High throughput generation and affinity maturation of humanized antibody

Abstract

Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

What is claimed is:  
     
         1 . A method for screening a library of humanized antibody in yeast, comprising: 
 expressing a library of humanized antibodies in yeast cells;    expressing a specific target protein in the yeast cells expressing the humanized antibodies; and    selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the humanized antibody to the target protein.    
     
     
         2 . The method of  claim 1 , wherein a member of the library of humanized antibody contains one or more CDRs of a non-human antibody grafted into a human antibody framework.  
     
     
         3 . The method of  claim 1 , wherein the library of humanized antibody are generated by mutagenizing a chimeric antibody that contains one or more CDRs of a non-human antibody grafted into a human antibody framework.  
     
     
         4 . A method for screening a library of humanized antibody in vitro, comprising: 
 expressing a library of humanized antibodies in vitro;    displaying the library of humanized antibodies on ribosome;    contacting the ribosome displaying the library of humanized antibodies with a target antigen; and    selecting those ribosomes that bind to the target antigen,    
     
     
         5 . The method of  claim 4 , wherein the target antigen is immobilized to a substrate.  
     
     
         6 . The method of  claim 4 , wherein the target antigen is fluorescence labeled.  
     
     
         7 . A method for screening a library of humanized antibody in vitro, comprising: 
 transcribing a library of cDNA encoding a library of humanized antibodies in vitro to produce a library of mRNA;    contacting the library of mRNA with a peptidyl acceptor linker to produce a library of modified mRNA;    translating the library of modified mRNA in vitro to produce a library of humanized antibody, wherein the translated humanized antibody is covalently linked to the modified mRNA by the peptidyl acceptor linker; and    contacting the library of humanized antibodies linked with the modified mRNA with a target antigen; and    selecting the humanized antibody that bind to the target antigen,    
     
     
         8 . The method of  claim 7 , wherein the peptidyl acceptor linker is puromycin.