Antibody Libraries

• US 20100099103A1
• Filed: 09/30/2009
• Published: 04/22/2010
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Memmott, John E., Kutskova, Yuliya A., Hsieh, Chung-Ming
• Granted Patent: US 8,404,445 B2
• Time in Patent Office: Days
• US Class Current: 435/6.14
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/1018 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention features improved in vitro RNA display libraries to allow reliable expression and selection of scFv antibody molecules from expression libraries. The scFv antibody libraries of the invention contain an optimized, shortened inter-domain linker that improves expression scFv antibody expression. The scFv antibody libraries also include short nucleic acid barcodes that allow for identification of individual library clones, libraries or subsets thereof. Primers for generating, amplifying and spectratyping the scFv antibody libraries of the invention are also provided.

Claims

1. An oligonucleotide consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs:

1-14, 19-42, and 58-210.

5. A nucleic acid library for expression of single chain antibodies (scFv), said library comprising a repertoire of sequences encoding heavy chain variable domains and light chain variable domains, wherein each member of said library contains an open reading frame comprising a heavy chain variable domain, a light chain variable domain, and a linker region, and wherein said library is generated using one or more oligonucleotides of claim 1.

6. The library of claim 5, wherein the linker region encodes less than 20 amino acids.

7. The library of claim 5, wherein the linker region encodes 15 amino acids.

8. The library of claim 5, wherein each member of said library further comprises a promoter operably linked to the open reading frame.

9. The library of claim 8, wherein said promoter is a promoter selected from the group consisting of T7, SP6, and T3.

10. The library of claim 9, wherein said promoter is a T7 promoter.

11. The library of claim 5, wherein each member of said library further comprises a 5′

untranslated region (5′

UTR) capable of enhancing transcription of a gene to which it is operably linked.

12. The library of claim 11, wherein said 5′

UTR is a Tobacco Mosaic Virus 5′

UTR or active fragment thereof.

13. The library of claim 5, wherein each member of said library further comprises a polyadenine sequence.

14. The library of claim 5, wherein each member of said library further comprises a nucleic acid barcode.

15. The library of claim 14, wherein said nucleic acid barcode comprises 8 nucleotides.

16. The library of claim 5, wherein each member of said library further comprises a nucleic acid sequence encoding an epitope tag.

17. The library of claim 16, wherein the epitope tag is a FLAG tag.

18. The library of claim 16, wherein said nucleic acid sequence is part of the linker region of the scFv.

19. The library of claim 5, wherein each member of said library further comprises a nucleic acid sequence encoding an antibody constant region or fragment thereof.

20. The library of claim 5, wherein each member of said library further comprises a ribosome pause sequence.

21. The library of claim 5, wherein each member of said library further comprises a peptide acceptor.

22. The library of claim 21, wherein the peptide acceptor is covalently attached via a linker comprising a Psoralen C6 molecule.

23. The library of claim 22, wherein the linker is 5′

(Psoralen C6) 2′

Ome (U AGC GGA UGC) XXX XXX CC (Puromycin), wherein X is a triethylene glycol linker or PEG-150 and CC is a DNA backbone.

24. A method of producing a nucleic acid library for expression of single chain antibodies (scFv) comprising:

providing a nucleic acid composition, wherein at least a portion of the nucleic acids in said composition comprise at least one open reading frame encoding an antibody variable domain; and

,

amplifying a plurality of antibody variable domains using one or more oligonucleotides of claim 1.

2. An oligonucleotide consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs:

14-16, and 43-57.

25. A method of spectratyping a nucleic acid comprising at least one open reading frame encoding an antibody variable domain comprising:

providing a nucleic acid composition, wherein at least a portion of the nucleic acids in said composition comprise at least one open reading frame encoding an antibody variable domain; and

,

amplifying the CDR3 regions of said variable domains using one or more oligonucleotides of claim 2.

3. An oligonucleotide consisting of a nucleic acid sequence as set forth in SEQ ID NOs:

17 or 18.

4. Use of any of the sequences in the preceding claims for library amplification, library reverse transcription, and/or library spectratyping.