Single chain multivalent binding protein compositions and methods

Patent metadata

Field	Value
Publication number	US-9458244-B2
Application number	US-201314141500-A
Country	US
Kind code	B2
Filing date	Dec 27, 2013
Priority date	Dec 28, 2012
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Provided are protein, nucleic acid, and cellular libraries of single chain multivalent binding proteins (e.g., scDVD and scDVDFab molecules) and methods of using these of these libraries for the screening of single chain multivalent binding proteins using cell surface display technology (e.g., yeast display).

First claim

We claim:

1. A diverse library of binding proteins comprising a polypeptide chain having the general formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a first heavy chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VH2 is a second heavy chain variable domain, X2 is a linker, VL1 is a first light chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VL2 is a second light chain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding sites, and wherein the amino acid sequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently vary within the library.

2. A diverse library of binding proteins comprising a polypeptide chain having the general formula (VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first antibody light chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VL2 is a second antibody light chain variable domain, X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VH2 is a second antibody heavy chain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding sites, and wherein the amino acid sequences of VL1, X1, VL2, X2, VH1, X3, and/or VH2 independently vary within the library.

3. The diverse library of claim 1 , wherein each binding proteins further comprises a cell surface anchoring moiety linked to the N or C terminus.

4. The diverse library of claim 3 , wherein the anchoring moiety is a cell surface protein.

5. The diverse library of claim 3 , wherein the anchoring moiety is Aga2p.

6. The diverse library of claim 1 , wherein the polypeptide chain is a scDVD.

7. The library of claim 1 , wherein the amino acid sequence of at least one CDR of VH1, VH2, VL1 or VL2 independently varies within the library.

8. The library of claim 1 , wherein the amino acid sequence of HCDR3 of VH1, VH2 independently vary within the library.

9. The library of claim 1 , wherein the amino acid sequence of HCDR1 and HCDR2 of VH1 or VH2 independently vary within the library.

10. The library of claim 1 , wherein the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH1 or VH2 independently vary within the library.

11. The library of claim 1 , wherein the amino acid sequence of HCDR3 of VL1 or VL2 independently vary within the library.

12. The library of claim 1 , wherein the amino acid sequence of HCDR1 and HCDR2 of VL1 or VL2 independently vary within the library.

13. The library of claim 1 , wherein the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VL1 or VL2 independently vary within the library.

14. The library of claim 1 , wherein X1 independently varies within the library and wherein X1 is selected from the amino acid sequences set forth in FIG. 2 .

15. The library of claim 1 , wherein X2 independently varies within the library and wherein X2 is (G 4 S)n, where n=1-10 (SEQ ID NO: 53).

16. The library of claim 1 , wherein X3 independently varies within the library and wherein X3 is selected from the amino acid sequences set forth in FIG. 2 .

17. The library of claim 1 , wherein the library of binding proteins share at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 amino acid sequence identity with a reference binding protein.

18. The library of claim 1 , wherein VH1 and VH2 of the reference binding protein specifically bind to different antigens.

19. A library of transformed host cells, expressing the diverse library of binding proteins of claim 1 .

20. The library of transformed host cells of claim 19 , wherein the binding proteins are anchored on the cell surface.

21. The library of transformed host cells of claim 19 , wherein the binding proteins are anchored on the cell surface through Aga1p.

22. The library of transformed host cells of claim 19 , wherein the host cells are eukaryotic.

23. The library of transformed host cells of claim 22 , wherein the host cells are yeast.

24. The library of transformed host cells of claim 22 , wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe and Yarrowia lipolytica.

25. The library of transformed host cells of claim 22 , wherein the yeast is Saccharomyces cerevisiae.

26. A method of selecting a binding protein that specifically binds to a target antigen, the method comprising:
a) providing a diverse library of transformed host cells expressing the diverse library of binding proteins of claim 1 ;
b) contacting the host cells with the target antigen; and
c) selecting a host cell that bind to the target antigen, thereby identifying a binding protein that specifically binds to a target antigen.

27. A method of selecting a binding protein that specifically binds to a first and a second target antigen simultaneously, the method comprising:
a) providing a diverse library of transformed host cells expressing the diverse library of binding proteins of claim 1 ;
b) contacting the host cells with the first and second target antigen; and
c) selecting a host cell that bind to the first and second target antigen, thereby identifying a binding protein that specifically binds to a first and a second target antigen simultaneously.

28. The method of claim 26 , wherein host cells that bind to the first and/or second antigen are selected by Magnetic Activated Cell Sorting using magnetically labeled antigen.

29. The method of claim 26 , wherein host cells that bind to the first and/or second antigen are selected by Fluorescence Activated Cell Sorting using fluorescently labeled antigen.

30. The method of claim 26 , further comprising isolating the binding protein-encoding polynucleotide sequences from the host cells selected in step (c).

31. A method of producing a binding protein, comprising expressing in a host cell a binding protein that was selected using the methods of claim 26 .

Assignees

• Abbvie Inc
• Abbvie Inc

Inventors

• Benatuil Lorenzo
• Hsieh Chung-Ming

Classifications

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K16/22

  against growth factors {; against growth regulators} · CPC title

• C07K16/241

  Tumor Necrosis Factors · CPC title

• C40B40/10

  Libraries containing peptides or polypeptides, or derivatives thereof · CPC title

• C07K16/468 Primary

  Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies · CPC title