Single domain binding molecule
The present invention provides a single domain specific binding molecule having the structure FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 in which the Framework Regions FW1, FW2, FW3a, FW3b, and FW4, the Complementarity Determining Regions CDR1 and CDR3, and the Hypervariable Regions HV2, and HV4 have amino acid sequences as defined which provide a high affinity anti-human serum albumin (HSA) binding domain.
1. A polypeptide comprising:
A) a single variable domain (VNAR) comprising the Framework regions FW1, FW2, FW3a, FW3b, and FW4, in which:
i) FW1 comprises TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28) or ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67),
ii) FW2 comprises TYWYQQKPGS (SEQ ID NO: 29) or TCWYQQKPGK (amino acids 34-43 of SEQ ID NO: 67),
iii) FW3a comprises GRYSESVN (SEQ ID NO: 30),
iv) FW3b comprises FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31) or FTLTISSLQPEDFATYYCGL (amino acids 65-84 of SEQ ID NO: 67), and
v) FW4 comprises GAGTKVEIK (SEQ ID NO: 32) or CGQGTKVEIK (amino acids 103-112 of SEQ ID NO: 67); or
B) a VNAR comprising framework regions FW1, FW2, FW3a, FW3b, and FW4 in which:
i) FW1 comprises a sequence having at least 50% identity to TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28) or ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67),
ii) FW2 comprises a sequence having at least 50% identity to TYWYQQKPGS (SEQ ID NO: 29) or TCWYQQKPGK (amino acids 34-43 of SEQ ID NO: 67),
iii) FW3a comprises a sequence having at least 50% identity to GRYSESVN (SEQ ID NO: 30),
iv) FW3b comprises a sequence having at least 50% identity to FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31) or FTLTISSLQPEDFATYYCGL (amino acids 65-84 of SEQ ID NO: 67), and
v) FW4 comprises a sequence having at least 50% identity to GAGTKVEIK (SEQ ID NO: 32) or CGQGTKVEIK (amino acids 103-112 of SEQ ID NO: 67).
2. A polypeptide as claimed in claim 1 , wherein:
i) FW1 comprises ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67) or a sequence having at least 50% identity thereto,
ii) FW2 comprises TCWYQQKPGK (amino acids 34-43 of SEQ ID NO: 67) or a sequence having at least 50% identity thereto,
iii) FW3a comprises GRYSESVN (SEQ ID NO: 30) or a sequence having at least 50% identity thereto,
iv) FW3b comprises FTLTISSLQPEDFATYYCGL (amino acids 65-84 of SEQ ID NO: 67) or a sequence having at least 50% identity thereto, and
v) FW4 comprises CGQGTKVEIK (amino acids 103-112 of SEQ ID NO: 67) or a sequence having at least 50% identity thereto.
3. A polypeptide as claimed in claim 1 , wherein:
i) FW1 comprises TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28) or a sequence having at least 50% identity thereto,
ii) FW2 comprises TYWYQQKPGS (SEQ ID NO: 29) or a sequence having at least 50% identity thereto,
iii) FW3a comprises GRYSESVN (SEQ ID NO: 30) or a sequence having at least 50% identity thereto,
iv) FW3b comprises FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31) or a sequence having at least 50% identity thereto, and
v) FW4 comprises GAGTKVEIK (SEQ ID NO: 32) or a sequence having at least 50% identity thereto.
4. A polypeptide as claimed in claim 1 , which is humanized.
5. A polypeptide as claimed in claim 1 , in which:
FW1 consists of TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28) or ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67).
6. A fusion protein comprising a polypeptide as claimed in claim 1 .
7. A fusion protein as claimed in claim 6 , in which the polypeptide is fused to a biologically active protein.
8. A fusion protein as claimed in claim 7 , in which the biologically active protein is an immunoglobulin domain.
9. A fusion protein as claimed in claim 8 , in which the immunoglobulin domain is an immunoglobulin variable region.
10. A fusion protein as claimed in claim 8 , in which the immunoglobulin domain is an immunoglobulin constant region.
11. A fusion protein as claimed in claim 8 , in which the immunoglobulin domain is an immunoglobulin Fc domain.
12. A fusion protein comprising two or more polypeptides as claimed in claim 1 .
13. A fusion protein consisting of two or more polypeptides as claimed in claim 1 .
14. A process for the production of a polypeptide as in claim 1 , comprising the step of expressing a nucleic acid sequence encoding said polypeptide in a host cell.
15. The process as claimed in claim 14 , wherein the polypeptide is humanised.
16. A pharmaceutical comprising a polypeptide as claimed in claim 1 .
17. The pharmaceutical composition as claimed in claim 16 , further comprising at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable adjuvant, and a pharmaceutically acceptable buffer solution.