Antibody producing non-human mammals

Abstract

Described are transgenic, non-human animals comprising a nucleic acid encoding an immunoglobulin light chain, whereby the immunoglobulin light chain is human, human-like, or humanized. The nucleic acid is provided with a means that renders it resistant to DNA rearrangements and/or somatic hypermutations. In one embodiment, the nucleic acid comprises an expression cassette for the expression of a desired molecule in cells during a certain stage of development in cells developing into mature B cells. Further provided is methods for producing an immunoglobulin from the transgenic, non-human animal.

1 . A transgenic, non-human mammal comprising, at least in its B cell lineage, a nucleic acid sequence encoding at least an immunoglobulin light chain or heavy chain, wherein the nucleic acid sequence is provided with means for rendering it resistant to DNA rearrangements and/or somatic hypermutations. 
     
     
         2 . The transgenic, non-human mammal of  claim 1 , which is a rodent or mouse. 
     
     
         3 . The transgenic, non-human mammal of  claim 1 , wherein the nucleic acid sequence is integrated into the transgenic, non-human mammal's genome. 
     
     
         4 . The transgenic, non-human mammal of  claim 3 , wherein the integration is in a locus resistant to silencing. 
     
     
         5 . The transgenic, non-human mammal of  claim 4 , wherein the integration is in the Rosa-locus. 
     
     
         6 . The transgenic, non-human mammal of  claim 1 , wherein a light chain encoding nucleic acid is provided with a means for allowing expression of the nucleic acid essentially limited to cells of B cell lineage. 
     
     
         7 . The transgenic, non-human mammal of  claim 6 , wherein the light chain encoding nucleic acid is provided with a means for allowing expression of the light chain encoding nucleic acid predominantly during a certain stage of the development of B cells. 
     
     
         8 . The transgenic, non-human mammal of  claim 7 , wherein the means for allowing expression of the light chain encoding nucleic acid predominantly during a certain stage of the development of B cells comprises a promoter selected from the group consisting of CD19, CD20, μHC, VpreB1, VpreB2, VpreB3, λ5, Igα, Igβ, κLC, λLC, and BSAP (Pax5). 
     
     
         9 . The transgenic, non-human mammal of  claim 7 , wherein the means for allowing expression of the light chain encoding nucleic acid predominantly during a certain stage of the development of B cells for allowing expression of the light chain encoding nucleic acid predominantly during a certain stage of the development of B cells comprises a cre-lox system. 
     
     
         10 . The transgenic, non-human mammal of  claim 1 , wherein the nucleic acid sequence encodes a light chain able to pair with at least two different heavy chains encoded by the transgenic, non-human mammal. 
     
     
         11 . The transgenic, non-human mammal of  claim 1 , wherein at least one endogenous loci encoding an endogenous light chain is functionally silenced. 
     
     
         12 . The transgenic, non-human mammal of  claim 1 , wherein endogenous κ light chain locus is functionally silenced. 
     
     
         13 . The transgenic, non-human mammal of  claim 1 , wherein the nucleic acid sequence comprises a human vκ sequence. 
     
     
         14 . The transgenic, non-human mammal of  claim 13  wherein the light chain encoding sequence is a germline-like sequence. 
     
     
         15 . The transgenic, non-human mammal of  claim 14 , wherein the light chain encoding sequence is a germline sequence. 
     
     
         16 . The transgenic, non-human mammal of  claim 15 , wherein the germline sequence is based on O12. 
     
     
         17 . The transgenic, non-human mammal of  claim 1  wherein the light chain encoding nucleic acid comprises in 5′ to 3′ direction: a vκ promoter, a human leader, a human V gene, optionally a MoEκi enhancer, a rat constant region (κ), and optionally a MoEκ3′ enhancer or a truncated MoEκ3′ enhancer. 
     
     
         18 . A transgenic, non-human animal which has been provided with an expression cassette for expressing a desired proteinaceous molecule in cells during a certain stage of development in cells developing into mature B cells, wherein the expression cassette comprises:
 means for preventing silencing of expression of the desired proteinaceous molecule after introduction into a host cell, and   means for timing expression of the desired proteinaceous molecule with the desired developmental stage of the host cell.   
     
     
         19 . The transgenic, non-human animal of  claim 18 , wherein the means for timing expression of the desired proteinaceous molecule with the desired developmental stage of the host cell is a promoter of which the activity is essentially limited to the certain stage of development. 
     
     
         20 . The transgenic, non-human animal of  claim 18 , wherein the certain stage starts at a stage immediately preceding or coinciding with the onset of expression of light chain molecules by the cells at a certain stage of development into a mature B cell. 
     
     
         21 . The transgenic, non-human animal of  claim 19 , wherein the promoter is selected from the group consisting of CD19 promoter, Ig-alpha promoter, Ig-beta promoter, μhc promoter, vk promoter, an analog of any thereof, and a homolog of any thereof. 
     
     
         22 . The transgenic, non-human animal of  claim 19 , wherein the promoter drives the expression of a cre-like protein. 
     
     
         23 . The transgenic, non-human animal of  claim 22  wherein the sequence encoding the desired proteinaceous molecule is activated by cre-like protein's action. 
     
     
         24 . A transgenic, non-human animal that has been provided with a set of nucleic acids that are expression cassettes, wherein a first nucleic acid comprises a promoter driving expression of a cre-like protein, and the second nucleic acid comprises a sequence encoding a desired proteinaceous molecule under control of a constitutive promoter which can be activated by a cre-like protein's action. 
     
     
         25 . The transgenic, non-human animal of  claim 23 , wherein activation is achieved by removal of a “stop”. 
     
     
         26 . The transgenic, non-human animal of  claim 18 , wherein the desired proteinaceous molecule is a polypeptide chain of an immunoglobulin. 
     
     
         27 . The transgenic, non-human animal of  claim 26 , wherein the polypeptide chain of an immunoglobulin is a light chain. 
     
     
         28 . The transgenic, non-human animal of  claim 27 , wherein the sequence encoding the polypeptide chain of an immunoglobulin is rendered essentially incapable of rearrangement. 
     
     
         29 . The transgenic, non-human animal of  claim 27 , wherein the light chain is a germline-like light chain. 
     
     
         30 . The transgenic, non-human animal of  claim 29 , wherein the germline-like light chain is O12. 
     
     
         31 . The transgenic, non-human animal of  claim 28 , wherein rearrangement is prevented by absence of elements at least partially responsible for somatic hypermutation or MoEκi enhancer. 
     
     
         32 . The transgenic, non-human animal of  claim 18 , wherein the means for prevention of silencing are means for insertion into a locus in the genome of the host cell that is resistant to silencing. 
     
     
         33 . The transgenic, non-human animal of  claim 32 , wherein the means for insertion are means for homologous recombination into a site resistant to silencing. 
     
     
         34 . The transgenic, non-human animal of  claim 32 , wherein the locus is the rosa-locus. 
     
     
         35 . A transgenic, non-human animal that has been provided with an expression cassette comprising, in 5′-3′ direction, a vκ promoter, a human leader, a human V gene, optionally a MoEκi enhancer, a rat constant region (κ), and, optionally, a MoEκ3′ enhancer or a truncated MoEκ3′ enhancer. 
     
     
         36 . A method for producing a desired antibody, the method comprising:
 exposing the transgenic non-human mammal of  claim 1  to an antigen such that an antibody response is induced, and   isolating the antibodies specific for the antigen so as to produce the desired antibody.   
     
     
         37 . A method for producing a desired antibody, the method comprising:
 exposing the transgenic non-human mammal of  claim 1  to an antigen such that an antibody response is induced,   isolating cells producing such antibodies, and   culturing and, optionally, immortalizing the isolated cells and harvesting desired antibodies.   
     
     
         38 . A method for producing a desired antibody, the method comprising:
 exposing the transgenic non-human mammal of  claim 1  to an antigen such that an antibody response is induced,   isolating a nucleic acid encoding at least part of such an antibody,   inserting nucleic acid encoding at least part of such an antibody into an expression cassette, and   expressing the desired antibody in a host cell.   
     
     
         39 . A progeny of the transgenic, non-human mammal of  claim 1 , wherein the progeny comprises, at least in its B-cell lineage, a heavy- or light chain encoding sequence together with a means that renders the encoding sequence resistant to DNA rearrangements and/or somatic hypermutations. 
     
     
         40 . A progeny of the transgenic, non-human animal of  claim 18 , wherein the progeny comprises an expression cassette for expressing a desired proteinaceous molecule in cells during a certain stage of development in cells developing into mature B cells. 
     
     
         41 . A process for producing a cell, the process comprising:
 isolating a cell from the transgenic, non-human animal of  claim 1 ,   wherein the isolated cell comprises a heavy or light chain encoding sequence together with a means for rendering the heavy or light chain encoding sequence resistant to DNA rearrangements and/or somatic hypermutations.   
     
     
         42 . A process for producing a cell, the process comprising
 isolating a cell from the transgenic, non-human animal of  claim 18 ,   wherein the isolated cell comprises an expression cassette for expressing a desired proteinaceous molecule in cells during a certain stage of development in cells developing into mature B cells.   
     
     
         43 . A transgenic, non-human mammal comprising:
 a nucleic acid sequence encoding an immunoglobulin light chain, wherein the nucleic acid sequence comprises
 means for rendering the nucleic acid sequence resistant to DNA rearrangements and/or somatic hypermutations, and 
 a promoter selected from the group consisting of CD19, CD20, μHC, VpreB1, VpreB2, VpreB3, λ5, Igα, Igβ, κLC, λLC, and BSAP (Pax5) 
   wherein the nucleic acid sequence is integrated into the transgenic, non-human mammal's genome in the Rosa-locus thereof.   
     
     
         44 . The transgenic, non-human mammal of  claim 43 , wherein the immunoglobulin light chain encoding sequence is a human vκ sequence. 
     
     
         45 . The transgenic, non-human mammal of  claim 43 , wherein the immunoglobulin light chain encoding nucleic acid comprises in 5′ to 3′ direction:
 a vκ promoter,   a human leader,   a human V gene,   a MoEκi enhancer,   a rat constant region (κ), and   a MoEκ3′ enhancer or a truncated MoEκ3′ enhancer.