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 non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric heavy chains, having a non-human mammal constant region and a human variable region,   wherein the insertion of the human DNA is made between the non-human mammal constant region and the last, 3′, non-human mammal J region, and   wherein the mammal comprises an insertion of the complete VJC human light chain region.   
     
     
         2 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   wherein the insertion of the human DNA is made between the non-human mammal constant region and the last, 3′, non-human mammal J region, and   wherein the mammal comprises an insertion of the complete VJC human light chain region.   
     
     
         3 . A non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region;   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human mammal constant region and a human variable region   wherein the inserted human IgH VDJ region comprises, in germline configuration, all of the V, D and J regions and intervening sequences from a human.   
     
     
         4 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region   wherein the inserted human IgH VDJ region comprises, in germline configuration, all of the V, D and J regions and intervening sequences from a human.   
     
     
         5 . A non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human mammal constant region and a human variable region;   
       or a non-human mammal whose genome comprises
 (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and 
 (b) optionally one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant; 
 wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human mammal constant region and a human variable region 
 wherein the non-human mammal genome into which DNA is inserted comprises endogenous V(D)J regions which have not been deleted 
 and wherein the insertion of the human heavy chain DNA is made between the non-human mammal constant region and the last, 3′, non-human mammal J region. 
 
     
     
         6 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region;   or a non-human mammal cell whose genome comprises   (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and   (b) optionally one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region   wherein the non-human mammal genome into which DNA is inserted comprises endogenous V(D)J regions which have not been deleted, and   wherein the insertion of the human heavy chain DNA is made between the non-human mammal constant region and the last, 3′, non-human mammal J region.   
     
     
         7 . A non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region;   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human mammal constant region and a human variable region or   
       a non-human mammal whose genome comprises
 (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and 
 (b) optionally one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant; 
 wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human mammal constant region and a human variable region 
 wherein the non-human mammal genome is modified to prevent expression of the native (fully host species specific) antibodies by inversion of all or part of the non-human mammal VDJ region, or VJ region. 
 
     
     
         8 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region;   
       or a non-human mammal cell whose genome comprises
 (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and 
 (b) optionally one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region 
 wherein the non-human mammal genome is modified to prevent expression of the native (fully host species specific) antibodies by inversion of all or part of the non-human mammal VDJ region, or VJ region. 
 
     
     
         9 . A non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric heavy chains, having a non-human mammal constant region and a human variable region,   wherein the mammal is a mouse and the insertion of the human heavy chain DNA is made in a mouse genome between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12 (co-ordinates refer to NCBIM37 for the mouse genome, relating to mouse strain C57BL/6J) or at equivalent position in another non-human mammal genome.   
     
     
         10 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   wherein the mammal is a mouse and the insertion of the human heavy chain DNA is made in a mouse genome between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12; (co-ordinates refer to NCBIM37 for the mouse genome, relating to mouse strain C57BL/6J) or at equivalent position in another non-human mammal genome.   
     
     
         11 . A non-human mammal whose genome comprises:
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies, or chimaeric heavy chains, having a non-human mammal constant region and a human variable region,   wherein the human IgH VDJ region comprises nucleotides 105,400,051 to 106,368,585 from human chromosome 14 (co-ordinates refer to NCBI36 for the human genome, ENSEMBL Release 54), or an equivalent human region from another human.   
     
     
         12 . A non-human mammal cell whose genome comprises
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region and   wherein the human IgH VDJ region comprises nucleotides 105,400,051 to 106,368,585 from human chromosome 14 (co-ordinates refer to NCBI36 for the human genome, ENSEMBL Release 54), or an equivalent human region from another human.   
     
     
         13 . The cell or mammal according to  claim 7 , wherein the insertion of the human DNA is made between the non-human mammal constant region and the last, 3′ non-human mammal J region. 
     
     
         14 . The cell or non-human mammal according to  claim 7 , wherein the mammal is a mouse or the cell is a mouse cell and wherein the insertion of the human heavy chain DNA is made in a mouse genome between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12, such as between co-ordinates 114,667,089 and 114,667,090 (co-ordinates refer to NCBI m37, for the mouse C57BL/6J strain), or at equivalent position in another non-human mammal genome. 
     
     
         15 . The cell or non-human mammal according to  claim 7 , wherein the insertion of the human heavy chain DNA is made between positions 114,667,090 and 114,667,091. 
     
     
         16 - 49 . (canceled) 
     
     
         50 . A method for producing a non-human mammal or cell, the method comprising inserting into a non-human mammal cell genome;
 (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and   (b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; respectively, the insertion being such that the non-human mammal is able to produce a repertoire of chimaeric antibodies or heavy or light antibody chains having a non-human mammal constant region and a human variable region, wherein steps (a) and (b) can be carried out in either order and each of steps (a) and (b) can be carried out in a stepwise manner or as a single step.   
     
     
         51 . A method for producing a non-human mammal or cell, the method comprising inserting into a non-human mammal cell genome;
 (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and   (b) optionally one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant;   
       the insertion being such that the non-human mammal is able to produce a repertoire of chimaeric antibodies or heavy or light antibody chains having a non-human mammal constant region and a human variable region, wherein steps (a) and (b) can be carried out in either order and each of steps (a) and (b) can be carried out in a stepwise manner or as a single step. 
     
     
         52 . The method according to  claim 50 , wherein the non-human mammal genome is then modified to prevent expression of the native (fully host species specific) antibodies in the mammal, optionally by inversion of all or part of the non-human mammal VDJ or VJ region, optionally by insertion of one or more site specific recombinase sites into the genome and then use of these sites in recombinase-mediated excision or inversion of all or a part of the non-human mammal Ig locus. 
     
     
         53 - 54 . (canceled) 
     
     
         55 . The method according to  claim 50 , wherein the insertion process commences at a site where an initiation cassette has been inserted into the genome of an ES cell providing a unique targeting region. 
     
     
         56 - 57 . (canceled) 
     
     
         58 . The method according to  claim 50 , wherein the method comprises the steps of
 (1) insertion of DNA forming an initiation cassette (also called a landing pad herein) into the genome of a cell;   (2) insertion of a first DNA fragment into the insertion site, the first DNA fragment comprising a first portion of a human DNA and a first vector portion containing a first selectable marker or generating a selectable marker upon insertion;   (3) optionally removal of part of the vector DNA;   (4) insertion of a second DNA fragment into the vector portion of the first DNA fragment, the second DNA fragment containing a second portion of human DNA and a second vector portion, the second vector portion containing a second selectable marker, or generating a second selectable marker upon insertion;   (5) removal of any vector DNA to allow the first and second human DNA fragments to form a contiguous sequence; and   (6) iteration of the steps of insertion of a part of the human V(D)J DNA and vector DNA removal, as necessary, to produce a cell with all or part of the human VDJ or VJ region sufficient to be capable of generating a chimaeric antibody in conjunction with a host constant region,   wherein the insertion of at least one DNA fragment uses site specific recombination.   
     
     
         59 - 69 . (canceled) 
     
     
         70 . A mouse whose genome comprises an insertion of human IgH VDJ DNA between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12, such as between co-ordinates 114,667,089 and 114667090, the insert comprising nucleotides 105,400,051 to 106,368,585 from human chromosome 14 (co-ordinates refer to NCBI36 for the human genome and NCBI m37, for the mouse C57BL/6J strain, or equivalent coordinates in another human chromosome 14 sequence or in another mouse genome respectively), the insertion being upstream of the host non-human mammal constant region such that the mouse is able to produce a repertoire of chimaeric heavy chains having a non-human mammal constant region and a human variable region, wherein the mammal also comprises an insertion of the complete VJC human light chain region such that a fully human lambda or kappa human antibody chain may be generated which is able to form an antibody with a chimaeric heavy chain. 
     
     
         71 . A mouse whose genome comprises an insertion of human IgH VDJ DNA between co-ordinates 114,667,090 and 114,667,091 of mouse chromosome 12, the insert comprising or consisting of nucleotides 105,400,051 to 106,368,585 from human chromosome 14 (co-ordinates refer to NCBI36 for the human genome and NCBI m37 for the mouse C57BL/6J strain, or equivalent coordinates in another human chromosome 14 sequence or in another mouse genome respectively), the insertion being upstream of the mouse constant region such that the mouse is able to produce a repertoire of chimaeric heavy chains having a mouse constant region and a human variable region, wherein the mouse also comprises an insertion of the complete VJC human light chain region such that a fully human lambda or kappa human antibody chain may be generated which is able to form an antibody with a chimaeric heavy chain. 
     
     
         72 . A mouse whose genome comprises an insertion of human IgH VDJ DNA between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12, where co-ordinates refer to NCBI m37, for the mouse C57BL/6J strain, or an insertion at an equivalent position in another mouse strain, the insert comprising or consisting of nucleotides 106,328,951-107,268,444 from human chromosome 14, where co-ordinates refer to the GRCH37/hg19 sequence database for humans, or the same nucleotides from an equivalent position in another human chromosome 14 sequence, the insertion being upstream of the host mouse constant region such that the mouse is able to produce a repertoire of chimaeric heavy chains having a mouse constant region and a human variable region, wherein the mouse also comprises an insertion of the complete VJC human light chain region which is functional to generate a fully human lambda or kappa human antibody chain which forms an antibody with a chimaeric heavy chain. 
     
     
         73 . (canceled) 
     
     
         74 . A non-human mammal or a cell whose genome comprises a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of a host non-human mammal light chain constant region, such that the cell or mammal is able to express a chimaeric antibody chain. 
     
     
         75 . A non-human mammal or a cell whose genome comprises a plurality of human Ig light chain V regions, and one or more human J regions upstream of a host non-human mammal heavy chain constant region, such that the cell or mammal is able to express a chimaeric antibody chain. 
     
     
         76 . (canceled) 
     
     
         77 . A transgene encoding a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of a host non-human mammal light chain constant region. 
     
     
         78 . A transgene encoding a plurality of human Ig light chain V regions, and one or more human light chain J regions upstream of a host non-human mammal heavy chain constant region. 
     
     
         79 . A polynucleotide landing pad sequence, the polynucleotide comprising nucleic acid regions homologous to regions of a target chromosome to allow for insertion by homologous recombination into the target chromosome, and comprising a nucleic acid site which permits recombinase-driven insertion of a nucleic acid into the landing pad, wherein the polynucleotide sequence comprises one or more of:
 (i) a non-human non-mouse sequence, such as a rat switch sequence, optionally the sequence of SEQ ID no 1;   (ii) in a 5′ to 3′ direction, a mouse Eμ sequence, a non-human, non-murine switch, and mouse Cμ;   (iii) a 3′ homology arm having the sequence of SEQ ID no 6.   
     
     
         80 - 83 . (canceled) 
     
     
         84 . A non-human cell or non-human mammal comprising:
 a fully human lambda locus comprising substantially all the lambda VJC regions from a human,   a chimaeric kappa locus comprising substantially all human kappa VJ regions operatively linked to a non-human host kappa constant region and   a chimaeric heavy chain locus, having a human VDJ region operatively linked to a non-human host heavy chain constant region.   
     
     
         85 . A chimaeric antibody comprising a human variable region and a non-human vertebrate or mammal (optionally a rat or mouse) constant region (optionally a C gamma or C mu),
 wherein the antibody is encoded by a nucleotide sequence corresponding to the nucleotide sequence of a chimaeric heavy chain locus of a cell (optionally a B-cell, ES cell or hybridoma), the locus comprising a non-human vertebrate or mammal constant region nucleotide sequence and a rearranged VDJ nucleotide sequence produced by the in-vivo rearrangement of a human V segment, a human D segment and a human J segment, the V region being selected from one of a V1-3 region, V2-5 region, V4-4 region, V1-2 region or V6-1 region from a human, optionally a V1-3 or V6-1 segment.   
     
     
         86 - 94 . (canceled) 
     
     
         95 . A chimaeric antibody comprising a human variable region and a non-human vertebrate or mammal (optionally a rat or mouse) constant region (optionally a light chain constant region), wherein the antibody is obtainable from a mammal (optionally a rat or mouse) whose genome comprises an antibody chain locus comprising a germline human kappa V1-8 and germline human kappa J1 sequence, and wherein the antibody is obtainable by in vivo recombination in said mammal of the V1-8 and J1 sequences and wherein the antibody has a variable region sequence which is different from that which is encoded by germline human kappa V1-8 and germline human kappa J1 sequences 
     
     
         96 . (canceled) 
     
     
         97 . A chimaeric antibody comprising a human variable region and a non-human vertebrate or mammal (optionally a rat or mouse) constant region (optionally a light chain constant region), wherein the antibody is obtainable from a mammal (optionally a rat or mouse) whose genome comprises an antibody chain locus comprising a germline human kappa V1-6 and germline human kappa J1 sequence, and wherein the antibody is obtainable by in vivo recombination in said mammal of the V1-6 and J1 sequences and wherein the antibody has a variable region sequence which is different from that which is encoded by germline human kappa V1-6 and germline human kappa J1 sequences. 
     
     
         98 . (canceled) 
     
     
         99 . A chimaeric antibody comprising a human variable region and a non-human (optionally a rat or mouse) constant region (optionally a C gamma or C mu or a C kappa), wherein the antibody is obtainable from a mammal (optionally a rat or mouse) whose genome comprises an antibody chain locus comprising a germline human kappa V1-5 and germline human kappa J1 sequence, and wherein the antibody is obtainable by in vivo recombination in said mammal of the V1-5 and J1 sequences. 
     
     
         100 . A chimaeric antibody comprising a human variable region and a non-human (optionally a rat or mouse) constant region (optionally a C gamma or C mu or a C kappa), wherein the antibody is obtainable from a mammal (optionally a rat or mouse) whose genome comprises an antibody chain locus comprising a germline human kappa V1-5 and germline human kappa J4 sequence, and wherein the antibody is obtainable by in vivo recombination in said mammal of the V1-5 and J4 sequences. 
     
     
         101 - 106 . (canceled) 
     
     
         107 . The method according to  claim 58 , wherein the initiation cassette is inserted between the mouse J4 and C alpha exons. 
     
     
         108 . (canceled) 
     
     
         109 . A non-human vertebrate (optionally a mouse or rat) or non-human vertebrate cell (optionally a mouse or rat cell) having a genome that
 (i) comprises a transgenic antibody chain locus capable of expressing an antibody chain comprising a human variable region (optionally following antibody gene rearrangement); and   (ii) is inactivated for endogenous non-human vertebrate antibody chain expression; wherein the transgenic locus comprises   (iii) a DNA sequence comprising a plurality of human antibody variable region gene segments inserted between endogenous antibody variable region gene segments and an endogenous antibody constant region, whereby endogenous antibody chain expression is inactivated.   
     
     
         110 - 115 . (canceled) 
     
     
         116 . A method of making a non-human vertebrate cell (optionally a mouse or rat cell), the method comprising
 (a) providing a non-human ES cell whose genome comprises an endogenous antibody chain locus comprising endogenous antibody variable region gene segments and an endogenous antibody constant region; and   (b) making a transgenic antibody chain locus by inserting into said endogenous locus a DNA sequence comprising a plurality of human antibody variable region gene segments between said endogenous antibody variable region gene segments and said endogenous constant region, so that the human antibody variable region gene segments are operably connected upstream of the endogenous constant region,   whereby a non-human vertebrate ES cell is produced that is capable of giving rise to a progeny cell in which endogenous antibody expression is inactivated and wherein the progeny is capable of expressing antibodies comprising human variable regions; and   (c) optionally differentiating said ES cell into said progeny cell or a non-human vertebrate (eg, mouse or rat) comprising said progeny cell.   
     
     
         117 - 133 . (canceled) 
     
     
         134 . A mouse or mouse cell comprising inverted endogenous heavy chain gene segments (eg, VH, D and JH, such as the entire endogenous heavy chain VDJ region) that are immediately 3′ of position 119753123, 119659458 or 120918606 on an endogenous mouse chromosome 12, wherein the mouse comprises a transgenic heavy chain locus comprising a plurality of human VH gene segments, a plurality of human D segments and a plurality of human JH segments operably connected upstream of an endogenous constant region (eg, C mu) so that the mouse or cell (optionally following differentiation into a B-cell) is capable of expressing an antibody comprising a variable region comprising sequences derived from the human gene segments. 
     
     
         135 . (canceled) 
     
     
         136 . A cassette for inversion and inactivation of endogenous non-human vertebrate (eg, mouse or rat) antibody chain gene segments, the segments being part of an antibody chain locus sequence on a chromosome of a non-human vertebrate (eg, mouse or rat) cell (eg, ES cell) wherein the sequence is flanked at its 3′ end by a site-specific recombination site (eg, lox, rox or frt),
 the cassette comprising a nucleotide sequence encoding an expressible label or selectable marker and a compatible site-specific recombination site (eg, lox, rox or frt) flanked by a 5′ and a 3′ homology arm, wherein the homology arms correspond to or are homologous to adjacent stretches of sequence in the cell genome on a different chromosome or on said chromosome at least 10 mb away from the endogenous gene segments. 
 
     
     
         137 . A cassette for inversion and inactivation of endogenous mouse antibody heavy chain gene segments, the segments being part of a heavy chain locus sequence on chromosome 12 of a mouse cell (eg, ES cell) wherein the sequence is flanked at its 3′ end by a site-specific recombination site (eg, lox, rox or frt),
 the cassette comprising a nucleotide sequence encoding an expressible label or selectable marker and a compatible site-specific recombination site (eg, lox, rox or frt) flanked by a 5′ and a 3′ homology arm, wherein (i) the 5′ homology arm is mouse chromosome 12 DNA from coordinate 119753124 to coordinate 119757104 and the 3′ homology arm is mouse chromosome 12 DNA from coordinate 119749288 to 119753123; or (ii) the 5′ homology arm is mouse chromosome 12 DNA from coordinate 119659459 to coordinate 119663126 and the 3′ homology arm is mouse chromosome 12 DNA from coordinate 119656536 to 119659458. 
 
     
     
         138 . A method of inactivating gene segments of an endogenous antibody locus, the method comprising
 (i) Providing a non-human vertebrate cell (eg, an ES cell, eg, a mouse ES cell) whose genome comprises an antibody chain locus comprising endogenous variable region gene segments;   (ii) Targeting a site-specific recombination site to flank the 3′ of the 3′-most of said endogenous gene segments;   (iii) Targeting a second site-specific recombination site at least 10 mb away from said endogenous gene segments, the second site being compatible with the first site inverted with respect to the first site;   (iv) Expressing a recombinase compatible with said sites to effect site-specific recombination between said sites, thereby inverting and moving said gene segments away from said locus, wherein the endogenous gene segments are inactivated; and   (v) Optionally developing the cell into a progeny cell or vertebrate (eg, mouse or rat) whose genome is homozygous for the inversion.   
     
     
         139 . A mouse or mouse cell whose genome comprises an inversion of a chromosome 12, wherein the inversion comprises inverted endogenous heavy chain gene segments (eg, VH, D and JH, such as the entire endogenous heavy chain VDJ region); wherein the mouse comprises a transgenic heavy chain locus comprising a plurality of human VH gene segments, a plurality of human D segments and a plurality of human JH segments operably connected upstream of an endogenous constant region (eg, C mu) so that the mouse or cell (optionally following differentiation into a B-cell) is capable of expressing an antibody comprising a variable region comprising sequences derived from the human gene segments; and wherein the inversion is (i) an inversion of mouse chromosome 12 from coordinate 119753123 to coordinate 114666436; (ii) an inversion of mouse chromosome 12 from coordinate 119659458 to coordinate 114666436; or (iii) an inversion of mouse chromosome 12 from coordinate 12091806 to coordinate 114666436. 
     
     
         140 . The cell or mammal according to  claim 8 , wherein the insertion of the human DNA is made between the non-human mammal constant region and the last, 3′ non-human mammal J region. 
     
     
         141 . The cell or non-human mammal according to  claim 8 , wherein the mammal is a mouse or the cell is a mouse cell and wherein the insertion of the human heavy chain DNA is made in a mouse genome between co-ordinates 114,667,090 and 114,665,190 of mouse chromosome 12, such as between co-ordinates 114,667,089 and 114,667,090 (co-ordinates refer to NCBI m37, for the mouse C57BL/6J strain), or at equivalent position in another non-human mammal genome. 
     
     
         142 . The cell or non-human mammal according to  claim 8 , wherein the insertion of the human heavy chain DNA is made between positions 114,667,090 and 114,667,091. 
     
     
         143 . The method according to  claim 51 , wherein the non-human mammal genome is then modified to prevent expression of the native (fully host species specific) antibodies in the mammal, optionally by inversion of all or part of the non-human mammal VDJ or VJ region, optionally by insertion of one or more site specific recombinase sites into the genome and then use of these sites in recombinase-mediated excision or inversion of all or a part of the non-human mammal Ig locus. 
     
     
         144 . The method according to  claim 51 , wherein the insertion process commences at a site where an initiation cassette has been inserted into the genome of an ES cell providing a unique targeting region. 
     
     
         145 . The method according to  claim 51 , wherein the method comprises the steps of
 (1) insertion of DNA forming an initiation cassette (also called a landing pad herein) into the genome of a cell;   (2) insertion of a first DNA fragment into the insertion site, the first DNA fragment comprising a first portion of a human DNA and a first vector portion containing a first selectable marker or generating a selectable marker upon insertion;   (3) optionally removal of part of the vector DNA;   (4) insertion of a second DNA fragment into the vector portion of the first DNA fragment, the second DNA fragment containing a second portion of human DNA and a second vector portion, the second vector portion containing a second selectable marker, or generating a second selectable marker upon insertion;   (5) removal of any vector DNA to allow the first and second human DNA fragments to form a contiguous sequence; and   (6) iteration of the steps of insertion of a part of the human V(D)J DNA and vector DNA removal, as necessary, to produce a cell with all or part of the human VDJ or VJ region sufficient to be capable of generating a chimaeric antibody in conjunction with a host constant region,   wherein the insertion of at least one DNA fragment uses site specific recombination.   
     
     
         146 . The method according to claim  155 , wherein the initiation cassette is inserted between the mouse J4 and C alpha exons.