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 antigen-specific antibody comprising a human immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region and a human IgH chain constant region, said antigen-binding fragment thereof comprising said human IgH chain variable region, the method comprising:
 (A) providing a cell comprising nucleic acid encoding said human IgH heavy chain of said antibody, or a cell comprising nucleic acid encoding said human IgH chain variable region,   wherein said IgH chain variable region is of a transgenic mouse contacted with said antigen,   wherein the germline of said mouse comprises a homozygous immunoglobulin heavy chain (IgH) locus,   wherein said homozygous IgH locus comprises 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, one or more human JH gene segments and human intronic DNA contiguous with mouse intronic DNA and located at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous CH gene segment,   wherein said C region comprises mouse Cμ and said human variable region gene segments are operably linked to said C region to permit rearrangement and expression of IgM heavy chain polypeptide comprising a human variable region and a mouse μ constant region,   wherein in said transgenic mouse expression of IgH heavy chains comprising endogenous mouse variable regions is reduced or prevented,   wherein said mouse comprises IgH-VDJCμ transcripts encoding said chimeric IgM polypeptide and wherein said IgM comprises human CDR-H3, and said IgH-VDJCμ transcripts comprise transcripts encoding human variable region CDR-H3 lengths of 19 to 22 amino acids, and   wherein the mean frequency of said transcripts encoding CDR-H3 lengths selected from the group consisting of 18 and 19 amino acids present in said IgH-VDJC μ transcripts of said mouse is between 5% and 10%, and   (B) expressing said antibody from said cell comprising nucleic acid encoding said human IgH chain of said antibody, or expressing said fragment from said cell comprising nucleic acid encoding said human IgH chain variable region.   
     
     
         2 . The method of  claim 1 , wherein in said mouse germline said human intronic DNA is contiguous with said mouse intronic DNA at a human DNA/mouse DNA joinder point and wherein said one or more human JH gene segments comprises a 3′-JH gene segment contiguous with said human intronic DNA, wherein said 3′-JH gene segment is less than 2 kb upstream of said joinder point. 
     
     
         3 . The method of  claim 2 , wherein in said mouse germline said 3′-JH gene segment is less than 1 kb upstream of said joinder point. 
     
     
         4 . The method of  claim 1 , wherein said IgH human variable region of said antibody or said fragment comprises a CDR-H3 length of 19 to 22 amino acids. 
     
     
         5 . The method of  claim 1 , wherein in said mouse germline said enhancer is Eμ. 
     
     
         6 . The method of  claim 5 , wherein in said mouse germline said Eμ is mouse Eμ. 
     
     
         7 . The method of  claim 2 , wherein in said mouse germline said 3′-JH gene segment comprises a human JH6 gene segment. 
     
     
         8 . The method of  claim 2 , wherein in said mouse germline said human intronic DNA comprises a truncated human JC intron and said mouse intronic DNA comprises a truncated mouse JC intron. 
     
     
         9 . The method of  claim 8 , wherein in said mouse germline said truncated mouse JC intron comprises mouse 129 strain DNA. 
     
     
         10 . The method of  claim 9 , wherein in said mouse germline said truncated mouse JC intron comprises mouse mouse 129Sv strain DNA. 
     
     
         11 . The method of  claim 1 , wherein said mouse germline further comprises all or part of mouse heavy chain variable region away from said heavy chain constant region, absent from said germline, or inverted with respect to said heavy chain constant region to render expression of Ig heavy chains comprising a mouse variable region reduced or prevented. 
     
     
         12 . The method of  claim 11 , wherein said mouse is capable of breeding to subsequent generation mice comprising a germline comprising a homozygous IgH locus comprises 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, one or more human JH gene segments and human intronic DNA contiguous with mouse intronic DNA and located at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous CH gene segment. 
     
     
         13 . The method of  claim 2 , wherein said mouse is functional to produce antibody isotypes IgM and IgG specific for an antigen, said antibody isotype heavy chain comprising a human heavy chain variable region. 
     
     
         14 . The method of  claim 1 , the method further comprising
 recovering a substance selected from the group consisting of: said antibody or antigen-binding fragment thereof; said nucleic acid encoding said human IgH chain variable region; and said cell expressing said antibody or antigen-binding fragment thereof.   
     
     
         15 . The method of  claim 1 , further comprising the step of providing a pharmaceutical composition comprising said antigen-specific antibody or anigen-binding fragment thereof and a pharmaceutically acceptable carrier or excipient. 
     
     
         16 . The method of  claim 1 , further comprising providing said antibody or antigen-binding fragment thereof to a human subject. 
     
     
         17 . The method of  claim 1 , wherein said antibody is a monoclonal antibody, a domain antibody or a neutralizing antibody, or antigen binding fragment thereof. 
     
     
         18 . The method of  claim 1 , wherein said cell is a first cell, and wherein said human IgH chain variable region is obtained from a second cell comprising nucleic acid encoding said human IgH chain variable region. 
     
     
         19 . The method of  claim 2 , wherein said second cell is selected from the group consisting of: a B cell of a transgenic mouse contacted with said antigen; a cell comprising nucleic acid encoding said human IgH chain variable region and a mouse IgH constant region; a hybridoma expressing said human IgH chain variable region; a plurality of cells comprising nucleic acid encoding human IgH chain variable regions; and an immortalised cell comprising nucleic acid encoding said human IgH chain variable region. 
     
     
         20 . The method of  claim 1 , wherein in said mouse germline said unrearranged human IgH VH gene segments comprise human V6-1, V11-1-1, V1-2, V111-2-1, V1-3, V4-4 and V2-5 gene segments. 
     
     
         21 . The method of  claim 1 , wherein in said mouse germline said unrearranged human IgH VH gene segments comprise all of the human V, D and J gene segments. 
     
     
         22 . The method of  claim 1 , wherein said IgH-VDJCμ transcripts encoding a human variable region comprise a CDR-H3 length of 14-19 amino acids. 
     
     
         23 . The method of  claim 22 , wherein the frequency of transcripts encoding a CDR-H3 length of 19 amino acids in said IgH-VDJCμ transcripts of said mouse is 5%. 
     
     
         24 . The method of  claim 22 , wherein the frequency of transcripts encoding a CDR-H3 length of 19 amino acids in said IgH-VDJCμ transcripts of said mouse is less than transcripts encoding a CDR-H3 length of 17 amino acids and more than transcripts encoding a CDR-H3 length of 20 amino acids. 
     
     
         25 . The method of  claim 22 , wherein the frequency of transcripts encoding a CDR-H3 length of 19 amino acids in said IgH-VDJCμ transcripts of said mouse is less than transcripts encoding a CDR-H3 length of 17 amino acids. 
     
     
         26 . The method of  claim 22 , wherein the mean frequency of transcripts encoding CDR-H3 lengths between 14-19 amino acids in said IgH-VDJCμ transcripts of said mouse is between 5% and 10%.