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

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.

1 . An antibody that binds the CD30 antigen, wherein said antibody comprises:
 a) a heavy chain comprising a heavy chain variable region and a constant heavy region, wherein said variable heavy region is selected from the group consisting of:
 i) the sequence of SEQ ID NO:223; 
 ii) the sequence of SEQ ID NO:226; 
 iii) the sequence of SEQ ID NO:227; 
 iv) the sequence of SEQ ID NO:228; and 
   b) a light chain comprising a light variable region and a light constant region, wherein said light variable region is selected from the group consisting of:
 i) the sequence of SEQ ID NO:220; 
 ii) the sequence of SEQ ID NO:224; and 
 iii) the sequence of SEQ ID NO:225; 
   
       wherein the numbering of the variable region is according to the Kabat numbering system. 
     
     
         2 . The antibody according to  claim 1 , wherein said variable light chain is the sequence of SEQ ID NO:224. 
     
     
         3 . The antibody according to  claim 1  or  2 , wherein said variable heavy chain is the sequence of SEQ ID NO:227. 
     
     
         4 . The antibody according to  claim 3 , wherein said heavy chain constant region comprises a substitution at position selected from the group consisting of 239, and 332, wherein number of the constant region is according to the EU index. 
     
     
         5 . The antibody according to  claim 4 , wherein said substitution comprises 332E. 
     
     
         6 . The antibody according to  claim 4 , wherein said heavy chain constant region comprises 239D and 332E. 
     
     
         7 . An antibody that binds the CD30 antigen, wherein said antibody comprises:
 a) a heavy chain variable region comprising:
 i) a first CDR comprising amino acids 31-35 of SEQ ID NO:227; 
 ii) a second CDR comprising amino acids 50-66 of SEQ ID NO:227; 
 iii) a third CDR comprising amino acids 99-106 of SEQ ID NO:227; and 
   b) a light chain variable region comprising:
 i) a first CDR comprising amino acids 24-38 of SEQ ID NO:224; 
 ii) a second CDR comprising amino acids 54-60 of SEQ ID NO:224; 
 iii) a third CDR comprising amino acids 93-101 of SEQ ID NO:224; 
   
       wherein the numbering of the variable region is according to the Kabat numbering system. 
     
     
         8 . A polynucleotide encoding the heavy and light chains of a human monoclonal antibody that competes for binding to human CD30 with an antibody comprising the heavy chain FR1 through FR4 amino acids sequences of SEQ ID NO:227 and the light chain FR1 through FR4 amino acid sequence of SEQ ID NO:224. 
     
     
         9 . An expression vector comprising the polynucleotide of  claim 7 . 
     
     
         10 . A mammalian host cell line comprising the polynucleotide of  claim 7 . 
     
     
         11 . 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;   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.