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 is: 
     
         1 . A method of obtaining nucleic acid encoding the human variable region of an antigen-specific antibody or antigen binding fragment thereof, the method comprising identifying and/or copying nucleic acid encoding a human variable heavy chain region of said antigen-specific antibody and/or nucleic acid encoding a human variable light chain region of said antigen-specific antibody from a B cell or hybridoma cell thereof, of a mouse contacted with said antigen,
 wherein the germline of said mouse comprises:
 (i) an IgH locus comprising one or more human variable heavy (VH) gene segments, one or more human D gene segments, and one or more human joining heavy (JH) gene segments at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous IgH C gene segment, and/or 
 (ii) an IgL locus comprising four or more human variable light (VL) gene segments and one or more human joining light (JL) gene segments at an endogenous IgL locus upstream of an IgL constant region comprising an endogenous IgL C gene segment, 
   wherein said IgH locus comprises in 5′ to 3′ orientation said one or more human VH gene segments, said one or more human D gene segments, and said one or more human JH gene segments, an enhancer, and said C region,
 wherein said intronic DNA comprises in 5′ to 3′ orientation JC intronic DNA of a human IgH locus and JC intronic DNA of a mouse IgH locus, wherein said one or more J H  gene segments comprises a 3′ human J H  gene segment, wherein said 3′ human J H  gene segment is less than 2 kb upstream of said mouse JC intronic DNA, wherein said human JC intronic DNA comprises human JC intronic DNA contiguous in a human IgH locus with said 3′ human J H  gene segment, 
 wherein said mouse JC intronic DNA comprises less than a complete JC intron of a mouse IgH locus, 
 wherein in the germline of the mouse, said human gene segments in each said Ig locus of (i) and (ii) are unrearranged and operably linked to a constant gene segment thereof so that the mouse is capable of producing an antibody heavy chain comprising a human variable domain generated by recombination of a said one or more human J H  gene segments with a said one or more D gene segments and a said one or more V H segments, and an antibody light chain comprising a human variable domain generated by recombination of a said human VL gene segment with a said one or more human JL segments, 
   wherein said one or more human VH gene segments of the IgH locus of (i) comprise one or more human VH gene segments selected from the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH13-13*01, VH3-7*01, VH3-20*d01 and VH3-9*01; and wherein said four or more human VL gene segments of the IgL chain locus of (ii) comprise four or more human Vκ gene segments selected from the group consisting of VK4-1*01, VK2-28*01, VK1D-13*d01, VK1-12*01, VK1D-12*02, VK3-20*01, VK1-17*01, VK1D-39*01, VK3-11*01, VK1 D-16*01 and VK1-9*d01.   
     
     
         2 . The method according to  claim 1 , further comprising modifying the nucleic acid of said B cell or hybridoma cell thereof, of said mouse contacted with said antigen such that said antigen-specific antibody encoded by said nucleic acid comprises a human constant region operably linked to said human VH and/or human VL domain, the method comprising replacing the nucleic acid encoding the endogenous constant region of the antigen-specific antibody or fragment thereof, with nucleic acid encoding a human constant region, thereby producing a fully humanised antibody or antigen binding fragment thereof. 
     
     
         3 . The method according to  claim 1 , wherein said one or more human JH gene segments of the heavy chain locus comprises human JH2*01 and/or human JH6*02. 
     
     
         4 . The method according to  claim 1 , wherein at least one of said one or more human VH gene segments is selected from the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH 1-3*01, VH3-13*01, VH3-7*01 and VH3-20*d01. 
     
     
         5 . The method according to  claim 1 , wherein the antigen is a multi-subunit human protein, a bacterial cytotoxin or a protein expressed as a transmembrane protein on human cells. 
     
     
         6 . The method according to  claim 1 , wherein the antibody or antigen binding fragment thereof specifically binds a human target selected from: proprotein convertase PC9, proprotein convertase subtilisin kexin-9 (PCSK9), CD126, IL-4, IL-4 receptor, IL-6, IL-6 receptor, IL-13, IL-18 receptor, Erbb3, cell ASIC1, ANG2, GDF-8, angiopoietin ligand-2, delta-like protein ligand 4, immunoglobulin G1, PDGF ligand, PDGF receptor or NGF receptor, toxin A or toxin B of  Clostridium  di/ficile, relaxin, CD48, Cd20, glucagon receptor, protease activated receptor 2, TNF-Like ligand 1A (TL1A), angiopoietin related-2 (AR-2), angiopoietin-like protein 4, RANKL, angiopoietin-like protein 3 (ANGPTL3), delta-like ligand 4 (DLL4), big endothelin-1 (ET-1), activin A, receptor tyrosine kinases, for example human AR-1 and tyrosine kinase with Ig and EGF homology domains (TI E) and TIE-2 receptor. 
     
     
         7 . A method according to of  claim 1 ,
 (a) wherein the heavy chain of the isolated antibody is encoded by a VH gene segment selected from the group consisting of human VH3-23*04 and human VH3-9*01,   (b) wherein the heavy chain of the isolated antibody is encoded by a JH gene segment selected from the group consisting of human J H6*02, human J H6*01, J H2*01 and JH3*02,   (c) wherein the light chain of the isolated antibody is encoded by a VK gene segment selected from the group consisting of human VK1 D-12*02, human VK1-12*01, human VK2-28*01 and human VK4-1*01,   (d) wherein the light chain of the isolated antibody is encoded by a JK gene segment selected from the group consisting of human JK2*01 and human JK4*01.   
     
     
         8 . The method of  claim 1 , wherein the cell is a B-cell encoding an antibody that binds said antigen. 
     
     
         9 . The method of  claim 2 , wherein the nucleic acid is comprised by a host cell selected from a CHO, HEK293, Cos or yeast cell. 
     
     
         10 . The method of  claim 9 , wherein the antibody or antigen binding fragment thereof encoded by said nucleic acid is produced by expression from a CHO cell. 
     
     
         11 . The method of  claim 1 , further comprising formulating the nucleic acid with a pharmaceutically acceptable diluent, carrier, excipient or a drug, thereby producing a pharmaceutical composition. 
     
     
         12 . The method of  claim 11 , further comprising packaging said pharmaceutical composition in a sterile container. 
     
     
         13 . The method of  claim 12 , wherein said sterile container is selected from the group consisting of a vial, a tube, an Intravenous bag and a syringe. 
     
     
         14 . The method of  claim 12 , wherein said packaging comprising a label or instructions comprises a medicament batch number and/or a marketing authorization number, further optionally an EMA or FDA marketing authorization number. 
     
     
         15 . The method of  claim 1 , wherein the antibody or antigen binding fragment encoded by said nucleic acid comprises
 (a) a human heavy chain variable domain that is a recombinant of a human VH gene segment selected from the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01, VH3-20*d01 and VH3-9*01, a human J H gene segment and a human D gene segment; and/or   (b) a human light chain variable domain is a recombinant of a human VL gene segment selected from the group consisting of VK4-1*01, VK2-28*01, VK1 D-13*d01, VK1-12*01, VK1 D-12*02, VK3-20*01, VK1-17*01, VK1 D-39*01, VK3-11*01, VK1 D-16*01 and V 1-9*d01 and a human J gene segment.   
     
     
         16 . The method of  claim 15 , wherein the human JH gene segment of claim  18 (a) is selected from the group consisting of JH2*01, JH6*02, JH6*01 and JH3*02. 
     
     
         17 . The method of  claim 15 , wherein the human JL gene segment of claim  18 (b) is selected from the group consisting of JK4*01 and JK2*01. 
     
     
         18 . The method of  claim 15 , wherein the antibody or antigen binding fragment thereof encoded by the nucleic acid comprises a heavy chain variable domain recombinant of  claim 19  and a light chain variable domain recombinant of  claim 20 . 
     
     
         19 . The method of  claim 15 , wherein the antibody or antigen binding fragment thereof encoded by the nucleic acid comprises a human heavy chain variable domain recombinant of VH3-23*04 and JH2*01 and a human light chain variable domain recombinant of VK4-1*01 and JK2*01. 
     
     
         20 . The method of  claim 15 , wherein the antibody or antigen binding fragment thereof encoded by the nucleic acid comprises a human heavy chain variable domain recombinant of VH3-7*01 and JH6*02 and a human light chain variable domain recombinant of VK2-28*01 and JK4*01. 
     
     
         21 . The method of  claim 1 , said mouse being functional to form rearranged human VH, D and JH gene segments and to express mRNA transcripts encoding chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse Cμ region, wherein said mouse comprises IgH mRNA transcripts comprising IgH-VDJCμ transcripts comprising rearranged human heavy chain V, D, and J gene segments and mouse Cμ and encoding chimeric IgH polypeptides, wherein each IgH-VDJCμ transcript encodes a human variable region comprising a CDR-H3, wherein said IgH-VDJCμ transcripts comprise transcripts encoding a human variable region comprising a CDR-H3 length of 17 amino acids and transcripts encoding human variable region comprising a CDR-H3 length of 18 amino acids, wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17 and 18 amino acids present in said IgH-VDJCμ transcripts of said mouse is between 5% and 10%.