Super humanized antibodies

Abstract

Disclosed herein are methods for humanizing antibodies based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, preferably germline antibody gene segments. Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences. The subset members may be further ranked by amino acid similarity between the human and the non-human CDR sequences. Top ranking human sequences are selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies. Chimeric antibodies made according to the method are also disclosed.

1 - 28 . (canceled) 
     
     
         29 . A humanized antibody that binds to human CD28, the humanized antibody comprising
 an antibody variable region containing at least two CDR sequences of a non-human antibody fused adjacent to variable framework sequences, the framework sequences having no more than 10 amino acid residues that differ from framework sequences in a human antibody variable region sequence having at least two human CDR sequences with the same canonical structure type as the non-human CDR sequences for at least two corresponding CDR positions between the variable region of the humanized antibody and the human antibody, wherein the non-human antibody comprises a light chain variable region designated SEQ ID NO:113 and a heavy chain variable region designated SEQ ID NO:114.   
     
     
         30 . (canceled) 
     
     
         31 . The humanized antibody of  claim 29  wherein the human variable region sequence is selected from the group consisting of V k , V λ , V H , J H , J k  and J λ  sequences. 
     
     
         32 . The humanized antibody of  claim 29  wherein the humanized antibody includes humanized antibody sequences for each of a variable light chain and a variable heavy chain. 
     
     
         33 . The humanized antibody of  claim 29  wherein the variable light chains and heavy chains of the humanized antibody are assembled to form a molecule selected from the group consisting of a Fab fragment, a (Fab)′ 2  molecule, and a single chain Fv molecule. 
     
     
         34 . The humanized antibody of  claim 29  further comprising a human antibody constant region domain to form a complete antibody. 
     
     
         35 . The method of  claim 29  wherein the human variable region sequence is a sequence from a human germline variable region fragment. 
     
     
         36 . The method of  claim 29  wherein the human variable regions sequence is a sequence from a human mature antibody. 
     
     
         37 . The humanized antibody of  claim 29  wherein the humanized antibody has a dissociation constant for its antigen of at least 10 8 M −1 . 
     
     
         38 . The humanized antibody of  claim 29  wherein the humanized antibody does not elicit an immune response when administered to a human. 
     
     
         39 - 46 . (canceled) 
     
     
         47 . The humanized antibody of  claim 29 , comprising a light chain variable region and a heavy chain variable region, each variable region containing three CDR sequences of the non-human antibody fused adjacent to variable framework sequences, the framework sequences having no more than 10 amino acid residues that differ from framework sequences in a human antibody variable region sequence having at least two human CDR sequences with the same canonical structure type as the non-human CDR sequences for at least two corresponding CDR positions between the variable region of the humanized antibody and the human antibody. 
     
     
         48 . The humanized antibody of  claim 29 , wherein the antibody variable region comprises a light chain variable region comprising three CDR sequences of the light chain of the non-human antibody and variable region framework sequences that differs by no more than 10 amino acid residues from variable region framework sequences encoded by human V K  B3, provided that position 34 by Kabat numbering of light chain CDR 1 may be alanine; and a heavy chain variable region comprising three CDR sequences of the heavy chain of the non-human antibody and variable region framework sequences having no more than 10 amino acid residues that differ from variable region framework sequences encoded by human V H  DP-45 and human JH4, provided that positions 60-65 by Kabat numbering in heavy chain CDR 2 may be residues from the corresponding positions of human DP-45. 
     
     
         49 . A humanized antibody of  claim 29  that comprises a light chain variable region designated SEQ ID NO:115 and a heavy chain variable region designated SEQ ID NO:116. 
     
     
         50 . In a method comprising humanizing a nonhuman antibody to produce a humanized antibody, the improvement wherein human antibody framework sequences for performing the humanizing are selected by comparing canonical CDR structure types of the non-human antibody with canonical CDR structure types of human germline antibody sequences without regarding to comparing the human antibody framework sequences with the framework sequences of the nonhuman antibody. 
     
     
         51 . In a humanized antibody, the improvement wherein variable region framework sequences of the humanized antibody are variable region framework sequences of human germline antibody sequences having at least two CDRs of the same canonical type as two corresponding CDRs in the humanized antibody.