Genetically Altered Anti-body Producing Cell Lines With Improved Antibody Characteristics

Abstract

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. Cells may be selected for expression of activation-induced cytidine deaminase (AID), stimulated to produce AID, or manipulated to express AID for further enhancement of hypermutability. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production.

1 . An isolated, hypermutable, antibody-producing cell produced in vitro by introducing into the antibody-producing cell a polynucleotide comprising a nucleic acid sequence encoding a PMS2 having an ATPase domain, wherein expression of said polynucleotide inhibits mismatch repair, wherein inhibition of mismatch repair stimulates expression of activation-induced cytidine deaminase, thereby generating a hypermutable antibody-producing cell. 
     
     
         2 . The cell of  claim 1 , wherein said polynucleotide encodes a mammalian PMS2 having an ATPase domain. 
     
     
         3 . The cell of  claim 1 , wherein said polynucleotide encodes a rodent PMS2 having an ATPase domain. 
     
     
         4 . The cell of  claim 1 , wherein said polynucleotide encodes a human PMS2 having an ATPase domain. 
     
     
         5 . The cell of  claim 1 , wherein said polynucleotide encodes a plant PMS2 having an ATPase domain. 
     
     
         6 . The cell of  claim 1  wherein said PMS2 is PMS2-134. 
     
     
         7 . The cell of  claim 6  wherein said PMS2-134 is encoded by the nucleic acid sequence SEQ ID NO:5, SEQ ID NO:33, or SEQ ID NO:37. 
     
     
         8 . A cell culture comprising the cell of  claim 1 . 
     
     
         9 . The cell of  claim 1 , wherein the polynucleotide is inactivated, thereby producing a genetically stable antibody-producing cell. 
     
     
         10 . A cell culture comprising the cell of  claim 9 . 
     
     
         11 . A hybridoma cell producing antibodies produced from in vitro immunized immunoglobulin-producing cells by:
 (a) combining cells capable of producing immunoglobulins with an immunogenic antigen in vitro;   (b) fusing said cells with myeloma cells to form parental hybridoma cells, wherein a polynucleotide comprising a nucleic acid sequence encoding a PMS2 having an ATPase domain is transfected into either said cells capable of producing immunoglobulins, said myeloma cells, or said parental hybridoma cells, wherein expression of said polynucleotide inhibits mismatch repair;   (c) selecting for mismatch repair-inhibited hybridoma cells exhibiting increased expression of activation-induced cytidine deaminase relative to mismatch repair-proficient hybridoma cells, wherein expression of activation-induced cytidine deaminase is stimulated by said inhibition of mismatch repair;   (d) incubating the selected mismatch repair-inhibited hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; and   (e) selecting hypermutated hybridoma cells that produce antibodies that specifically bind antigen,   
       thereby producing hybridoma cells producing antibodies from in vitro immunized immunoglobulin-producing cells. 
     
     
         12 . The hybridoma cell of  claim 11 , wherein said activation-induced cytidine deaminase is a mammalian activation-induced cytidine deaminase. 
     
     
         13 . The hybridoma cell of  claim 11 , wherein said activation-induced cytidine deaminase is a human activation-induced cytidine deaminase. 
     
     
         14 . The hybridoma cell of  claim 11 , wherein said activation-induced cytidine deaminase is a mouse activation-induced cytidine deaminase. 
     
     
         15 . The cell of  claim 11 , wherein said polynucleotide encodes a mammalian PMS2 having an ATPase domain. 
     
     
         16 . The cell of  claim 11 , wherein said polynucleotide encodes a rodent PMS2 having an ATPase domain. 
     
     
         17 . The cell of  claim 11 , wherein said polynucleotide encodes a human PMS2 having an ATPase domain. 
     
     
         18 . The cell of  claim 11 , wherein said polynucleotide encodes a plant PMS2 having an ATPase domain. 
     
     
         19 . The hybridoma cell of  claim 11 , wherein said PMS2 is PMS2-134. 
     
     
         20 . The hybridoma cell of  claim 19 , wherein said PMS2-134 is encoded by the nucleic acid sequence SEQ ID NO:5, SEQ ID NO:33, or SEQ ID NO:37. 
     
     
         21 . The hybridoma cell of  claim 20 , wherein said antibody-producing cell is a mammalian expression cell transfected with polynucleotides encoding immunoglobulin heavy and light chains.