Soluble CTLA4 mutant molecules
The present invention provides soluble CTLA4 mutant molecules which bind with greater avidity to the CD80 and/or CD86 antigen than wild type CTLA4 or non-mutated CTLA4Ig. The soluble CTLA4 molecules have a first amino acid sequence comprising the extracellular domain of CTLA4, where certain amino acid residues within the S25-R33 region and M97-G107 region are mutated. The mutant molecules of the invention may also include a second amino acid sequence which increases the solubility of the mutant molecule.
1. A CTLA4 mutant molecule which binds CD80 and/or CD86 comprising an extraceilular domain of CTLA4 as shown in SEQ ID NO: 8 beginning with alanine at position 26 or methionirie at position 27 and ending with aspartic acid at position 150, or a portion thereof, wherein in the extraceliular domain or portion thereof an alanine at position 55 is substituted with a tyrosine, and a leucine at position 130 is substituted with a glutamic acid.
2. A CTLA4 mutant molecule comprising:
(a) an amino acid sequence beginning with methionine at position 27 and ending with aspartic acid at position 150 of SEQ ID NO:4, or
(b) an amino acid sequence beginning with alanine at position 26 and ending with aspartic acid at position 150 of SEQ ID NO:4.
3. A CTLA4 mutant molecule comprising:
(a) an amino acid sequence beginning with methionine at position 27 and ending with aspartic acid at position 150 of SEQ ID NO:4 or a portion thereof that binds CD80 and/or CD86, or
(b) an amino acid sequence beginning with alanine at position 26 and ending with aspartic acid at position 150 of SEQ ID NO:4 or a portion thereof that binds CD80 and/or CD86.
4. The CTLA4 mutant molecule of claim 1 , 2 , or 3 further comprising an amino acid sequence which alters the solubility or affinity of the CTLA4 mutant molecule.
5. The CTLA4 mutant molecule of claim 4 , wherein the amino acid sequence which alters the solubility or affinity comprises an immunoglobulin.
6. The CTLA4 mutant molecule of claim 5 , wherein the immunoglobulin is an immunoglobulin constant region or portion thereof.
7. The CTLA4 mutant molecule of claim 6 , wherein the immunoglobulin constant region or portion thereof is mutated to reduce effector function.
8. The CTLA4 mutant molecule of claim 6 , wherein the immunoglobulin constant region comprises a hinge, CH2 and CH3 regions of an immunoglobulin molecule.
9. The CTLA4 mutant molecule of claim 6 , wherein the immunoglobulin constant region or portion thereof is a human or monkey immunoglobulin constant region.
10. A CTLA4 mutant molecule comprising:
(a) an amino acid sequence beginning with methionine at position 27 and ending with lysine at position 383 of SEQ ID NO:4, or
(b) an amino acid sequence beginning with alanine at position 26 and ending with lysine at position 383 of SEQ ID NO:4.
11. A CTLA4 mutant molecule consisting of:
(a) an amino acid sequence beginning with methionine at position 27 and ending with lysine at position 383 of SEQ ID NO:4, or
(b) an amino acid sequence beginning with alanine at position 26 and ending with lysine at position 383 of SEQ ID NO:4.
12. The CTLA4 mutant molecule of claim 1 , 2 , 3 , or 10 further comprising an amino acid sequence which permits secretion of the CTLA4 mutant molecule.
13. The CTLA4 mutant molecule of claim 12 , wherein the amino acid sequence which permits secretion comprises an oncostatin M signal peptide.
14. A CTLA4 mutant molecule comprising an amino acid sequence beginning with methionine at position 1 and ending with lysine at position 383 of SEQ ID NO:4.
15. A CTLA4 mutant molecule encoded by the nucleic acid molecule designated ATCC No. PTA-2104.
16. A CTLA4 mutant molecule comprising the entire extracellular domain of the CTLA4 mutant molecule encoded by the nucleic acid molecule designated ATCC No. PTA-2104.
17. A pharmaceutical composition comprising a CTLA4 mutant molecule of claim 1 , 2 , 3 , 10 , 11 , 15 , or 16 and a pharmaceutically acceptable carrier.
18. The CTLA4 mutant molecule of claim 5 , wherein the immunoglobulin comprises a hinge and any or all of the cysteine residues within the hinge are substituted with serine.
19. The CTLA4 mutant molecule of claim 18 , wherein a cysteine at position +156 is substituted with a serine, a cysteine at position +162 is substituted with a serine, and a cysteine at position +165 is substituted with a serine, as shown in SEQ ID NO:4.
20. The CTLA4 mutant molecule of claim 6 , wherein the immunoglobulin constant region or portion thereof is mutated to include a cysteine at position +156 substituted with a serine, a cysteine at position +162 substituted with a serine, a cysteine at position +165 substituted with a serine, and a praline at position +174 substituted with serine, as shown in SEQ ID NO:4.
21. The CTLA4 mutant molecule of claim 5 , wherein the immunoglobulin comprises an amino acid sequence which begins with glutamic acid at position +152 and ends with lysine at position +383, as shown in SEQ ID NO:4.
22. The CTLA4 mutant molecule of claims 1 , 2 , or 3 , further comprising a junction amino acid residue and an immunoglobulin, where the junction amino acid residue is located between the amino acid sequence which ends with aspartic acid at position +150 and the immunoglobulin.
23. The CTLA4 mutant molecule of claim 22 , wherein the junction amino acid residue is glutamine.
24. The CTLA4 mutant molecule of claims 1 , 2 , 3 , 10 or 11 , that has a slower dissociation rate from binding CD86than wild type CTLA4.
25. The CTLA4 mutant molecule of claims 1 , 2 , 3 , 10 , 11 , 15 , or 16 , that is soluble.
26. The CTLA4 mutant molecule of claims 1 , 2 , 3 , 10 , 11 , 15 , or 16 , that is substantially pure.
27. The CTLA4 mutant molecule of claims 1 , 2 , 3 , 10 , 11 , 15 , or 16 , wherein the CTLA4 mutant molecule is a dimer.
28. The CTLA4 mutant molecule of claims 1 , 2 , 3 , 10 , 11 , 15 , or 16 wherein the CTLA4 mutant molecule has a molecular mass of about 100 kilodaltons.
29. The CTLA4 mutant molecule of claim 28 , wherein the molecular mass is assessed by SDS-PAGE under non-reducing conditions.
30. The CTLA4 mutant molecule of claim 7 , wherein the immunoglobulin constant region comprises a hinge, CH2 and CH3 regions of an immunoglobulin molecule.
31. A pharmaceutical composition comprising a CTLA4 mutant molecule of claim 30 and a pharmaceutically acceptable carrier.
32. The CTLA4 mutant molecule of 30 , that is soluble.
33. The CTLA4 mutant molecule of claim 30 , that is substantially pure.
34. The CTLA4 mutant molecule of claim 30 , wherein the CTLA4 mutant molecule is a dimer.
35. The CTLA4 mutant molecule of claim 30 , wherein the CTLA4 mutant molecule has a molecular mass of about 100 kilodaltons.
36. The CTLA4 mutant molecule of claim 35 , wherein the molecular mass is assessed by SDS-PAGE under non-reducing conditions.
37. A pharmaceutical composition comprising a CTLA4 mutant molecule of claim 8 and a pharmaceutically acceptable carrier.
38. The CTLA4 mutant molecule of claim 8 that is soluble.
39. The CTLA4 molecule of claim 8 that is substantially pure.
40. The CTLA4 mutant molecule of claim 8 , wherein the CTLA4 mutant molecule is a dimer.
41. The CTLA4 mutant molecule of claim 8 , wherein the CTLA4 mutant molecule has a molecular mass of about 100 kilodaltons.
42. The CTLA4 mutant molecule of claim 41 , wherein the molecular mass is assessed by SDS-PAGE under non-reducing conditions.