Novel lowered affinity antibodies and uses therefor

Abstract

The present invention provides novel, rationally designed lowered affinity antibodies for use in various in vivo and in vitro applications. The antibodies of the present invention have variable domains that have been designed to reduce or eliminate the antigen binding activity of the parental antibody without altering the overall 3 dimensional antibody structure. Using the antibodies of the present invention in various assays allows researchers to distinguish effects that result from specific antigen-antibody interactions from other, non-specific antibody effects.

1 .- 20 . (canceled) 
     
     
         21 . An isolated OKT3 modified antibody or antigen binding fragment thereof, comprising:
 non-CDR regions having OKT3 wild-type sequence, and   CDR regions wherein no more than 3 of the CDR regions are mutated relative to the wild-type OKT3 sequence and the remaining CDR regions have wild-type OKT3 sequence,   each mutated CDR having substitutions characterized by little discernable structural change relative to the wild-type but resulting in lowered binding affinity to a CD3 antigen relative to the wild-type CDR;   wherein the OKT3 modified antibody or antigen binding fragment has a K D  greater than 10 −7  M.   
     
     
         22 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , having a K D  greater than 10 −6 M. 
     
     
         23 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein each mutated CDR has no more than 4 amino acid substitutions. 
     
     
         24 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRH1 comprises SEQ ID NO.: 2, and the antibody or antigen binding fragment further comprises at least one light chain CDR mutation. 
     
     
         25 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRH2 comprises SEQ ID NO.: 4. 
     
     
         26 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRH2 comprises SEQ ID NO.: 5, and the antibody or antigen binding fragment further comprises at least one light chain CDR mutation. 
     
     
         27 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRH3 comprises SEQ ID NO.: 7, and the antibody or antigen binding fragment further comprises at least one light chain CDR mutation. 
     
     
         28 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRH3 comprises SEQ ID NO.: 8, and the antibody or antigen binding fragment further comprises at least one light chain CDR mutation. 
     
     
         29 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRL1 comprises SEQ ID NO.: 10, and the antibody or antigen binding fragment further comprises at least one heavy chain CDR mutation. 
     
     
         30 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRL2 comprises SEQ ID NO.: 12, and the antibody or antigen binding fragment further comprises at least one heavy chain CDR mutation. 
     
     
         31 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , wherein CDRL3 comprises SEQ ID NO.: 14. 
     
     
         32 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , further comprising two mutated heavy chain CDRs and having variable region heavy chain sequence selected from the group consisting of: SEQ ID NO.: 30, SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ ID NO.: 33, SEQ ID NO.: 34, SEQ ID NO.: 35, SEQ ID NO.: 36 and SEQ ID NO.: 37. 
     
     
         33 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , further comprising three mutated heavy chain CDRs and having variable region heavy chain sequence selected from the group consisting of: SEQ ID NO.: 38, SEQ ID NO.: 39, SEQ ID NO.: 40 and SEQ ID NO.: 41. 
     
     
         34 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , further comprising two mutated light chain CDRs and having variable region light chain sequence selected from the group consisting of: SEQ ID NO.: 45, SEQ ID NO.: 46 and SEQ ID NO.: 47. 
     
     
         35 . The isolated OKT3 modified antibody or antigen binding fragment of  claim 21 , further comprising three mutated light chain CDRs and having variable region light chain sequence of: SEQ ID NO.: 48.