Methods for antibody engineering

Abstract

The invention provides a method for identifying positions of an antibody that can be modified without significantly reducing the binding activity of the antibody. In many embodiments, the method involves identifying a substitutable position in a parent antibody by comparing its amino acid sequence to the amino acid sequences of a number of related antibodies that each bind to the same antigen as the parent antibody. The amino acid at the substitutable position may be substituted for a different amino acid without significantly affecting the activity of the antibody. The subject methods may be employed to change the amino acid sequence of a CDR without significantly reducing the affinity of the antibody of the antibody, in humanization methods, or in other antibody engineering methods. The invention finds use in a variety of therapeutic, diagnostic and research applications.

1 - 36 . (canceled) 
     
     
         37 . A method of altering the amino acid sequence of a parent antibody, comprising:
 a) identifying a variation tolerant position in the amino acid sequence of said parent antibody by comparing the amino acid sequence of said parent antibody to the amino acid sequence of a related antibody that is clonally related to said parent antibody, wherein said parent antibody and said related antibody are derived from an ancestral antibody via affinity maturation; and   b) substituting the amino acid present at said variation tolerant position with a different amino acid to produce a modified antibody that binds the same antigen as said parent antibody but has an amino acid sequence that is different to the amino acid sequence of the parent antibody.   
     
     
         38 . The method of  claim 37 , wherein the amino acid of said variation tolerant position of said parent antibody is substituted by an amino acid present at a corresponding position in a related antibody. 
     
     
         39 . The method of  claim 37 , wherein the amino acid of said variation tolerant position of said parent antibody is substituted by an amino acid present at a corresponding position in a human antibody. 
     
     
         40 . The method of  claim 37 , wherein said substituting comprises making a conservative amino acid substitution. 
     
     
         41 . The method of  claim 37 , wherein said substituting comprises making a non-conservative amino acid substitution. 
     
     
         42 . The method of  claim 37 , wherein said method comprises identifying and substituting at least two variant tolerant positions in said parent antibody. 
     
     
         43 . The method of  claim 37 , further comprising testing said modified antibody for binding to said antigen. 
     
     
         44 . The method of  claim 37 , further comprising testing said modified antibody for more specific binding to said antigen relative to said parent antibody. 
     
     
         45 . The method of  claim 43 , wherein said testing is done by ELISA. 
     
     
         46 . The method of  claim 37 , wherein said variation tolerant position is in a CDR of said parent antibody. 
     
     
         47 . The method of  claim 37 , wherein said variation tolerant position is in a framework region of said parent antibody. 
     
     
         48 . The method of  claim 37 , wherein said parent antibody and said related antibody are derived from an ancestral antibody via affinity maturation in vivo. 
     
     
         49 . The method of  claim 37 , wherein said parent antibody and said related antibody are rabbit or mouse antibodies. 
     
     
         50 . The method of  claim 37 , further comprising:
 introducing a nucleic acid encoding said modified antibody into a cell;   culturing said cell to produce said modified antibody; and   isolating said modified antibody from said cell.   
     
     
         51 . The method of  claim 37 , wherein:
 said animal is a rabbit or a mouse; and   the amino acid of said variation tolerant position of said parent antibody is substituted by an amino acid present at a corresponding position in a human antibody.