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 heavy (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 comprises a homozygous immunoglobulin heavy chain (IgH) locus,   wherein said homozygous IgH locus comprises unrearranged human IgH variable region gene segments comprising Vs, Ds and Js at an endogenous locus operatively linked to an IgH constant (C) region comprising an endogenous C segment at an IgH locus;   wherein said homozygous IgH locus comprises in 5′ to 3′ transcriptional orientation said unrearranged human Vs, Ds and Js comprising a 3′ JH gene segment, a human/mouse chimeric DNA junction, an enhancer, and said operatively linked C region;   wherein said homozygous chimeric IgH locus comprises a chimeric J/C intron comprising human DNA downstream of and naturally contiguous with said 3′JH gene segment, said human DNA being contiguous with mouse J/C intronic DNA upstream of said enhancer, and wherein said human DNA joins said mouse J/C intronic DNA at said human/mouse chimeric junction within said J/C intron,   wherein DNA between said 3′ human JH segment and said human/mouse chimeric DNA junction is less than 2 kb,   said germline comprising all or part of mouse IgH variable region DNA; wherein said homozygous IgH locus of said mouse is capable of undergoing V, D, J joining; wherein said transgenic mouse is capable, upon stimulation with antigen, of producing antibody comprising a chimeric Ig heavy chain comprising a human IgH variable region; and wherein said transgenic mouse is capable of breeding with a second transgenic mouse, said second transgenic mouse having a germline with a homozygous IgH locus comprising unrearranged human IgH variable region gene segments operatively linked to an IgH constant (C) region comprising an endogenous C segment of an IgH locus to provide subsequent generation mice, wherein a said subsequent generation mouse comprises:
 (i) in its germline an homozygous IgH locus comprising unrearranged human IgH variable region gene segments operatively linked to an IgH constant (C) region comprising an endogenous C segment of an IgH locus, and 
 (ii) in its germline all or part of mouse IgH variable region DNA; and 
   wherein said IgH locus of said subsequent generation mouse is capable of undergoing V, D, J joining;   wherein said subsequent generation mouse is capable, upon stimulation with antigen, of producing antibody comprising a chimeric Ig heavy chain comprising a human IgH variable region; and   wherein said subsequent generation mouse is capable of breeding with a mouse having a germline with a homozygous IgH locus comprising unrearranged human IgH variable region gene segments operatively linked to an IgH constant (C) region comprising an endogenous C segment of an IgH locus to provide further subsequent generation mice, and   (B) expressing said antibody from said cell comprising a nucleic acid encoding said human IgL chain variable region and said human IgL chain constant region of said antibody, or expressing said antigen-binding fragment thereof from said cell comprising nucleic acid encoding said human IgL chain variable region of said antigen-binding fragment.   
     
     
         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 DNA between said 3′ human JH segment and said human/mouse chimeric DNA junction is less than 1 kb. 
     
     
         5 . The method of  claim 1 , wherein in said mouse germline said unrearranged human VH gene segments, unrearranged human DH gene segment(s), and unrearranged human JH gene segments are in the place of endogenous mouse Ig VH region segment(s) upstream of said enhancer. 
     
     
         6 . The method of  claim 1 , wherein in said mouse germline DNA said human intronic DNA comprises a truncated human JC intron and said mouse intronic DNA comprises a truncated mouse JC intron. 
     
     
         7 . The method of  claim 1 , wherein in said mouse germline said mouse J/C intronic DNA at said human/mouse chimeric junction and upstream of said enhancer comprises 129 mouse strain DNA and said enhancer comprises 129 strain Eμ. 
     
     
         8 . The method of  claim 1 , wherein in said mouse germline said constant region comprises mouse Cμ. 
     
     
         9 . The method of  claim 1 , wherein in said mouse germline said all or part of mouse heavy chain variable region DNA is away from its native position in an IgH locus and expression of Ig heavy chains comprising a mouse variable region is reduced or prevented. 
     
     
         10 . The method of  claim 10 , wherein in said mouse germline said all or part of mouse heavy chain variable region DNA 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 , 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. 
     
     
         13 . The method of  claim 1 , further comprising the step of providing said antibody or antigen-binding fragment thereof to a human subject. 
     
     
         14 . 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.   
     
     
         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, VII-1-1, V1-2, VIII-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.