Anti-α synuclein binding molecules
Provided are anti-human α-synuclein-specific binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof, as methods related thereto. Further provided are anti-human α-synuclein binding molecules which bind to specific N-terminal and C-terminal epitopes on human α-synuclein. The binding molecules described herein can be used in pharmaceutical and diagnostic compositions for α-synuclein targeted immunotherapy and diagnosis, respectively.
1. A polynucleotide linked to a heterologous nucleic acid, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and wherein the VH when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:26 binds to α-synuclein;
(b) a polynucleotide encoding an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and wherein the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:20 binds to α-synuclein;
(c) a polynucleotide encoding (i) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; and (ii) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively;
(d) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:15, wherein the VH when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO:26 binds to α-synuclein;
(e) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:20, wherein the VH when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO:26 binds to α-synuclein;
(f) a polynucleotide encoding an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:22, wherein the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:20 binds to α-synuclein;
(g) a polynucleotide encoding an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:26, wherein the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:20 binds to α-synuclein;
(h) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:15 and an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:22;
(i) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:15 and an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:26;
(j) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:20 and an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:22;
(k) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:20 and an immunoglobulin light chain or a fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:26;
(l) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:19;
(m) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:21;
(n) a polynucleotide encoding an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:27;
(o) a polynucleotide encoding an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:28;
(p) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:19, and an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:27;
(q) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising the nucleic acid sequence set forth in SEQ ID NO:19, and an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:28;
(r) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:21, and an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:27; and
(s) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:21, and an immunoglobulin light chain or a fragment thereof, comprising the nucleic acid sequence set forth in SEQ ID NO:28.
2. The polynucleotide of claim 1 , wherein the heterologous nucleic acid is a regulatory element.
3. The polynucleotide of claim 2 , wherein the regulatory element is a promoter, an enhancer, a ribosome binding site, or a transcription terminator.
4. The polynucleotide of claim 1 , wherein the heterologous nucleic acid is a secretory signal peptide.
5. The polynucleotide of claim 4 , wherein the secretory signal peptide is a mammalian signal peptide.
6. The polynucleotide of claim 5 , wherein the mammalian signal peptide is from tissue plasminogen activator.
7. A host cell comprising the polynucleotide of claim 1 .
8. The host cell of claim 7 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell.
9. A cDNA comprising a polynucleotide encoding a polypeptide comprising:
an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:16, 17, and 18, respectively, and wherein the VH when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:26 binds to α-synuclein; or
an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively, and wherein the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:20 binds to α-synuclein.
10. The cDNA of claim 9 , wherein the polypeptide comprises:
an immunoglobulin heavy chain or fragment thereof comprising a VH with the amino acid sequence set forth in SEQ ID NO:15;
an immunoglobulin heavy chain or fragment thereof comprising a VH with the amino acid sequence set forth in SEQ ID NO:20;
an immunoglobulin light chain or fragment thereof comprising a VL with the amino acid sequence set forth in SEQ ID NO:22; or
an immunoglobulin light chain or fragment thereof comprising a VL with the amino acid sequence set forth in SEQ ID NO:26.
11. An expression vector comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide comprising:
an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:16, 17, and 18, respectively, and wherein the VH when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:26 binds to α-synuclein; or
an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively, and wherein the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:20 binds to α-synuclein.
12. The expression vector of claim 11 , wherein the polypeptide comprises:
an immunoglobulin heavy chain or fragment thereof comprising a VH with the amino acid sequence set forth in SEQ ID NO:15;
an immunoglobulin heavy chain or fragment thereof comprising a VH with the amino acid sequence set forth in SEQ ID NO:20;
an immunoglobulin light chain or fragment thereof comprising a VL with the amino acid sequence set forth in SEQ ID NO:22; or
an immunoglobulin light chain or fragment thereof comprising a VL with the amino acid sequence set forth in SEQ ID NO:26.
13. The expression vector of claim 11 , wherein the expression vector is a plasmid, phage, or virus.
14. The expression vector of claim 13 , wherein the expression vector is a retrovirus.
15. The expression vector of claim 11 , wherein the heterologous promoter is a cytomegalovirus, simian virus 40, or retroviral promoter.
16. The expression vector of claim 11 , wherein the heterologous promoter is the cytomegalovirus immediate early promoter.
17. The expression vector of claim 11 , wherein the polypeptide comprises a signal peptide.
18. The expression vector of claim 17 , wherein the signal peptide is a heterologous signal peptide.
19. An expression vector comprising:
a first polynucleotide encoding a first polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising VH complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:16, 17, and 18, respectively; and
a second polynucleotide encoding a second polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively,
wherein the immunoglobulin heavy chain or fragment thereof when paired with the immunoglobulin light chain or fragment thereof forms an anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof, and
wherein the expression vector is a plasmid, phage, or virus.
20. The expression vector of claim 19 , wherein the expression vector is a retrovirus.
21. The expression vector of claim 19 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:15; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:22.
22. The expression vector of claim 19 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:20; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:26.
23. The expression vector of claim 22 , wherein the first polypeptide comprises a human IgG3 heavy chain constant region and the second polypeptide comprises a human kappa light chain constant region.
24. The expression vector of claim 22 , wherein the first polypeptide comprises a human IgG1 heavy chain constant region.
25. The expression vector of claim 19 , wherein:
the first polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:21; and
the second polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:27.
26. A host cell comprising:
a first expression vector comprising a first heterologous promoter operably linked to a first polynucleotide encoding a first polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising VH complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:16, 17, and 18, respectively; and
a second expression vector comprising a second heterologous promoter operably linked to a second polynucleotide encoding a second polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively,
wherein the immunoglobulin heavy chain or fragment thereof when paired with the immunoglobulin light chain or fragment thereof forms an anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof.
27. The host cell of claim 26 , wherein the first heterologous promoter is a cytomegalovirus, simian virus 40, or retroviral promoter and the second heterologous promoter is a cytomegalovirus, simian virus 40, or retroviral promoter.
28. The host cell of claim 26 , wherein the first heterologous promoter is the cytomegalovirus immediate early promoter and the second heterologous promoter is the cytomegalovirus immediate early promoter.
29. The host cell of claim 26 , wherein the first expression vector is a plasmid, phage, or virus and the second expression vector is a plasmid, phage, or virus.
30. The host cell of claim 29 , wherein the first expression vector is a retrovirus and the second expression vector is a retrovirus.
31. The host cell of claim 26 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:15; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:22.
32. The host cell of claim 26 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:20; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:26.
33. The host cell of claim 32 , wherein the first polypeptide comprises a human IgG3 heavy chain constant region and the second polypeptide comprises a human kappa light chain constant region.
34. The host cell of claim 32 , wherein the first polypeptide comprises a human IgG1 heavy chain constant region.
35. The host cell of claim 26 , wherein the host cell is a mammalian host cell.
36. The host cell of claim 26 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell, or a NSO cell.
37. A host cell comprising an expression vector comprising:
a first polynucleotide encoding a first polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising VH complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:16, 17, and 18, respectively; and
a second polynucleotide encoding a second polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively,
wherein the immunoglobulin heavy chain or fragment thereof when paired with the immunoglobulin light chain or fragment thereof forms an anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof, and
wherein the expression vector is a plasmid, phage, or virus.
38. The host cell of claim 37 , wherein the expression vector is a retrovirus.
39. The host cell of claim 37 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:15; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:22.
40. The host cell of claim 37 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:20; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:26.
41. The host cell of claim 40 , wherein the first polypeptide comprises a human IgG3 heavy chain constant region and the second polypeptide comprises a human kappa light chain constant region.
42. The host cell of claim 40 , wherein the first polypeptide comprises a human IgG1 heavy chain constant region.
43. The host cell of claim 37 , wherein the host cell is a mammalian host cell.
44. The host cell of claim 37 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell, or a NSO cell.
45. A method for preparing an anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof, the method comprising:
culturing the host cell of claim 26 in a cell culture; and
isolating the anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof from the cell culture.
46. The method of claim 45 , further comprising formulating the anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof into a sterile pharmaceutical composition suitable for administration to a human subject.
47. The method of claim 46 , wherein the pharmaceutical composition is suitable for intravenous or subcutaneous administration.
48. The method of claim 45 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:15; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:22.
49. The method of claim 45 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:20; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:26.
50. The method of claim 49 , wherein the first polypeptide comprises a human IgG3 heavy chain constant region and the second polypeptide comprises a human kappa light chain constant region.
51. The method of claim 49 , wherein the first polypeptide comprises a human IgG1 heavy chain constant region.
52. A method for preparing an anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof, the method comprising:
culturing the host cell of claim 37 in a cell culture; and
isolating the anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof from the cell culture.
53. The method of claim 52 , further comprising formulating the anti-human alpha-synuclein antibody or human alpha-synuclein-binding fragment thereof into a sterile pharmaceutical composition suitable for administration to a human subject.
54. The method of claim 53 , wherein the pharmaceutical composition is suitable for intravenous or subcutaneous administration.
55. The method of claim 52 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:15; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:22.
56. The method of claim 52 , wherein:
the VH consists of the amino acid sequence set forth in SEQ ID NO:20; and
the VL consists of the amino acid sequence set forth in SEQ ID NO:26.
57. The method of claim 56 , wherein the first polypeptide comprises a human IgG3 heavy chain constant region and the second polypeptide comprises a human kappa light chain constant region.
58. The method of claim 56 , wherein the first polypeptide comprises a human IgG1 heavy chain constant region.