Compositions comprising receptor-associated protein (RAP) variants specific for CR-containing proteins and uses thereof
View Patent ↗The present invention relates generally to receptor-selective variants of the low-density lipoprotein receptor-associated protein (RAP) and compositions thereof, methods of generating such variants and methods of using such receptor-selective RAP variant compositions for therapeutic purposes.
1. An isolated polypeptide comprising an amino acid sequence variant of the mature alpha-2-macroglobulin/low density lipoprotein receptor-related protein-associated protein 1 (RAP) set out in SEQ ID NO: 121, wherein said RAP variant binds selectively to a pair of complement-type repeats within a protein selected from the group consisting of very low density lipoprotein receptor (VLDLR), matriptase and CD320, with an affinity that is at least 3-fold greater than the affinity of wild-type RAP for the same pair of complement-type repeats, wherein the RAP variant consists of
(a) a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 121 and comprises mutations at at least two of the following positions: 251, 256, 257, 266 and 270, and optionally comprises a mutation at one or more of positions 175, 205, 213, 217, 226, 230, 232, 239, 246, 249, 251, 256, 257, 261, 266, 267, 268, 270, 273, 287, 290, 294, 296, 297, 298, 305, 312 or 313 of mature RAP; or
(b) a polypeptide having an amino acid sequence at least 90% identical to amino acids 200-323 of SEQ ID NO: 121 and comprises mutations at at least two of the following positions: 251, 256, 257, 266 and 270, and optionally comprises a mutation at one or more of positions 205, 213, 217, 226, 230, 232, 239, 246, 249, 251, 256, 257, 261, 266, 267, 268, 270, 273, 287, 290, 294, 296, 297, 298, 305, 312, or 313 of mature RAP.
2. The polypeptide of claim 1 wherein said RAP variant lacks amino acids 1-143 and 320-323 of a mature RAP.
3. A RAP variant of claim 1 missing at least 200 and up to 243 amino acids from the N-terminus.
4. The RAP variant of claim 3 further missing up to 11 amino acids from the C-terminus.
5. The RAP variant of claim 4 missing at least 4 amino acids from the C-terminus.
6. The RAP variant of claim 1 comprising a continuous portion of mature RAP that is (a) at least 71 amino acids in length and (b) comprises amino acids 256-270.
7. The RAP variant of claim 1 comprising a continuous portion of RAP d3 that is (a) at least 71 amino acids in length and (b) comprises amino acids 256-270.
8. The polypeptide of claim 7 wherein the variant contains a mutation at any one of positions 251, 256, 257, 266, 270, 279, 280, 296 or 305 of RAP.
9. The polypeptide of claim 1 wherein the variant binds selectively to a matriptase protein.
10. The polypeptide of claim 9 wherein the variant contains a mutation at any one of positions 251, 256, 257, 266, 270, or 280 of RAP.
11. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 251 of RAP.
12. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 256 of RAP.
13. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 14 of RAP.
14. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 266 of RAP.
15. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 270 of RAP.
16. The polypeptide of any one of claims 9 - 10 wherein the variant comprises a mutation at position 280 of RAP.
17. The polypeptide of claim 1 wherein the variant binds selectively to a VLDLR protein.
18. The polypeptide of claim 17 wherein the variant contains mutations at at least two positions selected from any one of positions 251, 256, 270 or 296 of RAP.
19. The polypeptide of any one of claims 17 - 18 wherein the variant comprises a mutation at position 251 of RAP.
20. The polypeptide of any one of claims 17 - 18 wherein the variant comprises a mutation at position 256 of RAP.
21. The polypeptide of any one of claims 17 - 18 wherein the variant comprises a mutation at position 270 of RAP.
22. The polypeptide of any one of claims 17 - 18 wherein the variant comprises a mutation at position 296 of RAP.
23. The polypeptide of claim 1 wherein the variant binds selectively to a CD320 protein.
24. The polypeptide of claim 23 wherein the variant contains a mutation at any one of positions 251, 256, 270, 279 or 305 of RAP.
25. The polypeptide of any one of claims 23 - 24 wherein the variant comprises a mutation at position 251 of RAP.
26. The polypeptide of any one of claims 23 - 24 wherein the variant comprises a mutation at position 256 of RAP.
27. The polypeptide of any one of claims 23 - 24 wherein the variant comprises a mutation at position 270 of RAP.
28. The polypeptide of any one of claims 23 - 24 wherein the variant comprises a mutation at position 279 of RAP.
29. The polypeptide of any one of claims 23 - 24 wherein the variant comprises a mutation at position 305 of RAP.
30. The polypeptide of claim 1 wherein the variant contains at least one additional mutation within positions 271-319 of RAP.
31. The polypeptide of claim 1 wherein the variant contains at least one mutation within positions 205-250 of RAP.
32. The polypeptide according to any one of claims 8 , 9 , 17 , or 23 , wherein any of the mutations comprises the replacement of an acidic amino acid with a basic amino acid.
33. The polypeptide of claim 32 , wherein said acidic amino acid is selected from the group consisting of D and E.
34. A polypeptide of claim 32 , wherein said basic amino acid is selected from the group consisting of K and R.
35. A polypeptide according to any one of claims 8 , 9 , 17 , or 23 , wherein any of the mutations comprises the replacement of a basic amino acid with an acidic amino acid.
36. A polypeptide of claim 35 , wherein said basic amino acid is selected from the group consisting of K and R.
37. A polypeptide of claim 35 , wherein said acidic amino acid is selected from the group consisting of D and E.
38. A polypeptide according to any one of claims 8 , 9 , 17 , or 23 , wherein any of the mutations comprises the replacement of an amino acid selected from the group consisting of A, C, D, E, G, I, K, L, M, N, P, Q, R, S, T, and V with an amino acid selected from the group consisting of F, Y, W, and H.
39. The polypeptide of any one of claims 1 , 7 , 8 , 9 , 17 , or 23 that comprises a mutation at three or more of the following positions: 175, 205, 213, 217, 226, 230, 232, 239, 246, 249, 251, 256, 257, 261, 266, 267, 268, 270, 273, 287, 290, 294, 296, 297, 298, 305, 312, 313.
40. The polypeptide of any one of claims 1 , 7 , 8 , 9 , 17 , or 23 that comprises a mutation at three or more of the following positions: 205, 217, 249, 251, 256, 257, 266, 270, 294, 296, 297, 305.
41. The polypeptide of any one of claims 1 , 7 , 8 , 9 , 17 , or 23 that comprises three or more of the following mutations: R205S, S213T, E217K, L226M, H249Y, A230V, S232P, E239G, E246G, E251L, E251K, E251T, E251G, E251P, E251N, E251R, K256R, K256V, K256A, K256I, K256P, K256L, I266F, I266T, H257Y, Q261R, A267V, H268R, K270P, K270D, K270N, K270G, K270E, K270W, L271M, H273Y, D279Y, V283M, R287H, H290Y, H290L, E294V, R296L, T297I, K298R, K305T, K306M, S312F, G313D.
42. A polypeptide consisting of oligomeric combinations of polypeptides of any one of claims 1 , 7 , 8 , 9 , 17 , or 23 with binding multivalent binding properties defined by each polypeptide of the combination.
43. A compound comprising the polypeptide according to any one of claims 1 , 7 , 8 , 9 , 17 , or 23 conjugated to a diagnostic or therapeutic agent.
44. The compound of claim 43 , wherein the polypeptide and diagnostic or therapeutic agent are linked through a linker.
45. The compound of claim 44 , wherein said linker is a peptide linker.
46. The compound of claim 43 , wherein the therapeutic agent is selected from the group consisting of a glial cell-derived neuronal growth factor (GDNF), brain-derived neuronal growth factor (BDNF), neuronal growth factor (NGF), disintegrin and metalloproteinase domain 10 (ADAM10), a chaperone protein for LRPS/6 (MESD), cancer chemotherapeutic agents, protease inhibitors, pro-apoptotic molecules, autoimmune antigens, lysosomal enzymes, nanoparticles, glycoconjugates and nucleic acids.
47. A pharmaceutical composition comprising a compound of claim 43 in a pharmaceutically acceptable carrier, diluent or excipient.