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:
 expressing the antibody or antigen binding fragment thereof from a host cell comprising nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or comprising nucleic acid encoding said human IgH chain variable region of said fragment,   wherein said human IgH chain variable region of said antigen-specific antibody or antigen binding fragment is of a B cell, or a hybridoma thereof, of a transgenic mouse contacted with said antigen;   wherein said transgenic mouse comprises a germline 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 method of  claim 1 , wherein said human 3′ JH is less than 2 kb upstream of said human/mouse chimeric DNA junction in said mouse germline. 
     
     
         3 . The method 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 method 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 method of  claim 3 , 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 method of  claim 1 , further comprising obtaining B cells expressing an antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide comprising a human heavy chain variable (VH) region and a mouse constant (C) region, comprising isolating B cells expressing said antigen-specific antibody from said mouse contacted with antigen. 
     
     
         7 . The method of  claim 6 , further comprising the step of culturing said B cells, and isolating antigen-specific antibody from said cultured B cells. 
     
     
         8 . The method of  claim 6 , further comprising the step of isolating from said B cells nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide. 
     
     
         9 . The method of  claim 8 , further comprising the step of replacing in said nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide, the portion encoding said mouse C region with nucleic acid encoding a human C region, thereby forming a nucleic acid encoding said human VH region and said human C region. 
     
     
         10 . The method of  claim 6 , further comprising isolating an antibody that binds said antigen, comprising
 (i) immortalizing said isolated B cells expressing said antigen-specific antibody or progeny thereof, optionally producing hybridomas therefrom; and   (ii) isolating an antibody expressed by the immortalized B cells of (i).   
     
     
         11 . The method of  claim 1 , wherein said mouse is naive. 
     
     
         12 . The method 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. 
     
     
         13 . The method of  claim 12 , wherein said plurality of unrearranged human VH gene segments comprises all functional human VH gene segments. 
     
     
         14 . The method according to  claim 1 , 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.   
     
     
         15 . The method of  claim 1 , wherein said antigen comprises a vaccine. 
     
     
         16 . A method of providing an antigen-specific polypeptide comprising a human IgH variable region, an antigen-specific antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide or said variable region and/or a biological sample comprising said polypeptide, the method comprising:
 isolating from a transgenic mouse one or more of said polypeptide, said antigen-specific antibody comprising said polypeptide, said cell producing said polypeptide, said nucleic acid encoding said polypeptide or said variable region and/or said biological sample comprising said polypeptide,   wherein prior to said isolating said transgenic mouse has been contacted with an antigen under conditions sufficient to generate antibody specific to said antigen,   wherein prior to said contacting with said antigen said transgenic mouse has a germline 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.   
     
     
         17 . The method of  claim 16 , wherein said human 3′ JH is less than 2 kb upstream of said human/mouse chimeric DNA junction. 
     
     
         18 . The method of  claim 16 , 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%. 
     
     
         19 . The method of  claim 18 , 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. 
     
     
         20 . The method of  claim 16 , 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. 
     
     
         21 . The method of  claim 16 , further comprising obtaining B cells expressing an antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide comprising a human heavy chain variable (VH) region and a mouse constant (C) region, comprising contacting the mouse of  claim 1  with an antigen, and isolating B cells expressing said antigen-specific antibody from said mouse contacted with antigen. 
     
     
         22 . The method of  claim 21 , further comprising the step of culturing said B cells, and isolating antigen-specific antibody from said cultured B cells. 
     
     
         23 . The method of  claim 21 , further comprising the step of isolating from said B cells nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide. 
     
     
         24 . The method of  claim 23 , further comprising the step of replacing in said nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide, the portion encoding said mouse C region with nucleic acid encoding a human C region, thereby forming a nucleic acid encoding said human VH region and said human C region. 
     
     
         25 . The method of  claim 21 , further comprising isolating an antibody that binds said antigen, comprising
 (i) immortalizing said isolated B cells expressing said antigen-specific antibody or progeny thereof, optionally producing hybridomas therefrom; and   (ii) isolating an antibody expressed by the immortalized B cells of (i).   
     
     
         26 . The method of  claim 16 , wherein said mouse is naïve. 
     
     
         27 . The method of  claim 16 , 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. 
     
     
         28 . The method of  claim 27 , wherein said plurality of unrearranged human VH gene segments comprises all human VH gene segments. 
     
     
         29 . The method according to  claim 16 , 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.   
     
     
         30 . The method of  claim 16 , wherein said antigen comprises a vaccine.