Methods for producing recombined antibodies

• US 6,576,467 B1
• Filed: 11/21/2000
• Issued: 06/10/2003
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): Park, Hankyel T.
• Application Number:
  US09/718,764
• Time in Patent Office: 931 Days
• Field of Search: 435/440, 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• 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 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• 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

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Claims

1. A method of producing a polynucleotide encoding an antibody chain that specifically binds to a predetermined antigen, the method comprising:

(a) denaturing a population of polynucleotide segments encoding CDR regions;

(b) annealing the denatured population of CDR encoding polynucleotides;

(c) incubating the annealed CDR encoding polynucleotides in the presence of a polymerase, thereby forming a population of polynucleotides comprising recombinant polynucleotides encoding antibodies with novel combinations of CDR regions, which novel combinations of CDR regions are not present in an antibody encoded by the population of polynucleotide segments provided in step (a);

(d) repeating steps (a) through (c); and

(e) screening or selecting the population of polynucleotides to identify at least one polynucleotide that encodes an antibody chain that specifically binds to a predetermined antigen.

2. The method of claim 1, wherein at least 10% of the population of polynucleotides comprise recombinant polynucleotides with novel combinations of CDR regions.

3. The method of claim 1, comprising screening or selecting using a bacteriophage display system.

4. The method of claim 1, further comprising expressing the at least one polynucleotide that encodes an antibody chain that specifically binds to a predetermined antigen in a host cell.

5. The method of claim 4, wherein the antibody specifically binds with an affinity of at least about 10⁻

9 M.

6. The method of claim 4, wherein the antibody specifically binds with an affinity of at least about 10⁻

10 M.

7. The method of claim 1, further comprising synthesizing a whole antibody based on the sequence of the at least one polynucleotide that encodes an antibody chain that specifically binds to a predetermined antigen.

8. The method of claim 7, wherein the predetermined antigen is selected from the group consisting of a human cell surface antigen, a human biological macromolecule and a nonhuman disease associated macromolecule.

9. The method of claim 1, wherein the polynucleotide segments encoding CDR regions are generated by synthesizing a collection of sequences based on naturally occurring human CDR sequences.

10. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions comprises consensus sequences of naturally occurring CDR sequences.

11. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions includes at least about 100 unique heavy chain CDR sequences.

12. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions includes at least about 1000 unique heavy chain CDR sequences.

13. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions includes at least about 10,000 unique heavy chain CDR sequences.

14. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions includes CDR sequences with other than naturally occurring residues.

15. The method of claim 1, wherein the population of polynucleotides segments encoding CDR regions includes CDR sequences having at least 40 percent positional sequence identity to known CDR sequences.

16. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions further comprises diverse adjacent framework regions.

17. The method of claim 1, performed reiteratively with the population of segments encoding CDR regions in a subsequent round of shuffling being obtained from selected polynucleotides from a previous iteration.

18. The method of claim 1, wherein the screening comprises screening for recombinant nucleic acids encoding antibodies with a plurality of epitope specificities.

19. The method of claim 1, wherein selected polynucleotides are shuffled with polynucleotide sequences encoding neutral antibody framework sequences having insubstantial functional effect on antigen binding in a subsequent iteration.

20. The method of claim 1, wherein the population of polynucleotide segments encoding CDR regions are produced by PCR amplification.

21. The method of claim 20, wherein the PCR amplification is performed on mRNA or cDNA from antibody-producing cells.

22. The method of claim 1, wherein the population of polynucleotides encoding CDR regions includes a plurality of oligonucleotides encoding different forms of each of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3.

23. The method of claim 20, wherein the plurality of oligonucleotides are synthesized on an oligonucleotide synthesizer.

24. The method of claim 1, wherein the recombinant nucleic acids encode single chain antibodies in which VL and VH regions are linked by a peptide linker.

25. An antibody comprising a novel combination of heavy CDR regions or a novel combination of light chain CDRs.

26. An antibody comprising a novel combination of heavy CDR regions and a novel combination of light chain CDRs.

27. A library of recombinant DNA sequences encoding antibodies produced by the steps of:

(a) providing a population of DNA fragments comprising at least one polynucleotide encoding all or part of an antibody;

(b) recursively recombining the DNA fragments in vitro or in vivo to form a pool of recombined polynucleotides encoding antibodies or subportions thereof, which pool of polynucleotides encoding antibodies or subportions thereof comprises a library of recombinant DNA sequences.

28. The library of claim 27, comprising providing homologous DNA fragments encoding all or part of at least one antibody.

29. The library of claim 27, comprising providing homologous DNA fragments encoding at least one antibody variable region.

30. The library of claim 27, wherein the library of recombinant DNA sequences comprises at least one recombined polynucleotide encoding an antibody.

31. An antibody with a desired property produced by the steps of:

(a) providing a population of DNA fragments comprising at least one polynucleotide encoding all or part of an antibody;

(b) recursively recombining the DNA fragments in vitro or in vivo to form a pool of recombined antibody polynucleotides; and

(c) identifying at least one recombined antibody polynucleotide encoding an antibody with a desired property.

32. The antibody of claim 31, wherein the population of DNA fragments comprises homologous DNA fragments encoding all or part of at least one antibody.

33. The antibody of claim 31, wherein the population of DNA fragments comprises homologous DNA fragments encoding at least one antibody variable region.

34. The antibody of claim 31, wherein the desired property is specificity for a predetermined antigen.

35. The antibody of claim 31, wherein the desired property is increased or decreased binding affinity.

36. The antibody of claim 31, wherein the desired property is reduced immunogenicity.

37. The antibody of claim 31, wherein the desired property is selected from the group consisting of:

inhibition of binding with a compound other than the predetermined epitope, stimulation of a metabolic pathway, inhibition of a metabolic pathway, signaling activity, activity as a messenger molecule, stimulation of cellular activity, inhibition of cellular activity, altered immunogenicity and catalytic activity.