (SEQ ID NO: 34).
30. The isolated antibody or antigen-binding fragment of claim 29, wherein the CDR-H2 sequence is selected from the group consisting of SEQ ID NOs 30, 39, 41 and 43.
31. The isolated antibody or antigen-binding fragment of claim 29 or 30, wherein the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 30, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 39, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 41, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 43, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34.
32. The isolated antibody or antigen-binding fragment of any one of claims 29 to 31, comprising a V having at least 85% sequence identity to any one of SEQ ID NOs 27, 35, 37, 38, 40, 42, 44, 45, and 46HAnd (4) sequencing.
33. The isolated antibody or antigen-binding fragment of claim 32, wherein the VHThe sequence has at least 90% sequence identity with SEQ ID NO. 27.
34. The isolated antibody or antigen-binding fragment of claim 33, wherein the VHThe sequence has at least 95% sequence identity with SEQ ID NO 27.
35. The isolated antibody or antigen-binding fragment of claim 34, wherein the VHThe sequence comprises SEQ ID NO 27.
36. The isolated antibody or antigen-binding fragment of any one of claims 29 to 35, comprising a V having at least 85% sequence identity to SEQ ID No. 28 or SEQ ID No. 36LAnd (4) sequencing.
37. The isolated antibody or antigen-binding fragment of claim 36, wherein the VLThe sequence has at least 90% sequence identity with SEQ ID NO 28.
38. The isolated antibody or antigen-binding fragment of claim 37, wherein the VLThe sequence has at least 95% sequence identity with SEQ ID NO 28.
39. The isolated antibody or antigen-binding fragment of claim 38, wherein the VLThe sequence comprises SEQ ID NO 28.
40. The isolated antibody or antigen-binding fragment of claim 36, wherein the antibody or antigen-binding fragment comprises:
(a) v comprising SEQ ID NO 27HSequences and V comprising SEQ ID NO 28LA sequence; or
(b) V comprising SEQ ID NO 35HSequences and V comprising SEQ ID NO 36LA sequence; or
(c) V comprising SEQ ID NO 37HSequences and V comprising SEQ ID NO 36LA sequence; or
(d) V comprising SEQ ID NO 38HSequences and V comprising SEQ ID NO 36LA sequence; or
(e) V comprising SEQ ID NO 40HSequences and V comprising SEQ ID NO 36LA sequence; or
(f) V comprising SEQ ID NO 42HSequences and V comprising SEQ ID NO 36LA sequence; or
(g) V comprising SEQ ID NO 44HSequences and V comprising SEQ ID NO 36LA sequence; or
(h) V comprising SEQ ID NO 45HSequences and V comprising SEQ ID NO 36LA sequence; or
(i) V comprising SEQ ID NO 46HSequences and V comprising SEQ ID NO 36LAnd (4) sequencing.
41. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof comprises:
(a) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 4, 12 and 29;
(b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 5, 13, 25, 30, 39, 41 and 43;
(c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 6, 14, 17 and 31;
(d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 7, 23 and 32;
(e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 8 and 33; and
(f) a CDR-L3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 9,18 and 34.
42. The isolated antibody or antigen-binding fragment of claim 41, wherein the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 6, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(e) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(f) CDR-H1 comprising the amino acid sequence SEQ ID NO. 12, CDR-H2 comprising the amino acid sequence SEQ ID NO. 13, CDR-H3 comprising the amino acid sequence SEQ ID NO. 14, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(g) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 30, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(h) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 39, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(i) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 41, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(j) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 43, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(k) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9.
43. The isolated antibody or antigen-binding fragment of claim 41 or 42, comprising a heavy chain variable region having at least 85% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79.
44. The isolated antibody or antigen-binding fragment of any one of claims 41 to 43, comprising a light chain variable region having at least 85% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, 28, and 36.
45. The isolated antibody or antigen-binding fragment of any one of claims 41 to 44, wherein the antibody or antigen-binding fragment comprises:
(a) v having at least 85% sequence identity to SEQ ID NO 2HSequences and V having at least 85% sequence identity to SEQ ID NO 3LA sequence; or
(b) V having at least 85% sequence identity to SEQ ID NO 10HSequences and V having at least 85% sequence identity to SEQ ID NO 11LA sequence; or
(c) V having at least 85% sequence identity to SEQ ID NO 15HSequences and V having at least 85% sequence identity to SEQ ID NO 16LA sequence; or
(d) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(e) Has at least 85% sequence with SEQ ID NO. 21V of column identityHSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(f) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(g) V having at least 85% sequence identity to SEQ ID NO 79HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(h) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(i) V having at least 85% sequence identity to SEQ ID NO 26HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(j) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(k) V having at least 85% sequence identity to SEQ ID NO 26HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(l) V having at least 85% sequence identity to SEQ ID NO 27HSequences and V having at least 85% sequence identity to SEQ ID NO 28LA sequence; or
(m) V having at least 85% sequence identity to SEQ ID NO 35HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(n) V having at least 85% sequence identity to SEQ ID NO:37HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(o) V having at least 85% sequence identity to SEQ ID NO:38HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(p) V having at least 85% sequence identity to SEQ ID NO 40HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(q) V having at least 85% sequence identity to SEQ ID NO:42HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(r) V having at least 85% sequence identity to SEQ ID NO:44HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(s) V having at least 85% sequence identity to SEQ ID NO:45HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(t) V having at least 85% sequence identity to SEQ ID NO 46HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(u) V having at least 85% sequence identity to SEQ ID NO:24HSequences and V having at least 85% sequence identity to SEQ ID NO 68LAnd (4) sequencing.
46. An isolated antibody or antigen-binding fragment thereof that specifically binds to trigger receptor 2(TREM2) expressed by human myeloid-lineage cells, wherein the antibody or antigen-binding fragment thereof recognizes an epitope that is the same as or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of: clone CL0020306, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7 and clone CL 0020123-8.
47. The isolated antibody or antigen-binding fragment of claim 46, wherein the antibody or antigen-binding fragment recognizes an epitope that is the same or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of: clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7 and clone CL 0020123-8.
48. The isolated antibody or antigen-binding fragment of claim 47, wherein the antibody or antigen-binding fragment recognizes one or more of the following in SEQ ID NO: 1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residue 126-129(VEVL (SEQ ID NO: 72)).
49. The isolated antibody or antigen-binding fragment of claim 46, wherein the antibody or antigen-binding fragment recognizes an epitope that is the same or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of: clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307 and clone CL 0020306.
50. The isolated antibody or antigen-binding fragment of claim 49, wherein the antibody or antigen-binding fragment recognizes amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO: 69)).
51. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of one or more of SEQ ID NO: 1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residue 126-129(VEVL (SEQ ID NO: 72)).
52. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO: 69)).
53. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 52, wherein the antibody or antigen-binding fragment thereof reduces the level of soluble TREM2 protein (sTREM 2).
54. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 53, wherein the antibody or antigen-binding fragment thereof enhances TREM2 activity.
55. The isolated antibody or antigen-binding fragment thereof of claim 54, wherein the antibody or antigen-binding fragment thereof enhances phagocytosis or enhances migration, differentiation, function, or survival of myeloid cells, microglia, or macrophages.
56. The isolated antibody or antigen-binding fragment thereof of claim 55, wherein the antibody or antigen-binding fragment thereof enhances microglial function without increasing neuroinflammation.
57. The isolated antibody or antigen-binding fragment thereof of claim 54, wherein the antibody or antigen-binding fragment thereof enhances Syk phosphorylation.
58. The isolated antibody or antigen-binding fragment thereof of claim 57, wherein the antibody or antigen-binding fragment thereof enhances Syk phosphorylation in the presence of a TREM2 ligand.
59. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 58, wherein the antibody or antigen-binding fragment thereof exhibits cross-reactivity to cynomolgus monkey TREM2 protein.
60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a monoclonal antibody.
61. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a chimeric antibody.
62. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a humanized antibody.
63. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a fully human antibody.
64. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antigen-binding fragment is Fab, F (ab')2scFv, or bivalent scFv.
65. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-64 and a pharmaceutically acceptable carrier.
66. An antibody or antigen-binding fragment thereof that competes for binding to a human TREM2 protein with the isolated antibody of any one of claims 1-64.
67. A kit, comprising:
the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 64 or the pharmaceutical composition of claim 65; and
instructions for its use.
68. A method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject the isolated antibody or antigen-binding fragment thereof of any one of claims 1-64 or the pharmaceutical composition of claim 65.
69. The method of claim 68, wherein the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, primary age-related tauopathy, Progressive Supranuclear Palsy (PSP), frontotemporal dementia with parkinsonism associated with chromosome 17, dementia with silvery particles, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis/Parkinson's disease-dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle calcification, Down's syndrome, familial dementia of great Britain, familial dementia of Danish type, GSS's disease, globular gliosis, Gualopt-Parkinson's disease, Gualopt PSP, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with globuloid change (HDLS), Huntington's disease, inclusion body myositis, multiple-system atrophy, myotonic dystrophy, Nara-Hara disease, dementia predominantly with neurofibrillary tangles, Niemann-pick disease type C, globus pallidus-pontine-substantia nigra degeneration, Parkinson's disease, pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle-only dementia.
70. A method of reducing the level of sTREM2 in a subject having a neurodegenerative disease, the method comprising administering to the subject the isolated antibody or antigen-binding fragment thereof of any one of claims 1-64 or the pharmaceutical composition of claim 65.
71. A method of enhancing TREM2 activity in a subject having a neurodegenerative disease, the method comprising administering to the subject the isolated antibody or antigen-binding fragment thereof of any one of claims 1-64 or the pharmaceutical composition of claim 65.
Background
The triggering receptor 2 expressed by myeloid cells (TREM2) is a transmembrane receptor that is expressed on microglia and is believed to play a role in regulating phagocytosis, cell survival, and proinflammatory cytokine production. Mutations in TREM2 have been identified in neurodegenerative diseases including Alzheimer's disease, nakai disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. In addition, altered levels of soluble TREM2(sTREM2) in cerebrospinal fluid have been reported in patients with alzheimer's disease or frontotemporal dementia and having a TREM2 mutation.
There remains a need for therapeutic agents that modulate TREM2 activity or sTREM2 levels.
Disclosure of Invention
In one aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to trigger receptor 2(TREM2) expressed by human myeloid cells is provided. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:
(a) a CDR-H1 sequence comprising sequence GFSIEDFYIH (SEQ ID NO: 29);
(b) CDR-H2 sequence comprising the sequence W-I-D-P-E-beta6-G-β8-S-K-Y-A-P-K-F-Q-G (SEQ ID NO:47), wherein6Is N or Q and beta8Is D or E;
(c) a CDR-H3 sequence comprising sequence HADHGNYGSTMDY (SEQ ID NO: 31);
(d) a CDR-L1 sequence comprising sequence HASQHINVWLS (SEQ ID NO: 32);
(e) a CDR-L2 sequence comprising the sequence KASNLHT (SEQ ID NO: 33); and
(f) a CDR-L3 sequence comprising sequence QQGQTYPRT (SEQ ID NO: 34).
In some embodiments, the CDR-H2 sequence is selected from SEQ ID NOs 30, 39, 41 and 43.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 30, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 39, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 41, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 43, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34.
In some embodiments, the antibody or antigen-binding fragment comprises a V having at least 85% sequence identity to any one of SEQ ID NOs 27, 35, 37, 38, 40, 42, 44, 45, and 46HAnd (4) sequencing. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO. 27. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO 27. In some embodiments, VHThe sequence comprises SEQ ID NO 27. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO 42. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO 42. In some embodiments, VHThe sequence comprises SEQ ID NO 42. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO 45. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO 45. In some embodiments, VHThe sequence comprises SEQ ID NO 45.
In some embodiments, the antibody or antigen binding fragment comprises a V having at least 85% sequence identity to SEQ ID NO 28 or SEQ ID NO 36LAnd (4) sequencing. In some embodiments, VLThe sequence has at least 90% sequence identity with SEQ ID NO 28. In some embodiments, VLThe sequence has at least 95% sequence identity with SEQ ID NO 28. In some embodiments, VLThe sequence comprises SEQ ID NO 28. In some embodiments, VLThe sequence has at least 90% sequence identity with SEQ ID NO: 36. In some embodiments, VLThe sequence has at least 95% sequence identity with SEQ ID NO: 36. In some embodiments, VLThe sequence comprises SEQ ID NO 36.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) v comprising SEQ ID NO 27HSequences and V comprising SEQ ID NO 28LA sequence; or
(b) V comprising SEQ ID NO 35HSequences and V comprising SEQ ID NO 36LA sequence; or
(c) V comprising SEQ ID NO 37HSequences and V comprising SEQ ID NO 36LA sequence; or
(d) V comprising SEQ ID NO 38HSequences and V comprising SEQ ID NO 36LA sequence; or
(e) V comprising SEQ ID NO 40HSequences and V comprising SEQ ID NO 36LA sequence; or
(f) V comprising SEQ ID NO 42HSequences and V comprising SEQ ID NO 36LA sequence; or
(g) V comprising SEQ ID NO 44HSequences and V comprising SEQ ID NO 36LA sequence; or
(h) V comprising SEQ ID NO 45HSequences and V comprising SEQ ID NO 36LA sequence; or
(i) V comprising SEQ ID NO 46HSequences and V comprising SEQ ID NO 36LAnd (4) sequencing.
In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:
(a) CDR-H1 sequence comprising the sequence G-F-T-F-T-alpha6-F-Y-M-S (SEQ ID NO:48), wherein alpha6Is D or N;
(b) CDR-H2 sequence comprising the sequence V-I-R-N-beta5-β6-N-β8-Y-T-β11-β12-Y-N-P-S-V-K-G (SEQ ID NO:49), wherein5Is K or R; beta is a6Is A or P; beta is a8Is G or A; beta is a11Is A or T; and beta12Is G or D;
(c) CDR-H3 sequence comprising the sequence gamma1-R-L-γ4-Y-G-F-D-Y (SEQ ID NO:50), wherein γ1Is A or T; and gamma is4Is T or S;
(d)CDR-L1 sequence comprising the sequence Q-S-S-K-S-L-L-H-S-delta10-G-K-T-Y-L-N (SEQ ID NO:51), wherein delta10Is N or T;
(e) a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and
(f) CDR-L3 sequence comprising the sequence Q-Q-F-L-E-phi6-P-F-T (SEQ ID NO:52), wherein phi6Is Y or F.
In some embodiments, the CDR-H1 sequence is selected from any one of SEQ ID NOs 4 and 12. In some embodiments, the CDR-H2 sequence is selected from any one of SEQ ID NOs 5, 13, and 25. In some embodiments, the CDR-H3 sequence is selected from any one of SEQ ID NOs 6, 14, and 17. In some embodiments, the CDR-L1 sequence is selected from any one of SEQ ID NOs 7 and 23. In some embodiments, the CDR-L3 sequence is selected from any one of SEQ ID NOs 9 and 18.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(e) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 6, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(f) CDR-H1 comprising the amino acid sequence SEQ ID NO. 12, CDR-H2 comprising the amino acid sequence SEQ ID NO. 13, CDR-H3 comprising the amino acid sequence SEQ ID NO. 14, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(g) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9.
In some embodiments, the antibody or antigen-binding fragment comprises a V having at least 85% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, and 79HAnd (4) sequencing. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO. 15. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO. 15. In some embodiments, VHThe sequence comprises SEQ ID NO 15. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO. 24. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO. 24. In some embodiments, VHThe sequence comprises SEQ ID NO 24. In some embodiments, VHThe sequence has at least 90% sequence identity with SEQ ID NO. 79. In some embodiments, VHThe sequence has at least 95% sequence identity with SEQ ID NO. 79. In some embodiments, VHThe sequence comprises SEQ ID NO 79.
At one endIn some embodiments, the antibody or antigen-binding fragment comprises a V having at least 85% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, and 68LAnd (4) sequencing. In some embodiments, VLThe sequence has at least 90% sequence identity with SEQ ID NO 16. In some embodiments, VLThe sequence has at least 95% sequence identity with SEQ ID NO 16. In some embodiments, VLThe sequence comprises SEQ ID NO 16. In some embodiments, VLThe sequence has at least 90% sequence identity with SEQ ID NO. 22. In some embodiments, VLThe sequence has at least 95% sequence identity with SEQ ID NO. 22. In some embodiments, VLThe sequence comprises SEQ ID NO 22. In some embodiments, VLThe sequence has at least 90% sequence identity with SEQ ID NO 68. In some embodiments, VLThe sequence has at least 95% sequence identity with SEQ ID NO 68. In some embodiments, VLThe sequence comprises SEQ ID NO 68.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) v comprising SEQ ID NO 15HSequences and V comprising SEQ ID NO 16LA sequence; or
(b) V comprising SEQ ID NO 19HSequences and V comprising SEQ ID NO 20LA sequence; or
(c) V comprising SEQ ID NO 21HSequences and V comprising SEQ ID NO 20LA sequence; or
(d) V comprising SEQ ID NO 19HSequences and V comprising SEQ ID NO 22LA sequence; or
(e) V comprising SEQ ID NO 79HSequences and V comprising SEQ ID NO 22LA sequence; or
(f) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 20LA sequence; or
(g) V comprising SEQ ID NO 26HSequences and V comprising SEQ ID NO 20LA sequence; or
(h) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 22LA sequence; or
(i) V comprising SEQ ID NO 26HSequences and V comprising SEQ ID NO 22LA sequence; or
(j) V comprising SEQ ID NO 2HSequences and V comprising SEQ ID NO 3LA sequence; or
(k) V comprising SEQ ID NO 10HSequences and V comprising SEQ ID NO 11LA sequence; or
(l) V comprising SEQ ID NO 24HSequences and V comprising SEQ ID NO 68LAnd (4) sequencing.
In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:
(a) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 4, 12 and 29;
(b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 5, 13, 25, 30, 39, 41 and 43;
(c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 6, 14, 17 and 31;
(d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 7, 23 and 32;
(e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 8 and 33; and
(f) a CDR-L3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 9,18 and 34.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 6, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(e) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(f) CDR-H1 comprising the amino acid sequence SEQ ID NO. 12, CDR-H2 comprising the amino acid sequence SEQ ID NO. 13, CDR-H3 comprising the amino acid sequence SEQ ID NO. 14, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9;
(g) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 30, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(h) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 39, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(i) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 41, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(j) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 43, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(k) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9.
In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region having at least 85% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region having at least 85% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, 28, 36, and 68.
In some embodiments, the antibody or antigen-binding fragment comprises:
(a) v having at least 85% sequence identity to SEQ ID NO 2HSequences and V having at least 85% sequence identity to SEQ ID NO 3LA sequence; or
(b) V having at least 85% sequence identity to SEQ ID NO 10HSequences and V having at least 85% sequence identity to SEQ ID NO 11LA sequence; or
(c) V having at least 85% sequence identity to SEQ ID NO 15HSequences and V having at least 85% sequence identity to SEQ ID NO 16LA sequence; or
(d) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 20LSequence of(ii) a Or
(e) V having at least 85% sequence identity to SEQ ID NO 21HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(f) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(g) V having at least 85% sequence identity to SEQ ID NO 79HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(h) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(i) V having at least 85% sequence identity to SEQ ID NO 26HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(j) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(k) V having at least 85% sequence identity to SEQ ID NO 26HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(l) V having at least 85% sequence identity to SEQ ID NO 27HSequences and V having at least 85% sequence identity to SEQ ID NO 28LA sequence; or
(m) V having at least 85% sequence identity to SEQ ID NO 35HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(n) V having at least 85% sequence identity to SEQ ID NO:37HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(o) V having at least 85% sequence identity to SEQ ID NO:38HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence;or
(p) V having at least 85% sequence identity to SEQ ID NO 40HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(q) V having at least 85% sequence identity to SEQ ID NO:42HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(r) V having at least 85% sequence identity to SEQ ID NO:44HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(s) V having at least 85% sequence identity to SEQ ID NO:45HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(t) V having at least 85% sequence identity to SEQ ID NO 46HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(u) V having at least 85% sequence identity to SEQ ID NO:24HSequences and V having at least 85% sequence identity to SEQ ID NO 68LAnd (4) sequencing.
In some embodiments, the epitope recognized by an antibody or antigen binding fragment thereof that specifically binds to TREM2 is the same as or substantially the same as the epitope recognized by an antibody clone selected from the group consisting of: clone CL0020306, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7 and clone CL 0020123-8.
In some embodiments, the epitope recognized by the antibody or antigen binding fragment thereof is the same or substantially the same as the epitope recognized by an antibody clone selected from the group consisting of: clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7 and clone CL 0020123-8. In particular embodiments, the antibody or antigen binding fragment recognizes one or more of the following epitopes in SEQ ID NO: 1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residue 126-129(VEVL (SEQ ID NO: 72)). In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of one or more of the following epitopes in SEQ ID NO: 1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residue 126-129(VEVL (SEQ ID NO: 72)). In some embodiments, the epitope recognized by the antibody or antigen binding fragment thereof is the same or substantially the same as the epitope recognized by an antibody clone selected from the group consisting of: clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307 and clone CL 0020306. In a particular embodiment, the antibody or antigen binding fragment recognizes amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO: 69)). In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO: 69)).
In some embodiments, an antibody or antigen-binding fragment as disclosed herein reduces the level of soluble TREM2 protein (sTREM 2). In some embodiments, an antibody or antigen-binding fragment as disclosed herein binds to soluble TREM2 protein (sTREM2) in healthy human CSF or cynomolgus monkey CSF with better potency compared to a reference antibody. In some embodiments, the reference antibody is represented by a combination of sequences selected from the group consisting of: 73 and 74; 75 and 76 for SEQ ID NO; and SEQ ID NOS 77 and 78. In some embodiments, the potency assay is performed essentially as described in example 11. In some embodiments, an antibody or antigen-binding fragment as disclosed herein enhances TREM2 activity. In some embodiments, the antibody or antigen-binding fragment thereof enhances phagocytosis or enhances migration, differentiation, function, or survival of myeloid cells, microglia, or macrophages. In some embodiments, the antibody or antigen binding fragment thereof enhances microglial function without increasing neuroinflammation. In some embodiments, the antibody or antigen binding fragment thereof enhances Syk phosphorylation. In some embodiments, the antibody or antigen binding fragment thereof enhances Syk phosphorylation in the presence of a TREM2 ligand. In some embodiments, the antibody or antigen-binding fragment thereof exhibits cross-reactivity with cynomolgus monkey TREM2 protein.
In some embodiments, an antibody or antigen binding fragment as disclosed herein is a monoclonal antibody. In some embodiments, an antibody or antigen-binding fragment as disclosed herein is a chimeric antibody. In some embodiments, an antibody or antigen-binding fragment as disclosed herein is a humanized antibody. In some embodiments, an antibody or antigen-binding fragment as disclosed herein is a fully human antibody. In some embodiments, an antibody or antigen binding fragment as disclosed herein is a Fab, F (ab')2scFv, or bivalent scFv.
In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes for binding to a human TREM2 protein with an isolated anti-TREM 2 antibody as disclosed herein.
In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment as disclosed herein that specifically binds to TREM2 and a pharmaceutically acceptable carrier.
In another aspect, the present disclosure provides a kit comprising: an antibody or antigen-binding fragment as disclosed herein or a pharmaceutical composition comprising an anti-TREM 2 antibody or antigen-binding fragment that specifically binds to TREM 2; and instructions for use thereof.
In another aspect, the present disclosure provides a method of treating a neurodegenerative disease in a subject. In some embodiments, the method comprises administering to the subject an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein or a pharmaceutical composition comprising an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein.
In some embodiments, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, primary age-related tauopathies, Progressive Supranuclear Palsy (PSP), frontotemporal dementia with parkinsonism associated with chromosome 17, dementia with silvery particles, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle calcification, Down syndrome, familial English dementia, familial Danish dementia, GSS's disease (Gerstmann-Straussler-Scheinker disease), Globulinopathy, Gualoputida parkinsonism with dementia (Guarel parkinsonism), Guader P PSP, Waller-Splender disease (Halley Splending) and Creutzfeldt-Jakob disease, Hereditary diffuse leukoencephalopathy with globuloid changes (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, dementia with predominant neurofibrillary tangles, Niemann-Pick disease type C (Niemann-Pick disease type C), pallido-ponto-nigrostriatosis (pallido-nigrograft degeneration), parkinson's disease, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle-only dementia.
In yet another aspect, the present disclosure provides methods of reducing the level of sTREM2 in a subject having a neurodegenerative disease. In some embodiments, the method comprises administering to the subject an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein or a pharmaceutical composition comprising an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein.
In yet another aspect, the present disclosure provides methods of enhancing TREM2 activity in a subject having a neurodegenerative disease. In some embodiments, the method comprises administering to the subject an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein or a pharmaceutical composition comprising an anti-TREM 2 antibody or antigen-binding fragment as disclosed herein.
Drawings
Figure 1 includes representative flow cytometry histograms representing binding of an exemplary anti-TREM 2 antibody to surface TREM2 on HEK cells expressing TREM 2.
Figure 2 includes a representative dose-response curve of activation of pSyk signaling by an exemplary anti-TREM 2 antibody in primary human macrophages. Filled black circles (●) represent anti-TREM 2 antibody, and open white circles (°) represent isotype controls.
Fig. 3A and 3B include representative dose response curves for activation of pSyk signal in human iPSC microglia after pretreatment with an exemplary anti-TREM 2 antibody for 5 minutes (fig. 3A) or 24 hours (fig. 3B), followed by administration of lipid vesicles and assessment of liposome response in the cells.
Figure 4 includes representative dose response curves for NFAT-luciferase reporter activity generated in response to stimulation with an exemplary anti-TREM 2 antibody in Jurkat NFAT cells expressing human TREM2/DAP 12. Filled black circles (●) represent anti-TREM 2 antibody, and open white circles (°) represent isotype controls.
Figure 5 shows a representative dose-response curve for cell survival in human macrophages in response to treatment with an exemplary anti-TREM 2 antibody.
Figure 6 shows representative soluble TREM2 levels (sTREM2) as a function of anti-TREM 2 antibody concentration for an exemplary anti-TREM 2 antibody.
FIG. 7 is a bar graph indicating the mean pHrodo fluorescence intensity per cell in human macrophages treated with an exemplary anti-TREM 2 antibody.
Fig. 8A is a representative microscope image of lipid accumulation in iPSC microglia treated with myelin followed by incubation with an exemplary anti-TREM 2 antibody or isotype control.
Fig. 8B is a representative bar graph of nile red staining (indicative of lipid accumulation) of iPSC microglia imaged in fig. 8A.
Fig. 8C-8F include bar graphs showing quantitative levels of cholesterol ester species (fig. 8C and 8E) and triacylglycerol lipid species (fig. 8D and 8F) in iPSC microglia cells treated with myelin followed by incubation with an exemplary anti-TREM 2 antibody. Fig. 8E and 8F represent data for iPSC microglia including a myelin elution step prior to incubation with an exemplary anti-TREM 2 antibody.
Fig. 9 includes a representative mouse plasma pharmacokinetic profile of an exemplary anti-TREM 2 antibody.
FIGS. 10A and 10B include graphs showing injection into TREM2cDNA KI (huTrem 2)KI/KI) Bar graph of the change in total soluble TREM2(sTREM2) (fig. 10A) and antibody binding TREM2 (fig. 10B) of exemplary anti-TREM 2 antibodies in mice in mouse plasma.
Fig. 11A and 11B include dose-response binding curves of exemplary humanized and sequence optimized anti-TREM 2 antibodies in HEK cells to human TREM 2.
Fig. 12A and 12B include dose response curves for activation of pSyk signal by exemplary humanized and sequence optimized anti-TREM 2 antibodies in HEK293-H6 cells.
Figure 13 shows a dose response curve for cell survival in human macrophages in response to treatment with an exemplary humanized and sequence optimized anti-TREM 2 antibody.
Fig. 14A and 14B include dose response curves in iPSC microglia in response to lipid clearance resulting from treatment with exemplary humanized and sequence optimized anti-TREM 2 antibodies.
Detailed Description
I. Introduction to the design reside in
TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages and osteoclasts. Without being bound by a particular theory, it is believed that upon ligand binding, TREM2 forms a signaling complex with the transmembrane adapter protein, DNAX activating protein 12(DAP12), which in turn is tyrosine phosphorylated by protein kinase SRC. The activated TREM2/DAP12 signaling complex is thought to mediate intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase. TREM2/DAP12 signaling regulates the activity of cells such as microglia and macrophages, e.g., phagocytosis, cell growth and survival, proinflammatory cytokine secretion and migration. TREM2 undergoes regulated intramembrane proteolysis, in which the membrane associated full length TREM2 is cleaved by metalloprotease ADAM10 into the fraction of sTREM2 shed from the cell and the membrane-retained C-terminal fragment further degraded by gamma secretase. Altered levels of sTREM2 have been reported in patients with Alzheimer's disease or frontotemporal dementia and having a TREM2 mutation. In addition, TREM2 mutations are associated with altered function such as impaired phagocytosis and reduced microglial function.
As detailed in the examples section below, antibodies have been generated that specifically bind to human TREM2 and modulate one or more downstream functions of the TREM2/DAP12 signaling complex. Accordingly, in one aspect, the disclosure provides anti-TREM 2 antibodies and antigen binding fragments thereof. Accordingly, in one aspect, the present disclosure provides anti-TREM 2 antibodies and antigen binding portions thereof.
In some embodiments, an anti-TREM 2 antibody enhances TREM2 activity, e.g., enhances phagocytosis or enhances differentiation, function, migration or survival of myeloid lineage cells, microglia or macrophages. Thus, in another aspect, methods of enhancing TREM2 activity, e.g., in a subject having a neurodegenerative disease, are provided.
In some embodiments, the anti-TREM 2 antibody reduces sTREM2 shedding. Thus, in another aspect, methods of reducing the level of sTREM2 in a subject, e.g., having a neurodegenerative disease, are provided.
Definition of
As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and the like.
As used herein, the terms "about" and "approximately" when used to modify an amount specified in a numerical value or range indicate that the numerical value and reasonable deviation from the stated value, e.g., ± 20%, ± 10%, or ± 5% as known to those skilled in the art, are within the intended meaning of the stated value.
As used herein, the term "TREM 2 protein" refers to trigger receptor 2 expressed by myeloid lineage cells encoded by the gene TREM 2. As used herein, "TREM 2 protein" refers to the native (i.e., wild-type) TREM2 protein of any vertebrate, such as, but not limited to, humans, non-human primates (e.g., cynomolgus monkeys), rodents (e.g., mice, rats), and other mammals. In some embodiments, the TREM2 protein is a human TREM2 protein having the sequence identified in UniprotKB accession number Q9NZC2(SEQ ID NO: 1).
As used herein, the term "anti-TREM 2 antibody" refers to an antibody that specifically binds to a TREM2 protein (e.g., human TREM 2).
As used herein, the term "antibody" refers to a protein having an immunoglobulin fold that specifically binds to an antigen via its variable region. The term includes intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" also includes antibody fragments that retain binding specificity via their variable regions, including but not limited to Fab, F (ab')2Fv, scFv, and bivalent scFv. Antibodies may contain light chains that are classified as kappa or lambda. Antibodies may contain heavy chains classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.
Exemplary immunoglobulin (antibody) building blocks include tetramers. Each tetramer is composed primarily of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25kD) and one "heavy chain" (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (V)H) These are referred to as the light chain and the heavy chain, respectively.
The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is derived from a germline variable (V) gene, a diversity (D) gene, or a joining (J) gene (rather than from constant (C μ and C δ) gene segments) and confers specificity for the antibody to bind to an antigen. Typically, an antibody variable region comprises four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".
The term "complementarity determining regions" or "CDRs" refers to the three hypervariable regions in each chain which interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding of the antibody to an epitope of the antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also commonly identified by the chain in which the particular CDR is located. Thus, VHThe CDR3 or CDR-H3 is located in the variable region of the heavy chain of an antibody in which the CDR is present, while the VLThe CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of an antibody in which the CDR is present.
The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework regions of the antibody are the combined framework regions of the constitutive light and heavy chains used to position and align the CDRs in three-dimensional space. The framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBASE 2" germline variable gene sequences for human and mouse sequences.
The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, such as Kabat, Chothia, international imminogenetics database (IMGT), AbM, and observed antigen contacts ("contacts"). In some embodiments, the CDR is determined according to a Contact definition. See MacC allum et al, J.mol.biol.,262:732-745 (1996). In some embodiments, the CDRs are determined by Kabat, Chothia, and/or Contact CDR definitions.
The terms "antigen-binding portion" or "antigen-binding fragment" are used interchangeably herein and refer to the ability of an antibody to retain specific binding to an antigen (e.g., a TREM2 protein) via its variable regionOne or more fragments of (a). Examples of antigen binding fragments include, but are not limited to, Fab fragments (consisting of VL, V)HMonovalent fragment consisting of the CL and CH1 domains), F (ab')2Fragments (bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region), single chain fv (scFv), disulfide-linked fv (dsFv), Complementarity Determining Regions (CDR), VL(light chain variable region) and VH(heavy chain variable region).
The term "epitope" refers to a region or region of an antigen to which a CDR of an antibody specifically binds and may comprise some amino acids or portions of some amino acids, for example 5 or 6 or more, for example 20 or more amino acids or portions of those amino acids. For example, where the target is a protein, the epitope may comprise contiguous amino acids (e.g., a linear epitope) or amino acids from different portions of the protein that are brought into proximity with each other by protein folding (e.g., a discontinuous or conformational epitope). In some embodiments, an epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).
As used herein, the phrase "recognizing an epitope" as used with respect to an anti-TREM 2 antibody means that the antibody CDRs interact or specifically bind with an antigen (i.e., a TREM2 protein) at the epitope or a portion of the antigen containing the epitope.
As used herein, the term "multispecific antibody" refers to an antibody comprising two or more different antigen-binding portions, wherein each antigen-binding portion comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two or more different antigens via its variable region. The term "bispecific antibody" as used herein refers to an antibody comprising two different antigen-binding portions, wherein each antigen-binding portion comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two different antigens via its variable region.
"monoclonal antibody" refers to an antibody which is produced by a single clone or single cell line of cells and consists of or consists essentially of antibody molecules of identical primary amino acid sequence.
"polyclonal antibody" refers to antibodies obtained from a heterogeneous population of antibodies, wherein different antibodies in the population bind to different epitopes of an antigen.
"chimeric antibody" refers to an antibody molecule in which constant regions or portions thereof are altered, replaced, or exchanged such that the antigen binding site (i.e., variable region, CDR, or portions thereof) is linked to a constant region having a different or altered class, effector function, and/or species, or in which variable regions or portions thereof are altered, replaced, or exchanged by variable regions having a different or altered antigen specificity (e.g., CDR and framework regions from a different species). In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human). Methods for producing chimeric antibodies are described in the art.
A "humanized antibody" is a chimeric immunoglobulin of non-human origin (e.g., murine) that contains minimal sequences derived from the non-human immunoglobulin outside the CDRs. In general, a humanized antibody will comprise at least one (e.g., two) antigen-binding variable region in which the CDR regions correspond substantially to those of a non-human immunoglobulin and the framework regions correspond substantially to those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin sequence. Methods for humanizing antibodies are known in the art.
A "human antibody" or "fully human antibody" is an antibody having human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, the antibody is produced by a human cell, by a non-human animal that utilizes a human antibody library (e.g., a transgenic mouse genetically engineered to express human antibody sequences), or by a phage display platform.
The term "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding portion thereof) that binds with greater affinity, greater avidity, and/or is more reactive than it does to another epitope or non-target compound (e.g., a structurally different antigen)A large duration of binding to the epitope or target in the sample. In some embodiments, an antibody (or antigen-binding portion thereof) that specifically binds to an epitope or target is an antibody (or antigen-binding portion thereof) that binds to the epitope or target with at least 5-fold greater affinity, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater than other epitopes or non-target compounds. As used herein, the terms "specifically binds," "specifically binds to," or is "specific for" a particular epitope or target can be, for example, by having the following equilibrium dissociation constant K for the epitope or target to which it bindsDThe molecule of (a): 10-4M or less, e.g. 10-5M、10-6M、10-7M、10-8M、10-9M、10-10M、10-11M or 10-12And M. The skilled person will recognize that an antibody that specifically binds to a target from one species (e.g., a TREM2 protein) may also specifically bind to an ortholog of the target (e.g., a TREM2 protein).
The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g., between an antibody (or antigen-binding portion thereof) and an antigen. Thus, for example, the term can refer to a 1:1 interaction between an antibody (or antigen-binding portion thereof) and an antigen, unless the context indicates otherwise or is otherwise evident from the context. Binding affinity can be measured by the equilibrium dissociation constant (K)D) To quantify, the equilibrium dissociation constant is referred to as the dissociation rate constant (k)dTime of day-1) Divided by the association rate constant (k)aTime of day-1M-1)。KDCan be determined by measuring the kinetics of complex formation and dissociation, for example, using the following method: surface Plasmon Resonance (SPR) methods, e.g. BiacoreTMA system; kinetic exclusion assays, e.g.And biolayer interferometry (e.g., usingOctet platform). As used herein, "binding affinity" includes not only formal binding affinities, such as those reflecting a 1:1 interaction between an antibody (or antigen-binding portion thereof) and an antigen, but also apparent affinities, the Ks of whichDValues are calculated and may reflect affinity binding.
As used herein, the term "cross-reactive" refers to the ability of an antibody to bind to an antigen other than the antigen from which the antibody is produced. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from another species in addition to the antigen from which the antibody was produced. As a non-limiting example, an anti-TREM 2 antibody as described herein and raised against a human TREM2 peptide can exhibit cross-reactivity with a TREM2 peptide or protein from a different species (e.g., monkey or mouse).
The term "isolated" as used with respect to a nucleic acid or protein (e.g., an antibody) means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in nature. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein (e.g., an antibody) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.
The term "amino acid" refers to both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Naturally occurring alpha-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring alpha-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "amino acid analogs" refers to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.
The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The amino acid polymer can comprise an intact L-amino acid, an intact D-amino acid, or a mixture of L-amino acids and D-amino acids.
As used herein, the term "protein" refers to a dimer (i.e., two) or multimer (i.e., three or more) of a polypeptide or single-chain polypeptide. Single chain polypeptides of a protein may be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.
The terms "polynucleotide" and "nucleic acid" interchangeably refer to a chain of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any matrix that can be incorporated into a strand by a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides encompassed herein include single-and double-stranded DNA, single-and double-stranded RNA, and hybrid molecules having a mixture of single-and double-stranded DNA and RNA.
The terms "conservative substitution" and "conservative mutation" refer to an alteration that results in the substitution of one amino acid with another amino acid that can be classified as having similar characteristics. Examples of classes of conservative amino acid groups defined in this way may include: "charged/polar group" includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups comprising: a "positively charged group" comprising Lys, Arg and His; a "negatively charged subset" comprising Glu and Asp; and "polar subgroups," comprising Asn and gin. In another example, the aromatic or cyclic group can be subdivided into subgroups that include: a "nitrogen ring subset" comprising Pro, His, and Trp; and "phenyl subgroups", comprising Phe and Tyr. In another other example, the aliphatic group can be subdivided into subgroups, such as "aliphatic nonpolar subgroups," comprising Val, Leu, Gly, and Ala; and an "aliphatic less polar subset" comprising Met, Ser, Thr and Cys. Examples of classes of conservative mutations include amino acid substitutions for amino acids within the above subgroups, e.g. but not limited toWithout limitation: substitution of Arg with Lys or vice versa such that a positive charge can be maintained; substitution of Glu for Asp or vice versa, such that a negative charge can be maintained; substitution of Ser for Thr or vice versa, such that free-OH can be maintained; and substitution of Asn with Gln or vice versa, such that free-NH can be maintained2. In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acids, for example in the active site, to maintain hydrophobicity.
In the context of two or more polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more percent, within a specified region, when compared and aligned for maximum correspondence over a comparison window or specified region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
With respect to sequence comparison of polypeptides, typically one amino acid sequence serves as a reference sequence to which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or maximum alignment using known algorithms using publicly available software. Such programs include the BLAST program, ALIGN-2(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters for alignment to achieve maximum alignment can be determined by one skilled in the art. For sequence comparison of polypeptide sequences for the purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two protein sequences with default parameters is used.
As used herein, the terms "subject," "individual," and "patient" refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the subject, individual or patient is a human.
The term "treating" and similar terms are used herein generally to refer to obtaining a desired pharmacological and/or physiological effect. "treating" can refer to any indicia of success in treating or ameliorating a neurodegenerative disease (e.g., alzheimer's disease or another neurodegenerative disease described herein), including any objective or subjective parameter, such as remission, improved survival of a patient, increased survival time or survival rate, alleviation of symptoms or making a disease more tolerable to a patient, slower rate of regression or debilitation, or improving the physical or mental health of a patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters. The therapeutic effect can be compared to an individual or group of individuals who have not received treatment, or to the same patient at different time points before or during treatment.
The term "pharmaceutically acceptable excipient" refers to inactive pharmaceutical ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.
As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (e.g., an antibody as described herein) is an amount of the agent that treats, alleviates, eliminates, or reduces the severity of a disease symptom in a subject. A "therapeutic amount" of an agent (e.g., an antibody as described herein) can improve patient survival, increase survival time or survival, reduce symptoms, make an injury, disease or disorder (e.g., a neurodegenerative disease) more tolerable, slow the rate of regression or decline, or improve physical or mental health of a patient.
The term "administering" refers to a method of delivering an agent, compound or composition to a desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, transdermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the antibody as described herein is administered intravenously.
The term "control" or "control value" refers to a reference value or a baseline value. Suitable controls can be determined by one skilled in the art. In some cases, the control value may be determined relative to a baseline within the same subject or experiment, e.g., a sTREM2 measurement obtained prior to treatment with an anti-TREM 2 antibody may be the control value for a post-treatment measurement of a sTREM2 level in the same subject. In other instances, the control value can be determined relative to a control subject (e.g., a healthy control or a disease control) or an average value in a population of control subjects (e.g., a healthy control or a disease control, e.g., a population of 10, 20, 50, 100, 200, 500, 1000 or more control subjects), e.g., a measurement of the subject's sTREM2 level at baseline or after treatment can be compared to a healthy control value.
anti-TREM 2 antibody
In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to a TREM2 protein is provided. In some embodiments, the antibody specifically binds to a human TREM2 protein. In some embodiments, the anti-TREM 2 antibody is selective for TREM2 relative to other TREM-like receptors (e.g., TREM 1).
In some embodiments, the anti-TREM 2 antibody is an antibody comprising one or more Complementarity Determining Regions (CDRs), heavy chain variable regions, and/or light chain variable region sequences as disclosed herein. In some embodiments, the anti-TREM 2 antibody comprises one or more CDRs, heavy chain variable region, and/or light chain variable region sequences as disclosed herein and further comprises one or more functional features as disclosed herein, for example, an antibody that enhances TREM2 activity (e.g., enhances phagocytosis or enhances migration, differentiation, function, or survival of cells such as myeloid cells, microglia, or macrophages) or an antibody that reduces the level of sTREM 2.
anti-TREM 2 antibody sequences
In some embodiments, the anti-TREM 2 or antigen-binding fragment thereof comprises a heavy chain sequence or portion thereof and/or a light chain sequence or portion thereof derived from any one of the following anti-TREM 2 antibodies described herein: clone CL0020306, clone CL0020188, clone CL0020307 and clone CL 0020123. The amino acid sequences of the CDRs, heavy chain variable regions and light chain variable regions of these clones are set forth in the informal sequence listing. In some embodiments, the anti-TREM 2 antibody is a chimeric antibody. In some embodiments, the anti-TREM 2 antibody is a humanized and/or affinity matured antibody.
In some embodiments, the anti-TREM 2 antibody comprises one or more CDRs selected from the group consisting of:
(a) a heavy chain CDR1(CDR-H1) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs 4, 12 and 29 or having at most two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs 4, 12 and 29;
(b) a heavy chain CDR2(CDR-H2) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs 5, 13, 25, 30, 39, 41 and 43 or having at most two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs 5, 13, 25, 30, 39, 41 and 43;
(c) a heavy chain CDR3(CDR-H3) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs 6, 14, 17 and 31 or having at most two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs 6, 14, 17 and 31;
(d) a light chain CDR1(CDR-L1) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs 7, 23 and 32 or having at most two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs 7, 23 and 32;
(e) a light chain CDR2(CDR-L2) sequence having at least 90% sequence identity to the amino acid sequence of either of SEQ ID NOs 8 and 33 or having at most two amino acid substitutions relative to the amino acid sequence of either of SEQ ID NOs 8 and 33; and
(f) a light chain CDR3(CDR-L3) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs 9,18 and 34 or having at most two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs 9,18 and 34.
In some embodiments, the anti-TREM 2 antibody comprises two, three, four, five, or all six of (a) - (f). In some embodiments, the anti-TREM 2 antibody comprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some embodiments, the anti-TREM 2 antibody comprises CDR-L1 of (d), CDR-L2 of (e), and CDR-L3 of (f). In some embodiments, a CDR with up to two amino acid substitutions has one amino acid substitution relative to a reference sequence. In some embodiments, a CDR with up to two amino acid substitutions has two amino acid substitutions relative to a reference sequence. In some embodiments, up to two amino acid substitutions are conservative substitutions.
In some embodiments, the anti-TREM 2 antibody comprises one or more CDRs selected from the group consisting of:
(a) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 4, 12 and 29;
(b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 5, 13, 25, 30, 39, 41 and 43;
(c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 6, 14, 17 and 31;
(d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs 7, 23 and 32;
(e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs 8 and 33; and
(f) a CDR-L3 sequence comprising the amino acid sequence of any one of SEQ ID NOs 9,18 and 34.
In some embodiments, the anti-TREM 2 antibody comprises two, three, four, five, or all six of (a) - (f). In some embodiments, the anti-TREM 2 antibody comprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some embodiments, the anti-TREM 2 antibody comprises CDR-L1 of (d), CDR-L2 of (e), and CDR-L3 of (f).
In some embodiments, the anti-TREM 2 antibody comprises:
(a) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 6, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(b) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(c) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 5, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(d) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(e) CDR-H1 comprising the amino acid sequence SEQ ID NO. 4, CDR-H2 comprising the amino acid sequence SEQ ID NO. 25, CDR-H3 comprising the amino acid sequence SEQ ID NO. 17, CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 18; or
(f) CDR-H1 comprising the amino acid sequence SEQ ID NO. 12, CDR-H2 comprising the amino acid sequence SEQ ID NO. 13, CDR-H3 comprising the amino acid sequence SEQ ID NO. 14, CDR-L1 comprising the amino acid sequence SEQ ID NO. 7, CDR-L2 comprising the amino acid sequence SEQ ID NO. 8, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 9; or
(g) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 30, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(h) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 39, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(i) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 41, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34; or
(j) CDR-H1 comprising the amino acid sequence SEQ ID NO. 29, CDR-H2 comprising the amino acid sequence SEQ ID NO. 43, CDR-H3 comprising the amino acid sequence SEQ ID NO. 31, CDR-L1 comprising the amino acid sequence SEQ ID NO. 32, CDR-L2 comprising the amino acid sequence SEQ ID NO. 33 and CDR-L3 comprising the amino acid sequence SEQ ID NO. 34.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79. In some embodiments, anti-TREM 2 comprises a heavy chain variable region comprising any one of the amino acid sequences SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, 28, 36, and 68. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising any one of the amino acid sequences SEQ ID NOs 3, 11, 16, 20, 22, 28, 36, and 68.
In some embodiments, the anti-TREM 2 antibody comprises: a heavy chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46 and 79, and a light chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 3, 11, 16, 20, 22, 28, 36 and 68. In some embodiments, the resistance TREM2 comprises: a heavy chain variable region comprising any one of amino acid sequences SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79, and a light chain variable region comprising any one of amino acid sequences SEQ ID NOs 3, 11, 16, 20, 22, 28, 36, and 68.
In some embodiments, the anti-TREM 2 antibody comprises:
(a) v having at least 85% sequence identity to SEQ ID NO 2HSequences and V having at least 85% sequence identity to SEQ ID NO 3LA sequence; or
(b) V having at least 85% sequence identity to SEQ ID NO 10HSequences and V having at least 85% sequence identity to SEQ ID NO 11LA sequence; or
(c) V having at least 85% sequence identity to SEQ ID NO 15HSequences and V having at least 85% sequence identity to SEQ ID NO 16LA sequence; or
(d) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(e) V having at least 85% sequence identity to SEQ ID NO 21HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(f) V having at least 85% sequence identity to SEQ ID NO 19HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(g) V having at least 85% sequence identity to SEQ ID NO 79HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(h) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 20LA sequence; or
(i) V having at least 85% sequence identity to SEQ ID NO 26HSequence and at least 85% sequence identity with SEQ ID NO 20A linear VLA sequence; or
(j) V having at least 85% sequence identity to SEQ ID NO 24HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(k) V having at least 85% sequence identity to SEQ ID NO 26HSequences and V having at least 85% sequence identity to SEQ ID NO 22LA sequence; or
(l) V having at least 85% sequence identity to SEQ ID NO 27HSequences and V having at least 85% sequence identity to SEQ ID NO 28LA sequence; or
(m) V having at least 85% sequence identity to SEQ ID NO 35HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(n) V having at least 85% sequence identity to SEQ ID NO:37HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(o) V having at least 85% sequence identity to SEQ ID NO:38HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(p) V having at least 85% sequence identity to SEQ ID NO 40HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(q) V having at least 85% sequence identity to SEQ ID NO:42HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(r) V having at least 85% sequence identity to SEQ ID NO:44HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(s) V having at least 85% sequence identity to SEQ ID NO:45HSequences and V having at least 85% sequence identity to SEQ ID NO 36LA sequence; or
(t) V having at least 85% sequence identity to SEQ ID NO 46HSequence and at least 85% sequence identity to SEQ ID NO 36Sexual VLA sequence; or
(u) V having at least 85% sequence identity to SEQ ID NO:24HSequences and V having at least 85% sequence identity to SEQ ID NO 68LAnd (4) sequencing.
In some embodiments, the anti-TREM 2 antibody comprises one or more sequences encompassed by the consensus sequences disclosed herein. As a non-limiting example, a consensus sequence can be identified by aligning heavy or light chain sequences (e.g., CDRs) from antibodies of the same (or similar) germline. In some embodiments, the consensus sequence can be generated from antibodies containing sequences having the same (or similar) length and/or having at least one highly similar CDR (e.g., highly similar CDR 3). In some embodiments, such sequences in these antibodies may be compared and compared to identify conserved amino acids or motifs (i.e., where changes in sequence may alter protein function) and/or regions where changes in sequence occur (i.e., where changes in sequence are unlikely to significantly affect protein function). Alternatively, consensus sequences can be identified by aligning the heavy or light chain sequences (e.g., CDRs) of antibodies that bind to the same or similar (e.g., overlapping) epitopes to identify conserved amino acids or motifs (i.e., regions in which changes in the sequence can alter protein function) and regions in which changes occur in the sequence alignment (i.e., regions in which changes in the sequence are unlikely to significantly affect protein function). In some embodiments, one or more consensus sequences in the antibody can be identified that recognize the same or similar epitope as the anti-TREM 2 antibody as disclosed herein. Exemplary consensus sequences include SEQ ID NOs 47-52. In consensus sequences SEQ ID NOs: 47-52, capital letters indicate amino acid residues that are absolutely conserved in the aligned sequences (e.g., the aligned CDR sequences), while "X" or Greek letters (e.g., "α", "β", "γ", "δ", "ε" or) Indicates amino acid residues that are not absolutely conserved within the aligned sequences. It will be understood that when an amino acid is selected for insertion at a position marked by an "X" or Greek letter, in some embodiments, the amino acid is selected from the aligned sequencesThose amino acids visible at the corresponding positions in the columns.
Clones CL0020123 and variants of CL0020123
In some embodiments, the anti-TREM 2 antibody or antigen-binding fragment thereof comprises:
(a) a CDR-H1 sequence comprising sequence GFSIEDFYIH (SEQ ID NO: 29);
(b) CDR-H2 sequence comprising the sequence W-I-D-P-E-beta6-G-β8-S-K-Y-A-P-K-F-Q-G (SEQ ID NO:47), wherein6Is N or Q and beta8Is D or E;
(c) a CDR-H3 sequence comprising sequence HADHGNYGSTMDY (SEQ ID NO: 31);
(d) a CDR-L1 sequence comprising sequence HASQHINVWLS (SEQ ID NO: 32);
(e) a CDR-L2 sequence comprising the sequence KASNLHT (SEQ ID NO: 33); and
(f) a CDR-L3 sequence comprising sequence QQGQTYPRT (SEQ ID NO: 34).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H2 sequence selected from SEQ ID NOs 30, 39, 41, and 43.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs 27, 35, 37, 38, 40, 42, 44, 45, and 46. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising any one of the amino acid sequences SEQ ID NOs 27, 35, 37, 38, 40, 42, 44, 45, and 46.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs 28 and 36. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising any one of the amino acid sequences SEQ ID NOs 28 and 36.
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising amino acid sequence SEQ ID No. 29, a CDR-H2 sequence comprising amino acid sequence SEQ ID No. 30, a CDR-H3 sequence comprising amino acid sequence SEQ ID No. 31, a CDR-L1 sequence comprising amino acid sequence SEQ ID No. 32, a CDR-L2 sequence comprising amino acid sequence SEQ ID No. 33, and a CDR-L3 sequence comprising amino acid sequence SEQ ID No. 34.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 27. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 27.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 28. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 28.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 27 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 28. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 27 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 28.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 29, 30, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 27. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 28.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 29, 30, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 27 and a light chain variable region comprising amino acid sequence SEQ ID NO 28).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising amino acid sequence SEQ ID No. 29, a CDR-H2 sequence comprising amino acid sequence SEQ ID No. 43, a CDR-H3 sequence comprising amino acid sequence SEQ ID No. 31, a CDR-L1 sequence comprising amino acid sequence SEQ ID No. 32, a CDR-L2 sequence comprising amino acid sequence SEQ ID No. 33, and a CDR-L3 sequence comprising amino acid sequence SEQ ID No. 34.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 42. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:42 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 42 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 36.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 29, 43, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 42. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 29, 43, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 42 and a light chain variable region comprising amino acid sequence SEQ ID NO 36).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising amino acid sequence SEQ ID No. 29, a CDR-H2 sequence comprising amino acid sequence SEQ ID No. 41, a CDR-H3 sequence comprising amino acid sequence SEQ ID No. 31, a CDR-L1 sequence comprising amino acid sequence SEQ ID No. 32, a CDR-L2 sequence comprising amino acid sequence SEQ ID No. 33, and a CDR-L3 sequence comprising amino acid sequence SEQ ID No. 34.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 45. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:45 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 45 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 36.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 29, 41, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 45. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 29, 41, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 45 and a light chain variable region comprising amino acid sequence SEQ ID NO 36).
Variants of clones CL0020188, CL0020306, CL0020307, and CL0020188
In some embodiments, the anti-TREM 2 antibody or antigen-binding fragment thereof comprises:
(a) CDR-H1 sequence comprising the sequence G-F-T-F-T-alpha6-F-Y-M-S (SEQ ID NO:48), wherein alpha6Is D or N;
(b) CDR-H2 sequence comprising the sequence V-I-R-N-beta5-β6-N-β8-Y-T-β11-β12-Y-N-P-S-V-K-G (SEQ ID NO:49), wherein5Is K or R; beta is a6Is A or P; beta is a8Is G or A; beta is a11Is A or T; and beta12Is G or D;
(c) CDR-H3 sequence comprising the sequence gamma1-R-L-γ4-Y-G-F-D-Y (SEQ ID NO:50), wherein γ1Is A or T; and gamma is4Is T or S;
(d) CDR-L1 sequence comprising the sequence Q-S-S-K-S-L-L-H-S-delta10-G-K-T-Y-L-N (SEQ ID NO:51), wherein delta10Is N or T;
(e) a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and
(f) CDR-L3 sequence comprising the sequence Q-Q-F-L-E-phi6-P-F-T (SEQ ID NO:52), wherein phi6Is Y or F.
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence selected from SEQ ID NOs 4 and 12. In some embodiments, the anti-TREM 2 antibody comprises a CDR-H2 sequence selected from SEQ ID NOs 5, 13, and 25. In some embodiments, the anti-TREM 2 antibody comprises a CDR-H3 sequence selected from SEQ ID NOs 6, 14, and 17. In some embodiments, the anti-TREM 2 antibody comprises a CDR-L1 sequence selected from SEQ ID NOs 7 and 23. In some embodiments, the anti-TREM 2 antibody comprises a CDR-L3 selected from SEQ ID NOs 9 and 18.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID nos. 2, 10, 15, 19, 21, 24, 26, and 79. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising any one of the amino acid sequences SEQ ID NOs 2, 10, 15, 19, 21, 24, 26, and 79.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs 3, 11, 16, 20, 22, and 68. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising any one of the amino acid sequences SEQ ID NOs 3, 11, 16, 20, 22, and 68.
Variants of clones CL0020188 and CL0020188
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID No. 4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID No. 5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID No. 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID No. 7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID No. 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID No. 18.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 15. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 15 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 16. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 4, 5, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 15. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 7, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 16.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 5, 17, 7, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 15 and a light chain variable region comprising amino acid sequence SEQ ID NO 16).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID No. 4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID No. 5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID No. 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID No. 23, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID No. 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID No. 18.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 79. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:79 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 79 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 4, 5, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 79. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDRs 1-3 having the amino acid sequences of SEQ ID NOs 23, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 4, 5, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 79 and a light chain variable region comprising amino acid sequence SEQ ID NO 22).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID No. 4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID No. 25, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID No. 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID No. 23, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID No. 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID No. 18.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 24 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 4, 25, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 24. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDRs 1-3 having the amino acid sequences of SEQ ID NOs 23, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 22.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 4, 25, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 24 and a light chain variable region comprising amino acid sequence SEQ ID NO 22).
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID No. 4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID No. 25, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID No. 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID No. 7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID No. 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID No. 9.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 68. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 68.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 68. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 24 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 68.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 4, 25, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 24. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID No. 68.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 4, 25, 17, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 24 and a light chain variable region comprising amino acid sequence SEQ ID NO 68).
Clone CL0020306
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID No. 4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID No. 5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID No. 6, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID No. 7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID No. 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID No. 9.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 2. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 3. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 3.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 2 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 3. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 3.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 4, 5, and 6, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 2. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences of SEQ ID NOs 7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 3.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 4, 5, 6, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO:2 and a light chain variable region comprising amino acid sequence SEQ ID NO: 3).
Clone CL0020307
In some embodiments, the anti-TREM 2 antibody comprises a CDR-H1 sequence comprising amino acid sequence SEQ ID No. 12, a CDR-H2 sequence comprising amino acid sequence SEQ ID No. 13, a CDR-H3 sequence comprising amino acid sequence SEQ ID No. 14, a CDR-L1 sequence comprising amino acid sequence SEQ ID No. 7, a CDR-L2 sequence comprising amino acid sequence SEQ ID No. 8, and a CDR-L3 sequence comprising amino acid sequence SEQ ID No. 9.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 10. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 11. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:10 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11. In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 10 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 11.
In some embodiments, the anti-TREM 2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having amino acids SEQ ID NOs 12, 13, and 14, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 10. In some embodiments, the anti-TREM 2 antibody comprises a light chain variable region comprising light chain CDR1-3 having amino acid sequences SEQ ID NOs 7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID No. 11.
In some embodiments, the anti-TREM 2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising amino acid sequences SEQ ID NOs 12, 13, 14, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising amino acid sequence SEQ ID NO 10 and a light chain variable region comprising amino acid sequence SEQ ID NO 11).
Binding characteristics of anti-TREM 2 antibodies
In some embodiments, an antibody that specifically binds to a TREM2 protein as described herein binds to TREM2 expressed on a cell (e.g., a primary cell or cell line endogenously expressing TREM2, e.g., human macrophages, or a primary cell or cell line engineered to express TREM2, e.g., as described in the examples section below). In some embodiments, an antibody that specifically binds to a TREM2 protein as described herein binds to a purified or recombinant TREM2 protein or portion thereof or to a chimeric protein comprising TREM2 or a portion thereof (e.g., a Fc-fusion protein comprising TREM2 or a Fc-fusion protein comprising a TREM2 extracellular domain).
In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with one or more other TREM2 proteins of another species. In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with cynomolgus monkey ("cyno") TREM2 protein. In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with a mouse TREM2 protein. In some embodiments, the anti-TREM 2 antibody exhibits cross-reactivity with human TREM2, cynomolgus monkey TREM2, and mouse TREM 2.
Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore)TM(GE Healthcare, Piscataway, NJ)), a power exclusion assay (e.g.,) Flow cytometry, Fluorescence Activated Cell Sorting (FACS), biolayer interferometry (e.g., Octet)TM(Forte Bio, Inc., Menlo Park, CA)) and Western blot analysis. In some embodiments, binding affinity and/or cross-reactivity is determined using ELISA. Methods for performing ELISA assays are known in the art and are also described in the examples section below. In some embodiments, Surface Plasmon Resonance (SPR) is used to determine binding affinity, binding kinetics, and/or cross-inversionsAnd (4) stress response. In some embodiments, binding affinity, binding kinetics, and/or cross-reactivity are determined using a kinetic exclusion assay. In some embodiments, binding affinity, binding kinetics, and/or cross-reactivity are determined using biolayer interferometry.
Epitopes recognized by anti-TREM 2 antibodies
In some embodiments, the epitope recognized by the anti-TREM 2 antibody by human TREM2 is the same or substantially the same as the epitope recognized by an antibody clone as described herein. As used herein, the term "substantially identical," as used with respect to an epitope recognized by an antibody clone as described herein, means that the epitope recognized by the anti-TREM 2 antibody is the same as, within, or nearly the same as (e.g., has at least 90% sequence identity to, or has one, two, or three amino acid substitutions, e.g., conservative substitutions, relative to, the epitope), or has significant overlap with (e.g., has at least 50%, 60%, 70%, 80%, 90%, or 95% overlap with) the epitope recognized by the antibody clone as described herein.
In some embodiments, the anti-TREM 2 antibody recognizes an epitope of human TREM2 that is the same or substantially the same as the epitope recognized by an antibody clone selected from the group consisting of: clone CL0020306, clone CL0020188, clone CL0020307 and clone CL 0020123.
In some embodiments, an anti-TREM 2 antibody binds within the TREM2 stalk region of an epitope of human TREM 2. In some embodiments, the anti-TREM 2 antibody recognizes an epitope of human TREM2 that comprises, is within, or consists of residue 129-172 or residue 131-169 of SEQ ID NO: 1. In some embodiments, the anti-TREM 2 antibody recognizes an epitope of human TREM2 that comprises, is within, or consists of residues 129-148 of SEQ ID NO: 1. In some embodiments, the anti-TREM 2 antibody recognizes an epitope of human TREM2 that comprises, is within, or consists of amino acid residues 143-149 of SEQ ID NO: 1. In some embodiments, an anti-TREM 2 antibody is an agonist within the TREM2 stalk region that activates TREM2/DAP12 signaling (e.g., by inducing phosphorylation of a kinase, such as Syk) and binds to an epitope of human TREM 2. In some embodiments, an anti-TREM 2 antibody binds to the TREM2 stalk region of an epitope of human TREM2 and inhibits cleavage of TREM2 by a protease (e.g., ADAM 17).
In some embodiments, an anti-TREM 2 antibody binds within the TREM2Ig variable (IgV) domain of an epitope of human TREM 2. In some embodiments, an anti-TREM 2 antibody is an agonist of a TREM2IgV domain that activates TREM2/DAP12 signaling (e.g., by inducing phosphorylation of a kinase, such as Syk) and binds to an epitope of human TREM 2. In some embodiments, an anti-TREM 2 antibody binds to an epitope of human TREM2 that comprises or consists of one or more of: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)) of SEQ ID NO:1, (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)) of SEQ ID NO:1, and (iii) amino acid residue 126-129(VEVL (SEQ ID NO:72)) of SEQ ID NO: 1.
Functional characterization of anti-TREM 2 antibodies
In some embodiments, an anti-TREM 2 antibody (e.g., an antibody as disclosed having one or more CDRs, heavy chain variable region, and/or light chain variable region sequences) plays a role in one or more TREM2 activities as disclosed herein. For example, in some embodiments, an anti-TREM 2 antibody is an antibody that modulates sTREM2 protein levels (e.g., sTREM2 levels that shed from the cell surface into an extracellular sample), modulates recruitment or phosphorylation of kinases that interact with the TREM2/DAP12 signaling complex (e.g., Syk kinase), and/or modulates one or more activities downstream of the signaling complex (e.g., phagocytosis, cell growth, cell survival, cell differentiation, cytokine secretion, or cell migration). In some embodiments, an anti-TREM 2 antibody as disclosed herein binds to soluble TREM2 protein (sTREM2) in healthy human CSF or cynomolgus monkey CSF with better potency than a reference antibody. In some embodiments, the reference antibody is represented by a combination of sequences selected from the group consisting of: 73 and 74; 75 and 76 for SEQ ID NO; and SEQ ID NOS 77 and 78. In some embodiments, the potency assay is performed essentially as described in example 11.
In some embodiments, an anti-TREM 2 antibody enhances one or more TREM2 activities (e.g., those described herein) induced by the ligand. In some embodiments, the ligand is a lipid ligand. Examples of TREM2 lipid ligands include, but are not limited to, 1-palmitoyl-2- (5' -oxo-pentanoyl) -sn-glycerol-3-phosphocholine (POVPC), 2-arachidonic acid glycerol (2-AG), 7-ketocholesterol (7-KC), 24(S) hydroxycholesterol (24OHC), 25(S) hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), Acylcarnitine (AC), alkylacylglycerophosphocholine (PAF), alpha-galactosylceramide (KRN7000), bis (monoacylglycerol) phosphate (BMP), Cardiolipin (CL), ceramide-1-phosphate (C1P), Cholesterol Esters (CE), Cholesterol Phosphate (CP), diacylglycerol 34:1(DG 34:1) Diacylglycerol 38:4(DG 38:4), diacylglycerol pyrophosphate (DGPP), dihydroceramide (DhCoer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), Free Cholesterol (FC), galactosylceramide (GalCer), galactosylceramide (GalSo), ganglioside GM1, ganglioside GM3, glucosyl ganglioside (GlcSo), Hank's Balanced Salt Solution (Hank's Balanced Salt Solution, HBSS), Kdo 2-lipid A (KLA), lactosylceramide (LacCer), Lysoalkylacylglycerophosphocholine (LPAF), lysophosphatidic acid (LPA), Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE), Lysophosphatidylglycerol (LPG), phosphatidylinositol (LPI), Lysosphingomyelin (LSM), Lysophosphatidylserine (LPS), N-acyl-phosphatidylethanolamine (NAPE), N-acyl-serine (NSer), oxidized phosphatidylcholine (oxPC), palmitic acid-9-hydroxy-stearic acid (PAHSA), Phosphatidylethanolamine (PE), phosphatidylethanol (PEtOH), Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylglycerol (PG), Phosphatidylinositol (PI), Phosphatidylserine (PS), sphinganine-1-phosphate (Sa1P), Sphingomyelin (SM), sphingosine-1-phosphate (So1P), and cerebroside sulfate.
Regulation of sTREM2 shedding
In some embodiments, the anti-TREM 2 antibody alters the sTREM2 protein level in the sample, e.g., the sTREM2 level that is shed from the cell surface into the extracellular sample. In some embodiments, an anti-TREM 2 antibody reduces sTREM2 levels.
In some embodiments, an anti-TREM 2 antibody reduces sTREM2 levels if the amount of sTREM2 in the treated sample is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to a control value. In some embodiments, an anti-TREM 2 antibody reduces sTREM2 levels if the amount of sTREM2 in the treated sample is reduced at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a control value. In some embodiments, the control value is the amount of sTREM2 in an untreated sample (e.g., a supernatant from TREM 2-expressing cells that have not been treated with an anti-TREM 2 antibody, or a sample from a subject that has not been treated with an anti-TREM 2 antibody) or a sample treated with an appropriate non-TREM 2 binding antibody.
In some embodiments, sTREM2 shedding is measured using a sample comprising a fluid, such as blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises cerebrospinal fluid. In some embodiments, the sample comprises supernatant from a cell culture (e.g., supernatant from a primary cell or cell line endogenously expressing TREM2, e.g., human macrophage, or a primary cell or cell line engineered to express TREM2, e.g., as described in the examples section below).
In some embodiments, the level of sTREM2 in the sample is measured using an immunoassay. Immunoassays are known in the art and include, but are not limited to, Enzyme Immunoassays (EIAs), such as enzyme multi-factor immunoassays (EMIAs), enzyme-linked immunosorbent assays (ELISAs), Microparticle Enzyme Immunoassays (MEIAs), Immunohistochemistry (IHC), immunocytochemistry, Capillary Electrophoresis Immunoassays (CEIA), Radioimmunoassays (RIA), immunofluorescence, chemiluminescent immunoassays (CL), and electrochemiluminescent immunoassays (ECL). In some embodiments, the sTREM2 levels are measured using an ELISA assay. In some embodiments, sTREM2 levels are measured using an ELISA assay as described in the examples section below.
Modulation of kinase recruitment or phosphorylation
In some embodiments, an anti-TREM 2 antibody induces phosphorylation of a kinase that interacts with the TREM2/DAP12 signaling complex (such as, but not limited to, Syk, ZAP70, PI3K, Erk, AKT, or GSK3 b). In some embodiments, an anti-TREM 2 antibody induces phosphorylation of a kinase that interacts with the TREM2/DAP12 signaling complex without blocking binding of a native TREM2 ligand. In some embodiments, an anti-TREM 2 antibody enhances phosphorylation of a kinase interacting with the TREM2/DAP12 signaling complex induced by a TREM2 ligand (e.g., a lipid ligand). In some embodiments, the anti-TREM 2 antibody induces or enhances phosphorylation of Syk. In some embodiments, an anti-TREM 2 antibody induces or enhances Syk phosphorylation if the level of Syk phosphorylation in a sample treated with the anti-TREM 2 antibody increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to a control value. In some embodiments, an anti-TREM 2 antibody induces Syk phosphorylation if the level of Syk phosphorylation in a sample treated with the anti-TREM 2 antibody increases at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a control value. In some embodiments, the control value is the level of Syk phosphorylation in an untreated sample (e.g., a sample comprising TREM2 expressing cells that have not been treated with an anti-TREM 2 antibody or a sample from a subject that has not been treated with an anti-TREM 2 antibody), or a sample that has been treated with a TREM2 ligand other than an anti-TREM 2 antibody, or a sample treated with an appropriate non-TREM 2 binding antibody.
To detect and/or quantify phosphorylation (e.g., Syk phosphorylation) in a sample, in some embodiments, an immunoassay is used. In some embodiments, the immunoassay is an Enzyme Immunoassay (EIA), an enzyme multi-factor immunoassay (EMIA), an enzyme-linked immunosorbent assay (ELISA), a particulate enzyme immunoassay (MEIA), Immunohistochemistry (IHC), immunocytochemistry, Capillary Electrophoresis Immunoassay (CEIA), Radioimmunoassay (RIA), immune assay (ia), immune assay (e.g., a bead, or a bead, or a combination thereofFluorescence, chemiluminescence immunoassay (CL) or electrochemiluminescence immunoassay (ECL). In some embodiments, a homogeneous assay using amplified luminescence proximity (amplified luminescence proximity) is used (a)PerkinElmer Inc.) to detect and/or quantify phosphorylation.
In some embodiments, phosphorylation is measured using a sample comprising one or more cells, e.g., one or more TREM2 expressing cells (e.g., a primary cell or cell line endogenously expressing TREM2, e.g., a human macrophage or iPSC-derived microglia cell, or a primary cell or cell line engineered to express TREM2, e.g., as described in the examples section below). In some embodiments, the sample comprises a fluid, such as blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises a tissue (e.g., lung, brain, kidney, spleen, neural tissue, or skeletal muscle) or a cell from such a tissue. In some embodiments, the sample comprises an endogenous fluid, tissue, or cell (e.g., from a human or non-human subject).
Regulation of phagocytosis
In some embodiments, an anti-TREM 2 antibody enhances phagocytosis of dead cell debris, tissue debris, amyloid β particles, or foreign substances. In some embodiments, an anti-TREM 2 antibody enhances phagocytosis without blocking binding of a native TREM2 ligand. In some embodiments, an anti-TREM 2 antibody enhances phagocytosis induced by a TREM2 ligand (e.g., a lipid ligand). In some embodiments, an anti-TREM 2 antibody enhances phagocytosis if the level of phagocytosis in a sample treated with the anti-TREM 2 antibody increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to a control value. In some embodiments, an anti-TREM 2 antibody enhances phagocytosis if phagocytosis is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more in a sample treated with the anti-TREM 2 antibody compared to a control value. In some embodiments, the control value is the level of phagocytosis in an untreated sample, a sample that has been treated with a TREM2 ligand instead of an anti-TREM 2 antibody, or a sample treated with an appropriate non-TREM 2 binding antibody.
In some embodiments, phagocytosis is measured using a phagocytosis assay with a labeled matrix. Phagocytosis assays are known in the art. In some embodiments, a sample comprising cells endogenously expressing TREM2, such as human macrophages or microglia, is subjected to a phagocytosis assay. In some embodiments, a phagocytosis assay is performed on a sample comprising cells that have been engineered to express TREM 2. In some embodiments, phagocytosis is measured using a human macrophage phagocytosis assay as described in the examples section below.
Modulation of cell differentiation, function, migration and survival
In some embodiments, the anti-TREM 2 antibody enhances cell migration, cell survival, cell function, or cell differentiation (e.g., with respect to myeloid cells, macrophages, and microglia, including iPSC-derived microglia and disease-associated microglia). Disease-associated microglia and methods for detecting disease-associated microglia are described in Keren-Shaul et al, Cell,2017,169: 1276-. In some embodiments, an anti-TREM 2 antibody enhances cell migration of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, an anti-TREM 2 antibody enhances cell survival of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, an anti-TREM 2 antibody enhances cellular function of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, the anti-TREM 2 antibody enhances cell differentiation of one or more cell types (e.g., myeloid lineage cells, macrophages, or microglia). In some embodiments, an anti-TREM 2 antibody enhances migration, survival, function, and/or differentiation of myeloid lineage cells. In some embodiments, an anti-TREM 2 antibody enhances macrophage migration, survival, function, and/or differentiation. In some embodiments, an anti-TREM 2 antibody enhances migration, survival, function, and/or differentiation of microglia. In some embodiments, the anti-TREM 2 antibody enhances microglial activation. In some embodiments, an anti-TREM 2 antibody enhances migration, survival, function, and/or differentiation of disease-associated microglia. In some embodiments, an anti-TREM 2 antibody enhances cell migration, cell survival, cell function, or cell differentiation without blocking binding of a native TREM2 ligand. In some embodiments, an anti-TREM 2 antibody enhances cell migration, cell survival, cell function, or cell differentiation induced by a TREM2 ligand (e.g., a lipid ligand).
In some embodiments, an anti-TREM 2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with the anti-TREM 2 antibody increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to a control value. In some embodiments, an anti-TREM 2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with the anti-TREM 2 antibody increases at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a control value. In some embodiments, the control value is the level of activity (e.g., migration, survival, function, or differentiation) in an untreated sample (e.g., a sample that has not been treated with anti-TREM 2 antibody), a sample that has been treated with a TREM2 ligand other than anti-TREM 2 antibody, or a sample that has been treated with an appropriate non-TREM 2 binding antibody.
In some embodiments, cell migration is measured using a chemotaxis assay. Chemotaxis assays are known in the art. In some embodiments, a cell migration assay (e.g., a chemotaxis assay) is performed on a sample comprising cells endogenously expressing TREM2, such as human macrophages. In some embodiments, a cell migration assay (e.g., a chemotaxis assay) is performed on a sample comprising cells that have been engineered to express TREM 2. In some embodiments, cell migration is measured using a human macrophage chemotaxis assay as described in the examples section below.
In some embodiments, cell viability assay is used to measure cell survival. Cell viability assays are known in the art. In some embodiments, a cell survival assay (e.g., a cell viability assay) is performed on a sample comprising cells endogenously expressing TREM2, such as human macrophages. In some embodiments, a cell survival assay (e.g., a cell viability assay) is performed on a sample comprising cells that have been engineered to express TREM 2. In some embodiments, cell survival is measured using a human macrophage viability assay as described in the examples section below.
In some embodiments, cell function is measured using a functional assay appropriate for the cell. For example, in some embodiments, macrophage function is assessed using a phagocytosis assay (e.g., as described in the examples section below).
In some embodiments, cell differentiation is measured by assessing the ability of a cell endogenously expressing TREM2 to differentiate. For example, in some embodiments, cell differentiation is measured by assessing the ability of macrophages to differentiate from monocytes (e.g., as described in the examples section below).
In some embodiments, the activation of microglia is measured in vivo. In some embodiments, microglial activation is measured using TSPO-PET imaging. TSPO-PET imaging methods are known in the art.
In some embodiments, the anti-TREM 2 antibody enhances microglial function without increasing neuroinflammation. The level of neuroinflammation can be determined by measuring the level of a cytokine (e.g., an inflammatory cytokine), such as, but not limited to TNF- α, IL-1 β, IL-6, IL-1ra, TGF β, IL-15, or IFN- γ. In some embodiments, cytokine levels are measured using an immunoassay, such as an Enzyme Immunoassay (EIA), an enzyme-multifactorial immunoassay (EMIA), an enzyme-linked immunosorbent assay (ELISA), a particulate enzyme immunoassay (MEIA), Immunohistochemistry (IHC), immunocytochemistry, a Capillary Electrophoresis Immunoassay (CEIA), a Radioimmunoassay (RIA), immunofluorescence, a chemiluminescent immunoassay (CL), or an electrochemiluminescence immunoassay (ECL).
Preparation of antibodies
In some embodiments, the antibodies are prepared by immunizing one or more animals (e.g., mice, rabbits, or rats) with an antigen or antigen mixture for inducing an antibody response. In some embodiments, an antigen or antigen mixture is administered in combination with an adjuvant (e.g., freund's adjuvant). After the initial immunization, one or more subsequent booster injections of the one or more antigens may be administered to increase antibody production. After immunization, antigen-specific B cells are harvested, for example, from spleen and/or lymphoid tissue. To generate monoclonal antibodies, B cells are fused to myeloid lineage cells and subsequently screened for antigen specificity. Methods of making antibodies are also described in the examples section below.
Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Alternatively, phage or yeast display techniques can be used to identify antibodies and Fab fragments that specifically bind to a selected antigen. Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens. The antibody may also be a heteroconjugate, e.g., two covalently linked antibodies or immunotoxins.
Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a hybridoma or CHO cell expression system. Many such systems are widely available from commercial suppliers. In the case of antibodies comprising VHAnd VLIn an embodiment of the zone, VHAnd VLThe region may be a single vector, e.g.expressing the unit as a dicistronic or non-cistronic sequenceIs expressed under the control of a promoter. In other embodiments, VHAnd VLThe regions may be expressed using separate vectors. V as described hereinHOr VLThe region may optionally comprise a methionine at the N-terminus.
In some embodiments, the antibody is a chimeric antibody. Methods for making chimeric antibodies are known in the art. For example, chimeric antibodies can be made in which an antigen binding region (heavy chain variable region and light chain variable region) from one species, e.g., a mouse, is fused to an effector region (constant domain) of another species, e.g., a human. As another example, "class switch" chimeric antibodies can be made in which the effector region of the antibody is replaced with an effector region of a different immunoglobulin class or subclass.
In some embodiments, the antibody is a humanized antibody. Generally, non-human antibodies are humanized in order to reduce their immunogenicity. Humanized antibodies typically comprise one or more non-human (e.g., derived from mouse variable region sequences) variable regions (e.g., CDRs) or portions thereof and possibly some non-human framework regions or portions thereof, and also comprise one or more constant regions derived from human antibody sequences. Methods for humanizing non-human antibodies are known in the art. Transgenic mice or other organisms such as other mammals can be used to express humanized or human antibodies. Other methods of humanizing antibodies include, for example, variable domain surface reforming, CDR grafting, graft Specificity Determining Residues (SDR), guided selection, and framework shuffling.
As an alternative to humanization, fully human antibodies can be generated. By way of non-limiting example, transgenic animals (e.g., mice) can be generated that, once immunized, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulins. For example, it has been described that homozygous deletion of the antibody heavy chain Junction (JH) gene in chimeric and germline mutant mice results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into the germline mutant mice will result in the production of human antibodies upon antigen challenge. As another example, human antibodies can be produced by hybridoma-based methods, for example, by using primary human B cells for generating cell lines that produce human monoclonal antibodies.
Human antibodies can also be produced using phage display or yeast display techniques. In phage display, variable heavy and variable light chain gene libraries are amplified and expressed in phage display vectors. In some embodiments, the antibody library is a natural library amplified from a human source. In some embodiments, the antibody library is a synthetic library made by cloning heavy and light chain sequences and recombining to generate a larger pool of antibodies with different antigen specificities. Phage typically display antibody fragments (e.g., Fab fragments or scFv fragments) which are then screened for binding to the antigen of interest.
In some embodiments, antibody fragments (e.g., Fab ', F (ab')2、scFv、VHOr VHH). Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of the intact antibody. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab '-SH fragments can be recovered directly from E.coli cells and chemically coupled to form F (ab')2And (3) fragment. According to another method, F (ab')2The fragments can be isolated directly from the recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art.
In some embodiments, the antibody or antibody fragment is conjugated to another molecule, such as polyethylene glycol (pegylated) or serum albumin, to provide an extended half-life in vitro.
In some embodiments, a multispecific, e.g., bispecific antibody is provided comprising an anti-TREM 2 antibody (or antigen-binding fragment thereof) as described herein. Multispecific antibodies are antibodies that have binding specificity for at least two different sites. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) has binding specificity to TREM2 and binding specificity to at least one other antigen. In some embodiments, a multispecific antibody (e.g., bispecific antibody) binds to two different TREM2 epitopes. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) is capable of inducing TREM2 aggregation at the cell surface. An illustrative method for measuring receptor aggregation using confocal FRET microscopy is described in Wallarbe et al, Biophys.J.,2003,85: 559-. Methods of making multispecific antibodies (e.g., bispecific antibodies) include, but are not limited to, recombinant co-expression of two pairs of heavy and light chains in a host cell, "knob-in-hole" engineering, intramolecular trimerization, and fusion of an antibody fragment to the N-terminus or C-terminus of another antibody, e.g., a tandem variable domain.
V. nucleic acids, vectors and host cells
In some embodiments, an anti-TREM 2 antibody as disclosed herein is prepared using recombinant methods. Accordingly, in some aspects, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding any one of the anti-TREM 2 antibodies as described herein (e.g., any one or more of the CDRs, heavy chain variable region, and light chain variable region described herein); vectors comprising such nucleic acids; and introducing the nucleic acid for replication of the antibody-encoding nucleic acid and/or a host cell expressing the antibody.
In some embodiments, the polynucleotide (e.g., an isolated polynucleotide) comprises a nucleotide sequence encoding an antibody or antigen-binding portion thereof as described herein (e.g., as described in the section entitled "anti-TREM 2 antibody sequence" above). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more of the amino acid sequences (e.g., CDR, heavy chain or light chain sequences) disclosed in the following informal sequence listing. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence disclosed in the following informal sequence listing (e.g., a CDR, heavy chain, or light chain sequence). In some embodiments, a polynucleotide as described herein is operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.
Suitable vectors containing polynucleotides or fragments thereof encoding the antibodies of the disclosure include cloning vectors and expression vectors. Although the cloning vector chosen may vary depending on the host cell intended for use, useful cloning vectors are generally self-replicating, may have a single target of a particular restriction endonuclease, and/or may carry a gene that may be used as a marker for selecting clones containing the vector. Examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK +) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and many other cloning vectors are available from commercial suppliers, such as BioRad, Strategene, and Invitrogen.
Expression vectors are typically replicable polynucleotide constructs containing a nucleic acid of the present disclosure. The expression vector may replicate in the host cell, either in episomal form or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), and any other vector.
Suitable host cells for cloning or expressing a polynucleotide or vector as described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell. In some embodiments, the host cell is a human cell, such as a Human Embryonic Kidney (HEK) cell.
In another aspect, methods of making an anti-TREM 2 antibody as described herein are provided. In some embodiments, the method comprises culturing a host cell as described herein (e.g., a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).
Methods of treatment using anti-TREM 2 antibodies
In another aspect, methods of treatment using an anti-TREM 2 antibody as disclosed herein (e.g., an anti-TREM 2 antibody as described in section III above) are provided. In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, methods of modulating one or more TREM2 activities (e.g., in a subject having a neurodegenerative disease) are provided.
In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, primary age-related tauopathy, Progressive Supranuclear Palsy (PSP), frontotemporal dementia with parkinsonism associated with chromosome 17, dementia with silvery particles, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis/Parkinson's disease-dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle calcification, Down's syndrome, familial dementia of great Britain, familial dementia of Danish type, GSS's disease, globular gliosis, Gualopt-Parkinson's disease, Gualopt PSP, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with globuloid change (HDLS), Huntington's disease, inclusion body myositis, multiple-system atrophy, myotonic dystrophy, Nara-Hara disease, dementia predominantly with neurofibrillary tangles, Niemann-pick disease type C, globus pallidus-pontine-substantia nigra degeneration, Parkinson's disease, pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle-only dementia. In some embodiments, the neurodegenerative disease is alzheimer's disease. In some embodiments, the neurodegenerative disease is na-haar disease. In some embodiments, the neurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is parkinson's disease. In some embodiments, the method comprises administering to the subject an isolated antibody or antigen-binding fragment thereof that specifically binds to a human TREM2 protein (e.g., an anti-TREM 2 antibody as described herein) or a pharmaceutical composition comprising an anti-TREM 2 antibody as described herein.
In some embodiments, an anti-TREM 2 antibody (or antigen-binding portion thereof or pharmaceutical composition) as described herein is used to treat a neurodegenerative disease characterized by a mutation of TREM 2. In some embodiments, the neurodegenerative disease characterized by a mutation in TREM2 is alzheimer's disease, for example, alzheimer's disease characterized by a mutation in R47H of TREM 2.
In some embodiments, methods of modulating one or more TREM2 activities in a subject (e.g., in a subject having a neurodegenerative disease) are provided. In some embodiments, the method comprises modulating the level of sTREM 2; modulating recruitment or phosphorylation of a kinase (e.g., Syk kinase) that interacts with the TREM2/DAP12 signaling complex; regulating phagocytosis (e.g., phagocytosis of cell debris, amyloid beta particles, etc.); modulating cell migration (e.g., migration of myeloid cells, macrophages, microglia and disease-related microglia); and/or modulating cell differentiation (e.g., with regard to myeloid cells, macrophages, microglia, and disease-related microglia). In some embodiments, methods are provided for enhancing one or more TREM2 activities in a subject having a neurodegenerative disease. In some embodiments, methods of reducing the level of sTREM2 in a subject having a neurodegenerative disease are provided. In some embodiments, a method of modulating one or more TREM2 activities in a subject comprises administering to the subject an isolated antibody or antigen-binding portion thereof that specifically binds to a human TREM2 protein (e.g., an anti-TREM 2 antibody as described herein) or a pharmaceutical composition comprising an anti-TREM 2 antibody as described herein.
In some embodiments, the subject to be treated is a human, e.g., a human adult or a human child.
In some embodiments, methods of reducing plaque accumulation in a subject having a neurodegenerative disease are provided. In some embodiments, the method comprises administering to the subject an antibody or pharmaceutical composition as described herein. In some embodiments, the subject has alzheimer's disease. In some embodiments, the subject is an animal model of neurodegenerative disease (e.g., a 5XFAD or APP/PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and/or Tau imaging, for example using Positron Emission Tomography (PET) scanning. In some embodiments, administration of the anti-TREM 2 antibody reduces plaque accumulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a baseline value (e.g., the level of plaque accumulation in the subject prior to administration of the anti-TREM 2 antibody).
In some embodiments, a therapeutically effective amount or dose of an anti-TREM 2 antibody is administered to the subject. Daily dosage ranges of from about 0.01mg/kg to about 500mg/kg, or from about 0.1mg/kg to about 200mg/kg, or from about 1mg/kg to about 100mg/kg, or from about 10mg/kg to about 50mg/kg may be used. However, the dosage can vary according to several factors, including the chosen route of administration, formulation of the composition, patient response, severity of the condition, subject weight, and the judgment of the prescribing physician. The dosage may be increased or decreased over time as required by the individual patient. In some cases, a low dose is initially administered to the patient and then increased to an effective dose that the patient can tolerate. Determination of an effective amount is well within the capability of those skilled in the art.
The route of administration of the anti-TREM 2 antibody as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, enteral, ocular, or aural delivery, or any other method known in the art. In some embodiments, the antibody is administered orally, intravenously, or intraperitoneally.
In some embodiments, the anti-TREM 2 antibody (and optionally another therapeutic agent) is administered to the subject for an extended period of time, e.g., for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days or longer.
Pharmaceutical composition and kit
In another aspect, pharmaceutical compositions and kits are provided comprising an antibody that specifically binds to a human TREM2 protein. In some embodiments, the pharmaceutical compositions and kits are for treating neurodegenerative diseases. In some embodiments, the pharmaceutical compositions and kits are for modulating (e.g., enhancing or inhibiting) one or more TREM2 activities, e.g., Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are for modulating (e.g., reducing) the level of sTREM 2.
Pharmaceutical composition
In some embodiments, a pharmaceutical composition comprising an anti-TREM 2 antibody or antigen-binding fragment thereof is provided. In some embodiments, the anti-TREM 2 antibody is an antibody described in section III above or an antigen-binding fragment thereof.
In some embodiments, the pharmaceutical composition comprises an anti-TREM 2 antibody as described herein and further comprises one or more pharmaceutically acceptable carriers and/or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating agent that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well known in the art.
In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical or subcutaneous administration. A pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds, for example, to stabilize the composition or to increase or decrease absorption of the active agent. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextran; antioxidants, such as ascorbic acid or glutathione; a chelating agent; a low molecular weight protein; compositions that reduce the clearance rate or hydrolysis of the active agent; or an excipient; or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and formulations thereof are well known in the art.
The pharmaceutical compositions described herein may be manufactured in a manner known to those skilled in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, burying or lyophilizing processes. The following methods and excipients are exemplary only and are in no way limiting.
For oral administration, the anti-TREM 2 antibody can be formulated by combining it with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by: the compound is mixed with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, if desired after adding suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrazolidone (PVP). If desired, disintegrating agents may be added, for example, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
The anti-TREM 2 antibody can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. For injection, one or more compounds may be formulated into a formulation by: dissolving, suspending or emulsifying the one or more compounds in an aqueous or non-aqueous solvent (e.g., vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol); and, if necessary, conventional additives such as solubilizers, isotonizing agents, suspending agents, emulsifiers, stabilizers and preservatives. In some embodiments, the compounds may be formulated in aqueous solutions, such as in physiologically compatible buffers (e.g., Hanks 'solution, Ringer's solution, or physiological saline buffer). Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
In some embodiments, the anti-TREM 2 antibody is prepared for delivery in a sustained release, controlled release, extended release, timed release, or delayed release formulation, for example, in a semipermeable matrix of a solid hydrophobic polymer containing the active agent. Various types of sustained release materials are established and well known to those skilled in the art. Current extended release formulations include film coated tablets, multiparticulate or spherulite systems, matrix technologies using hydrophilic or lipophilic materials, and wax based tablets with pore forming excipients. Sustained release delivery systems may release the compound over a time course of hours or days, e.g., 4, 6, 8, 10, 12, 16, 20, 24 hours or more, depending on their design. In general, sustained release formulations may be prepared using naturally occurring or synthetic polymers, such as polymeric vinyl pyrazolidinones, e.g., polyvinyl pyrazolidinone (PVP); a carboxyvinyl hydrophilic polymer; hydrophobic and/or hydrophilic hydrocolloids, such as methylcellulose, ethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose; and carboxypolymethylene.
Typically, pharmaceutical compositions for in vivo administration are sterile. Sterilization may be accomplished according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.
The dosage and desired drug concentration of the pharmaceutical compositions of the present disclosure may vary depending on the particular use contemplated. Determination of the appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described in section VI above.
Medicine box
In some embodiments, a kit is provided comprising an anti-TREM 2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-TREM 2 antibody is an antibody described in section III above or an antigen-binding fragment thereof.
In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises an anti-TREM 2 antibody as described herein and further comprises one or more additional therapeutic agents for treating a neurodegenerative disease, such as alzheimer's disease. In some embodiments, the therapeutic agent is an agent (e.g., an antidepressant, dopamine agonist, or antipsychotic) for treating a cognitive or behavioral symptom of a neurodegenerative disease. In some embodiments, the therapeutic agent is a neuroprotective agent (e.g., carbidopa/levodopa, anticholinergics, dopaminergic agents, monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, glutamatergic agents, Histone Deacetylase (HDAC) inhibitors, cannabinoids, caspase inhibitors, melatonin, anti-inflammatory agents, hormones (e.g., estrogens or progestins), or vitamins).
In some embodiments, the kit comprises an anti-TREM antibody as described herein and further comprises one or more reagents for measuring the level of sTREM 2. In some embodiments, the kit comprises an anti-TREM antibody as described herein and further comprises one or more reagents for measuring TREM2 activity (e.g., for measuring Syk phosphorylation).
In some embodiments, the kit further comprises instructional materials containing the instructions for practicing the methods described herein (i.e., the protocol) (e.g., instructions for using the kit to perform a method of treatment as described in section VI above). While illustrative materials typically include writing or printing materials, they are not limited to such materials. This disclosure encompasses any medium capable of storing such instructions and conveying the instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. Such media may include an internet website that provides such illustrative material.
VIII example
The invention will be described in more detail with the aid of specific embodiments. The following examples are provided for illustrative purposes only and are not intended to limit the invention in any way.
Example 1 Generation and initial characterization of anti-TREM 2 antibodies
Mouse Fc fused human TRERecombinant expression and purification of M2ECD
The extracellular domain (residues 19-172) of human TREM2(UniProtKB ID-Q9NZC2) was subcloned into a pRK vector with secretion signals from mouse IgG kappa chain V-III, amino acids 1-20(UniProtKB ID-P01661) were subcloned in the N-terminal region, and a mouse Fc tag was subcloned between TREM2ECD and Fc with the C-terminal region of GGGGS (SEQ ID NO: 64).
Expi293F was usedTMExpression System kit purified plasmids were transfected into Expi293F according to manufacturer's instructionsTMIn cells (Thermo Fisher). To inhibit maturation of N-linked glycans and reduce glycosylation heterogeneity, the high mannose glucosidase I inhibitor kifanosine (Sigma) was added to the culture medium at a concentration of 1 μ g/mL immediately after transfection. Transfected cells at 6% CO2Incubate under humidified atmosphere in a rotary shaker (Infors HT Multitron) at 125rpm and 37 ℃. Expifeacmine was administered 16 hours after transfectionTM293 transfection enhancers 1 and 2 were added to the cells and the media supernatant was harvested 96 hours post transfection. The clear supernatant was supplemented with EDTA-free protease inhibitor (Roche) and stored at-80 ℃.
For rhTREM2-Fc separation, the clarified media supernatant was loaded on a HiTrap MabSelect SuRe protein a affinity column (GE Healthcare Life Sciences) and washed with 200mM arginine and 137mM succinate buffer pH 5.0. The fusion protein was eluted in 100mM QB citrate buffer pH 3.0 and 50mM NaCl. Immediately after elution, 1M Tris-HCl buffer pH 8.0 was added to the protein solution to neutralize the pH. Protein aggregates were separated by Size Exclusion Chromatography (SEC) on a Superdex 200 incrasase 10/300 GL column (GE Healthcare Life Sciences). SEC mobile phase buffer is maintained at 20mM Tris-HCl pH 8.0, 100mM NaCl and 50mM arginine, which is also the protein storage buffer. All chromatography steps were performed on AKTA pure or AKTA Avant system (GE Healthcare Life Sciences).
Recombinant expression and purification of His-tagged TREM2ECD
The extracellular domain (residues 19-172) of TREM2(UniProtKB-Q9NZC2) was subcloned into the pRK vector with secretion signal from mouse Ig kappa chain V-III, amino acids 1-20(UniProtKB ID-P01661) were subcloned in the N-terminal region, and 6X-His tag (SEQ ID NO:65) was subcloned in the C-terminal region. Insert was verified by sequencing and maxi prep plasmid purification was performed.
Expi293F was usedTMExpression System kit purified plasmids were transfected into Expi293F according to manufacturer's instructionsTMIn cells (Thermo Fisher). Transfected cells at 6% CO2Incubate under humidified atmosphere in a rotary shaker (Infors HT Multitron) at 125rpm and 37 ℃. Expifeacmine was administered 16 hours after transfectionTM293 transfection enhancers 1 and 2 were added to the cells and the media supernatant was harvested 96 hours post transfection.
Harvest media was supplemented with 1M imidazole pH 8.0 to 10mM final concentration and with Nalgene with pore size of 0.4 micronTM Rapid-FlowTMDisposable filtration unit (Thermo Fisher) filtration. Mixing HisPurTMNi-NTA resin (Thermo Fisher) was washed with MQ water and equilibrated with loading buffer (20mM Tris pH 8.0, 150mM NaCl and 10mM imidazole). Affinity purification was performed using a gravity flow method. The harvested medium was loaded onto the resin and the non-specifically bound proteins were washed with loading buffer supplemented with 50mM and 100mM imidazole. The bound His-tagged TREM2 extracellular domain was eluted with 20mM Tris pH 8.0, 150mM NaCl and 200mM imidazole. The eluted proteins were concentrated using an Amicon 10kDa concentrator and further purified by gel filtration chromatography using the AKTA Avant system (GE Healthcare Life Sciences). The proteins were loaded onto a HiLoad Superdex 20016/600 (GE Healthcare Life Sciences) column equilibrated in 1 × PBS and eluted and fractionated using 1 × PBS as running buffer. The eluted fractions were analyzed by electrophoresis on Polyacrylamide (PAGE) gels under denaturing and native conditions. The eluted fractions were further characterized by analytical size exclusion chromatography and complete protein amount determination. The results from PAGE and analytical characterization were used to pool the highly glycosylated protein fractions and these fractions were aliquoted and stored at-80 ℃.
Production of antibodies
Rodents (mice and rats) are immunized with rhTREM2-Fc immunogen or BWZ cells expressing full length Trem2 receptor using standard protocols. Titers were measured throughout the immunization using sera collected at different time points. Detection of antigen-specific immune responses was performed using flow cytometry using rhTREM2-Fc immunogen and live BWZ cells expressing full-length TREM 2. Selection criteria for candidate antibodies include rodent antibody production and specificity of binding to TREM2, as detected by flow cytometry. Antibody secreting cells are isolated from animal immune tissues including spleen, lymph nodes and bone marrow.
Single cell suspensions were analyzed to determine the binding characteristics of the secreted antibodies. Antibody secreting cells are loaded into microfluidic devices and isolated in nanoliter volume reaction chambers to enable detection of secreted antibodies using fluorescence and microscopy assays based on bright field images (see, e.g., U.S. patent No. 9,188,593). Binding assays were performed that involved detecting binding of the antibody to the antigen-coated beads, detecting binding of the soluble fluorescently labeled antigen to the antibody immobilized on the beads, and detecting binding of the antibody to the cell surface expressed antigen. Cell surface expression of antigens includes recombinant and native forms of the antigens presented on the cell surface.
Image analysis is used to identify chambers that exhibit a positive fluorescent signal, indicating the presence of single cells that produce antibodies with the desired characteristics, and the chamber contents are recovered and lysed in 384-well plates (see, e.g., U.S. patent No. 10,087,408). The single cell lysates were then subjected to RT-PCR to amplify the heavy and light chain variable region sequences. The resulting amplicons were then sequenced to determine the cDNA sequences of the paired heavy and light chain variable regions from the selected single cells. The resulting sequences were examined manually and analyzed to determine sequence diversity and somatic hypermutation. Sequences were selected for expression based on screening data and sequence diversity. The expressed antibodies were tested to confirm antigen binding specificity.
Primary screening for anti-TREM 2 antibodies
Primary screening of antibodies was performed in HEK293 cells expressing TREM2, wild type ipscs and TREM2 knock-out ipscs as follows.
1. Screening for TREM2 binding in HEK cells expressing TREM2
HEK293 cell lines stably expressing human TREM2/DAP12 were generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12 and DAP12 alone, respectively. Stably expressing clones were selected and cell surface TREM2 expression was assessed by flow cytometry. APC-bound rat anti-human/mouse TREM2 monoclonal antibody (R & D, cat # MAB17291) was used to detect surface TREM2 expression. The clone exhibiting the highest expression level of wild-type TREM2 was selected and designated "HEK 293-H6". Clones stably expressing DAP12 were analyzed by western blotting and the selected clone was named "HEK 293-DAP12# 1".
HEK293 overexpressing human TREM2(HEK293-H6) and HEK293 overexpressing GFP (B5) were harvested by 0.05% trypsin and incubated at 37 ℃ for 2 hours. After incubation, cells were centrifuged and washed twice with FACS buffer (PBS + 0.5% BSA). Mixing the cells in each cell line 106The density of/mL was resuspended in FACS buffer with human Trustain FcX solution (Biolegend, cat. No. 422302). Mixed cell lines were seeded at 200,000 cells/well in 96-well round bottom plates and incubated for 20 minutes at room temperature. After incubation, cells were centrifuged and incubated with an approximately 0-200nM dose-titrated anti-TREM 2 antibody on ice for 45 minutes. After incubation, cells were centrifuged and washed three times with FACS buffer. The cells were then incubated with a secondary antibody (Alexa Fluor 647 AffiniPure F (ab')2 fragment goat anti-human IgG (H + L), Jackson ImmunoResearch Laboratories, Cat. No. 109-. After incubation, cells were washed three times with FACS buffer, resuspended in 100 μ Ι of FACS buffer, and analyzed by flow cytometry (BD facscan II, San Jose, CA) to obtain 30,000 events for each sample. The mean fluorescence intensity of each cell was calculated by FlowJo software and used to generate a dose-response binding curve.
FIG. 1 shows representative results for exemplary antibodies that bind to the cell surface receptor TREM2 in HEK293-H6 cells.
For TREMAssessment of activation of 2-dependent pSyk signaling
TREM 2-dependent activation of pSyk signaling was measured in human macrophages or HEK293-H6 cells using a commercial AlphaLisa assay from Perkin-Elmer.
For all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, lipid vesicles were prepared within two weeks of the experiment as follows: 7mg DOPC (1, 2-dioleoyl-sn-glycero-3-phosphocholine) and 3mg POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were combined in chloroform in glass vials and in N2Drying under gas flow for 1-2 hours or until complete drying. The lipid mixture was resuspended in 1mL HBSS (final lipid concentration of about 10mg/mL) and vortexed for 2-3 minutes. The lipid suspension was then extruded using an Avanti micro extruder built with one 100-nm pore size membrane to form small unilamellar vesicles at 10 mg/mL.
1. Administration of antibodies in cells
The day before the assay, human macrophages or HEK293-H6 cells were seeded at 100,000 cells/well or 40,000 cells/well, respectively, onto poly D-lysine coated 96-well plates. The antibody was diluted into PBS starting at 300nM and continued with 10-point serial dilution titration, with 3-fold dilution between each point. For the antagonist dose response curve, lipid vesicles containing 70% DOPC and 30% POPS were also included in the antibody/PBS mixture at a final concentration of 1 mg/mL. Cells were washed 3 times with HBSS using a Biotek 405/406 plate washer, followed by addition of 50 μ L of antibody/PBS (with or without vesicles) per well using a Hamilton Nimbus liquid processor. The plates were then transferred to a 37 ℃ incubator for 5 minutes. The liposome/antibody solution was removed by tapping the plate and 40 μ L of lysis buffer (Cell Signaling Technologies, CST) containing 1 μ M PMSF was added using a liquid processor. Lysates were then frozen at-80 ℃ or immediately assayed with the AlphaLisa assay.
Human macrophages were prepared for the following assay. Human monocytes were isolated from fresh blood according to the RosetteSep human monocyte enrichment cocktail protocol (Stemcell Technologies, REF # 15068). Isolated monocytes were washed with wash buffer (PBS + 2% FBS) and resuspended in 10mL ACK lysis buffer (thermo fisher Scientific, catalog No. a10492) to lyse red blood cells. 20mL of wash buffer was added to stop cell lysis, and the samples were centrifuged and washed again with medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin-streptomycin). Human monocytes were then differentiated into macrophages in the presence of 50ng/mL human recombinant M-CSF (Gibco, Cat. PHC9501) in medium in 250-mL flasks. Fresh human M-CSF was incorporated on day 3 and human macrophages were subsequently harvested on day 5 and used for the assay.
AlphaLisa assay
Cell lysates were assayed for pSyk using the standard protocol of the Perkin Elmer pSyk AlphaLisa kit. Briefly, 10 μ Ι _ of lysate/well was transferred to a white opaque 384-well optiplate (perkin elmer). Next, 5 μ L of Acceptor Mix (working solution containing Acceptor beads) was added per well, followed by sealing with a foil seal and incubation at room temperature for 1 hour. Subsequently, 5 μ L of Donor Mix (working solution containing Donor beads) was added to each well under reduced light conditions. Plates were sealed again and incubated at room temperature for 1 hour. Finally, the plates were read using the AlphaLisa settings on a Perkin Elmer EnVision plate reader.
Figure 2 shows a representative anti-TREM 2 antibody dose response curve for activation of pSyk signaling in primary human macrophages. Filled black circles (●) represent anti-TREM 2 antibody, and open white circles (°) represent isotype controls. Each curve represents the average of three independent experiments, and EC50The values are provided in table 1 below. The results indicate that anti-TREM 2 antibody was able to activate TREM2-DAP12 ITAM signaling in primary human macrophages.
Liposome response assay in iPSC microglia
TREM2 agonist antibodies and phosphatidylserine-containing liposomes activate pSyk via TREM 2. To understand the effect of anti-TREM 2 antibody on Syk signaling in the presence of liposomes, iPSC microglia were pretreated with anti-TREM 2 antibody, followed by assessment of liposome response in the cells.
Prior to the assay, ipscs were first differentiated into Hematopoietic Progenitor Cells (HPCs) using a commercially available kit (STEMdiff hematomimetic kit from StemCell Technologies). HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14-21 days. Once the floating cells in the co-culture were mainly identified as mature microglia (> 80%), the microglia were used for the assay.
Two days prior to the assay, human iPSC microglia cells were seeded at 30,000 cells/well in poly D-lysine coated 96-well plates. The antibody was diluted at 100nM into a medium containing IMDM, 10% Hyclone FBS and 1% penicillin-streptomycin, and the antibody solution was administered to the cells for 24 hours or at 37 ℃ for 5 minutes. Subsequently, the cells were washed once with HBSS and then 1mg/mL of lipid vesicles containing 70% DOPC and 30% POPS were administered at 37 ℃ for 5 minutes. The liposome solution was removed by tapping the plate and 30 μ L of lysis buffer (Cell Signaling Technologies, CST) containing 1 μ M PMSF was added. Lysates were then frozen at-80 ℃ or immediately assayed with the AlphaLisa assay. Cell lysates were assayed for pSyk as described above using the standard protocol of the Perkin Elmer pSyk AlphaLisa kit.
Figures 3A and 3B show activation of pSyk signal in human iPSC microglia cells incubated with anti-TREM 2 antibody, followed by administration of lipid vesicles and assessment of liposome response in the cells. White bars indicate incubations performed using PBS as a control instead of lipid vesicles. Data represent mean and standard error of 2-7 independent experiments. Fig. 3A shows data for iPSC microglia pretreated with antibody for 5 minutes, and fig. 3B shows data for iPSC microglia pretreated with antibody for 24 hours. The results show that pretreatment of human iPSC microglia with anti-TREM 2 antibody resulted in an increase in phospho Syk signal elicited by liposomes compared to isotype controls, suggesting that anti-TREM 2 antibody does not interfere with lipid activation that would otherwise enhance pSyk signaling in cells.
Human TREM2 NFAT reporter assay
Human TREM2/DAP 12-expressing Jurkat NFAT cell lines were generated as follows. Jurkat NFAT reporter cells were infected with lentiviral vectors expressing human TREM2 and DAP12 and cultured in RPMI containing 10% Hyclone FBS and 1% avidin/streptomycin. Stably expressing clones were selected in the presence of puromycin and Zeocin. Cell surface TREM2 expression was assessed by flow cytometry using biotinylated anti-TREM 2 antibodies (SEQ ID NOS: 66 and 67). The clone showing the highest wild type TREM2 expression level was selected and designated as hTrem2/NFAT Jurkat reporter cell for use in the assays described below.
The day before assay, 96-well plates were pre-coated with anti-TREM 2 antibody or isotype control titrated at 0-500nM dose (45 μ L/well, 12 points total) and incubated overnight at 4 ℃. After overnight incubation, pre-coated plates were washed twice with PBS and then loaded with hTrem2/NFAT Jurkat reporter cells (10) in 200 μ L fresh medium (RPMI with 10% Hyclone FBS and 1% avidin/streptomycin)6Individual cells/well). Plates were incubated at 37 ℃ for 24 hours, after which 50 μ L/well of quantlucia solution was added to each well and mixed well. For analysis, 20 μ L of solution was removed from each well and transferred to a 384-well white plate for signal measurement by a luminometer (Perkin Elmer Envision).
FIG. 4 includes a representative anti-TREM 2 antibody dose-response curve for activating NFAT as measured by detection of reporter luciferase, and activated EC50The values are provided in table 1 below. The results in fig. 4 demonstrate that the candidate anti-TREM 2 antibody is capable of inducing NFAT activation and sufficient downstream signaling to activate a transcriptional response relative to an isotype control.
Survival assay for human macrophages
Human monocytes were isolated according to the RosetteSep human monocyte enrichment cocktail protocol (Stemcell Technologies, Cat. No. 15068). Isolated monocytes were washed with wash buffer (PBS + 2% FBS) and resuspended in 10mL of ACK lysis solution (thermo fisher Scientific, catalog No. a10492) to lyse red blood cells. 20mL of wash buffer was added to stop lysis. The cell suspension is centrifuged and incubated with medium (RPMI1640+ 10% FBS + penicillin/streptomycin). The cells were treated with 106The density of individual cells μ L/mL was resuspended in culture medium and used in the survival assay described below.
The day before assay, 96-well plates were pre-coated with anti-TREM 2 antibody or isotype control titrated at 0-200nM dose (45 μ L/well, 12 points total) and incubated overnight at 4 ℃. After overnight incubation, the pre-coated plates were washed twice with PBS and then loaded with human monocytes (10) in the presence of low concentration human M-CSF (5ng/mL, Gibco, catalog number PHC9501)5Individual cells/well). After 5 days at 37 ℃, the medium was aspirated and 100 μ L PBS +100 μ L Celltiter-glo medium (Promega, catalog No. G7571) was added to each well. After incubation for 10 minutes, the cell culture medium was transferred to a multiwell plate compatible for photometric use, and the luminescence of the cell viability was recorded.
FIG. 5 shows a representative anti-TREM 2 antibody dose-response curve for cell survival in human macrophages under low M-CSF conditions, and surviving EC50The values are provided in table 1 below. The results indicate that TREM2 agonist antibodies have sufficient receptor activation capacity to induce a transcriptional response for modulating cell function and promoting survival of human macrophages under M-CSF conditions.
Biacore kinetic measurements of antibodies
Using surface plasmon resonance (Biacore)TM8K instrument) to measure the affinity of anti-TREM 2 antibodies to human and cynomolgus TREM2 ECD. anti-TREM 2 antibody was captured on Biacore series S CM5 sensor chip (GE Healthcare Life Sciences, catalog No. 29149604) using human Fab capture kit (GE Healthcare Life Sciences, catalog No. 28958325). Serial 3-fold dilutions of recombinant human or cynomolgus TREM2 were injected at a flow rate of 30 μ L/min. Antibody binding was monitored for 300 seconds followed by antibody dissociation for 600+ seconds in HBS-EP + running buffer (GE Healthcare Life Sciences, Cat. No. BR 100669). The binding reaction was corrected by subtracting the RU value from the blank flow cell. Using konAnd koffThe kinetic analysis was performed with a simultaneously fitted 1:1Languir model. KDThe binding value is provided belowIn table 1.
TABLE 1 in vitro characterization of antibodies
NB no detectable binding
ND: not determined
Example 2 modulation of soluble TREM2 levels and phagocytosis behavior in human macrophages
Soluble TREM2 dose response assay in human macrophages
Human macrophages were produced as described above. The day before the assay, human macrophages were seeded at 100,000 cells/well in poly D-lysine coated 96-well plates. The antibody was diluted in human macrophage medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids and 1% penicillin-streptomycin), starting at 300nM and continuing with 10-point serial dilution titration, with 3-fold dilution between points. Cells were given the antibody and incubated for 24 hours. After incubation with antibody, the plates were centrifuged to remove debris and the supernatant was collected for soluble TREM2 measurement.
The solubility TREM2 was measured as follows. Briefly, MSD small spot streptavidin plates (Meso Scale Discovery) were coated with biotinylated anti-hTREM 2 monoclonal antibody (R & D Systems) overnight at 4 ℃. Plates were then blocked with 3% BSA/TBST for 1 hour at room temperature. Samples and standards were prepared with SDS-containing buffer by heating to 95 ℃ for 5 minutes. The prepared samples and standards were diluted in assay plates with 3% BSA/TBST 1:10 after blocking. TREM2-His protein diluted in 3% BSA/TBST was used as a standard for absolute quantification. After two hours incubation at room temperature, plates were washed with TBST. The primary detection antibody was diluted with sulfo-labeled goat anti-human TREM2(R & D Systems) with 3% BSA/TBST, added to the plate, and incubated for one hour at room temperature. After washing with TBST, MSD plates were developed using 2x MSD read buffer T, followed by detection using MSD Sector plate reader. MSD values were converted to absolute amounts of sTREM2 by fitting a standard curve using Prism 7.0 software (Graphpad). Modulation of TREM2 shedding is expressed as the ratio of soluble TREM2 from cells incubated with test anti-TREM 2 antibody normalized to soluble TREM2 from cells not cultured in media with specific anti-TREM 2 antibody.
Figure 6 shows representative soluble TREM2 levels (sTREM2) as a function of anti-TREM 2 antibody concentration. The results indicate that anti-TREM 2 antibodies were able to reduce sTREM2 levels in human macrophages in a dose-dependent manner after overnight treatment.
Phagocytosis assay in human macrophages
Human macrophages were produced as described above. Two days prior to the assay, human macrophages were seeded at 80,000 cells/well in poly D-lysine coated 96-well plates. The antibody was diluted at 100nM into medium containing RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids and 1% penicillin-streptomycin. The antibody solution was then administered to the cells at 37 ℃ for 24 hours. The nuclei and membranes were then stained for 10 minutes, after which 5. mu.g/mL of pHrodo-myelin was added. The cells were then incubated at 37 ℃ for 4 hours. The pHrodo fluorescence of each cell was measured on a high volume confocal microscope (Opera Phoenix) and the fluorescence intensity was quantified on the instrument software.
pHrodo-myelin is prepared by purifying myelin from wild type C57Bl/6 mouse brain (Jackson Laboratories) using the method described by Safaiyan et al (2016, Nature Neuroscience 19(8): 995-998). After purification, myelin was resuspended in PBS and adjusted to 1mg/mL Protein concentration using DC Protein Assay kit 2(BioRad, cat # 5000112). Myelin was labeled with pHrodo-Red using a micro labeling kit (ThermoFisher, Cat. No. P35363) according to the manufacturer's instructions. Excess labeling was removed by precipitating myelin at 10,000g for 5 minutes, removing the supernatant, and repeating these steps 3-5 times.
Figure 7 shows representative results of phagocytosis assays in human macrophages. Myelin phagocytosis is measured by detecting and quantifying the pHrodo fluorescence in microscopic images of macrophages treated with TREM2 and comparing the measurements to those of isotype controls. The results show that human macrophages treated with exemplary TREM2 agonist antibodies increased pHrodo-myelin phagocytosis relative to isotype controls, suggesting that anti-TREM 2 antibodies can beneficially clear myelin debris from cells.
Example 3 modulation of lipid accumulation in iPSC microglia
Lipid storage assay
Prior to the assay, ipscs were first differentiated into Hematopoietic Progenitor Cells (HPCs) using a commercially available kit (STEMdiff hematomimetic kit from StemCell Technologies). HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14-21 days. Once the floating cells in co-culture were mainly identified as mature microglia (> 80%), the microglia were used for the assay.
Cells (iPSC microglia, 30,000 cells/well) were seeded into PDL-coated 96-well plates in complete serum medium. After 24 hours at 37 ℃, purified unlabeled myelin (50 μ g/mL final concentration, purified from wild type C57Bl/6 mouse brain (Jackson Laboratories) using the method described in Safaiyan et al (2016, Nature Neuroscience 19(8): 995-. After lipid treatment for 24 hours at 37 ℃, anti-TREM 2 antibody or RSV control was spiked into the wells until the final concentration was 100 nM. The cells were incubated at 37 ℃ for an additional 48-72 hours and then harvested or imaged. For myelin elution experiments, myelin was removed after a24 hour incubation period and replaced with antibody-containing media for subsequent 24-48 hour incubations.
For nile red imaging, the supernatant was removed and the cells were incubated at 37 ℃ for 30 minutes in live cell imaging buffer (Life Technologies, cat # a14291DJ) containing 1 μ M nile red (ThermoFisher, cat # N1142) and 1 drop/mL Nucblue (ThermoFisher, cat # R37605). After the incubation period, the staining solution was removed and the cells were fixed in 4% paraformaldehyde. The cells were then imaged on an Opera Phoenix high volume confocal imager using Alexa 568 and DAPI illumination settings. The liquid spots were analyzed using a spot discovery algorithm using the Harmony software supplied with the instrument. Figure 8A includes representative microscope images of iPSC microglia treated with vehicle or myelin (50 μ g/mL final concentration) for 24 hours followed by 72 hours incubation with isotype control or an exemplary anti-TREM 2 antibody (CL 0020123). Fig. 8B is a representative bar graph of the same anti-TREM 2 antibody used in the microscope image of fig. 8A. Nile red staining was quantified by the total spot intensity of each cell and the data is shown as the mean and standard deviation of three technical replicates in different fields of the same microscopy sample.
For lipidomic analysis, cells were washed once with PBS while kept on ice. A 70 μ L volume of 9:1 methanol in water solution containing 1:100 internal standard was added to the cells in the 96-well plate. The plates were stirred on a shaker at 4 ℃ and 1200rpm for 20 minutes and then centrifuged at 300x g for 5 minutes. A50. mu.L sample of the supernatant was transferred to an LCMS vial and kept at-80 ℃ until analyzed on the instrument.
Liquid levels were analyzed by liquid chromatography coupled to an electrospray mass spectrometer (QTRAP 6500+, Sciex, Framingham, MA, USA) (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 5. mu.L of the sample was injected into a BEH C181.7 μm, 2.1X 100mm column (Waters Corporation, Milford, Massachusetts, USA) at 55 ℃ using a flow rate of 0.25 mL/min. For positive ionization mode, mobile phase a consisted of 60:40 acetonitrile/water (v/v) and 10mM ammonium formate + 0.1% formic acid; mobile phase B consisted of 90:10 isopropanol/acetonitrile (v/v) and 10mM ammonium formate + 0.1% formic acid. For negative ionization mode, mobile phase a consisted of 60:40 acetonitrile/water (v/v) and 10mM ammonium acetate; mobile phase B consisted of 90:10 isopropanol/acetonitrile (v/v) and 10mM ammonium acetate. The gradient is programmed as follows: 0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, within 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B. Electrospray ionization was performed in positive or negative ion mode using the following settings: curtain gas under 30; a collision gas disposed at a median value; ion spray voltage at 5500 (positive mode) or 4500 (negative mode); temperatures at 250 ℃ (positive mode) or 600 ℃ (negative mode); an ion source gas 1 at 50; ion source gas 2 at 60. Data acquisition was performed in multiple reaction monitoring mode (MRM) using analyst1.6.3(Sciex), using the following parameters: dwell time (msec) and impact energy (CE); a Declustering Potential (DP) at 80; inlet potential (EP) at 10 (positive mode) or-10 (negative mode); and a collision cell exit potential (CXP) at 12.5 (positive mode) or-12.5 (negative mode). The fluid was quantified using a mixture of non-endogenous internal standards. The liquids were identified based on their retention times and the MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA).
Fig. 8C and 8D show the levels of Cholesteryl Ester (CE) (fig. 8C) and Triacylglycerol (TAG) lipid species (fig. 8D) as detected by mass spectrometry in cell lysates of iPSC microglia cells treated with exemplary anti-TREM 2 antibody for 72 hours after 24 hours myelin treatment. Fig. 8E and 8F show the levels of Cholesterol Ester (CE) (fig. 8E) and Triacylglycerol (TAG) lipid species (fig. 8F) in cell lysates of iPSC microglia subjected to myelin elution experiments using an exemplary anti-TREM 2 antibody, as detected by mass spectrometry. The LC/MS data generated in fig. 8C-8F were normalized to internal standards for CE data and to myelin + isotype control for each individual lipid species of TAG data.
Lipid accumulation in iPSC microglia was induced by myelin treatment, reflected by increased neutral lipid staining (nile red) and by LC/MS for detection of specific lipid species in cell lysates. The data shown in figures 8A-8F generally indicate that treatment of iPSC microglia with the exemplary anti-TREM 2 antibody after myelin challenge reduced accumulation of lipid species as indicated by a reduction in neutral lipid staining in the cells and by a reduction in CE and TAG lipid species measured by LC/MS. The reduction in lipid levels caused by antibody treatment was observed at different time points ranging from 24 hours to 72 hours. To eliminate the possibility of a reduction in lipid levels by blocking lipid uptake, a myelin elution experiment was performed in which myelin was removed prior to addition of anti-TREM 2 antibody. Figure 8F shows that anti-TREM 2 antibody also reduced lipid levels relative to isotype control in iPSC microglia with myelin elution prior to antibody treatment.
Example 4 functional epitope partitioning of antibodies
TREM2 antibody epitope small region (bin) was determined by competitive binding on TREM2 protein. Epitope partitioning experiments were performed on a cartera LSA instrument using the classical sandwich epitope partitioning configuration method at 25 ℃. All test antibodies were immobilized onto HC30M chips by amine coupling. The test antibody is then subjected to multiple cycles of sandwich competitive binding. Each cycle consists of injection of antigen (His-tagged TREM2ECD) to immobilized antibody followed by injection of analyte antibody to immobilized antibody. At the end of each cycle, the surface immobilized with antibody was regenerated by injection of a low pH buffer (pH 3) containing 1.25M NaCl. Epitope binding data were evaluated by cartera software to create competition matrices and epitope partitions. The results are provided in table 2.
Two agonist domains were identified by epitope partitioning of the anti-TREM 2 antibody: (1) a stalk-binding agonist and (2) an IgV domain-binding agonist. Antibodies with the same region demonstrate the same function, e.g., inhibition of TREM2-DAP12pSyk activation by lipid ligands (antagonist antibodies), pSyk activation using individual antibodies (stalk binding agonists, IgV domain binding agonists).
TABLE 2 anti-TREM 2 antibody cell labeled by functional class
Cell 1
Cell 2
Stalk agonists
IgV agonists
CL0020188
CL0020123
Example 5 binding of antibodies to TREM2 Stem peptide
Binding of TREM2 antibodies to human and mouse TREM2 stalk region peptides was assessed. The test peptides included: (1) the full-length stalk region (amino acids 129-172 of human TREM2, UniProtKB Q9NZC 2; amino acids 131-169 of mouse TREM2, UniProtKB Q99NH8) and (2) a truncated stalk peptide containing an ADAM10/17 cleavage site (amino acids 149-163 of human/mouse TREM 2). Binding of the antibody to TREM2 stalk peptide was detected using a standard sandwich ELISA. Briefly, 96-well half-field ELISA plates were coated with streptomycin overnight at 4 ℃. The following day, biotinylated TREM2 stalk peptide diluted in 1% BSA/PBS was added to the plate and incubated for 1 hour. Antibodies diluted with 1% BSA/PBS were then added and incubated for 1 hour. Antibodies bound to the peptide were detected using anti-human kappa-HRP secondary antibody (Bethyl Laboratories, Inc.) diluted with 1% BSA/PBS. By reaction with detection reagents (One-step TMB Ultra, Thermo) and by standard spectrophotometry instrumentsThe plate was assayed by measuring the absorbance at 450nm (A450). The results are provided in table 3 below. Data are presented as normalized values (fold against background, where background is isotype control).
TABLE 3 binding of anti-TREM 2 antibodies to TREM2 nectaride
Table 3 depicts the human and mouse stalk region peptide-antibody binding interactions that support the epitope zoning data in table 2. Based on the data in Table 3, the sites at which certain anti-TREM 2 antibodies appear to bind correspond to amino acids 129-148 of the TREM2 extracellular stem region.
The ability of anti-TREM 2 antibodies to inhibit the cleavage of TREM2 nectin by ADAM17 was also analyzed using a fluorescence polarization assay. First with an assay buffer (25mM Tris pH 7.5, 2.5. mu.M ZnCl) with streptomycin20.005% Brij-35) preparing TREM2 petide. anti-TREM 2 antibody was then preincubated with TREM2 stalk peptide for 30 minutes at room temperature. After the pre-incubation period, ADAM17 (R) was added&D systems, cat # 930-ADB) and incubated with the peptide for 20 hours at 37 ℃. The next day, the samples were further diluted with assay buffer and transferred to black light-tight 384-well plates. The fluorescence polarization was then measured on a Perkin Elmer EnVision plate reader. Fluorescence polarization of TREM2 depsipeptide preincubated with anti-TREM 2 antibody was compared to that of full-length TREM2 depsipeptide and enzyme control (full-length TREM2 depsipeptide with ADAM 17).
TREM2 stalk binding antibodies significantly enhanced fluorescence polarization, indicating partial inhibition of stalk peptide cleavage by ADAM17 (clones CL0020141, CL0020188, CL0020313, CL 0020308). IgV-binding antibody CL0020107 does not bind TREM2 stalk region peptide and therefore does not show an effect on peptide cleavage in a fluorescence polarization assay.
Example 6 pharmacokinetic analysis of anti-TREM 2 antibodies
Pharmacokinetic profiles of anti-TREM 2 antibodies were evaluated in mice. C57BL/6J mice were purchased from Jackson Laboratory (stock No. 000664), 2 months old, and used for 7 day Pharmacokinetic (PK) and target engagement studies. Mice homozygous for 3-month-old human Trem2cDNA KI (huTrem 2) were usedKI/KI) A24 hour target engagement study was performed. The generation and propagation of human Trem2cDNA KI mice is described below.
Human Trem2cDNA Generation of KI mouse model
Human TREM2cDNA KI mice (huTrem 2) were generated as followsKI/KI). The human Trem2cDNA-pA sequence was inserted at the start site of the mouse Trem2 endogenous ATG. Insertion of human Trem2cDNA-pA results in substitution of exon 1 sequence of mouse Trem2, which allows expression of human Trem2cDNA driven by endogenous mouse promoter and disrupts endogenous smallExpression of murine Trem 2. Generation of huTrem2 Using homologous recombination in a C57BL/6 genetic backgroundKI/KIA mouse.
For humem 2KI/KITargeting vector, long homology arm (LA) extended about 3.6kb upstream of the 5 'end of the human Trem2cDNA-pA sequence and short homology arm (SA) extended about 2.3kb 3' downstream of the Neo cassette flanking the FRT. Both the long and short homology arms were amplified from the C57BL/6BAC clone (RP23:358G22) and then subcloned into an approximately 2.4kb pSP72(Promega) backbone vector containing the ampicillin selection cassette. The hUBS-gb2 FRT-flanked neomycin cassette was inserted just downstream of the hTRem2-pA cassette, resulting in a targeting vector of approximately 13.6kb in size. Mu.g of the targeting vector was linearized with restriction enzyme Not I (New England Biolabs) and subsequently transfected into FLP C57Bl/6(B6) embryonic stem cells (ES) by electroporation. After selection with the G418 antibiotic, surviving clones were amplified for PCR analysis to identify positive recombinant ES clones. The primer sequences used for PCR screening (SEQ ID NO: 53-5'-AGG AAT GTG GGG AGC ACG GAG-3' and SEQ ID NO: 54-5'-TGC ATC GCA TTG TCT GAG TAG GTG-3') amplified a 2.81-kb fragment containing a region from the bghpA element downstream of the short homology arm (SA) outside the 3' region. Five clones were identified as positive and selected for further amplification. The Neo cassette was removed by the Flp transgene during ES clonal amplification.
Genomic DNA extracted from five positive clones was first characterized by sequencing analysis. The 1.19-kb product was amplified and sequenced by primers (SEQ ID NO:55 ═ 5'-ACC CTA GTC CTG ACT GTT GCT C-3'; SEQ ID NO:56 ═ 5'-TAT AGG AAC TTC GCGACA CGG ACA C-3') to confirm the 5 'genome/neo cassette adaptor region and the 3' KI cassette adaptor region. The sequencing results confirmed the introduction of the human Trem2cDNA-pA sequence into all five clones.
Five positive clones were further characterized by Southern blot (Southern Blotting) analysis using probes targeting the short and long arms. ES cell genomic DNA digested with Ssp I and Bam HI was hybridized to short-arm and long-arm probes, respectively. All five ES clones were confirmed to carry the corrected homologous recombination event in both the long and short arms. The primer sequence for amplifying the short-arm probe (658bp) was (SEQ ID NO: 57-5 ' -ACA GGA GGG ACC TAC CTT CAG 3 '; SEQ ID NO: 58-5 ' -GCC TGC CTT TCA GAG ACC TCA GTC-3). The primer sequence for amplifying the long-arm probe (681bp) is (SEQ ID NO: 59-5'-CCT CTC CGG CTG CTC ATC TTA CTC-3'; SEQ ID NO: 60-5'-GTC TCT CAG CCC TGG CAG AGT TTG-3').
All five ES cell clones were then injected into C57BL/6 blastocysts. Pups from one clone demonstrated germline transmission by PCR genotyping. Primers used for genotyping were (SEQ ID NO:61 ═ pr1:5'-CGC CTA CCC TAG TCC TGA CTG TTG-3', SEQ ID NO:62 ═ pr2:5'-AAA GCC TAC AGC ATC CTC ACC TC-3', and SEQ ID NO:63 ═ pr3:5'-GCA TCA TGG GGT TGT AGA TTC CG-3'). The PCR product for pr1/pr2 in wild type was 658 bp. The PCR product for pr1/pr3 on the KI allele was 469 bp.
Antibody administration and plasma/CSF Collection
For PK analysis, C57BL/6J mice were administered 10mg/kg anti-TREM 2 antibody or control IgG by Intravenous (IV) tail vein injection (administration volume was about 200 μ L/mouse, n ═ 3 per group). Blood samples were collected at 1 hour, 24 hours, 4 days and 7 days post-dose. Blood samples were collected at the first three time points via submandibular bleeding by using a 3-mm lancet (golden rod animal lancet). Final and final blood samples were collected by cardiac puncture on day 7. Blood was collected in EDTA tubes (Sarstedt Microvette 500K3E, cat No. 201341102), slowly inverted for mixing, and centrifuged at 4 ℃. The plasma (top) layer was transferred to a 1.5-mL Eppendorf tube and stored at-80 ℃ until analysis.
For the 24-hour target engagement study, the mouse surrogate anti-TREM 2 antibody was tested using C57BL/6J mice, and huTrem2 was usedKI/KIMice tested human anti-TREM 2 antibodies. Mice were given 100mg/kg anti-TREM 2 antibody or control IgG (dosing volume of about 200 μ L/mouse, n ═ 5 per group) by Intravenous (IV) tail vein injection. Blood samples were collected 24 hours prior to dosing to determine the TREM2 baseline level. Final blood and CSF samples were collected 24 hours after dosing. Plasma formulations were prepared as described above. To collect CSF samples, a sagittal incision was made in the back of the skull and the subcutaneous tissue and muscle were separated to expose the large cistern. Piercing using pre-stretched glass capillariesThe pool was enlarged and CSF samples were collected. The CSF was then centrifuged at 4 ℃ to remove blood residues and CSF supernatant was transferred to 0.5-mL Low Protein lobond Eppendorf tubes (Eppendorf, catalog No. 022431064) for storage at-80 ℃ until analysis.
Analysis of in vivo plasma levels of anti-TREM 2 antibody
For anti-TREM 2 antibody PK analysis, total antibody concentration in mouse plasma was quantified using a general human anti-Fc sandwich ELISA. 384 well MaxiSorp plates were coated with 1 μ g/mL anti-huFc donkey polyclonal antibody (Jackson Immunoresearch) overnight. anti-huFc donkey antibody (Jackson Immunoresearch) conjugated to HRP was added as detection reagent after incubation with plasma diluted 1:2,000 or 1:20,000 in assay buffer (PBST, 1% BSA). Standard curves for each individual antibody from 2nM to 2.7pM were generated using five-parameter logistic regression using 3-fold dilutions.
Figure 9 shows representative mouse PK profiles for certain anti-TREM 2 antibodies. Antibody clearance (CL [ mL/day/kg ]) for each representative antibody over a 7 day period is provided and compared to a normal effector-free isotype control. Each antibody shown in figure 9 exhibited comparable clearance relative to the isotype control.
In vivo target engagement: sTREM2 plasma levels
To measure soluble TREM2(sTREM2) plasma levels, human TREM2cDNA KI mice (huTrem 2) were madeKI /KI) Hemorrhages were then treated intravenously with 100mg/kg of test anti-TREM 2 antibody or isotype control. Mice were bled 24 hours after dosing. Plasma was obtained from blood samples and evaluated in the MSD assay performed as follows. MSD SECTOR plates were coated with 1. mu.g/mL capture antibody (R + D anti-TREM 2 antibody, cat # MAB17291-100) diluted in PBS and incubated overnight at 4 ℃. The sample wells were blocked with undiluted MSD blocker a for 1 hour. Plasma samples were diluted with 25% MSD blocking agent A1: 20 in Tris Buffered Saline (TBST) containing 0.05% Tween-20 and added to each sample well on the plate, which was then incubated at room temperature for 2 hours. The detection antibody (MSD sulfo-labeled goat anti-human antibody, cat # R32AJ-1, 1: 100)0) Add to each sample well and incubate the plate at room temperature for 1 hour. TBST washing was performed for each sample well using a Biotek plate washer. Detection reagents (MSD read buffer) were added and measured using an MSD Meso Sector S600 reader to obtain the results of sTREM2 binding to antibody.
FIGS. 10A and 10B are shown in huTrem2KI/KITotal sTREM2 levels (fig. 10A) and antibody binding sTREM2 levels (fig. 10B) of exemplary anti-TREM 2 antibodies in plasma. Data were normalized to pre-dose sTREM2 baseline levels for total sTREM2 and binding sTREM2 assays. The results indicate that total circulating sTREM2 levels did not significantly affect changes in mice treated with anti-TREM 2 antibody compared to isotype control 24 hours after treatment with the antibody, indicating that total circulating sTREM2 levels were unaffected at the early time point after antibody administration. In contrast, antibody binding sTREM2 levels were higher in mice injected with anti-TREM 2 antibody relative to isotype control.
Example 7 sequence optimization and humanization of anti-TREM 2 antibodies
Exemplary anti-TREM 2 antibodies were sequence optimized and humanized, followed by characterization of binding kinetics and binding specificity.
Sequence optimization was performed by searching within the CDR sequences for residues susceptible to chemical modification (e.g., asparagine deamidation motif (NG), aspartate isomerization motif (DS), and potentially oxidized residues (tryptophan (W) and methionine (M)) and making amino acid substitutions with conserved and germline residues to remove such sequence libraries.
The results of analyzing the binding characteristics of the humanized and sequence optimized variants of antibody CL0020188 are provided in table 4. The NG motif in the CL0020188CDR-H2 sequence (SEQ ID NO:5) and the CDR-L1 sequence (SEQ ID NO:7) was modified, grafted to human framework regions, and analyzed. Table 4 provides K as measured by Biacore in HEK293-H6 cellsDValues and EC as measured by a dose titration binding assay50The value is obtained.
TABLE 4 binding characteristics of sequence optimized and humanized variants of CL0020188
Cloning
hVH
hVL
KD
EC50
CL0020188-1
NG/graft
NG/graft
2.3nM
0.42nM
CL0020188-2
NG/3m
NG/graft
3.4nM
0.26nM
CL0020188-3
NG/graft
TG/graft
6.8nM
0.64nM
CL0020188-4
NG/3m
TG/graft
4.8nM
0.44nM
CL0020188-5
NA/graft
NG/graft
5.1nM
0.45nM
CL0020188-6
NA/3m
NG/graft
4.0nM
0.31nM
CL0020188-7
NA/graft
TG/graft
10nM
0.68nM
CL0020188-8
NA/3m
TG/graft
7.3nM
0.51nM
Parent strain
9.5nM
0.44nM
3m＝VHA24G/L45P/V48L in (1)
As shown in Table 4, humanized and sequence optimized clones of CL00201088 showed affinity values for hTREM2 similar to the parent antibody (K)D9.5nM) as measured by Biacore. This is consistent with the cell binding results in HEK293-H6 cells shown in Table 4. With parent antibody (EC)500.44nM), the humanized and sequence optimized clone showed comparable and sub-nanomolar affinity for TREM2 expressed in HEK293-H6 cells. In summary, the results indicate comparable binding kinetics between the parent antibody and the humanized and sequence optimized variants.
The results of analyzing the binding characteristics of the humanized and sequence optimized variants of antibody CL0020123 are provided in table 5. The NG and DS motifs in the CL0020123CDR-H2 sequence (SEQ ID NO:30) were modified, grafted to human framework regions, and analyzed. Table 5 provides K as measured by Biacore in HEK293-H6 cellsDValues and EC as measured by a dose titration binding assay50The value is obtained.
TABLE 5 binding characteristics of sequence optimized and humanized variants of CL0020123
1m＝VHR71A in (1)
2m＝VHV67A/R71A in (1)
With the parent antibody (K)D0.10nM), the humanized and sequence optimized clone showed about 4-fold higher K binding to hTREM2DValues as measured by Biacore. On the other hand, humanized and sequence optimized clones showed comparable and sub-nanomolar affinities for TREM2 in a dose-titration cell binding assay of HEK293-H6 cells.
Example 8 in vitro characterization of sequence optimized, humanized anti-TREM 2 antibodies
Exemplary sequence optimized and humanized anti-TREM 2 antibodies (example 7) were evaluated by in vitro methods as described in examples 1 and 3. Assessing TREM2 binding of the antibody in HEK cells expressing TREM2, TREM 2-dependent pSyk signaling in HEK-H6 cells, the ability to promote survival of human macrophages, and the ability to modulate lipid accumulation in iPSC microglia. Fig. 11 to 14 show the results for representative anti-TREM 2 antibodies. anti-TREM 2 antibody showed good cell binding in assays using HEK293-H6 cells expressing TREM2 (EC50 values of 0.34nM (fig. 11A) and 0.08nM (fig. 11B) — anti-TREM 2 antibody also activated pSyk phosphorylation in HEK293-H6 cells expressing TREM2 (fig. 12A and 12B) — in addition, anti-TREM 2 antibody induced macrophage survival, with better survival activity observed for CL0020188 variant antibody (fig. 13) — finally, anti-TREM 2 antibody demonstrated the ability to reduce lipid accumulation in myelin-treated iPSC microglia (fig. 14A and 14B).
Example 9 sequence optimization of mouse PK of humanized anti-TREM 2 antibody
The pharmacokinetic profile of certain sequence optimized, humanized anti-TREM 2 antibodies (example 7) was evaluated in wild type mice in analogy to the 7-day Pharmacokinetic (PK) study described in example 6. Each dose group contained n-3 mice. Table 6 provides the PK profile of an exemplary sequence optimized and humanized anti-TREM 2 antibody (example 7).
TABLE 6 mouse PK Properties of anti-TREM 2 antibodies
Example 10 sequence optimization of cynomolgus monkey PK with humanized anti-TREM 2 antibody
The pharmacokinetic profile of the anti-TREM 2 antibody was evaluated in natural cynomolgus monkeys. Natural cynomolgus monkeys 2-4 years old (approximately 2-3kg body weight) were injected with anti-TREM 2 antibody via intravenous bolus injection. The administered doses included 3mg/kg and 25mg/kg, with n-3 monkeys per dose group. Blood samples (approximately 1mL) were collected before dosing and at 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours, and at 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, and 28 days after dosing. The samples were frozen at about 5 ℃ and then centrifuged to obtain plasma, and the obtained plasma was kept on dry ice and then stored at-70 ℃ until analysis. Plasma samples were analyzed for anti-TREM 2 antibody levels as follows.
For anti-TREM 2 antibody PK analysis, total antibody concentration in monkey plasma was quantified using a general anti-human IgG sandwich electrochemiluminescence immunoassay (ECLIA) on a Meso Scale Discovery (MSD) platform. Briefly, 1% casein-based PBS blocking buffer (Thermo Scientific, MA) was added to MSD GOLD 96-well microspottom streptomycin-coated microtiter plates (Meso Scale Discovery, MD) and incubated for about 1 h. After plate blocking and washing steps, 0.5 μ g/mL of biotinylated anti-human _ IgG goat antibody (southern biotech, AL) in working solution was added to the assay plate and allowed to incubate for 1-2 h. After the incubation and washing steps, the plasma test sample (i.e., the sample with the humanized antibody against TREM2) was added to the assay plate and incubated for 1-2 h. Note that the test samples must be diluted 1:100 in assay Minimum Required Dilution (MRD), in 0.5% casein-based PBS assay buffer (Thermo Scientific, MA) to give the final 1% plasma matrix, which is then added to the assay plates. After capture of anti-TREM 2 antibody analyte and washing steps, 0.4 μ g/mL of secondary ruthenated (SULFO-TAG) anti-human IgG goat antibody (Meso Scale Discovery, MD) in working solution was added to the assay plate and incubated for about 1 h. Finally, after the incubation and washing steps, assay Read Buffer (1X MSD Read Buffer T) was added to the assay plate to generate the assay sample signal. The sample signal readings from the MSD plate reader are in the form of Electrochemiluminescence (ECL) signals and are expressed in ECL units (ECLU). All assay reaction steps described previously were performed at ambient temperature and with shaking on a plate shaker (as appropriate). The assay has a dynamic calibration standard range of 19.5-2500 ng/mL (or 0.195-25 ng/mL at 1:100 post MRD) with 8 standard points serially diluted at 1:2 plus a blank plasma sample. Plasma sample concentrations were calculated back from the assay calibration standard curve fitted using weighted four-parameter non-linear logistic regression. Table 7 provides the Pharmacokinetic (PK) profile of an exemplary sequence optimized and humanized anti-TREM 2 antibody (example 7).
TABLE 7 cynomolgus monkey PK Properties of anti-TREM 2 antibodies
CL0020188 showed similar low clearance levels between different dose levels and a linear pharmacokinetic profile in cynomolgus monkeys. In addition, there were no clinical pathology findings associated with administration of the variants in cynomolgus monkeys (data not shown).
Example 11 comparison of anti-TREM 2 antibodies
The affinity of anti-TREM 2 antibodies to human TREM2 and cynomolgus monkey TREM2 was measured by Biacore (described in example 1). The potency of anti-TREM 2 antibody in CSF samples of healthy human volunteers (Innovative Research) and healthy cynomolgus monkey CSF samples (world precursors) was measured by MSD assay. The potency of each antibody was determined by its EC 50. Briefly, MSD GOLD 96 well small spot streptavidin plates (MSD, cat # L45SA) coated with capture antibody (biotinylated goat anti-human TREM2, R & D Systems, cat # BAF1828) were incubated with biofluid samples diluted 1:3 in assay buffer (25% (v/v) MSD blocking agent a (MSD, cat # R93BA-a), 75% (v/v) TBST) for one hour at room temperature. After washing the wells with TBST, sulfo-labeled anti-TREM 2 antibody was added to the wells of the plate in serial dilutions (4 x dilution between 11 points) and incubated for one hour at room temperature. The wells were diluted with TBST and MSD read buffer (MSD, catalog No. R92TC-3) was added to the wells. The signal from the sample was measured using the MSD Meso sensor S600 apparatus. EC50 values were determined for each antibody by four parameter variable slope nonlinear regression. Reference antibodies #1 and #2 correspond to 4C5 and 6E7 described in WO 2018/195506. Reference antibody #3 corresponds to AL2p-58 described in WO 2019/028292. The variable regions of reference antibody #1 are represented by SEQ ID NOs 73 and 74. The variable regions of reference antibody #2 are represented by SEQ ID NOs 75 and 76. The variable regions of reference antibody #3 are represented by SEQ ID NOs 77 and 78. The results are provided in table 8.
As shown in table 8, CL0020188 and CL0020123 variants as disclosed herein have stronger affinity for human TREM2 relative to the reference antibody and bind more effectively to sTREM2 in CSF samples isolated from healthy human volunteer subjects. In addition, the CL0020188 variant has a stronger affinity for cynomolgus monkey TREM2 and binds more efficiently to sTREM2 in CSF samples isolated from healthy cynomolgus monkey subjects relative to the reference antibody. This is demonstrated by the relative amount of antibody required to achieve a half maximal effective concentration (EC50) to bind a given amount of sTREM2 under the same conditions. As shown in table 8, reference antibodies #1, #2 and #3 required higher relative amounts to reach EC50 compared to CL0020188 and CL0020123 antibodies.
TABLE 8 comparison of the Properties of anti-TREM 2 antibodies
Example 12 epitope mapping of anti-TREM 2 antibodies
To identify the epitope of the anti-TREM 2 antibody at peptide level, deuterium-exchanged (HDX) mass spectrometry was used. Recombinant human TREM2 alone or in admixture with anti-TREM 2 antibody was incubated with deuterium oxide-labeled buffer (50mM sodium phosphate, 100mM sodium chloride, pH 7.0) for 0, 60, 600 and 3600 seconds at 20 ℃. The hydrogen/deuterium exchange was quenched by the addition of an equal volume of 4M guanidine hydrochloride, 0.85M TCEP buffer (final pH 2.5). The quenched sample was then subjected to pepsin/protease XIII digestion and LC-MS analysis. Briefly, quenched samples were injected into a packed pepsin/protease XIII column (2.1 × 30mm) maintained at 20 ℃ and Q active-containing column was usedTMThe resulting peptides were analyzed by an HF-Hybrid Quadrupole-Orbitrap Mass spectrometer (Thermo Fisher) coupled to a UPLC-MS system from Waters acquisition UPLC (Waters Corporation). The peptides were isolated on a 50mm x 1mm C8 column maintained at-6 ℃ using a gradient from 2% to 31% solvent B (solvent B: 0.2% formic acid in acetonitrile; solvent a: 0.2% formic acid in water) over 16.5 minutes. Mass spectra were recorded only in MS mode. HDX Workbench (Pascal et al 2012.Journal of The American Society for Mass Spectrometry 23:1512-1521) was used to process raw materialMS data. Deuterium levels were calculated using the average mass difference at t0 between the deuterated peptide and its native form. Peptide identification was performed by searching the MS/MS data for the human TREM2 sequence using Mascot software (Matrix Science). The mass tolerances of the precursor and product ions were 10ppm and 0.02Da, respectively.
Based on HDX mass spectrometry results, variants of CL0020188 and CL0020188 bind to amino acid residues 143-149(FPGESES (SEQ ID NO:69)) of human TREM2(SEQ ID NO:1), while variants of CL0020123 and CL0020123 bind to (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107(TLRNLQPHDAGL (SEQ ID NO:71)) and (iii) amino acid residues 126-129(VEVL (SEQ ID NO:72)) of human TREM 2.
IX. informal sequence listing
Sequence listing
<110> Danali pharmaceutical Co., Ltd (DENALI THERAPEUTIC INC.)
<120> anti-TREM 2 antibodies and methods of use thereof
<130> 102342-003310PC-1173316
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<151> 2019-02-20
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100 105 110
Met Val Thr Val Ser Ser
115
<210> 16
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 16
Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly
1 5 10 15
Glu Ser Ala Ser Ile Thr Cys Gln Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Ser Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe
85 90 95
Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 17
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 17
Ala Arg Leu Thr Tyr Gly Phe Asp Tyr
1 5
<210> 18
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 18
Gln Gln Phe Leu Glu Tyr Pro Phe Thr
1 5
<210> 19
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 20
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 20
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Gln Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe
85 90 95
Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 21
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 22
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 22
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Gln Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Thr Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe
85 90 95
Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 23
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 23
Gln Ser Ser Lys Ser Leu Leu His Ser Thr Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 24
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 25
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 25
Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro Ser
1 5 10 15
Val Lys Gly
<210> 26
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 26
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Leu
35 40 45
Ser Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 27
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 27
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 28
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 28
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Thr Val Thr Ile Thr Cys His Ala Ser Gln His Ile Asn Val Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asp His Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 29
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 29
Gly Phe Ser Ile Glu Asp Phe Tyr Ile His
1 5 10
<210> 30
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 30
Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 31
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 31
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr
1 5 10
<210> 32
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 32
His Ala Ser Gln His Ile Asn Val Trp Leu Ser
1 5 10
<210> 33
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 33
Lys Ala Ser Asn Leu His Thr
1 5
<210> 34
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 34
Gln Gln Gly Gln Thr Tyr Pro Arg Thr
1 5
<210> 35
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 35
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 36
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln His Ile Asn Val Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 37
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 37
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 38
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 38
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 39
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 39
Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 40
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 40
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 41
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 41
Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 42
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 42
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 43
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 43
Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 44
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 44
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 45
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 46
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 46
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 47
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (6)..(6)
<223 >/substitution = "Gln"
<220>
<221> VARIANT
<222> (8)..(8)
<223 >/substitution = "Glu"
<220>
<221> SITE
<222> (1)..(17)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 47
Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 48
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (6)..(6)
<223 >/substitution = "Asn"
<220>
<221> SITE
<222> (1)..(10)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 48
Gly Phe Thr Phe Thr Asp Phe Tyr Met Ser
1 5 10
<210> 49
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (5)..(5)
<223 >/substitution = "Arg"
<220>
<221> VARIANT
<222> (6)..(6)
<223 >/substitution = "Pro"
<220>
<221> VARIANT
<222> (8)..(8)
<223 >/substitution = "Ala"
<220>
<221> VARIANT
<222> (11)..(11)
<223 >/substitution = "Thr"
<220>
<221> VARIANT
<222> (12)..(12)
<223 >/substitution = "Asp"
<220>
<221> SITE
<222> (1)..(19)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 49
Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro Ser
1 5 10 15
Val Lys Gly
<210> 50
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (1)..(1)
<223 >/substitution = "Thr"
<220>
<221> VARIANT
<222> (4)..(4)
<223 >/substitution = "Ser"
<220>
<221> SITE
<222> (1)..(9)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 50
Ala Arg Leu Thr Tyr Gly Phe Asp Tyr
1 5
<210> 51
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (10)..(10)
<223 >/substitution = "Thr"
<220>
<221> SITE
<222> (1)..(16)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 51
Gln Ser Ser Lys Ser Leu Leu His Ser Asn Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 52
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<220>
<221> VARIANT
<222> (6)..(6)
<223 >/substitution = "Phe"
<220>
<221> SITE
<222> (1)..(9)
<223 >/Note = "residues of variation given in sequence relative to position of variation
Residues in notes No preference "
<400> 52
Gln Gln Phe Leu Glu Tyr Pro Phe Thr
1 5
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 53
aggaatgtgg ggagcacgga g 21
<210> 54
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 54
tgcatcgcat tgtctgagta ggtg 24
<210> 55
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 55
accctagtcc tgactgttgc tc 22
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 56
tataggaact tcgcgacacg gacac 25
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 57
acaggaggga cctaccttca g 21
<210> 58
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 58
gcctgccttt cagagacctc agtc 24
<210> 59
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 59
cctctccggc tgctcatctt actc 24
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 60
gtctctcagc cctggcagag tttg 24
<210> 61
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 61
cgcctaccct agtcctgact gttg 24
<210> 62
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 62
aaagcctaca gcatcctcac ctc 23
<210> 63
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic primer "
<400> 63
gcatcatggg gttgtagatt ccg 23
<210> 64
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 64
Gly Gly Gly Gly Ser
1 5
<210> 65
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthesis of 6XHis tag "
<400> 65
His His His His His His
1 5
<210> 66
<211> 140
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 66
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Ser Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ser Asp Pro Thr Thr Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Val Arg Thr Ser Gly Thr Gly Asp Tyr Trp Gly Gln Gly Thr Ser Leu
100 105 110
Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala
115 120 125
Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr
130 135 140
<210> 67
<211> 140
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 67
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn
20 25 30
Asn Gly Asn Thr Phe Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Thr
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
130 135 140
<210> 68
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 68
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Gln Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro
35 40 45
Pro Lys Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Phe
85 90 95
Leu Glu Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 69
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 69
Phe Pro Gly Glu Ser Glu Ser
1 5
<210> 70
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 70
Gly Glu Lys Gly Pro Cys Gln Arg Val
1 5
<210> 71
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 71
Thr Leu Arg Asn Leu Gln Pro His Asp Ala Gly Leu
1 5 10
<210> 72
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 72
Val Glu Val Leu
1
<210> 73
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 73
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Val Gly Val Pro Leu Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Arg
85 90 95
Asn Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 74
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 74
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly His Ser Phe Thr Asn Tyr
20 25 30
Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gln Arg Thr Phe Tyr Tyr Asp Ser Ser Gly Tyr Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 75
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 75
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asp Ser Phe Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 76
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 76
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys
85 90 95
Ala Arg Gln Arg Thr Phe Tyr Tyr Asp Ser Ser Asp Tyr Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 77
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 77
Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr Arg Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 78
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 78
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Gln
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45
Gly Arg Ile Tyr Pro Gly Gly Gly Asp Thr Asn Tyr Ala Gly Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Leu Arg Asn Gln Pro Gly Glu Ser Tyr Ala Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 79
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic polypeptide "
<400> 79
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Leu
35 40 45
Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 80
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 80
Asn Gly Asp Ser
1
<210> 81
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 81
Gln Gly Asp Ser
1
<210> 82
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 82
Asn Gly Glu Ser
1
<210> 83
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> sources
<223 >/comment = "description of artificial sequence: synthetic peptide "
<400> 83
Gln Gly Glu Ser
1
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