Methods of generating variant proteins with increased host string content

• US 7,930,107 B2
• Filed: 10/31/2007
• Issued: 04/19/2011
• Est. Priority Date: 12/04/2003
• Status: Expired due to Fees

• Current Assignee: Xencor, Inc.
• Original Assignee: Xencor, Inc.
• Inventors: Lazar, Gregory Alan, Hammond, Philip W., Desjarlais, John R.
• Primary Examiner(s): Zhou; Shubo (Joe)
• Application Number:
  US11/981,794
• Publication Number:
  US 20080167449A1
• Time in Patent Office: 1,266 Days
• Field of Search: None
• US Class Current: 702/19
• 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 host species antibody variable region as compared to a non-host species antibody variable region, comprising:

A. aligning said non-host species antibody variable region sequence with two or more naturally occurring host species antibody variable region sequences;

B. defining mutational constraints in said non-host species antibody variable region sequence,

C. performing until no sequence segment substitution has a favorable string impact on host species string content,

i. comparing, for an effect on host species string content, one or more sequence segments of each of said host species antibody variable region sequences to the structurally corresponding sequence segment of said non-host species antibody variable region sequence; and

,

ii. substituting one sequence segment of said non-host species antibody variable region sequence with a structurally corresponding sequence segment of one of said two or more host species antibody variable region sequences, wherein said substituted sequence segment increases host species string content,

wherein said variant host species antibody variable region has increased host species string content as compared to said non-host species antibody variable region,

wherein said comparing uses a string window size that is greater than or equal to 9, and,

wherein at least one framework region of said variant host species antibody variable region includes a first sequence segment substitution from a first naturally occurring antibody variable region sequence from said two or more naturally occurring host species antibody variable region sequences, and a second sequence segment substitution from a second naturally occurring antibody variable region sequence from said two or more naturally occurring host species antibody variable region sequences,

where said host species string content (HSC) is measured by an equation functionally equivalent to;

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

3. The method according to claim 1, wherein one of the sequence segment substitutions is not the structurally corresponding sequence segment from a consensus of homologous natural sequences.

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

5. The method according to claim 1, wherein more than one variant host species antibody variable region is generated from said method to form a variant host species antibody variable region set.

6. The method according to claim 1, wherein said variant host species antibody variable region set comprises at least two variant host species antibody variable regions that differ by more than about 5 amino acids.

7. The method according to claim 1 wherein said string window size is nine amino acids in length.

8. A method of generating a variant host species antibody variable region as compared to a non-host species antibody variable region, comprising:

A. aligning said non-host species antibody variable region sequence with two or more naturally occurring host species antibody variable region sequences;

B. defining mutational constraints in said non-host species antibody variable region sequence,

C. performing until no sequence segment substitution has a favorable string impact on host species string content,

i. comparing, for an effect on host species string content, one or more sequence segments of each of said host species antibody variable region sequences to the structurally corresponding sequence segment of said non-host species antibody variable region sequence; and

ii. substituting one sequence segment of said non-host species antibody variable region sequence with a structurally corresponding sequence segment of one of said two or more host species antibody variable region sequences, wherein said substituted sequence segment increases host species string content relative to said non-host species antibody variable region,

wherein said comparing uses a string window size that is greater than or equal to 9 and less than or equal to 20, and,

where said host species string content (HSC) is measured by an equation functionally equivalent to;

9. The method according to claim 8, wherein one of said sequence segment substitutions is not the corresponding sequence segment from the most homologous natural sequence.

10. The method according to claim 8, wherein one of the sequence segment substitutions is not the structurally corresponding sequence segment from a consensus of homologous natural sequences.

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

12. The method according to claim 8, wherein more than one variant host species antibody variable region is generated from said method to form a variant host species antibody variable region set.

13. The method according to claim 12, wherein said variant host species antibody variable region set comprises at least two variant host species antibody variable regions that differ by more than about 5 amino acids.

14. The method according to claim 8, wherein said string window size is nine amino acids in length.