Antibody production

Abstract

A non-human mammal containing an endogenous lambda light chain gene locus, an endogenous kappa light chain gene locus and an endogenous heavy chain gene locus, each of which can re-arrange so that immunoglobulin heavy and light chain genes are formed and expressed in B-cells following antigen challenge but said loci have been mutated so that the ability to form functional immunoglobulin tetramers comprising re-arranged heavy and light chains produced from said mutated loci has been substantially reduced or eliminated.

1 . A non-human mammal containing an endogenous lambda light chain gene locus, an endogenous kappa light chain gene locus and an endogenous heavy chain gene locus, each of which can re-arrange so that immunoglobulin heavy and light chain genes are formed and expressed in B-cells following antigen challenge but said loci have been mutated so that the ability to form functional immunoglobulin tetramers comprising re-arranged heavy and light chains produced from said mutated loci has been substantially reduced or eliminated. 
     
     
         2 . The non-human mammal of  claim 1 , wherein at least one of the endogenous lambda light chain gene locus, the endogenous kappa light chain gene locus and the endogenous heavy chain gene locus has been mutated by the introduction of a frame shift mutation, a polypeptide-encoding sequence and/or one or more stop codons into the at least one endogenous locus. 
     
     
         3 . The non-human mammal of  claim 2 , wherein the mutation is an insertion of less than 50 nucleotides. 
     
     
         4 . The non-human mammal of  claim 1 , wherein the expression of at least one of the endogenous lambda light chain gene locus, the endogenous kappa light chain gene locus and the endogenous heavy chain gene locus has been substantially reduced by elimination of part or all of the LCR in the at least one locus. 
     
     
         5 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous kappa light chain gene locus. 
     
     
         6 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous lambda light chain gene locus. 
     
     
         7 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous kappa light chain gene locus and the endogenous lambda light chain gene locus. 
     
     
         8 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous heavy chain gene locus. 
     
     
         9 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous kappa light chain gene locus and in the endogenous heavy chain gene locus. 
     
     
         10 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous lambda light chain gene locus and in the endogenous heavy chain gene locus. 
     
     
         11 . The non-human mammal of  claim 2  or  claim 3 , in which the introduction is in the endogenous lambda light chain gene locus, in the endogenous kappa light chain gene locus and in the endogenous heavy chain gene locus. 
     
     
         12 . The non-human mammal of  claim 1 , wherein there is an introduction of a frame shift mutation, a polypeptide-encoding sequence, and/or one or more stop codons in the at least one endogenous kappa light chain gene locus, or an insertion of less than 50 nucleotides in the at least one endogenous kappa light chain gene locus and there is a partial or complete LCR deletion in the endogenous lambda light chain gene locus. 
     
     
         13 . The non-human mammal of  claim 1 , in which the endogenous heavy chain gene locus is mutated such that heavy chain gene rearrangement, mRNA transcription and protein synthesis occurs but that B-cell activation is blocked. 
     
     
         14 . The non-human mammal of  claim 1  comprising a transgene comprising one or more heterologous kappa light chain gene loci and associated B-cell specific regulatory elements. 
     
     
         15 . The non-human mammal of  claim 1  comprising a transgene comprising one or more heterologous lambda light chain gene loci and associated B-cell specific regulatory elements. 
     
     
         16 . The non-human mammal of  claim 14  or  claim 15 , wherein the transgene comprising a heterologous light chain gene locus comprises a dominant selective marker gene. 
     
     
         17 . The non-human mammal of  claim 1  comprising a transgene comprising one or more heterologous heavy chain gene loci and associated B-cell specific regulatory elements. 
     
     
         18 . The non-human mammal of  claim 17  comprising two or more transgenes comprising two or more different heterologous heavy chain gene loci and associated B-cell specific regulatory elements. 
     
     
         19 . The non-human mammal of  claim 17  or  claim 18 , wherein the one or more transgene comprising a heterologous heavy chain gene locus comprises a dominant selective marker gene. 
     
     
         20 . The non-human mammal of  claim 17  or  claim 18 , in which each heterologous heavy chain gene locus comprises a CTCF binding site. 
     
     
         21 . The non-human mammal of  claim 14  or  claim 17  comprising a transgene comprising a heterologous kappa light chain gene locus and a transgene comprising one or more heterologous heavy chain loci. 
     
     
         22 . The non-human mammal of  claim 15  or  claim 17  comprising a transgene comprising a heterologous lambda light chain gene locus and a transgene comprising one or more heterologous heavy chain loci. 
     
     
         23 . The non-human mammal of any one of  claims 14 ,  15 , or  17  comprising a transgene comprising a heterologous kappa light chain gene locus, a transgene comprising a lambda light chain gene locus and a transgene comprising one or more heterologous heavy chain gene loci. 
     
     
         24 . The non-human mammal of any one of  claims 14 ,  15 , or  17 , wherein each heterologous locus incorporates a cognate LCR. 
     
     
         25 . The non-human mammal of any one of  claims 14 ,  15 , or  17 , wherein each heterologous locus is a human locus. 
     
     
         26 . The non-human mammal of any one of  claims 14 ,  15 , or  17 , wherein each heterologous locus is a hybrid locus comprising variable regions and constant regions derived from more than one species. 
     
     
         27 . The non-human mammal of  claim 26 , wherein each heterologous locus is a hybrid locus comprising human variable regions and rat or murine constant regions. 
     
     
         28 . The non-human mammal of any one of  claims 14 ,  15 , or  17 , comprising groups of transgenes comprising different groups of different heterologous heavy chain gene loci, wherein each group of transgenes comprises a different dominant selective marker gene. 
     
     
         29 . The non-human mammal of any one of  claims 14 ,  15 , or  17 , comprising transgenes comprising heterologous light chain loci and transgenes comprising heterologous heavy chain loci, wherein transgenes comprising heterologous light chain loci and transgenes comprising heterologous heavy chain loci each comprise a different dominant selective marker gene. 
     
     
         30 . The non-human mammal of  claim 1 , which is a rodent. 
     
     
         31 . The non-human mammal of  claim 30 , which is a mouse. 
     
     
         32 . A method of producing an antigen-specific heterologous monoclonal antibody comprising:
 (a) immunising a non-human transgenic mammal of any  claim 16  or  claim 19 , with an antigen;   (b) preparing hybridomas or immortalised B-cell lines each of which produces a monoclonal antibody from the B-cells of the immunised transgenic mammal;   (c) optionally selecting at least one hybridoma or immortalised B-cell line expressing the heterologous antibody by use of the dominant selective marker genes present in the transgenes comprising the heterologous immunoglobulin light chain and heavy chain loci; and   (d) selecting at least one hybridoma or immortalised B-cell line which produces an antibody which binds specifically to the antigen.   
     
     
         33 . A method of deriving a mammalian, preferably human, antibody from a hybrid antibody comprising:
 (a) carrying out the method of  claim 32 , wherein the transgenes are hybrid;   (b) selecting at least one hybridoma or immortalised B-cell line which produces an antibody which binds specifically to the antigen and comprises V H  and V L  binding domains of the species of choice;   (c) cloning and sequencing the V H  and V L  domains;   (d) recloning selected sequences comprising the V H  and V L  binding domain coding sequences with constant effectors domains of choice from the same species; and   (e) co-expressing the recloned sequences encoding heavy and light chain polypeptides of the desired species using an expression vector in a cell type of choice.   
     
     
         34 . A method for the production of the non-human mammal of  claim 1  comprising mutating the endogenous heavy chain gene locus, the endogenous kappa light chain gene locus, and, optionally, the endogenous lambda light chain gene locus in a mammalian progenitor cell and producing a mammal from said progenitor cell, wherein the mutation is such that, in the mammal, each locus can re-arrange so that immunoglobulin heavy and light chain genes are formed and expressed in B-cells following antigen challenge but the ability to form functional immunoglobulin tetramers comprising re-arranged heavy and light chains produced from said mutated loci has been substantially reduced or eliminated. 
     
     
         35 . The method of  claim 34 , wherein the progenitor cell is a non-human embryonic stem cell. 
     
     
         36 . The method of  claim 34  or  claim 35 , wherein the non-human mammal is a rodent. 
     
     
         37 . The method of  claim 34  or  claim 35 , wherein the non-human mammal is a mouse. 
     
     
         38 . (canceled)