Anti-TIGIT Antibodies and Uses Thereof

Abstract

The present application relates to anti-TIGIT antibodies or antigen binding fragments thereof, nucleic acid encoding the same, therapeutic compositions thereof, and their use to enhance T-cell function to upregulate cell-mediated immune responses and for the treatment of T cell dysfunctional disorders, such as tumor immunity, for the treatment of infectious diseases and cancer.

Claims

1. An isolated heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein: (a) the HVR-H1 sequence is GYTFTX.sub.1YP; (b) the HVR-H2 sequence is INTNTGNP; (c) the HVR-H3 sequence is ARX.sub.2GX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13; further wherein: X.sub.1 is S or A; X.sub.2 is V or T; X.sub.3 is G or Y; X.sub.4 is Y, S or F; X.sub.5 is S, G or T; X.sub.6 is V, S or G; X.sub.7 is D, Y or P; X.sub.8 is E, D or Y; X.sub.9 is Y or W; X.sub.10 is A, F or S; X.sub.11 is F or D; X.sub.12 is D or P; X.sub.13 is V, I or absent.

2. The polypeptide of claim 1 wherein X.sub.1 is S or A; X.sub.2 is V or T; X.sub.3 is G or Y; X.sub.4 is Y or S; X.sub.5 is S or G; X.sub.6 is V or S; X.sub.7 is D or Y; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A or F; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V or I.

3. The polypeptide of claim 1 wherein X.sub.1 is S; X.sub.2 is V or T; X.sub.3 is G; X.sub.4 is Y; X.sub.5 is S or G; X.sub.6 is V; X.sub.7 is D or Y; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V or I.

4. The polypeptide of claim 1 wherein X.sub.1 is S; X.sub.2 is V; X.sub.3 is G; X.sub.4 is Y; X.sub.5 is S; X.sub.6 is V; X.sub.7 is D; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V.

5. The polypeptide of any one of claims 1-4 further comprising variable region heavy chain framework sequences HC-FR1, HC-FR2, HC-FR3 and HC-FR4, juxtaposed between the HVRs, thus forming the sequence of the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4).

6. The polypeptide of claim 5 wherein the heavy chain framework sequences are derived from human consensus framework sequences.

7. The polypeptide of claim 5 wherein the heavy chain framework sequences are derived from human germline framework sequences.

8. The polypeptide of claim 5 wherein one or more of the heavy chain framework sequences is the following: TABLE-US-00032 HC-FR1 is QVQLVQSGSELKKPGASVKVSCKAS; HC-FR2 is MNWVRQAPGQGLEWMGW; HC-FR3 is TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYC; HC-FR4 is WGQGTLVTVSS.

9. The polypeptide of any one of claims 5-8 further comprising at least a C.sub.H1 domain.

10. The polypeptide of claim 9 further comprising a C.sub.H2 and a C.sub.H3 domain.

11. The isolated heavy chain polypeptide of any one of claims 1-10 in combination with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein: (a) the HVR-L1 sequence is QGISSY; (b) the HVR-L2 sequence is AAS; (c) the HVR-L3 sequence is X.sub.14QX.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20; further wherein X.sub.14 is Q, G or H; X.sub.15 is L, V or T; X.sub.16 is N, S, I or M; X.sub.17 is S, R or F; X.sub.18 is Y or R; X.sub.19 is P or L; X.sub.20 is T or A.

12. The polypeptide of claim 11 wherein X.sub.14 is Q or G; X.sub.15 is L or V; X.sub.16 is N or S; X.sub.17 is S or R; X.sub.18 is Y; X.sub.19 is P; X.sub.20 is T.

13. The polypeptide of claim 11 wherein X.sub.14 is Q; X.sub.15 is L; X.sub.16 is S; X.sub.17 is S; X.sub.18 is Y; X.sub.19 is P; X.sub.20 is T.

14. The polypeptide of any of claims 11-13 further comprising variable region light chain framework sequences LC-FR1, LC-FR2, LC-FR3 and LC-FR4, juxtaposed between the HVRs, thus forming the sequence of the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

15. The polypeptide of claim 14 wherein the light chain framework sequences are derived from human consensus framework sequences.

16. The polypeptide of claim 14 wherein the light chain framework sequences are derived from human germline framework sequences.

17. The polypeptide of claim 14 wherein the light chain framework sequences are kappa light chain sequences.

18. The polypeptide of claim 14 wherein one or more of the light chain framework sequences is the following: TABLE-US-00033 LC-FR1 is DIQLTQSPSFLSASVGDRVTITCRAS; LC-FR2 is LAWYQQKPGKAPKLLIY; LC-FR3 is TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC; LC-FR4 is FGGGTKVEIK.

19. The polypeptide of any one of claims 14-18 further comprising a C.sub.L domain.

20. An isolated anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain comprises an HVR-H1, HVR-H2 and HVR-H3, wherein further: (i) the HVR-H1 sequence is GYTFTX.sub.1YP; (ii) the HVR-H2 sequence is INTNTGNP; (iii) the HVR-H3 sequence is ARX.sub.2GX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13; (b) the light chain comprises an HVR-L1, HVR-L2 and HVR-L3, wherein further: (iv) the HVR-L1 sequence is QGISSY; (v) the HVR-L2 sequence is AAS; (vi) the HVR-L3 sequence is X.sub.14QX.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20; wherein further X.sub.1 is S or A; X.sub.2 is V or T; X.sub.3 is G or Y; X.sub.4 is Y, S or F; X.sub.5 is S, G or T; X.sub.6 is V, S or G; X.sub.7 is D, Y or P; X.sub.8 is E, D or Y; X.sub.9 is Y or W; X.sub.10 is A, F or S; X.sub.11 is F or D; X.sub.12 is D or P; X.sub.13 is V, I or absent; X.sub.14 is Q, G or H; X.sub.15 is L, V or T; X.sub.16 is N, S, I or M; X.sub.17 is S, R or F; X.sub.18 is Y or R; X.sub.19 is P or L; X.sub.20 is T or A.

21. The antibody or antibody fragment of claim 20 wherein X.sub.1 is S or A; X.sub.2 is V or T; X.sub.3 is G or Y; X.sub.4 is Y or S; X.sub.5 is S or G; X.sub.6 is V or S; X.sub.7 is D or Y; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A or F; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V or I; X.sub.14 is Q or G; X.sub.15 is L or V; X.sub.16 is N or S; X.sub.17 is S or R; X.sub.18 is Y; X.sub.19 is P; X.sub.20 is T.

22. The antibody or antibody fragment of claim 20 wherein X.sub.1 is S; X.sub.2 is V or T; X.sub.3 is G; X.sub.4 is Y; X.sub.5 is S or G; X.sub.6 is V; X.sub.7 is D or Y; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V or I; X.sub.14 is Q; X.sub.15 is L; X.sub.16 is S; X.sub.17 is S; X.sub.18 is Y; X.sub.19 is P; X.sub.20 is T.

23. The antibody or antibody fragment of claim 20 wherein X.sub.1 is S; X.sub.2 is V; X.sub.3 is G; X.sub.4 is Y; X.sub.5 is S; X.sub.6 is V; X.sub.7 is D; X.sub.8 is E; X.sub.9 is Y; X.sub.10 is A; X.sub.11 is F; X.sub.12 is D; X.sub.13 is V; X.sub.14 is Q; X.sub.15 is L; X.sub.16 is S; X.sub.17 is S; X.sub.18 is Y; X.sub.19 is P; X.sub.20 is T.

24. The antibody or antibody fragment of claim 20, wherein (a) the HVR-H1 sequence is GYTFTSYP, (b) the HVR-H2 sequence is INTNTGNP, (c) the HVR-H3 sequence is ARVGGYSVDEYAFDV; and wherein (d) the HVR-L1 sequence is QGISSY, (e) the HVR-L2 sequence is AAS, (f) the HVR-L3 sequence is QQLSSYPT.

25. The antibody or antibody fragment of any of claims 20-24 further comprising: (a) variable region heavy chain framework sequences HC-FR1, HC-FR2, HC-FR3 and HC-FR4, juxtaposed between the HVRs, thus forming the sequence of the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and (b) variable region light chain framework sequences LC-FR1, LC-FR2, LC-FR3 and LC-FR4, juxtaposed between the HVRs, thus forming the sequence of the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

26. The antibody or antibody fragment of claim 25 wherein the framework sequences are derived from human consensus framework sequences.

27. The antibody or antibody fragment of claim 25 wherein the framework sequences are derived from human germline framework sequences.

28. The antibody or antibody fragment of claim 25 wherein one or more of the framework sequences is the following: TABLE-US-00034 HC-FR1 is QVQLVQSGSELKKPGASVKVSCKAS; HC-FR2 is MNWVRQAPGQGLEWMGW; HC-FR3 is TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYC; HC-FR4 is WGQGTLVTVSS.

29. The antibody or antibody fragment of claim 25 wherein one or more of the framework sequences is the following: LC-FR1 sequence is DIQLTQSPSFLSASVGDRVTITCRAS; LC-FR2 sequence is LAWYQQKPGKAPKLLIY; LC-FR3 sequence is TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC; LC-FR4 sequence is FGGGTKVEIK.

30. The antibody or antibody fragment of claim 25 wherein: (a) the variable heavy chain framework sequences are the following: TABLE-US-00035 (i) HC-FR1 is QVQLVQSGSELKKPGASVKVSCKAS; (ii) HC-FR2 is MNWVRQAPGQGLEWMGW; (iii) HC-FR3 is TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYC; (iv) HC-FR4 is WGQGTLVTVSS; and (b) the variable light chain framework sequences are the following: (i) LC-FR1 sequence is DIQLTQSPSFLSASVGDRVTITCRAS; (ii) LC-FR2 sequence is LAWYQQKPGKAPKLLIY; (iii) LC-FR3 sequence is TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC; (iv) LC-FR4 sequence is FGGGTKVEIK.

31. An isolated anti-TIGIT antibody or antigen binding fragment thereof, having the HC-FR and LC-FR sequences of claim 30, selected from the following: i) an antibody, wherein the HVR-H1, HVR-H2, HVR-H3 sequences are selected from one of the ID's shown in Table 2, and wherein (a) the HVR-L1 sequence is QGISSY, (b) the HVR-L2 sequence is AAS, (c) the HVR-L3 sequence is QQLNSYPT; ii) an antibody wherein the HVR-L1, HVR-L2, HVR-L3 sequences are selected from one of the ID's shown in Table 3, and wherein (a) the HVR-H1 sequence is GYTFTSYP, (b) the HVR-H2 sequence is INTNTGNP, (c) the HVR-H3 sequence is ARVGGYSVDEYAFDV; or iii) an antibody chosen from Table 4.

32. The antibody or antibody fragment of any one of claims 25-31 further comprising at least a C.sub.H1 domain.

33. The antibody or antibody fragment of claim 32 further comprising a C.sub.H2 and a C.sub.H3 domain.

34. The antibody or antibody fragment of any one of claims 25-33 further comprising a C.sub.L domain.

35. The antibody of claim 34, wherein the constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

36. The antibody of claim 35 wherein the constant region is IgG1.

37. The antibody or antibody fragment of any one of the preceding claims which is a fully human antibody.

38. An isolated anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region sequence and a light chain variable region sequence, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence: TABLE-US-00036 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNVRQAPGQGLEWMGWI NTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVGG YSVDEYAFDVWGQGTLVTVSS, and (b) the light chain sequence has at least 85% sequence identity to the light chain sequence: TABLE-US-00037 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLSSYPTFGGG TKVEIK.

39. The antibody or antigen binding fragment of claim 38, wherein the sequence identity is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or is 100%.

40. The antibody or antigen binding fragment of claim 39, wherein the sequence identity is 100%.

41. An isolated anti-TIGIT antibody wherein the heavy chain is: TABLE-US-00038 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARV GGYSVDEYAFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG, and (b) the light chain is: TABLE-US-00039 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLSSYPTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.

42. The antibody of any one of claims 20-41 wherein the antibody is capable of binding to human and cynomolgus monkey TIGIT.

43. The antibody of any one of claims 20-20 wherein the antibody is capable of blocking the interaction between human, or cynomolgus monkey TIGIT and the respective human, or cynomolgus monkey PVR.

44. The antibody of any of claims 20-43 wherein the antibody binds to human TIGIT with a K.sub.D of 10×10.sup.−9 M or less.

45. An isolated anti-TIGIT antibody or antigen binding fragment thereof which binds to a functional epitope comprising residues Q53, T55, Y113 and P114 of human TIGIT.

46. The isolated anti-TIGIT antibody or antigen binding fragment of claim 45 wherein the functional epitope further comprises residues Q56, N70, and H111 of human TIGIT.

47. An isolated anti-TIGIT antibody or antigen binding fragment thereof which binds to a conformational epitope comprising residues T51, A52, Q53, T55, Q56, N70, D72, H111, T112, Y113, P114, and G116 of human TIGIT.

48. An isolated anti-TIGIT antibody or antigen binding fragment thereof wherein the antibody cross-competes for binding to TIGIT with an antibody or antigen binding fragment of any of claims 20-42.

49. A pharmaceutical composition comprising the anti-TIGIT antibody or antigen binding fragment of any of claims 20-48 and at least one pharmaceutically acceptable carrier.

50. An isolated nucleic acid encoding a polypeptide of any one of claims 1-41.

51. An isolated nucleic acid encoding the light chain or a heavy chain sequence of an anti-TIGIT antibody or antigen binding fragment of any one of claims 20-41.

52. An isolated nucleic acid encoding the heavy chain according to claim 41, which nucleic acid has the following sequence: TABLE-US-00040 ATGGAAACAGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCG GCTCCACAGGCCAGGTGCAGCTGGTGCAGTCCGGCTCCGAGCTGAAGAA ACCCGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTC ACCTCCTACCCCATGAACTGGGTGAGGCAGGCTCCTGGCCAGGGACTGG AGTGGATGGGCTGGATCAACACCAACACCGGCAACCCTACCTACGCCCA GGGCTTCACCGGCAGGTTCGTGTTCTCCCTGGACACCAGCGTGTCCACC GCCTACCTGCAGATCTCCTCCCTGAAGGCCGAGGACACCGCCGTGTACT ACTGCGCCAGGGTGGGAGGCTACTCCGTGGACGAGTACGCCTTCGACGT GTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGCTAGCACCAAGGGC CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC GGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCACGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT.

53. An isolated nucleic acid encoding the light chain according to claim 41, which nucleic acid has the following sequence: TABLE-US-00041 ATGAGGGCCCTGCTGGCTAGACTGCTGCTGTGCGTGCTGGTCGTGTCCG ACAGCAAGGGCGACATCCAGCTGACCCAGTCCCCCTCCTTCCTGTCCGC TTCCGTGGGCGACAGGGTGACCATCACTTGTCGTGCCTCCCAGGGCATC TCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGC TGCTGATCTACGCCGCTTCCACACTGCAGTCCGGCGTGCCCTCCAGGTT TTCCGGATCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCTGTCCTCCTACC CCACCTTCGGCGGCGGCACAAAGGTGGAGATCAAGCGTACGGTGGCTGC ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT.

54. A vector comprising the nucleic acid of any of claims 50-53.

55. A host cell comprising the vector of claim 54.

56. The host cell of claim 55 which is eukaryotic.

57. The host cell of claim 56 which is mammalian.

58. The host cell of claim 57 which is a Chinese Hamster Ovary (CHO) cell, preferably CHO-K1SV.

59. A process for making an anti-TIGIT antibody or antigen binding fragment thereof comprising culturing the host cell of any one of claims 55-58 under conditions suitable for the expression of the vector encoding the anti-TIGIT antibody or antigen binding fragment, and recovering the antibody or fragment.

60. A method of treating cancer comprising administering to a subject in need thereof an effective amount of an anti-TIGIT antibody of any one of claims 20-48, or the pharmaceutical composition of claim 49, which induces antibody dependent cell-mediated cytotoxicity (ADCC).

61. A method of treating cancer comprising administering to a subject in need thereof an effective amount of an anti-TIGIT antibody of any one of claims 20-48, or the pharmaceutical composition of claim 49.

62. The method of claim 60 or 61 wherein the cancer is selected from the group consisting of: breast, lung, colon, ovarian, melanoma, bladder, kidney, liver, salivary, stomach, gliomas, thyroid, thymic, epithelial, head and neck cancers, gastric and pancreatic cancer.

63. A method of treating a T-cell dysfunctional disorder comprising administering a therapeutically effective amount of an anti-TIGIT antibody of any one of claims 20-48 or the pharmaceutical composition of claim 47, to a subject in need thereof.

64. The method of claim 63, wherein the T-cell dysfunctional disorder is tumor immunity.

65. The method of claim 64, wherein the tumor immunity results from a cancer selected from the group consisting of: breast, lung, colon, ovarian, melanoma, bladder, kidney, liver, salivary, stomach, gliomas, thyroid, thymic, epithelial, head and neck cancers, gastric and pancreatic cancer.

66. The method of any one of claims 60-65, wherein the method further comprises the application of a treatment regimen selected from the group consisting of: radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, adjuvant therapy, neoadjuvant therapy, hormonal therapy, angiogenesis inhibition, palliative care.

67. The method of any one of claims 60-66, further comprising the administration of at least one anti-cancer agent.

68. A kit of parts comprising the pharmaceutical composition of claim 49 and a package insert comprising instructions for using the pharmaceutical composition for the treatment according to any one of claims 60-66.

69. A kit of parts comprising the pharmaceutical composition of claim 49, at least one further anti-cancer agent, and a package insert comprising instructions for using the at least one anti-cancer agent in combination with the pharmaceutical composition for the treatment according to any one of claims 60-67.

Description

DESCRIPTION OF THE FIGURES

[0230] FIG. 1

[0231] Graph showing the results of a competitive ELISA assessing the abilities of the anti-TIGIT antibodies to block the binding of TIGIT to CD155.

[0232] FIG. 2

[0233] A rendering of the crystal structures of anti-TIGIT Fabs bound to human TIGIT (gray), with the heavy chain in black and the light chain in light gray: A. 3963H03; B. 3966C11. C. 3964A06; D. 7729G05; E. 7728B03; F. 3963H03-12.

[0234] FIG. 3

[0235] A rendering of the crystal structure of Fab 3963H03-12 overlayed with a rendering of the crystal structure of TIGIT in complex with PVR (Protein Data Bank entry 3UDW) showing that 3963H03-12 overlaps the binding site of PVR on TIGIT. The surface of PVR is rendered in dark gray. The light chain of 3963H03-12 is shown in light gray and the heavy chain shown in dark gray.

[0236] FIG. 4

[0237] Human TIGIT ECD crystal structure with mutagenized residues contacting 3963H03 shown in sticks. The residues were colored according to the change of binding affinity upon mutating to alanine or glycine. (dark grey: >3 kcal/mol; medium grey >2 kcal/mol; light grey <0.7 kcal/mol).

[0238] FIG. 5

[0239] A summary of kinetic binding affinities of anti-TIGIT 3963H03-12 to TIGIT mutants is shown. Binding KDs highlighted by the loss of binding affinity upon mutation (ΔΔG). Positions where mutation causing significant binding energy loss was highlighted in three different shades, indicating the magnitude of loss. Binding affinities stronger than KD=0.1 nM were reported as <0.1 as it is beyond the instrument measuring range. NB indicates no binding. Standard deviation was reported if more than one experiment was performed.

[0240] FIG. 6

[0241] The change of binding affinity to TIGIT mutants, converted from kinetic affinity data, is shown for anti-TIGIT 3963H03-12. The binding affinity ΔG was calculated from KD with the equation ΔG=ln(KD)*RT. The change of binding affinity ΔΔG is the difference of binding affinity between mutant and parental TIGIT. If the KD for either variant is stronger than 0.1 nM, the ΔΔG is not calculated and it is indicated as ND (not determined).

[0242] FIG. 7

[0243] Graphs showing the results of cell based binding assays using CHO-S cells expressing the human TIGIT (A) or cynomolgus monkey TIGIT (B) extracellular domains. Anti-TIGIT antibodies were tested at varying concentrations and binding was measured by flow cytometry.

[0244] FIG. 8

[0245] Blockade of functional TIGIT/CD155 interaction. Blockade of TIGIT/CD155 interaction was measured in the presence of a range of concentrations of anti-TIGIT or isotype control antibody with cell-based Jurkat reporter assay (Promega CS198801). Sequence optimized 3963H03-12, parental 3963H03 and an isotype control were tested. Data was plotted and curve fitting and EC50 value calculations were performed with GraphPad Prism program. RLU, relative luciferase units.

[0246] FIG. 9

[0247] Graphs showing ADCC activities of anti-TIGIT antibodies 3963H03 and 3963H03-12 using as targets CHO-S cells expressing the human TIGIT extracellular domain.

[0248] FIG. 10

[0249] Graph showing the complement dependent cytotoxicity (CDC) of 3963H03-12, using as the targets .sup.51Cr-labelled CHO-S cells expressing the human TIGIT extracellular domain

[0250] FIG. 11

[0251] Graph demonstrating that anti-TIGIT antibodies, A06, C11, D08, H03, enhanced IFNγ production in a T cell activation assay using anti-CD3 and anti-CD28.

[0252] FIG. 12

[0253] Graph demonstrating that anti-TIGIT H03 antibody reversed CD155-mediated CD8+ T cell suppression by increasing IFNγ production in a CD8+ T cell activation assay using anti-CD3.

[0254] FIG. 13

[0255] Graphs demonstrating the binding of H03-12 to human (A) and cynomolgus monkey (B) CD3+ T cells.

[0256] FIG. 14

[0257] Graphs demonstrating dose dependent target occupancy of H03-12 in human whole blood (A) and cynomolgus monkey spleen cells (B).

[0258] FIG. 15

[0259] Graphs demonstrating that H03-12 dose-dependently blocked TIGIT/CD155 (A) and TIGIT/CD112 (B) interaction.

[0260] FIG. 16

[0261] Graphs demonstrating the setup of a FRET-based TIGIT/CD226 blocking assay (A) and dose-dependent inhibition of TIGIT/CD226 interaction by 3963H03-12 (B).

[0262] FIG. 17

[0263] Graph demonstrating dose-dependent activity of 3963H03-12 in the two-way MLR assay.

[0264] FIG. 18

[0265] Graph demonstrating dose-dependent activity of 3963H03-12 in the one-way MLR allo assay.

[0266] FIG. 19

[0267] Graphs demonstrating that 3963H03-12 enhanced NK cell activation in NK cell-mediated killing assays using P815.hCD155 cells (A) and MDA-MB-231 GFP/Luc cells (B).

[0268] FIG. 20

[0269] Graph demonstrating blocking potency of 3963H03-12 and 3963H03-12-muIgG2c on the binding of muCD155 and muCD112 to CHO-S-huTIGIT cells.

[0270] FIG. 21

[0271] Pharmacokinetic evaluation of 3963H03-12-muIgG2c in B-huTIGIT knock-in mice bearing MC38 tumors

[0272] FIG. 22

[0273] Graphs demonstrating anti-tumor efficacy of 3963H03-12-muIgG2c in MC38 colon carcinoma model (A), GL261 glioblastoma model (B), Hepa 1-6 hepatocellular carcinoma model (C) and 3LL lung carcinoma model (D) in B-huTIGIT knock-in mice

[0274] FIG. 23

[0275] Graphs showing the dose-dependent anti-tumor efficacy of 3963H03-12-muIgG2c in the MC38 tumor model in B-huTIGIT knock-in mice. Average and individual tumor volumes are plotted for each treatment group in addition to median survival in days.

[0276] FIG. 24

[0277] Graphs demonstrating that effector competent 3963H03-12-muIgG2c, but not effector null 3963H03-12-muIgG1(D265A), had anti-tumor efficacy in the MC38 model (A) or Hepa 1-6 model (B) in B-huTIGIT knock-in mice

[0278] FIG. 25

[0279] Graphs showing results of combination treatment with 3963H03-12-muIgG2c and avelumab in MC38 tumor model in B-huTIGIT knock-in mouse comparing both average and individual tumor volumes for each treatment group in addition to median survival in days.

[0280] FIG. 26

[0281] Results of combination treatment with 3963H03-12-muIgG2c and bintrafusp alfa in MC38 tumor model in B-huTIGIT knock-in mouse. Both averaged and individual tumor volumes demonstrate enhanced anti-tumor efficacy with the combination treatment compared to either monotherapy. Prolonged survival is also observed with the combination relative to either monotherapy.

[0282] FIG. 27

[0283] Results of re-challenge studies performed on MC38 tumor-bearing B-huTIGIT knock-in mice that showed complete tumor regression after combination treatment with 3963H03-12-muIgG2c and either avelumab or bintrafusp alfa. Tumor volumes are shown of naïve mice compared to cured mice.

EXPERIMENTAL SECTION

[0284] The working examples presented below are intended to illustrate particular embodiments of the invention and are not intended to limit the scope of the specification or the claims in any way.

[0285] 1. Selection and Improvement of Antibodies

[0286] To generate fully human monoclonal antibodies to TIGIT, OmniRats (Open Monoclonal Technologies, Inc./Ligand Pharmaceutical Inc.) were immunized with the recombinant extracellular domain (ECD) of human TIGIT (Sino Biological Inc, Cat. 10917-H08H) using a Repetitive Immunization at Multiple Sites strategy (also known as RIMMS). Eight to twelve week old rats were immunized biweekly for six times with recombinant TIGIT protein emulsified with complete Freund's Adjuvant (Sigma-Aldrich, Cat. F5881) for the first injection followed by incomplete Freund's Adjuvant (Sigma-Aldrich, Cat. F5506) for the remaining injections. The serum immune response was monitored by ELISA against the immunogen. Briefly, a 96-well clear flat bottom plate (Thermo Scientific, Cat. 439454) was coated with human TIGIT protein (Sino Biological Inc, Cat. 10917-H08H) at 4° C. overnight. Plates were washed with PBS/0.05% Tween 20 and incubated with 3% BSA (Sigma, Cat. A3912-100G) for 2 hours at room temperature. Serially diluted serum samples were added into the plates and incubated 1 hour at room temperature. The plates were then incubated with 1:5000 diluted horse radish peroxidase-conjugated goat anti-rat IgG Fc fragment (Jackson ImmunoResearch, Cat. 112-036-071) for 1 hour. The color was developed with 100 ul of tetramethyl benzidine hydrochloride (TMB) substrate (BioFx, Cat. TMBW-1000-01) and stopped with the addition of 50 ul of 2N sulfuric acid (Sigma Aldrich, Cat. 320501-500). The absorbance at 450 nm was read using a SpectraMax M5 (Molecular Devices).

[0287] Single B cell sorting was performed from the lymphocytes collected from blood and/or spleen and/or lymph nodes from immunized rats with high serum immune response. In short, cells were incubated with anti-rat CD32 (clone D34-485, BD Biosciences) for 5 minutes followed by human TIGIT protein (R&D, cat. #7898-TG) for 1 hour at 4° C. Cells were then washed and incubated with a mixture of FITC-conjugated mouse anti-rat IgM (clone MRM-47, Biolegend), PE-Cy7-conjugated mouse anti-rat CD45R (clone HIS 24, eBioscience), and APC-conjugated mouse anti-His (clone AD1.1.10R, R&D) antibodies for 30 minutes at 4° C. Single TIGIT+ B cells were sorted into each well of a 96 well plate containing 4 ul lysis buffer (0.1M DTT, 40 U/ml Rnase Inhibitor, Invitrogen, Cat #10777-019) on BD FACS Aria III flow cytometer. Plates were sealed with Microseal ‘F’ Film (BioRad) and immediately frozen on dry ice before storage at −80° C.

[0288] Ig V-gene cloning from single sorted B cell was performed with a protocol modified from Tiller et al., 2008, J Imm Methods 329. In brief, total RNA from single sorted B cells was reverse transcribed in a final volume of 14 μl/well in the original 96-well sorting plate with nuclease-free water (Invitrogen, Cat #AM9935) using final amounts/concentrations of 150 ng random hexamer primer (pd(N)6, Applied Biosystems, P/N N808-0127) and 50U Superscript III reverse transcriptase (Invitrogen, Cat #18080-044) following the manufacturer's protocol. Primers (not listed) were modified based on previous publications (Wardemann et al, Science 2003 301:1374-1377) and/or designed by examining published Ig gene segment nucleotide sequences from IMGT®, the international ImMunoGeneTics information system (http://www.imgt.org; (Lefranc et al., 2009) and NCBI (http://www.ncbi.nlm.nih.gov/igblast/) databases. Human Igh, Igk and Igl V gene transcripts were amplified independently by two rounds of nested (Igh, Igk and Igl) PCR starting from 3.5 μl of cDNA as template. All PCR reactions were performed in 96-well plates in a total volume of 40 μl per well using AccuPrime Taq DNA Polymerase High Fidelity kit, (Invitrogen, Cat #. 12346-094) following the manufacturer's protocol. The first round of PCR was performed at 95° C. for 2 min followed by 40 cycles of 94° C. for 30 s, 50° C. for 30 s, 72° C. for 40 s, and final incubation at 72° C. for 5 min.

[0289] Nested second round PCR was performed with 5 μl of unpurified first round PCR product at 95° C. for 2 min followed by 5 cycles of 94° C. for 30 s, 42° C. for 30 s, 72° C. for 45 s, and then 50 cycles of 94° C. for 30 s, 55° C. for 30 s, 72° C. for 45 s, and final incubation at 72° C. for 5 min. PCR products were cloned into IgG expression vectors for Ig expression and functional screening.

[0290] A total of 860 TIGIT+ B cells were isolated by single cell fluorescence-activated cell sorting. Immunoglobulin VH and VL regions were PCR amplified from cDNA prepared from the individual lysed B cells. Paired VH and VL regions were obtained from 388 B cell lysates and cloned into IgG expression vectors for expression, and biochemical characterization, and DNA sequencing.

[0291] Hit optimization candidates were selected based on the potency to block binding of CD155 to TIGIT and the ability to bind to both human TIGIT and cynomolgus monkey TIGIT. Binding to TIGIT was originally determined by ELISA and to TIGIT expressing cells by FACS, and later quantified by Biacore. Eighty-three clones were confirmed by ELISA and flow cytometry assays as human and cynomolgus monkey TIGIT (Novoprotein cat. No. CP02) cross-reactive cell binders. Thirty of these clones blocked the TIGIT:CD155 interaction. Four candidates, 3963H03, 3964A06, 3965D08, and 3966C11 (also abbreviated as H03, A06, D08, and C11, respectively) fitted the predefined profile and ultimately 3963H03 was chosen for sequence optimization. The goals of the sequence optimization were to replace non-germline residues in the variable region frameworks with germline residues and to improve the manufacturability by removing sequence motifs potentially prone to post-translational modification.

[0292] The heavy and light chain amino acid sequences of 3963H03 are as follows:

TABLE-US-00014 Heavy chain: (SEQ ID NO: 22) EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMN WRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFS LDTSVSTAYLQISSLKAEDTAVYYCARVGGYSVDE YAFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG Light chain: (SEQ ID NO: 23) AIRLTQSPSFLSASVGDRVTITCRASQGISSYLAW YQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCQQLNSYPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0293] Yeast Display AFM and Sequence Optimization

[0294] 3963H03 heavy and light chain CDR3 regions were subjected to parsimonious mutagenesis and used to construct yeast display libraries for obtaining affinity matured variants. Two libraries were constructed initially: (1) a mutagenized L3-CDR light chain library paired with the parental heavy chain, and (2) a mutagenized H3-CDR heavy chain library paired with the parental light chain. The L3-CDR and H3-CDR libraries were screened separately by FACS for 2 rounds to select for the top 5-10% binders having a higher signal than a yeast clone expressing parental 3963H03. The mutagenized light chains and heavy chains were isolated from pools resulting from the selections and transformed into yeast to make new libraries having reduced complexities. From these, mating libraries were made and screened for up to three rounds to isolate the top 1-0.1% high affinity binders by FACS. Selected and validated clones with higher affinities were subcloned into a mammalian expression vector for further validation by BIACORE™ affinity analysis. Two candidates, 7729G05 and 7728B03, demonstrated affinities for human and cynomolgous monkey TIGIT in the picomolar KD range, and were selected for further studies.

[0295] An assessment of the variable region sequences of 3963H03 identified two non-germline amino acid residues in the light chain variable region framework and two non-germline amino acids in the heavy chain variable region framework. Additionally, a methionine residue within the heavy chain framework 4 was identified which could potentially oxidize overtime, and a deamidation motif was identified in the light chain CDR3. Based on these analyses, a series of sequence designs were generated in which the potentially problematic amino acids were replaced with either the germline-associated amino acid at that position or, in the case of the germline methionine, with a biophysically conservative leucine substitution. The amino acid substitutions are shown in Table 1

TABLE-US-00015 TABLE 1 Amino acid substitution variants of 3963H03 heavy and light chain V regions. Heavy chain positions Light chain positions IMGT numbering 1, 2 123 IMGT numbering 1 3 108 114 Kabat numbering H1, H2 H108 Kabat numbering LI L3  92  93 Sequential numbering 1, 2 117 Sequential numbering 1 3  92  93 Region FR1 FR4 Region FR1 FR1 CDR3 CDR3 Parent ID EI M Parent ID A R N S Germline ID QV L Germline ID D Q N S Substitution Designs QV L Substitution Designs D Q NSQ ASQ VH1.00 (parental) EI M VL1.00 (parental) A R N S VH1.01 QV M VL1.01 D Q N S VH1.02 EI L VL1.02 D Q S S VH1.03 QV L VL1.03 D Q Q S VL1.04 D Q N A VL1.05 D Q N Q

[0296] The sequence optimized variant designated H03-12, consisting of VH1.03 (E1Q, I2V, M117L, sequential numbering) and VL1.02 (A1D, R3Q, N92S, sequential numbering), was selected as the lead candidate based on having the most favorable substitutions, good productivity in CHO cells, and activity equivalent to, or better than, the parental molecule in the binding and functional assays.

[0297] 1.1 Variant Identification by NGS and SPR

[0298] 3963H03-related B cell sequences were expanded through Next Generation Sequencing (NGS) technology. Briefly, from the same lymph node tissue used to clone 3963H03, about 5×10.sup.5 TIGIT-specific B cells and plasma cells were collected using FACS through bulk sorting. Total RNA was isolated to generate the NGS library. Following cDNA synthesis, IGVH7-4 and IGKV1-9 (relevant to the 3963H03 hit) gene-specific primers were used for RT-PCR isolation of the 3963H03 specific IgH and IgK B cell V-region sequences; these were subcloned into expression vectors for IgG antibodies. 73 VH sequences related to 3963H03 with unique CDR sequences (Table 2) were paired with the parental light chain of 3963H03 and expressed as IgG in Expi293F cells. Similarly, 20 VK sequences related to 3963H03 with unique CDR sequences (Table 3) were paired with the parental heavy chain of 3963H03 and expressed as IgG in Expi293F cells. Culture supernatants for these 93 IgGs, as well as 39603H03 parental, were collected, diluted 1:10, and the kinetic off-rates of binding to human TIGIT were measured by Surface Plasmon Resonance (SPR) using a GE Healthcare Biacore 4000 instrument as follows. Goat anti-human Fc antibody (Jackson Immunoresearch Laboratories #109-005-098) was first immobilized on a BIAcore carboxymethylated dextran CM5 chip using direct coupling to free amino groups following the procedure described by the manufacturer. Antibodies were then captured on the CM5 biosensor chip. Binding measurements were performed using the running HBS-EP+ buffer. Two-fold dilution series of his-tagged human TIGIT, with starting concentrations between 100 nM and 10 nM, were injected at a flow rate of 30 μl/min at 25° C. Dissociation rates (koff, s-1) were calculated using a simple 1:1 Langmuir binding model (Biacore Evaluation Software). The measured koff are shown in Table 2 and Table 3, revealed both slower and faster koff among the new variants.

[0299] 1.2 High Affinity Variant Identification by Pairwise Additivity of Heavy Chains and Light Chains.

[0300] We hypothesized that k.sub.off gains or losses would be pair-wise additive, as illustrated mathematically in Equation 1.

[00001]$\begin{array}{cc}{k}_{\mathrm{off}\left({\mathrm{VH}}_i,{\mathrm{VL}}_j\right)}=\frac{k_{\mathrm{off}\left({\mathrm{VH}}_i,{\mathrm{VL}}_{\mathrm{nat}}\right)}}{k_{\mathrm{off}\left({\mathrm{VH}}_{\mathrm{nat}},{\mathrm{VL}}_{\mathrm{nat}}\right)}}\times \frac{k_{\mathrm{off}\left({\mathrm{VH}}_{\mathrm{nat}},{\mathrm{VL}}_j\right)}}{k_{\mathrm{off}\left({\mathrm{VH}}_{\mathrm{nat}},{\mathrm{VL}}_{\mathrm{nat}}\right)}}.& \mathrm{Equation}1\end{array}$

[0301] Using Equation 1, the off-rates of the antibodies in Tables 2 and 3 were used to predict the activity of variant heavy or light chains paired with other variant light or heavy chains. 15 NGS identified variant pairs were predicted to have improved binding affinity. These 15 were expressed and the kinetic off-rate characterized by SPR. Three of these were within 1.5 fold of 3963H03 while 12 variants had improved affinity of between 2 and 4.7 fold relative to 3963H03 as predicted (Table 4). Overall, this strategy together with the previous one allowed for identification of variants with a range of activities, including many with improved off-rate relative to the initial hit 3963H03, of which the sequence was used as a probe to generate the library.

TABLE-US-00016 TABLE 2 73 VH sequences with unique CDRs related to 3963H03 were identified by NGS. These were paired with the light chain of 3963H03 and the koff was measured. koff (second ID HVR-H1 HVR-H2 HVR-H3 1) 3963H03 GYTFTSYP INTNTGNP ARVGGYSVDEYAFDV 6.3E−04 VH D04H01 GYTFTSYA INTNTGNP ARVGGYSGYDYAFDI 8.3E−03 D04H02 GYTFTSYA INTNTGNP ARVGGYSGYDYAFDV 7.0E−03 D04H09 GYTFTSYP INTNTGNP ARVGGYSGYDYAFDI 1.4E−03 D04H10 GYTFTSYP INTNTGNP ARVGGYSGYDYAFDV 1.4E−03 D04H11 GYTFTSYP INTNTGNP ARVGGYGGYDYAFDI 5.1E−03 D04H12 GYTFTSYP INTNTGNP ARVGGYGVYDYAFDV 4.3E−03 D04H13 GYTFTSYP INTNTGNP ARVGGYIVYDYAFDV 3.7E−04 D04H15 GYTFTSYP INTNTGNP ARVGGYGGYDYAFDV 6.1E−03 D04H16 GYTFTSYP INTNTENP ARVGGYGGYDYAFDI 6.6E−03 D04H17 GYTFTAYA INTNTGNP ARVGGYGGYDYAFDI 4.2E−03 D04H18 GYTFTAYP INTNTGNP ARVGGYSGYDYAFDI 1.2E−03 D04H19 GYTFTAYP INTNTGNP ARVGGYSVNDYAFDI 4.8E−04 D04H20 GYTFTAYP INTNTGNP ARIGGYSVNDYAFDI 1.6E−03 D04H23 GYTFTTYP INTNTGNP ARVGGYSGYDYAFDI 2.6E−03 D04H24 GYTFTTYP INTNTGNP ARVGGYGVYDYAFDV 7.9E−03 D04H25 GYTFTTYP INTNTENP ARVGGYGGYDYAFDV 2.8E−02 D04H26 GYTFASYP INTNTGNP ARVGGYGGYDYAFDI 6.0E−02 D04H27 GYTLTSYP INTNTGNP ARVGGYGGYDYAFDF 4.9E−03 D04H28 GYTLTSYP INTNTGNP ARVGGYGGHDYAFDI 6.4E−03 D04H29 GYIVTSYA INTNTGNP ARVGGYSGYDYAFDI 3.2E−03 D04H36 GYTFTAYA INTNTGNP ARVGGYGVYDYAFDI 9.7E−03 D04M07 GYTFTSYA INTNTGNP ARVGGYSGFDYAFDI 6.4E−03 D04M11 GYTFTSYP INTNTGNP ARVGGYSGYDYGFDI 2.8E−03 D04M12 GYTFTSYP INTNTGNP ARVGGYSGNDYAFDI 2.3E−03 D04M21 GYTFTSYP INTNTGDP ARVGGYSGYDYAFDI 1.0E−03 D04M22 GYTFTSYP INTNTANP ARVGGYSGYDYAFDI 1.6E−03 D04M27 GYTFTSFP INTNTGNP ARVGGYSGYDYAFDI 2.2E−03 D04M28 GYTFTSFP INTNTGNP ARVGGYSGYDYAFDM 2.5E−03 D04M29 GYTFTSFP INTNTGNP ARVGGYGGYDYAFDI 4.9E−03 D04M30 GYTFTSFP INTNTGNP ARVGGYGGSDYAFDI 9.1E−03 D04M32 GYTFTNYA INTNTGNP ARVGGYSGYDYAFDI 4.9E−03 D04M33 GYTFTNYP INTNTGNP ARVGGYSGYDYAFDI 1.9E−03 D04M35 GYTFTYYP INTNTGNP ARVGGYSGYDYAFDI 1.1E−03 D04M36 GYTFASYP INTNTGNP ARVGGYSGYDYAFDI 1.3E−03 D04M37 GYTFISYP INTNTGNP ARVGGYSGYDYAFDI 1.3E−03 D04M39 GYTFPSYP INTNTGNP ARVGGYSGSDYAFDI 2.3E−03 D04M41 GYTFSSYP INTNTGNP ARVGGYSGYDYAFDI 1.4E−03 D04M42 GYTFSSYP INTNTGNP ARVGGYGGYDYAFDI 3.8E−03 D04M44 GYTFSSYP INTNTGNP ARVGGYGVYDYAFDI 4.3E−03 D04M47 GYTFSSYP INTNTGNP ARVGGYGGDDYAFDI 2.8E−02 D04M49 GYAFTSYP INTNTGNP ARVGGYSGYDYAFDI 1.2E−03 D04M51 GYAFTTYP LNTNTGNP ARVGGYGGYDYAFDI 3.6E−03 D04M52 GYAFSTYA INTNTGNP ARVGGYGGYDYAFDI 3.6E−03 D04M53 GYAFSTYA LNTNTGNP ARVGGYGGYDYAFDI 4.3E−03 D04M54 GYIFTSYP INTNTGNP ARVGGYSGYDYAFDI 1.3E−03 D04M55 GYSFTSYP INTNTGNP ARVGGYGGYDYAFDI 3.1E−03 D04M56 GYSFTDYP INTNTGNP ARVGGYSGYDYAFDI 1.8E−03 D04M58 GDTFRSYP INTNTGNP ARVGGYGGYDYAFDI 6.0E−03 D04M59 GFTFTSYP INTNTGNP ARVGGYSGYDYAFDF 4.5E−03 D04M60 GNTFTSYP INTNTGNP ARVGGYSGYDYAFDI 4.3E−03 D04M61 GYTFTNYP INTNTGNP ARVGGYSGYDYSFDI 2.0E−03 D04M64 GYSFTSYP INTNTGNP ARVGGYSGYDYAFDI 1.1E−03 D04M65 GYSFTNYP INTNTGNP ARVGGYSGYDYAFDI 1.3E−03 D04M66 GYTFTTYP INTNTGNP ARVGGYSAYDYAFDI 1.7E−03 H03H01 GYTFTSYA INTNTGNP ARVGGYSVYDYAFDI 3.5E−03 H03H02 GYTFTSYP INTNTGNP ARVGGYSVYDYAFDI 6.2E−04 H03H03 GYTFTSYP INTNTGNP ARVGGYSVYDYAFDA 7.5E−04 H03H04 GYTFTSYP INTNTGNP ARVGGYSVYDYAFDV 6.0E−04 H03H06 GYTFTSYP INTNTGNP ARVGGYSVYDYASDV 4.2E−03 H03H08 GYTFTSYP INTNTGNP ARVGGYSVDDYAFDV 5.7E−04 H03H15 GYTFTAYA INTNTGNP ARVGGYSVYDYAFDI 2.8E−03 H03H16 GYTFTAYP INTNTGNP ARVGGYSVYDYAFDI 6.4E−04 H03H17 GYTFTAYP INTNTGNP ARVGGYSVYDYAFDV 5.5E−04 H03H18 GYTFTAYP INTNTGNS ARVGGYSVYDYAFDI 8.6E−04 H03H19 GYTFTAYP INTNTGSP ARVGGYSVYDYAFDI 5.0E−04 H03H22 GYTFTTYA INTNTGNP ARVGGYSVYDYAFDI 5.7E−03 H03H23 GYTFTTYP INTNTGNP ARVGGYSVYDYAFDI 1.1E−03 H03H24 GYTFTTYP INTNTGNP ARVGGYSVYDYAFDV 1.0E−03 H03M02 GYAFSAYA LNTNTGNP ARVGGYSVYDYAFDI 2.5E−02 H03M03 GYSFTNYP INTNTGNP ARVGGFSDYDYAFDI 1.9E−03 H03M04 GSTFTSYP INTNTGNP ARVGGYSAYDYAFDI 5.5E−03 H03M05 GYTFTNYA INTNTGNP ARVGGYSDYDYAFDI 1.4E−02 H03M07 GYTFTSYP INTNTGNP ARVGGYSDYDYAFDI 1.4E−03

TABLE-US-00017 TABLE 3 20 VK sequences with unique CDRs related to 3963H03  were identified by NGS. These were paired with the heavy chain of 3963H03 and the koff was measured.. ID HVR-H1 HVR-H2 HVR-H3 koff (second 1) 3963H03 VK QGISSY AAS QQLNSYPT 6.3E−04 H03K02 QGISSY AAS QQLNSYLT 1.7E−03 H03K03 QGISSY AAS QQLNGYLT 2.5E−03 H03K04 QAISSY AAS QQLNGYLT 1.6E−03 H03K05 QGISSY AAS QQLNNYLT 2.0E−03 H03K06 QGISSY GAS QQLNGYPT 2.7E−04 H03K07 QGISSY GAS QQLNSYPT 3.4E−04 H03K08 QVISSY AAS QQLNSYPT 3.6E−04 H03K09 QGISSY AAS QQLNSYPL 5.6E−04 H03K10 QGISSY AAS QQLNSYPP 8.8E−04 H03K11 QGISSY AAS QQLNGYPT 2.8E−04 H03K12 QGISSY AAS QQLNGSPT 3.5E−04 H03K15 QGISSS AAS QQLNSYPT 3.4E−04 H03K24 QGIPSY AAS QQLNSYPT 3.6E−04 H03K25 QAISSY AAS QQLNSYPT 4.6E−04 H03K26 QGISSY AAS QQPNGYLT 4.2E−04 H03K29 QGISTY AAS QQLNSYLT 1.6E−03 H03K30 QGINSY AAS QQLNSYPT 3.4E−04 H03K31 QAISSY AAS QQPNGYLT 6.5E−04 H03K34 QGISSY AAS QQLNSYPH 6.1E−04 H03K35 QGISSY AAS QQLNSYIT 6.2E−04

TABLE-US-00018 TABLE 4 Novel pairings of VH & VK sequences related to 3963H03 identified by NGS and predicted to have a slower koff ID of heavy ID of light koff (second.sup.−1) of from Table 2 from Table 3 novel variant D04H13 H03K06 1.5E−04 D04H13 H03K08 1.3E−04 D04H13 H03K11 1.7E−04 D04H13 H03K15 2.1E−04 D04H13 H03K30 1.6E−04 H03H08 H03K06 1.4E−04 H03H08 H03K11 2.7E−04 H03H08 H03K12 2.7E−04 H03H08 H03K15 2.3E−04 H03H08 H03K19 3.1E−04 H03H08 H03K21 3.1E−04 H03H08 H03K26 6.3E−04 H03H08 H03K30 5.3E−04 H03H12 H03K06 2.1E−04 H03H24 H03K06 4.6E−04

[0302] 2. Manufacturing and Purification

[0303] 2.1 Bioproduction, Clarification and Purification

[0304] The antibody H03-12 as disclosed was produced from CHO-K1SV cells.

TABLE-US-00019 3963H03-12 heavy chain: (SEQ ID NO: 18) QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMN WWRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVF SLDTSVSTAYLQISSLKAEDTAVYYCARVGGYSVD EYAFDVWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. 3963H03-12 light chain: (SEQ ID NO: 19) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAW YQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCQQLSSYPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC.

[0305] Cells were grown in proprietary-CHO fed-batch growth media supplemented with glucose at 37° C. The cultures were fed with a mixture of proprietary feed components on days 3, 5, 7 and 10 days post inoculation.

[0306] Crude conditioned media from the bioreactor runs were clarified using 2.2 m2 Millistak+ Pod D0HC (Millipore MD0HC10FS1) and 1.1 m2 Millistak+ Pod X0HC (Millipore #MX0HC01FS1) filters, followed by terminal filtration with a Millipore Opticap XL3 0.5/0.2 μm filter (Millipore #KHGES03HH3).

[0307] The antibody was then purified using standard methods and formulated in 10 mM Histidine, 5 mM Methionine, 8% Trehalose, pH 5.5 with 0.05% Tween 20.

[0308] The antibody can be stored in phosphate buffer, pH adjusted with NaCl as isotoning agent.

[0309] 3. Biochemical and Biological Characterization

[0310] 3.1 Biacore Binding Affinity and Specificity

[0311] Binding affinities of anti-TIGIT hit candidate antibodies to human TIGIT and cynomolgus monkey TIGIT were measured by Surface Plasmon Resonance (SPR) using a GE Healthcare Biacore 4000 instrument and a GE Healthcare Biacore T200 instrument as follows. Goat anti-human Fc antibody (Jackson Immunoresearch Laboratories #109-005-098) was first immobilized on BIAcore carboxymethylated dextran CM5 chip using direct coupling to free amino groups following the procedure described by the manufacturer. Antibodies were then captured on the CM5 biosensor chip to achieve approximately 200 response units (RU). Binding measurements were performed using the running HBS-EP+ buffer. 2-fold dilution series with starting concentration between 100 nM and 10 nM of His-tagged human and cynomolgus monkey (cyno) TIGIT proteins were injected at a flow rate of 30 μl/min at 25° C. Association rates (kon, M-1 s-1) and dissociation rates (koff, s-1) were calculated using a simple 1:1 Langmuir binding model (Biacore Evaluation Software). The equilibrium dissociation constant (KD, M) was calculated as the ratio of koff/kon. Affinity of candidates 3963H03, 3963H03-12, 3964A06, 3965D08, and 3966C11 for binding to human TIGIT ranged from 2.5 to 10 nM and affinity for binding to cyno TIGIT ranged from 0.8 to 8.7 nM (Table 5).

TABLE-US-00020 TABLE 5 Biacore affinity measurements for anti-TIGIT hit candidates from OmniRat Antibody Human TIGIT Cyno TIGIT ID ka(M-1 sec-1) kd(sec-1) KD(M) KD(nM) ka(M-1 sec-1) kd(sec-1) KD(M) KD(nM) 3963H03 1.21E+05 4.07E−04 3.37E−09    3.4   1.47E+05 2.13E−04 1.45E−09   1.5 .sup.  3964A06 8.91E+04 2.33E−04 2.62E−09    2.6   1.09E+05 1.22E−04 1.11E−09   1.1 .sup.  3965D08 4.82E+05 4.97E−03 1.03E−08   10.3   6.53E+05 5.65E−03 8.66E−09   8.7 .sup.  3966C11 1.08E+05 2.74E−04 2.53E−09    2.5   1.18E+05 1.67E−04 1.41E−09   1.4 .sup.  3963H03-12 1.66E+05 6.70E−04 4.05E−09    4.05  1.60E+05 1.33E−04 8.29E−10   0.83.sup.  7728B03 3.25E+05 2.15E−05 6.61E−11    0.07  3.03E+05 1.57E−05 5.19E−11   0.05.sup.  7729G05 7.67E+05 1.33E−05 1.73E−11  <0.051 5.81E+05 1.23E−05 2.11E−11 <0.05.sup.1 .sup.1Affinity measurement is below the sensitivity of the Biacore instrument (50 pM)

[0312] Table 6a shows the CDR sequences of the candidate antibodies described herein.

TABLE-US-00021 TABLE 6a Anti-TIGIT CDR's of different anti TIGIT candidates HVR-H1 HVR-H2 HVR-H3 HVR-L1 HVR-L2 HVR-L3 H03 GYTFTSYP INTNTGNP ARVGGYSVDEYAFDV QGISSY AAS QQLNSYPT H03-12 GYTFTSYP INTNTGNP ARVGGYSVDEYAFDV QGISSY AAS QQLSSYPT A06 GYTFTAYP INTNTGNP ARVGGYSVYDYAFDI QGISSY AAS QQLNSYPT D08 GYTFTSYP INTNTGNP ARTGYSGSYYWFDP- QGISSY AAS QQLNSYLT C11 GYTFTSYP INTNTGNP ARVGGYGGYDYAFDI QGISSY AAS QQLNSYPT B03 GYTFTSYP INTNTGNP ARTGGYSVDEYSFDI QGISSY AAS HQTIFRPT G05 GYTFTSYP INTNTGNP ARVGGFTVPEYAFDI QGISSY AAS* GQVMRYPA

[0313] Table 6b shows deviations in the framework region sequences as compared to antibody

TABLE-US-00022 TABLE 6B Anti-TIGIT FR’s of different anti TIGIT candidates, as compared to H03-12 VL region residue position VH region residue position FR1 FR2 FR4 FR1 FR3 FR4 1 3 46 99 102 103 1 2 60 117 H03 A R L G K V E I Y M A06 D Q L G K V E V Y M D08 D R F Q R L E V Y L C11 D Q L G K L E I N M H03-12 D Q L G K V Q V Y L B03 D Q L G K V Q M Y T G05 D R L G K V Q V Y T
framework region amino acids as well as the constant regions corresponds to those of H03-12.

[0314] The full variable region sequences are provided in the following:

TABLE-US-00023 A06-VH (SEQ ID NO: 24) EVQLVQSGSELKKPGASVKVSCKASGYTFTAYPMNWVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVG GYSVYDYAFDIWGQGTMVTVSS A06-VL (SEQ ID NO: 25) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPTFGGG TKVEIK C11-VH (SEQ IN NO: 26) EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGLEWMGW INTNTGNPTNAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVG GYGGYDYAFDIWGQGTMVTVSS C11-VL (SEQ IN NO: 27) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPTFGGG TKLEIK H03-VH (SEQ ID NO: 28) EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVG GYSVDEYAFDVWGQGTMVTVSS H03-VL (SEQ ID NO: 29) AIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPTFGGG TKVEIK D08-VH (SEQ ID NO: 30) EVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTG YSGSYYWFDPWGQGTLVTVSS D08-VL (SEQ ID NO: 31) DIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYLTFGQG TRLEIK B03-VH (SEQ ID NO: 32) QMQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTG GYSVDEYSFDIWGQGTTVTVSS B03-VL (SEQ ID NO: 33) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQTIFRPTFGGG TKVEIK G05-VH (SEQ ID NO: 34) QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGLEWMGW INTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVG GFTVPEYAFDIWGQGTTVTVSS G05-VL (SEQ ID NO: 35) DIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCGQVMRYPAFGGG TKVEIK

[0315] 3.2 Selectivity

[0316] 3963H03, 3964A06, 3965D08, 3966C11 and derivatives had no detectable binding to the related family member protein CD226 or the unrelated protein PD-L1 in ELISA EC50 assays.

[0317] For a more comprehensive evaluation of selectivity, a proprietary fixed-cell microarray technology was used by Retrogenix Ltd. (High Peak, UK) to screen variant 3963H03-12 for off-target binding to a library of 5647 human proteins, comprised predominantly of cell-surface membrane proteins. The library included most known immunoglobulin superfamily receptors that are related to TIGIT such as CD226, CD96, PVR, and NECTINs 1-4. The study was done in four phases: (1) a prescreen to determine background levels and the optimal test antibody concentration for screening, (2) the primary screen for 3963H03-12 binding to fixed HEK293 cells expressing 5647 proteins, (3) a confirmation/specificity screen done by re-expressing putative hits in HEK293 cells and testing binding of 3963H03-12 to the fixed cells along with an isotype control, and (4) further validation by expressing the specific hits in live HEK293 cells and analyzing binding to both 3963H03-12 and the isotype control by flow cytometry.

[0318] Eleven binders were identified in the primary screen with intensities ranging from very weak to strong. All eleven were confirmed as binders in the confirmation/specificity secondary screen. The strong binders included the 3963H03-12 target protein TIGIT. Six of the eleven primary binders were also bound by one of the control antibodies and were classified as non-specific binders. These included Fc gamma receptors that either bind the primary antibody Fc or the secondary antibody directly. One binder was very weak and too close to background to consider significant, leaving the four final binders: TIGIT (Genbank accession NM_173799.3), TMEM25 isoform 1 (Genbank accession NM_032780.3), HAVCR2 (Genbank accession BC063431.1), and Cyclin G Associated Kinase (GAK, Genbank accession B0008668). GAK is an intracellular protein that did not bind 3963H03-12 when expressed in live HEK293 cells, and thus was invalidated. TMEM25 isoform 1 and HAVCR2 are transmembrane proteins and were scored as weak binders to 3963H03-12 in the fixed cell screens, then subsequently shown to have low level interactions on live transfected cells with 4.3-fold and 3.0-fold higher median fluorescence than 3963H03-12 binding to vector-only transfected HEK293 cells. The isotype control antibody had binding to TMEM25 isoform 1 and HAVCR2 transfected HEK293 cells slightly lower than 3963H03-12, with 1.4-fold and 1.9-fold higher median fluorescence than isotype control binding to vector-only transfected HEK293 cells, which was similar to the binding to cells transfected with the intracellular protein GAK. In contrast, 3963H03-12 demonstrated 130-fold higher median fluorescence for binding to TIGIT-transfected HEK293 cells compared to vector-only transfected HEK293 cells. Altogether, these results show 3963H03-12 binds selectively to TIGIT.

[0319] 3.3 ELISA Based TIGIT:CD155 Competition Assay

[0320] The ability of anti-TIGIT antibodies and a control antibody to compete with the binding of biotinylated human TIGIT-Fc chimera to human CD155-Fc chimera was determined by a competitive ELISA. FIG. 1 shows representative competition curves for the test antibodies. The results demonstrated that the anti-TIGIT antibodies 3963H03, 3964A06, 3965D08, 3966C11 efficiently block the interaction of TIGIT and CD155 with IC50 s of 0.8-1.2 nM.

[0321] The following assay protocol was used:

[0322] 1. 96-well plates were coated with 2.5 μg/ml rhCD155-Fc (Sino Biologicals; Cat #10109H02H) at 50 μl/well and incubated overnight at 4° C.

[0323] 2. Rinsed wells 3 times with PBS, 0.05% Tween, 200 μl/well.

[0324] 3. Blocked wells with 200 μl of 1% BSA in PBS, for 1 h at room temperature.

[0325] 4. Rinsed wells 3 times with PBS, 0.05% Tween, 200 μl/well.

[0326] 5. Mixed 75 μl 1 mg/ml human TIGIT-Fc-biotin (R&D Systems cat. No. 7898-TG biotinylated at EMD-Serono) with 75 μl test or control antibodies in a 1:3 dilution series (166.7 to 0.08 nM) and added 50 μl each to duplicate wells. Incubated 2 hours at room temperature.

[0327] 6. Rinsed wells 3 times with PBS, 0.05% Tween, 200 μl/well.

[0328] 7. For detection streptavidin-HRP conjugate (Millipore; Cat #18-152) was added, 100 μl/well at a 1:200 dilution; and incubated 30 min at room temperature.

[0329] 8. Rinsed wells 3 times with PBS, 0.05% Tween, 200 μl/well.

[0330] 9. Added 1-Step™ Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific Cat #34028), 100 μl/well and incubated 1-2 min at room temperature.

[0331] 10. Added 100 μl 2N sulfuric acid to each well.

[0332] 11. Measured ODs at 450 nm and 630 nm on ELISA plate reader.

[0333] 3.4 Structural and Functional TIGIT Epitope Mapping

[0334] a). Co-Crystallization of TIGIT with Fab Fragments of the Present Invention

[0335] Crystal structures of the complex of human TIGIT ECD and various Fab fragments of the antibodies in the present invention were determined to identify the interacting amino acids between human TIGIT and the antibody variable region. Human TIGIT was expressed in E. coli inclusion bodies, refolded, and purified by affinity and size exclusion chromatography. The Fab fragments were expressed with His-tags in Expi293F cells and purified by affinity chromatography according to standard methods. The 1:1 complex of TIGIT and each Fab fragment was formed and purified by gel filtration chromatography yielding a homogenous protein complex with a purity greater than 95%. The solution containing the complex was concentrated and standard techniques of high throughput vapor diffusion crystallization screening were applied.

[0336] Crystals of the 3963H03 Fab in complex with human TIGIT were grown by mixing 0.75 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 24.57 mg/mL) with 0.5 μl reservoir solution (0.2 M Ammonium citrate pH 7.0, 20% PEG 3350) and 0.25 μl seed stock at 20° C. using sitting drop vapor diffusion method. Crystals of 3963H03-12 Fab in complex with human TIGIT were grown by mixing 0.5 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 20.26 mg/mL) with 0.3 μl reservoir solution (0.15 M sodium citrate, 0.1 M Bis-Tris 8.5, 22% PEG 3350) and 0.2 μl seed stock at 20° C. using sitting drop vapor diffusion method. Crystals of 3964A06 Fab in complex with human TIGIT were grown by mixing 0.75 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 18.66 mg/mL) with 0.5 μl reservoirs solution (0.2 M Sodium Formate, 20% PEG 3350) and 0.25 μl seed stock at 20° C. using sitting drop vapor diffusion method. Crystals of 3966C11 Fab in complex with human TIGIT were grown by mixing 0.5 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 27 mg/mL) with 0.5 μl reservoirs solution (16% PEG 4000, 5%-10% Isopropanol, 0.1 M Hepes pH 7.5) at 20° C. using sitting drop vapor diffusion method. Crystals of 7728B03 Fab in complex with human TIGIT were grown by mixing 0.75 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 22.35 mg/mL) with 0.5 μl reservoirs solution (25% PEG 3350, 0.1M Tris pH 8.5) and 0.25 μl seed stock at 20° C. using sitting drop vapor diffusion method. Crystals of 7729G05 Fab in complex with human TIGIT were grown by mixing 0.3 μl protein solution (50 mM Tris-HCl pH 7.5, 200 mM NaCl, at 21 mg/mL) with 0.2 μl reservoirs solution (0.1 M Phosphate/citrate, 40% v/v Ethanol, 5% w/v PEG 1000) and 0.1 μl seed stock at 20° C. using sitting drop vapor diffusion method.

[0337] Crystals were flash-frozen and measured at a temperature of 100 K. The X-ray diffraction data have been collected at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) or at the Deutsches Elektronen-Synchrotron (Hamburg, Germany) using cryogenic conditions. Data were processed using the programmes XDS.

[0338] The structure of the complex was solved by molecular replacement using Phaser, version 2.5.7 (McCoy, A. J. et al. J. Appl. Cryst. (2007). 40, 658-674) using structures of human TIGIT (PDB ID: 3UCR) and a Fab (in-house structure) as search models. The structure was refined using Buster, version 2.11.6 (Bricogne, G. et al. Buster version 2.11.6 (2016) Cambridge, United Kingdom: Global Phasing Ltd.). All models including the final protein model were built using COOT version 0.8.1 (Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126-2132). All relevant data regarding data collection, data processing, structure refinement and structure quality can be found in Table 7 and Table 8.

TABLE-US-00024 TABLE 7 Data collection and processing statistics for TIGIT complexed with Fabs TIGIT:3963H03- TIGIT:3963H03 12 TIGIT:3964A06 X-ray Source PXI/X06SA PX11 (DESY2) PXII/X10SA (SLS1) (SLS1) Wavelength   0.99998   1.033227    1.000000 [Å] Detector Pilatus 6M Pilatus 6M Pilatus 6M Temperature  100  100   100 [K] Space Group P41212 P41212 C121 Cell: a; b; c; 109.39 107.96; 121.96; [Å] 109.39 107.96; 80.79; 230.74 231.50 126.86 α; β; γ; [°] 90.0; 90.0; 90.0 90.0; 90.0; 108.57 90.0 90.0 90.0 Resolution [Å] 2.41 (2.55-2.41)3 2.87 (2.92-2.87) 1.55 (1.55-1.64) Unique 53945 32153 167333 reflections Multiplicity  9.9 (7.7) 25.6 (27.4) 3.35 (3.16) Completeness 97.9 (88.9)  100 (99.8) 98.8 (97.0) [%] Rsym [%]4 15.6 (132)  2.6 (33.4)  6.3 (110) Rmeas [%]5 16.4 (140) 13.2 (176)  6.4 (117) Mean(I)/sd6 11.6 (0.9) 22.7 (2.3) 11.5 (0.97) TIGIT:3966C11 TIGIT:7728B03 TIGIT:7729G05 X-ray Source PXII/X10SA PXIII/X06DA PXI/X06SA (SLS1) (SLS1) (SLS1) Wavelength    1.000020    1.000000    0.999930 [Å] Detector Pilatus 6M Pilatus 6M Pilatus 6M Temperature   100   100   100 [K] Space Group C121 P212121 C121 Cell: a; b; c; 118.77 65.23 116.64 [Å] 87.96 73.00 68.29 126.86 117.98 262.66 α; β; γ; [°] 90.0 90.0 90.0 102.57 90.0 90.13 90.0 90.0 90.0 Resolution [Å] 1.73 (1.84-1.73) 1.35 (1.43-1.35)  1.9 (2.02-1.9) Unique 131029 123912 160020 reflections Multiplicity 3.33 (3.18) 6.45 (6.17) 3.17 (3.08) Completeness 98.7 (94.2) 99.8 (99.1) 98.2 (95.0) [%] Rsym [%]3  5.7 (143)  5.1 (200)  9.9 (90.5) Rmeas [%]4  5.8 (164)  4.6 (225) 10.6 (130) Mean(I)/sd5 11.3 (0.7) 17.6 (0.95)  6.9 (0.86) 1SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) 2values in parenthesis refer to the highest resolution [00002]$\begin{array}{c}3\mathrm{Rsym}=\frac{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}.Math.{\hat{I}}_h-I_{h,i}.Math.}{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}}\mathrm{with}{\hat{I}}_h=\frac{1}{n_h}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}\mathrm{where}{I}_{h,i}\mathrm{is}\mathrm{the}\\ \mathrm{intensity}\mathrm{value}\mathrm{of}\mathrm{the}\mathrm{ith}\mathrm{measurement}\mathrm{of}h\end{array}$[00003]$\begin{array}{c}4\mathrm{Rmeas}=\frac{\underset{h}{.Math.}\sqrt{\frac{n_h}{n_h-1}}\underset{i}{\overset{n_h}{.Math.}}.Math.{\hat{I}}_h-I_{h,i}.Math.}{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}}\mathrm{with}{\hat{I}}_h=\frac{1}{n_h}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}\mathrm{where}{I}_{h,i}\\ \mathrm{is}\mathrm{the}\mathrm{intensity}\mathrm{value}\mathrm{of}\mathrm{the}\mathrm{ith}\mathrm{measurement}\mathrm{of}h\end{array}$5calculated with independent reflections 1SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) 2Deutsches Elektronen-Synchrotron (Hamburg, Germany) 3values in parenthesis refer to the highest resolution [00004]$\begin{array}{c}4\mathrm{Rsym}=\frac{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}.Math.{\hat{I}}_h-I_{h,i}.Math.}{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}}\mathrm{with}{\hat{I}}_h=\frac{1}{n_h}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}\mathrm{where}{I}_{h,i}\mathrm{is}\mathrm{the}\\ \mathrm{intensity}\mathrm{value}\mathrm{of}\mathrm{the}\mathrm{ith}\mathrm{measurement}\mathrm{of}h\end{array}$[00005]$\begin{array}{c}5\mathrm{Rmeas}=\frac{\underset{h}{.Math.}\sqrt{\frac{n_h}{n_h-1}}\underset{i}{\overset{n_h}{.Math.}}.Math.{\hat{I}}_h-I_{h,i}.Math.}{\underset{h}{.Math.}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}}\mathrm{with}{\hat{I}}_h=\frac{1}{n_h}\underset{i}{\overset{n_h}{.Math.}}{I}_{h,i}\mathrm{where}{I}_{h,i}\\ \mathrm{is}\mathrm{the}\mathrm{intensity}\mathrm{value}\mathrm{of}\mathrm{the}\mathrm{ith}\mathrm{measurement}\mathrm{of}h\end{array}$6calculated with independent reflections

TABLE-US-00025 TABLE 8 Refinement Statistics for TIGIT.sup.1 TIGIT:3963H03- TIGIT:3963H03 12 TIGIT:3964A06 Resolution [Å] 48.92-2.41 97.84-2.87 46-62-1.55 Number of reflections 53945/2698 32072/1677 167299/8365 (working/test) Rcryst [%] 0.187 0.195 0.174 Rfree [%]2 0.228 0.231 0.202 DPI [σ(x, B.sub.avg)]3 0.218 0.322 0.075 Total number of atoms: Protein 8221 7194 8039 Water 194 — 1318 Deviation from ideal geometry:4 Bond lengths [Å] 0.010 0.009 0.01 Bond angles [°] 1.29 1.15 1.11 Ramachandran plot:5 Most favored regions [%] 94.7 95.5 97.9 Additional allowed regions 4.5 3.5 2.1 [%] Disallowed regions [%] 0.8 1 0 TIGIT:3966C11 TIGIT:7728B03 TIGIT:7729G05 Resolution [Å] 48.59-1.73 48.64-1.35 38.00-1.90 Number of reflections 131021/6552 123904/6196 157863/7715 (working/test) Rcryst [%] 0.202 0.184 0.209 Rfree [%]2 0.227 0.205 0.239 DPI [σ(x, B.sub.avg)]3 0.103 0.055 0.135 Total number of atoms: Protein 8183 4092 12255 Water 823 716 1334 Deviation from ideal geometry:4 Bond lengths [Å] 0.010 0.010 0.01 Bond angles [°] 1.11 1.15 1.2 Ramachandran plot:5 Most favored regions [%] 97.4 97.4 96.4 Additional allowed regions 2.6 2.6 3.3 [%] Disallowed regions [%] 0 0 0.3 .sup.1Values as defined by REFMAC5, without sigma cut-off 2Test-set contains 5% of measured reflections 3Diffraction-component precision index (DPI) is calculated according to Eq. 27 of CRUICKSHANK. D.W.J. (1999) ACTA CRYST D55, 583-601, where σ(x, B.sub.avg) = 1.0(N.sub.i/n.sub.obs).sup.1/2C.sup.−1/3R.sub.freed.sub.min 4Root mean square deviations from geometric target values 5Calculated with MOLPROBITY

[0339] The structures of the Fab format of anti-TIGIT antibodies 3963H03, 3963H03-12, 3966C11, 3964A06, 7729G05 and 7728B03 in complex with the TIGIT ECD were solved with resolution of 2.41, 2.87, 1.73, 1.6, 1.9, and 1.35 Å respectively.

[0340] The complexes have nearly identical folding, as displayed in FIG. 2 with average RMSD of 0.79 Å on non-hydrogen atoms of the antigen and antibody variable region. The structures show that each Fab binds to a region on TIGIT that will sterically interfere with PVR binding. Indeed, FIG. 3 provides an overlay of the TIGIT:3963H03-12 co-crystal structure with the available TIGIT:PVR co-crystal structure (Protein Data Bank entry 3UDW) showing significant overlap of PVR and the Fab.

[0341] The crystal structures of human TIGIT ECD with anti-TIGIT Fab complexes were used to identify the epitope of the anti-TIGIT Fabs on TIGIT. Contact residues are defined as residues of TIGIT with a non-hydrogen atom within 3.8 angstroms of a non-hydrogen atom of the Fab. Distances were measured from the final crystallographic coordinates using the BioPython package. Contacts present in all complexes of the asymmetric unit of each crystal structure are reported in Table 9. The interaction surface on TIGIT by the Fabs was formed by several continuous and discontinuous (i.e. noncontiguous) sequences: namely residues Met23, Thr51, Ala52, Gln53, Thr55, Gln56, Asn70, Ala71, Asp72, His111, Thr112, Tyr113, Pro114, Asp115, Gly116, or Thr117 as detailed in Table 9. These residues form the exemplary three-dimensional conformational epitope that is recognized by the anti-TIGIT Fabs described in this invention.

TABLE-US-00026 TABLE 9 Contacts present in all complexes of the asymmetric unit of each crystal structure. Interactions between human TIGIT and antigen binding region of antibodies described in the invention. The antibody residues are numbered based upon their linear amino acid sequence. Corresponding chains are labeled (“H” for heavy chain, “L” for light chain). TIGIT residues shown here have at least one non-hydrogen atom within 3.8 Å to an non-hydrogen atom in the antibody. TIGIT 3963H03 3963H03-12 3966C11 3964A06 7729G05 7728B03 Met23 Arg93.L Thr51 Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Asn52.H Asn52.H Asn52.H Asn52.H Asn52.H Asn52.H Asn57.H Asn57.H Asn57.H Asn57.H Thr59.H Ala52 Asn52.H Asn52.H Asn52.H Asn52.H Asn52.H Gln53 Thr30.H Thr30.H Thr30.H Thr30.H Thr30.H Thr30.H Ser31.H Pro33.H Pro33.H Pro33.H Pro33.H Pro33.H Pro33.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Asn52.H Asn52.H Asn52.H Asn52.H Asn52.H Asn52.H Thr53.H Thr53.H Thr53.H Thr53.H Thr53.H Thr53.H Asn54.H Asn54.H Asn54.H Asn54.H Asn54.H Asn54.H Thr55 Tyr102.H Tyr102.H Tyr102.H Tyr102.H Phe102.H Tyr102.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Gln56 Tyr102.H Tyr102.H Tyr102.H Tyr102.H Phe102.H Tyr102.H Ser103.H Thr103.H Asn70 Tyr102.H Tyr102.H Tyr102.H Phe102.H Tyr102.H Ala71 Ser31.H Ser31.H Tyr32.H Tyr32.H Phe102.H Asp72 Thr28.H Ser31.H Tyr32.H Tyr102.H Tyr102.H Tyr102.H Tyr102.H His111 Tyr102.H Tyr102.H Tyr102.H Tyr102.H Phe102.H Tyr102.H Ser103.H Ser103.H Ser103.H Ser103.H Thr103.H Ser103.H Val104.H Val104.H Val104.H Val104.H Val104.H Val104.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Thr112 Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr113 Pro33.H Pro33.H Pro33.H Pro33.H Pro33.H Pro33.H Asn35.H Asn35.H Asn35.H Asn35.H Asn35.H Asn35.H Trp47.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Val91.L Thr91.L Pro95.L Pro95.L Pro95.L Pro95.L Pro95.L Val99.H Val99.H Val99.H Val99.H Val99.H Thr99.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Tyr107.H Phe109.H Phe109.H Phe109.H Phe109.H Phe109.H Phe109.H Pro114 Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Trp50.H Val91.L Met92.L Ile92.L Ser93.L Ser93.L Arg93.L Phe93.L Asp115 Arg93.L Phe93.L Gly116 Ile92.L Val104.H Val104.H Val104.H Val104.H Val104.H Val104.H Thr117 Val104.H Val104.H Val104.H Val104.H

[0342] b) Mutagenesis

[0343] The contribution to anti-TIGIT antibody binding energy for contact residues on the TIGIT ECD was assessed by mutation of selected residues to alanine. Positions where the parental residue was alanine or proline were replaced with glycine. The loss of binding energy upon mutation indicates the importance of the parental residue to binding. In total, 11 human TIGIT variants with a point mutation to alanine or glycine were designed. The mutants were expressed in E. coli and purified with affinity and size exclusion chromatography. Binding kinetics to antibody 39631H03-12 were characterized using surface plasmon resonance (SPR). Binding hotspots, or residues that contribute most to the binding energy (Wells. J. A., PNAS 93, 1-6, 1996), were identified as those that did not meet a threshold binding signal at 100 nM antigen. Furthermore, the affinity of the antibody for wild-type and each mutant was determined and used to calculate the contribution of each epitope residue to the binding energy.

[0344] A diagram of the TIGIT ECD structure with the mutagenized residues shown in sticks, and shaded according to the change in affinity, is shown in FIG. 4. In addition, the results are summarized in Table 10 below, where 11 point mutants of TIGIT were compared to wild-type TIGIT antigen for antibody binding. SPR (Biacore) was used to perform a kinetic study allowing determination of kinetic rate constants (k.sub.a and k.sub.d). Briefly, goat polyclonal anti-human Fc antibody was chemically coupled to a CM5 chip. 3963H03-12 antibody was injected next and captured by the polyclonal antibody. Buffer was used to wash out unbound antibody until the baseline RU stabilized. Antigen (wild-type or mutant human TIGIT ECD) was next injected at a fixed concentration for 3 minutes and the association was recorded. Buffer was injected for a further 3 minutes and dissociation was observed. The antigens were injected at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM. Between each cycle, the chip was regenerated with low pH buffer and fresh 3963H03-12 was captured prior to injecting the next concentration of antigen. The rate constants were determined by iterative fitting of the data to a 1:1 binding model by an algorithm that minimizes Chi-squared. The equilibrium dissociation constant (K.sub.D) was calculated as the ratio of the kinetic constants and the change in the Gibbs free energy of binding of mutant relative to wild-type TIGIT (ΔΔG.sub.mut) was derived from the ratio of the wild-type and mutant K.sub.D's. The free energy changes are highlighted according to destabilization of antibody-antigen binding; “**”: >3 kcal/mol destabilization (binding hotspots); “*”: >0.7 kcal/mol. According to this analysis, amino acids marked with “**” or “*” are part of the functional epitope. NB denotes no binding. The temperature midpoint of fluorescently monitored thermal denaturation is given for the wild type and mutant proteins. The wild type TIGIT and all its mutants show monodisperse on size exclusion chromatography (SEC). For K.sub.D, the mean and standard deviation is given where n>1.

TABLE-US-00027 TABLE 10 Epitope mapping through analysis of 3963H03-12 binding affinity to TIGIT mutants ΔΔG.sub.mut Mutation (kcal/mol) K.sub.D (nM) T.sub.1/2 (° C.) huTIGIT  0.00 2.08 +/− 0.11 59.0 T51A −0.10 1.77 +/− 0.55 54.0 Q53A >3** NB 54.7 T55A >3** NB 52.3 Q56A  1.65* 33.90 +/− 1.23  50.7 N70A  1.61* 31.47 +/− 8.73  53.4 A71G  0.72* 7.02 +/− 0.62 54.4 H111A  1.60* 30.90 +/− 0.30  48.2 Y113A >3** NB 53.0 P114G >3** NB 51.8 D115A  1.14* 14.38 +/− 1.06  51.5 T117A −0.17 1.58 +/− 0.33 52.7

[0345] It was important to confirm that the lack of binding to 3963H03-12 of the Q53A, T55A, Y113A and P114G point mutants was indeed due to loss of hotspot residues and not to global unfolding of the antigen. The structural integrity of the mutated proteins was confirmed using a fluorescence monitored thermal unfolding assay in which the protein is incubated with a dye that is quenched in aqueous solution but fluoresces when bound by exposed hydrophobic residues. As the temperature increases, thermal denaturation of the protein exposes the hydrophobic core residues, and this can be monitored by an increase in fluorescence of the dye. The data were fit to equation 2 (adapted from Bullock, A. N. et al. Thermodynamic stability of wild-type and mutant p53 core domain. PNAS 94, 14338-14342 (1997)) to determine the temperature at the inflection point of the curve (T.sub.1/2).

[00006]$\begin{array}{cc}F=\frac{\left\{\mathrm{Fi}+\beta i*T+\left(\left(\mathrm{Fmax}+\beta \max *T\right)*e^{\left[m*\left(T-T1/2\right)\right]}\right)\right\}}{1+e^{\left[m*\left(T-T1/2\right)\right]}}& \mathrm{Equation}2\end{array}$

[0346] Mutants of Q53A, T55A, Y113A and P114G displayed minimal destabilization of the antigen indicated by a small decrease in the T.sub.1/2 of fluorescence monitored unfolding (Table 10). This confirms Q53, T55, Y113 and P114 are true binding hotspots for 3963H03-12. The structural integrity of most of the other mutant proteins was also confirmed by this method (Table 10). The observation that most mutant proteins behaved similarly to wild type on analytical size exclusion chromatography provides further support for native structure of mutant antigen proteins.

[0347] 3.5 EC50 Measured by Direct FACS Binding Assay

[0348] The dose dependent binding ability of 3963H03 to the target on the cell surface was confirmed by flow cytometry. It efficiently binds to human TIGIT ECD expressed on the CHO-S cell surface with an EC50 of 4.7 nM and to cynomolgus monkey TIGIT ECD expressed on the CHO-S cell surface with an EC50 of 3.6 nM (Table 11 and FIG. 8). The assays qualitatively described the dose dependent binding characteristics of the anti-TIGIT antibody.

TABLE-US-00028 TABLE 11 EC50 binding of anti-TIGIT antibody to cells expressing human TIGIT ECD or cyno TIGIT ECD measured by flow cytometry Cell binding EC50 (nM) Antibody CHO-S-hTIGIT CHO-S-cynoTIGIT 3963H03 4.7 3.6 Isotype control NA NA

[0349] 3.6 TIGIT Jurkat Reporter Assay

[0350] 3963H03 along with its sequence optimized variant, 3963H03-12, were tested in a cell based TIGIT/CD155 Blockade Bioassay (Promega Cat. No. CS198801) using the protocol supplied by the manufacturer. The assay is comprised of human Jurkat cells expressing recombinant human TIGIT with a luciferase reporter gene driven by the IL2 promoter, co-cultured with CHO-K1 cells expressing human CD155 and a T-cell receptor activator. The B-cell cloning hit 3963H03 and its sequence optimized variant 3963H03-12, formatted with IgG1 and kappa constant regions, had similar EC50 s ranging from 6.3 to 12.5 ug/ml (FIG. 8 and Table 12).

TABLE-US-00029 TABLE 12 Cell-based TIGIT/CD155 Blockade assay with sequence optimized and affinity matured anti-TIGIT antibody variants EC50 (ug/ml) Max RLU (potency) 3963H03 6.296 2560 3963H03-12 12.5 2384 Isotype control No activity 575

[0351] 3.7 Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)

[0352] The ADCC activities of anti-TIGIT 3963H03 and its sequence optimized variant 3963H03-12 were tested using stably transfected CHO-S-hTIGIT target cells and donor effector cells with heterozygous FcγRIIIa 158V/158F allotype using standard Chromium release assay. Briefly, CHO-S-hTIGIT cells were first labeled with .sup.51Cr for 45 min, then incubated for 15 min at 37° C. with 4-fold serial dilutions of anti-TIGIT antibodies at the starting concentration of 33 nM. Effector cells were added at the ratio of 1:100 and incubated for 4 hours at 37° C. Cells were transferred to Lumaplate 96 well DryPlates, dried overnight and radioactivity was measured using a gamma counter. The percent lysis was calculated as the ratio of ((Count-Spont)/(100% Lysis-Spont))×100 where Spont is the radioactivity counted with the CHO-S-hTIGIT cells alone (in the absence of antibody and effector cells) and 100% lysis was calculated by lysing the CHO-S-hTIGIT cells with detergent. The example assay shown was performed with effector cells from three donors with the allotype V/F. Both antibodies tested in this example assay induced ADCC of the CHO-S-hTIGIT target cells, with EC50 ranging from 0.026 to 0.1 nM (Table 13) and a similar percent maximal cell lysis of approximately 20-30% (FIG. 9).

TABLE-US-00030 TABLE 13 ADCC activity of anti-TIGIT antibodies, EC50 (nM) EC50, nM Antibody Donor 1102-7215 Donor 1005-4464 Donor 1106-0557 3963H03 0.069 0.028 0.080 3963H03-12 0.100 0.026 0.059

[0353] 3.8. Complement Dependent Cytotoxicity (CDC) Activity

[0354] For CDC assay, CHO-S-human TIGIT ECD cells were first labeled with .sup.51Cr for 45 min, then incubated for 15 min at 37° C. with 4-fold serial dilutions of anti-TIGIT antibodies at the starting concentration of 20,000 ng/ml. Previously CDC-qualified normal human serum complement was added at 1:10 dilution and incubated for 2 hours at 37° C. Cells were transferred to Lumaplate 96-well counting plates, dried overnight and radioactivity was measured using MicroBeta2 counter (Perkin Elmer). The percent lysis was calculated as the ratio of ((Count-Spont)/(100% Lysis-Spont))×100 where Spont is the radioactivity counted with the CHO-S-huTIGIT cells alone (in the absence of antibody and complement) and 100% lysis was calculated by lysing the labelled CHO-S-human TIGIT ECD cells with detergent. FIG. 10 shows an assay performed with CHO-S-human TIGIT ECD target cells and 3963H03-12, demonstrating that this antibody is capable of mediating CDC activity.

[0355] 3.9 T Cell Activation Assay

[0356] When treated with anti-CD3 and anti-CD28 antibodies, T cells in human PBMCs were activated. Co-treatment of antagonistic anti-TIGIT antibodies could block TIGIT inhibitory signaling and as a result potentially further enhance T cell activation, measured by IFNγ production. Human PBMC were stimulated with 0.5 ng/ml anti-CD3 OKT3 and 20 ng/ml anti-CD28 for 48 hours in the presence of anti-TIGIT antibodies or human IgG1 isotype control (20 μg/ml). IFN-γ in culture supernatant was measured by ELISA. PBMCs from 4 different donors (1003, 1579, 1059, 1558) were tested. Anti-TIGIT antibodies (A06, C11, D08, H03) enhanced IFNγ production as shown in FIG. 11. Anti-TIGIT H03 was shown to more consistently enhance IFNγ production than A06, C11 and D08.

[0357] 3.10 CD8+ T Cell Antagonistic Assay

[0358] The binding of CD155 to TIGIT triggers inhibitory signaling into CD8+ T cells and co-treatment with an antagonistic anti-TIGIT antibody could block TIGIT/CD155 interaction and as a result enhance T cell activation, measured by IFNγ production. 96-well cell culture plates were co-coated with anti-CD3 (OKT3, 2 μg/ml) and recombinant CD155-Fc (2 μg/ml). Freshly isolated human CD8+ T cells were added and cultured for 4 days in the presence of 10 μg/ml soluble anti-TIGIT antibody or human IgG1 isotype control. IFNγ production in the supernatant was measured by ELISA. Anti-TIGIT 3963H03 reversed CD155-mediated T cell suppression and as a result increased IFNγ production as shown in FIG. 12.

[0359] 3.11 Primary Cell Binding Assays

[0360] The ability of 3963H03-12 to bind to TIGIT expressed on the surface of human and cynomolgus monkey primary T cells was determined by flow cytometry. Human or cyno PBMCs were incubated with serial dilutions (1:3) of 3963H03-12 and the binding of anti-TIGIT antibody to CD3+ T cells was detected by anti-hIgG APC (1:1000). Flow cytometry analysis was carried out using BD-Calibur. CD3+ T cells were gated, and the mean florescence intensities (MFI) and percent APC staining of the parent population were determined. 3963H03-12 bound to primary human and cynomolgus monkey T cells in a dose-dependent manner with an EC50 of 85.2±28.8 ng/mL (0.6±0.2 nM) and 132.2±29.2 ng/mL (0.8±0.2 nM), respectively, as shown in FIG. 13.

[0361] 3.12 Target Occupancy (TO) Assays

[0362] The target occupancy of anti-TIGIT 3963H03-12 on CD3+ T cells was measured via flow cytometry using human whole blood and cynomolgus monkey spleen cells. Serial dilutions of anti-TIGIT were incubated with human or cynomolgus monkey samples for 1 hour, and the unoccupied TIGIT on CD3+ primary T cells was measured by flow cytometry with biotinylated anti-TIGIT (3963H03-12). Flow cytometry analysis was performed using a BD-Calibur gated on CD3+ cells and analyzed as follows. Percentage of target occupancy (TO %) was calculated using the formula, TO (%)=(1−(Dt−Ct)/(D0−C0))*100, where Dt=Percentage of TIGIT staining, Ct=Percentage of isotype control staining at a certain concentration of anti-TIGIT, D0=Percentage of TIGIT staining, and C0=Percentage of isotype control staining in the absence of anti-TIGIT. 3963H03-12 was shown to efficiently saturate target on both human (FIG. 14A) and cynomolgus monkey (FIG. 14B) T cells. The average EC50 from 9 human donors was 239.8±168.04 ng/mL (1.6±1.1 nM) and EC50 from 6 cyno donors was 92.7±21.6 ng/mL (0.6±0.1 nM).

[0363] 3.13 Cell Based TIGIT/CD155 and TIGIT/CD112 Blocking Assays

[0364] To evaluate the ability of anti-TIGIT 3963H03-12 to block the interaction of TIGIT with its ligands CD155 and CD112, a blocking assay was performed using CHO-S engineered cells stably expressing human TIGIT (CHO-S-hTIGIT cell line #4-60). CHO-S-human TIGIT cells were incubated with serial dilutions (1:3) of 3963H03-12 before biotinylated human CD112-Fc or human CD155-Fc (2 μg/mL final concentration) was added. The interaction of CD155/TIGIT or CD112/TIGIT was detected by streptavidin-APC (1:1000). 3963H03-12 dose-dependently blocked the interaction of TIGIT with CD155 (FIG. 15A) and CD112 (FIG. 15B), with an IC50 of 165.0±39.7 ng/mL (1.1±0.3 nM) and 410.6±315.5 ng/mL (2.8±2.1 nM), respectively.

[0365] 3.14 Cell Based TIGIT/CD226 Blocking Assay

[0366] TIGIT receptors expressed on cell surface interact with CD226 and disrupt CD226 homo-dimers that are important for CD226 stimulatory function. Blocking with 3963H03-12 reduces CD226 and TIGIT interactions and potentially leads to increased co-stimulatory signaling by CD226. A FRET assay was designed to measure the interaction between TIGIT and CD226 and the effect of 3963H03-12 on this interaction (FIG. 16A). CHO-CD226 cells were generated by transfection of CHO cells with CD226/SNAP tag plasmid using Lipofectamine 3000 (Invitrogen, L3000-015) and subsequent selection of stably expressing CD226 cells with 250 μg/ml of Hygromycin B (Invitrogen, 10687010). CHO-CD226 cells seeded in white 96-well plates (Greiner Bio-One, 655083) were transfected with 0.1 μg/well TIGIT/HA tag plasmid using Lipofectamine 3000 and incubated with 3963H03-12 or isotype control Abs at concentrations of 10, 1 and 0.1 μg/ml for 24 hours. After that cells were washed with Tag-lite labeling medium (Cisbio, 7SEC30K), then stained with 1 μM SNAP-Red acceptor (Cisbio, SSNPREDE) at 37° C.-5% C02 incubator for 1 hour. Next, cells were washed three times and incubated with 1.6 nM anti-HA-TB cryptate donor (Cisbio, 610HATTA) at room temperature for 2 hours. The FRET signal was recorded at 665 nm and 615 nm for 150 μs after excitation at 320 nm and 60 μs delay using an Envision Plate Reader (Perkin Elmer, Xcite Multilabel Reader). FRET intensity was calculated as (Emission 665 nm/Emission 615 nm from TIGIT-transfected cells)−(Emission 665 nm/Emission 615 nm from mock-transfected cells). Percent of FRET normalized to isotype control was calculated as (FRET intensity for TIGIT-transfected Cho.CD226 cells blocked with anti-TIGIT Abs)/(FRET intensity for TIGIT-transfected Cho.CD226 cells blocked with Isotype control Abs)*100. The quantification of inhibition of FRET signal which measures TIGIT and CD226 interaction by 3963H03-12 demonstrated that 3963H03-12 blocked TIGIT/CD226 interaction (FIG. 16B).

[0367] 3.15 Allogenic Two-Way MLR (Mixed Lymphocyte Reaction) Assay

[0368] In a two-way MLR assay with PBMCs from two unrelated donors, responder (effector T) undergo activation and proliferation in response to the major histocompatibility antigen (MHC Class I and II) differences between the responder cells and stimulator (target) cells in both donors. Co-treatment with a functional antagonist checkpoint inhibitor (CPI) antibody further potentiates T cell activation, measured by IFNγ production. PBMCs from two different human donors were co-cultured at 1:1 ratio and treated with serial dilutions of 3963H03-12 or isotype control for 2 days. Immune cell activation was evaluated by measuring IFN-γ in the supernatant. Results from 7 assays with 7 different donor pairs were plotted together as fold charges over isotype control at 1 ng/mL which was set to 1. 3963H03-12 dose-dependently enhanced IFN-γ production, with an EC50 of 158.9±185.0 ng/mL (1.1±1.2 nM) (FIG. 17).

[0369] 3.16 Allogenic One-Way MLR (Mixed Lymphocyte Reaction) Assay

[0370] In a one-way MLR assay with cells from two unrelated donors, responder (effector T) cells undergo activation and proliferation in response to the major histocompatibility antigen (MHC Class I and II) differences between the responder cells and stimulator (target) cells. Co-treatment with a functional antagonist checkpoint inhibitor (CPI) antibody further potentiates T cell activation, measured by IFNγ production. Irradiated MDA-MB-231 tumor cells were co-cultured with PBMCs from a human donor for 7 days using IL-2 (R&D Systems, IL-010) to induce allogenic reactive T cell expansion. These cells (effector cells) were then harvested and co-cultured at a 2:1 E:T ratio with freshly irradiated MDA-MB-231 cells (target cells) and co-treated with anti-TIGIT and/or anti-PD-L1 (avelumab) antibodies. T cell activation was evaluated by measuring IFN-γ in the supernatant. Co-cultured cells were treated with serial dilutions of 3963H03-12 or isotype control. Results from 2 assays were plotted together as fold charges over isotype control at 1 ng/mL which was set to 1. 3963H03-12 dose-dependently enhanced Allo-antigen specific T cell activation, with an EC50 of 136.9±114.6 ng/mL (0.9±0.8 nM) (FIG. 18). For combination studies, co-cultured cells were treated with serial dilutions of avelumab combined with 10 μg/mL of isotype control or H03-12. Combination of H03-12 with avelumab further enhanced IFNγ production (FIG. 19B).

[0371] 3.17 NK Cell Killing Assay

[0372] The ability of 3963H03-12, to enhance NK-mediated tumor cell killing by blocking TIGIT/CD155 interaction was demonstrated using a P815 cell line modified to express human CD155. NK cells were co-cultured with P815.hCD155 cells in the presence of 10 μg/mL of 3963H03-12 or IgG1 isotype control antibody. Tumor cell death was monitored by measuring green signal (Caspase-3/7) using IncuCyte system for 4.5 hours. Cell killing was monitored in four fields, p values for two-way ANOVA comparison between IgG1 control treated and anti-TIGIT antibody treated: p<0.00005 (****). 3963H03-12 increased tumor cell killing up to 2-fold compared with isotype control (FIG. 19A). The ability of anti-TIGIT, H03-12, to enhance NK-mediated tumor cell killing was further demonstrated using breast cancer MDA-MB-231 cell line expressing GFP-reporter. NK cells were co-cultured with MDA-MB-231 GFP/Luc cells in the presence of 10 μg/mL of anti-TIGIT H03-12 or IgG1 control antibody. Tumor cell killing was monitored by measuring GFP signal using IncuCyte system. GFP signal at each time point was normalized to 0-time point. Cell killing was monitored in four fields, p values for two-way ANOVA comparison between IgG1 control and anti-TIGIT H03-12 antibody: p<0.00005 (****), p<0.005 (**). Significant increase of NK-mediated tumor cells death by anti-TIGIT H03-12 was detected from 2.5 to 12.5 hours (FIG. 19B).

[0373] 4. In Vivo Activity

[0374] 4.1 Blocking Assay of 3963H03-12 and 3963H03-12-muIgG2c on the Binding of Mouse CD155 (muCD155) and Mouse CD112 (muCD112) to CHO-s-huTIGIT Cells

[0375] To evaluate the efficacy of 3963H03-12 in vivo, a version of 3963H03-12 with a murine immunoglobulin (3963H03-12-muIgG2c) was developed.

[0376] The ability of 3963H03-12-muIgG2c and 3963H03-12 to block the interaction of TIGIT with its ligands muCD155 and muCD112 was evaluated with a flow cytometry-based binding assay using CHO-S engineered cells stably expressing human TIGIT (CHO-S-hTIGIT cell line). Pre-incubation with 3963H03-12-muIgG2c and 3963H03-12, but not the isotype control, led to reduced binding of muCD155-Fc to CHO-hTIGIT cells. 3963H03-12-muIgG2c and 3963H03-12 both dose-dependently blocked the interaction of TIGIT with muCD155 with an IC50 of 290.7 ng/mL (1.994 nM) and 499.3 ng/mL (3.450 nM), respectively (FIG. 22). 3963H03-12-muIgG2c and 3963H03-12 also both dose-dependently blocked the interaction of TIGIT with muCD112 with an IC50 of 1189 ng/mL (8.155 nM) and 1678 ng/mL (11.593 nM), respectively (FIG. 20).

[0377] 4.2 Pharmacokinetic Evaluation of 3963H03-12-muIgG2c in B-huTIGIT Knock-In Mice Bearing MC38 Tumor

[0378] The PK of 3963H03-12-muIgG2c was measured in MC38 tumor-bearing B-huTIGIT knock-in mice developed by Biocytogen. In this model, mouse TIGIT has been replaced with human TIGIT via extreme genome editing (EGE). After a single ip administration, 3963H03-12-muIgG2c peak plasma concentration was measured at the 24 hours in all dose groups. After Cmax, different PK profiles were observed in the three dose groups. Slow monophasic elimination was observed in the high dose group, while fast monophasic elimination was observed in the low dose group. Biphasic elimination was observed in the intermediate dose group, with a slow concentration decrease up to 168 hours followed by a faster decrease up to the last quantifiable time point (336 hours). In line with the PK profile, the calculated terminal half-life was comparable for the low and intermediate dose groups and longer for the high dose group. The AUC 0-∞ increased higher than dose proportionally from 0.25 to 25 mg/kg with the AUC 0-∞ ratio values being 1.0:26.8:334 vs the actual dose ratio of 1:10:100. The increase was roughly dose proportional from 2.5 to 25 mg/kg, where the AUC 0-∞ ratio was 1.0:15.0 vs the actual dose ratio of 1:10 (FIG. 21)

[0379] 4.3 Anti-Tumor Efficacy of 3963H03-12-muIgG2c in the MC38, GL261, Hepa 1-6, and 3LL Tumor Models in B-huTIGIT Knock-In Mice

[0380] The anti-tumor efficacy of 3963H03-12-muIgG2c was evaluated in B-hTIGIT knock-in mice (C57BL/6 background). Female B-huTIGIT knock-in mice, 10 weeks old, were supplied by Biocytogen. The colon cancer cell line MC38, glioblastoma multiforme (GBM) cell line GL261, hepatocellular carcinoma (HCC) cell line Hepa 1-6 and lung cancer cell line 3LL were inoculated subcutaneously at the right upper flank. The inoculated cell amount was 1×10e6, 5×10e6, 2×10e6 and 5×10e6 respectively.

[0381] 3963H03-12-muIgG2c and anti-HEL-muIgG2c were administered at 25 mg/kg on days 0, 7, 14 via i.p. injection into tumor-bearing B-huTIGIT knock-in mice when tumor size reached 50-100 mm3. Tumor sizes were measured twice per week in three dimensions using a caliper, and the volume was expressed in mm3 using the formula: width×length×height×0.5236. The % TGI is defined as the formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the first day of treatment, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the first day of treatment. Two-way ANOVA was performed for significance analysis.

[0382] The results are shown in FIG. 22. Compared with the anti-HEL isotype control (binds to hen egg lysozyme), the TGI of 3963H03-12-muIgG2c was 63.5% in MC38 colon cancer (p<0.0001), 85.3% in GL261 GBM (p<0.0001), 85.7% in Hepa 1-6 liver cancer (p<0.0001) and 41.5% in 3LL lung cancer (p=0.0034). Taken together, these data suggest that 3963H03-12-muIgG2c showed anti-tumor efficacy in multiple tumor models, indicating the anti-tumor effect in extensive indications.

[0383] 4.4 Dose-Dependent Anti-Tumor Efficacy of 3963H03-12-muIgG2c in the MC38 Tumor Model in B-huTIGIT Knock-In Mice

[0384] The B-huTIGIT knock-in mice were used to evaluate the anti-tumor efficacy of 3963H03-12-muIgG2c. To optimize the best therapeutic dosage, 3963H03-12-muIgG2c was administered at 25, 5, 1, or 0.2 mg/kg on days 0, 7, 14 via i.p. injection into MC38 tumor-bearing B-huTIGIT knock-in mice. Methods for tumor inoculation and tumor size measurement were as same as described in section 4.3.

[0385] Compared with anti-HEL isotype control, 3963H03-12-muIgG2c at 25 mg/kg, 5 mg/kg, and 1 mg/kg induced significant tumor growth inhibition (TGI=63.5%, 41%, and 42.3%, respectively, and P<0.0001 for each of the three groups, day 30), and prolonged median survival (42, 37, and 38.5 days, respectively) relative to isotype control (31.5 days). Conversely, 0.2 mg/kg of 3963H03-12-muIgG2c did not show significant tumor growth inhibition (TGI=17.6%, P>0.05, day 30) relative to isotype control (FIG. 23).

[0386] Although there was not a significant difference in tumor volume between mice treated with 3963H03-12-muIgG2c at 5 mg/kg and those treated with 1 mg/kg (P>0.05, day 30), there was a significant decrease in tumor volume with both 5 mg/kg and 1 mg/kg doses relative to the 0.2 mg/kg dose of 3963H03-12-muIgG2c (P=0.0075 and P=0.0039, respectively, day 30). There was also a significant decrease in tumor volume with 25 mg/kg of 3963H03-12-muIgG2c relative to either 5 mg/kg or 1 mg/kg dose (P=0.0118 and P=0.0211, respectively, day 30). Taken together, these data suggest that 3963H03-12-muIgG2c had dose-dependent anti-tumor efficacy in this tumor model.

[0387] 4.5 Contribution of Antibody Fc-Mediated Effector Function to the Anti-Tumor Effector of 3963H03-12

[0388] The anti-tumor activity of 3963H03-12-muIgG2c and 3963H03-12-muIgG1(D265A) were compared in MC38 and Hepa 1-6 tumor-bearing mice in B-huTIGIT knock-in mice. Female B-huTIGIT knock-in mice, 10 weeks old, were supplied by Biocytogen. Each mouse was inoculated subcutaneously (sc) in the right flank with MC38 tumor cells (1×10e6) in 0.1 mL of PBS or Hepa 1-6 tumor cells (5×10e6) in 0.1 mL of PBS for tumor development. Mice were assigned to treatment groups using stratified randomization based on tumor volume when the average tumor volume reached approximately 50 mm3. There were 10 mice in each group. Mice were treated with anti-HELmuIgG2c (25 mg/kg) or 3963H03-12-muIgG2c (25 mg/kg) or 3963H03-12-muIgG1(D265A) (25 mg/kg) were given at days 0, 7, 14 via ip injection. Tumor size measurement and data analysis protocol were same as described in section 4.3.

[0389] To assess the role of antibody Fc-mediated effector function in the anti-tumor efficacy of 3963H03-12, MC38 and Hepa 1-6 tumor-bearing mice were treated with either effector competent 3963H03-12-muIgG2c or with effector null 3963H03-12-muIgG1(D265A). The immune effector functions of 3963H03-12-muIgG1(D265A) were abolished to reduce FcγR activation and Fc-mediated toxicity. 3963H03-12-muIgG1(D265A) shared many functional characteristics of 3963H03-12-muIgG2c, but it is an ‘effector-silent’ version and cannot induce cytotoxicity effector function. Effector competent 3963H03-12-muIgG2c treatment resulted in significant tumor inhibition in both MC38 and Hepa 1-6 model (TGI=46.82%, P<0.0001, day 24; and TGI=106.45%, P=0.0087, day 30, respectively) compared with isotype control, while the anti-tumor efficacy of effector null 3963H03-12-muIgG1(D265A) (TGI=−4.88% and TGI=−33.07% respectively) was significantly less than effector competent (P<0.0001, day 24 and p=0.0002, day 30, respectively) and not significantly enhanced relative to isotype control (FIG. 24). These results demonstrated that the Fc-mediated immune effector function plays an important role in the anti-tumor efficacy of 3963H03-12-muIgG2c.

[0390] 4.6. Combination Treatment with 3963H03-12-muIgG2c and Avelumab in the MC38 Tumor Model in B-huTIGIT Knock-In Mice

[0391] The anti-tumor efficacy of 3963H03-12-muIgG2c in combination with avelumab was also evaluated in MC38 tumor-bearing B-huTIGIT knock-in mice. Compared with anti-HEL+anti-PD-L1 isotype controls, 3963H03-12-muIgG2c and avelumab monotherapies all induced significant tumor growth inhibition (TGI=75.3%, and 56.7% respectively, P<0.0001 for each group relative to isotype control, day 27), and prolonged median survival (41 and 40.5 respectively) relative to isotype control (30 days) (FIG. 25). Tumor growth inhibition was further enhanced with the combination of 3963H03-12-muIgG2c with avelumab (TGI=90.5%) relative to 3963H03-12-muIgG2c (P=0.0028, day 27) and avelumab (P<0.0001, day 27) monotherapies. Combinations of 3963H03-12-muIgG2c with avelumab also prolonged median survival (55 days) (FIG. 25).

[0392] 4.6 Combination Treatment with 3963H03-12-muIgG2c and M7824 in the MC38 Tumor Model in B-huTIGIT Knock-In Mice

[0393] The anti-tumor efficacy of 3963H03-12-muIgG2c in combination with bintrafusp alfa (M7824) was also evaluated in MC38 tumor-bearing B-huTIGIT knock-in mice. Compared with anti-HEL+inactive anti-PD-L1 isotype controls, 3963H03-12-muIgG2c and M7824 monotherapies all induced significant tumor growth inhibition (TGI=75.3% and 63.3%, respectively, P<0.0001 for each group relative to isotype control, day 27), and prolonged median survival (41 and 42.5 days, respectively) relative to isotype control (30 days) (FIG. 26). Tumor growth inhibition was further enhanced with the combination of 3963H03-12-muIgG2c with M7824 (TGI=96.6%) relative to 3963H03-12-muIgG2c (P=0.0011, day 27) and M7824 (P<0.0001, day 27) monotherapies. Combinations of 3963H03-12-IgG2c with M7824 also prolonged median survival (55 days) (FIG. 26).

[0394] 4.7 Re-Challenge Study

[0395] Re-challenge studies were then performed on MC38 tumor-bearing B-huTIGIT knock-in mice that showed complete tumor regression for at least 3 months after 3963H03-12 muIgG2c and avelumab or bintrafusp alfa combination therapy. Mice that were ‘cured’ after 3963H03-12-muIgG2c and avelumab or bintrafusp alfa combination therapy from multiple studies (n=2, n=4 respectively) were re-challenged with MC38 tumor cells in the opposite side of the initial injection. None of these mice developed tumors (0/2 or 0/4 mice, respectively, 0%) for at least 36 days, whereas naïve B-huTIGIT knock-in mice (n=10) injected with MC38 cells all developed tumors (10/10, 100%) (see FIG. 27). These results suggested that 3963H03-12-muIgG2c and avelumab or bintrafusp alfa combination treatment conferred a tumor antigen specific long-term protective immunity in B-huTIGIT knock-in mice.

[0396] 4.5 Combination Treatment with 3963H03-12-muIgG2c and Avelumab in the MC38 Tumor Model in B-huTIGIT Knock-In Mice

[0397] The anti-tumor efficacy of 3963H03-12-muIgG2c in combination with avelumab was also evaluated in MC38 tumor-bearing B-huTIGIT knock-in mice. Compared with anti-HEL+inactive anti-PD-L1 isotype controls, 3963H03-12-muIgG2c and avelumab monotherapies all induced significant tumor growth inhibition (TGI=75.3%, and 56.7% respectively, P<0.0001 for each group relative to isotype control, day 27), and prolonged median survival (41 and 40.5 respectively) relative to isotype control (30 days) (FIG. 26). Tumor growth inhibition was further enhanced with the combination of 3963H03-12-muIgG2c with avelumab (TGI=90.5%) relative to 3963H03-12-muIgG2c (P=0.0028, day 27) and avelumab (P<0.0001, day 27) monotherapies. Combinations of 3963H03-12-muIgG2c with avelumab also prolonged median survival (55 days) (FIG. 25).

[0398] 4.6 Combination Treatment with 3963H03-12-muIgG2c and M7824 in the MC38 Tumor Model in B-huTIGIT Knock-In Mice

[0399] The anti-tumor efficacy of 3963H03-12-muIgG2c in combination with bintrafusp alfa (M7824) was also evaluated in MC38 tumor-bearing B-huTIGIT knock-in mice. Compared with anti-HEL+inactive anti-PD-L1 isotype controls, 3963H03-12-muIgG2c and M7824 monotherapies all induced significant tumor growth inhibition (TGI=75.3% and 63.3%, respectively, P<0.0001 for each group relative to isotype control, day 27), and prolonged median survival (41 and 42.5 days, respectively) relative to isotype control (30 days) (FIG. 26). Tumor growth inhibition was further enhanced with the combination of 3963H03-12-muIgG2c with M7824 (TGI=96.6%) relative to 3963H03-12-muIgG2c (P=0.0011, day 27) and M7824 (P<0.0001, day 27) monotherapies. Combinations of 3963H03-12-IgG2c with M7824 also prolonged median survival (55 days) (FIG. 26).

[0400] 4.7 Re-Challenge Study

[0401] Re-challenge studies were then performed on MC38 tumor-bearing B-huTIGIT knock-in mice that showed complete tumor regression for at least 3 months after 3963H03-12 muIgG2c and avelumab or bintrafusp alfa combination therapy. Mice that were ‘cured’ after 3963H03-12-muIgG2c and avelumab or bintrafusp alfa combination therapy (n=2, 1 respectively) were re-challenged with MC38 tumor cells in the opposite side of the initial injection. None of these mice developed tumors (0/2 or 0/1 mice, respectively, 0%) for at least 36 days, whereas naïve B-huTIGIT knock-in mice (n=10) injected with MC38 cells all developed tumors (10/10, 100%) (see FIG. 27). These results suggested that 3963H03-1-muIgG2c and avelumab or bintrafusp alfa combination treatment conferred a tumor antigen specific protective immunity in B-huTIGIT knock-in mice.

TABLE-US-00031 Sequence Listing SEQ ID NO. Sequence Description  1 MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQC Amino acid HLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPG sequence of human LGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHG TIGIT ARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLR RKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDY FNVLSYRSLGNCSFFTETG  2 QVQLVQSGSELKKPGASVKVSCKAS HC-FR1  3 MNVWRQAPGQGLEWMGW HC-FR2  4 TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYC HC-FR3  5 WGQGTLVTVSS HC-FR4  6 QGISSY HVR-L1  7 AAS HVR-L2  8 QQLSSYPT HVR-L3  9 DIQLTQSPSFLSASVGDRVTITCRAS LC-FR1 10 LAWYQQKPGKAPKLLIY LC-FR2 11 TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LC-FR3 12 FGGGTKVEIK LC-FR4 13 GYTFTSYP HVR-H1 14 INTNTGNP HVR-H2 15 ARVGGYSVDEYAFDV HVR-H3 16 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL HC variable region EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED sequence of an anti- TAVYYCARVGGYSVDEYAFDVWGQGTLVTVSS TIGIT antibody 17 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK LC variable region LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ sequence of an anti- LSSYPTFGGGTKVEIK TIGIT antibody 18 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL HC of antibody H03-12 EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED produced from TAVYYCARVGGYSVDEYAFDVWGQGTLVTVSSASTKGPSVFPLAP CHO-K1SV cells SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 19 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK LC of antibody H03-12 LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ produced from LSSYPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN CHO-K1SV cells NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 ATGGAAACAGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTG Isolated nucleic acid CCCGGCTCCACAGGCCAGGTGCAGCTGGTGCAGTCCGGCTCCGAG encoding the HC of CTGAAGAAACCCGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCC an anti-TIGIT GGCTACACCTTCACCTCCTACCCCATGAACTGGGTGAGGCAGGCT antibody CCTGGCCAGGGACTGGAGTGGATGGGCTGGATCAACACCAACACC GGCAACCCTACCTACGCCCAGGGCTTCACCGGCAGGTTCGTGTTC TCCCTGGACACCAGCGTGTCCACCGCCTACCTGCAGATCTCCTCC CTGAAGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGTGGGA GGCTACTCCGTGGACGAGTACGCCTTCGACGTGTGGGGCCAGGGC ACCCTGGTGACCGTGTCCTCCGCTAGCACCAAGGGCCCATCGGTC TTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCACGGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCCCCGGGT 21 ATGAGGGCCCTGCTGGCTAGACTGCTGCTGTGCGTGCTGGTCGTG Isolated nucleic acid TCCGACAGCAAGGGCGACATCCAGCTGACCCAGTCCCCCTCCTTC encoding the LC of CTGTCCGCTTCCGTGGGCGACAGGGTGACCATCACTTGTCGTGCC an anti-TIGIT TCCCAGGGCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCC antibody GGCAAGGCCCCCAAGCTGCTGATCTACGCCGCTTCCACACTGCAG TCCGGCGTGCCCTCCAGGTTTTCCGGATCCGGCTCCGGCACCGAG TTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCAGCTGTCCTCCTACCCCACCTTCGGCGGCGGC ACAAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTC ATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA AAGAGCTTCAACAGGGGAGAGTGT 22 EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL HC of 3963H03 EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARVGGYSVDEYAFDVWGQGTMVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 23 AIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK LC of 3963H03 LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ LNSYPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 24 EVQLVQSGSELKKPGASVKVSCKASGYTFTAYPMNVWRQAPGQGL A06-VH EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARVGGYSVYDYAFDIWGQGTMVTVSS 25 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK A06-VL LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ LNSYPTFGGGTKVEIK 26 EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL C11-VH EWMGWINTNTGNPTNAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARVGGYGGYDYAFDIWGQGTMVTVSS 27 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK C11-VL LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ LNSYPTFGGGTKLEIK 28 EIQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL H03-VH EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARVGGYSVDEYAFDVWGQGTMVTVSS 29 AIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK H03-VL LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ LNSYPTFGGGTKVEIK 30 EVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL D08-VH EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARTGYSGSYYWFDPWGQGTLVTVSS 31 DIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK D08-VL FLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ LNSYLTFGQGTRLEIK 32 QMQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNWVRQAPGQGL B03-VH EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARTGGYSVDEYSFDIWGQGTTVTVSS 33 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK B03-VL LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQ TIFRPTFGGGTKVEIK 34 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYPMNVWRQAPGQGL G05-VH EWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARVGGFTVPEYAFDIWGQGTTVTVSS 35 DIRLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPK G05-VL LLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCGQ VMRYPAFGGGTKVEIK 36 GYTFTX.sub.1YP HVR-H1 consensus X.sub.1 is S or A 37 ARX.sub.2GX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13 HVR-H3 consensus X.sub.2 is V or T X.sub.3 is G or Y X.sub.4 is Y, S or F X.sub.5 is S, G or T X.sub.6 is V, S or G X.sub.7 is D, Y or P X.sub.8 is E, D or Y X.sub.9 is Y or W X.sub.10 is A, F or S X.sub.11 is F or D X.sub.12 is D or P X.sub.13 is V, I or absent 38 X.sub.14QX.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20 HVR-L3 consensus X.sub.14 is Q, G or H X.sub.15 is L, V o r T X.sub.16 is N, S, I or M X.sub.17 is S, R, or F X.sub.18 is Y or R X.sub.19 is P or L X.sub.20 is T or A 39 QQLNSYPT HVR-L3 embodiment