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 obtaining an antigen specific antibody or antigen binding fragment thereof, said antibody comprising a human immunoglobulin light (IgH) chain, wherein said human IgH chain comprises a human IgH chain variable region and a human IgH chain constant region, and said fragment comprising a human IgH chain variable region, the method comprising:
 (A) providing a cell comprising a nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or a cell comprising a nucleic acid encoding said human IgH chain variable region of said antigen-binding fragment,   wherein said human IgH chain variable region is of a transgenic mouse contacted with said antigen;   wherein the germline of said mouse has a genome with a homozygous chimeric immunoglobulin heavy chain (IgH) locus,   
       wherein said homozygous chimeric IgH locus comprises unrearranged human IgH variable region gene segments at an endogenous mouse heavy chain IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous CH gene segment;
 wherein said human variable region gene segments in said chimeric IgH locus are operably linked to said C region at a human/mouse chimeric junction within the JC intron of said chimeric IgH locus; 
 wherein said homozygous chimeric IgH locus comprises in 5′ to 3′ transcriptional orientation:
 (i) unrearranged human immunoglobulin heavy chain (IgH) variable region (VH) DNA comprising human IgH V gene segments, human D gene segments and human JH gene segments comprising a human 3′JH gene segment, 
 (ii) a chimeric J/C intron comprising human DNA downstream of and naturally contiguous with said human 3′ JH gene segment, which is contiguous with mouse JC intronic DNA, and 
 (iii) said enhancer and said C region, 
 
 wherein said human 3′ JH is less than 1 kb upstream of said chimeric junction, 
 wherein DNA between said chimeric junction and said enhancer comprises mouse 129 strain JC intronic DNA; 
 wherein said enhancer is a mouse 129 strain μ enhancer; 
 wherein said transgenic mouse is functional to form rearranged human VH, DH and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse C region, and wherein said transgenic mouse is capable, upon stimulation with antigen, of producing an antibody comprising a chimeric Ig heavy chain comprising a human IgH variable region and said C region; and 
 wherein the genome of said transgenic mouse comprises all or part of the endogenous mouse IgH variable region, and is capable of breeding to produce subsequent generation mice having in their germline an IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of an IgH constant (C) region comprising an endogenous C gene segment of an IgH locus, and 
 (B) expressing the antibody or antigen binding fragment thereof, from a cell, wherein the cell comprises a nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or expressing the antigen binding fragment from a cell, wherein the cell comprises a nucleic acid encoding said human IgH chain variable region. 
 
     
     
         2 . The method of  claim 1 , wherein said cell is a first cell, and wherein said human IgH chain variable region is obtained from a second cell comprising nucleic acid encoding said human IgH chain variable region, and wherein said second cell is of a transgenic mouse contacted with said antigen. 
     
     
         3 . The method of  claim 2 , wherein said second cell is selected from the group consisting of: a B cell of a transgenic mouse contacted with said antigen; a cell comprising nucleic acid encoding said human IgH chain variable region and a mouse IgH constant region; a hybridoma expressing said human IgH chain variable region; a plurality of cells comprising nucleic acid encoding human IgH chain variable regions; and an immortalised cell comprising nucleic acid encoding said human IgH chain variable region. 
     
     
         4 . The method of  claim 1 , wherein in said mouse germline said 3′-most JH gene segment comprises a human JH6 gene segment. 
     
     
         5 . The method of  claim 1 , wherein in said mouse germline DNA said human DNA downstream of said human 3′JH gene segment comprises a truncated human JC intron and said mouse JC intronic DNA comprises a truncated mouse JC intron. 
     
     
         6 . The method of  claim 1 , wherein in said mouse germline said 129 strain DNA is 129Sv. 
     
     
         7 . The mouse of  claim 1 , wherein said C region comprises a mouse Cμ gene segment. 
     
     
         8 . The mouse of  claim 7 , wherein said mouse Cμ gene segment comprise mouse 129 strain DNA. 
     
     
         9 . The mouse of  claim 1 , wherein said human DNA contiguous with said human 3′JH gene segment comprises 400 base pairs of human JC intron DNA. 
     
     
         10 . The method of  claim 1 , wherein in said germline said all or part of mouse heavy chain variable region is inverted with respect to said heavy chain constant region. 
     
     
         11 . The method of  claim 1 , wherein said mouse is functional to produce antibody isotypes IgM and IgG specific for said antigen each isotype comprising a human heavy chain variable region. 
     
     
         12 . The method of  claim 1 , the method further comprising
 recovering a substance selected from the group consisting of: said antibody or antigen-binding fragment thereof; said nucleic acid encoding said human IgH chain variable region; and said cell expressing said antibody or antigen-binding fragment thereof.   
     
     
         13 . The method of  claim 1 , further comprising the step of providing a pharmaceutical composition comprising said antigen-specific antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or excipient. 
     
     
         14 . The method of  claim 1 , further comprising the step of providing said antibody or antigen-binding fragment thereof to a human subject. 
     
     
         15 . The method of  claim 1 , wherein said antigen-specific antibody or antigen binding fragment thereof is a monoclonal antibody, domain antibody, and/or a neutralizing antibody. 
     
     
         16 . The method of  claim 1 , wherein in said mouse germline said unrearranged human IgH VH gene segments comprise human V6-1, V11-1-1, V1-2, V111-2-1, V1-3, V4-4 and V2-5 gene segments. 
     
     
         17 . The method of  claim 1 , wherein in said mouse germline said unrearranged human IgH VH gene segments comprise all of the human V, D and J gene segments.