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.

What is claimed is: 
     
         1 . A transgenic mouse having a germline comprising a homozygous chimeric immunoglobulin heavy and light chain loci,
 said chimeric immunoglobulin heavy chain locus comprising unrearranged human IgH Vs, Ds and J gene segments at an endogenous IgH locus upstream of and operatively linked to a heavy chain constant region comprising an endogenous C gene segment of a mouse IgH locus,   said chimeric IgH locus comprising in 5′ to 3′ orientation said unrearranged human IgH Vs, Ds and J gene segments, a chimeric JC intron, and said heavy chain constant region,   wherein said chimeric JC intron comprises a truncated human JC intron contiguous with a truncated mouse JC intron,   said chimeric immunoglobulin light chain locus comprising in 5′ to 3′ orientation unrearranged human Ig Vκs and Jκ gene segments, a chimeric kappa JC intron, an enhancer and a constant region comprising an endogenous Cκ gene segment, wherein said chimeric kappa JC intron comprises a truncated human Igκ JC intron contiguous with a truncated mouse Igκ JC intron, said truncated mouse Igκ comprises 782 bp of mouse intron DNA between said contiguous human Igκ JC intron and said enhancer.   wherein said transgenic mouse is capable of producing an antibody comprising a chimeric heavy chain comprising a human IgH variable region and a mouse IgH constant region and chimeric kappa light chain comprising a human Igκ variable region and a mouse Ig κ constant region upon stimulation with antigen.   
     
     
         2 . The transgenic mouse of  claim 1 , wherein said truncated mouse JC intron comprises mouse Eμ. 
     
     
         3 . The transgenic mouse of  claim 2 , wherein said human JH gene segment is less than 1 kb upstream of the chimeric junction. 
     
     
         4 . The transgenic mouse of  claim 2 , wherein said mouse JC intronic DNA comprises mouse 129 strain DNA. 
     
     
         5 . The transgenic mouse of  claim 4 , wherein said mouse 129 strain in 129Sv strain. 
     
     
         6 . The transgenic mouse of  claim 2 , wherein said mouse Eμ is the only μ enhancer within said chimeric JC intron. 
     
     
         7 . The transgenic mouse of  claim 1 , wherein said endogenous C segment comprises a mouse Cμ segment. 
     
     
         8 . The transgenic mouse of  claim 7 , wherein said transgenic mouse is functional to form rearranged human VH, D and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse Cμ region. 
     
     
         9 . A method of producing a transgenic mouse, the method comprising the steps of inserting into a transgenic mouse germline:
 (a) a plurality of human IgH V gene segments, human IgH D gene segments, and human IgH J gene segments at an endogenous IgH locus upstream of and operatively linked to a heavy chain constant region comprising an endogenous CH gene segment of a mouse IgH locus, to form a chimeric IgH locus which comprises in 5′ to 3′ orientation,   said plurality of human IgH V gene segments, D gene segments and J gene segments, a chimeric JC intron comprising a truncated human JC intron contiguous with a truncated mouse JC intron, and said IgH constant region; and   (b) human Ig light chain kappa V gene segments and human Ig light chain kappa J gene segments at an endogenous Ig kappa locus upstream of a mouse kappa constant region comprising an endogenous C kappa gene, to form a chimeric Ig locus which comprises in 5′ to 3′ orientation, said human Kappa V gene segments and kappa J gene segments, a chimeric kappa JC intron, an enhancer and a constant region comprising an endogenous Cκ gene segment, wherein said chimeric kappa JC intron comprises a truncated human Igκ JC intron contiguous with a truncated mouse Igκ JC intron, said truncated mouse Igκ comprises 782 bp of mouse intron DNA between said contiguous human Igκ JC intron and said enhancer, the insertion being such that the transgenic mouse is capable of producing a plurality of polypeptides selected from the group consisting of chimaeric antibodies, heavy chain polypeptides and kappa chain polypeptides having a mouse constant region and a human variable region,   
       wherein steps (a) and (b) can be carried out in either order or each of steps (a) and (b) can be carried out in a stepwise manner or as a single step. 
     
     
         10 . A method of making a mouse cell, the method comprising
 (a) providing a mouse ES cell whose genome comprises endogenous immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgL) loci; and   (b)
 (i) making a chimeric Ig heavy chain locus by inserting into said endogenous IgH locus, nucleic acid comprising a plurality of human IgH V gene segments, human IgH D gene segments, human IgH J gene segments upstream of and operatively linked to a constant region comprising an endogenous CH gene segment of a mouse IgH locus, to form a chimeric IgH locus which comprises in 5′ to 3′ orientation, 
 said plurality of human IgH V gene segments, D gene segments and J gene segments, a chimeric JC intron comprising a truncated human JC intron contiguous with a truncated mouse JC intron, and said IgH constant region; 
 (ii) inserting at a said endogenous IgL locus a nucleic acid comprising a chimeric immunoglobulin light chain locus comprising in 5′ to 3′ orientation unrearranged human Ig Vκs and Jκ gene segments, a chimeric kappa JC intron, an enhancer and a constant region comprising an endogenous Cκ gene segment, wherein said chimeric kappa JC intron comprises a truncated human Igκ JC intron contiguous with a truncated mouse Igκ JC intron, said truncated mouse Igκ comprises 782 bp of mouse intron DNA between said contiguous human Igκ JC intron and said enhancer; 
   whereby a mouse ES cell is produced that is capable of giving rise to a progeny cell in which endogenous antibody expression is inactivated and wherein said progeny cell is capable of expressing antibodies comprising human IgH variable regions and human IgL variable regions; and   (c) differentiating said ES cell into said progeny cell or a transgenic mouse comprising said progeny cell.   
     
     
         11 . The method of  claim 10 , wherein said mouse cell comprises nucleic acid encoding a human immunoglobulin variable region and said method further comprising the steps of:
 providing the nucleic acid encoding said human immunoglobulin variable region and said human constant region.   
     
     
         12 . A method of obtaining B cells expressing an antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide, comprising contacting the mouse of  claim 1  with an antigen, and isolating B cells expressing said antigen-specific antibody from said immunized mouse. 
     
     
         13 . The method of  claim 10 , wherein said mouse cell comprises nucleic acid encoding a human immunoglobulin variable region and said method further comprising the steps of:
 isolating nucleic acid encoding said human immunoglobulin variable region.   
     
     
         14 . The method of  claim 13 , further comprising the steps of:
 expressing the antibody from said nucleic acid, wherein said nucleic acid encodes an antibody comprising a human variable region and a constant region.   
     
     
         15 . The method of  claim 11 , further comprising the steps of:
 expressing the antibody from said nucleic acid, wherein said nucleic acid encodes an antibody comprising said human variable region and said human constant region.   
     
     
         16 . The transgenic mouse of  claim 7 , wherein said transgenic mouse is functional to form rearranged human VH, D and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse Cμ region. 
     
     
         17 . A method for producing an antibody or antibody heavy or light chain specific to a desired antigen, the method comprising contacting the transgenic mouse as claimed in  claim 1  with a desired antigen and recovering the antibody or antibody heavy or light chain or a fragment thereof and/or recovering a cell producing the antibody or heavy or light chain thereof. 
     
     
         18 . A method for producing a humanised antibody or antibody chain comprising carrying out the method according to  claim 17  and further comprising replacing the constant region of the recovered antibody or antibody chain with a human constant region. 
     
     
         19 . The method of  claim 18 , wherein said humanization is performed by engineering of the nucleic acid encoding the antibody or antibody chain. 
     
     
         20 . The mouse of  claim 1 , wherein said truncated human intronic DNA comprises human DNA upstream of coordinate 106,368,585 of human chromosome 14.