Nucleic acids encoding binding members to TNF alpha
The present invention relates to anti-TNF alpha binding members and in particular to monovalent, high potency TNF alpha-binding antibody fragments being highly stable and soluble. Such binding members may be used in the treatment of inflammatory and other diseases as well as in diagnostics. Also provided are related nucleic acids, vectors, cells, and compositions.
1. An isolated nucleic acid molecule comprising a sequence encoding an antibody or fragment thereof having a binding specificity to TNF alpha, the antibody or fragment thereof comprising
(i) the variable heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences as set forth in SEQ ID Nos: 6, 7 and 8; and
(ii) the variable light chain CDR-L1, CDR-L2 and CDR-L3 sequences as set forth in SEQ ID Nos: 3, 4 and 5.
2. An isolated vector comprising the sequence of the nucleic acid molecule according to claim 1 .
3. An isolated host cell comprising the nucleic acid molecule of claim 1 .
4. An isolated host cell comprising the vector of claim 2 .
5. A composition comprising the nucleic acid molecule of claim 1 and further a carrier, diluent or excipient.
6. A composition comprising the vector of claim 2 and further a carrier, diluent or excipient.
7. A composition comprising the host cell of claim 3 and further a carrier, diluent or excipient.
8. A method of producing an antibody or fragment thereof, the method comprising:
cultivating the host cell of claim 3 under conditions adequate for recombinant protein expression, thereby allowing the antibody or fragment thereof to be expressed;
recovering the antibody or fragment thereof; and
optionally purifying the antibody or fragment thereof.
9. A method of producing an antibody or fragment thereof, the method comprising:
(a) contacting a cell-free expression system with the nucleic acid molecule of claim 1 , thereby forming a reaction mixture;
(b) allowing transcription and translation of the nucleic acid molecule to occur in said reaction mixture;
(c) recovering the antibody or fragment thereof produced thereby; and
(d) optionally purifying the antibody or fragment thereof from the reaction mixture.
10. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof has an IC 50 with regard to human TNF alpha of lower than 50 pM.
11. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof is humanized.
12. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof comprises
(i) a variable light chain as set forth in SEQ ID No. 1; and/or
(ii) a variable heavy chain as set forth in SEQ ID No. 2.
13. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof further comprises a linker sequence.
14. The isolated nucleic acid molecule of claim 13 , wherein the antibody or fragment thereof comprises SEQ ID No. 9.
15. The isolated nucleic acid molecule of claim 1 , wherein the variable heavy chain of the antibody or fragment thereof further comprises at least one of the following residues:
(i) Serine (S) at heavy chain amino acid position 12 (according to AHo numbering);
(ii) Serine (S) or Threonine (T) at heavy chain amino acid position 103 (according to AHo numbering); and/or
(iii) Serine (S) or Threonine (T) at heavy chain amino acid position 144 (according to AHo numbering).
16. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof is
(i) a Fab, a Fab′, a F(ab)′ 2 , a scFv, or a Fv fragment, or;
(ii) a full-length immunoglobulin molecule.
17. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof is monovalent or multivalent, wherein the binding member is optionally bispecific.
18. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof remains at least 93% monomeric after incubation for 1 week at 37° C. at a concentration of 10 mg/ml PBS pH7.2.
19. The isolated nucleic acid molecule of claim 1 , wherein the antibody or fragment thereof is a monovalent antibody or fragment thereof.
20. The isolated nucleic acid molecule of claim 19 , wherein the antibody or fragment thereof is a scFv.
21. The isolated nucleic acid molecule of claim 13 , wherein the linker sequence is the sequence set forth in SEQ ID No: 10.
22. The isolated nucleic acid molecule of claim 17 , wherein the antibody or fragment thereof is a diabody, a single-chain diabody or a tandem scFv.