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.

What is claimed: 
     
         1 . A method of providing a transgenic mouse ES cell comprising unrearranged human IgH VH, D, and JH gene segments in its genome, the method comprising serially inserting a plurality of human DNA fragments comprising unrearranged human IgH variable region gene segments into the genome of a mouse ES cell, comprising the following steps:
 inserting into a mouse ES cell genome at a position upstream of an endogenous IgH constant (C) gene segment a first human DNA fragment comprising a human DJH fragment comprising unrearranged human D gene segments and human JH gene segments, thereby to provide a mouse ES cell comprising a genome comprising said inserted human DJH fragment; and   inserting into a mouse ES cell genome comprising said inserted human DJH fragment a second human DNA fragment comprising a human VH fragment comprising unrearranged human IgH V gene segments such that said inserted human DJH fragment and said human VH fragment form a contiguous inserted human DNA comprising unrearranged human IgH VH, D, and JH gene segments, thereby to provide a transfected mouse ES cell comprising in its genome said unrearranged human IgH VH, D, JH gene segments positioned upstream of, and in functional arrangement with, said endogenous IgH C gene segment.   
     
     
         2 . The method of  claim 1 , further comprising the steps of:
 a) generating a transgenic mouse from said transfected mouse ES cell;   b) providing a subsequent generation mouse of said transgenic mouse of step (a), said subsequent generation mouse comprising in its genome unrearranged human IgH VH, D, and JH gene segments positioned upstream of, and in functional arrangement with, an endogenous IgH C gene segment;   c) subjecting said subsequent generation mouse to an antigen; and   d) isolating an antibody specific to said antigen or an IgH polypeptide comprising a human IgH variable region from said mouse of step (c).   
     
     
         3 . The method of  claim 1 , further comprising the steps of:
 a) generating a transgenic mouse from said transfected mouse ES cell;   b) providing a subsequent generation mouse of said transgenic mouse of step (a), said subsequent generation mouse comprising in its genome unrearranged human IgH VH, D, and JH gene segments in functional arrangement with an endogenous IgH C gene segment;   c) subjecting said subsequent generation mouse to an antigen; and   d) isolating a cell from said mouse of step (c), wherein said cell produces antibody specific to said antigen, said antibodycomprising a human IgH variable region.   
     
     
         4 . The method of  claim 1 , further comprising the steps of:
 a) generating a transgenic mouse from said transfected mouse ES cell;   b) providing a subsequent generation mouse of said transgenic mouse of step (a), said subsequent generation mouse comprising in its genome unrearranged human IgH VH, D, and JH gene segments in functional arrangement with an endogenous IgH C gene segment;   c) subjecting said subsequent generation mouse to an antigen; and   d) isolating from a cell of the mouse of step (c) a nucleic acid encoding a said human IgH variable region, wherein said cell produces antibody specific for said antigen, said antibody comprising said human IgH variable region.   
     
     
         5 . The method of  claim 4 , further comprising the steps of combining in a cell said nucleic acid encoding said human IgH variable region with nucleic acid encoding a human IgH constant region to provide nucleic acid encoding a polypeptide comprising said human IgH variable region and said human IgH constant region, thereby providing a cell comprising nucleic acid encoding and expressing said polypeptide. 
     
     
         6 . The method of  claim 5 , further comprising the step of: isolating said expressed polypeptide. 
     
     
         7 . The method of  claim 1 , further comprising the step of: modifying the genome of a mouse ES cell comprising said inserted human DJH fragment (from step (i)) or the genome of a mouse ES cell comprising said contiguous human DNA (from step (ii) to prevent expression of native mouse IgH chains, wherein said modifying is by recombinase-mediated excision or inversion of all or a part of the mouse IgH V region gene segments, thereby to provide a modified transfected mouse ES cell. 
     
     
         8 . The method of  claim 1 , further comprising performing subsequent serial insertions of human VH fragments into the genome of said transfected mouse ES cell. 
     
     
         9 . The method of  claim 8 , wherein insertion of a human DNA fragment comprising a human VH fragment is performed using 5 or more serial insertions. 
     
     
         10 . The method of  claim 8 , wherein said transfected mouse ES cell comprises at least 316 kb of contiguous inserted human DNA. 
     
     
         11 . The method of  claim 10 , wherein said transfected mouse ES cell comprises at least 940 kb of contiguous inserted human DNA. 
     
     
         12 . The method of  claim 1 , wherein said serial insertion of said first human DNA fragment commences at a site where a unique targeting region is present in the genome; and wherein said insertion of said first human DNA fragment is effected into said unique targeting region. 
     
     
         13 . The method of  claim 12 , wherein insertion at said unique targeting region is made by homologous recombination. 
     
     
         14 . The method of  claim 12 , wherein one or more insertion events utilises site specific recombination. 
     
     
         15 . The method of  claim 1 , wherein said serial insertions of human VH, D, and JH gene segments is in the mouse genome between coordinates 114,667,091 and 114,665,190 of mouse chromosome 12, wherein said coordinates refer to NCBI m37, April 2007 ENSEMBL Release 55.37h for the mouse C57BL/6J strain. 
     
     
         16 . The method of  claim 1 , wherein said inserted human DNA comprises human VH, D, and JH gene segments from 2 or more different human genomes. 
     
     
         17 . The method of  claim 1 , wherein after each said inserting step, the resulting transfected mouse ES cell is cultured. 
     
     
         18 . The method of  claim 1 , wherein said inserted human DJH fragment comprises a plurality of human D gene segments and a plurality of human JH gene segments. 
     
     
         19 . The method of  claim 1 , wherein said inserting step (i) or step (ii) replaces unrearranged mouse D gene segments and mouse JH gene segments with said unrearranged human D gene segments and human JH gene segments. 
     
     
         20 . The method of  claim 18 , wherein said inserted human DJH fragment comprises a human VH gene segment. 
     
     
         21 . The method of  claim 1 , wherein said inserted human DJH fragment is a subset of said human DNA fragment. 
     
     
         22 . The method of  claim 1 , wherein said inserted human VH fragment is a subset of said second human DNA fragment. 
     
     
         23 . A method of providing a cell expressing a polypeptide comprising a human IgH variable region and a human IgH constant region, the method comprising:
 a) providing a modified transgenic mouse ES cell comprising unrearranged human IgH VH, D, and JH gene segments in its genome, the method comprising serially inserting a plurality of human DNA fragments comprising unrearranged human IgH variable region gene segments into the genome of a mouse ES cell, comprising the following steps:
 i) inserting into a mouse ES cell genome a first human DNA fragment comprising a human DJH fragment comprising unrearranged human D gene segments and human JH gene segments, thereby providing an inserted human DJH fragment positioned upstream of an endogenous IgH constant (C) gene segment; and 
 ii) inserting into a mouse ES cell genome comprising said inserted human DJH fragment a second human DNA fragment comprising a human VH fragment comprising unrearranged human IgH V gene segments such that said human DJH fragment and said human VH fragment form a contiguous inserted human DNA comprising unrearranged human IgH VH, D, and JH gene segments, thereby providing said unrearranged human IgH VH, D, JH gene segments in functional arrangement with said endogenous IgH C gene segment; 
 iii) modifying amouse ES cell genome comprising said human DJH fragment (from step (i)) or amouse ES cell genome comprising said contiguous inserted human DNA (from step (ii)) to prevent expression of native mouse IgH chains, wherein said modification is by recombinase-mediated excision or inversion of all or a part of the mouse IgH variable region gene segments, thereby to provide a modified transfected mouse ES cell; 
   b) generating a transgenic mouse from said modified transfected mouse ES cell;   c) providing a subsequent generation mouse of said transgenic mouse of step (b), said subsequent generation mouse comprising in its genome unrearranged human IgH VH, D, and JH gene segments in functional arrangement with an endogenous IgH C gene segment;   d) subjecting said subsequent generation mouse to an antigen;   e) isolating from a cell of the mouse of step (d) a nucleic acid encoding a said human IgH variable region, wherein said cell produces antibody specific for said antigen, said antibody comprising said human IgH variable region; and   f) combining in a cell said nucleic acid encoding said human IgH variable region with nucleic acid encoding a human IgH constant region to provide nucleic acid encoding a polypeptide comprising said human IgH variable region and said human IgH constant region, thereby providing a transfected cell comprising nucleic acid encoding and expressing said polypeptide.   
     
     
         24 . The method of  claim 23 , further comprising the step of isolating a composition comprising a biological sample selected from the group consisting of: said cell of step (f), said nucleic acid of step (f) encoding said polypeptide and said expressed polypeptide of step (f). 
     
     
         25 . A method of providing a cell expressing a polypeptide comprising a human IgH variable region and a human IgH constant region:
 a) providing a transgenic mouse ES cell comprising unrearranged human IgH VH, D, and JH gene segments in its genome, the method comprising serially inserting a plurality of human DNA fragments comprising unrearranged human IgH variable region gene segments into the genome of a mouse ES cell, comprising the following steps:
 i) inserting into a mouse ES cell genome a first human DNA fragment comprising a human DJH fragment comprising unrearranged human D gene segments and human JH gene segments, thereby providing an inserted human DJH fragment positioned upstream of an endogenous IgH constant (C) gene segment; 
 ii) inserting into a mouse ES cell genome comprising said inserted human DJH fragment a second human DNA fragment comprising a human VH fragment comprising unrearranged human IgH V gene segments such that said human DJH fragment and said human VH fragment form a contiguous inserted human DNA comprising unrearranged human IgH VH, D, and JH gene segments; and 
 iii) performing subsequent serial insertions of human VH fragments into the genome of a mouse ES cell of step (ii), each subsequent serial insertion forming a longer contiguous inserted human DNA, thereby to provide a transfected mouse ES cell comprising in its genome said unrearranged human IgH VH, D, JH gene segments in functional arrangement with said endogenous IgH C gene segment; 
   b) generating a transgenic mouse from said transfected mouse ES cell of step (iii);   c) providing a subsequent generation mouse of said transgenic mouse of step (b), said subsequent generation mouse comprising in its genome unrearranged human IgH VH, D, and JH gene segments in functional arrangement with an endogenous IgH C gene segment;   d) subjecting said subsequent generation mouse to an antigen; and   e) isolating from a cell of the mouse of step (d) a nucleic acid encoding a said human IgH variable region, wherein said cell produces antibody specific for said antigen comprising said human IgH variable region; and   f) combining in a cell said nucleic acid encoding said human IgH variable region with nucleic acid encoding a human IgH constant region to provide nucleic acid encoding a polypeptide comprising said human IgH variable region and said human IgH constant region, thereby providing a transfected cell comprising nucleic acid encoding and expressing said polypeptide.   
     
     
         26 . The method of  claim 25 , further comprising the step of isolating a composition comprising a biological sample selected from the group consisting of: said cell of step (f), said nucleic acid of step (f) encoding said polypeptide, and said expressed polypeptide of step (f).