Suppression of b-cell apoptosis in transgenic animals expressing humanized immunoglobulin

Abstract

The invention provides a novel approach to increase immunoglobulin expression in non-human transgenic animals. For instance, the invention provides a method to increase humanized immunoglobulin production in animals genetically engineered to express one or several human or humanized immunoglobulin transloci. This can be done by overexpressing the apoptosis inhibitor, i.e. a rabbit bcl-2, whose expression is driven by a B-cell specific promoter specifically in the B-cell of the animal, thereby enhancing the survival of B-cells. This invention further relates to a method for selectively enhancing the survival of exogenous B-cells, that is B-cells expressing any immunoglobulin transgene locus, over the survival of endogenous B-cells that do not express the transgene locus. Selectivity is achieved by expressing the apoptosis-inhibitor only within exogenous B-cells, that is, by coupling exogenous immunoglobulin expression with apoptosis inhibitor expression. This latter method allows for increased expression and production of the transgene encoded product(s) over the endogenously produced immunoglobulin of the transgenic animal. The invention also provides a novel apoptosis-inhibitor, rabbit bcl-2.

1 - 25 . (canceled) 
     
     
         26 . A transgenic expression construct comprising a transgene encoding a fusion-protein comprising polypeptide sequences in the following order:
 a) an immunoglobulin or immunoglobulin chain;   b) a self-cleaving peptide;   c) an apoptosis inhibitor; and optionally,   d) a protease cleavage site between a) and b).   
     
     
         27 . The transgenic expression construct of  claim 26  wherein said protease cleavage site is selected from the group consisting of sites of aspartic proteases, cysteine proteases, metalloproteases, serine proteases and threonine proteases. 
     
     
         28 . The transgenic expression construct of  claim 26  wherein said protease cleavage site is the furin cleavage site. 
     
     
         29 . The transgenic expression construct of  claim 26  wherein said immunoglobulin fragment is a fragment of a human(ized) immunoglobulin heavy and/or light chain. 
     
     
         30 . The transgenic expression construct of  claim 26  wherein said self-cleaving peptide is derived from viral 2A/2B or 2A-like/2B peptides. 
     
     
         31 . The transgenic expression construct of  claim 30  wherein said virus is selected from the group consisting of the picornaviridae virus family, the equine rhinitis A (ERAV) virus family, the picornavirus-like insect virus family and from the type C rotavirus family. 
     
     
         32 . The transgenic expression construct of  claim 31  wherein said virus is selected from the group consisting of the foot and mouth disease virus (FMDV), the equine rhinitis A (ERAV) virus, and the Thosea asigna virus (TaV). 
     
     
         33 . The transgenic expression construct of  claim 26  wherein said apoptosis inhibitor is selected from the group consisting of bcl-2, caspase-9-DN mutants, baculovirus p35, caspase-9S, crmA, z-VAD-fink, z-DEVD-fmk, B-D-fmk, z-YVAD-fmk, Bcl-xL, Mc1-1, XIAP, TIAP, IUAP, NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1, HIAP2, MIHA, MalB, MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon, BRUCE, MLIAP, SODD and FLIP and variants thereof. 
     
     
         34 . The transgenic expression construct of  claim 26  wherein said apoptosis inhibitor is mammalian bcl-2. 
     
     
         35 . The transgenic expression construct of  claim 34  wherein said mammalian bcl-2 is selected from the group consisting of human bcl-2, mouse bcl-2 and rabbit bcl-2. 
     
     
         36 . An isolated host cell transformed with the transgenic expression construct of  claim 26 . 
     
     
         37 - 47 . (canceled)