Fusion proteins comprising P3 of bacteriophage
The invention relates to agents and to pharmaceutical compositions for reducing the formation of amyloid and/or for promoting the disaggregation of amyloid proteins. The compositions may also be used to detect amyloid.
1. A nucleic acid sequence encoding a fusion protein comprising an amyloid binding fragment of g3p and an Fc fragment of an immunoglobulin, wherein the fusion protein comprises:
(a) amino acids 21-506 of SEQ ID NO:9;
(b) amino acids 22-506 of SEQ ID NO:9;
(c) amino acids 23-506 of SEQ ID NO:9;
(d) amino acids 21-505 of SEQ ID NO:9;
(e) amino acids 22-505 of SEQ ID NO:9;
(f) amino acids 23-505 of SEQ ID NO:9;
(g) amino acids 21-506 of SEQ ID NO:11;
(h) amino acids 22-506 of SEQ ID NO:11;
(i) amino acids 23-506 of SEQ ID NO:11;
(j) amino acids 21-505 of SEQ ID NO:11;
(k) amino acids 22-505 of SEQ ID NO:11;
(l) amino acids 23-505 of SEQ ID NO:11;
(m) amino acids 21-509 of SEQ ID NO:13;
(n) amino acids 22-509 of SEQ ID NO:13;
(o) amino acids 23-509 of SEQ ID NO:13;
(p) amino acids 21-508 of SEQ ID NO:13;
(q) amino acids 22-508 of SEQ ID NO:13;
(r) amino acids 23-508 of SEQ ID NO:13;
(s) amino acids 21-528 of SEQ ID NO:31;
(t) amino acids 22-528 of SEQ ID NO:31;
(u) amino acids 23-528 of SEQ ID NO:31;
(v) amino acids 21-527 of SEQ ID NO:31;
(w) amino acids 22-527 of SEQ ID NO:31;
(x) amino acids 23-527 of SEQ ID NO:31; or
(y) a mutant or variant that is at least 95% identical to the amino acid sequence of any one of (a)-(x) and is capable of binding to amyloid.
2. The nucleic acid sequence of claim 1 , wherein the amyloid binding fragment of g3p in (y) has up to 5 amino acid substitutions as compared to the amyloid binding fragment in any one of (a)-(x).
3. The nucleic acid sequence of claim 1 , wherein the sequence further encodes a signal sequence.
4. The nucleic acid sequence of claim 3 , wherein the signal sequence is amino acids 1-20 of SEQ ID NO:9.
5. The nucleic acid sequence of claim 1 , selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:32.
6. A vector comprising the nucleic acid sequence of claim 1 .
7. An isolated host cell comprising the vector of claim 6 .
8. The host cell of claim 7 , wherein the host cell is selected from an insect cell, a fungal cell, a bacterial cell, an animal cell line, and a transgenic animal cell.
9. The host cell of claim 7 , wherein the host cell is selected from the group consisting of a Pichia cell, a Saccharomyces cell, an Aspergillus cell, an E. coli cell, an NSO cell, a CHO cell, a CHO-derived cell, a HEK293 cell, a HEK293-derived cell, a COS cell, and a HeLa cell.
10. The host cell of claim 9 , wherein the host cell is a CHO cell, a CHO-derived cell, a HEK293 cell, or a HEK293-derived cell.
11. The host cell of claim 10 , wherein the host cell is a CHO cell or a CHO-derived cell.
12. A method of making a fusion protein encoded by the nucleic acid sequence of claim 1 , comprising expressing the fusion protein encoded by the vector of claim 6 in the host cell of claim 9 , and isolating the expressed protein.
13. A nucleic acid sequence encoding a fusion protein comprising an amyloid binding fragment of g3p, wherein the fusion protein comprises:
(a) amino acids 21-238 of SEQ ID NO:9;
(b) amino acids 22-238 of SEQ ID NO:9;
(c) amino acids 23-238 of SEQ ID NO:9;
(d) amino acids 21-238 of SEQ ID NO:11;
(e) amino acids 22-238 of SEQ ID NO:11;
(f) amino acids 23-238 of SEQ ID NO:11;
(g) amino acids 21-238 of SEQ ID NO:13;
(h) amino acids 22-238 of SEQ ID NO:13;
(i) amino acids 23-238 of SEQ ID NO:13;
(j) amino acids 21-296 of SEQ ID NO:31;
(k) amino adds 22-296 of SEQ ID NO:31;
(l) amino acids 23-296 of SEQ ID NO:31; or
(m) a mutant or variant that is at least 95% identical to the amino acid sequence of any one of (a)-(l) and is capable of binding to amyloid.
14. The nucleic acid sequence of claim 13 , wherein the fusion protein further comprises an Fc fragment of an immunoglobulin.
15. A vector comprising the nucleic acid sequence of claim 13 .
16. An isolated host cell comprising the vector of claim 15 .
17. The host cell of claim 16 , wherein the host cell is selected from an insect cell, a fungal cell, a bacterial cell, an animal cell line, and a transgenic animal cell.
18. The host cell of claim 16 , wherein the host cell is selected from the group consisting of a Pichia cell, a Saccharomyces cell, an Aspergillus cell, an E. coli cell, an NSO cell, a CHO cell, a CHO-derived cell, a HEK293 cell, a HEK293-derived cell, a COS cell, and a HeLa cell.
19. The host cell of claim 18 , wherein the host cell is a CHO cell, a CHO-derived cell, a HEK293 cell, or a HEK293-derived cell.
20. The host cell of claim 19 , wherein the host cell is a CHO cell or a CHO-derived cell.
21. A method of making a fusion protein encoded by the nucleic acid sequence of claim 13 , comprising expressing the fusion protein encoded by the vector of claim 15 in the host cell of claim 18 , and isolating the expressed protein.
22. A method of treating a patient for a disease or condition by administering to the patient a fusion protein comprising an amyloid binding fragment of g3p and an Fc fragment of an immunoglobulin, wherein the fusion protein comprises:
(a) amino acids 21-506 of SEQ ID NO:9;
(b) amino acids 22-506 of SEQ ID NO:9;
(c) amino acids 23-506 of SEQ ID NO:9;
(d) amino acids 21-505 of SEQ ID NO:9;
(e) amino acids 22-505 of SEQ ID NO:9;
(f) amino acids 23-505 of SEQ ID NO:9;
(g) amino acids 21-506 of SEQ ID NO:11;
(h) amino acids 22-506 of SEQ ID NO:11;
(i) amino acids 23-506 of SEQ ID NO:11;
(j) amino acids 21-505 of SEQ ID NO:11;
(k) amino acids 22-505 of SEQ ID NO:11;
(l) amino acids 23-505 of SEQ ID NO:11;
(m) amino acids 21-509 of SEQ ID NO:13;
(n) amino acids 22-509 of SEQ ID NO:13;
(o) amino acids 23-509 of SEQ ID NO:13;
(p) amino acids 21-508 of SEQ ID NO:13;
(q) amino acids 22-508 of SEQ ID NO:13;
(r) amino acids 23-508 of SEQ ID NO:13;
(s) amino acids 21-528 of SEQ ID NO:31;
(t) amino acids 22-528 of SEQ ID NO:31;
(u) amino acids 23-528 of SEQ ID NO:31;
(v) amino acids 21-527 of SEQ ID NO:31;
(w) amino acids 22-527 of SEQ ID NO:31;
(x) amino acids 23-527 of SEQ ID NO:31; or
(y) a mutant or variant that is at least 95% identical to the amino acid sequence of any one of (a)-(x) and is capable of binding to amyloid; and
wherein the disease or condition is selected from SAA amyloidosis, cystatin C, hereditary Icelandic syndrome, senility, multiple myeloma, kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), scrapie, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA7), Huntington's disease, dentatorubral-pallidoluysian atrophy, spinal and bulbar muscular atrophy, hereditary cerebral amyloid angiopathy, familial amyloidosis, frontotemporal lobe dementia, British/Danish dementia, and familial encephalopathy.