Chimaeric Surrogate Light Chains (SLC) Comprising Human VpreB

Abstract

The present invention relates inter alia to improvements in the production of chimaeric antibodies in non-human transgenic vertebrates such as mice and rats bearing one or more chimaeric antibody transgenes. In particular, the invention provides for improved non-human vertebrates and cells in which VpreB has been species-matched with the variable region of the chimaeric antibodies. Also, embodiments also provide for species-matching of the entire surrogate light chain for efficient pairing with chimaeric heavy chains during B-cell development in vivo in a non-human transgenic vertebrate setting.

What is claimed is: 
     
         1 . A method for producing an antibody specific for a selected antigen, said antibody comprising an immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region,
 the method comprising isolating from a cultured cell expressing said IgH chain or fragment thereof comprising said human IgH chain variable region, one or more of the group consisting of:   (i) said IgH chain or fragment thereof comprising said human IgH chain variable region; and   (ii) nucleic acid encoding said IgH chain or fragment thereof comprising said human IgH chain variable region;   wherein said human IgH chain variable region is of a transgenic mouse contacted with said selected antigen or epitope thereof, for a time and under conditions which permit the antigen-contacted mouse to produce antibody specific for said antigen;   wherein said transgenic mouse comprises an IgH chain transgene, the IgH transgene comprising   (a) one or more human VH gene segments, one or more human D gene segments and one or more human JH gene segments operably connected upstream of a constant region gene so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and a constant region;   the genome further comprising   (i) a human VpreB gene capable of expressing a human VpreB, and   (ii) a mouse or rat λ5 gene;   wherein the transgenic mouse is capable of expressing a chimaeric surrogate light chain comprising human VpreB and mouse or rat λ5 for pairing with the heavy chain.   
     
     
         2 . The method of  claim 1 , wherein the constant region is a mouse constant region. 
     
     
         3 . The method of  claim 1 , wherein the constant region and the λ5 gene are mouse constant region and mouse λ5 gene, respectively. 
     
     
         4 . The method of  claim 2 , wherein the transgenic mouse is a strain selected from the group consisting of a 129-derived mouse strain, a C57Bl/6 derived mouse strain and a JM8 derived mouse strain. 
     
     
         5 . The method of  claim 2 , wherein the constant region is of a mouse 129 strain constant region or of a C57Bl/6 strain constant region, and the λ5 gene is a mouse 129 strain or a mouse C57Bl/6 strain. 
     
     
         6 . The method of  claim 1 , wherein the constant region gene and the λ5 gene are endogenous genes of said transgenic mouse. 
     
     
         7 . The method of  claim 1 , wherein the genome is homozygous for said transgene, human VpreB gene and mouse or rat λ5 gene. 
     
     
         8 . The method of  claim 1 , wherein endogenous antibody heavy chain expression in said mouse is inactive. 
     
     
         9 . The method of  claim 1 , wherein endogenous antibody light chain expression in said mouse is inactive. 
     
     
         10 . The method of  claim 1 , wherein the heavy chain transgene is devoid of a CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         11 . The method of  claim 1 , wherein the heavy chain transgene is devoid of a gamma CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         12 . The method of  claim 1 , wherein the heavy chain transgene is devoid of a mu CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         13 . The method of  claim 1 , wherein the constant region is a mu constant region. 
     
     
         14 . The method of  claim 1 , wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1. 
     
     
         15 . The method of  claim 1 , wherein said genome does not comprise a non-human vertebrate species VpreB1 and/or VpreB2 gene. 
     
     
         16 . The method of  claim 1 , wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene. 
     
     
         17 . The method of  claim 16 , wherein non-functional V and/or J gene segments are omitted. 
     
     
         18 . The method of  claim 16 , wherein the lambda V region or VJ region comprises a human VpreB gene and its associated promoter. 
     
     
         19 . The method of  claim 16 , wherein the insertion is an insertion into an antibody light chain locus. 
     
     
         20 . The method of  claim 16 , wherein the insertion replaces endogenous Vλ in whole or in part. 
     
     
         21 . The method of  claim 1 , wherein the human VpreB gene is not within the endogenous VpreB-λ5 locus. 
     
     
         22 . The method of  claim 1 , wherein the expression of the human VpreB gene is under endogenous control. 
     
     
         23 . The method of  claim 1 , wherein the human VpreB gene is operably linked to one or more DNaseI hypersensitive sites. 
     
     
         24 . The method of  claim 1 , wherein the human VpreB gene is present in the genome in any one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies. 
     
     
         25 . The method of  claim 1 , wherein all λ5 sequences in the genome are mouse λ5 sequences. 
     
     
         26 . The method of  claim 22 , wherein the human VpreB gene is operably linked to an endogenous promoter. 
     
     
         27 . The method of  claim 1 , wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain variable gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         28 . The method of  claim 1 , wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         29 . The method of  claim 1 , wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of one or more or all heavy chain variable region gene segments selected from the group consisting of VH6-1, VH1-3, VH3-7, VH1-8, VH3-9, VH3-11, JH1 and JH6, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         30 . The method of  claim 27 , wherein use of one or more or all of VH6-1, VH1-3, VH3-9 and JH1 is higher in the repertoire of said mouse than in the repertoire of the control. 
     
     
         31 . The method of  claim 27 , wherein use of one or more or all of VH3-7, VH1-8, VH3-11 and JH6 is lower in the repertoire of said mouse than in the repertoire of the control. 
     
     
         32 . The method of  claim 4 , wherein said transgenic mouse is a C57Bl/6—129/Sv hybrid strain. 
     
     
         33 . The method of  claim 13 , wherein said mu constant region is an endogenous mu constant region. 
     
     
         34 . The method of  claim 16 , wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and all the J regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene. 
     
     
         35 . The method of  claim 19 , where the insertion is an insertion into an endogenous lambda locus upstream of the endogenous lambda constant region. 
     
     
         36 . The method of  claim 26 , wherein said endogenous promoter is an endogenous VpreB promoter (e.g., a VpreB1 promoter). 
     
     
         37 . The method of  claim 28 , wherein the transgenic mouse expresses a repertoire of Ig heavy chain variable regions that differs by a probability of less than 0.05 in a binomial distribution test, from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         38 . The method of  claim 1 , wherein the genome of said transgenic mouse comprises
 (a) said antibody heavy chain transgene; and   (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene; wherein all of the V, D and J in said transgenes are human V, D and J; wherein endogenous antibody heavy and light chain expression is inactive.   
     
     
         39 . The method of  claim 38 , wherein the genome of said transgenic mouse is homozygous for said heavy and light chain transgenes. 
     
     
         40 . A method of promoting B cell development in a non-human vertebrate comprising an immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region,
 wherein said non-human vertebrate is a mouse or a rat,   wherein said non-human vertebrate comprises an IgH chain transgene, the IgH transgene comprising one or more human VH gene segments, one or more human D gene segments and one or more human JH gene segments operably connected upstream of a constant region gene so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and a constant region, optionally wherein the genome is homozygous for said IgH chain transgene;   
       the method comprising:
 (i) inactivating endogenous heavy chain expression in said vertebrate; 
 (ii) providing in the genome of said vertebrate:
 (a) a human VpreB gene capable of expressing a human VpreB, and 
 (b) a mouse or rat λ5 gene; 
 
 wherein the transgenic mouse is capable of expressing a chimaeric surrogate light chain comprising human VpreB and mouse or rat λ5, 
 wherein the transgenic mouse is capable of expressing a pre-B-cell receptor comprising said chimaeric surrogate light chain and an IgH mu chain comprising said human variable region, 
 wherein said pre-B-cell receptor promotes B cell development of cells bearing said IgH mu chain in said vertebrate. 
 
     
     
         41 . The method of  claim 40 , wherein said non-human vertebrate comprising an IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain is a mouse constant region. 
     
     
         42 . The method of  claim 40 , wherein said non-human vertebrate comprising an IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain and the λ5 gene are mouse constant region and mouse λ5 gene, respectively. 
     
     
         43 . The method of  claim 41 , wherein said mouse is a strain selected from the group consisting of a 129-derived mouse strain, a C57Bl/6 derived mouse strain and a JM8 derived mouse strain. 
     
     
         44 . The method of  claim 41 , wherein the constant region of said antibody heavy chain is of a mouse 129 strain constant region or of a C57Bl/6 strain constant region, and the λ5 gene is a mouse 129 strain or a mouse C57Bl/6 strain. 
     
     
         45 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the constant region gene and the λ5 gene are endogenous genes of said transgenic mouse. 
     
     
         46 . The method of  claim 40 , wherein the genome is homozygous for said transgene, human VpreB gene and mouse or rat λ5 gene. 
     
     
         47 . The method of  claim 1 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein endogenous antibody heavy chain expression in said mouse is inactive. 
     
     
         48 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein endogenous antibody light chain expression in said mouse is inactive. 
     
     
         49 . The method of  claim 40 , wherein the heavy chain transgene is devoid of a CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         50 . The method of  claim 40 , wherein the heavy chain transgene is devoid of a gamma CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         51 . The method of  claim 40 , wherein the heavy chain transgene is devoid of a mu CHI gene segment and the genome comprises no functional antibody light chain locus. 
     
     
         52 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain is a mouse mu constant region. 
     
     
         53 . The method of  claim 40 , wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1. 
     
     
         54 . The method of  claim 40 , wherein said genome does not comprise a non-human vertebrate species VpreB1 and/or VpreB2 gene. 
     
     
         55 . The method of  claim 40 , wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene. 
     
     
         56 . The method of  claim 55 , wherein non-functional V and/or J gene segments are omitted. 
     
     
         57 . The method of  claim 55 , wherein the lambda V region or VJ region comprises a human VpreB gene and its associated promoter. 
     
     
         58 . The method of  claim 55 , wherein the insertion is an insertion into an antibody light chain locus. 
     
     
         59 . The method of  claim 55 , wherein the insertion replaces endogenous Vλ in whole or in part. 
     
     
         60 . The method of  claim 40 , wherein the human VpreB gene is not within the endogenous VpreB-λ5 locus. 
     
     
         61 . The method of  claim 40 , wherein the expression of the human VpreB gene is under endogenous control. 
     
     
         62 . The method of  claim 40 , wherein the human VpreB gene is operably linked to one or more DNaseI hypersensitive sites. 
     
     
         63 . The method of  claim 40 , wherein the human VpreB gene is present in the genome in any one of 2, 3, 4, 5, 6, 7, 8 9 or 10 copies. 
     
     
         64 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein all λ5 sequences in the genome are mouse λ5 sequences. 
     
     
         65 . The method of  claim 61 , wherein the human VpreB gene is operably linked to an endogenous promoter. 
     
     
         66 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the heavy chain variable region repertoire of a control mouse in the proportion expressed as a percentage of use of heavy chain variable gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         67 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         68 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of one or more or all heavy chain variable region gene segments selected from the group consisting of VH6-1, VH1-3, VH3-7, VH1-8, VH3-9, VH3-11, JH1 and JH6, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         69 . The method of  claim 66 , wherein use of one or more or all of VH6-1, VH1-3, VH3-9 and JH1 is higher in the repertoire of said mouse than in the repertoire of the control. 
     
     
         70 . The method of  claim 66 , wherein use of one or more or all of VH3-7, VH1-8, VH3-11 and JH6 is lower in the repertoire of said mouse than in the repertoire of the control. 
     
     
         71 . The method of  claim 43 , wherein said transgenic mouse is a C57Bl/6-129/Sv hybrid strain. 
     
     
         72 . The method of  claim 52 , wherein said mu constant region is an endogenous mu constant region. 
     
     
         73 . The method of  claim 55 , wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and all the J regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene. 
     
     
         74 . The method of  claim 58 , where the insertion is an insertion into an endogenous lambda locus upstream of the endogenous lambda constant region. 
     
     
         75 . The method of  claim 65 , wherein said endogenous promoter is an endogenous VpreB promoter (e.g., a VpreB1 promoter). 
     
     
         76 . The method of  claim 67 , wherein the transgenic mouse expresses a repertoire of Ig heavy chain variable regions that differs by a probability of less than 0.05 in a binomial distribution test, from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. 
     
     
         77 . The method of  claim 40 , wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the genome of said transgenic mouse comprises
 (a) said antibody heavy chain transgene; and   (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene; wherein all of the V, (D) and J in said transgenes are human V, (D) and J;   wherein endogenous antibody heavy and light chain expression is inactive.   
     
     
         78 . The method of  claim 77 , wherein the genome of said transgenic mouse is homozygous for said heavy and light chain transgenes.