V-like domain binding molecules and polynucleotides encoding therefor
The present invention relates to binding moieties comprising at least one monomeric V-like domain (VLD) derived from a non-antibody ligand, the at least one monomeric V-like domain being characterised in that at least one CDR loop structure or part thereof is modified or replaced such that the solubility of the modified VLD is improved when compared with the unmodified VLD.
1. An isolated polynucleotide encoding a monomeric non-antibody ligand V-like domain (VLD) comprising within the VLD at least one CDR loop structure or part thereof that is modified or replaced such that
(i) the size of the CDR loop structure or part thereof is increased by at least one amino acid residue when compared with the corresponding CDR loop structure or part thereof in an unmodified VLD; and/or
(ii) the modification or replacement results in formation of a disuiphide bond within or between one or more of the CDR loop structures, wherein the CDR loop structure is a surface polypeptide loop structure corresponding to a complementarity determining region of an antibody V-domain, and wherein the non-antibody ligand is selected from the group consisting of CTLA-4, CD28 and ICOS.
2. The polynucleotide according to claim 1 , wherein the size of the CDR loop structure or part thereof is increased by at least two amino acid residues.
3. The polynucleotide according to claim 1 , wherein the size of the CDR loop structure or part thereof is increased by at least six amino acid residues.
4. The polynucleotide according to claim 1 , wherein the size of the CDR loop structure or part thereof is increased by at least nine amino acid residues.
5. The polynucleotide according to claim 1 , wherein the binding affinity of the modified VLD is altered when compared with the unmodified VLD.
6. The polynucleotide according to claim 5 , wherein the affinity of the modified VLD to at least one natural ligand of the unmodified VLD is reduced.
7. The polynucleotide according to claim 1 , wherein the binding specificity of the modified VLD is different than that of the unmodified VLD.
8. The polynucleotide according to claim 1 , wherein the non-antibody ligand is CTLA-4.
9. The polynucleotide according to claim 1 , wherein one or more of the CDR loop structure(s) or part(s) thereof is replaced with a binding determinant derived from a non-antibody polypeptide.
10. The polynucleotide according to claim 9 , wherein the binding determinant is derived from somatostatin or haemagglutinin.
11. The polynucleotide according to claim 1 , wherein one or more of the CDR loop structure(s) or parts thereof is replaced with one or more CDR loop structures derived from an antibody or antibodies.
12. The polynucleotide according to claim 11 , wherein the antibody or antibodies are derived from a rat, mouse, human, camel, llama or shark.
13. The polynucleotide according to claim 1 , linked to a diagnostic reagent.
14. The polynucleotide according to claim 13 , wherein the diagnostic reagent is selected from the group consisting of streptavidin, biotin, a radioisotope, a dye marker and an imaging reagent.
15. A vector comprising the polynucleotide according to claim 1 .
16. An isolated host cell transformed with the vector as claimed in claim 15 .
17. The host cell according to claim 16 , wherein the cell is a bacterial cell.
18. A method of producing a binding moiety, the method comprising culturing the host cell as claimed in claim 16 under conditions enabling expression of the binding moiety, thereby producing a binding moiety.
19. The method according to claim 18 , further comprising recovering the binding moiety.
20. The method according to claim 18 , wherein the binding moiety is unglycosylated.