CHIMERIC ANTIGEN RECEPTOR

Abstract

The present invention is directed to a nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising (a) an anti-CLEC14A binding domain, (b) a transmembrane domain and (c) an intracellular signalling domain; wherein said anti-CLEC14A binding domain is capable of binding to the C-type lectin domain of CLEC14A. The invention further provides a chimeric antigen receptor encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a cell comprising the nucleic acid molecule, vector or CAR and the therapeutic use of the nucleic acid, vector or cell, particularly in treating CLEC14A expressing conditions.

Claims

1. A nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising (i) an anti-CLEC14A binding domain, (ii) a transmembrane domain and (iii) an intracellular signalling domain; wherein said anti-CLEC14A binding domain is capable of binding to the C-type lectin domain of CLEC14A.

2. The nucleic acid molecule of claim 1 wherein said anti-CLEC14A binding domain inhibits the interaction between CLEC14A and MMRN2.

3. The nucleic acid molecule of claim 1 or 2 wherein said anti-CLEC14A binding domain binds to residues 97-108 of CLEC14A, wherein CLEC14A has the amino acid sequence as set out in SEQ ID NO. 1.

4. The nucleic acid molecule of claim 1, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 213 or a variant thereof having one, two or three amino acid substitutions.

5. The nucleic acid molecule of claim 1 or 4, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising a light chain CDR3 having an amino acid sequence of SEQ ID NO. 215 or a variant thereof having one, two or three amino acid substitutions.

6. The nucleic acid molecule of any one of claim 1, 4 or 5, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of: (a) a heavy chain CDR1 having an amino acid sequence of (S/T) SYW (UM) (E/H) (SEQ ID NO. 150), GYTF (S/T) SYW (SEQ ID NO. 151) or a variant thereof having one, two or three amino acid substitutions, (b) a heavy chain CDR2 having an amino acid sequence of WIG (E/A) I (L/Y) PG (S/N) (G/S) (S/D) T (N/S) (SEQ ID NO. 152), I (L/Y) PG (S/N) (G/S) (S/D) T (SEQ ID NO. 153) or a variant thereof having one, two or three amino acid substitutions, and/or (c) a heavy chain CDR3 having an amino acid sequence of (A/T) (R/H) (G/X) (G/X) (D/X) Y (D/Y) (E/G) (E/S) (Y/D) Y (V/A/L) MD (SEQ ID NO. 154), (A/T) (R/H)(G/X) (G/X)(D/X) Y (D/Y) (E/G) (E/S) (Y/D) Y (V/A/L) MDY (SEQ ID NO. 155) or a variant thereof having one, two or three amino acid substitutions, wherein X is no amino acid residue.

7. The nucleic acid molecule of any one of claims 1 or 4 to 6, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a light chain CDR1 having an amino acid sequence of S/X S/X S Y M/L Y/H WY (SEQ ID NO. 156), SSVS Y/S S/X Y/X (SEQ ID NO. 157) or a variant thereof having one, two or three amino acid substitutions, (b) a light chain CDR2 having an amino acid sequence of L L/W IY D/S TSNLA (SEQ ID NO. 158), D/S TS or a variant thereof having one, two or three amino acid substitutions and/or (c) a light chain CDR3 having an amino acid sequence of Q/H Q W/Y S/H S/R Y/S P L/R (SEQ ID NO. 160), Q/H Q W/Y S/H S/R Y/S P L/R T F/X (SEQ ID NO. 161) or a variant thereof having one, two or three amino acid substitutions, wherein X is no amino acid.

8. The nucleic acid molecule of any one of claims 1 to 7 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 207 or a variant thereof having one, two or three amino acid substitutions.

9. The nucleic acid molecule of any one of claims 1 to 8, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 32, SEQ ID NO. 44, or a variant thereof having one, two or three amino acid substitutions, (b) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 33, SEQ ID NO. 45, or a variant thereof having one, two or three amino acid substitutions and/or (c) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 116, SEQ ID NO. 118, SEQ ID NO. 34, SEQ ID NO. 46, SEQ ID NO. 100, SEQ ID NO. 102, or a variant thereof having one, two or three amino acid substitutions.

10. The nucleic acid molecule of any one of claims 1 to 9 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising a light chain CDR3 having an amino acid sequence of SEQ ID NO. 208 or a variant thereof having one, two or three amino acid substitutions.

11. The nucleic acid molecule of any one of claims 1 to 10, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO. 35, SEQ ID NO. 47, or a variant thereof having one, two or three amino acid substitutions, (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO. 36, DTS, or a variant thereof having one, two or three amino acid substitutions, and/or (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO. 37, SEQ ID NO. 49, or a variant thereof having one, two or three amino acid substitutions.

12. The nucleic acid molecule of any one of claims 1 to 11, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 116, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 32, 33 and/or 116, (b) SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 118, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 44, 45 and/or 118, (c) SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 100, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 32, 33 and/or 100, (d) SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 102, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 44, 45 and/or 102, (e) SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 32, 33 and/or 34; or (f) SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 44, 45 and/or 46.

13. The nucleic acid molecule of any one of claims 1 to 12, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 35, 36 and/or 37, or (b) SEQ ID NO. 47, DTS and SEQ ID NO 0.49, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID NO. 47, 49 and/or DTS.

14. The nucleic acid molecule of any one of claims 1 to 13 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 116, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 32, 33, 116, 35, 36 and/or 37; (b) SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 118, SEQ ID NO. 47, DTS and SEQ ID NO. 49, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 44, 45, 118, 47, 49 and/or DTS (c) SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 100, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 32, 33, 100, 35, 36 and/or 37; (d) SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 102, SEQ ID NO. 47, DTS, and SEQ ID NO. 49, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 44, 45, 102, 47, 49 and/or DTS; (e) SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 37, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 32, 33, 34, 35, 36 and/or 37; or (f) SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, DTS, and SEQ ID NO. 49, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 44, 45, 46, 47, 49 and/or DTS.

15. The nucleic acid molecule of any one of claims 1 or 4 to 7, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 216 or a variant thereof having one, two or three amino acid substitutions, (b) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 217 or a variant thereof having one, two or three amino acid substitutions, and/or (c) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 218 or a variant thereof having one, two or three amino acid substitutions.

16. The nucleic acid molecule of any one of claim 1, 4 to 7 or 15 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 64, SEQ ID NO.76, or a variant thereof having one, two or three amino acid substitutions, (b) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 65, SEQ ID NO. 77, or a variant thereof having one, two or three amino acid substitutions, and/or (c) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 78, or a variant thereof having one, two or three amino acid substitutions.

17. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 or 16 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO. 219 or a variant thereof having one, two or three amino acid substitutions, (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO. 220 or a variant thereof having one, two or three amino acid substitutions, and/or (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO. 221 or a variant thereof having one, two or three amino acid substitutions.

18. The nucleic acid molecule of any one of claims 1, 4 to 7 or 15 to 17 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising at least one of (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO. 67, SEQ ID NO. 79 or a variant thereof having one, two or three amino acid substitutions, (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO. 68, STS, or a variant thereof having one, two or three amino acid substitutions, and/or (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO. 69, SEQ ID NO. 81, or a variant thereof having one, two or three amino acid substitutions.

19. The nucleic acid molecule of any one of claims 1, 4 to 7 or 15 to 18 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 66 or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 64, 65 and/or 66; or (b) SEQ ID NO. 76, SEQ ID NO. 77 and SEQ ID NO. 78 or a variant thereof having one, two and/or three amino acid substitutions in any one or more of SEQ ID Nos 76, 77 and/or 78.

20. The nucleic acid molecule of any one of claims 1, 4 to 7 or 15 to 19 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO. 67, SEQ ID NO. 68 and SEQ ID NO. 69 or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 67, 68 and/or 69; or (b) SEQ ID NO. 79, STS and SEQ ID NO. 81 or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 79, 81 and/or STS.

21. The nucleic acid molecule of any one of claims 1, 4 to 7 or 15 to 20 wherein said anti-CLEC14A binding domain has an amino acid sequence comprising (a) SEQ ID NO 64, SEQ ID NO. 65, SEQ ID No. 66, SEQ ID NO. 67, SEQ ID NO 0.68 and SEQ ID NO. 69 or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 64, 65, 66, 67, 68 and/or 69; or (b) SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, STS and SEQ ID NO. 81 or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 76, 77, 78, 79, 81 and/or STS.

22. A nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising (i) an anti-CLEC14A binding domain (ii) a transmembrane domain and (iii) an intracellular signalling domain, wherein said anti-CLEC14A binding domain comprises at least one of (a) a heavy chain CDR having the amino acid sequence of SEQ ID NO. 167 or a variant thereof having one, two or three amino acid substitutions, (b) a heavy chain CDR having the amino acid sequence of SEQ ID NO. 168 or a variant thereof having one, two or three amino acid substitutions, (c) a heavy chain CDR having the amino acid sequence of SEQ ID NO. 169 or a variant thereof having one, two or three amino acid substitutions, (d) a light chain CDR having the amino acid sequence of SEQ ID NO. 129 or a variant thereof having one, two or three amino acid substitutions, (e) a light chain CDR having the amino acid sequence of SEQ ID NO. 68 or a variant thereof having one, two or three amino acid substitutions, and/or (f) a light chain CDR having the amino acid sequence of SEQ ID NO. 130 or a variant thereof having one, two or three amino acid substitutions.

23. The nucleic acid molecule of claim 1 or 22, wherein said anti-CLEC14A binding domain has an amino acid sequence comprising SEQ ID NO. 129, SEQ ID NO. 68 and SEQ ID NO. 130, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 129, 68 and/or 130 and/or comprising SEQ ID NO. 167, SEQ ID NO. 168 and SEQ ID NO. 169, or a variant thereof having one, two or three amino acid substitutions in any one or more of SEQ ID Nos 167, 168 and/or 169.

24. The nucleic acid molecule of any one of claims 1 to 14 wherein said anti-CLEC14A binding domain comprises (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 56, SEQ ID NO. 104, SEQ ID NO. 106 or SEQ ID NO. 121, or a variant thereof having at least 80% identity thereto, and/or (b) a light chain variable region comprising an amino acid sequence of SEQ ID NO. 57, SEQ ID NO. 105, SEQ ID NO. 107 or SEQ ID NO. 122, or a variant thereof having at least 80% identity thereto.

25. The nucleic acid molecule of any one of claims 1, 4 to 7 or 15 to 21 wherein said anti-CLEC14A binding domain comprises (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 88 or SEQ ID NO. 90, or a variant thereof having at least 80% identity thereto, and/or (b) a light chain variable region comprising an amino acid sequence of SEQ ID NO. 89 or SEQ ID NO. 91, or a variant thereof having at least 80% identity thereto.

26. The nucleic acid molecule of claim 22 or 23, wherein said anti-CLEC14A binding domain comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 173 or a variant thereof having at least 80% identity thereto and/or a light chain variable region comprising an amino acid sequence of SEQ ID NO. 133 or a variant thereof having at least 80% identity thereto.

27. The nucleic acid molecule of any one of claims 1 to 26 wherein said anti-CLEC14A binding domain comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs.

28. The nucleic acid molecule of any one of claim 1 to 14, 24 or 27, wherein said anti-CLEC14A binding domain comprises a scFv comprising an amino acid sequence of SEQ ID NO. 58, SEQ ID NO. 112 or SEQ ID NO. 125, or an amino acid sequence having at least 80% identity thereto.

29. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 to 21 or 27 wherein said CLEC14A binding domain comprises a scFv comprising an amino acid sequence of SEQ ID NO. 96, or an amino acid sequence having at least 80% identity thereto.

30. The nucleic acid molecule of any one of claim 1 to 14, 24, 27 or 28 wherein said polynucleotide comprises at least one of the following nucleotide sequences: (a) SEQ ID NO. 38, SEQ ID NO. 50 or a variant thereof having one, two or three nucleotide substitutions, (b) SEQ ID NO. 39, SEQ ID NO. 51, or a variant thereof having one, two or three nucleotide substitutions, and/or (c) SEQ ID NO. 40, SEQ ID NO. 52, SEQ ID NO. 101, SEQ ID NO. 103, SEQ ID NO. 117, SEQ ID NO. 120 or a variant thereof having one, two or three nucleotide substitutions.

31. The nucleic acid molecule of any one of claim 1 to 14, 24, 27, 28 or 30 wherein said polynucleotide comprises at least one of the following nucleotide sequences: (a) SEQ ID NO. 41, SEQ ID NO. 53 or a variant thereof having one, two or three nucleotide substitutions, (b) SEQ ID NO. 42, GACACATCC or a variant thereof having one, two or three nucleotide substitutions and/or (c) SEQ ID NO. 43, SEQ ID NO. 55 or a variant thereof having one, two or three nucleotide substitutions.

32. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 to 21, 27 or 29 wherein said polynucleotide comprises at least one of the following nucleotide sequences: (a) SEQ ID NO. 82 or a variant thereof having one, two or three nucleotide substitutions, (b) SEQ ID NO. 83 or a variant thereof having one, two or three nucleotide substitutions, and/or (c) SEQ ID NO. 84 or a variant thereof having one, two or three nucleotide substitutions.

33. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 to 21, 27, 29 or 32 wherein said polynucleotide comprises at least one of the following nucleotide sequences (a) SEQ ID NO. 85 or a variant thereof having one, two or three nucleotide substitutions, (b) AGCACATCC or a variant thereof having one, two or three nucleotide substitutions, and/or (c) SEQ ID NO. 87 or a variant thereof having one, two or three nucleotide substitutions.

34. The nucleic acid molecule of any one of claim 1 to 14, 24, 27, 28, 30 or 31 wherein said polynucleotide comprises (a) SEQ ID NO. 59, SEQ ID NO. 108, SEQ ID NO. 110 or SEQ ID NO. 123, or a variant thereof having from one to ten nucleotide substitutions, and/or (b) SEQ ID NO. 60, SEQ ID NO. 109, SEQ ID NO. 111 or SEQ ID NO. 124, or a variant thereof having from one to ten nucleotide substitutions.

35. The nucleic acid molecule of any one of claim 1 to 14, 24, 27, 28, 30, 31 or 34, wherein said polynucleotide comprises SEQ ID No. 61, SEQ ID NO. 113 or SEQ ID NO. 126, or a sequence having at least 80% identity thereof.

36. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 to 21, 27, 29, 32 or 33 wherein said polynucleotide comprises (a) SEQ ID NO. 92, SEQ ID NO. 94 or a variant thereof having from one to ten nucleotide substitutions, and/or (b) SEQ ID NO. 93, SEQ ID NO. 95 or a variant thereof having from one to ten nucleotide substitutions.

37. The nucleic acid molecule of any one of claim 1, 4 to 7, 15 to 21, 27, 29, 32, 33 or 36 wherein said polynucleotide comprises SEQ ID NO. 97 or a sequence having at least 80% identity thereto.

38. The nucleic acid molecule of any one of claims 1 to 37 wherein said transmembrane domain is derived from CD8 alpha or from CD28, and preferably comprises the amino acid sequence of SEQ ID NO. 146 or a sequence having at least 95% identity thereto.

39. The nucleic acid molecule of any one of claims 1 to 38 wherein said intracellular signalling domain is derived from CD3 zeta, and preferably comprises the amino acid sequence of SEQ ID NO. 148 or a sequence having at least 95% identity thereto.

40. The nucleic acid molecule of any one of claims 1 to 39 wherein said CAR comprises at least one costimulatory domain, preferably selected from any one of CD28, 4-1BB, OX40, ICOS, DAP10, CD27, CD30, CD40, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C, and/or B7-H3.

41. The nucleic acid molecule of claim 40, wherein said transmembrane domain and said costimulatory domain are derived from CD28.

42. The nucleic acid molecule of any one of claims 1 to 41 wherein said CAR comprises a leader sequence, preferably an oncostatin M leader sequence of SEQ ID NO. 135, a CD8a leader sequence encoded by SEQ ID NO. 162 or a sequence having at least 95% identity thereto, and/or wherein said CAR comprises a hinge or spacer, preferably derived from CD8alpha.

43. The nucleic acid molecule of claim 1 wherein said CAR (a) comprises an amino acid sequence of SEQ ID NO. 62, SEQ ID NO. 98, SEQ ID NO. 114 or SEQ ID NO. 127 or a sequence having at least 80% identity thereto, and/or (b) is encoded by a polynucleotide sequence comprising a nucleotide sequence of SEQ ID NO. 63, SEQ ID NO. 99, SEQ ID NO. 115 or SEQ ID NO. 128 or a sequence having at least 80% identity thereto.

44. The nucleic acid molecule of any one of claims 1 to 29 wherein said nucleic acid molecule is RNA.

45. A chimeric antigen receptor (CAR) encoded by a nucleic acid molecule of any one of claims 1 to 44.

46. A vector comprising the nucleic acid molecule of any one of claims 1 to 43, and optionally comprising a nucleic acid molecule comprising a polynucleotide sequence encoding a T cell receptor molecule.

47. The vector of claim 46 wherein said vector is a viral vector, preferably a gamma retroviral vector.

48. The vector of claim 46 or 47, wherein said vector further comprises a nucleotide sequence encoding a suicide gene, a cytokine, a dominant-negative TGF? receptor and/or a CD34 molecule, preferably a truncated CD34 molecule

49. A cell comprising a nucleic acid of any one of claims 1 to 44, a CAR of claim 45 or a vector of any one of claims 46 to 48, and optionally comprising a nucleic acid molecule or vector comprising a polynucleotide sequence encoding a T cell receptor molecule.

50. The cell of claim 49, wherein said cell is an immune cell, preferably a T-cell or a natural killer cell.

51. The cell of claim 49 or 50 wherein said cell comprises a further polynucleotide or vector encoding a suicide gene, a dominant-negative TGF? receptor or a cytokine.

52. A nucleic acid molecule of any one of claims 1 to 44, a vector of any one of claims 46 to 48 or a cell of any one of claims 49 to 51 for use in therapy.

53. A method of combating or treating disease comprising the step of administering a nucleic acid molecule of any one of claims 1 to 44, a vector of any one of claims 46 to 48 or a cell of any one of claims 49 to 51 to a subject in need thereof.

54. The nucleic acid molecule of any one of claims 1 to 44, the vector of any one of claims 46 to 48 or the cell of any one of claims 49 to 51 for use in treating a condition associated with expression of CLEC14A.

55. A method of treating a condition associated with expression of CLEC14A comprising the step of administering a nucleic acid molecule of any one of claims 1 to 44, a vector of any one of claims 46 to 48 or a cell of any one of claims 49 to 51 to a subject in need thereof.

56. Use of a nucleic acid molecule of any one of claims 1 to 44, a vector of any one of claims 46 to 48 or a cell of any one of claims 49 to 51 for use in the manufacture of a medicament for treating a condition associated with CLEC14A expression.

57. The nucleic acid molecule, vector or cell for use according to claim 54, the method of claim 55 or the use of claim 56, wherein said condition associated with expression of CLEC14A is a CLEC14A expressing solid tumour, or tumour angiogenesis.

58. The nucleic acid molecule, vector or cell for use according to claim 54, the method of claim 55 or the use of claim 56, wherein said nucleic acid molecule, vector or cell is for administration separately, simultaneously or sequentially in combination with one or more therapeutic agents, preferably (i) an anti-cancer agent, more preferably tirapazamine; (ii) a nucleic acid molecule comprising a polynucleotide sequence encoding a TCR molecule; (iii) a vector comprising a nucleic acid molecule of (ii); or (iv) a cell comprising a nucleic acid molecule of (ii) or a vector of (iii).

59. The nucleic acid molecule, vector or cell for use, the method or the use of claim 58, wherein the TCR molecule binds to WT1, preferably to HLA A2/RMFPNAPYL.

60. The nucleic acid molecule, vector or cell for use, the method or the use of claim 59, wherein the TCR molecule comprises an alpha chain and a beta chain, wherein the alpha chain comprises CDR1alpha of SEQ ID NO. 190, CDR2 alpha of SEQ ID NO. 191 and CDR3 alpha of SEQ ID NO. 192 or 193, and wherein the beta chain comprises CDR1 beta of SEQ ID NO. 194, CDR2 beta of SEQ ID NO. 195 and CDR3 beta of SEQ ID NO. 196 or 197, or a variant thereof wherein one or more of the CDRs comprise one, two or three amino acid substitutions, wherein said TCR molecule is capable of binding to an HLA A2/RMFPNAPYL complex.

61. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 44, a vector according to any one of claims 46 to 48 or a cell according to any one of claims 49 to 51 and optionally comprising a nucleic acid molecule comprising a polynucleotide sequence encoding a TCR, a vector comprising a nucleic acid molecule comprising a polynucleotide sequence encoding a TCR or a cell comprising a nucleic acid molecule comprising a polynucleotide encoding a TCR or a vector comprising a nucleic acid molecule comprising a polynucleotide encoding a TCR.

Description

[0481] The invention will now be described in more detail by reference to the following Examples and Figures:

[0482] FIG. 1 shows the polypeptide sequence of human CLEC14A from Genbank Accession No. NP_778230 (SEQ ID NO. 1) (FIG. 1A); cDNA of human CLEC14A from Genbank Accession No NM_175060 (SEQ ID NO. 2) (FIG. 1B) and coding region of human CLEC14A cDNA from positions 348-1820 of NM_175060 (SEQ ID NO. 3).

[0483] FIG. 2 shows a graph of the relative expression of CLEC14A in HUVECs and other primary cells. CLEC14A was expressed specifically in endothelial cells (HUVECs) and not in human aortic smooth muscle cells (HASMC), human lung fibroblasts (MRC50, human bronchial epithelial cells (HBE), hepatocytes, or peripheral blood mononuclear cells (PBMC).

[0484] FIG. 3 shows graphical results of a HUVEC scratch wound healing assay with anti-CLEC14A monoclonal antibody CRT-3 showing a retardation of wound closure.

[0485] FIG. 4 shows analysis of tubule formation assays with CLEC14A antibody treated HUVECs. HUVECs were treated with 20 ?g/ml CRT2, 3 or 4 or mouse IgG isotype control. Images of tubules were taken at 16 hours and analysed for total tubule length, number of junctions, number of branches, branch length, number of meshes and total mesh area. Data shown represents three experiments with five data points analysed for each. Error bars show SEM. *p,0.05. **p<0.01.

[0486] FIG. 5 shows siRNA knockdown of CLEC14A which reveals a role for CLEC14A in endothelial sprouting. FIG. 5A shows siRNA duplex targeting of CLEC14A can efficiently knockdown CLEC14A mRNA expression in HUVEC, as determined by qPCR. Relative expression was determined by normalising expression to flotilin2. FIG. 5B shows knockdown of CLEC14A at the protein level by Western blot analysis. Tubulin was used as a loading control. FIG. 5C shows representative images of sprout outgrowth after 16 hours for control or clec14a targeted siRNA treated HUVEC. FIG. 5D shows quantitation of sprouts for 27 spheroids (9 spheroids from 3 cords) for control and CLEC14A knockdown HUVEC; Mann-Whitney statistical test p<0.001. FIG. 5E shows representative images of sprout outgrowth after 24 hours for mixed control (green) and clec14a targeted siRNA treated HUVEC (red). FIG. 5F shows quantitation of the percentage of tip and stalk cells derived from control (CON) and CLEC14A knockdown (KD) HUVEC; two-way ANOVA statistical test with Bonferroni post-tests ***=p<0.001, ns=not significant.

[0487] FIG. 6 shows that loss of CLEC14A inhibits sprouting in vitro and in vivo. FIG. 6A shows a schematic diagram of clec14a gene in C57BL/6 (clec14a +/+) or C57BL/6.sup.(Clec14atm1(KOMP)Vlcg) (clec14a ?/?) mice. FIG. 6B shows quantitative PCR analysis of cDNA generated from three clec14a +/+ mice (white bars) and three clec14a ?/? mice (black bars) for the 5 untranslated region (UTR), coding sequence (CDS) and 3 UTR of clec14a. Relative expression was determined by normalising expression to flotilin2. FIG. 6C shows Western blot analysis of CLEC14A protein expression in lung lysates from clec14a +1+ and clec14a ?/? mice using polyclonal antisera against murine CLEC14A. Tubulin was used as a loading control. FIG. 6D shows representative images of the aortic ring sprouting assay from clec14a +/+ and clec14a ?/? mice. FIG. 6E shows quantitation of tubes formed per ring, and FIG. 6F shows quantitation of the maximal distance migrated by an endothelial tube from the aortic ring, data from 48 rings per genotype, 6 mice for each genotype; Mann-Whitney statistical test p<0.001. FIG. 6G shows representative images of haematoxylin and eosin stained sections of sponge implant from clec14a +1+ and clec14a ?/? mice, sections at the centre of the sponge were analysed. FIG. 6H shows quantitation of cellular invasion into the sponge implants shown in FIG. 6G; Mann-Whitney statistical test p<0.05. FIG. 6I shows quantitation of vessel density; Mann-Whitney statistical test p<0.001. FIG. 6J shows sections of liver and sponge tissue stained with x-gal from clec14a ?/? mice, counterstained with haematoxylin and eosin.

[0488] FIG. 7 shows loss of CLEC14A inhibits tumour growth. FIG. 7A shows Lewis lung carcinoma (LLC) tumour growth in clec14a +/+(black line with dots) and clec14a ?/? (black line with squares) mice; two-way ANOVA statistical analysis, *=p<0.05, **=p<0.01, ***=p<0.001. FIG. 7B shows representative images of LLC tumours. FIG. 7C shows endpoint tumour weight for 7 clec14a +1+(dots) and 7 clec14a ?/? (squares) mice; Mann-Whitney statistical test p<0.001. FIG. 7D shows representative images of immunofluorescent staining of LLC tumour sections stained for murine CD31. FIG. 7E shows quantitation of vessel density and FIG. 7F shows percentage endothelial coverage from clec14a +/+ and clec14a ?/? mice; Mann-Whitney statistical test p<0.0001. FIG. 7G shows sections of liver and LLC tumour tissue from clec14a ?/? mice stained with x-gal, counterstained with haematoxylin and eosin.

[0489] FIG. 8 shows MMRN2 binds to CLEC14A. In FIG. 8A 20 ?g CLEC14A-ECD-Fc or Fc was used to precipitate interacting partners. Precipitates and HUVEC lysates were separated on an SDS-PAG and blotted for MMRN2 (top panel) or CLEC14A-ECD-Fc (bottom panel). In FIG. 8B CLEC14A was immunoprecipitated from HUVEC lysates using polyclonal antisera against CLEC14A. Immunoprecipitates were analysed by Western blot for MMRN2 (top panel) and CLEC14A (bottom panel).

[0490] FIG. 9 shows MMRN2-CLEC14A interaction blocking antibody inhibits tumour growth. FIG. 9A shows results where mice injected with LLC were treated with 100 ?g injections of mIgG1 (black line with dots; n=7) or C4 antibody (black line with squares; n=7); two-way ANOVA statistical analysis, **=p<0.01, ***=p<0.001. FIG. 9B shows representative images of LLC tumours. FIG. 9C shows endpoint tumour weight for 7 mIgG1 treated mice (dots) and 7 C4 antibody treated mice (squares); Mann-Whitney statistical test p<0.001. FIG. 9D shows representative images of immunofluorescent staining of LLC tumour sections stained for murine CD31. FIG. 9E shows quantitation of vessel density and FIG. 9F shows percentage endothelial coverage from mice treated with mIgG1 or C4 antibody; Mann-Whitney statistical test p<0.001.

[0491] FIG. 10 shows that CLEC14A monoclonal antibodies C1, C4 and C5 block CLEC14A-MMRN2 interaction. Human CLEC14A-ECD-Fc was bound to protein A beads, blocked in 20% FCS and then incubated with each blocking condition. This was then added to lysates of HEK293T cells overexpressing full-length human MMRN2 with a His tag. Pre-incubating with CRT1, CRT4 and CRT5 decreased the levels of MMRN2 pulled down by CLEC14A-ECD-Fc.

[0492] FIG. 11 shows that MMRN2 directly binds to either the C-type lectin or sushi domain of CLEC14A under non-reduced conditions. HEK293T cells were mock transfected or transfected with pCS2 vectors containing CLEC14A wild type (WT) or constructs with each major domain deleted (?) with an N-terminal HA tag. Upon far western blotting with MMRN2 protein lysate, binding can be seen in all mutants except those missing the C-type lectin domain (CTLD) or the sushi domain. An anti-HA blot was included to show all mutant proteins were expressed.

[0493] FIG. 12 shows protein sequences of the chimeric proteins Chimera 5 and Chimera 6.

[0494] FIG. 13 shows analysis of the binding of CRT antibodies using flow cytometry. All constructs have a C-terminus GFP tag so green cells were gated and stained red. All CRT antibodies bind to CLEC14A wild type with a C terminal GFP tag expressed in HEK293T cells. None of the CRT antibodies bind to wild type thrombomodulin with GFP tag expressed in HEK293T cells.

[0495] FIG. 14 shows an alignment of CLEC14A regions 1-42 of CD141; CLEC14A regions 97-108 of CD141; and CLEC14A regions 122-142 of CD141.

[0496] FIG. 15 shows that Chimera 5 (CTLD of thrombomodulin, rest CLEC14A) is not recognised by any of the CRT antibodies except a slight shift in fluorescence with CRT2. Chimera 6 (Sushi of thrombomodulin to ensure correct folding of CTLD of CLEC14A) results in binding of all CRT antibodies except CRT2.

[0497] FIG. 16 shows flow cytometry analysis of binding by antibodies CRT1-5 when Residues 97-108 were swapped with corresponding regions from thrombomodulin. This resulted in correct folding as CRT2 and CRT3 can still bind. However CRT1, CRT4 and CRT5 cannot recognise this mutant suggesting this to be the binding region.

[0498] FIG. 17 shows the reduced tumour weight of lung carcinoma when a CRT-4 antibody drug conjugate treatment is employed. FIG. 17A shows the results when 1 million Lewis lung carcinoma cells were injected subcutaneously into the right flank of mice and allowed to grow to a visible size. 1 mg/kg of antibody CRT4-ADC (right image) or control B12-ADC (left image) was administered through tail vein injections. Mice were observed for an hour and culled 24 hours later. All organs were dissected and fixed. n=1. FIG. 17B shows end point tumour weights of antibody drug conjugate treatments. There was a significant difference between the wet weights of CRT4-ADC treatment group when compared with B12-ADC treatment group. Mann Whitney test p=0.0317. Error bars SEM, n=5. Data pooled from two separate experiments of the same method.

[0499] FIG. 18 shows the internalisation of a CRT-3 antibody drug conjugate and the effect of this on a tumour 24 hours after administration. FIG. 18A shows the internalisation of CRT-3 antibody drug conjugate in HUVECs where fluorescent imaging shows the localisation of the CRT-3 after 0 and 90 minutes and FIG. 18B shows the cytotoxicity measured by Cell Titre Glo luminescent cell viability assay in HUVEC treated CRT-3-ADC (IC50=137.6 ng/ml). FIG. 18C shows extensive haemorrhage at the site of the tumour in the CRT3-ADC treated mouse and not in a control, demonstrating tumour-specific disruption of angiogenesis.

[0500] FIG. 19 shows a retroviral CAR vector (based on pMP71) that co-expresses a truncated CD34 marker gene and an scFv fragment/CD3 zeta chain chimeric receptor (FIG. 19A). Expression is driven from the LTR promoter and the 2A peptide linker ensures equimolar expression of both the CD34 and the CAR. Second generation CAR constructs included the CD28 costimulatory domain. FIG. 19B shows CD34 staining analysed by flow cytometry demonstrating successful transduction of T cells using retroviral constructs that co-express a CLEC14A-specific CAR. First generation CARs based on the antibodies CRT3 and CRT5 are referred to as CRT3.z and CRT5.z respectively. Second generation CARs based on the antibodies CRT3 and CRT5 are referred to as CRT3.28z and CRT5.28z respectively. Note equivalent expression was seen in CD4 and CD8 T cell subsets (data not shown). FIG. 19C shows cells stained directly for expression of CAR using CLEC14A-Fc (% values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone).

[0501] FIG. 20 shows the ability of T cells transduced to express 1.sup.st or 2.sup.nd generation CARs based on antibodies CRT3 or CRT5 or mock-transduced (control) T cells to respond to CLEC14A expressed either as (A) plate-bound recombinant Fc fusion protein, (B) expressed on engineered CHO cells, or (C) expressed on human umbilical vein endothelial cells (HUVECs) which naturally express CLEC14A when grown in static culture. T cell response was measured using an ELISA for interferon gamma production. Data shown are representative of that obtained from 3-7 repeat experiments. T cells were adjusted to equalise the frequency of transgene expressing cells. All histograms show mean response+SD.

[0502] FIG. 21 shows further in vitro functional testing of CLEC14A-specific CAR-transduced T cells. T cells transduced to express 1.sup.st or 2.sup.nd generation CARs based on antibodies CRT3 or CRT5, or mock-transduced (control) T cells, were tested for their ability to respond to CLEC14A in the following functional assays: (A) Cytotoxicity, using CHO cells engineered to express human CLEC14A (having subtracted background levels of lysis of CHO alone (control cells)). Data shown are representative of 5 repeat experiments. (B) Proliferation, using CFSE-labelled CAR-transduced T cells we measured the proliferation of CAR+ (CD34+) and CAR? (CD34?) cell subsets when co-cultured for 4 days with HUVECs. Data shown are representative of 2 repeat experiments. (C) The response of (CLEC14A-specific CAR-transduced T cells to both human and mouse CLEC14A was assessed using interferon gamma release. T cells were adjusted to equalise the frequency of transgene expressing cells. Data shown are representative of 6 repeat experiments. All histograms show mean response+SD.

[0503] FIG. 22 shows in vitro functional testing of T cells transduced with a third CLEC14A-specific CAR based on antibody CRT1. T cells transduced to express 1.sup.st or 2.sup.nd generation CARs based on antibody CRT1, or mock-transduced (control) T cells, were tested for their ability to respond to CLEC14A expressed on (A) engineered CHO cells or (B) human umbilical vein endothelial cells (HUVECs) which naturally express CLEC14A when grown in static culture. T cell response was measured using an ELISA for interferon gamma production. (C) Cytotoxicity, using CHO cells engineered to express human CLEC14A (having subtracted background levels of lysis of CHO alone (control cells)). Data shown are representative of that obtained from at least 3 repeat experiments. T cells were adjusted to equalise the frequency of transgene expressing cells. All histograms show mean response+SD.

[0504] FIG. 23 shows toxicity testing in vivo using healthy C57/BL6 mice injected with CLEC14A-specific CAR-transduced mouse T cells. T cells transduced to express 1.sup.st or 2.sup.nd generation CARs based on antibodies CRT3 or CRT5, or mock-transduced (control) T cells, were injected into the tail vein of healthy C57BL6 mice that had previously been irradiated (4 Gy) to aid T cell engraftment. T cells were derived from a C57BL6 congenic strain (BoyJ) which carry the marker CD45.1. 20 million T cells (containing 4 million engineered T cells) were infused per mouse. Mice were monitored for the next 45 days and showed no visible signs of toxicity. (A) Body weights increased normally during this time. (B) Weekly tail bleeds demonstrated that the infused CD45.1+ T cells persisted for at least five weeks post infusion and throughout this time they comprised at least 30% of the total circulating T cell pool (note over time the host's own T cells recover). (C) Staining the same samples for CD34 as well as CD45.1 demonstrated that the proportion of engineered (CD34+) T cells relative to the total infused T cells population remained relatively constant during this time. (D) At the end of the experiment, splenocytes were harvested from a CAR-treated mouse and CD34+ cells were isolated using immunomagnetic bead selection. Testing these cells immediately ex vivo demonstrated that they were still capable of responding to both human and mouse CLEC14A as measured by ELISA for interferon gamma release. Graphs A-C show mean+SEM. Graph D shows mean+SD.

[0505] FIG. 24 shows toxicity testing in vivo using healthy C57/BL6 mice injected with CLEC14A-specific CAR-transduced mouse T cells. At the end of the experiment mice were culled and major organs harvested. Histological examination revealed no evidence of pathology.

[0506] FIG. 25 shows the anti-tumour response of CLEC14A-specific CAR-transduced mouse T cells when injected into mice carrying Lewis Lung carcinoma tumours. C57BL6 mice were injected subcutaneously with Lewis Lung Carcinoma cells (1 million cells/mouse) and 4 days later mice received 4 Gy total body irradiation to aid T cell engraftment. T cells transduced to express 2.sup.nd generation CARs based on antibodies CRT3 or CRT5, or mock-transduced (control) T cells, were then injected into the tail vein. Mice received a total of 20 million T cells (CD8:CD4=5:2) with CRT3.28z and CRT5.28z expressed on 2.2 and 1.4 million of these cells. Tumour growth was then monitored using (A) Bioluminescence or (B) Calipers.

[0507] FIG. 26 shows the anti-tumour response of CLEC14A-specific CAR-transduced mouse T cells when injected into mice carrying Lewis Lung carcinoma tumours. At the end of the experiment tumours were excised and weighed (A). Histological analysis demonstrated that tumours from mice treated with the CARs showed significantly reduced vascular density (B, staining for MECA-32) and greater levels of vascular leakage (C, staining for fibrinogen).

[0508] FIG. 27 shows the anti-tumour response of CLEC14A-specific CAR-transduced mouse T cells when injected into RipTag2 mice (where the rat insulin promoter (RIP) directs expression of the SV40 Large T antigen transgene (TAg) to beta cells of the pancreatic islets). Tumours arise at around 10 weeks of age and usually result in the death of the animal by approx. 14 weeks. Mouse T cells expressing a second generation CAR based on CRT5 (or mock-transduced control cells) were infused into 10 week old riptag2 mice. Mock-treated mice were culled at 14 weeks of age and tumour size measured. CAR-treated mice were culled 2 weeks later (16 weeks old) and again tumour size was measured. Results demonstrated a highly significant inhibition of tumour size in mice treated with CAR-transduced T cells (A) compared with mock-transduced T cell-treated animals. There was also some evidence of a survival benefit, since 12 mice were treated in each of the study, and of those that received mock-transduced T cells, 4 died before 14 weeks of age, whereas all 12 mice treated with CAR-transduced T cells were alive at 14 weeks of age, and all but two of them were still alive two weeks later. Data show tumour size in individual mice. Horizontal line indicates mean tumour size+SEM. Note untreated mice were not irradiated.

[0509] FIG. 28 shows confocal imaging of RIP-Tag2 tumours 4 weeks after intravenous injection of CAR-T cells. The imaging shows that CD34+(CAR-transduced) T cells accumulate in the tumours.

[0510] FIG. 29 shows histological analysis of RipTag2 tumours treated with CAR- (or Mock-) engineered T cells. FIG. 29A shows that vascular density is reduced in CAR-T cell treated tumours as indicated by staining for the endothelial marker MECA32. FIG. 29B shows a summary of vascular density in CAR- vs Mock T cell treated tumours. FIGS. 29C and D show that mice treated with CAR-transduced T cells display an increase in apoptotic vessels in the tumour as indicated by caspase 3 staining. FIGS. 29 E and F show that mice treated with CAR-transduced T cells display a decrease in fibrinogen staining in the tumour vasculature.

[0511] FIG. 30 shows further characterisation of CLEC14A expression in human tumour tissue arrays calculating the percentage of vessels within a tumour that expressed CLEC14A. n=number of cases studied for each cancer type. Each case is represented by a circle, with horizontal lines showing mean percentage values+SEM for each cancer type.

[0512] FIG. 31 shows the transduction of T-cells with CRT4 CAR.

[0513] FIG. 32 shows that both first and generation CRT1 CAR T cells can mediate cell lysis of CHO cells expressing CLEC14A.

[0514] FIG. 33 shows the proliferative activity of T cells expressing first and second generation CRT-1 CAR constructs.

[0515] FIG. 34 shows the interferon gamma release activity of T cells expressing first and second generation CRT-1 CAR constructs in response to CHO cells expressing humanCLEC14A (having subtracted the response to CHO cells alone). Data show the mean conc. of IFN? (+SEM) produced from 7 repeat experiments.

[0516] FIG. 35 shows the interferon gamma release activity of T cells expressing first and second generation CRT-1 CAR constructs in response to Human or mouse CLEC14A-Fc fusion proteins (or Fc protein alone).

[0517] FIG. 36 shows retroviral TCR gene transfer into human T cells and TCR expression in human PBMC after transduction. Peripheral blood lymphocyte activation was carried out using anti-CD3 antibodies, IL-7 and IL-2, followed by transduction with retroviral vectors (encoding a TCR specific for WT1) three days later. At day 6, TCR expression was monitored using TCR-V-beta 2.1 antibodies. The percentage of un-manipulated human T cells expressing V-beta 2.1 was shown using mock transduced T cells. Both CD8 negative and CD8 positive T cells after transduction had an increased percentage of V-beta 2.1 cells.

[0518] FIG. 37 shows an expansion of CD8 positive T cells expressing V-beta 2.1 is achieved by repeated stimulation of TCR transduced T cells with T2 cells presenting the WT126 peptide. Thus an increase of CD8+-Vb2.1+ T cells occurs after antigen stimulation.

[0519] FIG. 38 shows that HLA-A2/pWT126 tetramers and TCR-transduced T cells stain together.

[0520] FIG. 39 shows that T cells transduced with the TCR are able to kill T2 cells presenting the WT126 peptide but not T2 cells presenting the pWT235 peptide. The transduced T cells also further kill HLA-A2 BV173 leukaemia cells which endogenously express WT1. Thus, TCR transduced bulk T cells show pWT126-specific killing activity.

[0521] FIG. 40 shows that HLA-A2 positive T2 cells presenting the WT126 peptide are killed by purified TCR-transduced CD8 positive T cells but that T2 cells coated with the WT235 peptide are not killed. Further, CD8 positive transduced T cells also kill HLA-A2 BV173 leukaemia cells which endogenously express WT1. Thus, transduced CD8+ T cells show pWT126-specific killing activity. (Keyunfilled square=T2+pWT235, filled diamond=T2+pWT126, unfilled circle=BV173).

[0522] FIG. 41 shows that a small amount of purified CD4 positive transduced T cells stain together with HLA-A2/pWT126 tetramers.

[0523] FIG. 42 shows that HLA-A2 positive T2 cells presenting the WT126 peptide are killed by purified TCR-transduced CD4 positive T cells but that T2 cells coated with the WT235 peptide are not killed. Further, CD4 positive transduced T cells also kill HLA-A2 BV173 leukaemia cells which endogenously express WT1.

[0524] FIG. 43 shows that IFN-? is produced by the purified TCR-transduced CD8 positive cells after stimulation with the HLA-A2 positive T2 cells coated with the WT126 but not by equivalent cells coated with the pWT235 peptide. IFN-? is also produced by the CD8 positive transduced T cells after stimulation with HLA-A2 positive BV173 leukaemia cells which express WT1 endogenously. Thus, TCR transduced CD8+ T cells show pWT126-specific IFN? production.

[0525] FIG. 44 shows that the CRT5 CAR does not impede healing of a skin wound in tumour bearing mice.

[0526] FIG. 45 shows PDAC tumour volumes 3 weeks after treatment with control (n=5) or CRT5 CAR (with CD28 costimulatory domain) expressing cells.

[0527] FIG. 46 shows CRT1, 3 and 5 CAR (with CD28 costimulatory domain) T cell response to titrated concentrations of human and mouse recombinant CLEC14A.

[0528] FIG. 47 shows the design of constructs which encode CARs with different costimulatory domains; 1) tCD34-F2A-scFv-CD28 TM-CD28 signal-CD3zeta, 2) tCD34-F2A-scFv-CD8 TM-4-1BB signal-CD3 zeta, 3) tCD34-F2A-scFv-CD8 TM-OX40 signal-CD3 zeta, 4) tCD34-F2A-scFv-CD28 TM-CD28 signal-4-1BB signal-CD3zeta, 5) tCD34-F2A-scFv-CD28 TM-CD28 signal-OX40 signal-CD3 zeta, 6) tCD34-F2A-scFv-CD8 TM-4-1BB signal-OX40 signal-CD3zeta. The tCD34 is included to identify successfully transduced cells and thus constructs may exclude this and F2A. A hinge or spacer region may additionally be included e.g. one from CD8a.

[0529] FIG. 48 shows the results of a cytotoxicity assay with CRT1, 3 and 5 CARs vs mouse endothelial cells expressing CLEC14A (FIG. 48A). The results of a proliferative assay for CRT 1, 3 and 5 CARs are shown in FIG. 48B.

[0530] FIG. 49 shows the functional testing of CRT3 CAR T cells comprising different costimulatory domains and the IFNgamma production in response to titrated numbers of CHO cells expressing human CLEC14A.

[0531] FIG. 50 shows orthotopic PDAC tumour volumes 3 weeks after treatment with mock (n=5) or CRT5 CAR (CD28 costimulatory domain) T cells (n=8) (p=0.022; Mann Whitney).

[0532] FIG. 51 shows the IFN gamma release by CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells after incubated with 293 or SEND cells engineered to express CLEC14A chimera (A1human CLEC14A with mouse intracellular domain, B1human CLEC14A with mouse transmembrane and intracellular domains, huCLEChuman CLEC14A). Cytotoxicity data are shown in FIG. 51B for the CAR T cells after incubation with SEND cells.

[0533] FIG. 52 shows a schematic of a suitable vector to generate RNA for electroporation by in vitro transcription.

[0534] FIG. 53 shows constructs which encode CARs which can be used to transduce murine T cells. The constructs comprise transmembrane, costimulatory and intracellular signalling sequences from murine proteins (see SEQ ID NOs 227-232). The constructs may further comprise a hinge or spacer domain from murine CD8?.

[0535]

TABLE-US-00001 TABLE1 showingsequences SEQ Descriptionof IDNO sequence Sequence 1 CLEC14A MRPAFALCLLWQALWPGPGGGEHPTADRAGCSASGACY polypeptide SLHHATMKRQAAEEACILRGGALSTVRAGAELRAVLALLRA GPGPGGGSKDLLFWVALERRRSHCTLENEPLRGFSWLSS DPGGLESDTLQWVEEPQRSCTARRCAVLQATGGVEPAG WKEMRCHLRANGYLCKYQFEVLCPAPRPGAASNLSYRAP FQLHSAALDFSPPGTEVSALCRGQLPISVTCIADEIGARWD KLSGDVLCPCPGRYLRAGKCAELPNCLDDLGGFACECAT GFELGKDGRSCVTSGEGQPTLGGTGVPTRRPPATATSPV PQRTWPIRVDEKLGETPLVPEQDNSVTSIPEIPRWGSQST MSTLQMSLQAESKATITPSGSVISKFNSTTSSATPQAFDSS SAVVFIFVSTAVVVLVILTMTVLGLVKLCFHESPSSQPRKES MGPPGLESDPEPAALGSSSAHCTNNGVKVGDCDLRDRAE GALLAESPLGSSDA 2 CLEC14AcDNA CTCCTCTTGCTCTAAGCAGGGTGTTTGACCTTCTAGTCG ACTGCGTCCCCTGTACCCGGCGCCAGCTGTGTTCCTGA CCCCAGAATAACTCAGGGCTGCACCGGGCCTGGCAGC GCTCCGCACACATTTCCTGTCGCGGCCTAAGGGAAACT GTTGGCCGCTGGGCCCGCGGGGGGATTCTTGGCAGTT GGGGGGTCCGTCGGGAGCGAGGGCGGAGGGGAAGGG AGGGGGAACCGGGTTGGGGAAGCCAGCTGTAGAGGGC GGTGACCGCGCTCCAGACACAGCTCTGCGTCCTCGAGC GGGACAGATCCAAGTTGGGAGCAGCTCTGCGTGCGGG GCCTCAGAGAATGAGGCCGGCGTTCGCCCTGTGCCTCC TCTGGCAGGCGCTCTGGCCCGGGCCGGGCGGCGGCG AACACCCCACTGCCGACCGTGCTGGCTGCTCGGCCTCG GGGGCCTGCTACAGCCTGCACCACGCTACCATGAAGCG GCAGGCGGCCGAGGAGGCCTGCATCCTGCGAGGTGGG GCGCTCAGCACCGTGCGTGCGGGCGCCGAGCTGCGCG CTGTGCTCGCGCTCCTGCGGGCAGGCCCAGGGCCCGG AGGGGGCTCCAAAGACCTGCTGTTCTGGGTCGCACTGG AGCGCAGGCGTTCCCACTGCACCCTGGAGAACGAGCCT TTGCGGGGTTTCTCCTGGCTGTCCTCCGACCCCGGCGG TCTCGAAAGCGACACGCTGCAGTGGGTGGAGGAGCCC CAACGCTCCTGCACCGCGCGGAGATGCGCGGTACTCCA GGCCACCGGTGGGGTCGAGCCCGCAGGCTGGAAGGAG ATGCGATGCCACCTGCGCGCCAACGGCTACCTGTGCAA GTACCAGTTTGAGGTCTTGTGTCCTGCGCCGCGCCCCG GGGCCGCCTCTAACTTGAGCTATCGCGCGCCCTTCCAG CTGCACAGCGCCGCTCTGGACTTCAGTCCACCTGGGAC CGAGGTGAGTGCGCTCTGCCGGGGACAGCTCCCGATC TCAGTTACTTGCATCGCGGACGAAATCGGCGCTCGCTG GGACAAACTCTCGGGCGATGTGTTGTGTCCCTGCCCCG GGAGGTACCTCCGTGCTGGCAAATGCGCAGAGCTCCCT AACTGCCTAGACGACTTGGGAGGCTTTGCCTGCGAATG TGCTACGGGCTTCGAGCTGGGGAAGGACGGCCGCTCTT GTGTGACCAGTGGGGAAGGACAGCCGACCCTTGGGGG GACCGGGGTGCCCACCAGGCGCCCGCCGGCCACTGCA ACCAGCCCCGTGCCGCAGAGAACATGGCCAATCAGGGT CGACGAGAAGCTGGGAGAGACACCACTTGTCCCTGAAC AAGACAATTCAGTAACATCTATTCCTGAGATTCCTCGAT GGGGATCACAGAGCACGATGTCTACCCTTCAAATGTCC CTTCAAGCCGAGTCAAAGGCCACTATCACCCCATCAGG GAGCGTGATTTCCAAGTTTAATTCTACGACTTCCTCTGC CACTCCTCAGGCTTTCGACTCCTCCTCTGCCGTGGTCTT CATATTTGTGAGCACAGCAGTAGTAGTGTTGGTGATCTT GACCATGACAGTACTGGGGCTTGTCAAGCTCTGCTTTCA CGAAAGCCCCTCTTCCCAGCCAAGGAAGGAGTCTATGG GCCCGCCGGGCCTGGAGAGTGATCCTGAGCCCGCTGC TTTGGGCTCCAGTTCTGCACATTGCACAAACAATGGGGT GAAAGTCGGGGACTGTGATCTGCGGGACAGAGCAGAG GGTGCCTTGCTGGCGGAGTCCCCTCTTGGCTCTAGTGA TGCATAGGGAAACAGGGGACATGGGCACTCCTGTGAAC AGTTTTTCACTTTTGATGAAACGGGGAACCAAGAGGAAC TTACTTGTGTAACTGACAATTTCTGCAGAAATCCCCCTTC CTCTAAATTCCCTTTACTCCACTGAGGAGCTAAATCAGA ACTGCACACTCCTTCCCTGATGATAGAGGAAGTGGAAGT GCCTTTAGGATGGTGATACTGGGGGACCGGGTAGTGCT GGGGAGAGATATTTTCTTATGTTTATTCGGAGAATTTGG AGAAGTGATTGAACTTTTCAAGACATTGGAAACAAATAG AACACAATATAATTTACATTAAAAAATAATTTCTACCAAAA TGGAAAGGAAATGTTCTATGTTGTTCAGGCTAGGAGTAT ATTGGTTCGAAATCCCAGGGAAAAAAATAAAAATAAAAA ATTAAAGGATTGT 3 CLEC14Acoding ATGAGGCCGGCGTTCGCCCTGTGCCTCCTCTGGCAGGC region GCTCTGGCCCGGGCCGGGCGGCGGCGAACACCCCACT GCCGACCGTGCTGGCTGCTCGGCCTCGGGGGCCTGCT ACAGCCTGCACCACGCTACCATGAAGCGGCAGGCGGC CGAGGAGGCCTGCATCCTGCGAGGTGGGGCGCTCAGC ACCGTGCGTGCGGGCGCCGAGCTGCGCGCTGTGCTCG CGCTCCTGCGGGCAGGCCCAGGGCCCGGAGGGGGCT CCAAAGACCTGCTGTTCTGGGTCGCACTGGAGCGCAGG CGTTCCCACTGCACCCTGGAGAACGAGCCTTTGCGGGG TTTCTCCTGGCTGTCCTCCGACCCCGGCGGTCTCGAAA GCGACACGCTGCAGTGGGTGGAGGAGCCCCAACGCTC CTGCACCGCGCGGAGATGCGCGGTACTCCAGGCCACC GGTGGGGTCGAGCCCGCAGGCTGGAAGGAGATGCGAT GCCACCTGCGCGCCAACGGCTACCTGTGCAAGTACCAG TTTGAGGTCTTGTGTCCTGCGCCGCGCCCCGGGGCCG CCTCTAACTTGAGCTATCGCGCGCCCTTCCAGCTGCAC AGCGCCGCTCTGGACTTCAGTCCACCTGGGACCGAGGT GAGTGCGCTCTGCCGGGGACAGCTCCCGATCTCAGTTA CTTGCATCGCGGACGAAATCGGCGCTCGCTGGGACAAA CTCTCGGGCGATGTGTTGTGTCCCTGCCCCGGGAGGTA CCTCCGTGCTGGCAAATGCGCAGAGCTCCCTAACTGCC TAGACGACTTGGGAGGCTTTGCCTGCGAATGTGCTACG GGCTTCGAGCTGGGGAAGGACGGCCGCTCTTGTGTGA CCAGTGGGGAAGGACAGCCGACCCTTGGGGGGACCGG GGTGCCCACCAGGCGCCCGCCGGCCACTGCAACCAGC CCCGTGCCGCAGAGAACATGGCCAATCAGGGTCGACGA GAAGCTGGGAGAGACACCACTTGTCCCTGAACAAGACA ATTCAGTAACATCTATTCCTGAGATTCCTCGATGGGGAT CACAGAGCACGATGTCTACCCTTCAAATGTCCCTTCAAG CCGAGTCAAAGGCCACTATCACCCCATCAGGGAGCGTG ATTTCCAAGTTTAATTCTACGACTTCCTCTGCCACTCCTC AGGCTTTCGACTCCTCCTCTGCCGTGGTCTTCATATTTG TGAGCACAGCAGTAGTAGTGTTGGTGATCTTGACCATGA CAGTACTGGGGCTTGTCAAGCTCTGCTTTCACGAAAGC CCCTCTTCCCAGCCAAGGAAGGAGTCTATGGGCCCGCC GGGCCTGGAGAGTGATCCTGAGCCCGCTGCTTTGGGCT CCAGTTCTGCACATTGCACAAACAATGGGGTGAAAGTC GGGGACTGTGATCTGCGGGACAGAGCAGAGGGTGCCT TGCTGGCGGAGTCCCCTCTTGGCTCTAGTGATGCATAG 4 humanCLEC14A TAGTAGGAATTCGAGAGAATGAGGCCGGCGTTCGCCCT fwd G 5 humanCLEC14A AGAACCGCGGCCGCTGGAGGAGTCGAAAGCCTGAGGA rev GT 6 murineCLEC14A TAGTAGGAATTCGAGAGAATGAGGCCAGCGCTTGCCCT fwd G 7 murineCLEC14A CTACTAGCGGCCGCTCGTGGAAGAGGTGTCGAAAGT rev 8 humanCLEC14A TAGTAGTTAATTAAGAGAGAATGAGGCCGGCGTTC fwd 9 murineCLEC14A TAGTAGTTAATTAAGAGAGAATGAGGCCAGCGCTT fwd 10 humanFcrev CTACTAGTTTAAACTCATTTACCCGGAGACAGGGA 11 MMRN2fwd CCGGACCGGTCAGGCTTCCAGTACTAGCC 12 MMRN2rev CGGGGTACCGGTCTTAAACATCAGGAAGC 13 5UTRfwd TTCCTTTTCCAGGGTTTGTG 14 5UTRrev GCCTACAAGGTGGCTTGAAT 15 CDSfwd AAGCTGTGCTCCTGCTCTTG 16 CDSrev TCCTGAGTGCACTGTGAGATG 17 3 UTRfwd CTGTAGAGGGCGGTGACTTT 18 3 UTRrev AGCTGCTCCCAAGTCCTCT 19 mACTBfwd CTAAGGCCAACCGTGAAAAG 20 mACTBrev ACCAGAGGCATACAGGGACA 21 CD141residues MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALY 1-42 PGP 22 CD141residues QLPPGCGDPKRL 97-108 23 CD141residues TSYSRWARLDLNGAPLCGPL 122-142 24 CLEC14A ERRRSCHTLENE residues97-108 25 CD141CTLD MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYP aminoacid GPATFLNASQICDGLRGHLMTVRSSVAADVISLLLNGDGG VGRRRLWIGLQLPPGCGDPKRLGPLRGFQWVTGDNNTSY SRWARLDLNGAPLCGPLCVAVSAAEATVPSEPIWEEQQC EVKADGFLCEF 26 Chimera5GFP MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALY fusionaminoacid PGPATFLNASQICDGLRGHLMTVRSSVAADVISLLLNGDG C-typelectin GVGRRRLWIGLQLPPGCGDPKRLGPLRGFQWVTGDNNT domainofCD141 SYSRWARLDLNGAPLCGPLCVAVSAAEATVPSEPIWEEQ (bold),CLEC14A QCEVKADGFLCEFQFEVLCPAPRPGAASNLSYRAPFQLH (non-bold,non- SAALDFSPPGTEVSALCRGQLPISVTCIADEIGARWDKLSG italics),GFP DVLCPCPGRYLRAGKCAELPNCLDDLGGFACECATGFEL (italics) GKDGRSCVTSGEGQPTLGGTGVPTRRPPATATSPVPQRT WPIRVDEKLGETPLVPEQDNSVTSIPEIPRWGSQSTMSTL QMSLQAESKATITPSGSVISKFNSTTSSATPQAFDSSSAVV FIFVSTAVVVLVILTMTVLGLVKLCFHESPSSQPRKESMGP PGLESDPEPAALGSSSAHCTNNGVKVGDCDLRDRAEGAL LAESPLGSSDALQSTVPRARDPPVATMVSKGEELFTGVVPI LVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP VPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDG NILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRD HMVLLEFVTAAGITLGMDELYK 27 Chimera6GFP MRPAFALCLLWQALWPGPGGGEHPTADRAGCSASGAC fusionaminoacid YSLHHATMKRQAAEEACILRGGALSTVRAGAELRAVLAL ofCLEC14A,with LRAGPGPGGGSKDLLFWVALERRRSHCTLENEPLRGFS substitutedsushi WLSSDPGGLESDTLQWVEEPQRSCTARRCAVLQATGGV ofCD141 EPAGWKEMRCHLRANGYLCKYHFPATCRPLAVEPGAAA (underlined)and AAVSITYGTPFAARGADFQALPVGSSAAVAPLGLQLMCTA GFPinitalics PPGAVQGHWAREAPGACPGRYLRAGKCAELPNCLDDLG GFACECATGFELGKDGRSCVTSGEGQPTLGGTGVPTRRP PATATSPVPQRTWPIRVDEKLGETPLVPEQDNSVTSIPEIP RWGSQSTMSTLQMSLQAESKATITPSGSVISKFNSTTSSA TPQAFDSSSAVVFIFVSTAVVVLVILTMTVLGLVKLCFHESP SSQPRKESMGPPGLESDPEPAALGSSSAHCTNNGVKVGD CDLRDRAEGALLAESPLGSSDALQSTVPRARDPPVATMVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI ELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS ALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK 28 MMRN2amino MILSLLFSLGGPLGWGLLGAWAQASSTSLSDLQSSRTPGV acid WKAEAEDTGKDPVGRNWCPYPMSKLVTLLALCKTEKFLIH SQQPCPQGAPDCQKVKVMYRMAHKPVYQVKQKVLTSLA WRCCPGYTGPNCEHHDSMAIPEPADPGDSHQEPQDGPV SFKPGHLAAVINEVEVQQEQQEHLLGDLQNDVHRVADSLP GLWKALPGNLTAAVMEANQTGHEFPDRSLEQVLLPHVDTF LQVHFSPIWRSFNQSLHSLTQAIRNLSLDVEANRQAISRVQ DSAVARADFQELGAKFEAKVQENTQRVGQLRQDVEDRLH AQHFTLHRSISELQADVDTKLKRLHKAQEAPGTNGSLVLAT PGAGARPEPDSLQARLGQLQRNLSELHMTTARREEELQY TLEDMRATLTRHVDEIKELYSESDETFDQISKVERQVEELQ VNHTALRELRVILMEKSLIMEENKEEVERQLLELNLTLQHLQ GGHADLIKYVKDCNCQKLYLDLDVIREGQRDATRALEETQ VSLDERRQLDGSSLQALQNAVDAVSLAVDAHKAEGERAR AATSRLRSQVQALDDEVGALKAAAAEARHEVRQLHSAFAA LLEDALRHEAVLAALFGEEVLEEMSEQTPGPLPLSYEQIRV ALQDAASGLQEQALGWDELAARVTALEQASEPPRPAEHL EPSHDAGREEAATTALAGLARELQSLSNDVKNVGRCCEAE AGAGAASLNASLDGLHNALFATQRSLEQHQRLFHSLFGNF QGLMEANVSLDLGKLQTMLSRKGKKQQKDLEAPRKRDKK EAEPLVDIRVTGPVPGALGAALWEAGSPVAFYASFSEGTA ALQTVKFNTTYINIGSSYFPEHGYFRAPERGVYLFAVSVEF GPGPGTGQLVFGGHHRTPVCTTGQGSGSTATVFAMAELQ KGERVWFELTQGSITKRSLSGTAFGGFLMFKT 29 MMRN2 ATGATCCTGAGCTTGCTGTTCAGCCTTGGGGGCCCCCT nucleotide GGGCTGGGGGCTGCTGGGGGCATGGGCCCAGGCTTCC AGTACTAGCCTCTCTGATCTGCAGAGCTCCAGGACACCT GGGGTCTGGAAGGCAGAGGCTGAGGACACCGGCAAGG ACCCCGTTGGACGTAACTGGTGCCCCTACCCAATGTCC AAGCTGGTCACCTTACTAGCTCTTTGCAAAACAGAGAAA TTCCTCATCCACTCGCAGCAGCCGTGTCCGCAGGGAGC TCCAGACTGCCAGAAAGTCAAAGTCATGTACCGCATGG CCCACAAGCCAGTGTACCAGGTCAAGCAGAAGGTGCTG ACCTCTTTGGCCTGGAGGTGCTGCCCTGGCTACACGGG CCCCAACTGCGAGCACCACGATTCCATGGCAATCCCTG AGCCTGCAGATCCTGGTGACAGCCACCAGGAACCTCAG GATGGACCAGTCAGCTTCAAACCTGGCCACCTTGCTGC AGTGATCAATGAGGTTGAGGTGCAACAGGAACAGCAGG AACATCTGCTGGGAGATCTCCAGAATGATGTGCACCGG GTGGCAGACAGCCTGCCAGGCCTGTGGAAAGCCCTGC CTGGTAACCTCACAGCTGCAGTGATGGAAGCAAATCAAA CAGGGCACGAGTTCCCTGATAGATCCTTGGAGCAGGTG CTGCTACCCCACGTGGACACCTTCCTACAAGTGCATTTC AGCCCCATCTGGAGGAGCTTTAACCAAAGCCTGCACAG CCTTACCCAGGCCATAAGAAACCTGTCTCTTGACGTGGA GGCCAACCGCCAGGCCATCTCCAGAGTCCAGGACAGTG CCGTGGCCAGGGCTGACTTCCAGGAGCTTGGTGCCAAA TTTGAGGCCAAGGTCCAGGAGAACACTCAGAGAGTGGG TCAGCTGCGACAGGACGTGGAGGACCGCCTGCACGCC CAGCACTTTACCCTGCACCGCTCGATCTCAGAGCTCCAA GCCGATGTGGACACCAAATTGAAGAGGCTGCACAAGGC TCAGGAGGCCCCAGGGACCAATGGCAGTCTGGTGTTGG CAACGCCTGGGGCTGGGGCAAGGCCTGAGCCGGACAG CCTGCAGGCCAGGCTGGGCCAGCTGCAGAGGAACCTC TCAGAGCTGCACATGACCACGGCCCGCAGGGAGGAGG AGTTGCAGTACACCCTGGAGGACATGAGGGCCACCCTG ACCCGGCACGTGGATGAGATCAAGGAACTGTACTCCGA ATCGGACGAGACTTTCGATCAGATTAGCAAGGTGGAGC GGCAGGTGGAGGAGCTGCAGGTGAACCACACGGCGCT CCGTGAGCTGCGCGTGATCCTGATGGAGAAGTCTCTGA TCATGGAGGAGAACAAGGAGGAGGTGGAGCGGCAGCT CCTGGAGCTCAACCTCACGCTGCAGCACCTGCAGGGTG GCCATGCCGACCTCATCAAGTACGTGAAGGACTGCAAT TGCCAGAAGCTCTATTTAGACCTGGACGTCATCCGGGA GGGCCAGAGGGACGCCACGCGTGCCCTGGAGGAGACC CAGGTGAGCCTGGACGAGCGGCGGCAGCTGGACGGCT CCTCCCTGCAGGCCCTGCAGAACGCCGTGGACGCCGT GTCGCTGGCCGTGGACGCGCACAAAGCGGAGGGCGAG CGGGCGCGGGCGGCCACGTCGCGGCTCCGGAGCCAA GTGCAGGCGCTGGATGACGAGGTGGGCGCGCTGAAGG CGGCCGCGGCCGAGGCCCGCCACGAGGTGCGCCAGCT GCACAGCGCCTTCGCCGCCCTGCTGGAGGACGCGCTG CGGCACGAGGCGGTGCTGGCCGCGCTCTTCGGGGAGG AGGTGCTGGAGGAGATGTCTGAGCAGACGCCGGGACC GCTGCCCCTGAGCTACGAGCAGATCCGCGTGGCCCTG CAGGACGCCGCTAGCGGGCTGCAGGAGCAGGCGCTCG GCTGGGACGAGCTGGCCGCCCGAGTGACGGCCCTGGA GCAGGCCTCGGAGCCCCCGCGGCCGGCAGAGCACCTG GAGCCCAGCCACGACGCGGGCCGCGAGGAGGCCGCC ACCACCGCCCTGGCCGGGCTGGCGCGGGAGCTCCAGA GCCTGAGCAACGACGTCAAGAATGTCGGGCGGTGCTGC GAGGCTGAGGCCGGGGCCGGGGCCGCCTCCCTCAACG CCTCCCTTGACGGCCTCCACAACGCACTCTTCGCCACT CAGCGCAGCTTGGAGCAGCACCAGCGGCTCTTCCACAG CCTCTTTGGGAACTTCCAAGGGCTCATGGAAGCCAACG TCAGCCTGGACCTGGGGAAGCTGCAGACCATGCTGAGC AGGAAAGGGAAGAAGCAGCAGAAAGACCTGGAAGCTCC CCGGAAGAGGGACAAGAAGGAAGCGGAGCCTTTGGTG GACATACGGGTCACAGGGCCTGTGCCAGGTGCCTTGG GCGCGGCGCTCTGGGAGGCAGGATCCCCTGTGGCCTT CTATGCCAGCTTTTCAGAAGGGACGGCTGCCCTGCAGA CAGTGAAGTTCAACACCACATACATCAACATTGGCAGCA GCTACTTCCCTGAACATGGCTACTTCCGAGCCCCTGAG CGTGGTGTCTACCTGTTTGCAGTGAGCGTTGAATTTGGC CCAGGGCCAGGCACCGGGCAGCTGGTGTTTGGAGGTC ACCATCGGACTCCAGTCTGTACCACTGGGCAGGGGAGT GGAAGCACAGCAACGGTCTTTGCCATGGCTGAGCTGCA GAAGGGTGAGCGAGTATGGTTTGAGTTAACCCAGGGAT CAATAACAAAGAGAAGCCTGTCGGGCACTGCATTTGGG GGCTTCCTGATGTTTAAGACCTGA 30 siRNAduplexD1 GAACAAGACAATTCAGTAA 31 siRNAduplexD2 CAATCAGGGTCGACGAGAA CRT1Version(V1)-complementaritydeterminingregions 32 CRT1V1HC SSYWIE CDR1 33 CRT1V1HC WIGEILPGSGSTN CDR2 34 CRT1V1HC ARGGDYDEEYYVMD CDR3 35 CRT1V1LC SYMYWY CDR1 36 CRT1V1LC LLIYDTSNLA CDR2 37 CRT1V1LC QQWSSYPL CDR3 38 CRT1V1HC AGTAGCTACTGGATAGAG CDR1 (nucleotide) 39 CRT1V1HC TGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAAT CDR2 (nucleotide) 40 CRT1V1HC GCAAGAGGGGGGGATTACGACGAAGAATACTATGTCAT CDR3 GGAC (nucleotide) 41 CRT1V1LC AGTTACATGTACTGGTAC CDR1 (nucleotide) 42 CRT1V1LC CTCCTGATTTATGACACATCCAACCTGGCT CDR2 (nucleotide) 43 CRT1V1LC CAGCAGTGGAGTAGTTACCCGCTC CDR3 (nucleotide) CRT1Version2(V2)-Complementaritydeterminingregions 44 CRT1V2HC GYTFSSYW CDR1 45 CRT1V2HC ILPGSGST CDR2 46 CRT1V2HC ARGGDYDEEYYVMDY CDR3 47 CRT1V2LC SSVSY CDR1 CRT1V2LC DTS CDR2 49 CRT1V2LC QQWSSYPLT CDR3 50 CRT1V2HC GGCTACACATTCAGTAGCTACTGG CDR1 (nucleotide) 51 CRT1V2HC ATTTTACCTGGAAGTGGTAGTACT CDR2 (nucleotide) 52 CRT1V2HC GCAAGAGGGGGGGATTACGACGAAGAATACTATGTCAT CDR3 GGACTAC (nucleotide) 53 CRT1V2LC TCAAGTGTAAGTTAC CDR1 (nucleotide) CRT1V2LC GACACATCC CDR2 (nucleotide) 55 CRT1V2LC CAGCAGTGGAGTAGTTACCCGCTCACG CDR3 (nucleotide) CRT1heavyandlightvariableregions,scFv,CARs 56 CRT1HCprotein MAEVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVK QRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNT AYMQLSSLTSEDSAVYYCARGGDYDEEYYVMDYWGQGT SVTV 57 CRT1LCprotein QIVLTQSPAIMSASPGEKVTMTCSASSSV-- SYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG AGTKLELKR 58 CRT1ScFv MAEVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVK protein QRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNT AYMQLSSLTSEDSAVYYCARGGDYDEEYYVMDYWGQGT SVTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG AGTKLELKRAAA 59 CRT1HC ATGGCCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCT nucleotide GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCT GAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGA TTACGACGAAGAATACTATGTCATGGACTACTGGGGTCA AGGAACCTCAGTCACTGTC 60 CRT1LC CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA nucleotide TCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG CTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCC AGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAA CCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTG GGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGG AGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG AGTAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAGCTGAAACGT 61 CRT1ScFv ATGGCCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCT nucleotide GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCT GAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGA TTACGACGAAGAATACTATGTCATGGACTACTGGGGTCA AGGAACCTCAGTCACTGTCTCCTCAGGTGGAGGCGGTT CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAAAT TGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAA GTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGAT CCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGG CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGC TGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAG TTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAACGTGCGGCCGCA 62 CRT1CAR1 MGVLLTQRTLLSLVLALLFPSMA protein SMAEVQLQQSGAELMKPGASVK ISCKATGYTFSSYWIEWVKQRP GHGLEWIGEILPGSGSTNYNEK FKGKATFTADTSSNTAYMQLSS LTSEDSAVYYCARGGDYDEEYY VMDYWGQGTSVTVSSGGGGSG GGGSGGGGSQIVLTQSPAIMSA SPGEKVTMTCSASSSVSYMYWY QQKPGSSPRLLIYDTSNLASGV PVRFSGSGSGTSYSLTISRMEA EDAATYYCQQWSSYPLTFGAGT KLELKRAAAIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGP SKPFWVLVVVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRR GRDPEMGGKPQRRKNPQEGLY NELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDA LHMQALPPR 63 CRT1CAR1 ATGGGCGTGCTGCTGACCCAGAGGACCCTGCTGAGCCT nucleotide GGTGCTGGCCCTGCTGTTTCCATCTATGGCATCGATGG CCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCTGATG AAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTAC TGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAA GCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGA TTTTACCTGGAAGTGGTAGTACTAATTACAATGAGAAGTT CAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCA ACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAG GACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGATTA CGACGAAGAATACTATGTCATGGACTACTGGGGTCAAG GAACCTCAGTCACTGTCTCCTCAGGTGGAGGCGGTTCA GGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAAATTG TTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAG GGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGT GTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATC CTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGC TTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTG GGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCT GAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGT TACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCT GAAACGTGCGGCCGCAATTGAAGTTATGTATCCTCCTCC TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCA TGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCC CGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTG GCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGG CTCCTGCACAGTGACTACATGAACATGACTCCCCGCCG CCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC CACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCG GGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAG AACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAG GCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACG CCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAAAAG CTTAACACGAGCCA CRT3Version1(V1)-complementaritydeterminingregions 64 CRT3V1HC TSYWMH CDR1 65 CRT3V1HC WIGAIYPGNSDTS CDR2 66 CRT3V1HC TH---YYGSDYAMD CDR3 67 CRT3V1LC SSSYLHWY CDR1 68 CRT3V1LC LWIYSTSNLA CDR2 69 CRT3V1LC HQYHRSPR CDR3 70 CRT3V1HC ACCAGCTACTGGATGCAC CDR1 (nucleotide) 71 CRT3V1HC TGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGC CDR2 (nucleotide) 72 CRT3V1HC ACACATTACTACGGTAGTGACTATGCTATGGAC CDR3 (nucleotide) 73 CRT3V1LC AGTTCCAGTTACTTGCACTGGTAC CDR1 (nucleotide) 74 CRT3V1LC CTCTGGATTTATAGCACATCCAACCTGGCT CDR2 (nucleotide) 75 CRT3V1LC CCACCAGTATCATCGTTCCCCACGG CDR3 (nucleotide) CRT3Version2-complementaritydeterminingregions 76 CRT3V2HC GYTFTSYW CDR1 77 CRT3V2HC IYPGNSDT CDR2 78 CRT3V2HC THYYGSDYAMDY CDR3 79 CRT3V2LC SSVSSSY CDR1 CRT3V2LC STS CDR2 81 CRT3V2LC HQYHRSPRT CDR3 82 CRT3V2HC GGCTACACCTTTACCAGCTACTGG CDR1 (nucleotide) 83 CRT3V2HC ATTTATCCTGGAAATAGTGATACT CDR2 (nucleotide) 84 CRT3V2HC ACACATTACTACGGTAGTGACTATGCTATGGACTAC CDR3 (nucleotide) 85 CRT3V2LC TCAAGTGTAAGTTCCAGTTAC CDR1 (nucleotide) CRT3V2LC AGCACATCC CDR2 (nucleotide) 87 CRT3V2LC CACCAGTATCATCGTTCCCCACGGACG CDR3 (nucleotide) CRT3heavyandlightchainvariableregions,scFvandCARs 88 CRT3HCV1 MAEVQLQQSGTVLARPGASVKM SCKASGYTFTSYWMHWVKQRP GQGLEWIGAIYPGNSDTSYNQK FKGKAKLTAVTSTSTAYMELSS LTNEDSAVFYCTHYYGSDYAMD YWGQGTSVTISSG 89 CRT3LCV1 QIVLTQSPAIMSASLGERVTMT CTASSSVSSSYLHWYQQKPGSS PKLWIYSTSNLASGVPARFSGS GSGTSYSLTISSMEAEDAATYY CHQYHRSPRTFGGGTKLEIKRA A 90 CRT3HCV2 MAEVQLQQSGTVLARPGASVKM SCKASGYTFTSYWMHWVKQRP GQGLEWIGAIYPGNSDTSYNQK FKGKAKLTAVTSTSTAYMELSS LTNEDSAVFYCTHYYGSDYAMD YWGQGTSVTV 91 CRT3LCV2 QIVLTQSPAIMSASLGERVTMT CTASSSVSSSYLHWYQQKPGSS PKLWIYSTSNLASGVPARFSGS GSGTSYSLTISSMEAEDAATYY CHQYHRSPRTFGGGTKLEIKRA AA 92 CRT3HCV1 ATGGCCGAGGTCCAGCTGCAGCAGTCTGGGACTGTGCT nucleotide GGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGC AAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGAT TGGCGCTATTTATCCTGGAAATAGTGATACTAGCTACAA CCAGAAGTTCAAGGGCAAGGCCAAACTG ACTGCAGTCACATCCACCAGCACTGCCTACATGGAGCT CAGCAGCCTGACAAATGAGGACTCTGCGGTCTTT TACTGTACACATTACTACGGTAGTGACTATGCTATGGAC TACTGGGGTCAAGGAACCTCAGTCACTGTCTCC TCA 93 CRT3LCV1 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA nucleotide TCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAG CTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCA GAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCAC ATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTG GCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCA GCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAC CAGTATCATCGTTCCCCACGGACGTTCGGTGGAGGCAC CAAGCTGGAAATCAAACGTGCGGCCGC 94 CRT3HCV2 ATGGCCGAGGTCCAGCTGCAGCAGTCTGGGACTGTGCT nucleotide GGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGG CTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGC GCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAG AAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATC CACCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAA ATGAGGACTCTGCGGTCTTTTACTGTACACATTACTACG GTAGTGACTATGCTATGGACTACTGGGGTCAAGGAACC TCAGTCACTGTC 95 CRT3LCV2 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA nucleotide TCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAG CTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCA GAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCAC ATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTG GCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCA GCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAC CAGTATCATCGTTCCCCACGGACGTTCGGTGGAGGCAC CAAGCTGGAAATCAAACGT 96 CRT3scFv MAEVQLQQSGTVLARPGASVKM SCKASGYTFTSYWMHWVKQRP GQGLEWIGAIYPGNSDTSYNQK FKGKAKLTAVTSTSTAYMELSS LTNEDSAVFYCTHYYGSDYAMD YWGQGTSVTVSSGGGGSGGGG SGGGGSQIVLTQSPAIMSASLG ERVTMTCTASSSVSSSYLHWYQ QKPGSSPKLWIYSTSNLASGVP ARFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPRTFGGGTK LEIKRAAA 97 CRT3scFv ATGGCCGAGGTCCAGCTGCAGCAGTCTGGGACTGTGCT nucleotide GGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGG CTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGC GCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAG AAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATC CACCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAA ATGAGGACTCTGCGGTCTTTTACTGTACACATTACTACG GTAGTGACTATGCTATGGACTACTGGGGTCAAGGAACC TCAGTCACTGTCTCCTCAGGTGGAGGCGGTTCAGGCGG AGGTGGCTCTGGCGGTGGCGGATCGCAAATTGTTCTCA CCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAAC GGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGT TCCAGTTACTTGCACTGGTACCAGCAGAAGCCAGGATC CTCCCCCAAACTCTGGATTTATAGCACATCCAACCTGGC TTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTG GGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCT GAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGT TCCCCACGGACGTTCGGTGGAGGCACCAAGCTGGAAAT CAAACGTGCGGCCGCA 98 CRT3CAR3 MGVLLTQRTLLSLVLALLFPSMA SMAEVQLQQSGTVLARPGASVK MSCKASGYTFTSYWMHWVKQR PGQGLEWIGAIYPGNSDTSYNQ KFKGKAKLTAVTSTSTAYMELS SLTNEDSAVFYCTHYYGSDYAM DYWGQGTSVTVSSGGGGSGGG GSGGGGSQIVLTQSPAMSASL GERVTMTCTASSSVSSSYLHWY QQKPGSSPKLWIYSTSNLASGV PARFSGSGSGTSYSLTISSMEA EDAATYYCHQYHRSPRTFGGGT KLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPS KPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRG RDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDAL HMQALPPR 99 CRT3CAR3 atgggcgtgctgctgacccagaggaccctgctgagcctggtgctggccctgctgttt nucleotide ccatctatggcatcgatggccgaggtccagctgcagcagtctgggactgtgctggc aaggcctggggcttcagtgaagatgtcctgcaaggcttctggctacacctttaccag ctactggatgcactgggtaaaacagaggcctggacagggtctggaatggattggc gctatttatcctggaaatagtgatactagctacaaccagaagttcaagggcaaggc caaactgactgcagtcacatccaccagcactgcctacatggagctcagcagcctg acaaatgaggactctgcggtcttttactgtacacattactacggtagtgactatgctat ggactactggggtcaaggaacctcagtcactgtctcctcaggtggaggcggttcag gcggaggtggctctggcggtggcggatcgcaaattgttctcacccagtctccagca atcatgtctgcatctctaggggaacgggtcaccatgacctgcactgccagctcaagt gtaagttccagttacttgcactggtaccagcagaagccaggatcctcccccaaact ctggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtggcagt gggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgc cacttattactgccaccagtatcatcgttccccacggacgttcggtggaggcaccaa gctggaaatcaaacgtgcggccgcaattgaagttatgtatcctcctccttacctaga caatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtcca agtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtc ctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaag aggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggg cccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatc gctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggc cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttt tggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaa ggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggc acgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccct tcacatgcaggccctgccccctcgctaataaaagcttaacacgagcca CRT4Version1(V1)-complementaritydeterminingregions 32 CRT4V1HC SSYWIE CDR1 33 CRT4V1HC WIGEILPGSGSTN CDR2 100 CRT4V1HC ARGGDYDEEYYLMD CDR3 35 CRT4V1LC SYMYWY CDR1 36 CRT4V1LC LLIYDTSNLA CDR2 37 CRT4V1LC QQWSSYPL CDR3 38 CRT4V1HC AGTAGCTACTGGATAGAG CDR1 (nucleotide) 39 CRT4V1HC TGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAAT CDR2 (nucleotide) 101 CRT4V1HC GCGAGAGGGGGGGATTACGACGAAGAATACTATCTCAT CDR3 GGAC (nucleotide) 41 CRT4V1LC AGTTACATGTACTGGTAC CDR1 (nucleotide) 42 CRT4V1LC CTCCTGATTTATGACACATCCAACCTGGCT CDR2 (nucleotide) 43 CRT4V1LC CAGCAGTGGAGTAGTTACCCGCTC CDR3 (nucleotide) CRT4Version2(V2)-Complementaritydeterminingregions 44 CRT4V2HC GYTFSSYW CDR1 45 CRT4V2HC ILPGSGST CDR2 102 CRT4V2HC ARGGDYDEEYYLMDY CDR3 47 CRT4V2LC SSVSY CDR1 CRT4V2LC DTS CDR2 49 CRT4V2LC QQWSSYPLT CDR3 50 CRT4V2HC GGCTACACATTCAGTAGCTACTGG CDR1 (nucleotide) 51 CRT4V2HC ATTTTACCTGGAAGTGGTAGTACT CDR2 (nucleotide) 103 CRT4V2HC GCGAGAGGGGGGGATTACGACGAAGAATACTATCTCAT CDR3 TAC (nucleotide) 53 CRT4V2LC TCAAGTGTAAGTTAC CDR1 (nucleotide) CRT4V2LC GACACATCC CDR2 (nucleotide) 55 CRT4V2LC CAGCAGTGGAGTAGTTACCCGCTCACG CDR3 (nucleotide) CRT4heavyandlightchainvariableregions,scFvandCARs 104 CRT4V1HC MAQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NRRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN TAYMQLSSLTSEDSAVYYCARGGDYDEEYYLMDYWGQGT TLTVSS 105 CRT4V1LC QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPG SSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAED AATYYCQQWSSYPLTFGAGTKLEIKRAA 106 CRT4V2HC MAQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NRRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN TAYMQLSSLTSEDSAVYYCARGGDYDEEYYLMDYWGQGT TLTV 107 CRT4V2LC QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPG SSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAED AATYYCQQWSSYPLTFGAGTKLEIKRAAA 108 CRT4V1HC ATGGCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCT (nucleotide) GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAACCGGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAATACAGCCTACATGCAACTCAGCAGCCTCACATCT GAGGACTCTGCCGTCTATTACTGTGCGAGAGGGGGGGA TTACGACGAAGAATACTATCTCATGGACTACTGGGGTCA AGGCACCACTCTCACAGTCTCCTCA 109 CRT4V1LC CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA (nucleotide) TCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG CTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCC AGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAA CCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTG GGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGG AGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG AGTAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAAATCAAACGTGCGGCCGC 110 CRT4V2HC ATGGCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCT (nucleotide) GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAACCGGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAATACAGCCTACATGCAACTCAGCAGCCTCACATCT GAGGACTCTGTCGTCTATTACTGTGCGAGAGGGGGGGA TTACGACGAAGAATACTATCTCATGGACTACTGGGGTCA AGGCACCACTCTCACAGTC 111 CRT4V2LC CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA (nucleotide) TCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG CTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCC AGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAA CCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTG GGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGG AGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG AGTAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAAATCAAACGT 112 CRT4scFv MAQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NRRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN TAYMQLSSLTSEDSVVYYCARGGDYDEEYYLMDYWGQGT TLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG AGTKLEIKRAAA 113 CRT4scFv ATGGCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCT (nucleotide) GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAACCGGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAATACAGCCTACATGCAACTCAGCAGCCTCACATCT GAGGACTCTGTCGTCTATTACTGTGCGAGAGGGGGGGA TTACGACGAAGAATACTATCTCATGGACTACTGGGGTCA AGGCACCACTCTCACAGTCTCCTCAGGTGGAGGCGGTT CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAAAT TGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAA GTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGAT CCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGG CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGC TGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAG TTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAA TCAAACGTGCGGCCGCA 114 CAR4CRT4 MGVLLTQRTLLSLVLALLFPSMA SMAQVQLQQSGAELMKPGASVK ISCKATGYTFSSYWIEWVNRRP GHGLEWIGEILPGSGSTNYNEK FKGKATFTADTSSNTAYMQLSS LTSEDSVVYYCARGGDYDEEYY LMDYWGQGTTLTVSSGGGGSG GGGSGGGGSQIVLTQSPAIMSA SPGEKVTMTCSASSSVSYMYWY QQKPGSSPRLLIYDTSNLASGV PVRFSGSGSGTSYSLTISRMEA EDAATYYCQQWSSYPLTFGAGT KLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPS KPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRG RDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDAL HMQALPPR 115 CAR4CRT4 atggcccaggttcagctgcagcagtctggagctgagctgatgaagcctggggcct (nucleotide) cagtgaagatatcctgcaaggctactggctacacattcagtagctactggatagagt gggtaaaccggaggcctggacatggccttgagtggattggagagattttacctgga agtggtagtactaattacaatgagaagttcaagggcaaggccacattcactgcag atacatcctccaatacagcctacatgcaactcagcagcctcacatctgaggactct gtcgtctattactgtgcgagagggggggattacgacgaagaatactatctcatgga ctactggggtcaaggcaccactctcacagtctcctcaggtggaggcggttcaggcg gaggtggctctggcggtggcggatcgcaaattgttctcacccagtctccagcaatc atgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgt aagttacatgtactggtaccagcagaagccaggatcctcccccagactcctgattta tgacacatccaacctggcttctggagtccctgttcgcttcagtggcagtgggtctggg acctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattact gccagcagtggagtagttacccgctcacgttcggtgctgggaccaagctggaaat caaacgtgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaa gagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatt tcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgcta tagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcagg ctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgc aagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagt gaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccag ctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaaga gacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaacc ctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctac agtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcc tttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgca ggccctgccccctcgctaataa CRT5Version1(V1)-complementaritydeterminingregions 32 CRT5V1HC SSYWIE CDR1 33 CRT5V1HC WIGEILPGSGSTN CDR2 116 CRT5V1HC ARGGDYDEEYYAMD CDR3 35 CRT5V1LC SYMYWY CDR1 36 CRT5V1LC LLIYDTSNLA CDR2 37 CRT5V1LC QQWSSYPL CDR3 38 CRT5V1HC AGTAGCTACTGGATAGAG CDR1 (nucleotide) 39 CRT5V1HC TGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAAT CDR2 (nucleotide) 117 CRT5V1HC GCAAGAGGGGGGGATTACGACGAAGAATACTATGCTAT CDR3 GGAC (nucleotide) 41 CRT5V1LC AGTTACATGTACTGGTAC CDR1 (nucleotide) 42 CRT5V1LC CTCCTGATTTATGACACATCCAACCTGGCT CDR2 (nucleotide) 43 CRT5V1LC CAGCAGTGGAGTAGTTACCCGCTC CDR3 (nucleotide) CRT5Version2(V2)-complementaritydeterminingregions 44 CRT5V2HC GYTFSSYW CDR1 45 CRT5V2HC ILPGSGST CDR2 118 CRT5V2HC ARGGDYDEEYYAMDY CDR3 47 CRT5V2LC SSVSY CDR1 CRT5V2LC DTS CDR2 49 CRT5V2LC QQWSSYPLT CDR3 50 CRT5V2HC GGCTACACATTCAGTAGCTACTGG CDR1 (nucleotide) 51 CRT5V2HC ATTTTACCTGGAAGTGGTAGTACT CDR2 (nucleotide) 120 CRT5V2HC GCAAGAGGGGGGGATTACGACGAAGAATACTATGCTAT CDR3 GGACTAC (nucleotide) 53 CRT5V2LC TCAAGTGTAAGTTAC CDR1 (nucleotide) CRT5V2LC GACACATCC CDR2 (nucleotide) 55 CRT5V2LC CAGCAGTGGAGTAGTTACCCGCTCACG CDR3 (nucleotide) CRT5heavyandlightchainvariableregions,scFvandCARs 121 CRT5HC MAEVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NQRPGHGLEWIGEILPGSGST NYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCAR GGDYDEEYYAMDYWGQGTSVTL 122 CRT5LC QIVLTQSPAIMSASPGEKVTMTCSASSSV-- SYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDGATYYCQQWSSYPLTF GAGTKLELKR 123 CRT5HC ATGGCCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCT (nucleotide) GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAATCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCT GAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGA TTACGACGAAGAATACTATGCTATGGACTACTGGGGTCA AGGAACCTCAGTCACCCTC 124 CRT5LC CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA (nucleotide) TCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG CTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCC AGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAA CCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTG GGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGG AGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG AGTAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAGCTGAAACGT 125 CRT5scFv MAEVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN TAYMQLSSLTSEDSAVYYCARGGDYDEEYYAMDYWGQG TSVTLSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGE KVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGV PVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTF GAGTKLELKRAAA 126 CRT5scFv ATGGCCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCT (nucleotide) GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAATCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCT GAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGA TTACGACGAAGAATACTATGCTATGGACTACTGGGGTCA AGGAACCTCAGTCACCCTCTCCTCAGGTGGAGGCGGTT CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAAAT TGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAA GTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGAT CCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGG CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGC TGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAG TTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAACGTGCGGCCGCA 127 CAR5CRT5- MGVLLTQRTLLSLVLALLFPSM oncostatinleader ASMAEVQLQQSGAELMKPGASV (bold),scFv KISCKATGYTFSSYWIEWVNQR CRT5(italics), PGHGLEWIGEILPGSGSTNYNE CD28-CD3zeta KFKGKATFTADTSSNTAYMQLS restofsequence SLTSEDSAVYYCARGGDYDEEY YAMDYWGQGTSVTLSSGGGGS GGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMYW YQQKPGSSPRLLIYDTSNLASG VPVRFSGSGSGTSYSLTISRME AEDAATYYCQQWSSYPLTFGAG TKLELKRAAAIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGP SKPFWVLVVVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRR GRDPEMGGKPQRRKNPQEGLY NELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDA LHMQALPPR 128 CAR5CRT5 atgggcgtgctgctgacccagaggaccctgctgagcctggtgctggccctgctgttt (nucleotide) ccatctatggcatcgatggccgaggttcagcttcagcagtctggagctgagctgatg aagcctggggcctcagtgaagatatcctgcaaggctactggctacacattcagtag ctactggatagagtgggtaaatcagaggcctggacatggccttgagtggattggag agattttacctggaagtggtagtactaattacaatgagaagttcaagggcaaggcc acattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctga catctgaggactctgccgtctattactgtgcaagagggggggattacgacgaaga atactatgctatggactactggggtcaaggaacctcagtcaccctctcctcaggtgg aggcggttcaggcggaggtggctctggcggtggcggatcgcaaattgttctcaccc agtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagt gccagctcaagtgtaagttacatgtactggtaccagcagaagccaggatcctcccc cagactcctgatttatgacacatccaacctggcttctggagtccctgttcgcttcagtg gcagtgggtctgggacctcttactctctcacaatcagccgaatggaggctgaagat gctgccacttattactgccagcagtggagtagttacccgctcacgttcggtgctggg accaagctggagctgaaacgtgcggccgcaattgaagttatgtatcctcctccttac ctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacaccttt gtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtg gagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgagga gtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcag cctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagc agggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgca gagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataag atggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggca aggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacg acgcccttcacatgcaggccctgccccctcgctaataaaagcttaacacgagcca CRT2complementaritydeterminingregions 129 CRT2LCCDR1 SYMHWF 68 CRT2LCCDR2 LWIYSTSNLA 130 CRT2LCCDR3 QQRSSYPL 131 CRT2LCCDR1 AGTTACATGCACTGGTTC (nucleotide) 74 CRT2LCCDR2 CTCTGGATTTATAGCACATCCAACCTGGCT (nucleotide) 132 CRT2LCCDR3 CAGCAAAGGAGTAGTTACCCCCTC (nucleotide) 133 CRT2LC EIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGT SPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDA ATYYCQQRSSYPLTFGAPGKLELKRAA 134 CRT2LC GAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA (nucleotide) TCTCCAGGGGAGAAGGTCACC ATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCAC TGGTTCCAGCAGAAG CCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCC AACCTGGCTTCTGGAGTCCCT GCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTC TCTCACAATCAGCCGAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGT AGTTACCCCCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAACGTGCGGCCGC Others 135 OncostatinM MGVLLTQRTLLSLVLALLFPSMAS leadersequence 136 CAR5-CRT5: MPRGWTALCLLSLLPSGFMSLD truncatedCD34 NNGTATPELPTQGTFSNVSTNV pluspeptide2A SYQETTTPSTLGSTSLHPVSQH linker(bold),scFv GNEATTNITETTVKFTSTSVITS CRT5(italics), VYGNTNSSVQSQTSVISTVFTT CD28-CD3zeta, PANVSTPETTLKPSLSPGNVSD restofsequence LSTTSTSLATSPTKPYTSSSPIL SDIKAEIKCSGIREVKLTQGICL EQNKTSSCAEFKKDRGEGLARV LCGEEQADADAGAQVCSLLLA QSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQ DVASHQSYSQKTLIALVTSGAL LAVLGITGYFLMNRRSWSPTGE RLELEPVDRVKQTLNFDLLKLA GDVESNPGPGNMGVLLTQRTLL SLVLALLFPSMASMAEVQLQQS GAELMKPGASVKISCKATGYTF SSYWIEWVNQRPGHGLEWIGEI LPGSGSTNYNEKFKGKATFTAD TSSNTAYMQLSSLTSEDSAVYY CARGGDYDEEYYAMDYWGQGT SVTLSSGGGGSGGGGSGGGGS QIVLTQSPAIMSASPGEKVTMT CSASSSVSYMYWYQQKPGSSP RLLIYDTSNLASGVPVRFSGSG SGTSYSLTISRMEAEDAATYYC QQWSSYPLTFGAGTKLELKRAA AIEVMYPPPYLDNEKSNGTIIHV KGKHLCPSPLFPGPSKPFWVLV VVGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKF SRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGG KPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR 137 CAR5-CRT5: atgcctcgcggctggacagccctgtgcctgctgtctctgctgccatccggct truncatedCD34 tcatgagcctggataataacggcacagccaccccagagctgcctacacag pluspeptide2A ggcaccttcagcaatgtgtccacaaacgtgagctatcaggagaccacaac linker(bold),scFv cccttctaccctgggatccacaagcctgcaccccgtgtctcagcacggcaa CRT5(italics), cgaagccaccaccaacatcaccgagaccacagtgaagtttacctccacct CD28-CD3zeta ctgtgattacctctgtgtacggaaatacaaactccagcgtgcagtctcagac (nucleotide)rest atctgtgatctccacagtgtttacaacacctgccaatgtgtccaccccagaga ofsequence caaccctgaagcccagcctgtctcctggaaatgtgtccgatctgtctaccac ctccaccagcctggccacctctcccaccaagccctatacctcctcttctccc atcctgagcgatatcaaagccgagatcaaatgcagcgggattcgggaagt gaaactgacacagggcatctgcctggaacagaataagacatccagctgcg ccgagtttaagaaagatagaggagagggactggccagggtgctgtgtggc gaagagcaggccgacgccgatgccggcgcccaggtgtgttccctgctgct ggcccagtctgaggtgcgcccccagtgcctgctgctggtgctggccaatcg gacagaaattagcagcaagctgcagctgatgaaaaaacaccagagcgatc tgaaaaagctgggcatcctggactttaccgagcaggacgtggcctctcacc agagctacagccagaaaacactgatcgccctggtgaccagcggagccct gctggccgtgctgggcatcaccggatatttcctgatgaataggcgcagctg gagccccaccggcgagcggctggagctggagcctgtcgaccgagtgaa gcagaccctgaactttgatctgctgaagctggccggcgacgtggagtccaa ccccgggccagggaatatgggcgtgctgctgacccagaggaccctgctg agcctggtgctggccctgctgtttccatctatggcatcgatggccgaggttcag cttcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctg caaggctactggctacacattcagtagctactggatagagtgggtaaatcagagg cctggacatggccttgagtggattggagagattttacctggaagtggtagtactaatt acaatgagaagttcaagggcaaggccacattcactgcagatacatcctccaaca cagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattactgtg caagagggggggattacgacgaagaatactatgctatggactactggggtcaag gaacctcagtcaccctctcctcaggtggaggcggttcaggcggaggtggctctggc ggtggcggatcgcaaattgttctcacccagtctccagcaatcatgtctgcatctccag gggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgtactgg taccagcagaagccaggatcctcccccagactcctgatttatgacacatccaacct ggcttctggagtccctgttcgcttcagtggcagtgggtctgggacctcttactctctcac aatcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagta gttacccgctcacgttcggtgctgggaccaagctggagctgaaacgtgcggccgc aattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccatt atccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagc ccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacag tggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgacta catgaacatgactccccgccgccccgggcccacccgcaagcattaccagcccta tgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagc gcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaa tctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccc tgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtac aatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaa aggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagta cagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgct aataa 138 CAR1CRT1: MPRGWTALCLLSLLPSGFMSLD truncatedCD34 NNGTATPELPTQGTFSNVSTNV pluspeptide2A SYQETTTPSTLGSTSLHPVSQH linker(bold),scFv GNEATTNITETTVKFTSTSVITS CRT1(italics), VYGNTNSSVQSQTSVISTVFTT CD28-CD3zeta, PANVSTPETTLKPSLSPGNVSD restofsequence LSTTSTSLATSPTKPYTSSSPIL SDIKAEIKCSGIREVKLTQGICL EQNKTSSCAEFKKDRGEGLARV LCGEEQADADAGAQVCSLLLA QSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQ DVASHQSYSQKTLIALVTSGAL LAVLGITGYFLMNRRSWSPTGE RLELEPVDRVKQTLNFDLLKLA GDVESNPGPGNMGVLLTQRTLL SLVLALLFPSMAS MAEVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVK QRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNT AYMQLSSLTSEDSAVYYCARGGDYDEEYYVMDYWGQGT SVTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG AGTKLELKRAAAIEVMYPPPYLDNEKSN GTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYR SRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDP EMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQ ALPPR 139 CAR1CRT1: atgcctcgcggctggacagccctgtgcctgctgtctctgctgccatccggct truncatedCD34 tcatgagcctggataataacggcacagccaccccagagctgcctacacag pluspeptide2A ggcaccttcagcaatgtgtccacaaacgtgagctatcaggagaccacaac linker(bold),scFv cccttctaccctgggatccacaagcctgcaccccgtgtctcagcacggcaa CRT1(italics), cgaagccaccaccaacatcaccgagaccacagtgaagtttacctccacct CD28-CD3zeta, ctgtgattacctctgtgtacggaaatacaaactccagcgtgcagtctcagac restofsequence atctgtgatctccacagtgtttacaacacctgccaatgtgtccaccccagaga (nucleotide) caaccctgaagcccagcctgtctcctggaaatgtgtccgatctgtctaccac ctccaccagcctggccacctctcccaccaagccctatacctcctcttctccc atcctgagcgatatcaaagccgagatcaaatgcagcgggattcgggaagt gaaactgacacagggcatctgcctggaacagaataagacatccagctgcg ccgagtttaagaaagatagaggagagggactggccagggtgctgtgtggc gaagagcaggccgacgccgatgccggcgcccaggtgtgttccctgctgct ggcccagtctgaggtgcgcccccagtgcctgctgctggtgctggccaatcg gacagaaattagcagcaagctgcagctgatgaaaaaacaccagagcgatc tgaaaaagctgggcatcctggactttaccgagcaggacgtggcctctcacc agagctacagccagaaaacactgatcgccctggtgaccagcggagccct gctggccgtgctgggcatcaccggatatttcctgatgaataggcgcagctg gagccccaccggcgagcggctggagctggagcctgtcgaccgagtgaa gcagaccctgaactttgatctgctgaagctggccggcgacgtggagtccaa ccccgggccagggaatatgggcgtgctgctgacccagaggaccctgctg agcctggtgctggccctgctgtttccatctatggcatcg ATGGCCGAGGTTCAGCTTCAGCAGTCTGGAGCTGAGCT GATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGG CTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGG TAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAG AAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCC TCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCT GAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGGGGA TTACGACGAAGAATACTATGTCATGGACTACTGGGGTCA AGGAACCTCAGTCACTGTCTCCTCAGGTGGAGGCGGTT CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAAAT TGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAA GTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGAT CCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGG CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGC TGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAG TTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAACGTGCGGCCGCAaattgaagttatgtatcctcctccttacctaga caatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtcca agtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtc ctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaag aggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggg cccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatc gctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggc cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttt tggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaa ggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggc acgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccct tcacatgcaggccctgccccctcgctaataa 140 CAR3CRT3: MPRGWTALCLLSLLPSGFMSLD truncatedCD34 NNGTATPELPTQGTFSNVSTNV pluspeptide2A SYQETTTPSTLGSTSLHPVSQH linker(bold),scFv GNEATTNITETTVKFTSTSVITS CRT3(italics), VYGNTNSSVQSQTSVISTVFTT CD28-CD3zeta, PANVSTPETTLKPSLSPGNVSD restofsequence LSTTSTSLATSPTKPYTSSSPIL SDIKAEIKCSGIREVKLTQGICL EQNKTSSCAEFKKDRGEGLARV LCGEEQADADAGAQVCSLLLA QSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQ DVASHQSYSQKTLIALVTSGAL LAVLGITGYFLMNRRSWSPTGE RLELEPVDRVKQTLNFDLLKLA GDVESNPGPGNMGVLLTQRTLL SLVLALLFPSMASMAEVQLQQS GTVLARPGASVKMSCKASGYTF TSYWMHWVKQRPGQGLEWIGAI YPGNSDTSYNQKFKGKAKLTAV TSTSTAYMELSSLTNEDSAVFY CTHYYGSDYAMDYWGQGTSVT VSSGGGGSGGGGSGGGGSQIV LTQSPAIMSASLGERVTMTCTA SSSVSSSYLHWYQQKPGSSPKL WIYSTSNLASGVPARFSGSGSG TSYSLTISSMEAEDAATYYCHQ YHRSPRTFGGGTKLEIKRAAAIE VMYPPPYLDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVV GGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKP QRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR 141 CAR3CRT3: ATGCCTCGCGGCTGGACAGCCCTGTGCCTGCTGTCTCT truncatedCD34 GCTGCCATCCGGCTTCATGAGCCTGGATAATAACGGCA pluspeptide2A CAGCCACCCCAGAGCTGCCTACACAGGGCACCTTCAG linker(bold),scFv CAATGTGTCCACAAACGTGAGCTATCAGGAGACCACA CRT3(italics), ACCCCTTCTACCCTGGGATCCACAAGCCTGCACCCCGT D28-CD3zeta, GTCTCAGCACGGCAACGAAGCCACCACCAACATCACC restofsequence GAGACCACAGTGAAGTTTACCTCCACCTCTGTGATTAC (nucleotide) CTCTGTGTACGGAAATACAAACTCCAGCGTGCAGTCTC AGACATCTGTGATCTCCACAGTGTTTACAACACCTGCC AATGTGTCCACCCCAGAGACAACCCTGAAGCCCAGCC TGTCTCCTGGAAATGTGTCCGATCTGTCTACCACCTCC ACCAGCCTGGCCACCTCTCCCACCAAGCCCTATACCTC CTCTTCTCCCATCCTGAGCGATATCAAAGCCGAGATCA AATGCAGCGGGATTCGGGAAGTGAAACTGACACAGGG CATCTGCCTGGAACAGAATAAGACATCCAGCTGCGCC GAGTTTAAGAAAGATAGAGGAGAGGGACTGGCCAGGG TGCTGTGTGGCGAAGAGCAGGCCGACGCCGATGCCGG CGCCCAGGTGTGTTCCCTGCTGCTGGCCCAGTCTGAG GTGCGCCCCCAGTGCCTGCTGCTGGTGCTGGCCAATC GGACAGAAATTAGCAGCAAGCTGCAGCTGATGAAAAA ACACCAGAGCGATCTGAAAAAGCTGGGCATCCTGGAC TTTACCGAGCAGGACGTGGCCTCTCACCAGAGCTACA GCCAGAAAACACTGATCGCCCTGGTGACCAGCGGAGC CCTGCTGGCCGTGCTGGGCATCACCGGATATTTCCTGA TGAATAGGCGCAGCTGGAGCCCCACCGGCGAGCGGCT GGAGCTGGAGCCTGTCGACCGAGTGAAGCAGACCCTG AACTTTGATCTGCTGAAGCTGGCCGGCGACGTGGAGT CCAACCCCGGGCCAGGGAATATGGGCGTGCTGCTGAC CCAGAGGACCCTGCTGAGCCTGGTGCTGGCCCTGCTG TTTCCATCTATGGCATCGATGGCCGAGGTCCAGCTGCA GCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACC AGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACA GGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAG TGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCA AACTGACTGCAGTCACATCCACCAGCACTGCCTACATG GAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTT TTACTGTACACATTACTACGGTAGTGACTATGCTATGGA CTACTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAG GTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG CGGATCGCAAATTGTTCTCACCCAGTCTCCAGCAATCAT GTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCA CTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGT ACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATT TATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCTCG CTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCAC AATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTA CTGCCACCAGTATCATCGTTCCCCACGGACGTTCGGTG GAGGCACCAAGCTGGAAATCAAACGTGCGGCCGCAAAT TGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAA GAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCT TTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTT TTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCT ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAA GCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAG CCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGT ACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCC ACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGCTAATAA 142 CAR4CRT4: MPRGWTALCLLSLLPSGFMSLD truncatedCD34 NNGTATPELPTQGTFSNVSTNV pluspeptide2A SYQETTTPSTLGSTSLHPVSQH linker(bold),scFv GNEATTNITETTVKFTSTSVITS CRT4(italics), VYGNTNSSVQSQTSVISTVFTT CD28-CD3zeta, PANVSTPETTLKPSLSPGNVSD restofsequence LSTTSTSLATSPTKPYTSSSPIL SDIKAEIKCSGIREVKLTQGICL EQNKTSSCAEFKKDRGEGLARV LCGEEQADADAGAQVCSLLLA QSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQ DVASHQSYSQKTLIALVTSGAL LAVLGITGYFLMNRRSWSPTGE RLELEPVDRVKQTLNFDLLKLA GDVESNPGPGNMGVLLTQRTLL SLVLALLFPSMAS MAQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWV NRRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN TAYMQLSSLTSEDSVVYYCARGGDYDEEYYLMDYWGQGT TLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG AGTKLEIKRAAAIEVMYPPPYLDNEKSN GTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYR SRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDP EMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQ ALPPR 143 CAR4CRT4: ATGCCTCGCGGCTGGACAGCCCTGTGCCTGCTGTCTCT truncatedCD34 GCTGCCATCCGGCTTCATGAGCCTGGATAATAACGGCA pluspeptide2A CAGCCACCCCAGAGCTGCCTACACAGGGCACCTTCAG linker(bold),scFv CAATGTGTCCACAAACGTGAGCTATCAGGAGACCACA CRT4(italics), ACCCCTTCTACCCTGGGATCCACAAGCCTGCACCCCGT CD28-CD3zeta, GTCTCAGCACGGCAACGAAGCCACCACCAACATCACC restofsequence GAGACCACAGTGAAGTTTACCTCCACCTCTGTGATTAC (nucleotide) CTCTGTGTACGGAAATACAAACTCCAGCGTGCAGTCTC AGACATCTGTGATCTCCACAGTGTTTACAACACCTGCC AATGTGTCCACCCCAGAGACAACCCTGAAGCCCAGCC TGTCTCCTGGAAATGTGTCCGATCTGTCTACCACCTCC ACCAGCCTGGCCACCTCTCCCACCAAGCCCTATACCTC CTCTTCTCCCATCCTGAGCGATATCAAAGCCGAGATCA AATGCAGCGGGATTCGGGAAGTGAAACTGACACAGGG CATCTGCCTGGAACAGAATAAGACATCCAGCTGCGCC GAGTTTAAGAAAGATAGAGGAGAGGGACTGGCCAGGG TGCTGTGTGGCGAAGAGCAGGCCGACGCCGATGCCGG CGCCCAGGTGTGTTCCCTGCTGCTGGCCCAGTCTGAG GTGCGCCCCCAGTGCCTGCTGCTGGTGCTGGCCAATC GGACAGAAATTAGCAGCAAGCTGCAGCTGATGAAAAA ACACCAGAGCGATCTGAAAAAGCTGGGCATCCTGGAC TTTACCGAGCAGGACGTGGCCTCTCACCAGAGCTACA GCCAGAAAACACTGATCGCCCTGGTGACCAGCGGAGC CCTGCTGGCCGTGCTGGGCATCACCGGATATTTCCTGA TGAATAGGCGCAGCTGGAGCCCCACCGGCGAGCGGCT GGAGCTGGAGCCTGTCGACCGAGTGAAGCAGACCCTG AACTTTGATCTGCTGAAGCTGGCCGGCGACGTGGAGT CCAACCCCGGGCCAGGGAATATGGGCGTGCTGCTGAC CCAGAGGACCCTGCTGAGCCTGGTGCTGGCCCTGCTG TTTCCATCTATGGCATCGATGGCCCAGGTTCAGCTGCA GCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAG TGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTA GCTACTGGATAGAGTGGGTAAACCGGAGGCCTGGACAT GGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGG TAGTACTAATTACAATGAGAAGTTCAAGGGCAAGGCCAC ATTCACTGCAGATACATCCTCCAATACAGCCTACATGCA ACTCAGCAGCCTCACATCTGAGGACTCTGTCGTCTATTA CTGTGCGAGAGGGGGGGATTACGACGAAGAATACTATC TCATGGACTACTGGGGTCAAGGCACCACTCTCACAGTC TCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTG GCGGTGGCGGATCGCAAATTGTTCTCACCCAGTCTCCA GCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTG GTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGA TTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTC GCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTC ACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTA TTACTGCCAGCAGTGGAGTAGTTACCCGCTCACGTTCG GTGCTGGGACCAAGCTGGAAATCAAACGTGCGGCCGCA AATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAG AAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACAC CTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTG CTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC AGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTAT AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCC TGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCTAATAA 144 TruncatedCD34 MPRGWTALCLLSLLPSGFMSLD pluspeptide2A NNGTATPELPTQGTFSNVSTNV linker SYQETTTPSTLGSTSLHPVSQH GNEATTNITETTVKFTSTSVITS VYGNTNSSVQSQTSVISTVFTT PANVSTPETTLKPSLSPGNVSD LSTTSTSLATSPTKPYTSSSPIL SDIKAEIKCSGIREVKLTQGICL EQNKTSSCAEFKKDRGEGLARV LCGEEQADADAGAQVCSLLLAQ SEVRPQCLLLVLANRTEISSKLQ LMKKHQSDLKKLGILDFTEQDV ASHQSYSQKTLIALVTSGALLAV LGITGYFLMNRRSWSPTGERLE LEPVDRVKQTLNFDLLKLAGDV ESNPGPGNMGVLLTQRTLLSLV LALLFPSMAS 145 TruncatedCD34 ATGCCTCGCGGCTGGACAGCCCTGTGCCTGCTGTCTCT pluspeptide2A GCTGCCATCCGGCTTCATGAGCCTGGATAATAACGGCA linker(nucleotide CAGCCACCCCAGAGCTGCCTACACAGGGCACCTTCAGC AATGTGTCCACAAACGTGAGCTATCAGGAGACCACAAC CCCTTCTACCCTGGGATCCACAAGCCTGCACCCCGTGT CTCAGCACGGCAACGAAGCCACCACCAACATCACCGAG ACCACAGTGAAGTTTACCTCCACCTCTGTGATTACCTCT GTGTACGGAAATACAAACTCCAGCGTGCAGTCTCAGAC ATCTGTGATCTCCACAGTGTTTACAACACCTGCCAATGT GTCCACCCCAGAGACAACCCTGAAGCCCAGCCTGTCTC CTGGAAATGTGTCCGATCTGTCTACCACCTCCACCAGCC TGGCCACCTCTCCCACCAAGCCCTATACCTCCTCTTCTC CCATCCTGAGCGATATCAAAGCCGAGATCAAATGCAGC GGGATTCGGGAAGTGAAACTGACACAGGGCATCTGCCT GGAACAGAATAAGACATCCAGCTGCGCCGAGTTTAAGA AAGATAGAGGAGAGGGACTGGCCAGGGTGCTGTGTGG CGAAGAGCAGGCCGACGCCGATGCCGGCGCCCAGGTG TGTTCCCTGCTGCTGGCCCAGTCTGAGGTGCGCCCCCA GTGCCTGCTGCTGGTGCTGGCCAATCGGACAGAAATTA GCAGCAAGCTGCAGCTGATGAAAAAACACCAGAGCGAT CTGAAAAAGCTGGGCATCCTGGACTTTACCGAGCAGGA CGTGGCCTCTCACCAGAGCTACAGCCAGAAAACACTGA TCGCCCTGGTGACCAGCGGAGCCCTGCTGGCCGTGCT GGGCATCACCGGATATTTCCTGATGAATAGGCGCAGCT GGAGCCCCACCGGCGAGCGGCTGGAGCTGGAGCCTGT CGACCGAGTGAAGCAGACCCTGAACTTTGATCTGCTGA AGCTGGCCGGCGACGTGGAGTCCAACCCCGGGCCAGG GAATATGGGCGTGCTGCTGACCCAGAGGACCCTGCTGA GCCTGGTGCTGGCCCTGCTGTTTCCATCTATGGCATCG 146 CD28 IEVMYPPPYLDNEKSNGTIIHVK GKHLCPSPLFPGPSKPFWVLVV VGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRS 147 CD28nucleotide AATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAG AAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACAC CTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTG CTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC AGCCTATCGCTCC 148 CD3zeta RVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPE MGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQA LPPR 149 CD3zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT (nucleotide) ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAG AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACA CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC TAATAA 150 Consensus S/TSYWI/ME/H sequencefor CRT1,3,4and5 V1heavychain CDR1 151 Consensus GYTFS/TSYW sequencefor CRT1,3,4and5 V2heavychain CDR1 152 Consensus WIGE/AIL/YPGS/NG/SS/DTN/S sequencefor CRT1,3,4and5 V1heavychain CDR2 153 Consensus IL/YPGS/NG/SS/DT sequencefor CRT1,3,4and5 V2heavychain CDR2 154 Consensus A/TR/HG/XG/XD/XYD/YE/GE/SY/DYV/A/LMD sequencefor CRT1,3,4and5 V1heavychain CDR3 155 Consensus A/TR/HG/XG/XD/XYD/YE/GE/SY/DYV/A/LMDY sequencefor CRT1,3,4and5 V2heavychain CDR3 156 Consensus S/XS/XSYM/LY/HWY sequencefor CRT1,3,4and5 V1lightchain CDR1 157 Consensus SSVSY/SS/XY/X sequencefor CRT1,3,4,and5 V2lightchain CDR1 158 Consensus LL/WIYD/STSNLA sequencefor CRT1,3,4and5 V1lightchain CDR2 Consensus D/STS sequencefor CRT1,3,4and5 V2lightchain CDR2 160 Consensus Q/HQW/YS/HS/RY/SPL/R sequencefor CRT1,3,4and5 V1lightchain CDR3 161 Consensus Q/HQW/YS/HS/RY/SPL/RTF/X sequencefor CRT1,3,4and5 V2lightchain CDR3 119 Nucleotide ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT sequencefor CCTTCTCCTGTCACTGGTTATCACCCTTTACTGC CD8? transmembrane domain 162 Nucleotide ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGC sequenceof CTTGCTGCTCCACGCCGCCAGGCCG CD8? leader 163 CH2CH3hinge DPAEPKSPDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKKDPK 164 Nucleotidefor AAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCC CH2CH3hinge AGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGGTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAAGGGCCGGCCGCT 165 CD8? hinge FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACD 166 ShortenedIgG AEPKSPDKTHTCP hinge 159 Linker KDPK 86 OX40(CD134) RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI co-stimulatory domain 80 Nucleotidefor4- AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA 1BB CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT costimulatory GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG domain ATGTGAACTG 54 Nucleotidefor TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTG CD28 CTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG costimulatory GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA domain CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC AGCCTATCGCTCC 48 Nucleotidefor AGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCC OX40 CTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGA costimulatory GCAGGCCGACGCCCACTCCACCCTGGCCAAGATC domain CRT2heavychainCDRsandsequences 167 Heavychain GFTFNTYA CDR1 168 Heavychain IRSKSNNYAT CDR2 169 Heavychain VREGVYYYGSSGYYAMDY CDR3 170 Ntheavychain GGTTTCACCTTCAATACCTATGCC CDR1 171 Ntheavychain ATAAGAAGTAAAAGTAATAATTATGCAACA CDR2 172 Ntheavychain GTGAGAGAAGGGGTTTATTACTACGGTAGTAGTGGGTACTATGCTATGGACTAC CDR3 173 Heavychain MAEVQGVESGGGLVQPKGSLKLSCAAS GFTFNTYAMHWVCQAPGKGLEWVARI RSKSNNYATYYADSVKDRFTISRDDSQS MLYLQMNNLKTEDTAMYYCVREGVYY YGSSGYYAMDYWGQGTSVTVSSG 174 Ntheavychain GACGCTTATCGATGGCCGAGGTGCAGGGGGTGGAGTCTGGTGGAGGATTGGTGCA GCCTAAAGGATCATTGAAACTCTCATGTGCCGCCTCTGGTTTCACCTTCAATACC TATGCCA TGCACTGGGTCTGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAG AAGTAAAAGTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGATTC ACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACCTGA AAACTGAGGACACAGCCATGTATTACTGTGTGAGAGAAGGGGTTTATTACTACGG TAGTAGTGGGTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC TCCTCAGGT 175 CRT2scFv MAEVQGVESGGGLVQPKGSLKLSCAAS GFTFNTYAMHWVCQAPGKGLEWVARI RSKSNNYATYYADSVKDRFTISRDDSQS MLYLQMNNLKTEDTAMYYCVREGVYY YGSSGYYAMDYWGQGTSVTVSSGGGGGS GGGGSGGGGSEIVLTQSPAIMSASPGEKVTITCSASSSVS YMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTS YSLTISRMEAEDAATYYCQQRSSYPLTFGAPGKLELKRAA 176 CRT2scFv GACGCTTATCGATGGCCGAGGTGCAGGGGGTGGAGTCTGGTGGAGGATTGGTGCA Nucleotide GCCTAAAGGATCATTGAAACTCTCATGTGCCGCCTCTGGTTTCACCTTCAATACC TATGCCA TGCACTGGGTCTGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAG AAGTAAAAGTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGATTC ACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACCTGA AAACTGAGGACACAGCCATGTATTACTGTGTGAGAGAAGGGGTTTATTACTACGG TAGTAGTGGGTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC TCCTCAGGT TCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTG GCGGTGGCGGATCG GAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCA TCTCCAGGGGAGAAGGTCACC ATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCAC TGGTTCCAGCAGAAG CCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCC AACCTGGCTTCTGGAGTCCCT GCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTC TCTCACAATCAGCCGAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGT AGTTACCCCCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAACGTGCGGCCGC CodonoptimisedscFvnucleotidesequences(hu-codonoptimisedforexpressionin humancells,mu-codonoptimisedforexpressioninmurinecells) 177 CRT1scFvhu ATGGCAGAGGTGCAGCTGCAGCAGAGCGGAGCAGAGCTGATGAAGC CAGGAGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTATACATTCA GCTCCTACTGGATCGAGTGGGTGAAGCAGCGGCCTGGCCACGGCCTG GAGTGGATCGGAGAGATCCTGCCAGGCAGCGGCTCCACCAACTATAA TGAGAAGTTCAAGGGCAAGGCCACCTTTACAGCCGACACCTCTAGCAA CACAGCCTACATGCAGCTGTCCTCTCTGACAAGCGAGGATTCCGCCGT GTACTATTGCGCCAGGGGCGGCGACTATGATGAGGAGTACTATGTGA TGGACTACTGGGGCCAGGGCACCTCCGTGACCGTGAGCAGCGGcGGA GGcGGCAGCGGAGGAGGAGGCTCCGGCGGCGGCGGCTCTCAGATCG TGCTGACCCAGAGCCCAGCAATCATGTCTGCCAGCCCAGGAGAGAAG GTGACCATGACATGTTCCGCCTCTAGCTCCGTGAGCTACATGTATTGGT ATCAGCAGAAGCCCGGCTCTAGCCCTCGGCTGCTGATCTATAGAACCT CCAATCTGGCATCTGGCGTGCCCGCAAGGTTCTCCGGCTCTGGCAGCG GCACCTCCTACTCTCTGACCATCGGCACAATGGAGGCCGAGGATGCCG CCACATACTATTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGCGC CGGCACAAAGCTGGAGATCAAGCGCGCGGCCGCA 178 CRT1scFvmu ATGGCTGAGGTGCAGCTGCAGCAGTCCGGAGCTGAGCTGATGAAGCC AGGCGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTACACATTCAG CTCCTACTGGATCGAGTGGGTGAAGCAGAGGCCTGGCCACGGACTGG AGTGGATCGGAGAGATCCTGCCAGGCAGCGGCAGCACCAACTACAAC GAGAAGTTCAAGGGCAAGGCTACCTTTACAGCCGACACCTCTAGCAAC ACAGCTTACATGCAGCTGTCCTCTCTGACAAGCGAGGATAGCGCCGTG TACTACTGCGCCAGGGGCGGAGACTACGATGAGGAGTACTACGTGAT GGACTACTGGGGCCAGGGAACCTCTGTGACCGTGAGCAGCGGAGGA GGAGGAAGCGGCGGAGGAGGCAGCGGAGGAGGAGGATCTCAGATC GTGCTGACCCAGAGCCCAGCTATCATGTCTGCCAGCCCCGGCGAGAAG GTGACCATGACATGTAGCGCCTCTAGCTCCGTGTCCTACATGTACTGGT ATCAGCAGAAGCCCGGATCTAGCCCTAGGCTGCTGATCTACAGAACAT CCAACCTGGCTTCTGGCGTGCCCGCTCGGTTCTCCGGCTCTGGAAGCG GCACCTCCTACTCTCTGACCATCGGCACAATGGAGGCTGAGGATGCCG CTACATACTACTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGAGC CGGCACAAAGCTGGAGATCAAGCGCGCGGCCGCA 179 CRT2scFvhu ATGGCAGAGGTGCAGGGAGTGGAGAGCGGAGGCGGCCTGGTGCAGC CTAAGGGCTCCCTGAAGCTGTCTTGCGCCGCCAGCGGCTTCACCTTTAA CACATATGCAATGCACTGGGTGTGCCAGGCACCAGGCAAGGGCCTGG AGTGGGTGGCACGGATCAGAAGCAAGTCCAACAATTATGCCACCTACT ATGCCGACAGCGTGAAGGATAGGTTCACAATCTCCCGCGACGATTCTC AGAGCATGCTGTACCTGCAGATGAACAATCTGAAGACCGAGGACACA GCCATGTACTATTGCGTGCGGGAGGGCGTGTACTATTACGGCAGCTCC GGCTATTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGT GTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGGCGGCGGC GGCAGCGAGATCGTGCTGACCCAGTCCCCAGCAATCATGTCCGCCTCT CCAGGAGAGAAGGTGACCATCACATGCTCCGCCTCCTCTAGCGTGTCT TATATGCACTGGTTCCAGCAGAAGCCCGGCACCTCTCCTAAGCTGTGG ATCTACAGCACATCCAATCTGGCATCCGGCGTGCCCGCAAGGTTTTCTG GCAGCGGCTCCGGCACCTCTTATAGCCTGACAATCAGCCGGATGGAG GCAGAGGACGCAGCAACCTATTACTGTCAGCAGAGATCCTCTTACCCT CTGACCTTTGGCGCCGGCACAAAGCTGGAGCTGAAGCGCGCGGCCGC A 180 CRT2scFvmu ATGGCTGAGGTGCAGGGAGTGGAGAGCGGAGGAGGCCTGGTGCAGC CTAAGGGCTCCCTGAAGCTGTCTTGCGCCGCTAGCGGATTCACCTTTAA CACATACGCTATGCACTGGGTGTGCCAGGCTCCAGGAAAGGGCCTGG AGTGGGTGGCCAGGATCAGAAGCAAGTCCAACAACTACGCTACCTACT ACGCCGACAGCGTGAAGGATCGGTTCACAATCTCCCGCGACGATTCTC AGAGCATGCTGTACCTGCAGATGAACAACCTGAAGACCGAGGACACA GCTATGTACTACTGCGTGCGGGAGGGCGTGTACTACTACGGCAGCTCC GGATACTACGCTATGGACTACTGGGGACAGGGCACCTCCGTGACAGT GTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGGAGGCGGA GGCAGCGAGATCGTGCTGACCCAGTCTCCAGCTATCATGTCCGCCTCT CCCGGCGAGAAGGTGACCATCACATGCTCCGCCTCCTCTAGCGTGTCT TACATGCACTGGTTCCAGCAGAAGCCCGGCACCTCTCCTAAGCTGTGG ATCTACAGCACATCCAACCTGGCTAGCGGAGTGCCCGCTCGGTTTTCT GGAAGCGGCTCCGGAACCTCTTACAGCCTGACAATCTCCAGGATGGA GGCTGAGGACGCCGCTACATACTACTGTCAGCAGAGATCCTCTTACCC TCTGACCTTTGGCGCCGGAACAAAGCTGGAGCTGAAGCGCGCGGCCG CA 181 CRT3scFvhu ATGGCCGAGGTGCAGCTGCAGCAGTCTGGCACCGTGCTGGCCAGGCC CGGAGCAAGCGTGAAGATGTCCTGCAAGGCCTCTGGCTACACCTTCAC AAGCTATTGGATGCACTGGGTGAAGCAGCGCCCAGGACAGGGCCTGG AGTGGATCGGAGCAATCTACCCCGGCAACTCCGACACCTCTTATAATC AGAAGTTCAAGGGCAAGGCCAAGCTGACAGCCGTGACCTCTACAAGC ACCGCCTACATGGAGCTGAGCAGCCTGACCAACGAGGATAGCGCCGT GTTTTATTGCACACACTACTATGGCTCCGACTACGCTATGGACTATTGG GGCCAGGGCACCTCCGTGACAGTGTCTAGCGGAGGAGGAGGCAGCG GAGGAGGAGGCTCCGGCGGCGGCGGCTCTCAGATCGTGCTGACCCAG AGCCCTGCCATCATGTCCGCCTCTCTGGGCGAGCGGGTGACAATGACC TGTACAGCCTCCTCTAGCGTGTCCTCTAGCTACCTGCACTGGTATCAGC AGAAGCCCGGCTCCTCTCCTAAGCTGTGGATCTACAGCACCTCCAATCT GGCATCCGGCGTGCCTGCAAGGTTCTCTGGCAGCGGCTCCGGCACCTC TTACAGCCTGACAATCAGCAGCATGGAGGCAGAGGACGCAGCAACAT ACTATTGTCACCAGTATCACCGGAGCCCAAGAACCTTTGGCGGCGGCA CAAAGCTGGAGATCAAGCGGGCGGCCGCA 182 CRT3scFvmu ATGGCCGAGGTGCAGCTGCAGCAGTCTGGCACCGTGCTGGCTCGGCC CGGAGCTAGCGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCAC AAGCTACTGGATGCACTGGGTGAAGCAGCGCCCAGGACAGGGCCTGG AGTGGATCGGCGCCATCTACCCCGGAAACTCCGACACCTCTTACAACC AGAAGTTCAAGGGCAAGGCTAAGCTGACAGCCGTGACCTCTACAAGC ACCGCTTACATGGAGCTGAGCAGCCTGACCAACGAGGATAGCGCCGT GTTTTACTGCACACACTACTACGGCTCCGACTACGCTATGGATTACTGG GGACAGGGCACCTCCGTGACAGTGTCTAGCGGAGGAGGAGGAAGCG GCGGAGGcGGCAGCGGAGGAGGAGGATCTCAGATCGTGCTGACCCA GTCTCCTGCTATCATGTCCGCCTCTCTGGGCGAGAGGGTGACAATGAC CTGTACAGCCTCCTCTAGCGTGTCCTCTAGCTACCTGCACTGGTATCAG CAGAAGCCCGGCTCCTCTCCTAAGCTGTGGATCTACAGCACCTCCAACC TGGCTTCCGGAGTGCCTGCTCGGTTCTCTGGAAGCGGCTCCGGAACCT CTTACAGCCTGACAATCAGCAGCATGGAGGCTGAGGACGCCGCTACAT ACTACTGTCACCAGTACCACAGGAGCCCAAGAACCTTTGGCGGAGGCA CAAAGCTGGAGATCAAGAGGGCGGCCGCA 183 CRT4scFvhu ATGGCACAGGTGCAGCTGCAGCAGAGCGGAGCAGAGCTGATGAAGC CAGGAGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTATACATTCA GCTCCTACTGGATCGAGTGGGTGAACAGACGGCCCGGCCACGGCCTG GAGTGGATCGGAGAGATCCTGCCAGGCAGCGGCTCCACCAACTATAA TGAGAAGTTCAAGGGCAAGGCCACCTTTACAGCCGACACCTCTAGCAA TACAGCCTACATGCAGCTGTCCTCTCTGACAAGCGAGGATTCCGTGGT GTACTATTGCGCCAGGGGCGGCGACTATGATGAGGAGTACTATCTGAT GGACTACTGGGGCCAGGGCACCACACTGACCGTGAGCAGCGGAGGA GGAGGCAGCGGAGGAGGAGGCTCCGGCGGCGGCGGCTCTCAGATCG TGCTGACACAGTCCCCAGCAATCATGTCTGCCAGCCCAGGAGAGAAGG TGACCATGACATGTTCCGCCTCTAGCTCCGTGAGCTACATGTATTGGTA TCAGCAGAAGCCCGGCTCTAGCCCTAGGCTGCTGATCTATGACACCTC CAACCTGGCATCTGGCGTGCCCGTGCGCTTCTCCGGCTCTGGCAGCGG CACCTCCTACTCTCTGACAATCAGCCGGATGGAGGCAGAGGATGCAGC AACCTACTATTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGCGCC GGCACAAAGCTGGAGATCAAGCGGGCGGCCGCA 184 CRT4scFvmu ATGGCTCAGGTGCAGCTGCAGCAGTCCGGAGCTGAGCTGATGAAGCC AGGCGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTACACATTCAG CTCCTACTGGATCGAGTGGGTGAACAGGAGGCCCGGCCACGGACTGG AGTGGATCGGAGAGATCCTGCCAGGCAGCGGCAGCACCAACTACAAC GAGAAGTTCAAGGGCAAGGCTACCTTTACAGCCGACACCTCTAGCAAC ACAGCTTACATGCAGCTGTCCTCTCTGACAAGCGAGGATTCCGTGGTG TACTACTGCGCCAGGGGCGGAGACTACGATGAGGAGTACTACCTGAT GGACTACTGGGGCCAGGGAACCACACTGACCGTGAGCAGCGGAGGA GGAGGAAGCGGCGGAGGAGGCAGCGGAGGAGGAGGATCTCAGATC GTGCTGACACAGTCTCCAGCTATCATGTCTGCCAGCCCCGGCGAGAAG GTGACCATGACATGTAGCGCCAGCAGCAGCGTGAGCTACATGTACTG GTATCAGCAGAAGCCCGGATCTAGCCCTCGGCTGCTGATCTACGACAC CTCCAACCTGGCTTCTGGCGTGCCCGTGCGCTTCTCCGGCTCTGGAAGC GGCACCTCCTACTCTCTGACAATCAGCAGGATGGAGGCTGAGGATGCC GCTACATACTACTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGAG CCGGCACAAAGCTGGAGATCAAGAGGGCGGCCGCA 185 CRT5scFvhu ATGGCAGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGATGAAGCC AGGAGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTATACATTCAG CTCCTACTGGATCGAGTGGGTGAACCAGCGCCCTGGCCACGGCCTGG AGTGGATCGGAGAGATCCTGCCAGGCAGCGGCTCCACCAACTATAAT GAGAAGTTCAAGGGCAAGGCCACCTTTACAGCCGACACCTCTAGCAAT ACAGCCTACATGCAGCTGTCCTCTCTGACAAGCGAGGATTCCGCCGTG TACTATTGCGCCAGAGGCGGCGACTATGATGAGGAGTACTATGCTATG GACTACTGGGGCCAGGGCACCTCTGTGACCCTGAGCAGCGGAGGAGG AGGCAGCGGcGGAGGAGGCTCCGGCGGCGGCGGCTCTCAGATCGTG CTGACCCAGAGCCCAGCAATCATGTCTGCCAGCCCAGGAGAGAAGGT GACCATGACATGTAGCGCCTCTAGCTCCGTGTCCTACATGTATTGGTAT CAGCAGAAGCCCGGCTCTAGCCCTCGGCTGCTGATCTATGACACCTCC AACCTGGCCTCTGGCGTGCCCGTGAGATTCTCCGGCTCTGGCAGCGGC ACCTCCTACTCTCTGACAATCAGCAGGATGGAGGCCGAGGATGCCGCC ACATACTATTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGCGCCG GCACAAAGCTGGAGCTGAAGAGGGCGGCCGCA 186 CRT5scFvmu ATGGCTGAGGTGCAGCTGCAGCAGTCCGGAGCTGAGCTGATGAAGCC AGGCGCCTCTGTGAAGATCAGCTGTAAGGCCACCGGCTACACATTCAG CTCCTACTGGATCGAGTGGGTGAACCAGCGCCCTGGCCACGGACTGG AGTGGATCGGAGAGATCCTGCCAGGCAGCGGCAGCACCAACTACAAC GAGAAGTTCAAGGGCAAGGCTACCTTTACAGCCGACACCTCTAGCAAC ACAGCTTACATGCAGCTGTCCTCTCTGACAAGCGAGGATAGCGCCGTG TACTACTGCGCCAGGGGCGGAGACTACGATGAGGAGTACTACGCTAT GGACTACTGGGGCCAGGGAACCTCTGTGACCCTGAGCAGCGGAGGAG GAGGAAGCGGCGGAGGAGGCAGCGGAGGAGGAGGATCTCAGATCGT GCTGACCCAGAGCCCAGCTATCATGTCTGCCAGCCCCGGCGAGAAGGT GACCATGACATGTAGCGCCAGCAGCAGCGTGAGCTACATGTACTGGT ATCAGCAGAAGCCCGGATCTAGCCCTAGGCTGCTGATCTACGACACCT CCAACCTGGCCTCTGGCGTGCCCGTGAGATTCTCCGGCTCTGGAAGCG GCACCTCCTACTCTCTGACAATCAGCCGGATGGAGGCTGAGGATGCCG CTACATACTACTGCCAGCAGTGGTCCTCTTACCCTCTGACCTTTGGAGC CGGCACAAAGCTGGAGCTGAAGCGGGCGGCCGCA TCRmoleculesequences 187 HumanTCR YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD alphachain MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVE constantregion KSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 188 HumanTCRbeta EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGK chainconstant EVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQ region FYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDSRG 189 VVT1epitope RMFPNAPYL 190 CDR1alpha SSYSPS 191 CDR2alpha YTSAATL 192 CDR3alpha VVSPFSGGGADGLT 193 CDR3alpha SPFSGGGADGLT 194 CDR1beta DFQATT 195 CDR2beta SNEGSKA 196 CDR3beta SARDGGEG 197 CDR3beta RDGGEGSETQY 198 HumanTCR MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLF alphachain WYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMS (WT1) DAAEYFCVVSPFSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSD KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF RILLLKVAGFNLLMTLRLWSS 199 HumanTCRbeta MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWY chain(WT1) RQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPE DSSFYICSARDGGEGSETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEIS HTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 200 HumanTCR MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLF alphachain WYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMS (WT1)withThr48 DAAEYFCVVSPFSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSD toCys KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNK substitution SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF RILLLKVAGFNLLMTLRLWSS 201 HumanTCRbeta MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWY chain(WT1)with RQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPE Ser57toCys DSSFYICSARDGGEGSETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEIS substitution HTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 202 WT1235-243 CMTWNQMNL epitope 203 Framework FGKGTHLIIQP sequence 204 Framework FGPGTRLLVL sequence 205 Consensus ARGGDYDEEYY(A/V/L)MD sequenceof CRT5,1and4 Variant1,heavy chainCDR3 (SEQIDNos116, 34and100) 206 Consensus ARGGDYDEEYY(A/V/L)MDY sequenceof CRT5,1and4 Variant2,heavy chainCDR3 (SEQIDNos118, 46and102) 207 Consensusof ARGGDYDEEYY(A/V/L)MD(Y/X) CRT5,1and4 Variants1and2, heavychain CDR3(SEQID Nos116,34,100, 118,46,102) 208 Consensusof QQWSSYPL(T/X) CRT5,1and4 Variants1and2, lightchainCDR3 (SEQIDNos37 and49) 209 Consensusof ((GYTF)/X)SSYW((IE)/X) CRT5,1and4 Variants1and2, heavychain CDR1(SEQID Nos32and34) 210 Consensusof ((WIGE)/X)ILPGSGST(N/X) CRT5,1and4 Variants1and2, heavychain CDR2(SEQID NOs33and45) 211 Consensusof ((GYTF)/X)(S/T)SYW(((I/M)(E/H))/X) CRT5,1,4and3 Variants1and2 heavychains CDR1(SEQID NOs150and 151) 212 Consensusof (((WIG)(E/A))/X)I(L/Y)PG(S/N)(G/S)(S/D)T((N/S)/X)) CRT5,1,4and3 Variants1and2 heavychain CDR2(SEQID NOs152and 153) 213 Consensusof (A/T)(R/H)(G/X)(G/X)(D/X)Y(D/Y)(E/G)(E/S)(Y/D)Y(V/A/L)MD CRT5,1,4and3 (Y/X) Variants1and2 heavychains CDR3(SEQID NOs154and 155) 214 Consensusof ((L(L/W)IY)/X)(D/S)TS((NLA)/X) CRT5,1,4and3 Variants1and2 lightchainCDR2 215 Consensusof (Q/H)Q(W/Y)(S/H)(S/R)(Y/S)P(L/R)((TF)/X) CRT5,1,4and3 Variants1and2 lightchainCDR3 216 Consensusof ((GYTF)/X)TSYW((MH)/X) CRT3Variants1 and2heavy chainCDR1 (SEQIDNOs64 and76) 217 Consensusof ((WIGA)/X)IYPGNSDT(S/X) CRT3Variants1 and2heavy chainCDR2 (SEQIDNOs65 and77) 218 Consensusof THYYGSDYAMD(Y/X) CRT3Variants1 and2heavy chainCDR3 (SEQIDNOs66 and78) 219 Consensusof ((SSV)/X)SSSY((LHWY)/X) CRT3Variants1 and2lightchain CDR1(SEQID NOs67and79) 220 Consensusof ((LWIY)/X)STS((NLA)/X) CRT3Variants1 and2lightchain CDR2(SEQID NOs68andSTS) 221 Consensusof HQYHRSPR(T/X) CRT3Variants1 and2lightchain CDR3(SEQID NOs69and81) 222 Nucleotide actactaccaagccagtgctgcgaactccctcacctgtgcaccctaccgggacatc sequence tcagccccagagaccagaagattgtcggccccgtggctcagtgaaggggaccg encodingHinge gattggacttcgcctgtgatatttacatctgggcacccttggccggaatctgcgtggc and ccttctgctgtccttgatcatcactctcatctgctaccacaggagccga transmembrane regionsofmouse CD8? 223 Nucleotide aatagtagaaggaacagactccttcaaagtgactacatgaacatgactccccgga sequence ggcctgggctcactcgaaagccttaccagccctacgcccctgccagagactttgca encodingmouse gcgtaccgcccc intracellular signalling sequencesfrom mouseCD28 224 Nucleotide aaatggatcaggaaaaaattcccccacatattcaagcaaccatttaagaaga sequence ccactggagcagctcaagaggaagatgcttgtagctgccgatgtccacag encodingmouse gaagaagaaggaggaggaggaggctatgagctg 4-1BBdomain 225 Nucleotide agagcaaaattcagcaggagtgcagagactgctgccaacctgcaggaccccaa sequence ccagctctacaatgagctcaatctagggcgaagagaggaatatgacgtcttggag encodingmouse aagaagcgggctcgggatccagagatgggaggcaaacagcagaggaggagg CD3zetachain aacccccaggaaggcgtatacaatgcactgcagaaagacaagatggcagaag cctacagtgagatcggcacaaaaggcgagaggcggagaggcaaggggcacg atggcctttaccagggtctcagcactgccaccaaggacacctatgatgccctgcat atgcagaccctggccc 226 Nucleotide cggaaggcttggagattgcctaacactcccaaaccttgttggggaaacagcttcag sequence gaccccgatccaggaggaacacacagacgcacactttactctggccaagatc encodingmouse OX40domain 227 Nucleotide ggaggcaccaagctggaaatcaaacgtgcggccgcaactactaccaagccagtgctgcgaa sequence ctccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcccc encodingmurine gtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcacccttgg CD8? hingeand ccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagccg transmembrane aaatagtagaaggaacagactccttcaaagtgactacatgaacatgactccccggaggcct regions,CD28 gggctcactcgaaagccttaccagccctacgcccctgccagagactttgcagcgtaccgcccc intracellular agagcaaaattcagcaggagtgcagagactgctgccaacctgcaggaccccaaccagctct signallingdomain acaatgagctcaatctagggcgaagagaggaatatgacgtcttggagaagaagcgggctcg andCD3? ggatccagagatgggaggcaaacagcagaggaggaggaacccccaggaaggcgtataca intracellular atgcactgcagaaagacaagatggcagaagcctacagtgagatcggcacaaaaggcgaga signallingdomain ggcggagaggcaaggggcacgatggcctttaccagggtctcagcactgccaccaaggacac ctatgatgccctgcatatgcagaccctggcccctcgctaataaaagcttaacacgagccatag atagaataaaag 228 Nucleotide ggaggcaccaagctggaaatcaaacgtgcggcgcaactactaccaagccagtgctgcgaa sequence ctccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcccc encodingmurine gtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcacccttgg CD8? hingeand ccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagccg transmembrane aaaatggatcaggaaaaaattcccccacatattcaagcaaccatttaagaagaccactgga domains,4-1BB gcagctcaagaggaagatgcttgtagctgccgatgtccacaggaagaagaaggaggagga intracellular ggaggctatgagctgagagcaaaattcagcaggagtgcagagactgctgccaacctgcagg signallingdomain accccaaccagctctacaatgagctcaatctagggcgaagagaggaatatgacgtcttgga andCD3? gaagaagcgggctcgggatccagagatgggaggcaaacagcagaggaggaggaaccccc intracellular aggaaggcgtatacaatgcactgcagaaagacaagatggcagaagcctacagtgagatcg signallingdomain gcacaaaaggcgagaggcggagaggcaaggggcacgatggcctttaccagggtctcagca ctgccaccaaggacacctatgatgccctgcatatgcagaccctggcccctcgctaataaaag cttaacacgagccatagatagaataaaag 229 Nucleotide ggaggcaccaagctggaaatcaaacgtgcggccgcaactactaccaagccagtgctgcgaa sequence ctccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcccc encodingmurine gtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcacccttgg CD8? hingeand ccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagccg transmembrane acggaaggcttggagattgcctaacactcccaaaccttgttggggaaacagcttcaggaccc domains,OX40 cgatccaggaggaacacacagacgcacactttactctggccaagatcagagcaaaattcag intracellular caggagtgcagagactgctgccaacctgcaggaccccaaccagctctacaatgagctcaatc signallingdomain tagggcgaagagaggaatatgacgtcttggagaagaagcgggctcgggatccagagatgg andCD3? gaggcaaacagcagaggaggaggaacccccaggaaggcgtatacaatgcactgcagaaa intracellular gacaagatggcagaagcctacagtgagatcggcacaaaaggcgagaggcggagaggcaa signallingdomain ggggcacgatggcctttaccagggtctcagcactgccaccaaggacacctatgatgccctgc atatgcagaccctggcccctcgctaataaaagcttaacacgagccatagatagaataaaag 230 Nucleotide ggaggcaccaagctggaaatcaaacgtgcggccgcaactactaccaagccagtgctgcgaa sequence ctccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcccc encodingmurine gtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcacccttgg CD8? hingeand ccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagccg transmembrane aaatagtagaaggaacagactccttcaaagtgactacatgaacatgactccccggaggcct domains,CD28 gggctcactcgaaagccttaccagccctacgcccctgccagagactttgcagcgtaccgcccc and4-1BB aaatggatcaggaaaaaattcccccacatattcaagcaaccatttaagaagaccactggag intracellular cagctcaagaggaagatgcttgtagctgccgatgtccacaggaagaagaaggaggaggag signalling gaggctatgagctgagagcaaaattcagcaggagtgcagagactgctgccaacctgcagga domainsand ccccaaccagctctacaatgagctcaatctagggcgaagagaggaatatgacgtcttggag CD3? intracellular aagaagcgggctcgggatccagagatgggaggcaaacagcagaggaggaggaaccccca signallingdomain ggaaggcgtatacaatgcactgcagaaagacaagatggcagaagcctacagtgagatcgg cacaaaaggcgagaggcggagaggcaaggggcacgatggcctttaccagggtctcagcac tgccaccaaggacacctatgatgccctgcatatgcagaccctggcccctcgctaataaaagct taacacgagccatagatagaataaaag 231 Nucleotide ggaggcaccaagctggaaatcaaacgtgcggccgcaactactaccaagccagtgctgcgaa sequence ctccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcccc encodingmurine gtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcacccttgg CD8? hingeand ccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagccg transmembrane aaatagtagaaggaacagactccttcaaagtgactacatgaacatgactccccggaggcct domains,CD28 gggctcactcgaaagccttaccagccctacgcccctgccagagactttgcagcgtaccgcccc andOX40 cggaaggcttggagattgcctaacactcccaaaccttgttggggaaacagcttcaggacccc intracellular gatccaggaggaacacacagacgcacactttactctggccaagatcagagcaaaattcagc signalling aggagtgcagagactgctgccaacctgcaggaccccaaccagctctacaatgagctcaatct domainsand agggcgaagagaggaatatgacgtcttggagaagaagcgggctcgggatccagagatggg CD3? intracellular aggcaaacagcagaggaggaggaacccccaggaaggcgtatacaatgcactgcagaaag signallingdomain acaagatggcagaagcctacagtgagatcggcacaaaaggcgagaggcggagaggcaag gggcacgatggcctttaccagggtctcagcactgccaccaaggacacctatgatgccctgca tatgcagaccctggcccctcgctaataaaagcttaacacgagccatagatagaataaaag 232 Nucleotide Ggaggcaccaagctggaaatcaaacgtgcggccgcaactactaccaagccagtgctgcga sequence actccctcacctgtgcaccctaccgggacatctcagccccagagaccagaagattgtcggcc encodingmurine ccgtggctcagtgaaggggaccggattggacttcgcctgtgatatttacatctgggcaccctt CD8? hingeand ggccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctgctaccacaggagc transmembrane cgaaaatggatcaggaaaaaattcccccacatattcaagcaaccatttaagaagaccactg domains,4-1BB gagcagctcaagaggaagatgcttgtagctgccgatgtccacaggaagaagaaggaggag andOX40 gaggaggctatgagctgcggaaggcttggagattgcctaacactcccaaaccttgttgggga intracellular aacagcttcaggaccccgatccaggaggaacacacagacgcacactttactctggccaaga signalling tcagagcaaaattcagcaggagtgcagagactgctgccaacctgcaggaccccaaccagct domainsand ctacaatgagctcaatctagggcgaagagaggaatatgacgtcttggagaagaagcgggct CD3? intracellular cgggatccagagatgggaggcaaacagcagaggaggaggaacccccaggaaggcgtata signallingdomain caatgcactgcagaaagacaagatggcagaagcctacagtgagatcggcacaaaaggcga gaggcggagaggcaaggggcacgatggcctttaccagggtctcagcactgccaccaaggac acctatgatgccctgcatatgcagaccctggcccctcgctaataaaagcttaacacgagccat agatagaataaaag
In the above table the designation of an amino acid as X indicates that no amino acid may be present at that position.

EXAMPLES

Example 1: Experimental Studies on CLEC14A

Materials and Methods

HUVEC Preparation and Culture

[0536] Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords donated by the UK National Health Service after informed consent of the donors. Cords were dissected from placentas and the vein was washed in sterile PBS to remove blood. 1 mg/ml of collagenase diluted in M199 medium (Sigma) was injected into the vein and then incubated at 37? C. for 20 minutes to detach the endothelial cells. HUVECs were collected by washing in M199 complete medium containing 10% FCS, 10% large vessel endothelial cell growth supplement (TCS Cell Works), and 4 mM L-glutamine, and plated on 0.1% Type 1 gelatin from porcine skin (Sigma) coated dishes.

Primary Cells Source

[0537] Human aortic smooth muscle cells (HASMC) and human bronchial epithelial cells (HBE) were purchased from TCS Cell Works. Human lung fibroblasts (MRCS) were obtained from Cancer Research UK Central Services. Human peripheral blood mononuclear cells (PBMCs) were obtained from the Institute of Cancer Studies at the University of Birmingham. Hepatocytes were a gift from Professor David Adams, School of Immunity and Infection, University of Birmingham.

HUVEC Immunofluorescence

[0538] HUVECs were grown in glass micro-well chambers (Nunc) fixed in ice-cold methanol, washed with PBST blocked in 10% FCS 3% BSA in PBST. Cells were then stained with CLEC14A antibody following the same protocol used for paraffin embedded sections or co-stained with 5 ?g/ml mouse monoclonal IgG antibody against human VE-cadherin, kindly donated by Professor Maria Grazia Lampugnani, Firc Institute for Molecular Oncology, Milan. Sections staining were analyzed with a 510 laser scanning confocal microscope (Carl Zeiss).

Scratch Wound Healing Assay with CLEC14A Monoclonal Antibodies

[0539] A scratch with a 10 ?l pipette tip was made in confluent HUVECs. New medium containing 1 ?g/ml or 10 ?g/ml of a monoclonal CLEC14A antibody raised in mice against the extracellular domain of CLEC14A was applied. Chemokinetic migration of HUVECs was assessed by acquiring images of wound closure at time zero, 4, 6, 12 hours with a Leica DM 1000 light microscope and USB 2.0 2M Xli camera. The open area of the wound was quantitated using Image J software.

Immunofluorescence on Paraffin Embedded Tissues

[0540] Immunofluorescence was performed on paraffin embedded normal and cancer human tissue collection obtained from Cancer Research UK histology service and on cancer and normal tissue arrays (Superbiochips) (Data not shown). Human common cancers 1 (MA2) including 10 cores of each of the following carcinoma: stomach, oesophagus, lung, colon/rectum, thyroid and kidney, and common cancers 2 (MB3) including 10 cores of each of the following carcinomas: breast, liver, bladder, ovarian, pancreas and prostate were used. Two additional control arrays of matching adjacent normal tissues were also analysed. After removal of paraffin, tissues were rehydrated and microwaved for 3 minutes on medium power in citrate buffer pH6 for antigen retrieval. Sections were blocked in PBST containing 10% FCS and 3% BSA. Sections were probed with 10 ?g/ml of sheep IgG primary polyclonal antibody against the extracellular domain of human CLEC14A (R&D system) and 15 ?g/ml of FITC conjugated rabbit IgG secondary anti-sheep polyclonal antibody (Zymax). Vessel endothelial cells were stained with 20 ?g/ml of Ulex europeaus agglutinin I (UEAI) conjugated with rhodamine (Vector labs). Slides were permanently mounted with prolong gold anti-fade reagent with DAPI (Invitrogen) to counterstain cell nuclei. Section staining was analysed using a 510 laser scanning confocal microscope (Carl Zeiss).

Preparation of Monoclonal Antibodies

[0541] The antigens used for the preparation of monoclonal antibodies were murine CLEC14A-Fc (CM) and human CLEC14A-Fc (CH), optionally conjugated with adjuvant protein (AP). These four antigens (CM, CH, CM-AP, CH-AP) were used for mice immunisation using the following protocol:

TABLE-US-00002 Day Operation 0 Pre-immune sample taken Immunisation of 100 ?g of antigen in complete Freunds adjuvant (foot pads) 14 Immunisation of 100 ?g of antigen in incomplete Freunds adjuvant (foot pads) 17 Test bleed 18 Popliteal lymph node harvest for fusion
Sera were tested by ELISA against three antigens: CM, CH and Fc. A non-immune serum was taken as a negative control.
The fusion protocol was as follows:
(1) Popliteal lymph nodes were harvested from the immune mice and homogenised.
(2) Cells were washed with warm DMEM.
(3) Cells were mixed with sp2/0 myeloma cells.
(4) The mixture was centrifuged (1000 g)
(5) The pellet was suspended in 50% PEG 1500 and incubated for 1 min.
(6) The suspension was slowly diluted with warm DMEM.
(7) Suspension was centrifuged (1000 g).
(8) Cells were seeded into plates with peritoneal macrophages.
(9) Cells were cultivated at 37? C. and 5% CO.sub.2

[0542] More than 500 HAT-resistant hybridoma clones from each mouse were obtained. All of the clone supernatants were tested twice with 4 days interval by ELISA against three absorbed antigens (CM, CH and Fc). Testing resulted in 5 clones (all subclass IgG1) that reacted with both CM and CH and did not react with Fc. All positives were cloned 2-4 times by the limiting dilution method, propagated in culture flasks and injected into mice for ascites. Three clones were derived as a result of immunisation with CLEC14a human (CH), one clone (CRT-3) was the result of immunisation with CLEC14a human-AP (CH-AP), and one clone (CRT-2) was the result of immunisation with CLEC14a mouse-AP (CM-AP).

Tubule Formation Assays

[0543] HUVECs were treated with 20 ?g/ml of CRT2, CRT3 or CRT4 or IgG isotype control. Images of the tubules were taken at 16 hours and were analysed for total tubule length, number of junctions, number of branches, branch length, number of meshes and total mesh area. The experiments were repeated three times, with five data points analysed per experiment.

Results

[0544] FIG. 2 is a graph showing the relative expression of CLEC14A in HUVECs and other primary cells. CLEC14A was expressed specifically in endothelial cells. This confirms our previous finding that CLEC14A was endothelial-specific (Herbert et al, 2008).

[0545] The ability of CLEC14A monoclonal antibodies to inhibit angiogenesis was examined. Scratch wound healing assays using monoclonal antibodies were carried out. As shown in FIG. 3, when HUVECs were treated with 10 ?g/ml of monoclonal antibody CRT-3, 25% of the wound area remained open at 12 h compared to 13% in the control. These results show that CLEC14A antibodies, have an inhibitory effect on endothelial cell migration. Endothelial cell migration is an essential feature of angiogenesis. Accordingly, this assay provides evidence that a CLEC14A antibody, inhibits angiogenesis.

[0546] Further, the tubule formation assays showed that the number and total length of branches was significantly increased by treatment with CRT4 and that CRT4 also significantly reduced the number of meshes per filed. These results suggest that CRT4 does not affect tube formation but that it affects the connection of tubes. This is evidenced by the increased number and length of branches, indication that the tubules are less well interconnected. CRT2 and CRT3 treatment produced a significant reduction in tubule length and the number of junctions and CRT2 also significantly reduced the mesh area per field. Thus these assays provide further evidence that distinct CLEC14A antibodies inhibit angiogenesis (albeit by having differing effects on tube formation.

[0547] The expression of CLEC14A in sections of solid tumours and normal tissue was examined using CLEC14A-specific probes (data not shown). CLEC14A expression was seen in the blood vessels in all tumour tissues analysed. Ovarian, bladder, liver, breast, kidney and prostate tumours were strongly positive for CLEC14A expression, whereas stomach, oesophagus, lung, colon, rectal, pancreatic and thyroid tumour tissues showed a lower level of specific CLEC14A expression. CLEC14A expression was not detected in any of the corresponding normal control (non-tumour) tissues. Accordingly, it has been demonstrated that CLEC14A is specifically expressed in tumour vasculature.

Example 2: Blocking CLEC14A-MMRN2 Binding Inhibits Sprouting Angiogenesis and Tumour Growth

Materials and Methods

Reagents

[0548] For Western blotting and immunoprecipitation; primary antibodies: sheep polyclonal anti-human CLEC14A (R&D systems), mouse monoclonal anti-human Tubulin (Sigma), mouse polyclonal anti-human MMRN2 (Abnova); secondary antibodies: goat polyclonal anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Dako), donkey polyclonal anti-sheep IgG conjugated to HRP (R&D systems). For immunofluorescence; primary antibodies: rabbit polyclonal anti-murine PECAM (Santa Cruz); secondary antibodies: donkey polyclonal anti-rabbit conjugated to Alexa Fluor488 (Invitrogen). For flow cytometry; primary antibodies: mouse monoclonal anti-HA tag (CRUK), mouse monoclonal anti-CLEC14A (C2, C4 described below); secondary antibodies: goat polyclonal anti-mouse IgG conjugated to Alexa Fluor488 (Invitrogen).

Plasmids

[0549] For protein production; lentiviral plasmids psPAX2 (lentiviral packaging; Addgene), pMD2G (Envelope plasmid; Addgene) and pWPI hCLEC14A-ECD-Fc (lentiviral mammalian expression plasmid containing IRES-EGFP; Addgene) were used. pWPI hCLEC14A-Fc and mCLEC14A-Fc was generated by initial PCR subcloning from clec14a IMAGE clone (Origene) into pcDNA3-Fc plasmid. The primers used were as follows: human CLEC14A fwd 5TAGTAGGAATTCGAGAGAATGAGGCCGGCGTTCGCCCTG3 (SEQ ID NO: 4); human CLEC14A rev5AGAACCGCGGCCGCTGGAGGAGTCGAAAGCCTGAGGAGT3 (SEQ ID NO: 5); murine CLEC14A fwd5TAGTAGGAATTCGAGAGAATGAGGCCAGCGCTTGCCCTG3 (SEQ ID NO: 6; murine CLEC14A rev5CTACTAGCGGCCGCTCGTGGAAGAGGTGTCGAAAGT3 (SEQ ID NO: 7). EcoR1 and Not1 restriction sites were used to insert CLEC14A. A further round of PCR subcloning was performed to transfer the CLEC14A-Fc fusion into pWPI. The primers used were as follows: human CLEC14A fwd5TAGTAGTTAATTAAGAGAGAATGAGGCCGGCGTTC3 (SEQ ID NO: 8); murine CLEC14A fwd5TAGTAGTTAATTAAGAGAGAATGAGGCCAGCGCTT3 (SEQ ID NO: 9); human Fc rev5CTACTAGTTTAAACTCATTTACCCGGAGACAGGGA3 (SEQ ID NO: 10). For this step, Pac1 and Pme1 restriction sites were used.

[0550] MMRN2 mammalian expression plasmid was constructed by PCR cloning from mmrn2 IMAGE clone (Thermo) into pHL-Avitag3, using the following primers: fwdCCGGACCGGTCAGGCTTCCAGTACTAGCC (SEQ ID NO: 11); revCGGGGTACCGGTCTTAAACATCAGGAAGC (SEQ ID NO: 12). Age1 and Kpn1 restriction enzymes were used.

Cell Culture

[0551] Human Umbilical Vein Endothelial Cells were isolated as described previously. Umbilical cords were obtained from Birmingham Women's Health Care NHS Trust with informed consent. HUVECs were used between passages 1-6 and were cultured in M199 complete medium (cM199) containing 10% fetal calf serum (PAA), 1% bovine brain extract, 90 ?g/ml heparin, and 4 mM L-glutamine, 100 U/ml penicillin and 100 ?g/ml streptomycin (Invitrogen) and were seeded on plates coated in 0.1% type 1 gelatin from porcine skin. HEK293T cells were cultured in DMEM (Sigma) complete medium (cDMEM) containing 10% fetal calf serum (PAA), 4 mM L-glutamine, 100 U/ml penicillin and 100 ?g/ml streptomycin (Invitrogen).

[0552] SiRNA transfections in HUVEC were performed as previously described. Lentivirus was produced in HEK293T cells by transient transfection with the lentiviral packaging, envelope and expression plasmids above. Plasmids were incubated in OptiMEM (Invitrogen) with polyethylenimine (36 ?g/ml) at a 1:4 ratio for 10 minutes at room temperature prior to adding to HEK293T cells in cDMEM. Media supernatant was used to transduce fresh HEK293T cells. GFP positive HEK293T cells were sorted and used for protein production. Expression of MMRN2 in HEK293T cells was achieved by polyethylenimine transient transfection as above using pHL-Avitag3 hMMRN2.

Quantitative PCR

[0553] cDNA was prepared using the High-Capacity cDNA Archive kit (Applied Biosystems), from 1 ?g of extracted total RNA. qPCR reactions were performed with Express qPCR supermix (Invitrogen) on a RG-3000 (Corbett/Qiagen, Manchester, UK) thermocycler. Primers for human clec14a and flotillin-2 were as previously described. Primers for murine clec14a 5 UTR, CDS and 3 UTR and murine beta-actin, are as follows: 5UTR fwdTTCCTTTTCCAGGGTTTGTG (SEQ ID NO: 13); 5 UTR revGCCTACAAGGTGGCTTGAAT (SEQ ID NO: 14); CDS fwdAAGCTGTGCTCCTGCTCTTG (SEQ ID NO: 15; CDS revTCCTGAGTGCACTGTGAGATG (SEQ ID NO: 16); 3 UTR fwdCTGTAGAGGGCGGTGACTTT (SEQ ID NO 17); 3 UTR revAGCTGCTCCCAAGTCCTCT (SEQ ID NO: 18); mACTB fwdCTAAGGCCAACCGTGAAAAG (SEQ ID NO: 19); mACTB revACCAGAGGCATACAGGGACA (SEQ ID NO: 20). Relative expression ratios were calculated according to the efficiency adjusted mathematical model.

Western Blotting and Immunoprecipitation

[0554] Whole cell protein lysates were made and co-immunoprecipitation experiments were performed as previously described, except protein was extracted from 2?10.sup.7 HUVECs. For initial isolation of CLEC14A interacting proteins 5 ?g CLEC14A-Fc or an equimolar amount of hFc was used. For endogenous immunoprecipitation experiments 0.4 ?g anti-CLEC14A antibody or sheep IgG was used. For blocking experiments 5 ?g CLEC14A-Fc or hFc were bound to protein G beads overnight in PBS. Beads were blocked for 5-6 hours in PBS containing 20% FCS (PAA). Bound CLEC14A-Fc or hFc protein was blocked with increasing concentrations of mIgG, C2 or C4 in binding buffer overnight. Lysates from MMRN2 transfected HEK293T cells were then incubated overnight with the bead complexes before washing and analysing by Western blot. Standard protocols were used for Western blotting and SDS-PAGE. Primary antibodies were used as indicated in the text with corresponding HRP conjugated secondary antibodies.

Flow Cytometry

[0555] Cells were detached with cell dissociation buffer (Invitrogen), rinsed in PBS before incubation in blocking buffer (PBS, 3% BSA, 1% NaN.sub.3) for 15 minutes. Subsequent staining using 10 ?g/ml anti-HA tag (CRUK), 10 ?g/ml anti-CLEC14A (C2, C4 described below), as primary antibodies, in blocking buffer for 30 minutes. Cells were rinsed in PBS and stained with goat polyclonal anti-mouse IgG conjugated to Alexa Fluor488 (Invitrogen) in blocking buffer. Data (15,000 events/sample) were collected using a FACSCalibur apparatus (Becton Dickinson, Oxford, UK), and results were analysed with Becton Dickinson Cell Quest software.

HUVEC Spheroid Sprouting Assay and In Vitro Matrigel Tube Forming Assay

[0556] Generation of HUVEC spheroids and induction of endothelial sprouting in a collagen gel was performed as previously described, using 1000 HUVECs per spheroid. Quantification was performed 16 hours after embedding. To quantify sprout growth the number of sprouts were counted, the cumulative sprout length and the maximal sprout length was assessed. For two colour sprouting experiments, HUVECs were pre-labelled with orange and green CellTracker dyes (Invitrogen). After 24 hours spheroids were fixed in 4% formaldehyde and mounted with Vectorshield (Vector labs). Slides were imaged with an Axioskop2 microscope and AxioVision SE64 Re14.8 software (Zeiss, Cambridge, UK).

For the Matrigel tube forming assays 1.4?10.sup.5 HUVECs were seeded onto 70 ?l basement membrane extract (Matrigel, BD Bioscience, Oxford, UK) in a 12 well plate. After 16 hours, images were taken of 5 fields of view per well using a Leica DM IL microscope (Leica, Milton Keynes, UK) with a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, Tex., USA) at 10? magnification. Images were analysed with the Angiogenesis analyser plugin for Image J (Carpentier G. et al., Angiogenesis Analyzer for ImageJ. 4th ImageJ User and Developer Conference proceedings) and available at the NIH website (http://imagej.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt).

Protein Production

[0557] Culture media (CM) from CLEC14A-Fc expressing HEK293T cells was collected. CM was flowed over a HiTrap protein A HP column (GE healthcare, Amersham, UK) and protein eluted using a 0-100% gradient of 100 mM sodium citrate (pH 3) before neutralising with 1 M Tris base. Fractions were run on a SDS-PAG and assessed for protein purity and specificity by Coomassie staining and Western blotting. Fractions containing similar concentrations of protein were combined and dialysed in PBS prior to functional assays.

Monoclonal Antibody Generation

[0558] Mouse monoclonal antibodies were commercially prepared by Serotec Ltd (Oxford, UK) using the following protocol to break tolerance supplied by us. Purified mouse CLEC14A-Fc fusion protein was given at 50 ?g in Freunds complete adjuvant subcutaneously. Two weeks later mice were given another 50 ?g subcutaneously but this time in Freunds adjuvant. Mice were culled and spleens harvested for fusion two weeks later.

Generation of clec14a ?/? Mice

[0559] Mice were housed at the Birmingham Biomedical Services Unit (Birmingham, UK). C57BL/6N VGB6 feeder-dependent embryonic stem cells containing the CLEC14A deletion cassette (Clec14atm1(KOMP)Vlcg; project ID VG10554) were procured from the Knockout Mouse Project (University of California, Davis, USA). The Transgenic Mouse Facility at the University of Birmingham generated chimeric mice by injection of embryonic stem cells into albino C57BL/6 mice and were bred to C57BL/6 females to generate mice heterozygous for the cassette. Animal maintenance had appropriate

Home Office Approval and Licensing

Aortic Ring and Murine Subcutaneous Sponge Angiogenesis Assay

[0560] Aortas were isolated and processed for aortic ring assays in collagen. Tube/sprout outgrowth, maximal endothelial migration and total endothelial outgrowth was quantitated. Themurine subcutaneous sponge angiogenesis assay was performed as previously described, with slight modification. Male C57 black mice were implanted with a subcutaneous sterile polyether sponge disc (10?5?5 mm) under the dorsal skin of each flank at day 0. 100 ?l bFGF (40 ng/ml; R&D systems) was injected through the skin directly into the sponges every other day for 14 days. Sponges were excised on day 14, fixed in 10% formalin, and paraffin embedded. Sections were stained with haematoxylin and eosin, sponge cross-sections were taken using a Leica MZ 16 microscope (Leica, Milton Keynes, UK) with a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, Tex., USA) at ?1 magnification for cellular invasion analysis. Images captured by Leica DM E microscope (Leica, Milton Keynes, UK) at 40? magnification were analysed for vessel density. Vessel counts were assessed in five fields per section per sponge. All animal experimentation was carried out in accordance with Home Office License number PPL 40/3339 held by RB.

Tumour Implantation Assays

[0561] 10.sup.6 Lewis lung carcinoma cells were injected subcutaneously into the flank of male mice at 8-10 weeks of age. Tumour growth was monitored by daily calliper measurements and after two-four weeks growth, tumour mass was determined by weight, fixed in 4% PFA, paraffin embedded and serial sections cut at 6 ?m.

Immunofluorescence and X-Gal Staining

[0562] Immunofluorescence staining and X-Gal staining were performed using methods known in the art.

Results

CLEC14A Regulates Sprouting Angiogenesis In Vitro

[0563] CLEC14A has previously been shown to be involved in endothelial migration and tube formation in vitro. To investigate the role of CLEC14A in sprouting angiogenesis in vitro, HUVEC spheroids were generated from HUVECs treated with siRNA targeting clec14a or a non-complementary siRNA duplex. Knockdown of clec14a expression was confirmed at the mRNA level by qPCR with an average reduction of 74% across three experiments (FIG. 5A) and at the protein level by Western blot analysis of protein extracts probed with an anti-CLEC14A polyclonal antisera (FIG. 5B). VEGF induced sprouting from CLEC14A knockdown spheroids was impaired, knockdown spheroids produced on average 6.9 sprouts per spheroid, compared to 13.2 for control cells (FIGS. 5C and 5D). To determine the role of CLEC14A in tip/stalk cell formation, control HUVECs and knockdown HUVECs were stained either red or green and mixed, prior to spheroid formation and induced sprouting (FIG. 5E). Knockdown of CLEC14A reduced the percentage of cells at the tip position (33%) compared to control cells (67%), however, there was no effect on the percentage of stalk cells that were derived from CLEC14A knockdown HUVECs (FIG. 5F). These data suggest CLEC14A has a role in sprout initiation and migration.

CLEC14A Regulates Sprouting Angiogenesis In Vivo

[0564] Previously published data for CLEC14A has demonstrated its role in endothelial biology in vitro, however its in vivo role has not been reported. To investigate the role of CLEC14A in vivo and ex vivo, mice were generated to replace the clec14a coding sequence with a lacZ reporter (FIG. 6A). Breeding of heterozygotes (clec14a ?/+) produced equal proportions of male and female mice (49.5%/50.5% respectively) and a Mendelian ratio of wildtype:heterozygote:homozygote mice (26.4%:47.2%:26.4% respectively). As clec14a is an endothelial-restricted gene, aortas were isolated from clec14a +1+ and clec14a ?/?mice. Extracted cDNA was analysed by qPCR and confirmed loss of the clec14a coding region but expression of the 5 and 3 untranslated regions were retained (FIG. 6B). Loss of CLEC14A at the protein level was also confirmed by Western blot analysis of lung tissue lysates (FIG. 6C).

[0565] To confirm the role of CLEC14A in sprouting angiogenesis in multicellular three dimensional co-culture, aortas were isolated, cut into rings and embedded in collagen. Cellular outgrowth was stimulated by VEGF and monitored over 7 days before end-point quantitation of endothelial sprouting. Again, loss of CLEC14A impaired endothelial sprout outgrowth and migration (FIG. 6D). Aortic rings from wildtype mice produced over double the number of tubes compared to that observed for CLEC14A knockout mice (30.6 tubes compared to 13.4 tubes respectively) (FIG. 6E). In addition, the maximum migration, which is defined by the furthest distance migrated away from each aortic ring, was also reduced in knockout cultures (FIG. 6F). To assess whether CLEC14A has a similar function in vivo, sponge barrels were implanted subcutaneously into CLEC14A knockout mice. Cellular infiltration and neo-angiogenesis were stimulated using bFGF injections into the sponge every two days for two weeks. Macroscopic analysis of sponge sections stained with haematoxylin and eosin revealed impaired infiltration of cells into the sponge in clec14a ?/? animals (FIGS. 6G and 6H). In addition, vascularity was significantly reduced (p<0.01) for clec14a ?/? animals (FIG. 6I). To confirm the endothelial cells lining the neoangiogenic vessels express clec14a in this model, sponges and livers from CLEC14A KO mice were stained with x-gal. Strong x-gal staining was observed on blood vessels within the sponge compared to matched liver sections (FIG. 6J). From these data we can conclude that mouse CLEC14A expression regulates endothelial migration and angiogenic sprouting in vivo, as well as in vitro, and CLEC14A is upregulated on sprouting endothelium.

CLEC14A Promotes Tumour Growth

[0566] CLEC14A expression is found highly up-regulated on human tumour vessels compared to vessels from healthy tissue, suggesting that cancer therapies could be targeted against CLEC14A. Therefore, to investigate whether loss of CLEC14A effects tumour growth we used the syngeneic Lewis lung carcinoma (LLC) model. For this 1?10.sup.6 LLC cells were injected subcutaneously into the right flank of either clec14a +/+ or clec14a ?/? mice. Tumour growth was impaired in the clec14a ?/? mice compared to clec14a +1+ littermates (FIG. 7A). This was confirmed by three independent experiments. Excised tumours taken from clec14a ?/? mice were smaller in size (FIG. 7B) and smaller in weight (FIG. 7C) than clec14a +1+ littermates. To determine whether the vascular density within these tumours was also effected, tissue sections were stained with an anti-CD31 antibody. Analysis shows a reduced density of discrete vessels (FIGS. 7D and 7E) and reduced percentage endothelial coverage (FIG. 7F). Furthermore, x-gal staining of tumour and liver sections taken from clec14a ?/? mice reveals high expression of clec14a on both mature vessels, with erythrocyte filled lumens (FIG. 3G, black arrows), and immature microvessels within the tumour (FIG. 7G, red arrows), confirming clec14a is upregulated on tumour vessels.

Identification and Confirmation of CLEC14A-MMRN2 Interaction

[0567] To identify potential binding partners for the extracellular domain for CLEC14A, we first purified CLEC14A extracellular domain protein tagged with human Fc. This protein or Fc alone was incubated with HUVEC whole cell lysates and precipitated using protein A agarose beads. The precipitated proteins were then washed and separated on a SDS-PAG. Seven gel regions were excised, digested and analysed by mass spectrometry. The most abundant protein identified was MMRN2 with 12 peptides (11 unique), and no peptides in the corresponding control pulldown fraction. Western blot analysis of the precipitates confirmed the presence of MMRN2 in the CLEC14A-ECD-Fc pull-down and was not detected in the Fc alone pull-down (FIG. 8A). To further confirm this interaction, endogenous CLEC14A was immunoprecipitated from HUVEC whole cell lysates. Western blot analysis confirmed MMRN2 co-precipitation in the CLEC14A precipitate but was not detected in the IgG control (FIG. 8B).

Development and Validation of CLEC14A Monoclonal Antibodies

[0568] To further our understanding of CLEC14A, we next produced cross-species reactive antibodies. To enable this, murine CLEC14A protein with a human Fc tag was expressed in HEK293T cells and purified on a protein A column. Mice were then immunised with 50 ?g mCLEC14A with complete Freund's adjuvant to break tolerance. Clones were screened for activity against human CLEC14A or human Fc. To confirm the clones could recognise cell bound CLEC14A, HEK293T cells overexpressing HA-CLEC14A were stained with clone C2 or C4 or a monoclonal HA tag antibody. FACs analysis shows increased fluorescence for each of the antibodies in the HA-CLEC14A overexpressing cells compared to control transfected cells (data not shown). To confirm that antibodies recognise the endogenous form of CLEC14A, these clones were used to stain HUVEC treated with control or clec14a targeted siRNAs. Control HUVEC were stained strongly by clone C2 and C4 and this staining was reduced to isotype control levels by knockdown of CLEC14A (data not shown). These results confirmed the specificity of the CLEC14A monoclonal antibodies.

[0569] To determine whether the C2 and C4 clones bind to the same region of CLEC14A, HUVECs were pre-treated with BSA, C2 or C4 antibody prior to C2-FITC staining. C2 incubation blocked C2-FITC staining effectively, but C4 had little effect. The same pre-treatment was repeated prior to C4-FITC staining. C2 antibody did not affect C4-FITC staining however, HUVECs pre-treated with C4 showed reduced binding of C4-FITC. From these results we can conclude that C2 and C4 bind to discrete regions of CLEC14A.

A CLEC14A Monoclonal Antibody Blocks CLEC14A-MMRN2 Binding

[0570] To determine whether either of these CLEC14A monoclonal antibodies could inhibit the binding of MMRN2 to CLEC14A, CLEC14A-ECD-Fc was pre-incubated with increasing concentrations of mIgG1, or C2, or C4, prior to incubation with lysates from HEK293T cells overexpressing MMRN2. Precipitates were then separated and probed for MMRN2 or CLEC14A-ECD-Fc. MMRN2 binding was observed for CLEC14A-ECD-Fc precipitates blocked with mIgG1 or C2 but no MMRN2 binding was observed in the C4 blocked precipitates. This confirms that the C4 but not the C2 monoclonal antibody blocks MMRN2 binding to CLEC14A. (Data not shown)

CLEC14A-MMRN2 Blocking Antibody Inhibits Tumour Growth

[0571] Mice with LLC tumours were injected intraperitoneally twice per week with 10 ?g C4 or mIgG1 (control) for the duration of the experiment. Tumour growth was slowed for mice treated with C4 antibody compared to the control, mIgG1, treatment group (FIG. 9A). Tumours from the C4 treated mice were smaller in size (FIG. 9B) and weight (FIG. 9C) than control animals. Again we examined the vascular density within these tumours. Tissue sections were stained with an anti-CD31 antibody and fluorescent analysis revealed a reduced density of discrete vessels (FIGS. 9D and 9E) and the percentage endothelial coverage (FIG. 9F), suggesting that CLEC14A binding to MMRN2 is an important functional component of tumour induced angiogenesis.

Discussion

[0572] CLEC14A is one of a small group of endothelial genes that contribute to tumour angiogenesis in multiple tumour types. Here we demonstrate that through loss of CLEC14A, tumour growth is inhibited in vivo (FIG. 7). A similar phenotype has also been observed for other tumour endothelial markers, such as TEM8, Endoglin, Galectin, ELTD1, and Endosialin and this demonstrates the importance of these tumour endothelial expressed genes in vascularisation and tumour growth.

[0573] Upregulation of CLEC14A has been observed in human tumours and murine models of pancreatic and cervical cancer which supports the findings that clec14a expression is upregulated on tumour vessels in the LLC model (FIG. 7). CLEC14A has been shown to regulate multiple aspects of endothelial biology including adhesion, migration, tube formation, and the results show that it is also important for sprouting angiogenesis in vitro and in vivo (FIGS. 5 and 6). We can infer that this role of CLEC14A is through endothelial-endothelial interactions or endothelial-extracellular matrix interactions, because in vitro HUVEC sprouting is perturbed by CLEC14A knockdown, suggesting the presence of other cell types is dispensable. We also observed for the first time upregulation of clec14a expression on neoangiogenic vessels in the subcutaneous sponge assay (FIG. 6). This is expected as newly formed endothelial sprouts have been modelled to experience extremely low shear stress (0.2 Pa) from the 4.2 ?m of the bifurcation point to the tip of the sprout, and clec14a expression is known to be upregulated by low shear stress.

[0574] The interaction of CLEC14A with MMRN2 has been shown through pulldown of proteins from HUVEC lysates using the extracellular domain of CLEC14A, as well as co-immunoprecipitation of the endogenous proteins (FIG. 8). Through the generation and validation of CLEC14A monoclonal antibodies, we identified two antibodies that bind to discrete regions of CLEC14A (C2 and C4) and have shown that the C4 but not the C2 clone blocks the interaction of CLEC14A with MMRN2. To probe the function of the CLEC14A-MMRN2 interaction, we used the C4 antibody in Matrigel tube forming assays and found an increase in branching and decrease in evolved meshes. It was further found that the CLEC14A-MMRN2 interaction is important for tumour growth (FIG. 9), C4 treatment recapitulated tumour growth and reduced tumour vascularity as seen in clec14a ?/? mice (FIG. 7). Although in this example no ligand or mode of activity was identified, this is the first time that CLEC14A and a specific extracellular interaction has been shown to be important for tumour growth, and suggests a hitherto avenue into new anti-angiogenic therapies.

Example 3 CLEC14A Monoclonal Antibodies C1, C4 and C5 Block CLEC14A-MMRN2 Interaction

[0575] To determine which CLEC14A monoclonal antibodies could inhibit the binding of MMRN2 to CLEC14A, CLEC14A-ECD-Fc was pre-incubated with increasing concentrations of mIgG1, or CR1-5, prior to incubation with lysates from HEK293T cells overexpressing MMRN2. Precipitates were then separated and probed for MMRN2 or CLEC14A-ECD-Fc. MMRN2 binding was observed for CLEC14A-ECD-Fc precipitates blocked with mIgG1 or C2 and C3 but no MMRN2 binding was observed in the C1, 4 and 5 blocked precipitates. This confirms that antibodies C1, 4 and 5 bind CLEC14a on an epitope that is distinct from the one that C2 and 3 monoclonal antibodies bind and thus specifically block the MMRN2 interaction with CLEC14A.

Example 4 Mapping of MMRN2 Binding Domain and CRT Antibodies

1) MMRN2 Binds to Either the CTLD or SUSHI Domain or CLEC14a

[0576] The binding of MMRN2 to CLEC14A was narrowed down to the CTLD or SUSHI domain of CLEC14A. It is likely that without the CTLD or SUSHI domain present in the domain deletions, CLEC14A is not properly folded resulting in it no longer binding to MMRN2 (Or the CRT antibodies). This was found out using deletion constructs of CLEC14A far Western blotted with MMRN2 shown in FIG. 11.

2) CRT Antibodies Bind to CTLD Domain of CLEC and not SUSHI

[0577] To further determine whether the CTLD or SUSHI was the binding domain and to ensure correct folding Chimeric constructs of CLEC14A were made with CTLD or SUSHI domains swapped with those of thrombomodulin (also known as CD141)a type 14 CTLD family member which does not bind to MMRN2.

[0578] The sequences of Chimera 5 (CLEC14A with CTLD of CD141) and Chimera 6 (CLEC14A with SUSHI of CD141) are shown in FIG. 12.

[0579] Binding of CRT antibodies was analysed using flow cytometry. All constructs have a C-terminus GFP tag so green cells were gated and stained red. All CRT antibodies bind WT CLEC14A andas expectednone binds to WT CD141 (FIG. 13). In addition, none of the antibodies bound to Chimera 5 (except slight binding by CRT2) and all of the antibodies bind to Chimera 6 (except CRT2) (FIG. 13). This confirms that the binding site of the antibodies CRT1, 3, 4 and 5 and MMRN2 are within the C type lectin domain. It is possible that CRT2 binds on a region between the CTLD and sushi domain.

3) CRT Antibodies that Block MMRN Interaction do not Bind to the Regions Specified in WO 2013/187724 but to a Region that Includes aa 97-108 of CLEC14a CTLD

[0580] To further determine the binding region of the antibodies and MMRN2, chimeric loop constructs were made. This was based on the structural predictions of CLEC14A CTLD and also the regions that the WO2013/187724 antibodies bind to.

TABLE-US-00003 CLEC14Awithregions1-42ofCD141 CD141sequence- (SEQIDNO.21) MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGP CLEC14Awithregions97-108ofCD141 CD141sequence- (SEQIDNO.22) QLPPGCGDPKRL CLEC14Awithregions122-142ofCD141 CD141sequence- (SEQIDNO.23) TSYSRWARLDLNGAPLCGPL

[0581] The alignment is shown in FIG. 12. Unfortunately 1-42 and 122-142 chimeras did not fold correctly. This is thought due to the fact they are present on the cell surface (stain positive for CLEC14A polyclonal antibodies, but they do not stain for any of the C antibodies not even C2.

[0582] However the 97-108 chimera does bind C2 and C3 showing that this mutant is correctly folded. This mutant does not bind MMRN2 or C1, 4 or 5 (which are the antibodies thought to block the CLEC14A-MMRN2 interaction) (FIG. 15). Therefore we conclude that the binding domain is dependent upon the loop containing the following residues: ERRRSCHTLENE (SEQ ID NO: 24).

[0583] Residues 97-108 were swapped with corresponding regions from thrombomodulin. This resulted in correct folding as C2 and C3 can still bind (FIG. 16). However C1, C4 and C5 cannot recognise this mutant suggesting this to be the binding region.

[0584] This experiment has been repeated three times with the same result.

Example 5Antibody Drug Conjugate Tumour Data

[0585] Wild type male C57BL6 mice aged between 6-8 weeks were subcutaneously injected with 1?10?6 Lewis lung carcinoma (LLC) cells in the right flank. Once tumours reached a palpable size, mice were randomly assigned to each treatment group, B12-ADC, or C4-ADC/CRT3-ADC. Mice received two intravenous injections into the tail vein one week apart of 1 mg/kg. One week after final injection mice were culled, tumours were excised and wet weights were measured. The data is shown in FIGS. 17A and B.

[0586] HUVECs were treated with CRT-3 ADC and fluorescent imaging was carried out to determine the localisation of CRT-3 after 0 and 90 minutes. The results are shown in FIG. 18A. Further, the cytotoxicity was measured using a Cell Titre Glo luminescent cell viability assay and the results are shown in FIG. 18B. As described above, 1 million Lewis lung carcinoma cells were injected subcutaneously into the right flank of 2 mice and allowed to grow to a visible size. 1 mg/kg of CRT-3-ADC or B12-ADC (control) was administered through tail vein injections. The mice were observed for an hour and culled 24 hours later.

Results

[0587] The results shown in FIG. 18A demonstrates that the CRT3-ADC are internalised. Further treatment with CRT3-ADC for 24 hours had no effect on the overall health of the mouse. Extensive haemorrhage at the site of the tumour was observed only in the CRT3-ADC treated mouse and not the control, demonstrating tumour-specific disruption of angiogenesis.

Example 6CAR Construction and Experiments

Materials and Methods

Generation of CAR Constructs

[0588] Hybridomas expressing CLEC14A-specific monoclonal antibodies that cross react with human and mouse forms of the protein were obtained as described in Noy et al (Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth. Oncogene. 2015). Gene constructs encoding an scFv were then isolated from each of the mouse hybridomas by RT-PCR using degenerate primer sets designed to amplify all mouse V-gene families as previously described in Hawkins et al (Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines. Blood. 1994; 83(11):3279-88.

[0589] The scFv genes were then subcloned into two previously described CAR vectors pMP71.tCD34.2A.CD19? and pMP71.tCD34.2A.CD19.IEV? (Cheadle et al, J. Immunol., 2014, 192(8), 3654-65) as a ClaI, NotI fragment, replacing the CD19-specific scFv region. These vectors were originally constructed using the MP71 retroviral expression plasmid (a kind gift from C. Baum, Hannover) and coexpressed a truncated CD34 marker gene (Fehse et al, Mol Ther., 2000; 105 Pt 1: 448-56).

Transduction of Human and Mouse T-Cells

[0590] To generate recombinant retrovirus for transducing human T cells, Phoenix amphotropic packaging cells were transfected with an MP71 retroviral vector and pCL ampho (Imgenex) using FuGENE HD (Roche) according to the manufacturer's instructions. Recombinant retrovirus for transducing mouse T cells was generated in the same way but using Phoenix ecotropic packaging cells and pCL eco. Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation on lymphoprep (Axis Shield, Oslo, Norway). PBMCs were pre-activated for 48 hours using anti-CD3 antibody (OKT3, eBioscience; 30 ng/ml), anti-CD28 antibody (R&D Systems; 30 ng/ml) and interleukin-2 (IL2; 300U/ml; Chiron, Emeryville, Calif.) using standard medium (RPMI1640 (Sigma) containing 10% foetal bovine serum (FBS; PAA, Pasching Austria), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 ?g/ml streptomycin) plus 1% human AB serum (TCS Biosciences, Buckingham, UK). Transduction of mouse T cells was conducted using mouse splenocytes pre-activated for 48 hours with concanavalin A (2 ug/ml; Sigma) and mouse interleukin 7 (1 ng/ml; eBioscience) in standard medium. Preactivated human and mouse T cells were subsequently transduced (or mock-transduced with conditioned supernatant from non-transfected phoenix cells) by spinfection in retronectin (Takara)-coated plates according to the manufacturer's instructions. Human T cells were then cultured in standard medium plus 1% human AB serum with IL2 (100 U/ml). After spinfection, mouse T cells were cultured for 24 hrs in standard medium with IL2 (100 U/ml), then purified using lymphoprep (Axis Shield). Where indicated, transduced cells were enriched by immunomagnetic selection using anti-CD34 microbeads (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Studies with human donors were approved by the National Research Ethics Service Committee West Midlands (Solihull) and all donors gave written informed consent

Cell Lines and Recombinant Proteins

[0591] Phoenix A or E, CHO and Lewis lung carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS; PAA, Pasching Austria), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. CHO cells had been transduced with the pWPI vector (Addgene) expressing full length human CLEC14A (or vector alone). Human umbilical vein endothelial cells (HUVECs) were isolated as described previously using umbilical cords obtained from Birmingham Women's Health Care NHS Trust with informed consent and with ethical approval of the south Birmingham research ethics committee. HUVECs were maintained in M199 complete medium containing 10% FBS, 4 mM L-glutamine, 10% large vessel endothelial cell growth supplement (TCS Cellworks) and cultured in plates coated with 0.1% type 1 gelatin from porcine skin (Sigma). Human and murine CLEC14A proteins with a human Fc tag were expressed in HEK293T cells and purified on a protein A column as described in Noy et al (supra)

SiRNA Knockdown of CLEC14A

[0592] Transfection with siRNA was performed as previously described (Armstrong et al, Arteriosclerosis, thrombosis and vascular biology, 2008, 28(9): 1640-6) using the following siRNA duplexes: D1-GAACAAGACAATTCAGTAA (SEQ ID NO. 30) and D2-CAATCAGGGTCGACGAGAA (SEQ ID NO. 31) (EuroGentec, Liege, Belgium).

Flow Cytometry

[0593] HUVECs were trypsinised and stained for 1 hr on ice with CLEC14A-specific mouse monoclonal antibodies described above (10 ug/ml) or IgG1 isotype control (Dako) in 5% normal goat serum/PBS. Cells were washed and bound antibody detected by incubating with R-PE-conjugated goat-anti mouse antibody (Serotec). Dead cells were identified by staining with propidium iodide. Human T-cells were washed with PBS and stained with Live/Dead Fixable Violet Dead Cell Stain Kit (Life Technologies) for 20 mins in the dark. Cells were then washed with flow buffer (0.5% w/v BSA+2 mM EDTA in PBS; pH7.2) and stained with anti human CD4 (PE-conjugated), anti human CD8 (FITC-conjugated) (all from BD Pharmingen) and anti-human CD34 (Pe-Cy5) (BioLegend) for 30 mins on ice in the dark. Alternatively rather than staining for CD34, CAR expression was detected directly by firstly blocking cells with human Fc fragment (10 ug/ml), then incubating them with 10 ug/ml recombinant human CLEC14A-Fc fusion protein (or Fc control) followed by sheep anti CLEC14A polyclonal antibody (R&D systems, 10 ug/ml). Finally cells were stained with FITC-conjugated rabbit anti-sheep antibody (Invitrogen, diluted 1:10). All incubations were conducted for 1 hour on ice.

[0594] When staining mouse T cells from heparinized tail bleeds they were first subject to red blood cell lysis using BD Pharm lyse (Becton Dickinson) before staining as described above but using anti mouse CD4-FITC, CD8-PE and CD45.1 (PE-Cy7 conjugated) (all BD Biosciences). Cells were analyzed using a BD LSR II flow cytometer and FlowJo software (TreeStar Inc, Ashland, Oreg.).

CFSE Labelling

[0595] T-cells were washed twice with PBS and incubated with 2.5 ?M Carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at 37? C. The labelling reaction was quenched by addition of RPMI-1640 containing 10% FBS. Cells were washed, resuspended in standard medium plus 1% human AB serum and IL2 (10 IU/ml) at 1.5?10.sup.6 cells/ml and added to wells containing HUVECs to give a T-cell:HUVEC ratio of 10:1. After 5 days incubation at 37? C./5% CO.sub.2, cells were analysed by flow cytometry as described above using anti-human CD34 (Pe-Cy5).

IFN? Release Assay

[0596] Stimulator cells (2.5?10.sup.4/well) were co-cultured in triplicate with CD34+ CAR-T-cells at responder:stimulator ratios indicated. Alternatively 2?10.sup.4 CD34+ CAR-T cells were incubated in wells precoated with recombinant protein (1 ug/ml). Cells were incubated at 37? C./5% CO.sub.2 in 100 ?l/well of standard medium supplemented with IL2 (25U/ml). After 18 hours, culture supernatant was tested for secreted IFN? using an ELISA (Pierce Endogen, Rockford, Ill.) according to the manufacturer's instructions.

Cytotoxicity Assays

[0597] Chromium release assays have been described in detail previously. They were set up at known effector:target ratios (1250 targets/well) and harvested after 7.5 hours.

In Vivo Experiments

Toxicity Testing

[0598] Six to eight week old C57BL6 mice (Charles River Laboratories) received 4 Gy total body irradiation (TBI). Eighteen hours later, each mouse was injected into the tail vein with 2?10.sup.7 CAR- or Mock-transduced T cell preparations from CD45.1+ congenic BoyJ mice. Mice were monitored for signs of toxicity and immune monitoring was conducted by weekly tail bleeds. Mice were eventually culled 45 days later and major organs removed for histological analysis.

RipTag2 Transgenic Mouse Tumour Model

[0599] Generation of RIP-Tag2 mice as a model of pancreatic islet cell carcinogenesis has been previously reported (Hanahan et al, Nature, 1985, 315 (6015), 115-122). RIP-Tag2 mice were maintained on a C57BL/6J background (The Jackson Laboratory). Cryopreserved CAR-transduced and mock transduced T cells were thawed, washed and 15 million T cells/mouse injected intravenously into the tail vein on a single occasion into 12-week old mice that had been conditioned with 4 Gy TBI the day before. From 12 weeks of age, all RIP-Tag2 mice received 50% sugar food (Harlan Teklad) to relieve hypoglycaemia induced by the insulin-secreting tumours. Total tumour burden in culled CAR-T cell-treated mice was quantified at 16 weeks of age using calipers to measure individually excised macroscopic tumours (>1 mm.sup.3) using the formula: volume=a?b.sup.2?0.52, where a and b represent the longer and shorter diameter of the tumour, respectively. The volumes of all tumours from each mouse were added to give the total tumour burden per animal. There are no age-matched control comparisons for the 16-week CAR-treated mice, since untreated RIP-Tag2 mice do not survive to 16 weeks, and thus the comparison was made to 14-week old Mock-treated mice.

Lewis Lung Carcinoma (LLC) Mouse Model

[0600] 6-8 week old female C57BL6 mice were inoculated subcutaneously on the flank with 10.sup.6 LLC cells. Three days later mice received 4 Gy TBI and 18 hrs after this each mouse was injected into the tail vein with 2?10.sup.7 CAR or Mock T cell preparations from CD45.1+ congenic BoyJ mice. Tumour growth was measured with calipers (using the formula: volume=length?width.sup.2?0.5) and bioluminescence imaging (IVIS Spectrum, Caliper Life Sciences). Immune monitoring was conducted by weekly tail bleeds.

All procedures with RipTag2 mice were approved by the Ethics Committee of the University of Turin, and by the Italian Ministry of Health, in compliance with international laws and policies. All other mouse studies were performed with appropriate UK Home Office approval.

Tissue Preparation and Immunofluorescence Analysis

[0601] Tissues from mouse experiments were embedded in OCT (Bio Optica), frozen in dry ice and stored at ?80? C. Tissue preparation and histology analysis were carried out as described (24) with the following primary antibodies: purified rat monoclonal anti-panendothelial cell antigen (550563, clone Meca32, BD Pharmingen, USA), diluted 1:100; rabbit monoclonal anti-cleaved caspase 3 (asp175, clone 5A1, Cell Signaling, USA), diluted 1:100; rabbit polyclonal anti-Fibrinogen (A0080, Dako), diluted 1:100; and rabbit monoclonal anti-CD34 (ab174720, Abcam) diluted 1:50; sheep polyclonal anti-CLEC14A (AF4968, R&D) diluted 1:50. After incubation and washing, samples were incubated with secondary antibodies anti Rabbit Alexa Fluor-488 and Alexa Fluor-555; anti Rat Alexa Fluor-488 and Alexa Fluor-555; and anti Sheep Alexa Fluor-488 (Molecular Probes) and counterstained with DAPI Nucleic Acid Stain (Invitrogen). To detect CAR-transduced T cells tissues were stained with rabbit monoclonal anti-CD34 (ab174720, Abcam) diluted 1:50 in PBS. After incubation and washing, samples were stained with anti Rabbit Alexa Fluor-555 (Molecular Probes) and counterstained with DAPI.

[0602] Human tumour tissue arrays (SuperBiochips Inc., Seoul, Korea) were stained using sheep polyclonal anti-CLEC14A (AF4968, R&D systems) diluted 1:20 and Ulex europaeus agglutinin I conjugated to rhodamine (Vectorlabs, UK) for 1 hour, followed by anti sheep FITC antibody (10 ?g/ml, Invitrogen, UK).

[0603] For analysis of RipTag2 tumour tissue, the surface area occupied by vessels was quantified through the ImageJ software as the area occupied by Meca32-positive structures, compared with the total tissue area visualised by DAPI. For each animal, the total vessel area of at least four field/images was quantified. To determine the amount of fibrinogen extravasation (red channel) in each image, we drew a region of interest (ROI) close to each blood vessel (Meca32, green channel), and then quantified the mean fluorescence intensity (MFI) of red and green channels using the Leica Confocal Software Histogram Quantification Tool. In order to normalize the vessel number values obtained, we calculated the ratio between red and green channel MFI; values are expressed as percentage of red-green co-staining. To determine the expression levels of caspase 3 (green channel) in each analysed image, we considered 5 random ROIs of the same size. Then we measured the MFI of the green channel, and we normalized the values by comparing caspase 3-stained area with the total cells present in the tissue area. At least 10 images of five mice per treatment group were analyzed for each sample. Tissue from RipTag2 mice were analyzed using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems). All other tissues were analysed using an Axiovert 100M laser scanning confocal microscope (Carl Zeiss, Welwyn Garden City, UK).

Statistical Analysis

[0604] Statistical analyses of data were conducted using the tests indicated and GraphPad Prism software. A p value <0.05 was considered significant.

Results

[0605] CAR constructs have been successfully made using CRT1, 3, 4, and 5 and the expression of these CAR constructs on cells has been demonstrated (see FIGS. 19B and C, FIG. 22 and FIG. 31). Thus, retroviral CAR vectors (based on pMP71) that co-express a truncated CD34 marker gene and an scFv fragment/CD3 zeta chain chimeric receptor were generated. Expression is driven from the LTR promoter and the 2A peptide linker ensures equimolar expression of both the CD34 and the CAR. Second generation CAR constructs included the CD28 costimulatory domain. As shown in FIG. 19B, CD34 staining shows successful transduction of T cells with the vectors comprising CRT3 and 5 (both first and second generation). Similar results can be sheen for cells stained directly for expression of CAR using CLEC14A-Fc (% values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone).

[0606] Further, the data in FIG. 20 show that T-cells transduced to express a first or second generation CAR based on antibodies CRT3 or 5 can respond to CLEC14A in vitro. Results are shown for plate bound protein, protein expressed on engineered CHO cells or expressed in HUVECs, where IFN? production can be seen by T cells transduced with the CARs in all three experiments, in response to the CLEC14A present.

[0607] CARs based on antibodies CRT3 and 5 were tested for their ability to induce cytotoxicity in CHO CLEC14A expressing cells (FIG. 21A), where an increase in the % specific lysis can be seen in cells exposed to T cells with CAR CRT3/5. Further, the proliferation of CD34+ T-cells when cultured with HUVECs shows that both CRT3 and 5 first and second generation CAR T cells proliferate under such culture conditions (i.e. due to exposure to CLEC14A expressing HUVECs). Thus, CRT3/5 CAR T cells are able to respond to CLEC14A expressing cells, resulting in the proliferation of the T-cells and specific lysis of the cells.

[0608] A CAR based on CRT1 antibody also shows activity against CLEC14A expressing targets. Particularly, second generation CRT1 CAR T cells were shown to respond to CLEC14A expressed on CHO cells engineered to express CLEC14A and HUVECs (measured by IFN? release). Further, first and second generation CRT1 CAR T cells were shown to induce specific lysis in CHO CLEC14A expressing cells (see FIG. 22). FIGS. 32 to 35 further show the T-cell response to CLEC14A when transduced with CRT1-CAR and the % lysis induced by such transduced T-cells.

[0609] First or second generation CRT3 and 5 CAR T cells were injected into C57/BL6 mice to determine the toxicity of the CAR T cells to healthy mice. Mice were monitored for 45 days and showed no visible signs of toxicity. FIG. 23 shows that the body weights of the mice increased during this time, and weekly tail bleeds indicate that the CAR T cells persisted for at least 5 weeks after injection and that they comprised at least 30% of the total circulating T cell population during this time. Splenocytes harvested at the end of the experiment were still capable of responding to CLEC14A (both mouse and human). Further, as shown in FIG. 24, histological analysis of several tissues (brain, heart, lung, liver, colon and kidney) showed no pathology for mice infused with second generation CRT3/5 CAR T cells.

[0610] The anti-tumour effect of second generation CARs based on CRT3 or CRT5 antibodies was tested in C57BL6 mice which had previously been injected with 1 million Lewis Lung Carcinoma cells. T cells transduced with the CAR constructs were injected into the tail veins of the mice (20 million T cells) and tumour growth was monitored. As can be seen from FIG. 25, mice injected with either CRT3 or 5 second generation CAR T cells had tumours of smaller volumes than control mice, demonstrating the anti-tumour effect of the CAR T cells. Further, FIG. 26 shows a reduced mean tumour weight, reduced vascular density and an increased level of vascular leakage in mice injected with either CRT3 or 5 second generation CART cells.

[0611] Second generation CRT5 CAR T cells were injected into RIP-Tag2 mice, where the rat insulin promoter directs expression of the SV40 Large T antigen transgene to beta cells of the pancreatic islets, resulting in tumours at around 10 weeks of age and death by week 14. As can be seen in FIG. 27, tumour size was significantly reduced in the CAR T cell injected mice compared to untreated or mock T-cell control mice. Further, FIG. 28 shows that CAR transduced T cells accumulate in RIP-Tag2 tumours 4 weeks after intravenous injection.

[0612] Histological analysis of RipTag2 tumours from mice treated with CAR engineered T cells showed that vascular density is reduced, apoptotic vessels are increased and fibrinogen staining is decreased compared to mice treated with Mock T cells (FIG. 29).

Example 7Functionally Active TCR Specific for the WT1 Derived Peptide pWT126 (RMFPNAPYL)

[0613] A TCR has been cloned that is specific for a peptide RMFPNAPYL (WT126) of the Wilms Tumour antigen-1 (WT1) which is presented by HLA-A2 class I molecules. The WT1 transcription factor is expressed in various human malignancies, including leukaemia, breast cancer, colon cancer, lung cancer, ovarian cancer and other. The CTL (from which the TCR was cloned) show killing activity against human cancer cells that express WT1, but not against normal human cells that express physiological levels of WT1.

[0614] The therapeutic goal was to equip patient T cells with this potent and specific killing activity by transfer of the genes encoding the TCR. For this, TCR genes have been inserted into retroviral vectors and it has been demonstrated that gene transduced human T cells show killing activity against WT1 expressing human cancer and leukemia cell lines. The specificity profile of this CTL line has been described in several research papers and can be summarized as: (1) Killing of HLA-A2-positive targets coated with the WT1-derived peptide pWT126 (Gao et al (2000) Blood 95, 2198-2203); (2) Killing of fresh HLA-A2-positive leukaemia cells expressing WT1 (Gao et al (2000) Blood 95, 2198-2203); (3) Killing of HLA-A2-positive leukemia CFU progenitor cells (Gao et al (2000) Blood 95, 2198-2203; Bellantuono et al (2002) 100, 3835-3837); (4) Killing of HLA-A2-positive leukaemia LTC-IC stem cells (Bellantuono et al (2002) Blood 100, 3835-3837); (5) Killing of HLA-A2-positive NOD/SCID leukaemia initiating cells (Gao et al (2003) Transplantation 75, 1429-1436); and (6) No killing of normal HLA-A2-positive NOD/SCID engrafting hematopoietic stem cells (Gao et al (2003) Transplantation 75, 1429-1436). It has now been shown that human T cells transduced with the WT1-specific TCR display similar specificity as the CTL line from which the TCR was cloned.

[0615] The data described in detail in the legends to FIGS. 36 to 43 indicate that TCR gene transfer into human T cells is feasible and that it leads to the surface expression of the introduced TCR chains. The recipient T cells show killing activity against HLA-A2-positive targets coated with the pWT126 peptide. The TCR-transduced T cells also kill human tumour cells expressing WT1 endogenously. In addition, the transduced T cells produce IFN-? in an HLA-A2-restricted, peptide-specific fashion. Finally, the transferred TCR can function in CD4-positive helper T cells. These CD4-positive T cells show HLA-A2-restricted, antigen-specific killing activity and antigen-specific cytokine production (not shown). This indicates that TCR gene transfer can be used to confer HLA class 1-restricted antigen-specific effector function to both CD8-positive and CD4-positive human T cells.

Methodology

Cell Lines

[0616] T2 is a transporter associated with antigen processing (TAP)-deficient human HLA-A2+ cell line that can be efficiently loaded with exogenous peptides. The BV173 cell line was established from the peripheral blood of a male patient with CML. All cells were cultured in RPMI plus 10% FCS at 37? C.

Synthetic Peptides and HLA-A2/Peptide Complex Tetramer

[0617] pWT126 (RMFPNAPYL) and pWT235 (CMTWNQMNL) are HLA-A2 binding peptides derived from human WT1. pWT126 was dissolved in PBS and pWT235 was dissolved in DMSO before diluting in PBS to give a concentration of 2 mM.

Retroviral TCR Constructs and Transduction of TCR Genes

[0618] The WT1-specific TCR alpha and beta genes were isolated from the allo-restricted pWT126-specific human CTL line 77. To clone the TCR genes, total RNA was extracted from CTL line 77, and reverse transcribed into cDNAs. cDNAs were amplified using a consensus primer that binds to both variable alpha and beta genes in combination with a set of constant primers. The isolated TCR Valpha or V beta gene was then cloned into pMP71 retroviral vector using the NotI and EcoRI restriction sites.

[0619] 2?10.sup.6 amphotropic packaging cells were seeded into a T25 flask and 24 hours later were transiently transfected with retroviral TCR constructs using calcium phosphate precipitation. In preparation for transduction, PBMCs were activated using anti-CD3 antibody and IL-2 for 2 days. Activated T cells (3?10.sup.6) were then resuspended in 3 ml of normal growth medium plus 3 ml of virus supernatant harvested from transfected packaging cells and plated in 6-well plates costed with fibronectin. Plates were incubated at 37? C. at 5% CO2 and 24 to 48 hours after transduction, expression of TCR transgenes was carried out.

IFN?-Secretion Assays

[0620] TCR-transduced T cells (5?10.sup.4) were stimulated with 5?104 leukaemia cells or peptide-coated T2 cells (1:1 ratio) in triplicate in a 96-well plate. After 24 hours incubation, the supernatant was harvested and tested in an interferon ? enzyme linked immunosorbent assay (ELISA) using a human IFN? determination kit (AMS Biotechnology).

Example 8: Selection and Treatment of a Patient

[0621] Peripheral blood monocyte cells (PBMCs) are taken from an HLA-A2-positive patient who has a WT1-expressing malignancy. The PBMCs are activated with anti-CD3/CD28 antibodies added to the culture or on beads for 3 days and then transduced with TCR encoding retroviral particles as described in Example 1. At day 5 we can demonstrate that transduced CD4 and CD8 T cells express the introduced TCR. At day 6 we can demonstrate antigen-specific activity of the transduced T cells. At day 6 the transduced T cells are reinfused into the patient.

Example 9: Wound Healing in Tumour Bearing Mice

[0622] At day 0, mice were injected subcutaneously with 1?10.sup.6 Lewis Lung carcinoma cells and on day 3 were irradiated with 4 Gy. On day 3 mice were injected intravenously with 13?10.sup.6 Mock (n=7) or CAR T cells (n=7) (CRT5 CAR). Cells were 31% CD4+(93% CD34+) and 43% CD8+(62% CD34+). Wound healing was observed for 7 days.

[0623] The results in FIG. 44 show that mouse T cells expressing the CRT5 CAR did not impede healing of a skin wound in tumour bearing mice compared to similar mice treated with mock-transduced T cells.

Example 10: CRT5 CAR in a PDAC Mouse Model

[0624] K-RasG12D; Ink4a/Arf?/?; p53R172H cells are injected into the pancreas of syngeneic immunocompetent mice to generate the PDAC mouse model which is a mouse model of pancreatic adenocarcinoma. The staining of tumours from this mouse model has shown CLEC14A expression on the majority of tumour vessels. Treatment of PDAC mice with CRT5 CAR T cells (where the CAR comprises a costimulatory domain from CD28) results in significant tumour control (FIG. 45) 3 weeks after treatment. Results from an additional experiment can be seen in FIG. 50.

Example 11: Titration of CRT1, 3 and 5 Against CLEC14A

[0625] CLEC14A was expressed as an Fc fusion protein for incubation with CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells. All CAR-T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. The results can be seen in FIG. 46 where it is shown that all of the tested CAR T cells respond well to CLEC14A (data shown are means of triplicate cultures+SD).

Example 12: CRT1, 3 and 5 CAR T Cell Cytotoxicity and Proliferation Assay

[0626] A cytotoxicity study was carried out using CRT1, 3 and 5 CAR (with CD28 costimulatory domain) T cells. The T cells were diluted with Mock T cells to equalise for transduction efficiencies and were incubated with mouse endothelial cells expressing human CLEC14A. The results are shown in FIG. 48A which demonstrate that all three tested CARs can mediate cytotoxicity. The data shown are means of triplicate cultures+SD.

[0627] Further, a proliferation assay was carried out (CFSE labelling) with CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells stimulated with plate-bound recombinant CLEC14A-Fc fusion proteins. All the CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies and the results can be seen in FIG. 48B, where all three tested CARs were capable of proliferating after stimulation. The data shown are for CD8+ T cells only but similar data were obtained for CD4+ T cells.

Example 13: CARs with Different Costimulatory and Transmembrane Regions

[0628] The following CARs have been cloned and engineered into T cells from a single donor using a retroviral vector:

[0629] 1) CRT3-CD28 TM-CD28 costim signal-CD3 (CRT3.28z)

[0630] 2) CRT3-CD8 TM-4-1BB costim signal-CD3 (CRT3.BBz)

[0631] 3) CRT3-CD28 TM-CD28 and 4-1BB costim signals-CD3 (CRT3.28BBz)

[0632] 4) CRT3-CD28 TM-CD28 and OX40 costim signals-CD3 (CRT3.28Oxz)

[0633] 5) CRT3-CD8 TM-4-1BB and OX40 costim signals-CD3 (CRT3.BBOxz)

[0634] All constructs generated transduced well into T cells. The function of the different constructs was assessed in vitro, analysing cytokine production, cytotoxicity and proliferative response (see FIG. 49). Cytokine release indicated strong antigen specific responses especially by T cells expressing CRT3.28z and CRT3.28Oxz. Cytokine production was analysed by measuring IFNgamma production in response to titrated numbers of CHO cells expressing human CLEC14A (or vector only control). All CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. Data shown are means of triplicate cultures+SD. Cytotoxic activity was measured against mouse endothelial cells (SEND) engineered to express CLEC14A and the proliferative response was measured following stimulation with plate-bound recombinant CLEC14A-Fc fusion proteins (data not shown).

Example 14: Determination of Cytokine Release from CAR T Cells Following Stimulation with Chimeric CLEC14A

[0635] Chimeric forms of CLEC14A that contain the human sequence but with the transmembrane and/or intracellular domains of mouse origin were expressed in 293 and SEND cells. These cells were sorted using GFP co-expressed from a lentiviral vector to equalise for CLEC expression and then tested using CAR T cells (CRT1, 3 and 5 with CD28 costimulatory domain). The release of IFN gamma was measured after incubation of the CAR T cells with both the 293 and SEND cells. The results can be seen in FIG. 51. Additionally, the cytotoxicity of the T cells when incubated with the CLEC14A chimera expressing SEND cells was determined. All CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. Data shown are means of triplicate cultures.

[0636] As can be seen from FIG. 51, all of the tested CRT1, 3 and 5 CAR T cells result in the release of IFN gamma from 293 and SEND cells expressing either human CLEC14A (huCLEC), human CLEC14A with mouse intracellular domain (A1) and human CLEC14A with mouse transmembrane and intracellular domain (B1).