Methods and compositions for polypeptide engineering

• US 6,406,855 B1
• Filed: 11/22/2000
• Issued: 06/18/2002
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Stemmer, Willem P. C.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/717,419
• Time in Patent Office: 573 Days
• Field of Search: 435/6, 435/69.1, 435/69.51, 435/69.52, 435/91.2, 435/91.1, 435/196, 435/440, 530/350, 436/501, 424/85.2, 424/85.4, 536/23.1
• US Class Current: 435/6.14
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 14/56 IFN-alpha
• 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 15/1034 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 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• 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: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Claims

1. A method of identifying a cytokine variant with at least one desired property, the method comprising:

(a) providing a mixture of nucleic acid subsequences of two or more parental polynucleotides, wherein each parental polynucleotide differs from at least one other parental polynucleotide in at least one nucleotide and encodes at least one cytokine or fragment thereof;

(b) extending one or more of the nucleic acid subsequences with at least one polymerase to produce one or more recombined polynucleotides that each encode one or more cytokine variants;

(c) expressing the one or more recombined polynucleotides to provide the one or more cytokine variants;

(d) screening or selecting the one or more cytokine variants to identify at least one cytokine variant with the at least one desired property;

(e) recovering at least one recombined polynucleotide encoding the at least one cytokine variant identified in step (d); and

,

(f) repeating (a)-(d) using the at least one recombined polynucleotide recovered in step (e) as at least one of the two or more parental polynucleotides of a repeated step (a).

2. The method of claim 1, wherein the nucleic acid subsequences of step (a) are provided in single-stranded form.

3. The method of claim 1, wherein in step (a) the nucleic acid subsequences are provided by fragmenting the two or more parental polynucleotides using one or more of:

sonication, shearing, nuclease digestion, DNase digestion, RNase digestion, or restriction enzyme digestion.

4. The method of claim 1, wherein the nucleic acid subsequences are from 5 bp to 5 kb in size.

5. The method of claim 1, wherein at least one of the two or more parental polynucleotides comprises a mutagenized variant.

6. The method of claim 1, wherein the two or more parental polynucleotides of step (a) encode different cytokines.

7. The method of claim 1, wherein at least one of the two or more parental polynucleotides of step (a) encodes a human cytokine.

8. The method of claim 1, wherein at least one of the two or more parental polynucleotides of step (a) encodes a cytokine that is a therapeutic protein.

9. The method of claim 1, wherein the one or more cytokine variants of step (c) comprise chimeras of different cytokines.

10. The method of claim 1, wherein the one or more cytokine variants of step (c) comprise a library of cytokine variants, which library is expressed in a display format.

11. The method of claim 1, wherein the at least one desired property of the at least one cytokine variant identified in step (d) is selected from the group consisting of:

an improved specificity or affinity for a receptor or a ligand relative to cytokines encoded by the two or more parental polynucleotides, an improved agonist activity relative to cytokines encoded by the two or more parental polynucleotides, an improved pharmaceutical property relative to cytokines encoded by the two or more parental polynucleotides, an improved shelf life relative to cytokines encoded by the two or more parental polynucleotides, an increased stability or resistance to proteases relative to cytokines encoded by the two or more parental polynucleotides, and an improved half life in serum relative to cytokines encoded by the two or more parental polynucleotides.

12. The method of claim 1, wherein at least one of the two or more parental polynucleotides of step (a) encodes IL2 and wherein the at least one cytokine variant identified in step (d) is at least one IL2 variant that stimulates only a high affinity or a low affinity IL2 receptor.

13. The method of claim 1, wherein the at least one recombined polynucleotide recovered in step (e) comprises a library of recombinant polynucleotides.

14. The at least one cytokine variant identified in claim 1.

15. The method of claim 1, further comprising derivatizing the one or more cytokine variants with at least one half life extending chemical moiety prior to step (d).

16. The method of claim 1, further comprising derivatizing the at least one cytokine variant identified in step (d) with at least one half life extending chemical moiety.

17. The method of claim 1, wherein at least one of the two or more parental polynucleotides of step (a) encodes at least one interleukin or at least one interferon.

18. The method of claim 17, wherein the at least one interleukin is selected from the group consisting of:

IL2, IL3, IL4, IL7, IL9, and IL15.

19. The method of claim 1, wherein the at least one recombined polynucleotide recovered in step (e) is subjected to mutagenesis to provide at least one mutagenized polynucleotide.

20. The method of claim 19, wherein the at least one mutagenized polynucleotide is used in step (f) as the at least one of the two or more parental polynucleotides of the repeated step (a).

21. The method of claim 1, wherein at least one of the two or more parental polynucleotides of step (a) comprises a murine IL1-β

gene or a human IL1-β

gene.

22. A method of identifying a cytokine variant with at least one desired property, the method comprising:

(a) providing a mixture of nucleic acid subsequences of two or more parental polynucleotides, wherein each parental polynucleotide differs from at least one other parental polynucleotide in at least one nucleotide and encodes at least one cytokine or fragment thereof;

(b) extending one or more of the nucleic acid subsequences with at least one ligase to produce one or more recombined polynucleotides that each encode one or more cytokine variants;

(c) expressing the one or more recombined polynucleotides to provide the one or more cytokine variants;

(d) screening or selecting the one or more cytokine variants to identify at least one cytokine variant with the at least one desired property;

(e) recovering at least one recombined polynucleotide encoding the at least one cytokine variant identified in step (d); and

,

(f) repeating (a)-(d) using the at least one recombined polynucleotide recovered in step (e) as at least one of the two or more parental polynucleotides of a repeated step (a).

23. The method of claim 22, wherein in step (a) the nucleic acid subsequences are provided by fragmenting the two or more parental polynucleotides using one or more of:

sonication, shearing, nuclease digestion, DNase digestion, RNase digestion, or restriction enzyme digestion.

24. The method of claim 22, wherein the nucleic acid subsequences are from 5 bp to 5 kb in size.

25. The method of claim 22, wherein at least one of the two or more parental polynucleotides comprises a mutagenized variant.

26. The method of claim 22, wherein the two or more parental polynucleotides of step (a) encode different cytokines.

27. The method of claim 22, wherein at least one of the two or more parental polynucleotides of step (a) encodes a human cytokine.

28. The method of claim 22, wherein at least one of the two or more parental polynucleotides of step (a) encodes a cytokine that is a therapeutic protein.

29. The method of claim 22, wherein the one or more cytokine variants of step (c) comprise chimeras of different cytokines.

30. The method of claim 22, wherein the one or more cytokine variants of step (c) comprise a library of cytokine variants, which library is expressed in a display format.

31. The method of claim 22, wherein the at least one desired property of the at least one cytokine variant identified in step (d) is selected from the group consisting of:

an improved specificity or affinity for a receptor or a ligand relative to cytokines encoded by the two or more parental polynucleotides, an improved agonist activity relative to cytokines encoded by the two or more parental polynucleotides, an improved pharmaceutical property relative to cytokines encoded by the two or more parental polynucleotides, an improved shelf life relative to cytokines encoded by the two or more parental polynucleotides, an increased stability or resistance to proteases relative to cytokines encoded by the two or more parental polynucleotides, and an improved half life in serum relative to cytokines encoded by the two or more parental polynucleotides.

32. The method of claim 22, wherein at least one of the two or more parental polynucleotides of step (a) encodes IL2 and wherein the at least one cytokine variant identified in step (d) is at least one IL2 variant that stimulates only a high affinity or a low affinity IL2 receptor.

33. The method of claim 22, wherein the at least one recombined polynucleotide recovered in step (e) comprises a library of recombinant polynucleotides.

34. The at least one cytokine variant identified in claim 22.

35. The method of claim 22, further comprising derivatizing the one or more cytokine variants with at least one half life extending chemical moiety prior to step (d).

36. The method of claim 22, further comprising derivatizing the at least one cytokine variant identified in step (d) with at least one half life extending chemical moiety.

37. The method of claim 22, wherein at least one of the two or more parental polynucleotides of step (a) encodes at least one interleukin or at least one interferon.

38. The method of claim 37, wherein the at least one interleukin is selected from the group consisting of:

IL2, IL3, IL4, IL7, IL9, and IL15.

39. The method of claim 22, wherein the at least one recombined polynucleotide recovered in step (e) is subjected to mutagenesis to provide at least one mutagenized polynucleotide.

40. The method of claim 39, wherein the at least one mutagenized polynucleotide is used in step (f) as the at least one of the two or more parental polynucleotides of the repeated step (a).

41. The method of claim 22, wherein at least one of the two or more parental polynucleotides of step (a) comprises a murine IL1-β

gene or a human IL1-β

gene.