Animal Models and Therapeutic Molecules

A method of providing an immunized transgenic mouse expressing an antigen-specific polypeptide comprising a human IgH variable region (VH), antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide and/or a biological sample comprising one or more of said polypeptide, said cell and said nucleic acid, wherein said transgenic mouse is for the purposes of isolating said human VH, said polypeptide, said nucleic acid, said biological sample, the method comprising immunizing a naïve transgenic mouse with said antigen such that said mouse expresses said polypeptide, said antibody comprising said polypeptide, said cell producing said polypeptide, said nucleic acid encoding said polypeptide and/or said biological sample comprising said polypeptide, thereby providing said immunized transgenic mouse;

• wherein said transgenic mouse comprises a germline with a homozygous chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments positioned within the JC intron of the endogenous IgH locus, upstream of a 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;
• 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 one or more human IgH V gene segments, one or more human D gene segments and one or more human JH gene segments comprising a human JH6 gene segment,
• (ii) a chimeric JC intron comprising human DNA downstream of and naturally contiguous with a said human JH gene segment, mouse DNA and an enhancer, wherein said chimeric JC intron comprises mouse 129 strain DNA upstream of said enhancer, wherein the 3′ end of said one or more human JH gene segments is less than 2 kb upstream from said chimeric junction;
• (iii) said C region;
• wherein said homozygous chimeric IgH locus is functional to undergo human IgH gene segment rearrangement,
• wherein said transgenic mouse is functional to form rearranged human VH, D and JH gene segments and to express mRNA transcripts encoding chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse Cμ region, and wherein said immunized mouse forms rearranged human VH, D and JH gene segments, and expresses mRNA transcripts encoding said antigen-specific polypeptide,
• wherein prior to immunization said transgenic mouse comprises IgH mRNA transcripts comprising IgH-VDJCμ transcripts comprising rearranged human heavy chain V, D, and J gene segments and mouse Cμ and encoding chimeric IgH polypeptides, wherein each IgH-VDJCμ transcript encodes a human variable region comprising a CDR-H3, wherein said IgH-VDJCμ transcripts comprise transcripts encoding a human variable region comprising a CDR-H3 length of 17 amino acids and transcripts encoding human variable region comprising a CDR-H3 length of 18 amino acids, wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17 and 18 amino acids present in said IgH-VDJCμ transcripts of said mouse is between 5% and 10%.

A method of immunizing a mouse comprising

• (a) immunizing a naïve transgenic mouse with an antigen such that the immunized mouse produces antibody specific for said antigen, said antibody comprising an IgH polypeptide comprising a mouse constant region and a human variable region;
• wherein said transgenic mouse comprises a germline with a homozygous chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments positioned within the JC intron of the endogenous IgH locus, upstream of a 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;
• 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 one or more human IgH V gene segments, one or more human D gene segments and one or more human JH gene segments comprising a human JH6 gene segment,
• (ii) a chimeric JC intron comprising human DNA downstream of and naturally contiguous with a said human JH gene segment, mouse DNA and an enhancer, wherein said chimeric JC intron comprises mouse 129 strain DNA upstream of said enhancer, wherein the 3′ end of said one or more human JH gene segments is less than 2 kb upstream from said chimeric junction;
• (iii) said C region;
• wherein said homozygous chimeric IgH locus is functional to undergo human IgH gene segment rearrangement,
• wherein said naïve transgenic mouse is functional to form rearranged human VH, D and JH gene segments and to express mRNA transcripts encoding a chimaeric IgH chain comprising a human VH region and a mouse Cμ region, and wherein said immunized mouse comprises rearranged human VH, D and JH gene segments, and expresses mRNA transcripts encoding said IgH polypeptide,
• wherein said naive transgenic mouse comprises IgH mRNA transcripts comprising IgH-VDJCμ transcripts comprising rearranged human heavy chain V, D, and J gene segments and mouse Cμ and encoding a chimeric IgM chain,
• wherein each IgH-VDJCμ transcript encodes a human variable region comprising a CDR-H3,
• wherein said IgH-VDJCμ transcripts comprise transcripts encoding a human variable region comprising a CDR-H3 length of 17 amino acids and transcripts encoding human variable region comprising a CDR-H3 length of 18 amino acids,
• wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17 and 18 amino acids present in said IgH-VDJCμ transcripts of said mouse is between 5% and 10%.