Methods of generating variant proteins with increased host string content and compositions thereof

• US 20060008883A1
• Filed: 12/03/2004
• Published: 01/12/2006
• Est. Priority Date: 12/04/2003
• Status: Active Grant

• Current Assignee: Xencor, Inc.
• Original Assignee: Xencor, Inc.
• Inventors: Lazar, Gregory Alan, Desjarlais, John R., Hammond, Philip W.
• Granted Patent: US 7,657,380 B2
• Time in Patent Office: Days
• US Class Current: 435/69.700
• 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 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

The present invention relates to novel methods for generating variant proteins with increased host string content, and proteins that are engineered using these methods.

Claims

1. A method of generating a variant protein for a host as compared to a parent protein, comprising:

A. comparing said parent protein sequence with two or more natural protein sequences from said host species;

B. analyzing one or more amino acid strings of said parent protein sequence with a corresponding amino acid string of each natural protein sequence; and

,

C. substituting one or more amino acids of said parent protein sequence with a corresponding amino acid string of a natural protein sequence on an amino acid string by amino acid string basis.

D. wherein said variant protein has increased host string content as compared to said parent protein, and,

E. wherein said substituted amino acids include a first substitution in a first string from a first natural protein, and a second substitution in a second string from a second natural protein.

2. The method according to claim 1, wherein said substituted amino acid is a consensus of two or more natural proteins.

3. The method according to claim 1, wherein said host species is human.

4. The method according to claim 1, wherein said parent protein family is murine.

5. The method according claim 1, wherein said natural protein sequences are germline sequences.

6. The method according to claim 1, wherein one of the substitutions is not the corresponding residue from the most homologous natural sequence.

7. The method according to claim 5, wherein one of the substitutions is not the corresponding residue from a consensus of homologous natural sequences.

8. The method according to claim 1, wherein one of the substitutions is made at a position that is not surface exposed.

9. The method according to claim 1, wherein said variant protein is an antibody.

10. The method according to claim 9, wherein one of the substitutions is not in a CDR.

11. The method according to claim 9, wherein one of the substitutions is made at a position that is part of the VH/VL interface.

12. The method according to claim 9, wherein said variant antibody comprises an affinity for an antigen no lower than about two-fold less than said parent protein.

13. The method according to claim 9, wherein said variant antibody comprises an affinity for an antigen of about two-fold more than said parent protein.

14. The method according to claim 9, wherein said variant protein comprises a framework region identity as compared to a natural protein sequence of less than about 85%.

15. The method according to claim 9, wherein said variant protein comprises a framework region homogeneity of less than about 0.6.

16. The method according to claim 9, wherein said variant antibody comprises no substitutions within about 5 Angstroms of a CDR residue.

17. The method according to claim 9, wherein said antibody has modulated effector function as compared to said parent antibody.

18. The method according to claim 17, wherein said effector function is ADCC.

19. The method according to claim 1, wherein more than one variant protein is generated from said method to form a variant protein set.

20. The method according to claim 19, wherein said variant protein set comprises at least two variant proteins that differ by more than about 5 amino acids.

21. The method according to claim 19, further synthesizing at least one variant protein.

22. The method according to claim 1, wherein said analyzing one or more amino acid strings is not applied to a CDR region.

23. The method according to claim 1, wherein said variant protein has at least one improved property is selected from a group consisting of solubility, stability, expression, affinity, immunogenicity, activity and functionality.

24. The method according to claim 1, wherein said analyzing one or more amino acid strings includes theoretical binding to MHC agretopes.

25. The method according to claim 24, wherein said string is nine amino acids in length.

26. A computer readable memory to direct a computer to function in a specified manner comprising:

A. a comparison module to compare a parent protein sequence and two or more host natural protein sequences;

B. an analyzing module to provide a comparison between a parent protein sequence and one or more natural protein sequences;

C. a substitution module to replace one or more amino acids said parent protein sequence with a corresponding amino acid string of a natural protein sequence on an amino acid by amino acid basis,

D. wherein said variant protein has increased host string content as compared to said parent protein.

27. A method of generating a variant protein for a host as compared to a parent protein, comprising:

A. comparing said parent protein sequence with two or more natural protein sequences;

B. substituting amino acids in said parent protein sequence with amino acids in one or more natural protein sequences, wherein a substitution increases the sequence identity of said variant protein to a natural protein sequence as compared to said parent protein within a string,

C. wherein said variant protein has increased host string content as compared to said parent protein.

28. A method of generating a variant protein for a host as compared to a parent protein, comprising:

A. comparing said parent protein sequence with two or more natural protein sequences from said host species;

B. analyzing one or more amino acid strings of said parent protein sequence with a corresponding amino acid string of each natural protein sequence; and

,

C. substituting one or more amino acids of said parent protein sequence with a corresponding amino acid string of a natural protein sequence on an amino acid string by amino acid string basis,

D. wherein said variant protein has increased host string content.

29. The method according to claim 28, wherein said substituted amino acid is a consensus of two or more natural proteins.

30. The method according to claim 28, wherein said host species is human.

31. The method according to claim 28, wherein said parent protein is murine.

32. The method according to claim 28, wherein said natural protein sequences are germline sequences.

33. The method according to claim 28, wherein one of the substitutions is not the corresponding residue from the most homologous natural sequence.

34. The method according to claim 33, wherein one of the substitutions is not the corresponding residue from a consensus of homologous natural sequences.

35. The method according to claim 28, wherein one of the substitutions is made at a position that is not surface exposed.

36. The method according to claim 28, wherein said variant protein is an antibody.

37. The method according to claim 36, wherein one of the substitutions is not in a CDR.

38. The method according to claim 36, wherein one of the substitutions is made at a position that is part of the VH/VL interface.

39. The method according to claim 36, wherein said variant antibody comprises an affinity for an antigen no lower than about two-fold less than said parent protein.

40. The method according to claim 36, wherein said variant antibody comprises an affinity for an antigen of about two-fold more than said parent protein.

41. The method according to claim 36, wherein said variant protein comprises a framework region identity as compared to a natural protein sequence of less than about 85%.

42. The method according to claim 36, wherein said variant protein comprises a framework region homogeneity of less than about 0.6.

43. The method according to claim 36, wherein said variant antibody comprises less than about 28 amino acid substitutions, as compared to a parent protein.

44. The method according to claim 36, wherein said variant antibody comprises no substitutions within about 5 Angstroms of a CDR residue.

45. The method according to claim 36, wherein said antibody has modulated effector function as compared to said parent antibody.

46. The method according to claim 45, wherein said effector function is ADCC.

47. The method according to claim 28, wherein more than one variant protein is generated from said method to form a variant protein set.

48. The method according to claim 47, wherein said variant protein set comprises at least two variant proteins that differ by more than about 5 amino acids.

49. The method according to claim 47, further synthesizing at least one variant protein.

50. The method according to claim 28, wherein said strings are not applied to a CDR region.

51. The method according to claim 28, wherein said variant protein has at least one improved property is selected from a group consisting of solubility, stability, expression, affinity, immunogenicity, activity and functionality.

52. The method according to claim 28, wherein said analyzing one or more amino acid strings includes theoretical binding to MHC agretopes.

53. The method according to claim 52, wherein said string is nine amino acids in length.