CTLA-4 protein variants
Protein variants of CTLA-4, which show increased activity compared to wild-type in a cell assay and which exhibit increased stability. Such variants are useful for treatment of disorders whereby attenuation of the T cell response would be beneficial.
1. A CTLA-4 polypeptide variant having improved potency or stability compared to the CTLA-4 polypeptide of SEQ ID NO: 8 comprising 4 to 10 amino acid substitutions compared to the CTLA-4 polypeptide of SEQ ID NO: 8 wherein 4 to 10 of the amino acid substitutions are selected from the following table:
Position
Amino acid residue
17
S, V, T
26
P
28
S
29
N
43
G
46
S
49
W, Y
54
V
59
S
61
S
62
P
66
T
70
I
71
F, L
81
R
86
V, T
94
E
wherein said 4 to 10 amino acid substitutions include those at amino acid positions 26, 59, 71, and 94, wherein said improved potency is at least a 2-fold reduction in IC 50 in an assay of IL-2 production using T-cells activated by B-cells, and wherein said improved stability is at least 10% residual binding calculated as the binding signal to B7-1 or B7-2 in the presence of 10 mM DTT divided by the binding signal to B7-1 or B7-2 in the absence of DTT, multiplied by 100.
2. The CTLA-4 polypeptide variant according to claim 1 , wherein the amino acid substitutions comprise a set of mutations selected from:
(i) I17S, S26P, G28S, L59S, S71L, K94E;
(ii) S26P, M54V, L59S, S71F, K94E;
(iii) I17V, S26P, K29N, L59S, I66T, S71F, M86V, K94E;
(iv) S26P, L59S, T70I, S71F, M86T, K94E;
(v) S26P, C49W, L59S, L62P, S71F, Q81R, K94E;
(vi) I17T, S26P, C49Y, L59S, F61S, S71F, K94E; and
(vii) S26P, S43G, T46S, L59S, S71F, Q81R, K94E.
3. The CTLA-4 polypeptide variant according to claim 2 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 7, 5, and 6.
4. A composition comprising a CTLA-4 polypeptide variant according to claim 1 and a pharmaceutically acceptable excipient.
5. A fusion protein comprising at least the extracellular domain of a CTLA-4 polypeptide variant according to claim 1 fused to a further polypeptide.
6. A fusion protein according to claim 5 , wherein said further polypeptide comprises an immunoglobulin portion.
7. A method of treating rheumatoid arthritis in an individual, comprising administering to the individual a CTLA-4 variant according to claim 6 .
8. The CTLA-4 polypeptide variant according to claim 1 wherein the T-cells are Jurkat T cells and the B-cells are Raji B-cells.
9. The CTLA-4 polypeptide variant according to claim 1 which is further mutated by substituting Tyr for Ala at amino acid position 30 and substituting Glu for Leu at amino acid position 105.
10. A CTLA-4 polypeptide variant having improved potency compared to the CTLA-4 polypeptide of SEQ ID NO: 8 comprising 4 to 10 amino acid substitutions compared to the CTLA-4 polypeptide of SEQ ID NO: 8 wherein 4 to 10 of the amino acid substitutions are selected from the following table:
Position
Amino acid residue
17
S, V
26
P
28
S
29
N
54
V
59
S
66
T
70
I
71
F, L
86
V, T
94
E
wherein said 4 to 10 amino acid substitutions include those at amino acid positions 26, 59, 71, and 94, and wherein said improved potency is at least a 2-fold reduction in IC 50 in an assay of IL-2 production using T-cells activated by B-cells.
11. The CTLA-4 polypeptide variant according to claim 10 , wherein the amino acid substitutions comprise a set of mutations selected from:
(i) I17S, S26P, G28S, L59S, S71L, K94E;
(ii) S26P, M54V, L59S, S71F, K94E;
(iii) 117V, S26P, K29N, L59S, 166T, S71F, M86V, K94E; and
(iv) S26P, L59S, T70I, S71F, M86T, K94E.
12. The CTLA-4 polypeptide variant according to claim 11 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, and 4.
13. A CTLA-4 polypeptide variant having improved stability compared to the CTLA-4 polypeptide of SEQ ID NO: 8 comprising 4 to 10 amino acid substitutions compared to the CTLA-4 polypeptide of SEQ ID NO: 8 wherein 4 to 10 of the amino acid substitutions are selected from the following table:
Position
Amino acid residue
17
T
26
P
43
G
46
S
49
W, Y
59
S
61
S
62
P
71
F
81
R
94
E
wherein said 4 to 10 amino acid substitutions include those at amino acid positions 26, 59, 71, and 94, and wherein said improved stability is at least 10% residual binding calculated as the binding signal to B7-1 or B7-2 in the presence of 10 mM DTT divided by the binding signal to B7-1 or B7-2 in the absence of DTT, multiplied by 100.
14. The CTLA-4 polypeptide variant according to claim 13 , wherein the amino acid substitutions comprise a set of mutations selected from:
(i) S26P, C49W, L59S, L62P, S71F, Q81R, K94E;
(ii) I17T, S26P, C49Y, L59S, F61S, S71F, K94E; and
(iii) S26P, S43G, T46S, L59S, S71F, Q81R, K94E.
15. The CTLA-4 polypeptide variant according to claim 14 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 5, and 6.
16. A method of making a CTLA-4 polypeptide variant having improved potency or stability compared to the CTLA-4 polypeptide of SEQ ID NO: 8 comprising:
making 5 or more amino acid substitutions to the human CTLA-4 of SEQ ID NO: 8, where the amino acid positions substituted are selected from positions 17, 26, 28, 29, 43, 46, 49, 54, 59, 61, 62, 66, 70, 71, 81, 86, and 94; and
testing the CTLA-4 variant for improved potency and/or stability, wherein said improved potency is at least a 2-fold reduction in IC 50 in an assay of IL-2 production using T-cells activated by B-cells, and wherein said improved stability is at least 10% residual binding calculated as the binding signal to B7-1 or B7-2 in the presence of 10 mM DTT divided by the binding signal to B7-1 or B7-2 in the absence of DTT, multiplied by 100.
17. A method of making a CTLA-4 polypeptide variant having improved potency or stability compared to the CTLA-4 polypeptide of SEQ ID NO: 9 comprising:
making 5 or more amino acid substitutions to the human CTLA-4 of SEQ ID NO: 9, where the amino acid positions substituted are selected from positions 17, 26, 28, 29, 43, 46, 49, 54, 59, 61, 62, 66, 70, 71, 81, 86, and 94; and
testing the CTLA-4 variant for improved potency and/or stability, wherein said improved potency is at least a 2-fold reduction in IC 50 in an assay of IL-2 production using T-cells activated by B-cells, and wherein said improved stability is at least 10% residual binding calculated as the binding signal to B7-1 or B7-2 in the presence of 10 mM DTT divided by the binding signal to B7-1 or B7-2 in the absence of DTT, multiplied by 100.
18. A method of identifying or obtaining a CTLA-4 variant with improved potency and/or stability compared with a parent CTLA-4 polypeptide, wherein said parent CTLA-4 polypeptide is selected from the group consisting of a human wild-type CTLA-4 having the amino acid sequence of SEQ ID NO: 8, a CTLA-4 variant having the amino acid sequence of SEQ ID NO: 9, and a CTLA-4 variant with one amino acid difference compared to SEQ ID NO: 8, the method comprising:
mutating nucleic acid encoding the parent CTLA-4 polypeptide to provide one or more nucleic acids with sequences encoding one or more CTLA-4 variants having altered amino acid sequences at five or more of the following positions in the amino acid sequence of said parent CTLA-4 polypeptide: 17, 26, 28, 29, 43, 46, 49, 54, 59, 61, 62, 66, 70, 71, 81, 86 and 94;
expressing the nucleic acid or nucleic acids to produce the CTLA-4 variant or variants;
testing the CTLA-4 variant or variants thus produced for improved potency and/or stability compared with said parent CTLA-4 polypeptide, wherein said improved potency is at least a 2-fold reduction in IC 50 in an assay of IL-2 production using T-cells activated by B-cells, and wherein said improved stability is at least 10% residual binding calculated as the binding signal to B7-1 or B7-2 in the presence of 10 mM DTT divided by the binding signal to B7-1 or B7-2 in the absence of DTT, multiplied by 100 and wherein, optionally, a library or diverse population of CTLA-4 variants with altered amino acid sequences is produced and tested.
19. A method of making a CTLA-4 variant with improved potency compared with a parent CTLA-4 polypeptide, wherein said parent CTLA-4 polypeptide is human wild-type CTLA-4 having the amino acid sequence of SEQ ID NO: 8, the method comprising:
providing a mutation at two or more vernier positions selected from 26, 28, 71, and 94 in the amino acid sequence of SEQ ID NO: 8, wherein said mutation enhances the ability of the CTLA-4 variant to bind a B7 molecule compared with said parent CTLA-4 polypeptide.