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 transgenic mouse having a genome comprising a homozygous immunoglobulin heavy chain (IgH) locus comprising a plurality of unrearranged human IgH V gene segments, D gene segments and JH gene segments at an endogenous IgH locus, said human gene segments join mouse DNA at a human/mouse chimeric DNA junction within the JC intron of said IgH locus downstream of said human JH gene segments and upstream of an enhancer and a constant (C) region comprising an endogenous IgH C gene segment;
 wherein said homozygous 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 JC intronic DNA downstream of and naturally contiguous with said human 3′ JH gene segment, which is contiguous with mouse JC intronic DNA, wherein said mouse JC intronic DNA comprises DNA of an IgH locus which, in its native position in a non-transgenic mouse, is naturally contiguous with and upstream of a mouse Cμ enhancer, and 
 (iii) said enhancer and said C region, 
   wherein said enhancer of (iii) is a mouse μ enhancer;   wherein said plurality of unrearranged human V gene segments, D gene segments and J gene segments in said IgH locus are operatively linked to said C region such that said IgH locus of said mouse is capable of undergoing V, D, J joining of said unrearranged human IgH variable region gene segments,   such that said transgenic mouse is capable, upon stimulation with an antigen, of producing a plurality of antibodies specific for said antigen comprising a chimeric Ig heavy chain comprising a human VH region and a mouse C region;   wherein expression of mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region is inactive in said mouse.   
     
     
         2 . The mouse of  claim 1 , wherein said human 3′ JH is less than 2 kb upstream of said human/mouse chimeric DNA junction. 
     
     
         3 . The mouse of  claim 1 , wherein said mouse JC intronic DNA comprises DNA of an IgH locus of a 129 strain mouse which, in its native position in a non-transgenic mouse, is naturally contiguous with and upstream of a mouse Cμ enhancer. 
     
     
         4 . The mouse of  claim 1 , wherein said mouse comprises IgH-VDJCμ transcripts encoding IgH polypeptides comprising CDR-H3 lengths of 17, 18 and 19 amino acids, wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17, 18 and 19 amino acids is between 5% and 10%. 
     
     
         5 . The mouse of  claim 4 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths selected from the group consisting of: 20, 21, and 22 amino acids. 
     
     
         6 . The mouse of  claim 1 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 19 and 20 amino acids. 
     
     
         7 . The mouse of  claim 1 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 20 and 21 amino acids. 
     
     
         8 . The mouse of  claim 1  wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 21 and 22 amino acids. 
     
     
         9 . The mouse of  claim 4 , wherein each said IgH mRNA transcript comprises a transcript region complementary to DNA comprising a rearranged human JH segment, and wherein transcripts comprise regions complementary to rearranged human JH4 and rearranged human JH6 gene segments. 
     
     
         10 . The mouse of  claim 1 , wherein said mouse is naïve. 
     
     
         11 . The mouse of  claim 1 , wherein said plurality of unrearranged human D gene segments and said plurality of unrearranged human JH gene segments comprises all functional human D (D) gene segments and all functional human heavy chain J (JH) gene segments. 
     
     
         12 . The mouse of  claim 11 , wherein said plurality of unrearranged human VH gene segments comprises all functional human VH gene segments. 
     
     
         13 . A transgenic mouse having a genome comprising a homozygous immunoglobulin heavy chain (IgH) locus comprising a plurality of unrearranged human IgH V gene segments, D gene segments and JH gene segments at an endogenous IgH locus, said human gene segments join mouse DNA at a human/mouse chimeric DNA junction downstream of said human JH gene segments and upstream of a mouse Cμ enhancer and a constant (C) region comprising an endogenous IgH C gene segment;
 wherein said homozygous 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, and 
 (ii) said mouse Cμ enhancer and said C region, 
 
 wherein said plurality of unrearranged human V gene segments, D gene segments and J gene segments in said IgH locus are operatively linked to said C region such that said IgH locus of said mouse is capable of undergoing V, D, J joining of said unrearranged human IgH variable region gene segments, 
 such that said transgenic mouse is capable, upon stimulation with an antigen, of producing a plurality of antibodies specific for said antigen comprising a chimeric Ig heavy chain comprising a human VH region and a mouse C region; 
 wherein said genome comprises all or part of the mouse IgH variable region DNA in a position that is upstream of its native chromosomal position, wherein expression of mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region is inactive in said mouse, 
 and wherein said transgenic mouse is capable of breeding with a second transgenic mouse, said second transgenic mouse having a genome comprising a homozygous IgH locus comprising unrearranged human IgH variable region gene segments at an endogenous IgH locus upstream of and operably linked to a constant (C) region comprising an endogenous C segment of an IgH locus, to provide subsequent generation mice comprising in their genome a homozygous IgH locus comprising unrearranged human IgH variable region gene segments at an endogenous IgH locus upstream of a constant (C) region comprising an endogenous C segment of an IgH locus and operably linked to the C region. 
 
     
     
         14 . The mouse of  claim 13 , wherein said human 3′ JH is less than 2 kb upstream of said human/mouse chimeric DNA junction. 
     
     
         15 . The mouse of  claim 13 , wherein said mouse comprises IgH-VDJCμ transcripts encoding IgH polypeptides comprising CDR-H3 lengths of 17, 18 and 19 amino acids, wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17, 18 and 19 amino acids is between 5% and 10%. 
     
     
         16 . The mouse of  claim 15 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths selected from the group consisting of: 20, 21 and 22 amino acids. 
     
     
         17 . The mouse of  claim 13 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 19 and 20 amino acids. 
     
     
         18 . The mouse of  claim 13 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 20 and 21 amino acids. 
     
     
         19 . The mouse of  claim 13 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 21 and 22 amino acids. 
     
     
         20 . The mouse of  claim 15 , wherein each said IgH mRNA transcript comprises a transcript region complementary to DNA comprising a rearranged human JH segment, and wherein transcripts comprise regions complementary to rearranged human JH4 and rearranged human JH6 gene segments. 
     
     
         21 . The mouse of  claim 13 , wherein said mouse is naïve. 
     
     
         22 . The mouse of  claim 13 , wherein said plurality of unrearranged human D gene segments and said plurality of unrearranged human JH gene segments comprises all human D (D) gene segments and all human heavy chain J (JH) gene segments. 
     
     
         23 . The mouse of  claim 22 , wherein said plurality of unrearranged human VH gene segments comprises all human VH gene segments.