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 the antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in the methods.

We claim: 
     
         1 . A method of obtaining one or more of:
 i) a nucleic acid encoding an antigen-specific antibody comprising a heavy chain comprising a human IgH variable region and a human heavy chain constant region,   ii) a cell comprising said nucleic acid,   iii) a cell expressing said antibody,   iv) said antigen-specific antibody and   v) a biological sample comprising said i) nucleic acid, said ii) cell, said iii) cell, and/or said iv) antibody,   A) providing a nucleic acid encoding said human IgH variable region, wherein said human IgH variable region is of a transgenic mouse contacted with said antigen,
 wherein said transgenic mouse comprises in its germline a homozygous chimeric immunoglobulin heavy chain (IgH) locus, 
 wherein said chimeric IgH locus comprises unrearranged human IgH V gene segments, D gene segments and J gene segments at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous CH gene segment, 
 wherein said human IgH V gene segments, D gene segments and J gene segments are operably linked to said C region 
 wherein said unrearranged human IgH V gene segments, D gene segments and J gene segments comprise all the functional human D and JH gene segments and human V gene segments Vh2-5, Vh7-4-1, Vh4-4, Vh1-3, Vhf-2 and Vh6-1, and are operably joined to said C region via a chimeric JC intron comprising human JC intronic DNA and mouse JC intronic DNA, 
 wherein said human JH gene segments comprise a human 3′ JH gene segment, wherein said 3′ JH gene segment is less than 2 kb upstream of the mouse JC intronic DNA of said chimeric JC intron, 
 wherein bone marrow B cells of said mouse comprise more pro-B cells than pre-B cells, wherein said pro B cells express CD43 Med  B220 Med  and said pre-B cells express CD43 low  B220 high , 
 wherein said transgenic mouse further comprises rearranged human VH, D and JH gene segments, and expresses chimeric Ig heavy chain polypeptide encoded by a rearranged VHDJH gene; 
   B) performing one or more of steps a)-e):   a. operatively linking nucleic acid encoding said human IgH variable region with nucleic acid encoding said human heavy chain constant region, thereby obtaining nucleic acid encoding said antibody;   b. transferring said nucleic acid of step a) to a cell, thereby obtaining a cell comprising nucleic acid encoding said antigen-specific antibody;   c. expressing antibody comprising a heavy chain comprising said human IgH variable region and said human heavy chain constant region from said cell of step b), thereby obtaining a cell expressing said antibody and thereby obtaining a biological sample comprising said antibody;   d. isolating said antibody from said cell of step c), thereby obtaining antigen-specific antibody and thereby obtaining a biological sample comprising said antibody; and/or   e. isolating nucleic acid encoding said antibody from said cell of step b) or said cell of step c), thereby obtaining nucleic acid encoding said antibody.   
     
     
         2 . The method of  claim 1 , wherein the mouse JC intronic DNA of said chimeric JC intron upstream of said enhancer of said chimeric IgH locus of said transgenic mouse comprises mouse 129 strain or C57BL/6 strain DNA. 
     
     
         3 . The method of  claim 1 , wherein the transgenic mouse expresses a normal relative proportion of serum IgG1, IgG2a, IgG2b and IgM antibodies. 
     
     
         4 . The method of  claim 1 , wherein the transgenic mouse expresses (i) serum IgG1 at a concentration of about 25-350 μg/ml; (ii) serum IgG2a at a concentration of about 0-200 μg/ml; (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and (iv) serum IgM at a concentration of about 50-300 μg/ml; or (i) serum IgG1 at a concentration of about 10-600 μg/ml; (ii) serum IgG2a at a concentration of about 0-500 μg/ml; (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and (iv) serum IgM at a concentration of about 50-700 μg/ml; as determined by Ig capture on a plate followed by incubation with anti-mouse isotype-specific labeled antibodies and quantification of Ig using the label. 
     
     
         5 . The method of  claim 1 , wherein the transgenic mouse produces a normal proportion or percentage of mature splenic B-cells and/or a normal proportion or percentage of bone marrow B-cell progenitor cells. 
     
     
         6 . The method of  claim 1 , wherein said human 3′ JH gene segment of said transgenic mouse comprise human JH6, wherein said JH6 is less than 2 kb upstream of the mouse JC intronic DNA of said chimeric JC intron. 
     
     
         7 . The method of  claim 2 , wherein the mouse JC intronic DNA of said chimeric JC intron upstream of said enhancer of said chimeric IgH locus of said transgenic mouse comprises mouse 129 strain. 
     
     
         8 . The method of  claim 1 , wherein said germline comprises all or part of mouse heavy chain variable region inverted with respect to the heavy chain constant region of said transgenic mouse. 
     
     
         9 . The method of  claim 1 , wherein said germline comprises all or part of mouse heavy chain variable region away from the heavy chain constant region of said transgenic mouse. 
     
     
         10 . The method of  claim 1 , wherein said germline comprises all, part or none of the mouse heavy chain variable region of said transgenic mouse. 
     
     
         11 . The method of  claim 1 , wherein said transgenic mouse expresses serum antibodies comprising immunoglobulin heavy chains IgG1, IgG2b and IgM. 
     
     
         12 . The method of  claim 11 , wherein said transgenic mouse expresses serum antibodies further comprising immunoglobulin heavy chain IgG2a. 
     
     
         13 . The method of  claim 1 , wherein less than 10% of IgH chains of said transgenic mouse comprise a mouse variable region. 
     
     
         14 . The method of  claim 13 , wherein said transgenic mouse does not express endogenous IgH heavy chain. 
     
     
         15 . The method of  claim 1 , wherein 75% of IgH chains of said transgenic mouse comprise a mouse variable region. 
     
     
         16 . The method of  claim 1 , wherein both endogenous heavy chain loci are inactive to express endogenous heavy chain polypeptide; said transgenic mouse comprises intact endogenous lambda loci to express endogenous lambda light chain polypeptide; and said transgenic mouse comprises endogenous kappa loci which are inactive to express endogenous kappa light chain polypeptide. 
     
     
         17 . The method of  claim 1 , wherein said antigen comprises a vaccine. 
     
     
         18 . The method of  claim 1 , wherein said endogenous Ig CH segment is selected from the group consisting of mouse Cμ, mouse Cγ, mouse Cδ, and mouse Cα. 
     
     
         19 . The method of  claim 1 , wherein said human JC intronic DNA comprises a 400 bp DNA segment of human intronic DNA. 
     
     
         20 . The method of  claim 1 , said transgenic mouse being an offspring of 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 an endogenous 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. 
     
     
         21 . The method of  claim 19 , wherein the germline of said transgenic mouse comprises all or part of a mouse IgH variable region. 
     
     
         22 . The method of  claim 20 , wherein the germline of said parent mouse comprises all or part of a mouse IgH variable region. 
     
     
         23 . The method of  claim 1 , wherein said mouse JC intronic DNA of said transgenic mouse comprises mouse 129 strain DNA upstream of said enhancer. 
     
     
         24 . The method of  claim 1 , wherein said transgenic mouse is 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 an endogenous Ig constant region (C) gene segment, wherein the germline of said subsequent generation mouse comprises all or part of a mouse IgH variable region. 
     
     
         25 . The method of  claim 10  wherein said all or part of the endogenous IgH variable region of said transgenic mouse is inverted and is positioned at a telomeric locus. 
     
     
         26 . The method of  claim 1 , wherein the 3′ end of said one or more human JH gene segments is less than 1 kb upstream of the mouse JC intronic DNA of said chimeric JC intron. 
     
     
         27 . The method of  claim 18 , wherein said endogenous Ig C gene segment is mouse Cμ, and said enhancer is mouse μ enhancer. 
     
     
         28 . The method of  claim 1 , further comprising humanizing the C region of the antibody or IgH chain, comprising (a) recovering the antibody or cells producing the antibody and (b) replacing the non-human mammal constant region with a human constant region, using protein or DNA engineering. 
     
     
         29 . The method of  claim 23 , said mouse JC intronic DNA comprising mouse 129Sv strain DNA. 
     
     
         30 . The method of  claim 24 , wherein said transgenic mouse comprises all or part of an endogenous mouse IgH variable region and is capable of breeding with another said transgenic mouse to produce said subsequent generation mouse. 
     
     
         31 . The method of  claim 30 , wherein said subsequent generation mouse is capable of breeding with a mouse having a germline comprising said homozygous IgH locus comprising unrearranged human IgH variable region gene segments operatively linked to an IgH constant (C) region comprising an endogenous C segment of said IgH locus to provide further subsequent generation mice.