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 method for producing a biological sample selected from the group consisting of: an antibody specific for a selected antigen and comprising an Igκ polypeptide comprising a human Vκ region, said Igκ polypeptide comprising said human Vκ region, said human Vκ region, a cell expressing said antigen-specific antibody, a cell expressing said Igκ polypeptide comprising said human Vκ region, nucleic acid encoding said antigen-specific antibody, nucleic acid encoding said Igκ polypeptide comprising said human Vκ region, and nucleic acid encoding said human Vκ region, the method comprising the step of:
 (a) contacting a transgenic mouse with an antigen, 
 said transgenic mouse having a germline comprising 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 Igκ 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 by of mouse intron DNA, and wherein said chimeric JC intron comprises less than the complete mouse JC intron; 
 
 wherein said recombinant Igκ locus of said mouse is capable of undergoing V, D, J joining of unrearranged human Igκ variable region gene segments, and said mouse is capable of expressing Igκ polypeptide comprising a human Vκ region and a mouse Cκ region, and 
 (b) isolating from said mouse of step (a) or a cell of said mouse of step (a) said biological sample. 
 
     
     
         2 . The method of  claim 1 , wherein said isolated biological sample comprises said Igκ polypeptide comprising said human Vκ region and said Igκ polypeptide comprises a polypeptide constant region encoded by a C gene segment of said constant region. 
     
     
         3 . The method of  claim 2 , further comprising the step of providing a fully human Igκ polypeptide by replacing said constant region with a human constant region. 
     
     
         4 . The method of  claim 2 , wherein said C gene segment comprises said endogenous C gene segment. 
     
     
         5 . The method of  claim 1 , wherein said isolated biological sample comprises a cell which is a B cell expressing said Igκ polypeptide comprising said human Vκ region, or a B cell expressing said antigen-specific antibody, and said method further comprises the step of preparing a hybridoma from said B cell. 
     
     
         6 . The method of  claim 5 , wherein said method further comprises the step of:
 isolating nucleic acid from said hybridoma, wherein said nucleic acid encodes a polypeptide selected from the group consisting of: said human Vκ region, said Igκ polypepide comprising said human Vκ region, or said antigen-specific antibody comprising said Igκ polypeptide.   
     
     
         7 . The method of  claim 6 , further comprising combining nucleic acid encoding a human Vic region with nucleic acid encoding a human constant region, thereby producing a nucleic acid encoding a human Igκ polypepide. 
     
     
         8 . The method of  claim 7 , further comprising inserting said nucleic acid encoding said fully human Igκ polypepide into the genome of a cell, thereby providing a cell capable of expressing said human Igk polypeptide. 
     
     
         9 . The method of  claim 8 , further comprising the step of:
 expressing said human Igκ polypepide from said cell, and optionally isolating the expressed polypeptide.   
     
     
         10 . The method of  claim 7 , further comprising the step of:
 expressing said human Igκ polypepide from said nucleic acid, and optionally isolating the expressed polypeptide.   
     
     
         11 . The method of  claim 1 , wherein said isolated biological sample comprises a cell which is a B cell expressing said Igκ polypeptide comprising said human Vκ region, or a B cell expressing said antigen-specific antibody, and said method further comprises the step of isolating nucleic acid from said B-cell, wherein said nucleic acid encodes said human Vκ region, said Igκ polypepide comprising said human Vκ region, or said antigen-specific antibody comprising said Igκ polypeptide. 
     
     
         12 . The method of  claim 11 , further comprising combining nucleic acid encoding said human Vκ region with nucleic acid encoding a human constant region, thereby producing a nucleic acid encoding a human Igκ polypepide. 
     
     
         13 . The method of  claim 12 , further comprising the step of:
 expressing said human Igκ polypepide from said nucleic acid, and optionally isolating the expressed polypeptide.   
     
     
         14 . The method of  claim 1 , wherein said isolated biological sample comprises nucleic acid encoding said human Vκ region, and said method further comprises the step of:
 combining said nucleic acid encoding said human Vκ region polypeptide to nucleic acid encoding a human C region polypeptide to produce nucleic acid encoding a fully human polypeptide comprising said human Vκ region and a human C region. 
 
     
     
         15 . The method of  claim 14 , further comprising the step of:
 expressing said fully human polypeptide.   
     
     
         16 . The method of  claim 15 , further comprising isolating said fully human polypeptide and combining said polypeptide with a human heavy chain polypeptide to provide an antibody. 
     
     
         17 . The method of  claim 1 , wherein said antigen is a human antigen. 
     
     
         18 . The method of  claim 1 , further comprising, after said contacting step, the step of contacting said mouse with said antigen a second time. 
     
     
         19 . The method of  claim 1 , wherein said 3′ human Jκ gene segment is human Jκ5, 
     
     
         20 . The method of  claim 1 , wherein said enhancer is a mouse Eiκ enhancer. 
     
     
         21 . The method of  claim 1 , said human Jκ gene segments comprising Jκ1, Jκ2, Rκ3, Jκ4, and Jκ5 gene segments located at an endogenous Igκ locus and operatively linked to said enhancer and an Igκ constant (C) region comprising an endogenous Cκ gene segment. 
     
     
         22 . The method of  claim 1 , wherein said chimeric Igκ locus is homozygous. 
     
     
         23 . The method of  claim 22 , wherein in said transgenic mouse expression of Igκ light chains comprising endogenous mouse variable regions is reduced or prevented. 
     
     
         24 . The method of  claim 1 , wherein said human Vκ gene segments comprise two or more segments selected from the group consisting of: Vκ1-9, Vκ3-7, Vκ1-6, Vκ1-5, Vκ2-4, Vκ7-3, Vκ5-2 and Vκ4-1. 
     
     
         25 . The method of  claim 1 , wherein the distance from said 3′ human Jκ gene segment to said chimeric junction is less than 1 kb. 
     
     
         26 . The method of  claim 1 , wherein DNA between said chimeric junction and said enhancer comprises mouse 129 strain JC intronic DNA. 
     
     
         27 . The method of  claim 1 , said mouse further comprising a recombinant (IgH) locus comprising in 5′ to 3′ transcriptional orientation unrearranged human IgH variable region gene segments, an enhancer, and a C region comprising an endogenous CH gene segment, wherein said human variable region gene segments are operatively linked to said enhancer and said constant region;
 wherein said recombinant IgH locus of said mouse is capable of undergoing V, D, J joining of unrearranged human IgH variable region gene segments and said mouse is capable of expressing chimeric IgH polypeptide comprising a VH region; 
 
     
     
         28 . The method of  claim 27 , wherein said mouse is homozygous for said recombinant IgH locus and said germline comprises all or part of mouse IgH variable region DNA away from said constant region and said mouse is non-functional to express fully mouse Ig comprising a mouse VH region and a mouse CH region;
 and wherein said transgenic mouse is capable of breeding with a second transgenic mouse to produce subsequent generation transgenic mice,   said second transgenic mouse having a germline comprising a recombinant homozygous IgH locus comprising unrearranged human IgH variable region gene segments upstream of a constant (C) region comprising an endogenous C segment of an IgH locus,
 wherein a said subsequent generation transgenic mouse comprises a germline comprising 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 by of mouse intron DNA, and wherein said chimeric JC intron comprises less than the complete mouse JC intron; 
 
   wherein said recombinant Igκ locus of said mouse is capable of undergoing V, J joining of unrearranged human Igκ variable region gene segments, and said mouse is capable of expressing Igκ polypeptide comprising a human Vκ region and a mouse Cκ region.