High affinity Melan-A T cell receptors

1. An isolated T-cell receptor (TCR) having the property of binding to AAGIGILTV (SEQ ID NO:43) -HLA-A*0201 and comprising:

a TCR α chain variable domain having:

SEQ ID NO:1 or

SEQ ID NO:1 except that one or more of amino acids 28D, 29R, 30G, 31S, 49M, 51I, 53S, 54N, 72Y, 94V, 95A, 96G, 97K, 98S, and 99T is mutated; and

a TCR β chain variable domain having:

SEQ ID NO:2; or

SEQ ID NO:2 except that one or more of amino acids 45L, 51S, 52V, 53G, 54I, 76I, 100G, 101T, 102G, 103E, 104L, and 105F are mutated, said TCR having a K D for the said AAGIGILTV (SEQ ID NO:43)-HLA-A*0201 complex of less than or equal to 3 μM and/or an off-rate (k off ) of 1×10 −3 S − 1 or slower,

wherein the α and β chain variable domains of the TCR are not simultaneously unmutated SEQ ID NO:1 and unmutated SEQ ID NO:2, respectively.

2. A TCR as claimed in claim 1 comprising one or more alpha chain variable region mutations corresponding to 28D→F, 28D→Y, 28D→S, 28D→N, 29R→Q, 29R→L, 29R→I, 29R→F,30G→H, 31S→A, 49M→I, 51I→T, 53S→R, 54N→E, 72Y→H, 94V→D, 94V→P, 94V→S, 94V→L, 94V→N, 95A→G, 95A→S, 95A→E, 9G6→N, 96G→P, 96G→V, 96G→M, 96G→L, 96G→R, 97K→R, 97K→Y, 97K→V, 97→L, 97K→H, 97K→G, 97K→I, 97K→P, 98S→L, 98S→M, 98S→R, 99T→L or 99T→R using the numbering shown in SEQ ID NO:1.

3. A TCR as claimed in claim 1 comprising one or more beta chain variable region mutations corresponding to 45L→P, 51S→Y, 51S→F, 51S→W, 52V→G, 53G→P, 54I→F, 54→Y, 7I6→V, 100G→N, 101T→M, 101T→L, 101T→V, 102G→S, 102G→N, 102G→T, 103E→G, 104L→W, 105F→S, 105F→A, 105F→Q, 105F→D, or 105F→E using the numbering shown in SEQ ID NO:2.

4. A TCR as claimed in claim 1 comprising one of the alpha chain variable region amino acid sequences of SEQ ID NOS:11 to 24 or 47 to 53.

5. A TCR as claimed in claim 1 comprising one of the beta chain variable region amino acid sequences of SEQ ID NOS:54 to 67.

6. A TCR as claimed in claim 1 comprising an alpha and beta chain variable region pairing selected from the group consisting of SEQ ID NOS:11 and 2; SEQ ID NOS:12 and 2; SEQ ID NOS:13 and 2; SEQ ID NOS:14 and 2; SEQ ID NOS:15 and 2; SEQ ID NOS:16 and 2; SEQ ID NOS:17 and 2; SEQ ID NOS:18 and 2; SEQ ID NOS:19 and 2; SEQ ID NOS:20 and 2; SEQ ID NOS:21 and 2; SEQ ID NOS:22 and 2; SEQ ID NOS:23 and 2; SEQ ID NOS:24 and 2; SEQ ID NOS:47 and 2; SEQ ID NOS:48 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:50 and 2; SEQ ID NOS:51 and 2; SEQ ID NOS:52 and 2; SEQ ID NOS:53 and 2; SEQ ID NOS:11 and 54; SEQ ID NOS:11 and 55; SEQ ID NOS:11 and 56; SEQ ID NOS:11 and 57; SEQ ID NOS:11 and 58; SEQ ID NOS:11 and 59; SEQ ID NOS:11 and 60; SEQ ID NOS:11 and 61; SEQ ID NOS:11 and 62; SEQ ID NOS:11 and 63; SEQ ID NOS:11 and 64; SEQ ID NOS:11 and 65; SEQ ID NOS:11 and 66; and SEQ ID NOS:11 and 67.

7. A TCR as claimed in claim 1 further comprising the alpha chain constant region amino acid sequence shown in SEQ ID NO:25 and/or one of the beta chain amino acid constant region sequences shown in SEQ ID NOS:26 and 27.

8. A TCR as claimed in claim 1 which is a dimeric T cell receptor (dTCR) or a single chain T cell receptor (scTCR).

9. A TCR as claimed in claim 1 wherein the TCR is associated with at least one polyalkylene glycol chain(s).

10. A TCR as claimed in claim 1 associated with a therapeutic agent or detectable moiety.

11. A TCR as claimed in claim 10 wherein the therapeutic agent or detectable moiety is covalently linked to the C terminus of one or both TCR chains.

12. A TCR as claimed in claim 10 associated with a therapeutic agent which is an immune effector molecule.

13. A TCR as claimed in claim 12 wherein the immune effector molecule is a cytokine.

14. A TCR as claimed in claim 12 wherein the immune effector molecule is IL-2, or a functional variant or fragment thereof.

15. A multivalent TCR complex comprising at least two TCRs as claimed in claim 1 .

16. A pharmaceutical composition comprising:

a TCR as claimed in claim 1 .

17. A recombinant T-cell receptor (TCR) having the property of binding to AAGIGILTV (SEQ ID NO:43)-HLA-A*0201 and comprising:

a TCR α chain variable domain having:

SEQ ID NO:1 or

SEQ ID NO:1 except that one or more of amino acids 28D, 29R, 30G, 31S, 49M, 51I, 53S, 54N, 72Y, 94V, 95A, 96G, 97K, 98S, and 99T is mutated; and

a TCR β chain variable domain having:

SEQ ID NO:2; or

SEQ ID NO:2 except that one or more of amino acids 45L, 51S, 52V, 53G, 54I, 76I, 100G, 101T, 102G, 103E, 104L, and 105F are mutated, said TCR having a K D for the said AAGIGILTV (SEQ ID NO:43)-HLA-A*0201 complex of less than or equal to 3 μM and/or an off-rate (k off ) of 1×10 −3 S − 1 or slower,

wherein the α and β chain variable domains of the TCR are not simultaneously unmutated SEQ ID NO:1 and unmutated SEQ ID NO:2, respectively.

18. A TCR as claimed in claim 17 comprising one or more alpha chain variable region mutations corresponding to 28D→F, 28D→Y, 28D→S, 28D→N, 29R→Q, 29R→L, 29R→I, 29R→F, 30G→H, 31S→A, 49M→I, 51I→T, 53S→R, 54N→E, 72Y→H, 94V→D, 94V→P, 94V→S, 94V→L, 94V→N, 95A→G, 95A→S, 95A→E, 9G6→N, 96G→P, 96G→V, 96G→M, 96G→L, 96G→R, 97K→R, 97K→Y, 97K→V, 97K→L, 97K→H, 97K→G, 97K→I, 97K→P, 98S→L, 98S→M, 98S→R, 99T→L or 99T→R using the numbering shown in SEQ ID NO:1.

19. A TCR as claimed in claim 17 comprising one or more beta chain variable region mutations corresponding to 45L→P, 51S→Y, 51S→F, 51S→W, 52V→G, 53G→P, 54I→F, 54→Y, 7I6→V, 100G→N, 101T→M, 101T→L, 101T→V, 102G→S, 102G→N, 102G→T, 103E→G, 104L→W, 105F→S, 105F→A, 105F→Q, 105F→D, or 105F→E using the numbering shown in SEQ ID NO:2.

20. A TCR as claimed in claim 17 comprising one of the alpha chain variable region amino acid sequences of SEQ ID NOS:11 to 24 or 47 to 53.

21. A TCR as claimed in claim 17 comprising one of the beta chain variable region amino acid sequences of SEQ ID NOS:54 to 67.

22. A TCR as claimed in claim 17 comprising an alpha and beta chain variable region pairing selected from the group consisting of SEQ ID NOS:11 and 2; SEQ ID NOS:12 and 2; SEQ ID NOS:13 and 2; SEQ ID NOS:14 and 2; SEQ ID NOS:15 and 2; SEQ ID NOS:16 and 2; SEQ ID NOS:17 and 2; SEQ ID NOS:18 and 2; SEQ ID NOS:19 and 2; SEQ ID NOS:20 and 2; SEQ ID NOS:21 and 2; SEQ ID NOS:22 and 2; SEQ ID NOS:23 and 2; SEQ ID NOS:24 and 2; SEQ ID NOS:47 and 2; SEQ ID NOS:48 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:50 and 2; SEQ ID NOS:51 and 2; SEQ ID NOS:52 and 2; SEQ ID NOS:53 and 2; SEQ ID NOS:11 and 54; SEQ ID NOS:11 and 55; SEQ ID NOS:11 and 56; SEQ ID NOS:11 and 57; SEQ ID NOS:11 and 58; SEQ ID NOS:11 and 59; SEQ ID NOS:11 and 60; SEQ ID NOS:11 and 61; SEQ ID NOS:11 and 62; SEQ ID NOS:11 and 63; SEQ ID NOS:11 and 64; SEQ ID NOS:11 and 65; SEQ ID NOS:11 and 66; and SEQ ID NOS:11 and 67.

23. A TCR as claimed in claim 17 further comprising the alpha chain constant region amino acid sequence shown in SEQ ID NO:25 and/or one of the beta chain amino acid constant region sequences shown in SEQ ID NOS:26 and 27.

24. A TCR as claimed in claim 17 which is a dimeric T cell receptor (dTCR) or a single chain T cell receptor (scTCR).

25. A TCR as claimed in claim 17 wherein the TCR is associated with at least one polyalkylene glycol chain(s).

26. A TCR as claimed in claim 17 associated with a therapeutic agent or detectable moiety.

27. A TCR as claimed in claim 26 wherein the therapeutic agent or detectable moiety is covalently linked to the C terminus of one or both TCR chains.

28. A TCR as claimed in claim 26 associated with a therapeutic agent which is an immune effector molecule.

29. A TCR as claimed in claim 28 wherein the immune effector molecule is a cytokine.

30. A TCR as claimed in claim 28 wherein the immune effector molecule is IL-2, or a functional variant or fragment thereof.

31. A multivalent TCR complex comprising at least two TCRs as claimed in claim 17 .

32. A pharmaceutical composition comprising:

a TCR as claimed in claim 17 .

33. An engineered T-cell receptor (TCR) having the property of binding to AAGIGILTV (SEQ ID NO:43)-HLA-A*0201 and comprising:

a TCR α chain variable domain having:

SEQ ID NO:1 or

SEQ ID NO:1 except that one or more of amino acids 28D, 29R, 30G, 31S, 49M, 51I, 53S, 54N, 72Y, 94V, 95A, 96G, 97K, 98S, and 99T is mutated; and

a TCR β chain variable domain having:

SEQ ID NO:2; or

SEQ ID NO:2 except that one or more of amino acids 45L, 51S, 52V, 53G, 54I, 76I, 100G, 101T, 102G, 103E, 104L, and 105F are mutated, said TCR having a K D for the said AAGIGILTV (SEQ ID NO:43)-HLA-A*0201 complex of less than or equal to 3 μM and/or an off-rate (k off ) of 1×10 −3 S − 1 or slower,

wherein the α and β chain variable domains of the TCR are not simultaneously unmutated SEQ ID NO:1 and unmutated SEQ ID NO:2, respectively.

34. A TCR as claimed in claim 33 comprising one or more alpha chain variable region mutations corresponding to 28D→F, 28D→Y, 28D→S, 28D→N, 29R→Q, 29R→L, 29R→I, 29R→F, 30G→H, 31S→A, 49M→I, 51I→T, 53S→R, 54N→E, 72Y→H, 94V→D, 94V→P, 94V→S, 94V→L, 94V→N, 95A→G, 95A→S, 95A→E, 9G6→N, 96G→P, 96G→V, 96G→M, 96G→L, 96G→R, 97K→R, 97K→Y, 97K→V, 97K→L, 97K→H, 97K→G, 97K→I, 97K→P, 98S→L, 98S→M, 98S→R, 99T→L or 99T→R using the numbering shown in SEQ ID NO:1.

35. A TCR as claimed in claim 33 comprising one or more beta chain variable region mutations corresponding to 45L→P, 51S→Y, 51S→F, 51S→W, 52V→G, 53G→P, 54I→F, 54→Y, 7I6→V, 100G→N, 101T→M, 101T→L, 101T→V, 102G→S, 102G→N, 102G→T, 103E→G, 104L→W, 105F→S, 105F→A, 105F→Q, 105F→D, or 105F→E using the numbering shown in SEQ ID NO:2.

36. A TCR as claimed in claim 33 comprising one of the alpha chain variable region amino acid sequences of SEQ ID NOS:11 to 24 or 47 to 53.

37. A TCR as claimed in claim 33 comprising one of the beta chain variable region amino acid sequences of SEQ ID NOS:54 to 67.

38. A TCR as claimed in claim 33 comprising an alpha and beta chain variable region pairing selected from the group consisting of SEQ ID NOS:11 and 2; SEQ ID NOS:12 and 2; SEQ ID NOS:13 and 2; SEQ ID NOS:14 and 2; SEQ ID NOS:15 and 2; SEQ ID NOS:16 and 2; SEQ ID NOS:17 and 2; SEQ ID NOS:18 and 2; SEQ ID NOS:19 and 2; SEQ ID NOS:20 and 2; SEQ ID NOS:21 and 2; SEQ ID NOS:22 and 2; SEQ ID NOS:23 and 2; SEQ ID NOS:24 and 2; SEQ ID NOS:47 and 2; SEQ ID NOS:48 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:49 and 2; SEQ ID NOS:50 and 2; SEQ ID NOS:51 and 2; SEQ ID NOS:52 and 2; SEQ ID NOS:53 and 2; SEQ ID NOS:11 and 54; SEQ ID NOS:11 and 55; SEQ ID NOS:11 and 56; SEQ ID NOS:11 and 57; SEQ ID NOS:11 and 58; SEQ ID NOS:11 and 59; SEQ ID NOS:11 and 60; SEQ ID NOS:11 and 61; SEQ ID NOS:11 and 62; SEQ ID NOS:11 and 63; SEQ ID NOS:11 and 64; SEQ ID NOS:11 and 65; SEQ ID NOS:11 and 66; and SEQ ID NOS:11 and 67.

39. A TCR as claimed in claim 33 further comprising the alpha chain constant region amino acid sequence shown in SEQ ID NO:25 and/or one of the beta chain amino acid constant region sequences shown in SEQ ID NOS:26 and 27.

40. A TCR as claimed in claim 33 which is a dimeric T cell receptor (dTCR) or a single chain T cell receptor (scTCR).

41. A TCR as claimed in claim 33 wherein the TCR is associated with at least one polyalkylene glycol chain(s).

42. A TCR as claimed in claim 33 associated with a therapeutic agent or detectable moiety.

43. A TCR as claimed in claim 42 wherein the therapeutic agent or detectable moiety is covalently linked to the C terminus of one or both TCR chains.

44. A TCR as claimed in claim 42 associated with a therapeutic agent which is an immune effector molecule.

45. A TCR as claimed in claim 44 wherein the immune effector molecule is a cytokine.

46. A TCR as claimed in claim 44 wherein the immune effector molecule is IL-2, or a functional variant or fragment thereof.

47. A multivalent TCR complex comprising at least two TCRs as claimed in claim 33 .

48. A pharmaceutical composition comprising:

a TCR as claimed in claim 33 .

49. A soluble TCR comprising an alpha chain and beta chain pairing selected from the group consisting of: SEQ ID NOS:29 and 10; SEQ ID NOS:68 and 10; SEQ ID NOS:70 and 10; SEQ ID NOS:29 and 71; SEQ ID NOS:29 and 72; SEQ ID NOS:29 and 73; SEQ ID NOS:29 and 74; SEQ ID NOS:29 and 75; SEQ ID NOS:29 and 76; SEQ ID NOS:29 and 77; SEQ ID NOS:29 and 78; and SEQ ID NOS:29 and 79.