Animal Models and Therapeutic Molecules

Abstract

The invention discloses methods for the generation of chimaeric human—non-human antibodies and chimaeric antibody chains, antibodies and antibody chains so produced, and derivatives thereof including fully humanised antibodies; compositions comprising said antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in said methods.

1 . A method of isolating a polypeptide comprising a human immunoglobulin (Ig) variable region of an antigen-specific antibody, and/or nucleic acid encoding a polypeptide comprising said human variable region, the method comprising:
 isolating from a cell comprising nucleic acid encoding and/or producing said polypeptide comprising said human Ig variable region
 (i) said polypeptide comprising said human Ig variable region and/or 
 (ii) said nucleic acid encoding said human Ig variable region, 
 wherein said human variable region is of a transgenic mouse having a genetically altered germline comprising:
 (a) a homozygous chimeric immunoglobulin heavy chain locus comprising human IgH variable (V) region DNA at an endogenous IgH locus upstream of a constant region comprising an endogenous mouse heavy chain constant (CH) gene segment, the human IgH V region DNA comprising unrearranged human IgH variable (VH) segments, unrearranged human D segments and unrearranged human heavy chain J (JH)s segments; and 
 (b) mouse IgH variable (V) region DNA, 
 wherein said transgenic mouse is inoperative to express mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region; 
 wherein said transgenic mouse when contacted with said antigen expresses antigen-specific antibody comprising a chimeric heavy chain Ig polypeptide comprising said human variable region. 
 
   
     
     
         2 . The method of  claim 1 , wherein said mouse IgH variable (V) region DNA is upstream of said unrearranged human IgH variable (VH) segments. 
     
     
         3 . The method of  claim 1 , wherein said isolated nucleic acid encodes a polypeptide comprising said human Ig variable region and a mouse constant region, and wherein said isolated polypeptide of step (b) comprises said human Ig variable region and a mouse constant region. 
     
     
         4 . The method of  claim 1 , said cell being a B-cell. 
     
     
         5 . The method of  claim 4 , further comprising the step of preparing a hybridoma from said B cell to thereby provide a B cell hybridoma. 
     
     
         6 . The method of  claim 1 , wherein said isolated nucleic acid encodes a polypeptide comprising said human Ig variable region and a human constant region, and wherein said isolated polypeptide of step (b) comprises said human Ig variable region and a human constant region. 
     
     
         7 . The method of  claim 1 , wherein said isolated nucleic acid encodes a polypeptide comprising said human Ig variable region and a constant region, and the method further comprises the step of: expressing the polypeptide from said nucleic acid. 
     
     
         8 . The method of  claim 1 , wherein said isolated nucleic acid encodes an antibody specific for said antigen and comprising said human Ig variable region, and said method comprises: isolating said antibody. 
     
     
         9 . The method of  claim 8 , further comprising the step of subjecting said isolated antibody to affinity maturation to provide an affinity maturated antibody. 
     
     
         10 . The method of  claim 1 ,
 wherein said isolated nucleic acid encodes antigen-specific antibody comprising said human Ig variable region and a constant region, and the method further comprises the step of: expressing the antibody from said nucleic acid.   
     
     
         11 . The method of  claim 4 , said cell being a B-cell, wherein said B cell expresses antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide. 
     
     
         12 . The method of  claim 1 , wherein said mouse IgH variable (V) region DNA is upstream of said unrearranged human IgH variable (VH) segments. 
     
     
         13 . The method of  claim 1 , wherein the germline of said transgenic mouse further comprises:
 a recombinant immunoglobulin light chain kappa (Igκ) locus, said locus comprising unrearranged human Igκ variable region gene segments at an endogenous locus upstream of an enhancer and a constant (C) region comprising an endogenous Cκ gene segment;
 said recombinant Igκ locus comprising in 5′ to 3′ transcription orientation:
 (i) human Vκ gene segments and human Jκ gene segments, wherein said Jκ gene segments comprise a 3′ human Jκ gene segment, 
 (ii) a chimeric JC intron comprising human JC intronic DNA and mouse JC intronic DNA and comprising said enhancer, and 
 (iii) said C region, 
 wherein said human Ig κ variable region gene segments are operatively linked to said constant region, wherein the distance from said 3′ human Jκ gene segment to said mouse intronic DNA is less than 2 kb and wherein the distance from said human intronic DNA to said enhancer comprises 782 bp of mouse intronic DNA; 
 
 wherein said recombinant Ig κ locus of said mouse is capable of undergoing V J joining of unrearranged human Ig κ variable region gene segments, 
   and said mouse is capable of expressing Ig κ polypeptide comprising a human V κ region and a mouse C κ region,   wherein said antigen-specific antibody further comprises a chimeric kappa chain polypeptide comprising a human kappa variable region.   
     
     
         14 . The method of  claim 13 , further comprising the step of isolating from said isolated cell nucleic acid encoding said human kappa variable region. 
     
     
         15 . The method of  claim 14 , wherein said isolated nucleic acid encoding said human Ig kappa variable region also encodes a constant region. 
     
     
         16 . The method of  claim 15 , wherein said constant region is a mouse constant region. 
     
     
         17 . The method of  claim 15 , wherein said constant region is a human constant region. 
     
     
         18 . A method of isolating a cell that comprises nucleic acid encoding an antigen-specific antibody comprising an immunoglobulin (Ig) polypeptide chain comprising a human variable region, and produces said antigen-specific antibody, the method comprising:
 isolating a cell from a plurality of cells wherein said cell comprises said nucleic acid   encoding said antigen-specific antibody comprising said human variable region, and   produces said antigen-specific antibody comprising said human variable region;
 wherein said human variable region is of a transgenic mouse having a genetically altered germline comprising:
 (i) a homozygous chimeric immunoglobulin heavy chain locus comprising human IgH variable (V) region DNA at an endogenous IgH locus upstream of a constant region comprising an endogenous heavy chain mouse constant (CH) gene segment, the human IgH V region DNA comprising unrearranged human IgH variable (VH) segments, unrearranged human D segments and unrearranged human heavy chain J (JH) segments; and 
 (ii) mouse IgH variable (V) region DNA, 
 wherein said transgenic mouse is inoperative to express mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region; 
 wherein said transgenic mouse when contacted with said antigen expresses antigen-specific antibody comprising a chimeric heavy chain polypeptide comprising said human variable region. 
 
   
     
     
         19 . The method of  claim 18 , wherein said mouse IgH variable (V) region DNA is upstream of said unrearranged human IgH variable (VH) segments. 
     
     
         20 . The method of  claim 18 , wherein said cell is a hybridoma cell. 
     
     
         21 . The method of  claim 18 , wherein said cell is a B-cell. 
     
     
         22 . The method of  claim 21 , wherein said B cell expresses antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide. 
     
     
         23 . The method of  claim 21 , further comprising the step of preparing a hybridoma from said B cell to thereby provide a B cell hybridoma. 
     
     
         24 . The method of  claim 18 , wherein said cell nucleic acid encoding said antibody encodes an Ig polypeptide chain comprising said human variable region and a mouse constant region and said cell produces said antibody comprising said Ig polypeptide chain comprising said human variable region and a mouse constant region. 
     
     
         25 . The method of  claim 18 , wherein said cell nucleic acid encoding said antibody encodes an Ig polypeptide chain comprising said human variable region and a human constant region and said cell produces said antibody comprising said Ig polypeptide chain Ig polypeptide chain comprising said human variable region and human constant region. 
     
     
         26 . The method of  claim 18 , further comprising the step of isolating from said isolated cell of step (b) nucleic acid encoding said human variable region. 
     
     
         27 . The method of  claim 26 , wherein said isolated nucleic acid encoding said human Ig variable region also encodes a constant region. 
     
     
         28 . The method of  claim 27 , wherein said constant region is a mouse constant region. 
     
     
         29 . The method of  claim 27 , wherein said constant region is a human constant region. 
     
     
         30 . The method of  claim 18 , wherein the germline of said transgenic mouse further comprises:
 a recombinant immunoglobulin light chain kappa (Igκ) locus, said locus comprising unrearranged human Igκ variable region gene segments at an endogenous locus upstream of an enhancer and a constant (C) region comprising an endogenous Cκ gene segment;
 said recombinant Igκ locus comprising in 5′ to 3′ transcription orientation:
 (i) human Vκ gene segments and human Jκ gene segments, wherein said Jκ gene segments comprise a 3′ human Jκ gene segment, 
 (ii) a chimeric JC intron comprising human JC intron DNA contiguous with mouse JC intron DNA at a chimeric junction and comprising said enhancer, and 
 (iii) said C region, 
 said chimeric junction being internal in said chimeric JC intron of said recombinant Igκ locus such that said human Ig κ variable region gene segments are operatively linked to said constant region, wherein the distance from said 3′ human Jκ gene segment to said chimeric junction is less than 2 kb and wherein the distance from said chimeric junction to said enhancer comprises 782 bp of mouse intron DNA, and wherein said chimeric JC intron comprises less than the complete mouse JC intron; 
 
   wherein said recombinant Ig κ locus of said mouse is capable of undergoing V J joining of unrearranged human Ig κ variable region gene segments,   and said mouse is capable of expressing Ig κ polypeptide comprising a human V κ region and a mouse C κ region,   wherein said antigen-specific antibody further comprises a chimeric kappa chain polypeptide comprising a human kappa variable region.   
     
     
         31 . The method of  claim 30 , further comprising the step of isolating from said isolated cell of step (b) nucleic acid encoding said human kappa variable region. 
     
     
         32 . The method of  claim 31 , wherein said isolated nucleic acid encoding said human Ig kappa variable region also encodes a constant region. 
     
     
         33 . The method of  claim 32 , wherein said constant region is a mouse constant region. 
     
     
         34 . The method of  claim 32 , wherein said constant region is a human constant region.