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 for obtaining an antigen specific antibody or antigen binding fragment thereof, said antigen-specific antibody comprising a human immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region and a human IgH chain constant region, said antigen-binding fragment comprising a human IgH chain variable region, the method comprising:
 expressing the antibody from a cell comprising nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or expressing said fragment from a cell comprising nucleic acid encoding said human IgH constant region,   wherein said human IgH chain variable region is of a transgenic mouse contacted with said antigen;   wherein the germline of said transgenic mouse comprises a homozygous chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments at an endogenous mouse heavy chain locus upstream of an enhancer and constant (C) region comprising an endogenous IgH C 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 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 
 and 
 (iii) said enhancer and said constant region 
   wherein said human 3′JH is less than 2 kb upstream of said chimeric junction,   wherein DNA between said chimeric junction and said enhancer comprises mouse JC intronic DNA;   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 antibody specific for said antigen comprising a chimeric Ig heavy chain comprising a human IgH variable region and said C region; and
 wherein the germline 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 upstream of an IgH constant C region comprising an endogenously-located CH gene segment. 
   
     
     
         2 . The method of  claim 1 , said method further comprises the step of:
 recovering said antibody or antigen-binding fragment thereof, said cell expressing said antibody or antigen-binding fragment there, and/or recovering nucleic acid encoding said human VH region.   
     
     
         3 . The method of  claim 1 ,
 wherein said cell is a first cell, and wherein said nucleic acid encoding 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.   
     
     
         4 . The method of  claim 1 , wherein in said mouse germline said human 3′JH is less than 1 kb upstream of said chimeric junction. 
     
     
         5 . The method of  claim 1 , wherein in said mouse germline said unrearranged human heavy chain V region DNA comprises gene segments V6-1, V11-1-1, V1-2, V111-2-1, V1-3, V4-4 and V2-5 gene segments. 
     
     
         6 . The method of  claim 1 , wherein in said mouse germline said unrearranged human IgH VH gene segments comprise all of human V, D and J gene segments. 
     
     
         7 . The method of  claim 1 , wherein in said mouse germline said mouse JC intron comprises mouse 129 strain DNA. 
     
     
         8 . The method of  claim 2 , further comprising the step of: expressing the antibody from said nucleic acid, wherein said nucleic acid encodes an antibody comprising said human variable region. 
     
     
         9 . The method of  claim 3 , 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. 
     
     
         10 . The method of  claim 3 , wherein all or part of said endogenous mouse IgH locus is inactivate. 
     
     
         11 . The method of  claim 10 , wherein expression of IgH heavy chains comprising endogenous mouse variable and constant regions in said transgenic mouse is reduced or prevented. 
     
     
         12 . 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. 
     
     
         13 . The method of  claim 1 , wherein in said mouse germline said human DNA downstream of and naturally contiguous with said human 3′ JH gene segment comprises a truncated human JC intron and said mouse intronic DNA comprises a truncated mouse JC intron. 
     
     
         14 . The method of  claim 1 , further comprising providing a pharmaceutical composition comprising said antigen-specific antibody or anigen-binding fragment thereof and a pharmaceutically acceptable carrier or excipient. 
     
     
         15 . The method of  claim 1 , further comprising providing said antibody or antigen-binding fragment thereof to a human subject. 
     
     
         16 . The method of  claim 1 , said antigen-specific antibody or antigen binding fragment thereof is a monoclonal antibody, a domain antibody or a neutralizing antibody, or antigen binding fragment thereof. 
     
     
         17 . The method of  claim 1 , wherein in said mouse germline said enhancer comprises a Eμ enhancer. 
     
     
         18 . The method of  claim 17 , wherein in said mouse germline said mouse enhancer is a mouse Eμ enhancer. 
     
     
         19 . The method of  claim 1 , wherein said in said mouse germline said 3′ JH gene segment comprises a human JH6 gene segment. 
     
     
         20 . The method of  claim 18 , wherein said mouse Eμ enhancer comprises mouse 129 strain DNA. 
     
     
         21 . The method of  claim 1 , wherein in said mouse germline said C region comprises a plurality of CH gene segments endogenous to said transgenic mouse.