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 providing an antigen-specific polypeptide comprising a human IgH variable region, antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide and/or a biological sample comprising said polypeptide, the method comprising providing a transgenic mouse immunized with an antigen and expressing said polypeptide,
 isolating from said transgenic immunized mouse one or more of 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,   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%.   
     
     
         2 . 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%. 
 
     
     
         3 . 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%.   
     
     
         4 . The method according to  claim 3 , further comprising one or more steps of the group consisting of:
 (a) isolating nucleic acid encoding a said antibody, said IgH polypeptide, and/or a human VH region thereof,   (b) isolating said antibody, said IgH polypeptide, and/or a human VH region thereof,   (c) isolating a cell comprising said nucleic acid of step (a)   (d) isolating a cell comprising said antibody, said IgH polypeptide, and/or a human VH region thereof, and   (e) isolating a biological sample comprising nucleic acid encoding and/or a cell comprising said human VH region.   
     
     
         5 . The method of  claim 1 , wherein said antigen comprises a vaccine. 
     
     
         6 . The method of  claim 2 , wherein said antigen comprises a vaccine. 
     
     
         7 . The method of  claim 1 , wherein said transgenic mouse germline comprises a light chain locus comprising human immunoglobulin light chain V gene segments capable of expressing immunoglobulin comprising a human light chain variable region. 
     
     
         8 . The method of  claim 2 , wherein said transgenic mouse germline comprises a light chain locus comprising human immunoglobulin light chain V gene segments capable of expressing immunoglobulin comprising a human light chain variable region. 
     
     
         9 . The method of  claim 1 , wherein said endogenous Ig C segment is selected from the group consisting of mouse Cμ, mouse Cγ, mouse Cδ, and mouse Cα. 
     
     
         10 . The method of  claim 1 , wherein said human DNA which is downstream and naturally contiguous with said human JH comprises a 400 bp DNA segment of human DNA naturally-associated and contiguous with and located immediately 3′ to a human JH6 segment. 
     
     
         11 . The method of  claim 1 , said transgenic mouse being produced by breeding together parent mice, each of whose germline comprises a homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned at an endogenous IgH locus upstream of an enhancer and a mouse constant region C gene segment, said human IgH gene segments comprising unrearranged human IgH variable (VH) gene segments, unrearranged human D (D) gene segments and unrearranged human IgH (JH) gene segments. 
     
     
         12 . The method of  claim 1 , wherein said transgenic mouse is male and capable of producing a subsequent generation mouse, said subsequent generation mouse comprising in its germline a homozygous IgH locus comprising unrearranged human IgH gene segments positioned at an endogenous IgH locus upstream of an enhancer and a mouse Ig constant region (C) gene segment, wherein the germline of said subsequent generation mouse comprises all or part of a mouse IgH variable region. 
     
     
         13 . The method of  claim 10 , wherein said transgenic mouse is male and homozygous for a said Ig heavy chain (IgH) locus and wherein the germline of said transgenic mouse comprises all or part of a mouse IgH variable region. 
     
     
         14 . The method of  claim 11 , wherein said parent mouse is male and homozygous for said transgenic IgH locus and wherein the germline of said parent mouse comprises all or part of a mouse IgH variable region. 
     
     
         15 . The method of  claim 1 , wherein said mouse 129 strain is 129Sv. 
     
     
         16 . The method of  claim 1 , wherein said transgenic mouse is male. 
     
     
         18 . The method of  claim 1 , wherein all or part of the endogenous IgH variable region of said transgenic mouse is inverted. 
     
     
         19 . The method of  claim 18  wherein said all or part of the endogenous IgH variable region of said transgenic mouse is inverted and is positioned at a telomeric locus. 
     
     
         20 . The method of  claim 1 , wherein the 3′ end of said one or more human JH gene segments is less than 1 kb from said chimeric junction. 
     
     
         21 . The method of  claim 1 , wherein said mouse comprises IgH-VDJCμ transcripts encoding chimeric 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%. 
     
     
         22 . The method of  claim 1 , wherein said mouse further comprises IgH-VDJCμ transcripts encoding chimeric IgH polypeptide comprising CDR-H3 lengths selected from the group consisting of: 20, 21, and 22 amino acids. 
     
     
         23 . The method of  claim 9 , wherein said endogenous Ig C segment is mouse Cμ, and said enhancer is mouse μ enhancer. 
     
     
         24 . The method of  claim 5 , wherein said antibody comprises said human VH region and said human VL region. 
     
     
         25 . The method of  claim 6 , wherein said antibody comprises said human VH and said human VL region. 
     
     
         26 . The method of  claim 2 , wherein said antibody, said antibody chain or said VH region has a HCDR3 having a length of 17-22 amino acids. 
     
     
         27 . The method of  claim 1 , wherein said antibody, said antibody chain or said VH region has a HCDR3 having a length of 17-22 amino acids. 
     
     
         28 . The method of  claim 3 , wherein said antibody has a HCDR3 having a length of 17-22 amino acids. 
     
     
         29 . The method of  claim 1 , further comprising humanizing the C region of the antibody or IgH chain. 
     
     
         30 . The method of  claim 2 , further comprising humanizing the C region of the antibody or IgH chain.