Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

Abstract

The present invention describes transgenic animals with human(ized) immunoglobulin loci and transgenes encoding human(ized) Igα and/or Igβ sequences. Of particular interest are animals with transgenic heavy and light chain immunoglobulin loci capable of producing a diversified human(ized) antibody repertoire that have their endogenous production of Ig and/or endogenous Igα and/or Igβ sequences suppressed. Simultaneous expression of human(ized) immunoglobulin and human(ized) Igα and/or Igβ results in normal B-cell development, affinity maturation and efficient expression of human(ized) antibodies.

1 . A transgene construct encoding a chimeric Igα subunit of the BCR, said chimeric Igα subunit comprising:
 (a) an intracellular domain sequence and a transmembrane domain sequence of a non-human, Igα polypeptide sequence; and,   (b) a polypeptide having at least 85% sequence identity to the extracellular domain of human Igα of SEQ ID NO.: 1.   
     
     
         2 . A transgene construct encoding a chimeric Igβ subunit of the BCR, said chimeric Igβ subunit comprising:
 (a) an intracellular domain sequence and a transmembrane domain sequence of a non-human, Igβ subunit; and,   (b) a polypeptide having at least 85% sequence identity to the extracellular domain of the human Igβ of SEQ ID NO.:7.   
     
     
         3 . The transgene constructs of  claim 1 , wherein said non-human Igα polypeptide sequence includes bovine (SEQ ID NO: 2); murine (SEQ ID NO: 3); canine (SEQ ID NO: 4); primate ((SEQ ID NO: 5); rabbit (SEQ ID NO: 6) or other non-human sequences. 
     
     
         4 . The transgene constructs of  claim 2 , wherein said non-human Igβ polypeptide sequence includes canine (SEQ ID NO: 8); rat (SEQ ID NO: 9); bovine (SEQ ID NO: 10); murine (SEQ ID NO: 11); chicken (SEQ ID NO: 12); or other non-human sequences. 
     
     
         5 . A non-human transgenic animal comprising:
 (a) a transgene construct encoding either a full-length, human Igα subunit of SEQ ID NO.: 1 or the chimeric Igα subunit of  claim 1 ; and/or,   (b) a transgene construct encoding either a full-length, human Igβ subunit of SEQ ID NO.: 7 or the chimeric Igβ subunit of  claim 2 ; and,   (c) a transgene construct encoding a human(ized) immunoglobulin locus; wherein the resultant transgene products combine to form a human(ized) B-cell receptor complex.   
     
     
         6 . The non-human transgenic animal of  claim 5  wherein the expression of any endogenous Ig production and/or Igα and/or Igβ subunits of the non-human transgenic animal has been substantially reduced. 
     
     
         7 . The non-human transgenic animal of  claim 5  wherein said animal is selected from a group consisting of rabbit, mouse, rat, pig, sheep, goat, bird, horse, donkey and cow. 
     
     
         8 . The non-human transgenic animal of  claim 5  which is a rabbit. 
     
     
         9 . An isolated human(ized) immunoglobulin from the non-human transgenic animal of  claim 5 , wherein said human(ized) immunoglobulin is either an antibody or an antibody fragment. 
     
     
         10 . The isolated human(ized) immunoglobulin of  claim 9 , wherein said antibody is a polyclonal or a monoclonal antibody. 
     
     
         11 . The isolated human(ized) immunoglobulin of  claim 9 , wherein said antibody fragment is a fragment of a polyclonal or a monoclonal antibody. 
     
     
         12 . The isolated human(ized) immunoglobulin of  claim 9 , wherein said antibody or antibody fragment is labeled. 
     
     
         13 . The isolated human(ized) immunoglobulin of  claim 9 , wherein said antibody or antibody fragment is fused to a toxin to form an immunotoxin or is coupled to a therapeutic agent. 
     
     
         14 . The isolated human(ized) immunoglobulin of  claim 9 , wherein said antibody or antibody fragment is fused to a heterologous amino acid sequence. 
     
     
         15 . The isolated human(ized) immunoglobulin of  claim 11 , wherein said antibody fragment is an Fc, Fv, Fab, Fab′ or F(ab′) 2  fragment. 
     
     
         16 . An isolated B-cell from said non-human transgenic animal of  claim 5 , wherein said B-cell expresses either the native human Igα subunit or a chimeric Igα subunit and/or either the native human Igβ subunit or the chimeric Igβ subunit, and the human(ized) immunoglobulin locus. 
     
     
         17 . The isolated B-cell of  claim 16  wherein said B-cell is immortalized. 
     
     
         18 . The isolated B-cell of  claim 16  or  17  wherein said B-cell is derived from a rabbit. 
     
     
         19 . An antibody preparation comprising the antibody or the antibody fragment of  claim 9 . 
     
     
         20 . A pharmaceutical composition comprising the antibody preparation of  claim 19  in a mixture with a pharmaceutically acceptable ingredient. 
     
     
         21 . The pharmaceutical composition of  claim 20  comprising a monoclonal antibody or a fragment thereof. 
     
     
         22 . The pharmaceutical composition of  claim 20  comprising one or a plurality of polyclonal antibodies or fragments thereof. 
     
     
         23 . A method for producing human(ized) antibodies in a non-human animal comprising:
 (a) introducing and expressing a transgene construct encoding either a native human Igα subunit or a chimeric Igα subunit, and/or a transgene construct encoding either a native human Igβ subunit or a chimeric Igβ subunit into said non-human animal; and,   (b) introducing and expressing a transgene construct encoding a human(ized) immunoglobulin locus into said non-human animal;   (c) subjecting said animal to an antigenic stimulus; and,   (d) isolating human(ized) antibodies from said animal.   
     
     
         24 . The method for producing human(ized) antibodies of  claim 23 , wherein said antibody is a polyclonal or a monoclonal antibody. 
     
     
         125 . The method for producing human(ized) antibodies of  claim 23 , wherein said antibody fragment is a fragment of a polyclonal or a monoclonal antibody. 
     
     
         26 . The method for producing human(ized) antibodies of  claim 23 , wherein said antibody or antibody fragment is labeled. 
     
     
         27 . The method for producing human(ized) antibodies of  claim 23 , wherein said antibody or antibody fragment is fused to a toxin to form an immunotoxin or is coupled to a therapeutic agent. 
     
     
         28 . The method for producing human(ized) antibodies of  claim 23 , wherein said antibody or antibody fragment is fused to a heterologous amino acid sequence. 
     
     
         29 . A method for producing a non-human animal expressing human(ized) antibodies comprising:
 (a) introducing and expressing a transgene construct encoding either a native human Igα subunit or a chimeric Igα subunit and/or a transgene construct encoding either a native human Igβ subunit or a chimeric Igβ subunit into the B-cell of said non-human animal; and,   (b) introducing and expressing a transgene construct encoding a human(ized) immunoglobulin locus into said non-human animal;
 wherein the resultant transgene products combine to form a human(ized) B-cell receptor complex. 
   
     
     
         30 . The method for producing a non-human animal expressing human(ized) antibodies of  claim 27  wherein, said animal creates antibody diversity by gene conversion and/or somatic hypermutation. 
     
     
         31 . The method for producing a non-human animal expressing human(ized) antibodies of  claim 27  wherein, said animal is a rabbit.