Targeted replacement of a gene without endogenous and selectable residual sequences

Abstract

Transgenic mice that produce high levels of humanized antibodies are described. Targeted gene replacement exchanges constant regions of the mouse immunoglobulin heavy and light chain genes with human genes, either through conventional gene targeting, or by use of the bacteriophage-derived Cre-loxP recombination system. The transgenic animals undergo antibody affinity maturation, and a class switch from the native immunoglobulin to the humanized form.

What is claimed is:  
     
         1 . A method for replacement of a gene or gene segment in the cell of a nonhuman mammal with a homologous gene or a homologous gene segment of another mammal, the method comprising: 
 transfecting an embryonic stem cell with a recombination vector having a selectable marker;    selecting said transfected cells for the presence of said marker gene;    selecting for cells having targeted recombination by at least one of PCR and Southern blotting;    injecting said selected cells into blastocysts of said nonhuman mammal;    transferring said blastocytes to surrogate mothers,    wherein said endogenous gene or endogenous gene segment is functionally replaced in one step by the homologous gene or homologous gene segment in the recombination event.    
     
     
         2 . A method according to  claim 1 , wherein said introduced gene or gene segment is human and said nonhuman mammal is a rodent.  
     
     
         3 . A method according to  claim 1 , wherein said homologous gene or gene segment encodes a protein selected from the group consisting of proteins of the immune system; proteins of the nervous system; virus receptors; proteins of the blood-forming system; and proteins of the support tissue.  
     
     
         4 . A method according  claim 1 , wherein said homologous gene or gene segment encodes a protein selected from the group consisting of antibody, T-cell receptor, cytokine, cytokine receptor, MHC antigen, adhesion molecule and signal-mediating molecule.  
     
     
         5 . A method according to  claim 4 , wherein said homologous gene or gene segment encodes a human antibody.  
     
     
         6 . A method according to  claim 1 , wherein said recombination vector is a replacement vector containing the gene or gene segment to be introduced; sequences homologous to the sequences flanking the endogenous DNA segment to be replaced; a marker gene selected from neomycin and hygromycin; and additional functional gene sequences.  
     
     
         7 . A method according to  claim 6 , wherein said functional gene sequences are recognition sequences for a non-mammalian recombinase.  
     
     
         8 . A method according to  claim 1 , wherein said recombination vector is pTZ 2 -CκN (PA−)(DSM 7211).  
     
     
         9 . A method according to  claim 7 , wherein said marker gene and said sequences homologous to the sequences flanking the endogenous DNA segment are removed by means of said recombinase recognition sequences.  
     
     
         10 . A recombination vector comprising a gene or gene segment to be introduced; sequences homologous to the sequences flanking the endogenous DNA segment to be replaced; a marker gene selected from neomycin and hygromycin; and additional functional gene sequences.  
     
     
         11 . A recombination vector according to  claim 10 , wherein said functional gene sequences are recognition sequences for a non-mammalian recombinase.  
     
     
         12 . A method for producing a transgenic nonhuman mammal, the method comprising: 
 transfecting an embryonic stern cell with a recombination vector according to  claim 10;     selecting said transfected cells for the presence of said marker gene;    selecting for cells having targeted recombination by at least one of PCR and Southern blotting;    injecting said selected cells into blastocysts of said nonhuman mammal;    transferring said blastocytes to surrogate mothers,    mating the chimeric offpspring of said mothers; and    selecting progeny of said mating for the presence of said introduced gene or gene segment.    
     
     
         13 . A method according to  claim 12 , wherein said transgenic non-human mammal is a mouse.  
     
     
         14 . A transgenic, nonhuman mammal obtained according to the method of  claim 12 .  
     
     
         15 . A method of producing a gene product, the method comprising: 
 isolating said gene product from a transgenic animal according to  claim 14 .    
     
     
         16 . A method for producing humanized antibodies, the method comprising: 
 isolating said humanized antibodies from a transgenic animal according to  claim 14 , wherein said introduced gene or gene segment encodes at least a portion of a human antibody.    
     
     
         17 . A method for producing viral proteins, the method comprising: 
 isolating said viral proteins from a transgenic animal according to  claim 14 , wherein said introduced gene or gene segment encodes a viral protein.    
     
     
         18 . A transgenic mouse having a genome comprising: 
 at least one human immunoglobulin constant region gene functionally replacing an analogous mouse gene, wherein humanized antibodies comprising a human constant region joined to a mouse variable region are produced at high levels in response to antigen; and wherein said humanized antibodies undergo somatic hypermutation.    
     
     
         19 . A transgenic mouse according to  claim 18 , wherein said at least one human immunoglobulin constant region is present on both chromosomes.  
     
     
         20 . A transgenic mouse according to  claim 19 , wherein said humanized antibodies are produced at levels of at least 50 μg/ml in serum.  
     
     
         21 . A transgenic mouse according to  claim 20 , wherein said at least one human immunoglobulin constant region gene is Cγ1.  
     
     
         22 . A transgenic mouse according to  claim 18 , wherein said at least one human immunoglobulin constant region gene is Cκ, and wherein humanized antibodies are produced at levels of at least 500 μg/ml.  
     
     
         23 . A transgenic mouse according to  claim 21 , further comprising a human Cκ gene functionally replacing the analogous mouse gene on both chromosomes, wherein said human Cκ and said human Cγ chains form an antibody molecule in vivo.  
     
     
         24 . A method of producing humanized antibodies in response to an antigen, the method comprising: 
 immunizing a mouse according to  claim 18  with an antigen;    collecting antibodies from said mouse;    selecting for antibodies comprising a human constant region.    
     
     
         25 . A method according to  claim 24 , wherein said human constant region is Cγ1.  
     
     
         26 . A method according to  claim 24 , wherein said human constant region is Cκ.  
     
     
         27 . A method according to  claim 24 , wherein said antibodies comprise human Cκ and human Cγ1 chains.  
     
     
         28 . A method of producing an antigen specific monoclonal humanized antibody, the method comprising: 
 immunizing a mouse according to  claim 18  with all antigen;    immortalizing B cells from said immunized mouse;    growing said clones of said immortalized B cells;    screening said immortalized B cells for production of antigen specific humanized antibodies;    collecting antibodies from said immortalized B cells.    
     
     
         29 . A method according to  claim 28 , wherein said human constant region is Cγ1.  
     
     
         30 . A method according to  claim 28 , wherein said humanized antibodies further comprise a human Cκ region.