Transgenic animals for producing specific isotypes of human antibodies via non-cognate switch regions

Abstract

The present invention provides fully human antibodies in a transgenic animal of a desired isotype in response to immunization with any virtually any desired antigen. The human immunoglobulin heavy chain transgene in the foregoing animals comprises a human constant region gene segment comprising exons encoding the desired heavy chain isotype, operably linked to switch segments from a constant region of a different heavy chain isotype, i.e., a non-cognate switch region. Said additional constant region segment comprises a switch region and human constant region coding segment, wherein the constant region coding segment is operably linked to a switch region that it is not normally associated with, i.e., a non-cognate switch region. In the transgenes of the invention, the non-cognate switch region may be a switch region from a different species than the constant region coding segment. The switch region and membrane exons of the invention may comprise a human gamma-2 constant region and the secreted constant region exons are from a human gamma-1 or a human gamma-4 constant region.

We claim:  
     
         1 . A transgene comprising a DNA fragment comprising a DNA sequence identical to the DNA sequence of human chromosome 14 from the D segment genes of the human immunoglobulin heavy chain locus, continuing through the J segment genes and the constant region genes through Cμ of that locus, wherein said DNA fragment is operably linked to at least one human V segment gene, and wherein said DNA fragment further is operably linked to an additional constant region, said additional constant region comprising a mouse Cγ1 switch region and human constant region coding exons.  
     
     
         2 . The transgene according to  claim 1 , wherein said human constant region coding exons encode a human constant region selected from the group consisting of: a human gamma constant region, a human alpha constant region and a human epsilon constant region.  
     
     
         3 . The transgene according to  claim 2 , wherein said human constant region coding exons encode a human gamma constant region.  
     
     
         4 . The transgene according to  claim 3 , wherein said human gamma constant region is a gamma-1 constant region.  
     
     
         5 . The transgene according to  claim 4  which is the yH2Bm yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         6 . The transgene according to  claim 3 , wherein said human gamma constant region is a gamma-2 constant region.  
     
     
         7 . The transgene according to  claim 3 , wherein said human gamma constant region is a gamma-3 constant region.  
     
     
         8 . The transgene according to  claim 3 , wherein said human gamma constant region is a gamma-4 constant region.  
     
     
         9 . The transgene according to  claim 8  which is the yH2Cm yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         10 . The transgene according to  claim 2 , wherein said human constant region coding exons encode a human alpha constant region.  
     
     
         11 . The transgene according to  claim 10 , wherein said human alpha constant region is an alpha-1 constant region.  
     
     
         12 . The transgene according to  claim 10 , wherein said human alpha constant region is an alpha-2 constant region.  
     
     
         13 . The transgene according to  claim 2 , wherein said human constant region coding exons encode a human epsilon constant region.  
     
     
         14 . The transgene according to  claim 1 , wherein said DNA fragment is operably linked to a plurality of human VH genes.  
     
     
         15 . The transgene according to  claim 14 , wherein said DNA fragment is operably linked to at least 50% of the human germline VH genes.  
     
     
         16 . The transgene according to  claim 14 , wherein said DNA fragment is operably linked to at least 40 different human VH genes.  
     
     
         17 . The transgene according to  claim 14 , wherein said DNA fragment is operably linked to a sufficient number of different human VH genes so that the transgene is capable of encoding at least 1×10 5  different functional human immunoglobulin heavy chain sequence combinations, without taking into account junctional diversity or somatic mutation events.  
     
     
         18 . The transgene according to  claim 14 , wherein the number of human VH genes is sufficient to produce at least 50% of the B-cell population of a wild-type mouse in a transgenic mouse containing the transgene.  
     
     
         19 . A transgene comprising a DNA fragment comprising a DNA sequence identical to the DNA sequence of human chromosome 14 from the D segment genes of the human immunoglobulin heavy chain locus, continuing through the J segment genes and the constant region genes through Cμ of that locus, wherein said DNA fragment is operably linked to at least one human V segment gene, and wherein said DNA fragment further is operably linked to am additinal constant region, said additional constant region comprising a human switch region and human constant region coding exons, wherein said human switch region and said human constant region coding exons are from different isotypes.  
     
     
         20 . The transgene according to  claim 19 , wherein the human switch region is a human Cγ2 switch region.  
     
     
         21 . The transgene according to  claim 20 , wherein said human constant region coding exons encode a human constant region selected from the group consisting of: a human gamma-1 constant region, a human gamma-3 constant region, a human gamma-4 constant region, a human alpha-1 constant region, a human alpha-2 constant region and a human epsilon constant region.  
     
     
         22 . The transgene according to  claim 21 , wherein said human constanat region coding exons encode a human gamma-1 constant region.  
     
     
         23 . The transgene according to  claim 22  which is the yHG1 yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         24 . The transgene according to  claim 21 , wherein said human constanat region coding exons encode a human gamma-4 constant region.  
     
     
         25 . The transgene according to  claim 24  which is the yHG4 yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         26 . The transgene according to  claim 19 , wherein said DNA fragment is operably linked to a plurality of human VH genes.  
     
     
         27 . The transgene according to  claim 19 , wherein said DNA fragment is operably-linked to at least 50% of the human germline VH genes.  
     
     
         28 . The transgene according to  claim 19 , wherein said DNA fragment is operably linked to at least 40 different human VH genes.  
     
     
         29 . The transgene according to  claim 19 , wherein said DNA fragment is operably linked to a sufficient number of different human VH genes so that the transgene is capable of encoding at least 1×10 5  different functional human immunoglobulin heavy chain sequence combinations, without taking into account junctional diversity or somatic mutation events.  
     
     
         30 . The transgene according to  claim 19 , wherein the number of human VH genes is sufficient to produce at least 50% of the B-cell population of a wild-type mouse in a transgenic mouse containing the transgene.  
     
     
         31 . A transgene comprising a DNA fragment comprising a DNA sequence identical to the DNA sequence of human chromosome 14 from the D segment genes of the human immunoglobulin heavy chain locus, continuing through the J segment genes and the constant region genes through Cμ of that locus, wherein said DNA fragment is operably linked to at least one human V segment gene, and wherein said DNA fragment further is operably linked to an additional constant region, said additional constant region comprising a human switch region, human CH1, C hinge , CH2 and CH3 exons and human membrane exones, wherein said human switch region and said human membrane exons are from the same isotype and the human CH1, C hinge , CH2 and CH3 exons are from a different isotype than said human switch region and said human membrane exons.  
     
     
         32 . The transgene according to  claim 31 , wherein said human switch region and said human membrane exons are human gamma-2 sequences.  
     
     
         33 . The transgene according to  claim 32 , wherein said human CH1, C hinge , CH2 and CH3 exons encode a human constant region selected from the group consisting of: a human gamma-1 constant region, a human gamma-3 constant region, a human gamma-4 constant region, a human alpha-1 constant region, a human alpha-2 constant region and a human epsilon constant region.  
     
     
         34 . The transgene according to  claim 33 , wherein the human CH1, C hinge , CH2 and CH3 exons encode a human gamma-1 constant region.  
     
     
         35 . The transgene according to  claim 34  which is the yHG1/2 yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         36 . The transgene according to  claim 31 , wherein the human CH1, C hinge , CH2 and CH3 exons encode a human gamma-4 constant region.  
     
     
         37 . The transgene according to  claim 36  which is the yHG4/2 yeast artificial chromosome (YAC) having ATCC accession number ______.  
     
     
         38 . The transgene according to  claim 31 , wherein said DNA fragment is operably linked to a plurality of human VH genes.  
     
     
         39 . The transgene according to  claim 31 , wherein said DNA fragment is operably linked to at least 50% of the human germline VH genes.  
     
     
         40 . The transgene according to  claim 31 , wherein said DNA fragment is operably linked to at least 40 different human VH genes.  
     
     
         41 . The transgene according to  claim 31 , wherein said DNA fragment is operably linked to a sufficient number of different human VH genes so that the transgene is capable of encoding at least 1×10 5  different functional human immunoglobulin heavy chain sequence combinations, without taking into account junctional diversity or somatic mutation events.  
     
     
         42 . The transgene according to  claim 31 , wherein the number of human VH genes is sufficient to produce at least 50% of the B-cell population of a wild-type mouse in a transgenic mouse containing the transgene.  
     
     
         43 . The transgene according to any one of claims  1 ,  19  or  31 , further comprising a mouse 3′ enhancer.  
     
     
         44 . The transgene according to  claim 43 , wherein said mouse 3′ enhancer is an approximately 0.9 kb core fragment of a mouse germline 3′ enhancer.  
     
     
         45 . The transgene according to  claim 43 , wherein said mouse 3′ enhancer is an approximately 4 kb fragment of of a mouse germline 3′ enhancer.  
     
     
         46 . The transgene according to  claim 43 , wherein said mouse 3′ enhancer is a locus control region.  
     
     
         47 . An embryonic stem (ES) cell comprising a transgene according to any one of claims  1 - 46 .  
     
     
         48 . The embryonic stem (ES) cell according to  claim 47 , which is a mouse ES cell.  
     
     
         49 . A transgenic non-human animal and its progeny, wherein the somatic and germ cells comprise a transgene according to any one of claims  1 - 46 .  
     
     
         50 . The transgenic non-human animal and progeny according to  claim 49 , further comprising a human immunoglobulin light chain transgene.  
     
     
         51 . The transgenic non-human animal and progeny according to  claim 49  or  50 , wherein the animal is a mouse.  
     
     
         52 . The transgenic non-human animal and progeny according to  claim 49  or  50 , further comprising inactivated endogenous immunoglobulin heavy and light chain loci.  
     
     
         53 . The transgenic non-human animal according to  claim 52 , wherein the animal is a mouse.  
     
     
         54 . A method for producing a transgenic non-human animal and its progeny whose somatic and germ cells comprise a transgene according to any one of claims  1 - 46  and that produces high affinity, fully human antibodies of a desired isotype specific for an-antigen of interest following immunization with the antigen of interest, comprising: 
 (a) introducing said transgene into an embryonic stem cell;  
 (b) producing from the embryonic stem cell a transgenic non-human animal whose somatic and germ cells contain said transgene; and  
 (c) breeding said transgenic non-human animal as needed to produce a transgenic non-human animal and its progeny that produces high affinity, fully human antibodies of a desired isotype specific for an antigen of interest following immunization with the antigen of interest.  
 
     
     
         55 . The method according to  claim 54 , wherein the transgenic non-human animal is a mouse.  
     
     
         56 . A method for producing a high-affinity, fully human antibody of a desired isotype, wherein said antibody is specific for an antigen of interest, comprising contacting a transgenic, non-human animal according to any one of claims  49 - 53  with the antigen of interest to induce antibody production in the B-cells of said animal and collecting said antibody.  
     
     
         57 . An antibody producing B-cell harvested from a transgenic non-human animal according to any one of claims  49 - 53  immunized with an antigen of interest.  
     
     
         58 . The B-cell according to  claim 57  which has been immortalized.  
     
     
         59 . The method according to  claim 56 , wherein the antibody is collected from the blood stream of said transgenic non-human animal.  
     
     
         60 . The method according to  claim 56 , wherein the antibody is collected from an immortalized B-cell according to  claim 58 .  
     
     
         61 . The method according to  claim 56 , wherein said antibody is collected from a host cell transfected with DNA isolated from a B-cell according to  claim 57.