Proteins belonging to the Bcl-2 family and fragments thereof, and their use in cancer patients
The present invention relates to proteins belonging to the Bcl-2 family and peptides fragments thereof for use in pharmaceutical compositions. The disclosed proteins and peptide fragments are in particularly useful in vaccine compositions for treatment of cancer. The invention furthermore relates to methods of treatment using said compositions. It is also an aspect of the invention to provide T-cells and T-cell receptors specifically recognising the disclosed proteins and peptide fragments.
1. A vaccine composition comprising an immunogenically active peptide wherein the peptide consists of at the most 15 amino acids and comprises SEQ ID NO:8 or a sequence which differs from SEQ ID NO:8 by one or two amino acid substitutions.
2. The composition of claim 1 , wherein the vaccine composition when administered to a cancer patient, is capable of eliciting an immune response against the cancer disease.
3. The composition of claim 1 , wherein the vaccine composition, when administered to a cancer patient where a Bcl-2 protein family member is expressed, is capable of eliciting an immune response against the cancer disease.
4. The vaccine composition of claim 1 , wherein the peptide is a fragment of Bcl-2.
5. An isolated immunogenically active peptide, wherein the peptide consists of at the most 15 amino acids and comprises SEQ ID NO:8 or a homologue thereof differing from SEQ ID NO:8 by one or two amino acid substitutions.
6. The peptide according to claim 5 , wherein the peptide is a fragment of Bcl-2.
7. The peptide according to claim 5 that is capable of eliciting a cellular immune response in a cancer patient.
8. The peptide according to claim 5 , which is an MHC Class I-restricted peptide having at least one of the following characteristics:
(i) capable of binding to the Class I HLA molecule to which it is restricted at an affinity as measured by the amount of the peptide that is capable of half maximal recovery of the Class I HLA molecule (C 50 value) which is at the most 50 μl as determined by the assembly binding assay as described herein,
(ii) capable of eliciting INF-γ-producing cells in a PBL population of a cancer patient at a frequency of at least 1 per 10 4 PBLs as determined by an ELISPOT assay, and/or
(iii) capable of in situ detection in a tumor tissue of CTLs that are reactive with the epitope peptide.
9. The peptide of claim 8 having a C 50 value, which is at the most 30 μM.
10. The peptide of claim 8 having a C 50 value, which is at the most 20 μM.
11. The peptide of claim 8 , which is restricted by a MHC Class I HLA-A molecule.
12. The peptide of claim 11 , which is restricted by a MHC Class I HLA species selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.
13. The peptide of claim 8 , which is restricted by HLA-A2.
14. The peptide according to claim 5 , which comprises PLFDFSWLSL (SEQ ID NO:8).
15. The peptide of claim 8 , which is restricted by a MHC Class I HLA-B molecule.
16. The peptide of claim 15 , which is restricted by a MHC Class I HLA-3 species selected from the group consisting of HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.
17. The peptide of claim 5 , which is a decapeptide.
18. The peptide according to claim 5 comprising, for each specific HLA allele, any of the amino acid residues as indicated in the following table:
HLA
C-
allele
Position 1
Position 2
Position 3
Position 5
Position 6
Position 7
terminal
HLA-A1
T, S
D, E
L
Y
HLA-A2
L, M
V
L, V
HLA-A3
L, V, M
F, Y
K, Y, F
HLA-
V, I, F, Y
M, L, F, Y, I
K, R
A11
HLA-
I, Y
W, I
A23
HLA-
Y
I, V
F
I, L, F
A24
HLA-
M, A, T
I
W
A25
HLA-
E, D
V, T, I, L, F
I, L, V
Y, F
A26
HLA-
E, D
V, A, L
A, R
A28
HLA-
E
Y, L
A29
HLA-
Y, L, F, V
Y
A30
HLA-
L, M, F, Y
R
A31
HLA-
I, L
W
A32
HLA-
Y, I, L, V
R
A33
HLA-
V, L
R
A34
HLA-
E, D
T, V
R, K
A66
HLA-
E, D
T, V
R, K
A68
HLA-
V, T, A
V, L
A69
HLA-
T
V, L
A74
HLA-B5
A, P
F, Y
I, L
HLA-B7
R, A
P
L, F
HLA-B8
K
K, R
L
HLA-
R, K
L, V
B14
HLA-
Q, L, K, P,
F, Y, W
B15
H, V, I, M,
(B62)
S, T
HLA-
L, V
B17
HLA-
R
Y, K, F, L
B27
HLA-
P
I, L, M, Y
B35
HLA-
D, E
I, L, M
B37
HLA-
H
D, E
F, L
B38
HLA-
R, H
L, F
B39
HLA-
E
F, I, V
L, V, A, W,
B40
M, T, R
(B60, 61)
HLA-
L, P
Y, L
B42
HLA-
E
F, Y, W
B44
HLA-
M, I, L, V
Y, F
B46
HLA-
Q, K
L
B48
HLA-
A, P, G
F, Y, I, V
B51
HLA-
Q
F, Y
I, V
B52
HLA-
P
W, F, L
B53
HLA-
P
B54
HLA-
P
A, V
B55
HLA-
P
A, V
B56
HLA-
A, T, S
F, W, Y
B57
HLA-
A, T, S
F, W, Y
B58
HLA-
P
L
B67
HLA-
R
P
B73
HLA-
A, L
L
Cw1
HLA-
A, L
F, Y
Cw2
HLA-
A, L
L, M
Cw3
HLA-
Y, P, F
L, M, F, Y
Cw4
HLA-
Y
L, Y, F, Y
Cw6
HLA-
Y
L, I,
Cw8
HLA-
A, L
L, V
Cw16.
19. The peptide according to claim 5 that is capable of eliciting INF-γ-producing cells in a PBL population of a cancer patient at a frequency of at least 10 per 10 4 PBLs.
20. The peptide according to claim 5 which is capable of eliciting INF-γ-producing cells in a PBL population of a patient having a cancer disease where a protein belonging to the Bcl-2 protein family is expressed.
21. The peptide of claim 20 where the cancer disease is selected from the group consisting of a haematopoietic malignancy, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.
22. The vaccine composition according to claim 1 comprising a peptide which is an isolated immunogenically active peptide derived from a protein belonging to the Bcl-2 protein family.
23. The vaccine composition of claim 22 wherein said peptide has a C 50 value which is at the most 30 μl.
24. The vaccine composition according to claim 1 where the vaccine elicits the production in a vaccinated patient of effector T-cells having a cytotoxic effect against the cancer cells.
25. The vaccine composition according to claim 1 further comprising an immunogenic protein or peptide fragment selected from a protein or peptide fragment not belonging to or derived from the Bcl-2 protein family.
26. The vaccine composition of claim 25 where the protein or peptide fragment not belonging to or derived from the Bcl-2 protein family is a protein involved in regulation of cell apoptosis or a peptide fragment derived therefrom.
27. The vaccine composition of claim 25 where the immunogenic protein or peptide fragment selected from a protein or peptide fragment not belonging to or derived from the Bcl-2 protein family is survivin or a peptide fragment thereof.
28. The vaccine composition of claim 25 where the immunogenic protein or peptide fragment selected from a protein or peptide fragment not belonging to or derived from the Bcl-2 protein family is ML-IAP or a peptide fragment thereof.
29. The vaccine composition according to claim 1 , wherein the composition comprises an adjuvant.
30. The vaccine composition according to claim 29 , wherein the adjuvant is selected from the group consisting of bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants and imidazochinilines.
31. The vaccine composition according to claim 1 , wherein the vaccine composition further comprises antigen presenting cells comprising the peptide of claim 1 .
32. The vaccine composition according to claim 31 , wherein the antigen presenting cell is a dendritic cell.
33. The vaccine composition according to claim 1 , wherein the composition comprises a liposome.
34. The vaccine composition according to claim 1 , furthermore comprising a T-cell stimulatory polypeptide.
35. The vaccine composition according to claim 34 , wherein the T-cell stimulatory polypeptide is selected from the group consisting of B7.1, ICAM-1 and LFA-3.
36. A kit-of-parts comprising the vaccine composition according to claim 34 , and a further anti-cancer agent.
37. The kit-of-parts according to claim 36 , wherein the anti-cancer agent is an antibody.
38. The kit-of-parts according to claim 37 , wherein the anti-cancer agent is a cytokine.
39. A composition for ex vivo or in situ diagnosis of the presence in a cancer patient of T cells in PBL or in tumor tissue that are reactive with a Bcl-2 protein family member, the composition comprising a peptide according to claim 5 .
40. A diagnostic kit for ex vivo or in situ diagnosis of the presence in a cancer patient of T cells in PBL or in tumor tissue that are reactive with a Bcl-2 protein family member, the kit comprising a peptide according to claim 5 .
41. A complex of a peptide according to claim 5 and a Class I HLA molecule or a fragment of such molecule.
42. The complex of claim 41 which is monomeric.
43. The complex of claim 41 which is multimeric.
44. The peptide of claim 5 which consists of SEQ ID NO:8.
45. The vaccine composition of claim 1 , wherein the peptide is capable of raising a BC12-specific T-cell response.
46. The peptide of claim 14 which is a fragment of Bcl-2.
47. The peptide of claim 5 which comprises a sequence which differs from SEQ ID NO:8, if at all, by a single amino acid substitution.
48. The peptide of claim 5 , which comprises a sequence which differs from SEQ ID NO:8 only at residues other than residues 2, 6 and 10 of SEQ ID NO:8.
49. The peptide of claim 5 , which comprises a sequence which, if it differs from SEQ ID NO:8, is such that the residue corresponding to residue 2 of SEQ ID NO:8 is L or M, and the residue corresponding to residue 10 of SEQ ID NO:8 is L or V.
50. The peptide of claim 5 , which, if it differs from SEQ ID NO:8, at least one such difference is that the residue corresponding to residue 6 of SEQ ID NO:8 is V.
51. The peptide of claim 47 , which comprises a sequence which differs from SEQ ID NO:8 only at residues other than residues 2, 6 and 10 of SEQ ID NO:8.
52. The peptide of claim 47 , which comprises a sequence which, if it differs from SEQ ID NO:8 is such that the residue corresponding to residue 2 of SEQ ID NO:8 is L or M, and the residue corresponding to residue 10 of SEQ ID NO:8 is L or V.
53. The peptide of claim 5 , which, if it differs from SEQ ID NO:8, said difference is that the residue corresponding to residue 6 of SEQ ID NO:8 is V.