Trimeric collagen scaffold antibodies
A collagen scaffold domain, including a collagenous or collagen-like domain, which directs self-trimerization is provided. The collagen scaffold domain can be fused to one or more heterologous domains, such as an antibody domain. Methods for generating and using the scaffold domains and fusion proteins are also provided.
1. A trimeric soluble antibody comprising three polypeptides, wherein each polypeptide comprises:
(a) a collagen scaffold domain comprising (G-P-P) 10 (SEQ ID NO: 20),
wherein G is glycine, P is proline, and at least 6 of the G-P-P repeats are hydroxyprolinated at the second proline; and
(b) an antibody domain that binds to a ligand;
wherein the polypeptide does not comprise a separate trimerization domain.
2. The trimeric soluble antibody of claim 1 , wherein the ligand for the trimeric soluble antibody is human epidermal growth factor receptor.
3. The trimeric soluble antibody of claim 1 , wherein the ligand for the trimeric soluble antibody is human CD3.
4. The trimeric soluble antibody of claim 1 , wherein the ligand for the trimeric soluble antibody is human TNF-α.
5. The trimeric soluble antibody of claim 2 , wherein the antibody is a bispecific antibody.
6. The trimeric soluble antibody of claim 1 , wherein the polypeptide further comprises a coding sequence for a marker polypeptide.
7. The trimeric soluble antibody of claim 6 , wherein the marker polypeptide is a luciferase polypeptide or a green fluorescent polypeptide.
8. The trimeric soluble antibody of claim 1 , wherein each polypeptide comprises two antibody domains.
9. The trimeric soluble antibody of claim 1 , wherein each polypeptide further comprises an affinity tag.
10. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises the sequence of a single chain antibody.
11. The trimeric soluble antibody of claim 1 , wherein the first, second, and third polypeptides are substantially identical.
12. The trimeric soluble antibody of claim 1 , wherein the antibody domain is obtained by phage display screening.
13. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises the sequence of a single chain antibody and further comprises a hinge region and a coding sequence for luciferase.
14. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises the sequence of an immunoglobulin.
15. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises a chimeric scFv-Fc antibody which comprises the sequence of a single chain antibody and a constant region (Fc).
16. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises the sequence of an Fab domain and further comprises a hinge region.
17. The trimeric soluble antibody of claim 16 , wherein the ligand for the trimeric soluble antibody is human CD3.
18. The trimeric soluble antibody of claim 1 , wherein the antibody domain comprises the sequence of an Fab domain and further comprises a hinge region and a coding sequence for luciferase.
19. A trimeric protein complex comprising three polypeptides, wherein each polypeptide comprises:
(a) a self-trimerizing triple helix coil scaffold domain comprising (G-P-P) (SEQ ID NO: 20), wherein G is glycine, P is proline, and at least 6 of the G-P-P repeats are hydroxyprolinated at the second proline, and wherein the self-trimerizing scaffold domain does not comprise a separate trimerization domain; and
(b) a first heterologous domain in-frame fused to one end of the scaffold domain, wherein the first heterologous domain is a binding domain selected from the group consisting of an antibody domain, a ligand binding domain, a ligand, a receptor, and a proteoglycan, or which is a fluorescent protein or an enzymatic domain.
20. The protein complex of claim 19 , wherein each polypeptide further comprises a second heterologous domain in-frame fused to the other end of the scaffold domain, wherein the second heterologous domain is a binding domain selected from the group consisting of an antibody domain, a ligand binding domain, a ligand, a receptor, and a proteoglycan, or which is a fluorescent protein or an enzymatic domain; wherein the triple helix coil scaffold domains of the three polypeptides interact with each other to form a trimeric protein complex.
21. The protein complex of claim 19 , wherein the heterologous domain is an antibody domain.
22. The protein complex of claim 21 , wherein the scaffold domain is in-frame fused to the amino terminus of the antibody domain.
23. The protein complex of claim 21 , wherein the scaffold domain is in-frame fused to the carboxy terminus of the antibody domain.
24. The protein complex of claim 21 , wherein the antibody domain comprises one or more complementarity-determining regions of an immunoglobulin.
25. The protein complex of claim 24 , wherein the antibody domain comprises the sequence of an antigen-binding fragment.
26. The protein complex of claim 25 , wherein the antigen-binding fragment specifically binds to CD3.
27. The protein complex of claim 25 , wherein the antigen-binding fragment specifically binds to epidermal growth factor receptor (EGFR).
28. The protein complex of claim 26 , wherein the antigen-binding fragment comprises a single chain antibody.
29. The protein complex of claim 21 , wherein each polypeptide further comprises a second heterologous domain comprising a second antibody domain.
30. The protein complex of claim 29 , wherein the first antibody domain comprises a first single chain antibody that specifically binds to CD3.
31. The protein complex of claim 29 , wherein the second antibody domain comprises a second single chain antibody that specifically binds EGFR.
32. The protein complex of claim 30 , wherein the second antibody domain comprises a second single chain antibody that specifically binds EGFR.
33. The protein complex of claim 20 , wherein the second heterologous domain of at least one of the polypeptides further comprises an enzymatic domain or a fluorescent protein.
34. The protein complex of claim 20 , wherein the three polypeptides are substantially identical.
35. A trimeric soluble antibody comprising three polypeptides, wherein each polypeptide comprises:
(a) a collagen scaffold domain comprising GSP(GPP) 10 GPSSGG (SEQ ID NO: 13); and
(b) an antibody domain;
wherein the polypeptide does not comprise a separate trimerization domain.